NOVEL ORGANIC NlTRATES AS POSSIBLE NEUROPROTECTANTS IN AN hV VITRO MODEL OF S'I'ROKE IN THE RAT HPPOCAMPUS

Allison Elizabeth Clarke

A thesis submitted to the Department of Pharmacology and Toxicology in conformity with the requirements for the degree of Master of Science

Queen's University Kingston, Ontario, Canada

May, 2001

Copyright O Allison Elizabeth Clarke, 2001

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Allison Elizabeth Clarke: Novel Organic Nitrates as Possible Neuroprotectants in an In Vitro Mode1 of Stroke in the Rat Hippocampus. M.Sc. Thesis, Queen's University, Kingston, Ontario, Canada, May, 2001.

Novel organic nitrates are a group of established nitric oxide donors based on the chemical structure of glyceryl trinitrite (GTN). It has been previously suggested that nitnc oxide can potentially play a neuroprotective role in ischemia due to its ability to: inhibit ~ a ' + influx through the N-methyl-D-aspartate (NMDA) receptor, act as an antioxidant and increase cGMP levels in the neuron. A group of investigators examining the neuroprotective properties of the secreted f o m of amyloid precursor protein (sAPPu) have discovered a protein kinase G (PKG) dependent. cGMP mediated mechanism. They have postulated that this neuroprotection is due to: activation of K' channels, inhibition of the NMDA receptor and enhancement of glucose and glutamate uptake into synaptic compartrnents. This thesis tested the hypothesis that novel organic nitrates are neuroprotective in an in vitro model of stroke possibly due to a cGMP mediated mechanism. The first objective was to establish the in vitro model of stroke with respect to testing of known neuroprotectants such as hypothermia and determining an appropriate length of insult. A half hour insult time was chosen because it caused a subrnavimal increase in lactate dehydrogenase (LDH) release. LDH release was used as a marker of ce11 viability. The induction of hypothermia during the ischemic insult completely protected the hippocarnpal slices from the in vitro iscliemic insult. The in vitro ischemic insult involved low[Oz] and low[glucose] in the incubation buffer. The second objective was to determine if the novel organic nitrates had any neuroprotective properties and if any observed neuroprotection was dependent upon cGMP generation. Three novel organic nitrates, GT-091, GT-094 and GT-310 were significantly protective against low[02] and low[glucose]. Similarly, the cGMP analogue, dibutyql cGMP was also neuroprotective in the same model suggesting that the neuroprotection observed with the novel organic nitrates may be due to a cGMP mediated mechanism. Unexpectantly, the neuroprotection provided by GT-094 could not be attenuated by CO-application of 1H- [ 1,2,4]oxadiazolo[4,3-a]quinoxalin- 1 -one (ODQ), a guanylyl cyclase inhibitor. Additionally, GT-094 was unable to increase cGMP levels in hippocampal slices d e r hypoxialhypoglycemia as assessed by a cGMF radioirnrnunoassay. These results indicate that the neuroprotective mechanism of GT-094 does not involve cGMP generation. Interestingly, CAMP could mimic the neuroprotection observed with cGMP. A protein kinase A ( P U ) or PKG inhibitor could not attenuate the neuroprotective effects of CAMP and cGMP, respectively. In summary, these findings suggest GT-094 and the cyclic nucleotides are exerting neuroprotection by two separate and independent mechanisms. Furthemore, these results indicate that the cyclic nucleotides are acting by a pathway that does not involve PKA or PKG activation.

1 would like to take this opportunity to thank my supervisor Dr. Roland J. Boegman for al1 his guidance and insight in the completion of this thesis project. 1 would also like to thank Dr. James N. Reynolds for al1 his invaluable help. 1 would like to acknowledge and thank Lihua Xue and Diane Andenon for generously providing me with some of the data contained in this thesis. Their help is greatly appreciated. Lihua Xue completed the LDH study on Sin4 chloride CO-administered with ODQ as well as the cresyl violet staining with the hippocampal slices treated with Sin-1 chlonde and GT-094. Diane Anderson provided me with the RIA data on GT-094 and ODQ. 1 would also like to thank the other members of the GoBang team: Dr. Jhamandas. Dr. Bennett, Dr. Thatcher, Margo Poklewska-Koziell and Adrian Nicolescu.

GoBang Therapeutics and Queen's Medical Discoveries primarily financed this research. Queen's School of Graduate Studies provided personal funding.

This thesis is dedicated to my farnily.

TABLE OF CONTENTS

Abstrac t

Acknowledgrnents

Table of Contents

List of Figures

List of Abbreviations and Symbols

1 INTRODUCTION

1.1 S tatement of Research Problem 1.2 Ischemic Ce11 Damage

1.2.1 NMDA Receptor Antagonists 1.2.2 Metabotropic Glutamate Receptors

1.3 Nitric Oxide

1.3.1 Nitric Oxide Synthase Antagonists 1.3.2 Nitric Oxide and Neurotoxicity 1 -3.3 Peroxynitrite 1.3.4 Nitrk Oxide Production and Neuronal Outcome 1.3.5 Nitric Oxide and Neuroprotection 1.3.6 Niûic Oxide and NMDA Receptor Inhibition

Antioxidant Properties of Nitric Oxide

cGMP and Neurotoxicity cGMP and Neuroprotection cGMP and P-Arnyloid Precursor Protein Glutamate and Glucose Uptake Inhibition of the NMDA Receptor Potassium Channels

5 Cyclic P {ucleotide Gated Ion ChanneIs 1.6 Phosphodiesterases and Ischemia 1.7 CAMP 1.8 Guanine Nucleotide Exchange Factors 1.9 Novel Organic Nitrates 1.10 In Vitro Mode1 of kchernia

Page . . 11

S . .

111

v

vii

ix

1

1 3

5 7

8

10 11 12 12 12 14 15

18

18 19 20 21 2 1 22

23 23 25 26 27 29

1.1 1 Research Rationale, Hypothesis and Objectives

2 METHODS AND MATERIALS

2.1 Chemical Solutions 2.2 Experimental Animals 2.3 Tissue Isolation 2.4 LDH Assay 2.5 Protein Determination 2.6 cGMP Radioimmunoassay 2.7 Cresyl Violet Staining 2.8 Data Analysis

3 RESULTS

3.1 Time Course of LDH Release 3.2 Temperature and LDH Release 3.3 Conventional NO Donors 3.4 Novel Organic Nitrates 3.5 Synthetic cGMP Analogues

3 S. 1 Dibutyryl cGMP and Rp-8-pCPT-cGMP

3.6 Synthetic CAMP Analogues

3.6.1 Dibutyryl CAMP and H-89

3.7 Dibutyiyl CAMP and Forskolin 3.8 ODQ 3.9 cGMP Radioirnmunoassay 3.10 Cresyl Violet Staining

4 DISCUSSION

4.1 Future Research Directions

Re ferences

Vita

LIST OF FIGURES

1.1 Sumrnary of hypoxic/hypoglycemic injury

1.2 A schematic depicting NO production by NOS

1.3 Summary of the neurotoxic properties of NO

1.4 Sumrnary of the neuroprotective properties of NO

1.5 Chemical structure and proposed biotransformation of the novel organic nitrates

3.1 Rat hippocarnpal slices exposed to differing lengths of hypoxiahypoglycemia

3.2 Rat hippocampal slices exposed to hypothermie conditions

3.3 Rat hippocampal slices treated with Sin-1 chlonde

3.4 Rat hippocarnpal slices treated with NO-exhausted Sin-1 chloride

3.5 Rat hippocarnpal slices treated with GSNO

3.6 Rat hippocampal slices treated with GT-09 1

3.7 Rat hippocampal slices treated with GT-094

3.8 Rat hippocampal slices treated with GT-3 10

3.9 Hippocarnpal slices treated with 1 mM 8-bromo-cGMP

3.10 Rat hippocarnpal slices treated with dibutyryl cGMP

3.1 1 Rat hippocampal slices treated with cGMP

3.12 Rat hippocarnpal slices treated with 8-pCPT-cGMP

3.13 Rat hippocampal slices treated with dibutyryl cGMP and Rp-8-pCPT-cGMP

3.14 Rat hippocarnpal slices treated with dibutyryl CAMP

Page 6

3.15 Rat hippocampal slices treated with I m M 8-bromo-c AMP

3.16 Rat hippocampal slices treated with dibutyryI CAMP and H-89

3.1 7 Rat hippocampal slices treated with 50pM fonkolin and 100pM dibutyryl cGMP

3.1 8 Rat hippocampal slices treated with 50pM GT-094 and 0.5pM ODQ

3.19 Rat hippocampal slices treated with Sin- 1 chloride and ODQ

3.20 Rat hippocarnpal slices treated with ODQ

3.2 1 Concentration of cGMP in rat hippocampal slices

3.22 Rat hippocampal slices stained with cresyl violet

4.1 Proposed mechanism of action of the novel organic nitrates

4.2 Proposed mechanism of neuroprotection mediated by cGMP

LIST OF ABBREVIATIONS AND SYMBOLS

a

AP AMPA

ANOVA P BSA ca2+ CaM CaMK II CAMP CBF cGMP CNS CPT CAMP

CREB DHPG DMSO eNOS Epac FAD ~ e ' * FMN g GEF GT GTN GTP GSNO H-89

HNE Hz02 ICP iNOS in vitro in vivo

alpha arnyloid beta peptide a-arnino-3-hydroxy-5-methyl-4-isoxazole propionate analysis of variance beta bovine s e m albumin calcium ion calmodulin calcium-calmodulin-dependent protein kinase II cyclic adenosine 3'5 ' -monophosphate cerebral blood flow cyclic guanosine 3 ' 5 '-monophosphate centnl nervous system 8-(4-c hlorop heny1thio)-adenosine 3 ' : 5 ' -cyclic- monophosphate cyclic AMP-responsive element binding protein 3'5-dihydroxyphenylglycine dimethyl sulfoxide endothelial nitric oxide exchange protein directly activated by cyclic AMP flavin adenine dinucleotide ferrous iron flavin mononucleo tide

gram guanine nucleo tide exchange factor(s) GoBang Therapeutics glyceryl trinitrite guanosine triphosphate S-nitrosogIutathione N-[2-(p-bromocinnarnylamino)ethyl] -5- isoquinolinesul fonamide 4-hydroxynonenal hydrogen peroxide intracranial pressure inducible nitric oxide in glass in the living body potassium ion activation constant; concentration required for half- maximal activation potassium chloride potassium phosphate, monobasic

KREB

L LDH L-NAME M

min MK-80 I

rnmo 1 n N? Na' NaCl NADH NADPH Na.iiC03 NaOH NMDA NMDA-R NNA nNOS NO NOS 02 of- ODQ ONOO' PARS PBS PDE

inhibition constant; concentration required for half- maximal inhbtion modified Krebs-Henseleit bicarbonate solution kilogram(s) lipophilicity defined as the extrapolated capacity factor for 100% water in isocratic reversed-phase HPLC ii tre(s) lactate dehydrogenase bf-nitro-L-arginine methyl ester molar milligram(s) magnesium ion metabotropic glutamate receptor magnesium sulfate 8-para-chlorop henylthio-cGMP microgram(s) micrometre(s) minute (+)-MK-80 1 maieate rnillilitre(s) rnillimolar. millimole(s) number of determinations molecular nitrogen sodium ion sodium chloride nicotinamide adenine dinucleotide reduced form nicotinamide adenine dinucleotide phosphate sodium bicarbonate sodium hydroxide N-methyl-D-aspartate N-methyl-D-aspartate receptor N-a-ni tro-L-arginine neuronal niûic oxide nitnc oxide nitric oxide synthase molecular oxygen superoxide anion 1 H-[1,2,4]oxadiazolo[4,3-alquinoxalin- 1 -one peroxynitrite poly(ADP-ribose) synthase phosphate buffered saline phosphodiesterase(s)

PKA PKC PKG PLAz pmol PSD-95 RIA ROS Rp-8-pCPT-cGMP

sAPPa 4C3HPG SEM sGC Sin- 1 SOD T m VSCC XO O C

Y0

+ a3

negative base 10 logar-ithrn of hydrogen ion concentration protein kinase A protein kinase C protein kinase G phospholipase Ar picornole(s) postsynaptic density protein-95 radioimrnunoassay reactive oxygen species (Rp)-8-@ara-chlorophenyIthio)guanosine-3 ' ,Y- cyclic rnonophosphorothioate secreted form of amyloid precursor protein s-4-carbox y-3 -hydroxy-phenylgl ycine standard enor of the mean soluble guanylyl cyclase 3-morpholino-sydnonimine superoxide dismutase tetrahydrobiopterin voltage-dependent calcium channels xanthine oxidase degrees centigrade (Celsius) percent plus or minus registered trademark

1. rnTRODUCTION

1.1 Statement of the Research Problem

Stroke is an inhibition of blood flow to the brain usually occuring as a result of

a blood clot. Each year, stroke claims the lives of 15,000 people and severely

debilitates another 350,000 individuals (Dr. Tony Hakimj , Neuroscience Stroke

institute, Neuroscience Seminar). During stroke glucose and oxygen are unable to

gain access to the brain, which sets the stage for cellular energy store depletion and

ce11 death. The consequences of stroke are magnified because many individuals

misinterpret the symptoms of stroke, which include headache, nausea, diuiness,

blurred vision and muscle weakness, ofien causing them to wait hours before they

seek medical attention. Treatrnents have focused on removing the blood clot, either

surgically or with dnigs such as tissue plasminogen activator. Although this does

improve outcorne, these treatments are only effective when administered within two

houn after the Srst signs of stroke. At present there are no clinically available

treatments to inhibit neuronal ce11 loss after the stroke. The problem will become

more acute as the baby boomers approach middle age. Novel treaments need to be

explored in order to arrest this growing medical problem.

Excessive release of glutamate and overactivation of ionotropic glutamate

receptos such as the N-rnethyl-D-aspartate (NMDA) receptor have been postulated to

be the underlying mechanism of neuronal ce11 death due to ischemia. Calcium influx

through the NMDA receptor activates nitric oxide synthase (NOS), which produces

nitric oxide frorn the conversion of L-arginine to L-cimilline (see review: Yun H. et

al., 1996). Several physiological processes such as vascular smooth muscle relaxation

(Huang et al., 1995), long term potentiation (Wu et al., 1997, see review: Hawkins et

al., 1998), and even neurotoxicity (Almeida et al., 1998, Panahian et ai., 1996) have

been associated with the production of nitric oxide. The role of nittic oxide in

ischemic ce11 damage is controversial. It has been hypothesized that niûic oxide rnay

act as an neurotoxin due io its ability to inhibit mitochondrial enzymes (Cassina et al.,

1996, Stadler et al., 199 1, Lizasoain et al., 1996), to cause DNA damage (2hang et al.,

1994) and to react with superoxide anion to form peroxynitrite (Endres et al., 1998).

Al1 of these events would increase cellular damage due to ischemia.

Convenely, nitric oxide has also been s h o w to inhibit the NMDA receptor

(Manzoni et al., 1992), react with lipid radicals to form stable nitroso-compounds

(Rubbo et al., 1994) and increase the production of cGMP. These functions of nitric

oxide al1 have the potential to be neuroprotective. The NMDA receptor is

continuously activated in ischemic conditions due to the accumulation of glutamate in

the synaptic cleR. Thus, inhibition of the NMDA receptor is expected to improve

neuronal outcome following an ischemic insult. Additionally, in the reperfusion

penod following ischemia, there is increased production of oxygen radicals which

leads to lipid peroxidation. Nitnc oxide's ability to react with lipid radicals to fom

stable compounds may inhibit lipid peroxidation and be beneficial in the treatment of

isc hemia.

Studies examining the role of cGMP in neuronal cell death have found

conflicting results. Some investigators have ascertained that treatment with cGMP

potentiates cell injury (Montoliu et al., 1 999, Yonghong et al., 1997). Alternatively,

cGMP has also demonstrated neuroprotective properties (Moro et al.. 1998,

Garthwaite et al., 1988, Yoshioka et al., 2000, Mattson et al., 1999, Furukawa et al.,

1998, Barger et al., 1995, Furukawa et al., 1996). A group of researchers studying the

neuroprotective properties of the secreted fom of amyloid precursor protein observed

that the neuroprotection provided with the secreted form of amyloid precursor protein

could be rnimicked by synthetic cGMP analogues and be attenuated by a PKG

inhibitor (Mattson et al., 1999, Furukawa et al., 1998, Barger et al., 1995, Furukawa et

al., 1996). They suggested that this neuroprotection is due to activation of K'

channels (Fumkawa et al., 1996), inhibition of the NMDA receptor (Furukawa et al.,

1998) or increased uptake of glutamate and glucose into synaptic compartments

(Mattson et al., 1999).

GT-015 is part of a group of novel organic nitrates, which are synthetic nitric

oxide donors, based on the chernical smicture of glyceryl trinitrate (GTN). GT-O15

was neuroprotective in an in vitro model of ischemia using hippocampal brain slices

and in an in vivo middle cerebral artery occlusion model. in the itz vitro model, it was

found that inhibition of guanylyl cyclase attenuated the neuroprotection of GT-0 1 5

indicating that the neuroprotection observed with GT-015 was due to generation of

cGMP (Clarke et al., 2000). Therefore it was proposed that the effects of GT-015 are

due to production of cGMP. possibly by one of the aforementioned mechanisms.

The research hypothesis explored in this thesis postulates that novel organic

nitrates, which are established nitric oxide donors, are neuroprotective in an in vitro

model of stroke due to a cGMP-PKG mediated mechanism.

1.2 Ischemic Ceii Damage

In the initial stages of ischemic ce11 loss, there is a massive decrease in

energy stores. This occurs within 1 to 2 minutes of the cessation of blood flow

(Martin et al. 1994). This enormous decrease in energy reserves leads to neuronal

damage. Low levels of ATP contribute to alterations in ion homeostasis by inhibiting

the N~'/K' ATPase and activating the K' ATPase present on the neuronal membrane.

This results in an early rise in extracellular levels of K', which in tum causes

membrane depolarization. Using intracellular recordings in striatal q iny neurons

exposed to glucose and oxygen deprivation, Calabresi and colleagues (1999), found

that increases in ~ a ' and cal' levels mirrored membrane depolarization. This

produces a dramatic rise in intracellular ~ a ' and cal' levels which further disnipts ion

homeostasis.

Glutamate, released by reversal of Na'/ glutamate transporter and ~ a "

dependent exocytosis, has been postulated to be the major player in ischemic ce11

death (Longuemare et al., 1 995, Kimura et al., 1998). This process has been termed

glutamate excitotoxicity. Excessive release of glutamate overactivates several

glutamatergic receptors and in particular, the ionotropic NMDA receptor, which has

been associated with neurotoxicity. Calcium influx through the NMDA receptor leads

to a toxic accumulation of ca2' in the neuron. Ellrén and colleagues (1989)

discovered that NMDA toxicity in pyramidal cells of the immature hippocampus was

~ a " dependent as accessed by rnorpholo@cal analysis and LDH release.

Additionally, this group found that NMDA toxicity was only partially ~ a " dependent

in granule cells, suggesting that the requirement for ca2' in NMDA neurotoxicity

diffee among ce11 types. Another scientific group examining cytosolic ~ a " levels as

determined by confocal fluorescent microscopy, has suggested that the early nse in

~ a ' + levels are due to NMDA activation and that the sustained levels of cal' are due

to reversa1 of the mitochondrial2~a~-~a" exchanger (Zhang et al., 1999). Regardless

of its source, toxic levels of cal' can cause a number of destmctive events.

The activation and upregulation of potentially neurotoxic enzymes such as

calpain, proteases, and nNOS by ca2' can lead to neuronal ce11 death. Calpain and

proteases aid in the degradation of intracellular proteins. The enzyme nNOS produces

nitric oxide, which can be toxic under certain circumstances. Mitochondrial injury

occun dunng ischemia as a result of lactic acidosis, increased production of reactive

oxygen species and activation of ~ a " regulated proteolytic enzymes such as calpain

(see review: Fiskum et al., 1997). The mitochondria normaily buffer increased ca2'

levels by energy dependent sequestration. However, damage to the mitochondria

caused by ischemia impairs this process making neurons more susceptible to ~ a "

mediated injury (Sciamanna et al., 1 992). in addition, mitochondrial respiration

becomes more sensitive to Ca'' inhibition. This contributes to further energy

depletion of the neuron. It has been suggested that Ca" influx can lead to either

apoptosis or necrosis, depending on the energy charge of that cell. Further, Tenneti

and colleagues (1998) found that caspase inhibitors were unable to arrest the

disturbance in mitochondnal membrane potential that takes place after exposure to

NMDA receptor activation. This indicates that mitochondrial membrane potential

detenoration may be an early event in both necrosis and apoptosis.

The cascade of events that take place in ischemia can result in apoptosis or

necrosis depending on the energy charge of the cell. Necrosis, which is charactenzed

by neuronal swelling and membrane breakdown, occurs when energ stores are

compietely depleted. However, if cellular energy levels are adequate, apoptosis rnay

occur. Membrane potential disruption of the mitochondria causes the opening of the

membrane transition pore and reiease of apoptotic initiators such as cytochrome C.

Apoptosis leads to the shrinking of the cytoplasm and formation of apoptotic bodies.

Figure 1.1 depicts the events that take place in ischemia. The hypothesis that

glutamate excitotoxicity is one of the key incidents in ischemia is supported by the

following experiment.

1.2.1 NMDA Receptor Antagonists

An in vitro study, exarnining the neuropmtective effects of NMDA receptor

antagonists, found that a combination of NMDA and AMPA receptor antagonists

Neuronal Ce11 Damage as a Result of Ischemia

1 L~issue ATP Therobic Glycolysil & w

) t ~ i s s u e Lactate d~ i s sue pH T c e T N ~ ' ~ Tc1-i 1 1

1 Neuronal Depolarkation 1

d r i vaiionctivation of non-NMDA-~

activation LROS Formation Proteolysis

I DNA DamagelFragmentatior . PARP Activation

L

w

Energy Depletion Lipid Peroxidation L

Ce11 Death

Activation of Neuroimrnune Response

Activation of: PLA2 Xanthine Oxidase PKC CaMK II Calcineunn A Calpain I&ii Endonucleases

Figure 1 -1 : Summary of hypoxic/hypoglycemic injury. Adapted from Samdani et al., 1997. CBF indicates cerebral blood flow; VSCC, voltage-dependent calcium channels; NMDA-R, N-methyl-D-aspartate receptor, PLA2, phospholipase Az; PKC, protein kinase C; CaMK II, calcium-calmodulin-dependent protein kinase II; ROS ; PARS; poly(ADP-ribose) synthase; reactive oxygen species and [CE, interleukin- l B converting enzyme.

resulted in alrnost complete protection against an in vitro ischemic insult in

hippocampal brain slices. Additionally, it was discovered that omitting caZ' kom the

buffer mimicked the protection that was observed with the NMDA and AMPA

receptor antagonists. This indicates that ca2' influx through ionotropic glutamate

receptors in ischemia may be one of the key events responsible for ischemic damage

(Anas et al., 1999).

1.2.2 ~Metabotropic Glutamate Receptors

Interestingly, unlike ionotropic glutamate receptors, it has been shown that

metabotropic glutamate receptors play a protective role against excitotoxic or

hypoxic/hypoglycemic injury (Bussion et al., 1995, Bruno et al.. 1995, Schroder et al.,

1999, Srnall et al., 1996, Pivi et al., 1996). Metabotropic glutamate receptors are

coupled to G-proteins. Group 1 metabotropic receptors, mGluRl and mGluR5, are

posi tively coupled to phospholipase C, whereas group 2 metabotropic receptors.

mGluR2-3, and group 3 receptors, mGIuR4-6-7-8, are negatively coupled to adenylyl

cyclase. These studies suggest that decreasing CAMP Ievels and activating PKC are

protective against ischemic injury. Smali and colleagues ( 1 996), discovered that ten

minutes of oxygen and glucose deprivation in rat hippocampal slices resulted in a six

fold increase in CAMP levels and an approximately 50% decrease in PKC activity.

They also observed that pretreatment with a PKC activator significantly protected the

slices against the hypoxic/hypoglycemic insult, whereas pretreatment with an adenylyl

cyclase activator did not, indicating that PKC activity is protective against

hypoxic/hypoglycemic injury. Additionally, Bussion and colleagues ( 1999,

discovered that a selective agonist for group 2 metabotropic receptors, s-4-carboxy-3-

hydroxy-phenylglycine (4C3HPG), decreased an NMDA-induced increase in CAMP

levels. The protective effects observed with 4C3HPG following exposure to NMDA

could be attenuated by the addition of 8-(4-chloropheny1thio)-adenosine 3'5'-cyclic-

monophosphate (CPT CAMP) to the ce11 culture media. The authors propose that

decreasing CAMP levels through activation of group 2 metabotropic receptors may be

protective against excitotoxicity associated with oxygen glucose deprivation.

Activation of group 1 metabotropic recepton has also been affiliated with

neuroprotection (Schroder et al., 1999, Pivi et al., 1996). Schroder and colleagues

(1999), observed that protection with the group 1 metabotropic agonist, 3,5-

dihydroxyphenylglycine (DHPG), only occurred when the hippocarnpal slices were

exposed to the drug pnor to the hypoxic/hypoglycemic insult. This protection could

aIso be attenuated by CO-application with a PKC inhibitor. This suggested that PKC

might be involved in protective mechanisms in hypoxic/hypoglycemic injury, but only

if activated pnor to the injury. These studies with metabotropic glutamate receptor

agonists may provide ches as ro what second messenger systems are affected by

ischemic damage and what needs to be conected in order to decrease impairment.

1.3 Xitric Oxide

Activation of the NMDA receptor by glutamate stimulates neuronal nitric

oxide synthase (nNOS), which in tum increases nitric oxide levels in the neuron.

Nitnc oxide synthase converts L-arginine into L-ciûulline in a ~a~'1calrnodulin

dependent manner in the presence of oxygen and NADPN. Representation of the

production of NO from NOS is displayed in figure 1.2. Under conditions, when the

level of L-arginine is rate-limiting NOS is able to produce superoxide anion as well as

nitric oxide (Heinzel et al., 1992). Sirnilarly, when the cofactor tetrahydrobioptenn

( T B ) is absent, the toxic species hydrogen peroxide (H2O2) and superoxide anion are

also fonned by NOS (Heinzel et al., 1992; Pou et al., 1992). Therefore, in situations

such as ischemia, when substrates are lirnited, NOS is able to form superoxide anion

NO Production by nNOS

HOOC NADPH FAD FMN HEME TBH? - . .6

. HOOC NADPH F A D FMN HEME TBH? - NH2

Reductase domain Oxygenase domain

Figure 1.2: A diagrüni depicting NO production by NOS. Adüpted from ladecola, 1997. Abbreviations: CaM, calrnodulin; Cyt c, cytochrome oxidase; FAD, flavin adenine dinucleotide; FMN, flaviii mononucleotide; NADPH, nicotinamide adenine dinucleotide phosphate; NO, nitric oxide; NOS, nitric oxide synthase; TBH, tetrahydrobiopterin.

and H202, which could contribute to neurotoxicity. There are three different isoforms

of niûic oxide synthase, neuronal NOS (nNOS), endothelial NOS (eNOS) and

inducible NOS (iNOS). Both nNOS and eNOS are constitutively expressed whereas

iNOS is produced by macrophages in times of irnmunological challenge (see review:

Iadecola, 1997). The NMDA receptor is directly coupled to nNOS by postsynaptic

density protein -95 (PSD-95). In a study examining the role that PSD-95 may play in

neurotoxicity, it was suggested that PSD-95 coupling to nNOS might be responsible

for the toxicity observed with NMDA receptor activation (Sattler et al., 1999).

Suppression of PSD-95 protein in cultured cortical neurons was found to attenuate the

toxicity associated with NMDA receptor activation. Additionally, it was observed

that treatment of the cultured cortical neurons with nitric oxide donors restored

neurotoxicity. These results indicate that nitric oxide may be a mediator of ischemic

damage.

1.3.1 Nitric Oxide Synthase Antagonists

Studies examining generalized antagonism of NOS following ischemic

damage have found increases in infarct volume, no change in infarct volume or a

decrease in infarct volume (Yamamoto et al., 1992; Hamada et al., 1995; Dawson et

al., 1992; Nowicki et al., 1991; Huang et al., 1994, Panahian et al., 1996). In an

attempt to explain these conflicting results investigators have hypothesized that some

isoforms of NOS are protective while other isoforms are destructive. The use of

general NOS antagonists such as N-w-nitro-L-arginine (NNA) and p-nitro-L-

arginine methyl ester (L-NAME), which simultaneously block eNOS and nNOS,

resdted in an increase in infarct volume (Hammada et a1.,1995; Yamamoto et al.,

1992). It has been suggested that eNOS is beneficial following ischemia due to its

ability to increase collateral blood flow to the infarct area and that nNOS is

detrimental because it is able to dramatically increase NO levels in the neuron

(Samdani et al., 1997). The use of knockout iriice has partially resolved this conflict.

Two studies looking at mice lacking nNOS and cerebral ischemia have found that

deficient nNOS mice have significantly Iess brain damage than wild-type mice. Brain

darnage was accessed by infarct volume and by qualitative grading and ce11 counting

in the CA1 region of the hippocampus. No behavioral differences were observed

behveen the two groups (Huang et al., 1994; Panahian et al., 1996). This further

supports the notion that the negative eRects of nitnc oxide in ischemia are due to

activation of nNOS and that eNOS may be beneficial in decreasing cerebral brain

damage.

1.3.2 Nitric Oxide and Neurotoxicity

It has been suggested that the toxicity associated with increased nitric oxide

levels is due to energy depletion (Almeida et al., 1998; Brorson et al., 1999), DNA

damage (Endres et al., 1998) and oxidative stress (see review: Gross & Wolin, 1995).

Nitric oxide was also s h o w to deplete cellular ATP levels in cultured hippocampal

neurons (Brorson et al., 1999). in a ce11 culture study, it was discovered that

glutamate exposure inhibited succinate-cytochrome C reductase and cytochrome C

oxidase. A NMDA receptor antagonist or a NOS inhibitor couid reverse these effects,

indicating that nitric oxide is able to disrupt mitochondnal energy production

following glutamate neurotoxicity (Almeida et al., 1998). Nitric oxide is able to

inhibit complex 1, II (Stadler et al., 199 1) and reveaibly inhibit cytochrome C oxidase

of the electron transport chain (Cassina and Radi, 1996). Mitochondrial aconitase of

the tricarboxylic acid cycle is also inhibited by NO (Stadler et al.. 1991). This

decreases the production of AïP, which further contributes to the energy depletion

experienced in ischemia.

1.3.3 Peroxynitrite

It has also been suggested that the neurotoxic properties of nitric oxide stems

fiom its reaction with superoxide anion to form the powerful oxidant peroxynitrite.

Peroxynitrite is able to cause DNA damage, which results in the subsequent activation

of poly(ADP-ribose) synthase (PARS). The ATP dependent process by which PARS

repain DNA, depletes energy reserves increasing the cell's susceptibility to damage.

In an i11 vivo study conducted by Endres and colleagues (1998), there were

sipificantly lower levels of PARS immunostaining in nNOS knockout mice as

compared to wild-type mice. Additionally, it was observed that peroxynitrite, but not

various nitic oxide donors, activated PARS irt vitro. It was also found that ce11 loss

induced by exposure to peroxynitrite bi vitro could be attenuated by CO-application of

PARS inhibitors. This indicates that some of the toxicity associated with nitric oxide

is a result of peroxynitmrite formation and activation of PARS. The neurotoxic effects

of NO are sumrnarized in figure 1.3.

1.3.4 Nitric Oxide Production and Neuronal Outcome

In a clinical snidy it was found that higher levels of nitric oxide following the

onset of stroke were associated with early neurological deterioration and poor

outcome at three months. Levels of nitric oxide were determined by measuring the

amount of nitrates and nitrites in the patient's blood in the first 24 houe following the

onset of symptoms. Early neurological deterioration was defined as a fa11 of one or

more points on the Canadian Stroke Scale within the first 48 hours (Castillo et al.,

2000). This clinical investigation demonstrated that nitric oxide levels are correlated

with a greater neurological damage.

1.3.5 Nitric Oxide and Neuroprotection

Alternatively, nitric oxide has been found to be neuroprotective in several

models of neurotoxicity (Fernandez-Tome et al., 1999; Zhang et al., 1994; Vidwans et

al., 1999). In a middle cerebral artery occlusion (MCA) mode1 of ischemia, infusion

of nitric oxide donors following injury decreased infarct volume and improved

cerebral blood flow and EEG amplitude (Zhang et al., 1994). Using an irr vitro mode1

of neuronal injury, Femandez-Tome and colleagues ( 1999) demonstrated that nitric

oxide donors could protect against hydrogen peroxide (HzOz) induced damage. The

protection afforded by the nitric oxide donor could be attenuated with ODQ, a

puanylyl cyclase inhibitor, and Rp-8-pCPT-cGMP, a protein kinase G inhibitor. This

suggests that the neuroprotective effects of nitric oxide donors may be due to a cGMP

mechanism. Vidwans and colleagues ( 1999) observed that various nitric oxide donors

could decrease ca2' accumulation and NMDA neurotoxicity in a cortical ce11 culture

system. Since NMDA receptor antagonists could mimic the actions of the nitric oxide

donors, they suggested that the nitric oxide donors might be inhibiting the NMDA

receptor. They also proposed that the NO donating character of the NO donor may

determine whether that agent is neuroprotective.

1.3.6 Nitric Onde and NMDA Receptor Inhibition

It has been postulated that NO may have neuroprotective properties due to its

ability to donate a nitrosonium ion to the NMDA receptor and downregulate its

activity (Lipton et al., 1993). Lipton and colleagues (1993) hypothesized that the

neurotoxic/neuroprotective properties of nitnc oxide are dependent upon the redox

environment of the neuron. Conditions that favor the formation of a nitrosonium ion

(NO+) Iead to S-nitrosylation reactions and have the potential to be protective.

Donation of a nitrosonium ion to the redox modulatory site of the NMDA receptor by

a S-nitrosylation reaction inhibits ~ a " 80w through the receptor. A nitrosonium ion-

thiol group reaction at the redox modulatory site downregulates the NMDA receptor,

which blocks caZC influx into the neuron. Altematively, conditions that favor the

formation of peroxynitrite from nitric oxide and superoxide anion can be toxic to the

neuron. Peroxynitrite causes DNA damage and further depletes energy stores by

activating PARS as discussed in section 1.2.3. Studies investigating the properties of

NO donors have demonstrated that NO is able to inhibit the NMDA receptor. The NO

donor, 3-morpholino-sydnonimine (Sin-1), was able to inhibit NMDA receptor

activation and increases in intracellular ca2' in a ceIl culture system (Mazoni et al.,

1992). incubation of Sin- 1 with hemoglobin blocked the effects of Sin-1, indicating

that the actions of Sin-1 were due to nitric oxide generation and inhibition of the

NMDA receptor. It has also been shown that the NO donor, nitroglycerin, is able to

inhibit the NMDA receptor. It has been suggested that nitroglycerin reacts with thiol

groups present on the redox modulatory site to produce nitric oxide, which results in

the formation of a disulfide bond and subsequent downregulation of the NMDA

receptor (Lipton et al., 1993; Lei et al., 1992).

Nitric oxide is also able to block the NMDA receptor at an alternative site.

This is suggested by the observation that including metal ion-chelators in the

extracellular media could attenuate the actions of nitric oxide. Thus the inhibition of

the NMDA receptor at this alternative site by nitric oxide requires divalent ions (Fagni

et al., 1995). In surnmary, nitic oxide may be able to inhibit ~ a " flow through the

NMDA receptor by acting at the redox modulatory site or by interacting with divalent

ions at an alternative site. Both of these actions can potentially limit ischemic damage

due to stroke by inhibiting ca2' influx through the NMDA receptor.

1.3.7 Antioltidant Properties of Nitric Oxide

It has been posnilated that antioxidants are beneficial in the reperfûsion period

following ischemic injury due to their ability to interact with oxygen free radicals to

forrn stable compounds. It has been hypothesized that nifric oxide may be able to act

as an antioxidant based on its physio-chemical properties. Nitric oxide is a nitrogen-

centered fiee radical and is extremely reactive in the cell. Rauhala and coIIeagues

(1998) proposed that nitric oxide is able to react with peroxyl lipid radical produced

from lipid peroxidation to fom a stable nitroso-compound (LOO + NO + LOO-

NO). It has also been demonstrated by Kanner and colleagues (1991) that nitric oxide

can inhibit the initiation reaction of lipid peroxidation by reacting with ferrous iron.

Ferrous iron interacts with hydrogen peroxide in the Fenton reaction to initiate lipid

peroxidation. This is particularly relevant in the reperfusion period of ischemia when

lipid peroxidation is taking place at an increased rate.

Studies both in vivo and in vitro have shown that nitric oxide is able to inhibit

lipid peroxidation (Rauhala et al., 1998; Rubbo et al., 1994; Rauhala et al., 1996;

Kanner et al., 1991). Using both techniques it was observed that S-nitrosothiol was

able to protect dopaminergic neurons against oxidative stress. The in vivo mode1

consisted of measunng fluorescent products of lipid peroxidation in brain

hornogenates of anirnals that were previously infùsed with ferrous citrate, with or

without S-nitrosoglutathione (GSNO). In the substantia nigra, GSNO and NO were

able to significantly decrease lipid peroxidation. This effect could not be mimicked

by photodegraded GSNO, GSH or GSSG indicating that the inhibition of lipid

peroxidation was due to production of NO (Rauhala et al., 1996). Additionally, it was

shown that GSNO was able to significantly decrease lipid peroxidation in brain

homogenates exposed to ferrous citrate as indicated by fluorescent end products.

They suggested that this effect was mediated by the ability of nitric oxide to interact

with peroxyl lipid radicals (Rauhala et al., 1998). By measuring hydroxyl radical

generation as an indication of lipid peroxidation, it was observed that nitnc oxide-

myoglobin was able to inhibit the formation of ferryl myoglobin, which initiates lipid

peroxidation. The production of ferryl myoglobin results fiom a reaction between

meûnyoglobin and oxymyoglobin with hydrogen peroxide. These results demonstrate

that nihic oxide may be able to inhibit the initiation of lipid peroxidation since it

hinders the formation of ferryl myoglobin. (Kamer et al., 199 1).

In a liposomal rn~del, it was found that nitric oxide was able to either increase

or decrease lipid peroxidation depending on its concentration. It was demonstrated

that at concentrations of nitric oxide equimolar to that of Oi', XO-dependent lipid

peroxidation was stimulated in a liposome model. However, when the rate of nilric

oxide production exceeded that of Or'- , lipid peroxidation was inhibited. It was also

demonstrated by mass spectrornetry that nitric oxide was able to form nitrito-, nitro-,

nitrosoperoxo-, andlor nitrated lipid oxidation adducts which are termination products

of lipid peroxidation (Rubbo et al., 1994). This study rnight help explain the

conflicting neuroprotective/neurotoxic effects observed with nitric oxide. This theory

suggests that if the concentration of nitric oxide is lower than that of hydroxyl

radicals, nitric oxide increases ischemic injury by stimulating lipid peroxidation.

Nitic oxide could also exacerbate ischemic injury by interacting with superoxide

anion and forming the powerful oxidant peroxynitrite. Superoxide anion

concentrations are increased in ischemic injury due to uncoupling of the electron

transport chain present in the mitochondria. Altematively, if nitric oxide was present

at concentrations exceeding that of hydroxyl radicals, it could terminate the

propagation of lipid peroxidation by forming stable nitroso-compounds. This study

demonstrates that nitnc oxide may be beneficial in the treatrnent of ischemic injury

due to its ability to inhibit lipid peroxidation, if present at the proper concentration.

These findings also indicate that increasing the concentration of NO may be rnost

beneficial during the reperfùsion period when lipid peroxidahon is occurring at an

increased rate.

1.4 cGMP

Nitric oxide is also able io influence second messenger pathways by

interacting with the heme moiety present on guanylyl cyclase, which stimulates its

activity. Guanylyl cyclase produces cGMP fiom GTP. Like nitric oxide, the role of

cGMP in ischemia is controveaial. Some investigators have found that cGMP

contributes to neuronal ce11 death (Li et al., 1997; Montoliu et al., 1999), while others

have observed neuroprotective effects of cGMP against glutamate excitotoxicity and

beta amyloid toxicity (Keller et al., 1998; Garthwaite et al., 1988; Furukawa et al.,

1996; Barger et al., 1995; Fumkawa et al., 1998; Mattson et al., 1999; Yoshioka et al.,

2000; Moro et al., 1998).

1.4.1 cGMP and Neurotoxicity

Two studies examining glutamate excitotoxicity in ce11 culture systems have

conciuded that cGMP potentiates ce11 death. One group found that membrane

permeable analogues of cGMP increased ce11 death in cortical and hippocampal

neurons exposed to glutamate. This effect could be attenuated by soluble guanylyl

cyclase inhibition. They postulated that the neurotoxic properties of cGMP involved

activation of a ~ a " ion channel, since inhibition of soluble guanylyl cyclase

arneliorated increases in ca2' levels and cGMP analogues elevated ~ a " levels (Li et

al., 1997). Interestingly, another goup of scientists observed that increasing

intracellular levels of cGMP induced ce11 death whereas extracellular elevations in

cGMP were neuroprotective against glutamate excitotoxicity. However, the

mechanisms involved in this neuroprotective pathway mediated by extracellular

cGMP are unknown. This may partly resolve the neuroprotective/neurotoxic

controversy surrounding cGMP. Perhaps the localization of cGMP is important in

determining whether cGMP is protective or toxic to the neuron (Montoliu et al.,

1999).

1.4.2 cGMP and Neuroprotection

Altematively, a number of studies have found cGMP to be neuroprotective

against a vanety of neuronal insults (Moro et al., 1998. Garthwaite et al.. 1988, Keller

et al., 1998, Yoshioka et al., 2000). In one study, it was observed that 1H-

[1,2,4]oxadiarolo[4,3,-a]quinoxalin-1 -one (ODQ), a guanylyl cyclase inhibitor, dose-

dependently increased cell death in primary cortical neurons exposed to Sin-1 in the

presence of superoxide dismutase. Sin4 is toxic in these conditions due to enhanced

production of H t02 mediated by the enzyme superoxide dismutase. Superoxide

dismutase forms H202 and molecular oxygen from two superoxide anions and two H+.

The cGMP analogue, 8-bromo-cGklP, was able to reverse the toxicity associated with

ODQ. Therefore it was concluded that cGMP plays a neuroprotective role in H202

toxicity (Moro et al., 1998).

Similarly, in a study examining the role of cGMP in excitotoxicity, it was

found that guanylyl c yclase activators, phoqhodiesterase inhibitors and synthetic

cGMP analogues were neuroprotective. Conversely, inhibiting guanylyl cyclase with

vanous guanylyl cyclase inhibitors was neurodestntctive. Since the toxicity

associated with guanylyl cyclase inhibition resernbled that observed with oxygen

radical generatos, it was proposed that cGMP might be able to limit oxidative damage

(Garthwaite et al., 1988). Oxygen radicals are also able to activate guanylyl cyclase.

Therefore, it was hypothesùed that cGMP may be acting in a negative feedback

fashion by protecting neurons against oxidative stress (Mitta1 et al., 1982). It was

suggested that this protection afforded by cGMP is due to direct scavenging of lipid

radicals or transcription of endogenous proteins involved in oxygen radical inhibition.

Cyciic nucleotides were also shown to significantly decrease ce11 loss in PC6 cells and

cultured hippocampal neurons exposed to 4-hydroxynonenal (HNE). HNE increases

free radical formation, thus promohng lipid peroxidation. The results from this study

indicate that cyclic nucleotides are able to inhibit lipid peroxidation (Keller et al.,

1998). The findings fiom these studies suggest that cGMP may be able to protect

neurons fiom oxidative stress.

Yoshioka and colleagues (2000) have proposed a PKG-cGMP mediated

mechanism of neuroprotection. They found that synthetic cyclic nucleotides and

phosphodiesterase inhibitors increased cGMP concentrations and significantly

decreased kainate induced cal' influx in oligodendroglial-like cells (OLC). In

addition, they discovered that an activator of PKG, 8-(4-ch1orophenylthioI)-

guanosine-3',5'-monophosphate, could protect the oligodendrogIial ceil from

excitotoxicty in a similar marner. Western blot analysis revealed that PKG I P was

translated in OLCs exposed to a PKG activator or protein phosphatase 1 and 2A

inhibitors (Yoshioka et al., 2000). These results indicate that a PKG mechanism may

be responsible for decreasing ca2+ influx mediated by kainate receptor activation.

This would protect neurons from excitotoxicity.

1.4.3 cGMP and &AmyIoid Precursor Protein

Investigatoa researching the neuroprotec tive properties of the secreted fom of

amyloid precursor protein (sAPPa) have suggested a PKG mediated mechanism of

neuroprotection (Mattson et al., 1999; Furukawa et al.. 1997; Barger et al., 1995;

Funikawa et al., 1996). The neuroprotection observed in these studies could be

mimicked by synthetic cGMP analogues and be inhibited by a PKG inhibitor,

suggesting that cGMP and PKG are involved in the underlying mechanism of

neuroprotection afforded by sAPPa.

1.4.4 Glutamate and Glucose Uptake

It has been proposed that a cGMPlPKG mechanism enhances glutamate and

glucose uptake into synaptic compartments. Glucose and glutamate transport was

impaired in cortical synaptosomes exposed to ~e ' - and amyloid P-peptide (AP)

(Mattson et al., 1999). This could be beneficial in ischemia when excessive amounts

of glutamate are being released into the neuronal synapse. If glutamate is taken up

into synaptic compartrnents this would decrease the levels of glutamate present in the

synapse and perhaps prevent excitotoxicity. Glutamate excitotoxicity has been

suggested to be the main mechanism involved in neuronal ceil loss in

isc hemic/reperfùsion injury.

1.4.5 Inhibition of the W A Receptor

It has also been suggested that cGMP mediated activation of PKG rnay inhibit

the NMDA receptor. It was discovered that sAPPa and cyclic nucleotide analogues

were able to decrease ca2' levels in cultured hippocampal and cortical neurons after

exposure to glutamate. A PKG inhibitor could block this effect indicating that the

decrease in intracellular ca2' levels observed with cGMP and sAPPa were due to

PKG activation (Barger et al., 1995). Additionally, it was found that sAPPa and

cGMP analogues could attenuate glutamate and NMDA currents produced in cultured

hippocampal neurons. Data was obtained from whole ce11 patchîlamp recordings.

Protein kinase G inhibitors reversed the effects of sAPPa and cGMP analogues on

NMDA and glutamate induced currents. Further, they found that a phosphatase

inhibitor, okadaic acid, also blocked the effects of sAPPa and cGMP analogues. It

was proposed that PKG may upregulate or activate a phosphatase that would

dephosphorylate the NMDA receptor, decreasing its open probability (Furukawa et

al., 1997). The notion that cGMP may be able to decrease currents generated from

the NMDA receptor indicates that cGMP may be part of an endogenous mechanism to

downregulate the NMDA receptor when it becomes excessively active. The NMDA

receptor increases the production of NO, which activates guanylyl cyclase and

subsequently increases the level of cGMP in the neuron. Thecefore, cGMP may be

able to inhibit the NMDA receptor in an attempt to control its activation.

Manipulating this endogenous system by increasing cGMP may be protective in

disorders caused by glutamate excitotoxicity such as ischemia.

1.4.6 Potassium Channels

Using whole ce11 perforated patch and single channel patch-clamp techniques

it was observed that sAPPa hyperpolarized hippocampal neurons by activating K'

charnels. This effect could be mimicked by cGMP analogues and blocked by PKG

inhibitors. It was found that phosphatase inhibitors could attenuate the

hyperpolanzation observed with sAPPa (Funikawa et al., 1996). Thus APPa is able

to activate K' channels and hyperpolarize the membrane by means of a

dephosphorylation reaction. It was also found that sAPPa is able to decrease ~ a "

levels in the neuron. Hyperpolarization of the neuron causes a decline in ca2+. These

results indicate that augmenting cGMP levels may be protective in ischemia due to its

ability to offset increases in ca2' levels.

In summary, shidies have shom that activation of PKG can protect neurons by

three mechanisrns: increased uptake of glucose and glutamate into synaptosomes and

activation of a phosphatase which downregulates the NMDA receptor or activates K'

channels. Al1 of these events have the potential to be protective in ischemia where ion

homeostasis and glutamate regulation are disrupted. The potential neuroprotective

mechanisrns of nitric oxide are illustrated in figure 1 -4.

1.5 Cyclic Nucleotide Activated Ion ChanneIs

There are alternative mechanisrns by which cGMP may be exerting its effects

in the neuron. Cyclic GMP may be activating cyclic nucleohde activated ion charnels

or inhibiting phosphodiesterases (PDEs). Cyclic nucleotide activated ion channels are

a group of recently characterized ion channels present in most tissues. They are

activated by the cyclic nucleotides, cGMP and CAMP, and allow passage of Na', K'

and ca2' into the neuron. These channels depolarize the neuronal membrane, which

lead to increases in cytosolic ca2' (Dzeja et al., 1999). However, it is unlikely that the

neuroprotective effects observed with cGMP are due to activation of cyclic nucleotide

activated ion channels since activation of these channels increase ca2+ levels in the

cell.

1.6 Phosphodiesterases and Ischemia

Phosphodiesterase inhibitors increase the levels of cGMP and cAMP by

blocking the breakdown of cGMP and cAMP into ScGMP and YcAMP. Studies

have shown that post-ischemic treatrnent with a PDE inhibitor rnay be neuroprotective

through its ability to increase the concentration of CAMP in the neuron. in one of

these studies, the dnig roliprarn was exarnined in a four-vesse1 occlusion mode1 of

ischemia. Rolipram is an inhibitor of PDEr, a phosphodiesterase specific for the

breakdown of CAMP, thus it increases the Ievels of cAMP in the neuron. This agent

has been used in clinical irials as an antidepressant (Bertolino et al., 1988). In this

study, roliprarn was administered six hours after a 20 minute ischernic insult and was

continued once daily for seven days. Neuronal damage was accessed four weeks

following injury by counting the number of s u ~ v i n g neurons in the hippocampus and

Neuroprotective Properties of NO

1 Nitric Oxide 1

1 Inhibit the NMDA 1 Receptor I .

-Donation of a nitrosoniuni ion at the redox niodulatory site ai the NMDA receptor. -Interaction with divülent ions and inhibition of the NMDA receptor at an alternative site.

-Interaction wiih lipid radicals to fonii stable nitroso-conipounds. -1nteract with ferrous ions to iiiliibit the initiation of lipid peroxidatioii.

lncrease Production of cGMP

-cGMP-mediated protection from oxidative stress. -A cGMP-PKG mechanisni: a) Enhniice glucose/glutamate uptake into synaptic compartments. b) Inhibit the NMDA receptor. c) Activate Kt channels.

Figure 1.4: Summary of the neuroprotective properties of NO. Abbreviations: PKG, protein kinase G ; NMDA, N-methyl-D-aspartate.

striatum. It was found that post-ischemic treatment with rolipram could significantly

increase neuronal survival in the hippocampus and striatum (Block et al., 1997).

Furthemore, it was demonstrated that rolipram could improve leaming and memory

after cerebral ischemia. Leaming and memory outcomes were accessed by a 3-panel

runway paradigm. Post-ischemic treatment with rolipram could significantly improve

behavioural outcome following four-vesse1 occlusion (Imanishi et al., 1997). These

results suggest that CAMP may play a neuroprotective role in ischemic injury.

1.7 CAMP

It has been suggested that cAMP may play a role in neuronal survival. It was

found that, in the absence of tropic factors. spiny motor neurons were able to survive

in conditions of elevated CAMP levels in a ce11 culture system (Hanson et al., 1998).

AdditionaIIy, it was observed that decreased CAMP binding occurred in regions of the

hippocampus, such as the CA1 region, that are susceptible to ischemic damage.

Cyclic AMP binding was determined by measunng the levels of radiolabelled cAMP

present in the hippocampal slices (Tanaka et al., 2000). These studies indicate that

CAMP may be involved in mechanisms associated with neuroprotection.

Cyclic AMP may be exerting its effects through the activation of the cyclic

AMP-responsive element binding protein (CREB). Phosphorylation of CREB at

serM3 causes CREB to become active. A number of protein kinases such as protein

kinase A ( P M ) and ~a~'/calmodulin-dependent protein kinases are able to

phosphorylate CREB. It has been suggested that activation of CREB can be

neuroprotective (Tanaka et al., 2000; Walton et al., 1999; Hu et al., 1999). It was

observed that there was a greater amount of phospho-CREB in the dentate granule

cells in the hippocampus following 15 minutes of ischemia than in the CA1 pyramidal

cells (Hu et al., 1999). Because dentate granule cells are more resistant to ischemia,

the increased levels of phospho-CREB in the dentate granule cells may be part of a

neuroprotective mechanism. In a cell culture system, using PCL2 cells, it was found

that CREB phosphorylation increased ce11 survival (Walton et al., 1999). Further,

there was more CREB phosphorylation in neurons that showed no histological signs

of damage, suggesting that CREB phosphorylation may protect neurons from

ischemia. A voltage-sensitive ca2'/Na' channel blocker could not attenuate CREB

phosphorylation, indicating that CREB was phosphorylated by PKA as opposed to

~a~'lcalrnodu1in dependent kinases which are dependent upon Ca" for their activation

(Tanaka et al., 2000).

Altematively, it was observed that there was no potentiation of ischemic

injury in CREB knockout mice (Hata et al., 1998). It is possible that this lack of

potentiation may be due to compensatory mechanisms in the CREB knockout mice.

Therefore one cannot conclusively declare that CREB phosphorylation does not play

an important role in ischemic injury. The evidence thus far supports the hypothesis

that CREB phosphorylation is protective in ischemia. The reason for this is that

CREB phosphorylation is responsible for the transcription of a number of genes such

as bcl-2. Mc11 and bdif (Bonni et al., 1999; Riccio et al., 1999; Walton et al., 2000),

which may play neuroprotective roles against isc hemic injury.

1.8 Guanine Nucleotide Exchange Factors

A novel CAMP mediated mechanism that is independent of PKA activation has

been recently discussed in the literature. The mechanism involved guanine nucleotide

exchange factors (GEFs). GEFs increase the dissociation of GDP from small

GTPase's such as Rap 1 to allow the binding of GTP, which is subsequently

hydrolyzed. An exchange factor, Epac, is directly activated by CAMP in a manner

similar to that of PKA. Epac interacts with the guanine nucleotide binding protein,

Rap-1 (see review: Zwartlauis et al., 1999). The precise role of GEFs remains

unclear, however, they have been suggested to be involved in cell proliferation

(Altschuler et al., 1998), ce11 differation (York et al., 1998) and in ceIl cycle control.

It will be interesting to discover their precise tunction and determine whether they

play a significant role in ischemia.

1.9 Novel Organic Nitrates

Novel organic nitrates are a goup of established NO donors that are denved

fiom the chemical structure of glyceryl trinitrite (GTN). GTN has previously been

s h o w to decrease neurotoxicity in both in vitro and in vivo rodent models (Sathi et

al., 1993, Lei et al., 1992). Treatment with GTN for 36h before and 48h after an

ischemic insult significantly decreased infarct size as calculated fiom MR images.

The isc hemic insult was induced by photothrombosis or by bilateral carotid ligation.

In the in vivo studies, blood pressure dropped initially afler application of GTN and

renirned to normal within 90 minutes (Sathi et al., 1993). GTN was also protective

against NMDA excitotoxicity in a brain slice rnodel (Lei at al., 1992). These studies

demonstrate that G R I has the potential to be therapeutically useful in the treatment of

ischemia.

in place of the rhird nitrate present in GTN, novel organic nitrates have a

substituted phenol group attached to the glyceryl backbone by a disulfide bond.

Evidence suggests that novel organic nitrates spontaneously release nitric oxide in the

presence of thiol groups (Zavonn et al., 2001). The chemical structure of GTN and

the novel organic nitrates, as well as the proposed biotransformation, of the novel

organic nitrates are depicted in figure 1 S.

A major disadvantage of using nitric oxide donors in the in vivo treatment of

ischemia is the vasodilation that occurs. This generalized vasodilation would decrease

Novel Organic Nitrates

- ONO,

GTN Novel Organic Nitrates

S- S- Ph

E S- S- Ph

OH ONO, PhSH (aq.) +NO

pH 7.4 ONO, ONO,

+ PhSSPh + Other Products

Figure 1.5: Chemical structures of GTN as compared to the novel organic nitrates. Proposed biotransformation of the novel organic nitrates. Adapted from Zavorin et al., 200 1. Abbreviations: Ph, phenol; NO, nitric oxide.

blood flow to the brain and perhaps exacerbate injury. However, the vasodilatory

effects of the novel organic nitrates are ten times less potent than that of GTN

(Bennett et al., 2000). Thus, these agents would be more efficacious clinically than

GTN as they do not produce generalized relaxation of blood vessels, which would

decrease blood perfusion of the brain.

GT-015, a novel organic nitrate, was neuroprotective in both in vivo and in

vitro models of neurotoxicity (Clarke et al.. 2000). The neuroprotection obsemed

with GT-015 could be attenuated by the CO-application of ODG, a guanylyl cyclase

inhibitor, indicating that the effects of GT-0 15 were cGMP dependent. Another novel

organic nitrate, GT-715, was able to improve spatial leaming in scopolamine-impaired

male rats. Leaming was measured using the Moms water maze. GT-715 was also

observed to have a greater activation potential for soluble guanylyl cyclase (sGC) in

hippocampal homogenate in the presence of 1mM L-cysteine than GTN as assayed by

a cGMP radioimmunoassay. Because GT-7 15 had a high activation potential for sGC.

it was proposed that GT-715 improves leaming by a cGMP-mediated mechanism

(Smith et al., 2000).

1.10 In Vitro iModel of Ischemia

An in vitro model consisting of hippocarnpal brain slices was chosen for the

evaluation of organic nitrates due to the fact that agents could be investigated quickly

and easily. There are a number of inherent advantages in using this model. The

extemal environment of the neuron is controlled by the investigator, which facilitates

manipulations such as drug administration and changes in temperature. Further, it is

ideal for a mechanistic study as extemal influences are minirnized. Slices frorn the

same animal can be used for various manipulations, which is advantageous, as they al1

possess the same expenmental history. Anesthetics do not have to be used for tissue

preparation and brain slices maintain some integrity. A major disadvantage of in vitro

models, however, is the extent that they represent the in vivo situation. Therefore, any

discovenes that are made in vitro should also be replicated in an in vivo mode1 (Schurr

et al., 1986).

The rrisynaptic circuit is maintained in the hippocampas brain slice, which

makes it an ideal tissue preparation. The circuitry in the hippocampus is highly

glutarnatergic that is important because ischemic damage is associated with glutamate

excitotoxicity. Additionally, the hippocarnpus has been implicated in learning and

memory, which are two functions that are adversely affected by ischecis.

1.1 1 Research Rationale, Hypothesis and Objectives

Nitric oxide could potentially play a neuroprotective role in ischemic injury by

inhibiting ~ a " flow through the NMDA receptor (Lipton et al., 1993), by acting as an

antioxidant (Rauhala et al., 1998 & 1996; Rubbo et al., 1994; Kanner et al., 1991) and

by increasing production of cGMP. Investigators studying sAPPa have proposed a

cGMPIPKG mediated mechanisrn of neuroprotection. They suggested that this

neuroprotection is due to inhibition of NMDA receptor (Funikawa et al., 1997; Barger

et al., 1995), enhanced uptake of glucose and glutamate (Mattson et al., 1999) and

activation of K+ channels (Furukawa et al., 1996). Ln addition, GT-015, a novel nitric

oxide donor, displayed neuroprotective properties in both in vivo and in vitro models

of ischemia. It was hypothesized that this neuroprotection was cGMP dependent since

it could be attenuated by a guanyIy1 cyclase inhibitor in vitro (Clarke et al., 2000).

The goal of this thesis was to test the hypothesis that novel organic nitrates,

which are established nitric oxide donors, are neuroprotective in an in vitro stroke

mode1 by a cGMP-PKG mediated mechanism. in order to test this hypothesis the

following objectives were established:

1) To define the in vitro model of ischemia with respect to duration of ischemia and

neuroprotection with hypothemia.

2) To evaluate several novel organic nitrates in the in vitro model of ischemia for

neuroprotective properties. To determine if any observed neuroprotection is due to

generation of cGMP by examining whether the neuroprotection could be mimicked by

synthetic cGMP analogues or be attenuated by CO-application of ODQ or PKG

inhibitors.

2. MATERIALS AND METHODS

2.1 ChemicaI Solutions

Sodium chloride (NaCl), potassium chloride (KCI), potassium phosphate

(KH2POr), calcium chloride (CaCI?), magnesium sulfate (MgS04), sodium

bicarbonate (NaHC03), glucose, sucrose, NADH, pymvate, dibutyryl cGMP,

dibutyryl cAMP,8-bromo-cGMP and dimethyl sui foxide (DMSO) were al1 purchased

fiom Sigma Chemical Co. (St. Louis, MO). 8-bromo-cGMP, H-89, ODQ and Sin4

chloride were obtained fiom Tocris (Ballwin, MO). 8-pCPT-cGMP and Rp-8-pCPT-

cGMP were purchased fiom Calbiochem (San Diego, Califomia). The reagents and

the standard, bovine semm albumin (BSA) for the dye-binding assay were purchased

from Bio-Rad Labotatories (Mississauga, Ont.). Forskolin and 8-bromo-CAMP were

generously given to us fiom Dr.Maurice, a professor fiom the Deparûnent of

Pharmacology and T o x i c o l o ~ at Queen's University. GSNO was produced and

generously given to us From Dr. Gin, a postdoctoral student from the Department of

Pharmacology and Toxicology at Queen's University. Came1 hair fine paint brushes

were purchased fiom Wallacks (Kingston. Ont.). The fiozen tissue embedding media,

the superfiost microscope slides, Hemo-De and acetic acid were purchased fiom

Fisher Scientific (Nepean, Ont.). Ethanol was obtained fiom Commercial Alcohols

Inc. (Brampton, Ont.). Al1 the novel organic nitrates were synthesized in the

Department of Chemistry at Queen's University (Kingston, Ont.). Al1 aqueous

solutions were made with deionized water purchased from Aquaterra Corporation

(Mississauga,Ont.).

2.2 Experimental Animais

Adult male Sprague-Dawley rats weighng beween 225-250% were purchased

from Charles River Canada inc. (St. Constant, Quebec). Anirnals were housed in

pairs with free access to food and water and were exposed to a 12hrs lighvdark cycle.

The Queen's University Animal Care Cornmittee approved the experimental protocol.

The animals were cared for according to the pnnciples and guidelines of the Canadian

Council on Animal Care.

2.3 Tissue Isolation

Male Sprague-DawIey rats were euthanized, without prior anesthetic, by

decapitation. The brain was then quickly excised and placed in ice-cold sucrose

substituted Krebs' ( 1 18mM sucrose, 4.7 m M KCI, 1.2 mM KH2PO~, 1.3 mM CaClt,

1.2 mM MgSOr, 25mM NaHCO], and lOmM glucose). The hippocampus was

dissected on an ice filled petri dish and transverse slices of 400pm were made with a

Mcnwain Tissue Chopper. Slices were separated in a petri dish filled with sucrose

substituted Kreb's solution with came1 hair fine paint brushes. The slices were

allowed to equilibrate with the Kreb's solution ( 1 18mM NaCl, 4.7mM KCl, 1 .ZmM

KH2PO4, 1 -3mM CaClr, 1.2 mM MgSO~,25rnM NaHC03 and 1 OmM glucose) for 1 h

pnor to the expenment. The slices were placed on a strainer in a beaker with standard

Kreb's solution and bubbled 95% 02/5% N2. The slices were separated into a control

group and a low[02]/low[glucose] group. The slices that were part of the control

group were subdivided into smaller groups of 3-4 slices each and were placed into

vials with 2mL of standard Kreb's solution and bubbled 95% 02/5% Nz. These vials

were placed in a water bath heated to 37OC. Al1 of the remaining slices undenvent an

in vitro ischemic insult. ïhey were placed in a beaker with Kreb's solution which had

glucose substituied with equimolar sucrose to maintain proper osmolarity and bubbled

95% Nd 5% O2 for 1/2 h. A half hour insult time was chosen because it was

previously shown to induce a submaximal increase in lactate dehydrogenase (LDH)

release. This will be discussed in more detail in section 3.1. Slices were then

subdivided into groups of 3-4 slices and divided into control and treatrnent groups.

Treatment groups received the specified concentration of dmg immediately following

the low[Oz]Aow[glucose] insult. The organic nitrates, Rp-8-pCPT-cGMP, 8-pCPT-

cGMP, H-89 and ODQ were dissolved in dirnethyl sulfoxide (DMSO). Sin-1

chloride, GSNO, cGMP, dibutyryl CAMP, the synthetic cGMP analogues dibutyryl

cGMP and 8-bromo-cGMP were dissolved in deionized water. Drug vehicle in equal

concentrations was also included in the control groups. Slices were then placed into

vials with 2mL standard Kreb's solution, bubbled 95% 02/5% Nz and drug or vehicle

for a 4h reperfusion penod. These vials were also placed in a water bath prewarmed

to 37°C.

2.4 LDH Assay

At the end of the 4h reperfusion penod, l . lmL of the incubation buffer was

assayed for LDH release. LDH release was used as a measure of ce11 viability. LDH

is a cytosolic enzyme involved in glycolysis that is released when the celiular

membrane is damaged. The amount of LDH was determined by incubating the

samples with 2OOpL of 1 AlmM NADH for 5-10 minutes at 25°C. Adding ZOOPL of

11.5mM pyruvate to start the reaction then accessed LDH activity. Phosphate buffer

(O. I M) was used to make the aqueous solutions of NADH and pynivate. NADH was

measured on a Beckrnan 500 spectrorneter with a winUV enzyme kinetics program at

an absorbance of 340nm. The decline of NADH that took place over a 1 minute

period was used as an indication of the amount of LDH present. LDH converts

pynivate into lactate using one equivalent of NADH. Therefore, by measunng the

decline of NADH one can calculate the amount of LDH available. The results were

standardized to protein content of each vial.

2.6 Protein Determination

Following the removal of 1. l mL of the incubation buffer for LDH analysis, the

remainder of the incubation buffer was discarded. In order to digest the tissue, ImL

of 1N sodium hydroxide (NaOH) was added to the slices. The amount of protein was

determined spectrophotometricly using bovine semm albumin as the standard in a

protein dye binding assay based on the Bradford method (Bio-Rad Laboratones, inc.,

Mississauga, Ont.).

2.7 cGMP Radioimmunoassay

After the hypoxic/hypoglycemic insult the slices were equilibrated for 1 Smin,

prior to the administration of the therapeutic agents. The supematant was discarded.

The slices were centnfuged and then frozen in liquid nitrogen 3 minutes afier the

administration of drug or vehicle. The slices were hornogenized with 600pL of 6%

TCA and were centrifbged at 2200 rpm for 20 minutes. The supematant was frozen

for subsequent radioimmunoassay (RIA) analysis. The pellet was digested in 1mL of

NaOH for later protein detemination.

On the day of the RIA analysis, 50pL of IN hydrogen chloride (HCL) was

added to the slices. Water saturated diethyl ether was then used to extract the TCA

from the slices. After the K A extmction. 5OpL of I N NaOH was added to neutralize

the HCL as well as 50pL 1M sodium acetate pH 4. The samples were acetylated with

20pL triethylamine and lOpL acidic anhydride to enhance sensitivity.

A series of standards were made using cGMP concentrations in the fentamol

range. Radiolabelled cGMP (I%GMP) was added to the samples, standards, non-

specific binding, total and blank tubes. Antibody specific to cGMP was added to the

sarnples, standards and blank tubes. The following day, gamma globulin was added to

every tube except the total tube and 100% isopropyl alcohol was added to every tube.

The gamma globulin was used to precipitate out the antibody-radiolabelled cGMP

complexes from solution. Al1 tubes were centrifbged at 2000 rpm for ljmin. to

separate the gamma globulin-antibody-radiolabelled cGMP complexes from the

isopropyl alcohol. The isopropyl alcohol was poured out and the samples were read

using a BeckrnanB gamma counter.

The levels of cGMP were deterrnined fiom the cGMP standard curve that was

prepared. The detection of cGMP was based on the cornpetitive aspect of the binding

of cGMP and radiolabelled cGMP to the antibody. This depicts an inverse

relationship benveen the amount of cGMP present and the gamma counts detected.

2.8 Cresyl Violet Staining

After 1 .1 mL of the incubation bu ffer was taken From the hippocampal slices for

LDH analysis, a 4% (w/v) paraformaldehyde solution was added to the slices. The

following day the slices were placed in a 20% (wh) sucrose solution for another 24h.

At this time the slices were kozen ont0 a chuck using fiozen tissue embedding media.

The slices were then cut into 40nm sections using a Richert-Jung Cryocut 1800. The

40nm slices were immediately placed onto microscope slides. The tissue was first

deparaffinized by placing slices in a glass container containing Hemo-De for 2

consecutive periods of 5 minutes. The tissue was then rehydrated using a graded

ethanol series: 100% rthanol for 2 periods of 5 minutes, 95% ethanol for 2 periods of

5 minutes, 80% ethanol for 1 period of 2 minutes, 70% ethanol for 1 period of 2

minutes, 50% ethanol for 1 period of 2 minutes and distilled water for 1 period of 5

minutes. The slices were then stained with the cresyl violet stain (0.5g cresyl violet,

300mL of distilled water, 30mL of t .OM Na acetate, 170rnL of 1 .OM acetic acid) for 1

to 2 minutes. The slices were then destained in distilled water for 5 minutes. Placing

sIices in 70% ethanol for 1 minute, 95% ethanoVacetic acid for 1 to 2 minutes, 95%

ethanol for 1 minute and 100% ethanol for 2 periods of 2 minutes dehydrated the

tissue. The slices were then clanfied by placing them in Hemo-De for 2 minutes and

covealiped. The slides were viewed using a microscope.

2.9 Data Analysis

Al1 the data are presented as group means f SEM. Data from the LDH release

are expressed as enzyme units per milligram protein. An enzyme unit is defined as the

amount of LDH that is able to reduce I p o l of pymvate to lpmol of lactate in one

minute. The data obtained hom the cGMP radioimmunoassay is expressed as

picomol cGMP per milligram protein. Because slices from the same animal were

exposed to al1 conditions, a repeated-measures one-way Anova was used to determine

if any of the groups were statistically different (Px0.05). A Banlett's test for

heterogeneity of variance was completed pnor to the Anova. A Newman-Keuls post

hoc test was then used to determine which groups were statistically different.

3. RESULTS

3.1 Time Course of LDH Release

LDH release was examined as a function of time. This data is presented in figure

3.1. Differing lengths of low[O~]/low[glucose], 1 jmin., 30min, JSmin, and 60 min. were

analyzed for LDH release. A LDH time course was done in order to determine an

appropriate insult tirne. It was observed that increasing the duration of the

Iow[Oz]/low[glucose] insult linearly increased the arnount of LDH release up to

15minutes. AAer this, LDH release plateaued. The 30min. insult time was chosen for

subsequent expenments because it produced a submavimal increase in LDH release.

3.2 Temperature and LDH Release

Decreasing the temperature dunng conditions of low[02]/Iow[glucose] was used

as a positive control. These results are presented in figure 3.2. Lowing the temperature

has previously been s h o w to be neuroprotective in in vitro and in vivo ischemic models

(Tanimoto et al., 1987; Barone et al., 1997). It has been suggested that hypothemia is

protective due to its ability to lower the metabolic rate and conserve energy stores.

Hypothemia has also been used as a clinical treatment in people who suffered severe

ischemic stroke of the middle cerebral artery. Within 14 hours of the stroke, the induction

of hypothermia for 18-72 hours with cooling blankets and cold washings took place. It

was found that hypothermia decreased intracranial pressure (ICP) and improved sumival

from the stroke. n i e only major side effect was the occurrence of pneumonia upon

rewarming. This indicates that hypothemia might be usehl clinically (Schwab et al.,

1998). In our study, the temperature was lowered to 30°C during the

low[02]/low[glucose] insult and the reperfûsion penod to produce a hypothermie state in

Rat Hippocampal Slices Exposed to Differing Lengths

of Low[02]/Low[Glucose]

I I I I O 15 30 45 60

Length of lschemia (minutes)

Figure 3.1 : Hippocampal slices were exposed to varyi ng lengths of low[02]/low[glucose], n= 12.

Rat Hippocampal Slices Exposed to Hypotherrnic

Conditions

Figure 3.2: lschemia @ 30*C refers to a 30min. ischemic insult and 4h reperfusion period at 30*C. Groups with different letters are significantly different from one another, n=5 for each group.

the hippocampal brain slices. Lowering the temperature completely attenuated rises in

LDH release fiom hypoxia and hypoglycemia. LDH release fiom the slices exposed to

hypothermia was the same as slices that had not undergone any insult. Hypothermia

provided approximately 96% protection from ischemia. This result established that the in

vitro mode1 of ischemia was efficient since it replicated what others have previously

documented (Tanimoto et al., 1987; Barone et al., 1997; Schwab et al., 1998).

3.3 Conventional NO Donors

in order to determine whether NO has the potential to be protective in the

treatrnent of ischemia, NO donors were administered at the beginning of the reperfusion

period following a low[Oz]ilow[glucose] insult. These results are presented in figures

3.3-3.5. The NO donon Sin-1 chloride and GSNO were chosen due to their stability and

relatively long half-lives in solution. There was some concem with Sin-1 chloride as it

has been shown to spontaneously release superoxide anion as well as NO (Feelisch et al.,

1989). This may increase the formation of peroxynitnte, which is associated with

neurotoxicity. For this reason, a second NO donor, GSNO was also examined. It was

f o n d that Sin4 chlonde significantly protected the hippocampal brain slices from

ischemia in a dose dependent rnanner. Maximum effects were observed at a

concentration of 250pM. Sin-1 chloride afforded approximately 60% protection as

compared to controls (Fig. 3.3). To verify that the results observed with Sin-1 chloride

were due ro NO generation and not to any metabolites, NO exhausted Sin4 chloride was

also studied. It has previously been s h o w that a solution of Sin- 1 chloride stored at room

temperature for more than 12 hours no longer releases NO (DrAeynolds: persona1

communication). Therefore, Sin4 chloride that had been in solution at room temperature

Rat Hippocampal Slices Treated Wth Sin-1 Chloride

Figure 3.3: Groups with different letters are significantly different from one another. The number of experiments repeated is indicated in ( ).

for at l e s t 12 hours was used. NO exhausted Sin4 chlonde had no significant effect on

LDH release (Fig. 3.4). These results indicate that the protective effects observed with

Sin-1 chloride are due to NO generation. The NO donor, GSNO, also significantly

decreased LDH release from hypoxic/hypoglycemic slices. hterestingly, lOOpM of

GSNO was unable to protect the brain slices against ischemia, yet 50pM of GSNO did

(Fig. 3.5). The results obtained with GSNO differ from Sin4 chlonde in that greater

concentrations of GSNO did not provide any neuroprotection.

3.4 Novel Organic Nitrates

Several organic nitrates were studied for neuroprotective effects. The following

agents were examined, GT-091, GT-094 and GT-310. The chemical structure of these

agents is similar to GTN. However, they differ from GTN in that they contain a

substituted phenol group in place of the third nitrate. These results are presented in

figures 3.6-3.8. Al1 dmgs were tested at an initial concentration of ZOOpM since this dose

of the novel organic nitrates has previously been s h o w to be neuroprotective in in vitro

studies (Clarke et al., 2000). Toxicity was observed in the slices when the DMSO

concentration was greater than 0.1%. For this reason al1 experiments where DMSO had

to be used, the DMSO concentration was limited to less than or equal to 0.1%. The same

arnount of DMSO was also included in the control groups to exclude the possibility that

the DMSO was masking any neuroprotective effects of the agents being studied.

The novel organic nitrate, GT-091, produced maximal neuroprotective effects at

al1 doses examined (Fig. 3.6). The GT-09 1 group was not significantly different from the

control group, which did not undergo an ischemic insult. hterestingly, the LDH release

observed with the GT-091 group was lower than that of the control group, which

Rat Hippocampal Slices Treated With NO-Exhausted

Sin4 Chloride

Figure 3.4: Groups with different letters are significantly different from one another. Sin-1-NO Ex stands for Sin-1 chloride that is NO exhausted. Sin-1 chloride was administered at a concentration of 250uM, n=3 for al1 groups.

Rat Hippocampal Slices Treated With GSNO

control ischemia

Figure 3.5: Groups with different letters are significantly different from one another, ~ ~ 0 . 0 5 , n=3 for al1 groups.

Rat Hippocampal Slices Treated With GT-091

control ischemia 5OPM 10OPM 20OPM

Figure 3.6: Groups with different letters are significantly different from one another, pc0.05, n=3 for al1 groups.

theoretically should have shown no darnage. This suggests that GT-091 can protect

against some of the darnage that occurs as a result of tissue isolation and brain slice

preparation.

GT-094 was also able to significantly decrease LDH release From the

low[Oz]/low[glucose] control (Fig. 3.7). This effect was dose dependent with maximum

effects being observed at 50pM. GT-094 was unable to decrease LDH levels to control

values, yet GT-094 was still 83% protective as compared to controis.

GT-3 10 was synthesized as a congener of GTN and Trolau, an antioxidant. This

was done in order to combine any neuroprotective effects of NO with that of an

antioxidant. Treatment with GT-3 10 significantly protected the hippocampal brain slices

from ischemia by approxirnately 55%. Maximum effects were observed at a

concentration of 100pM and the effects of GT-3 10 were dose-dependent (Fig. 3.8).

3.5 Synthetic cGMP Analogues

Previous studies have shown that the novel organic nitrate, GT-015 is able to

increase cGMP. In order to elucidate if this was how the novel organic nitrates were

causing neuroprotection, a nurnber of synthetic cGMP analogues were analyzed for

neuroprotective properties. The goal of these expenments was to determine whether the

synthetic cGMP analogues could mimic the neuroprotection observed with the novel

organic nitrates. Three different synthetic analogues, 8-bromo-cGMP, dibutyryl cGMP

and 8-pCPT-cGMP were studied. They were chosen due to their differences in cell

membrane permeability and phosphodiesterase sensitivity. It was undesirable to miss any

neuroprotective actions of cGMP due to susceptibility to breakdown by

Rat Hippocampal Slices Treated With GT-094

control ischemia 5OPM

Figure 3.7: Groups with different letters are significantly different from one another, pc0.05, n=3 for al1 groups.

Rat Hippocampal Slices Treated With GT3lO

control hypoxia 25pM 5OPM 100CiM

Figure 3.8: Groups with different letters are significantly different from one another, pc0.01, n=5 for al1 groups.

phosphodiesterases or an inability to cross the membrane. These results are presented in

figures 3.9-3.12.

8-Brorno-cGMP is a synthetic cGMP analogue that is more resistant to breakdown

by PDEs than cGMP. Furthemore, 8-bromo-cGMP has a greater activation potential for

PKG than cGMP. ùiitially, 8-bromo-cGMP appeared to completely protect the

hippocarnpal brain slices from ischemic injury. However, it was discovered that this

apparent protection as exhibited through a reduction in LDH activity was actually due to a

direct inhibition of the LDH enzyme itself. This was observed when 8-bromo-cGMP was

purchased From Sigma as opposed to Tocns. 8-Bromo-cGMP purchased from Sigma did

not produce any significant neuroprotective effects (Fig. 3.9). it was later determined that

the neuroprotective effects observed with 8-bromo-cGMP were due to direct inhibition of

the LDH enzyme. Al1 the dmgs used in the preparation of this thesis were subsequently

tested for any interference with the LDH assay.

Dibutyryl cGMP is a membrane penneable synthetic cGMP analogue. It was

observed that dibutyryl cGMP was able to significantly protect the brain slices from

low[Oz]/low[glucose] in a dose dependent manner. This partial protection was observed

to be approximately 37% as compared to controls (Fig. 3.10). These results demonstrate

that cGMP may be involved in a neuroprotective mechanisrn against ischemic injury.

Cyclic GMP was also exarnined. Since cGMP is unable to pass the ce11

membrane, it was used to determine whether dibutyryl cGMP is involved in an

intracellular or extracellular rnechanisrn of neuroprotective. No signi ficant

neuroprotection was observed with c G W (Fig. 3.1 1). This indicates that the effects

observed with dibutyryl cGMP are due to an intracellular mechanism.

Hippocampal Slices Treated with 1 mM 8-BromocGMP

Figu ire 3.9: A. 8-bromo- cGMP was purchased from Tocris ~uokson Inc. B. &bromcGMP was purchased from Sigma-Aldrich Co. Groups with different letters are significantly different from one another. lhe number of experiments repeated are indicated in ( ).

Rat Hippocampal Slices Treated With Dibutyryl cGMP

Figure 3.10: Groups ~4th different letters are significantly different from one another, pc0.05. The number of experiments repeated are indicated in ( ).

Rat Hippocampal Slices Treated With cGMP

control ischemia

Figure 3.11: Groups with different letters are significantly different from one another, ~ ~ 0 . 0 5 , n=3 for al1 groups.

8-pCPT-cGMP was analyzed for neuroprotective properties. This cGMP analogue

differs fiom 8-bromo-cGMP and dibutyryl cGMP in that it is highly resistant to

breakdown by PDEs. It is also membrane penneable and a potent activator of PKG. 8-

pCPT-cGMP was unable to significantly protect the hippocampal slices from

low[Oz]/low[glucose] (Fig. 3.12). This suggests that PKG activation is not involved in a

neuropro tec tive mec hanism.

3.5.1 Dibutyryl cGMP and Rp-8-pCPT-cGMP

A PKG inhibitor, Rp-8-pCPT-cGMP, was CO-applied with dibutyryl cGMP to the

hippocampal slices at the beginning of the reperfusion period in order to determine

whether the protection observed with dibutyryl cGMP was PKG dependent. These results

are shown in figure 3.13. Rp-8-pCPT-cGMP (7.5pM) was unable to attenuate the

protection observed with dibutyryl cGMP. This concentration of Rp-8-pCPT-cGMP was

previously s h o w to inhibit PKG activation (Vaandrager et al., 1997). Rp-8-pCPT-cGMP

has a K, of 0.5pM for PKG and a K, of 8.3pM For PKA (Butt et al., 1994). Thus at a

concentration of 7.5pM, Rp-8-pCPT-cGMP should completely inhibit PKG and slightly

inhibit PKA showing moderate selectivity for PKG. Therefore it was concluded that the

effects of dibutyyl cGMP were not due to a PKG mediated mechanism.

3.6 Synthetic CAMP Analogues

An alternative mechanisrn of cGMP could involve inhibition of

phosphodiesterases, which would increase the intracellular levels of cGMP and CAMP. In

order to determine if increasing the CAMP levels would be protective against ischemic

injury the synthetic CAMP analogue, dibutyryl CAMP was

Rat Hippocampal Slices Treated Wth 8-p-CPT-cGMP

control ischemia 40OPM 20OPM 10OPM

Figure 3.12: Groups with different letters are significantly different from one another, pc0.05, n=3 for al1 groups.

Rat Hippocampal Slices Treated Wth Dibutyryl cGMP

and Rp-8-pCPT-cGM P

control ischemia

Figure 3.13: DB stands for di butyryl cGMP (1 00pM) and Rp stands for Rp-8-pCPT-cGMP (7.5pM). Groups with different letters are significantly different from one another, ~ ~ 0 . 0 5 , n=3 for al1 groups.

examined. Concentrations similar to that of dibutyryl cGMP were used. These results are

shown in figure 3.14. Dibutyryl CAMP dose-dependently protected the slices against

ischemic damage. Maximum protection of 58% as compared to controls was observed at

200pM demonstrating that CAMP rnay also be involved in a neuroprotective mechanism.

The synthetic CAMP analogue, 8-bromo-CAMP was also studied for

neuroprotective properties. These results are presented in figure 3.15. No significant

protection was observed with 8-bromo-CAMP. Similar to the synthetic cGMP analogues,

these differing results may be due to the distinctive properties of the two synthetic

analogues. 8-bromo-CAMP may be more susceptible to breakdown by

phosphodiesterases or it may be less able to diffûse across the ce11 membrane as compared

to dibutyryl CAMP.

3.6.1 Dibutyryl CAMP and H-89

In order to determine if the neuroprotective effects of dibutyryl CAMP were due to

PKA activation, H-89, a potent PKA inhibitor, was CO-applied with dibutyryl CAMP at the

beginning of the reperfusion period. At a concentration of 7.5pM, PKA should have been

effectively inhibited in our expenmental mode1 since H-89 has a K, of 0.048 f 0.008pM

(Chijiwa et al., 1990). These results are presented in figure 3.16. It was observed that H-

89 was unable to attenuate the effects of dibutyryl CAMP. H-89 did not have any

significant protective effects against low[02]/low[glucose] on its own. These results

propose that a P U mediated mechanism of neuroprotection is not responsible for the

effects of CAMP. ln addition, these results demonstrate that the neuroprotective

properties of cGMP are not due to PKA activation.

3.7 Dibutyryl cGMP and Forskoiin

LDH Release Fold lncrease above Control (Eulmg

protein)

Rat Hippocampal Slices Treated With 8-Bromo-CAM P

control ischemia

Figure 3.15: Groups with different letters are significantly different from one another, ~ ~ 0 . 0 5 , n=4 for al1 groups.

Rat Hippocampal Slices Treated With Dibutyryl cAMP

and H-89

Figure 3.16: DcAMP stands for di butyryl cAMP (50pM), H-89 is present at 7.5pM. Groups with different letters are significantly different from one another, p<0.05, n=3 for al1 groups.

in order to more clearly define the relationship between CAMP and cGMP,

dibutyryl cGMP and fonkolin were studied in combination against oxygen/glucose

deprivation. Forskolin is a potent activator of adenylyl cyclase and increases the

concentration of CAMP in the cell. If CAMP and cGMP are acting through two separate

mechanisms, the effects of raising the intracellular levels of CAMP and cGMP would be

expected to be additive. Similarly, the effect of raising the intracellular levels of CAMP

and cGMP would be potentiated, if the two cyclic nucleotides were causing

neuroprotection by the same mechanism. These results are presented in figure 3.17. It

was observed that dibutyryl cGMP and fonkolin significantly decreased LDH release

from the low[Oz]/low[glucose] control group both independently and together. However,

the protective effects of the two agents on their own and together were not significantly

different fiom one another. No potentiation or additive actions were seen with the

administration of these two agents. This may be due to the dose of dibutyryl cGMP and

forskolin that were studied. These doses may already be producing maximum

neuroprotective effects. If maximal doses are being used then no potentiation of effects

will be obsetved. However, if the two agents were acting by different mechanisms then

additive effects would still be expected to occur. Therefore these results would indicate

that cGMP and CAMP are acting through a cornmon mechanism.

3.8 ODQ

It is hypothesized that the effects of GT-094 are due to activation of guanylyl

cyclase and generation of cGMP. If the effects of GT-094 are dependent upon cGMP

production a guanylyl cyclase inhibitor, ODQ, should be able to attenuate the effects of

GT-094. GT-094 was CO-applied with ODQ (0.5pM) at the start of the reperfusion

Rat Hippocampal Slices Treated with 50p M Forskolin and 100pM Dibutyryl cGMP

control ischemia For.

Figure 3.17: For. stands for forsko for d signi for al

in and DB stands butyryl cGMP. Groups with different letters are Ficantly different from one another, pc0.05, n=4 groups.

period. These results are presented in figure 3.18. It was observed that ODQ at this

concentration was unable to block the neuroprotective effects of GT-094. Additionally,

ODQ did not have any significant effects on LDH release at this concentration. Thus the

effects of GT-094 are not dependent upon cGMP generation.

To determine if the effects of Sin-l chloride (250pM) were dependent upon

cGMP production, ODQ (0.5pM) was CO-applied with Sin-l chloride at the beginning of

the reperfusion period. These results are presented in figure 3.19. Similar to GT-094,

ODQ could not block the neuroprotective effects of Sin-1 chloride. This indicates that

cGMP does not play a role in the neuroprotective mechanism provided by nitric oxide.

hterestingly, it was also determined that ODQ on its own was able to significantly

decrease LDH release from the low[O~]/low[glucose] control group. This effect was dose

dependent with approximately 32% protection being observed at a concentration of

10pM. The effects of ODQ were ameliorated at a concentration o10.5pM. These results

are presented in figure 3.20. This suggests that ODQ is involved in a neuroprotective

mechanism. ODQ is able to inhibit guanylyl cyclase by interacting with a heme moiety

on that enzyme (Feelisch et al.. 1999). Therefore ODQ may be inhibiting a

neurodestructive enzyme or a toxic pathway by reacting with heme moieties.

3.9 cGMP Radioimmunoassay

Previous studies have stated that ODQ is able to inhibit guanylyl cyclase at a

concentration of 0.5uM (Tseng et al., 2000). Some NO donon are much more potent

than othen and require a higher concentration of ODQ to inhibit the activation of

guanylyl cyclase. A cGh4P radioirnrnunoassay was performed to verifj that ODQ at a

concentration of 1 pM was able to inhibit guanylyl cyclase activation mediated by GT-094

control

ischemia

GT-094

GT-094 + ODQ

ODQ

LDH Release (EUlmg prote in)

ci' CD V)

Rat Hippocam Treated Wth 2! Chloride and

al Slices 10pM Sin-1

0.5pM ODQ

Figure 3.19: Groups with different letters are significantly different from one another, pc0.05, n=3. Sin-1 stands for Sin-1 chloride.

50-

40-

30-

20 -

I O -

Rat Hippocampal Slices Treated With ODQ

Figure 3.20: Relative percent protection as compared to the controls in that particular experiment. The number of experiments repeated are indicated in ( ).

(50pM). These results are presented in figure 3.21. The cGMP levels observed in the

control grooups were similar to previous findings in our laboratory (Clarke et al., 2000).

Interestingly, GT-094 was unable to significantly increase cGMP to levels obtained in the

control group. The ischemic insult significantly decreased cGMP levels as compared to

the control group. Treatment with GT-094, GT-094 and ODQ did not significantly alter

cGMP levels from that observed in the low[0~]/low[glucose] group. These results

indicate that the effects of GT-094 are not due to cGMP generation since GT-094 is

unable to increase cGMP levels,

3.1 1 Cresyl Violet Staining

Hippocarnpal slices treated with GT-094 (100pM) and Sin- 1 chloride (250pM) were

stained to veriS, the LDH release data. Photomicrographs of the hippocampal slices are

displayed in figure 3.22. Sirnilar to the LDH release findings, 30 minutes of

oxygen/glucose deprivation causes marked neuronal degeneration in the CA 1 region of

the hippocampus. The CA1 region of the hippocampus was examined because it is the

area most vulnerable to ischemic damage. There was less neuronal darnage in

hippocarnpal slices treated with GT-094 and Sin- 1 chloride. These results are comparable

to the LDH release data.

Concentration of cGMP in Rat Hippocampa Slices

Figure 3.21: GT-094 was administered at a concentration of 50pM and ODQ was administered at a concentration of 1 PM. Groups with different letters are significantly different from one another, p<0.05.

1.0 DISCUSSION 'I

The purpose of this study was to examine the mechanisms behind preçiouslv

observed neuroprotection with novel orsanic nitrates (Clarke et al.. 2000). In order to

address this research problem an in \ ~ o model of stroke. usine hippocarnpal brain slices.

was chosen. The I I I i r r r o model has several advantages over the i r ~ W o model. .ln i)r

i*it~.o niodel is ideal for a mechanistic study as al1 esternal svnaptic connections are

severed. In addition. the external environment. which is under the control of the

investirrator. can be easily manipiilated. In order to be exposed to the brain slices. dmgs

are simplv included in the bathin? solution Only the membrane permeability of the

particular agent and the thickness of the slice need to be considered. Funher. unlike cell

culture systems. the trisynaptic circuit is maintained in brain slices preserving

communicati«n between ditrerent areas of the hippocampus. .A disadvantage of usins I r r

w r o models is the extent to which they replicate the corresponding 11, w i n situation.

Although this is a major limitation. especially when interpretins data. the information

provided from i t r i ~ o esperiments can be invaluable when t-ing to elucidate

physiolo@cal processes. An i i l \?/ru model was thourht to be more appropriate for the

purposes of this study. which was to determine if novel organic nitrates demonstrate

neuroprotective propenies by their ability to increase levels of cGMP.

In order to ascertain an appropriate lenpth of insult. a time course of

osygen/glucose deprivation. in relation to LDH releasr was completed. .A half hour

ischemic insult produced a sub-maximal increase in LDH release and was used for ail

subsequent experiments. It has been documented that 5-7 minutes of an in r i m ischemic

insult is suficient to cause damage io the CA1 pyramidal cells of the hippocampus.

Lon-er insuit times were required to cause sirnilar damase to the dentate granule cells of

the hippocampus. It has been observed that 30 minutes results in damage to vnaptic

transmission. protein synthesis. maintenance of ATP levels. cytoskeletal integrity and

neuronal morpholosy (see review: Lipton. 1999). This is similar to our findings where 15

minutes of i i t vitro ischemia caused an increase in LDH release. probably retlecting CAl

pvramidal cell death. and 30 minutes caused a sub-maximal increase in LDH release.

Hvpothemia has been found previouslv to act as a neuroprotectant in i l ) ilitro and iii r i ia

expenments (Tanimoto et al.. 1987). A clinical trial has been completed which exarnined

the potential of hypothermia in the treatment of stroke. I t was found that hypothermia

could decrease neurolo-,ical damage followiny stroke. however. some patients developed

pneumonia upon rewarming and this may limit the clinical usetùlness of hypothermia

(Schwab et al.. 1998) It has been hypothesized that the neuroprotective effects of

hvpothermia are due to its ability to decrease the metabolic rate and energ requirements

of the tissue This reduction in metabolic rate heips conserve energy stores. which are

massivelv depleted as a result of stroke (Ha-erdal et al.. 1975). In our studies.

hypothermia was used as a posirive control to validate the ut iwtv model of ischemia.

Lowering the tempernt~ire aforded complete protection a~ainst low[02]/low[glucose].

Since the previously documented neuroprotective etfects of hypothermia could be

replicated in our model. this su-gested that the model was appropriate for studyins asents

in ischemia. Similar to the esperiment conducted bv Tanimoto and colleagues ( 1987).

hypothermia was initiated dut-in~ the ischemic insult Interestin&. when hypothermia

was induced in the reperfusion period. no neuroprotective effects were observed. This

suggests that hypot hermia is protecrive againsi earlv irreversible damase caused bu

O-xygen/@ucose deprivation.

The second objective of this research project was to determine if established nitric

oxide donors were neuroprotective against oxyged~lucose deptivation. Two widely used

nitic oxide donors. Sin- l chloride and S-nitroso_elutathione (GSNO). were administered

dunng the reperfùsion period to determine whether nitric oxide has any neuroprotective

putential against ischemic injury. Hippocampal slices treated with Sin-1 chloride at the

beginnins of the reperhsion prriod were signiticantly protected from a

low[O:]/low[glucose] insult in a dose dependent manner In addition. NO-exhausted Sin-

I chlonde could not replicate the previously observed eîFects of Sin-1 chlonde. This

indicates that the actions of Sin-l çhloride are due to VO generation These tindin-s

replicate previous studirs. which have found Sin-l chloride to be protective in neuronal

injury Sin- 1 chlonde significantly decreased infarct volume caused by btCh occlusion

~vhen administered up to 60 minutes afier the insult (Zhang et al.. 1994) h o t h e r study

found that treatment with Sin4 chloride concurrently rvith NMDA protected murine

mised cortical cell cultures against NhIDh neiirotosicity The investisators suggested

that this neuroprntection \vas due to inhibition of NMD.4 induced ~ a ' currents (Vidwans

et al.. 1999).

In contrast. soine cell culture studies have found that Sin4 can produce neuronal

tosicity Neuronal death caused by Sin-l rsposure \vas potentiated in the presence of

superoside dismutase in cortical cultures. indicatina that the neurotoxic rffects observed

with Sin- 1 were a result of hvdrogen peroxide formation. This group of investisators also

observed that Sin- 1. up to concentrations of 500pXI. had no efect on neuronal viability

Interest in& application of synt het ic cGhP anaIoeues could reverse the neurotosicity

observed with Sin- 1 (Moro et ai.. 1998). This study demonstrates that the neurotosic

etfects of Sin4 are associated with hydrogen peroxide formation and sugests that the

neuroprotective effects were due to 'IO mediated cGlMP production. .Another studv

camed out in ceIl culture reported that the neurotoxic etiects with Sin-1 occur at

concentrations equal to or greater than ImiM (Lipton et al.. 1993). In addition. in Chinese

hamster V79 lung fibroblasts. ImM Sin-l potentiated cytotosicity induced by hydroren

peroide (Wink et al . 1996). These tindings are contrary to what was observed in our

esperiments. The touicity associated with Sin- l chloride reported above occurred at

concentrations higher than i00p11 In our study. Sin- l chloride was onlv used at

concentrations less than 500uXI. The diferent results observed with Sin- l chloride may

be a consequrnce of the concentration ~itilized Prrhaps at higher concentrations such as

Imkl there is a qeatrr probability of perouynitrite torrnation due to increased superoxide

anion production. In our study. only the beneficial etfects were observed with Sin4

chloride as indicated bv a decrease in LDH release

.A second nitric oside donor. G S 6 0 \vas used to ver@ that the neuroprotective

efkcts observed with Sin-1 chloride werr d u r to \O seneration GSNO is a congener of

nitric oside and glutathione that spontanrously releases nitric oside. Singh and

colleagues ( 1999) demonstrated that GSNO released nitric oxide in a linear fashion up to

6 h in a liposomal mode1 with SOD and hydrogen peroside in Chelex-treated phosphate

buff'er This suggests that GSNO will release nitric oside for the duration of the

repertiision period making it an ideal agent for our esperiments GSNO protected the

hippocampal slices hm hvpoxic/hypoglycemic i n j u ~ at a concent ration of SOp M.

Interestin& at the higher dose of 1OOpM. GSNO did not decrease LDH release from

that of the hyposic/hvpo~lvcemic control This is contra? to what kvas obsenred with

Sin- l chlonde. which produced dose dependent neuroprotective effect s wit h maximum

neuroprotection being observed at 50pM. .-\ possible reason for these codicting results

is that the nitric oxide donating abilitv differs between these two agents. GSNO may be a

more potent nitric oside donor with tosic concentrations of nitric oxide being obtained at

1 OOpM. With escessive concentrations of nitric oxide being produced at 1 OOpM. there

will be an increased probability of an interaction with superoxide anion and tosic effects

associated with the formation of perosynitrite Thus. these results support the hypothesis

that nitric oside has the potential to be neuroprotecti\~e if it is present in the appropriate

concentration range These findings also demonsrrate tliat nitric oside has the potential to

be neuroprotective adainst ischemic injun when adrninistered at the bqinning of the

repertùsion period

Novel organic nitrates are a group of established nitric oside donors derived from

the chemical structure of GTS GTN has previously been found to be neuroprotective in

/ i l w t n and in tri w o studies t Sathi et al . le9.3. Lei et al . 1992) GTN is cumposed of a

rrlyceryl backbone with three nitrate yroups attached by ester bonds The novrl orsanic b

nitrates difer in that rhes contain a substituted phenol group attached bv a disulfide bond

in place of the third nitrate The unique propenies of the novel organic nitrates are

thought to anse form this third group In some cases. this third group has been

synthesized to act as an antioxidant

Three novel orsanic nitrates were analyzed for neuroprotect ive propenies. GT-

09 1. GT-094 and GT-3 l O The novel organic nitrates were dissolved in DMSO. The

DMSO concentrations were equal to or less than O 1 O to avoid tosicity DMSO was also

included in the control and ischemic sarnples to control for the effects of DMSO on LDH

release The protectivr effects of GT-094 and GT-3 10 were dose dependent. Equal

protection tbas observed with GT-09 1 ar al1 concentrations attempted. The relative

degree of neuroprotection observed with the novel organic nitrates are as follows GT-

09 1 -GT-O94:GT-3 10.

-Alterations in the chemical formulation mav account for the different etfects

observed with the novel oreanic nitrates since changes in the '10 donating - ability or the

potency of the compound will alter its neuroprotective propenies. -411 of the novel

organic nitrates were tested at an original concentration of 200pX1 GT-O15 \+as

previously found to be neuroprotective at 100p51. which is why this concentration was

chosen for subseqiienr esperiments uith the novel organic nitrates (Clarke et al.. 2000)

Additionally. GT-3 10 \vas svnthesized with an alpha rocopherol goup. which is the pan

of vitamin E that is associated ivith its antiosidant tttfects Therefore the nruroprotective

propenies obsened with GT-3 1 O ma? be due to its ability to behave as an antiosidant. as

opposed t« a cGMP mrdiated mechanisni

Previous studies with GT-(1 I S and GT-7 15 have sugested that the novel orsanic

nitrates esen their neuroprotective etfect s t hrough a cGhlP mediated mechanism since

the neuroprotective etfects of GT-O 1 5 could be attenuated by the co-administration of

ODQ. a guanylyl cyclase inhibitor Funhermore it was observed that GT-O l i was able to

increase cGMP levels in ischemic hippocampal tissue as assayed bu a cGMP

radioimrnunoassay ( Clarke et al.. 1000) This provides hn her evidence that the

neuroprotective efects of GT-O15 are dependrnt upon the production of cGMP In

addition. GT-7 1 5 improved task-acquisition in scopolamine-treated male rats in the

Morris water maze This was found to be associated with a geater activation potential

for soluble ganylyl cyclase than GTN. Thesr findings support the notion that novel

organic nitrates can improve neuronal recovery following an insult by a cGMP mediated

mechanism ( Smith et al.. 2000).

In order to determine whether the neuroprotection observed with the novel organic

nitrates was due to cGMP seneration. a number of synthetic cGMP analogues were

rxplored. If the etfects observed with the novei O-anic nitrates could be mimicked by

the synthetic cGMP analogues this would suggest that the novel organic nitrates are

acting throush cGMP Three synthetic cGMP analoyues. dibuty-1 cGMP. 8-bromo-

cGhlP. S-pCPT-cGhIP and cGNP itself were studied These agents were chosen due to

ditferences in membrane permeability and susceptibilitv to phosphodiesterases Since

cGMP is unable to pass the cell membrane. this agent was administered to determine

whether the efects obsened with the synthetic cGMP analogues were due to an

intracellular or extracellular mechanisni Signitiçant neuruprotecti~~e etiects w r e

obsemed oniy witli the membrane permeable dibutyryl cGXlP The etfrcts of dibuirylyl

cGhlP were dose dependent with inasiniuin neuroprotection occurring at 5OuM This

indicates that the nruroprotective etfects were due to an intracellular mechanism as

opposed to estracellular

Our obsenbations uith dibutvd - - cGSlP. which point to a cGh1P mechanism of

ncuroprotecrion. are challen-ed by the rsperiments carried out with S-bromo-cGhIP and

S-pCPT-cGblP No neiiroprotection was observed with 8-bromo-cGMP or 8-pCPT-

cGhIP Thesr results ma! be due to the different physio-chrmical propenies of these

agents. S-Bromo-cGhIP is relatively resistant to breakdown by PDEs. It is also

moderatelv se1ectit.e tor PKG activation with Ki, values of I O-26nM h r PKG-Iu. 2 1 O-

1000nM for PKG-IP and 3 n i M for PKG II . The major limitation with S-bromo-cGMP is

that it is not very membrane permeable: it is only 2.5 fold more permeable than cGMP

Therefore. it is possible that 8-bromo-cGiW did not sain access to the cell's interior.

Like S-bromo-cGMP. S-pCPT-cGW is a potent activator of PKG and is resistant to

hvdrolvsis by PDEs The K., values for 8-pCPT-cGMP are comprisable to that of S-

bromo-cGMP. however. unlike 8-bromo-cGhlP. S-pCPT-cGMP is 56 fold more

membrane permeable than cGMP This would su-sest that S-pCPT-cGMP is able to

enter the interior of the neuron Cnlike the other tuo asents. dibutyyl cGhlP is

si~sceptible to breakdown bu PDEs and is a poor activator of PKG Dibutvnl cGhlP is

also brokrn down t» buty-yl-substituted cyclic nucleotides that ma' have etiects on signal

transduction pathways Siinilar to S-pCPT-cGhIP. dibutvryl cGMP is 62 fold more

membrane penneable than cGhlP (see review Scliuede et al.. 2000)

In conclusion. the lack of neuroprotective rtfects observed with S-bromo-cGMP

ma- be due to its inability to cross tlir neuronal membrane Our finding su-gest rhat the

neuroprotective properties of dibutvryl cGhlP are a result of an intracellular mechanism.

as the- coiild not be replicated by cGMP Sincr S-pCPT-cGhP and dibutvrvl - - cGMP are

borh able to cross the neiironal membrane. diferences in the physio-cltemical properties

of these agents ma! provide dues as to the neuroprotective pathway mediated by

dibutyl cGMP The major ditference betn ern these t w agents is that S-pCPT-cGMP is

a potent activator of PKG Thus the results from Our study would indicate that activation

of PKG does not plav a role in rhc neuroprotective mechanism mediated by dibutyryi

LGMP However. tùnher studies wouid have to be done to determine if this is the case

In addition. esperinients could have bren camed out to ensure that the effects of dibutyl

cGMP are not due to butyrate metabolites.

A number of studies esaminin- the neuroprotective role of the secreted tom of

amyloid precursor protein (SAPPU) have postulated a PKG mediated mechanism of

neuroprotection. The neuroprotection aforded bv SAPPU could be mimicked by

synthetic cGMP analogues and be attenuated bv a PKG inhibitor (Mattson et al.. 1999.

Barger et al.. 1995- Funikawa et al.. 1998. Funikawa et al. 1996). tt has been susrested

that this neuroprotection is due to activation of K' channeis. which leads to a decrease in

intracellular ~ a ' (Funikawa et al.. 19%). This would be beneficial in ischemia when the

neurons are ovrrloaded with ~ a ' I t Iias also been demonstrated that SAPPU is able to

inhibit the NMDA receptor b? a cGhlWPKG mechanism which bloclis C'a:' influx into

the neuron (Funikaw et al . 1 V)S. Baryr et al . 1995) Previoiis studies have

hypothesized that Nb1D.A antagmists may be iiseful in the treatment of ischemia becausr

rhe NMD.4 recrptor is overactivated in h~~po~ivhypu~lycernic conditions (Arias et al..

1 9 W Schurr et al . I C I W Theretbre. a cGMP/PKG rnediated rnechanism of NhlD.4

receptor inhibition Iias the potentiai to b r nruroprotective apainst ischemic injury Finally

it has been reponrd that SAPPU is respcinsible Ior inçrrased uptake of glucose and

dutaniate into synaptic companments and that this response is mediated by a cGMP!PKG - mechanism ( Jlattson et al.. 1 999) This can potentially be neuroproiective in ischemia

since glucose levels are drplaed and glutamate acciimulates in the synaptic cleti.

In summary. these studies indicate that cGMP mav plav a protective role in

ischemia by several PKG mediated mechanisms its ability to activate K channels

(Furukawa et al . 1996). its ability to inhibit the Nb1D.A receptor (Furuliawa et al.. 1998.

Barger et al.. 1995): and its ability to increase glucose and glutamate uptake into synaptis

compartments (Mattson et al. 1999) .Al1 of these mechanisms have the potential to

decrease injury caused by oygen/glucose deprivation.

-4 PKG inhibitor. Rp-8-pCPT-cGhIP was CO-administered with dibutvryl cGMP

in order to determine if the effects of diblityr-l cGMP were dependent upon PKG

activation. Treatment with Rp-8-pCPT-cGhIP did not attenuate rhe protective effects that

were observed with dibuty-yl cGhlP There were no signiticant differences between

treatment with dibutyryl cGhlP and treatment witti dibutvryl cGMP and Rp-8-pCPT-

cGXlP against neiironal dainage çaused by osygervglucose deprivation. Thus the

neuroprotective etfects of dibutyyl cGhlP are not dependent upon PKG activation.

This is in contrast to the finding from the sAPPn studies and çould be due to a

diffèrent esperimentai model iised in t heir stiidies The observations made in t heir

rsperiments were from synaptosonies (Jlattson et al . 1999) and hippocampal cell culture

systems ( Bargrr et al . 19%. Funikau.a et al . 1996 R: I W S

Since it has been proposed that the neuroprotective etiects of the novel organic

nitrates and Sin- l chloride are dependent iipon cGXlP generation (Xloro et al -1998). GT-

094. was CO-adrninistered with the guanylyl cyclase inliibitor. ODQ The neuroprotection

attorded bu GT-094. could not be attenuated bv the CO-administration of ODQ This

would indicate that the neiiroprotective propenies of GT-094 are not dependent iipon

production of cGMP Therefore. GT-094 could be actins bv an alternative mechanism

such as inhibition of the yL'X[D.A recrptor or bu acting as an antiosidant. In addition.

ODQ \vas not able to attenuate the protective etfects of Sin-1 chloride. indicating that the

ability of Sin4 chloride to increase survival of brain slices exposed to

low[02]/lo\v[~lucose] is not dependent upon cGbP seneration. Sin- l chlonde ma! be

providina neuroprotection by one of the mechanisms mentioned above for GT-O94

To detemine if ODQ was etfectively inhibiting guanvlvl cyclase activation and if

GT-094 was increasing cGMP concentrations in the slices. a radioimmunoassay for

cGMP \vas completed. It was obsened that GT-094 was unable to signiticantly increase

cGMP concentrat ions in slices exposed to osygemglucose deprivation. In addition. ODQ

did not fiirther decrease cGhIP concentrations froin the low[02]/low[glucose] controis.

This may be diie to the hc t that yanylyl cylase is not being activated by GT-094 It is

surprisins that GT-O94 !vas unable to inçrease çGMP levels above that of the

lo~v[02]/low[glitcose] contr~ils since it has previousiy been su-eested that the novel

orsanic nitrates are protectivr bu a cGhlP mrdiated rnrtchanism Therefore. the novel

orsaiiic nitrates ma- act by ditferent pathways dependin2 upon their specitic physio-

cliemical propenies Xeuroprotection atforded bu nitric oside donors rnay be due to their

ability to inhibit the Sh1D.A rrceptor (Lei a al . I 9 V ) or to act as antiosidants (Rubbo et

al . 1994) Since the rfects of GT-094 are n«t due to cGMP generation this tvould

indicate that GT-0W nia- be acting through an alternative mechanism such as inhibition

of the NMDA rrceptor or scavenging of lipid free radicals.

Even rhoiigh GT-091 ma" b r eserting its neuroprotective etTects by an alternative

inechanism. our results indicate that c G W plays a rule in protecrins neurons tioni

ischemia. In suppon of our tindings. a number of researchers have published data that

support the concept that the soluble giianylyl cyclaseicGhlP pathway is involved in

neuroprotectioii. I t was obsen-ed that giianylyl cyclase inhibitors increased neuronal cell

death in cerebellar slices esposed to escitatoc amino acids. The effects of the guanylyl

cvclase inhibit ors coiild be reversed by administration of synthetic cGMP analo, w e s or

phosphodiesterase inhibitors. It was susgested that cGMP ma? be part of a protective

mechanism agiainst o q e n free radicals since the tosicity observed rvith yanylyl cyclase

inhibitors was similar to that of oxvsen free radical generatins enzyme systerns such as

ducose osidase and sanithine oxidase. Glucose osidase and xanthine oxidase increase - production of the oxidants. hydrogen peroside and superoxide anion. Furthemore. the

addition of f ier radical scavenging enzymes. such as catalase and the reduction of the

partial pressure of osygen dccreased the neurotouicity observed with guanylyl cyclase

inhibitors (Ganhwaite et al . 1988) In addition. another study demonstrated that cyclic

nucleotides could protect neuronal PC6 crlls from lipid perosidation (Keller et al . 1998)

Thrse investisations indicate that cGMP can potentially decrease neuronal injury

associated wi t h free radical ~enrratiun

I t was recently discovered that ÇGMP is able to inhibit .AMP..\ ~enerated currents

by - 3S00 using whole-cell recordings in hippocampal neurons The inhibiton. actions of

cGMP coiild not hr niiniickrd by rstraçrllular perfiision of cGhlP. indicatins that the

ettects of cGhfP were a result of an intracell~ilar mechanism. .AiCIP.A inhibtion was not

dependent upon PKG activation (Lei et al . 2000) Similarly. in our study. dibutyryl

cGhlP protected the hippocampal neiirons bu X O o from an / I r ~ r o ischemic insult This

protection appears to be due to an intracellular pathivay as our results sould not be

replicated by the membrane impermeable cGMP Thrse resuirs suggest that cGMP ma!.

be esertino neuroprotective properties in our mode1 bv inhibition of the -4MP.A receptor

The second messenger. cGhlP. is also able to nctivate cyclic nucleotide gated

(CNG) cation channels ( Dzeja et al.. 1999) and inhibit PDEs (Maurice Rr Haslam. 1990)

The recently characterized CNG channeis allow the entry of cations upon activation by

intracellular cyclic nucleotides. The! were originally discovered in olfactory and

photoreceptor sensory neurons and were hypothesîzed to play an important role in

auditory and visual tùnctions however. the- have been found in other tissues. such as the

CA 1-3 hippocampal neurons (see re\;ie\v: Biel et al . 1999). Intuitively. it would not

appear that CNG channels would be protective in ischemia since thev cause increases in

intracellular ions. Excessive Ca" accumulation has been su-gested to be a major

mediator of neuronal injury. Therefore. tünher increases in Ca ' by activation of a CNG

channel would more likely potentiate neuronal darnage. as opposed to being protective in

ischemia.

Cyclic nucleotides are also able to inhibit PDEs. which are responsible for the

breakdown of cGhlP and c . A W in the cell Recent studies with rolipram. a PDE

inhibitor. have rrponed decreases in injury associated with ischemia (Block et al.. 1997.

[manishi et al.. 1097). Rolipram inhibits PDEJ, which is responsible for the breakdown of

cAX1P Therefore. treatinrnt ivi th rolipram increases the concentration of c.-\MP in the

nruron. I r a a s hund t h post-ischemic rreatrnrrit ivith rolipram decreased deticits of

working memory in rats caused by four-vesse1 occliision. Learning and memory were

accessed bu a 3-panel ninway paradigm (Imanishi et al.. 1997). Thus increasinp c.-\XIP

atirr an ischemic insult can be beneticial in treatin- the behavioral manifestations of

cerebral ischemia. I t was also obsensed that treatrnent with rolipram up to six hours atier

the ischemic insult decreased ce11 loss in the CA 1 region of the hippocampus and the

striatum. The ischemic insult was induced by tour-vesse1 occlusion for 20 minutes

(Block et al.. 1997). This is clinically relevant. since the majority of patients do not seek

rnedical attention until afier the stroke has occurred. At this time the only available

agents that are etfective in decreasins neuronal damape after ischemia are tissue

plasminogen activators (TP.4). Therefore novei therapeutics designed to increase

may be eficacious in improving neuronal outcome following stroke.

This hypot hesis was tùnher examined bu investigating the possible

neuroprotective propenies of the synthetic CAMP analogues. 8-bromo-CAMP and

dibutyryl CAMP Similar to the synthetic cGhIP analogues. 8-bromo-CAMP did not

protect the hippocampal neurons from neuronal injury However. dibuty-l CAMP was

able to sig~ificantlp decrease LDH release tiom the low[Ol]/low[glucose] slices in a dose

dependent manner Dibutyryl CAMP mimicked the neuroprotection obsened with

dibiitynj cGhlP. indicatins that c.UIP ma! be involved in a neuroprotective mechanism

Because dibuty-yl cGhlP protected the hippocampal slices from osy-eniglucose

deprivation and this wns not dependent upon PKG activation. it was hvpothesized that

dibutvnl - - cGXlP may be inhibitinp PDE; uliich is a cGMP regulated PDE responsible for

CAMP breakdow This would increase the c.A;LIP concentration in the neuron. Thus.

the neuroprotection atiorded by a cGhIP analo-iie may actually be mediated through

CAMP

If the neuroprotective etfects of dibutyryl cGhlP were due ta the inhibition of

PDE; and subsequrnt elrvati«n of c . N P concentrations. t tien forskolin might increase

the abiiit!: of dibut--1 cGMP to protect hippocampal slices from hyposia/hypogiycrmia.

If the etfects of dibutvryl cGXIP were potentiated by forskolin. this rvould indicate that

cGhlP and c.\XIP were producinr! - neuruprotective rKects by the same niechanism.

However. if the eKects of dibutyryl cGhfP were additive with that of forskolin. this woiild

suegest that cGhlP and CAMP \iere acting b'. t ~ k o separate and independent mechanisms

Forskolin is a direct activator of adenylyl cyclase and was chosen because it is able to

eficiently increase intracellular cXMP levels I Awad et al.. 1983 ). Equal neuroprotection

was observed against lo~~~[Ol]ilow[glucose] in hippocampal slices esposed to dibutyql

c G W or forskolin or the two agents together. These tindinss suggest that dibutvn-1 - -

cGMP and forskolin are producing maximum protection individually by a common

pathway and also indicate that cGhZP and cXiMP ma? be acting by the same mechanism

However. additional studies would have to be done to determine conclusivelv if cGMP

and CAMP are acting bv a murual pathwav

Work by Walton et al (1999). Hu et al (1999) and Hanson et al ( 1998) support

the idea that c. lXIP may be a mediator of neuronal suri-ivai Embryonic spinal motor

ncurons were able to sunive fm- 1 week in the absence of tropic factors when the levels

of c..\MP were elevated. Cyclic AhlP activates protrin kinase A I PKA). which is able to

phosphorplate the cylic responsivr element binding protein ICREB). a

transcription tàctor CREB promotes the transcription of a numbrr of proteins upon its

phospliorylation. Two studies I w e suggested that CREB phosphorylntion leads to the

transcription of proteins involveci in neuroprotection One study demonstrated that

increased levels of CREB çoiild inhibit apoptosis The- proposed that CREB might play

a neuroprotective role aeainst apoptotic cell death (Walton et al.. 1999) Fiinherrnore. it

\vas hund that follotving cerebral ischeniia in the rat. neuronal populations that are more

resistant to injury such as the dentate ganule çells of the hippocampus. had higher levels

of phospho-CREB ( Hii et ai.. 1999).

Thus. \ve administered a PKA inhibitor. H-89 with dibutvryl c . A W at the

be~inning of the repertùsion period. to determine if the eRects of dibuty-yl CAMP were

dependent upon PKA activation. H-89 did not attenuate the neuroprotective etrects of

dibutvwl - - c.LVP nor did it evhibit neuroprotective propenies on its own. demonstratinp

that PKA is not involved in a neurotosic cascade.

Because a PK.4 or PKG inhibitor did not block the actions of cGMP and CAMP.

this suagests that the neuroprotective properties of cGhW and crLMP are independent of

protein kinase activation. However. a CAMP-dependent mechanism. which does not

require PKA activation was described recently in the literature. Guanine nucleotide

exchange factors (GEF) are a goup of proteins that activate GTPase's by causing the

exchange of GDF with GTP (sec review Zwankruis and Bos. 1999). One GEF. Epac. is

activated by C A M P in a manner similar to that of PKA. Epac stimulates the activity of a

Rapl GTPase (Rooij et al. lïW3) Rapl has been irnplicated in a number of cellular

processes such as crll proliferation ~Altschuler et al . 1998) and cell differentiation (York

et al.. 1998) .\lthough the Rap l pathwü). has not been clearly definrd. the GEF's

represent a non-PKA mediated CAMP dependent mechanism Since protein kinase

inhibitors could not atteniiate the protective rtfects of dibutyryl c .WP and dibutyyl

cGMP. it is possible that cyclic nucleotides are acting ttirough a novel mechanism such as

GEF activation

In addition. c.\.JfP ma' be providins neiiriiprotrction by increasing adenosinr

concentrations. It has been supgested that adenosinr provides neuroprotection by

inhibitin- $tamate release and by otfsetting C'a' accumulation bv hyperpolarization of

the neuron ( ser reviw Scliiiben et al . 1997) Bot11 of these actions could potentiall!

protect neurons from ischeinic damage Augmenting adenosine receptor activation was

found to be protective in two i i i w r i models of ischeniia (Von Lubitz et al. 1999. Halle et

al.. 1907). .A selective adenosine Ai receptor ayonist. protected neurons in the stratum

pyramidale of the gerbil in a bilateral carotid occuliision model. su-gestin- that adenosine

plays a neuroprotective role in ischemia (\.'on Lubitz et al.. 1999) Enhancing adenosinr

.AI receptor binding in neonatal rats was also found

ischemia as rxhibited throush a decrease in the rveieht

(Halle et al.. 1097). These findings swgest ihat

to be protective against hyposia-

loss of the lefi cerebral hemisphere

adennsine receptor activation is

protective in ischemic injury This pathway represents another potential CAMP mediated.

PKX-independent neuroprotective mechanism

In summary. the resiilts from this study sugpest that the cyclic niicleotides and

novel organic nitrates are esening their neuroprotective etiects bv two independent

mechanisms ODQ was ina able to attenuate the etfects observed with GT-094 In

addition. GT-O94 coiild not increase c G W levels significantly above the

low[OZ]/low[glucosr] controls These findinss indicate that the neiiroprotective effects of

GT-094 are not due to cGhlP generation .Altrrnativrly, GT-094 may be providing

neuroprotection by inhibiting rit her lipid perovidation tir the NJ1D.A receptor

Interestingly. ODQ on its own prodciced dose dependent neuroprotection. The

ne~iroprotectiw rtfects of ODQ LLere not additive nith or potentiatrd b> GT-OW -4s

disçussed previously. this nia? be due t« the fact ttiat ODQ and GT-094 are actins bu a

common mechanisin and their effects ma'. b r masiinal resiilting in no potentiation of the

response Therefore. determining hou ODQ is protectin~ the hippocampal neurons ma?

provide clues as to how GT-(IL)-! is esening its neuroprotection. ODQ inhibits puanylyl

cyclase by interactins wirh its hrme nioiety (Tseng et a l . 1000) One study has

demonstrated that the rffects of ODQ are not specifc They found that ODQ w s able to

inhibit bioactivation of GTS and SSP as wll as nitric oside synthase in aonic

homogenates (Feelisch et al . 1999). while another group obsemed that ODQ was able to

interact with heme goups present in mvoglobin (We-ener et al. 1999). These studies

stron-lu sugaest that ODQ is able to react with other iron cornpleses in the ceIl besides

that of G-cyclase. Therefore. ODQ ma' be esening its neuroprotective etfects by

interacting with heme groups. which would inhibit the initiation of lipid perosidation.

Since lipid peroxidation occurs at an increased rate durin? the reperfusion penod. ODQ

may be protective by its abilitv to hinder the initiation ot'lipid peroxidation.

Similari';. CiT-O94 ma- also be able to interfere with the propagation of lipid

peroxidation in the neuron. I t has been denionstrated previously that nitric oxide is able

to interact witli lipid radicals to form stable nitroso-compounds which block the series of

rertctions involved in lipid peroxidation (Riibbo et al . 199-4) Uitric oside is also able to

interact with tèrrous cornpleses. which w«dd inhibit the initiation of lipid perosidation

(Kanner et al . 109 1 ) Xitric oside donors have been bund to be protective in botli ri1

i w o and 111 \n*o stiidies against iron-indiicrd lipid perosidation (Railhala et al . 1996 %

I9W) Thrse studies indicate that asents. such as GT-094. which are established nitriç

oside donors. are able t« arrcst lipid perinidation by formin? stable nitroso-compounds or

bu interacting with ferrous coinpieses inhibitin: their ability to initiate lipid perosidation

The novrl organic nitrates are brins administrred during the repertùsion period

when lipid perosidation is takinp place It is ivrll rstabiished that the rnetabolic stress

that occiirs as a result of ischemia sets the stage for production of superoside anion and

initiation of lipid perosidation durin2 the rcpertiision period The concentrations of

superoside anion are increased during the repertùsion period bl; purine metabolism (sec

review- hlcCord. 19S7). prostaglandin synthesis ( Kukreja et al.. 1986). and respiration

(see review Freeman et al.. 1982). Hydrosyl radical can also be produced from the

Fenton reaction. a reaction betiveen hydrogen peroside and ferrous iron containing

proteins Thrse species are able to initiate lipid perosidation (see review Watson ri al..

1989) Nitric oside is able to inhibit lipid perosidation by forming stable nitroso-

compounds or by interacting with ~ e - compounds (Rubbo et a!.. 1994: Kanner et al..

199 1). ODQ is also able to interact with ~ r " compounds thus hinderino the initiation of

lipid perosidation. Therefore it is possible that GT-O94 and ODQ are demonstratinr their

neuroprotective properties throiigh a common ability to act as antiovidants

Nitric oside is able to inhibit C'a' flow through the MD..\ receptor. This takes

place by donation of a nitrosoniiim ion frorn iiitric oside to the redos modulatory site on

the NMDA receptor forming a disulfide bridge. This results in decreased ca2 ' tlow

through the W I D . 4 receptor (Lipton et al . 10% Becnuse a substantial arnount of the

. injury caused by ischemia is a result OF ercessive Ca' levels. it is plausible that the novel

organic nitrates are eserting their neuroprotective eKects by restrictina ~ a " intlow

throiish the S V D A receptor .A st~idy that rsaminrd the rtfects of SXID.U.AMP.-!

receptor antagonism on an I I I i*rn-O ischemic insult administered these agents during the

insult (Arias et al . 1999) The rtr utro isctiemic insiilt consisted of 30 minutes of

hypogiycernia ccinciirrent with anosia diiring tlir Iast i - I O minutes of hypoglycernia.

However. the novel organic nitrates were al1 administered during the rrpertùsion pei-iod

when lipid peroxidation is occurrins .At this rime point. it ma! be too late to arresr

neuronal damage caused by XMDX receptor activation Therefore the novel orsanic

nitrates are more likely to esen their neuroprotecti\.e rtieçts by acting as antiosidanrs

In conclusion. the novel orsanic nitrates. GT-094. GT-3 10 and GT-09 1 eshibited

neuroprotection in an iir ivlti.o mode1 of stroke. The neuroprotective properties of GT-094

were not dependent upon cGMP activation. The fact that the effects of GT-094 could be

mimicked by ODQ sugpests that GT-094 may be acting as an antiosidant in our model

The cyclic nucleotides. cAMP and cGhfP were also neuroprotective in the same iir i ~ t a

model of stroke Neither PKA nor PKG activation mediated the neuroprotection afforded

by c.&VP or c G W respectivelv. Funhermore. the protective efects of c G W could be

mimicked bv cAiiIP. suggestin- a common mechanism of neuroprotection. These

findinps suggest that the neuroprotection obsened with the novel or-anic nitrates and the

cvclic nucleotides are a result of two separate and independent rnechanisms. Funher

studies need to be conducted to clearly detine the pathways involved in the

neuroprotective actions of the nowl orgmis nitrates and the cyclic nucleotides .A

summat-y of the proposed mechanisms of neuroprotection of the novel organic nitrates

and cyclic nucleotides are presentrd in figiires 4 1 and 1 2 respectively

The results iibtained rhus far suggest that nitnc ovide donors çan decreasr the

injury associated with stroke when administered post-ischemia This novel treatment

should be funher esplored in ordrr to masimizr its potential as a much needed

tlierapeutic in strokr

4.1 Future Resewcti Directions

Based on the findings of this thrsis projeçt. research should be conducted iii the

tollowin, ( r areas

( a ) i t is not iincciininon h r the 111 \ w o siti~ation tu differ from that of the r / r \ * r w

III order tu veri5 that the eKects t liat were obsewed rtr rrrm also take place ur

i w j . these dmgs \ b i l l also have ro be studied in an rir \ ~ i ~ j model The middle

cerebral a n e n occlusion (SICA) model is a ivtrll established rtr model for

analvzin! ischemic injury Tlierefore the neuroprotective efects of the novel

organic nitrates should alsri be st~idied in a SICA model

(b) it iras sug-ested that the novel organic nitrates are esening their

neuroprotective propenies by acting as antiosidants. These agents should be

studied in models of osidaiive stress to determine if the? have any antiosidant

propenies. There are a nurnber of spectrophotometnc assavs available that

could be used to address this problem.

Proposed Neuroprotective Pathway Mediated by Novel Organic Nitrates - --

~ A d r n i t i o ~ o f ~ o v e ~ Organic Nitrate 1

Biotransformed to NO in the presence of thiol groups.

Inhibition of lipid peroxidation by scavenging Iipid radicals to form stable ni troso-compounds or interacting with ferrous compounds inhibiting initiation of lipid peroxidation.

Donation of a nitrosonium ion to the redox regulatory site present on the NMDA receptor inhibiting Ca3 influx into the neuron.

Neuroprotection against

Figure 4.1 : Proposed mechanism of action of the novel organic nitrates.

Proposed Neuroprotective Pathway Mediated by Cyclic Nucleotides

1 Increase in intracellular cGMP 1

1 Block Ca2+ influx into the neuron

1

Activation of Epac, a 1 CAMP specific GEP. I

Inhibition of the AMPA receptor

Transcription of neuroprotective proteins

Inhibit lipid peroxidation

lnhibit NMDA-R, hyperpolarize the neuron.

Neuroprotection against ischemic inj ury

Increase iiitracellular CAMP

Figure 4.2: Proposed mechanism of neuroproiection mediated by cGMP. Abbreviations: AMPA, alpha-amino-3-hydroxy-5-methyl-4-isoxazole; PDE,, phosphodiesterase 3; Epac, exchange protein directly activated by CAMP; GEF, guanine nucleotide exchange factor; NMDA-R, N-rnethyl-D- aspartate receptor.

lncrease [Adenosine]

(c) Nitric oxide has also been sugeested to be able to inhibit ~ a ' - tlow through the

NMD.4 receptor (Manzoni et al.. 1991. Lipton et al.. 1993). In particular. the

nitric oside donor. GTS has previously been shown to be able to inhibit ~ a "

intlus caused bv NMDA receptor activation (Lei et al.. 1992). To address this

issiir electrophysiologicd studies should be done to determine if the novel

or-anic nitrates are able to inhibit ~ a ' tlow into the neuron as a result of

Nh1D.A receptor activation. C«rn-ersely. if the novel orsanic nitrates do not

7

iiitliience Ca- intlus rhis would provide hnher evidencr that the n o 4 orsanic

nitrates are actin- by an alternative inechanisiii.

( d ) The results froni tliis thesis s u g p t iliat the çvclic nuclrotides are rsening thrir

neuroprotrctive etfects independently of protein kinase activation. To

determine potential transcriptional targets of cyclic nucleotide treatnient. a

cDh.4 iiiiçroassay c l i p could be done \rith hippocampal tissue from a treatrd

and a control animal This would provide mechanistic dues as to how the

qclic nucleiitides are producing nruroprotection. This procedure ivould

identi% potential targets of the qclic nucleotides. which could t h r n be tùnher

esplored using traditional pharrnacolo~ical techniques

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