b?, · body Minor sources of CO include auto-oxidation of phenols, tlavonoids. and halomethanes....
Transcript of b?, · body Minor sources of CO include auto-oxidation of phenols, tlavonoids. and halomethanes....
SELECTIVE INHIBITION OF HEME OXYGENASE BY
iSIIETALLOP0RPHYRINS
b?,
SCOTT DAVID APPLETON
A thesis submitted to the Department of Pharmacology and
Toxicology in conformity with the requirements for
the degree of blaster of Science
Queen's University
Kingston, Ontario, Canada
September 1999
Copyright O Scott David Appletoo
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Abstract
Studies on the physiological role of heme oxygenase (HO) require an inhibitor
that will selectively inhibit HO activity without inhibiting any other function. particular1 y
without affecting the activity of either nitric oxide synthase (NOS) or soluble guanylyl
have previously been shown to inhibit HO activity, for their ability to inhibit HO without
inhibitins NOS or sGC activities. Measurement o i activity of HO in rat brain
microsomes and NOS in rat brain cytosol was conducted in samples incubatcd with
metalloporphyrins (0.15 to 50 M ) including zinc protoporphyrin I?<. ~ i n c
dcuteroporphyrin I?< ?,-I-bis ethylene glycol (ZnBG), chromium mrsoporphyrin IS
(CrMP). tin protoporphyin I?( and zinc N-methylprotoporphyrin IS. CrMP and ZnBG
were found to be the most selective inhibitors of HO activity; i.e. caused the greatest
inhibition of HO activity. 89 and 80%. respectively. without inhibition of NOS activity.
Based on these results, sGC activity in rat lung cytosol was determined in the presence of
CrMP or ZnBG (0.15 to 15 pM). ZnBG did not affect basal sGC activity. bur it did
potenriate S-nitroso-N-acetylpenicillamine (SNAP)-induced sGC activity. CrMP did not
affect either basal or SNAP-induced activity. It was concluded that of the five
metalloporphyrins studied. CrMP. at a concentration of 5 pM, was a selective inhibitor of
HO activity and was the most useful metalloporphynn For the conditions tested. Thus.
CrMP appears to be a valuable chemical probe in elucidating the physiological role of
HO.
Acknowledgements
There are several people that I would like to acknowledge for their contributions
towards this thesis. Although different in nature, all were greatly appreciated. To Dr.
Gerald S. Marks, I would like to thank him for his entertaining stories and helpful insight
Into the field of carbon monoxide research. To Dr. Kanji Nakatsu. for his attempts to
show me how to write and his careful ~uidance and concern for my future. I could not
have asked for better supervisors. I am also fortunate enough to have been blessed with
such wonderful parents and grandparents. who have selflessly sacrificed many things in
order for me to further my education. To Marc Chretien. for his work with the NOS
assays and the rest of the Nakatsu lab for providing me with some entertainment whilr 1
was writins this thesis. To Dr. Donald t-l. Maurice. for making the soluble yuany1yI
cyclase assay "child's play". To Dr. James F. Brien. for his guidance and his ability to
insight the "bloody obvious test". To Brian E. McLaughlin. for persevering through my
inane questions and his continual assistance throughout my project including teaching rnc
the HO assay and the value of proper controls. To the Adam's lab. for providing me with
a computer to work on and endless entertainment while writing my paper. Finally. I
would also like to thank the rest of the faculty and students in the Department of
Pharmacology for their friendship and support.
To papa, you will be "still kicking" in our hearts and our thoughts.
Table of Contents
Page
ABSTRACT ....................................................................................... I I
... ACKNOWLEDGEMENTS ..................................................................... 111
TABLE OF COPU'TENTS ........................................................................
LIST OF TABLES ................................................................................
.......................................................................... LIST OF FIGURES
................................................................ LIST OF ABBREVIATIONS
Chapter One GENERAL lNTRODUCTION ....................................................... A . Heme osygcnase ...................................................................
............................................ B . Suggested physiological roles of CO 1 . Suggested molecular mechanisms of action of CO ...................
.......................... 2 . Role of CO as a neuromodul;ttor in the CNS ........................... . 3 Role of CO as a modulator of vascular tone
...................................... 4 . Role of CO in other organ systems ..................................................................... C . Inhibition of HO
................................................. D . Statement of aims and objectives
Chapter Two SELECTIVE INHIBITION OF HEME OXYGENASE BY
............................................................ METALLOPORPHYRINS .......................................................................... A . Introduction
....................................... ................. B . Materials and Methods .. 26 ........................................................ 1 . Drugs and solutions 26
7 . Preparation of subcellular fractions of rat brain and lung ............ 27 3 . Measurement of HO enzymatic activity in microsomal fraction of rat
...................................................................... brain 29 4 . Measurement of NOS enzymatic activity in cytosolic fraction of rat
.......................................................................... brain 31 5 . Measurement of sGC enzymatic activity in cytosolic fraction of rat
.......................................................................... lung 32 ................................................................ . 6 Data Analysis 34
.............................................................. C . Results and Discussion 3 5
Chapter Three .............................................................. FUTURE DIRECTIONS 47
Page
........................................................................................ References 5 1
Vita ................................................................................................ 59
List of Tables
Page I . Comparison of rnetalloporphynn inhibition of HO.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 40
List of Figures
Pagc Figure 1 .1 The heme degradation pathway .................................................... 4
Figure 1.2 The endogenous production of NO ................................................ 7
Figure 1.3 Activation of sGC by NO and CO ................................................. 8
Figure 1.1 Chemical structureofYC-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1
Figure 1 . 5 Chemical structure of metalloporphyrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Figure 2.1 Inhibition of HO and NOS activity by CrMP and SnPP ........................ 36
Figure 2.2 Inhibition of HO and NOS activity by ZnBG and ZnPP ........................ 37
Figure 1.3 Inhibition of HO and NOS activity by ZnMePP ................................ ?S
Figure 1.4 Effects of ZnBG and CrMP on basal sGC activity .............................. 1 2
. . . . . . . . . . . . . . . . . . . Figure 2.5 Effects of ZnBG and CrMP on SNAP-induced sGC activiry 43
viii
List of Abbreviations
ABT
BSA
de rlovo
EDTA
e.g.
c2t id.
ET- 1
GTP
'H
HO
HO- 1
HO-2
HO-3
hr
irl viiro
1 -aminobenzotriazole
bovine serum albumin
carbon- 1 3
charybdotoxin
carbon monoxide
chromium mesoporphyrin IN
of new
ethylenediaminetetraacetic acid
exernpli gratia (for example)
st alia (and others)
endothelin- 1
guanosine 5'-triphosphate
tritium
heme oxygenase
heme oxygmase- 1
heme oxygenase-2
heme oxygenase-3
hour@)
id est (that is)
in glass
inside the living body
calcium-activated K channels
L-NAME
LPS
LTP
rn
M
min
MS I concentration
NANC
NMDA
NO
NOS
ODQ
PBS
sGC
S bIC
SNAP
SnMP
SnPP
TEA
VIZ.
w/v
Zd3G
ZnPP
il""-nitro-L-arginine methyl ester
lipopoly saccharide
long-term potentiat ion
milli
molar
minute(<\
maximum selective inhibitory concentration
non-adrenrrgic. non-cho lincrgic
N-methyl-D-aspartate
nitric oxide
nitric oxide synthasr
1 H-[l.2.4]-oxadiazolo[- 4.3 -u]qui nosa
phosphate buffered saline
soluble guanylyl cyclase
smooth muscle cell
S-ni troso-iV-acety lpenicillamine
tin mesoporphynn iX
tin protoporphynn IX
tetraethylammonium
narnel y
weight per volume
zinc deuteroporphyrin IX 2,4-bis ethylene glycol
zinc protoporphyrin IX
zinc N-methylprotoporphyrin IX
degrees Celcius
plus or minus
micro
Chapter One
GENERAL INTRODUCTION
In 199 1 , Dr. Gerald S. Marks and colleagues introduced to the scientific
cornmunitv the notion that carbon monoxide (CO) may possess a phvsiological role.
These investigators drew comparisons between carbon monoxide and another
endogenous. gaseous molecule that had been recently shown to have a physiological
etfect. namely nitric oxide (NO) (Furchgott and Jothianandan. 199 1 ) Marks L'I d
( 199 1 ) highlighted the similarity between the structures of NO and CO, but noted that CO
has geater chemical stability than NO. The presence of an unpaired electron in YO
compared with the absence of unpaired electron in CO underlies the relative stability of
C'O and its greater half-life relative to NO Since NO has recently been demonstrated as
a primary messenger. it then follows that CO may also act as a primary messenger as
well. The following criteria (modified from Goodman and Gilman (9"' edition)) must be
satistied lor a substance to be considered as a primary messen9t.r particularly as thrv
apply to CO.
1 Exogenous application of the pirimary messenger must mimic its proposed
biological eflect. Liu el trl (1992) mimicked CO-induced relasation in rabbit
aortic strips precontracted with phenylephrine by bubbling CO into the tissue bath
containing the tissue.
2 . .A mechanism for the termination of action of the primary messenger must esist
Beins a gaseous molecule. the main mechanism for its termination of action
locally is ditksion away from the target area. CO is eliminated from the body
through exhalation as demonstrated by several investigators including Horvath rt
'11. ( 1998) who measured and used exhaled CO as an index of HO activity i l l w o .
3 . Drugs that decrease the endogenous production of the primary messenger should
also decrease the biological effect it, \ivo. Several studies have used this criterion
to demonstrate a physiological role For CO, for example Zakhaq L.I d. (1996)
demcnstrated !hat CO played a role as a vasodilator using competitive inhibitors
for HO. \iz. metalloporphyrins, despite issues concerning their selectivity
-I There must be an endogenous source for the primary messenger and. as hlarks C I
t r l pointed out. heme ovyyenase (HO) produces CO Heme biodegradation is
catalvzed by HO and produces CO as one of its products
HO is a widespread enzyme. which leads to funher speculation as to the putatix
physiological roles of HO and its products. Imrnunohistochrmical studies have
demonstrated that HO is found in the endothelial and the adjacent medial laver of certain
blood vessels (hlarks c.1 trl.. 1997) The localization of HO in both endothelium and
smooth muscle lavers of blood vessels has been shown by Johnson c~ r r l ( 1999) to
provide a functional role in times of endothelial dyshnction where any physiological
effect of NO on vascular tone is removed. This conclusion is based on studies in which
HO-derived CO determined the vascular tone in vessels where the endothelial NOS was
blocked or removed by denuding the vessel of rndothelium. Thus, simulating endothelial
dysfunction, either by removing the endothelium or blocking one of the major
vasodilating factors (EDRF) involved in the equilibrium between depressors and
constrictors, allowed HO-derived CO from the smooth muscle layer to become the major
mediator of vasodilation.
.A. Heme oxygenase
There are several mechanisms by which carbon monoxide (CO) is produced in the
body Minor sources of CO include auto-oxidation of phenols, tlavonoids. and
halomethanes. photo-oxidation of organic compounds; and peroxidation of membrane
!iplds (Iluines. 1084. \Inines. ! Q Q 7 ) The major w u c e r ~ f CO i s heme degradation bv
heme ovygenase (HO) to form CO as one of the products The HO system. in general. is
responsible for the catabolism of heme in the presence of NADPH and 0: ro form
biliverdin I?(. iron (Fe), and carbon monoxide (CO) (Figure I 1 ) The products of this
system are thought to be biologically active Bilirubin. subsequently formed from
biliverdin by biliverdin reductase. is an antioxidant. Fe has been shown to regulate the
expression of certain genes. including that of nitric oxide svnthase (NOS) and ferritin.
and it has also been ~mplicated along with an excess of intracellular heme ( a pro-oxidant)
in endothelial cell damage (hlaines. 1997. Juckett d.. 1998) Fcrritin binds and stores
Fe in a form less apt to contribute to oxidative injurv in endothelial cells and in
coordination with HO provides protection and constitutes an adaptive response to excess
heme (Juckett rr ul., 19%). And finally, it has been proposed that the endogmously
formed carbon monoxide (CO) pliiys a role in the reylation of cell hnction and
communication (Marks rt d.. 199 I , Verma rf d.. 1993).
The enzyme responsible for heme degradation is HO. which is a
ubiquitous enzyme for which there are two well-identified isoforms: heme oxygenase- 1
(HO- I ) and heme oxvgenase-2 (HO-2). HO- I is an oxidative stress-inducible protein.
ADPH BILIVER DIN-IX
HEME OXYGENASE
C (P450) REDUCTASE
Figure I . I : The heme degradation pathway. HO degrades heme in the presence of ovvaen d - and NADPH to form iron (Fe). biliverdin IX. and CO HO requires NADPH- cytochrome c(P450) reductase as a cotactor.
which has also been referred to as heat shock protein 32 (HSP 3 2 ) , localized in the
endoplasrnic reticulum. I t has a wide distribution in the vasculature, but it is highlv
expressed in liver and spleen. HO- 1 expression is upregulated upon exposure to heme as
well as heavy metals ( ~ d " . CO' + ), trivalent arsenicals. heat shock. ischemia. GSH-
depletion, radiation. hypoxia, hyperouia. endotoxin. intlammatory cytokines.
prost3ylandins (PG.4:). hormones. and other cellular transformations and disease states
(hlainrs. 1097) HO- 1 is responsible for the marked increase in CO svnthrsis during
pathological conditions and based on its wide variety of inducers. it has been proposed
that HO- 1 may a play role in maintaining cellular llorneostasis.
HO-2 is the constitutive isozyrne and is localized in rnicrosornes where it is
responsible for CO production under normal physiological conditions. It is not induced
bv the factors that increase HO-1 espression. but it has been shown that HO-2 is induced
by adrenal glucoconicoids it is widelv distributed in endothelium and neurons. its
greatest expression being in the brain as well as other pans of the nenaus system HO-2
has been shown to have similar distribution patterns with both nitric oxide synthase
(NOS) and soluble guanylyl cyclase (sGC).
Recently. a third isozyrne has been identified. namely heme onygenase-3 (HO-3 )
(McCoubrey rt d.. 1997). It exhibits approximately 90% homology in amino acid
sequence to HO-2. but is the product of a different gene. HO-3 is a hemoprotein unlike
the other two isozymes and has relatively lower heme catalytic abilities. This third
isozyme is located in the spleen. liver. thymus. prostate. heart. kidnev. brain and testis
and its physiological role requires hrther characterization.
B. Suggested Physiologic;rl Roles of Carbon Monoxide
Manv of the proposed phvsiological roles for CO are similar to ones which ha\,e
been previously elucidated for NO. it was not too long ago that endothelial-derived
relaxing factor (EDRF) was introduced to the scientific community as nitric oxide (YO)
This gaseous. chemically labile molecule is produced endogenously by nitric oxide
svnthase (NOS). which converts L-arginine to L-citrulline and NO (Moncada cJr (11 .
199 1 ) (Figure 1 7 ) . The bodv of work published since the discovery of NO as EDRF has
been enormous and continues to grow Elucidation of other phvsiological roles of LO
outside of its hnctional contribution to vascular tone include its role as an antitrophic
factor for vascular structure. a retrograde messenger in long-term potentiation (LTP). an
inhibitor of platelet aggregation, a causative factor in platelet disaggregation. and a
neurotransmitter in the central nervous system (CN S).
Many of the biological roles proposed for NO parallel those of another
endogenously formed gaseous molecule. rrz. C O Both gaseous molecules can mediate a
physiological effect, such as blood vessel relaxation. by activation of soluble guanylyi
cyclase (sGC) (Figure 1.3) (Vedernikov rt d, 1989; Moncada ri tr / . 199 1. Furchgott and
Jothianandan. 199 1 ; Hussain et (11, . 1997). However. CO has n considerably lower
potency than NO as a vasodilator (Furchgott and Jothianandan, 199 1 ) .
It has recently been shown that, in the presence of a compound known as YC-l ,
CO can attain a level of activation of sGC similar to that achieved by N O Based on
oenous these observations, several investigators have postulated the existence of an endo,
YC-l -like substance that would allow CO to attain a great potency irr vico. Furthermore.
NO SYNTHASE s
co 0
H3
Lcitrulline
Figure 1.2: The endogenous production of NO. NOS is a ubiquitous enzyme responsible toi the production of endogenous NO it converts the guanidino group of L-arginine to the urea group in L-citrulline. A s a result of this alteration in the structure of L-aryinine. NO is formed along with L-citrulline.
I GUANYLYL CYCLASE I I m
GTP cG
Figure 1 . 3 Activation of sGC by NO and CO Both NO and CO are able to stimulate sGC via interaction with the heme moiety. which is the predominant mechanism through which these gaseous molecules mediate their physiological effects.
the original state of knowledge concerning CO potency was based on tissue bath studies
where aiiquots of CO saturated solution are added. The amount of CO contained within
each aliquot was determined theoretically and the potency of CO was interpolated from
these theoretical calculc?tions We measured the CO concentration in Krebs' medium
following the addition of CO-saturated solution to tissue baths by a gas chromatograph-
headspace method i t was found that the cnncentrarinn nf CO in I I W I C bath medium at
maslmum tissue relaxation was approximately one-third of that calculated based on
theory Thus, the potency of CO as a vasorelaxant is higher than previouslv beliebed to
be the case
8.1 Suggested klolecular hlechimisms of CO
CO and NO bind to the heme moiety of sGC, which produces an increase in the
conversion of guanosine 5'-triphosphate (GTP) to cvclic guanosine 3 '.Y-monophosphate
(cG\IP) The heme regulatory subunit is essential for activation of sGC by CO as
demonstrated by the inabiiity of CO to activate sGC where the heme moiety was absent
(Brune ei t i / . . 1990). The increase in intracellular cGMP levels begins a cascade of
cellular events that produces many of the physiological erects of CO and KO.
Preventing CO-mediated increases in cGhZP by inhibiting sGC activity blocks many of
the physiological eRects of CO such as vasorelauation. The ferrous heme in sGC
possesses diRerent properties to that of the heme found in hemoglobin in that it has an
extremely low affinity for oxygen binding. The difference in the potency of NO and CO
was thought to be in their relative abilities to break the bond between the heme Fe and a
proximal histidine in sGC When CO binds to the ferrous heme of sGC, it is unable to
cleave the Fe-His bond, but instead assumes a slightly bent conformation NO. however.
can rupture this bond. which is thought to account for its more effective activation of
sGC.
A compound known as YC-1, a benzyl indazole derivative, stimulates sGC at an
allosteric site independent of the heme moiety (Figure I -I) .As mentioned earlier. in the
presence of CO YC-I activates sGC to the wme specitic activitv as attained with T O
YC-I has no effect on the afinitv of CO for the heme of sGC. and also permits the
maximal activation of sGC bv CO it was postulated by Friebe cJr tr l ( 1 gC)b) that YC- I
acts through stabilization of the activated configuration of sGC. based on experimental
observations that YC-1 potentiation was independent of the mode of activation (NO. CO.
or protoporphvrin I?() One criticism of CO is that it is less potent than YO in activatmg
sGC resulting in a less substantial physiological erect However. the presence of an
endogenous Y C- l -like substance may allow more serious consideration to be given to the
biological roles of CO Despite its perceived shortcomings as an activator of sGC. CO
has been implicated in many biological roles
Wang rt trl utilized the rat tail artery as a model for studving the mechanisms
underlying CO-induced relaxation in the belief that this vessel possesses all three of the
cellular targets for CO mentioned below The first target is sGC where CO binds to the
heme moiety of the enzyme. Following sGC stimulation, the subsequent increase in
intracellular cGMP concentration produces several effects in smooth muscle cells
( SMCs). The elevated intracellu!ar cGMP levels inhibit inositol I .4,5-triphosphate (IP;)
formation, inhibit voltage-dependent calcium channels, and activate ~ a ' ' ATPase which
all result in a decrease in intracellular free calcium in SMCs (Lincoln rt a/ . . 1994). Wang
Figure 1.4: Chemical structure of YC- I This benzyl indazole derivative stimulates sGC at a site independent of the heme regulatory unit and is thought to act as an allosteric modulator of sGC acti~ity.
and colleagues determined that cGMP has a partial role in the signal transduction
pathway to induce vasorelasaion of rat tail artery since methylene blue. an inhibitor of
sGC. reduced but did not eliminate CO-induced vasorelaxation (Wang. 1998) CO-
induced elevation of cGMP was hrther supported by the observation that a membrane-
permeable inhibitor of cGW-dependent protein kinase (PKG), Rp-8-Br-cGklPS. also
reduced CO-induced mscrelauation c.f the rat tail artery In the rabbit aortic .;trip IRA<!
CO-induced usorelasation appears to be mediated exclusively by cGhlP since I H-
[ 1 .?.-I]-ouadiazolo[4.3-tr]quino.ralin- I -one (ODQ)(Hussain er tri . 1997). a more specific
inhibitor of sGC (Ganhwaite rr trl . 1995) completely inhibits CO induced relaxation in
this tissue
The plasma membrane of ShlCs have a high input resistance By activating m l y
a small number of K ' channels, hyperpolarization of the membrane could be achieved
This hyperpolarization results in inactivation of voltage-dependent calcium channels.
1.
inhibition of agonist-induced increase in [PI. and a decrease in Cam sensitiwty (Quast.
19%) This modulation of calcium-activated K - channels (Kc.,) by CO would account t'or
the decrease in muscle-relaxing effect of CO when precontractrd with KC1 relative to
relaxation seen with muscle precontracted with vasoactive hormones or neurotransmitters
(Utz and Ltllrich. I99 1 ).
Various vascular contraction studies by Wang (1998) showed that CO-induced
relaxation of rat tail artery was significantly reduced in the presence of
tetraethylammonium (TEA, 30 rnM) At high concentrations, TEA may interfere with
various types of K - channels, with the greatest effects on Kc, channels and delaved
outward rectit'ying K ' channels. Thus, studies using more specific K - channel blockers
would be required to hrther elucidate the roles of the different K* channel subtypes.
Charybdotoxin (ChTX) and apamin are used as selective inhibitors of high conductance
and low conductance Kc, channels, respectively. ChTX inhibited the CO-induced
vasorelasation in a similar manner to that of TEA. while aparnin had no effect Based on
these results. Wang suggests that high conductance Kc., channels are involved in the
mcchanicm for CO-induced vasnrelauari(-rn Finally. these workers aholishrd CO-induced
relaxation by concurrently blockin certain elements of the cGhtP pathway as wll as the
activation of high conductance Kc,, channels in vascular SMCs. From these studies it was
strongly suggested that both sGC and high conductance K ' channels are involved in
vasorelaxation mediated by CO.
Wane - t d ~ K e r hrther evidence to support the idea that CO-induced
vasorelaxation is mediated by the opening of high conductance Kc., channels in addition
to the activation of the cGhlP pathway in L ' S M Despite conflictins results from other
studies to determine the etlkcts of CO on Kc., channels. these investiyators den~onstrated
that CO. when applied cvtracellularly or intracellulariy, increased the open probability of
single high conductance Kc, channels in a concentration-dependent manner (Wang.
1998) This group postulated that Kc., channels may have modulatory sites that arc
sensitive to CO and that increasing CO concentrations would increase the probability that
these sites would be modified. Modification would entail an increased exposure of ca2 ' -
binding sites via conformational changes in the channel protein. it was also shown that
CO acts directly on Kc, channels independent of membrane-associated G proteins (Wang
r i nl.. 1997; White r i trl., 1993). Wang and Wu (1997) suggest that CO "increases the
open probability in a reversible fashion probably as a result of a relatively weak reaction
between CO and the imidazole group of histidine via the ':ormation of a hydrogen bond "
Due to the lack of permeability of the drugs used in this study. hnher experimentation is
required in order to determine the identit)i(ies) and location(s) of the residues involved in
the hnctioning of the channel.
Another mechanism bv which CO has been proposed to act is via the inhibition of
c!.tnchrome P450 (Coceani r ( ( I / , 198% Activation of this y t e m may produce
vasoconstricting substances, such as an arachidonic metabolite (Coceani r i d.. 198-1.
Harder r / trl. 1996) or endothelin-I (Coceani et d. 1996) I f the basal activitv of this
svstem contributes to the maintenance of vascular tone. it is hypothesized that mhibition
of cytochrorne P l 5 O by CO would result in a decrease in vascular tone or vasorelaxation
Coceani and co-workers demonstrated the rehat ion of lamb ductus arteriosus a i t h 1 -
aminobenzotriazole (ABT), a suicide substrate for monooxygenase. thus implicatins the
cytochrome P l S O based monooxygenase reaction mediating the production of endot helin-
1 (Coceani er d . . 1996; Coceani et d.1997). The role of the cytochrorne P1SO based
mechanism in the vascular et'Yect of CO has not been well characterized with the
exception of certain fetal vessels and, therefore. requires further investigation.
B.2 Role of CO as a neuromodulator in the CNS.
One role in which CO has been implicated is as a neuronal messenger. CO has
been shown to play a role in LTP, which is a proposed mechanism for learning and
memory formation, which occurs in the CAI region of the hippocampus. In fact. both
NO and CO are thought to act, either alone or in combination, as retrograde messengers
activating guanyiyl cyclase and, in turn, cGMP-dependent protein kinases. This is the
proposed mechanism by which CO and NO produce activity-dependent presvnaptic
enhancement durins LTP in the hippocampus (Hawkins et t r l . 1994) CO has been
shown to play a role in mechanical hyprralgesia along with mrtabotropic glutamate
receptors. similar to the roles which NO and the N-methyl-D-aspartate (NMDA) receptor
assume in thermal hyperalgesia (Aleller er t d , 1994) I t has also been demonstrated that
CO pr[rduce.; ctTect~ In the rat hvpothalamr~.; \\here i t may h n c t i o n ;I.; a nrr~rntransm~ttrr
in the modulation of neuroendocrine function. Thus, hemin, the substrate of HO. dose-
dependently inhibited the release of KCI-stimulated corticotropin-releasln_r hormone
(CRH) wlth no effects on basal CRH release (Pozzoli c3f d. 1994) CO has also been
indirectly shown to stimulate the release of gonadotropin releasing hormone (GnRH) In a
dose-dependent manner (Lamar rt trl . 1996).
When cobalt protoporphyrin 1X. an inducer of HO. was injected into the medial
nuclei of the rat hypothalamus. w i ~ h t loss occurred Based on these results. it has been
postulated that CO is involved in the signal transduction pathway in the medial
hypothalamus to suppress appetite (Marks. 1994)
B.3 Role of CO as a Modulator of Vascular Tone
CO mav play a physiological role in regulating vascular tone. CO-induced
vasorelaxation, as a result of a direct action on vascular smooth muscle, has been
demonstrated in a variety of isolated blood vessels from a number of species such as
porcine coronary meries (Graser rt c t l . . 1990), rabbit and rat thoracic aorta (Furchgott
and Jothianandan, 1991; Lin and McGrath, 1988), rat femoral and tail arteries
(Vedernikov rr d . . 1989; Wang rt nl., 1997), and the lamb ductus arteriosus (Coceani et
d.. 1984) CO dilates resistance vessels in the heart. lungs. and liver W a n ( 1999)
tested the effects of CO on the rat tail artery and considers it to be representative of
peripheral vascular tissue. This group demonstrated a concentration-dependent relaxation
bv CO in a rat tail artery precontracted with phenylephrine. This relaxation was not
mediated bv blockade of the a-adrenoreceptor as CO also relaxed U-16619
(prostaglandin L,, analog) i o n i r a c i ~ d tissuc, an agonisi that causes con:raction -:io
1 .
release of intraceilular Ca- The investigators reported an cndothelium-independent
sustained relaxation. which was reversed upon removal of CO.
The relaxation of blood vessels by CO is not uniform throughout the vasculature.
\'aqing degrees of vasorelusation are produced bv CO in ditferent ~clscular beds. In ;i
study by Brian cr d. ( 1994). the middle and basilar cerebral arteries in rabbits and dogs
were apparently not affected when CO was administered rtr w r o ( I to 300 phl) (Brian et
d. 1994). but were relaxed by NO However. in a later study LrfTler and co-wrkers
( 1999) using the cerebral microvasculature of newborn pigs demonstrated CO-induced
dilation of pial arterioles in the cerebral vascular bed. These investigators offer the
following reasons for the variation in response between the two preparations Brian d.
( 1994) examined the responses of major cerebral arteries whereas Lettler and colleagues
( 1999) utilize the arterioles that are among the smallest precapillary vessels on the brain
surface. It is important to recognize that blood vessel tone is controlled in the arterioles
and not in the large arteries. Second. the latter study was conducted on intact brain l tr
w o . whereas the former used a tissue bath apparatus with precontracted anery rings
Lefler r i a/. (1999) suggest that CO dilation is less functional under the ill ~ r - o
conditions employed by Brian et ( I / . and that underlying neurons in the i ~ r ~iw
preparation are possiblv contributing to the CO-induced vasodilation. Moreover. t h e
studv , . by Leffler rt d . involves newborn animals where the HO-CO system is
developmentally regulated. Finally. it is suggested that the species difference may
account for the divergent findings of the two studies since Brian rt t t l . studied dog and
rabbit cerebral arteries and LeMer ul used pigs.
There are several mechanistic evplanations for the ditferent etlkcts in the
vasculature bv NO and C O Firstly, it has been proposed that the cellular targets of CO
and NO may account for the differences. NO acts through sGC activation resulting in
elevation of cGMP content and in pan to hyperpolarization resulting from direct
interaction of NO with c a 2 ' -dependent K* channels (Bolotina rr t r i . . 1994) CO
stimulates sGC resulting in elevation of cGhlP content and mav have actions through the
cytochrome P450/endothelin- l system (Coceani er d., 1996) andior Kc , channels (Wang
c.t d . . 1997) depending on the vascular bed being esamined The differences in CO and
NO actions on vascular tone may also b e accounted for by their relative abilities to
activate sGC. On the basis of studies in the ductus arteriosus with methvlene blue. an
inhibitor of sGC of poor selectivity, CO was reported to produce an insignificant increase
in cGMP accumulation. It would be of interest to repeat this study with ODQ. a more
selective inhibitor of sGC (Garthwaite rt d.. 1995). On the other hand, sodium
nitroprusside (SNP), a NO donor, significantly stimulated sGC (Coceani rt d, 1996).
NO is more potent than CO in activating sGC, which also accounts for the varying
abilities of these two gaseous molecules to produce vasorelaxation.
CO has also been shown to play another role in modulating vascu
as a vasorelauant. In a recent review published by Johnson el a!. ( 1999),
liar tone. besides
CO is identified
as having a dual action. Endogenous CO production contributes to the functional
equilibrium between vnsoconstrictor and vasodilator factors. that determines a certain
level of blood pressure and its role in this equilibrium has been regarded as that of a
vasodilator. According to Johnson rt d. ( 1999), investigators have failed to differentiate
between the endothelium-dependent and -independent roles of endogenously formed CO
This gmup (Kozma rr ( I / , lq(JRl~) demonstrated CO-induced vasodilation as an
endothelium-independent event in isolated superfused rat gracillis first-order arterioles
denuded of endothelium. but when the endothelium was lef intact. a product of HO.
presumably CO (as these results were mimicked bv CO). elicited a vasoconstrictive
response Johnson LV '11 ( 1999) suggest that endothelium-dependent vasoconstriction is
either due to CO-induced release of a vasoconstrictive agent from the endothelium or to
CO-induced inhibition of NOS or to both. The latter possibility is supported by rvidmcr
tiom earlier studies where CO binds to NOS and inhibits NO (EDRF) production (White
and Marletta, 1992). Thus. removal of a vasodilating factor, such as EDRF. from the
equilibrium that contributes to blood pressure results in a vasoconstrictive effect.
Kozma r f id. ( 1997. 1998tr. 199Sh) have attempted to clarie the role of CO in
the vascuiature These investigators inhibited NOS in endothelium intact vessels by .V"-
nitro-L-aryinine methyl ester (L-NAME) pretreatment. When heme was added to the
tissue bath. CO was produced by the actions of HO resulting in vasodilation. The
vasodilation induced by heme addition was inhibited by HO inhibitors. These workers
suggest that heme-derived CO in the smooth muscle may mediate its vasodilatory effects
in a local manner and its vasodilatory effects may be buffered simultaneously by the
inhibition of the vasodilatory influences of endothelium-derived NO. Johnson el ol.
( 1999) express the need to acknowledge and clarify the potential interactions between the
CO and NO systems. Without the knowledge of the functional hemodynamic
significance of this interaction. accurate interpretations of results from future experiments
would be dificult.
CO has been proposed to assume an antitrophic role in the vasculaturr Under
! I \ ~ I Y ! C condition.;. it ha.: been ihown that CO conrrols the proliferation of vascular
SblCs (blorita d, 1997). I t was previously reported that CO. produced from HO- 1 in
hypoxic vascular SMCs. regulates the production of the growth factors. mdothelin-1
(ET-I ). and platelet-derived growth factor-p (PDGF-P) and decreases the proliferative
response of vascular S M C s to ET-1 (blorita and Kourembanas. 1095) CO has also been
shown to suppress E2F-1 expression, a prototype member of s family of transcription
kctors that participate in the control of cell cvcle progression. Taken together. these
reports susgest that CO. under certain pathophysiologic conditions. is responsible for not
only the immediate effects on vascular tone. but also producing more long-term
adaptations in vascular structure.
B.4 Role of CO in other organ systems.
It has been shown that CO is involved in intestinal neurotransmission using mice
with genomic deletions for HO-2 and nNOS resulting in the absence of these enzymes in
the rnyenteric ganglia. It was shown in these knockout mice that non-adrenergic. non-
cholinergic (NANC)-mediated intestinal relaxation and c 0 elevations induced by
electrical field stimulation were markedly reduced in both HO-7 and nNOS knockout
mice (Zakhary rt ul., 1997). These results indicate that NO and CO act as
neurotransmitters in the myenteric plexus and mediate their etrects via the cGhlP
pathway. Further studies with HO-2 knockout mice involving the bulbospongiosus
muscle, which mediates ejaculation and ejaculatory behavior, have demonstrated a
diminished retlex activity of this muscle (Burnett et id.. 1998) CO was concluded to be
the most plausible product of HO-2. that would account for the results obtained.
C. Inhibition o f HO
HO activity is inhibited by compounds that act as pseudosubstrates These
compounds are resistant to HO degradation and compete with heme (iron protoporphvrin
[X. FePP) for the hydrophobic heme pocket (Maines. 1997) These metalloporphyrins
ditfer from heme with respect to the metal cation associated with the porphyrin ring
and/or the substituents bound to the porphyrin ring (Figure 1.5) These variations from
heme affect their potency as HO inhibitors The hydrophobic pocket for which the
metalloporphyrins and heme compete. exhibits specificity for the porphyrin side chains
but not for the chrlated metal (hlaines. 1997). By definition of n competitive inhibitor.
the relative concentrations of heme and metalloporphyrins will determine the degree to
which HO is inhibited.
It was recently shown that a KO donor, in a dose-dependent manner. also
inhibited HO activity. It was suggested that the NO nitrosylates intracellular free heme
and prevents its degradation by heme oxygenase (Juckett r i d, 1998). This contributes
hrther to the close relationship that exists between the HO and NOS systems.
Metalloporphyrins are heme analogs that vary in the metal cation coordinately
bound to the njtrogen groups of the porphyrin ring as well as substituents of
Figure 1.5: Chemical structure of metalloporphyrins. Heme is a metalloporphyrin where the substituents RI and R2 are vinyl groups. For other metalloporphyrins. a different metal cation is coordinately bound to the porphyrin ring. For ZnPP: substitute Zn for Fe and RI= R2= CH=CH2; for SnPP: substitute Sn for Fe and RI= Rz= CH=CH2. for CrhlP substitute Cr for Fe and RI= R2= CHI-CH3; for ZnBG: substitute Zn for Fe and RI= R2= CHOH-CH20H; ZnMePP has the same chemical structure as ZnFP with the exception of a methyl group bound to one of the nitrogens in the porphyrin ring.
the porphyrin ring. These compounds have been used as pharmacological tools in the
past. but it has also been pointed out that they are produced endogenously under certain
physiological settings. Zinc protoporphyrin is formed when zinc replaces iron for
insertion into protoporphyrin, in a reaction catalyzed by ferrochelatase. i t has been
suggested that in iron deficiency states, zinc protoporphyrin will limit the amount of
l i m e catabolism
Endogenous zinc protoporphyrin production has also been hypothesized to play a
role. at least in pan. in the decrease in attention span and small decrements in IQ scores
seen in patients experiencing subclinical lead toxicity (Marks er i d . 19%) Production of
zinc protoporphyrin by ferrochelatase is increased in iron deficiency states as well as in
lead poisoning. The increased endogenous zinc protoporphyrin concentration has been
proposed to inhibit the formation of carbon monoxide by heme oxyrenase and thus
modulates LTP and memory. The inhibition of heme oxygenase in the hippocampus was
proposed to alter the neurophysiological phenomenon known as LTP. which might
account for the cognitive deficits observed in lead poisoned patients.
inhibitors of enzymatic activity are useful tools in establishing a physiological
role for specific enzymes. Thus. L-NAME has played an important role in elucidating the
biological roles of NOS (Moncada el ~ z l . . 1991). Metalloporphyrins have been shown to
inhibit HO. and their potency is affected by the metal cation associated with the
porphyrin ring as well by different ring substituents (Vreman rt dl . . 1993). Inhibition of
HO activity has been demonstrated for each of the metalloporphyrins used in this study.
specifically zinc protoporphyrin IX (ZnPP). tin protoporphyrin IX (SnPP), chromium
mesoporphyrin IX (CrbP) (Vreman rt 01.. 1993; Cook rt at., 1995; Marks rr d., 1997).
zinc deuteroporphyrin 1X 2.4-bis ethylene glycol (ZnBG) (Chernick r t d l . 1989; Vallier
e t d , 1991; Vreman et al., 1992) and zinc N-methylprotoporphyrin IX (ZnklePP) (De
\ latteis rt c r / . 1985) hletalloporphvrins have been used to test the hypothesis that CO
has a physiological role SnPP and ZnPP have been used to investigate a possible role for
CO as a vasodilator (Zakhary er id.. 1996) ZnPP (1-20 ph.1) also has been used to
demcnstr3te 3n apparent role for CO in LTP (Zhuo cf d.. P 3 ) and the inhibition of
depolarizat~on-induced glutamate release ( I O ~ L M ) by Shinornura ~ . r d. ( 19W)
Mrtalloporphyrins are also used in a clinical setting as HO inhibitors.
Hyperbilirubinemia remains the most frequent clinical problem pediatricians must deal
ivith during the newborn period. and under certain circumstances may cause severe brain
damage even in healthy term newborns. Certain metalloporphyrins have been exploited
therapeutically to decrease hyperbilirubinemia in the neonate (Qato and hlaines. P W .
L'alaes r.1 d.. 1994. Jlaninez ' I / . 1990) Tin rnrsop~rphyrin IS ( SnbIP) was recently
used to control severe hyperbilirubinemia in hll-term newborns SnhlP 1s a potent
inhibitor of HO activity and was administered by Martinez and co-workers to healthy
newborns to decrease hyperbilirubinemia. This pharmacological means of preventing the
development of severe hyperbilirubinemia eliminates the need for phototherapy and
reduces the length of time the infants are under clinical care. This method offers some
logical advantages over the current methods for controlling hyperbilirubinemia. such as
phototherapy and eschange transhsion, that are used only after the problem has become
severe.
Several investigators have shown that metalloporphyrins are not specific
inhibitors of HO, but also inhibit NOS and sGC (Luo and Vincent, 1994; kletfert tcr d..
l994; Grundemar and Ny. 1997) Based on these findings. the conclusions reached by
investigators using rnetalloporphyrins to establish a physiological role for CO in
biological systems, in which NOS and sGC are also active, have been criticized
In contrast, Zakhary el d. ( 1996) have reported that SnPP was 10 times more
potent in inhibiting HO-2 than NOS or sGC and based on this finding have used SnPP as
R selective agent to study CO-induced vasodilation
D. Statement of aims and objectives.
In the past. it has been demonstrated that HO can modulate selective cytochrornr
PJSO isozyme activity by degrading the heme moiety of selected PJjOs. NOS is a
molecule closely similar to P-150 and its heme moiety mav also be subject to dewadation - by HO Furthermore, it was recently observed that when HO was incubated together with
cycloosygenase, a hemoprotein. cyclooxygenase activity was decreased. Co-incubation
with SnhlP prevented the reaction The results were interpreted as showing that HO. or a
product derived from heme degradation played a role in modulatins the activity of
cvclooxygenase. Thus. the activities of NOS and sGC may be modulated by degradation
of their heme moieties by HO
In order to study the interactions between HO and sGC or NOS. a selective
metalloporphyrin inhibitor of HO is required. The hypothesis which we wish to
investigate is the following: "A metalloporphyrin when used at an appropriate
concentration will hnction as a selective inhibitor of HO " To test this hypothesis ive
compared a range of concentrations of five metalloporphyrins as inhibitors of both 30s
and HO. Two of the most selective inhibitors of HO were selected and the effects of
these two metalloporphyrins on basal and SNAP-induced sGC activity were assessed
Chapter Two
SELECTIVE INHIBITION OF HEME OXYGENASE BY METALLOPORPHYRINS
.A. lntrodrlction
Ln the present study. the objecti~c was to test five rnetalloporphvrins that haw
been shown previously to inhibit HO activity. for their ability to selectivelv inhibit HO
relative to UOS and sGC activities For each rneta1loporphl;rin. a concentration lvns
determined that inhibited HO activity, without inhibiting h O S activitv The two most
selective inhibitors. CrMP and ZnBG. were hrther studied to determine whether they
atyected basal or 3'-nitroso-IV-penicillamine (SNAP)-induced sGC activitv it was found
that CrMP. at a concentration of 5 phl, was a selective inhibitor of HO activity and
appeared to be the most usetLl HO inhibitor based on the studies conducted
B. Materials iind Methods
B. 1 Drugs and solutions.
Erhvlenediaminetetraacetic acid (EDTA) disodium salt. hemin. ethanoiamine.
bovine serum albumin (BS A), leupeptin. Amberlite IRP-69, L-N AbE. heparin. cGhlP.
NADPH and SNAP were obtained from Sigma Chemical Co (St Louis, hlO) Tris-HCI
and 3-isobutyl- l -methylxanthine were purchased from ICN Biomedicals Inc. (Costa
Mesa, CA). CrMP, ZnSG, ZnPP, SnPP, and ZnMePP were purchased from Porphyrin
Products inc. (Logan, UT). rUI other chemicals were at least reagent grade and were
obtained from BDH Inc. (Toronto, ON). Stock solutions of methemalbumin ( 1.5 mi1
hemin and 0.1 5 mM BSA) and of each of the five metalloporphyrins ( 1.0 mM) were
prepared as previously described (Vreman rt d., 1993). Briefly, hemin or
metalloporphyrin was dissolved in 0.5 ml of 10Y0 (wiv) ethanolamine. BSA dissolved in
1 ml of deionized water was added to the hemin solution only The volume was made up
to 7 ml and slowly adjusted to pH 7 4 with 1 h 1 HCl and vigorous stirring. The final
?:c!urne f i r each stmk solut io~ was adjusted to ir! ml with deionized water The
mrtalloporphy-in vehicle was prepared as above without the addition of any
metalloporphyrin. The methemalbumin and metalloporphyrin stock solutions were
prepared with the laboratory lights turned off and were stored at -20°C for up to 1 month.
B.2 Preparation of ~ ~ b ~ e l l ~ l i ~ r fractions of rat brain and lung.
Adult male Sprague-Dawley rats (300-3SOg) were obtained tiom Charles River
Canada inc (Montreal. Q C ) Rats were given ~ l t l lihitrrm access to Ralston Purina
Laboratory Chow (no. 500 I , Rrn's Feed and Supplies. Ltd.. Oekville. O N and water
All animals were cared for in accordance with the principles and guidelines of the
Canadian Council on Animal Care, and the experimental protocol was approved by the
Queen's C1niversity Animal Care Committee For measuring HO and NOS activity. each
rat was killed by decapitation. and its brain was excised and weighed For measuring
sGC activity, the rat was injected i . p. with heparin (3m& body weight) and then 45 min
later anaesthetized with sodium pentobarbital (93 mykg body weight). The lungs were
perfused with 40 ml of ice-cold phosphate buffered saline (PBS) by inserting a syringe
into the pulmonary artery through the right ventricle. The left atrium was cut to allow
outflow of perfusate. During perfusion, the lungs were continuously inflated and detlated
with an air-filled syringe inserted into the trachea. Perhsion was continued until the
lungs were cleared of all blood and appeared white in colour The lllngs were excised
and rinsed with I0 rnl ice-cold PBS.
:Llicro.somd i+acriori of Rar Hruirr jbr hletr.s~rri~rg HO dcti~ity . .A homo yenat e
( 1 ~ O , O . wiv) of brains pooled from four rats was prepared in ice-cold. HO homogenizing
buffer (20 mhl KH:PO.:, 1 3 mM KC! and n ! 0 mhl EDTA; adjusted to pH 7 4 at -W
with I M KOH) using a Potter-Elvehjern homogenizing system with a ~ef lon ' pestle The
n~icrosomal fraction of the rat brain hornogenate was obtained by centrifugation at 10.000
u g for 20 min at J'C. followed bv centrifugation of the supernatant at 100.000 K ,c for 60
nlin at 4°C. The 100.00ir u g pellet (microsomes) was resuspended in 100 mhl KH2P04
butfer (adjusted to pH 7 4 with I bl KOH) using a Potter-Elvehjern homogenizins svstem.
The rat brain microsomal fraction was divided into equal aliquots, placed into
rnicrocentrihge tubes. and stored at -80°C for up to two months Protein concentration
of the microsomal fraction was determined by the Biuret method (Gornall rr trl. 1949).
which was modified as described previouslv (Marks el trl, 1997).
( > w . s o l i ~ * brtrcrroti c ~ f k r t Brtrirr $)re ilt~.ccsriri~g IVOS d cririty. Brains from four
rats were pooled and homosenized in ice-cold, NOS homogenizing buffer (50 rnM
HEPES. I mhl EDTA and 10 pyrni leupeptin; adjusted to pH 7 4 at 4°C with I 0 41
NaOH) with a Potter-Elvehjem homogenizing system. in a ratio of 1 y tissue: I . I mi
homogenizing buffer. The cytosoiic fraction was obtained by centrihgation of the
homogenate at 10,000 u g and then 100,000 x g, as described above for preparation of the
microsomal fraction of rat brain. The 100,000 x ,g supernatant (cytosol) was divided into
equal aliquots, placed into microcentrifbge tubes, and stored at -80°C for up to two
months Protein concentration of the cytosolic fraction was determined by the Biuret
method as described above
(:~voso/ic Frtzctror~ oflitit l.iuig-sji)r.Llc.ti.sitrnig.s(;C'.4~~tir~it~~. Lungs from one rat
were homogenized in ice-cold sGC homogenizing buffer (50rnM Tris HCl (pH 7 1).
5mM MsCI:. 1 mM EDTA and 5mM benzamidine HCI) to produce a 20% (w/v)
homogenat r The cvtnsolic fraction \\as ohtamed hv di tfrrential wnlr ih~gat ion o f t he
hornogenate at 10,000 s g and then 100.000 x g, as described above for the preparation of
the microsomal fraction of rat brain. The 100,000 x g supernatant (cytosol) was divided
into equal aliquots. placed into microcentrifhge tubes, and stored at 4°C for less than 24
hours Protein concentrarion was determined using bicinchoninic acid (Smith ct d..
1985). which was obtained as pan of a protein assay kit from Pierce Chemical Co
(Rockfbrd. IL)
B.3 Measurement of HO enzymatic activity in microsomal fraction of rat brain.
HO activitv in the microsomal fraction of rat brain homoyenate was determined
by measuring the rate of CO formation during the NXDPH-dependent oxidation of heme
(Vreman and Stevenson, 1988) and modified as described by Cook et d. ( 19%) For
each ZnPP. CrMP, ZnBG, SnPP and ZnMePP sample, a reaction mixture consisting of
100 mhl KH2P04. 0.2 mg of microsomal protein and methemalbumin (final
concentration of 25 p M hemin and 2.5 ph.1 BSA), was pipetted into six 3.5-ml amber
glass vials (Chromatographic Specialties Inc., Brockville ON). The following
concentrations of metalloporphyrin were added to individual vials: 0 . I 5 . 0.5. I . 5 . 5 . 1
and 50 pM. Each vial was sealed with a silicon-~eflon' septum and a screw cap
(Chromatographic Specialties Inc.). and then was preincubated for 5 min in the dark at
3 7 O C . in a shaking water bath NADPH (0.5 mhl) then was added to each vial. the
headspace zas was displaced with CO-free air and the incubation was continued for
another 15 rnin. The reaction was stopped by placing the vial on pulverized dry ice t -
78°C). where it remained for 30 rnin until the headspace gas was analyzed. For each
metalloporphyrin reaction set. CO production was corrected for the CO produced in a
blank reaction vial that contained a rnetalloporphvrin concentration of 50 LN. but no
NADPH. To determine total HO activity in the microsornal fraction of rat brain. a
reaction vial was prepared that did not contain metalloporphyrin or vehicle (There was
no inhibition of HO activity 111 a reaction set containing rnetalloporphyrin vehicle
equivalent to a range of 0 15 to 50 pbl metalloporphyrin.)
CO in the headspace gas was quantitated by gas-solid chromatography using a
I3X molecular sieve as the stationary phase and a sprctrophotometric detector (RG.4-3:
Trace Analytical Inc.. Pvlenlo Park. CX) set at 254 nm to quantitate the Hg vapour formed
from the reaction of CO with HgO The headspace gas was injected onto the gas
chromatographic column via the carrier-gas stream of CO-free air (35 mlimin). ~bhich
was directed througl~ the headspace for 50 sec Analysis of the headspace continued over
the n e a I10 sec The amount of CO in the hradspace gas was determined by
interpolating the peak area of the chromatographic signal on the linear CO standard curve
( 10 - 170 pmol CO), which had a correlation coeficient of 0.999 (iV=9 determinations).
The rate of formation of CO in the microsomal fraction of rat brain hornogenate was
expressed as nmol CO formed/mg protein/ hr. NADPH-dependent formation of CO was
calculated by subtracting the value for CO produced in samples not containing NADPH
(blank) from the value for CO formed in samples containing NADPH.
B.4 Measurement of NOS enzymatic activity in cytosolic fraction of rat brain.
NOS activitv in the cytosolic fraction of rat brain hornogenate was determined
using a modification of an established procedure (Bredt and Snyder. 1990) that was
optimized tbr the hippocampus of the guinea pig (Brien et trl . , 1995. Kimura tv d..
1996). A 100-111 volume of reaction buffer (50 mhl HEPES. 1 mhl EDTA. 1 25 mR1
CaClz and 2 mM NADPH; adjusted to pH 7.4 at 37°C with 1.0 M NaOH) and a -3i-ld
volume of cytosol containing 0.525 mg protein were added to six test tubes. The
following metalloporphyrin concentrations. 0 15. 0.5. 1 5 , 5. 15 and 50 phi. were added
to individual test tubes in a volume of 100 p1 The samples were preincubated for 5 mi11
at 37°C. after which a S p I aliquot of an aqueous solution containing 35.000 dpm L-
["c] argir~ine (hew England Nuclear -- Xlandei. Guelph. OW) and I SO p?rl nun-
radiolabelled L-arginine was added. The samples were incubated for 15 min. and the
reaction was stopped by the addition of ice-cold 'stop' bufier (20 mM HEPES and 2 mhl
EDT.4: adjusted to pH 5 . 5 at 4°C with 1.0 M NaOH) Experimental blanks were
prepared by adding the 'stop' buffer to a sample containing reaction buffer, cytosolic
protein and a metalloporphyrin concentration of 50 pM. before the addition of L-['"c]
aryinine and incubation at 37°C for 1 5 min To determine total NOS activity. samples
were prepared that did not contain metalloporphyrin or vehicle. (There was no inhibition
of NOS activity in samples containing metalloporphyrin vehicle equivalent to a
concentration range of 0.15 to 50 pM metalloporphyrin.)
.Af er incubation. each sample was subjected to ion-exchange chromatography
using an Amberlire IRP-69 (sodium ion form) column (4-cm high. 0 4-crn diameter. and
0.75-ml volume) with a silanized glass wool plug .A 1 0-ml volume of deionized Lkatrr
was added to the column to elute L-[l"~]citrulline. The eluate was collected and mixed
with 10 ml of scintillation cocktail .A 35 -p I aliquot of L-[ ' ' '~lar~inine solution was
mixed with ! r? rnl scintillaticn cccktail to dewmine the tctal r3dicacti~ity 2dded :c a x ! ?
sample. Radioactivity was quantitated by liquid scintillation spectrometry. and the
radioactivity of the individual samples was corrected for experimental blank NOS
activity was espressed as nmol L-["c] citrulline formedimy proteini h r In a prelimman
experiment. incubation of rat brain cytosol with 1 m M L-NAME. an inhibitor of UOS.
1 .
inhibited Ca- -dependent formation of L-["c] citrulline in rat brain cvtosol by 97 b r
0 49.0 ( r ' V 4 )
B.5 Measurement of sGC enzymatic activity in cytosolic fraction of rat lung.
sGC activitv was measured in the cytosolic fraction of rat lung homoyenate using
a modification of the procedure described by Liu rr td . ( 1993). Rat lung cytosolic
fraction (25 p1) and 50 p1 of a reaction mixture ( 100 mM Tris-HCI (pH 7 4). 6 mhi
StgCIZ. 2 nihl hsobutyl- 1 -methyluanthine. I 0 mbt cGhlP. 1 mhl L-cyteine and 1 mg
BSNml) were added to each of five test tubes. The following metalloporphvrin
concentrations, 0.15, 0.5, 1.5, 5 and 15 pM, were added to individual test tubes in a final
volume of 10 pl. For the basal sGC activity experiments, the reaction was started by the
addition of 25 yl of an aqueous solution containing 500,000 dpm of ['HIGTP (New
England Nuclear-Mandel) and 200 pM non-radiolabelled GTP. The samples were
incubated at 30°C for 30 min and the reaction was stopped by adding 100 p1 of ice-cold
10% (w/v) trichloroacetic acid and putting the tube on ice. For the stimulated sGC
activitv experiments. the reaction was started by adding 100 phl SNAP. followed by 25
pI of ['HIGTP solution for a final volume of 120 p1. The reaction was carried out as
described for the basal sGC activity experiments. Experimental blanks were prepared by
a d d i r ~ ~ I-a1 lung cylusuiic ii-a1
metalloporphyrin concentrat
determine total sGC activity
rim that i ud been 'ooiittd for 5 min. ~,ttiiction nisrur t : and a
on of I S piLI ro a vial before starting the reaction. To
samples were prepared that did not contain
metelloporphvrin .After incubation was completed. approximately 1.000 dpm of
[ ' " C ] C G ~ I P (New England Nuclear-blandel) was added to each sample to provide an
index of column etficiency The precipitated proteins were removed from the acidified
sample by centrifugation at 11.000 s g for 3 min The supernatant was applied to a
column (4-cm high and 0 4-cm diameter) containing 0 5 5 of neutral alumina that was
equilibrated with 5*'0 (wiv) trichloroacet~c acid Each column was washed with I ml of
5% (wiv) trichloroacetic acid. followed by 3 ml of water and fmallv by 0.75 rnl of 200
m M sodium acetate (pH 6.0) [ 'H]CG~IP and ['' 'C~CGILLP were eluted with I ml of 200
mM sodium acetate (pH 6.0). The eluate was collected, mixed with 8.0 mi of
scintillation cocktail and quantitated by liquid scintillation spectrometry. Radioactivitv
measured for each sample was corrected for crossover of '"c radioactivity into the 'H
channel during liquid scintillation spectromeiry and for the recovery of ['"c]cG~.IP from
the column, which ranged from 60-70% The amount of cGMP formed during the
incubation was calculated from the specific radioactivity of the ['HIGTP (20 9 Ci/rnrnol)
and corrected for experimental blank sGC activity was expressed as prnol cGMP
formed/mg p r o t d h r ,
B.6 Data analysis.
The activity of HO, NOS and sGC following incubation with each concentration
of metalloporphyrin was expressed as percent of total activity. measured in the absence of
metnlloporphyrin. The data are presented as group means k SD of four tissue
preparations from ditierent afiimals. unless otherwise stated. Parametric statistical
analysis of the data was conducted by repeated-measures, one-way analvsis of variance
For a statistically sigificant I.' statistic (pc-0.05). a post hoc Newman-Keuls test was
conducted to determine which experimental groups were statistically different (p- O 0 5 )
C. Results and Discussion
The metalloporphyrins used in this study have been shown previously to inhibit
HO activity (De Matteis r i tr/. , 1985; Chernick rt d., 1989; Vreman ct LI/. . 1992. 1993 )
The use of metalloporphyrins in investigating a physiological role for HO has been
criticized because some metalloporphyrins have been shown also to inhibit %OS and sGC
activity (Luo and Vincmt. 1994: Meffert rr d.. 1994. Grundemar and %\iv. I W7) In
contrast. Zakhary L.1 (I/. ( 1996). in a study demonstrating HO-derived CO as a vasodilator.
reported a dose of SnPP that appeared to selectively inhibit HO relative to NOS Thus.
the potential exists for finding a metalloporphyrin and/or a metalloporphyrin
concentration that would inhibit HO activitv without inhibiting NOS or sGC activity i n
the present study. five metalloporphyrins were investigated to detrrnminr whether there is
a metalloporphyrin andlor a metalloporphyrin concentration that can be used as a
selective inhibitor of HO relative to NOS and sGC.
HO activity in rat brain rnicrosornes and NOS activity in ra
3.3 0.3 nmol CO formed!mg protrinihr (n=6) and 5 0 2 I . I nmo
t brain cytosol were
formedlmg proteidhr (n=4), respectively. .All five metalloporphyrins, viz. CrMP. ZnBG.
SnPP. ZnPP. and ZnMePP. inhibited both HO and NOS activity (Figures 2.1-2 3 )
However. there was a concentration for each metalloporphyrin. at and below which only
HO activity was inhibited. The metalloporphyrin vehicle did not affect HO or NOS
activity (data not shown).
CrMP
SnPP
I
0.1 I i'o 1 A0
Metalloporphyrin Concentration (pM)
Figure 2.1 : hrhihiliorr of HO tord NOS trcfivity hy C'nLP md 91PP. Concentration- response curves for HO (r ) and NOS (. ) activity in rat brain microsomes and cytosol. respectively, were obtained in the presence of CrMP and Sr.PP. The data are presented as group means k SD (N=4). The letter 'a' denotes the maximum selective inhibitory ( M I ) concentration at or below which there is selective inhibition of HO activity, with no effect on NOS activity; NOS activity for lower concentrations of metalloporphyrins were not ditierent from the MSI concentration. Group means with different letters are statistically different from each other, p<O.O5.
ZnBG
ZnPP
I
0.1 1 i o 160
Metalloporphyrin Concentration (pM)
Figure 3.1: I d ~ i h i t i o ~ t oj. H O turd ;VOS ~rclivi& hj. ZIIBG mid Z~rl'l'. Concentration- response curves for HO ( r ) and NOS ( . ) activity in rat brain microsomes and cytosol. respectively, were obtained in the presence of ZnBG and Z n P P The data are presented as group means + SD ( N = l ) . The letter 'a' denotes the maximum selective inhibitory (MSI) concentration at or below which there is selective inhibition of HO activitv. with no effect on NOS activity; NOS activity for lower concentrations of metalloporphyrins were not different from the MSI concentration. Group means with different letters are statistically different from each other, p<O.OS.
Metalloporphyrin Concentration (pM)
Figure 3. -3. l ~ i l ~ r h r r i o ~ i of HO toid W S i l ~ ~ r ~ r f y hrv ZtuLId'i'. Concentration-response curves for HO ( - ) and NOS ( m ) activity in rat brain microsomes and cytosol. respectively. were obtained in the presence of ZnhlePP The data are presented as group means i SD (X=l) The letter ' a ' denotes the maximum selective inhibitory (MI) concentration at or below which there is selective inhibition of HO activity with no effect on NOS activity; YOS activity for lower concentrations of metalloporphyrins \yere not different from the MSI concentration. Group means with different letters are statistically different from each other, p<0.05
In comparing the metalloporphyrins as selective inhibitors of HO. the first step
was to determine a maximum selective inhibitory (MSI) concentration for each
metalloporphyrin. at or below which there is inhibition of HO. with no effect on NOS or
sGC activitv (Table I ) The next step was to compare the percent inhibition of HO
~ctivity st h e M I concentration t'or each rnetallnpnrphvrin - \ l ~ > , the percent inhihition
of HO activity at concentrations N o i d and 10-fold lower than thc MSI concentration was
compared (Table I ) . This latter comparison was made because in an experiment. a
concentration lower than the MSI concentration, at which the metalloporphyrin becomes
non-selective. would normally be used. Based on these criteria. the inhibition of HO
activity was lowest and/or declined most rapidly with metalloporphyrin concentration fur
ZnMePP. ZnPP and SnPP (Table I ) Thus. it was concluded that CrMP and ZnBG at
concentrations at and below 5 pbl were selective inhibitors of HO activitv relative to
UOS activity in rat brain.
It is interesting to compare the above data (Figures 1.1-2 3 ) with that of bletfen cJr
d. ( 1994). These workers concluded that CrMP and ZnPP, but not tin mesoporphyrin or
ZnBG, inhibited NOS in the rat hippocampus Based on these findings. it was
emphasized that some metalloporphyrins are non-selective and would therefore not be
useful in biological studies involving CO. The concentration of metalloporphyrin used in
these studies ranged from 10 to 100 pM. while in the present study metalloporphyrin
concentrations from 0.15 to 50 pM were used. The results of Metfen r / d. ( 1994).
showing that CrMP and ZnPP inhibited NOS at concentrations of 10 pPvl and higher are
in agreement with the results of the present study. In contrast, it was found in this study
Xtetalloporphyrin " &IS1 inhibition of HO Activity (' O )
concent ration At blS1 .At 1/3 MSI .-it l l 10 \IS1 (PW concentration concentration concentration
ZnblePP 0.5 5 6 1 4 2 29 f 27.1 16 -C 20 9 "
Table 1 Comparison of metalloporphyrin inhibition of HO (N 4). " Maximum selective inhibitory (MSI) concentration, concentration at or below which there is selective inhibition of HO activity with no effect on NOS activity 11 Vote; the data for this concentration is not shown on the ZnhdePP graph in Figure 2. -3
that ZnBG inhibits NOS at concentrations higher than 5 pM. Tin mesoporphyrin was not
tested in the present s tudy The message that emerges in comparing the data is that to
achieve selectivity of metalloporphyrin-induced inhibition of HO versus NOS. the
concentration used is critically important and must be kept at or below 5 pM.
To characterize further the selectivit). of CrhlP and ZnBG as inhibitors of HO. the
c t k i t of the iuo rncitalloporphyrins on basal and SKIP-induccd ;GC actiiit; ;\as
determined for metalloporphyrin concentrations similar to those used for the study of HO
and NOS activity The basal and SNAP-induced sGC activities in the rat lung cytosol
were 12 1 i 56 (n=6) and 2059 2 82 1 (n=-3) pmol cGMP forrnedhg proteidhr.
respectively. Thus, the addition of 100 pM SNAP produced approximately a 1 Mold
increase in sGC activitv from basal level Neither CrMP nor ZnBG had any etfect on
basal sGC activity (Figure 2 4) at the concentrations tested Ho~tevrr . ZnBG elevated
SNAP- induced sGC activity (Figure ' S ) , which is consistent with reports from
investigators who found that certain rnetalloporphyrins. such as cobalt protoporphyrin.
enhance NO-induced sGC activity (Dierks r / trl.. 1997) CrMP had no rtfect on SNAP-
induced sGC activity for the concentration range tested, a range that included the %IS1
concentration of 5 pM, at and below which C r b P selectively inhibits HO activitv. with
no efTect on NOS activity Thus. CrhlP was found to be the most selective and useful
inhibitor of HO activity compared with NOS and sGC activities in rat brain and lung.
To our knowledge. no particular structural feature of metalloporphyrins has been
identified that allows the prediction of the efficacy of the compounds to inhibit HO or
NOS. For sGC, a mechanism to explain. at least in part, the interactions of
metalloporphyrins with this enzyme has been proposed by Serfass and Burstyn ( 1998).
Figure 2.4: Lflects qf ZttBC; mtd C'rhlP ujl Ekl.snl sCX' nczivitj. Concentration-response curves for basal (N=4) sGC activity were obtained for ZnBG ( - ) and CrMP ( m ) . The data are presented as group means k SD. Group means with different letters are statistically different (p<0.05) from other group means within the same concentration-response curve.
1401
120- w I-
> I-- 100- w
3 80-
601
* 40- s 20-
0
T T
-
I 1 1
0.1 I I 0 100
Metalloporphyrin Concentration (pM)
Metalloporphyrin Concentration (pM)
Figure 2.5 : Ii&fec~.s of ZnBG mid CrhlP or1 SIVA P-btdtrcrd sGC1 trctivil).. Concentration-response curves for SNAP-induced (N=3) sGC activity were obtained for ZnBG ( 7 ) and CrMP (. ). The data are presented as group means 2 SD. Group means with "a" are not statistically different (pC0.05) From each other. Group means labeled with "b" are statistically different @<0.05) to all other group means. Group means labeled with "c" are not different to means labeled with either -'a" or "b" within the sanle concentration-response curve.
These investigators postulate that a key requirement for sGC activation by
metalloporphyrins is the absence of a bond between a proximal protein-histidine and the
metal in the porphyrin. This proposed mechanism is based on the observation that
activation of sGC by NO has been attributed to binding of NO to heme iron with
concomitant breaking of a bond between a proximal protein-histidine and iron. In their
itudt. the n ~ ~ n i n ~ a l acti\ .ati(~n (3f vCrC hc. ZnPP i.; ilttrihtltect t o the likelihood that the h o n d
between a proximal histidine and Zn atom is intact On the other hand. the marked
activation of sGC by SnPP is attributed to the absence of a bond between a proximal
histidine and the Sn atom Activation of sGC by SnPP. as demonstrated by Serfass and
Burstvn (19%). provides funher rationale for selecting CrhlP from among the
metalloporphyrins tested, as the most selective metalloporphyrin to elucidate the
physiological role of HO
Luo and Vincent (199.1) and Grundemar and Uv ( 1997) have concluded that
ZnPP. SnPP and ZnBG cannot be used to establish a messenger role for CO This
comment is based on the fact that these metalloporphyrins inhibit sGC in addition to HO
Ho~ever . in their study, the concentration of these metalloporphyrins ranged from 10 to
100 pM for sGC inhibition. This conclusion requires reconsideration in light of our data
which demonstrates that concentrations of metalloporphyrins less than 10 pM can inhibit
HO activity without inhibiting hOS and sGC activities. Thus, carehl exploration of
concentration-response relationships with a variety of metalloporphyrins potentially can
lead to the identification of an appropriate selective inhibitor for the biological model
being used. This conclusion is reinforced by the results of Zakhary er tri. (1997). who
used HO-2 knockout mice and SnPP to demonstrate that CO plays a role in NANC
relaxation evoked by electrical field stimulation of mouse ileal segments in wild-tvpe
mice. SnPP partially inhibited NXNC relaxation. However. in mice where the gene for
HO-2 had been deleted, SnPP did not affect NANC transmission.
There is considerable interest in the use of metalloporphyrins to inhibit HO in the
treatment of juvenile jaundice (Valaes et 01.. 19%; Qato and Maines. 1985) CrbIP and
ZnBG appear to ire promising candidates hecause of their high potency to inhibit HO 3.;
demonstrated in the present and other studies (Vreman ~~r t r l . . 1908). good oral absorption
(Vallier el c t l . 1991; Vallier ~ j r d . 1993). resistance to metabolism by HO. and inability
to upregulate HO- 1 in cell culture (Zhang, Contag, Stevenson and Contag, personal
sornrnunicatian). >loreover. CrhlP has the additional advantages of nut distributing
across the blood-brain barrier and being photochemically inactive. .Although ZnBG is a
photosensitizer. the potential low doses required for therapeutic use, due to its high
potency as a HO inhibitor, may restrict its photoreactivity For the above reasons.
therapeutic and toxicological studies of CrklP and ZnBG are warranted.
In summary, of the five metalloporphyrins tested. CrMP and ZnBG inhibited HO
to the greatest extent at and below the concentration for which there was no measurable
inhibition of NOS activity Furthermore. CrhlP was found to have no erect on basal or
SNAP-induced sGC activity, unlike ZnBG, which enhanced SNAP-induced sGC activity
Thus, in this study, CrMP, at or below a concentration of 5 pM, was found to be a
selective inhibitor of HO relative to NOS and sGC, in rat brain and lung The hypothesis.
which we set out to test, was the following: "A metalloporphyrin when used at an
appropriate concentration will function as a selective inhibitor of HOW. Clearly the
hypothesis has been shown to be correct. In other studies using different biological
models, it will be necessary to determine the concentration of CrMP or other
rnetalloporphyrins that will selectively inhibit HO activity without inhibiting NOS and
sGC activities.
Chapter Three
FUTURE DlRECTIONS
.A common feature of biological regulatory systems is that they themselves are
subject to modification by multiple input signals These highly integrated h n c t ~ o n s are
controlled bv multiple variables in the case of respiration. for example. blood CO: is the
major consideration but pH also plays a role. In the autonomic nervous system. it has
long been established that the release of the major transmitters is subject to autoinhibiton'
feedback mediated bv presvnaptic acetylcholine and norepinephrine receptors but many
other substances act pres)maptically as well. As CO and NO act on similar regulatory
systems. a naturally arising question is whether there are interactions between NOMOS
and C O H O systems.
On theoretical considerations alone one could build a case for HO altering NOS
nctivitv because heme. the HO substrate. is an essential prosthetic group of \OS
Evidence contributing tc, the reciprocal regulaton: interactions be twen CO and NO has
been reported where endogenously derived NO may mediate the induction of HO-I in
vascular SMCs (Durante and Schafer, 1998) Thus. three structurally dissimilar NO
donors induced HO-I sene expression in vascular SbICs. Moreover. the same
investigators demonstrated that blockade of endogenous NO production also blocked the
induction of HO-1 gene expression in vascular SMCs (Durante rt a / . , 1997). Other
studies in the vascular endothelium led to similar results (Motterlini rr o/.. 1996. Yee rr
d., l996).
Further evidence in the field of immunology has been reported for the interaction
between HO and NOS. Macrophages use agents such as NO to defend the host organism
against invading microorganisms NO also evens effects against normal cells located
proximally to activated macrophages resulting in tissue damage and intlammation.
Turcanu rr d. ( 1998~1. 199Sh) have reported HO- 1 modulation of NOS in macrophages in
situation.; nt' widative .;tre.;.;% and suggest !!!at HO may XI as a 5hc.n-term anti-
inflammatory asent and limit tissue damage under circumstances in which YO
hyperproduction leads to toxic effects. These workers suggested that HO inhibits NOS
by degrading its heme cofactor and that it might also reduce c / e trow) NOS synthesis by
decreasing the cellular heme levels (Turcanu rt t r l . . l998h) This postulate is based on the
following Bone marrow-derived macrophages were treated with lipopolysaccharide
(LPS) in order to induce NOS and NOS activitv was assessed bv NO production. When
HO- I was induced in bone-derived niacrophagrs for 24 hrs prior to LPS treatment. UOS
activity, as assessed by NO production, was decreased. The anti-intlarnmatory effects of
HO- I have also been characterized in situations such as experimental arthritis and acute
pleurisy (Willis rt d. 1996). HO was also shown to assume a protecti\.e role in
xenograft endothelium during accommodation (Bach rt tzl. . 1997). Further evidence
contributing to the interaction between the HOKO and NOSMO systems has been
reported in the vascular endothelium it was shown by White and Marietta (1992) that
CO inhibits NOS activity by binding to the heme moiety of the enzyme.
Further evidence implicating a potential interaction between the HO and NOS
systems and a possible role for HO modulation of NOS lies in the marked similarities in
the location of HO and NOS. Both isozymes of HO (HO-I and HO-2) along with NOS
and sGC were shown to be co-localized in select neurons of the brain using
imrnunohistochemical analysis (Yincent and Maines, 1994) I t was found that many
neurons expressing HO-2 correspond to those known to express high levels of sGC ( a
hemoprotein) as well In other studies in blood vessels. HO-2 and NOS displayed the
same localization in endothelium and adventitial neurons: in the intestine. HO-2 and
neuronal W S were both Iocdized to the rnyenteric plexus Due to the cc-localiza!!c\n (3'
HO with these hemoprotein enzymes. HO is ranted access to other sources of heme
~vithin these cells and thus. provides further rationale for the hypothesis of HO
modulating NOS and sGC activity
In our present studies. CrMP (5ph.l) has been shown to inhibit HO wthout
afiectiny NOS or sGC This will facilitate testiny the hypothesis that HO may regulate
the activity of NOS and sGC by degrading the heme moieties of these enzymes. similar to
the manner in which HO has been shown to degrade the heme moiety of certain
cvtochrome P150 isozyrnes We propose ro accomplish this by co-incubating sGC or
NOS with HO and then measuring the change in enzymatic activity followiny HO
exposure in addition to the broken cell preparations described above, we would measure
the effects of 140 on NOS and sGC activity in intact tissues .A selective HO inhibitor is
required to demonstrate whether any eflects seen in NOS or sGC activity are in b c t due
to HO or some other aspect of the reactions.
One of the criticisms of CO as a signaling molecule is that it has a lower potency
than NO. However, it has been recently shown that CO, in the presence of YC- I . can
stimulate sGC to levels similar to that observed with NO. Thus, the search for an
endogenous YC-1 analog has become increasingly important to CO research in order to
redefine the physiological relevance of CO. Certain structural characteristics have been
sugested by Sharma et ~ 1 1 ( 1999) to be responsible for the stirnulatory actions of YC- 1
These workers attribute the sGC-activating abilities of YC-I to the fact that it has two
nitrogen groups, which. according to Sharma rt of. are responsible for coordinating with
ihe heme iron by donating a non-bonding pair of electrons This structural feature along
wi th its hydrophobic nature are prcposed to acccun! f ~ r .;GC ~timulatinn hy YC-I Both
biliverdin and bilirubin possess four nitrogen groups and are hydrophobic The
poseibilitv exists that either of these compounds may serve as an endogenous YC-1
analog and we propose to compare the effects of biliverdin and bilirubin with the rtfects
of YC- I on sGC in the presence and absence of CO
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