REGULATION OF CALCIUM STORES IN NORMAL AND DIABETIC ...
Transcript of REGULATION OF CALCIUM STORES IN NORMAL AND DIABETIC ...
REGULATION OF CALCIUM STORES IN NORMAL
AND DIABETIC ENDOTHELIAL CELLS
by
SHANKAR CHITTARANJAN SANKA, M.B.B.S.
A THESIS
IN
PHYSIOLOGY
Submitted to the Graduate Faculty of Texas Tech University Health Sciences Center
in Partial Fulfillment of the Requirements for the Degree of
MASTER OF SCIENCE
Advisory Committee
Raul Martinez-Zaguilan, Chairperson Narine Sarvazyan
Sandor Gyorke
Accepted
Dean of thj^/Graduate School of Biomedical Sciences Texas Tech University Health Sciences Center
December, 2000
"^ ^5 ACKNOWLEDGMENTS
/Jo . ^ o3
n ^ > I would like to take this opportunity to thank all the people responsible for making
my life a blessing. My parents, who were always patient, always believing, enough to let
me take a detour in life to become more "professionally qualified," thank you all your
support throughout the years in school. My uncle, aunt, my cousins, for all the support,
emotional, financial and physical, you will never comprehend how much your support
has meant to me all these years, and it will take me many lifetimes to repay all that you
have done for me. And last and not the least, my sisters and their husbands. They
listened to me crib, complain, cry, shout, and above all confide in them during frustrating
times, when I thought I would quit. They stood by me and showed me how important it
was to have siblings that cared for one another and even after all these years, I could tum
to them for anything and everything. My nephews and niece, who kept reminding me of
how beautiful the world is through their irmocent eyes, and all that I had to look forward
to in life.
Words cannot express what my mentor, Dr. Martinez-Zaguilan, has meant to me.
He is simply the most amazing person I have met. His motivation and energy never
ebbed. He will always be the "energizer buimy" and keeps going and going. His support
and direction have defined the future for me and I will never forget that. Maybe I will try
to a find way to repay him for all that he has done for me over the 2 years in his lab. I
would like to express gratitude to my committee (Drs. Gyorke and Sarvazyan) for being
patient with my erratic road to the completion of my master's.
ii
I am thankful for such a supportive lab, Gloria, who is simply the best, was an
epitome of guidance and support, Geraldine Tasby, who taught me to do calcium studies,
and all those who came and went, leaving me with good memories.
The 'Boys of Physiology," what can I say. Your support and friendship has meant
the world to me. I will always treasure your friendship. To all my friends who put up
with me in my "strange" times, thank you.
I came into the physiology department with one family and am leaving with two.
The Physiology family has meant a lot to me and all the other grad students. Thank you
all for the best two years of my life. Thank you Dr. Orem for having such a wonderful
family of physiologists with you.
And last but not the least, the Almighty God for watching me through all these
years, remembering me in times good and bad, even though I did not think it was
necessary to reciprocate the gesture. Please guide me for the rest of the journey.
Technical and Financial Support
I would like to acknowledge the technical support of Gloria Martinez, Geraldine
Tasby, and Defeng Luo. To Joe, who taught every single thing I know, this masters
would not happened without you. I owe you more than this one life. The work has been
supported by grants to RMZ from AHA (National) 9750558N and from the Texas Higher
Educafion Coordinafing Advanced Technology Program (ATP) #010674-034.
ni
TABLE OF CONTENTS
ACKNOWLEDGMENTS ii
ABSTRACT v
LIST OF FIGURES vii
CHAPTER
I. INTRODUCTION 1
Endothelial CeH Biology 1
Calcium Homeostasis -Overview 3
Pathophysiology of altered Ca ^ handling 5
Role of Endoplasmic/Sarcoplasmic Reticulum 5
Role of Mitochondria 8
Role of Nucleus 9
Role of Golgi Apparatus 10
Role of Endosomes and Lysosomes (E/L) 10
Rationale 11
Hypothesis 12
II. MATERIALS AND METHODS 13
Isolation of Microvascular Coronary Endothelial Cells... 13
CeU Culture 14
Buffers 14
Fluorescence Spectroscopy 15
iv
spectral Imaging Microscopy 15
Laser scanning Confocal Microscopy 18
Culture Preparation 18
In situ calibration of Ca ^ indicators 19
Study of Endosomal/Lysosomal Compartments 20
Immunocytochemistry 20
Data Analysis 21
Materials 21
III. RESULTS 22
Steady State cytosolic Ca ^ i[Ca^^f^') levels in normal and diabetic microvascular endothelial cells 22
Presence of Na^/Ca^^-exchanger in microvascular endothelial cells 22
[Ca^^P* studies in cell populations 26
[Ca^^]^^ studies in cell populations 29
Pharmacological studies in ceUs populations 30
Immunocytochemistry 33
Study of single vesicles 42
IV. DISCUSSION AND CONCLUSION 45
REFERENCES 50
V
ABSTRACT
Cytosolic Ca ^ ([Ca ]*' ) mediates many cellular ftinctions, e.g.. cell growth,
motility, secretion, etc. In many cell types, ion transport processes appear to be dependent
on metabolism of glucose for maximal activity. In certain cell types, a strict coupling
between glycolysis and the acfivity of Endoplasmic Reticulum Ca^"-ATPases (SERCA).
involved in regulating Ca ^ homeostasis, has been suggested. In diabetes, glucose
homeostasis is altered. We hypothesize that Ca ^ homeostasis in microvascular
endothelial cells from diabetic animals is altered due to a dysfunction of glycolysis
coupling the activity of SERCA. We further hypothesize that endosomal/lysosomal (E/L)
compartments exhibiting SERCA are involved in this dysfunction. Our data indicated that
agonist stimulation (ATP, vasopressin, angiotensin-II) elicited [Ca "] * increases
(independent of extracellular Ca ) that were larger in endothelial cells from diabetic than
from normal animals. Simultaneous measurements of [Ca ]' ' and Ca ^ in E/L
compartments ([Ca^^]^) using fluorescence spectroscopy, indicated that E/L
compartments released Ca ^ following agonist-stimulation. The magnitude of the Ca'*
release was significantly larger in microvascular endothelial cells from diabetic rats.
SERCA inhibitors elicited Ca ^ releases from E/L compartments in both normal and
diabetic models. The magnitude of the [Ca^^]^ release was however similar among
normal and diabetic cells. Immunocytochemical experiments demonstrated that 60% of
E/L compartments exhibited SERCA. These data indicate that (a) E/L compartments are
important for Ca ^ homeostasis in microvascular endothelial cells from both normal and
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diabetic models; (b) Ca ^ regulation in E/L compartments is different in cells from a
diabefic model, (c) the compartment involved in altered Ca'* homeostasis in diabetes is
unknown.
Vll
LIST OF FIGURES
1. Structure of macro and micro vessels 2
2. Overview of Ca ^ homeostasis in cells 4
3. Schematic illustration of a Spectral imaging microscope 16
4. Steady state [Ca l ^ levels in normal and diabetic microvascular endothelial cells 23
5. Microvascular endothelial cells exhibit Na /Ca^^ exchanger 25
6. The microvascular endothelial cells from a diabetic animal have greater increases in [Ca J' y' in response to ATP when compared their normal counterparts 27
7. The microvascular endothelial cells from a diabetic animal have greater increases in [Ca J' y* in response to vasopressin when compared their normal counterparts 28
8. Loading of fluorescent indicators to measure [Ca-"]'' ^ [Ca-*]^^, and (pH)' y simultaneously in endothelial ceUs 31
9. Increase in [Ca l' * is accompanied by decrease in [Ca-*]^^ 32
10. E/L compartments express functional SERCA 34
11. Microvascular endothelial cells express SERCA as demonstrated by immunocytochemistry 36
12. 60% of E/L compartments express SERCA 37
13. Microvascular endothelial cells express ryanodine receptors as demonstrated by immunocytochemistry 38
14. 70-80% of E/L compartments exhibit RyR 39
viu
15. Agonist induced Ca^' releases from E/L compartments is greater in normal ceUs compared to diabetic cells 41
16. Study of Single vesicles by Spectral Imaging Microscopy 44
IX
CHAPTER I
INTRODUCTION
Endothelial Cell Biologv
The endothelium lines the luminal side of a blood vessel, and acts as a signal
sensor and transducer. Because of its unique location, the endothelium is ideally situated
to sense changes within the circulation. It senses and reacts to changes in variety of
stimuli, including blood pressure, flow, sheer stress, and humoral factor concentrations
(Sage et al. 1991). These stimuli help the endothelium to maintain vascular tone and
structure through its communication with the vascular smooth muscle layer on the
outside. Through the presence of receptors, ion channels, and other membrane bound
structures, the endothelial cells function as sensors detecting specific changes in the
environment and helps maintain normal tone and structure of the blood vessels. It plays a
major role in vascular homeostasis by release of vasoactive compounds such as nitric
oxide (NO) and prostaclyclin.
The vascular smooth muscle is in close proximity with the endothelial cells
(Figure 1). The vascular endothelium releases diffusable factors that hyperpolarize hence
relax the vascular smooth muscle. The endothelial cells are capable of generating new
blood vessels, which enable wound healing and neovascularization, as in after an infarct.
This is particularly true in the coronary vasculature where the endothelial cells are of
utmost importance, as they are the only constituents of the microvasculature to the
cardiac muscle.
Calcium Homeostasis-Overview
The endothelial cells react to wide variety of ligands like brad>'kinin, adenosine
triphosphate (ATP), angiotensin-II, vasopressin, histamine, acetylcholine, etc. with an
increase in cytosolic Ca ^ ([Ca J ^ ) (Clapham et al. 1995). The increase in [Ca-^]'>' is
brought about by G-protein coupled receptors, that leads to activation of phospholipase-
C, and subsequent break down of phospho-inositol diphosphate (PIP2) into inositol
triphosphate(IP3) and diacyl glycerol (DAG). The IP3 causes the release of Ca-* from IP3
sensitive stores, and the DAG leads to activation of protein kinase C (Berridge et al.
1993; Mountain et al. 1999). So, it appears that Ca ^ plays an important role in the signal
transduction function of the endothelium. The cell maintains the [Ca^]'''" in the
nanomolar range for its normal physiological functions by striking a balance between the
entry and the exit of Ca " from the cytosol (Greger et al. 1989). The voltage and ligand
gated Ca ^ charmels control the entry of Ca ^ from outside the cell in response to
depolarization and ligands, respectively (Cheng et al. 1993). The IP3 sensitive and
ryanodine sensitive Ca ^ stores in compartments like the endoplasmic/sarcoplasmic
reticulum is another major source for increase in [Ca^ J ^ '(Mountain et al. 1999). The
Ca ^ pumps on the membranes of the intracellular organelles and on the plasma
membrane act to remove Ca- from the cytosol (Misquitta et al. 1998). An increase in the
[Ca- ]'' * is a common denominator for the release of the autocoids like bradykinin, since
both the phospholipase A2 (rate limiting enzyme for the PGI2 production) and the NO
synthase are Ca ^ dependent (Ren et al. 1998).
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Pathophvsiologv of Altered Ca-' Handling
Any disturbance in these homeostatic mechanisms leads to alteration in the Ca-'
handling by the cells. This could result in altered autocoid release mechanisms, as the}-
are Ca * dependent, and altered communication between the endothelium and the \ ascular
smooth muscle layer.
Alterations in the endothelial cell function lead to wide \ ariet>' of vascular
complications ranging from altered blood flow, atherosclerosis, stroke, neuropathy, to
retinopathy. The vascular abnormalities associated with diabetes, are said to be in part
due to altered endothelial cell function in diabetes (Mazzanti et al. 1990: Santilli et al.
1992; DCCT Research Group, 1993; Massry et al. 1997). Studies in cardiac myocytes
(Davidoff et al. 1997; Ren et al. 1998; Ha et al. 1999), arteries (Abebe et al. 1989).
diaphragm (Kimura et al. 1990), leukocytes (Akiba el al. 1995). platelets (Bellagatta et al.
1993) and endothelial cells from diabetic models, animal and human (Salvolini et al.
1999; Ribau et al. 2000). have shown that these cells exhibit altered Ca " homeostasis
with manifestations ranging from altered Ca ^ currents through voltage acti\ated
channels, to altered contractility. Altered Ca ^ homeostasis, in any of the cell models, can
explain the vascular complications of diabetes like diabetic neuropathy, diabetic
nephropathy, and diabetic retinopathy.
Role of Endoplasmic/Sarcoplasmic Reticulum
Studies done in a variety of cell types, ranging from vascular smooth muscle cell,
and endothelial cells to cardiac myocytes, platelets and leukocytes, ha\ e shown altered
Ca ^ homeostasis in cells isolated from a diabetic model. [Ca ]j levels in cardiac
myocytes have been shown to be elevated in diabetic rat myocytes (Noda et al. 1990). In
polymorpholeukocytes from a streptozotocin induced diabetic rat, there is increased
receptor mediated Ca ^ mobilization (Akiba et al. 1995).
As explained earlier in the overview of Ca ^ homeostasis, these differences in
behavior may be explained by dysftmction of any of the various compartments and
proteins that act as regulators of calcium. For example, studies done in cardiac myocytes
isolated from diabetic Wistar rats, the ER/SR Ca ^ content was shown to be decreased
(Lagadic-Gossmann et al. 1996), and the Na /K^ ATPase activity was reduced, and Ca ^
ATPase activity was increased in platelets from diabetic patients (Mazzanti et al. 1990).
The Golgi apparatus has also been shown to possess Ca * regulatory proteins.
The endoplasmic/sarcoplasmic reticulum is an important player in controlling the
release of Ca ^ into the cytoplasm and its sequestration from the cytoplasm. It plays a
principal role in maintaining the cytosolic Ca ^ ([Ca ]' ^ ) in the nanomolar range for
proper cell functioning. The entry of Ca ^ into the cytosol as a function of release from
the ER/SR occurs through a G-protein coupled process. Upon binding of ligands like
angiotensin-II, the phospholipase-C P gets activated which in tum causes the breakdown
of membrane bound phospholipid, phospho-inositol diphosphate (PIP2). The breakdown
products of PIP2 are inositol triphosphate (IP3) and diacyl glycerol (DAG). The IP3 that
is produced rapidly diffuses through the cytosol and causes release of Ca ^ from the IP3-
gated channels on the ER membrane. The Ryanodine receptor (RyR) mediated channels
are present in muscle cells that cause similar releases of Ca'* from the SR. The DAG in
tum activates an important serine/threonine kinase, protein kinase C (PKC), called so
because of its Ca * dependence. It is believed that the Ca- released from the IP3 sensitive
stores act in part with DAG to activate PKC.
The ER also plays an equally important role in the sequestration of Ca'^ from the
cytosol by the Sarcoplasmic/Endoplasmic reticulum Ca^^-ATPase (SERCA) (Benham et
al. 1989; Martinez-Zaguilan et al. 1996). This pump acts to pump Ca'^ into the ER/SR
from the cytosol. It has been shown o have dependence to the rate of glycolysis in cells
(Martinez-Zaguilan et al. 1996). The endothelial cells express various isoforms of this
SERCA pump (Mountain et al. 1999). Of all the known isoforms, only SERCA 2a and
SERCA 3 are expressed in endothelial cells (Anger et al. 1993).
In endothelial cells, apart from the IP3 induced Ca ^ release (IICR), Ca ^ is also
released from these compartments by a phenomenon called calcium induced calcium
release (CICR), (Mozhayeva et al. 1996). This Ca ^ release is usually from the RyR
sensitive stores and maybe present in conjimction with the other Ca- stores.
Most of the data about the behavior of ER and its role in Ca " homeostasis has
been acquired from cardiac myocytes and vascular smooth muscle cells. Some work has
been done in the ER and endothelial cells (Sasajima et al. 1997; Sedova et al. 2000) and
much more remains to be done towards explaining a definitive role of ER in endothelial
cells and Ca ^ homeostasis.
Role of Mitochondria
The mitochondria is also an important player in the Ca ^ homeostasis in a cell. It
possesses an elaborate system for transporting Ca- across their inner membrane (Gunter
et al. 1994). The influx of Ca ^ into the mitochondria occurs from regions of higher
concentrations to regions of lower concentrations. The earlier view that mitochondria
served as a sink for excess iCa^y^^ to protect the cell from high levels of (Ca )' ', it was
challenged by the demonstrations that when cytosolic Ca * pulses are produced in
response to hormones or neurotransmitters, mitochondria do not stabilize (Ca ) * (Gunter
et al. 1994; Babcock et al. 1997). However, Ca ^ is the only second messenger known to
affect mitochondria (Gunter et al. 1994). The entry of Ca ^ into the mitochondria utilizes
an intemally negative membrane potential and is not coupled to transport of any other
ion. The Ca ^ concentration dependence to this mechanism exhibits a second order
dependence and is believed to be a result of an external activation site that binds Ca-
This is a uniporter that is not only activated by Ca * but also by some antibiotics and
protamine. This uniporter transports many other divalent cations which competitively
inhibit the transport of Ca .
The efflux mechanisms are many. There is the Na^ independent Ca ^ efflux
mechanism. Then there is the Ca^ /2Na^ exchanger. Both these secondary transport
mechanisms differ in their tissue distribution (Gunter et al. 1994). The energy for driving
these transport systems comes from the electrochemical gradient of a co-transported or
exchanged ion (Babcock et al. 1997).
.'>+
8
Intramitochondrial free Ca ^ [(Ca )"""] concentration appears to be the most likeh'
candidate for an additional metabolic mediator in addition to ADP, P, and ATP.
Specifically [(Ca *)'"' ] is known to regulate the level of activation of Ca-"-sensitive
dehydrogenases (Denton et al. 1985; Hansford et al. 1985) and other Ca-"-sensitive
metabolic processes (Hansford et al. 1982; McCormack et al. 1990). Critical steps of Tri
Carboxylic Acid (TCA) cycle are regulated by [(Ca^*)"'''] (Denton et al. 1985; Hansford et
al. 1982; McCormack et al. 1990).
Role of Nucleus
Recently, the nucleus has emerged as a potential player in Ca " homeostasis in
cells (Meyer et al. 1995; Hsu et al. 1996; Carafoli et al. 1997). Several hypotheses have
been developed to explain Ca ^ movements into the nucleus. It handles Ca ^ by its
movement through the nuclear pore complex (NPC) requires two transport systems. First
is a GTPase which is also the pathway for nuclear import of proteins containing the basic
nuclear localization sequence. An altemative hypothesis is that of a GTP independent,
Ca * and calmodulin stimulated pathway. The transport across the nuclear envelope
through the NPC required an ATPase located on the outer leaflet of the envelope. This
ATPase was activated by phosphorylation via Protein Kinase A (PKA), and inhibited by
high Ca ^ concentrations. IP3 and IP4 receptors have also been demonstrated on the inner
surface of the nuclear envelope (Malviya et al. 1994), the former's role would be to
discharge the envelope Ca ^ into the nucleoplasm. This data suggests that Ca ^ enters the
envelope space either by ATP driven pump or by the IP4 receptor, whereas Ca-* exit
occurs via IP3 driven pathways.
Role of Golgi Apparatus
Studies have shovm sequestration of calcium into the Golgi apparatus in cultured
mammalian cells (Chandra et al. 1991). The Golgi apparatus is known to exhibit Ca-'-
transporting systems such as the Golgi Ca^*-ATPase, a SERCA, independent of the one
on the ER/SR (Pinton et al. 1998; Marchi et al. 1999) and the HVCa^" exchanger. As the
Golgi apparatus is an important organelle involved in secretion of various proteins, and
the fact that Ca-" plays an important role on the secretion of substances, Ca * depletion
blocked the cleavage of plasma proteins interfering with their exocytosis. Most of the
enzymes involved in the cleavage and the exocytotic events that are located in the golgi
or the trans-golgi network are Ca ^ dependent. Not surprisingly, altering the Ca-' pool in
the Golgi apparatus interferes with normal cell functioning (Oda et al. 1992).
Role of Endosomes and Lysosomes
The endosomes and lysosomes (E/L compartments) are involved in the recycling
and secretion of various proteins in cells. Ca-" plays an important role in this fusion and
secretion of secretory granules. Fast chelators of Ca-* inhibited the fusion of the
endosomes (Holroyd et al. 1999). In epithelial cells and fibroblasts, the ftision of the
lysosomes to the plasma membrane is Ca " dependent, and thence was the exoc\totic
events (Rodriguez et al. 1997). In fibroblasts, the endosomes and their endocytic
10
pathways are thought to be responsible for uptake of Ca-" into the cells, but are not
responsible for storing them for longer times (Gerasimenko et al. 1998). Therefore. e\en
though, the E/L compartments depend on Ca ^ for the completion of their functions in the
cell, no presence of Ca ^ handling machinery such as the SERCA pump or the ryanodine
receptor (RyR) have been demonstrated in any cell type.
Rationale
Studies done earlier in vascular smooth muscle cells have shown that the SERCA
activity is closely related to the rate of glycolysis in these cells (Martinez-Zaguilan et al.
1996). These studies used hypoglycemic conditions to mimic a diabetic condition and
showed that the SERCA activity was decreased . Because of the close relationship
between the vascular smooth muscle cells and endothelial cells in the macro circulation
and the lack of smooth muscle cells in the microcirculation, we decided to investigate
whether the altered rate of glycolysis could affect the Ca ^ homeostasis in microvascular
cells isolated from a rat model of spontaneous insulin dependent diabetes mellitus in BB
Wistar rats (Nakhooda et al. 1976, 1979; Meininger et al. 2000).
Our studies in vascular smooth cells demonstrated that the increases in [Ca- ] ' in
response to agonists were mostly due to Ca ^ release from intracellular stores, as they did
not differ in Ca-^-free media. The source could have been any of the intracellular
storehouses of Ca^ , i.e., ER, Golgi, mitochondria, and endosomes and lysosomes.
Further studies indicated that the E/L compartments played a major role in the Ca-
release. Because the pH in the E/L compartments is acidic (3.0-5.0), and as the
11
dissociation constants (Kd) of Ca^^-binding proteins typically decreases with increasing
pH, it si not surprising that the E/L compartments would exhibit high concentrations of
ionized Ca^ . This study extended our previous work in vascular smooth muscle cells.
Hypotheses
Based on the above mentioned studies and rationale, we came up with the
following hypotheses:
1. Endosomal and lysosomal (E/L) compartments are important subcellular compartments
for Ca ^ homeostasis in microvascular endothelial cells.
2. E/L compartments exhibit Ca ^ homeostatic mechanisms such as SERCA pumps, IP3
receptors and RyR receptors.
3. The re-uptake of Ca ^ by the SERCA pump on the E/L compartments, is decreased in
microvascular endothelial cells isolated from a diabetic animal.
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CHAPTER II
MATERIALS AND METHODS
Isolation of Coronary Microvascular Endothelial Cells
Microvascular endothelial cells were isolated from small coronary vessels of
Wistar (Bio Breeders) normal (BBNs) and spontaneously diabetic (BBDs) rat models by
methods described earlier (Nakhooda et al. 1976, 1979; Meininger et al. 2000). Briefly,
the rats weighing 200-300 g were given intraperitoneal injections of Heparin,
anesthetized and the hearts were surgically removed. The aorta was cannulated and
perfused with Joklik's medium that contained 0.1% dialyzed BSA and heparin (lU/ml).
After a 10 min perfusion, collagenase (0.7 mg/ml) was introduced, and the perfusate was
allowed to recirculate ca. 30 minutes. Ventricles were cur from the hearts, minced, and
placed in fresh collagenase-containing medium and shaken in a water bath for 10 min.
CaCl2 (50 mM) was added to the minced tissue and digestion with collagenase continued
for an additional 10 min. The cells were then dispersed, filtered, and diluted 1:4 with
Joklik's modified medium with 0.1% BSA, then allowed to settle in order to separate
myocytes (which are heavier) from microvascular endothelial cells. Micro\ ascular
endothelial cells were further purified by sequential filtration through a series of n>lon
screens obtaining a preparation free from smooth muscle cells and myocytes.
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Cell Culnire
The normal and diabetic microvascular endothelial cells were collected b\
centrifiigation and plated in culture dishes and grown in DMEM (ICN Biomedical, Inc.
Costa Mesa, CA) containing 2 mM glutamine, 24 mM NaHC03, 5 mM Glucose,
supplemented with 10% FBS (Gibco, Grand Island, N.Y.) under a 95% air-5% CO^
humidified environment at 37° C. Cells were plated on 9 X 22 mm rectangular cover
slips for fluorescence spectroscopy, and on 25 mm round coverslips for confocal and
spectral imaging microscopy.
Buffers
Cell Suspension Buffer (CSB) contained: 1.3 mM CaClj, 1 mM MgS04, 5.4 mM
KCl, 0.44 mM KH2PO4, 110 mM NaCl 0.35 mM NaH.P04. 5 mM Glucose, 2 mM
Glutamine and 20 mM HEPES, at a pH of 7.4 at 37° C. Ca^^-EGTA buffers of defined
composition were used to generate both in vitro and in situ calibration curves with Fura-2
(Martinez-Zaguilan et al. 1995). Corrections of Ca ^ :EGTA associations constants for
pH. temperature, and ionic strength were performed as described elsewhere. 0 Ca-*
contained: 110 mM KCl. 20 mM MOPS (pKa 7.0, 37° C), 10 mM KjH.EGTA. Calcium
sanirated buffer (CaEGTA) contained: 110 mM KCl, 20 mM MOPS, 10 mM KCaEGTA.
High K* buffer contained: 146 mM KCl, 5 mM glucose, 2 mM glutamine. 10 mM
HEPES (pK 7.4, 37° C), 10 mM MES (pK 6.0, 37°C), 10 mM Bicine (pKa 8.0, 37°C).
The rationale of selecting a distinct Ca'* buffer for in-situ titrations is that MOPS unlike
HEPES, MES and Bicine, exhibits the lowest affinity for calcium (Martinez-Zaguilan et
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al. 1995). Media pH was directly measured with a Beckman model 71 pH meter, using a
Coming glass combination electrode. The electrode was calibrated at two known pH
values using commercially prepared standards from VWR Scientific (San Francisco.
CA.). The buffers were further pH adjusted using fluorescent ratios using phenol red. In
vitro calibrations were carried out using the free acid forms of the dyes solubilized in
High K^ or in K/CaEGTA, as required. Fura-2 and SNARF-1 free acids were dissolved
in dimethyl sulfoxide as 1 mM stocks and maintained at -80°C in the dark until used.
Fluorescence Spectroscopy
The fluorescence studies in vitro and in cell populations were performed in a
temperature controlled stirred cuvette unit housed in a an SLM8000C (SLM, Urbana, IL)
at 37°C, using 4-nm band pass slits and an external rhodamine standard as reference. The
simultaneous measurements were taken using the parameters described previously
(Martinez-Zaguilan et al. 1991).
Spectral Imaging Microscope
Figure 3 shows the schematic representation of the major components required for
Spectral imaging microscopy. Briefly, an OLYMPUS IX 70 inverted microscope was
equipped with a 100 watt Hg lamp as an illumination source. Imaging optics included a
60X 1.4 NA OLYMPUS objective and a 5.4 X eyepiece to focus the cell image onto the
input slit of a grating monochromator (Aries 250IS/SM spectrograph, Chromex, Inc.
Albuquerque, NM). A grating blazed at 300 lines/inch provided a spectral bandwidth of
15
Eyepiece Spectrograph
Figure 3: Schematic representation of the Spectral Imaging Microscopy system. The specific excitation of each fluorophore is selected using a excitation filter (Ex. Filter) with a 100 watt mercury lamp (Hg Lamp) as the light source. The emission from the cells on the stage passes through the eyepiece into the spectrograph. The light entering the spectrograph is controlled by adjusting the entrance slit (Adj. Slit), where it analyzed the computer giving the spectra of the emissions captured.
16
200 nm. The spectral output from the grating was imaged onto a liquid cooled CCD
camera (Photometries Mod. CH350, Tucson, Arizona) equipped with a 512 by 512
element imaging chip that is 60% quantum efficient at 546 nm ( Techtronics, AZ). The
output image is composed of spectra acquired at multiple positions along the length of the
entrance slit (Figure 3). The spectral image occupies less than V^ of the serial register of
the imaging chip and for a single cell imaging it occupies a significantly lesser area.
Therefore, internal shifting of data allows for rapid sequential image acquisition.
Adequate signal can be obtained with as little as 2 msec exposure time, allowing
collection of many images per second. Read out of the fiill chip then requires 500 msec.
Spectral resolution is primarily dependent on the grating resolution, but also on the w idth
of the entrance slit. As slit width is increased, signal increases substantially while
spectral resolution suffers comparably less (Martinez-Zaguilan et al. 1996). Thus, spectral
resolution can be sacrificed for improved signal to noise ratio. In the current experiments
the entrance slit was set at 200 |am. Mapping of the wavelength to position on the CCD
chip was performed by reflecting light from the Hg lamp to the monochromator. The
grating was scanned to position the 546 line of the Hg lamp near one end of the output
spectrum and the doublet peak at 577/579 imaged near the center. The digitalized output
of the CCD camera was stored in a PC with 128 MB RAM. Image analysis was done on
a PC 300 MHz /128MB RAM using MAPS analysis software version 2.0 (Photometries.
Tucson, AZ) and further analyzed using MS Excel (Microsoft Co., Redmond. WA) and
Sigma Plot for Windows, version 5.0 (Jandel Scientific, San Rafael, CA).
17
Laser Scanning Confocal Microscope
Confocal Microscopy was performed with a Bio-Rad 1024 confocal microscope
(Chu et al. 1995). Endothelial cells loaded with Ca^^-green Dextran (10.000 M.W.) were
imaged using a 488 nm laser line of the 25 mW argon laser for excitation, and the
emission was collected at 530 nm using T1/T2 filter blocks with a single photo multiplier
tube. The acquisition of the data was done on a 0S2 based computer using the
Lasersharp® software, and further analyzed with MS Origin statistical software.
Culture Preparation
Confluent BBN and BBD cultures grown on rectangular coverslips were used for
the experiments in the SLM8000C fluorescence spectroscope. For spectral imaging and
laser scarming confocal microscopy, subconfluent cultures grown on 25 mm round
coverslips were used. The cells are washed three times with CSB. Subsequently, the
cells are incubated for 45 minutes at 37°C in a 5% CO2 atmosphere with 3 ml of CSB
containing 7 |LIM S N A R F - 1 / A M and 2 |LIM Fura-2/AM. The AM forms of these dyes are
lipophilic and cell permeant. Cellular esterases cleave the ester groups of these AMs to
yield the free acids which are more impermeant and therefore "trapped" within cells
(Tsein, RY 1989). After the 45 minute incubation the cells are transferred into CSB and
rinsed and incubated again at 37°C in CSB to allow complete hydrolysis of the imbound
dyes. At his point the cells are placed back to back in a holder/perfusion device, which
was subsequently inserted into the fluorometer cuvette (Giuliano et al. 1987). The
18
temperature was maintained at 37°C by keeping both the water jacket and the perftision
buffer at 37°C using a iso-temperature immersion circulator water bath (Lauda model
RM20, Brinkmarm Instruments, Westbury, NY).
For the spectral imaging and the laser scanning confocal microscopy,
subconfluent BBN and BBD cultures grown on 25 mm coverslips were mounted onto a
special chamber and placed in a special temperature controlled perfusion stage PDMI-2
(Medical Systems Inc., Greenvale, NY), with a buffer perfusion rate of 3 ml/min. This
was placed on the 1X70 Olympus inverted microscope for the experiment in either case.
In Situ Calibration of Ca " Indicators
In situ calibrations for Ca ^ indicators were carried out using K/CaEGTA buffers
supplemented with non-fluorescent Ca ^ ionophore 4Br-A23187 (5|iM) to collapse the
Ca ^ gradient, and 6.8 |iM nigericin plus 2 [iM valinomycin, to set pHex = pHin
(Martinez-Zaguilan et al. 1999 ). Calibrations were initiated at 0 Ca-* and a selected pH.
Ca ^ was increased by removal of buffer from the cells, washing with the next stock
media and then incubation in an aliquot of the next stock for 3 minutes prior to
acquisition of spectra. This process was repeated imtil spectra at 10-11 different Ca-'
concentrations were obtained. Once the maximum Ca ^ concentration at a specific pH
was attained, cells were titrated for pH as previously described (Martinez-Zaguilan et al.
1996). Sequential iterations were performed imtil a high pH was reached. Inclusion of
ionophores to collapse the pH gradient is important, since it is known that changes in
19
pHin may occur in some cell types upon 4Br-A23187 treatment. The data generated from
in situ calibrations were evaluated by analysis of the relation between Ca-* and the ratio
values as described by the following equation:
[Ca^ ] = Kd [(R- Rmin/Rmax-R)] equation [ 1 ]
where Kd is the apparent Fura-2 dissociation constant for Ca *, Rmax represents the Ca-*-
dye chelate and the Rmin is the ftilly Ca ^ free Fura-2 signal intensity.
Study of Endosomal/Lysosomal Compartments
Confluent rectangular coverslips were initially coloaded with SNARF-1/AM and
Fura-2/AM as described previously. A stock solution of Calcium Green Dextran 10,000
MW (5mg/ml) was prepared. 5 [i\ of this was dissolved in 45 fil for each cover slip to be
loaded. The 50 \i\ are placed on a piece of parafilm in a petri dish, and the coverslips are
placed cell side down on it for 45 minutes and incubated at room temperature covered in
aluminum foil.
Immunoc vtochemi stry
For this the microvascular endothelial cells from both the normal and diabetic
rats, were grown on round 18 mm cover slips. Briefly, the cells were fixed with a 4%
solution of paraformaldehyde for 15 minutes, which were then neutralized with 25 mM
solution of glycine. The cells were permeabilized with a 0.1% solution of triton before the
cells were incubated with the primary and the secondary antibodies towards the proteins
of interest. The cover slips were washed thoroughly with 0.05% solution of triton
20
(antibody wash) to wash away the unbound markers and minimize the background. The
cover slips were then mounted onto glass slides using Prolong Antifade (Molecular
Probes, Eugene OR) and fixed with commercially available clear nail paint. The
antibodies were purchased from commercial houses. (SERCA: Research Diagnostics,
Planders NJ; IP3: Calbiochem; Ryonidine BODIPY: Molecular Probes, Eugene, OR).
Data Analysis
The values from the in situ titrations for both pH and Ca ^ indicators were
routinely fitted using the simplex method and non-linear regression analysis employing
commercially available computer software (MINSQ, Micro Math Scientific Software,
Salt Lake City, UT). This type of analysis allows the estimation of in situ calibration
parameters (i.e. pK, Kd, Rmax and Rmin) needed to calculate pHin or [Ca-^]'" () . Data
are presented as group means ± SE unless otherwise indicated. Statistical analysis was
done using the student t-test and the analysis of the variance was calculated as needed.
Materials
Fluorescent compoimds were purchased from TEFLABS (Austin, TX) or
Molecular Probes Inc. (Eugene, OR). All other chemicals were of reagent grade and were
obtained commercial sources.
21
CHAPTER III
RESULTS
Steady State [Ca ]* ^ Levels in Microvascular Endothelial Cells from Normal and Diabetic models
Basal [Ca ]* ^ signals as measured by Fura-2 AM fluorescence indicate that there
was no significant difference between cells from normal [(13.3 ± 0.96 nM) (n = 51)] and
those from diabetic rats [(15.2 ± 0.95 nM) (n = 38)] (Figure 4).
Because simultaneous measurements of both pH* -' and [Ca-*]^^ were performed,
we also determined that the steady state pH' y levels are not significantly different for
microvascular endothelial cells from normal [7.434 ± 0.0231 (n = 6)] and diabetic models
[7.445 ± 0.0409 (n = 6)], respectively).
Role of Transmembrane Ca- Movement in the Ca ^ Homeostasis in
Microvascular Endothelial cells
The activity of the Na -Ca^^ exchanger has been demonstrated in many cell types
including endothelial cells (Carafoli et al. 1987, Sage et al. 1991). We therefore
evaluated the role of this exchanger for Ca ^ homeostasis. This can be easily done b\
removing extracellular [Na"] and evaluating its effect on the [Ca-']'y* regulation. The
removal of Na^ should result in an increase in [Ca l' ^ due to the reversal of the
exchanger (Hudson et al. 1998). Previous studies from our laboratory have shown that
human umbilical vein endothelial cells (HUVECs) responded to this maneuver with only
a small increase in Fura-2 fluorescence (Martinez-Zaguilan et al. 1996). This was
22
ri
U
20
15 -
10 -
5 -
0
Normal Diabetic
m' ^"^^5^
" i a t ^ -a^
Figure 4: Steady-state [Ca-"] ^ values are similar in microvascular endothelial cells from normal and diabetic rats.
23
associated with a sustained decrease in pH - following Na* removal, likeh to be
associated with inactivation of the ubiquitous Na /H* exchanger present in these cells
(Figure 5 A). Corrections for H* binding to Fura-2 indicated that the "apparent" increase
in Fura-2 signal was not due to a Ca-* increase, but rather due to H* binding to Fura-2
since corrections for pH effects on the Fura-2 signal indicated that there was no increase
in [Ca "]*"- (Figure 5 B). Because of this effect in HUVE cells we repeated similar
experiments in microvascular endothelial cells. Upon removal of Na*. these cells also
responded with an apparent increase in Fura-2 fluorescence, which was slow and
sustained (Figure 5 D). Interestingly, upon Na" removal, these cells responded b\ a rapid
acidification followed by a recovery (Figure 5 C). Following pH corrections for the Fura-
2 signal, we still saw an increase in the [Ca^"]^-\ leading us to conclude that these cells do
exhibit Na"/Ca^^ exchanger activity, and reiterating the fact the simultaneous
measurements for pH need to be along with Ca * measurements to correct for proton
binding in each case.
In excitable cells like the cardiac myocytes, removal of Na" causes an sudden and
steep rise in [Ca^^]"^ (Lee et al. 1987). This is not the case in microvascular endothelial
cells. The kinetics of the rise in [Ca *]*"- in response to Na" removal were too slow for the
Na*/Ca^^ exchanger to be playing an important role in these cells.
24
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25
Agonist Induced Increases in ICa-"]*' in Microvascular Endothelial Cells from a Diabetic Model as Compared to Normal Counterparts
Cells at confluency on rectangular coverslips were loaded with SNARF-1 and
Fura-2, then transferred to the fluorometer cuvette and then perfused with CSB at the rate
of 3 ml/min. After a steady pH/Ca^^ signal was obtained, the perfusate was exchanged
with CSB containing agonists (ATP in Figure 6 A & and vasopressin in Figure 6 B,
arrows). Signals from both the pH sensitive dye, SNARF-1, and the Ca-" indicator Fura-
2 were collected and converted to (pH)in and [Ca *]i, respectively. For the purpose of data
presentation, only pH corrected Ca ^ signals are presented. Notice that under steady state
conditions, the levels of [Ca l' y are similar in microvascular endothelial cells from
normal and endothelial models (13.3 ± 0.96, n = 51; vs. 15.2 ± 0.95 , n = 38; normal and
diabetic models, respectively). Because simultaneous measurements of (pH)j and [Ca-"]
were performed, we also determined that the steady state pW^^ levels were similar in
microvascular endothelial cells from normal and diabefic models (7.434 ± 0.0231, n = 6;
vs 7.4425 ± 0.0401, n = 6, for normal and diabetic model respectively). Following agonist
stimulation cells responded with a rapid and transient increase in [Ca ] y\ This effect is
seen in both normal and the diabetic microvascular endothelial cells, but the magnitude of
the [Ca l' y increase was greater in cells from the diabetic model than in the normal cells,
regardless of the agonist employed (Figure 6 & 7).
cvt
26
1000 -1
800 -
600
400
200
Diabetic
Normal
0 -»
5 min
1200
-T" 03
1000 •
800 •
600
400
200
0
Normal (n=6) Diabetic (n=6)
^ '• ^ \ l . ; ;-*«
m .2+ Basal Ca -free Peak
Figure 6: ATP induced increases in [Ca " ]* ' are greater in microvascular endothelial cells isolated from a diabetic animal. Microvascular endothelial cells grown to confluence on rectangular cover slips, were loaded with 2 mM Fura-2 AM, a Ca ^ sensitive probe. The cells are perfused with CSB at 3ml/min, and the perfusate is exchanged for CSB containing 1 mM ATP. The same experiments were repeated in Ca^^-free CSB. In the absence of extracellular Ca^ , the source of the Ca " spike had to be intracellular stores of Ca^ . The basal signal did not differ in regular CSB (basal) and in Ca ^ free-CSB (Ca^-'-free).
27
200 1
150
Vasopressin
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Diabetic
Normal
5 min
o •T"
CM
200
150 -
100 -
(0 Si 50
Normal (n=6) Diabetic (n=6)
Basal Ca^Mree Peak Figure 7: Vasopressin induced increases in [Ca ' ]' ' are greater in microvascular
endothelial cells isolated from a diabetic animal. Microvascular endothelial cells grown to confluence on rectangular cover slips, were loaded with 2 mM Fura-2 AM, a Ca2+ sensitive probe. The cells are perfused with CSB at 3ml/min, and the perfusate is exchanged for CSB containing 500 mM vasopressin. The same experiments were repeated in Ca2+-free CSB. In the absence of extracellular Ca2+, the source of the Ca * spike had to be intracellular stores of Ca^ . The basal signal did not differ in regular CSB (basal) and in Ca ^ free-CSB (Ca^^-free).
28
Role of Endosomal/Lysosomal Compartments in Ca-* Homeostasis of
Microvascular Endothelial Cells.
The handling of Ca ^ by the various intracellular compartments has been studied
extensively by various labs. The ER/SR plays an key role in regulating Ca-* homeostasis
by allowing Ca ^ to move between the cytosol and ER/SR (Golovina et al. 1997; Pinton
et al. 1998). In addifion, Ca ^ refilling/release also occurs in other organelles such as the
nucleus (Hsu et al. 1996; Carafoli et al. 1997), mitochondria (Gunter et al. 1992; Rizzuto
et al. 1992; Babcock et al. 1997), and the Golgi apparatus (Chandra et al. 1991; Oda et al.
1992; Pinton et al. 1998; Marchi et al. 1999; Miseta et al. 1999). Recently, the role of
endosomes/lysosomes in regulating Ca ^ refilling/release following agonist stimulation
has been suggested (Rodriguez et al. 1997; Gerasimenko et al. 1998; Holroyd et al. 1999).
As we wanted to investigate if a particular subcellular compartment may explain the
differences in Ca ^ handling between the normal and diabetic cells, we chose to
understand the role of the E/L compartments. To study specific intracellular
compartments requires that the fluorescent indicators be targeted to the compartment of
interest. It has been difficult to load intracellular compartments with Ca-" indicators, for
example, mitochondria (Rizzuto et al. 1993), ER/SR loading (Golovina et al. 1997),
Golgi apparatus (Pinton et al. 1998; Marchi et al. 1999; Miseta et al. 1999) etc. Therefore
we decided to evaluate a compartment that we can unequivocally assign. The E/L
compartments are a good target because fluorescent Ca-* indicators can be easily assigned
to these compartments by conjugating them to high molecular weight dextran. Hence we
29
decided to investigate these compartments for alterations in functional status in
microvascular endothelial cells isolated from a diabetic animal.
The E/L compartments were loaded as described in the methods section. Cells
were coloaded with Fura-2 AM for monitoring [Ca-'l'^" as described earlier. Figure 8 A
shows an image of a microvascular endothelial cell with distinct loading in the E/L
compartments with a Ca ^ fluorophore, Ca^^-Green Dextran (10,000 M.W.). Figure 8 B
shows the same cell with cytoplasmic loading of the Fura-2 AM. Figure 8 C shows a
tracing of the signals from the Fura-2 AM (which measures the [Ca^*]' ^, and the Ca-*-
Green Dextran (which measures the Calcium signal from the E/L compartments
([Ca^*]^^). Figure 9 shows that, upon stimulation with an agonist, we see an increase in
the Fura-2 signal, indicating an increase in the [Ca l' y which coincides with a sudden
drop in the Ca^^-Green signal, indicating a release from the E/L compartment.
The data indicates that E/L compartments are important for Ca " homeostasis in
microvascular endothelial cells.
Functional SERCA Pumps are Present on the E/L Compartments
In order to evaluate the mechanism involved in this release of Ca * from the E/L
compartments we employed a pharmacological approach. We evaluated the effects of
inhibitors to the sarcoplasmic/endoplasmic reticulum Ca-" ATPase (SERCA) which
regulates the refilling/release of the ER/SR in various cells and plays an important role in
Ca * homeostasis in cells. Thapsigargin (TG), cyclopiazic acid (CPA), and tetra-butyl
hydroquinone (BHQ) are wellknown SERCA inhibitors. As different cells exhibit
30
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Figure 9: Simultaneous measurement of [Ca ' ]'' ' and [Ca ""] ^ in cell populations. Confluent rectangular coverslips of microvascular endothelial cells are loaded first with Fura-2 AM by methods described in text. Then the same coverslips are inverted onto 50 |il of CSB containing 5|J,1 dextran conjugated Ca^^-green, face down for 30 minutes at room temperature. The above tracing shows that at least a part of the increase in [Ca " ]* ' in response to ATP was coming from the E/L compartments, as there is a simultaneous decrease in signal from the E/L compartments with the increase in [Ca '"]'' '.
32
different isoforms of the SERCA (Varadi et al. 1996 ), and have different sensitivities to
each of these drugs, we decided to evaluate the effect of all three of them on the Ca-*
handling by the E/L compartments.
Confluent rectangular cover slips of the microvascular endothelial cells were
loaded with Ca^^-green Dextran. Experiments were done to demonstrate of presence
functional SERCA pumps on the E/L compartments. Thapsigargin (TG), and cyclopiazic
Acid (CPA) were employed as they known inhibitors of SERCA pumps in the ER. As
seen in Figure 10 A, treating the cells with TG, there was no release of Ca " from the E/L
compartments. If TG and ATP were added together, they elicited a release of Ca-* from
the E/L compartments, which almost completely recovered to basal levels after washing
out the drugs. When CPA was added by itself (Figure 10 B), it elicited a release of Ca'"
from the E/L compartments, which was ftirther enhanced upon addition of ATP. Upon
washing away the drugs, there was a recovery of Ca * levels towards base line. When
vasopressin was added after treating with CPA (Figure 10 C), there was no further release
of Ca *. This could be attributed to the possibility that ATP and Vasopressin maybe
acting on different intracellular Ca ^ stores in cells. The same can be said about the
difference in the effects of TG and CPA on the SERCA pumps.
The E/L Compartments Express SERCA Pumps in Microvascular
Endothelial Cells
Immunohistochemical studies done with microvascular endothelial cells, indicate
that E/L compartments express Sarcoplasmic/endoplasmic Ca-"-ATPase pump (SERCA).
33
"r3 3 C
E LO
34
For this experiment, cells were loaded ovemight in media containing Texas red
conjugated Dextran dye which is readily taken up by the E/L compartments b\
endocytosis. Then the cells are fixed with primary antibodies for SERCA-2 and a
secondary fluorescent anfibody FITC. Figure 11 A shows the cell with its labeling for
Dextran conjugated Texas red dye in the E/L compartments. Figure 11 B shows the same
cells with the FITC conjugated SERCA-2 antibodies. The colocalization was calculated
by dividing cells into quadrants and counting the areas that expressed both the fluorescent
tags. It is depicted as the white dots. Figure 12 shows the histogram representation of
colocalizations. It was approximately 60%, i.e., 60% of the E/L compartments expressed
SERCA.
The E/L Compartments Express Ryanodine Receptors (RyR)
Similar protocol was followed to label the E/L compartments with TX-Red
Dextran dyes. The markers for the RyR are available in BODIPY form, which enables us
to stain the live cells. Figure 13 A shows a microvascular endothelial cell with E/L
compartmental loading with FITC-DEX. Figure 13 B shows the same cell stained with
TX-Red conjugated BODIPY form of antibody to the RyR. Pixel by pixel analysis of the
two images yielded the areas of co-localization, which is shown in Figure 14. About 70
% of the E/L compartments express the RyR.
35
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39
Microvascular Endothelial Cells Isolated from a Normal Rat Elicit Larger Releases from E/L Compartments upon Agonist stimulation
than Their Diabetic Counterparts
Rectangular coverslips with cells grown to confluenc> were loaded with Ca-'-
Green Dextran (10,000 M.W.) by ovemight incubafion of the cells with the fluorophore
in media. The excess dye was washed away with CSB. Upon agonist stimulation, the
decrease in [Ca^"]^ signal (release of Ca " from the E/L compartments) was compared
between microvascular endothelial cells from normal and diabetic rats (Figure 15). The
releases were greater in cells from normal animals than the ones isolated from a diabetic
model. Thence, the greater increase in (Ca-*) - seen in cells from a diabetic model upon
agonist stimulation was probably not due to increased releases from the E/L
compartments.
Since these compartments exhibit SERCA pumps, it would be logical to
investigate whether the reuptake of Ca-* by these compartments following agonist
stimulation is decreased in cells from a diabetic model. As mentioned before, there is an
altered rate of glycolysis in diabetes (Martinez-Zaguilan et al. 1996). And the acti\ity of
the SERCA pump is shown to be linked to the rate of glycolysis. Therefore it may be
logical to conclude that the altered rate of glycolysis in diabetes may be responsible for a
decreased rate of reuptake of Ca-* b\ the endosomes and lysosomes. which may explain
the greater increases in [Ca 'J' y* in response to agonists in microvascular endothelial cells
from a diabefic animal.
40
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41
Study of Single Vesicles in Microvascular Endothelial Cells using Spectral Imaging Microscopy
Using spectral imaging microscopy it is possible to study the E/L compartments
in single microvascular endothelial cells. Subconfluent 25 mm round coverslips with
cells are loaded with Ca^^-Green Dextran as described in the methods. They are then
washed and moimted onto the chamber that houses the cover slip. This is then placed in a
temperature controlled perfusion chamber PDMI-2 (Medical Systems Inc. Greenvale,
NY) which maintains the temperature at 37°C, while allowing us to perfuse media
through the chamber. Figure 16 A shows a zero-order image of a microvascular
endothelial cell loaded with dextran conjugated Ca^^-green with the slit width of the
spectrograph set at 2mm. Then, images are taken at a slit width of 200 |Lim (Figure 16 B).
This is done to minimize the area of interest to increase signal to noise ratio. Then the
emission filter is removed and the spectrograph and CCD are centered to capture an
image of the Ca^^-green emission coming out of each individual vesicle (Figure 16 C).
The software allows to study the spectral properties of the individual emissions as shown
in Figure 16 D. The cells are perfused with CSB at the rate of 3 ml/min and a baseline
signal is obtained. Cells are then perfused with CSB containing 1 mM ATP. The response
of the individual vesicles to the agonist was heterogenous (Figure 16 E). This may be
attributed to the fact that not vesicles may have the machinery to respond to stimulation
by agonists as explained by the fact that only 60-70% of the vesicles exhibited SERCA
pumps.
42
Further studies are needed to define the relative contribution of other Ca-"
homeostatic machinery on the E/L compartments, such as RyR, IP3 receptors, and Ca'
binding proteins such as calmodulin, calsequestrin, and calreticulin.
43
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44
CHAPTER IV
DISCUSSION AND CONCLUSION
Ca ^ homeostasis is altered in various cell types isolated from a diabetic animal
model and some human cells (Abebe et al. 1989; Epstein et al. 1989; Mazzanti et al.
1990; Pellagatta et al. 1993; Lagadic-Gossmann et al. 1996; Ren et al. 1998; Ha et al.
1999). However no studies have been done in microvascular endothelial cells from a
diabetic model, to investigate whether Ca ^ homeostasis is altered. The hypothesis that
Ca * homeostasis is altered in microvascular endothelial cells from a diabetic model was
investigated using novel approaches.
Using fluorescence spectroscopy, we have shown that the resting (Ca"")" * levels
were not different as compared to cells from normal animals. We then investigated the
various ports of Ca ^ entry into the cytosol of microvascular endothelial cells.
Experiments demonstrated that abrupt removal of Na^ from the perfusate activated the
Na*/Ca^^ exchanger and resulted in an significant increase in (Ca )' '", albiet minor, in
micro but not in macro vascular endothelial cells. Upon stimulation with KCl, which
would trigger entry of Ca ^ into the cytosol through voltage gated Ca ^ chaimels on the
plasma membrane, resulting in an increase in (Ca )*' *, had no effect in these cells. The
data indicate that the Ca" channels were not a major player in the Ca ^ homeostasis in
microvascular endothelial cells.
Upon treating these cells with agonists like ATP, vasopressin, angiotensin, and
BHQ, we noticed increases in (Ca^^)'' \ in both cells from normal and diabefic animal
45
model. The increases in (Ca )' ' were greater in cells from a diabetic model, irrespective
of the agonist used (cf. Figure 6). This lead us to hypothesize that the source of this
increase in (Ca )* ^ were intracellular stores of Ca'".
The study of intracellular stores was limited to the possibility of loading the
compartment of interest with a Ca ^ sensitive fluorophore. The most studied
compartments for Ca ^ homeostasis in cells, excitable and non excitable has been the ER
(Hurst et al. 1992). However, most of the studies were done using pharmacological
approaches where SERCA inhibitors and Ryanodine homologues were used to study
releases and uptake of Ca ^ by the ER/SR (Mazzanfi et al. 1990; Hurst et al. 1992). We
decided to investigate the role of the E/L compartments, as they were easy to load with
Ca ^ sensitive fluorophores conjugated with high molecular weight dextran, which the
cells took up by endocytosis, and ended up in these compartments.
Upon treating these cells with agonists like ATP and vasopressin, we elicited a
decrease in Ca ^ Green Dextran fluorescence, i.e., release of Ca ^ from these
compartments. Upon simultaneous loading of both the cytosol and the E/L compartments
with fluorophores, and treating them with agonists, the increase in Fura-2 Fluorescence
was preceded by a decrease in the Ca^^-Green signal, indicating that some of the increase
in (Ca )* ' was being contributed by the E/L compartments. This lead us to hypothesize
that the E/L compartments must have some Ca ^ handling machinery. We employed
pharmacological approaches to investigate whether the E/L compartments exhibited
SERCA. The use of thapsigargin, CPA and BHQ, showed that CPA and not TG caused
release of Ca ^ from the E/L compartments. This may be because that E/L compartments
46
exhibit different isoforms of the SERCA and ha\e different responses to the \arious
SERCA inhibitors. Further, the demonstration of SERCA machinery b>
immunocytochemistr}'. supports the pharmacological e\ idence towards the presence of
SERCA pumps on the E/L compartments. We have also demonstrated the presence of IP,
receptors and Ryanodine receptors on the E/L compartments and they exhibit 50% and 70
% co-localization Vvith dextran dyes. This further suggests the various other possibilities
of altered physiology the microvascular cells from a diabetic model may exhibit when
compared to cells from a normal rat. It remains to be investigated thoroughly.
The release of Ca"* from the E/L compartments in response to agonists was
greater in normal cells when compared to the release in diabetic cells. This is a
conundrum since the diabetic cells had a greater increase in [Ca 'l' Mn response to
agonists, suggesting that the greater increase was not due to greater release from the E'L
compartments in diabetic cells. We hypothesize that the re-uptake mechanisms for Ca-* in
the diabetic cells \'ia SERCA pumps is altered in diabetes. It has been shown before that
glucose causes the E/L compartments to take up glucose via the SERCA pump. We also
know that glucose metabolism is altered in diabetes that may lead to altered SERCA
activit}' in these cells. The decreased uptake of glucose in the diabetic cells may explain
the greater increase in [Ca" ] 'Mn these cells. The solution of this conundrum requires
further investigation.
The study in single vesicles and the heterogenous response to ATP by the
individual vesicles could be explained by extent of SERCA co-localization on these E/L
compartments. It can also be explained by possible differences in the extent of
47
expression of other Ca^^-handling proteins on these compartments. One might argue that
as these compartments are the recycling houses of the cell, it is not surprising to find all
these proteins of interest in these compartments. However we have demonstrated that the
SERCA pumps in these compartments are functional since they can be blocked using
inhibitors to the SERCA pumps. Preliminary experiments indicate that these
compartments do express ryanodine and IP3 receptors along with calreticulin and
calsequestrin. The fimctional importance of these proteins in E/L compartments needs to
be investigated since it could provide us with a ftirther insight as to why the E/L
compartments in microvascular endothelial cells isolated from a diabetic animal release
less Ca ^ upon agonist stimulation, yet have a greater increase in [Ca "] *.
The agonists cause release of Ca " from these compartments, coinciding with the
increase in [Ca^"]' ^ (as suggested by the increases in Fura-2 ratios), suggesting that at
least some of ihe increase in [Ca ]* ^ was coming from the E/L compartments.
Pharmacological studies revealed that cyclopiazic acid (CPA) and not thapsigargin (TG)
(known inhibitors of the SERCA pump) elicited releases of Ca ^ from the E/L
compartments.
Paradoxically, the agonists caused greater releases of Ca * from the E/L
compartments in microvascular endothelial cells from normal animals than in those from
diabetic animal models. This led us to hypothesize that, since the SERCA activit\ was
closely linked to the rate of glycolyis in cells (Martinez-Zaguilan et al. 1996), and
because glucose transport and metabolism is altered in diabetes, the microvascular
endothelial cells isolated from a diabetic model would exhibit decreased uptake of the
48
released Ca^", causing greater increases in [Ca "]" ' when compared to cells from a normal
animal.
To summarize, we have demonstrated that the E/L compartments are important in
the Ca^" homeostasis of microvascular endothelial cells. Diabetic cells respond to
agonists like ATP and vasopressin, with much larger increases in [Ca-*]''- uhen
compared to normal cells. The E/L compartments express SERCA, which were
demonstrated both by immimocytochemistry and by pharmacological means. The
conundrum was that the E/L compartments in diabetic cells release lesser Ca-" than their
normal counterparts. It is possible to study single vesicles using techniques like spectral
imaging and confocal microscopy. And finally, further studies need to be done to identif>
the compartment responsible for the greater increases in [Ca *]* ^ in diabetic cells in
response to agonists.
49
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57
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