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

VI

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).

d u G C/3

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o o -o c 0)

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T « OH

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T3 O . ^

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r C o Oi '-' T3

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H S "" c« O «5

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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.

12

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.

13

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

14

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 -»

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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

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Diabetic

Normal

5 min

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200

150 -

100 -

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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

N_

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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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