Effect of oxidant stress on calcium signaling in vascular endothelial cells.

Fret, Radical Biology & Medicine. Vol. 13, pp. 635-650, 1992 Printed in the USA. All rights reserved.

0891-5849/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.

Review Article EFFECT

OF OXIDANT STRESS ON CALCIUM SIGNALING IN VASCULAR ENDOTHELIAL CELLS

STEPHEN J. ELLIOTT, J. G A R Y MESZAROS, and WILLIAM P. SCHILLING Departments of Pediatrics and Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA

(Received 30 March 1992; Revised and Accepted 25 June 1992) A b s t r a c t - - T h e endothelial cell is recognized as a critical modulator of blood vessel tone and reactivity. This regulatory function of endothelial cells occurs via synthesis and release of diffusible paracrine substances which induce contraction or relaxation of adjacent vascular smooth muscle. In response to stimulation by blood-borne agonists such as bradykinin or histamine, the endothelial cell utilizes cytosolic ionic Ca 2+ as a trigger in the transduction of the stimulatory signal into a paracrine response. Considerable evidence has accumulated to indicate that various forms of biologically important oxidant stress alter vascular function in an endothelium-dependent manner. Further, oxidant stress is known to alter the mechanisms which govern Ca 2+ homeostasis in the endothelial cell. Recently, we have described a model in which the oxidant tert-butylhydroperoxide is utilized to examine the effects of oxidant stress on Ca2+-dependent signal transduction in vascular endothelial cells. In this model, three temporal phases are evident and consist of( 1) inhibition of the agonist-stimulated Ca 2+ influx pathway, (2) inhibition of receptor-activated release of Ca 2÷ from internal stores and elevation of resting cytosolic free Ca 2+ concentration, and (3) progressive increase in resting cytosolic Ca 2÷ concentration and loss of responsiveness to agonist stimulation. In this review, the mechanisms which characterize agonist-stimulated Ca 2÷ signaling in vascular endothelial cells, and the effects of oxidant stress on signal transduction, will be described. The mechanisms potentially responsible for oxidant-induced inhibition of Ca 2÷ signaling will be considered. Keywords--Endothelial cell, Signal transduction, Cytosolic Ca 2+, tert-butylhydroperoxide,

Oxidant stress

investigators in many different disciplines. An explosion of information in the field of endothelial cell biology followed the landmark discovery that the vasorelaxant action of acetylcholine is endothelium dependent. 1 Since that time, it has been learned that this effect is related to the action of nitric oxide, which is synthesized within, and released from, vascular endothelial cells. 2'3 Nitric oxide (NO) is derived from L-arginine via the action of NO synthase, 4-6 an enzyme which has been sequenced and cloned. 7 Using molecular biological techniques, NO synthase has been identified in a number of different tissues, suggesting that NO may play a ubiquitous role in regulation of cellular function. 7"8In addition to nitric oxide, endothelial cells produce and release several other paracrine factors, including endothelin, 9 prostaglandin I2 (Ref. 10),

INTRODUCTION

During the past decade, the physiological importance of the vascular endothelium has been recognized by Address correspondence to: Dr. Stephen J. Elliott, Department of Pediatrics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Stephen J. Elliott is a graduate of the University of Western Australia School of Medicine (1980). He received pediatric residency training at Case Western Reserve University, Cleveland, Ohio, followed by postdoctoral fellowship training in neonatology at Baylor College of Medicine, Houston, Texas. He is an Assistant Professor of Pediatrics and Molecular Physiology and Biophysics at Baylor College of Medicine. Dr. Elliott holds membership in the Oxygen Society, the American Physiological Society, and the Microcirculatory Society. He is the recipient of a Clinical Investigator Award from the National Heart, Lung and Blood Institute. His research interest is the study of oxidative injury and Ca 2+ signaling in vascular endothelial cells. J. Gary Meszaros graduated with a Bachelor of Science from College of Wooster, Ohio in 1989, where he was elected to the Sigma Xi Scientific Research Society. Currently, he is in graduate school in the Department of Physiology and Cell Biology at the University of Texas Health Science Center in Houston. William P. Schilling is a graduate of Chapman College, Orange, California (BS, Chemistry, 1974) and received his PhD in Pharmacology at the Medical University of South Carolina in 1981. He holds a faculty appointment as Associate Professor and Director of

the Graduate Program in Molecular Physiology and Biophysics at Baylor College of Medicine, Houston, Texas. Dr. Schilling is an Established Investigator of the American Heart Association. He is an active member of the Biophysical Society and serves on the Editorial Board of the American Journal of Physiology. His research focuses on the mechanisms responsible for translocation of Ca 2÷ between extracellular and endothelial subcellular compartments. 635

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and endothelium-derived hyperpolarization factor, ~l,x2 each of which may act to alter vascular smooth muscle tone. Other endothelium-derived substances include tissue plasminogen activator, ~3-~5 interleukin, '6''v von Willebrand factor, as platelet activating factor, ~9 platelet activator inhibitor, 2° and various growth factors. Thus, the endothelium is involved in the regulation of diverse physiological functions such as inflammation, platelet aggregation, thrombosis, fibrinolysis, angiogenesis, mechanoreception, and vasoreactivity. Various model systems, employed to investigate the effect of oxidative stress on blood vessel function, suggest that oxidant-induced changes in vascular reactivity reflect alterations in endothelium-dependent regulatory mechanisms. For example, exposure of rats to hyperoxia attenuates the relaxant effect of acetylcholine on precontracted pulmonary arteries, although no effect is observed in the action of agents which are direct-acting muscle relaxants. 2~ Perfusion of rabbit lungs with the membrane-permeant oxidant tertiary-butylhydroperoxide (tert-bu-OOH) results in a vasoconstrictive response which is inversely related to the content of the vasodilator prostaglandin 12 in the perfusate effluent. 22 Ischemia reperfusion in isolated perfused rat heart decreases the vasorelaxant response to acetylcholine yet does not affect action of the direct muscle relaxant nitroglycerin, 23 suggesting an endothelium-dependent mechanism. Production of H 2 0 2 (Ref. 24) and/or its reactive metabolites 25 by polymorphonuclear neutrophils upon interaction of these cells with vascular endothelial cells may also alter vasoreactivity. Neutrophils activated by phorbol myristate acetate alter endothelial production of prostaglandin 12 as does cell-free, enzyme-generated H 2 0 2 (Refs. 24 and 26) and reagent U 2 0 2 (Refs. 27 and 28). Thus, oxidant stress generated by a variety of different mechanisms influences vascular reactivity and tone through altered production, release, or effect o f e n d o thelium-derived paracrine substances. Despite these advances, the molecular mechanisms associated with oxidant-induced endothelial cell damage remain largely unknown. In various cell types, oxidants such as hyperoxia, peroxides, and free radical species may produce peroxidation of membrane lipids, 29'3° oxidation of protein sulfhydryls, 29'31-33 DNA scission, 34-36 activation of poly(ADP-ribose)polymerase, 29'34'37'3s rearrangement of microfilaments, 39'4° altered protein synthesis, 4~ and/ or reduction of membrane fluidity. 42'43 Considerable evidence indicates that, in addition to these effects, oxidant stress alters Ca 2+ homeostatic mechanisms within the vascular endothelial cell. 44-51 Since an increase in cytosolic free Ca 2+ concentration ([Ca2+]i)

appears to trigger release of paracrine factors involved in endothelial control ofvasoreactivity and permeability, oxidant-induced changes in Ca 2+ signaling could explain much of the observed pathophysiology of oxidative injury. Indeed, an early event associated with endothelial cell dysfunction in oxidative stress involves alteration in transmembrane signaling mechanisms. 44'45 It is important to note that the effects of oxidative stress on cell function undoubtedly are related to the specific reactive species involved and the particular cell type examined. This review will focus on how Ca 2+ signaling mechanisms in vascular endothelial cells may be altered under conditions of oxidative stress mediated by tert-bu-OOH. Ca2+ SIGNALING IN VASCULAR ENDOTHELIAL CELLS Increases in [Ca2+]i within stimulated vascular endothelial cells result in activation of various Ca2+-de pendent events, including release of endotheliumderived relaxation factor. In this regard, the Ca 2+ ionophore, A23187, mimics agonist stimulation and is a potent stimulus for endothelium-derived relaxation factor release. Development of the technique by which cells are loaded with the Ca2+-sensitive fluorescent probe, fura-2, has resulted in the ability to monitor [Ca2+]i during stimulation by agonists. 52-s8 The agonist-stimulated increase in [Ca2+]~ comprises two c o m p o n e n t processes: (l) influx of Ca 2+ from the extracellular space, and (2) release of Ca 2+ from intracellular stores. The response of cultured endothelial cells to bradykinin is typical ofagonist responses in many different cell types. Bradykinin produces a four- to fivefold rise in [Ca2+]i, which increases within seconds to a peak and subsequently declines to a sustained elevated level. 59 Whereas the sustained c o m p o n e n t is eliminated in the presence of extracellular La 3+ or in the absence of extracellular Ca 2+, the transient response is preserved under these conditions. Thus, the transient c o m p o n e n t reflects the release of Ca z+ from internal stores, whereas the sustained c o m p o n e n t reflects the influx of Ca 2+ from the extracellular space. It is now well established that the release of Ca 2+ from internal stores results from generation of inositol-l,4,5-trisphosphate (Ins(1,4,5)P3) from phosphatidylinositol4,5-bisphosphate via G-protein-dependent activation of phospholipase C. 55'60-65 The contribution of Ca/+ release via non-Ins(1,4,5)P3-dependent mechanisms or from Ins(1,4,5)P3-insensitive internal stores of endothelial cells remains uncertain. In contrast to the intracellular release of Ca 2+, the mechanism by which agonists stimulate influx of Ca 2+ from the extracellular space has yet to be identi-

Calcium signaling in oxidant stress fled. Although a n u m b e r of groups have reported agonist-induced ionic currents measured using the gigaseal patch-clamp technique in vascular endothelial cells, 66"67correlation between channel activity and the rise in [Ca2+]i has not been demonstrated. At the whole vessel level, release of endothelium-derived relaxation factor is unaffected by drugs known to modulate Ca 2+ channels. 68-72 In cultured endothelial cells, depolarizing concentrations of extracellular K + have no effect on [Ca2+]i (Ref. 59), dihydropyridine Ca 2+ channel antagonists have no effect on bradykininstimulated changes in [CaZ+]i (Refs. 53, 57-59), and whole cell current measurements have failed to provide evidence for the existence of voltage-operated Ca 2+ channels. 59'73-75 Together with very low specific dihydropyridine binding in cultured endothelial cells, it appears that agonist-induced changes in [Ca2+]~ are not related to voltage-activated Ca 2+ channels. Although not directly voltage-gated, the agoniststimulated Ca 2+ influx pathway conducts Ca 2+ according to that ion's electrochemical gradient, is inhibited by membrane depolarization, 76-85 conducts Ba 2+ (Ref. 76), and allows 45Ca 2+ to rapidly equilibrate with a large c o m p o n e n t of the total cellular Ca 2+ compartment. 76 Sensitivity to membrane potential, magnitude of flux, and Ba 2+ conductance are consistent with a channel mechanism. In this regard, bradykinin-induced inward Na + and Ca 2+ currents have been observed in clusters of electrically coupled endothelial cells, but such currents have not been observed in single cell recordings. 86 Another potential mechanism for Ca 2+ entry, that via Na+/Ca 2+ exchange, appears not to be involved since isosmotic replacement of extracellular Na + with N-methyl-D-glucamine fails to alter either the time course or magnitude of response of [Ca2+]i to bradykinin. 87 Several studies in nonexcitable cells have suggested that Ca 2+ influx is coupled to the level of Ca 2+ within the internal store. After depletion of the stores and removal of the agonist, the Ca 2+ influx pathway remains activated until the internal store is replenished with Ca 2+ (Refs. 88-92). In various cell types, thapsigargin, a selective inhibitor of endoplasmic reticular Ca2+-ATPase, stimulates Ca 2+ influx. A similar effect on Ca 2+ influx in endothelial cells is produced by the Ca2+-ATPase inhibitors, 2,5-di(tert)-butylhydroquinone and cyclopiazonic acid. 93,94 Recent evidence suggests that the Ca 2+ influx pathway activated by depletion of internal Ca 2+ stores may share identity with that activated by agonists. 93 Both pathways are inhibited by La 3+, membrane depolarization, and the synthetic organic, SKF 96365, and both are insensitive to known modulators of voltage-gated Ca 2+ channels. These similarities raise the possibility that even in

637

the absence of agonist stimulation, depletion of Ins(1,4,5)P3-sensitive internal Ca 2+ stores activates the agonist-sensitive Ca 2+ influx pathway in cultured vascular endothelial cells. In summary, agonist-activated Ca 2+ influx does not occur via a classical voltage-gated Ca 2÷ channel, via Na+/Ca 2+ exchange, or via a channel activated by the rise in [Ca2+]i. The coupling between receptor activation and the Ca 2+ influx pathway requires the stimulation of phosphoinositide hydrolysis a n d / o r release o f Ca 2+ from the Ins(1,4,5)P3-sensitive internal store. Although some mechanisms involved in Ca 2+ signaling are yet to be defined, agonist-induced elevation of [Ca2+]i is important for release of paracrine factors which influence vascular reactivity and permeability and, as discussed later, is altered under conditions of oxidative stress. EFFECT OF OXIDANT STRESS ON Ca2+ SIGNALING Oxidant stress influences vascular reactivity via alterations in production, release, or effect of endothelium-derived paracrine factors. To determine whether oxidants alter receptor-mediated Ca 2+ signaling o f the vascular endothelial cell, we examined the effect of tert-bu-OOH on bradykinin-stimulated changes in [Ca2+]i of bovine aortic and calf pulmonary artery endothelial cells. 44'4j Direct measurement of Ca 2+ flux is ultimately required to understand the molecular details of oxidant-induced alterations in Ca 2+ homeostasis, and toward this end, we examined the effect of tert-bu-OOH on basal and agonist-stimulated 45Ca2+ uptake and efflux.46 Additionally, the effect of the peroxide on basal and agonist-stimulated 86Rb+ effiux was determined, since 86Rb+ flux is an indirect estimate of [Ca2+]i and reflects activity of Ca2+-dependent K + channels. Tert-bu-OOH is a desirable model oxidant for several reasons. 95 First, its lipid solubility affords rapid delivery to the cytosolic milieu and, unlike hydrogen peroxide, it is not a substrate for catalase. Second, this peroxide produces an oxidative stress that is well characterized. In hepatocytes, for example, metabolism of tert-bu-OOH is via the glutathione redox system. When the capacity of this system to metabolize tert-bu-OOH is compromised or exceeded, radical decay may give rise to tert-butoxyl (RO ") and/or tert-butylperoxyl (ROO ") radicals, 96'97 which may oxidize m e m b r a n e lipids or interact with critical sulfhydryl groups on cellular proteins. Third, the glutathione system is amenable to interventions which can increase or decrease the effect of tert-buOOH. The activities ofglutathione reductase or glutathione peroxidase may be respectively modified by 1,3-bis(2-chloroethyl)-l-nitrosourea (BCNU) or by

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the availability of selenium. Likewise, the levels of cellular reduced glutathione (GSH) can be reduced by inhibition ofgamma-glutamyl-synthetasewith buthionine sulfoximine, or cellular thiol status can be modified by exogenous supplemention with dithiothreitol or N-acetylcysteine. Hence, the level of oxidant stress may be modified by manipulation of the antioxidant capacity of the cell and/or the a m o u n t or time of oxidant exposure. Three temporal phases characterize the effect of tert-bu-OOH on bradykinin-stimulated Ca 2+ signaling in vascular endothelial cells. At a sublytic concentration of 0.4 mM, tert-bu-OOH initially inhibits the agonist-stimulated Ca 2+ influx pathway, with no alteration in basal (resting) [Ca2+]i . Later, receptor-activated release of Ca z+ from internal stores is inhibited and resting [Ca2+]i is elevated. Finally, basal [Ca2+]i progressively rises, and cytosolic Ca 2+ becomes unresponsive to agonist stimulation. The elevation in [Ca2+]i observed during the third phase is associated with activation of CaZ+-dependent K + channels, an effect which leads to significant depletion of cellular K + (Ref. 46). Net cellular Na + increases concomitantly with the decrease in cellular K + content. 98 Elevated intracellular Na + is in turn associated with increased activity of plasmalemmal Na + pump activityfl8 These effects oftert-bu-OOH occur before loss of membrane integrity and before any apparent compromise of cellular energy stores and will be discussed in greater detail in the following sections.

Phase I: Inhibition of the bradykinin-stimulated Ca 2+ influx path way The effect of the agonist, bradykinin, on [Ca2+]~ of cells incubated with tert-bu-OOH for 0, 1,2, and 3 h is shown in Fig. 1. The initial phase of oxidant stress is characterized by a decrease in the peak response of [Ca2+]i to bradykinin and a more rapid return of [Ca2+]i toward basal levels following stimulation (Fig. I B). This result could be explained by inhibition of agonist-stimulated Ca 2+ influx and/or inhibition of Ca 2+ release from internal stores. To address these two possibilities, the response of oxidant-treated cells to bradykinin was determined in the absence of extracellular Ca 2+ (Fig. 2). In oxidant-treated cells examined using this protocol, neither basal [Ca2+]i nor the bradykinin-stimulated increase in [Ca2+]i is altered following incubation with tert-bu-OOH for 30-60 min. However, the increase in [Ca2+]i observed upon readdition of Ca 2+ to the external buffer is significantly diminished, consistent with the conclusion that an early effect of oxidant stress in endothelial cells is inhibition of the agonist-stimulated Ca 2+ influx pathway.

To obtain further support for this contention, we examined the effect of tert-bu-OOH on agonist-stimulated 45Ca2+ uptake and demonstrated that incubation of endothelial cells with tert-bu-OOH for l h results in inhibition of bradykinin-stimulated 45Ca2+ uptake. 46 The actual mechanism by which inhibition of agonist-stimulated Ca 2+ influx occurs during this initial phase of oxidant stress is unknown. Free radicals may directly modify the protein responsible for Ca 2+ entry, or the coupling mechanisms between receptor stimulation and Ca 2÷ influx may be altered. At least in part, inhibition of influx may be related to membrane depolarization. In this regard, during the initial phase of incubation of cells with tert-bu-OOH, membrane potential changes from -60.7 _+0.9 mV to -27.6 _+ 2.9 mV (Ref. 99). The role of resting potential in the response of cells to oxidant stress and its role in attenuation of agonist-induced Ca 2÷ influx is presently being defined. Finally, isotope flux studies demonstrate that uptake under basal (unstimulated) conditions is elevated during the initial phase of oxidant stress, an effect not observed during measurement of [Ca2+]i. Thus, increased basal Ca 2+ uptake must be matched by Ca 2+ sequestration mechanisms during this period.

Phase H: Inhibition of receptor-activated Ca 2+ release from internal stores and elevated basal [Ca2+]i There are three lines of evidence that subsequent to inhibition of Ca z+ influx, oxidant stress inhibits receptor-activated release of Ca 2+ from internal stores. First, incubation of cells with tert-bu-OOH for 2 h results in further inhibition of the bradykinin-stimulated rise in [Ca2+]i (Fig. 1C), an effect also observed in cells suspended in Ca2+-free/ethylene glycol-bis(3aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) buffer (Fig. 2C). Second, bradykinin-stimulated 45Ca2+ efflux from isotope-loaded cells is inhibited by more than 50%. 46 Stimulation of 45CaZ+ efflux by receptor-activation reflects release of Ca 2+ from Ins(l,4,5)P3-sensitive intracellular stores. 63 Upon release into the cytosol, Ca 2+ leaves the cell via the action of plasmalemmal CaZ+-ATPase. Bradykinin increases unidirectional efllux of Ca :+ from calf pulmonary artery endothelial cells by approximately 12-fold. The inhibitory effect of tert-bu-OOH on agonist-stimulated Ca 2+ efllux is consistent with decreased receptor-activated Ca 2+ release and/or decreased plasmalemmal CaZ+-ATPase activity. Alternatively, oxidant stress may inhibit the endoplasmic reticular Ca/+ pump and cause depletion of the agonist-sensitive intracellular pool, thus limiting Ca 2+ mobilization. To further investigate these possibili-

Calcium signaling in oxidant stress

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TIME (minutes) Fig. 1. Effect of/ert-bu-OOH on bradykinin-stimulated changes in [Ca2+]i. Calf pulmonary artery endothelial cells loaded with fura-2 were incubated at 37°C with 0.4 mM tert-bu-OOH for 0 (A), 60 (B), 120 (C), and 180 (D) min before measurement of fluorescence. Bradykinin (50 nM) was added at the time indicated by the arrow. Reprinted with permission from: Elliott, S. J.; Schilling, W. P. Carmustine augments the effects oftert-butyl-hydroperoxide on calcium signaling in cultured pulmonary artery endothelial cells. Journal q/Biological Chemistry. 265:103-107; 1990. Copyright 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

ties, we examined the effect oftert-bu-OOH on bradykinin-stimulated production of phosphatidylinositol bisphosphate (PIP2) metabolites. The results of these studies provide additional evidence that oxidant stress inhibits receptor-activated Ca 2+ release. The cell-associated pool of PIP 2 was radiolabeled using [3H]myo-inositol.l°°'l°l Following a 48-h equilibration period, extracellular label was removed, and the cells were incubated with tert-bu-OOH (0.4 mM) for various times. Cells were stimulated with bradykinin, and metabolites of PIP 2 hydrolysis were extracted using anion-exchange chromatography and quantitatively analyzed via standard liquid scintillation technique. As shown in Figs. 3 and 4, tert-bu-OOH inhibited bradykinin-stimulated production of inositol trisphosphate in a time-dependent manner. After incubation periods of 1 and 2 h with the oxidant, production of inositol trisphosphate was decreased by 37% and 80%, respectively. Accumulation of inositol bisphosphate was similarly inhibited by tert-bu-OOH in a time-dependent manner. Thus, via measurement of cytosolic Ca 2+ concentration, activity of Ca 2+ flUX pathways, and bradykinin-stimulated production of inositol trisphosphate, it appears that tert-bu-OOH inhibits agonist-stimulated C a 2+ release from internal stores. In agreement with these results is the finding that tert-bu-OOH inhibits hormonally and nonhormonally induced increases in total inositol polyphos-

phate in rat hepatocytes.102 However, in porcine pulmonary artery endothelial cells labeled with [t4C] or [3H]inositol, brief incubation (10 min) with H20 2 (12.5 mM) increases the fraction of label comigrating with lysophosphatidylinositol, phosphatidylinositol bisphosphate, and inositol polyphosphate under basal (unstimulated) conditions.I°3 Together, these findings suggest that the effect of oxidant stress on phospholipid metabolism may vary with the particular oxidant employed or with the cell type examined. As noted, inhibition of endothelial cell Ca2+-ATP ase following incubation with tert-bu-OOH may contribute to inhibition of agonist-stimulated 45Ca2+ efflux. During the second phase of oxidant stress, basal [Ca2+]i is elevated whether measured in the absence or presence of extracellular Ca 2+ (Figs. 1C and 2C), a finding which suggests that the mechanism(s) which extrudes Ca 2+ from the cytosol is impaired. Active Ca 2+ pumps at the endoplasmic reticulum and plasmalemma serve to maintain cytosolic Ca 2+ homeostasis. At the endoplasmic reticulum, Ca2+-ATPase acts to maintain a store of releaseable Ca 2+, and pump inhibition will result in depletion of storage pools. In other cell types, such as erythrocytes, inhibition of Ca2+-ATPase activity is observed on incubation with tert-bu-OOH, 1°4'1°5 while in the rat hepatocyte, tertbu-OOH decreases Ca 2÷ sequestration into microsomal fractions 1°6 and inside-out plasmalemmal vesi-

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TIME ( m i n u t e s ) Fig. 2. Effect of tert-bu-OOH on bradykinin-stimulated changes in [Ca2+]~determined in Ca2+-Free/EGTA buffer. Calf pulmonary artery endothelial cells loaded with fura-2 were incubated at 37°C with 0.4 mM tert-bu-OOH for 0 (A), 60 (B), 120 (C), and 180 (D) min before measurement of fluorescence in CaZ+-free/EGTA buffer. Bradykinin (50 nM) and CaCI2 (2.0 mM) were added at the times indicated by the arrows. Reprinted with permission from: Elliott, S. J.; Schilling, W. P. Carmustine augments the effects oftert-butyl-hydroperoxide on calcium signaling in cultured pulmonary artery endothelial cells. Journal of Biological Chemistry, 265:103-107; 1990. Copyright 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

cles. 3~ Vascular endothelial cells incubated with tertbu-OOH clearly retain the majority of their Ca 2+ (Ref. 46) and display inhibited bradykinin-stimulated 45Ca2+ efflux, results which together are consistent with inhibition of cell membrane Ca2+-ATPase. It is possible, however, that under normal conditions, Ca 2* may flux directly from storage pools to the cell exterior via connections directly linking endoplasmic reticulum to cell membrane. Uncoupling of such junctions may contribute to inhibition of agoniststimulated Ca 2+ efflux under conditions of oxidant stress.

Phase III: Loss of responsiveness to agonists and sustained elevation of [Ca2+]i The third phase of oxidant stress is characterized by failure of [Ca2+]i to respond to stimulation by bradykinin (Figs. 1D and 2D). This observation is not surprising, since inhibition of bradykinin-stimulated Ca z+ uptake and efllux has been observed in parallel flux studies.46 Furthermore, incubation of endothelial cells with tert-bu-OOH for 3 h results in loss of cellular ability to maintain Ca 2+ homeostasis; basal [Ca2+]i is increased to approximately 200 nM and progressively rises with time (Fig. 1D). Prolonged incubation with tert-bu-OOH does not cause generalized loss of mem-

brane integrity within the time flame examined since a large inward Ca 2+ gradient continues to be maintained. It is possible, however, that continued incubation with the oxidant beyond 3 h may eventually result in decreased cell viability and decreased levels of ATP. There is substantial evidence that oxidative injury is associated with depletion of high energy compounds. H20 z acts to release [3H]purines from porcine aortic endothelial cells, 27 and the same oxidant decreases ATP levels in P388Dl cells ~°7 and endothelial cells. 39,~°8'~°9 However, the relationship between decreased energy charge and cell injury is complex and remains unclear.~° For example, the iron chelator, deferoxamine, and the xanthine oxidase inhibitors, allopufinol and oxypurinol, each prevent H20 2mediated cytotoxicity in endothelial cells as determined by 51Cr release yet do not protect against oxidant-associated decreases in ATp.1 ~ In the present model, availability of high-energy intermediates does not seem to be significantly compromised after incubation of calf pulmonary endothelial cells with tertbu-OOH, and for the following reasons the changes in Ca 2+ signaling do not appear to be directly linked to depletion of ATP. First, the cells are able to maintain a large Ca 2+ gradient across the plasmalemma. Second, unidirectional ouabain-sensitive 86Rb+ influx, a measure of Na + pump activity, is stimulated by treat-


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Time (s) Fig. 3. Effectof tert-bu-OOHon inositol polyphosphate (IP) signaling in bradykinin-stimulated vascular endothelial cells. Calf pulmonary artery endothelial cells were incubated for 48 h with [3H]-myo-inositol,prior to washing with serum-free medium and incubation in the absence (O) or presence (0) of tert-bu-OOH(0.4 mM) for 1 h. At the completion of the incubation period with tert-bu-OOH,cellswere stimulated with bradykinin (50 nM) (t = 0) for 0, 15, 30, or 90 s in the presenceof l 0 mM LiCl. The reaction was terminated by rapid aspiration of medium and precipitation of cellular proteins by addition of ice-cold perchloric acid at each timepoint. Elution ofinositol monophosphate (A), inositol bisphosphate (B), inositol trisphosphate (C), and total IP (D) fractions from the supernatant was performed via increasing concentrations of NH4F, in the presence of Na2B4OT/EDTAor formic acid using anion-exchange chromatography. Fractions were quantitated using standard liquid scintillation counting technique. Values are plotted as fold-changeover control basal production for each moiety. Data represent means +_SEM (n = 3). Where not shown, SEM was smaller than symbol size employed.

m e n t of the cells with tert-bu-OOH for 3 h, 98 suggesting that even after prolonged incubation with the oxidant, A T P appears to be sufficiently available to support increased activity of the Na + p u m p . Third, tert-bu-OOH does not alter cell-associated 3H-adenine (Ref. 98), an estimate of the A T P pool size. t12 Thus, rather than loss o f high-energy substrate, inhibition of CaE+-ATPase in endothelial cells treated with tert-bu-OOH is probably linked to a direct effect on the p u m p protein, such as oxidation of critical sulfhydryl groups. 31'1°5'1t3 DEPENDENCE ON EXTRACELLULAR OXIDANT Incubation of endothelial cells with tert-bu-OOH results in altered Ca 2÷ signaling in a time-dependent manner, as described earlier. T o determine whether the temporal effects of tert-bu-OOH on signaling depend on continuous presence of the oxidant, cells were incubated with tert-bu-OOH before resuspension in the absence or presence of the oxidant. Because tert-bu-OOH is readily permeable across the cell m e m b r a n e , 95 progressive inhibition of Ca 2+ signaling, despite removal of the oxidant from the extracellular space, would suggest that after entry into the cell, tert-

b u - O O H is either trapped within an intracellular c o m p a r t m e n t or is converted to a long-lasting metabolite which itself is responsible for the observed changes in signal transduction. In this regard, tert-bu-OOH is metabolized predominantly via glutathione peroxidase to tert-butylalcohol, although radical decay with formation of tert-butoxyl ( R O ' ) and/or tert-butoxyperoxyl (ROO") moities is possible if glutathione-dependent metabolism is compromised. 96'97 Alternatively, free radical species might initiate and propagate chain reactions such as lipid peroxidation, the effects of which might not b e c o m e apparent until later timepoints, or tert-bu-OOH m a y produce damage to a critical biochemical pathway which triggers events leading to apoptosis. In this regard, Deuticke et al. reported induction of progressive erythrocytic m e m brane damage over several hours, measured by stilbenedisulfonate-insensitive C1- permeability and colloid-osmotic lysis, following a 15-min pulse incubation with tert-bu-OOH (2 mM). 114 Thus, a process of irreversible cell injury m a y be initiated after only short incubations with oxidant but m a y not be apparent or measurable during the initial phases. Cessation of progressive inhibition o f Ca 2+ signaling upon rem o v a l of tert-bu-OOH would suggest that the inhibi-

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Time (s) Fig. 4. Effect of tert-bu-OOH on inositol polyphosphate (IP) signaling in bradykinin-stimulated vascular endothelial cells. Production ofinositol monophosphate (A), inositol bisphosphate (B), inositol trisphosphate (C), and total IP (D) in bradykininstimulated calf pulmonary artery endothelial cells under control (O) conditions and after treatment with tert-bu-OOH (0.4 mM) (O) for 2 h. Experimental method was as described in legend to Fig. 3. Comparison of profiles in panel C with those of Fig. 3C demonstrate the time-dependent effect of tert-bu-OOH. Data represent means _+ SEM (n - 3). Where not shown, SEM was smaller than symbol size employed.

tory changes are due to action of a free parent molecule or a short-lived metabolite. Cells were incubated with tert-bu-OOH (0.4 mM) for 20 min prior to resuspension in the absence or presence of the oxidant. Bradykinin-stimulated changes in [Ca2+]i were determined at 20 min, 1 h, and 2 h (interval times of 0, 40, and 100 min, respectively). Compared to a control value of 4.3 _+ 0.2 (n = 18), bradykinin stimulated a fold-change in [Ca2+]i of 3.3 + 0.1 (n = 12;p < .02) in cells treated with tert-buOOH for 20 min and measured immediately after the incubation period. No further decrease in bradykininstimulated fold-change was observed in cells measured either at 1 h or 2 h (Fig. 5A). In contrast, in cells reincubated with tert-bu-OOH after the initial incubation period, the bradykinin-stimulated fold-change in [Ca2+]i progressively and significantly decreased, reaching 2.6 _+0.1 at 1 h and 1.6 + 0.1 at 2 h (each, p < .002 compared to control). This decrease in foldchange was comprised of two components: (1) elevated basal [Ca2+]i, which increased from 70.3 + 6.1 nM at the 20-min timepoint to 104 + 8 nM at 2 h, and (2) decreased bradykinin-stimulated peak [Ca2+]i , which declined from 236 + 16 nM to 170 _+ 14 nM through the same period. Inhibition of bradykinin-stimulated Ca 2+ influx, determined by the ratio of plateau phase [Ca2+]~ to basal [Ca2+]i , depended on continued presence oftertbu-OOH in the extracellular space (Fig. 5B). In continuous presence of the oxidant, plateau phase [Ca2+]i/

basal [Ca2+]i progressively decreased, whereas after removal of tert-bu-OOH at the 20-rain timepoint, no significant decrease in agonist-stimulated influx of Ca 2+ was observed through 2 h. Thus, although the inhibitory effects initially observed upon incubation of endothelial cells with tertbu-OOH are sustained during the time frame of experimental measurement, they progress only slightly following removal of the oxidant from the incubation medium. These results are in contrast to progressive inhibition of Ca 2+ signaling observed when cells are continuously incubated with tert-bu-OOH, and they suggest that inhibition depends, at least in part, on the presence of extracellular tert-bu-OOH. It is possible that following its removal from the extracellular space, tert-bu-OOH diffuses from the cell down its chemical gradient, thus decreasing intracellular content of the oxidant. It is clear, however, that a stable injury persists after removal of extracellular oxidant, since reversal to normal signaling is not observed, at least within the time frame examined. These results suggest that the time-dependent effects of tert-buOOH cannot solely be accounted for by a long-lasting intracellular metabolite or propagation of a chain reaction. ROLE OF GLUTATHIONE REDUCI'ASE

Glutathione peroxidase-dependent metabolism of tert-bu-OOH utilizes the sulfhydryl moiety ofglutathi-


643

A 4-

300

o rn

+

~2o

f'4 O O

v

I

C

°--

c~ c o cO

B 37

I

-o 0 b_

&2 A p( + p( ++ p( r-'7

o o I O_

0.02 0.002 0.0001 vs lira

* p( 0.02 ,,,,, p< 0.0001

(n=18)

(n=12)

(n=6)

(n=6)

60 120 Time at measurement (min)

Control

20

Fig. 5. Role of extracellular tert-bu-OOH on oxidant-induced inhibition of bradykinin-stimulated changes in [Ca2+]i. (A) Bradykinin-stimulated fold-change in [Ca2+]~ (peak [Ca2+]dbasal [Ca2+]i and (B) bradykinin-stimulated Ca 2+ influx (plateau phase [Ca2+]i/basal [Ca2+]i) were determined in control cells (solid bar) and in cells incubated with tert-bu-OOH (0.4 mM) for 20 min followed by resuspension in the absence (cross-hatched bars) or presence (open bars) of 0.4 mM tert-bu-OOH. Cells were stimulated via addition of bradykinin (50 nM). Fluorescence measurements were performed at 20, 60, and 120 min. Data represent means ___SEM. Responses obtained immediately after the initial 20 min incubation period with tert-bu-OOH (i.e., at 20 min) have been grouped together (n - 12). Control responses did not vary between timepoints and for clarity are grouped together (n = 18).

one (GSH), which provides reducing equivalents for reduction of oxidant to tert-butylalcohol. 9s Regeneration of GSH from oxidized glutathione depends on the action of glutathione reductase, an enzyme that can be specifically inhibited by the nitrosourea, BCNU. Various studies have employed BCNU to demonstrate the protective importance of GSH in oxidant-induced cell injury. ~15-119We employed BCNU in an effort to further understand the response of endothelial cell antioxidant defense mechanisms and to determine the role ofglutathione reductase in tert-buOOH-induced cell i n j u r y . 45'46 In vascular endothelial cells treated with BCNU (75 uM) alone, basal [Ca2+]i is increased by a small but significant amount when compared to control ( 129 +_ 10 nM vs. 103 ___6 nM), suggesting that BCNU and/or inhibition of glutathione reductase alters the cellular mechanism associated with the steady state between Ca 2+ influx and efflux. 45 The increase in basal [Ca2+]i

is evident even when measurements are performed in Ca2+-free/EGTA buffer, suggesting that BCNU may inhibit the plasmalemmal Ca 2÷ pump which acts to remove Ca 2÷ from the cytosolic compartment. The nitrosourea does not, however, alter the biphasic response of [Ca2+]i to bradykinin stimulation. Thus, the signaling mechanisms associated with receptor stimulation and responsible for the rise in [Ca2+]i appear to be intact in cells treated solely with BCNU. Next, we examined the effect of BCNU on cells subsequently treated with tert-bu-OOH. Whereas incubation of endothelial cells with tert-bu-OOH for a short time (30 min) only slightly attenuates the response to bradykinin, incubation of cells with BCNU followed by tert-bu-OOH (30 min) dramatically inhibits both bradykinin-stimulated release of Ca 2÷ from internal stores and influx of Ca 2÷ from the extracellular space. BCNU clearly accelerates the time course of effect of tert-bu-OOH on Ca 2÷ signaling in

S.J. ELLIOTT el al.

644

endothelial cells, such that the profile of Ca 2+ signaling in cells preincubated with the nitrosourea and treated with tert-bu-OOH for 30 min is virtually identical to that observed in cells incubated with tert-buOOH alone for 2 h. In 45Ca2+ flux studies, incubation of cells with BCNU followed by tert-bu-OOH inhibited bradykinin-stimulated Ca 2+ uptake by more than twice that of either agent alone. 46 Thus, the effect of tert-bu-OOH is clearly dependent on the glutathione redox system, since the action of the oxidant on Ca 2+ signaling is markedly enhanced when glutathione reductase is inhibited by BCNU. EXOGENOUS SULFHYDRYLS

Under certain conditions, supplementation with exogenous sulfhydryl groups, either in the form ofglutathione itself or surrogate sulfhydryl-containing compounds such as dithiothreitol or N-acetylcysteine, may protect against oxidant stress. ~2o,121For example, in rabbit renal proximal tubules, supplemental glutathione protects against tert-bu-OOH-induced injury, as determined by malondialdehyde production and cellular contents of K + and ATP. ~z2 The intracellular cysteine delivery agent, L-2-oxothiazolidine-4-carboxylate, increases the glutathione content of cultured calf pulmonary artery endothelial cells and protects against loss of cell viability induced by enzyme-generated H202 and measured by release of 5'Cr (Ref. 120). Energy-dependent Ca R+ pumps which extrude Ca 2+ from the cytosol either into internal stores or across the cell membrane possess thiol-containing amino acids susceptible to oxidation or disulfide exchange.123 In this regard, the function of plasmalemreal CaZ+-ATPase in hepatocyte cell membrane vesicles is sensitive to oxidation of key sulfhydryl groups, 3~ an effect which can be prevented by dithiothreitol. We sought to determine whether the sulfhydryl-containing compounds, dithiothreitol and Nacetylcysteine, offer protection against oxidant-induced changes in endothelial cell Ca 2+ signaling. A protective effect of these compounds against oxidantinduced inhibition of Ca 2+ signaling would suggest a sulfhydryl-dependent mechanism linking tert-buOOH-induced oxidant stress with inhibition of the signal transduction pathway. Initial studies were performed to determine whether sulfhydryls alone altered signaling behavior in vascular endothelial cells. Compared to control cells in which basal [Ca2+]i was 68 + 3 nM (n = 4), cells incubated with 0.2 mM or 2 mM dithiothreitol had basal [Ca2+]i values of 97 _+ 10 nM (p < .02) and 87 + 6 nM (p < .02), respectively. Bradykinin-stimulated peak [Ca2+]i and [Ca2+]i during the subsequent plateau phase were not signifi-

Table I. Effect of Dithiothreitol Preincubation on Changes in Cytosolic Free Calcium Concentration ([Ca2+]~) Induced by tert-Butylhydroperoxide (tert-bu-OOH)

Condition Control Dithiothreitol, tert-bu-OOH tert-bu-OOH

Basal [Ca2+], (nM) 69 _+ 12 87 + 4* 112 _+ 4

Bradykinin-stimulated fold-change in [Ca2+]i 3.9 + 0.3 2.2 +_ 0.1** 1.7 _+ 0.1

Fura-2-1oaded cells were preincubated in the absence or presence of 0.2 m M dithiothreitol (20 min) prior to washing and incubation with 0.4 m M tert-bu-OOH (2 h). Basal values represent those recorded immediately prior to addition of bradykinin (50 nM). Fold-change was determined from basal [Ca2+]~ and the peak value of [Ca2+]~ upon stimulation with bradykinin. Values are means _+ SEM (n - 6). * p < .01 compared to tert-bu-OOH alone. ** p < .02 compared to tert-bu-OOH alone.

cantly different from control. Similar results were observed in cells incubated with N-acetylcysteine. To determine whether sulfhydryl agents alter the effects of tert-bu-OOH, cells were preincubated in the absence or presence of dithiothreitol (0.2 mM or 2.0 mM) or N-acetylcysteine (1 mM) prior to incubation with tert-bu-OOH (0.4 mM) for 2 h. Dithiothreitol partially prevented the tert-bu-OOH-induced increase in basal [Ca2+]i and acted to preserve the bradykininstimulated fold-change in [Ca2+]i (Table 1). These effects were similarly observed in cells preincubated with 2 mM dithiothreitol or Noacetylcysteine. These findings suggest that sulfhydryl supplementation partially prevents oxidant-induced inhibition of bradykinin-stimulated Ca 2+ signaling, and they provide evidence that oxidant-induced changes in endothelial cell signal transduction are at least in part related to cellular thiol status. It has been known for many years that reactive sulflaydryl reagents alter Ca 2+ homeostasis of skeletal muscle sarcoplasmic reticulum, 124 and recently, Trimm et al. 125 demonstrated that Cu2+/cysteine-induced Ca 2+ efflux from sarcoplasmic reticulum occurs via formation of a mixed disulfide between the exogenous mercaptan and a critical sulfhydryl on the putative transmembrane channel protein in a process that may be reversed by dithiothreitol. Similar effects mediated by reactive disulfide compounds and reversed by dithiothreitol have been documented in Ca 2+ release from cardiac sarcoplasmic reticulum. Z26 Less information is available regarding potentially critical sulfhydryl groups on Ca 2+ flux pathways in vascular endothelial cells, particularly the Ins( 1,4,5)P3-sensitive Ca 2+ channel of endoplasmic reticulum. By analogy, however, a mouse Purkinje cell


Ins(l,4,5)P3 receptor and the skeletal muscle ryanodine receptor have similar amino acid sequences, ~z7 raising the possibility that the Ins(1,4,5)P3 receptor and/or the Ca 2÷ release channel of vascular endothelial cell endoplasmic reticulum may be susceptible to direct modification by thiols. In adrenal cortical microsomes, dithiothreitol decreases affinity of the Ins(1,4,5)P3 receptor for that messenger, with no change in the number of binding sites, whereas the alkylating agent, N-ethylmaleimide, decreases the number of binding sites but does not significantly alter binding affinity.~28 In human platelet internal membranes, the sulfhydryl reagents, AgCI and pchloromercuribenzoate, inhibit Ins(1,4,5)P3-induced Ca 2+ release, an effect reversed by dithiothreitol. ~29 Recently, Rooney et al.~3°demonstrated that pretreatment of intact hepatocytes with tert-bu-OOH (0.75 mM) results in inhibition of Ins(l,4,5)P3-induced Ca 2+ release following cell permeabilization. Using the same protocol, inhibition of Ca 2+ reuptake was noted following Ins(1,4,5)Ps-induced release. In contrast to these effects observed after pretreatment of intact hepatocytes with tert-bu-OOH, no similar effect was observed when the oxidant was added after permeabilization, suggesting that some other intracellular component is additionally necessary for tert-buOOH to produce changes in Ins(1,4,5)P3-mediated Ca z+ flux. In this regard, the effects of tert-bu-OOH could be mimicked by oxidized glutathione (GSSG), albeit using a high concentration (2 mM), raising the possibility that formation of mixed disulfides may ultimately be the mechanism by which inhibition of Ins(l,4,5)P3-induced Ca 2+ signaling occurs. The exogenous sulfhydryl-containing compounds used in our studies of vascular endothelial cells may attenuate oxidant-induced elevation of[Ca2+]i by limiting oxidation of ATP-dependent Ca 2+ pumps at either the surface membrane or at the endoplasmic reticulum. However, the mechanism underlying the interaction of exogenous thiol-containing compounds and intracellular Ca 2+ homeostasis remains uncertain. Although dithiothreitol in concentrations of 0.2 and 2 mM limits oxidant-induced increases in basal [Ca2+]i, a different result is obtained using a concentration of 20 mM. Under this condition, basal [Ca2+]i was 161 + 27 nM in tert-bu-OOH-treated cells preincubated with dithiothreitol (20 mM) compared to 108 _+8 nM observed in tert-bu-OOH-treated cells preincubated with vehicle. In cells pretreated with 20 mM dithiothreitol but not exposed to tert-bu-OOH, basal [Ca2+], was 112 +_4 nM compared to 68 _+3 nM for untreated control cells. Thus, in contrast to the findings observed using lower concentrations of the sulfhydryl, dithiothreitol had no protective effect on oxidant-in-

645

duced inhibition of Ca 2+ signaling, suggesting that dithiothreitol may itself engage in sulfhydryl exchange and alter critical -SH groups of membrane Ca 2+ channels and/or pumps. It is also possible that intracellular levels of GSSG and/or GSH could modulate channel function, and in this regard, disulfide compounds such as 2,2'dithiodipyridine which are considered to be more reactive than GSSG act to induce release of Ca 2+ from cardiac sarcoplasmic reticulum. ~26 At present, the molecular targets which result in altered signal transduction when vascular endothelial cells are subjected to oxidant stress are being identified. Within the cell, it is likely that compartmentalization of reactive moieties, antioxidant defense mechanisms (e.g., glutathione peroxidase), and intracellular target proteins or enzymes (e.g., CaZ+-ATPase) contributes to the determination of the ultimate functional outcome for the cell. POSSIBLE MECHANISMS

We have employed information from the aforementioned studies to identify possible pathways involved in oxidant-induced inhibition of Ca 2+ signaling in vascular endothelial cells. Figure 6 identifies proposed mediators which might be involved in the observed changes in agonist-stimulated Ca z+ signaling. As discussed earlier, although Ca 2+ influx probably occurs via a plasmalemmal channel, neither the channel nor the underlying regulatory mechanisms have been identified. Thus, it is uncertain which, if any, intracellular second messengers transduce the agonist signal to the putative channel. Nevertheless, it is clear from our own studies that tert-bu-OOH inhibits the agonist-stimulated Ca 2+ influx pathway as an early event in the cell injury process. This may occur via a direct effect on the pathway, wherein tert-buOOH elicits a conformational change in the channel protein. Alternatively, tert-bu-OOH may interrupt agonist-receptor coupling or receptor-G protein coupling, or may inhibit the generation of a critical second messenger which normally activates Ca 2+ influx. Although we have preliminary evidence that tert-buOOH depolarizes vascular endothelial cells, 99 it is uncertain whether depolarization is etiologically linked to oxidant-induced inhibition of Ca 2+ influx. The temporal effects of tert-bu-OOH on Ca 2+ signaling are clearly accelerated in the presence of BCNU-induced inhibition of glutathione reductase. This phenomenon may reflect the action of GSSG, which may accumulate in the intracellular compartment if not exported to the cell exterior. Thiol status, including glutathione redox status, may influence the release of Ca 2+ from Ins(1,4,5)P3-sensitive stores, al-

S.J. ELLIOTT et al.

646

Ca2+

Ag ~ R >

tert-bu-OOH

PLC (2)

R ~'~~-I~'J{ 2)

~)

jf'tL~

~~ ~".~

/

~R( tert-bu-OOH ~

Ca2*

~I~l~ R"

'nst'~?~(~ T R-SG

"® _ ~ P I I I

J~ G~I--'~'-ABCNU ~ 2 ~ terl-bu-OH GSSG/ \ ~ ~/E~=~,~ NADPH t ~

a'-

f

Ca2+

Ca2•

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~ ~

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Fig. 6. Representation of possible pathways involved in inhibition of Ca 2÷ signaling by ten-bu-OOH. Coupling of an agonist (Ag) with plasmalemmal receptor (R) stimulates dissociation of the a subunit from cell membrane GTP-binding protein, leading to activation of phospholipase C, which catalyzes hydrolysis of phosphotidylinositol bisphosphate (PIP2) to inositol trisphosphate (InsP3) and diacylglycerol (DAG). Activation of InsP3 receptors (IR) at the endoplasmic reticulum activates release of Ca 2÷ into the cytosol. The membrane-permeant organic, tert-butylhydroperoxide (tert-bu-OOH), enters the cell, where it is reduced by cytosolic glutathione peroxidase (GPX) to tert-butylalcohol. In the same step, glutathione (GSH) is oxidized to its dimeric form (GSSG). Reduction of GSSG occurs via gluathione reductase (GR). Inhibition of the agonist-stimulated Ca 2÷ influx pathway may occur via an effect on the membrane receptor ( 1), via an effect on the coupling between receptor stimulation and activation of the influx pathway (2), or via a direct effect on the putative channel (3). See text for evidence that tert-bu-OOH inhibits agonist-stimulated production of InsP~. Enhancement of oxidant-induced inhibition of signaling by 1,3-his (2-chloroethyl)-lnitrosourea (BCNU) appears to reflect either direct or indirect effects of GSSG and/or NADP redox status (see text for discussion). Metabolism of tert-bu-OOH to radical species such as tert-butoxyl, tert-butylperoxyl, or other moieties may occur and ultimately may be responsible for tert-bu-OOH-induced changes in vascular endothelial cell signal transduction. For clarity, potential radical metabolites (R °) have not been individually depicted.

though this mechanism has not been demonstrated in vascular endothelial cells. Full consideration of the sensitivity of mitochondrial Ca 2÷ stores to changes in pyridine redox status is beyond the scope of this review, but it is of note that shifts in reduced nicotinamide adenine dinucleotide (NADH)/nicotinamide adenine dinucleotide (NAD ÷) balance toward the oxidized form result in inhibition of mitochondrial Ca 2÷ flux in other cell systems such as rat hepatocytes. 131.132 Thus, extrapolation of these effects to vascular endothelial cells allows for the possibility of glutathione and/or pyridine redox status to contribute to regulation of Ca 2+ flux pathways in these cells. Metabolism of tert-bu-OOH may result in production of various radical species, especially if the glutathione system is compromised. Potential metabolic products of tert-bu-OOH have not been fully characterized in vascular endothelial cells, although there is evidence that in other systems tert-butoxyl and tertbutylperoxyl radicals may be produced. 95 In this regard, the biological effects of tert-bu-OOH within the

cell may be etiologically related to the action of these radical products rather than to that of the parent molecule. Although radical derivatives oftert-bu-OOH can initiate lipid peroxidation chain reactions within the plasmalemma, preliminary evidence suggests that this particular mechanism does not mediate tert-buOOH-induced inhibition of Ca 2+ signaling. 133

CONCLUSION

Oxidative stress produces time-dependent changes in Ca z+ signaling within vascular endothelial cells. These changes appear to occur early in the pathophysiological process of cell injury and may significantly contribute to altered vascular function in a number of disease states. Future directions in this field should focus on identification of the specific metabolites and chemical interactions which are responsible for the observed changes at the cellular and tissue levels, since the physiologic and biophysical outcome is


likely related to both the responsible oxidant moiety and the target cell type. Acknowledgement--This work was supported by grants from the American Heart Association (AHA--Texas Atfiliate 89G-190; 91R-190; and AHA--National 900946) and from the National Institutes of Health (HL-44119). This work was performed during the tenure of an NHLBI Clinical Investigatorship (HL-02595) awarded to S. J. Elliott and an AHA Established lnvestigatorship awarded to W. P. Schilling.

18. 19.

20. 21.

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