Suppression of Ca2+ Oscillations in Cultured Rat Hepatocytes by Chemical Hypoxia*

The model of “chemical hypoxia” with KCN plus iodoacetic acid mimics the ATP depletion and reductive stress of hypoxia. Here, we examined the effects of chemical hypoxia on cytosolic free Na+ and Ca” in single cultured rat hepatocytes by multiparameter digitized video microscopy and ratio imaging of sodium- binding furan indicator (SBFI) and Fura-2. Intracellular Na+ increased from about 10 mM to more than 100 mM after 20 min of chemical hypoxia, whereas cytosolic free Ca2+ remained virtually unchanged. In normoxic hepatocytes, phenylephrine (50 KM) and Arg-vasopressin (20-40 nM) induced Ca2+ oscillations in 70 and 40% of cells, respectively. These Ca2+ oscillations were suppressed after one spike following the onset of chemical hypoxia. Phenylephrine and vasopressin also increased inositol phosphate formation by 22 and 147%, respectively. This effect was suppressed by KCN plus iodoacetate. Intracellular acidosis is char- acteristic of chemical hypoxia. Intracellular acidosis induced by 40 mM Na-acetate suppressed Ca” oscilla- tions but did not inhibit hormone-induced inositol phosphate formation. Cytosolic alkalinization also suppressed Ca2+ oscillations. However, prevention of in- tracellular acidosis with monensin (10 WM) did not prevent

The model of "chemical hypoxia" with KCN plus iodoacetic acid mimics the ATP depletion and reductive stress of hypoxia. Here, we examined the effects of chemical hypoxia on cytosolic free Na+ and Ca" in single cultured rat hepatocytes by multiparameter digitized video microscopy and ratio imaging of sodiumbinding furan indicator (SBFI) and Fura-2. Intracellular Na+ increased from about 10 mM to more than 100 mM after 20 min of chemical hypoxia, whereas cytosolic free Ca2+ remained virtually unchanged. In normoxic hepatocytes, phenylephrine (50 KM) and  induced Ca2+ oscillations in 70 and 40% of cells, respectively. These Ca2+ oscillations were suppressed after one spike following the onset of chemical hypoxia. Phenylephrine and vasopressin also increased inositol phosphate formation by 22 and 147%, respectively. This effect was suppressed by KCN plus iodoacetate. Intracellular acidosis is characteristic of chemical hypoxia. Intracellular acidosis induced by 40 mM Na-acetate suppressed Ca" oscillations but did not inhibit hormone-induced inositol phosphate formation. Cytosolic alkalinization also suppressed Ca2+ oscillations. However, prevention of intracellular acidosis with monensin (10 WM) did not prevent suppression of Ca2+ oscillations during chemical hypoxia. Mitochondrial depolarization with uncoupler did not change free Ca2+ levels during chemical hypoxia, indicating that mitochondria do not regulate free Ca2+ during chemical hypoxia. From these results, we conclude: 1) chemical hypoxia does not block Na+ influx across the plasma membrane; 2) Chemical hypoxia inhibits hormone-stimulated Ca2+ flux pathways across cellular membranes by two different mechanisms: (a) by ATP depletion, which disrupts hormonemyo-inositol 1,4,5-triphosphate coupling, and ( b ) by intracellular acidosis, which inhibits myo-inositol 1,4,6-triphosphate-stimulated Caz+ release from intracellular stores; 3) during ATP depletion by chemical hypoxia, mitochondria do not take up Ca2+ to maintain cytosolic free Ca2+ at low concentrations. and cell surface blebbing was well developed (1-3). Several hypotheses might explain why cytosolic free Ca2+ does not increase after ATP depletion. First, a generalized blockade of ion movements through the plasma membrane and other membranes may occur, as has been described for mitochondrial membranes during hypoxic stress to hepatocytes (5, 6). Second, specific calcium flux pathways across cellular membranes may become inhibited due to ATP depletion and intracellular acidosis (7) during hypoxic injury. Third, the mitochondrial membrane potential persists after ATP depletion in hypoxic and anoxic hepatocytes (1,5). Thus, mitochondrial uptake of Ca2+ may maintain cytosolic free Ca2+ at a constant level during hypoxia.
Mammalian cells typically are bathed in extracellular fluid containing upwards of 120 mM Na'.
By contrast, cytosolic Na+ concentration is about 10 mM. This Na' gradient is important for many biological processes, including nutrient uptake, action potentials, regulation of other intracellular ions, and so on. The concentration gradient of Na+ is maintained by the Na+,K+-ATPase of the plasma membrane, which drives ATP-dependent Na+ efflux from the cell. Therefore, ATP depletion should inhibit the Na" pump and lead to an increase of intracellular Na+, unless Na' influx is blocked. Recently, a new fluorescent indicator, SBFI,' has been described which is suitable for digital imaging of intracellular Na+ (8). Thus, to determine whether a generalized blockade of ion movements was produced after ATP depletion, we used this new fluorescent indicator to measure cytosolic Na' during chemical hypoxia.

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(11-13). Ca2+ oscillations presumably are caused by movement of free Ca2+ across cellular membranes into and out of the cytosol (14-16). Ca2+ oscillations represent highly regulated and coordinated cellular events. Thus, to test the hypothesis that chemical hypoxia was causing inhibition of specific Ca2+ flux pathways, we studied the effects of hypoxic stress on hormone-induced Ca2+ oscillations.
ATP-dependent Ca2+ pumps in the plasma membrane and endoplasmic reticulum maintain cytosolic free Ca'+ in the range of 100 to 200 nM. Although isolated mitochondria accumulate Ca2+ avidly, the K , for this process is too large for mitochondria to regulate free Ca2+ in situ (17). In pathological states, however, mitochondria may take up large amounts of Ca2+ (18). Thus, a role for mitochondria in Ca2+ homeostasis during cell injury cannot be excluded. Accordingly, as mitochondrial Ca2+ uptake is driven by the mitochondrial membrane potential, a final aim of this study was t o determine the effect of mitochondrial depolarization on Ca2+ homeostasis during chemical hypoxia.
Here, we show that chemical hypoxia inhibits oscillations of free Ca2+. Chemical hypoxia also inhibits the increase of inositol phosphate formation induced by the calcium-mobilizing hormone agonists phenylephrine and vasopressin. The findings indicate that ATP depletion and acidosis during hypoxia inhibit Ca2+ release from intracellular stores by separate mechanisms. Na+ movement into cells is not prevented, and mitochondria do not play a role in maintaining Ca2+ homeostasis during hypoxic insult.

MATERIALS AND METHODS
Hepatocyte Isolation and Culture-Hepatocytes from Sprague-Dawley rats (200-300 g) were isolated by collagenase digestion as described previously (2). Isolated hepatocytes were diluted to 0.5 X lo6 cells/ml in Waymouth's medium MB752/1, containing 5% fetal calf serum, 2 mM glutamine, 10 nM dexamethasone, and 100 nM insulin. For experiments in which inositol phosphate formation was measured, hepatocytes were diluted to 2 X lo6 cells/ml. Aliquots of 1 ml were cultured on collagen-coated 22-mm square glass coverslips inside 35 X 10-mm Petri dishes, as described (7). Hepatocytes were used after 21-30 h of culture in humidified 5% C02, 95% air at 37 "C.
Measurement of Cytosolic Free Nu+, Ca", and pH by Multiparameter Digitized Video Microscopy-Hepatocyte cultures were incubated for 60 min with SBFI acetoxymethyl ester (5 p M ) for measurement of cytosolic free Na+ concentration, for 30 min with Fura-2 acetoxymethyl ester (3 p M ) for measurement of cytosolic free Ca", or for 30 min with BCECF acetoxymethyl ester (5 p~) for measurement of pH,. Subsequently, coverslips were washed three times with KRH buffer and mounted in a chamber on the stage of a Zeiss IM35 inverted fluorescence microscope (Thornwood, NY). Cytosolic free Na+ was measured by ratio imaging of SBFI fluorescence excited at 340 and 380 nm. In some experiments, SBFI fluorescence was also imaged at an excitation wavelength of 365 nm. Intracellular free Ca2' concentration was measured by ratio imaging of Fura-2 fluorescence excited at 340 and 365 nm. SBFI and Fura-2 fluorescence was imaged through a 395-nm dichroic reflector and a 420-nm long pass filter. Cytosolic pH was measured by ratio imaging of BCECF fluorescence excited at 440 and 490 nm. BCECF fluorescence was imaged through a 510-nm dichroic reflector and a 530-nm long pass filter. Excitation light was provided by a 75 watt Xenon lamp for Fura-2 and SBFI, and a 100 watt mercury lamp for BCECF. Excitation light was passed through an interference and neutral density filter wheel assembly to select wavelength and intensity under computer control. A shutter, also under computer control, automatically blocked the excitation light source between measurements. A low-light intensified siliconintensified target video camera (model 66, MTI-Dage, Michigan City, IN) collected fluorescent images, which were fed to a Sun 3/110 workstation (Mountain View, CA) for frame averaging, background subtraction, and ratioing. Ratio images during Ca2+ oscillations were collected every 10 s, unless otherwise noted.
Calibration of SBFI, Fura-2, and BCECF Signals-Attempts to calibrate cellular SBFI signals by imaging SBFI fluorescence in standard solutions through the microscope optics yielded erroneous results. This procedure often produced estimates of cellular Na+ that were less than zero. This problem may result from changes of the properties of SBFI brought about by the intracellular environment (e.g. changes of viscosity) (19). Therefore, intracellular Na+ concentration was calibrated in situ by the method of Harootunian et al. (19), with minor modifications. SBFI-loaded cells were incubated with 4 p~ gramicidin D, 10 p M monensin, and 10 p M nigericin to equilibrate Na' between the cytoplasm and the extracellular solution ( Fig. 9, Miniprint). The bathing solution was then changed to a Na+free gluconate solution (120 mM K-gluconate, 1 mM KHzPO~, 1.2 mM MgSO.,, 2 mM CaC12, and 25 mM HEPES, pH 7.4) containing ionophores. By substitution of Na-gluconate for K-gluconate, Na+ concentration was increased in increments from 0 to 120 mM. A standard relating the 340380 fluorescence ratio to Na+ was thus established. In other experiments, excitation and emission spectra for SBFI were determined with a Perkin-Elmer Cetus 850-40 fluorescence spectrophotometer (Nonvalk, CN). Spectra were uncorrected for lamp artifacts. Fura-2 and BCECF signals were calibrated as reported previ-Measurement of Inositol Phosphate Production-Inositol phosphate production was measured as previously described (20) with minor modifications. After 4 h of culture, 5 pCi of my~-[~HH]inositol/ ml were added to the culture medium. After another 16-20 h, the culture medium was changed to KRH containing 20 mM LiCI. The hepatocytes were incubated for 7 min under air at 37 "C, and various chemicals and hormones were then added. After another 15 min, the medium was removed by aspiration and replaced with 0.5 ml of cold 5% perchloric acid. The perchloric acid was removed and each dish was washed with 0.5 ml of H20, which was combined with the perchloric acid fraction. subsequently, 20 pl of 100 mM EDTA and 2 ml of octylamine/freon (1:1, v/v) were added, vortexed, and allowed to settle at room temperature.
[3H]Inositol phosphates in the upper phase were separated by anion-exchange chromatography on 1-ml Dowex AG1-X8 columns in the formate form. The samples were applied, and the columns were washed with 10 ml of H20 and 8 ml of 50 mM ammonium formate. Inositol mono-, bis-, tri-, and tetraphosphates were eluted with 8 ml of 1.2 M ammonium formate and 0.1 M formic acid. The eluate was added to 20 ml of scintillation mixture (15 g of diphenyloxazole and 0.375 g of p-bis[o-phenylstryllbenzene in 2 liters of Triton X-100 and 2 liters of toluene ously (3,7).

Effect of Chemical Hypoxia on Ca2+ Oscillations Induced by Phenylephrine and Vasopressin-
The rapid rise of Na' during chemical hypoxia, together with the absence of a change of free Ca2+ as reported previously (1, 3), suggested a relatively specific regulation of ionic Ca2+ in ATP-depleted hepatocytes. Accordingly, using ratio imaging of Fura-2 fluorescence, we investigated the response of control and ATP-depleted hepatocytes to the Ca'+-mobilizing hormone agonists, phenylephrine and arg-vasopressin. Phenylephrine (50 p~) elicited transient elevations of cytosolic Ca'+, which showed considerable heterogeneity. In about 30% of hepatocytes, repetitive Ca'+ spikes were observed (Fig. L4). These spikes were sustained Portions of this paper (including "Results," Figs. 9-14, Table IV, and Footnote 3) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press. for 5 min or more. The amplitude of oscillations was 30-250 nM, and frequency was 0.5-2 per minute. In about 40% of hepatocytes, damped oscillations of Ca2+ were observed which ceased after 1-3 beats (Fig. 1B). In the remaining 30% of hepatocytes, the increase of Ca2+ after phenylephrine was small (<20 nM) (Fig. IC). Ca2+ oscillations were also observed in about 40% of hepatocytes after addition of 20 nM Argvasopressin, but the oscillations typically were damped within 8 min (Fig. 2 ) .
After addition of KCN plus iodoacetate, oscillations induced by phenylephrine ceased after one spike (Fig. 3A). When phenylephrine was added 1 min after KCN plus iodoacetate to hepatocytes previously shown to oscillate, a single Ca2+ spike, often of diminished magnitude, was observed (Fig. 3B). When phenylephrine was added after 3.3 min, a brief small increase of Ca2+ occurred (Fig. 3C). After 5 min, no increase of Ca2+ was observed (Fig. 3 0 ) . Chemical  Inhibition of phenylephrine-induced CaZ+ oscillations by chemical hypoxia. Cytosolic free Ca" was measured in single cultured hepatocytes as described in Fig. 1. 50 p~ phenylephrine (%e) and 2.5 mM KCN plus 0.5 mM iodoacetate (ZAA) were added where indicated. In panels B-D, hepatocytes were selected that oscillated after an earlier addition of 50 p~ Phenylephrine. Subsequently, the cells were washed with KRH to remove hormone agonist.
After more than 15 min of recovery, the cells were then exposed to KCN plus iodoacetate prior to readdition of phenylephrine. Four experiments typical of more than 20. Inhibition of vasopressin-induced Ca2+ oscillations by chemical hypoxia. Cytosolic free Ca2+ was measured in single cultured hepatocyte, as described in Fig. 1, after addition of 20 nM Arg-vasopressin (Vas) followed by 2.5 mM KCN plus 0.5 mM iodoacetate (ZAA). The cell was selected for its oscillatory response to vasopressin. One experiment typical of three.
hypoxia also stopped oscillations induced by vasopressin (Fig.  4). Thus, chemical hypoxia led rapidly to suppression of Ca2+ mobilization by both phenylephrine and vasopressin.
Effect of Changes of pHi on Calcium Oscillations-Chemical hypoxia causes a rapid decrease of pHi, due largely to proton release during ATP hydrolysis (7, see Fig. 11). Since this decrease of pHi might account for suppression of Ca2+ mobilization in ATP-depleted cells, the effect of intracellular acidosis on phenylephrine-induced Ca2+ oscillations was examined. Na-acetate (40 mM) was added 3 min after 50 p M phenylephrine to decrease pHi. After the addition, one Ca2+ spike was observed, and subsequently Ca" oscillations by phenylephrine were suppressed (Fig. 5A). In parallel measurements we measured pHi by ratio imaging of BCECF fluorescence. Na-acetate produced a 0.3-0.5 unit decrease of pHi within 30 s. pHi gradually returned toward base line over the next several min (Fig. 5B).
We also examined the effect of intracellular alkalinization. Alkalinization with 20 mM NH,Cl inhibited Ca2+ oscillations sharply (Fig. 6A). Parallel measurements of pHi showed that addition of 20 mM NH&1 increased pHi by 0.6-0.7 units (Fig.  6B).
Effect of Chemical Hypoxia on Ca2+ Oscillations during pHi Clamping-To further investigate the hypothesis that inhibition of Caz+ oscillations during chemical hypoxia is due to cytosolic acidosis, we measured Ca2+ oscillations in cultured hepatocytes treated with 10 p~ monensin in modified KRH buffer containing 105 mM choline and 10 mM Na+. Under these conditions, pHi is clamped to pH,, even during chemical hypoxia, because of monensin-catalyzed Na+/H+ exchange (7). Phenylephrine produced Ca2+ oscillations in monensintreated cells (Fig. 7). However, contrary to expectation, these Ca2+ oscillations were fully and rapidly inhibited by KCN plus iodoacetate. Effect of Chemical Hypoxia and Intracellular Acidosis on Inositol Phosphate Formation-To investigate whether ATP depletion was suppressing hormone-induced inositol phosphate formation and thereby inhibiting Ca2+ release from intracellular Ca2+ stores, inositol phosphate formation was measured during metabolic inhibition with cyanide plus iodoacetate (Table I)  Cultured hepatocytes were labeled with my~-[~H]inositol as described under "Materials and Methods." Subsequently, cells were incubated with (chemical hypoxia) or without (normoxia) 2.5 mM KCN and 0.5 mM iodoacetate. After 5 min, 50 p~ phenylephrine, 40 mM vasopressin, or no hormone agonist was added. Samples were then taken after another 10 min for measurement of inositol phosphate formation. Inositol phosphate formation is expressed as percent f S.E. of the average control value (2782 dpm) from three to six experiments/group.  Cultured hepatocytes labeled with myo-[3H]inositol were incubated with 2.5 mM KCN, 0.5 mM iodoacetate (IAA), KCN plus iodoacetate, or no inhibitor and subsquently exposed to 40 nM vasopressin or no hormone agonist as described in Table I. Samples were then taken for inositol phosphate determination. Inositol phosphate formation is expressed as percent f S.E. of the average control value (2120 dpm) from three experiments per group. However, when KCN plus iodoacetate were added 5 min before the hormone agonists, the increase of inositol phosphate formation was completely suppressed (Table I).  showed that pretreatment of hepatocytes with sulfhydryl reagents such as p-chloromercuribenzoic acid, diamine, and N-ethylmaleimide inhibited the increase of inositol phosphates induced by hormone agonists. Therefore, we investigated the possibility that the inhibitory effect of chemical hypoxia was due to thiol alkylation by iodoacetate. Iodoacetate in the absence of KCN only slightly inhibited the vasopressin-induced increase of inositol phosphate formation, whereas KCN alone produced inhibition almost as great as KCN and iodoacetate together (Table 11).
To investigate whether intracellular acidosis inhibited hormone-induced inositol phosphate formation, the effect of Naacetate on inositol phosphate formation was determined. Naacetate (40 mM) at a concentration that caused intracellular acidification and suppression of Ca2+ oscillations had no effect either on basal inositol phosphate formation or on inositol phosphate formation in response to phenylephrine and vasopressin (Table 111). Thus, acidic pHi did not prevent inositol phosphate formation in response to either phenylephrine or vasopressin.

DISCUSSION
This study was designed to evaluate mechanisms of ion homeostasis in ATP-depleted cells. Previously, cytosolic free Ca2+ did not increase in cultured hepatocytes after metabolic inhibition with cyanide and iodoacetate, a model of ATP depletion and reductive stress that we call chemical hypoxia (1)(2)(3). Accordingly, our specific goals were to determine 1)

Effect of intracellular acidosis on inositol phosphate formation
Cultured hepatocytes were labeled with my~- [~H]inositol and incubated without (normal) or with (acidotic) 40 mM Na-acetate for 1 min to lower pH,. Subsequently, 50 pM phenylephrine, 40 mM vasopressin, or no hormone agonist was added. After 10 min, samples were taken for inositol phosphate determination as described under "Materials and Methods." Inositol phosphate formation is expressed as percent f S.E. of the average control value (3347 dpm) from three experiments/group. whether ATP depletion caused a generalized blockade of ion movements across cellular membranes; 2) whether specific hormone-stimulated flux pathways for Ca2+ were inhibited during chemical hypoxia; and 3) whether mitochondrial membrane potential-dependent Ca2+ uptake contributed to free Ca2+ homeostasis in ATP-depleted cells. Nu+ Fluxes after ATP Depletion-After addition of metabolic inhibitors, cytosolic free Na+, as measured with the fluorescent probe SBFI, increased almost immediately and approached extracellular concentrations after 20 min. Hepatocytes acidify during chemical hypoxia (7), and SBFI fluorescence is pH-dependent (19). Therefore, we determined the Kd of SBFI for Na+ as a function of pH, and corrected our intracellular Na+ measurements for actual pHi, as measured by BCECF ratio imaging. Ouabain also increased free Na+ measured by SBFI, consistent with the hypothesis that the increase of Na+ after chemical hypoxia was due to inhibition of the Na+,K+-ATPase pump of the plasma membrane. Berger and co-workers (22) have described a similar increase of total cell Na' in anoxic hepatocytes measured by x-ray microanalysis. Taken together, these results indicate that Na+ rapidly gains access to ATP-depleted hepatocytes. Ca2+ Oscillations in Cultured Hepatocytes-In agreement with the recent report by Rooney and co-workers (13), phenylephrine and vasopressin stimulated oscillations of cytosolic free Ca2+ in 40-70% of primary 1-day cultured hepatocytes. However, in some hepatocytes, the Ca2+ response to these hormones was slight, even though high concentrations of phenylephrine (50 pM) and vasopressin (20 p M ) were employed. In addition, Ca2+ oscillations were usually damped within 10 min. This contrasts with previous studies that described Ca2+ oscillations lasting more than 30 min in freshly isolated hepatocytes (11,12). Fura-2 concentration in our cells is estimated to be about 50 p~ (23), which is much below the concentration of high affinity Ca2+-binding sites inside cells (24). Thus, Ca2+ buffering by Fura-2 does not explain the absence of hormone sensitivity observed in some cells. A decline of hormone receptors after culturing is a possible explanation (25).
Ca2+-mobilizing hormones increase cytosolic free Ca2+ by releasing Ca2+ from non-mitochondrial stores, either from a portion of the endoplasmic reticulum (17,(26)(27)(28)(29) or from newly described organelles called calciosomes (30,31). During Ca2+ oscillations, each individual spike results from Ca2+ release from these intracellular stores. Data presented by Kawanishi et al. (12) suggest that Ca2+ influx from the extracellular medium regulates the frequency of oscillations by influencing the rate of refilling of the intracellular stores. In the present experiments, KCN and iodoacetate not only stopped Ca2+ oscillations (Fig. 3A), but also suppressed the first spike of Ca2+ (Fig. 3, B -D ) . Chemical hypoxia did not deplete intracellular Ca2+ stores, as KCN plus iodoacetate, in the absence of added hormone agonists, caused no transient increase of cytosolic free Ca2+. Therefore, it seems most likely that chemical hypoxia suppresses Ca2+ oscillations by inhibiting Ca2+ release from Ca2+ stores.
Effect of ATP Depletion and Intracellular Acidosis on Ca2+ Oscillations and Inositol Phosphate Formution-Chemical hypoxia rapidly induces ATP depletion and intracellular acidosis (2,7). In this study, intracellular acidosis induced by Naacetate in the absence of ATP depletion suppressed Ca2+ oscillations. However, Ca2+ oscillations were also suppressed during chemical hypoxia when pHi was clamped to pH, using monensin. Thus, intracellular acidosis is not the only factor contributing to the suppression of Ca2+ oscillations by chemical hypoxia. Notably, clamping of pH, to physiologic pH, did not influence the amplitude or frequency of the Ca2+ oscillations themselves. Thus, oscillations of pHi were not providing beat-to-beat initiation of Ca2+ oscillations. Nevertheless, since intracellular acidosis and alkalosis both suppressed Ca2+ oscillations, pHi may play a significant role in regulation of Ca2+ oscillations.
The monensin experiments were performed in cholinesubstituted buffer, in which the ratio of intracellular to extracellular Na+ was approximately one to one. In this buffer, cytosolic free Ca2+ did not increase after chemical hypoxia. Thus, Ca2+ homeostasis in ATP-depleted cells is not linked to the Na' gradient as, for example, by Na+/Ca2+ exchange.
We evaluated in detail the effect of changes of pHi on free Ca2+ measurements by Fura-2 ratio imaging. As pHi decreased during chemical hypoxia, the Kd of Fura-2 for Ca2+ increased by about 30%. Even after correction for this pH-induced change of Kd, the increase of free Ca2+ after ATP depletion was small and much less than that caused by hormones. This interpretation is supported by experiments with monensin. With monensin, pHi was clamped to pH, and Kd was constant, but the increase of Ca2+ was small and slow after ATP depletion, despite the fact that monensin accelerates the onset of lethal injury (4,7).

Inhibition of Inositol Phosphate Formation after Chemical
Hypoxia-IP3 and IP, are second messengers for hormoneinduced Ca2+ release from intracellular stores (32,33). In the present study, KCN and iodoacetate strongly suppressed hormone-stimulated inositol phosphate formation. This finding can also explain suppression of Ca2+ oscillations by chemical hypoxia. Inhibition of inositol phosphate synthesis was not due to intracellular acidosis, as intracellular acidification with Na-acetate in the absence of ATP depletion did not inhibit inositol phosphate synthesis. Protein thiol alkylation by iodoacetate also could not account for inhibition, as iodoacetate alone did not suppress inositol phosphate formation. Lack of Contribution of Mitochondrial Ca" Uptake to Ca2+ Homeostasis-During anoxia and chemical hypoxia, mitochondria remain polarized for many minutes after ATP depletion, although the mechanism for sustaining this membrane potential remains obscure (1,5). Perhaps mitochondria assist in cytosolic Ca2+ homeostasis by membrane potentialdependent Ca2+ accumulation. However, protonophoric uncoupler (carbonyl cyanide m-chlorophenylhydrazone) added after the onset of chemical hypoxia did not increase cytosolic Ca2+. Thus, mitochondrial uptake of Ca2+ does not appear important for cytosolic Ca2+ homeostasis during chemical kypoxia. Similarly, carbonyl cyanide m-chlorophenylhydrazone did not increase cytosolic free Ca2+ in normoxic cells. These findings challenge the assumption that cellular mitochondria normally contain stores of Ca2+ that can be released as free Ca2+ after mitochondrial depolarization. Rather, our  workers, (38, 39), the coupling of receptors for ca*+-mobilizing hormones to the hydrolysis of PIP, to IP3 is through a guanine nucleotidebinding regulatory protein (GJ. During chemical hypoxia, intracellular GTP concentration decreases because of ATP depletion. As a consequence, Gp is inactivated leading to uncoupling of the hormone (HI-receptor (RH) complex and phospholipase C (PLC). Thus, IP, formation from PIP, decreases, and Ca2+ release from intracellular storage sites is suppressed. Chemical hypoxia also induces intracellular acidosis, which decreases the binding of IP3 to its receptor ( R I P 3 ) and inhibits Ca2+ release through the Caz+ channel. data support recent in vitro estimations that free calcium in the mitochondrial matrix is the same or less than cytosolic free Ca2+ (34)(35)(36). This conclusion is also supported by recent studies showing that cytosolic free Ca2+ does not increase in cultured hepatocytes after mitochondrial depolarization with HgClz in Ca2'-free medium (23).
Scheme of Ca2+ Flux Pathways during Chemical Hypoxia-Our working hypothesis for the effects of chemical hypoxia on cellular Caz+ homeostasis is summarized in Fig. 8. Hormone-induced release of Ca" from intracellular stores is inhibited by at least two mechanisms. First, ATP depletion and accompanying GTP depletion causes G-protein inactivation and uncoupling of the linkage between the hormonereceptor complex and activation of phospholipase C. As a consequence, hormone binding to receptor does not lead to IP3 formation and subsequent IP3-mediated Ca2+ release from calciosomes. The acidosis of hypoxia contributes to suppression of Ca2+ oscillations by a separate mechanism. Acidosis does not suppress IP3 formation; rather it inhibits Ca2+ release from calciosomes induced by IP3. The mechanism for this inhibition may be decreased binding of IP3 to its receptor at acidotic pH, as described by . Thus, chemical hypoxia blocks multiple pathways regulating intracellular Ca2+ flux.