Calcium mobilization from the intracellular mitochondrial and nonmitochondrial stores of the rat cerebellum

BRAIN RESEARCH Brain Research 718 (1996) 151-158

ELSEVIER

Research report

Calcium mobilization from the intracellular mitochondrial and nonmitochondrial stores of the rat cerebellum Wei-Cherng

Huang, Sheau-Huei

Chueh

*

Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan Accepted 10 January 1996

Abstract Two major intracellular Ca 2+ stores, the mitochondrial and nonmitochondrial (microsomes) fractions isolated from rat cerebellum, exhibited a Ca 2+ concentration and ATP-dependent Ca 2+ accumulation. The maximal Ca 2+ accumulation in mitochondria was higher than in microsomes, but the affinity of the mitochondria for C a 2+ w a s lower. In this study, C a 2+ accumulation within the mitochondria was energized by ATP hydrolysis. Thus, the protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and the F1F0 ATP synthase inhibitor, oligomycin, blocked C a 2+ accumulation and induced the discharge of the entrapped Ca 2+ in the mitochondria, whereas the metabolic inhibitor, rotenone, affected neither the C a 2+ accumulation nor discharge. On the other hand, the uniporter inhibitor, ruthenium red, blocked the mitochondrial accumulation of Ca 2+, but did not cause the discharge of preloaded Ca 2+. In addition, arachidonic acid (AA), sphingosylphosphorylcholine (SPC) and sphingosine (SPH) elicited the dose-dependent release of Ca 2+ from microsomal stores. Although the magnitudes of the Ca 2+ release induced by AA, SPC or SPH were all dependent on the presence of extravesicular C a 2+ at concentrations ranging from 0.0l to 0.1 /~M Ca 2+, only the AA- and SPC-evoked Ca 2+ releases were insensitive to temperature. The mitochondria were more sensitive than the microsomes to the AA induced release of accumulated Ca 2+. Our results indicate the existence of multiple intracellular Ca 2+ stores in nerve cells which can be released by various Ca 2+ mediators. Keywords: Ca 2+ mobilization; Intracellular Ca 2+ pool; Mitochondria; Arachidonic acid; Sphingosylphosphorylcholine; Sphingosine

1. Introduction Both the A T P - d e p e n d e n t C a 2+ p u m p and the N a + / C a 2+ exchanger are localized in the plasma membrane and play major roles in maintaining the low resting Ca z+ level in neural cells [.15]. In addition to Ca 2+ extrusion across the plasma membrane, it is clear that in most ceils Ca 2+ can be sequestered and controlled by intracellular organelles [3]. The endoplasmic reticulum is believed to be the major organelle that stores Ca 2+ via a Ca 2+ pump [25]. Such intracellular Ca 2+ sequestration has great relevance to receptor coupled Ca z+ signalling pathways. Thus, inositol 1,4,5-trisphosphate (IP 3) links the signal received at the plasma membrane to intemal Ca 2+ release [1]. Many studies have recently shown that mitochondrial Ca 2+ accumulation may also serve as a Ca 2+ buffer [11,18]. In addition to IP 3, several compounds have been identified as Ca 2+ mediators. First, GTP may convey Ca 2+ from

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IP3-recruitable pools to IP3-releasable pools, thereby modulating the size of IP3-releasable pools [14]. Second, cyclic A D P ribose has been proposed as a second messenger to release Ca 2+ through the ryanodine-sensitive Ca 2+ channel [29]. Third, sphingosine (SPH) can profoundly affect cytosolic Ca 2+ levels by either stimulating Ca 2+ influx or triggering Ca 2+ release [4,9,13,16,19,20,28,34]. Finally, arachidonic acid ( A A ) can induce the release of Ca 2+ from intracellular nonmitochondrial Ca 2+ pools [33]. Thus, except for GTP, all these compounds act as second messengers to trigger Ca 2 + release from intracellular Ca 2+ stores. Many reports have shown that Ca 2+ influx can increase after agonist stimulation to replenish the depleted Ca 2+ stores a n d / o r prolong the intracellular Ca 2 + signal [26]. In the current study, we compared two distinct Ca 2+ accumulating organelles, the mitochondrial and microsomal fractions, isolated from 6 - 9 - d a y - o l d rat cerebellum. W e characterized the influence of various inhibitors on mitochondrial Ca 2+ movement and various Ca 2+ mediators on microsomal Ca 2+ release. W e found that the mitochondria were more sensitive than the microsomes to

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the AA induced release of accumulated Ca 2+. Ruthenium red inhibited Ca 2÷ uptake, but did not induce the discharge of entrapped Ca 2÷ within the mitochondria.

2. Materials and methods 2.1. Preparation of membrane vesicles

Membrane vesicles including the mitochondrial and nonmitochondrial (microsomes) fractions were prepared from the cerebellum of 7-9-day-old Sprague-Dawley rats by the following procedure. All procedures after dissection of the cerebella were undertaken at 4°C. The isolated cerebella were homogenized (20 strokes, loose-fitting Dounce type homogenizer) in 10 volumes of cold homogenizing buffer containing 0.32 M sucrose, 5 mM HEPES, 2 mM MgC12, and 0.1 mM phenylmethanesulphonyl fluoride, pH 7.4. The homogenate was initially centrifuged at 800 × g for 5 rain and the collected supernatant was further centrifuged at 10000 × g for 10 min. The pellet (mitochondria enriched fraction) was resuspended in suspension buffer containing 0.32 M sucrose, 2 mM MgC12, 5 mM HEPES, 0.1 mM phenylmethanesulphonyl fluoride, and 0.5 mM EDTA, pH 7.4 at a protein concentration of 6-8 m g / m l (referred to in this report as mitochondria) whereas the supernatant was centrifuged at I00000 × g for 1 h. The resulting pellet (enriched in plasma membranes and endoplasmic reticulum) was resuspended in suspension buffer at a protein concentration of 10 m g / m l (referred to as microsomes). All membrane vesicles were rapidly frozen in liquid nitrogen and stored at - 9 0 ° C in 500 /zl aliquots. 2.2. Ca 2 + transport experiments with membrane vesicles

After thawing at room temperature, membrane vesicles (mitochondria or microsomes) were diluted with 1 ml buffer containing 140 mM KC1, 10 mM NaC1, 2.5 mM MgC12 and 10 mM HEPES, pH 7.0 (intracellular buffer) and centrifuged at 100000 × g for 1 h. The pellet was resuspended in intracellular buffer at a concentration of 4 mg/ml. Uptake of Ca 2+ was initiated by adding 20 /zl aliquots of membrane vesicles into 200 /xl intracellular buffer containing 1 mM ATP and various concentrations of free Ca 2+ buffered in 50 /~M total 45Ca2+/CaC12 (4 /~Ci/ml) containing the required amount of EGTA as previously described [5,10,15] either with or without mitochondrial inhibitors. After incubation at 37°C for the indicated times, 45Ca2+ uptake was terminated by adding 4 ml ice-cold intracellular buffer containing 1 mM LaC13 followed by rapid filtration through Whatman G F / C glass fiber filters. After four more washes, the radioactivity remaining on the filters was counted, and the amount of Ca 2+ remaining within membrane vesicles was thereby deduced. In experiments measuring the influence of vari-

ous reagents on the ability of membrane vesicles to retain Ca 2+ (or release Ca2+), reagents were added individually after Ca 2÷ accumulation within membrane vesicles had reached a steady state level at the indicated free Ca 2+ concentration. After an appropriate time, the Ca 2+ remaining within the membrane vesicles was determined as described above. Basal uptake without ATP addition was measured under each condition unless otherwise stated. Basal values were subtracted to give the ATP-dependent Ca 2+ uptake values shown in the figures. All experiments were carried out at least three times in triplicate with similar results, and the data presented are mean values + S.D. from n different preparations. 2.3. Culture of NGl08-15 cells

Neuroblastoma × glioma NG108-15 cells obtained from Dr. M. Nirenberg (National Institutes of Health, MD) were cultured in Dulbecco's modified Eagle's medium (with high glucose) supplemented with 10% fetal bovine serum, 100 /zM hypoxanthine, 1 /zM aminopterin and 16 /zM thymidine and incubated in a humidified atmosphere of 5% CO2/95% air at 37°C as previously described [6]. 2.4. Ca 2+ transport experiments with permeabilized NG108-15 cells

Permeabilization of NG108-15 cells was performed as previously described [2,5]. Briefly, cells were permeabilized by incubating cells (at a density of 5 × 105 cells/ml) with 0.005% saponin in intracellular buffer at 37°C for 10 min with gentle shaking. Cells were then washed twice and suspended in intracellular buffer. The intracellular Ca 2÷ transport (uptake and release) activities within saponin-permeabilized cells were measured under conditions similar to those described above for membrane vesicles. 2.5. Trypan blue exclusion assay

After harvesting, cells were resuspended in a buffer containing 150 mM NaC1, 5 mM KCI, 1 mM MgCI 2, 5 mM glucose, and 10 mM HEPES, pH 7.4 (designated loading buffer). Aliquots of cells (1 × 105 cells) were mixed with the indicated concentrations of AA, SPH or sphingosylphosphorylcholine (SPC) in a total volume of 100 /M loading buffer. Exclusion of trypan blue was observed under a phase-contrast microscope after 5 min, using 0.5% trypan blue in loading buffer. 2.&Ma~Ha~

Dulbecco's modified Eagle's medium, fetal bovine serum and hypoxanthine/aminopterin/thymidine were purchased from Life Technologies, Inc. (Grand Island, NY). Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), rotenone, oligomycin, ruthenium red, ATP,

W.-C. Huang, S.-H. Chueh / Brain Research 718 (1996) 151-158

GTP, phenylmethanesulphonyl fluoride, AA, SPH, SPC and caffeine were obtained from Sigma Chemical Co. (St. Louis, MO). Thapsigargin was supplied by Research Biochemicals International (Natick, MA). IP 3 was purchased from Boehringer Mannheim (Mannheim, Germany). 45Ca2+ and cyclic ADP ribose were obtained from Amersham Corp. All other chemicals were of analytical grade and were obtained from Merck.

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3. Results

The relationship between free Ca 2÷ concentration and ATP-dependent Ca 2+ accumulating activities within two major intracellular Ca 2÷ accumulation organelles are shown in Fig. 1. Microsomes prepared from rat cerebellum accumulated Ca 2+ via a Ca 2+ pump in an ATP-dependent manner (Fig. la). Thus, Ca 2÷ accumulation simply depended on Ca 2+ concentration with an ECs0 value of 0.12 __+0.01 /xM, n --- 12. Saturation of the Ca 2÷ accumulation activity was complete at approximately 0.3 /~M Ca2÷; higher Ca 2÷ concentrations did not result in any significant change in the maximal Ca 2÷ accumulation of 4.3 --+0.22 n m o l / m g , n = 12. The Ca 2÷ accumulation process was virtually unaffected by mitochondrial inhibitors including 10 /xM rotenone, 10 /zM oligomycin, 10 /zM FCCP and 10 ~ M ruthenium red (Fig. la). These 10

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Fig. 2. Effects of various inhibitors on mitochondrial Ca 2+ accumulation. ATP-dependent Ca 2+ accumulation was initiated by addition of aliquots of mitochondria to intraceUular buffer containing 1 mM ATP, 3 /zM free Ca 2+, either without ((3) or with mitochondrial inhibitors including 2 mM spermine (zx), 10 /xM rotenone ( * ) , 10 /zM oligomycin ( 0 ) , 10 /xM FCCP (I2) or 10 p~M ruthenium red (11). At the indicated times, 45Ca2+ accumulation in each group was determined. Mitochondrial inhibitors were added either simultaneously with mitochondria (a) or after steady state Ca 2+ accumulation within mitochondria has been reached (b). Results represent the mean + S.D. (n = 5 different preparations).

results indicate that the microsomal fraction employed in this study was enriched in nonmitochondrial organelles. Thus, further experiments using microsomes did not include mitochondrial inhibitors. We further characterized the significance of the mitochondria in Ca 2+ homeostasis in nerve terminals. Ca 2+ accumulation within mitochondria also displayed Ca 2+ concentration dependency and saturable activities. The ECs0 value for Ca 2+ and the maximal Ca 2+ accumulation were 0.56 +- 0.03 /zM and 9.9,-+0.57 n m o l / m g , n = 12, respectively. The same mitochondrial inhibitors described above when added together only blocked approximately 71% of the mitochondrial Ca 2+ accumulation (Fig. lb). Because mixed mitochondrial inhibitors could not totally block the mitochondrial Ca 2÷ accumulation activity, we further investigated the influence of individual inhibitors on the mitochondrial Ca z+ transport. As shown in Fig. 2a, mitochondria accumulated Ca 2÷ in the presence of 3 /zM free Ca 2÷. FCCP (10 /zM), oligomycin (10 /zM), rotenone (10/xM), ruthenium red (10 ~ M ) or spermine (2 mM) differentially inhibited Ca 2÷ accumulation; the Ca 2÷ remaining within mitochondria after 15 min was 35 +2.5%, 32 +- 2.8%, 80 +- 6.1%, 38 5: 2.4% and 84 +- 7.4% (n = 5) of the control group, respectively. We also examined the effect of the inhibitors on the ability of mitochondria to retain accumulated Ca 2+. Mitochondria were preloaded with Ca z÷ before application of the inhibitors. If

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the drugs only block Ca 2+ accumulation, they should not affect the previously loaded stores. Ruthenium red and rotenone did not cause Ca 2+ leakage from mitochondria once Ca 2÷ had been accumulated within the matrix, whereas oligomycin and FCCP caused the discharge of entrapped Ca 2÷ (Fig. 2b). Our data indicate that FCCP and oligomycin not only blocked the mitochondrial Ca 2÷ accumulation, but also caused the discharge of preloaded Ca 2÷, while rotenone affected neither accumulation nor discharge of Ca 2÷. Ruthenium red, on the other hand, inhibited Ca 2÷ accumulation but did not cause Ca 2÷ release from mitochondria. The intracellular nonmitochondrial Ca 2+ stores play a pivotal role in the receptor-mediated Ca 2÷ signalling pathways. We next determined the effects of various modulators on Ca 2+ release from microsomes. After Ca 2+ accumulation reached a steady state level, 93 + 1.7% of the accumulated Ca 2÷ could be released by l0 /zM A23187 within 5 rain, while only 4 + 0.2% of Ca 2÷ was released during the same period in control buffer (Fig. 3). This result suggests the presence of a large Ca 2+ concentration gradient within an intravesicular compartment. Fig. 3 also demonstrates that IP3 (10 /zM), GTP (10 /zM), cyclic ADP ribose (10 /zM) and caffeine (10 mM) were ineffective at inducing the release of Ca 2+ from microsomes, whereas AA (40 tzM), SPC (10 tz), SPH (10/~M) and TG (1 /zM) caused the release of 81 +4.4%, 25 __+2.1%, 47 __+4.6% and 41 _ 4.5% (n = 4) of the accumulated Ca 2÷, respectively. Addition of 3% polyethylene glycol was previously found to be required to observe GTP-mediated Ca 2÷ release from microsomes prepared from N1E115 neuroblastoma cells [30]. In the current study, even in the presence of 3% polyethylene glycol, no significant Ca 2+ release was induced by 10 /xM GTP (data not shown). However, 3 /,M IP3 triggered the release of approximately 30% of microsomal Ca 2+ from microsomes that were prepared from 250-300 g adult rats (Fig. 4). Fig. 5 demonstrates the concentration dependence of AA, SPC or SPH in mediating Ca 2+ release from microsomes. The ECs0 values for AA, SPC and SPH were 35 _ 4.6, 18 + 2.1 and 11 + 2.5/xM (n = 3), respectively. To further confirm that AA, SPC and SPH did not exert a membrane detergent-like effect, we examined their influence on trypan blue exclusion in neuroblastoma × glioma hybrid NG108-15 cells. As shown in Fig. 5, 60 ~ M AA did not cause membrane leakiness, while SPC and SPH at concentrations greater than 300 /zM increased the general permeability of the plasma membrane. Our results indicate that the effects of SPC and SPH on Ca 2÷ release could be due, at least in part, to non-specific permeability changes of the memrane since they both caused trypan blue entry into the cells. In addition to stimulating Ca 2+ release from microsomes, AA also exhibited a similar effect on mitochondrial Ca 2÷ stores. Mitochondria displayed 10-fold higher sensitivity to the AA induced release of accumulated Ca 2÷

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Fig. 3. Effects of putative intracellular Ca 2+ releasing mediators on Ca 2+ accumulation within microsomes. Ca 2+ accumulation within microsomes was allowed in the presence of 0.1 /xM free Ca 2+ and 1 mM ATP in intracellular buffer for 15 min. Buffer, 10 /xM A23187, 40 /~M AA, 10 /*M cyclic ADP ribose, 10 /xM GTP, 10 /xM IP3, 10 /*M SPC, 10 tzM SPH, 1 /xM thapsigargin (TG) or 10 mM caffeine was then added as indicated. Ca/+ remaining within microsomes was determined 5 rain later and is expressed as % of total accumulation. The total accumulation of Ca 2+ before the addition of any mediators was 1.9+0.02 nmol/mg. Results are mean + S.D. (n = 4 different preparations).

compared to the microsomes. The ECs0 value for AA was 4.2 + 0.8 /~M (n = 3) in mitochondria (Fig. 6). We reassessed the effect of AA on mitochondrial Ca 2÷ efflux using permeabilized NG108-15 cells. We have previously shown that Ca 2+ was only taken up by nonmitochondrial Ca 2+ stores, presumable endoplasmic reticulum, at Ca 2+ concentrations below 0.1 /xM in permeabilized NG108-15 cells, whereas Ca 2÷ was mainly sequestered into mitochondria at a Ca 2÷ concentration of 3 /~M in the presence of 1 /zM TG [5]. In permeabilized NG108-15 cells, mitochondira still exibited a higher organelle-specific potency to the AA induced discharge of accumulated Ca 2+ (ECs0 value of 3.9 + 0.7/,M, n = 3). Fig. 7 demonstrates the effect of extravesicular Ca 2+ concentration on the AA-, SPC- or SPH-evoked Ca 2÷ release from microsomes. The magnitudes of the Ca 2÷ release induced by AA, SPC or SPH were proportional to free Ca 2+ concentrations from 0.01 to 0.1 /xM, while Ca 2+ release remained unaltered at Ca 2+ concentrations higher than 0.1 /zM. Thus, cytosolic Ca 2+ concentration modulates AA-, SPC- or SPH-evoked Ca 2÷ release from microsomal Ca 2+ stores. To further understand the mechanism of Ca 2÷ release from microsomes induced by AA, SPC and SPH, the

W.-C. Huang, S.-H. Chueh / Brain Research 718 (1996) 151-158

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Ti me (mini Fig. 4. IP3-evoked Ca 2+ release from microsomes isolated from adult rat cerebella. Calcium accumulation within microsomes isolated from adult rat cerebella was allowed for 15 min in the presence of 1 mM ATP and 0.03 /xM free Ca 2+. After Ca 2+ accumulation attained steady state, buffer ( O ) or 10/xM IP3 ( 0 ) was added as t = 0. At the indicated times, Ca 2+ remaining within microsomes was determined. Results are mean 5: S.D. (n = 3 different preparations).

Fig. 6. Arachidonic acid induced Ca 2+ release from mitochondria. Aliquots of mitochondria (1.1 /xg) ( O ) or saponin-permeabilized NG10815 cells (1.9 ~g) ( 0 ) were first incubated with 1 mM ATP and 3 /xM free Ca 2+ for 15 min. For permeabilized NG108-15 cells, 1 /zM thapsigargin was also included in the incubation mixture. Indicated concentrations of AA were then added and the Ca 2÷ remaining within mitochondria in either group was measured after another 5 min. Data are mean 5: S.D. (n = 3 different preparations).

temperature dependence of Ca 2+ release was investigated. As shown in Fig. 8, when Ca 2+ release was determined at 37°C, 87, 66 and 68% of accumulated Ca 2+ was released

in response to 5 0 / z M AA, 30 /zM SPC and 3 0 / x M SPH, respectively. Reduction of the temperature from 37°C to 4°C did not alter the action of AA, while it reduced

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W.-C. Huang, S.-H. Chueh / Brain Research 718 (1996) 151-158

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Ca 2+ mobilization also prevents hypercalcemia in the cytosol during the Ca 2+ pulses evoked by hormones or neurotransmitters or by depolarization [11,18]. In the current study, we prepared two discrete Ca 2÷ accumulation organelles, mitochondria and microsomes, from rat cerebellum. The mitochondria displayed a 2-fold higher Ca 2+ accumulation capacity and a lower affinity for Ca 2+ compared with the microsomes (Fig. 1). Similar results have been previously found for other cell types [2,5,27]. Perhaps more important, we found that AA could trigger Ca 2+ release from both microsomal and mitochondrial stores (Figs. 5 and 6). Ca 2+ accumulation within microsomes was insensitive to mitochondrial inhibitors including FCCP, oligomycin, rotenone and ruthenium red indicating their nonmitochondrial origin (Fig. la), while the same inhibitors blocked the mitochondrial Ca 2+ accumulation by about 71% (Fig. lb). The incomplete inhibition of Ca 2+ accumulation within the mitochondrial fraction by mitochondrial inhibitors indicates that either part of this fraction is resistant

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4. Discussion Intracellular Ca 2+ stores comprise two distinct organelles of mitochondrial and nonmitochondrial origin. The nonmitochondrial stores, likely the endoplasmic reticulum, release stored Ca 2+ in response to IP 3 via surface IP 3 receptors [l]. The physiological role of mitochondrial Ca 2+ stores is uncertain. It has been suggested that the mitochondria might not contribute to the regulation of cytosolic Ca 2+ concentration under normal resting conditions because of their low affinity for Ca 2+ although they have a large capacity to accumulate Ca 2+ [7]. Recently, evidence has shown that Ca 2+ undergoes continuous cyclic movement across the mitochondrial inner membrane. The uniporter of the mitochondria utilizes an internally negative membrane potential to accumulate Ca 2+, whereas Na÷-independent efflux is driven by the pH gradient and Na+-dependent efflux exchanges Ca 2+ with Na ÷. The primary role of mitochondfial Ca 2+ mobilization is to activate the Ca2+-sensitive metabolic enzymes and to control the matrix Ca 2+ concentration [17]. Mitochondrial

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Fig. 8. Temperature modification of AA-, SPC- or SPH-evoked Ca 2+ release from microsomes. Accumulation of Ca 2+ within microsomes took place at 37°C in 1 mM ATP and 0.1 /zM free Ca 2+ ( 0 ) . After uptake, microsomes were either maintained for a further 5 rain at 37°C (a-c), or were cooled during this same period to 4°C (d-f). Ca 2+ release then proceeded at these respective temperatures for the indicated times after the addition of buffer ( zx), 50/xM AA ( • ) (a,d), 30 /xM SPC ( • ) (b,e) or 30 /xM SPH ( A ) (c,f). Results represent mean +S.D. ( n = 3 different preparations).

W.-C. Huang, S.-H. Chueh / Brain Research 718 (1996) 151-158

to these inhibitors or that nonmitochondrial organelles were also present in this fraction. The inhibitor resistant mitochondrial fraction displayed an ECs0 value for Ca 2÷ (0.25 /zM) that was 2-fold higher than the ECs0 value in the microsomes (0.12 /xM) (Fig. la,b) suggesting a nonmicrosomal origin. It is possible that an internal negative membrane potential still existed in the mitochondria in the presence of the mixed mitochondrial inhibitors and under this residual membrane potential, the influx of Ca 2÷ was faster than its efflux causing incomplete inhibition of Ca 2÷ accumulation. Under normal cellular conditions, the electrochemical proton gradient in mitochondria is formed by the operation of the electron transport chain of substrate oxidation. ADP is phosphorylated to ATP when protons flux back to the matrix via FIF 0 ATP synthase. Four different types of mitochondrial inhibitors were used in the current study including the F l F0 ATP synthase inhibitor oligomycin, the metabolic inhibitor rotenone, the protonophore FCCP, and the uniporter inhibitor ruthenium red. The membrane potential mediated Ca 2+ accumulation is energized by ATP hydrolysis to pump protons out of mitochondria in the current study. Thus, Ca 2÷ uptake was inhibited by the F~F0 ATP synthase inhibitor, the uniporter inhibitor and the protonophore, while it was resistant to the metabolic inhibitor rotenone which does not alter the mitochondrial membrane potential (Fig. 2a). On the other hand, unlike the F~F0 ATP synthase inhibitor or protonophore which dissipate the pH gradient, preloaded Ca 2÷ within mitochondria was not released in response to the addition of the uniporter or metabolic inhibitors (Fig. 2b). Although the IP3 receptor was first cloned from rat cerebellar Purkinje cells [12,23], its expression on postnatal day 7 is less than 25% of the maximal expression attained on postnatal day 28 regardless of the subtype of IP3 receptor [24]. This might explain why IP3 did not trigger Ca 2÷ release from microsomes prepared from 7-9day-old rats in the current study (Fig. 3). Indeed, when we used microsomes prepared from adult rats, IP3-evoked Ca 2÷ release was observed (Fig. 4). The accumulated Ca 2÷ in the microsomes was partially released by the addition of IP3 suggesting that part of the microsomal fraction was derived from organelles that are IP3 insensitive. Receptor-operated phospholipase A 2 activation causes AA release. It is well-established that the AA metabolites including leukotrienes, thromboxanes and prostaglandins, modulate many cellular functions. Many studies have shown that AA itself is involved in stimulating Ca 2÷ influx [31]. There is also evidence that AA triggers Ca 2÷ release from 1P3-sensitive intracellular Ca 2+ stores [33]. In the current study, we found that in addition to microsomes, AA also stimulated Ca 2÷ release from mitochondria with even greater potency (Fig. 6). The possibility that the sensitivity of mitochondria to AA might have been due to contamination with the plasma membrane or endoplasmic

157

reticulum was ruled out by the higher affinity of the mitochondria for AA compared with the microsomes. Furthermore, a similar effect was observed when permeabilized cells were used to eliminate the possibility of membrane contamination. The ubiquitous effect of AA on Ca 2+ release raises the question of whether it exerts a membrane detergent-like effect. The results of trypan blue exclusion experiments and extravesicular Ca 2+ concentration dependency preclude this possibility. The temperature insensitivity of the AA-evoked Ca 2÷ release suggests a channel-like rather than a carrier-like mechanism. Metabolites of sphingolipids have recently been shown to affect a wide variety of biological processes. In Swiss 3T3 fibroblasts, sphingosine 1-phosphate (SPP), SPH and SPC stimulated DNA synthesis and cell proliferation [8]. SPP further activates the mitogen-activated protein kinase signalling pathway through a pertussis toxin sensitive G protein [32]. In addition, many studies have demonstrated that SPH, SPP and SPC are important in the regulation of Ca 2÷ homeostasis. Using permeabilized DDTIMF-2 smooth muscle cells, Ghosh et al. first found that SPH and SPC induced Ca 2+ release from intracellular Ca 2÷ stores [13]. Since then, a number of other studies have confirmed that SPC, SPH and SPP can induce Ca 2÷ mobilization in various cell types including Swiss 3T3 fibroblasts [16], rat basophilic leukemia cells [20], parotid acinar cells [28], pancreatic acinar cells [34], brain microsomes [9], human dermal fibroblasts [4] and human endothelial cells [19]. Many studies have shown that SPP, converted from SPH by sphingosine kinase, was the active species evoking Ca 2+ mobilization. Using SPC as a soluble surrogate for SPH, an mRNA for a SPC-gated Ca 2+ channel has been identified [19], the size of which (1.5-2.0 kb) was distinct from the IP3 receptor ( > 8 kb) [12] and the ryanodine receptor (16 kb) [21]. In the current study, SPH induced Ca 2+ release only at 37°C whereas SPC induced Ca 2+ release at both 37°C and 4°C, suggesting that the enzymatic conversion product of SPP was the active species inducing Ca 2÷ release. On the other hand, the differential dependence on temperature might indicate that SPH and SPC stimulate Ca 2+ release via distinct mechanisms. In permeabilized rat pancreatic acinar cells, SPC induced Ca 2+ release from intracellular Ca 2÷ stores, while SPP did not [34]. Other evidence has suggested that SPC and SPP have distinct signalling pathways. Thus, SPC selectively enhanced AA release whereas SPP selectively stimulated phospholipase C and D and inhibited adenylyl cyclase although both increased cytosolic Ca 2÷ levels in Swiss 3T3 fibroblasts [8,16,22]. In addition, SPC, lysosphingomyelin, is structurally similar to detergents. Thus, SPC increases general cell permeability at lower concentrations than SPH, while it is less potent in releasing Ca 2÷ than SPH (Fig. 5). Our results further indicate that the effect of SPC and SPH on Ca 2÷ release is dependent on the extravesicular Ca 2+ concentration (Fig. 7) suggesting that SPH and SPC may act as intracellular Ca 2+ mediators in addi-

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tion to IP3 [I], cyclic ADP ribose [29], and arachidonic acid (see above).

[17]

Acknowledgements

[18]

This work was supported by a grant from the National Science Council, Republic of China (NSC85-2331-B016093).

[19]

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