Calcium stimulates intramitochondrial cholesterol transfer in bovine adrenal glomerulosa cells.

In adrenal glomerulosa cells, angiotensin II (Ang II) stimulates aldosterone synthesis through rises of cytosolic calcium ([Ca2+]c). The rate-limiting step in this process is the transfer of cholesterol to the inner mitochondrial membrane, where it is converted to pregnenolone by the P450 side chain cleavage enzyme. The aim of the present study was to examine the effect of changes in [Ca2+]c and of Ang II on intramitochondrial cholesterol distribution. Freshly prepared bovine zona glomerulosa cells were submitted to a cytosolic Ca2+ clamp (600 nM) or stimulated with Ang II (10 nM). Mitochondria were isolated and subfractionated into outer membranes (OM), inner membranes (IM), and contact sites (CS). Cholesterol content was determined by the cholesterol oxidase assay. Stimulation of intact cells with Ca2+ led to a marked decrease in cholesterol content of OM (to 54 +/- 24% of controls, n = 5) and to a concomitant increase of cholesterol in CS and IM (to 145 +/- 14%, n = 5). When glomerulosa cells were exposed to Ang II, a marked increase of cholesterol in CS occurred (to 172 +/- 16% of controls, n = 5). No significant changes were detected in OM cholesterol, suggesting a stimulation of cholesterol supply to the mitochondria in response to Ang II. Cycloheximide specifically and significantly reduced Ca2+-activated cholesterol transfer to CS and IM. In conclusion, our data indicate that one of the main functions of the Ca2+ messenger is to increase cholesterol supply to the P450 side chain cleavage enzyme by enhancing endogenous intermembrane cholesterol transfer to a mitochondrial site containing the enzymes responsible for the initial steps of the steroidogenic cascade.

The crucial role of the Ca 2ϩ messenger in the acute regulation of aldosterone production in adrenal glomerulosa cells is well recognized (4,11,12). However, the sites of action and the specific molecular targets of calcium along the complex steroidogenic cascade are poorly defined. A first direct indication of the involvement of the adrenal glomerulosa mitochondria as targets for the Ca 2ϩ messenger has been obtained when our laboratory has shown using permeabilized bovine glomerulosa cells that changes of ambient Ca 2ϩ within the range of the physiological cytosolic concentrations are able to activate aldosterone production and that this effect can be prevented by ruthenium red, a blocker of the mitochondrial Ca 2ϩ uniport (13). Moreover, recent work has allowed us to narrow the potential target domain for Ca 2ϩ to the very early steps of steroidogenesis (14), which occur inside the mitochondria.
Indeed, the acute response of steroidogenic cells to hormone stimulation involves the mobilization of cholesterol from intracellular lipid droplets to the mitochondrial inner membrane, where the first enzymatic step of steroidogenesis, namely the conversion of cholesterol to pregnenolone by the cytochrome P450 scc , occurs (15). The rate-limiting and hormonally regulated step in this process is the delivery of cholesterol from the outer to the inner mitochondrial membrane. This step is known to require de novo protein synthesis. Studies from several laboratories have shown that a family of hormone-induced and cycloheximide-sensitive 30-kDa mitochondrial proteins, described in different steroidogenic cell types (16 -20), play a crucial role in the acute regulation of steroid synthesis. Recently, the steroidogenic acute regulatory (StAR) protein has been proposed as an essential mediator of the acute steroidogenic response. Cholesterol transfer is believed to be facilitated by contact sites that occur between the outer and the inner mitochondrial membranes during the import of the StAR protein precursor into the mitochondria (20). This hypothesis is strengthened by many observations showing that mitochondrial contact sites are involved in phospholipid and protein import into the mitochondria (21,22).
Although the regulation of the cholesterol transfer steps has been almost exclusively investigated in response to elevated cAMP levels in adrenal fasciculata cells, many questions remain unanswered concerning the possible role of Ca 2ϩ in the regulation of cholesterol mobilization in adrenal glomerulosa cells. The aims of the present study were firstly to determine whether the changes in intracellular calcium concentration triggered by activators of steroidogenesis in glomerulosa cells are accompanied by concomitant changes in cholesterol distribution in mitochondrial membranes and secondly to examine the effect of the inhibitor of protein translation, cycloheximide (CHX), on the calcium-mediated cholesterol transfer from the outer to the inner mitochondrial membrane.
Calcium Clamping of Bovine Adrenal Glomerulosa Cells-After having been washed in Krebs-Ringer buffer, glomerulosa cells were Ca 2ϩclamped as reported elsewhere (14) in the presence of 2 M ionomycin, 1 mM total extracellular Ca 2ϩ , and 0.2 mM EGTA, in order to achieve an intracellular [Ca 2ϩ ] c of 600 nM. 500 M of aminogluthetimide (AMG) was included in the incubation medium to avoid cholesterol side chain cleavage by the cytochrome P450 scc located in the inner mitochondrial membrane. At the end of a 2-h incubation period at 37°C, the cells were sedimented at 200 ϫ g for 15 min. All subsequent operations were conducted at 4°C, in buffers containing 500 M AMG.
Isolation of Mitochondria and Preparation of Submitochondrial Fractions-Glomerulosa cells were homogenized with a Potter-Elvehjem homogenizer (1200 rpm, 35 strokes) in a 5 mM Tris-HCl buffer, pH 7.4, containing 275 mM sucrose. The homogenate was centrifuged at 200 ϫ g for 15 min to remove large debris and nuclei. Further centrifugation of the supernatant at 10,000 ϫ g for 10 min yielded the mitochondria. The mitochondrial pellet was washed twice at 8,000 ϫ g with the same buffer.
Submitochondrial particles were prepared as described elsewhere (24). The washed mitochondrial pellets were exposed to a swelling procedure by incubation in 10 mM sodium phosphate buffer, pH 7.4, for 20 min (final protein concentration, ϳ1 mg/ml), followed by the addition of 61.5% sucrose in order to obtain a 0.45 M sucrose medium. After a 20-min incubation period, 10-ml portions were mildly sonicated (3 ϫ 30 s), using a probe sonicator (Branson Sonifier 250). The suspension was centrifuged at 8,000 ϫ g to remove unbroken mitochondria. The supernatant was collected and centrifuged again at 150,000 ϫ g for 90 min. The pellet containing the submitochondrial membrane fraction was resuspended in 10 mM sodium phosphate buffer, pH 7.4, containing 0.45 M sucrose (final protein concentration, 5 mg/ml) using a Teflon homogenizer. The membrane suspension (1-2 mg of protein) was loaded onto a linear (15-50%) sucrose density gradient (10 ml) and centrifuged for 20 h at 100,000 ϫ g. Subsequently, the gradients were divided into 500-l fractions that were assayed for marker enzyme activities. Protein was quantified using the Bio-Rad protein micro assay and bovine serum albumin as a standard.
Cholesterol Determination-The cholesterol content of each submitochondrial fraction of the gradient was determined by a coupled cholesterol oxidase-peroxidase assay with cholesterol as a standard (27). Aliquots of the fractions (200 l) were transferred to glass tubes. To each sample, 20 l of 20 mM cholate and 1% Triton X-100 in 100 mM potassium phosphate buffer, pH 7.4, were added, followed by the addition of 25 l of 95% ethanol. The reaction mixture containing potassium phosphate buffer (100 mM), pH 7.4, cholesterol oxidase, peroxidase, and p-hydroxyphenylacetic acid was then added to each fraction in a final volume of 1 ml. Assay tubes were incubated for 1 h at 37°C. Cholesterol oxidase generates H 2 O 2 , and peroxidase catalyzes the reaction of H 2 O 2 with p-hydroxyphenylacetic acid to yield a stable fluorescent product. The fluorescence was measured in a Jasco CAF-110 fluorimeter (excitation, 325 nm; emission, 405 nm).
Aldosterone Measurements-The aldosterone content of the incubation medium was measured by radioimmunoassay using a commercially available kit (Diagnostic Systems Laboratories, Webster, TX).
Analysis of Data-Results are expressed as the means Ϯ S.E. The mean values were compared by analysis of variance using Fisher's test. A value of p Ͻ 0.05 was considered as statistically significant.

Characterization of the Submitochondrial Fractions-Mem-
branes of osmotically lysed mitochondria isolated from glomerulosa cells in which [Ca 2ϩ ] c had been previously clamped for 2 h at either low levels (Ͻ100 nM, control cells) or at high levels (600 nM) were separated into various fractions by continuous sucrose density gradient centrifugation as described under "Experimental Procedures." The protein distribution in the gradient under each condition is shown in Fig. 1A. Fig. 1B illustrates the separation of mitochondrial membranes from control (low [Ca 2ϩ ] c ) glomerulosa cells. The activity profile of specific mitochondrial marker enzymes in the gradient led to the characterization of three distinct membrane populations. A first population with the lowest density (fractions 5-8) showed the highest monoamine oxidase activity, which is specific of the outer mitochondrial membranes. The population of membranes with the highest density (fractions 13-15) exhibits the highest content of cytochrome c oxidase activity, as expected for the inner mitochondrial membranes. In addition, a third mem-FIG. 1. Separation of submitochondrial membranes by density gradient centrifugation. Bovine adrenal glomerulosa cells were submitted for 2 h to a Ca 2ϩ clamp in the presence of 500 M AMG as described under "Experimental Procedures." Submitochondrial particles were prepared on a 15-50% sucrose density gradient as described. The protein content (A) and the activities of mitochondrial marker enzymes (B) were determined in duplicate in each fraction of the gradient. The activity profiles are representative of eight independent experiments. MAO, monoamine oxidase; COX, cytochrome c oxidase; NDP-K, nucleoside-diphosphate kinase. Ordinate units are pmol deaminated tryptamine/min/mg protein for monoamine oxidase, nmol oxidized cytochrome c/min/mg protein for cytochrome c oxidase, and nmol ADP/min/mg protein for NDP kinase. brane population of intermediate density (fractions 9 -12) possessing both monoamine oxidase and cytochrome c oxidase activities displayed the highest nucleoside-diphosphate kinase activity, which is characteristic of mitochondrial intermembrane contact sites. A similar profile of mitochondrial membrane marker enzymes was obtained after fractionation of mitochondria isolated from high Ca 2ϩ -clamped cells; fractions 5, 11, and 14 contained the bulk of monoamine oxidase, NDP kinase, and cytochrome c oxidase activities, respectively (data not shown).
Calcium Is a Potent Stimulator of Intramitochondrial Cholesterol Transfer-We have previously shown in bovine adrenal zona glomerulosa cells that the calcium-ionophore, ionomycin, can be effectively used at low concentration to clamp the cytosolic free Ca 2ϩ concentration, [Ca 2ϩ ] c , at various physiological levels (50 -1000 nM) (14). This submicromolar [Ca 2ϩ ] c stimulates the early mitochondrial steps of steroidogenesis, namely pregnenolone formation from cholesterol side chain cleavage, as well as aldosterone synthesis (14).
Cholesterol content was determined in the various fractions of the sucrose gradient. In Fig. 2, the total cholesterol content in each submitochondrial fraction from high Ca 2ϩ -clamped cells has been expressed as a percentage of the cholesterol content measured in the corresponding submitochondrial fractions from control cells. The stimulation of intact glomerulosa cells with Ca 2ϩ led to a marked decrease of cholesterol content in the outer mitochondrial membranes (fraction 5, corresponding to the peak of monoamine oxidase activity: 54 Ϯ 24% of controls, n ϭ 5) with a concomitant increase in contact sites (fraction 11, corresponding to the peak of NDP kinase activity: 145 Ϯ 14% of controls) and a less pronounced augmentation in the inner mitochondrial membranes (fraction 14, corresponding to the peak of cytochrome c oxidase activity: 125 Ϯ 5% of controls).
These results demonstrate that Ca 2ϩ activates mitochondrial endogenous cholesterol transfer from the outer membrane to the contact sites and inner membrane, a process that is accompanied by an increased aldosterone production. Indeed, the aldosterone output measured in Ca 2ϩ -clamped cells incubated in the absence of AMG amounted to 345 Ϯ 42% of controls (n ϭ 8).
Ca 2ϩ -stimulated Cholesterol Transfer from the Outer to the Inner Membrane Is Sensitive to Cycloheximide-Earlier reports have indicated that ACTH-activated cholesterol transport to the mitochondrial inner membrane of steroidogenic cells is blocked by inhibitors of protein synthesis such as CHX, with a resulting ACTH-induced accumulation of cholesterol in the outer mitochondrial membrane (15,28). Moreover, it has been reported that the increase in pregnenolone and aldosterone synthesis triggered by Ang II is inhibited by the same compound (29), suggesting that at the mitochondrial level, both hormones operate through similar mechanisms. We have therefore tested whether CHX may also inhibit Ca 2ϩ -stimulated intramitochondrial cholesterol transfer in glomerulosa cells. Fig. 3 shows that when CHX was added concomitantly with Ca 2ϩ , the outer membrane fractions 5 and 6 contained 82 Ϯ 4.4 and 82 Ϯ 4.9% of the cholesterol content of their respective control fractions, as compared with only 68.3 Ϯ 4.8% and 67 Ϯ 3.6%, respectively, when the Ca 2ϩ clamp was performed in the absence of CHX (p Ͻ 0.05, n ϭ 4). By contrast, CHX significantly reduced Ca 2ϩ -activated cholesterol transfer to contact sites (fractions 11-12) and inner membranes (fractions 13-14) (Fig. 3). Cycloheximide similarly prevented Ang II-induced cholesterol transfer to contact sites and inner membranes (data not shown).
Ang II Stimulates Exogenous Cholesterol Transport to Mitochondria, with a Concomitant Transfer of Endogenous Cholesterol to Contact Sites-In order to test whether Ang II-mediated cholesterol mobilization in mitochondria is similar to that triggered by the cytosolic Ca 2ϩ clamp, glomerulosa cells were incubated for 2 h in the presence of 10 nM Ang II and 500 M AMG. Submitochondrial membranes were prepared and analyzed as above. No change in protein profile of sucrose gradients could be observed upon Ang II stimulation (data not shown). Fig. 4 illustrates the distribution of cholesterol content in the submitochondrial fractions of glomerulosa cells exposed to Ang II. Firstly, the hormone induced a pronounced increase of cholesterol content in the contact site-enriched fractions (fractions 11 and 12, 172 Ϯ 16 and 169 Ϯ 6% of controls, respectively, n ϭ 5). In separate experiments, we have observed a significant (28 Ϯ 5%, n ϭ 5) increase in total mitochondrial cholesterol of Ang II-stimulated-cells. Secondly, no significant changes were detected in the outer mitochondrial membranes (fractions 5 and 6, 83 Ϯ 6 and 80 Ϯ 11% of controls, respectively, n ϭ 5). Thirdly, no increase in cholesterol content was observed in the inner membrane fractions (fractions 13 and 14, 109 Ϯ 13 and 76 Ϯ 22% of controls, respectively, n ϭ 5). Interestingly, a subpopulation of the inner membranes revealed a significant decrease in cholesterol content, as compared with the respective control fractions (fractions 15, 16, and 17, 64 Ϯ 4, 52 Ϯ 6, and 57 Ϯ 13% of controls, respectively, n ϭ 5). DISCUSSION In the present study, we took advantage of the cytosolic Ca 2ϩ clamp technique to investigate the Ca 2ϩ sensitivity of intramitochondrial cholesterol transfer in bovine glomerulosa cells. Recently, using the ionomycin-mediated Ca 2ϩ clamp, our laboratory has provided a first direct demonstration that [Ca 2ϩ ] c in the submicromolar range stimulates aldosterone synthesis in intact glomerulosa cells (14). Because Ca 2ϩ affects the formation of pregnenolone, an early mitochondrial step of aldosterone production (14), one could expect that one or several target(s) of the Ca 2ϩ messenger are located within the mitochondria.
The overall rate-limiting step of the steroidogenic cascade is the transport of cholesterol from a presteroidogenic pool in the outer membrane to a steroidogenic pool in the inner membrane (15,28,30). The regulation by ACTH of cholesterol distribution in adrenal mitochondria has been studied by several groups using mitochondrial disruption to yield outer and inner membranes (28,31,32). The hormone has been shown to activate cholesterol supply to the P450 scc enzyme in the inner membrane. However, restricting the separation to two major fractions may fail to uncover additional, functionally relevant membranous structures. We therefore fractionated bovine adrenal glomerulosa mitochondria into outer membranes, inner membranes, and intermembrane contact sites. The latter fraction contains marker enzymes for both membranes, in addition to NDP kinase activity (Fig. 1), which has been shown to be specific of mitochondrial contact sites in several tissues (33).
The data presented here show that endogenous cholesterol transfer from the outer to the inner mitochondrial membrane and contact sites is substantially stimulated by physiological levels of cytosolic Ca 2ϩ . The supply of cholesterol to the P450 scc appears thus to be a Ca 2ϩ -sensitive step in the early steroidogenic pathway. This observation is in agreement with the data recently reported by Kowluru and colleagues (34), showing that Ca 2ϩ stimulates the metabolism of endogenous cholesterol to pregnenolone in rat adrenal mitochondria. Our previous studies have demonstrated that the P450 scc enzyme is located in the contact sites, in addition to being in the inner membrane of bovine adrenocortical mitochondria (24). Interestingly, the present data indicate that the Ca 2ϩ -induced increase in cholesterol content is greater in the contact sites than in the inner membrane (Fig. 2). One interpretation could be that the P450 scc located in the contact sites is more active than the P450 scc of the inner membrane. Indeed, contact sites are known to be enriched in cardiolipin, a phospholipid that enhances the affinity of the P450 scc for cholesterol (35), although other explanations may be envisaged. Moreover, our results are in agreement with the data reported by Stevens et al. (36), suggesting that in addition to the outer and inner membrane pools, a third pool of steroidogenic cholesterol may be found in contact sites. On the other hand, Ca 2ϩ induces direct contacts between the outer and the inner mitochondrial membranes, presumably by promoting a nonbilayer configuration leading to membrane fusion (37)(38)(39). Such an increase in the number of contact sites could result in an increase in the rate of cholesterol transfer.
Recently, Clark and colleagues (40) have observed that the elevations of cytosolic calcium triggered by Ang II, K ϩ , and the Ca 2ϩ channel agonist, BayK8644, are accompanied by increases in the level of the 30-kDa StAR protein in the human H295R adrenocarcinoma cell line. StAR protein import into the mitochondria via contact sites is thought to be a crucial event promoting cholesterol transfer to the inner membrane (20). We have also observed an increase in StAR protein content of mitochondria isolated from Ca 2ϩ -clamped bovine glomerulosa cells. 2 Our results therefore lead us to conclude that the StAR protein participates in the Ca 2ϩ -induced cholesterol transfer in bovine glomerulosa mitochondria.
The above hypothesis is strengthened by the sensitivity of Ca 2ϩ -induced cholesterol transfer to cycloheximide, an inhibitor of protein translation that depletes a set of 30-kDa proteins involved in the activation of steroidogenesis (16). From our study, it appears that CHX partially inhibits Ca 2ϩ -stimulated cholesterol transfer from the outer membrane to the contact sites and inner membranes, suggesting that at least one part of the calcium-activated cholesterol transfer process requires protein synthesis. This finding is in agreement with the results reported by Kowluru et al. (34), suggesting that two intramitochondrial cholesterol transfer processes are mediated by Ca 2ϩ . One of these mechanisms involves mitochondrial membrane sites accessible to activation by Ca 2ϩ and GTP (presumably the sites of StAR-mediated cholesterol transfer). In the presence of CHX, this process is blocked, leading to redistribution of cholesterol to other sites involving mitochondrial membrane contact sites resulting from Ca 2ϩ -induced matrix swelling.
Several conclusions can be drawn from the experiments with Ang II stimulation. The lack of a decrease of cholesterol content in the outer membranes can be explained by a stimulation of cholesterol supply to the mitochondria in response to Ang II. Furthermore, a striking feature of the present study was the marked increase of cholesterol content in the contact sites, as compared with inner membranes, when glomerulosa cells were challenged with Ang II. This finding suggests that the hormone markedly enhances cholesterol availability in regions where the intermembrane space barrier is abolished. Interestingly, a subpopulation of the inner membranes even showed a decrease of cholesterol content, suggesting either a possible diffusion of cholesterol to specific sites in the inner membrane or a more unlikely selective loss of cholesterol during the fractionation procedure of mitochondria. Although we have no clear explanation at the present time for this result, it is worth mentioning that contact sites may be preferential sites for cholesterol transfer and metabolism, because they are enriched in the phospholipid cardiolipin, which promotes nonbilayer structures and enhances the affinity of P450 scc for cholesterol (35). One could therefore speculate that in addition to activating cholesterol transfer from the outer to the inner membrane, Ang II may also affect cholesterol movement within the inner membrane.
In conclusion, using the ionomycin-mediated cytosolic Ca 2ϩ clamp, we have shown that Ca 2ϩ itself is able to activate cholesterol transfer from the outer to the inner mitochondrial membrane and to intermembrane contact sites in bovine glomerulosa cells, a process that occurs even in the absence of cholesterol metabolism to pregnenolone. In fact, cholesterol flux could be even more important if steroidogenesis were allowed to proceed. We also demonstrate that Ang II, a Ca 2ϩmobilizing hormone, markedly increases cholesterol content in contact sites. Our results strongly suggest that one of the main functions of the Ca 2ϩ messenger under hormonal stimulation is to increase cholesterol supply to the P450 scc enzyme by enhancing intermembrane cholesterol transfer, thus promoting the activation of the subsequent steroidogenic cascade.