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Instituto Teófilo Hernando, Department de Farmacología y Terapeútica, Facultad de Medicina, Universidad Autónoma de Madrid, E-28029, Madrid, Spain
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Secretory Process Research Program, Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada, K1H 8M
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Abstract |
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(Received 9 March 2004;
accepted after revision 2 May 2004;
first published online 7 May 2004)
Corresponding author J.-M. Trifaró: Secretory Process Research Program, Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5. Email: jtrifaro{at}uottawa.ca
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Introduction |
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Calcium ions play a pivotal role, acting at more than one level in the cascade of events leading to exocytosis. A rise in the cytosolic Ca2+ concentration ([Ca2+]c) triggers exocytosis and this increase in Ca2+ is, depending of the type of stimulus, due either to Ca2+ entering the cell through specific channels or to Ca2+ being released from intracellular stores such as the endoplasmic reticulum (Kuba, 2000). Another component of the intracellular buffering machinery is the mitochondria (Duchen, 1999), which have emerged as important players in the intracellular regulation of Ca2+ levels (Friel & Tsien, 1994; Park et al. 1996; Herrington et al. 1996; Babcock et al. 1997; Montero et al. 2000).
Activation of Ca2+ channels or Ca2+ release from the endoplasmic reticulum triggers fast millimolar mitochondrial Ca2+ transients that modulate chromaffin cell secretion (Giovannucci et al. 1999; Montero et al. 2000) as well as secretion from PC12 cells (Taylor et al. 2000). Exposure of chromaffin cells to protonophores abolished mitochondrial Ca2+ uptake and potentiated stimulated secretion; this led to the conclusion that mitochondria could regulate the availability of Ca2+ to the secretory machinery, and hence secretion (Montero et al. 2000). However, how this modulation is exerted is unknown. The purpose of the present investigation was to elucidate the mechanisms involved in the potentiation of secretion when the mitochondrial Ca2+ sequestration is interrupted by a protonophore. Here we demonstrate that, in chromaffin cells, the potentiation of the secretory response observed upon the collapse of the mitochondrial transmembrane electrochemical potential is accompanied by PKC and myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylation and is mediated through cortical F-actin disassembly. These effects were inhibited by PKC blockers.
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Methods |
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Phorbol 12-myristate, 13-acetate (PMA), rotenone, oligomycin A, chelerythrine chloride, and carbonyl cyanide m-chlorophenyl hydrazone (CCCP) were purchased from Sigma Chemical Co. (St Louis, MO, USA); latrunculin B and cytochalasin D were obtained from Calbiochem (San Diego, CA, USA); rhodamine phalloidin and fura-2 and 1,2-bis(2-amino-phenoxy)-ethane-N,N,N'N'-tetra-acetic acid tetrakis (acetoxymethyl ester) (BAPTA AM) were purchased from Molecular Probes Inc. (Eugene, Oregon, USA). PMA, CCCP and chelerythrine were prepared in DMSO and kept as 10–2 M stock solutions at –20°C. Dilutions were freshly prepared on the day of the experiment, and final concentrations of DMSO were 0.1% or less in each case. Rabbit polyclonal phospho-PKC (pan) and phospho-MARCKS (ser 152/156) antibodies were obtained from Cell Signalling Technology Inc. (Beverley, MA, USA) and mouse monoclonal antibody against tubulin was purchased from Sigma Chemical Co.
Isolation and culture of chromaffin cells
Bovine adrenal glands were obtained from a local slaughterhouse. Chromaffin cells were isolated by adrenal medulla digestion with collagenase, according to the procedure of Trifaró & Lee (1980) with some modifications (Moro et al. 1990). Our preparations were enriched in adrenaline-containing cells. Cells were suspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum, 10 µM cytosine arabinoside, 10 µM fluorodeoxyuridine, 50 IU ml–l penicillin, and 50 µg ml–l streptomycin. Cells (5 x 106 in 10 ml DMEM) were plated in 5 cm diameter Petri dishes and kept in a water-saturated incubator at 37°C, in a 5% CO2–95% air atmosphere, and used 3–5 days thereafter. The culture medium was replaced by serum-free DMEM 24 h later, and then every 2 days.
On-line measurement of catecholamine release from bovine chromaffin cells
Cells were scraped off carefully from the bottom of the Petri dish with a rubber policeman, and centrifuged at 800 g for 10 min. The cell sediment was resuspended in 200 µl of Krebs-Hepes (composition (mM): NaCl 144; KCl 5.9; MgCl2 1.2; glucose 11; Hepes 10; pH 7.4); the concentration of CaCl2 varied depending on the protocol. Cells were introduced to a jacketed microchamber for their superfusion at 37°C. The superfusion rate was 2 ml min–l. The liquid flowing from the superfusion chamber reached an electrochemical detector (model Metrohn AG CH-9100 Hersau) placed just at the outlet of the microchamber that monitored, ‘on line’ under the amperometric mode, the amount of catecholamines secreted (Borges et al. 1986). Cells were stimulated to secrete with short pulses (1–5 s) of high K+ Krebs-Hepes solution controlled by electrovalves. Depolarizing concentrations of K+ were applied in the presence or absence of different test compounds when cells were being superfused with Krebs-Hepes solution (see Results for further details).
Measurement of changes of [Ca2+]c in fura-2-loaded bovine chromaffin cells
Chromaffin cells were loaded with fura-2 by incubating them with fura-2 AM (4 µM) for 60 min at 37°C in Krebs-Hepes solution (pH 7.4) containing (mM): NaCl 144; KCl 5.9; MgCl2 1.2; CaCl2 2.4; sodium Hepes 10; glucose 10. The loading incubation was terminated by washing the coverslip containing the attached cells several times with Krebs-Hepes solution. The coverslip containing the fura-2-loaded cells was placed in a chamber mounted on the stage of a Nikon Diaphot inverted microscope. The chamber was continuously perfused at room temperature (22 ± 2°C) with Krebs-Hepes solution. Once selected, an individual cell was locally superfused with various solutions that were changed using electronically driven miniature solenoid valves coupled to a multibarrel concentration-clamp device, the common outlet of which was placed within 100 µm of the cell being explored. The flow rate was smaller than 1 ml min–1 and was regulated by gravity to achieve complete replacement of the cell surroundings within less than 1 s. This allowed the precise application of depolarizing pulses of K+ of 5 s duration.
The fluorescence of fura-2 in single cells was measured with the photomultiplier-based system described by Neher (1989), which produces a spatially averaged measure of the [Ca2+]c. Fura-2 was excited with light alternating between 360 and 390 nm, using a Nikon 40 x fluorite objective. Emitted light was transmitted through a 425 nm dichroic mirror and a 500–545 nm barrier filter before being detected by the photomultiplier. [Ca2+]c was calculated from the ratios of the light emitted when the dye was excited by the two alternating excitation wavelengths (Grynkiewicz et al. 1985).
Protein phosphorylation
A PKC phosphorylation assay was performed according to the manufacturer's instructions (Cell Signalling Technology Inc.). Briefly, 1 x 106 chromaffin cells were incubated with Krebs-Hepes buffer in the absence (control) or presence of PKC inhibitors and then 2 µM CCCP in Krebs-Hepes buffer was added for 90 s. When PKC inhibitors were used, cells were pre-exposed to 1 µM of RO31-8220, staurosporine or chelerythrine for 8.5 min and then incubated with 2 µM CCCP for 90 s. The reaction was stopped by addition of 1 x SDS Sample Buffer (62.5 mM Tris, pH 6.8, 2% w/v SDS, 20% glycerol, 50 mM DTT, 0.5 mM EDTA, 0.5 mM EGTA), supplemented with the protease inhibitors phenymethylsulphonyl fluoride (0.5 mM), leupeptin (10 µg ml–1) and aprotinin (10 µg ml–1), as well as phosphatase inhibitors sodium fluoride (1 mM) and sodium orthovanadate (0.1 mM), followed by boiling for 5 min. Samples, each containing an equal amount of protein, were separated by SDS-PAGE and then electrotransferred onto a nitrocellulose membrane for analysis of PKC phosphorylation by Western blotting. Primary antibodies were rabbit polyclonal phospho-PKC (pan) antibody (1: 500), phospho-MARCKS (ser 152/156) rabbit polyclonal antibody (1: 500) and mouse monoclonal anti-tubulin antibody (1: 2000). Following incubation with the corresponding goat (anti-rabbit or anti-mouse) horseradish peroxidase conjugated IgG (1: 3000) (Bio-Rad Laboratories, Hercules, CA, USA), the membranes were incubated in electrochemiluminescence (ECL) Western blotting detection reagents (Amersham, Oakville, Canada). Chemiluminescence-emitting signals were detected by Hyperfilm ECL. Multiple exposures of each set of samples were carried out and autoradiographs were scanned. The density and area of the bands were calculated with the Scion Image Beta-3b software (Scion Corporation, Frederick, MD, USA). Values obtained for each phospho-PKC band were normalized on the basis of the tubulin band intensity (loading control) to correct for minor variations in loading.
Fluorescence microscopy
Chromaffin cells were plated on collagen-coated coverslips contained within plastic Petri dishes at a density of 3 x 105 cells per 35 mm dish. Cells were rinsed with Krebs-Hepes solution. Cells were incubated for different periods of time with Krebs-Hepes solution and in the absence or presence of different compounds; then they were stimulated by high K+, fixed in 3.7% formaldehyde and processed for fluorescence microscopy (Lee & Trifaró, 1981). Chromaffin cells were stained with rhodamine phalloidin (a probe for filamentous actin; 1.25 U ml–1) and only changes in the cortical actin network of chromaffin cells were recorded and analysed. Coverslips were thoroughly washed with phosphate-buffered saline (PBS). Finally coverslips were rinsed with PBS and mounted in glycerol–PBS (1: 1). Slides were viewed with a Leitz Ortholux fluorescence microscope and photographs were taken with Kodak-Tri-X pan films (400 ASA) (Vitale et al. 1991). To study the effect of several treatments on cortical F-actin disassembly, 100 single rounded chromaffin cells per coverslip (usually 6–8 coverslips per experimental condition) were examined. Each cell was classified as having either a ‘continuous’ or ‘discontinuous’ cortical rhodamine (F-actin) fluorescent ring (Vitale et al. 1991). The percentage of chromaffin cells showing cortical F-actin disassembly (discontinuous rhodamine fluorescent ring) was calculated for each experimental condition. To avoid personal bias, code numbers were given to each coverslip. The cells were examined and classified without knowing whether they were from control or treated preparations. The codes were revealed to identify the experimental conditions used only after all results were recorded (single-blind design).
Video-enhanced image processing
Quantitative analysis of cortical rhodamine fluorescence (F-actin) was performed by using a Hamamatsu Photonic KK Argus-50/CL Image Processor (Hamamatsu Photonic Systems, Bridgewater, NJ, USA). The fluorescence microscope was coupled to the video camera (Carl Zeiss, TV3M model), which was connected to the Argus 50-Image processor. Video camera control parameters (i.e. gain, offset and sensitivity) were set up to obtain a clear image of the cell on the monitor and a fluorescence intensity of 250 (arbitrary units) in the cortical region of the cell. The three-dimensional graphic analysis represents the coordinates of the equatorial plane of the cell as the X and Y axes and the fluorescence intensity of this plane as the Z axis (Vitale et al. 1995).
Data analyses and statistics
Data are presented as means ± standard errors of the mean (S.E.M.) and were analysed by one-way ANOVA. Scheffe's test was used to determine the level of significance of differences between groups. Results shown in Figs 4 and 6 were analysed by Student's t test.
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Results |
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Recent published work has demonstrated that short exposure (90 s) of chromaffin cells to 2 µM of the protonophore cyanide m-chlorophenyl hydrazone (CCCP) potentiated the secretory response to a short depolarizing pulse of K+ (Montero et al. 2000). The strong potentiating effect on K+ depolarization-evoked amine release observed with CCCP treatment was similar to that detected when cells were exposed to 100 nM phorbol ester (PMA; Fig. 1A and D). It is known that potentiation of the chromaffin cell secretory response by PMA is mainly due to a disruption of the cortical F-actin networks allowing the movements of chromaffin vesicles to release sites on the plasma membrane (Vitale et al. 1995). Another way to disrupt cortical F-actin networks is through direct interactions with actin filaments, as was the case for latrunculin B or cytochalasin D treatments (Fig. 1B–D). However, in this situation, the potentiation of high K+-evoked amine release was smaller than that observed with either CCCP or PMA.
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These experiments were performed in fura-2-loaded, single chromaffin cells, at room temperature. Figure 4A shows the original [Ca2+]c traces obtained in an example cell. After the [Ca2+]c reached a stable baseline (at about 100 nM concentration) a K+ pulse (70 mM K+, 5 s duration) caused a sharp rise of [Ca2+]c that declined quickly to reach basal levels in about 1 min. The cell was subsequently superfused with cytochalasin D (2 µM), which did not change the basal [Ca2+]c. Then a second pulse of K+ was added, followed by the application of CCCP (2 µM), which produced a slow rise of [Ca2+]c that reached a plateau at around 200 nM and returned quickly to the basal level upon washing out CCCP.
Similar experiments were performed in other cells to test the effect of PMA. For instance, Fig. 4B shows a cell that was first challenged with K+ and subsequently with CCCP. Note that, again, CCCP caused a mild and sustained elevation of the [Ca2+]c; note also that PMA (100 nM) did not change the basal levels of [Ca2+]c. An example of a third protocol is shown in Fig. 4C. Note that CCCP produced its typical [Ca2+]c elevation effect. When the cell was superfused with chelerythrine (100 µM), there was no change in the basal levels of Ca2+. When applied in the presence of chelerythrine, CCCP produced an elevation of the [Ca2+]c that was slightly higher. However, averaged results of four experiments show no changes in the kinetic parameters observed for the CCCP-evoked changes in [Ca2+]c, i.e. peak amplitude, time to peak and area of the Ca2+ curve, which represents the total Ca2+ accumulated in the cytosol during the CCCP challenge. Note that chelerythrine did not modify either the basal [Ca2+]c or the kinetic parameters of the [Ca2+]c elevation elicited by CCCP.
Effect of CCCP on chromaffin cell cortical F-actin networks
The chromaffin cell possesses a cortical network of filamentous actin underneath the plasma membrane which is disrupted by cholinergic receptor stimulation, cell depolarization or PMA treatment (Vitale et al. 1991, 1995). Therefore, one of the effects of PKC activation responsible for the potentiation of the secretory response is the disassembly of cortical filamentous-actin networks (Vitale et al. 1995), an effect mediated through the phosphorylation of MARCKS (Rosé et al. 2001). Treatment of chromaffin cells for 90 s with 2 µM CCCP produced cortical F-actin disassembly as evaluated by fluorescence microscopy of rhodamine–phalloidin (a probe for filamentous actin) stained cells (Fig. 5A and B). The fluorescence images of cortical F-actin disassembly obtained were quite similar to those observed after 100 nM PMA or high K+ treatment of chromaffin cells (Vitale et al. 1995). Moreover, the disruption in the cortical F-actin fluorescence ring produced by CCCP was inhibited by treatment with 1 µM chelerythrine (Fig. 5A and B), suggesting the involvement of PKC in this process. Similarly to PKC stimulation, CCCP-induced cortical F-actin disassembly was not dependent on extracellular Ca2+. CCCP-induced F-actin disassembly was observed in 76.9 ± 3.2% (n = 400) of the cells in the presence of extracellular Ca2+, and in 74.1 ± 1.8% (n = 600) of the cells in Ca2+-free medium. Moreover, F-actin disassembly in Ca2+-free medium in response to CCCP was inhibited by 98.8 ± 6.6% (n = 600) by preincubation of cells for 30 min with 50 µM BAPTA AM.
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Potentiating by CCCP of high K+ -evoked catecholamine output and cortical F-actin disassembly was measured in the presence or absence of increasing concentrations of chelerythrine, staurosporine or RO31-8220 (Fig. 6A–C), three PKC inhibitors. The three blockers produced a concentration-dependent inhibition of both responses, suggesting the involvement of PKC in CCCP potentiation of these two high K+-evoked effects. Moreover each blocker produced the same degree of inhibition for both catecholamine release and F-actin disassembly (Fig. 6A–C). Thus, as expected each inhibitor showed similar IC50 values for catecholamine output and F-actin disassembly. IC50 values were (µM): chelerythrine, 0.35 and 0.46, staurosporine, 0.22 and 0.20, RO31-8220, 0.013 and 0.016 for catecholamine output and F-actin disassembly, respectively.
Effect of CCCP on PKC and MARCKS phosphorylation
Phosphorylation of PKC is an indication of its activation (Keranen et al. 1995). Therefore, the effect of 90 s CCCP exposure on chromaffin cell PKC phosphorylation was measured by Western blotting using an antibody specific for phosphorylated PKC. Under these experimental conditions, a 50% increase in PKC phosphorylation was observed (Fig. 6D and E). As expected, CCCP-evoked PKC phosphorylation was inhibited by the three PKC blockers.
Furthermore, experiments from our laboratory have shown that cortical F-actin disassembly induced by PKC activation is mediated by phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) (Rosé et al. 2001), a PKC substrate. Unphosphorylated MARCKS cross-links actin filaments whereas phosphorylated MARCKS decreases F-actin cross-linking and promotes F-actin disassembly (Hartwig et al. 1992; Rosé et al. 2001). An increase in MARCKS phosphorylation was observed 90 s following CCCP exposure (Fig. 6F and G) and, again, this effect was abolished by PKC inhibitors. MARCKS phosphorylation was similar in the presence (70 ± 9.5% increase in phosphorylation, n = 8) or absence (61 ± 8.2%, n = 6) of extracellular Ca2+. Moreover, the increase in MARCKS phosphorylation produced by CCCP in Ca2+-free medium was inhibited 100 ± 8% (n = 6) by 30 min preincubation with 50 µM BAPTA AM.
Specificity of CCCP effects: Is mitochondria the only target for CCCP?
It has been suggested that CCCP may have other intracellular targets such as the endoplasmic reticulum (ER) and therefore the possibility exists that the effects observed here with CCCP were the result of Ca2+ being released from the ER. To test this possibility, the ER Ca2+ store was depleted by 30 min treatment with a combination of 10 mM caffeine, 10 µM ryanodine and 1 µM thapsigargin. This treatment releases ER Ca2+, leaves the ryanodine receptor channel of the ER in a low conductance open state (Alonso et al. 1999) and, in addition, blocks Ca2+ uptake into the ER. Under these conditions, the CCCP potentiating effect on high K+-evoked secretory responses was maintained (Fig. 7) whereas the secretory response to caffeine was abolished (data not shown). Furthermore, when mitochondrial respiration and ATP production were blocked by treatment with a combination of 4 µM rotenone and 3 µM oligomycin A, a potentiation of the high K+-evoked secretion was also observed (Fig. 8A and D). As with the CCCP-evoked potentiation of depolarization-evoked amine release, pretreatment with 100 nM PMA significantly reduced the rotenone and oligomycin A-induced potentiation (Fig. 8B and D) and, in addition, 1 µM chelerythrine produced a complete blockade (Fig. 8C and D), suggesting the involvement of PKC in the process. Cortical F-actin cytoskeleton disassembly was also observed with this combined treatment (Fig. 8E). The fluorescence images of cortical F-actin disassembly obtained following rotenone–oligomycin A treatment were quite similar to those observed upon CCCP treatment. Moreover, the effects of rotenone–oligomycin A on the cytoskeleton were blocked by 1 µM chelerythrine (Fig. 8E).
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Discussion |
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As demonstrated here, other treatments such as exposure to cytochalasin D, latrunculin B or phorbol esters (PMA) also potentiated the secretory response to a depolarizing pulse of K+. However, exposure to these substances did not modify resting intracellular Ca2+ levels (Fig. 4). It is known that PMA potentiates chromaffin cell responses to different secretagogues (Burgoyne & Norman, 1984; Pocotte et al. 1985; Brocklehurst et al. 1985; Burgoyne et al. 1988; Knight et al. 1988; Terbush et al. 1988; Bittner & Holz, 1990; Tachikawa et al. 1990; Vitale et al. 1992, 1995; Smith et al. 1998; Rosé et al. 2001). It is also known that PMA treatment does not induce secretion but disrupts chromaffin cell cortical F-actin networks and increases both the number of secretory vesicles at the subplasmalemmal zone (release-ready vesicle pool) and the initial rate of exocytosis in response to stimulation (Vitale et al. 1995). Indeed, membrane capacitance studies showed, in PMA-treated cells, an increased number of vesicles fusing with the plasma membrane during cell depolarization (Vitale et al. 1995; Smith et al. 1998). The chromaffin cell cortical F-actin network acts as a barrier to the secretory vesicles by blocking their movement toward the plasma membrane (Trifaró et al. 1982, 1985; Cheek & Burgoyne, 1986; Burgoyne & Cheek, 1987; Sontag et al. 1988; Burgoyne et al. 1989). Cell stimulation is accompanied by a focal transient disassembly of the cortical F-actin network (Cheek & Burgoyne, 1986; Vitale et al. 1991). This allows the movement of additional vesicles from the reserve pool to release sites at the plasma membrane (Vitale et al. 1995).
Cytochalasin D is a fungal toxin that depolymerizes F-actin and disrupts actin filaments (Brenner & Korn, 1979; Flanagan & Lin, 1980; Schliwa, 1982) and latrunculin B is a unique marine sponge toxin that also disrupts actin microfilaments, but in this case, by blocking actin polymerization (Spector et al. 1983). These two toxins did not cause secretion on their own but, as demonstrated here, they potentiated the secretory response. It is therefore quite possible that the potentiating effect of the toxins was due to the movement of secretory vesicles to the subplasmalemma area prior to the depolarizing stimulus; this being the result of the disruption of cortical F-actin (Gil et al. 2000). Perfusion of chromaffin cells with a combination of cytochalasin D and CCCP produced a summation of effects in the potentiation of high K+ secretory responses (Fig. 2), suggesting the involvement of different mechanisms. On the other hand, the combination of PMA and CCCP did not show summation of effects, although responses to stimulation were larger than those in the presence of either substance (Fig. 2). This observation suggested to us that PMA and CCCP elicited potentiation of the secretory response through the same final common pathway. Indeed, when the CCCP pulse was preceded by a PMA pulse the potentiating effect of CCCP was completely abolished (Fig. 3), suggesting, again, a common pathway for these effects. The effect of PMA on secretion is the result of PKC stimulation (Vitale et al. 1992, 1995) since the effect is blocked by PKC inhibitors (Vitale et al. 1992). Chelerythrine, staurosporine and RO31-8220, three known inhibitors of PKC, also blocked the potentiating effect of CCCP (Fig. 6), suggesting the involvement of PKC in the effects elicited by this compound. Indeed, exposure to CCCP for 90 s increased PKC and MARCKS phosphorylation, effects blocked by the three PKC inhibitors. Published work from our laboratory has also demonstrated that MARCKS, a PKC substrate, mediates, at least in part, the effect of PKC on secretion (Rosé et al. 2001). In view of these observations, immunocytochemical studies with rhodamine-labelled phalloidin, a probe for filamentous actin, indicated, as expected, that exposure of chromaffin cells to CCCP induced cortical F-actin disassembly which was inhibited by the three PKC blockers (Fig. 6), suggesting, again, the involvement of PKC in this process. Cortical F-actin disassembly was also observed during the exposure of resting cells to CCCP. Under these conditions, no increase in secretion was observed. These results are similar to those obtained with PMA, a substance which evoked cortical F-actin disassembly without inducing secretion (Vitale et al. 1995). The increase in cellular Ca2+ observed upon CCCP treatment was of such magnitude as to trigger the mechanism responsible for cortical F-actin disassembly. In this regard, it has been suggested that an increase in cytosolic Ca2+ might signal the mechanism involved in supplying vesicles to release sites (Von Rüden & Neher, 1993; Kamiya & Zucker, 1994). It can be concluded from the present results that CCCP potentiation of the secretory responses was, as with PMA treatment, the result of translocation of secretory vesicles from the reserve pool to the subplasmalemma area (release-ready vesicle pool) in preparation for exocytosis. As a site of action for these effects, mitochondria were demonstrated to be responsible for the potentiation of the secretory response observed in the presence of CCCP, even when the release of Ca2+ from the endoplasmic reticulum was blocked by previous treatment with a cocktail of caffeine, ryanodine and thapsigargin. In addition, exposure of chromaffin cells to a combination of rotenone and oligomycin A, two other mitochondrial inhibitors that do not affect Ca2+ mobilization from the endoplasmic reticulum, also caused a potentiation of secretion. The combination of rotenone and oligomycin A not only potentiated the secretory response to high K+, but also evoked chromaffin cell cortical F-actin disassembly, effects that were also inhibited by chelerythrine (Fig. 8). The inhibition by chelerythrine suggests, again, the involvement of PKC in this process. Two pathways are known to be involved in the control of chromaffin cell cortical F-actin networks during secretion (Trifaró et al. 2000). These are the Ca2+–scinderin and the PKC–MARCKS pathway. The Ca2+–scinderin pathway uses Ca2+ that enters the cell through voltage-dependent channels during depolarization induced by nicotinic receptor stimulation (Trifaró et al. 2000) whereas the PKC pathway seems to be activated during either the release of Ca2+ from the endoplasmic reticulum (Zhang et al. 1995; Trifaró et al. 2000; Rosé et al. 2001) or the increase in cell Ca2+ observed when mitochondria Ca2+ uptake is inhibited, as in this case with CCCP treatment.
In conclusion, our findings suggest that the clearance of cytosolic Ca2+ by mitochondria during chromaffin cell stimulation limits the extent of the exocytotic response by preventing excessive stimulation of PKC and enhanced cortical F-actin disassembly. Thus excessive PKC activation would increase the number of secretory vesicles at release sites followed by an increased and perhaps unnecessary secretory response.
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P < 0.01 versus high K+ or rotenone–oligomycin A.