Maintenance of quantal size and immediately releasable granules in rat chromaffin cells by glucocorticoid

Jianhua Xu,1,2,* Kim San Tang,1,* Van B. Lu,1 Chandana P. Weerasinghe,1 Amy Tse,1,2 and Frederick W. Tse1,2

1Department of Pharmacology and 2Centre for Neuroscience, University of Alberta, Edmonton, Alberta, Canada

Submitted 22 October 2004 ; accepted in final form 26 May 2005


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 ABSTRACT
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Glucocorticoid is reported to regulate catecholamine synthesis and storage. However, it is not clear whether the actual amount of catecholamine released from individual granules (quantal size, Q) in mature chromaffin cells is affected by glucocorticoid. Using carbon fiber amperometry, we found that dexamethasone did not affect mean cellular Q or the proportional release from different populations of granules in rat chromaffin cells cultured for 1 day in a serum-free defined medium. After two extra days of culture in the defined medium, there was a rundown in mean cellular Q, and it was associated with a shift in the proportional release from the different granule populations. This phenomenon could not be rescued by serum supplementation but could be prevented by dexamethasone via an action that was independent of changes in voltage-gated Ca2+ channel (VGCC) density. Using simultaneous measurements of membrane capacitance and cytosolic Ca2+ concentration, we found that for cells cultured in defined medium dexamethasone enhanced the exocytotic response triggered by a brief depolarization (50 ms) without affecting the VGCC density or the fast exocytotic response triggered via flash photolysis of caged Ca2+. Thus glucocorticoid may regulate the number of immediately releasable granules that are in close proximity to a subset of VGCC. Because chromaffin cells in vivo are exposed to high concentrations of glucocorticoid, our findings suggest that the paracrine actions of glucocorticoid maintain the mean catecholamine content in chromaffin cell granules as well as the colocalization of releasable granules with VGCCs.

catecholamines; paracrine action; exocytosis; calcium channels


THE LOCAL BLOOD CIRCULATION within the adrenal gland exposes the chromaffin cells in the medulla to high concentrations of glucocorticoid that are secreted by the cortical cells. This paracrine interaction is suggested to contribute significantly to the regulation of catecholamine release from chromaffin cells (21, 35). For example, in mouse chromaffin cells, differences in voltage-gated Ca2+ channel (VGCC) subtypes and their contributions to exocytosis have been reported between cultured cells and adrenal slices (1). The expression pattern of VGCCs in cultured bovine chromaffin cells was found to be different from that in the adrenal medullae, and the loss of glucocorticoid was partially responsible for these differences (5). Indeed, glucocorticoid has been shown to increase VGCC density in cultured rat and porcine chromaffin cells (14, 15). The increase in extracellular Ca2+ entry via VGCCs can in turn lead to an enhancement in the triggering of Ca2+-dependent exocytosis of catecholamine-containing granules from the readily releasable pool or the mobilization of additional granules (13, 23, 49). Besides changing VGCC density, glucocorticoid may influence catecholamine secretion via other mechanisms. In rat PC-12 cells, glucocorticoid treatment dramatically enhanced Ca2+-dependent exocytosis, and this enhancement was partially attributed to an increase in coupling between VGCCs and secretory granules (11). In addition, glucocorticoid was reported to affect key enzymes involved in catecholamine biosynthesis (21). In rat PC-18 cells (41) and chromaffin cells in long-term (30 day) culture (42), glucocorticoid enhanced the activity of tyrosine hydroxylase, the rate-limiting enzyme in the synthesis of L-3,4-dihydroxyphenylalanine (L-DOPA), which is the precursor of all catecholamines. In bovine chromaffin cells cultured for 6–21 days, glucocorticoid increased the activity of the enzyme phenylethanolamine N-methyltransferase (PNMT), which converts norepinephrine to epinephrine (6, 24). For cells in long-term cultures, in which both tyrosine hydroxylase and PNMT were reported to be downregulated (6, 10, 21, 24), the effect of glucocorticoid on these enzymes might be expected to become more prominent. On the other hand, for mature chromaffin cells in short-term culture, where much catecholamine is already synthesized and stored in granules, it is not clear whether glucocorticoid can have any influence on the total amount of catecholamines stored in individual granules of chromaffin cells. Therefore, in this study, we examined whether glucocorticoid could affect quantal size (Q) in rat chromaffin cells kept in short-term culture for 1 or 3 days.

Other than changes in catecholamine synthesis, Q can also be affected by other glucocorticoid-mediated mechanisms [particularly cytosolic Ca2+ concentration ([Ca2+]i); Ref. 7]. In calf chromaffin cells, the modulation of Ca2+ entry via VGCCs has been reported to alter Q by greater than twofold (12). Furthermore, an increase in Ca2+ entry via VGCCs may also increase Q via secondary changes in the pH of cellular compartments (32). Because glucocorticoid can increase VGCC density in chromaffin cells, it is important to separate the effects of increased VGCC density on Q from those mediated by other mechanisms. Therefore, in this study, we used two different culture media (a defined medium and a serum-supplemented medium). For cells cultured with serum supplementation, treatment with the glucocorticoid agonist dexamethasone increased VGCC density, as previously reported in cultured rat and guinea pig chromaffin cells (14, 15). On the other hand, for cells cultured in defined medium, VGCC density was not affected by dexamethasone. By comparing the actions of dexamethasone on Q in cells cultured with the two different culture media, we could determine whether the action of dexamethasone was related to changes in VGCC density. Because dexamethasone did not affect VGCC density in cells cultured in defined medium, we also examined whether dexamethasone could affect exocytosis via mechanisms that were independent of changes in overall VGCC density.


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Cell preparation. Male Sprague-Dawley rats (200–250 g) were euthanized with halothane in accordance with the standards of the Canadian Council on Animal Care. This procedure was reviewed and approved by the Health Sciences Animal Policy and Welfare Committee of the University of Alberta. Adrenal chromaffin cells were isolated as described previously (40, 52). Cells were maintained in standard culture conditions, either in a defined medium [MEM supplemented with 1% (vol/vol) insulin-transferrin-selenium A; all from GIBCO, Grand Island, NY] or in a serum-supplemented medium (DMEM supplemented with 10% horse serum; GIBCO). Both culture media were supplemented with 50 U/ml penicillin G and 50 µg/ml streptomycin (GIBCO). Recordings were performed on cells maintained in culture for 1 or 3 days.

Chemicals and solutions. Indo-1 and difluoro-indo-1 (indo-1FF) were purchased from TEF Labs (Austin, TX). BAPTA, ATP, DM-nitrophen, and NG-monomethyl-L-arginine (L-NMMA) were from Calbiochem (San Diego, CA). All other chemicals were purchased from Sigma-Aldrich (Oakville, ON, Canada). The standard bath solutions contained (in mM) 150 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 8 glucose, and 10 Na-HEPES (pH 7.4). For measurement of membrane capacitance, the pipette solution contained (in mM) 135 Cs-aspartate, 10 tetraethylammonium (TEA)-Cl, 20 Cs-HEPES, 3 MgCl2, 2 Na2ATP, 0.3 GTP, and 0.1 indo-1 (pH 7.4). In these experiments, the CaCl2 in the standard bath solution was increased to 10 mM, and TTX (0.5 µM) and apamin (0.4 µM) were added to block the voltage-gated Na+ current and the small-conductance Ca2+-activated K+ current (SK), respectively. In experiments involving measurement of VGCC density, the pipette solution also contained 10 mM BAPTA. In experiments in which [Ca2+]i was elevated via flash photolysis of caged Ca2+, the pipette solution contained (in mM) 70 Cs-aspartate, 40 Cs-HEPES, 20 TEA-Cl, 0.1 Na4GTP, 0.1 indo-1FF, and 6.5–7.2 DM-nitrophen (~80–90% saturated with Ca2+) (pH 7.4).

[Ca2+]i measurement. [Ca2+]i was measured fluorometrically with the Ca2+ indicator indo-1 (in experiments involving depolarization-triggered exocytosis) or indo-1FF (in experiments involving flash photolysis of caged Ca2+). The Ca2+ indicator was dialyzed into the cell via the whole cell patch pipette. Details of the instrumentation and procedures of the [Ca2+]i measurement were as described previously (45). In experiments involving flash photolysis of caged Ca2+, UV flash from a modified XF-10 xenon flash lamp (Hi-Tech, Salisbury, UK) was delivered to the cell via a fused silica focusing lens that replaced the microscope's condenser (46). To raise [Ca2+]i to different levels, we varied the intensity of the UV flash or the concentration of the Ca-DM-nitrophen.

Electrophysiology. In experiments involving detection of quantal catecholamine release from individual cells, carbon fiber (tip diameter 7 µm) amperometry (8, 51, 56) was used as described in our previous studies (40, 52). Chromaffin cells were stimulated by bath application of a high-K+ concentration ([K+]; 50 mM) extracellular solution (equimolar replacement of NaCl in the standard bath solution by KCl) (52). In all experiments, we collected data for 5 min while extracellular [K+] was raised. To minimize the slight variation in the sensitivity of individual carbon fiber electrodes, each electrode was used only twice for one control cell and one cell that received the experimental manipulation (in random order). The amount of catecholamine released from individual granules (Q) was calculated from the time integral of individual amperometric events. The criteria used to select the amperometric events for analysis were described in detail previously (40). For experiments involving measurement of secretion from the entire cell, exocytosis was monitored as increases in membrane capacitance ({Delta}Cm) resulting from addition of granule membrane to cell membrane (26, 30). Single chromaffin cells were voltage clamped at a direct current (DC) holding potential of –80 mV (corrected for junction potential) with the whole cell gigaseal technique (18). {Delta}Cm was measured with a software-based phase-sensitive detector [Pulse Control (20) during depolarization-triggered exocytosis (53)] or with a dual-phase lock-in amplifier [during flash photolysis experiments (44)].

Statistical analyses. All mean values in the text and figures are given as means and SD. When comparing distributions of the cube root of quantal size (Q) with fitted Gaussian (see Fig. 1) or between cumulative frequency histograms of Q of amperometric events collected from cells of different treatment groups (see Fig. 3), Kolmogorov-Smirnov (K-S) statistics (Mini Analysis Program version 5.24, Synaptosoft, Decatur, GA, or GB stat version 8.0, Dynamic Microsystem, Silver Spring, MD) were used. Any difference with P < 0.05 in the K-S test was considered to be statistically significant (see Figs. 1 and 3). In all other analyses, an independent Student's t-test for two populations was used to determine whether there was any statistical difference between the mean Q values from individual cells in the treatment groups and those from control cells. Any difference with P < 0.05 in the Student's t-test was considered statistically significant (see Figs. 2, 4, and 5).



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Fig. 1. Distribution of cube root of quantal size (Q) of individual amperometric events deviated significantly from a single Gaussian, but the distribution of cellular mean Q did not. A: distributions of events collected from cells cultured in defined medium for 1 day (1,522 events from 18 cells; i) and for 3 days (1,979 events from 20 cells; ii), for 1 day with dexamethasone (1,841 events from 20 cells; iii), and for 3 days with dexamethasone (iv). B: distributions of events from cells cultured in serum-supplemented medium for 1 day (2,925 events from 22 cells; i) and for 3 days (4,437 events from 20 cells; ii), for 1 day with dexamethasone (2,704 events from 21 cells; iii), and for 3 days with dexamethasone (4,602 events from 18 cells; iv). Note that each distribution deviated significantly from a single Gaussian [solid line; *P < 0.05 in Kolmogorov-Smirnov (K-S) test]. Inset of each plot shows the distribution of the Q of individual cells in the same treatment group (obtained by averaging the amperometric events collected from individual cells). Note that the distribution of the cellular mean Q values did not deviate from a single Gaussian (solid line; P > 0.05 in K-S test).

 


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Fig. 3. Dexamethasone prevented the shift in the proportional release of the granules in defined medium during rundown. Plots of the cumulative frequency histogram of Q for all amperometric events collected from control cells cultured for 1 or 3 days (A and D), cells cultured for 1 day in control condition or in dexamethasone (B and E), and cells cultured for 3 days in control condition or in dexamethasone (C and F) are shown. A–C: cells cultured in defined medium. D–F: cells cultured in serum-supplemented medium. For comparison, the mean Q values in each plot (except B) were matched by scaling (with a fixed percentage) the Q value of all amperometric events from control 1-day cells. Note that in both defined (A) and serum-supplemented (D) culture medium, there was a shift in the proportional release (denoted by thin arrows) after 2 extra days of culture. Thick arrows indicate the region that was critical for K-S test. Dexamethasone treatment for 1 day did not affect the proportional release in cells cultured in either medium (B and E). In defined medium, dexamethasone completely prevented the change in proportional release (C). For cells cultured with serum-supplemented medium, dexamethasone only partially prevented the shift in proportional release. *Statistically significant at indicated P value.

 


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Fig. 2. Time-dependent rundown in Q was prevented by dexamethasone. Mean Q of cells cultured in defined medium (A) and in serum-supplemented medium (B) in the absence (control) or presence of dexamethasone (1 µM). *Statistically significant differences from relevant control cells cultured for 1 day. Note that dexamethasone treatment for 1 day caused no significant effect, but dexamethasone for 3 days prevented the time-dependent rundown of Q in both culture media. Number of cells for each treatment is shown in parentheses. C: action of dexamethasone on quantal size was concentration dependent. Mean Q of cells cultured with 1, 10, or 100 nM or 1 µM dexamethasone for 3 days in serum-supplemented medium or defined medium is shown. *Statistically significant differences from the relevant control cells, which were cultured for 3 days.

 


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Fig. 4. Action of dexamethasone on the voltage-gated Ca2+ channel (VGCC) density of rat chromaffin cells. Current-voltage relationship of Ca2+ current (ICa) from chromaffin cells cultured in the absence (control, {bullet}) or presence ({circ}) of 1 µM dexamethasone. A andB: data from cells cultured in serum-supplemented medium for 1 or 3 days. C andD: data from cells cultured in defined medium for 1 or 3 days. ICa was evoked by depolarization (50 ms) to different membrane potentials (Vm) in the presence of 10 mM extracellular Ca2+. VGCC density of obtained by normalizing ICa to an individual cell's initial membrane capacitance (Cm). Each data point is the average of 7–20 cells. *Statistically significant differences between dexamethasone-treated cells and relevant controls. For clarity, error bars include only the negative values of SD.

 


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Fig. 5. Dexamethasone increased only the amplitude of the exocytotic response to a brief depolarization. A: representative simultaneous measurement of cytosolic Ca2+ concentration ([Ca2+]i) and changes in membrane capacitance ({Delta}Cm) in a chromaffin cell cultured for 1 day in defined medium. The cell was held at –80 mV (direct current), and a train of depolarization (to +10 mV, 15 steps at 3 Hz) was applied. {Delta}C1, increase in Cm after the termination of the first depolarization; {Delta}Ctotal, maximum increase in Cm after the termination of the entire train of depolarizations. [Ca2+]i was monitored with indo-1. B: the following parameters were compared between cells incubated in the absence or presence of dexamethasone (1 µM) for 1 or 3 days in a defined medium: basal [Ca2+]i (measured 10–30 s before the train of depolarizations), peak [Ca2+]i rise (elicited by the train of depolarizations), VGCC density [ICa/Cm, the amplitude of ICa elicited during the first depolarization pulse normalized to initial cell membrane capacitance], {Delta}C1/Cm (amplitude of exocytotic response elicited by the first depolarization), and {Delta}Ctotal/Cm (maximum amplitude of exocytotic response elicited by the train of depolarizations). All values shown were normalized to those of time-matched controls. The experimental protocol was as described in A. *Statistically significant differences from the relevant time-matched control cells.

 

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Dexamethasone prevented rundown in mean Q after 3 days of culture. To determine the amount of catecholamine released from single granules, we used carbon fiber amperometry (8, 51) to detect quantal release of catecholamine from rat chromaffin cells. All three catecholamines in chromaffin cells, dopamine, norepinephrine, and epinephrine, oxidize to yield two electrons per molecule by amperometry (8). Therefore, the time integral of each "amperometric event" (i.e., the quantal charge) provides a reliable estimate of the total amount of catecholamine released from the fusion of one granule (i.e., Q). Our previous study (52) showed that when rat chromaffin cells were depolarized by a bath application of 50 mM [K+], the activation of VGCCs typically elevated [Ca2+]i to 0.5–1 µM and triggered Ca2+-dependent quantal release of catecholamine. The same experimental approach was used in the present study. Because our goal was to examine the action of glucocorticoid, in our first series of experiments we used the following procedures to minimize the contamination of extraneous glucocorticoid in cell culture. First, the adrenal cortex was completely removed from the medulla during cell preparation, thus reducing the presence of cortical cells in the culture (<0.5%). Second, isolated chromaffin cells were kept in a defined serum-free culture medium, thus avoiding exposure to any glucocorticoid that might be present in the serum.

Figure 1A, i and ii, shows the distributions of Q generated by pooling individual amperometric events collected from different cells cultured for 1 or 3 days in defined serum-free culture medium. It should be noted that compared with the control cells cultured for 1 day (Fig. 1A,i), the distribution of Q of the amperometric events from control cells cultured for 3 days (Fig. 1A,ii) was shifted to the left, reflecting a rundown in quantal size during the two extra days of culture. However, a quantitative comparison between the treatment groups was complicated by the presence of multiple populations of granules in rat chromaffin cells. In a previous study (40), we analyzed a large number (10,000–50,000) of amperometric events from rat chromaffin cells and found that the distribution of Q could be reasonably described by the summation of three Gaussians, suggesting the presence of at least three populations of granules, each with a different modal Q. We have also shown (40) that the rundown in Q was associated with a shift in the proportional release from the different populations of granules. Because of the much lower number of amperometric events (1,500–4,500) in the present study, it was not possible to fit the distribution of Q with multiple Gaussians reliably to examine the contribution of the different populations of granules. Although each distribution of Q of individual amperometric events in Fig. 1 appeared to have a smooth bell shape, K-S statistics showed that they all deviated significantly from a single Gaussian (P < 0.05 in K-S test). In general, there were too many events with large values of Q (>0.85 pC) and too few events with small values of Q (<0.2 pC). In our previous study (40), we showed that the distribution of the Q of individual cells (generated by averaging the Q values of all amperometric events collected from the same cell) in the same treatment group (60–277 cells) could be well described by a single Gaussian. In the present study, although the number of cells (18–22) was much smaller, the distribution of the mean Q from individual cells in each treatment group (Fig. 1A, insets) did not deviate significantly from a single Gaussian (P > 0.05 in K-S test). The advantage of grouping the amperometric events according to cells has been explored in depth by Colliver et al. (9), and this method allowed us to compare the average value of the mean Q of individual cells between different treatment groups with simple parameter statistics (Fig. 2). As shown in Fig. 2A, the mean value of Q in cells cultured for 1 day in defined medium was 0.63 pC (SD 0.06; n = 18 cells). The mean Q for cells cultured for 3 days in defined medium was 0.53 pC (SD 0.11; n = 20 cells). Thus, compared with cells cultured for 1 day, the mean Q of cells cultured for 3 days in defined medium was reduced by ~16%, reflecting a decrease of ~40% of mean Q (Fig. 2A).

Because the rundown in Q was observed in cells cultured in serum-free defined medium, we first examined whether the rundown could be rescued with serum supplementation. The above-described experiments were repeated with cells cultured in a medium supplemented with 10% horse serum (Fig. 1B). Similar to the cells cultured in defined medium, the Q distribution of amperometric events (Fig. 1B, i and ii) could not be described by a single Gaussian (P < 0.0001 in K-S test), but the distribution of the mean Q from individual cells in each treatment group (of Fig. 1B, i and ii, insets) did not deviate significantly from a single Gaussian (P > 0.05 in K-S test). Note that the leftward shift in the Q distribution of amperometric events was still observed in cells cultured in serum-supplemented medium for 3 days (Fig. 1B,ii). Mean Q of cells cultured for 1 day in serum-supplemented medium was 0.58 pC (SD 0.09; n = 22 cells), which was not significantly different from that of cells cultured for the same duration in the defined medium (Fig. 2B). Note that in cells cultured for 3 days in serum-supplemented medium, mean Q was reduced by ~17% to 0.48 pC (SD 0.06; n = 20 cells), reflecting a ~42% decrease in Q (Fig. 2A). This reduction in Q was almost identical in magnitude to the rundown observed in cells cultured for the same duration in the defined medium (Fig. 2A). Thus the rundown in Q in chromaffin cells could not be rescued by serum supplementation.

To examine whether this rapid rundown in Q in isolated chromaffin cells was related to the loss of the paracrine actions of glucocorticoid during culture, we measured Q of chromaffin cells cultured in defined medium supplemented with dexamethasone (1 µM), a glucocorticoid agonist. We found that the Q distribution for cells cultured for 1 day in defined medium supplemented with dexamethasone (Fig. 1A,iii) was similar to that of the control cells cultured in defined medium for 1 day (Fig. 1A,i). However, the Q distribution for cells cultured for 3 days in defined medium supplemented with dexamethasone (Fig. 1A,iv) was shifted to the right compared with the control cells cultured for 3 days (Fig. 1A,ii). A similar result was obtained for cells cultured with dexamethasone in serum-supplemented medium (Fig. 1B). Compared with control cells cultured for 3 days in serum-supplemented medium (Fig. 1B,ii), the Q distribution for cells cultured with dexamethasone was shifted to the right (Fig. 1B,iv). Mean Q values for cells treated with dexamethasone (1 µM) for 1 or 3 days in defined medium were similar to that of the control cells cultured for 1 day but significantly larger than that of control cells cultured for 3 days in defined medium (Fig. 2A). Figure 2B shows that for cells cultured with dexamethasone and serum supplement for 1 day, mean Q was 0.64 pC (SD 0.12; n = 21 cells), which was not statistically different from the control cells [0.58 pC (SD 0.09); n = 22 cells]. For cells cultured with dexamethasone in serum-supplemented medium for 3 days, mean Q was 0.56 pC (SD 0.08; n = 18 cells). Although this value was significantly smaller than the mean Q value of cells cultured with dexamethasone and serum supplement for 1 day, it was not significantly different from that of the control cells cultured in serum-supplemented medium for 1 day. The overall results in Fig. 2, A and B, indicated that dexamethasone (1 µM) did not cause any significant increase in mean cellular Q after 1 day in culture but prevented the rundown of mean Q (by ~40%) that would otherwise occur with the two extra days of culture. In addition, this effect of dexamethasone could be observed in the defined medium as well as in the presence of serum supplementation.

Because dexamethasone (1 µM) prevented the rundown of the cellular mean value of Q with or without serum supplementation, we examined whether this effect of dexamethasone had similar concentration dependence in the two culture media. For this series of experiments, chromaffin cells were incubated with different concentrations of dexamethasone for 3 days in defined medium or serum-supplemented medium. Mean Q values of cells from this experiment are shown in Fig. 2C. In cells cultured in serum-supplemented medium, the mean Q values of cells cultured with 1 or 10 nM dexamethasone were 0.50 pC (SD 0.13; n = 27 cells) and 0.49 pC (SD 0.06; n = 16 cells), respectively. These values are similar to those of the control cells [0.49 pC (SD 0.07); n = 78 cells]. For cells cultured with 100 nM dexamethasone in serum-supplemented medium for 3 days, mean Q was 0.55 pC (SD 0.08; n = 21 cells), similar to the increase in Q by 1 µM dexamethasone (Fig. 2). Similarly, in cells cultured with defined medium (Fig. 2C), dexamethasone at 1 µM, but not at 10 nM, was effective in increasing Q. The above results suggested that ~1 µM dexamethasone was required to maintain Q in chromaffin cells when cultured in defined medium or serum-supplemented medium.

Dexamethasone prevented change in proportional release of different populations of granules after 3 days of culture in defined medium. In a previous study (40), we showed that the phenomenon of rundown in Q after 3 days of culture in defined medium was associated with a shift in the proportional release from the different granule populations. Although the result from Fig. 2 suggested that dexamethasone could prevent the decrease in mean cellular Q during culture, it was not clear whether dexamethasone prevented the shift in the proportional release from the different granule populations. Because the sample size in the current study was too small for fitting multiple Gaussians to dissect out the contribution from the different granule populations, we used the following analysis to address this issue. This analysis is based on the assumption that when there is no change in the proportional release from the different granule populations the cumulative frequency histograms of Q distributions of all the amperometric events collected from the two groups of cells (with the same value in mean Q) should be similar (according to K-S statistics). We first pooled the amperometric events collected from all the cells in the same treatment group. The fraction of amperometric events with different values of Q (bin size of 0.05 pC) was then plotted as a cumulative frequency histogram. Because the majority of the amperometric events have Q values <1.2 pC (Fig. 1), the cumulative fraction of events approached the value of 1 at Q = 1.2. For clarity, all cumulative frequency histograms are shown as lines instead of bar graphs. Figure 3A shows the cumulative histograms obtained from control cells cultured in defined medium for 1 or 3 days. In this plot, the Q value of individual amperometric events from control day 1 cells was scaled down (by 37%) such that their mean Q matched the mean Q value of control day 3 cells. Thus, if the rundown in culture was solely a result of uniform decrease in the Q value of every granule by 37%, the two cumulative frequency histograms should be similar. It should be noted, however, that the two histograms were significantly different (P = 0.025 in K-S test). For control cells cultured for 3 days in defined medium, there was an increase in the proportional release of granules with medium values of Q (~0.4–0.7 pC; critical for K-S test) and a reduction in the proportional release from granules with small Q (<0.3 pC) and large Q (>0.7 pC). To examine whether dexamethasone altered the proportional release of granules in cells cultured in defined medium, we compared the cumulative frequency histogram of Q of control 1-day cells to that obtained from dexamethasone-treated 1-day cells (Fig. 3B). It should be noted that even without any scaling for mean Q, the two cumulative frequency histograms were not significantly different (P = 0.33 in K-S test). Thus, in cells cultured with defined medium, dexamethasone treatment for 1 day did not have any significant effect on the proportional release (Fig. 3B). A comparison between the cumulative frequency histogram of control 1-day cells (scaled down by 13%) in defined medium and that obtained from dexamethasone-treated 3-day cells in defined medium shows that there was no significant difference (P = 0.46 in K-S test; Fig. 3C). Thus, for cells cultured in defined medium, if the relatively small (~13%) uniform decrease in Q of every granule was compensated, dexamethasone indeed prevented the shift in proportional release during the two additional days of culture. A similar comparison was made to the cells cultured with serum-supplemented medium (Fig. 3, D–F). Figure 3D shows that for cells cultured in serum-supplemented medium, there was also a shift in the proportional release of granules for cells cultured for 3 days, but the shift was different from that observed for cells cultured in defined medium (Fig. 3A). It should be noted that in cells cultured for 3 days in the serum-supplemented medium, there was a reduction in the proportional release of granules with Q < 0.5 pC and an increase in the contribution from granules with large modal Q (Q > 0.5 pC). Figure 3E shows that the cumulative histogram from cells treated with dexamethasone for 1 day was not significantly different from that of the time-matched controls (scaled up by 41%). Thus, similar to the observation for cells cultured in defined medium (Fig. 3B), dexamethasone treatment for 1 day in serum-supplemented cells did not affect the proportional release. To examine whether dexamethasone also prevented the shift in proportional release in cells cultured with serum-supplemented medium, we compared the cumulative frequency histogram of control 1-day cells (scaled down by 22%) with the histogram from dexamethasone-treated 3-day cells (Fig. 3F). For cells treated with dexamethasone, the shift in proportional release was less compared with the control 3-day cells (Fig. 3D), but the proportional release of the dexamethasone-treated 3-day cells was still significantly different from the control 1-day cells (P = 0.0002 in K-S test). Thus, for cells cultured in serum-supplemented medium, dexamethasone only partially prevented the shift in proportional release during culture.

Prevention of mean Q rundown by dexamethasone could not be mimicked by inhibitor of nitric oxide synthase. In addition to its action as a glucocorticoid agonist, dexamethasone has also been reported to be an inhibitor of nitric oxide (NO) synthase (NOS) (34). In primary culture of bovine chromaffin cells, immunoreactivity of NOS was found in the majority of chromaffin cells (39). The action of NO on chromaffin cells is controversial. Inhibitors of NOS have been reported to enhance (39, 43) or reduce (47) catecholamine release in cultured bovine chromaffin cells, as well as to inhibit the activity of tyrosine hydroxylase in rat adrenal medulla (25). A recent study in bovine chromaffin cells showed that NO slowed down the emptying of catecholamine-containing granules during exocytosis (27). Interestingly, in the same study, acute incubation with NOS inhibitors or NO scavengers caused a reduction in Q. Therefore, we examined whether 3 days of treatment with a potent NOS inhibitor (L-NMMA; 300 µM), affected Q of rat chromaffin cells in defined medium. We found that the mean Q value for cells treated with L-NMMA for 3 days was 0.65 pC (SD 0.09; n = 20 cells), which was not significantly different from that obtained from the time-matched controls [0.66 pC (SD 0.07); n = 20 cells]. Thus the NOS inhibitor L-NMMA could not mimic the effect of dexamethasone in preventing the rundown of Q.

Action of dexamethasone on preventing mean Q rundown was unrelated to upregulation of VGCC density. In rat chromaffin cells, a previous study showed that 48-h exposure to dexamethasone (1 µM) could double the amplitude of the VGCCs (14). This raised the possibility that the regulation of Q by dexamethasone observed in our study might be partially related to an increase in extracellular Ca2+ entry. To address this possibility, we examined the effect of dexamethasone on VGCC density under our experimental conditions. In this series of experiments, a single rat chromaffin cell was recorded with the whole cell patch-clamp technique. The cell was held at –80 mV, and voltage was stepped in 10-mV increments to +70 mV for 50 ms. The density of VGCC was obtained by normalizing the peak amplitude of the Ca2+ current (ICa) elicited at different potentials to the whole cell capacitance of individual cells. Figure 4, A and B, shows the current-voltage relationship of the VGCC from cells cultured for 1 or 3 days in serum-supplemented medium. It should be noted that the mean VGCC density of the control cells increased by approximately twofold after 3 days of culture in the serum-supplemented medium. In contrast, for cells cultured in defined medium for 1 day, the VGCC density was approximately twofold that of those cultured in serum-supplemented medium for the same duration (Fig. 4C). However, by the third day of culture in defined medium, VGCC density was reduced by ~50% (Fig. 4D). Thus, depending on the culture medium (serum supplemented or defined), the VGCC density in chromaffin cells could be up- or downregulated by approximately twofold. Because the rundown in Q was similar for cells cultured in either medium, it was unlikely that the rundown in Q during culture was correlated to the variations in VGCC density. To investigate further whether the prevention of Q rundown by dexamethasone was related to the enhancing action of dexamethasone on VGCC density, we examined the effect of dexamethasone on VGCC density under our culture conditions. Consistent with a previous finding (14), we found that dexamethasone (1 µM) treatment resulted in an enhancement of VGCC density in cells cultured for 1 or 3 days in serum-supplemented medium (Fig. 4, A and B). However, for cells cultured in defined medium, dexamethasone neither affected the density of VGCC nor prevented the rundown in VGCC density (Fig. 4, C and D). Because dexamethasone prevented the rundown in mean Q for cells cultured with either medium (Fig. 1), this result suggested that this action of dexamethasone was independent of its effect on VGCC density.

Dexamethasone increased number of granules in immediately readily releasable pool. Our finding that dexamethasone did not affect the overall VGCC density in chromaffin cells cultured in defined medium (Fig. 4C) offered a unique opportunity to investigate whether glucocorticoid could affect Ca2+-dependent exocytosis via mechanisms that were independent of changes in overall VGCC density. In this series of experiments, we compared depolarization-triggered exocytosis in chromaffin cells that were cultured in the defined medium with or without dexamethasone (1 µM) for 1 or 3 days. Under this condition, neither Q (Fig. 1A) nor VGCC density (Fig. 4, C and D) was affected by dexamethasone. Single chromaffin cells were voltage clamped at –80 mV (DC), and a train of depolarization pulses (50 ms each to +10 mV, 15 pulses at 3 Hz) was applied. [Ca2+]i was monitored simultaneously with indo-1 fluorometry. An example of such an experiment on a control cell is shown in Fig. 5A. In this cell, the first depolarization (50 ms) elevated [Ca2+]i to ~1 µM and caused a small increase (~ 10 fF) in cell membrane capacitance ({Delta}C1). The subsequent depolarizations caused further elevations in [Ca2+]i (to >2 µM) and further increases in capacitance. After the termination of the train of depolarizations, [Ca2+]i gradually decayed to the basal level, and the capacitance increase reached a maximum ({Delta}Ctotal). The results of these experiments are summarized in Fig. 5B. It should be noted that in cells treated with dexamethasone for 1 day in defined medium, the mean amplitude of the exocytotic response triggered by the first depolarization (normalized to the initial whole cell capacitance, i.e., {Delta}C1/Cm) was 1.30% (SD 0.45; n = 9 cells), significantly larger than that of the time-matched controls ({Delta}C1/Cm = 0.72%, SD 0.4; n = 8 cells). In contrast, dexamethasone treatment did not affect the total exocytotic response triggered by the train of depolarizations ({Delta}Ctotal/Cm, value ~10% for both conditions). Consistent with our finding that dexamethasone did not affect VGCC density in cells cultured in defined medium (Fig. 4C), neither the Ca2+ influx triggered by the train of depolarizations [measured as VGCC density (ICa/Cm) of the first depolarization pulse] nor the peak [Ca2+]i measured at the end of the train of depolarizations was affected by dexamethasone (Fig. 5B). Note that basal [Ca2+]i was also not affected by dexamethasone. The dexamethasone-mediated enhancement of the exocytotic response triggered by the first depolarization could also be observed in cells cultured for 3 days in defined medium (Fig. 5B). For cells treated with dexamethasone (1 µM) for 3 days in defined medium, the mean amplitude of the exocytotic response triggered by the first depolarization was 0.78% (SD 0.29; n = 6 cells), significantly larger than that obtained from the time matched controls [0.29% (SD 0.21); n = 7 cells]. Overall, these results suggested that dexamethasone selectively increased the exocytotic response triggered by a single brief depolarization, and this enhancement was not related to any change in the overall magnitude of extracellular Ca2+ entry.

Previous studies in rat chromaffin cells (23, 49) showed that a brief depolarization could exhaust only a subset of readily releasable granules that were in close proximity to VGCCs (the immediately releasable pool). In view of this, the enhancement of the first depolarization-triggered exocytotic response by dexamethasone observed here may be related to one or more of the following mechanisms. The first mechanism is a general increase in the Ca2+ sensitivity for the triggering of exocytosis among the readily releasable granules. The second mechanism is an increase in the pool size of readily releasable granules. The third mechanism is an increase in the coupling of a subpopulation of readily releasable granules (e.g., immediately releasable granules) with at least a subset of VGCCs. To examine the first two possible mechanisms, we bypassed the activation of VGCC and triggered Ca2+-dependent exocytosis directly via flash photolysis of caged Ca2+. Previous studies in bovine chromaffin cells (31, 54) as well as rat pituitary cells (45, 46) showed that the size of the readily releasable pool of granules could be estimated from the amplitude of the fastest kinetic component of the exocytotic burst when [Ca2+]i was rapidly and uniformly elevated in the cell via flash photolysis of caged Ca2+. The Ca2+ dependence of this component could be obtained by documenting the initial rate (or rate constant) of exocytosis when [Ca2+]i was rapidly raised to different levels (19, 31). Using this experimental approach, we asked whether dexamethasone increased the number of granules and/or the Ca2+ sensitivity of the readily releasable pool in cells cultured in defined medium for 1 day. Figure 6A shows the exocytotic response in a rat chromaffin cell when [Ca2+]i was rapidly elevated via flash photolysis of a photolabile Ca2+ chelator (DM-nitrophen). In this example, the cell was cultured with 1 µM dexamethasone for 1 day in the defined medium. Shortly after the UV flash, [Ca2+]i rose rapidly to ~30 µM and triggered a rapid increase in Cm. This fast exocytotic burst was followed by rapid endocytosis and then a slow component of exocytosis (not shown in the time scale of Fig. 6A). A similar pattern of exocytosis was observed in control cells. We estimated the size of the readily releasable pool from the amplitude of the fast exocytotic burst, and the comparison between dexamethasone-treated cells and controls is shown in Fig. 6B. In this plot, the amplitude of the fast exocytotic burst was normalized to the cell initial Cm. It should be noted that with [Ca2+]i >20 µM, the mean amplitude of the fast exocytotic burst for the dexamethasone-treated cells was 2.4% (SD 0.7; n = 12), similar to that of the control cells [2.5% (SD 0.5); n = 6]. This finding suggests that dexamethasone did not affect the number of granules in the readily releasable pool. In Fig. 6C, we compared the initial rate of the exocytotic burst triggered at different elevations of [Ca2+]i and found that dexamethasone had no significant effect on this aspect. It should be noted that the [Ca2+]i in the flash photolysis experiment (Fig. 6) was at a higher range compared with the depolarization-triggered [Ca2+]i rise shown in Fig. 5. Similar to a previous study in bovine chromaffin cells (19), we found that when [Ca2+]i was uniformly elevated via flash photolysis of caged Ca2+ exocytosis could be reliably triggered at [Ca2+]i ≥4 µM. In contrast, exocytosis could be observed when depolarization raised average cytosolic [Ca2+] to ~1 µM (Fig. 5A). This discrepancy is likely due to the transient spatial Ca2+ gradient generated during activation of VGCCs. Because of the close proximity of VGCCs to at least some secretory granules (36), the local [Ca2+] near the secretory granules during our 50-ms voltage step is at least one order of magnitude higher (4) than the average cytosolic [Ca2+] (e.g., monitored by indo-1 fluorometry in Fig. 5). Thus the exocytosis triggered by the uniform elevation of [Ca2+] to the range of 5–20 µM (in Fig. 6) should be typical of the local [Ca2+] near the secretory granules during VGCC activation.



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Fig. 6. Dexamethasone did not increase the size or the Ca2+ sensitivity of the readily releasable pool of granules. A: [Ca2+]i elevation and exocytosis in a chromaffin cell cultured with dexamethasone for 1 day in defined medium when triggered via flash photolysis of caged Ca2+. After the UV flash, [Ca2+]i rose to ~30 µM, and it was accompanied by a fast exocytotic burst (rapid increase in Cm), reflecting the exhaustion of the readily releasable pool of granules. [Ca2+]i was measured with indo-1FF. B: amplitude of the fast exocytotic burst (normalized to initial cell Cm) from control cells and cells treated with dexamethasone. Each value shown was averaged from 2–6 cells. C: rate of exocytosis (normalized to the size of an average chromaffin cell of 4.78 pF) vs. [Ca2+]i. The fast exocytotic burst could be described by a second-order polynomial. The rate of exocytosis was estimated from the derivative of the polynomial at 20 ms after the delivery of the UV flash. Each value shown was averaged from 2–6 cells.

 

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Our results show that when chromaffin cells were maintained for short-term culture (between 1 and 3 days) in defined medium, the average amount of catecholamine released from individual chromaffin granules decreased dramatically (Fig. 2A). In our previous study (40), we showed that the rundown in Q when chromaffin cells were cultured in defined medium was associated with a shift in the proportion of release from at least three populations of granules, as well as a small reduction in the modal Q value of individual granule populations. In the current study, we have shown that serum supplementation could not prevent the decrease in Q and the shift in proportional release during rundown. In contrast, dexamethasone was able to prevent the rundown in mean cellular Q (Fig. 2A) as well as preventing the shift in proportional release (Fig. 3C) in cells cultured with defined medium. For cells cultured with serum supplementation for 3 days, dexamethasone could also restore the rundown in mean cellular Q (Fig. 2B) but only partially prevented the shift in proportional release (Fig. 3F). The inability of dexamethasone to fully prevent the shift in proportional release from cells cultured with serum supplementation was not clear. It should be noted, however, that the shift in proportional release for cells cultured for 3 days in serum-supplemented medium was more dramatic and the pattern of the Q distribution was also different from that observed in the defined medium (Fig. 3, A and D). Because serum contains multiple steroids as well as growth factors, it is possible that some of these factors may affect the proportional release and this shift could not be completely prevented by dexamethasone. One major difference between chromaffin cells in culture and in vivo was the drastic reduction of glucocorticoid that was secreted by the cortical cells. Because of the local blood circulation within the adrenal gland, chromaffin cells in the medulla are exposed to high concentrations of glucocorticoid released from the adrenal cortex (21, 35). Here we found that relatively high concentrations of dexamethasone (0.1–1 µM) were required to prevent the rundown in mean Q (Fig. 2C). This range of dexamethasone concentration is physiologically relevant, because the plasma level of the main glucocorticoid (cortisone) in rats was reported to reach ~1 µmol/l (22, 38). Thus our results suggest that the continued presence of a high level of glucocorticoid is essential for the maintenance of the amount of catecholamine stored in the granules of rat chromaffin cells.

Q of chromaffin cells can be influenced by multiple factors, including inhibition of NOS, extracellular Ca2+ entry, as well as catecholamine biosynthesis/storage (21). Although dexamethasone has been reported to be an inhibitor of NOS (34), we found that the effect of dexamethasone on Q in rat chromaffin cells could not be mimicked by the NOS inhibitor L-NMMA, suggesting that the involvement of NOS inhibition is unlikely. In PC-12 cells, dexamethasone treatment for 5–7 days was reported to double mean Q; however, this effect was accompanied by an approximately threefold increase in VGCC density (11, 55). Interestingly, for rat chromaffin cells cultured with serum-supplemented medium, dexamethasone treatment for 1 day caused an almost twofold increase in VGCC density (Fig. 4A). This increase in VGCC density was accompanied by ~40% increase in Q of individual amperometric events (Fig. 3E), although the change in mean cellular Q was statistically insignificant (Fig. 2B). This raised the possibility that the increase in VGCC-mediated extracellular Ca2+ entry in chromaffin cells cultured for 1 day in serum-supplemented medium contributed to the increase in Q via the mechanism suggested by Elhamdani et al. (12). On the other hand, for chromaffin cells cultured in defined medium, dexamethasone did not affect the VGCC density (Fig. 4, C and D). This may explain why dexamethasone did not cause any measurable change in Q in cells cultured in defined medium for 1 day (Figs. 2A and 3B). It should be noted, however, that even without any change in VGCC density dexamethasone was effective in preventing the rundown in Q as well as the shift in proportional release in cells cultured with defined medium (Figs. 2A and 3C). Therefore, we conclude that dexamethasone maintained Q via a mechanism that was independent of the regulation of VGCC density (thus extracellular voltage-gated Ca2+ entry). One possible explanation for the time-dependent rundown in Q of chromaffin cells in culture is a general reduction in catecholamine biosynthesis. For example, a recent study using intracellular patch electrochemistry reported that the concentration of catecholamines in the cytosol (i.e., the most metabolically active pool) of individual cultured chromaffin cells decayed with a time constant of ~4 days (29). In a previous study (40), we also found that, compared with cells cultured for 1 day, the total cellular catecholamine content in cells cultured for 3 days was reduced by ~25%. In view of the stimulatory action of dexamethasone on tyrosine hydroxylase, the rate-limiting enzyme for catecholamine biosynthesis, it is possible that dexamethasone maintains Q in chromaffin cells via its action on tyrosine hydroxylase. The importance of tyrosine hydroxylase activity in the maintenance of Q was shown in PC-12 cells, in which inhibition of tyrosine hydroxylase reduced Q by ~50% (33). In addition, dexamethasone may increase the storage of catecholamine in chromaffin cells via the enhanced expression of chromogranin (37), which is a main component of the dense core matrix where catecholamines are packaged. Thus an increase in biosynthesis and storage of catecholamine may underlie the prevention of Q rundown in rat chromaffin cells by dexamethasone.

Our study also showed that in rat chromaffin cells cultured in defined medium for 1 or 3 days dexamethasone enhanced the exocytotic response triggered by a brief (50 ms) depolarization ({Delta}C1/Cm; Fig. 5B). The average Cm of rat chromaffin cells was ~3 pF, and in control day 1 cells the 50-ms depolarization triggered a 0.72% increase in Cm (equivalent to 22 fF). Assuming that the exocytosis of a single granule contributes to ~1 fF (2), ~22 granules should be released during the brief depolarization. Because the tip diameter (7 µm) of the carbon fiber electrode is about half the diameter of rat chromaffin cells and the cell is roughly a hemisphere, the carbon fiber electrode can detect release from approximately one-twelfth of the entire cell surface. Assuming that the granules are uniformly released on the cell surface and all the granules contain catecholamine, the carbon fiber should be able to detect only 1 or 2 amperometric events when 22 granules were released. Consistent with this, in our simultaneous measurements of {Delta}Cm and amperometric events (unpublished observations), we found that in six of eight control cells the increase in Cm triggered by the first 50-ms voltage step during a train of depolarization was accompanied by at least one amperometric event. These observations suggest that at least a fraction of the small exocytotic response triggered by a brief depolarization (50 ms) was indeed associated with release of catecholamine-containing granules.

The enhancement of depolarization-triggered exocytotic response in bovine chromaffin cells and PC-12 cells has been reported to involve different stages of excitation-secretion coupling. For example, elevation of basal [Ca2+]i has been shown to increase the rate of mobilization of granules (50); activation of protein kinase C and/or Munc13 has been shown to increase the number of granules in the readily releasable pool (3, 16, 48); and increase in voltage-gated Ca2+ entry has been shown to enhance Ca2+-dependent exocytosis (11). Under our experimental condition of culturing rat chromaffin cells in a defined medium, dexamethasone did not affect the basal [Ca2+]i, the overall VGCC density (ICa/Cm), or the peak [Ca2+]i elicited by the train of depolarization compared with time-matched controls (Fig. 5B). Thus the enhancement of brief depolarization-triggered exocytotic response by dexamethasone could not be due to an increase in basal [Ca2+]i, overall voltage-gated Ca2+ entry, or mechanisms secondary to changes in [Ca2+]i. Similar to other endocrine cells, chromaffin cells possess different pools of releasable granules at various stages of the exocytotic pathway as well as a much larger depot pool of granules that can be mobilized into the releasable pools (31, 49). Because the total exocytotic response triggered by a train of depolarization involved granules that were in the releasable pool(s) as well as some mobilized from the depot pool (13, 23, 49) and dexamethasone did not affect the magnitude of total exocytosis ({Delta}Ctotal/Cm; Fig. 5B), it was unlikely that dexamethasone altered the mobilization of granules. In addition, dexamethasone had no significant effect on Q under this culture condition (Fig. 2A). Thus the increase in exocytotic response by dexamethasone could not be caused by an increase in the physical dimension [e.g., volume and surface area (17)] and therefore the Cm of individual granules that release catecholamines. Interestingly, we found that dexamethasone also did not affect the amplitude or the Ca2+ sensitivity of the burst of exocytotic response triggered via flash photolysis of caged Ca2+ (Fig. 6B). The fast exocytotic burst triggered via flash photolysis of caged Ca2+ reflected the exhaustion of the readily releasable pool of granules (19, 31). In both control and dexamethasone-treated cells (1 day in defined medium), the exhaustion of this pool contributed an average of ~2.5% increase in cell surface area (Fig. 6B). In contrast, a brief depolarization increased the cell surface area by 0.7% in control cells and by 1.3% in dexamethasone-treated cells (1-day culture). For control cells cultured for 3 days in defined medium, the amplitude of the exocytotic response triggered by the brief depolarization was reduced to 0.3% (probably in part related to the decrease in overall VGCC density). Nevertheless, dexamethasone treatment still increased the exocytotic response triggered by the short depolarization (0.8%; Fig. 5B). This result suggested that dexamethasone selectively increased the coupling of a subpopulation of readily releasable granules (the immediately releasable pool) with VGCCs, probably via colocalization at the level of molecular interactions.

A recent study on bovine chromaffin cells (5) reported that different types of {alpha}-subunits for VGCCs were either up- or downregulated in short-term (up to 4 days) cell culture (in a medium supplemented with fetal bovine serum). Most interestingly, the same study also found that dexamethasone (1 µM) selectively prevented the downregulation of the {alpha}-subunit (Cav2.3) for R-type VGCC, although the effect of dexamethasone in maintaining the current mediated by the R-type VGCC was found to be statistically insignificant. Previous reports have shown that, at least in mouse chromaffin cells, the R-type VGCC was linked to a rapid component of VGCC-triggered exocytosis (1, 28). Therefore, a possible explanation for our observation on the enhancement of the rapid burst of exocytosis from the small subpopulation of immediately releasable granules is that dexamethasone also upregulates a small number of colocalized R-type VGCCs in rat chromaffin cells. The relative contribution of R-type VGCCs to the overall VGCC density and exocytosis in rat chromaffin cells is not clear. The R-type VGCC was reported to contribute <10% of the overall VGCC density in cultured bovine chromaffin cells (5) and ~20% in cultured mouse chromaffin cells (1).

In summary, we found that glucocorticoid could affect catecholamine secretion from rat chromaffin cells via multiple mechanisms. Even under experimental conditions in which glucocorticoid did not cause any measurable enhancement of voltage-gated Ca2+ entry or VGCC density, the coupling of VGCCs to the immediately readily releasable granules as well as the amount of catecholamine released from individual granules were still increased. Thus the paracrine actions of glucocorticoid may be essential for the maintenance of efficient stimulus-secretion coupling in adrenal chromaffin cells.


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This work was supported by the Canadian Institute of Health Research, the Alberta Heritage Foundation for Medical Research (Senior Scholars to A. Tse and F. W. Tse, Summer Studentship to V. B. Lu), and the Government of Canada (Summer Career Placement funding to C. P. Weerasinghe).


    ACKNOWLEDGMENTS
 
We thank Dr. Alexander S. Clanachan for assistance in statistics and Drs. Andy K. Lee and William F. Dryden for critical comments.

Present address of J. Xu: National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.


    FOOTNOTES
 

Address for reprint requests and other correspondence: F. W. Tse, Dept. of Pharmacology, 9-70 Medical Sciences Bldg., Univ. of Alberta, Edmonton, AB, T6G 2H7, Canada (e-mail: fred.tse{at}ualberta.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* J. Xu and K. S. Tang contributed equally to this work. Back


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