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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catecholamines; paracrine action; exocytosis; calcium channels
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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METHODS |
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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 6.57.2 DM-nitrophen (8090% 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 (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).
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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RESULTS |
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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 (10,00050,000) 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 (1,5004,500) 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 (60277 cells) could be well described by a single Gaussian. In the present study, although the number of cells (1822) 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 (0.40.7 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, DF). 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 (
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 (
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.,
C1/Cm) was 1.30% (SD 0.45; n = 9 cells), significantly larger than that of the time-matched controls (
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 (
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 520 µM (in Fig. 6) should be typical of the local [Ca2+] near the secretory granules during VGCC activation.
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DISCUSSION |
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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 57 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 (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
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 (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 -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
-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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GRANTS |
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ACKNOWLEDGMENTS |
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Present address of J. Xu: National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.
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FOOTNOTES |
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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.
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