Skip to main content
NCBI home page
Endocrinology logoLink to Endocrinology
. 2008 Jun 26;149(10):4921–4927. doi: 10.1210/en.2007-1798

Nongenomic Glucocorticoid Effects on Activity-Dependent Potentiation of Catecholamine Release in Chromaffin Cells

Yong-Soo Park 1, Yoon Ha Choi 1, Choon-Ho Park 1, Kyong-Tai Kim 1
PMCID: PMC2734489  PMID: 18583423

Abstract

Adrenal medulla chromaffin cells are neuroendocrine and modified sympathetic ganglion cells. Catecholamines released from chromaffin cells mediate the fight-or-flight response or alert reaction against dangerous conditions. Here we report that short-term treatment with glucocorticoids, released from adrenal cortex cells in response to chronic stress, inhibits activity-dependent potentiation (ADP) of catecholamine release. First, short-term treatment with dexamethasone (DEX), a synthetic glucocorticoid, reduces ADP in a concentration-dependent manner (IC50 324.2 ± 54.5 nm). The inhibitory effect of DEX is not reversed by RU-486 treatment, suggesting that the rapid inhibitory effect of DEX on ADP of catecholamine release is independent of glucocorticoid receptors. Second, DEX treatment reduces the frequency of fusion between vesicles and plasma membrane without affecting calcium influx. DEX disrupts activity-induced vesicle translocation and F-actin disassembly, thereby leading to inhibition of the vesicle fusion frequency. Third, we provide evidence that DEX reduces F-actin disassembly via inhibiting phosphorylation and translocation of myristoylated alanine-rich C kinase substrate and its upstream kinase protein kinase Cε. Altogether, we suggest that glucocorticoids inhibit ADP of catecholamine release by decreasing myristoylated alanine-rich C kinase substrate phosphorylation, which inhibits F-actin disassembly and vesicle translocation.

THE ADRENAL MEDULLA functions as a neuroendocrine system to release catecholamine on cholinergic stimulation from the splanchnic sympathetic neurons. Chromaffin cells of the adrenal medulla are the main source for generating high levels of catecholamine, including adrenalin, noradrenalin, and dopamine, in the bloodstream, mediating the fight-or-flight responses as a defense mechanism against the dangerous environment (1).

The adrenal medulla is surrounded by the adrenal cortex, which is involved in the hypothalamic-pituitary-adrenal axis. Glucocorticoids including cortisol can be released from the adrenal cortex in response to chronic stress, affecting digestion, mood, and the immune system (2). The adrenal medulla and adrenal cortex are interwoven with capillary vessels and thereby interact with each other through their hormones (3).

The genomic effect of glucocorticoids has been well identified for gene expression in chromaffin cells. Glucocorticoids modulate the expression of specific genes via activation of glucocorticoid receptors (GRs), which act as transcription factors (2). In addition, the genomic effects of glucocorticoids have been implicated in the control of mRNA or protein stability as well as chromaffin cell differentiation (2). Typically the genomic effect of glucocorticoids results in increasing catecholamine release through promoting gene expression (2). However, the nongenomic effects of glucocorticoids on catecholamine release in chromaffin cells have been poorly understood. In humans, short-term treatment with glucocorticoids inhibits the level of catecholamine released from chromaffin cells in the blood stream (4,5). Despite the studies at the organism level, it has been mysterious why glucocorticoids cannot inhibit catecholamine release at the cellular level. Here we show that glucocorticoids inhibit catecholamine release induced by repetitive stimulation but not by a single stimulation.

We previously reported that repetitive stimulation of nicotinic acetylcholine receptor (nAChR) induces activity- dependent potentiation (ADP) of catecholamine release in chromaffin cells (6,7). Here we investigate the signaling mechanism by which glucocorticoids can inhibit ADP of catecholamine release. Glucocorticoids inhibit ADP without affecting calcium influx. Our data suggest that the rapid inhibitory effect of glucocorticoids on ADP is not dependent on the intracellular GR or membrane GR but mediated by blocking vesicle translocation and F-actin disassembly. We provide evidence that glucocorticoids lead to inhibition of F-actin disassembly by suppressing phosphorylation and translocation of protein kinase C (PKC)-ε and its downstream target myristoylated alanine-rich C kinase substrate (MARCKS).

Materials and Methods

Materials

Fura-2 pentaacetoxymethyl ester (fura-2/AM) was from Molecular Probes (Eugene, OR). 1,1-Dimethyl-4-phenylpiperazinium iodide (DMPP), dexamethasone (DEX), hydrocortisone, and RU-486 were purchased from Sigma (St. Louis, MO). DEX-BSA conjugate is from Steraloids Inc. (Newport, RI). Anti-PKC-ε and antisynaptotagmin monoclonal antibody were from Transduction Laboratories (Lexington, KY). Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-MARCKS polyclonal antibody was from Cell Signaling (Beverly, MA). The enhanced chemiluminescence (Supex) kit was from Neuronex (Pohang, Korea).

Preparation of bovine chromaffin cells

Chromaffin cells were isolated from the bovine adrenal gland medulla by two-step collagenase digestion as previously described (6). For amperometric measurement and calcium imaging, cells were grown on poly-d-lysine-coated glass coverslips at the density of 1 × 105 cells per 35-mm dish. The cells were maintained in DMEM/F-12 (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (Hyclone Laboratories, Logan, UT) and 1% antibiotics (Invitrogen). We finished experiments within 48 h after chromaffin cell preparation.

Amperometric measurement

Recordings of chromaffin cells, grown on poly-d-lysine-coated glass coverslips, were performed at room temperature as described previously (7). Chromaffin cells were buffered with amine-free solution containing 137.5 mm NaCl, 2.5 mm KCl, 2 mm CaCl2, 1 mm MgCl2, 10 mm d-glucose, and 10 mm HEPES (pH 7.3) titrated by NaOH. Carbon-fiber electrodes were fabricated from 8-μm-diameter carbon fibers (Alfa Aesar, Ward Hill, MA). A carbon-fiber electrode backfilled with 3 m KCl connected to the head stage was attached to a single cell. The amperometric current, generated by oxidation of catecholamines, was measured by using an axopatch 200B amplifier (Axon Instruments Inc., Foster City, CA) and operated in the voltage-clamp mode at a holding potential of + 650 mV. Amperometric signals were low-pass filtered at 100 Hz and sampled at 500 Hz. For data acquisition and analysis, pCLAMP 10 software (Axon Instruments) was used. Peak number and peak height of amperometric current were calculated by using IGOR software (Wave Metrics, Lake Oswego, OR). Solutions were exchanged by a local perfusion system that allows complete exchange of medium bathing the cells within 2 sec.

Intracellular free calcium concentration imaging

We performed calcium imaging of single cells with fura-2/AM, as described in detail elsewhere (6). We performed single-cell calcium imaging experiments with a monochromator-based spectrofluorometric system (DeltaScan Illumination System; DeltaScan Photon Technology International, Inc., Birmingham, NJ).

Total internal reflection fluorescence (TIRF)

TIRF microscopy was used to obtain evanescent field fluorescent images. Briefly, experiments were performed on an Olympus inverted microscope (model IX81; Olympus, Tokyo, Japan) with the Olympus Apo ×100 1.65 numerical aperture oil-immersion objective. Fluorescence was excited at 488 nm obtained from 10 mW multiline argon-ion laser with the TIRF slider and emitted through U-MNIB3 filter (Olympus). Images were taken with an intensified charge-coupled device camera (C9100; Hamamatsu, Hamamatsu City, Japan) and analyzed using MetaMorph7 (Molecular Devices, Sunnyvale, CA). Cells were incubated in 200 nm LysoTracker Green for 10 min to stain vesicles, as described in detail elsewhere (8). After stimulation with 3 μm DMPP, the average intensities of whole-cell evanescent field fluorescence were measured for 2 min at 3-sec intervals. The change of evanescent field fluorescence (EFF) was analyzed by subtracting the basal level of EFF intensity.

Recombinant adenoviruses

Adenovirus containing actin-green fluorescent protein (GFP) was provided by Dr. Ashworth (University of Maine, Orono, ME). Chromaffin cells were transfected with adenovirus (1 multiplicity of infection) for 24 h, as described previously (7).

Cell fractionation and immunoblotting

We performed cell fractionation and immunoblotting as previously described in detail (7). Cells stimulated as indicated were washed with chilled PBS. To separate the cell lysates into the cytosolic and membrane fraction, cells were suspended in buffer A [10 mm Tris-HCl (pH 7.4), 1 mm EDTA, 0.5 mm EGTA, 10 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 1 mm Na3VO4, 1 mm dithiothreitol, 2 mm ascorbic acid, 10 μg/ml pepstatin A, 10 μg/ml leupeptin, and 10 μg/ml aprotinin]. After removing cell debris and organelle by centrifuge at 3000 × g for 15 min, the supernatant was further centrifuged at 100,000 × g for 1 h, and the supernatant was saved as the cytosolic fraction. The pellet was then resuspended in buffer A and saved as the membrane fraction. Proteins were then separated by electrophoresis containing 0.1% sodium dodecyl sulfate and transferred to a nitrocellulose membrane, which was blocked with 5% nonfat dry milk in a solution of 20 mm Tris-HCl (pH 7.4), 140 mm NaCl, and 0.05% Tween 20.

Real-time imaging for F-actin disassembly

Cells were transfected with adenovirus expressing actin-GFP for monitoring F-actin dynamics. Green fluorescent imaging was investigated using an Olympus Fluoview FV1000 confocal laser system with an UPLSAPO ×60 Oil (numerical aperture: 1.35) objective. Real-time images (512 × 512 pixels) were acquired at 5-sec intervals for 3 min with a 2 μsec/pixel sampling speed.

Statistical analysis

All quantitative data are presented as means ± sem. Comparison between two groups were analyzed by two-tailed unpaired Student’s t test or Mann-Whitney test as indicated for individual experiments using Clampfit 10.2 (Axon Instruments). To compare results between more than three groups, we used a one-way ANOVA with post hoc Tukey’s honestly significant difference, and the Student-Newman-Keuls test. Statistical significance was set at P < 0.05.

Results

Glucocorticoids inhibit ADP of catecholamine release

Amperometry was used for real-time measurement of catecholamine release. We investigated the effect of glucocorticoids on the vesicle fusion frequency as well as the total amount of released catecholamine using amperometric measurement. As we previously reported (6,7), repetitive activation of nAChR gives rise to ADP of catecholamine release. ADP is the increase in neurotransmitter release induced by successive and repetitive stimulation. When chromaffin cells were stimulated three times with DMPP, a selective agonist of nAChR exerting cell depolarization, which provokes calcium influx, ADP of catecholamine release occurred (Fig. 1). Because glucocorticoids are released from the adrenal cortex and regulate adrenal medulla chromaffin cells, we examined whether glucocorticoids affect ADP with nongenomic manner in a short period of time. DEX, a synthetic glucocorticoid of steroid hormones, was used to study the nongenomic effect of glucocorticoids on ADP. Short-term DEX treatment (500 nm, 5 min) blocked ADP of catecholamine release without affecting the first stimulation-induced basal secretion (Fig. 1, A and B), suggesting that glucocorticoids inhibit the potentiation process. Cortisol (3 μm, 5 min) also inhibited ADP, showing the inhibitory role of glucocorticoids on ADP of catecholamine in the adrenal medulla chromaffin cells (Fig. 1, C and D). DEX can repress ADP in a concentration-dependent manner (IC50 324.2 ± 54.5 nm; Hill coefficient 1.5 ± 0.4) (Fig. 1E).

Figure 1.

Figure 1

Open in a new tab

Inhibitory effect of glucocorticoids on ADP of catecholamine release. A, Pretreatment with DEX attenuates ADP without inhibiting the first stimulation-induced exocytosis. Chromaffin cells were repetitively stimulated with 3 μm DMPP under pretreatment with vehicle (top; n = 8) or 500 nm DEX (bottom; n = 8) for 5 min. Shown is a representative trace recorded by amperometry. The duration of each stimulus was 20 sec and the interval time between stimuli was 2 min. B, Amperometric current generated by repetitive stimulation was individually integrated. Relative exocytosis is presented as a percentage value of the first stimulation-induced total catecholamine release obtained from vehicle-treated cells. C and D, Hydrocortisone also inhibits ADP of exocytosis without affecting the first stimulation-induced exocytosis. Chromaffin cells were repetitively stimulated with DMPP under pretreatment with vehicle (top; n = 5) or 3 μm hydrocortisone (bottom; n = 6) for 5 min. E, DEX inhibits ADP in a concentration-dependent manner, with a half-maximal inhibitory concentration (IC50) of 324.2 ± 54.5 nm and Hill coefficient of 1.5 ± 0.4 (n = 5–15). F, DEX blocks the increase in the spike frequency induced by the repetitive stimulation without affecting the first stimulation-induced spike frequency. The spike frequency represents the number of amperometric spikes per 1 sec. G, Inhibitory effect of DEX on ADP of catecholamine release is not reversed by the intracellular glucocorticoid receptor antagonist, RU-486 (RU). Cells were stimulated as described in A, after preincubation in the absence or presence of 10 μm RU for 1 h (n = 5–8; P < 0.01, one-way ANOVA test). Data presented in B, D, E, and F are means ± sem. *, P < 0.05; **, P < 0.01; ***, P < 0.005 (Mann-Whitney test).

We previously showed that ADP of catecholamine release can be attributed to the increase of vesicle translocation and vesicle fusion (7). The number of amperometric spikes (representing fused vesicles) increases upon repetitive stimulation with DMPP (7). The third DMPP stimulation increases the spike frequency, compared with the first stimulation (Fig. 1F). Short-term treatment with DEX (500 nm, 5 min) inhibited the increase in amperometric spike number (Fig. 1F) as well as the total amount of catecholamine (Fig. 1B) without affecting the first stimulation-induced exocytosis. These data suggest that glucocorticoids repress the repetitive stimulation-induced potentiation by reducing the number of fused vesicles but cannot inhibit the single stimulation-induced exocytosis.

Rapid glucocorticoid effects can be mediated by specific interaction with the cytosolic GR (9). To investigate whether rapid DEX-induced inhibition of ADP is GR dependent, RU-486 (glucocorticoid antagonist) was applied for 1 h before DEX treatment. Ten- or 50-μm RU-486 treatments failed to reverse the inhibitory effect of glucocorticoids (Fig. 1G and supplemental Fig. 1C, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org), indicating that the inhibitory effect of glucocorticoids on ADP is GR independent. Furthermore, we examined the involvement of membrane bound GR in the inhibitory effect of glucocorticoid by using membrane-impermeable DEX-BSA (BSA conjugate form of dexamethasone) (see supplemental Fig. 1B). Impermeable DEX-BSA (500 nm, 5 min) could not inhibit ADP, indicating that the inhibitory effect of glucocorticoid is not mediated by membrane bound GR or interaction with membrane. Altogether we suggest that short-term DEX treatment (5 min) suppresses ADP of catecholamine release by reducing the vesicle fusion frequency, which is independent on interactions with membrane or intracellular GR.

Lack of glucocorticoids effect on calcium influx

Because an intracellular calcium influx is known to regulate vesicle exocytosis, we tested whether short-term DEX treatment inhibits calcium influx. DEX treatment (500 nm, 5 min) did not significantly affect the single DMPP-induced calcium influx (Fig. 2A). To further examine whether DEX might inhibit calcium influx induced by repetitive stimulation, we performed calcium imaging experiments. As previously reported, three times repetitive stimulation with DMPP leads to a calcium influx with a constant peak level (6). Short-term DEX treatment could not inhibit the calcium influx induced by repetitive stimulation (Fig. 2C), indicating that short-term DEX treatment has no effect on the DMPP-induced calcium influx. Taken together, these results suggest that the short-term inhibitory effect of glucocorticoids on ADP is not associated with the decrease in calcium influx (Fig. 2) but the decrease in the vesicle fusion frequency (Fig. 1F).

Figure 2.

Figure 2

Open in a new tab

Glucocorticoids have no effect on calcium influx. A, DEX treatment cannot inhibit calcium influx induced by the single DMPP stimulation. Cells were exposed to 500 nm DEX for 5 min before 3 μm DMPP application. B, Calcium influx induced by repetitive stimulation with DMPP is not affected by DEX. Chromaffin cells were loaded with fura-2/AM and repetitively stimulated with 3 μm DMPP (indicated as bar) for 20 sec at 2-min intervals in the presence or absence [control; published in previous report (6)] of DEX. C, Calcium influx induced by repetitive stimulation with DMPP (n = 35) is presented as a percentage of the first stimulation-induced calcium influx. Error bars are means ± sem.

DEX treatment attenuates activity-induced vesicle translocation

Increase of the vesicle fusion frequency is ascribed to vesicle translocation (6,7). Because short-term DEX treatment reduced the vesicle fusion frequency, we further examined whether DEX treatment abolishes vesicle translocation. Vesicles were visualized using evanescent field objective-based TIRF microscopy to monitor the movement of large dense-core vesicles (LDCVs) in live cells. The evanescent wave selectively illuminates fluorescent dye within about 400 nm of the plasma membrane-coverslip interface. Evanescent field imaging provides real-time monitoring of vesicle translocation at or near the plasma membrane (10,11). In accordance with previous studies (8,11), evanescent field fluorescence imaging showed dynamic movement of lysotracker-stained vesicles (Fig. 3A). DMPP stimulation caused the elevation of EFF intensity (Fig. 3A), indicating an increase of vesicle translocation toward the plasma membrane. This enlargement of the vesicle pool size near the plasma membrane correlated with the augmentation of the number of amperometric spikes, showing that activity-induced vesicle translocation to the plasma membrane leads to an increase in the number of fused vesicles (Fig. 1F).

Figure 3.

Figure 3

Open in a new tab

Short-term treatment with DEX blocks activity-induced LDCV translocation. A, DMPP-induced nAChR activation leads to LDCV translocation to the plasma membrane in live cells. Shown are evanescent field fluorescent images of lysotracker-stained vesicles. Cells were stimulated with 3 μm DMPP for the indicated times. Ctrl, Control. B, Time course of the EFF signal evoked by DMPP stimulation in the absence (n = 12) or presence (n = 6) of 500 nm DEX. C, Preincubation with DEX for 5 min reduces DMPP-induced vesicle translocation. Mean change in fluorescence is presented. Data are means ± sem values. ***, P < 0.000001 (two-tailed unpaired Student’s t test).

Short-term treatment with DEX blocked the activity-induced increase in EFF (Fig. 3, B and C), as correlated with a reduction of the fused vesicle number (Fig. 1F). Therefore, we suggest that short-term treatment with DEX inhibits ADP of catecholamine release via attenuating activity-induced vesicle translocation.

Glucocorticoids inhibit F-actin disassembly induced by DMPP stimulation

As we previously proved, F-actin dynamics regulates ADP by controlling vesicle translocation and the vesicle fusion frequency (6,7). Inhibition of DMPP-induced F-actin disassembly decreases vesicle translocation and the frequency of fusion between vesicles and the plasma membrane (7). To verify that short-term DEX treatment inhibits DMPP-induced F-actin disassembly, F-actin dynamics was monitored using a confocal laser microscope. Cells were transfected with adenovirus expressing actin-GFP to analyze F-actin disassembly. As a control, DMPP stimulation causes F-actin disassembly within 30 sec after stimulation (Fig. 4A). Moreover, the cortical F-actin is selectively destroyed by DMPP stimulation, whereas the cytosolic GFP fluorescence remains unchanged, excluding the possibility of a fluorescence decay effect (Fig. 4B). Short-term treatment of DEX (500 nm, 5 min) or hydrocortisone (3 μm, 5 min) reduced cortical F-actin disassembly induced by DMPP stimulation (Fig. 4, C and 4D), suggesting that the rapid inhibitory effect of glucocorticoid on ADP of catecholamine release is mediated by inhibition of F-actin disassembly.

Figure 4.

Figure 4

Open in a new tab

Inhibition of DMPP-induced F-actin disassembly by short-term treatment with glucocorticoids. A, Cortical F-actin disassembly induced by DMPP stimulation. Line-scanning confocal imaging (noted as a white dotted line) reveals that the intensity of cortical F-actin decreases within 30 sec after DMPP stimulation. A.U., Arbitrary unit. B, DMPP stimulation selectively induces cortical F-actin disassembly. The time course of actin-GFP imaging shows dramatic F-actin disassembly in the cortical region (dotted line) without changes in the interior region (solid line). OD in region of interest (white rectangle) was normalized to initial fluorescence (100% F0) so that the disassembly of F-actin is represented as a decrease of fluorescence intensity. C, Short-term treatment with DEX inhibits DMPP-induced F-actin disassembly. Cells were stimulated with 3 μm DMPP (indicated as an arrow) after preincubation with DMSO vehicle (ctrl; n = 14) or 500 nm DEX (n = 24; P < 0.000001, two tailed unpaired Student’s t test) for 5 min. Shown are representative actin-GFP images upon DMPP stimulation in the presence or absence of DEX. D, Short-term treatment with hydrocortisone also inhibits DMPP-induced F-actin disassembly. Then 3 μm hydrocortisone were applied for 5 min (P < 0.05, two-tailed unpaired Student’s t test). Scale bar, 3 μm. Data are means ± sem values.

Glucocorticoids inhibit MARCKS phosphorylation and translocation into the cytosolic fraction

Next, the mechanisms were sought by which short-term DEX treatment blocks F-actin disassembly, which regulates vesicle translocation. It has been shown that phosphorylation of MARCKS regulates F-actin dynamics (12) and thereby affects ADP (7). Because DEX treatment blocked F-actin disassembly, which plays a crucial role in ADP, we examined whether short-term DEX treatment affects phosphorylation of MARCKS. DEX treatment (500 nm, 5 min) reduced the phospho-MARCKS level induced by DMPP stimulation (Fig. 5A). In addition, DEX treatment blocked translocation of PKC-ε, which can phosphorylate MARCKS in the membrane fraction (Fig. 5B) (7). We also validated that hydrocortisone (3 μm, 5 min) inhibited PKC-ε translocation (Fig. 5D). To further confirm that DEX treatment inhibits translocation of PKC-ε and thereby suppresses MARCKS phosphorylation, we investigated whether DEX treatment regulates MARCKS translocation into the cytosolic fraction. In line with down-regulation of MARCKS phosphorylation (Fig. 5A), DEX treatment inhibited MARCKS translocation from the membrane to the cytosolic fraction (Fig. 5C). We also confirmed that hydrocortisone inhibited MARCKS translocation (Fig. 5E). Taken together, these results suggest that short-term treatment of glucocorticoids inhibits MARCKS translocation into the cytoplasm by blocking MARCKS phosphorylation, thereby attenuating F-actin disassembly and vesicle translocation.

Figure 5.

Figure 5

Open in a new tab

Glucocorticoids regulate MARCKS phosphorylation and translocation into the cytosol. A, DEX blocks MARCKS phosphorylation induced by DMPP stimulation. Then 3 μm DMPP were applied in the absence or presence of 500 nm DEX for 5 min. Cell lysates were subjected to SDS-PAGE after stimulation and immunoblotted with anti-phospho-MARCKS antibody. Immunoblotting with the GAPDH antibody was performed for normalization. Immunoblotting was quantified by densitometry and normalized as an arbitrary unit (A.U.). B, DEX inhibits translocation of PKC-ε into the membrane fraction (Memb. frac.). Samples from the membrane fraction were immunoblotted with anti-PKC-ε antibody. C, Short-term treatment with DEX reduces translocation of MARCKS into the cytosolic fraction (Cyto. frac.). D, Hydrocortisone (3 μm, 5 min) also inhibits translocation of PKC-ε into the membrane fraction. Samples from the membrane fraction were immunoblotted with anti-PKC-ε antibody. E, Short-term treatment with hydrocortisone reduces translocation of MARCKS into the cytosolic fraction. Immunoblotting with the GAPDH and synaptotagmin antibodies was performed for normalization of the cytosolic and membrane fraction, respectively. Data are means ± sem values. *, P < 0.05 (Student’s t test).

Discussion

This study shows that short-term glucocorticoid treatment inhibits ADP of catecholamine release without affecting the single stimulation-induced exocytosis (Fig. 1, B and D) and suggest that glucocorticoids have the nongenomic effect on inhibition of ADP in a GR-independent manner (Fig. 1G and supplemental Fig. 1). In addition, glucocorticoids inhibit ADP by reducing the number of fused vesicles (Fig. 1F) and vesicle translocation (Fig. 3). Glucocorticoids suppress F-actin disassembly, which mediates vesicle translocation (Fig. 4C). Furthermore, glucocorticoids reduce F-actin disassembly by inhibiting MARCKS phosphorylation and translocation into the cytoplasm (Fig. 5). Altogether glucocorticoids block ADP of catecholamine release via decreasing PKC-ε-induced MARCKS phosphorylation, which leads to inhibition of F-actin disassembly and vesicle translocation.

It has been reported that the nongenomic effects of glucocorticoids mediate inhibition of ion channels such as nAChR, potassium channel, or voltage-gated calcium channel (13,14,15,16). However, in our experiments, short-term treatment with glucocorticoids did not affect the calcium influx evoked by nAChR activation in bovine chromaffin cells (Fig. 2), as correlated with the previous report (15). Accordingly, there have been no reports showing that short-term glucocorticoid treatment inhibits catecholamine release in chromaffin cells. Here we first describe that short-term treatment with glucocorticoids inhibits repetitive stimulation-induced potentiation of catecholamine release without affecting the calcium influx.

It is unlikely that the rapid inhibitory effect of glucocorticoids on ADP of catecholamine release is mediated by the genomic effect because glucocorticoids were treated for only 5 min. Glucocorticoids show the genomic effect by binding to the intracellular GR that, in turn, regulates gene expression. This process of the genomic effect typically takes at least 30–60 min (17).

Nongenomic effects can be mediated by: 1) specific interaction with the cytosolic GR, 2) specific interactions with membrane bound GR, or 3) nonspecific interactions with intracellular machinery (9,17,18). We show that the cytosolic GR is not involved in the inhibitory nongenomic effect on ADP because GR inhibitors (RU-486) cannot reverse the inhibitory effect of glucocorticoids (Fig. 1G and supplemental Fig. 1C). Also, treatment with impermeable DEX-BSA failed to block ADP (supplemental Fig. 1B), indicating that the inhibitory effect of glucocorticoid is not associated with membrane bound GR. In addition, treatment with DEX (less than 2 min preincubation) cannot reduce ADP (data not shown), whereas treatment with DEX (500 nm, 5 min preincubation) inhibits ADP. Altogether we suggest that the inhibitory effect of glucocorticoids might be mediated by nonspecific interactions with intracellular proteins, not by GR signaling. Several reports show that the nongenomic effect of glucocorticoids is GR independent in neurons (19,20,21). However, the molecular mechanisms by which the nongenomic effect of glucocorticoids can inhibit ADP of catecholamine release still remain to be elucidated.

The adrenal medulla and adrenal cortex are interwoven with capillary vessels to interplay each other through their hormones (3). Importantly, glucocorticoid level in the venous effluent or capillary vessels from the adrenal gland is 100-fold higher than in the peripheral circulation of blood in response to stress (22,23), indicating that glucocorticoid concentration in the adrenal medulla is much higher than in the blood. By this reason, we believe that the concentration of glucocorticoids applied in this report could be the physiological level in the adrenal medulla chromaffin cells.

It has been reported that an iv acute injection of glucocorticoids in healthy humans represses the level of catecholamine, thereby inhibiting the sympathetic nervous system (4,5). However, the signaling pathway by which inhibitory effect of glucocorticoids can be mediated has not proved at the cellular level. We suggest that glucocorticoids, released from the adrenal cortex in response to chronic stress, might inhibit the fight-or-flight response by repressing ADP of catecholamine release in the adrenal medulla chromaffin cells.

Supplementary Material

[Supplemental Data]
en.2007-1798_index.html (966B, html)

Acknowledgments

We are grateful to Ms. Kyung-Young Ji for the imaging experiments.

Footnotes

This work was supported by the Systems Biodynamics National Core Research Center and the Brain Korea 21 Program sponsored by the Korean Ministry of Education, Science, and Technology.

Disclosure Statement: Y.-S.P., Y.H.C., C.-H.P., and K.-T.K. have nothing to declare.

First Published Online June 26, 2008

Abbreviations: ADP, Activity-dependent potentiation; DEX, dexamethasone; DMPP, 1,1-dimethyl-4-phenylpiperazinium iodide; EFF, evanescent field fluorescence; fura-2/AM, fura-2 pentaacetoxymethyl ester; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; GR, glucocorticoid receptor; LDCV, large dense-core vesicle; MARCKS, myristoylated alanine-rich C kinase substrate; nAChR, nicotinic acetylcholine receptor; PKC, protein kinase C; TIRF, total internal reflection fluorescence.

References

  1. de Diego AM, Gandia L, Garcia AG 2007 A physiological view of the central and peripheral mechanisms that regulate the release of catecholamines at the adrenal medulla. Acta Physiol (Oxf) 192:287–301 [DOI] [PubMed] [Google Scholar]
  2. Hodel A 2001 Effects of glucocorticoids on adrenal chromaffin cells. J Neuroendocrinol 13:216–220 [DOI] [PubMed] [Google Scholar]
  3. Schinner S, Bornstein SR 2005 Cortical-chromaffin cell interactions in the adrenal gland. Endocr Pathol 16:91–98 [DOI] [PubMed] [Google Scholar]
  4. Dodt C, Keyser B, Molle M, Fehm HL, Elam M 2000 Acute suppression of muscle sympathetic nerve activity by hydrocortisone in humans. Hypertension 35:758–763 [DOI] [PubMed] [Google Scholar]
  5. Del Rio G, Velardo A, Menozzi R, Zizzo G, Tavernari V, Venneri MG, Marrama P, Petraglia F 1998 Acute estradiol and progesterone administration reduced cardiovascular and catecholamine responses to mental stress in menopausal women. Neuroendocrinology 67:269–274 [DOI] [PubMed] [Google Scholar]
  6. Park YS, Jun DJ, Hur EM, Lee SK, Suh BS, Kim KT 2006 Activity-dependent potentiation of large dense-core vesicle release modulated by mitogen-activated protein kinase/extracellularly regulated kinase signaling. Endocrinology 147:1349–1356 [DOI] [PubMed] [Google Scholar]
  7. Park YS, Hur EM, Choi BH, Kwak E, Jun DJ, Park SJ, Kim KT 2006 Involvement of protein kinase C-epsilon in activity-dependent potentiation of large dense-core vesicle exocytosis in chromaffin cells. J Neurosci 26:8999–9005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Becherer U, Moser T, Stuhmer W, Oheim M 2003 Calcium regulates exocytosis at the level of single vesicles. Nat Neurosci 6:846–853 [DOI] [PubMed] [Google Scholar]
  9. Buttgereit F, Scheffold A 2002 Rapid glucocorticoid effects on immune cells. Steroids 67:529–534 [DOI] [PubMed] [Google Scholar]
  10. Loerke D, Stuhmer W, Oheim M 2002 Quantifying axial secretory-granule motion with variable-angle evanescent-field excitation. J Neurosci Methods 119:65–73 [DOI] [PubMed] [Google Scholar]
  11. Johns LM, Levitan ES, Shelden EA, Holz RW, Axelrod D 2001 Restriction of secretory granule motion near the plasma membrane of chromaffin cells. J Cell Biol 153:177–190 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Arbuzova A, Schmitz AA, Vergeres G 2002 Cross-talk unfolded: MARCKS proteins. Biochem J 362:1–12 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Wagner PG, Jorgensen MS, Arden WA, Jackson BA 1999 Stimulus-secretion coupling in porcine adrenal chromaffin cells: acute effects of glucocorticoids. J Neurosci Res 57:643–650 [PubMed] [Google Scholar]
  14. Lovell PV, King JT, McCobb DP 2004 Acute modulation of adrenal chromaffin cell BK channel gating and cell excitability by glucocorticoids. J Neurophysiol 91:561–570 [DOI] [PubMed] [Google Scholar]
  15. Armstrong SM, Stuenkel EL 2005 Progesterone regulation of catecholamine secretion from chromaffin cells. Brain Res 1043:76–86 [DOI] [PubMed] [Google Scholar]
  16. Qiu J, Lou LG, Huang XY, Lou SJ, Pei G, Chen YZ 1998 Nongenomic mechanisms of glucocorticoid inhibition of nicotine-induced calcium influx in PC12 cells: involvement of protein kinase C. Endocrinology 139:5103–5108 [DOI] [PubMed] [Google Scholar]
  17. Cato AC, Nestl A, Mink S 2002 Rapid actions of steroid receptors in cellular signaling pathways. Sci STKE 2002:RE9 [DOI] [PubMed] [Google Scholar]
  18. Song IH, Buttgereit F 2006 Non-genomic glucocorticoid effects to provide the basis for new drug developments. Mol Cell Endocrinol 246:142–146 [DOI] [PubMed] [Google Scholar]
  19. Xiao L, Qi A, Chen Y 2005 Cultured embryonic hippocampal neurons deficient in glucocorticoid (GC) receptor: a novel model for studying nongenomic effects of GC in the neural system. Endocrinology 146:4036–4041 [DOI] [PubMed] [Google Scholar]
  20. Hinz B, Hirschelmann R 2000 Rapid nongenomic feedback effects of glucocorticoids on CRF-induced ACTH secretion in rats. Pharm Res 17:1273–1277 [DOI] [PubMed] [Google Scholar]
  21. Chen YZ, Qiu J 2001 Possible genomic consequence of nongenomic action of glucocorticoids in neural cells. News Physiol Sci 16:292–296 [DOI] [PubMed] [Google Scholar]
  22. Einer-Jensen N, Carter AM 1995 Local transfer of hormones between blood vessels within the adrenal gland may explain the functional interaction between the adrenal cortex and medulla. Med Hypotheses 44:471–474 [DOI] [PubMed] [Google Scholar]
  23. Jones MT, Hillhouse EW, Burden JL 1977 Dynamics and mechanics of corticosteroid feedback at the hypothalamus and anterior pituitary gland. J Endocrinol 73:405–417 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

[Supplemental Data]
en.2007-1798_index.html (966B, html)
en.2007-1798_1.pdf (19.8KB, pdf)
en.2007-1798_2.pdf (14.2KB, pdf)
en.2007-1798_3.pdf (9.5KB, pdf)

Articles from Endocrinology are provided here courtesy of The Endocrine Society

RESOURCES