Dehydroepiandrosterone Sulfate and Allopregnanolone Directly Stimulate Catecholamine Production via Induction of Tyrosine Hydroxylase and Se
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内分泌学杂志 2005年第8期
Departments of Pharmacology (I.C., A.G.), Clinical Chemistry (E.D., C.T., A.N.M.), and Biochemistry (L.V., C.S.), School of Medicine, University of Crete, Heraklion GR-711 10, Greece
Address all correspondence and requests for reprints to: Achille Gravanis, Department of Pharmacology, School of Medicine, University of Crete, Heraklion GR-711 10, Crete, Greece. E-mail: gravanis@med.uoc.gr.
Abstract
Adrenal cortical cells of zona reticularis produce the neuroactive steroids dehydroepiandrosterone (DHEA), its sulfate ester dehydroepiandrosterone sulfate (DHEAS), and allopregnanolone (ALLO). An interaction between zona reticularis and adrenal medulla has been postulated based on their close proximity and their interwoven borders. The aim of this paper was to examine in vitro the possible paracrine effects of these steroids on catecholamine production from adrenomedullary chromaffin cells, using an established in vitro model of chromaffin cells, the PC12 rat pheochromocytoma cell line. We have found the following: 1) DHEA, DHEAS, and ALLO increased acutely (peak effect between 10–30 min) and dose-dependently (EC50 in the nanomolar range) catecholamine levels (norepinephrine and dopamine). 2) It appears that the acute effect of these steroids involved actin depolymerization/actin filament disassembly, a fast-response cellular system regulating trafficking of catecholamine vesicles. Specifically, 10–6 M phallacidin, an actin filament stabilizer, completely prevented steroid-induced catecholamine secretion. 3) DHEAS and ALLO, but not DHEA, also affected catecholamine synthesis. Indeed, DHEAS and ALLO increased catecholamine levels at 24 h, an effect blocked by L-2-methyl-3-(-4hydroxyphenyl)alanine and 3-(hydrazinomethyl)phenol hydrochloride, inhibitors of tyrosine hydroxylase and L-aromatic amino acid decarboxylase, respectively, suggesting that this effect involved catecholamine synthesis. The latter hypothesis was confirmed by finding that DHEAS and ALLO increased both the mRNA and protein levels of tyrosine hydroxylase. In conclusion, our findings suggest that neuroactive steroids exert a direct tonic effect on adrenal catecholamine synthesis and secretion. These data associate the adrenomedullary malfunction observed in old age and neuroactive steroids.
Introduction
THE ZONA RETICULARIS cells of adrenal cortex produce the neuroactive steroids dehydroepiandrosterone (DHEA), its sulfate ester dehydroepiandrosterone sulfate (DHEAS), and allopregnanolone (ALLO) (1, 2, 3). DHEA- and ALLO-producing adrenocortical cells of zona reticularis and catecholamine-producing adrenomedullary cells are interwoven with each other, providing ample contact surfaces for paracrine interactions (4). The aim of this paper was to examine whether the neuroactive steroid products of zona reticularis exert paracrine effects on catecholamine production from adrenomedullary chromaffin cells. It should be noted that these neuroactive steroids, produced in the central nervous system (1, 5), modulate brain catecholamine production. Indeed, it has been shown that DHEAS potentiates NMDA-evoked norepinephrine secretion from rat hippocampal cells (4); whereas, in the mouse, DHEA prevents MPTP-induced dopamine depletion of striatal neurons (7). Furthermore, ALLO increases the dopaminergic response to morphine in the rat nucleus accumbens (8), whereas its depletion from rat cortex potentiates stress-induced cortical dopamine output (9).
Based on these findings, we have tested the possible direct effects of these neuroactive steroids on catecholamine production and secretion from adrenal chromaffin cells in vitro, using as a model the well-established rat pheochromocytoma cell line PC12 (10, 11, 12, 13). Specifically, we first examined the acute effect of DHEAS and ALLO on catecholamine (norepinephrine and dopamine) secretion from PC12 cells. Subsequently, we examined the effect of these neuroactive steroids on actin polymerization and stability of its filaments, a mechanism shown to control the trafficking of catecholamine-containing vesicles in adrenal chromaffin cells (14, 15) and in the PC12 cells (12). The secretory vesicles containing catecholamines are mainly located inside the cortical (subplasmalemmal) actin ring, which blocks them from reaching the exocytosis sites, a process requiring a transient actin depolymerization and filament disassembly (12, 16, 17). After that, we examined the effect of neuroactive steroids on catecholamine production by measuring their effects on catecholamine levels at 24 h and on tyrosine hydroxylase (TH) mRNA and protein levels, i.e. the rate-limiting factor of catecholamine biosynthesis. Our findings provide in vitro evidence that DHEAS and ALLO stimulate the production (synthesis and secretion) of catecholamines from PC12 cells, suggesting that zona reticulosa-deriving neurosteroids exert a continuous direct tonic effect of adrenal chromaffin cell production of catecholamines.
Materials and Methods
PC12 cell cultures
PC12 cells were obtained from Dr. M. Greenberg (Children’s Hospital, Boston, MA). They were cultured in flat-bottom wells (6-well plates of 9.5-cm2 surface area/well, or 24-well plates of 1.9-cm2 surface area/well; Costar Europe Ltd., Badhoevedorp, The Netherlands) at an initial concentration of 1.5 x 105 cells/cm2. The cells were grown at 5% CO2 and 37 C, in RPMI medium 1640 (Life Technologies, Inc., Gaithersburg, MD) containing 10 mM L-glutamine, 15 mM HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 10% horse serum, and 5% fetal calf serum (FCS) (both charcoal-stripped for removing endogenous steroids) and different steroids at various concentrations, diluted in ethanol. The final concentration of ethanol in each well, including controls, was 0.01%. The TH inhibitor L-2-methyl-3-(-4hydroxyphenyl)alanine (AMPT) and the L-aromatic amino acid decarboxylase (AACD) inhibitor 3-(hydrazinomethyl)phenol hydrochloride (NSD-1015) were purchased from Sigma-Aldrich Corp. (St. Louis, MO). All steroids were purchased from Steraloids (Newport, RI). Newly plated cells were cultured for 2 d in the growth medium described above, then they were left in serum-free medium supplemented with 0.1% Bovine Albumin Fraction V (BSA) (Sigma-Aldrich).
Measurement of catecholamines
Cells were grown in 6-well plates, coated with poly-L-lysine, at a concentration of 106 cells/well. Cells were incubated with neuroactive steroids for various time periods; for the short-term experiments, the incubation time ranged from 5–30 min and for the long-term, from 3–48 h. For dose-response experiments, PC12 cells were exposed to DHEA for 10 min and to DHEAS or ALLO for 30 min. One milliliter of supernatants was transferred to tubes containing 200 μl 0.1-M HCI for measurement of catecholamines. It should be noted that catecholamines are stable in acid environment.
Dopamine and norepinephrine were measured by a highly sensitive RIA (TriCat RIA, RE29395; IBL Immuno Biological Laboratories, Hamburg, Germany) as previously described (18, 19). In brief, catecholamines were converted to 3-methoxytyramine using COMT (freshly prepared every time) as enzyme and S-adenosyl-L-methionine as coenzyme and simultaneously acylated to N-acyl-3-methoxytyramine. After separation of the bound- from the free-labeled-antigen by precipitation and centrifugation (bounded 125I-labeled antigen was precipitated with a second antibody), the amount of bound radioactivity of the precipitates is measured in a -counter (1275 Minigamma; LKB Wallac, Uppsala, Sweden). The analytical sensitivity of the method was 30 pg/ml for dopamine and 22 pg/ml for norepinephrine, its intraassay coefficient of variation was 9.5% for dopamine and 7.8% for norepinephrine, and its interassay coefficient of variation was 16.7% for dopamine and 10.9% for norepinephrine. The cross-reactivity between dopamine and norepinephrine was less than 0.013%. No significant difference was found between the mean concentrations of catecholamines measured by the RIA method and by HPLC (18).
Western blot analysis
PC12 cell lysates were electrophoresed through a 12% SDS-polyacrylamide gel, then proteins were transferred to nitrocellulose membranes, which were processed according to standard Western blotting procedures, as previously described (13). To detect protein levels, membranes were incubated with the appropriate antibodies: TH (MAB318; Chemicon Int., Inc.; dilution, 1:1000), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sc-20357, V-18; Santa Cruz Biotechnology Inc., Santa Cruz, CA; dilution 1:200). Proteins were visualized using the ECL Western blotting kit (ECL; Amersham Biosciences, Little Chalfont, UK), and blots were exposed to Kodak X-Omat AR films. A PC-based Image Analysis program (Image Analysis, Inc., Ontario, Canada) was used to quantify the intensity of each band. To normalize for protein content, the blots were stripped and stained with anti-GAPDH antibodies; the concentration of the TH protein was normalized vs. GAPDH.
RT-PCR
Total RNA was extracted from PC12 cells using the Trizol Reagent (Invitrogen Life Technologies, Carlsbad, CA). One microgram of total RNA was reverse transcribed by the Thermo-Script RT-PCR System (Invitrogen Life Technologies) using random hexamers in a total vol of 20 μl. Two microliters of the RT product were used as a template, amplified by PCR using 2 mM MgCl2, one strength PCR buffer, 0.2 mM of sense and antisense primers, 0.2 mM deoxynucleotide triphosphates, and 2.5 U AmpliTaq Gold DNA polymerase (PerkinElmer ABD, Foster City, CA) in a final reaction vol of 50 μl. PCR was performed in a PerkinElmer DNA Thermal Cycler.
Primers for TH had the following sequence: 5'-TCGCCACAGCCCAAGGGCTTCAGAA-3' (sense), 5-CCTCGAAGCGCACAAAATAC-3 (antisense); and for GAPDH, 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' (sense), 5'-CATGTGGGCCATGAGGTCCACCAC-3' (antisense).
The oligonucleotides were synthesized by MWG Biotech AG (Munchen, Germany).
After RT, the cDNA product was amplified by PCR for 33 cycles. The cycle number 33 was chosen such that amplification of the products was in the linear range with respect to the amount of input cDNA. PCR for GAPDH was performed in parallel to assure good quality of RNA and cDNA preparations. Each cycle consisted of 60 sec at 92 C for denaturation, 120 sec at 53 C for annealing, and 180 sec at 72 C for extension (60 sec at 98 C, 90 sec at 55 C, and 150 sec at 72 C for GAPDH, respectively). Ten microliters of the amplified products (368 bp for TH and 983 bp for GAPDH) were separated on a 2% agarose gel and visualized by ethidium bromide staining.
Real-time PCR
For quantitation of TH mRNA, 1 μl cDNA was used together with the primers shown above in a 20-μl reaction, using SYBR green as a marker for DNA content, provided in the SYBRgreen master Mix (Applied Biosystems, Foster City, CA). Amplification was performed in an ABI-Prizm7000 Real-Time PCR for a maximum of 33 cycles as follows: 60 sec at 92 C, 120 sec at 53 C, 180 sec at 72 C. No by-products were present in the reaction, as indicated by the dissociation pattern provided at the end of the reaction and by agarose gel electrophoresis (data not shown). The amplification efficiency of the TH product was the same as the one of GAPDH, as indicated by the standard curves of amplification, allowing us to use the formula: fold increase = 2-(CtA–CtB).
Measurement of G/total actin ratio
PC12 cells were exposed to steroids for 5–60 min, then washed three times with ice-cold PBS and resuspended in cold lysis buffer containing 10 mM K2HPO4, 100 mM NaF, 50 mM KCl, 2 mM MgCl2, 1 mM EGTA, 0.2 mM dithiothreitol, 0.5% Triton X-100, and 1 M sucrose (pH 7.0). The monomeric (G) and total actin contents in cell extracts were determined using the DNaseI inhibition assay as described previously (20).
Immunofluorescence confocal microscopy
PC12 cells were grown on glass or chamber slides and treated with steroids for 5–30 min. Control cells were left untreated. At the end of the incubation period, cells were fixed in 2% (wt/vol) formaldehyde for 10min, permeabilized in 0.2% (wt/vol) Triton X-100 for 10 min, blocked in 1% FCS in PBS for 15 min, and then incubated overnight at 4 C with an actin antibody (sc-109; Santa Cruz Biotechnology Inc.) diluted 1:100 in PBS containing 1% FCS. Samples were washed with PBS and incubated for 1 h with fluorescein isothiocyanate-conjugated secondary antirabbit polyclonal antibody diluted 1/100. Cells were analyzed in a confocal laser-scanning microscope (Leica TCS-NT; Leica Microsystems Heidelberg GmbH, Heidelberg, Germany).
Statistical analysis
To compare results from independent experiments, the concentrations of noradrenaline, dopamine, and actin were normalized per total cellular protein measured by the Bradford Coomassie Brilliant Blue G250 method (Serva Feinbiochemika GmbH & Co., Heidelberg, Germany) using BSA as standard. For statistical evaluation of our data, we have used ANOVA, post hoc comparison of means followed by two multiple-comparison tests: the Fisher’s least-significance-difference and the Newman-Keuls test. For the data expressed as percent change over parallel controls, we have used the nonparametric Kruskal-Wallis test for several independent samples.
Results
DHEA, DHEAS, and ALLO stimulate catecholamine secretion
To assess the effectiveness of our in vitro system (PC12 cells), we have tested the effect of nicotine, a known inducer of catecholamine secretion. As shown in Fig. 1, nicotine at 10–6 M acutely increased (peaking at 10 min) dopamine and norepinephrine levels in the culture media.
FIG. 1. DHEA, DHEAS, and ALLO induce acutely the secretion of catecholamines. Nicotine at 10–6 M and DHEA, DHEAS, and ALLO at 10–7 M induced the secretion of dopamine and norepinephrine in the culture media of PC12 cells, an effect peaking between 5–30 min. Catecholamine levels were measured by specific RIAs, as described in Materials and Methods. Data are expressed as micrograms of dopamine or nanograms of norepinephrine per milligram of cellular proteins. Values represent mean ± SD of four separate experiments. *, P < 0.01, denotes significant statistical difference compared with parallel control cells.
PC12 cells were exposed to DHEA, DHEAS, and ALLO (10–7 M) for short periods of time (5–30 min), and the concentration of dopamine and norepinephrine in culture media was measured using a RIA, as described in Materials and Methods. All three steroids tested provoked a fast stimulation of catecholamine secretion, doubling their levels in the culture media (Fig. 1). The effect of DHEA was faster compared with the other two steroids, peaking at 10 min. DHEAS and ALLO increased catecholamine levels at 30 min. The effect of all three steroids was dose-dependent (Fig. 2). The EC50 values of DHEA, DHEAS, and ALLO for stimulation of dopamine secretion were 17 ± 1.9, 32 ± 3, and 15 ± 1.5 nM; and for stimulation of norepinephrine secretion, they were 26 ± 2.3, 72 ± 2.9, and 6 ± 1.2 nM (n = 3; P < 0.05). Conversely, testosterone and progesterone at 10–7 M were ineffective in PC12 cells (data not shown), an in vitro system not expressing functional androgen and progesterone receptors (13, 21, 22).
FIG. 2. Dose-response curves of neuroactive steroids on catecholamine secretion. A range of concentrations (10–11–10–5 M) of DHEA, DHEAS, and ALLO were used, and the concentration of dopamine and norepinephrine was measured in the culture media of PC12 cells, as per Fig. 1. Data are expressed as percentage of parallel controls, i.e. cells cultured in the absence of these steroids. Values represent mean ± SD of three separate experiments.
DHEAS and ALLO stimulate catecholamine production
We have also tested the effect of steroids for longer periods of time. More specifically, PC12 cells were incubated with DHEA, DHEAS, and ALLO (10–7 M) for 3–48 h, and the concentration of dopamine and norepinephrine in the culture media was measured. In contrast to its short-term effect, long-term DHEA did not significantly alter the levels of catecholamines (Figs. 3A and 4A). Conversely, incubation of PC12 cells with DHEAS or ALLO resulted in an increase of catecholamine levels, peaking at 36 h for dopamine and 24 h for norepinephrine. These long-term effects of DHEAS and ALLO suggest that these steroids, in addition to their acute effects on catecholamine secretion, may also affect the de novo production of catecholamines in PC12 cells. To test this hypothesis, we have measured the effects of neuroactive steroids in the absence and the presence of TH inhibitor AMPT or of AACD inhibitor NSD-1015, at 10–6 M. These enzymes hold a central role in the pathway of catecholamine de novo synthesis. Both inhibitors completely blocked the increase of catecholamine levels in the culture media of PC12 cells exposed for 3–48 h to 10–7 M DHEAS and ALLO (Fig. 3, B and C, for dopamine; and Fig. 4, B and C, for norepinephrine). These findings suggest that the long-term effects of DHEAS and ALLO on catecholamines are mainly exerted at the de novo production level.
FIG. 3. Effect of neuroactive steroids on dopamine production. PC12 cells were exposed to DHEA, DHEAS, and ALLO (10–7 M) for longer periods of time (3–48 h) in the absence or presence of the TH inhibitor AMPT or the AACD inhibitor NSD-1015, at 10–6 M. At the end of the incubation, dopamine levels were measured in the culture media. Data are expressed as nanograms per milligram of cellular protein. Values represent mean ± SD of four separate experiments. *, P < 0.01, denotes significant statistical difference compared with parallel controls, i.e. cells not exposed to the inhibitors.
FIG. 4. Effect of neuroactive steroids on norepinephrine production. PC12 cells were exposed to DHEA, DHEAS, and ALLO (10–7 M) for longer periods of time (3–48 h) in the absence or presence of the TH inhibitor AMPT or of the AACD inhibitor NSD-1015, at 10–7 M. At the end of the incubation, norepinephrine levels were measured in the culture media. Data are expressed as nanograms per milligram of cellular protein. Values represent mean ± SD of four separate experiments. *, P < 0.01, denotes significant statistical difference compared with parallel controls, i.e. cells not exposed to the inhibitors.
DHEA, DHEAS, and ALLO increase catecholamine secretion by acutely stimulating actin depolymerization and actin filament disassembly
Actin depolymerization and filament disassembly is required for catecholamine secretion because a subplasmalemmal actin mesh prevents their secretory vesicles from reaching their exocytosis sites (12, 16, 17). Based on these observations, we have tested whether DHEA, DHEAS, or ALLO affect catecholamine secretion in PC12 cells via the same mechanism. To test this hypothesis, we first examined the acute effects of DHEA, DHEAS, and ALLO on the ratio of G-monomeric to total cellular actin, an established marker of actin cytoskeleton dynamics (23). PC12 cells were cultured for short time periods (5–60 min) in control medium or in media containing the steroids at 10–7 M, then the ratio of G to total actin was measured, as previously described (12). All three steroids induced a rapid increase of the G/total actin ratio, reflecting actin depolymerization (Fig. 5A). DHEA and DHEAS were the most effective, increasing the G/total actin ratio within 5 min to 0.63 ± 0,01, compared with 0.46 ± 0.05 of parallel controls (n = 4; P < 0,001), or to 0.68 ± 0.03, compared with 0.59 ± 0.02 of the controls (n = 4; P < 0.05) for DHEA and DHEAS, respectively. Rising of G/total actin ratio by DHEA and DHEAS persisted for at least 60 min. ALLO significantly augmented G/total actin ratio within 5 min (0.63 ± 0.02, compared with 0.56 ± 0.03 of parallel controls; n = 4; P < 0.05); however, its effect was transient because it returned to basal levels within 15 min (Fig. 5A).
FIG. 5. Effects of neuroactive steroids on actin depolymerization (A) and actin filament disassembly (B). A, PC12 cells were cultured for short time periods (5–60 min) in control medium (in the absence of steroids) or in media containing DHEA, DHEAS, or ALLO at 10–7 M, then the ratio of G-monomeric (G) to total actin was measured, as described in Materials and Methods. Values represent mean ± SD of four separate experiments. *, P < 0.05, denotes significant statistical difference compared with controls. A, Confocal microscopy images of PC12 cell staining. PC12 cells were cultured for 10 min in control medium (in the absence of steroids) or in media containing DHEA, DHEAS, or ALLO at 10–7 M, then actin filaments were stained with rhodamine and observed under confocal laser microscopy. Section thickness was adjusted to 0.5 μm.
Figure 5B depicts the morphological alterations of PC12 cells exposed to the steroids. The confocal laser microscopy data supplement the results obtained by the measurement of the G/total actin ratio. Figure 5B depicts the 10-min steroid-mediated decrease in rhodamine-stained actin, confined mainly to the subplasmalemmal area beneath the cell membrane. In control cells (not exposed to any steroid), subplasmalemmal actin filaments were wider and more intense (Fig. 5B, Control).
The involvement of actin filaments in the effect of DHEA, DHEAS, and ALLO on catecholamine secretion was further supported by experiments using phallacidin, an actin filament stabilizer. PC12 cells were primed for 30 min with 10–6 M phallacidin; then DHEA, DHEAS, or ALLO were added to a final concentration of 10–7 M, and the incubation continued for an additional 10 min for DHEA and 30 min for DHEAS and ALLO. As shown in Fig. 6, phallacidin treatment almost completely prevented the stimulatory effect of DHEA, DHEAS, and ALLO on both dopamine and norepinephrine secretion. It should be noted that PC12 cells exposed to phallacidin alone did not have their basal catecholamine secretion altered (data not shown). Taken together, these findings support further the notion that actin microfilament redistribution plays a pivotal role in regulating the rapid catecholamine release, induced by the neuroactive steroids.
FIG. 6. Phallacidin prevents the stimulatory effect of neuroactive steroids on catecholamine secretion. PC12 cells were primed for 30 min in the presence of 10–6 M actin filament stabilizer phallacidin. Subsequently, DHEA, DHEAS, or ALLO was added to a final concentration of 10–7 M, and the incubation continued for an additional 10 min for DHEA and 30 min for DHEAS and ALLO. At the end of the incubation, the concentration of dopamine and norepinephrine was measured in the culture media. Data are expressed as percentage of parallel controls exposed to vehicles. Values represent mean ± SD of three separate experiments. *, P < 0.01, denotes significant statistical difference compared with parallel controls.
DHEAS and ALLO induce TH expression
As mentioned above, DHEAS and ALLO, in addition to their fast action, exhibited a long-term (up to 24 h) stimulatory effect on catecholamines, suggesting that they may also affect the de novo production of catecholamines. To test this hypothesis, we have studied their effects on TH protein and mRNA levels, i.e. the rate-limiting step of catecholamine biosynthesis. PC12 cells were incubated for 4–24 h in the absence or presence of DHEA, DHEAS, or ALLO at 10–7 M, and the protein levels of TH were measured in cell extracts by Western blot analysis. Levels of the TH protein were normalized against GAPDH levels. Results are depicted in Fig. 7. DHEA did not significantly affect TH levels at all time points tested. Conversely, DHEAS and ALLO increased TH levels in a time-dependent fashion, their effect starting as early as 2 h and peaking at 18 h.
FIG. 7. DHEAS and ALLO elevate the concentration of TH protein in cell lysates. PC12 cells were incubated for 4–24 h in the absence (control) or presence of DHEA, DHEAS, or ALLO at 10–7 M, and protein levels of TH were measured in cell extracts with Western blot analysis. Levels of the TH protein were normalized against GAPDH, presented as TH/GAPDH ratio. Values represent mean ± SD of three separate experiments. *, P < 0.01, denotes significant statistical difference compared with parallel controls.
Finally, we have assessed the effect of these steroids on TH mRNA levels, using a experimental protocol similar to that of TH protein. Levels of the TH mRNA were measured with semiquantitative RT-PCR and normalized against GAPDH mRNA levels. Results are depicted in Fig. 8A. As in the case of TH protein, DHEA did not alter TH mRNA levels at all time points tested, whereas DHEAS and ALLO increased TH mRNA levels in a time-dependent fashion. Their effect was detectable as early as 2 h, peaking at 6 h. More specifically, at 6 h, both DHEAS and ALLO increased the TH/GAPDH mRNA ratio by 4- to 5-fold. The maximal effect of DHEAS and ALLO on TH mRNA levels was further confirmed by real-time PCR. Indeed, at 6 h, DHEAS and ALLO resulted in a 4.84 ± 0.5 and 3.71 ± 0.8-fold (n = 3; P < 0.001) increase of TH mRNA levels, respectively, confirming the semiquantitative RT-PCR data (Fig. 8B).
FIG. 8. DHEAS and ALLO increase the mRNA levels of TH. A, RT-PCR data: PC12 cells were incubated for 2–24 h in the absence (control) or the presence of DHEA, DHEAS, or ALLO at 10–7 M, and the mRNA levels of TH were measured in cell extracts with RT-PCR analysis. Levels of the TH mRNA were normalized against GAPDH mRNA levels and are presented as TH/GAPDH ratio. Values represent mean ± SD of three separate experiments. *, P < 0.05, denotes significant statistical difference compared with controls. B, Real-time PCR data: PC12 cells were incubated for 6 h in the absence (control) or presence of DHEAS or ALLO at 10–7 M, and the mRNA levels of TH were measured in cell extracts with real-time PCR. Data are presented as fold increase compared with controls. Values represent mean ± SD of three separate experiments. *, P < 0.01, denotes significant statistical difference compared with controls. Rn, Normalized reporter signal.
Discussion
We have found that the neuroactive steroids DHEAS and ALLO stimulate the production of catecholamines from PC12 cells, a rat pheochromocytoma cell line used extensively as an in vitro model to study adrenal catecholamine production and secretion. The effect of these neuroactive steroids included stimulation of catecholamine secretion via an acute acceleration of actin filament depolymerization and induction of catecholamine synthesis via induction of TH expression. We consider that our findings have physiological relevance based on the anatomy of the human adrenal gland (rat adrenals do not produce DHEA). Indeed, the neuroactive steroid (DHEAS and ALLO)-producing human adrenocortical cells of zona reticularis and the catecholamine-producing adrenomedullary cells are interwoven, providing ample contact surfaces for potentially paracrine interaction (4). Adrenomedullary chromaffin cells can be found within all zones of the adrenal cortex, including zona reticularis. On the other hand, rims of reticularis cortical cells are present within the adrenal medulla. Our findings provide experimental in vitro evidence supporting the hypothesis of cortical-medullary interaction within the adrenals. Indeed, our data suggest that DHEA stimulates the secretion, whereas DHEAS and ALLO also stimulate the synthesis of catecholamines in our experimental model of PC12 cells. Our data indicate that the neuroactive steroids DHEAS and ALLO induce catecholamine secretion from PC12 cells in an acute and phallacidin-reversible manner, suggesting that these neuroactive steroids affect subplasmalemmal actin filament disassembly (24, 25). The subplasmalemmal actin filament ring represents a fast-response mechanism regulating the trafficking of secretory granules in several types of cells, including adrenomedullary cells (16, 17). Secretion of synaptic vesicles involves detachment from actin filaments and translocation toward their exocytosis sites. These steps require activation of severing proteins, causing transient filament disassembly. It should be noted that nicotine induces catecholamine secretion by provoking actin filament disassembly (14, 15). Furthermore, actin filament disruptors like cytochalasin promote basal and Ca2+-stimulated catecholamine secretion, whereas actin stabilizers like phallacidin inhibit it (25, 26, 27). Phallacidin binds polymerized-F-actin with high affinity, thus stabilizing the F-actin cytoskeleton by preventing depolymerization, an event necessary for the dynamic reordering of actin microfilaments. Ours is the first report on the effect of neuroactive steroids on subplasmalemmal actin filament disassembly. Showing that neuroactive steroids affect the actin cytoskeleton confirms and augments our data showing that these steroids exert a definite stimulatory effect on catecholamine secretion from adrenal chromaffin cells because the subcortical actin filament plays a central role in it. More specifically, our data show that DHEAS and ALLO acutely increase catecholamine secretion, their effect peaking at 10 min. The effect of these neuroactive steroids is preceded by an acute increase of monomeric G-actin, suggesting a rapid stimulatory effect on actin depolymerization and the subsequent destabilization of actin filaments. Our data on steroid actin filament destabilization were further confirmed by confocal laser microscopy, showing a steroid-mediated decrease in rhodamine-stained actin, confined to the subplasmalemmal area. Priming of PC12 cells with actin filament stabilizer phallacidin completely prevented the stimulatory effect of DHEAS and ALLO on norepinephrine and dopamine release from PC12 cells, suggesting that the effect of these steroids involves polymerized actin. The molecular mechanism by which these steroids modulate actin polymerization is yet not known. However, it should be noted that DHEA and pregnenolone have been recently reported to bind to several microtubule-associated proteins (28, 29). This observation points to a direct interaction between neuroactive steroids and actin-associated proteins. Furthermore, human ovarian granulosa cells exposed to DHEA contain fewer and less stable actin filaments (30). In our system, DHEAS and ALLO were highly potent in stimulating catecholamine secretion, with EC50 in the nanomole range, contrasting with the inhibitory effect of DHEAS on catecholamine secretion reported in bovine chromaffin cells, though at the suprapharmacological concentrations of 0.01–1 mM, known to alter, in a less specific manner, cellular organelles and cellular membranes (31). These findings considered together suggest that the effect of neuroactive steroids on catecholamine secretion may be cell type- and species-specific.
DHEAS and ALLO, but not DHEA, affected catecholamine synthesis in addition to their effect on catecholamine secretion. Indeed, these neuroactive steroids induced the expression of TH production, the rate-limiting enzyme of catecholamine biosynthesis as suggested by our RT-PCR, real-time PCR, and Western blot experiments, which showed that DHEAS and ALLO provoked a 4-fold increase of the concentration of its transcript and protein within 6 h and 8 h, respectively, pointing to a direct transcriptional effect on TH expression. In agreement with these data, the concentration of norepinephrine and dopamine in the culture media was significantly higher, compared with parallel control cells exposed only to the neuroactive steroid vehicles. Regulation of catecholamine production by DHEAS and ALLO was further supported by experiments with the specific inhibitors of TH and AACD, two key enzymes of catecholamine biosynthesis. Indeed, long-term effects of both steroids were completely blocked by TH inhibitor AMPT and AACD inhibitor NSD-1015. DHEA had no effect on TH expression, contrasting with a recent report showing with in situ hybridization an inhibitory effect of this steroid on TH mRNA levels in rat tuberoinfundibular neurons (32). However, in rat substantia nigra neurons, DHEA prevented the MPTP-induced TH mRNA decrease, measured by in situ hybridization (7). It thus appears that these neuroactive steroids, as in the case of catecholamine secretion, regulate catecholamine production and TH expression in a cell type-specific manner. DHEA was ineffective in increasing catecholamine levels at longer time periods. This finding suggests that DHEAS most probably represents the active form affecting catecholamine production, and that DHEA needs conversion to its sulfate ester to also become effective. The synthesis of sulfated DHEA is catalyzed by a cytosolic enzyme hydroxysteroid sulfotransferase (HST), which transfers the sulfonate moiety from the donor molecule 3'-phosphoadenosine 5'-phosphosulfate onto the 3-hydroxy acceptor site of DHEA. Previous studies have demonstrated the occurrence of HST-like activity in the nervous system of primates, rodents, and amphibians (33, 34, 35, 36). It is thus possible that adrenal medulla, an integral part of the peripheral nervous system, also expresses HST activity.
We believe that the possible physiological significance of our findings can be based on several published reports showing that, with advancing age, the intraadrenal and circulating levels of DHEAS decline in humans (37, 38, 39). Indeed, it has been calculated that by the age of 70, the circulating levels of DHEAS decrease by about 20% compared with young adults. It is of note that the release of epinephrine from the human adrenal medulla at rest was found to be lower in older men, 112 ng/ml compared with 248 ng/ml in younger men (40, 41). Furthermore, in younger men, the secretion of epinephrine doubles, or even triples, with mental stress, with isometric exercise, or dynamic exercise, compared with older men, who can master only 33% of the corresponding responses of younger men (40). We propose that the decline of DHEAS and ALLO production from zona reticularis of the adrenal cortex may affect catecholamine levels and the effectiveness of adrenal medulla to respond to sympathetic stimuli, particularly with advancing age. Our findings suggest that an intraadrenal paracrine regulatory loop is in action between adrenal neuroactive steroids and catecholamines that may be deregulated with advancing age. This hypothesis is supported by recent experimental findings in H295R human adrenocortical cells showing that synthetic catecholamine isoproterenol increases the secretion of DHEA dose-dependently (42). Similarly, isoproterenol stimulates DHEAS production from human fetal adrenocortical cells, in culture (43). Furthermore, in the central nervous system, neuroactive neurosteroids affect brain catecholamines. Thus, DHEAS potentiates NMDA-evoked norepinephrine secretion in rat hippocampal cells (6), whereas in the mouse, DHEA prevents MPTP-induced dopamine depletion in striatal neurons (7). In addition, ALLO increases the dopaminergic response to morphine in the rat nucleus accumbens (8), whereas its depletion from rat cortex potentiates stress-induced cortical dopamine output (9). Our findings supplement these reports, suggesting that DHEAS and ALLO may directly augment dopamine and norepinephrine levels in the brain, in addition to the above mentioned indirect effects, via a direct paracrine manner. Induction of dopamine or norepinephrine secretion and production by DHEAS and ALLO might contribute to the neuroprotective effects of these neurosteroids, further suggesting their involvement in the pathophysiology of neurodegenerative processes, such as Parkinson’s and Alzheimer’s disease (7, 44, 45).
The exact nature of the receptor systems mediating the stimulatory effect of DHEAS and ALLO on catecholamine production in chromaffin cells is unknown. In the central nervous system, DHEA up-regulates the NMDA receptors and/or down-regulates the GABAA receptors (1). Similarly, ALLO has been shown to affect GABAA receptors (1). In our experimental model, the effect of DHEA and ALLO should be independent of these receptor systems because PC12 cells do not express functional NMDA or GABAA receptors (46, 47). Generally, it is assumed that DHEA exerts its biological effects after its conversion into androgens or estrogens. In our in vitro model, we have previously shown that this is not the case, because no testosterone, estradiol, or estrone is detectable by immunoassays in the culture media of PC12 cells exposed to either DHEA or DHEAS (13). In addition, radiolabeled steroid binding assays have identified very low levels of intracellular estrogen receptors in cytosolic preparations of PC12 cells, whereas no progesterone or testosterone cytosolic receptors were detectable (13, 21, 22). The rapid onset of DHEAS and ALLO actions on catecholamine secretion in our model supports the hypothesis that these neuroactive steroids may use specific membrane receptors, although the participation of some yet-unidentified cytosolic receptors cannot be excluded. It should be mentioned here that specific membrane bindings sites have been recently reported for various steroids, including DHEA (48). These hypotheses are now under investigation.
Acknowledgments
We thank students Charoula Kontaki and Kostoula Troulinaki for their technical assistance.
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Address all correspondence and requests for reprints to: Achille Gravanis, Department of Pharmacology, School of Medicine, University of Crete, Heraklion GR-711 10, Crete, Greece. E-mail: gravanis@med.uoc.gr.
Abstract
Adrenal cortical cells of zona reticularis produce the neuroactive steroids dehydroepiandrosterone (DHEA), its sulfate ester dehydroepiandrosterone sulfate (DHEAS), and allopregnanolone (ALLO). An interaction between zona reticularis and adrenal medulla has been postulated based on their close proximity and their interwoven borders. The aim of this paper was to examine in vitro the possible paracrine effects of these steroids on catecholamine production from adrenomedullary chromaffin cells, using an established in vitro model of chromaffin cells, the PC12 rat pheochromocytoma cell line. We have found the following: 1) DHEA, DHEAS, and ALLO increased acutely (peak effect between 10–30 min) and dose-dependently (EC50 in the nanomolar range) catecholamine levels (norepinephrine and dopamine). 2) It appears that the acute effect of these steroids involved actin depolymerization/actin filament disassembly, a fast-response cellular system regulating trafficking of catecholamine vesicles. Specifically, 10–6 M phallacidin, an actin filament stabilizer, completely prevented steroid-induced catecholamine secretion. 3) DHEAS and ALLO, but not DHEA, also affected catecholamine synthesis. Indeed, DHEAS and ALLO increased catecholamine levels at 24 h, an effect blocked by L-2-methyl-3-(-4hydroxyphenyl)alanine and 3-(hydrazinomethyl)phenol hydrochloride, inhibitors of tyrosine hydroxylase and L-aromatic amino acid decarboxylase, respectively, suggesting that this effect involved catecholamine synthesis. The latter hypothesis was confirmed by finding that DHEAS and ALLO increased both the mRNA and protein levels of tyrosine hydroxylase. In conclusion, our findings suggest that neuroactive steroids exert a direct tonic effect on adrenal catecholamine synthesis and secretion. These data associate the adrenomedullary malfunction observed in old age and neuroactive steroids.
Introduction
THE ZONA RETICULARIS cells of adrenal cortex produce the neuroactive steroids dehydroepiandrosterone (DHEA), its sulfate ester dehydroepiandrosterone sulfate (DHEAS), and allopregnanolone (ALLO) (1, 2, 3). DHEA- and ALLO-producing adrenocortical cells of zona reticularis and catecholamine-producing adrenomedullary cells are interwoven with each other, providing ample contact surfaces for paracrine interactions (4). The aim of this paper was to examine whether the neuroactive steroid products of zona reticularis exert paracrine effects on catecholamine production from adrenomedullary chromaffin cells. It should be noted that these neuroactive steroids, produced in the central nervous system (1, 5), modulate brain catecholamine production. Indeed, it has been shown that DHEAS potentiates NMDA-evoked norepinephrine secretion from rat hippocampal cells (4); whereas, in the mouse, DHEA prevents MPTP-induced dopamine depletion of striatal neurons (7). Furthermore, ALLO increases the dopaminergic response to morphine in the rat nucleus accumbens (8), whereas its depletion from rat cortex potentiates stress-induced cortical dopamine output (9).
Based on these findings, we have tested the possible direct effects of these neuroactive steroids on catecholamine production and secretion from adrenal chromaffin cells in vitro, using as a model the well-established rat pheochromocytoma cell line PC12 (10, 11, 12, 13). Specifically, we first examined the acute effect of DHEAS and ALLO on catecholamine (norepinephrine and dopamine) secretion from PC12 cells. Subsequently, we examined the effect of these neuroactive steroids on actin polymerization and stability of its filaments, a mechanism shown to control the trafficking of catecholamine-containing vesicles in adrenal chromaffin cells (14, 15) and in the PC12 cells (12). The secretory vesicles containing catecholamines are mainly located inside the cortical (subplasmalemmal) actin ring, which blocks them from reaching the exocytosis sites, a process requiring a transient actin depolymerization and filament disassembly (12, 16, 17). After that, we examined the effect of neuroactive steroids on catecholamine production by measuring their effects on catecholamine levels at 24 h and on tyrosine hydroxylase (TH) mRNA and protein levels, i.e. the rate-limiting factor of catecholamine biosynthesis. Our findings provide in vitro evidence that DHEAS and ALLO stimulate the production (synthesis and secretion) of catecholamines from PC12 cells, suggesting that zona reticulosa-deriving neurosteroids exert a continuous direct tonic effect of adrenal chromaffin cell production of catecholamines.
Materials and Methods
PC12 cell cultures
PC12 cells were obtained from Dr. M. Greenberg (Children’s Hospital, Boston, MA). They were cultured in flat-bottom wells (6-well plates of 9.5-cm2 surface area/well, or 24-well plates of 1.9-cm2 surface area/well; Costar Europe Ltd., Badhoevedorp, The Netherlands) at an initial concentration of 1.5 x 105 cells/cm2. The cells were grown at 5% CO2 and 37 C, in RPMI medium 1640 (Life Technologies, Inc., Gaithersburg, MD) containing 10 mM L-glutamine, 15 mM HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 10% horse serum, and 5% fetal calf serum (FCS) (both charcoal-stripped for removing endogenous steroids) and different steroids at various concentrations, diluted in ethanol. The final concentration of ethanol in each well, including controls, was 0.01%. The TH inhibitor L-2-methyl-3-(-4hydroxyphenyl)alanine (AMPT) and the L-aromatic amino acid decarboxylase (AACD) inhibitor 3-(hydrazinomethyl)phenol hydrochloride (NSD-1015) were purchased from Sigma-Aldrich Corp. (St. Louis, MO). All steroids were purchased from Steraloids (Newport, RI). Newly plated cells were cultured for 2 d in the growth medium described above, then they were left in serum-free medium supplemented with 0.1% Bovine Albumin Fraction V (BSA) (Sigma-Aldrich).
Measurement of catecholamines
Cells were grown in 6-well plates, coated with poly-L-lysine, at a concentration of 106 cells/well. Cells were incubated with neuroactive steroids for various time periods; for the short-term experiments, the incubation time ranged from 5–30 min and for the long-term, from 3–48 h. For dose-response experiments, PC12 cells were exposed to DHEA for 10 min and to DHEAS or ALLO for 30 min. One milliliter of supernatants was transferred to tubes containing 200 μl 0.1-M HCI for measurement of catecholamines. It should be noted that catecholamines are stable in acid environment.
Dopamine and norepinephrine were measured by a highly sensitive RIA (TriCat RIA, RE29395; IBL Immuno Biological Laboratories, Hamburg, Germany) as previously described (18, 19). In brief, catecholamines were converted to 3-methoxytyramine using COMT (freshly prepared every time) as enzyme and S-adenosyl-L-methionine as coenzyme and simultaneously acylated to N-acyl-3-methoxytyramine. After separation of the bound- from the free-labeled-antigen by precipitation and centrifugation (bounded 125I-labeled antigen was precipitated with a second antibody), the amount of bound radioactivity of the precipitates is measured in a -counter (1275 Minigamma; LKB Wallac, Uppsala, Sweden). The analytical sensitivity of the method was 30 pg/ml for dopamine and 22 pg/ml for norepinephrine, its intraassay coefficient of variation was 9.5% for dopamine and 7.8% for norepinephrine, and its interassay coefficient of variation was 16.7% for dopamine and 10.9% for norepinephrine. The cross-reactivity between dopamine and norepinephrine was less than 0.013%. No significant difference was found between the mean concentrations of catecholamines measured by the RIA method and by HPLC (18).
Western blot analysis
PC12 cell lysates were electrophoresed through a 12% SDS-polyacrylamide gel, then proteins were transferred to nitrocellulose membranes, which were processed according to standard Western blotting procedures, as previously described (13). To detect protein levels, membranes were incubated with the appropriate antibodies: TH (MAB318; Chemicon Int., Inc.; dilution, 1:1000), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sc-20357, V-18; Santa Cruz Biotechnology Inc., Santa Cruz, CA; dilution 1:200). Proteins were visualized using the ECL Western blotting kit (ECL; Amersham Biosciences, Little Chalfont, UK), and blots were exposed to Kodak X-Omat AR films. A PC-based Image Analysis program (Image Analysis, Inc., Ontario, Canada) was used to quantify the intensity of each band. To normalize for protein content, the blots were stripped and stained with anti-GAPDH antibodies; the concentration of the TH protein was normalized vs. GAPDH.
RT-PCR
Total RNA was extracted from PC12 cells using the Trizol Reagent (Invitrogen Life Technologies, Carlsbad, CA). One microgram of total RNA was reverse transcribed by the Thermo-Script RT-PCR System (Invitrogen Life Technologies) using random hexamers in a total vol of 20 μl. Two microliters of the RT product were used as a template, amplified by PCR using 2 mM MgCl2, one strength PCR buffer, 0.2 mM of sense and antisense primers, 0.2 mM deoxynucleotide triphosphates, and 2.5 U AmpliTaq Gold DNA polymerase (PerkinElmer ABD, Foster City, CA) in a final reaction vol of 50 μl. PCR was performed in a PerkinElmer DNA Thermal Cycler.
Primers for TH had the following sequence: 5'-TCGCCACAGCCCAAGGGCTTCAGAA-3' (sense), 5-CCTCGAAGCGCACAAAATAC-3 (antisense); and for GAPDH, 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' (sense), 5'-CATGTGGGCCATGAGGTCCACCAC-3' (antisense).
The oligonucleotides were synthesized by MWG Biotech AG (Munchen, Germany).
After RT, the cDNA product was amplified by PCR for 33 cycles. The cycle number 33 was chosen such that amplification of the products was in the linear range with respect to the amount of input cDNA. PCR for GAPDH was performed in parallel to assure good quality of RNA and cDNA preparations. Each cycle consisted of 60 sec at 92 C for denaturation, 120 sec at 53 C for annealing, and 180 sec at 72 C for extension (60 sec at 98 C, 90 sec at 55 C, and 150 sec at 72 C for GAPDH, respectively). Ten microliters of the amplified products (368 bp for TH and 983 bp for GAPDH) were separated on a 2% agarose gel and visualized by ethidium bromide staining.
Real-time PCR
For quantitation of TH mRNA, 1 μl cDNA was used together with the primers shown above in a 20-μl reaction, using SYBR green as a marker for DNA content, provided in the SYBRgreen master Mix (Applied Biosystems, Foster City, CA). Amplification was performed in an ABI-Prizm7000 Real-Time PCR for a maximum of 33 cycles as follows: 60 sec at 92 C, 120 sec at 53 C, 180 sec at 72 C. No by-products were present in the reaction, as indicated by the dissociation pattern provided at the end of the reaction and by agarose gel electrophoresis (data not shown). The amplification efficiency of the TH product was the same as the one of GAPDH, as indicated by the standard curves of amplification, allowing us to use the formula: fold increase = 2-(CtA–CtB).
Measurement of G/total actin ratio
PC12 cells were exposed to steroids for 5–60 min, then washed three times with ice-cold PBS and resuspended in cold lysis buffer containing 10 mM K2HPO4, 100 mM NaF, 50 mM KCl, 2 mM MgCl2, 1 mM EGTA, 0.2 mM dithiothreitol, 0.5% Triton X-100, and 1 M sucrose (pH 7.0). The monomeric (G) and total actin contents in cell extracts were determined using the DNaseI inhibition assay as described previously (20).
Immunofluorescence confocal microscopy
PC12 cells were grown on glass or chamber slides and treated with steroids for 5–30 min. Control cells were left untreated. At the end of the incubation period, cells were fixed in 2% (wt/vol) formaldehyde for 10min, permeabilized in 0.2% (wt/vol) Triton X-100 for 10 min, blocked in 1% FCS in PBS for 15 min, and then incubated overnight at 4 C with an actin antibody (sc-109; Santa Cruz Biotechnology Inc.) diluted 1:100 in PBS containing 1% FCS. Samples were washed with PBS and incubated for 1 h with fluorescein isothiocyanate-conjugated secondary antirabbit polyclonal antibody diluted 1/100. Cells were analyzed in a confocal laser-scanning microscope (Leica TCS-NT; Leica Microsystems Heidelberg GmbH, Heidelberg, Germany).
Statistical analysis
To compare results from independent experiments, the concentrations of noradrenaline, dopamine, and actin were normalized per total cellular protein measured by the Bradford Coomassie Brilliant Blue G250 method (Serva Feinbiochemika GmbH & Co., Heidelberg, Germany) using BSA as standard. For statistical evaluation of our data, we have used ANOVA, post hoc comparison of means followed by two multiple-comparison tests: the Fisher’s least-significance-difference and the Newman-Keuls test. For the data expressed as percent change over parallel controls, we have used the nonparametric Kruskal-Wallis test for several independent samples.
Results
DHEA, DHEAS, and ALLO stimulate catecholamine secretion
To assess the effectiveness of our in vitro system (PC12 cells), we have tested the effect of nicotine, a known inducer of catecholamine secretion. As shown in Fig. 1, nicotine at 10–6 M acutely increased (peaking at 10 min) dopamine and norepinephrine levels in the culture media.
FIG. 1. DHEA, DHEAS, and ALLO induce acutely the secretion of catecholamines. Nicotine at 10–6 M and DHEA, DHEAS, and ALLO at 10–7 M induced the secretion of dopamine and norepinephrine in the culture media of PC12 cells, an effect peaking between 5–30 min. Catecholamine levels were measured by specific RIAs, as described in Materials and Methods. Data are expressed as micrograms of dopamine or nanograms of norepinephrine per milligram of cellular proteins. Values represent mean ± SD of four separate experiments. *, P < 0.01, denotes significant statistical difference compared with parallel control cells.
PC12 cells were exposed to DHEA, DHEAS, and ALLO (10–7 M) for short periods of time (5–30 min), and the concentration of dopamine and norepinephrine in culture media was measured using a RIA, as described in Materials and Methods. All three steroids tested provoked a fast stimulation of catecholamine secretion, doubling their levels in the culture media (Fig. 1). The effect of DHEA was faster compared with the other two steroids, peaking at 10 min. DHEAS and ALLO increased catecholamine levels at 30 min. The effect of all three steroids was dose-dependent (Fig. 2). The EC50 values of DHEA, DHEAS, and ALLO for stimulation of dopamine secretion were 17 ± 1.9, 32 ± 3, and 15 ± 1.5 nM; and for stimulation of norepinephrine secretion, they were 26 ± 2.3, 72 ± 2.9, and 6 ± 1.2 nM (n = 3; P < 0.05). Conversely, testosterone and progesterone at 10–7 M were ineffective in PC12 cells (data not shown), an in vitro system not expressing functional androgen and progesterone receptors (13, 21, 22).
FIG. 2. Dose-response curves of neuroactive steroids on catecholamine secretion. A range of concentrations (10–11–10–5 M) of DHEA, DHEAS, and ALLO were used, and the concentration of dopamine and norepinephrine was measured in the culture media of PC12 cells, as per Fig. 1. Data are expressed as percentage of parallel controls, i.e. cells cultured in the absence of these steroids. Values represent mean ± SD of three separate experiments.
DHEAS and ALLO stimulate catecholamine production
We have also tested the effect of steroids for longer periods of time. More specifically, PC12 cells were incubated with DHEA, DHEAS, and ALLO (10–7 M) for 3–48 h, and the concentration of dopamine and norepinephrine in the culture media was measured. In contrast to its short-term effect, long-term DHEA did not significantly alter the levels of catecholamines (Figs. 3A and 4A). Conversely, incubation of PC12 cells with DHEAS or ALLO resulted in an increase of catecholamine levels, peaking at 36 h for dopamine and 24 h for norepinephrine. These long-term effects of DHEAS and ALLO suggest that these steroids, in addition to their acute effects on catecholamine secretion, may also affect the de novo production of catecholamines in PC12 cells. To test this hypothesis, we have measured the effects of neuroactive steroids in the absence and the presence of TH inhibitor AMPT or of AACD inhibitor NSD-1015, at 10–6 M. These enzymes hold a central role in the pathway of catecholamine de novo synthesis. Both inhibitors completely blocked the increase of catecholamine levels in the culture media of PC12 cells exposed for 3–48 h to 10–7 M DHEAS and ALLO (Fig. 3, B and C, for dopamine; and Fig. 4, B and C, for norepinephrine). These findings suggest that the long-term effects of DHEAS and ALLO on catecholamines are mainly exerted at the de novo production level.
FIG. 3. Effect of neuroactive steroids on dopamine production. PC12 cells were exposed to DHEA, DHEAS, and ALLO (10–7 M) for longer periods of time (3–48 h) in the absence or presence of the TH inhibitor AMPT or the AACD inhibitor NSD-1015, at 10–6 M. At the end of the incubation, dopamine levels were measured in the culture media. Data are expressed as nanograms per milligram of cellular protein. Values represent mean ± SD of four separate experiments. *, P < 0.01, denotes significant statistical difference compared with parallel controls, i.e. cells not exposed to the inhibitors.
FIG. 4. Effect of neuroactive steroids on norepinephrine production. PC12 cells were exposed to DHEA, DHEAS, and ALLO (10–7 M) for longer periods of time (3–48 h) in the absence or presence of the TH inhibitor AMPT or of the AACD inhibitor NSD-1015, at 10–7 M. At the end of the incubation, norepinephrine levels were measured in the culture media. Data are expressed as nanograms per milligram of cellular protein. Values represent mean ± SD of four separate experiments. *, P < 0.01, denotes significant statistical difference compared with parallel controls, i.e. cells not exposed to the inhibitors.
DHEA, DHEAS, and ALLO increase catecholamine secretion by acutely stimulating actin depolymerization and actin filament disassembly
Actin depolymerization and filament disassembly is required for catecholamine secretion because a subplasmalemmal actin mesh prevents their secretory vesicles from reaching their exocytosis sites (12, 16, 17). Based on these observations, we have tested whether DHEA, DHEAS, or ALLO affect catecholamine secretion in PC12 cells via the same mechanism. To test this hypothesis, we first examined the acute effects of DHEA, DHEAS, and ALLO on the ratio of G-monomeric to total cellular actin, an established marker of actin cytoskeleton dynamics (23). PC12 cells were cultured for short time periods (5–60 min) in control medium or in media containing the steroids at 10–7 M, then the ratio of G to total actin was measured, as previously described (12). All three steroids induced a rapid increase of the G/total actin ratio, reflecting actin depolymerization (Fig. 5A). DHEA and DHEAS were the most effective, increasing the G/total actin ratio within 5 min to 0.63 ± 0,01, compared with 0.46 ± 0.05 of parallel controls (n = 4; P < 0,001), or to 0.68 ± 0.03, compared with 0.59 ± 0.02 of the controls (n = 4; P < 0.05) for DHEA and DHEAS, respectively. Rising of G/total actin ratio by DHEA and DHEAS persisted for at least 60 min. ALLO significantly augmented G/total actin ratio within 5 min (0.63 ± 0.02, compared with 0.56 ± 0.03 of parallel controls; n = 4; P < 0.05); however, its effect was transient because it returned to basal levels within 15 min (Fig. 5A).
FIG. 5. Effects of neuroactive steroids on actin depolymerization (A) and actin filament disassembly (B). A, PC12 cells were cultured for short time periods (5–60 min) in control medium (in the absence of steroids) or in media containing DHEA, DHEAS, or ALLO at 10–7 M, then the ratio of G-monomeric (G) to total actin was measured, as described in Materials and Methods. Values represent mean ± SD of four separate experiments. *, P < 0.05, denotes significant statistical difference compared with controls. A, Confocal microscopy images of PC12 cell staining. PC12 cells were cultured for 10 min in control medium (in the absence of steroids) or in media containing DHEA, DHEAS, or ALLO at 10–7 M, then actin filaments were stained with rhodamine and observed under confocal laser microscopy. Section thickness was adjusted to 0.5 μm.
Figure 5B depicts the morphological alterations of PC12 cells exposed to the steroids. The confocal laser microscopy data supplement the results obtained by the measurement of the G/total actin ratio. Figure 5B depicts the 10-min steroid-mediated decrease in rhodamine-stained actin, confined mainly to the subplasmalemmal area beneath the cell membrane. In control cells (not exposed to any steroid), subplasmalemmal actin filaments were wider and more intense (Fig. 5B, Control).
The involvement of actin filaments in the effect of DHEA, DHEAS, and ALLO on catecholamine secretion was further supported by experiments using phallacidin, an actin filament stabilizer. PC12 cells were primed for 30 min with 10–6 M phallacidin; then DHEA, DHEAS, or ALLO were added to a final concentration of 10–7 M, and the incubation continued for an additional 10 min for DHEA and 30 min for DHEAS and ALLO. As shown in Fig. 6, phallacidin treatment almost completely prevented the stimulatory effect of DHEA, DHEAS, and ALLO on both dopamine and norepinephrine secretion. It should be noted that PC12 cells exposed to phallacidin alone did not have their basal catecholamine secretion altered (data not shown). Taken together, these findings support further the notion that actin microfilament redistribution plays a pivotal role in regulating the rapid catecholamine release, induced by the neuroactive steroids.
FIG. 6. Phallacidin prevents the stimulatory effect of neuroactive steroids on catecholamine secretion. PC12 cells were primed for 30 min in the presence of 10–6 M actin filament stabilizer phallacidin. Subsequently, DHEA, DHEAS, or ALLO was added to a final concentration of 10–7 M, and the incubation continued for an additional 10 min for DHEA and 30 min for DHEAS and ALLO. At the end of the incubation, the concentration of dopamine and norepinephrine was measured in the culture media. Data are expressed as percentage of parallel controls exposed to vehicles. Values represent mean ± SD of three separate experiments. *, P < 0.01, denotes significant statistical difference compared with parallel controls.
DHEAS and ALLO induce TH expression
As mentioned above, DHEAS and ALLO, in addition to their fast action, exhibited a long-term (up to 24 h) stimulatory effect on catecholamines, suggesting that they may also affect the de novo production of catecholamines. To test this hypothesis, we have studied their effects on TH protein and mRNA levels, i.e. the rate-limiting step of catecholamine biosynthesis. PC12 cells were incubated for 4–24 h in the absence or presence of DHEA, DHEAS, or ALLO at 10–7 M, and the protein levels of TH were measured in cell extracts by Western blot analysis. Levels of the TH protein were normalized against GAPDH levels. Results are depicted in Fig. 7. DHEA did not significantly affect TH levels at all time points tested. Conversely, DHEAS and ALLO increased TH levels in a time-dependent fashion, their effect starting as early as 2 h and peaking at 18 h.
FIG. 7. DHEAS and ALLO elevate the concentration of TH protein in cell lysates. PC12 cells were incubated for 4–24 h in the absence (control) or presence of DHEA, DHEAS, or ALLO at 10–7 M, and protein levels of TH were measured in cell extracts with Western blot analysis. Levels of the TH protein were normalized against GAPDH, presented as TH/GAPDH ratio. Values represent mean ± SD of three separate experiments. *, P < 0.01, denotes significant statistical difference compared with parallel controls.
Finally, we have assessed the effect of these steroids on TH mRNA levels, using a experimental protocol similar to that of TH protein. Levels of the TH mRNA were measured with semiquantitative RT-PCR and normalized against GAPDH mRNA levels. Results are depicted in Fig. 8A. As in the case of TH protein, DHEA did not alter TH mRNA levels at all time points tested, whereas DHEAS and ALLO increased TH mRNA levels in a time-dependent fashion. Their effect was detectable as early as 2 h, peaking at 6 h. More specifically, at 6 h, both DHEAS and ALLO increased the TH/GAPDH mRNA ratio by 4- to 5-fold. The maximal effect of DHEAS and ALLO on TH mRNA levels was further confirmed by real-time PCR. Indeed, at 6 h, DHEAS and ALLO resulted in a 4.84 ± 0.5 and 3.71 ± 0.8-fold (n = 3; P < 0.001) increase of TH mRNA levels, respectively, confirming the semiquantitative RT-PCR data (Fig. 8B).
FIG. 8. DHEAS and ALLO increase the mRNA levels of TH. A, RT-PCR data: PC12 cells were incubated for 2–24 h in the absence (control) or the presence of DHEA, DHEAS, or ALLO at 10–7 M, and the mRNA levels of TH were measured in cell extracts with RT-PCR analysis. Levels of the TH mRNA were normalized against GAPDH mRNA levels and are presented as TH/GAPDH ratio. Values represent mean ± SD of three separate experiments. *, P < 0.05, denotes significant statistical difference compared with controls. B, Real-time PCR data: PC12 cells were incubated for 6 h in the absence (control) or presence of DHEAS or ALLO at 10–7 M, and the mRNA levels of TH were measured in cell extracts with real-time PCR. Data are presented as fold increase compared with controls. Values represent mean ± SD of three separate experiments. *, P < 0.01, denotes significant statistical difference compared with controls. Rn, Normalized reporter signal.
Discussion
We have found that the neuroactive steroids DHEAS and ALLO stimulate the production of catecholamines from PC12 cells, a rat pheochromocytoma cell line used extensively as an in vitro model to study adrenal catecholamine production and secretion. The effect of these neuroactive steroids included stimulation of catecholamine secretion via an acute acceleration of actin filament depolymerization and induction of catecholamine synthesis via induction of TH expression. We consider that our findings have physiological relevance based on the anatomy of the human adrenal gland (rat adrenals do not produce DHEA). Indeed, the neuroactive steroid (DHEAS and ALLO)-producing human adrenocortical cells of zona reticularis and the catecholamine-producing adrenomedullary cells are interwoven, providing ample contact surfaces for potentially paracrine interaction (4). Adrenomedullary chromaffin cells can be found within all zones of the adrenal cortex, including zona reticularis. On the other hand, rims of reticularis cortical cells are present within the adrenal medulla. Our findings provide experimental in vitro evidence supporting the hypothesis of cortical-medullary interaction within the adrenals. Indeed, our data suggest that DHEA stimulates the secretion, whereas DHEAS and ALLO also stimulate the synthesis of catecholamines in our experimental model of PC12 cells. Our data indicate that the neuroactive steroids DHEAS and ALLO induce catecholamine secretion from PC12 cells in an acute and phallacidin-reversible manner, suggesting that these neuroactive steroids affect subplasmalemmal actin filament disassembly (24, 25). The subplasmalemmal actin filament ring represents a fast-response mechanism regulating the trafficking of secretory granules in several types of cells, including adrenomedullary cells (16, 17). Secretion of synaptic vesicles involves detachment from actin filaments and translocation toward their exocytosis sites. These steps require activation of severing proteins, causing transient filament disassembly. It should be noted that nicotine induces catecholamine secretion by provoking actin filament disassembly (14, 15). Furthermore, actin filament disruptors like cytochalasin promote basal and Ca2+-stimulated catecholamine secretion, whereas actin stabilizers like phallacidin inhibit it (25, 26, 27). Phallacidin binds polymerized-F-actin with high affinity, thus stabilizing the F-actin cytoskeleton by preventing depolymerization, an event necessary for the dynamic reordering of actin microfilaments. Ours is the first report on the effect of neuroactive steroids on subplasmalemmal actin filament disassembly. Showing that neuroactive steroids affect the actin cytoskeleton confirms and augments our data showing that these steroids exert a definite stimulatory effect on catecholamine secretion from adrenal chromaffin cells because the subcortical actin filament plays a central role in it. More specifically, our data show that DHEAS and ALLO acutely increase catecholamine secretion, their effect peaking at 10 min. The effect of these neuroactive steroids is preceded by an acute increase of monomeric G-actin, suggesting a rapid stimulatory effect on actin depolymerization and the subsequent destabilization of actin filaments. Our data on steroid actin filament destabilization were further confirmed by confocal laser microscopy, showing a steroid-mediated decrease in rhodamine-stained actin, confined to the subplasmalemmal area. Priming of PC12 cells with actin filament stabilizer phallacidin completely prevented the stimulatory effect of DHEAS and ALLO on norepinephrine and dopamine release from PC12 cells, suggesting that the effect of these steroids involves polymerized actin. The molecular mechanism by which these steroids modulate actin polymerization is yet not known. However, it should be noted that DHEA and pregnenolone have been recently reported to bind to several microtubule-associated proteins (28, 29). This observation points to a direct interaction between neuroactive steroids and actin-associated proteins. Furthermore, human ovarian granulosa cells exposed to DHEA contain fewer and less stable actin filaments (30). In our system, DHEAS and ALLO were highly potent in stimulating catecholamine secretion, with EC50 in the nanomole range, contrasting with the inhibitory effect of DHEAS on catecholamine secretion reported in bovine chromaffin cells, though at the suprapharmacological concentrations of 0.01–1 mM, known to alter, in a less specific manner, cellular organelles and cellular membranes (31). These findings considered together suggest that the effect of neuroactive steroids on catecholamine secretion may be cell type- and species-specific.
DHEAS and ALLO, but not DHEA, affected catecholamine synthesis in addition to their effect on catecholamine secretion. Indeed, these neuroactive steroids induced the expression of TH production, the rate-limiting enzyme of catecholamine biosynthesis as suggested by our RT-PCR, real-time PCR, and Western blot experiments, which showed that DHEAS and ALLO provoked a 4-fold increase of the concentration of its transcript and protein within 6 h and 8 h, respectively, pointing to a direct transcriptional effect on TH expression. In agreement with these data, the concentration of norepinephrine and dopamine in the culture media was significantly higher, compared with parallel control cells exposed only to the neuroactive steroid vehicles. Regulation of catecholamine production by DHEAS and ALLO was further supported by experiments with the specific inhibitors of TH and AACD, two key enzymes of catecholamine biosynthesis. Indeed, long-term effects of both steroids were completely blocked by TH inhibitor AMPT and AACD inhibitor NSD-1015. DHEA had no effect on TH expression, contrasting with a recent report showing with in situ hybridization an inhibitory effect of this steroid on TH mRNA levels in rat tuberoinfundibular neurons (32). However, in rat substantia nigra neurons, DHEA prevented the MPTP-induced TH mRNA decrease, measured by in situ hybridization (7). It thus appears that these neuroactive steroids, as in the case of catecholamine secretion, regulate catecholamine production and TH expression in a cell type-specific manner. DHEA was ineffective in increasing catecholamine levels at longer time periods. This finding suggests that DHEAS most probably represents the active form affecting catecholamine production, and that DHEA needs conversion to its sulfate ester to also become effective. The synthesis of sulfated DHEA is catalyzed by a cytosolic enzyme hydroxysteroid sulfotransferase (HST), which transfers the sulfonate moiety from the donor molecule 3'-phosphoadenosine 5'-phosphosulfate onto the 3-hydroxy acceptor site of DHEA. Previous studies have demonstrated the occurrence of HST-like activity in the nervous system of primates, rodents, and amphibians (33, 34, 35, 36). It is thus possible that adrenal medulla, an integral part of the peripheral nervous system, also expresses HST activity.
We believe that the possible physiological significance of our findings can be based on several published reports showing that, with advancing age, the intraadrenal and circulating levels of DHEAS decline in humans (37, 38, 39). Indeed, it has been calculated that by the age of 70, the circulating levels of DHEAS decrease by about 20% compared with young adults. It is of note that the release of epinephrine from the human adrenal medulla at rest was found to be lower in older men, 112 ng/ml compared with 248 ng/ml in younger men (40, 41). Furthermore, in younger men, the secretion of epinephrine doubles, or even triples, with mental stress, with isometric exercise, or dynamic exercise, compared with older men, who can master only 33% of the corresponding responses of younger men (40). We propose that the decline of DHEAS and ALLO production from zona reticularis of the adrenal cortex may affect catecholamine levels and the effectiveness of adrenal medulla to respond to sympathetic stimuli, particularly with advancing age. Our findings suggest that an intraadrenal paracrine regulatory loop is in action between adrenal neuroactive steroids and catecholamines that may be deregulated with advancing age. This hypothesis is supported by recent experimental findings in H295R human adrenocortical cells showing that synthetic catecholamine isoproterenol increases the secretion of DHEA dose-dependently (42). Similarly, isoproterenol stimulates DHEAS production from human fetal adrenocortical cells, in culture (43). Furthermore, in the central nervous system, neuroactive neurosteroids affect brain catecholamines. Thus, DHEAS potentiates NMDA-evoked norepinephrine secretion in rat hippocampal cells (6), whereas in the mouse, DHEA prevents MPTP-induced dopamine depletion in striatal neurons (7). In addition, ALLO increases the dopaminergic response to morphine in the rat nucleus accumbens (8), whereas its depletion from rat cortex potentiates stress-induced cortical dopamine output (9). Our findings supplement these reports, suggesting that DHEAS and ALLO may directly augment dopamine and norepinephrine levels in the brain, in addition to the above mentioned indirect effects, via a direct paracrine manner. Induction of dopamine or norepinephrine secretion and production by DHEAS and ALLO might contribute to the neuroprotective effects of these neurosteroids, further suggesting their involvement in the pathophysiology of neurodegenerative processes, such as Parkinson’s and Alzheimer’s disease (7, 44, 45).
The exact nature of the receptor systems mediating the stimulatory effect of DHEAS and ALLO on catecholamine production in chromaffin cells is unknown. In the central nervous system, DHEA up-regulates the NMDA receptors and/or down-regulates the GABAA receptors (1). Similarly, ALLO has been shown to affect GABAA receptors (1). In our experimental model, the effect of DHEA and ALLO should be independent of these receptor systems because PC12 cells do not express functional NMDA or GABAA receptors (46, 47). Generally, it is assumed that DHEA exerts its biological effects after its conversion into androgens or estrogens. In our in vitro model, we have previously shown that this is not the case, because no testosterone, estradiol, or estrone is detectable by immunoassays in the culture media of PC12 cells exposed to either DHEA or DHEAS (13). In addition, radiolabeled steroid binding assays have identified very low levels of intracellular estrogen receptors in cytosolic preparations of PC12 cells, whereas no progesterone or testosterone cytosolic receptors were detectable (13, 21, 22). The rapid onset of DHEAS and ALLO actions on catecholamine secretion in our model supports the hypothesis that these neuroactive steroids may use specific membrane receptors, although the participation of some yet-unidentified cytosolic receptors cannot be excluded. It should be mentioned here that specific membrane bindings sites have been recently reported for various steroids, including DHEA (48). These hypotheses are now under investigation.
Acknowledgments
We thank students Charoula Kontaki and Kostoula Troulinaki for their technical assistance.
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