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Endocrinology Vol. 143, No. 5 1748-1760
Copyright © 2002 by The Endocrine Society

INTRACELLULAR SIGNAL SYSTEMS

Activation of 5-HT7 Receptor in Rat Glomerulosa Cells Is Associated with an Increase in Adenylyl Cyclase Activity and Calcium Influx through T-Type Calcium Channels

Sebastien Lenglet, Estelle Louiset, Catherine Delarue, Hubert Vaudry and Vincent Contesse

European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, Institut National de la Santé et Recherche Médicale Unité-413, Unité Affiliée Centre National de la Recherche Scientifique, University of Rouen, 76821 Mont-Saint-Aignan, France

Address all correspondence and requests for reprints to: Dr. Hubert Vaudry, European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, Institut National de la Santé et de la Recherche Médicale Unité-413, Unité Affiliée Centre National de la Recherche Scientifique, University of Rouen, 76821 Mont-Saint-Aignan, France. E-mail: . hubert.vaudry{at}univ-rouen.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Serotonin (5-HT) stimulates aldosterone secretion from the rat adrenal gland through 5-HT7 receptors. The aim of the present study was to investigate the transduction mechanisms associated with activation of 5-HT7 receptors in rat glomerulosa cells. The stimulatory effect of 5-HT on aldosterone secretion and cAMP formation was significantly reduced by the 5-HT7 receptor antagonist LY 215840. Pretreatment of cells with the adenylyl cyclase inhibitor SQ 22536 or the PKA inhibitor H-89 markedly attenuated the effect of 5-HT on aldosterone secretion. Conversely, type 2 and 4 phosphodiesterase inhibitors potentiated the 5-HT-induced stimulation of aldosterone secretion. Administration of 5-HT in the vicinity of cultured glomerulosa cells induced a slowly developing and robust increase in cytosolic calcium concentration ([Ca2+]i). The effect of 5-HT on [Ca2+]i was suppressed by mibefradil, a T-type calcium channel blocker. Patch-clamp studies confirmed that 5-HT activated a T-type calcium current. Mibefradil also induced a dose-dependent inhibition of 5-HT-induced aldosterone secretion. The sequence of events associated with activation of 5-HT7 receptors was investigated. The PKA inhibitor H-89 markedly attenuated both the [Ca2+]i response and the activation of T-type calcium current induced by 5-HT. In contrast, reduction of the calcium concentration in the incubation medium did not affect 5-HT- induced cAMP formation. Preincubation of glomerulosa cells with cholera toxin abolished the stimulatory effect of 5-HT on aldosterone secretion, but pertussis toxin had no effect. Taken together, these data demonstrate that, in rat glomerulosa cells, activation of native 5-HT7 receptors stimulates cAMP formation through a Gs{alpha} protein, which in turn provokes calcium influx through T-type calcium channels. Both the adenylyl cyclase/PKA pathway and the calcium influx are involved in 5-HT-induced aldosterone secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THERE IS NOW clear evidence that various neurotransmitters and neuropeptides released locally within the adrenal cortex participate in the regulation of corticosteroid secretion (1, 2). In particular, it has been shown that serotonin, 5-hydroxytryptamine (5-HT), produced by chromaffin and/or mast cells, acts as a paracrine factor stimulating the activity of adrenocortical cells (3). In frogs and humans, the effect of 5-HT on adrenocortical cells is mediated through a 5-HT4 receptor subtype (4, 5, 6, 7). In rat, although the stimulatory effect of 5-HT on the adrenal cortex has been documented more than 40 yr ago (8), the type of receptor involved in the action of 5-HT has long remained elusive. By combining pharmacological and molecular approaches, we have recently demonstrated that the corticotropic effect of 5-HT on rat glomerulosa cells is mediated through 5-HT7 receptors (9). It is worth noting that this was the first report describing the occurrence of 5-HT7 receptors in endocrine glands and demonstrating their involvement in the control of hormonal secretions.

The 5-HT7 receptor has been cloned in several species including Xenopus laevis (10), mouse (11), rat (12, 13, 14), guinea pig (15), and human (16), and the expression of the 5-HT7 gene has been described in brain and peripheral tissues (17). Alternative splicing of the 5-HT7 receptor transcript has the potential to generate at least four receptor isoforms that differ in the length of the C-terminal tail (17). To date, the transduction mechanisms associated with activation of 5-HT7 receptors have only been investigated in heterologous expression systems (12, 14, 18, 19, 20). These studies have established that recombinant 5-HT7 receptors are positively coupled to adenylyl cyclase (AC) via a Gs{alpha} protein. Coexpression of the human 5-HT7(a) receptor with various isoforms of AC in human embryonic kidney 293 cells has shown that this receptor activates not only AC5, a typical Gs{alpha}-sensitive AC, but also AC1 and AC8, two Ca2+/calmodulin-sensitive ACs (19).

The rat glomerulosa cell, which is the first endocrine cell type expressing 5-HT7 receptors that has been described so far (17), appears to be a valuable model in which to investigate the second messenger systems associated with activation of native 5-HT7 receptors. A clear advantage of this model is that the effect of 5-HT7 receptor stimulation on various transduction pathways can be easily correlated with the physiological response, i.e. the secretion of aldosterone.

In the present study, we have investigated the signal transduction mechanisms recruited during stimulation of 5-HT7 receptors in rat glomerulosa cells, and we have determined the respective roles of AC activation and calcium influx in the stimulatory effect of 5-HT on aldosterone secretion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and tissue preparation
Male and female Wistar rats weighing 250–350 g were maintained under controlled conditions of temperature (22 C) under an established photoperiod (lights on from 0700 h to 1900 h). Rats had free access to laboratory chow (UAR, Epinay-sur-Orge, France) and water. All manipulations were performed according to the recommendations of the French ethical committee and under the supervision of authorized investigators. The animals were killed by decapitation between 0830 h and 0930 h. The adrenal glands were quickly removed and dissected free of adherent fat. For perifusion experiments, the adrenal cortex, separated from the medulla, was sliced and preincubated in HBSS (130 mM NaCl, 3.5 mM KCl, 1.8 mM CaCl2, 0.5 mM MgCl2, 2.5 mM NaHCO3, 5 mM HEPES, supplemented with 1 g/liter BSA, 1 g/liter glucose, and 1% of the antimycotic/antibiotic solution). The HBSS was gassed with a 95% O2/5% CO2 mixture, and the pH was adjusted at 7.4.

Reagents
5-HT, 9-(2-tetrahydrofuryl)adenine (SQ 22536), 6-chloro-9-[(4-diethylamino)-1-methylbutyl]amino-2-methoxyacridine (quinacrine), 1,2- dimethoxy-N-methyl[1,3]benzodioxolo[5,6-c]phenanthridinium chloride (chelerythrine), 1-methyl-3-isobutylxanthine, 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP), 4-(3-butoxy-4-methoxy-benzyl)-imidazolidin-2-one (Ro 20-1724), dipyridamole, 1,4-dihydro-5-2[2-propoxyphenyl]-7H-1,2,3-triazolo[4,5-d] pyrimidine-7-one (zaprinast), erythro-9-[2-hydroxy-3-nonyl]adenine (EHNA), BSA, nifedipine, cholera toxin (CTX), pertussis toxin (PTX) and the substance-P-related peptides pGlu-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH2 (GPAnt-1) and Arg-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH2 (GPAnt-2A) were purchased from Sigma (St. Louis, MO). Nimodipine and (±)-1-(2,5-dimethoxy-4-iodophenyl)-2 aminopropane (DOI) were obtained from RBI (Bioblock Scientific, Illkirsch, France). N-[2-(p-bromocinnamyl-amino)ethyl]-5-isoquinoline-sulfonamide (H-89) was obtained from ICN Biomedicals, Inc. (Orsay, France). The cAMP RIA kit and [1,2,6,7-3H]aldosterone were purchased from Amersham International (Les Ulis, France). Indo-1-acetoxymethyl ester was purchased from Molecular Probes, Inc. (Eugene, OR). LY 215840 was kindly provided by Lilly Research Laboratories (Indianapolis, IN). Mibefradil (dihydrochloride) was a gift from Hoffman-LaRoche Inc. (Basel, Switzerland). Initial solutions were made in HBSS or dimethylsulfoxide (DMSO), depending on the solubility of each compound. The maximum final concentration of DMSO was set below 0.02%, a concentration that was found to have no effect on spontaneous or 5-HT-induced aldosterone secretion.

Perifusion experiments
The effects of 5-HT and/or test substances on aldosterone secretion were studied by means of a perifusion technique, as described previously (9). Briefly, slices of rat adrenal cortex were layered between several beds of Bio-Gel P2 (Bio-Rad Laboratories, Inc., Richmond, CA) into perifusion chambers (equivalent of two adrenal glands/chamber). The adrenal tissue was continuously perifused with gassed HBSS at a constant flow rate (300 µl/min) and temperature (37 C). The glands were allowed to stabilize for 5 h before any test substance was added to reach a steady-state level of aldosterone secretion. After stabilization, the mean secretion rate of aldosterone in basal conditions was 251 ± 14 pg/min per adrenal. Test compounds were dissolved in gassed HBSS immediately before use and infused into the columns at the same flow rate as the HBSS alone, by means of a multichannel peristaltic pump (Desaga, Heidelberg, Germany). Fractions of effluent perifusate were collected every 5 min (1.5 ml/fraction), and the tubes were immediately frozen until the aldosterone assay.

Aldosterone concentration was determined by RIA, without prior extraction, in 100- to 200-µl aliquots from each fraction of perifusate. The specificity characteristics of the RIA have been reported previously (21). The intra- and interassay coefficients of variation were lower than 4% and 10%, respectively. The recovery of the assay was 95.2% ± 4.4%.

Preparation of adrenal glomerulosa cells
The adrenal capsules were separated from the inner zones and washed in DMEM (Sigma) supplemented with 1% of the antimycotic/antibiotic solution. The tissues were gently stirred for 30 min at 37 C in culture medium containing collagenase (1 mg/ml) and deoxyribonuclease I (25 µg/ml) (Sigma) in a 95% O2/5% CO2 air atmosphere. The tissues were disaggregated by gentle aspiration with a sterile 10-ml pipette, and dispersed cells were filtered on a nylon sieve (100-µm mesh opening). The cells were harvested by centrifugation (100 x g, 10 min), and the pellet was suspended in DMEM medium supplemented with 5% FCS. The residual tissues were subjected to a second period of digestion/dispersion as described above. Glomerulosa cells were plated onto glass coverslips in 35-mm Petri dishes or in 15-mm 24-well culture dishes (500 µl medium/well) and grown at 37 C in a humidified incubator with an atmosphere of 5% CO2/air. Experiments were performed 5 h after seeding.

cAMP measurement
Glomerulosa cells grown in 24-well culture dishes were preincubated in HBSS containing 100 µM 1-methyl-3-isobutylxanthine for 30 min at 37 C in a 95% O2/5% CO2 air atmosphere. Drugs and/or 5-HT was added to the HBSS medium and the cells were incubated for 60 min. The duration of the incubation period was determined, based on a previous report showing steady-state stimulation of cAMP formation 60–120 min after the onset of 5-HT stimulation (22). The reaction was stopped by addition of 500 µl of 20% ice-cold trichloroacetic acid. After centrifugation (14,000 x g, 15 min, 4 C), the supernatant was recovered, washed with water-satured diethyl ether, and lyophilized. The concentration of cAMP contained in the dried extract was measured by using a commercial kit (Amersham International). The pellet was used for protein determination by the method of Lowry, using BSA as a standard.

Measurement of cytosolic calcium concentration
To determine the effect of test substances on intracellular calcium concentration ([Ca2+]i), single rat glomerulosa cells were monitored by microfluorimetry as previously described for human and frog adrenocortical cells (23, 24). Rat glomerulosa cells were incubated in the dark for 45 min at 37 C with 5 µM indo-1/AM in DMEM. The cells were washed twice with fresh medium and the Petri dish was fitted to the stage of a Diaphot inverted microscope Nikon (Melville, NY). The [Ca2+]i was monitored at 37 C using the microscope in the epifluorescence mode with an oil-immersion objective (X 100 CF Fluor series). Test substances were ejected in the vicinity of the cells by a pressure ejection system. The concentrations indicated are those contained in the ejection pipette, which is necessarily much higher than that occurring at the cell surface. The emitted fluorescence of indo-1, induced by excitation at 355 nm (Xenon lamp), was collected by two separate photometers (P1, Nikon) centered at 405 (for the Ca2+-complexed form) and 480 nm (for the Ca2+-free form). The 405 nm/480 nm ratio (R) was determined using an AS1-type acquisition card (Notocord Systems, Croissy-sur-Seine, France). All three signals (i.e. 405 nm, 480 nm, and R) were continuously recorded with the JAD-FLUO program (version 1.2, Notocord Systems). [Ca2+]i was calculated according to the equation of Grynkiewicz et al. (25): [Ca2+]i = Kd x ß[R-Rmin/Rmax-R] where Kd is the dissociation constant for indo-1 (250 nM), ß the ratio between the minimal and maximal [Ca2+]i at 480 nm, Rmin the fluorescence ratio obtained after incubation of cells for 90 min with 10 mM EGTA and 1 µM ionomycin, and Rmax the fluorescence ratio obtained after incubation of cells for 90 min with 10 mM CaCl2 and 1 µM ionomycin. For rat glomerulosa cells, the average values of Rmin, Rmax and ß were 0.090 ± 0.003 (n = 37), 0.839 ± 0.055 (n = 11), and 3.23 (n = 11), respectively.

Recording of calcium currents
Electrophysiological recordings were performed at room temperature on freshly dispersed rat glomerulosa cells by using the patch-clamp technique in the whole-cell configuration. The culture dishes were continuously superfused (2 ml/min) with an extracellular saline solution containing 140 mM NaCl, 1.2 mM CaCl2, 1.2 mM MgCl2, 4 mM KCl, 10 mM HEPES-NaOH, 5.5 mM glucose, pH 7.4. Calcium currents were recorded by using a high barium solution containing 150 mM tetraethylammonium chloride, 10 mM BaCl2, 10 mM HEPES-CsOH, 5.5 mM glucose, pH 7.4. Patch pipettes (3–5 M{Omega}) were filled with an intracellular saline solution containing 120 mM CsCl, 2 mM MgCl2, 10 mM EGTA, 2 mM ATP, 0.2 mM GTP, 10 mM HEPES-CsOH, pH 7.4. In some experiments, GPAnt-1 and GPAnt-2A were dissolved in the internal saline solution at a final concentration of 30 µM. Electric signals were amplified with an Axopatch 200B (Axon Instruments, Foster City, CA) at a holding potential of -80 mV. The currents were filtered at 5 kHz and sampled at 2 kHz with a Digidata 1200 interface and pClamp 6.02 software (Axon Instruments). Leak was subtracted by a P/4 protocol.

Data analysis
Each perifusion pattern was established as the mean profile of aldosterone secretion (±SE) calculated from at least three independent experiments. The concentration-response curves were fitted using the Prism program (GraphPad Software, Inc., San Diego, CA), and maximum response (Emax) and pEC50 values [negative logarithm of the 50% effective concentration (EC50), the agonist concentration producing 50% of the maximum aldosterone secretion] were derived from this analysis. To determine the affinity of the 5-HT7 receptor antagonist LY 215840, EC50 ratios from the 5-HT concentration-response curves on aldosterone secretion were calculated in the absence or presence of three increasing concentrations of the antagonist. The data were analyzed according to the procedure of Arunlakshana and Schild (26) to determine the pA2 value. The effect of LY 215840 on 5-HT-induced cAMP formation was also investigated. In this case, the apparent antagonist dissociation constant (pKB) for a single antagonist concentration was calculated using the method of Furchgott (27). Statistical significance was determined using unpaired t test, and comparisons among groups were performed by ANOVA, with post hoc Tukey’s test using the Instat version 3.01 program (GraphPad Software, Inc.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of the 5-HT7 receptor antagonist LY 215840 on 5-HT-induced aldosterone secretion and 5-HT-induced cAMP production
As previously described (9), graded concentrations of 5-HT (10 nM to 10 µM) induced a dose-dependent stimulation of aldosterone secretion from perifused rat adrenocortical slices with a mean pEC50 of 7.44 ± 0.03. The 5-HT7 receptor antagonist LY 215840 (1–31 nM) had no effect on basal aldosterone secretion (data not shown) but shifted the 5-HT response curve to the right in a concentration-dependent manner (Fig. 1Go). Schild analysis yielded linear regression with pA2 value of 8.85. The slope of the Schild regression (0.94 ± 0.15) was not significantly different from unity, suggesting competitive antagonism (Fig. 1Go, inset).



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  		Figure 1. Effect of the 5-HT7 receptor antagonist LY 215840 on 5-HT-induced stimulation of aldosterone secretion by perifused rat adrenocortical slices. Concentration-response curves showing the amplitude of the aldosterone secretion responses to 5-HT in the absence ({bullet}) or presence of 1 nM ({circ}), 10 nM ({triangleup}), or 31 nM ({triangledown}) LY 215840. Each curve represents the mean ± SE of five independent experiments. Inset, Schild plot revealed a slope of the Schild regression of 0.94. The x-axis intercept represents the pA2 value according to Arunlakshana and Schild (26 ). The number of independent experiments is indicated in parentheses.

 
Incubation of freshly dispersed rat glomerulosa cells with graded concentrations of 5-HT (10 nM to 10 µM) induced a dose-related increase in cAMP formation with a mean pEC50 of 7.60 ± 0.08 and a maximum stimulation of 46.6% ± 0.5% over basal level (Fig. 2Go). The 5-HT7 receptor antagonist LY 215840 (100 nM) shifted the concentration-response curve of 5-HT to the right yielding a mean pEC50 of 6.32 ± 0.21 (Fig. 2Go). The average pKB value of LY 215840 on 5-HT-induced cAMP formation was 8.26.



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  		Figure 2. Effect of the 5-HT7 receptor antagonist LY 215840 on 5-HT-induced stimulation of cAMP formation in rat glomerulosa cells. The effect of graded concentrations of 5-HT on cAMP production was studied in the absence ({bullet}) or presence ({circ}) of 100 nM LY 215840. The pKB value of LY 215840 was calculated according to Furchgott (27 ). Results are expressed as the percentage of the basal level of cAMP production. The mean basal level of cAMP production was 1.82 ± 0.02 pmol cAMP/105 cells/h. Results are the mean ± SE of six independent experiments performed in triplicate.

 
Effects of AC, PKA, and phosphodiesterase (PDE) inhibitors on 5-HT-induced aldosterone secretion
Prolonged infusion (30 min) of the AC inhibitor SQ 22536 or the PKA inhibitor H-89 had no effect on spontaneous aldosterone secretion (data not shown). In contrast, SQ 22536 (500 µM) induced a significant (P < 0.05) inhibition of the Emax of 5-HT on aldosterone secretion (Fig. 3AGo). Similarly, H-89 induced a significant (P < 0.001) and concentration-dependent inhibition of the Emax of 5-HT on aldosterone secretion (Fig. 3BGo). The possible involvement of cAMP or cGMP in 5-HT-induced aldosterone secretion was also investigated using selective PDE inhibitors (Fig. 3CGo). The nonspecific cAMP/cGMP PDE2 inhibitor EHNA (25 µM) significantly (P < 0.05) potentiated the stimulatory effect of 5-HT on aldosterone output. Similarly, the cAMP-specific PDE4 inhibitor Ro 20-1724 (20 µM) significantly (P < 0.05) enhanced the Emax of 5-HT. In contrast, the cGMP-specific PDE5,6 inhibitors zaprinast (10 µM) and dipyridamole (10 µM) had no effect on 5-HT-induced aldosterone secretion (Fig. 3CGo). Administration of the PKC inhibitor chelerythrine (10 µM) or the PLA2 inhibitor quinacrine (100 µM) did not affect 5-HT-induced aldosterone secretion (data not shown).



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  		Figure 3. Effect of AC, PKA, and PDE inhibitors on 5-HT-induced stimulation of aldosterone secretion by perifused rat adrenocortical slices. A, Effects of graded concentrations of the AC inhibitor SQ 22536 on aldosterone secretion stimulated by 1 µM 5-HT. The increase in aldosterone secretion induced by 1 µM 5-HT (100% of maximum secretion) was 979 ± 134 pg/min per adrenal. Results are the mean ± SE of three independent experiments. *, P < 0.05. B, Concentration-response curves showing the amplitude of 5-HT-induced aldosterone secretion in the absence ({bullet}) or presence of 10 µM ({circ}) or 30 µM ({triangleup}) of H-89. Each curve represents the mean ± SE of at least three independent experiments. ***, P < 0.001. C, Effects of a nonspecific cAMP/cGMP PDE inhibitor (EHNA), a cAMP-specific PDE inhibitor (Ro 20-1724) and two cGMP-specific PDE inhibitors (zaprinast, dipyridamole) on 5-HT-induced stimulation of aldosterone secretion. The increase in aldosterone secretion induced by 1 µM 5-HT (100% of maximum secretion) was 833 ± 40 pg/min per adrenal. Results are the mean ± SE of at least three independent experiments. *, P < 0.05.

 
Effect of 5-HT on [Ca2+]i
The mean [Ca2+]i in cultured rat glomerulosa cells under resting conditions was 88.5 ± 12.9 nM (14 cells). As previously reported (28), the cells did not exhibit any spontaneous oscillations. Application of 5-HT (10 µM) in the vicinity of glomerulosa cells induced a substantial increase in [Ca2+]i from 90.6 ± 18.3 nM to 403.4 ± 87.4 nM (10 cells). The [Ca2+]i response to 5-HT developed slowly, peaked only within 14 ± 2 min (10 cells), and then [Ca2+]i declined gradually (Fig. 4AGo). In contrast, application of 10 mM KCl on cultured glomerulosa cells induced an immediate increase in [Ca2+]i (which developed within only 1–2 sec) followed by a sustained plateau phase (Fig. 4AGo, inset). The potent and selective 5-HT2 receptor agonist DOI (10 µM) did not affect [Ca2+]i in glomerulosa cells, whereas 5-HT induced a robust increase in the same cells (Fig. 4BGo).



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  		Figure 4. Effect of 5-HT on [Ca2+]i in cultured rat glomerulosa cells. A, Typical profile illustrating the effect of a single application of 5-HT (10 µM, 30 sec). Inset, Application of KCl (10 mM, 30 sec) induced an immediate increase in [Ca2+]i. B, Typical profile illustrating the effects of a single application of the 5-HT2 receptor agonist DOI (10 µM, 30 sec) and 5-HT on the same cell (10 µM, 30 sec). Inset, No effect of DOI (10 µM, 30 sec) was observed for up to 6 min after application of the compound (Fig. 4BGo, inset). The arrows indicate the onset of administration of each test substance. The number of cells studied is indicated in parentheses.

 
To assess the possible involvement of extracellular calcium in 5-HT-induced [Ca2+]i rise, EGTA-supplemented medium was added before or at the peak of the calcium response. After a 10-min incubation period with 10 mM EGTA-containing DMEM (reducing the concentration of free extracellular calcium from 1.8 mM to 6 nM), the stimulatory effect of 5-HT on [Ca2+]i was totally suppressed (Fig. 5AGo). Addition of EGTA during the calcium response provoked a rapid decrease of 5-HT-evoked [Ca2+]i that returned to basal value within 1–2 min (Fig. 5BGo). Application of the T-type calcium channel blocker mibefradil (1 µM) also induced an immediate fall in the [Ca2+]i response induced by 5-HT (Fig. 5CGo), and the kinetics of the [Ca2+]i decline was very similar to that observed with EGTA (Fig. 5Go, B and C).



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  		Figure 5. Effect of EGTA and mibefradil on 5-HT-induced [Ca2+]i rise in cultured rat glomerulosa cells. A, Typical profiles illustrating the effect of a single application of 5-HT (10 µM, 30 sec) in normal DMEM (control) and 10 min after preincubation in EGTA (10 mM)-supplemented DMEM (EGTA). B, C, Typical profiles illustrating the effects of EGTA (10 mM, 30 sec) (B) or mibefradil (1 µM, 30 sec) (C) ejected during the peak calcium response induced by a single application of 5-HT (10 µM, 30 sec). The arrows indicate the onset of administration of each test substance. The number of cells studied is indicated in parentheses.

 
Effect of 5-HT on calcium currents
Electrophysiological recordings were performed to further characterize the type of calcium current activated by 5-HT in rat glomerulosa cells. Increasing voltage pulses from a holding potential of -80 mV evoked transient inward barium currents for depolarizations higher than -60 mV (Fig. 6AGo). Stronger depolarizations elicited faster rising and inactivating inward currents but did not activate sustained inward current. The current-voltage (I-V) relationships showed that the barium current reached a maximum amplitude at -30 mV (-137 ± 18 pA, 11 cells), and this current was abrogated by Ni2+ (40 µM, 6 min) or mibefradil (1 µM, 17 min) (Fig. 6BGo). As previously described (29), all cells studied displayed low voltage-activated calcium currents, which exhibited the typical biophysical and pharmacological properties of the T-type current. The absence of high voltage-activated L-type Ca2+ current was confirmed by testing the effect of L-type channel blockers. Perfusion of nifedipine (1 µM, 6 min) had no effect on the barium currents evoked by depolarizing pulses at -30 mV (Fig. 6CGo). Similarly, nimodipine (1 µM, 6 min) did not modify the barium currents evoked by depolarizing pulses at -30 mV or 0 mV (Fig. 6DGo). In contrast, mibefradil (1 µM, 6 min) (Fig. 6Go, C and D) or Ni2+ (40 µM, 6 min) (Fig. 6EGo) abrogated the inward barium currents.



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  		Figure 6. Effect of Ca2+ channel blockers on voltage-activated barium currents in rat glomerulosa cells. A, Superimposed Ba2+ currents elicited by increasing depolarizing pulses (-60 mV to +20 mV, 10-mV increments, 200 msec) from a holding potential of -80 mV. B, I-V relationships of the maximal Ba2+ currents shown in A before ({bullet}) and during superfusion with Ni2+ (40 µM, 6 min) ({triangleup}) or mibefradil (1 µM, 17 min) ({circ}). C, D, E, Superimposed current traces illustrating the effects of nifedipine (1 µM, 6 min) or mibefradil (1 µM, 6 min) (C), nimodipine (1 µM, 6 min) or mibefradil (1 µM, 6 min) (D), and Ni2+ (40 µM, 6 min) (E) on Ba2+ currents generated by depolarizing pulses from -80 mV to -30 mV (C–E, upper traces) and from -80 mV to 0 mV (D, E lower traces).

 
The effect of 5-HT (10 µM) was tested on the T-type calcium current expressed by rat glomerulosa cells (Fig. 7Go). Application of 5-HT (10 µM, 4 min) enhanced the amplitude of barium currents evoked by increasing voltage pulses more positive than -60 mV (Fig. 7AGo). The I-V relationships of the barium currents revealed that the stimulatory effect of 5-HT occurred without modification of the activation threshold and was maximum at -30 mV (212% ± 24%, 12 cells) (Fig. 7BGo). Kinetic analysis of the 5-HT-induced variation of barium currents elicited by voltage pulses at -30 mV showed that the maximum amplitude of the current occurred 12.5 ± 2.3 min after the application of 5-HT, and then the current gradually declined during the next 30 min (Fig. 7CGo). Perfusion of mibefradil (1 µM, 11 min) or Ni2+ (40 µM, 4 min) inhibited the stimulatory effect of 5-HT on the barium currents enhanced by 5-HT (Fig. 7DGo).



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  		Figure 7. Effect of 5-HT on the T-type calcium current in rat glomerulosa cells. A, Activation of barium currents by increasing depolarizing pulses from a holding potential of -80 mV in the absence (control) or presence of 10 µM 5-HT. B, I-V relationships of the maximal T-type calcium currents recorded before ({bullet}) and during superfusion with 10 µM 5-HT ({circ}) shown in A. C, Time course of the effect of 10 µM 5-HT on the maximal amplitude of the T-type calcium current. The number of cells studied is indicated in parentheses. D, Superimposed current traces illustrating the effects of mibefradil (1 µM, 11 min, left) or Ni2+ (40 µM, 4 min, right) on the stimulatory effect of 5-HT on the barium depolarizing pulses from -80 mV to -30 mV.

 
Involvement of calcium in aldosterone secretion evoked by 5-HT
The calcium dependence of the 5-HT-induced aldosterone secretion was investigated by modifying the extracellular calcium concentration ([Ca2+]e) in the HBSS. In normal HBSS ([Ca2+]e = 1.8 mM), 5-HT induced a 9-fold increase in aldosterone secretion from perifused adrenocortical slices (Fig. 8Go). Reduction of [Ca2+]e in the HBSS attenuated the Emax of 5-HT on aldosterone secretion in a concentration-dependent manner (Fig. 8Go). Reduction of [Ca2+]e from 1.8 to 1 mM decreased by 50% the Emax of 5-HT on aldosterone secretion. In Ca2+-free medium, a 6-fold decrease of 5-HT-induced aldosterone secretion was observed (Fig. 8Go). In contrast, reduction of [Ca2+]e did not significantly affect the potency of 5-HT on aldosterone secretion (Fig. 8Go).



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  		Figure 8. Effect of [Ca2+]e changes on 5-HT-induced stimulation of aldosterone secretion by perifused rat adrenocortical slices. Concentration-response curves showing the amplitude of the aldosterone secretion response to 5-HT in normal medium ([Ca2+]e = 1.8 mM) or in Ca2+-depleted perifusion solutions. The number of independent experiments is indicated in parentheses.

 
Administration of graded concentration of the T-type calcium channel blocker mibefradil (10-9 M to 10-5 M) induced a dose-dependent inhibition of the Emax of the 5-HT-evoked aldosterone secretion without affecting the potency of 5-HT (Fig. 9AGo). At a very low concentration (10-8 M), mibefradil reduced by 38% (P < 0.001) the stimulatory effect of 5-HT on aldosterone secretion and the 50% inhibitory concentration (IC50) value for mibefradil was 0.14 µM (Fig. 9BGo). A series of experiments similar to those presented in Fig. 9AGo was conducted with the L-type calcium channel blocker nifedipine. At concentrations of 10-7 and 10-6 M, nifedipine had no effect on 5-HT-induced aldosterone secretion. At a high concentration (10-5 M), nifedipine induced a 40% decrease of the maximal effect of 5-HT on aldosterone secretion (Fig. 9BGo). Neither mibefradil nor nifedipine induced any modification of basal aldosterone secretion (data not shown).



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  		Figure 9. Effect of voltage-sensitive calcium channel blockers on 5-HT-induced stimulation of aldosterone secretion by perifused rat adrenocortical slices. A, Concentration-response curves showing the amplitude of the secretory response to 5-HT in the absence (control) or presence of graded concentrations of the T-channel blocker mibefradil. The number of independent experiments is indicated in parentheses. B, Semilogarithmic plot comparing the effect of increasing concentrations of the T-type calcium channel blocker mibefradil and the L-type calcium channel blocker nifedipine on 5-HT-induced stimulation of aldosterone secretion. Experimental values were calculated from data similar to those presented in (A). The increase in aldosterone secretion induced by 1 µM 5-HT (100% of maximum secretion) was 923 ± 69 pg/min per adrenal. **, P < 0.01; ***, P < 0.001.

 
Involvement of the cAMP/PKA pathway in calcium influx induced by 5-HT
Reduction of [Ca2+]e from 1.8 mM (control condition) to 1 mM did not significantly affect the concentration-response curve of 5-HT on cAMP production (Fig. 10Go). Similarly, mibefradil (1 µM) did not impair the stimulatory effect of 5-HT on cAMP production (data not shown).



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  		Figure 10. Effect of [Ca2+]e changes on 5-HT-induced stimulation of cAMP formation in rat glomerulosa cells. Concentration-response curves showing the effect of 5-HT on cAMP production in normal medium ([Ca2+]e = 1.8 mM) or in Ca2+-depleted medium ([Ca2+]e = 1 mM). The mean basal level of cAMP production was 0.77 ± 0.04 pmol cAMP/105 cells/h. Results are the mean ± SE from three independent experiments performed in triplicate.

 
Microfluorimetric recordings showed that the stimulatory effect of 5-HT on [Ca2+]i was mimicked by 8-Br-cAMP (100 µM), a permeant analog of cAMP (Fig. 11AGo). Reciprocally, application of H-89 (10 µM) during the 5-HT-evoked calcium influx caused a rapid decline of [Ca2+]i (Fig. 11BGo). In contrast, H-89 had no effect on basal [Ca2+]i (data not shown). Patch-clamp recordings revealed that application of 8-Br-cAMP (100 µM) enhanced the amplitude of barium currents evoked by depolarizing pulses at -30 mV (Fig. 11CGo), whereas application of H-89 (10 µM) during the 5-HT-induced activation of the T-type calcium current caused a marked reduction of the amplitude of this current (Fig. 11DGo).



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  		Figure 11. Effect of 8-Br-cAMP and the PKA inhibitor H-89 on 5-HT-induced calcium influx in cultured rat glomerulosa cells. A, Microfluorimetric recording illustrating the effect of 8-Br-cAMP (100 µM, 30 sec) on [Ca2+]i. B, Microfluorimetric recording illustrating the effects of H-89 (10 µM, 1 min) ejected during the peak calcium response induced by a single application of 5-HT (10 µM, 30 sec). Dotted line, Typical profile illustrating the effect of a single application of 5-HT (10 µM, 30 sec) in a control experiment. The arrows indicate the onset of administration of each test substance. C, Time course of the effect of 8-Br-cAMP (100 µM, 5 min) on the amplitude of T-type calcium currents evoked by depolarizing pulses from -80 mV to -30 mV. D, Time course of the effect of H-89 (10 µM, 7 min) on the 5-HT (10 µM, 4 min)-induced activation of T-type calcium currents elicited by pulses from -80 mV to -30 mV ({circ}). The effect of 5-HT alone (10 µM, 4 min) is shown as a control ({bullet}). The number of cells studied is indicated in parentheses.

 
As described above, reduction of [Ca2+]e from 1.8 mM to 1 mM provoked a 46% decrease of aldosterone secretion induced by 5-HT, and infusion of 10 µM H-89 reduced by 34% the 5-HT-evoked aldosterone secretion (Fig. 12Go). Concomitant administration of 10 µM H-89 in 1 mM Ca2+- containing medium provoked a 69% decrease in 5-HT- induced aldosterone secretion, i.e. an effect that was significantly greater than each individual effect (Fig. 12Go).



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  		Figure 12. Effect of depletion of calcium in the incubation medium ([Ca2+]e) and application of the PKA inhibitor H-89 on 5-HT-induced stimulation of aldosterone secretion by perifused rat adrenocortical slices. The results are expressed as a percentage of the response induced by 5-HT in control conditions ([Ca2+]e = 1.8 mM in the absence of H-89). The increase in aldosterone secretion induced by 1 µM 5-HT (100% of maximum secretion) was 831 ± 46 pg/min per adrenal). Results are the mean ± SE of 3 to 11 independent experiments.

 
Effects of G protein toxins and blockers
Pretreatment of glomerulosa cells with PTX (1 µg/ml) for 5 h did not affect the spontaneous (Fig. 13AGo, inset) or the 5-HT-induced aldosterone secretion levels (Fig. 13AGo). In contrast, pretreatment of glomerulosa cells with CTX (1 µg/ml) for 5 h produced a robust increase (368% ± 54%) in basal aldosterone secretion (Fig. 13AGo, inset) and, in CTX-treated cells, 5-HT had no significant effect on aldosterone secretion (Fig. 13AGo).



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  		Figure 13. Effects of G protein toxins and blockers on 5-HT responses on rat glomerulosa cells. A, Effects of G protein toxins on 5-HT-induced stimulation of aldosterone secretion by dispersed rat glomerulosa cells. The effect of graded concentrations of 5-HT on aldosterone secretion was studied on cells preincubated for 5 h in the absence (control) or in the presence of 1 µg/ml CTX or 1 µg/ml PTX. The results are expressed as a percentage of basal aldosterone secretion in each condition. Inset, Basal levels of aldosterone secretion from cells preincubated for 5 h in the absence (control) or in the presence of CTX and PTX. The data are the mean ± SE of quadruplicate determinations from two independent experiments. B, Time course of the effect of 5-HT (10 µM, 5 min) on the T-type calcium current evoked by pulses from -80 mV to -30 mV recorded on different cells dialyzed with GPAnt-1 (30 µM, {circ}) or GPAnt-2A (30 µM, {bullet}). The number of cells studied is indicated in parentheses.

 
To further investigate the involvement of G proteins associated with 5-HT7 receptors in rat glomerulosa cells, specific G protein blockers were dialyzed through the patch pipette. Intracellular application of GPAnt-2A (30 µM), a Gq/11 protein antagonist (30), did not affect the 5-HT-induced T-type calcium currents (Fig. 13BGo, three cells). In contrast, application of GPAnt-1 (30 µM), a Go/i/s protein antagonist (31), abrogated the response to 5-HT (Fig. 13BGo, three cells).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have recently shown that the stimulatory effect of 5-HT on the rat adrenal gland is mediated through activation of 5-HT7 receptors (9). The present study now demonstrates that, in rat glomerulosa cells, activation of native 5-HT7 receptors stimulates AC activity via a CTX-sensitive G protein and calcium influx through T-type calcium channels.

Until recently, no specific ligands for the 5-HT7 receptor were available and the pharmacological characterization of the receptor involved in the effect of 5-HT on aldosterone secretion has been performed by using a series of nonselective agonists and antagonists (9). We now show that the newly developed high-affinity 5-HT7 receptor antagonist LY 215840 (17, 32) inhibits 5-HT-induced stimulation of aldosterone production from rat adrenocortical slices and cAMP formation in rat glomerulosa cells. The potency of LY 215840 to inhibit 5-HT-induced steroidogenesis (pA2 = 8.85) and 5-HT-induced cAMP accumulation (pKB = 8.26) were similar. The slight difference observed between these two values can be ascribed to the different approaches used, i.e. dynamic incubation of tissue slices for monitoring aldosterone secretion vs. static incubation of dispersed cells for measurement of cAMP formation. These two potency values were also in the same range as that reported for the blockage of 5-HT-evoked relaxation in canine coronary artery (pA2 = 8.3) (33). It is also interesting to note that besides the 5-HT7 receptor, several other serotonergic receptor subtypes are also positively coupled to the adenylyl cyclase signaling pathway, i.e. 5-HT4 and 5-ht6 receptors (34). However, the contribution of these two latter receptors to 5-HT-evoked stimulation of rat glomerulosa cells can be excluded inasmuch as: 1) the 5-HT4 receptor agonist zacopride is totally devoid of effect on cAMP production (data not shown) and 2) by using specific antibodies against the 5-ht6 receptor, we have not observed any 5-ht6-immunoreactive materiel in the zona glomerulosa (data not shown). Altogether, these data provide additional evidence for the presence of functional 5-HT7 receptors in rat glomerulosa cells and for their involvement in the stimulatory effect of 5-HT on cAMP production and aldosterone secretion.

Although several studies have demonstrated that activation of 5-HT7 receptors expressed in transfected cells stimulates AC activity, the pEC50 values for 5-HT on cAMP formation can vary over three orders of magnitude (from 6 to 9.25) depending on the cell line used for heterologous expression of recombinant receptors (13, 16, 18, 20). The wide variations in the potency of 5-HT can be accounted for by the differential expression of Gs{alpha} protein, AC isoforms and/or receptor level in the various cell lines (35). Very little information is currently available concerning the functional properties of native 5-HT7 receptors. The present study revealed that, in rat glomerulosa cells, 5-HT stimulates cAMP formation with a potency (pEC50 = 7.60 ± 0.08) very similar to that recently reported for 5-HT-induced cAMP formation in the guinea pig hippocampus (pEC50 = 7.70 ± 0.10) (36). However, in this latter model, the physiological significance of the presence of 5-HT7 receptors remains to be determined.

An advantage of glomerulosa cells as a model for studying the transduction mechanisms associated with the activation of 5-HT7 receptors is that the effects of 5-HT on second messenger systems can be easily correlated with the final response of the cells, i.e. aldosterone secretion. The fact that the potencies of 5-HT on aldosterone secretion and cAMP formation were closely related, suggested that cAMP is functionally involved in 5-HT-induced aldosterone secretion. To test this hypothesis, we have investigated the contribution of the AC/PKA pathway on 5-HT-evoked aldosterone secretion. The stimulatory effect of 5-HT was inhibited in a concentration-dependent manner by SQ 22536 (37) or H-89. Reciprocally, the effect of 5-HT on aldosterone secretion was potentiated by the cAMP-specific PDE4 inhibitor Ro 20-1724 and by the cAMP/cGMP PDE2 inhibitor EHNA. Previous studies have demonstrated that Ro 20-1724 potentiates the inhibition of porcine myometrial contractility induced by the 5-HT7 receptor activation (38) and that EHNA potentiates the increase in cAMP production induced by ACTH in the rat and human adrenal gland (39). Thus, the present data provide evidence that, in rat glomerulosa cells, 5-HT7 receptors are positively coupled to the AC/PKA pathway and that PDE2 and PDE4 are involved in the regulation of 5-HT-stimulated levels of cAMP.

We next investigated the possible involvement of calcium in the response of glomerulosa cells to 5-HT7 receptor activation. We first observed that application of 5-HT in the vicinity of cells induced a slowly developing but robust increase in [Ca2+]i. The profile of the [Ca2+]i response was very similar to that observed after administration of ACTH in rat, bovine, and human glomerulosa cells (28, 40). The observations that preincubation of rat glomerulosa cells with EGTA totally suppressed the stimulatory effect of 5-HT on [Ca2+]i, and that addition of EGTA to the incubation medium during the calcium wave rapidly and completely abolished the 5-HT-evoked Ca2+ response indicate that the increase in [Ca2+]i associated with 5-HT7 receptor activation can be ascribed only to calcium influx, as previously shown for ACTH receptors (28, 40).

Consistent with the stimulus-secretion coupling concept, the Emax of 5-HT on aldosterone secretion, but not the potency (pEC50), was highly dependent on [Ca2+]e. The type of channel responsible for the 5-HT-evoked calcium influx was investigated by using the microfluorimetry and patch-clamp approaches. The T-type calcium channel blocker mibefradil, which has been reported to inhibit T-type channels at concentrations 10-fold lower than that necessary for L-type channels inhibition (41, 42), totally abrogated the increase in [Ca2+]i induced by 5-HT. Mibefradil also caused a concentration-dependent inhibition of 5-HT-induced aldosterone secretion (IC50 {approx} 0.1 µM), whereas the L-type calcium channel blocker nifedipine, at concentrations up to 1 µM, was devoid of effect. The fact that mibefradil did not affect 5-HT-evoked cAMP formation (data not shown) indicates that mibefradil did not directly interact with the serotonergic receptor. Collectively, these observations suggested that the increase in [Ca2+]i induced by 5-HT in rat glomerulosa cells could be ascribed to activation of T-type calcium channel. To confirm this hypothesis, we have applied the patch-clamp technique in the whole-cell configuration to investigate the effect of 5-HT on T-type calcium current. In agreement with previous studies, which have demonstrated the occurrence of low voltage-activated (T-type) calcium channels in bovine (43) and human glomerulosa cells (44), we identified in rat glomerulosa cells the presence of rapidly inactivating low-voltage-activated calcium currents. We found that these currents displayed biophysical and pharmacological characteristics of T-type calcium currents (45), i.e. they were insensitive to nifedipine and nimodipine and highly sensitive to mibefradil and Ni2+.

Accordingly, in rat glomerulosa cells recorded in the whole-cell configuration, it has been recently demonstrated that the L-type currents run down and that the cells exclusively display a stable current exhibiting properties of the T-type current (29). We found that 5-HT markedly and reversibly enhanced the amplitude of the remaining current. In the presence of Ni2+ or mibefradil, the inward Ca2+ current induced by 5-HT was inhibited, thus confirming that 5-HT actually stimulates a T-type current. Consistent with this finding, it has been recently reported that the mRNA encoding the {alpha}1H subunit of T-type calcium channels is actively expressed in the zona glomerulosa of the rat adrenal gland (46). There is now accumulating evidence that T-type calcium channels are involved in the corticotropic effects of angiotensin II (47, 48), ACTH (47, 49), and K+ (47, 48, 50) in the adrenal gland of mammals. We have also previously demonstrated that 5-HT, acting through 5-HT4 receptors, stimulates T-type calcium channels in frog adrenocortical cells (24). The present data provide the first evidence that stimulation of 5-HT7 receptors is associated with activation of T-type calcium channels.

Because, in rat glomerulosa cells, 5-HT causes an increase in both cAMP formation and [Ca2+]i, the final goal of the present study was to determine the sequence of the transduction mechanisms associated with activation of 5-HT7 receptors. It has been previously reported that the mRNAs encoding the Ca2+/calmodulin-sensitive AC isoforms AC1, AC3, and AC8 are not expressed in the rat glomerulosa zone (51), suggesting that the stimulatory effect of 5-HT on AC activity cannot be accounted for by an increase in [Ca2+]i. In agreement with this hypothesis, we have found that 5-HT-evoked cAMP production was not impaired by reduction of [Ca2+]e, indicating that 5-HT7 receptors are coupled to a Ca2+-insensitive AC. Furthermore, the fact that CTX suppressed 5-HT-evoked stimulation of aldosterone secretion suggests that 5-HT7 receptor coupling to AC occurs via a Gs{alpha} protein. It is interesting to note that, in human embryonic kidney 293 cells cotransfected with the 5-HT7(a) receptor and specific AC isoforms, 5-HT is capable of activating the Ca2+/calmodulin-sensitive AC isoforms AC1 and AC8 through an increase of intracellular Ca2+ (19). These data indicate that the transduction mechanisms associated with 5-HT7 receptor activation in heterologous expression systems significantly differ from those occurring in cells naturally expressing 5-HT7 receptors.

In fact, several lines of evidence indicate that, in rat glomerulosa cells, the 5-HT-evoked stimulation of calcium influx is mediated through activation of the AC/PKA pathway. (1) The stimulatory effect of 5-HT on cAMP production was not significantly affected by reduction of [Ca2+]e from 1.8 to 1 mM, whereas, in the same experimental conditions, 5-HT-induced aldosterone secretion was reduced by 52%. Similarly, mibefradil did not impair the stimulatory effect of 5-HT on cAMP production but markedly inhibited 5-HT-evoked aldosterone output. These observations indicate that, in rat glomerulosa cells, activation of AC is not a calcium-dependent process and that calcium influx plays a crucial role in the transduction cascade downstream cAMP formation. (2) Administration of 8-Br-cAMP, a permeant analog of cAMP, mimicked the stimulatory effect of 5-HT on both [Ca2+]i and the T-type current. Consistently, although intracellular application of the Gq/11 protein antagonist GPAnt-2A (30) had no effect on the 5-HT-enhanced T-type current, dialysis of the Go/i/s protein antagonist GPAnt-1 (31) suppressed the effect of 5-HT on this current. (3) The intracellular loops of T-type calcium channels possess several PKA phosphorylation motifs (52). Indeed, stimulation of calcium influx by cAMP/PKA-dependent activation of T-type calcium channels has already been reported for 5-HT4 receptors in frog adrenocortical cells (24) and M3 muscarinic receptors in the fibroblast cell line NIH-3T3 (53). (4) Finally, the stimulatory effects of 5-HT on [Ca2+]i rise and T-type calcium current were both markedly attenuated by the PKA inhibitor H-89. The fact that administration of H-89 and reduction of [Ca2+]e provoked additive inhibitory effects on aldosterone secretion strongly suggests that the increase in [Ca2+]i induced by 5-HT is responsible, at least in part, for the stimulatory action of 5-HT on glomerulosa cells. Collectively, these observations indicate that activation of 5-HT7 receptors in rat glomerulosa cells stimulates the AC/PKA transduction pathway, which subsequently activates calcium influx through T-type calcium channels.

A proposed model illustrating the mechanism of action of 5-HT on rat glomerulosa cells is shown in Fig. 14Go. 5-HT, contained within chromaffin cells (54, 55) and mast cells (56) stimulate 5-HT7 receptors positively coupled to adenylyl cyclase through a CTX-sensitive G protein. The activation of a cAMP-dependent protein kinase causes a subsequent increase in calcium entry through T-type calcium channels. Both the stimulation of the AC/PKA pathway and the increase in [Ca2+]i are involved in 5-HT-evoked stimulation of aldosterone secretion.



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  		Figure 14. Schematic representation summarizing the mechanism of action of 5-HT in rat glomerulosa cells. Chromaffin cells and/or mast cells release 5-HT in the vicinity of glomerulosa cells. Activation of the 5-HT7 receptor increases adenylyl cyclase activity via a CTX-sensitive G protein. The resulting activation of PKA causes stimulation of calcium influx through T-type calcium channels.

 


   Acknowledgments
 
We thank Marjorie Gras, Huguette Lemonnier, and Gérard Cauchois for skillful technical assistance.


   Footnotes
 
This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale (U-413), European Institute for Peptide Research (IFRMP 23), and Conseil Régional de Haute-Normandie. S.L. was recipient of a doctoral fellowship from the Conseil Régional de Haute-Normandie.

Abbreviations: AC, Adenylyl cyclase; 8-Br-cAMP, 8-bromoadenosine cAMP; [Ca2+]i, cytosolic calcium concentration; CTX, cholera toxin; DMSO, dimethylsulfoxide; DOI, (±)-1-(2,5-dimethoxy-4-iodophenyl)-2 aminopropane; EC50, 50% effective concentration; EHNA, erythro-9- [2-hydroxy-3-nonyl]adenine; Emax, maximum response; GPAnt-1, substance-P-related peptide pGlu-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH2; GPAnt-2A, Arg-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH2; H-89, PKA inhibitor; 5-HT, serotonin; I-V, current-voltage; LY 215840, 5-HT7 receptor antagonist; PDE, phosphodiesterase; pEC50, negative logarithm of EC50; pKB, antagonist dissociation constant; PTX, pertussis toxin; R, photometers 405 nm/480 nm ratio; Ro 20-1724, 4-(3-butoxy-4-methoxy-benzyl)imidazolidin-2-one; SQ 22536, adenylyl cyclase inhibitor.

Received July 6, 2001.

Accepted for publication January 31, 2002.


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 Introduction
 Materials and Methods
 Results
 Discussion
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