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DUAL EFFECTS OF DOPAMINE IN RAT ADRENAL GLOMERULOSA

1100-1108

CELLS*

Nicole Gallo-Payet’, Lucie Chouinard’, Marie-Noelle Balestre’ and Gilles Guillon2 ’ Endocrine Service, Department of Medicine, Faculty of Medicine, University of Sherbrooke, Sherbrooke (Quebec) CANADA JlH 5N4 2 Centre CNRS-INSERM Pharmacologic-Endocrinologie, Rue de la Cardonille, 34094Montpellier Cedex, FRANCE Received

September

11,

1990

SUMMARY: The effects of dopamine(DA) on CAMP production and aldosteronesecretionwere comparedin freshly isolatedcells and in primary cultures of rat adrenal glomerulosacells.Under isolatedconditions,glomerulosacellsexhibited dopaminereceptorsfrom DA-l and DA-2 subclass, whereasin cultured conditions, where cells are very sensitive to their known stimuli, cells only exhibited dopamine receptors from the DA-l subclass.Moreover, unlike freshly isolated cells, dopamine stimulated both CAMP production and aldosterone secretion in 3-day cultured preparations. These effects were receptor specific since they were completely suppressedby Scherring 23390 (a specific DA-1 antagonist) and were unaffected by a B-adrenergicantagonist. As in vivo rat adrenal cortex contains DA, we discuss a possible involvement of this neurotransmitter in the regulation of aldosteronesecretion. 0 19’30 Academic PW55,1°C. The participation of dopamine(DA) in the multifactorial regulation of aldosteronesecretionis still an open question (1,2). It has been primary postulated that aldosterone secretion is under dopaminergicinhibition, sincebromocriptin (a dopamineagonist)inhibits and metoclopramide(a dopamineantagonist) stimulatesaldosteronesecretionin both human and rat (3,4,5). It has also been shown that DA inhibits the angiotensinII (AT II)-stimulating effect on steroid secretion (6,7,8). However, this role of DA is not accepted unanimouslysince Campbell et al. (9) Carey (10) and Lun et al. (11) do not confirm these results. In in vitro conditions, using isolated glomerulosacells,dopaminedoesnot modify the basalrate of aldosteronesecretion and, in some instances,either inhibits the AT II-stimulating effect (12,13) or fails to modify it (14,15,16,17). Conflicting reports have alsobeen publishedconcerningthe nature of dopaminereceptors in the zona glomerulosa. Both DA-1 receptors (positively coupled to adenylate cyclase) and DA-2 receptors (negatively coupled to adenylate cyclase) have been described in rat and bovine glomerulosacells(18,19,20,21).Basedon the observationsthat the concentration necessaryto block the AT II effect is very high comparedto the circulating level of DA, Fraser (1) and Campbell et al. (2,9) emphasizedcaution asto the importanceof DA in the regulation of aldosteronesecretion. *This work has been supported by MRC grants to N. Gallo-Payet and CNRS-INSERM grantsto G. Guillon. N. Gallo-Payet is a recipient of a developmentgrant of the Medical Research Council of Canada (MRCC). 0006-291X/90 Copyright All rights

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However, important DA concentrations have been found in the glomerulosa tissue (22,23), probably localized in recently described medullary rays (24). This presence could explain a paracrine role of dopamine in the modulation of aldosterone secretion. As previously shown by De Lean et al. (17) and Pratt et al. (25), high levels of DA, epinephrine and nor-epinephrine contained in vivo or in freshly isolated cells, may interfere with the specific responses of glomerulosa cells to a specific stimulus and may explain the discrepancies between results obtained from freshly isolated cells or after primary culture (25). To test this hypothesis, we have compared the effects of dopamine on CAMP production and on aldosterone secretion both in freshly isolated glomerulosa cells and on cells maintained three days in primary culture. We have observed that freshly isolated cells exhibit properties of DA-l and DA2 receptors while cultured cells exhibit only DA receptors from the DA-1 subclass.

MATERIALS

AND METHODS

Chemicals: The chemicals used in the present study were obtained from the following sources: aldosterone antiserum from Miles-Yeda Ltd. (Kiryat Weizmann, Rehovot, Israel); [3H]-aldosterone from New England Nuclear @chine, Quebec, Canada); [3H]-adenine from Amersham (Oakville, Ontario, Canada); dopamine, isoproterenol, epinephrine, ATP, CAMP and DNAse from Sigma (StLouis, MI, USA); Minimal Essential Medium (Eagle’s medium) (MEM) and collagenase from Gibco (Burlington, Ontario, Canada). Dopamine antagonists (Scherring, SCH23390 and (-) sulpiride) and the B-adrenergic antagonist -alperidol- were kindly provided by Dr J. Bockaert from the CCIPE, Montpellier, France and islet activating peptide (LAP) from List Laboratories (Campbell, CA, USA). The zonae glomerulosa were obtained from adrenal glands of Prenaration of domerulosa cells: female Long Evans rats weighing 200-250 g, and were isolated according to the method described in detail elsewhere (26). The successive steps of zona glomerulosa isolation and cell dissociation were performed in MEM Eagle medium (supplemented with 100 U/ml penicillin and 100 pg/ml streptomycin). After a 20 min incubation at 37°C in collagenase (2 mg/ml, 4 glomerulosa/ml) and DNAse (25 pg/ml), the cells were disrupted by gentle aspiration with a sterile 5 ml pipette, filtered and centrifuged for 10 min at 100 x g, and suspended in the appropriate medium. For studies using freshly isolated cells, red blood cells and broken cells were removed by centrifugation at 100 x g over a 60% Percoll barrier (Pharmacia Canada Inc). The cells were then suspended in MEM Eagle culture medium (containing 5.4 m K’ and 1.4 mM Ca*+) and preincubated for 2 hours at 37°C in a humidified atmosphere (95% air: 5% CO,). After this resting period, the cells were centrifuged at 100 x g for 10 min and resuspended in phosphate buffered saline (PBS). For studies using primary cultures, cells were suspended in MEM Eagle’s medium supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 pg/ml streptomycin, and plated in tissue culture dishes (35 mm in diameter for CAMP determinations) or multi-well plates (1.6 cm for steroid measurements) (Flow Laboratories, Mississauga, Ontario) at a density of approximately 7-10 x lo4 cells/multi-well or dish, respectively (27). The culture medium was changed every day and cells were used after three days in culture. At this time, cell density was approximately 1.5-2.0 x 16 cells/dish or multi-well plates. Incubations for steroid secretion measurements: These experiments were performed on cells maintained either two hours or three days in a minimum essential medium which maintained the differentiated status of the cells. Before the experiments, the medium of the cultured cells was aspirated and the cells were washed twice with cold phosphate-buffered saline (PBS-glucose): CaCl,, 0.44 mM; KCl, 3.5 mM; KH,PO,, 1.5 mM; MgCIZ, 0.5 mM; NaCl, 137 mM; Na,HPO,, 1 mM, HEPES, 5 mM, and 1 g/l glucose, pH 7.4. The cells were incubated in 1 ml consisting of 0.9 ml PBS-glucose supplemented with 0.5% BSA + 0.1 mg/ml bacitracin and 0.1 ml of stimuli. After a two-hour incubation at 37°C in 95% air: 5% CO, atmosphere, the medium was removed and kept at - 20°C until steroid measurements were performed. The cell suspensions were also washed in PBS-glucose, centrifuged at 100 x g for 10 min and resuspended at a concentration of 5 x IO4 cells/ml of PBS buffer and incubated for two hours as described above. After incubation, the tubes were centrifuged at 800 x g and the supernatants stored at -20°C. We have previously shown that 1101

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cultured cells were more sensitive to their stimuli than freshly isolated cells and that the ratio corticosteronefaldosterone was the same in both conditions (27,28). DA and DA antagonists were dissolved in water containing 3% glutathione to avoid oxydization during incubation. SCH 23390 was used to selectively inhibit the DA-1 receptor subtype, sulpiride to inhibit the DA-2 receptor subtype, and alperidol, the B-adrenergic components. Steroid analvsis: Aldosterone was determined by radioimmunoassay after extraction with methylene chloride, using a specific aldosterone antiserum and [3H - aldosterone, as described previously (26). The results are normalized to pg produced by 1 x 1CAcells during the two hour incubation. Cvclic AMP determination: Intracellular cyclic AMP production was determined by measuring the conversion of [3H]-ATP into [3H]-cyclic AMP, according to the method described by Weiss et al. (29). In short, cultured or isolated cells were incubated at 37°C in the same MEM Eagle culture media containing 2 @/ml [3H]-adenine. After 1 hour, the cultures were washed while the isolated cells were diluted and centrifuged. They were further incubated in PBS glucose containing 1 mM isobutyl methylxanthine (IBMX) for 1.5min at 37°C. The hormones or drugs were then added to the incubation medium for a further 15 min incubation period at 37°C. The reaction was ended by aspiration of the media (for the cultures) or by direct addition of 1 ml ice-cold 5% trichloroacetic acid in suspended cells. The cultured cells were scraped with a rubber policeman and 100 1.11of cold 5 mM (ATP-cyclic AMP) was added to the mixture. Cellular membranes were pelleted at 5000 g x 15 min and the supernatants were sequentially chromatographed on Dowex and alumina columns which allowed the separation of [3H]-adenine nucleotide (primarily [3H]-ATP) and [3H]-cyclic AMP (30). Cyclic AMP formation is expressed as: % conversion = ([3H]-cyclic AMP/[3H]-cyclic AMP + [‘HI-ATP) x 100 per 15 min. Data analvsis: Statistical analysis of the data was performed using the one-way analysis of variance (ANOVA). Homogeneity of variance was assessed by Bartlett’s test (31) and P values were obtained from Dunnett’s tables (32). The data are presented as the mean +. S.E.M. The ED,, is the concentration which produces the half-maximal response. Estimates of the ED,, for agonists were directly obtained from the dose-response curve.

RESULTS Figure 1A shows that in three-day cultured glomerulosa cells, DA induces a dose-dependent increase in CAMP production with an ED,, of 1.1 f 0.1 x 10dM and a maximal stimulation over basal level of 2.4 fold (from 0.099 2 0.002 to 0.235 2 0.02, n =7). This effect is completely blocked by 10”M SCH 23390, a specific antagonist of the DA-l brain receptors’ subclass. Figure 1C and D show that SCH 23390 induces a dose-dependent inhibition of the DA effect which is maximum at lO”M, while sulpiride does not significantly alter CAMP production induced by 10qM DA. Fig. 1B shows that DA stimulates aldosterone secretion at concentrations

slightly lower than that

required to stimulate CAMP production. This effect is dose-dependent with an ED,, of 3.3 ? 1.1 x 10.‘M (3 distinct determinations)

and the maximal stimulation over basal is 1.9 fold (from

25.4 2 3.6 to 49.4 of: 3.9pg/16 cells, n=7).

Moreover, SCH 23390 completely blocks the DA-

stimulated aldosterone secretion. We have compared the dose-response curves of DA alone, of DA + 10”M SCH 23390 and of DA + 10bM sulpiride, on CAMP production and aldosterone response in freshly isolated cells. In contrast to that observed in cultured glomerulosa cells, DA, up to a concentration of 10dM, does not modify CAMP accumulation nor aldosterone secretion. However, as illustrated in Fig. 2A and B, in the presence of SCH 23390, DA induces a dose-dependent

inhibition of both CAMP

accumulation and aldosterone secretion, with an ED,, around lo-‘M. At the concentration of 10. 4M, and in the presence of lOdM SCH 23390, DA induces a significant inhibition of 20% (n=3, P < 0.01) and 15% (n = 3, p < 0.02) for both CAMP production and Aldosterone secretion. On 1102

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Fre. - Effects of dopamine and dopamine antagonists on CAMP accumulation and aldosterone secretion in primary cultures of rat glomerulosa cells. Panels A and B: glomerulosa cells were incubated with increasing amounts of DA in the absence (+) or in the presence of (m)l PM of the specific DA-1 antagonist, SCH 23390. CAMP accumulation (A) or aldosterone secretion (B) were measured as described in the “Materials and Methods”. Results are the mean k*SEM of 7 distinct experiments, each done in triplicate. Significant difference from control values: p < 0.01, ** p < 0.001. Panels C and D: Cells were incubated with graded doses of the DA-l antagonist, SCH 23390 with (0) or without 10’M DA (0) or with graded doses of the specific DA-Z antagonist sulpiride with (A) or without 10dM DA (A). Results are the mean + SEM of triplicate determinations from one representative experiment (where no error bars are shown, they are contained within the symbol). Arrows indicate the concentration at which the ED,, are observed.

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Fie. - Effects of dopamine and dopamine antagonists on CAMP accumulation and aldosterone secretion in freshly isolated rat glomerulosa cells. Cells were prepared as described in “Materials and Methods” and were incubated with increasing amounts of DA in the absence (+) or in the presence (# of either 1 PM SCH 23390 or 1 MM sulpiride (A). CAMP accumulation (A) or aldosterone secretion (B) were measured as described earlier. Each point is the mean + SEM of 4 distinct experiments. Significant difference from control values: * p < 0.05. Arrows on the graphs indicate the concentration at which ED,, is observed. 1103

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Dual effects of dopamine in rat adrenal glomerulosa cells.

The effects of dopamine (DA) on cAMP production and aldosterone secretion were compared in freshly isolated cells and in primary cultures of rat adren...
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