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The CRF-related peptide sauvagine stimulates and the GABAB receptor agonist baclofen inhibits cyclic-AMP production in melanotrope cells of Xenopus laevis
Life Sciences, Vol. 48, pp. 1633-1637 Printed in the U.S.A.
Pergamon Press
THE CRF-RELATED PEPTIDE SAUVAGINE STIMULATES AND THE GABAe RECEPTOR AGONIST BACLOFEN INHIBITS CYCLIC-AMP PRODUCTION IN MELANOTROPE CELLS OF XENOPUS LAEVIS
B.G. Jenks, I.D. van Zoest, H.P. de Koning, H.J. Leenders and E.W. Roubos
Department of Animal Physiology, University of Nijmegen, Toernooiveld, 6525 ED Nijmegen, The Netherlands (Received in final form February 15, 1991) Summary
Release of ,-MSH from the pars intermedia melanotrope cells of Xenopus laevis is regulated by various classical neurotransmitters and neuropeptides. We have examined the effect of two of these regulatory substances, the neurotransmitter GABA and the CRF-related peptide sauvagine, on the adenylate cyclase system of the melanotrope cells. Sauvagine treatment, which stimulates cx-MSH release, lead to an elevation in the level of cyclic-AMP, an effect which was potentiated by cholera toxin. Treatment with baclofen, a GABAs receptor agonist, gave a pertussis toxinsensitive decrease in the cyclic-AMP level and an inhibition of c~-MSH release. We conclude that sauvagine stimulates ¢x-MSH secretion through activation of adenylate cyclase and that GABAs receptor activation inhibits secretion through inhibition of cyclic-AMP production. Baclofen treatment sensitized melanotrope cells to the stimulatory action of 8-bromo-cyclic-AMP on the secretion of ~x-MSH. This observation supports the conclusion that GABAs receptor activation inhibits cyclic-AMP production.
The physiological process of color change in amphibians is regulated by melanophore stimulating hormone, c~-MSH, secreted from the pars intermedia of the pituitary gland. This peptide, which is released in animals on a black background, stimulates dispersion of pigment in dermal melanophores thus causing darkening of the skin (1). The pars intermedia melanotrope cells of the amphibian Xenopus laevis are regulated, at least in part, through the adenylate cyclase system. We have previously shown that cyclic-AMP analogues stimulate the secretion of c~-MSH (2,3,4) and that the neurotransmitter dopamine, which inhibits c~-MSH secretion through a dopamine D2-type receptor (5), inhibits the production of cyclic-AMP in the melanotropes (6). This effect proved to be pertussis toxin-sensitive, which indicates the involvement of a Gi protein in the coupling of the Xenopus D2-receptor to adenylate cyclase. Melanotropo cells of Xenopus laevis are regulated by various factors of hypothalamic origin (7). Besides dopamine, there are several other a-MSH-secretagogues which might act via an action on adenylate cyclase. The neurotransmitter GABA inhibits ¢x-MSH secretion via activation of both GABA^ and GABAs receptors (8). While the GABA^ receptor mechanism likely involves direct ion channel activation, we have preliminary evidence that the GABAs receptor action in Xenopus 0024-3205/91 $3.00 +.00 Copyright (c) 1991 Pergamon Press plc
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Sauvagine and Baclofen on Cyclic-AMP
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melanotropes involves inhibition of adenylate eyclase (6), in keeping with evidence that, in other systems, GABAs receptors are negatively coupled to adenylate cyclase (9,10). The secretion of ¢xMSH is stimulated by sauvagine (11), an amphibian peptide structurally related to corticotropin releasing factor (CRF). There are several reports indicating that the general mechanism of action of CRF on effector systems involves stimulation of the production of cyclic-AMP (12,13,14,15). The purpose of the present study was to examine if the adenylate cyclase system of Xenopus melanotrope cells is regulated by sauvagine and/or by GABA8 receptors. To this end, the effects of sanvagine and baclofen (GABAB receptor agonist) on the cyclic-AMP levels in melanotrope cells were determined; furthermore, the effect of baclofen on the secretory response of melanotropes to cyclic-AMP treatment was assessed. Methods
Animals Xenopus laevis were bred in the laboratory. Young adult animal were used; three weeks prior to the experiments they were placed in black buckets and kept under constant illumination.
Isolated cell experiments Experiments involving cyclic-AMP measurements were conducted on isolated melanotrope cells in order to prevent possible bias in cyclic-AMP measurements due to the presence of pars nervosa tissue. The method for obtaining isolated melanotropes is similar to a reported procedure (16). Briefly, neurointermediate lobes were excised and cells were dispersed by incubating the lobes for 1 h in LI5 medium (Gibco), diluted 2:1 with water, and containing 5 mg/ml collagenase (type V, Sigma) and 10 mg/ml protease (type IX, Sigma). This was followed by 10 passes through a siliconized Pasteur's pipet. The suspension was filtered through a nylon filter (pore size 0.15 mm) to remove incompletely dissociated tissue fragments including the entire pars nervosa. The cells were washed three times with Ringer's solution and then distributed into siliconized glass vials. Agents to be tested for effects on MSH secretion and on cyclic-AMP levels were added in Ringer's solution containing 1 mM isobutyl-methyl-xanthine OBMX, Sigma). The agents tested, either alone or in various combinations (see Results), were: sauvagine (gift of Dr. J. Rivier, The Salk Institute, San Diego, CA), baclofen (Ciba-Geigy), cholera toxin (Sigma) and pertussis toxin (PHLS Center for Microbiology and Research, Salisbury, U.K.). The vials were incubated at 22"C for 20 min after which cells and media were separated by centrifugation and the cells were homogenized in 0.1N HCI. Samples of each medium and cell extract were submitted to a radioimmunoassay for c~-MSH, described previously (17), and to a radioimmunoassay for cyclic-AMP (Amersham RPA 509). Preliminary experiments established that IBMX treatment was necessary to obtain measurable amounts of cyclic-AMP and that the maximum level was reached within 20 min. The cyclic-AMP levels in the media reflected tissue levels but were usually 10-fold lower; all results shown are from media measurements.
Superfusion experiments Individual neurointermediate lobes were placed in a superfusion chamber; four such chambers were superfused simultaneously with Hepes-buffered amphibian Ringer's solution (pH 7.3)containing 2 g/l glucose, 1 mg/l ascorbic acid and 0.3 g/l bovine serum albumin (fraction V, Sigma). The flow rate was 1.5 ml/h and fractions of 7.5 min were collected; 8-bromo-3',5'-cyclic-adenosine monophosphate (Sigma) and/or baclofen were dissolved in Ringer's solution and introduced to the superfusion chambers according to the protocols given in the Results. The collected fractions were submitted to a radioimmunoassay for o~-MSH.
Statistics Statistical analysis was performed using the Mann-Whitney U-test. P-values <0.05 (two-tailed) were taken to indicate significance.
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Sauvagine and Baclofen on Cyclic-AMP
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Results
Sauvagine effect on cyclic-AMP levels Sauvagine, at 10Q¢I, gave a strong elevation in the level of cyclic-AMP and a small but significant increase in the amount of a-MSH present in the incubation medium (fig. la). At 10"M the peptide had no effect on either the cyclic-AMP level or ,-MSH secretion (fig. lb). This same concentration of sauvagine, when given in combination with cholera toxin, caused a significant increase in the level of cyclic-AMP; cholera toxin by itself had no effect on either the cyclic-AMP level or ~-MSH secretion (fig. lb).
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FIG. 1 The effects of c~-MSH-secretagogues and toxins on the level of cyclic-AMP (upper bars) and ,v-MSH release (lower bars) during incubations with isolated melanotrope cells of Xenopus. Abbreviations: C, control; S, sauvagine; Ct, cholera toxin; Pt Pertussis toxin; B, baclofen. The concentration of sauvagine was 106M in experiment (a) and 10SM in experiment (b); cholera toxin was 5 #g/ml. In experiment (c) the concentration of pertussis toxin was 500 ng/ml and that of baclofen 10SM. Results are expressed as percentage of the values obtained in control incubations; error bars indicate +s.e.m. Stars indicate where significant differences were found compared to control values (*, P<0.05;**, P<0.01;***, P<0.001).The number of incubations conducted for each group is indicated (n).
Baclofen effect on cyclic-AMP levels Treatment with baclofen significantly decreased both the cyclic-AMP level and o~-MSH secretion (fig. lc). Baclofen did not evoke these effects in the presence of pertussis toxin. The toxin itself induced a slight but nonsignificant increase in cyclic-AMP level and had no effect on c~-MSH secretion.
Cyclic-AMP effect on secretion Pulses of 8-bromo-cyclic-AMP resulted in only slight stimulations above the level of spontaneous secretion of a-MSH (about 10%, see caption of fig. 2). Baclofen, when given in superfusion, strongly inhibited ot-MSH secretion (fig. 2). Pulses of 8-bromo-cyclie-AMP given under the baclofen-inhibited condition induced relatively strong stimulations above the suppressed level of secretion (over 100%, see caption of fig. 2).
1636
Sauvaglne and Baclofen on Cycllc-AMP
Percent
Vol. 48, No. 17, 1991
Basal Release
160
baclofen
120
...... 80
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10 20 30 40 Superfusion Fraction Number
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FIG. 2 The effect of discrete pulses of 8-bromo-3',5'-cyclic-AMP (cAMP) on spontaneous secretion of e~-MSH and on secretion under baclofen-inhibited conditions during superfusion of neurointermediate lobes of Xenopus. The concentration of cyclicAMP was 6 mM and that of baclofen 3.3x10~M. Results are expressed as the mean percent basal secretion derived from four neurointermediate lobes and, for each lobe, the average ot-MSH value in the three superfusion fractions before the first pulse of cyclic-AMP was defined as 100% basal. Vertical bars indicate -s.e.m. CyclicAMP stimulated secretion 9.7+3.5% (first pulse) and 10.7±1.3% (second pulse) above spontaneous basal values and 191.3±25.4% (third pulse) and 108.0±15.2% (fourth pulse) above the baclofen-inhibited condition. For the purpose of these calculations the basal values were defined as the mean amount of a-MSH in the three fractions immediately preceding each cyclic-AMP pulse (filled squares). Discussion Previous studies, involving superfusion methods, have shown that sauvagine stimulates cxMSH secretion from Xenopus pars intermedia (11). The concentration of peptide used in the present study were based on these earlier experiments. Sauvagine, at 10~M, stimulated cx-MSH secretion and this stimulatory action was accompanied by a strong stimulatory effect on the levels of cyclic-AMP, thus suggesting that sauvagine stimulates secretion through stimulation of cyclic-AMP production. The stimulatory action of CRF on secretion in rat melanotropes (12,13) and corticotropes (14,15) has been shown to involve activation of adenylate cyclase. Our results would thus indicate that sauvagine, which is an amphibian peptide structurally related to mammalian CRF, is also functionally closely related to the mammalian peptide. The fact that cholera toxin potentiated the action of sauvagine on cyclic-AMP production indicates that in melanotropes of Xenopus, sauvagino-stimulated cyclic-AMP production involves activation of a G s protein. Baclofen is a specific GABAs agonist which inhibits c~-MSH release from Xenopus melanotrope cells (8). The inhibition of o~-MSH release observed in the present study was concomitant with a decreased level of cyclic-AMP, suggesting that baclofen acts on the melanotropes via an adenylate cyclase-coupled GABAs receptor. The observation that pertussis toxin diminished baclofen-induced inhibition of cyclic-AMP production supports the idea that an inhibitory Gl protein is involved. In the central nervous system GABAs receptors have been shown to function through inhibition of adenylate cyclase (9). The present results indicate that a GABAB receptor mechanism coupled to adenylate cyclase might also operate in a neuroendocrine cell. We have previously shown that 8-bromo-cyclic-AMP is far more potent in stimulating c~MSH secretion from intermediate lobes of Xenopus adapted to white-background than to black-
Vol. 48, No. 17, 1991
Sauvagine and Baclofen on Cyclic-AMP
1637
background (4) and others have even found that the secretory process of lobes from black-adapted
Xenopus is totally unresponsive to this cyclic-AMP analogue (18). A possible explanation for this phenomenon is that cyclic-AMP levels are already very high in the actively secreting melanotropes of black-adapted animals, thus making these cells particularly unresponsive to exogenous cyclicAMP. A direct test of this hypothesis, through measurement of physiological cyclic-AMP levels, has been precluded by the fact that IBMX treatment is necessary in order to obtain measurable amount of cyclic-AMP. We now show that baclofen treatment, which lowers cyclic-AMp levels, at the same time sensitizes melanotropes of black-adapted animals to exogenous cyclic-AMP, an observation in keeping with the conclusion that melanotrope cells of black-adapted animals have cyclic-AMP levels which support maximum or near maximum secretion. Altogether we conclude that cyclic-AMP is of physiological relevance for the regulation of c~-MSH release from the melanotrope cells of Xenopus laevis; the activity of the adenylate cyclase of these cells is regulated by both inhibitory and stimulatory mechanisms involving established o~-MSH secretagogues such as dopamine, GABA and the CRF-related peptide sauvagine. The physiological function of CRF-related peptides in the Xenopus pars intermedia might be to override tonic inhibition induced by dopaminergic or GABAergic mechanisms. Ackn0wledeement~ The authors thank Mr. P.M.J.M. Cruijsen and Ms. S. Lendi for technical assistance and Mr. R.J.C. Engels for animal care. This work was supported by a grant from the Foundation for Biological Research (BION), which is subsidized by the Netherlands Organization for Scientific Research (NWO), and by a grgant by the European Community (Contract No. ST25-O468-C). References 1.
2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
J.T. BAGNARA and M.E. HADLEY, Chromatophores and Color Change, Prentice-Hall, Englewood Cliffs NJ. (1973). B.G. JENKS, Frontiers of Hormone Research 4, 63-65 (1977). G.J.M. MARTENS, B.G. JENKS and A.P. VAN OVERBEEKE, Comp. Biochem. Physiol. 69¢, 75-82 (1981). B.M.L. VERBURG-VAN KEMENADE, B.G. JENKS and A.P. VAN OVERBEEKE, Gem Comp. Endocrinol. 63,471-480 (1986). B.M.L. VERBURG-VAN KEMENADE, M.C. TONON, B.G. JENKS and H. VAUDRY, Neuroendocrinology 44, 446-456 (1986). B.M.L. VERBURG-VAN KEMENADE, B.G. JENKS and A.J.H.M. HOUBEN, Life Sci. 40, 1859-1867 (1987). B.G. JENKS, B.M.L. VERBURG-VAN KEMENADE and G.J.M. MARTENS, Melanotropic Peptides: volume 1, Mac E. Hadley, (ed), 103-126, CRC Press, Boca Raton, Florida (1988). B.M.L. VERBURG-VAN KEMENADE, B.G. JENKS, F.J.A, LENSSEN and H. VAUDRY, Endocrinol. 120, 622-628 (1987). W.J. WOJCIK and N.H. NEFF, Mol. Pharmacol. 2__~,24-28 (1984). R.B. INNIS and G.K. AGHAJANIAN, Eur. J. Pharmacol. 143,195-204 (1987). B.M.L. VERBURG-VAN KEMENADE, B.G. JENKS, P.M.J.M. CRUIJSEN, A. DINGS, M.C. TONON and H. VAUDRY, Peptides 8, 1093-1100(1987). F. LABRIE, B. GAGNE and G. LEFEVRE, Life Sci. 31, 1117-1121 (1982). M.J. CRONIN, J.R. ZYSK and A.J. BAERTSCHI, Peptides 7,935-938 (1986), M. MEUNIER, G. LEFEVRE, G. DUMONT and F. LABRIE, Life Sci. 31, 2129-2136 (1982). F. LABRIE, B. GAGNE, G. LEFEVRE and G. MEUNIER, Mol. Cell. Endocrinol. 30, 347-351 (1983). B.M.L. VERBURG-VAN KEMENADE, B.G. JENKS and R.J.M. SMITS, Neuroendocrinology 46, 289-296 (1987). I.D. VAN ZOEST, P.S. HEIJMEN, P.M.J.M. CRUIJSEN and B.G. JENKS, Gen. Comp. Endocrinol. 76, 19-28 (1989). Y.P. LOH, B. MYERS, B. WONG, D.C. PARISH, M. LANG and M.E. GOLDMAN, J. Biol. Chem. 260, 8956-8963 (1985).
Pergamon Press
THE CRF-RELATED PEPTIDE SAUVAGINE STIMULATES AND THE GABAe RECEPTOR AGONIST BACLOFEN INHIBITS CYCLIC-AMP PRODUCTION IN MELANOTROPE CELLS OF XENOPUS LAEVIS
B.G. Jenks, I.D. van Zoest, H.P. de Koning, H.J. Leenders and E.W. Roubos
Department of Animal Physiology, University of Nijmegen, Toernooiveld, 6525 ED Nijmegen, The Netherlands (Received in final form February 15, 1991) Summary
Release of ,-MSH from the pars intermedia melanotrope cells of Xenopus laevis is regulated by various classical neurotransmitters and neuropeptides. We have examined the effect of two of these regulatory substances, the neurotransmitter GABA and the CRF-related peptide sauvagine, on the adenylate cyclase system of the melanotrope cells. Sauvagine treatment, which stimulates cx-MSH release, lead to an elevation in the level of cyclic-AMP, an effect which was potentiated by cholera toxin. Treatment with baclofen, a GABAs receptor agonist, gave a pertussis toxinsensitive decrease in the cyclic-AMP level and an inhibition of c~-MSH release. We conclude that sauvagine stimulates ¢x-MSH secretion through activation of adenylate cyclase and that GABAs receptor activation inhibits secretion through inhibition of cyclic-AMP production. Baclofen treatment sensitized melanotrope cells to the stimulatory action of 8-bromo-cyclic-AMP on the secretion of ~x-MSH. This observation supports the conclusion that GABAs receptor activation inhibits cyclic-AMP production.
The physiological process of color change in amphibians is regulated by melanophore stimulating hormone, c~-MSH, secreted from the pars intermedia of the pituitary gland. This peptide, which is released in animals on a black background, stimulates dispersion of pigment in dermal melanophores thus causing darkening of the skin (1). The pars intermedia melanotrope cells of the amphibian Xenopus laevis are regulated, at least in part, through the adenylate cyclase system. We have previously shown that cyclic-AMP analogues stimulate the secretion of c~-MSH (2,3,4) and that the neurotransmitter dopamine, which inhibits c~-MSH secretion through a dopamine D2-type receptor (5), inhibits the production of cyclic-AMP in the melanotropes (6). This effect proved to be pertussis toxin-sensitive, which indicates the involvement of a Gi protein in the coupling of the Xenopus D2-receptor to adenylate cyclase. Melanotropo cells of Xenopus laevis are regulated by various factors of hypothalamic origin (7). Besides dopamine, there are several other a-MSH-secretagogues which might act via an action on adenylate cyclase. The neurotransmitter GABA inhibits ¢x-MSH secretion via activation of both GABA^ and GABAs receptors (8). While the GABA^ receptor mechanism likely involves direct ion channel activation, we have preliminary evidence that the GABAs receptor action in Xenopus 0024-3205/91 $3.00 +.00 Copyright (c) 1991 Pergamon Press plc
1634
Sauvagine and Baclofen on Cyclic-AMP
Vol. 48, No. 17, 1991
melanotropes involves inhibition of adenylate eyclase (6), in keeping with evidence that, in other systems, GABAs receptors are negatively coupled to adenylate cyclase (9,10). The secretion of ¢xMSH is stimulated by sauvagine (11), an amphibian peptide structurally related to corticotropin releasing factor (CRF). There are several reports indicating that the general mechanism of action of CRF on effector systems involves stimulation of the production of cyclic-AMP (12,13,14,15). The purpose of the present study was to examine if the adenylate cyclase system of Xenopus melanotrope cells is regulated by sauvagine and/or by GABA8 receptors. To this end, the effects of sanvagine and baclofen (GABAB receptor agonist) on the cyclic-AMP levels in melanotrope cells were determined; furthermore, the effect of baclofen on the secretory response of melanotropes to cyclic-AMP treatment was assessed. Methods
Animals Xenopus laevis were bred in the laboratory. Young adult animal were used; three weeks prior to the experiments they were placed in black buckets and kept under constant illumination.
Isolated cell experiments Experiments involving cyclic-AMP measurements were conducted on isolated melanotrope cells in order to prevent possible bias in cyclic-AMP measurements due to the presence of pars nervosa tissue. The method for obtaining isolated melanotropes is similar to a reported procedure (16). Briefly, neurointermediate lobes were excised and cells were dispersed by incubating the lobes for 1 h in LI5 medium (Gibco), diluted 2:1 with water, and containing 5 mg/ml collagenase (type V, Sigma) and 10 mg/ml protease (type IX, Sigma). This was followed by 10 passes through a siliconized Pasteur's pipet. The suspension was filtered through a nylon filter (pore size 0.15 mm) to remove incompletely dissociated tissue fragments including the entire pars nervosa. The cells were washed three times with Ringer's solution and then distributed into siliconized glass vials. Agents to be tested for effects on MSH secretion and on cyclic-AMP levels were added in Ringer's solution containing 1 mM isobutyl-methyl-xanthine OBMX, Sigma). The agents tested, either alone or in various combinations (see Results), were: sauvagine (gift of Dr. J. Rivier, The Salk Institute, San Diego, CA), baclofen (Ciba-Geigy), cholera toxin (Sigma) and pertussis toxin (PHLS Center for Microbiology and Research, Salisbury, U.K.). The vials were incubated at 22"C for 20 min after which cells and media were separated by centrifugation and the cells were homogenized in 0.1N HCI. Samples of each medium and cell extract were submitted to a radioimmunoassay for c~-MSH, described previously (17), and to a radioimmunoassay for cyclic-AMP (Amersham RPA 509). Preliminary experiments established that IBMX treatment was necessary to obtain measurable amounts of cyclic-AMP and that the maximum level was reached within 20 min. The cyclic-AMP levels in the media reflected tissue levels but were usually 10-fold lower; all results shown are from media measurements.
Superfusion experiments Individual neurointermediate lobes were placed in a superfusion chamber; four such chambers were superfused simultaneously with Hepes-buffered amphibian Ringer's solution (pH 7.3)containing 2 g/l glucose, 1 mg/l ascorbic acid and 0.3 g/l bovine serum albumin (fraction V, Sigma). The flow rate was 1.5 ml/h and fractions of 7.5 min were collected; 8-bromo-3',5'-cyclic-adenosine monophosphate (Sigma) and/or baclofen were dissolved in Ringer's solution and introduced to the superfusion chambers according to the protocols given in the Results. The collected fractions were submitted to a radioimmunoassay for o~-MSH.
Statistics Statistical analysis was performed using the Mann-Whitney U-test. P-values <0.05 (two-tailed) were taken to indicate significance.
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Sauvagine and Baclofen on Cyclic-AMP
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Results
Sauvagine effect on cyclic-AMP levels Sauvagine, at 10Q¢I, gave a strong elevation in the level of cyclic-AMP and a small but significant increase in the amount of a-MSH present in the incubation medium (fig. la). At 10"M the peptide had no effect on either the cyclic-AMP level or ,-MSH secretion (fig. lb). This same concentration of sauvagine, when given in combination with cholera toxin, caused a significant increase in the level of cyclic-AMP; cholera toxin by itself had no effect on either the cyclic-AMP level or ~-MSH secretion (fig. lb).
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FIG. 1 The effects of c~-MSH-secretagogues and toxins on the level of cyclic-AMP (upper bars) and ,v-MSH release (lower bars) during incubations with isolated melanotrope cells of Xenopus. Abbreviations: C, control; S, sauvagine; Ct, cholera toxin; Pt Pertussis toxin; B, baclofen. The concentration of sauvagine was 106M in experiment (a) and 10SM in experiment (b); cholera toxin was 5 #g/ml. In experiment (c) the concentration of pertussis toxin was 500 ng/ml and that of baclofen 10SM. Results are expressed as percentage of the values obtained in control incubations; error bars indicate +s.e.m. Stars indicate where significant differences were found compared to control values (*, P<0.05;**, P<0.01;***, P<0.001).The number of incubations conducted for each group is indicated (n).
Baclofen effect on cyclic-AMP levels Treatment with baclofen significantly decreased both the cyclic-AMP level and o~-MSH secretion (fig. lc). Baclofen did not evoke these effects in the presence of pertussis toxin. The toxin itself induced a slight but nonsignificant increase in cyclic-AMP level and had no effect on c~-MSH secretion.
Cyclic-AMP effect on secretion Pulses of 8-bromo-cyclic-AMP resulted in only slight stimulations above the level of spontaneous secretion of a-MSH (about 10%, see caption of fig. 2). Baclofen, when given in superfusion, strongly inhibited ot-MSH secretion (fig. 2). Pulses of 8-bromo-cyclie-AMP given under the baclofen-inhibited condition induced relatively strong stimulations above the suppressed level of secretion (over 100%, see caption of fig. 2).
1636
Sauvaglne and Baclofen on Cycllc-AMP
Percent
Vol. 48, No. 17, 1991
Basal Release
160
baclofen
120
...... 80
. • .......... .:....
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10 20 30 40 Superfusion Fraction Number
!:!::!i:::: 50
FIG. 2 The effect of discrete pulses of 8-bromo-3',5'-cyclic-AMP (cAMP) on spontaneous secretion of e~-MSH and on secretion under baclofen-inhibited conditions during superfusion of neurointermediate lobes of Xenopus. The concentration of cyclicAMP was 6 mM and that of baclofen 3.3x10~M. Results are expressed as the mean percent basal secretion derived from four neurointermediate lobes and, for each lobe, the average ot-MSH value in the three superfusion fractions before the first pulse of cyclic-AMP was defined as 100% basal. Vertical bars indicate -s.e.m. CyclicAMP stimulated secretion 9.7+3.5% (first pulse) and 10.7±1.3% (second pulse) above spontaneous basal values and 191.3±25.4% (third pulse) and 108.0±15.2% (fourth pulse) above the baclofen-inhibited condition. For the purpose of these calculations the basal values were defined as the mean amount of a-MSH in the three fractions immediately preceding each cyclic-AMP pulse (filled squares). Discussion Previous studies, involving superfusion methods, have shown that sauvagine stimulates cxMSH secretion from Xenopus pars intermedia (11). The concentration of peptide used in the present study were based on these earlier experiments. Sauvagine, at 10~M, stimulated cx-MSH secretion and this stimulatory action was accompanied by a strong stimulatory effect on the levels of cyclic-AMP, thus suggesting that sauvagine stimulates secretion through stimulation of cyclic-AMP production. The stimulatory action of CRF on secretion in rat melanotropes (12,13) and corticotropes (14,15) has been shown to involve activation of adenylate cyclase. Our results would thus indicate that sauvagine, which is an amphibian peptide structurally related to mammalian CRF, is also functionally closely related to the mammalian peptide. The fact that cholera toxin potentiated the action of sauvagine on cyclic-AMP production indicates that in melanotropes of Xenopus, sauvagino-stimulated cyclic-AMP production involves activation of a G s protein. Baclofen is a specific GABAs agonist which inhibits c~-MSH release from Xenopus melanotrope cells (8). The inhibition of o~-MSH release observed in the present study was concomitant with a decreased level of cyclic-AMP, suggesting that baclofen acts on the melanotropes via an adenylate cyclase-coupled GABAs receptor. The observation that pertussis toxin diminished baclofen-induced inhibition of cyclic-AMP production supports the idea that an inhibitory Gl protein is involved. In the central nervous system GABAs receptors have been shown to function through inhibition of adenylate cyclase (9). The present results indicate that a GABAB receptor mechanism coupled to adenylate cyclase might also operate in a neuroendocrine cell. We have previously shown that 8-bromo-cyclic-AMP is far more potent in stimulating c~MSH secretion from intermediate lobes of Xenopus adapted to white-background than to black-
Vol. 48, No. 17, 1991
Sauvagine and Baclofen on Cyclic-AMP
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background (4) and others have even found that the secretory process of lobes from black-adapted
Xenopus is totally unresponsive to this cyclic-AMP analogue (18). A possible explanation for this phenomenon is that cyclic-AMP levels are already very high in the actively secreting melanotropes of black-adapted animals, thus making these cells particularly unresponsive to exogenous cyclicAMP. A direct test of this hypothesis, through measurement of physiological cyclic-AMP levels, has been precluded by the fact that IBMX treatment is necessary in order to obtain measurable amount of cyclic-AMP. We now show that baclofen treatment, which lowers cyclic-AMp levels, at the same time sensitizes melanotropes of black-adapted animals to exogenous cyclic-AMP, an observation in keeping with the conclusion that melanotrope cells of black-adapted animals have cyclic-AMP levels which support maximum or near maximum secretion. Altogether we conclude that cyclic-AMP is of physiological relevance for the regulation of c~-MSH release from the melanotrope cells of Xenopus laevis; the activity of the adenylate cyclase of these cells is regulated by both inhibitory and stimulatory mechanisms involving established o~-MSH secretagogues such as dopamine, GABA and the CRF-related peptide sauvagine. The physiological function of CRF-related peptides in the Xenopus pars intermedia might be to override tonic inhibition induced by dopaminergic or GABAergic mechanisms. Ackn0wledeement~ The authors thank Mr. P.M.J.M. Cruijsen and Ms. S. Lendi for technical assistance and Mr. R.J.C. Engels for animal care. This work was supported by a grant from the Foundation for Biological Research (BION), which is subsidized by the Netherlands Organization for Scientific Research (NWO), and by a grgant by the European Community (Contract No. ST25-O468-C). References 1.
2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
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