Hypothalamic α-melanocyte-stimulating hormone (α-MSH) is not under dopaminergic control

203

Brain Research, 423 (1987) 203-212

Elsevier BRE 12947

Hypothalamic a-melanocyte-stimulating hormone (a-MSH) is not under dopaminergic control C. Delbende 1, S. J6gou 1, D. Tranchand-Bunel 1, G. Pelletier2 and H. Vaudry 1 IGroupe de Recherche en Endocrinologie Mol~culaire, UnitdAlli~e ~ I'INSERM, VA C.N.R.S. 650, Facultddes Sciences, Universitd de Rouen, Mont-Saint-Aignan (France) and 2MRC Group in Molecular Endocrinology, le Centre Hospitalier de r Universitd Laval, Quebec (Canada)

(Accepted 12 March 1987) Key words." Melanocyte-stimulating hormone; fl-Endorphin; Proopiomelanocortin-containing neuron; Dopaminergic agonist;

Dopaminergic antagonist; Hypothalamus; High-performance liquid chromatography; Perifusion

A possible dopaminergic regulation of hypothalamic proopiomelanocortin (POMC)-containing neurons has been investigated in rats by means of in vivo and in vitro approaches. Acute or 3-weeks chronic in vivo treatments with the dopaminergic agonists apomorphine (1 mg/kg; s.c.) and 2-Br-a-ergocriptine (2.5 mg/kg; s.c.) or the dopaminergic antagonist haloperidol (0.15-3 mg/kg; i.p.) had no significant effect on the concentration of a-melanocyte-stimulating hormone (a-MSH) in two hypothalamic regions: arcuate nucleus (AN) and dorsolateral area (DLH). In the same way, chronic administration of the dopaminergic agonists or antagonist did not induce any change in hypothalamic contents of fl-endorphin, another peptide derived from POMC. Reverse-phase high-performance liquid chromatographic analysis revealed that acetic acid extracts of AN and DLH both contained two major forms of a-MSH-like peptides: deacetylated a-MSH and authentic a-MSH. The ratio between these two forms was not altered after acute haloperidoi treatment (3 rng/kg, i.p.). The possible effect of dopamine on the release of hypothalamic a-MSH was studied in vitro using perifused rat hypothalamic slices. Infusion of dopamine (10-7-10-5M) or its antagonist haloperidol (10-SM) had no effect on spontaneous a-MSH release from hypothalamic tissue. In addition, none of these drugs had any effect on potassium (50 mM)-induced a-MSH release. It is concluded that dopaminergic neurons are not involved in the regulation of synthesis, post-translational processing (acetylation) or release of hypothalamic a-MSH. INTRODUCTION Alpha-melanocyte stimulating hormone (a-MSH) is an acetyltridecapeptide amide which has been originally characterized in the pig pituitary, a - M S H derives from a larger precursor molecule called proopiomelanocortin (POMC), which can generate through proteolytic cleavage various regulatory peptides including corticotropin ( A C T H ) , lipotropins (,8and 7-LPH), melanotropins (a, fl- and y-MSH) and fl-endorphin 26'34. Immunoreactive and bioactive aMSH has been detected in the central nervous system of the rat 41. Three distinct groups of perikarya producing a-MSH-like peptides have been identified by immunocytochemistry. The first a - M S H neuronal system has been visualized in the arcuate nucleus;

these neurons which stain positively for various POMC-derived peptides such as A C T H , fl-endorphin and the N-terminal 16K fragment 33,44, send fibers in a number of brain regions including the anterior hypothalamus, septum, thalamus, amygdala, and mesencephalon 6,3°,4°. A second group of cells is located in the dorsolateral region of the hypothalamus; these cells specifically contain a - M S H s,45 and project to the cerebral cortex, striatum and hippocampus 9'3°'36. More recently, a third group of perikarya staining for a-MSH- and ACTH-like materials has been detected in the commissural nucleus; these latter neurons project towards the mesencephalon 19'47. At least two distinct forms of immunoreactive a - M S H have been characterized in the hypothalamus: authentic a - M S H and deacetylated a-

Correspondence: S. J6gou, Groupe de Recherche en Endocrinologie Mol6culaire, UA CNRS 650, Unit6 Alli6e ~ I'INSERM, Facult6 des Sciences, Universit6 de Rouen, 76130 Mont-Saint-Aignan, France.

0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

204

M S H 31'39. In a previous report, we have shown that, in the dorsolateral area of the rat hypothalamus, immunoreactive a-MSH has the same retention time as monoacetyl a-MSH during reverse phase HPLC analysis, whereas the arcuate nucleus produces both deacetylated a-MSH and authentic a-MSH ~5. Although many investigations have raised the possibility that brain a-MSH acts as a neurotransmitter or neuromodulator (see ref. 30 for review), little is known on the regulation of neuronal a-MSH. Recently, anatomical evidence for interactions between catecholamine- and POMC-containing neurons has been reported in the rat 1. In addition, a number of studies indicate that dopaminergic neurotransmission is involved in ACTH- or a-MSH-induced grooming behavior 7'37'46. Several in vivo studies have been performed to determine the effect of dopaminergic drugs on fl-endorphin levels in the brain. However the results obtained are conflicting. Some of the authors observed modifications of fl-endorphin contents, in particular in the hypothalamus, after haloperidol, sulpiride, 2-Br-a-ergocriptine or lisuride treatments u,22,27, whereas other authors did not find any effect of haloperidol on brain fl-endorphin levels 23. In the same way, the data concerning the effect of dopamine on the release of POMC-derived peptides by hypothalamic neurons are contradictory42'43. To our knowledge, the possible role of dopamine on the control of a-MSH synthesis and/or release by hypothalamic neurons has not been studied yet. We have therefore investigated in vivo the effect of dopaminergic agonists and antagonist on the concentration of a-MSH and fl-endorphin in the arcuate nucleus and dorsolateral hypothalamic area. Concurrently, we have used a perifusion technique for rat hypothalamic slices which has recently been developed in our laboratory 17, to investigate a possible control of dopamine on a-MSH release in vitro. MATERIALS AND METHODS Materials Drugs and their sources were as follows: dopamine hydrochloride and apomorphine hydrochloride (Sigma; U.S.A.), 2-Br-a-ergocriptine (CB 154: Sandoz; France), haloperidol (Janssen Le Brun; France). Synthetic a-MSH and deacetylated a-MSH were a generous gift from Drs. R. Andreatta and K. Scheibli

(Ciba-Geigy; Switzerland). The standard solution (KRB) used for perifusion contained: 12(/mM NaCI, 5 mM KC1, 2.6 mM CaCI 2, 1.2 mM KH2PO ~, 0.7 mM MgSO 4, 27.5 mM NaHCO 3 and was supplemented with glucose (1.8 g/l), bacitracin (0.03 g/l), bovine serum albumin (1 g/l). When KC1 was increased to 50 mM, the concentration of NaC1 was proportionally lowered to keep a constant osmolarity. Acetonitrile and trifluoroacetic acid (TFA) used for reversephase high-performance liquid chromatography (HPLC) were from Carlo-Erba (Italy). Animals Adult male Wistar rats, weighing 200-250 g, were used as tissue donors. They were housed in group cages (5 rats/cage) under controlled conditions of light (lights on 07.00-19.00 h) and temperature (24 °C). The animals were given laboratory chow and water ad libitum. In vivo studies For acute treatments, the animals were injected with either apomorphine (1 mg/kg, s.c.) or haloperidol (0.15, 0.3, 1 or 3 mg/kg, i.p.) between 09.00 and 11.00 h. Apomorphine and haloperidol were administered in 500/A vehicle (0.9% NaC1 supplemented with 0.1% ascorbic acid). Control animals were injected with 500 ¢tl vehicle (s.c. or i.p., respectively). The animals were killed 30 or 60 min after the injection. For chronic treatments, the rats were treated for 3 weeks with daily injections of apomorphine (1 mg/kg, s.c.), CB 154 (2.5 mg/kg, s.c.), or haloperidol (1 mg/kg, i.p.). Apomorphine and CB 154 were administered in 500 /.tl vehicle (distilled water supplemented with 8% ethanol and 0.1% ascorbic acid). Haloperidol was injected in 500 #1 0.9% NaC1 supplemented with 0.1% ascorbic acid. Control animals received daily injections of 500~tl vehicle (s.c. or i.p., respectively). The rats were sacrified 3 h after the last injection. Tissue extraction procedure Just after decapitation, the brain was quickly removed and the hypothalamus rapidly dissected out. The arcuate nucleus and dorsolateral area of the hypothalamus were isolated as previously described 15. These two regions were immediately immersed in

205 boiling 2 N acetic acid and maintained in a boiling water bath for 10 min. The tissues were then sonicated for 1 min. The homogenates were centrifugated (10,000 g; 4 °C) for 30 rain and the supernatants were collected and lyophilized. For chromatographic analysis, tissue samples from 4 to 10 animals were pooled together and prepared as described above.

Gel filtration and high-performance liquid chromatography fl-endorphin-like immunoreactivity (fl-endorphinLI) in whole hypothalamic extracts was characterized by gel exclusion chromatography using Sephadex G-75 column (1.5 x 90 cm) as described previously13. The column was calibrated with synthetic human fl-endorphin (fl-h endorphin), a-MSH and purified fl-h LPH. a-MSH-related peptides were resolved by reverse phase HPLC on a Dupont chromatograph equipped with a 870 pump module, a 8800 programmable gradient module coupled to a Zorbax C8 column (0.46 x 25 cm). The mobile phase was a mixture of acetonitrile: TFA: water adjusted to pH 2.4 using 25% ammoniac solution. Chromatography was routinely performed using a linear gradient of acetonitrile (25-30% in 0.1% TFA over 40 min) with a flow rate of 1 ml/min. Fractions were collected every 1 min, evaporated in a Speed Vac concentrator and radioimmunoassayed for a-MSH. The column was calibrated with synthetic a-MSH and deacetylated ctMSH and their sulfoxide forms, as previously described 3. In order to ascertain that no contamination of the hypothalamic extracts by the synthetic standards may occur, blank samples (injection of the solvent only) were systematically analyzed by HPLC and radioimmunoassayed before each experimental HPLC run. The concentrations of the different forms of a-MSH-LI separated by HPLC in AN and D H L extracts were compared in control rats and rats acutely treated with haloperidol (3 mg/kg, i.p.). Since absolute values varied between experiments, the data were expressed as follows: the amount of each form of a-MSH (sulfoxide, deacetylated a-MSH and authentic a-MSH) was calculated as the net area under each HPLC peak (corresponding to the amount of a-MSH-LI measured in 3 consecutive fractions) and expressed as a percentage of total a-MSH-LI in

the extract.

Perifusion of rat hypothalamic slices The perifusion apparatus employed for this study has been previously described in detail 17. The perifusion chamber was composed of a siliconized glass column (0.9 x 15 cm) delimited by two Teflon pestles. The hypothalamic slices were layered in Biogel P2 and perifused with KRB medium. Flow rate (160 /A/min), pH (7.4) and temperature (37 °C) were kept constant. The effluent perifusate was set apart every 3 min in polystyrene tubes, kept at 4 °C, and assayed for a-MSH in duplicate on the same day as the perifusion experiment. Potassium stimulations consisted in 9-min pulses of standard medium containing 50 mM KCI. Dopamine hydrochloride and haloperidol were tested for their ability to modify both basal and K ÷evoked a-MSH releases. These test substances were directly dissolved in gassed KRB medium containing either 5 mM KCI or 50 mM KC1 just before use and infused into the chamber for definite durations at the same flow rate, pH, and temperature as KRB medium alone. Perifusion patterns were established as the mean profiles of a-MSH release (+ S.E.M.) calculated over 3 or 4 independent experiments, a-MSH re-

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FRACTION NUMBER Fig. 1. Gel filtration and RIA quantification of fl-cndorphinlike pcptid¢ in rat hypothalamic extracts. Whole hypothalami from 10 rats w e r e homogenized in 2N acetic acid and chromatographied on a sephadex G-75 column (1.5 × 90 cm). Seventy fractions (1.8 mi each) were collected, stored at -20 °C and radioimmunoassayed in duplicate. The column was calibrated with purified fl-h LPH, synthetic fl-h endorphin and a-MSH (arrows).

206 des-No~-acet yl (X-MSH sulfoxide forms~

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Fig. 2. Reverse-phase HPLC analysis and RIA quantification of a-MSH-like peptides in rat hypothalamic extracts. The arcuate nuclei (AN) and dorsolateral hypothalamic areas (DLH) were sonicated in 2 N acetic acid, lyophilized and submitted to HPLC separation as indicated in Materials and Methods. The dotted line represents the gradient of acetonitrile used over 40 min. Forty fractions (1 ml each) were collected, evaporated in a Speed-Vac concentrator, and radioimmunoassayed in duplicate. Synthetic des-Na-acetyl a-MSH, a-MSH and their sulfoxide derivatives were chromatographied in the same conditions as the tissue extracts (arrows).

leased at any time was expressed as a percentage of the basal level. The reference levels were calculated for each experiment as the m e a n concentration of aMSH in the 4 fractions collected just before the administration of the first depolarizing solution of potassium.

Radioimmunoassays for a-MSH and fl-endorphin The concentrations of a - M S H - and fl-endorphinlike immunoreactivities were m e a s u r e d by means of double-antibody r a d i o i m m u n o a s s a y methods, as described elsewhere 14:6 except that r a d i o i o d i n a t e d synthetic a - M S H was purified by H P L C 17. The sensitivity thresholds of the a - M S H and fl-endorphin assays were 1 pg and 60 pg, respectively. The crossreactivities of a - M S H antibodies with synthetic human or porcine ACTHI_39 , ACTHl_24 and h u m a n or bovine fl-MSH were lower than 0.1%. Conversely, the a - M S H antiserum fully cross-reacted with deacetylated a - M S H and sulfoxide derivates of a - M S H

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Fig. 3. Effect of a single injection of haloperidol (0.15, 0.3 or 1 mg/kg), apomorphine (1 mg/kg) or vehicle (500/d saline) on the contents of a-MSH-LI in the arcuate nucleus (AN) and dorsolateral hypothalamus (DLH) of individual rats. Animals were sacrified 30 or 60 min after the injection. All values are expressed in nanogram of peptide per hypothalamic region (mean ___S.E.M.). The number of rats in each group is indicated under the columns. and des-Na-acetyl a - M S H . The antiserum read the sequence 10-11 of the a - M S H molecule. The antigenic determinant recognized by the fl-p endorphin antibodies was the sequence 7 2 - 8 7 of fl-LPH. This antiserum did not cross-react with y-p endorphin (0.006%). On a weight basis, cross-reactivity between fl-p e n d o r p h i n antiserum and fl-p L P H was 15%. Peptide concentrations were calculated with a Hewlett Packard m o d e C ( H P 85), from the p a r a m eters of standard curves linearized by means of the logit-log transformation. Statistical significance of differences between values was calculated using Student's t-test.

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The chromatographic profile of hypothalamic flendorphin-LI on Sephadex G-75 column is shown in Fig. 1. A major peak of fl-endorphin-LI (94% of total immunoreactivity) co-eluted with synthetic fl-h endorphin whereas a minor peak co-eluted with pudried fl-h LPH. The characterization of a-MSH-LI in acetic acid extracts of arcuate nucleus (AN) and dorsolateral hypothalamus (DLH) was carded out by combining reverse phase HPLC and radioimmunoassay. As shown in Fig. 2, three peaks of immunoreactivity were resolved by HPLC with the same retention times as des-Na-acetyl a-MSH, authentic a-MSH and their sulfoxide forms. In both arcuate nucleus and dorsolateral hypothalamic extracts, the major peak corresponded to deacetylated a-MSH (51-60% of total ctMSH-LI). Authentic a-MSH represented 29-36% of the total immunoreactivity while the sulfoxide forms were only minor components.

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The effects of haloperidol, apomorphine or CB 154 on the levels of a-MSH-like peptides in AN or D L H extracts are shown in Fig. 3 and 4. Acute haloperidol or apomorphine treatments did not impair the concentrations of a-MSH-LI in the two hypothalamic regions (Fig. 3). Similarly, chronic administrations of dopaminergic agonists (apomorphine or CB 154) or antagonist (haloperidol) did not alter aMSH-LI or fl-endorphin-LI in the AN or D L H (Fig. 4). Only a slight increase in fl-endorphin-LI concentrations was observed in D L H of rats chronically treated with apomorphine (1 mg/kg) (Fig. 4b). However, no significant differences between control and treated groups could be detected when the data were

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Fig. 4. Effect of chronic treatment with haloperidol (1 mg/kg), apomorphine (1 mg/kg), CB 154 (2.5 mg/kg), or vehicle (500/~1 saline or distilled water), during 3 weeks, on the contents of aMSH-LI (a) and fl-endorphin-LI (b) in the arcuate nucleus (AN) and dorsolateral hypothalamus (DLH) of rats. Peptide levels in whole rat hypothalamus are noted AN + DLH. Animals were sacrified 3 h after the last injection. The data are expressed in nanogram of peptide per hypothalamic region (mean + S.E.M.). The number of rats is indicated under each column. * P < 0.05 when compared to controls.

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The possible involvement of d o p a m i n e in the regulation of spontaneous and K+-evoked releases of aMSH was investigated in vitro, using perifused hypothalamic slices. Depolarizing concentrations of potassium (50 mM) were applied at the beginning and at the end of the perifusion experiments to make sure that a - M S H n e u r o n s were functioning properly. As n~4

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expressed as peptide levels in whole rat hypothalamic areas. The amounts of the 3 different forms of a - M S H - L I separated by H P L C analysis of hypothalamic extracts have been compared in control animals and in rats receiving acute haloperidol administration (Fig. 5). In control rats, the percentage of deacetylated aMSH was significantly greater than that of authentic a - M S H in both A N and D L H extracts. No modification of the distribution profile of the a - M S H - L I was observed in A N and D L H from haloperidol-treated rats, indicating that haloperidol treatment has no el-

Fig. 6. Effect of dopamine (10 -6 M) on basal (a) or K÷-evoked (b) release of a-MSH by perifused rat hypothalamic slices. After a 36-rain equilibration period, KCI (50 mM) was infused for 9 min. Slices were allowed to stabilize for 27 min, and dopamine (10 -6 M) was then infused for 24 min. In one series of experiments (b), 15 min after the onset of dopamine infusion, a second pulse of KCI (50 mM) was applied for 9 min. A third 9min pulse of KCI (50 mM) was applied 27 min after dopamine withdrawal, as a control. The data represent the mean (+ S.E.M.) of 3 and 4 independent experiments. The reference level of a-MSH release (100% basal level) was calculated for each experiment as the mean rate of a-MSH release during 12 min (4 consecutive fractions; O--C)) just preceding the infusion of the first pulse of KC1. The mean basal level (100%) of a-MSH release in the experiments were, respectively, 9.2 + 0.2 (a) and 6.1 + 2.2 (b) pg/min × 10 hypothalami.

209 TABLE I Effect of dopamine (10-7-10-5 M) and haloperidol (10-5 M) on basal and K +-evoked release of a-MSH by perifused rat hypothalamic slices

The same protocol as in Fig. 6 was applied to study the effect of various doses of dopamine and haioperidol on hypothalamicaMSH release. The data represent the mean (__-S.E.M.) of independent experiments. The number of experiments is given in parenthesis. The reference level of a-MSH release (100% basal level) was calculated for each experiment as the mean rate of a-MSH release during 18 rain (6 consecutive fractions) just precedingthe infusionof the test substance. Test substance

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shown in Fig. 6a, the infusion of dopamine (10-6 M) during 24 min did not modify the basal release of aMSH. As previously reported 17, 9-rain pulses of KCI (50 mM) significantly increased a-MSH release by hypothalamic slices (Fig. 6a). Infusion of dopamine for 24 rain did not alter KCl-induced stimulation of aMSH release (Fig. 6b). A series of experiments similar to those represented in Fig. 6 were conducted with various doses of dopamine (10-7-10 -5 M) and with a high dose of haloperidol (10 -5 M). As summarized in Table I, neither dopamine nor haloperidol had any effect on spontaneous or K ÷-induced a-MSH release. DISCUSSION The present study demonstrates that acute or chronic administrations of apomorphine or haloperidol have no effect on a-MSH contents in the arcuate nucleus and in the dorsolateral hypothalamic area of the rat. These findings suggest that the synthesis of POMC and the processing of this precursor molecule to generate a-MSH are not under dopaminergic con-

trol. The lack of dopaminergic regulation of the hypothalamic POMC system is consistent with the observation that fl-endorphin concentrations in hypothalamic extracts were not affected by chronic administration of apomorphine, CB 154 or haloperidol. The presence of significant amounts of fl-endorphin detected in the dorsolateral area of the hypothalamus is in apparent disagreement with histochemical studiess'45 which showed that a-MSH-containing perikarya located in the zona incerta do not stain for other POMC-derived peptides. However, in a previous study 15, we have provided an explanation for this discrepancy: we showed that colchicine treatment significantly reduced the concentration of fl-endorphin in the DLH, indicating that immunoreactive fl-endorphin measured in this diencephalic region is essentially contained in nerve fibers, most probably originating from the AN, and coursing through the DLH towards the thalamic periventricular nucleus, dorsal and ventral midbrain and/or dorsal brainstem 3°. Unlike our findings, several studies have suggested that hypothalamic fl-endorphin levels are reduced by chronic treatment with the dopaminergic agonists CB 154 or lisuride 22 and elevated by administration of dopaminergic antagonists such as haloperido111 or sulpiride 27, suggesting that synthesis of POMC-derived peptides in hypothalamic neurons and intermediate lobe melanotrophs 4,12 are similarly regulated by dopamine. However, more recent data have clearly shown that chronic injections of haloperidol do not affect hypothalamic fl-endorphin levels23. The discrepancy between these last findings (which totally agree with our present results) and those previously reported 11,22 does not appear to be attributable to differences in treatment protocols since, in all studies, haloperidol and CB 154 were found to alter fl-endorphin levels in the intermediate lobe of the pituitary. In a recent study, Roberts et al. showed that long-term treatment of rats with either haloperidol or CB 154 did not modify POMC mRNA levels in the hypothalamus, confirming that dopaminergic systems are not involved in the control of biosynthesis of neuronal POMC (Cited in ref. 23). The process of acetylation of a-MSH, which occurs on the N-terminal amino acid is a regulatory point for this peptide to achieve full biological activity. For instance, behavioral studies indicate that acetylated aMSH is more potent than the deacetylated molecule

210 in improving performance on visual discrimination task and in inducing grooming behavior 2s'3j. Thus, we have investigated the possible effect of haloperidoi treatment on acetylation of hypothalamic aMSH. As the a-MSH antiserum used in the present study does not discriminate between the various forms of a-MSH (authentic a-MSH, des-Na-acetyl a-MSH and their sulfoxide derivatives are equally potent in displacing antibody-bound [I~-5I]a-MSH), we have analyzed by HPLC a-MSH immunoreactive peptides in order to determine whether dopaminergic agents may affect the acetylation process. Our results show that, in AN and DLH extracts from control rats, the percentage of des-Na-acetyl a-MSH is significantly higher than those of authentic a-MSH and sulfoxide forms. This observation is consistent with that of Turner et al. 39 who also found a major peak of deacetylated a-MSH during HPLC purification of whole hypothalamic extracts. The sulfoxide derivatives of a-MSH and des-Na-acetyl a-MSH were probably artifactually generated during the acetic acid extraction procedure, since the methionine residue is highly susceptible to oxidation 2s. The fact that acute treatment with haloperidol did not modify the amounts of deacetylated and acetylated forms present in both hypothalamic regions indicates that dopaminergic neurons are not involved in the posttranslational processing of neuronal a-MSH, and particularly does not exert any control in the acetyltransferase activity which has been characterized in the rat brain by O'Donohue et al. 29. Our findings are consistent with those of Ham et al. 1° who demonstrated that chronic administration of haloperidol does not influence the processing (and particularly the acetylation) of fl-endorphin in the rat hypothalamus. in contrast it is interesting to mention that, at the intermediate lobe level, dopamine has been found to inhibit the acetyltransferase activity24 and to reduce the formation of acetylated a-MSH TM. Recent biochemical and anatomical studies suggest that a-MSH-like immunoreactivity contained in the lateral hypothalamic cell group is not authentic aMSH but an amidated peptide with carboxy-terminal homology to a-MSH 2°'35. The most prominent finding in these studies was the fact that an antiserum against the 4-10 region of a-MSH stained the arcuate nucleus POMC-neurons but failed to detect immunoreactive perikarya in the lateral region of the

rat hypothalamus. The a-MSH antiserum used in our study was directed against the COOH-terminal part of the a-MSH molecule 14. Thus this antiserum did not discriminate between authentic a-MSH and other related peptides which would differ from a-MSH by some determinant in the midregion. It should be noticed however that, in the present study, the aMSH immunoreactive material contained in the dorsolateral hypothalamus co-eluted with authentic aMSH, deacetylated a-MSH and their sulfoxide forms during HPLC analysis. Our data indicate therefore that the a-MSH-like peptide located in the lateral hypothalamus must be closely related, if not identical to pituitary/arcuate a-MSH. In a previous study, we have developed a perifusion technique for rat hypothalamic slices and we have characterized the release of a-MSH from hypothalamic tissue in vitro: we showed that a-MSH release is stimulated by depolarizing concentrations of potassium or by veratridine and that the evoked releases were calcium-dependent 17. In the present study, we have applied our in vitro perifusio n system to investigate the possible involvement of the dopaminergic system in the control of release of neuronal a-MSH. As reported in our previous study, successive potassium pulses induced a reproducible rise in hypothalamic a-MSH release which made it possible to compare, within the same perifusion experiment, the neurosecretory responses to different secretagogues in basal conditions or under depolarizing conditions. The results shown in Table I demonstrate that neither dopamine (10-7-10 -s M) nor haloperidol (10 -5 M) had any effect on the basal release of aMSH and that these drugs did not impair K+-evoked release of a-MSH. These latter results are in good agreement with our in vivo data indicating that acute treatments with apomorphine or haloperidol do not change hypothalamic a-MSH levels. Using hypothalamic synaptosomial preparations, Warberg et al. 43 have also examined the possible effect of dopamine on basal and K+-stimulated a-MSH releases and reached the same conclusion. In pituitary melanotrophs, dopamine is generally considered as the major a-MSH-release inhibiting factor2s'3s. Therefore, our results together with those of other investigators 43 provide evidence that distinct mechanisms regulate a-MSH release in hypothalamic neurons and pars intermedia cells. In contrast, Vermes et al. 42 re-

211 ported that dopamine caused an inhibition of fl-endorphin release. The difference between these series of data would suggest that dopamine exerts a selective control on a - M S H and fl-endorphin releases. However, the fact that a - M S H and fl-endorphin derive from the same precursor molecule 34 and are cosequestered in the same synaptic vesicles 2'32 strongly suggests that these two peptides are released concomitantly. It should be mentioned that some of the studies 42 were performed using incubated hypothalamic slices, a system where ultra-short feed-back of neuropeptides on their own release cannot be excluded. Indeed such a self-regulation of P O M C neurons by POMC-derived peptides is supported by immunocytochemical data showing synaptic contacts among P O M C neurons at the hypothalamic level 5,21. In conclusion, the present studies performed both in vivo and in vitro support the concept that dopami-

nergic neurons are not involved in the regulation of synthesis, processing and release of hypothalamic aMSH. These data indicate that the mechanisms controlling synthesis and release of neuronal a - M S H are fundamentally different from those previously demonstrated in the intermediate lobe of the pituitary.

REFERENCES

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ACKNOWLEDGEMENTS We are indebted to Drs. R. Andreatta and K. Scheibli (Ciba-Geigy) for their generous gift of synthetic des-Na-acetyl a - M S H and authentic a-MSH. This research was supported by grants from INS E R M (84-6020), D R E T (85-1406; 86-1164; 861375), M E N (86-1128), the Region of Haute Normandie and the Institut de Recherches Internationales S E R V I E R . We thank Miss S. Letellier for typing the manuscript.

212

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