D-2 dopamine receptor-mediated inhibition of pro-opiomelanocortin synthesis in rat intermediate lobe. Abolition by pertussis toxin or activators of adenylate cyclase.

Stimulation of the D-2 dopamine receptor inhibits pro-opiomelanocortin (POMC) synthesis in isolated rat intermediate lobe tissue. Intermediate lobe tissue was incubated in the absence or presence of various dopaminergic compounds, and then its capacity to incorporate [3H]tyrosine into POMC was tested. D-2 dopaminergic agonists caused a dose-dependent inhibition of POMC synthesis; the maximal inhibitory effect was approximately a 50% reduction in the amount of POMC synthesized. D-2 dopaminergic antagonists blocked the inhibitory effect of each agonist. Pretreatment of the tissue with pertussis toxin abolished the D-2 dopaminergic inhibition of POMC synthesis. The potency of pertussis toxin in abolishing the dopaminergic inhibition of POMC synthesis corresponded to its potency in abolishing the D-2 dopaminergic inhibition of adenylate cyclase activity. Cholera toxin, forskolin, and 8-bromo-cAMP, compounds that activate the cAMP pathway, enhanced the capacity of intermediate lobe tissue to synthesize POMC and counteracted the dopaminergic inhibition of POMC synthesis. Incubation of intermediate lobe tissue for 24 h with bromocriptine, a D-2 dopaminergic agonist, decreased the POMC mRNA content by 46% as determined by hybridization of RNA to a 32P-labeled probe. Incubation of intermediate lobe tissue with forskolin increased the level of POMC mRNA; incubation of the tissue with a combination of bromocriptine and forskolin also resulted in an increase in the level of POMC mRNA. It is proposed that Ni, the inhibitory guanyl nucleotide binding protein, and possibly adenylate cyclase mediate the dopaminergic inhibition of POMC synthesis.

Stimulation of the D-2 dopamine receptor inhibits pro-opiomelanocortin (POMC) synthesis in isolated rat intermediate lobe tissue. Intermediate lobe tissue was incubated in the absence or presence of various dopaminergic compounds, and then its capacity to incorporate [3H]tyrosine into POMC was tested. D-2 dopaminergic agonists caused a dose-dependent inhibition of POMC synthesis; the maximal inhibitory effect was approximately a 50% reduction in the amount of POMC synthesized. D-2 dopaminergic antagonists blocked the inhibitory effect of each agonist. Pretreatment of the tissue with pertussis toxin abolished the D-2 dopaminergic inhibition of POMC synthesis. The potency of pertussis toxin in abolishing the dopaminergic inhibition of POMC synthesis corresponded to its potency in abolishing the D-2 dopaminergic inhibition of adenylate cyclase activity. Cholera toxin, forskolin, and 8bromo-CAMP, compounds that activate the CAMP pathway, enhanced the capacity of intermediate lobe tissue to synthesize POMC and counteracted the dopaminergic inhibition of POMC synthesis. Incubation of intermediate lobe tissue for 24 h with bromocriptine, a D-2 dopaminergic agonist, decreased the POMC mRNA content by 46% as determined by hybridization of RNA to a "P-labeled probe. Incubation of intermediate lobe tissue with forskolin increased the level of POMC mRNA; incubation of the tissue with a combination of bromocriptine and forskolin also resulted in an increase in the level of POMC mRNA. It is proposed that Ni, the inhibitory guanyl nucleotide binding protein, and possibly adenylate cyclase mediate the dopaminergic inhibition of POMC synthesis.
The melanotroph of the intermediate lobe of the rat pituitary gland contains mRNA directing the synthesis of proopiomelanocortin (POMCl). POMC is processed into several * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The present study was designed to investigate the mechanism by which the D-2 dopamine receptor inhibits POMC synthesis in rat intermediate lobe tissue. The involvement of the D-2 dopamine receptor in the synthesis and processing of POMC has been less well characterized than its involvement in the inhibition of hormone release. Dopaminergic drugs (when administered to rats for 24 h or longer) change the following parameters related to POMC synthesis by the intermediate lobe: the content of mRNA directing the synthesis of POMC; the capacity of the tissue to synthesize POMC; and the capacity of the tissue to synthesize . Dopaminergic antagonists enhance these parameters while dopaminergic agonists decrease these parameters. Furthermore, the intermediate lobes of stalk-sectioned animals have an enhanced capacity to synthesize POMC compared to the intermediate lobes from sham-operated animals (38). Taken together, these observations suggest that the dopaminergic neurons innervating the intermediate lobe exert a tonic inhibitory influence on the functioning of the melanotrophs. The biochemical mechanisms involved in the dopaminergic inhibition of POMC synthesis have not been investigated.
Preparation and Treatment of Tissue-After decapitation of each rat (Sprague-Dawley, male, 300-350 g, Taconic Farms, Germantown, NY), intermediate lobe tissue was dissected from other pituitary tissue as previously described (12). Each intermediate lobe was collected in 1 ml of Eagle's Minimal Essential Medium fortified with 100,000 units/liter penicillin, 100 mg/liter streptomycin, HEPES (pH 7.4), and 0.25% bovine serum albumin (EMEM/BSA) equilibrated with a mixture of 95% air and 5% COZ. Each intermediate lobe was washed two times with 5 ml of EMEM/BSA and resuspended in 1 ml of Ham's FIO medium fortified with 2.5% fetal calf serum, 12.5% horse serum, 100,000 units/liter penicillin, and 100 mg/ml streptomycin equilibrated with a mixture of 95% air and 5% CO,. Each intermediate lobe was routinely incubated at 37 "C in an atmosphere of 95% air and 5% COz in a multiwell plate in the absence or presence of the indicated drugs. When the effect of pertussis toxin was investigated ( Assays-To determine the capacity of the intermediate lobe to synthesize POMC following various pretreatment protocols, intermediate lobe tissue was transferred to a Microfuge tube, centrifuged 5 s, and resuspended in 0.5 ml of EMEM/BSA without tyrosine (or without methionine, when appropriate) and incubated 30 min at 37 "C (38). The tissue was then centrifuged, resuspended in 200 pl of EMEM/BSA containing 40 pCi of l-[3H]tyrosine (specific activity, 54 Ci/mmol; final concentration 3.7 p M ) and incubated 30 min at 37 "C. At the end of the incubation, each tube was centrifuged for 5 s; the resulting supernatant fluid was discarded, and the tissue was washed twice with 1-ml aliquots of complete EMEM/BSA. Intermediate lobe tissue was resuspended in 200 pl of 2 N acetic acid containing 5 mg/ml bovine serum albumin and 0.17 mM phenylmethylsulfonyl fluoride and 0.17 mM iodoacetamide. This suspension was sonicated for 5 s and extracted overnight at 4 "C. Occasionally, [35S] methionine (specific activity, 400 Ci/mmol; final concentration, 0.8 p~) was substituted for [3H]tyrosine in the assay. Routinely, the newly synthesized protein was isolated by chromatography on commercially prepared CN columns (J. T. Baker). Each column was equilibrated with 600 pl of acetonitrile followed by 1.8 ml of a solution of triethylamine adjusted to pH 3.0 with phosphoric acid (TEAP); the final concentration of triethylamine was 1 mM. A 75-pl aliquot of the tissue extract was applied to the column. The column was washed with 1.8 ml of 1 mM TEAP and the newly synthesized POMC was eluted with a solution composed of 30% acetonitrile and 70% 20 mM triethylamine phosphate (pH 3.0). Validation of the column extraction procedure for isolation of newly synthesized POMC is presented in the Miniprint Supplement?
Samples eluted from the extraction column were occasionally lyophilized and subjected to a double antibody immunoprecipitation scheme (40) using B-endorphin antiserum as previously described (38). Samples from the extraction columns were also occasionally subjected to SDS-polyacrylamide gel electrophoresis on a 12% gel and the gels were subsequently exposed to x-ray film (41). Fig. 1 illustrates that the radiolabeled protein isolated by chromatography on the disposable extraction columns is composed predominantly of a 34,000-36,000-Da doublet. Four lines of evidence presented in the Miniprint Supplement suggest that this material is POMC.
Adenylate cyclase activity was determined as described previously (12). Intracellular and extracellular levels of IR-aMSH were determined by radioimmunoassay as previously described (16). h with CB154 caused a dosedependent inhibition in the capacity of the tissue to synthesize POMC (Fig. 2, left). Significant inhibition of POMC synthesis was obtained with incubations of 16 h or longer. For the sake of consistency with an earlier study (38), 24-h incubations were routinely used. The inhibitory effect of CB154 was blocked in a dose-dependent manner b y spiroperidol, a D-2 dopaminergic antagonist (Fig. 2, right). N-0434 and LY171555, two selective D-2 dopaminergic agonists (44, 46), also inhibited POMC synthesis in a dose-dependent manner; YM09151-2, a selective D-2 antagonist (47), blocked the inhibitory effect of either agonist (Fig. 2, left, inset).

inset).
Pertussis toxin abolished the ability of D-2 agonists to inhibit POMC synthesis. Intermediate lobe tissue was treated with a range of concentrations of pertussis toxin for 18 h; the tissue was then incubated for an additional 24 h in the absence or presence of 0.1 PM CB154; finally, the capacity of t h e tissue to synthesize POMC was determined (Fig. 3). Pertussis toxin caused a dose-dependent abolition of the dopaminergic inhibition of POMC synthesis. In a parallel experiment, pertussis toxin was also tested for its ability to abolish the dopaminergic inhibition of adenylate cyclase activity. Intermediate lobe tissue was treated with a range of concentrations of pertussis toxin for 18 h. Each treatment group was washed, homogenized, and tested in the adenylate cyclase assay in the absence or presence of dopamine. The potency of pertussis toxin in attenuating the dopaminergic inhibition of adenylate cyclase activity was similar t o the potency of pertussis toxin in attenuating the dopaminergic inhibition of POMC synthesis (Fig. 3, inset).
Compounds that activate the CAMP stimulatory pathway enhanced the capacity of the intermediate lobe to synthesize POMC. Intermediate lobe tissue was treated for 24 h with the indicated concentrations of either cholera toxin, forskolin, or 8-bromo-CAMP (Fig. 4). Each of these compounds caused a Portions of this paper (including Table A  The newly synthesized protein from each sample was isolated from disposable extraction columns as described under "Experimental Procedures." An aliquot of the sample of each group that contained radioactivity closest to the mean of that group was subjected to electrophoresis on a 12% SDS-polyacrylamide gel as described under "Experimental Procedures." The gel was exposed to x-ray film for 72 h. Migration of molecular weight markers (Bio-Rad, low molecular weight) is indicated at the right.
dose-dependent increase in the capacity of the tissue to synthesize POMC. In addition, forskolin was tested for its ability to counteract the dopaminergic inhibition of POMC synthesis. Intermediate lobe tissue was incubated for 24 h in the absence or in the presence of 0.1 p~ N-0434 and a range of concentrations of forskolin (Fig. 5). Forskolin reversed in a dose-dependent manner the dopaminergic inhibition of POMC synthesis. In a parallel experiment, forskolin increased intermediate lobe adenylate cyclase activity in both the absence and presence of a D-2 dopamine agonist (Fig. 5, inset); at a concentration of 1 pM, forskolin stimulated adenylate cyclase activity in the presence of N-0434. A higher concentration of forskolin caused a greater enhancement of adenylate cyclase activity but failed to cause a further enhancement of POMC synthesis. Cholera toxin and 8-bromo-CAMP also counteracted the dopaminergic inhibition of POMC synthesis ( Table I).
The intracellular and extracellular levels of IR-aMSH from intermediate lobe tissue treated for 24 h in the absence of drugs or in the presence of CB154, forskolin, or a combination of CB154 and forskolin are shown in Table 11. All drugtreated groups had elevated tissue levels of IR-aMSH compared to control levels. Treatment with CB154 alone decreased the level of extracellular IR-aMSH, whereas treatment with either forskolin alone or in combination with CB154 increased the level of extracellular IR-aMSH compared to the control level.
In three experiments, intermediate lobe tissue was treated for 24 h in the absence of drugs, or in the presence of CB154, forskolin, or a combination of CB154 and forskolin (Fig. 6). The level of POMC mRNA in each group was then quantified by hybridization with a radiolabeled probe derived from bovine tissue (see Miniprint Supplement). Tissue treated with CB154, forskolin, or a combination of CB154 and forskolin had 54,320, and 178% of the control level of POMC mRNA, respectively. The changes in amount of POMC mRNA were not the result of differences in the amount of tissue RNA, since equal amounts of total RNA were applied to each lane of the gel.

DISCUSSION
The present study presents evidence suggesting that the D-2 dopamine receptor in the intermediate lobe of the rat pituitary gland regulates the capacity of this tissue to synthesize POMC (44-49). The D-2 dopamine receptor in the intermediate lobe is associated with an inhibitory guanyl nucleotide-binding protein (termed Ni (19)) (12,13). Ni acts as a transducer converting stimulation of the D-2 receptor into inhibition of adenylate cyclase activity (19). Pertussis toxin uncouples Ni from the D-2 dopamine receptor and abolishes the receptor-mediated inhibition of adenylate cyclase activity (22). In the present study, pertussis toxin caused a dosedependent abolition of the dopaminergic inhibition of POMC synthesis. This finding strongly suggests that Ni and cAMP participate in the dopaminergic inhibition of POMC synthesis.
This study also presents evidence that N,, the stimulatory guanyl nucleotide binding protein (19), adenylate cyclase, and cAMP participate in the regulation of capacity of the inter- POMC synthesis in the absence of drugs is expressed as 100%; POMC synthesized on the presence of drugs is expressed as a per cent of control values. mediate lobe to synthesize POMC. The participation of N, in the regulation of POMC synthesis by the intermediate lobe is suggested by the ability of cholera toxin to increase POMC synthesis with approximately the same molar potency that it displays as a stimulant of adenylate cyclase (13, 49-56). The involvement of adenylate cyclase per se in the regulation of POMC synthesis is suggested by the ability of forskolin to increase POMC synthesis and to overcome the D-2 dopaminergic inhibition of POMC synthesis. The direct involvement of cAMP in the regulation of POMC synthesis is suggested by the ability of 8-bromo-CAMP to mimic the effects of cholera toxin (52). The involvement of adenylate cyclase in the dopaminergic inhibition of POMC synthesis is suggested by the ability of forskolin and cholera toxin to reverse the dopaminergic inhibiton of POMC synthesis at concentrations that reverses the dopaminergic inhibition of adenylate cyclase activity.
The findings of the present study provide evidence for the following model of the dopaminergic regulation of POMC synthesis in the rat melanotroph Stimulation of the D-2 receptor on the surface of the melanotroph alters the property of Ni so that intracellular GTP can activate Ni causing Ni to inhibit adenylate cyclase activity and thereby lower the intracellular concentration of CAMP. The diminished level of cAMP results in a decrease in the capacity of the cell to synthesize POMC. Diminished gene transcription provides one potential mechanism for decreasing POMC synthesis as a consequence of stimulating the D-2 receptor: in in vivo studies, CB154 caused parallel decreases in the content of POMC mRNA and in the capacity of the tissue to synthesize POMC (37, 38). In the current study (Fig. 6)  Changes in tissue levels of P O M C mRNA following treatment with CB154, forskolin, or a combination of C B 1 5 4 and forskolin. Intermediate lobe tissue (10 lobes/group) were incubated for 24 h in the absence of drugs, or in the presence of either CB154 (30 nM), forskolin (10 p~) , or a combination of CB154 (30 nM) and forskolin (10 p M ) as described under "Experimental Procedures." Total RNA was extracted, and tissue levels of POMC mRNA were determined by Northern blot analysis as described in text. The amount of POMC mRNA hybridized was quantified by cutting out the sections of the gel corresponding to the location of the bands on the x-ray film and determining the radioactivity of each section by liquid scintillation spectroscopy. This experiment was repeated three times. The actual amounts of radioactivity f S.E. in the three experiments were 740 f 18,396 f 16,2366 f 79, and 1316 f 37 for the control, CB154-treated, forskolin-treated, and CB154 plus forskolin-treated groups, respectively. Kb, kilobase. can be reversed by drugs either stimulating cAMP synthesis or mimicking the intracellular action of cAMP (57-60).
In vivo studies suggest that the dopamine receptor may regulate many aspects of the functioning of the intermediate lobe. Prolonged blockade of the dopamine receptor results in an elevation of acetyltransferase activity and and increase in the tissue concentrations of IRaMSH and B-endorphin, whereas prolonged stimulation of the dopamine receptor results in a decrease in the tissue concentration of IRaMSH and an atrophy of intermediate lobe tissue (38,61). Taken together, these observations suggest that activation of the dopamine receptor does not merely diminish transcription of the POMC gene but rather causes a coordinated diminution of numerous processes involved in the synthesis of the intermediate lobe peptides.
While this paper was being reviewed for publication, a similar study showing that dopamine inhibited pro-opiomelanocortin synthesis in the amphibian pituitary intermediate lobe appeared in this journal (68). Four linea of evidence suggest that the radiolabelled material eluted from the CN rapidly incorporates radiolabelled amino acids into POMC ( 3 ) . Second, when subjected to analysis br WLC, the bulk of the mateiial isolated from the CN dinosable exfracLIo~ column was found t o have the same elurion profile as POHC previously purified by W L C ( fig. I1 Right). lbird, h e n analyzed by electrophoresis, the bulk of the radiolabelled material isolated from the CN column occurred as a doublet with an apparent molecular weight of 34,000/36,000 DA (Fig. 1). Purrhermare, treatments of intermediate lobe tis-Q U~ that either decreased (CB154 rreatmenr) or sn increased (for&olin treatmeof) the decrease or increase in the relative intensity of the 34.000136.000 DA doublet (fie. 1 amount of radioactive material recovered from the CN column caused a nearly identical radioactivity of each seerion by liquid scintillation spectroscopy. b7 Affolfer and Reisine (67).
The construction of the POMC I@XA probe is described in a recent publication