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In mammals, pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors PAC1-R, VPAC1-R, and VPAC2-R play a role in various physiological processes, including proopiomelanocortin (POMC) and brain-derived neurotrophic factor (BDNF) gene expression. We have previously found that PACAP stimulates POMC gene expression, POMC biosynthesis, and alpha-MSH secretion in the melanotrope cell of the amphibian Xenopus laevis. This cell hormonally controls the process of skin color adaptation to background illumination. Here, we have tested the hypothesis that PACAP is involved in the regulation of Xenopus melanotrope cell activity during background adaptation and that part of this regulation is through the control of the expression of autocrine acting BDNF. Using quantitative RT-PCR, we have identified the Xenopus PACAP receptor, VPAC1-R, and show that this receptor in the melanotrope cell is under strong control of the background light condition, whereas expression of PAC1-R was absent from these cells. Moreover, we reveal by quantitative immunocytochemistry that the neural pituitary lobe of white-background adapted frogs possesses a much higher PACAP content than the neural lobe of black-background adapted frogs, providing evidence that PACAP produced in the hypothalamic magnocellular nucleus plays an important role in regulating the activity of Xenopus melanotrope cells during background adaptation. Finally, an in vitro study demonstrates that PACAP stimulates the expression of BDNF transcript IV.

Proopiomelanocortin (POMC) is synthesized predominantly in pituitary corticotrophs, melanotrophs, and arcuate hypothalamic neurons. Corticotroph-derived ACTH mediates basal and stress-induced glucocorticoid secretion, but it is uncertain whether POMC peptides produced in the brain also regulate the hypothalamic-pituitary-adrenal axis. To address this question, we generated neuron-specific POMC-deficient mice by transgenic (Tg) replacement of pituitary POMC in a global Pomc(-/-) background. Selective restoration of pituitary POMC prevented the adrenal insufficiency and neonatal mortality characteristic of Pomc(-/-) mice. However, adult Pomc(-/-)Tg/+ mice expressing the pituitary-specific transgene exhibited adrenal cortical hypertrophy, elevated basal plasma corticosterone, elevated basal but attenuated stress-induced ACTH secretion, and inappropriately elevated CRH expression in the hypothalamic paraventricular nucleus. In addition, Pomc(-/-)Tg/+, Pomc(+/-)Tg/+, and Pomc(+/-) mice, which all displayed varying degrees of elevated CRH, frequently developed melanotroph adenomas after 1 yr of age, whereas Pomc(-/-) mice, with maximal CRH expression and glucocorticoid disinhibition, developed corticotroph and melanotroph adenomas. These results indicate that neuronal POMC peptides are necessary to regulate CRH within physiological limits and that a chronic reduction or absence of hypothalamic POMC leads to trophic stimulation of pituitary cells directly or indirectly through elevated CRH levels.

Cushing's disease (CD) is a rare condition in which hypercortisolemia is secondary to excessive ACTH release from a pituitary corticotroph adenoma. CD is associated with significant morbidity and mortality, and a safe therapy that effectively targets the pituitary tumor is still lacking. Retinoic acid (RA) and dopamine agonists (DAs) have recently been considered as monotherapy in CD patients, and satisfactory results have been reported, albeit in a limited number of patients. Given the permissive role of RA on the dopamine receptor type-2 (DRD2), the aim of the present study was to see whether a combination of 9-cis RA and the DA bromocriptine (Br) might represent a possible treatment for CD. Here we show that 9-cis RA induces a functional DRD2 in the pituitary corticotroph cell line AtT20, and increases cell sensitivity to Br via a mechanism only partially related to corticotroph-to-melanotroph transdifferentiation. In addition, 9-cis RA and Br act synergistically to modulate cell viability, with favorable implications for clinical use. In nearly 45% of corticotropinoma-derived primary cultures, the combined administration of 9-cis RA and Br lowered the steady-state level of the ACTH precursor proopiomelanocortin (POMC) more efficiently than either of the drugs alone. In conclusion, the effects of a combination of 9-cis RA and Br on ACTH synthesis/secretion and cell viability in AtT20, and on POMC transcriptional activity in human corticotropinomas might represent a suitable starting point for assessing the potential of this treatment regimen for ACTH-secreting pituitary adenomas. This study thus has potentially important implications for novel therapeutic approaches to CD.

The frog pars intermedia is composed of a single population of endocrine cells directly innervated by gamma-aminobutyric acid (GABA)ergic nerve terminals. We have previously shown that GABA, acting through GABA(A) receptors, modulates both the electrical and secretory activities of frog pituitary melanotrophs. The aim of the present study was to take advantage of the frog melanotroph model to determine the relationship between the subunit composition and the pharmacological properties of native GABA(A) receptors. Immunohistochemical labeling revealed that in situ and in cell culture, frog melanotrophs were intensely stained with alpha2-, alpha3-, gamma2-, and gamma3-subunit antisera and weakly stained with a gamma1-subunit antiserum. Melanotrophs were also immunolabeled with a monoclonal antibody to the beta2/beta3-subunit. In contrast, frog melanotrophs were not immunoreactive for the alpha1-, alpha5-, and alpha6-isoforms. The effects of allosteric modulators of the GABA(A) receptor on GABA-activated chloride current were tested using the patch-clamp technique. Among the ligands acting at the benzodiazepine-binding site, clonazepam (EC50, 5 x 10(-9) M), diazepam (EC50, 10(-8) M), zolpidem (EC50, 3 x 10(-8) M), and beta-carboline-3-carboxylic acid methyl ester (EC50, 10(-6) M) were found to potentiate the whole cell GABA-evoked current in a dose-dependent manner. Methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (IC50, 3 x 10(-5) M) inhibited the current, whereas Ro15-4513 had no effect. Among the ligands acting at other modulatory sites, etomidate (EC50, 2 x 10(-6) M) enhanced the GABA-evoked current, whereas 4'-chlorodiazepam (IC50, 4 x 10(-7) M), ZnCl2 (IC50,5 x 10(-5) M), and furosemide (IC50,3 x 10(-4) M) depressed the response to GABA. PK 11195 did not affect the GABA-evoked current or its inhibition by 4'-chlorodiazepam. The results indicate that the native GABA(A) receptors in frog melanotrophs are formed by combinations of alpha2-, alpha3-, beta2/3-, gamma1-, gamma2-, and gamma3-subunits. The data also demonstrate that clonazepam is the most potent, and zolpidem is the most efficient positive modulator of the native receptors. Among the inhibitors, 4'-chlorodiazepam is the most potent, whereas ZnCl2 is the most efficient negative modulator of the GABA(A) receptors. The present study provides the first correlation between subunit composition and the functional properties of native GABA(A) receptors in nontumoral endocrine cells.

The effects of PGE2 on voltage-dependent Ca2+ channel currents were studied in dissociated rat melanotrophs by the whole-cell configuration of the patch-clamp technique. In about 90% of melanotrophs examined, PGE2 reversibly inhibited voltage-dependent Ba2+ currents elicited by voltage steps from a holding potential of −80 to 0 mV, with an ED50 of 68 nm. The maximum inhibition of Ba2+ currents by 1 μm PGE2 (35.3%) was comparable with that by the maximally effective concentration (100 nm) of dopamine. The EP1/EP3 PGE (EP) agonists, 17PT-PGE2 and sulprostone, and the EP2/EP3 agonist, misoprostol, mimicked the inhibition by PGE2, whereas the selective EP2 agonist, butaprostol, had little effect. The inhibition by PGE2 was partially, but significantly, reduced by the selective EP1 antagonist, SC-51322. The magnitude of the PGE2-induced inhibition of Ba2+ currents was greatly reduced by pretreatment with pertussis toxin, or by a depolarizing prepulse, to +80 mV, lasting for 50 msec. Although four distinct type...

The pituitary gland is a heterogeneous tissue comprised of several hormone secreting and supporting cells, most of which are targeted by estrogens. Estrogen-induced changes in the pituitary are presumably mediated via the classical estrogen receptor, ERα. However, a novel receptor, ERβ, and pituitary-specific truncated estrogen receptor products (TERPs) were recently identified. The objectives of this study were to examine the distribution of these receptors in the rat pituitary and compare their regulation by estradiol in Sprague-Dawley and the estrogen-sensitive Fischer 344 rats. Pituitary cryosections were subjected to immunocytochemistry for specific cell types, followed by in situ hybridization for ERα or ERβ. ERα was expressed by approximately 45% of the lactotrophs and melanotrophs, 35% of the corticotrophs and folliculo-stellate cells, and 25% of the gonadotrophs. The expression of ERβ showed a similar pattern but was generally lower than ERα. In two cell types, melanotrophs and gonadotrophs, ERβ ...

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported to stimulate melanotroph secretion, and PACAP-like immunoreactivity and expression of PACAP type I receptor messenger RNA have been identified in the pituitary pars intermedia (PI). The present study showed that PACAP messenger RNA is also expressed in the PI. To examine the mechanism of PACAP action in the PI, cytosolic Ca2+ concentrations ([Ca2+]i) and ionic currents were measured in acutely dissociated rat melanotrophs. In about 40% of the melanotrophs studied, PACAP induced an increase in [Ca2+]i, which was suppressed by extracellular Ca2+ removal; extracellular Na+ replacement; the blocker of L-type Ca2+ channels, nicardipine; or the secreto-inhibitory neurotransmitter, dopamine. The PACAP-induced [Ca2+]i increase was mimicked by activators of protein kinase A (PKA) and protein kinase C (PKC), Sp-diastereomer of cAMP and 1-oleoyl-2-acetyl-sn-glycerol, and was reduced by inhibitors of PKA and PKC, Rp-diastereomer of cAMP and staurosporine. Patch-clamp analysis revealed that PACAP caused inward currents with a reversal potential of -0.8 mV and facilitated voltage-dependent Ba2+ currents. It further revealed that PACAP-induced inward currents were mimicked by 1-oleoyl-2-acetyl-sn-glycerol and inhibited by staurosporine, and that Sp-diastereomer of cAMP facilitated Ba2+ currents. These results suggest that PACAP potentiates Ca2+ entry mechanisms of rat melanotrophs by activation of nonselective cation channels via PKC and facilitation of voltage-dependent Ca2+ channels via PKA.

The distribution of prodynorphin (proDyn) messenger RNA (mRNA) was examined in the rat pituitary using Northern and in situ hybridization analysis. Anterior pituitary gonadotrophs are known to express ProDyn, but the present study demonstrated that proDyn mRNA was also expressed in the intermediate lobe melanotrophs and was colocalized with POMC mRNA. The 2.6-kilobase proDyn transcript observed in the intermediate lobe was shown to be translatable by polysome analysis. Immunohistochemical studies showed dynorphin (Dyn)-like immunoreactivity in all intermediate lobe melanotrophs. Intermediate lobe proDyn gene expression was not regulated by dopamine, in contrast to intermediate lobe POMC mRNA levels, which were increased with haloperidol and decreased with bromocriptine treatment, as expected. A gender difference in ProDyn gene expression was noted, since intermediate lobes of male rats had nearly 2-fold higher proDyn mRNA levels than intermediate lobes of female rats. In contrast, no gender difference of intermediate lobe POMC mRNA levels were detected. ProDyn mRNA levels were up-regulated by 3- to 4-fold in the intermediate lobes of postpartum females as compared to pregnant or nonpregnant female rats, whereas POMC mRNA levels were unchanged, suggesting a role for intermediate lobe ProDyn in the postpartum period of the female rat. Although our results demonstrate proDyn and POMC coexpression in the pituitary intermediate lobe melanotrophs and show a differential regulational control for each gene in this tissue, the present data also strengthen the notion that proDyn is a precursor that has a role to play in reproductive functions.

Neuropeptide-Y (NPY) is present, along with dopamine and gamma-aminobutyric acid, in the neurons innervating the intermediate lobe of the pituitary gland of Xenopus laevis, and all three neurotransmitters have been shown to inhibit melanotroph secretion from isolated neurointermediate lobes. However, unlike dopamine and gamma-aminobutyric acid, NPY has been reported to be without inhibitory effect on secretion from dispersions of intermediate lobe cells. Moreover, binding studies have been taken as indicating that Xenopus melanotrophs lack NPY receptors, although such receptors appear to be present on folliculostellate cells. For these reasons, NPY has been considered to act indirectly on Xenopus melanotrophs; the putative intermediary is supposed to be the folliculo-stellate cell. However, the present experiments show that NPY does strongly inhibit melanotroph secretion from cells dispersed from Xenopus intermediate lobes. In addition, they demonstrate that NPY acts directly on individual Xenopus melanotrophs (immunohistochemically identified and under conditions that preclude any interaction between cells) to inhibit the intermittent rises in cytosolic free Ca (cytosolic Ca pulsing). From these observations, we conclude that NPY does act directly on melanotrophs of Xenopus.

The purpose of the experiments was to examine the behavior of cytosolic Ca2+ ([Ca2+]i) in individual pituitary melanotrophs and how that is affected by physiological ligands that inhibit or stimulate melanotroph secretion. Melanotrophs were dispersed from neurointermediate lobes of Xenopus laevis adapted to a black background, and [Ca2+]i was measured with fura-2. In basal (unstimulated) conditions, repetitive transient elevations in [Ca2+]i, not hitherto observed in any melanotrophs, were detected in 73% of the cells. These cytosolic Ca pulses occurred at fairly regular intervals (1-10 min) and lasted from a half to several minutes, during which [Ca2+]i rose several-fold. Pulsing was promptly and reversibly arrested by the secreto-inhibitory transmitters, dopamine, neuropeptide-Y (NPY), and gamma-aminobutyric acid (GABA), and also by quinpirole, muscimol, and baclofen. Pertussis toxin eliminated the responses to dopamine, NPY, and GABAB receptor activation, but spared responses to GABAA receptor activation. The responses to the physiological inhibitors and to the Ca channel blocker Ni were close to all or nothing; a mere doubling of an ineffective concentration commonly sufficed to arrest pulsing. Submaximal responses, seen over a narrow concentration range, involved a slowing of the pulsing. Cells not pulsing spontaneously were responsive to dopamine, GABA, and NPY and pulsed in response to the secretagogues CRF and TRH. They are suggested to be melanotrophs not actively secreting. The behavior of [Ca2+]i parallels secretory activity and strengthens the view that spontaneous secretion is driven by spontaneous influx of Ca ions and that secreto-inhibitory transmitters act by suppressing this influx and lowering [Ca2+]i. Cytosolic Ca pulsing may provide an efficient means of sustaining a high rate of spontaneous secretion.

We recently discovered that melanotrophs of Xenopus laevis exhibit spontaneous pulse-like rises in cytosolic free calcium ([Ca2+]i) and that this cytosolic Ca pulsing is inhibited by the secreto-inhibitory transmitters dopamine, gamma-aminobutyric acid, and neuropeptide-Y and stimulated by the secretagogues CRF and TRH. Here we provide evidence for the factors responsible for the individual cytosolic Ca pulse and the repetitive behavior. Isolated melanotrophs of Xenopus were loaded with fura-2, and fluorescence was recorded from perifused single cells to measure [Ca2+]i and assess the patency of divalent cation channels by Mn quenching of fluorescence. Cytosolic Ca pulsing was arrested by omission of Ca and by the Ca channel blockers Ni and Co, but was unaffected by tetrodotoxin. Mn (0.3 mM) caused phasic quenching, each "quench" being synchronous with the rising phase of a cytosolic Ca pulse. Quenching was blocked by Ni, but was unaffected by tetrodotoxin. When introduced during the course of an individual Ca pulse, Ni aborted the pulse and [Ca2+]i collapsed. Extracellular K+ (10 mM) or K channel block with tetraethylammonium stimulated Ca pulsing. Threshold concentrations of Ni slowed Ca pulsing without reducing the amplitude of the individual pulses. The overshoots in quenching and Ca pulsing observed after exposure to Ni, dopamine, or baclofen are interpreted as off responses. It is concluded that each cytosolic Ca pulse is attributable, probably quantitatively, to Ca influx during a spontaneous increase in Ca permeability independent of Na-mediated action potentials, and that the pacemaker for the repetitive phenomenon is voltage sensitive and may involve Ca currents active around basal membrane potential.

Prohormone convertases (PCs) are thought to represent the major proteinases involved in the biosynthetic processing of peptide hormone precursors to bioactive peptide products. The maturation of PC2 requires the aid of a helper protein, 7B2, in order for the zymogen to become an active enzyme species. The 7B2 and PC2 nulls should thus be functionally equivalent with regard to deficits in precursor processing. In this article, we have examined this proposition through the study of proopiomelanocortin (POMC) biosynthesis and granule content in both null models. RIA data indicate that both PC2 and 7B2 nulls lack pituitary alpha-MSH; interestingly, 7B2 nulls are still able to generate beta-endorphin from beta-lipotropin, whereas PC2 nulls contain little if any beta-endorphin. Labeling experiments demonstrate a build-up of POMC, high molecular weight intermediates, and intact ACTH, as well as the disappearance of alpha-MSH, in both null models. Electron microscopy of neurointermediate lobe melanotrophs reveals the presence of a significantly greater number of secretory granules in both 7B2 and PC2 nulls compared with wild-type controls. However, PC2 null melanotrophs contain twice as many granules as 7B2 null melanotrophs. Another difference between the two null models is a relatively enhanced accumulation of precursors in the PC2 null compared with the 7B2 null; these include not only PC2 substrates, but also presumed PC1 substrates. These data indicate that the two nulls are not phenotypically equivalent.

Nitric oxide (NO) is implicated in the regulation of various endocrine functions, but the effect of NO on GABAA receptor transmission has never been reported in endocrine cells. In the present study, we have investigated the effects of various agents acting on the NO transduction pathway on GABAA receptor function in frog pituitary melanotrophs. Histochemical studies using the NADPH-diaphorase reaction and immunohistochemical labeling with antibodies against neuronal NO synthase (nNOS) revealed that nNOS is expressed in the intermediate lobe of the pituitary and in cultured melanotrophs. Whole-cell patch-clamp recordings showed that the specific substrate of NOS l-arginine (l-Arg, 10−4m) or the NO donor sodium nitroprusside (10−5m) provoked a long-lasting inhibition of the current evoked by GABA (5 × 10−6m). The NOS inhibitor l-nitroarginine (10−5m) produced a biphasic effect, i.e. a transient decrease followed by a delayed increase of the GABA-evoked current amplitude. Similarly, the specific nNOS inhibitor 7-nitroindazole and the specific inducible NOS (iNOS) inhibitor aminoguanidine (10−5m each) provoked a transient depression of the current followed by a sustained potentiation. Formation of cGMP in neurointermediate lobes was enhanced by l-Arg (10−4m) and by the calcium-releasing agent caffeine (10−4m), and inhibited by the calmodulin (CaM)/Ca2+ complex blocker W7 (10−5m). The GABA-evoked current was potentiated by the guanylyl cyclase inhibitor ODQ (10−8–10−7m) and inhibited by the protein kinase G (PKG) activator 8pCPT-cGMP (3 × 10−7–3 × 10−5m). The present data indicate that NO, produced by a CaM/Ca2+-dependent NOS in frog melanotrophs, exerts an autocrine inhibitory effect on the GABA-evoked current. The action of NO on the GABAA receptor function is mediated through activation of the cGMP/PKG pathway.

Targeted tumorigenesis, using the POMC gene promoter ligated to the simian virus 40 large T antigen, generated transgenic mice with massive tumors of the intermediate lobe (IL) of the pituitary. Inoculation of nude mice with the IL tumor cells resulted in very large secondary tumors. As the IL from several species produces a potent PRL-releasing factor (PRF), it was of interest to determine whether IL tumors from these mice also contain PRF. The objectives were to 1) measure serum PRL levels in mice with IL tumors, 2) determine whether these tumors contain PRF and examine its chromatographic properties, and 3) analyze whether this PRF is related to POMC, its derivatives, or other PRL secretagogues. Serum PRL levels were 5- to 6-fold higher in transgenic than in control mice. Primary and secondary IL tumors were acid extracted and successively fractionated using Sephadex G-100 gel filtration and reverse phase and gel permeation HPLC. PRF activity was determined using short term incubation of tissue extracts or column fractions with GH3 cells. Crude tumor extracts exhibited a strong and dose-dependent PRF activity. Upon chromatography, the PRF activity from either primary or secondary tumors resolved into two classes of compounds: a big PRF with an estimated mol wt of 70-80 kilodaltons and two small, very hydrophobic peptides. The elution profiles of the three PRFs differed from those of beta-endorphin, alpha MSH, beta MSH, ACTH, TRH, oxytocin, angiotensin II, vasoactive intestinal polypeptide, or corticotropin-like intermediate peptide. In summary, we have identified an animal model with IL tumors that has hyperprolactinemia and overproduces PRF. Two classes of PRFs, big and small, were resolved which differ from POMC derivatives and known regulators of PRL release. These data suggest that PRF is produced by melanotrophs, but is not a product of the POMC gene. The IL tumors should provide an excellent source for the purification and structural elucidation of PRFs.

Cobalt (Co), nickel (Ni), manganese (Mn), cadmium (Cd), and lanthanum (La) are commonly used as calcium (Ca) channel blockers, but some of them, besides reducing Ca entry, also traverse Ca channels and can exert effects intracellularly that confound interpretation of functional responses. Because of this and our need to use Ca channel blockers in an ongoing analysis of Ca channel activity in the regulation of the cytosolic free Ca concentration ([Ca2+]i) and secretion in melanotrophs, we assessed whether the cations mentioned enter these cells. This was done by incorporating the fluorescence for changes that would signal the presence of the cations in the cytosol. In cell-free solution, where the probe and cations can interact freely, Mn, Co, and Ni all quench fluorescence, whereas Cd and La act in a Ca-like manner. When tested on fura-2-loaded melanotrophs in basal (unstimulated) conditions, Mn, Co, and Cd each yielded corresponding signals, thereby showing that they had penetrated the cells. By contrast, Ni caused no quenching of fluorescence even in melanotrophs exposed to 100 mM K+ to recruit additional Ca channels. Ni, therefore, did not penetrate the cells. However, as expected, Ni quenched fluorescence when given artificial access to the cytoplasm by ionomycin. Ni blocked spontaneous entry of Mn, Co, and Cd. It also lowered [Ca2+]i in unstimulated melanotrophs, consistent with blockade of spontaneous Ca entry. Like Ni, La lowered basal [Ca2+]i in unstimulated melanotrophs without penetrating the cells; however, unlike Ni, it penetrated when the melanotrophs were exposed to high potassium. We conclude that Ni is the most specific of the Ca channel blockers tested and that results obtained with Mn, Co, Cd, and La must be interpreted with reserve.

Intravenous infusion (14 nmol/min) and lateral cerebral ventricular injection (9 pmol) of endothelin-1 increased rates of glucose utilization (quantitative autoradiographic [14C]deoxyglucose method) by 75-219% in pars intermedia and distalis of the rat pituitary gland. In rats given intraventricular endothelin, glucose metabolism was also increased significantly in pars nervosa (+92%). Metabolic activation by central endothelin in pars intermedia and distalis was inhibited by intraventricular pretreatment with the dihydropyridine, nimodipine, indicating that endothelin stimulates energy metabolism and probably hormone secretion in melanotrophs and pars distalis cells via L-type calcium channels.

Stimulation of the release of alpha MSH from dispersed rat melanotrophs by L-isoproterenol, 8-bromo-cAMP, or cholera toxin requires calcium ion (Ca++) in the incubation medium; the stimulatory effect of each of these agents is attenuated by D-600, a Ca++ antagonist. In contrast, stimulation of the formation of cAMP by L-isoproterenol, isobutyl methylxanthine, or cholera toxin does not require Ca++ in the incubation medium. Ca++ diminishes the amount of cAMP formed by cholera toxin-treated melanotrophs. Ca++ inhibits adenylate cyclase activity and enhances cyclic nucleotide phosphodiesterase activity in cell-free homogenates of intermediate lobe tissue. A23187, a calcium ionophore, increases the accumulation of 45Ca by melanotrophs and enhances the release of alpha MSH. Furthermore, when tested upon cholera toxin-treated melanotrophs, A23187 potentiates the Ca++-induced inhibition of cAMP formation. The results indicate that Ca++ is essential for the release of alpha MSH, and that cAMP in some way enhances the effects of Ca++ upon the release process.

The role of dopaminergic and adrenergic innervation of the intermediate lobe of amphibian pituitary in the release of alpha MSH has been studied in vitro. Neurointermediate lobes of frog (Rana ridibunda Pallas) have been perifused in amphibian culture medium (ACM) for 5-7 h. alpha MSH released in the effluent perifusate was measured by means of a sensitive and specific RIA. No significant morphological alteration of neurointermediate lobe cells was observed during the perifusion experiment, even at the electron microscopic level. The existence of dopaminergic receptors, responsible for an inhibition of frog melanotrophs, was shown using the dopaminergic agonists apomorphine (10(-6) M) and bromo-2-ergocryptine (10(-8) and 10(-7) M), which initiated a marked reduction of alpha MSH secretion. The effect of apomorphine was obliterated by the dopaminergic antagonist haloperidol. Haloperidol itself induced a dose-related stimulation, and the monoamine oxidase inhibitor nialamide (4 x 10(-3) M) inhibited alpha MSH secretion. In addition, haloperidol led to a complete reversal of the inhibitory effect of nialamide on alpha MSH secretion. These results demonstrate the existence, in the parenchyme of the intermediate lobe, of dopaminergic nerve fibers that are functionally active. The beta-adrenergic agonist isoproterenol was responsible for a dose-related stimulation of alpha MSH secretion; the stimulatory effect was reversed by the beta-adrenergic antagonist propranolol. TRH is a potent stimulator of alpha MSH secretion in amphibians. Since haloperidol and propranolol did not abolish the stimulation of alpha MSH release induced by TRH, it appeared that TRH action was not mediated via an inhibition of dopamine release or via a stimulation of adrenergic nerve fibers.

The melanotroph, a polyhedral secretory cell with an ovoid smooth nucleus, is the primary cell type of the intermediate lobe (IL) of the rat pituitary. The melanotrophs are not uniform, but differ in the tinctorial properties of their cytoplasm; some cells appear distinctly darker, others lighter, and cells staining in intermediate shades are also found. In addition, in situ hybridization using proopiomelanocortin (POMC) probes shows an uneven distribution of POMC mRNA among melanotrophs, indicating that different cells maintain different levels of biosynthetic activity. Dopaminergic drugs known to alter the secretion of POMC-related peptides from the IL produced parallel changes in histological staining properties and the amount of POMC mRNA per cell, as determined by in situ hybridization. Acute bromocriptine treatment (6 h) produced a dramatic reduction in grain counts over melanotroph cytoplasm (to 10% of the control levels). A similar reduction persisted after chronic treatment. Six hours after a single haloperidol injection, the grain counts were 180% of control levels. After chronic haloperidol treatment, they were further elevated to 300% of control levels. Chronic bromocriptine and haloperidol treatment also changed the thickness of the IL. Bromocriptine reduced and haloperidol treatment increased the number of cell layers in the IL by changing the rate of cell proliferation. Thus, haloperidol treatment significantly increased and bromocriptine treatment significantly decreased the number of melanotrophs labeled by [3H]thymidine. The mitotic index followed the same trend. These results suggest that dopamine regulation of the IL acts by two different mechanisms: POMC gene expression and cellular proliferation. The change in POMC gene expression is the cell's first rapid response. The influence on the cell cycle appears after subchronic and chronic treatment.

When placed in short term (2-day) tissue culture, the melanotrophs from the intermediate lobe of the rat pituitary gland synthesize a proopiomelanocortin-like material (POMC-LM). Exposure of these cells to bromocriptine (CB 154), an agonist upon their D-2 dopamine receptor, reduces the synthesis of POMC-LM; spiroperidol, an antagonist of the D-2 receptor, prevents this effect of CB 154. Cultured melanotrophs secrete an alpha MSH-like material. The amount of this alpha MSH-like material, either stored intracellularly or secreted into the culture medium, can be quantified in a specific RIA; the material identified in this manner is designated immunoreactive alpha MSH (IR-alpha MSH). CB 154 inhibits the secretion of IR-alpha MSH from these cells. Either spiroperidol or 8-bromo-cAMP prevent this inhibitory effect of CB 154. The capacity of these cells to synthesize alpha MSH-like molecules and release them into the culture medium can be assessed by incubation in the presence of [3H]tyrosine, followed by immunoprecipitation with an antibody directed against alpha MSH. This newly synthesized immunoprecipitable material is designated immunoprecipitable alpha MSH (IP-alpha MSH) and should be distinguished from IR-alpha MSH. Both CB 154 and quinpirole, a selective D-2 agonist (but not SKF 38393, a selective D-1 agonist), inhibit the synthesis and secretion of IP-alpha MSH. YM-09151-2, a selective D-2 antagonist (but not SCH 23390, a selective D-1 antagonist), blocks the inhibitory effects of quinpirole. Several compounds affecting cAMP metabolism (cholera toxin, forskolin, 8-bromo-cAMP, and 3-isobutyl-1-methylxanthine) can also prevent the inhibitory effect of CB 154 on the synthesis of IP-alpha MSH. We conclude the following. The D-2 receptor in the intermediate lobe directly regulates the synthesis and secretion of IP-alpha MSH. cAMP can regulate either the synthesis of POMC-LM or the processing of this substance into alpha MSH-like peptides.