Alopecia areata - hyperactivity of the hypothalamic-pituitary-adrenal axis is a myth?

INTRODUCTION: Psychological stress is known to cause exacerbation of different skin pathologies including alopecia areata (AA). A hyperactivity of the hypothalamic-pituitary-adrenal axis (the HPA axis) in patients affected by AA (AA patients) was proposed to be a neuroendocrine response to stress. Still little is known about melanocyte-stimulating hormone (MSH) and cortisol production in AA settings. AIM: The aim of the study was to compare trends in a production of MSH and cortisol in patients with AA patients and healthy controls. MATERIAL AND METHODS: Plasma concentrations of free cortisol and MSH were measured in 43 AA patients (35.5 ± 10.6 years) and 37 healthy subjects (35.9 ± 10.5 years) selected from the Dermatology Outpatient Clinic at Medical University of Silesia in Katowice, Poland. Results were submitted to statistical analysis with Shapiro-Wilk W-test and subsequently nonparametric (Mann-Whitney U-test) or parametric (Student's t-test) statistics were performed. RESULTS: Mean plasma level of MSH was 5.39 ng/mL in AA patients and 5.71 ng/mL in healthy controls. The difference between groups was non-significant (P = 0.435), but the control group manifested higher values of MSH (Q75 = 13.6 ng/mL vs Q75 = 5.98 ng/mL) and this tendency was especially stronger in females. AA patients had greater mean plasma level of cortisol (157.63 ± 91.16 µg/L) than healthy controls (123.32 ± 71.28 µg/L); however, the difference between them was also non-significant (P = 0.063). No sex-dependent tendency to a greater production of cortisol was found. CONCLUSIONS: Expectations of disturbances in production of MSH and cortisol were not fulfilled. Neither MSH nor cortisol plasma levels appear to be clearly changed in AA patients.

The biosynthesis, intracellular transport, and packaging of melanocyte-stimulating peptides in the amphibian pars intermedia.

Experiments in which glycine-(3)H has been introduced into excised neurointermediate lobes of Xenopus laevis incubated in a modified Krebs-Ringer bicarbonate medium have shown that approximately 50% of the incorporated radioactivity is present in small peptides which have an electrophoretic mobility characteristic of the melanocyte-stimulating (MSH) peptides shown to be elaborated within the tissue. Based on these results and the demonstration that a discrete approximately 7 min pulse of the label can be introduced into the tissue, electron microscope radioautography has been employed to follow the subcellular events concerned with the synthesis, intracellular transport, and packaging of the labeled secretory product. Together, these studies indicate that the newly synthesized material arises in peptide form, rather than as part of a larger prohormone molecule, on the ribosomes of the rough endoplasmic reticulum within the parenchymal cells of the intermediate portion of the lobe. A proportion is then incorporated into and remains for an extended period within the intracisternal granules which are a feature of the rough endoplasmic reticulum within these cells in vitro Most ( approximately 60%) of the labeled secretory product, however, is transferred to the Golgi complex within 30 min and, within a further 10 min, becomes packaged into small ( approximately 200 mmicro) electron-opaque secretory granules. It is probable that under the conditions employed these granules represent the final intracellular location of secretory product before it is released

Induction of the intermediate pituitary by stress: synthesis and release of a nonopioid form of beta-endorphin.

beta-Endorphin in the intermediate lobe of the pituitary gland is posttranslationally modified to produce opioid inactive peptides. Whether these are metabolites or biologically relevant products has not been known. It was found that repeated stress induces increased biosynthesis and release of beta-endorphin-like substances from the intermediate lobe of rats and that opioid-inactive N-acetylated beta-endorphin-(1-31) is selectively made and liberated. The possible role of this nonopioid product and the selective release of peptide forms are discussed.

Cytosolic Ca2+, exocytosis, and endocytosis in single melanotrophs of the rat pituitary.

We have monitored cytosolic [Ca2+] with fura-2 and exocytosis by measuring the membrane capacitance, and we have studied the influence of cytosolic [Ca2+] on secretion in single endocrine cells. As in neurons, cytosolic Ca2+ is sufficient to trigger exocytosis. The rate of secretion grows with the fourth or fifth power of cytosolic [Ca2+], and paired stimuli reveal facilitation. Ca2+ influx through voltage-sensitive Ca2+ channels can stimulate secretion 1000-fold over the basal levels measured biochemically. Unlike neurons, however, melanotrophs continue to secrete for seconds afer a depolarizing pulse, while they extrude or sequester the Ca2+ that has entered through Ca2+ channels. Following episodes of secretion, pituitary cells can retrieve membrane with half-times around 30 s at 32 degrees C, even in the absence of cytosolic K+.

Central administration of insulin suppresses food intake in chicks.

Although the orexigenic action of peptide hormones such as ghrelin and growth hormone releasing peptide is different between chickens and mammals, the anorexigenic action of peptide hormones is similar in both species. For example, central administration of peptide hormones such as leptin, cholecystokinin or glucagon has been shown to suppress food intake behavior in chickens and mammals. Central administration of insulin suppresses food intake in mammals. However, the anorexigenic action of insulin in chickens has not yet been identified. In the present study, we investigated the effects of central administration of insulin on food intake in chicks. Intracerebroventricular administration of insulin in chicks significantly suppressed food intake. Central administration of insulin significantly upregulated mRNA levels of proopiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART) and corticotropin-releasing factor (CRF), but did not influence mRNA levels of neuropeptide Y and agouti-related protein in the hypothalamus. These results suggest that alpha-melanocyte stimulating hormone (alpha-MSH, an anorexigenic peptide from the post-translational cleavage of POMC), CART and CRF are involved in the anorexigenic action of insulin in chicks. Furthermore, central administration of alpha-MSH or CART significantly suppressed food intake. In addition, alpha-MSH significantly upregulated CRF mRNA expression, suggesting that the anorexigenic action of alpha-MSH is mediated by CRF. Our findings demonstrate that insulin functions in chicks as an appetite-suppressive peptide in the central nervous system and suggest that this anorexigenic action is mediated by CART, alpha-MSH and CRF.

Production of calcitonin, adrenocorticotropic hormone, and beta-melanocyte-stimulating hormone in tumors derived from amine precursor uptake and decarboxylation cells.

The tumor production of human calcitonin (CT) was examined by radioimmunoassay, and it was found that 50 of 85 (59%) tumor tissues collected at random contained immunoreactive CT. These tumors were grouped as to whether they were derived from the amine precursor uptake and decarboxylation (APUD) series. The group that was derived from APUD cells showed appreciable amounts of CT in 30 of 31 (97%) of these tumors or in 20 of 21 (95%) when the medullary carcinomas of the thyroid were excluded. However, of the non-APUD group of tumors only 20 of 54 (37%) were found to contain CT, so that the difference between these two groups was highly significant (p less than 0.001). Of the tumors with ectopic adrenocorticotropic hormone-melanocyte-stimulating hormone production, 12 of 14 were shown to contain CT. These data indicate that CT is a common product of the APUD tumors and that tumor production of CT is often associated with that of adrenocorticotropic hormone and beta-melanocyte-stimulating hormone.

Biosynthesis, processing, and control of release of melanotropic peptides in the neurointermediate lobe of Xenopus laevis.

The neurointermediate lobes of dark-adapted toads Xenopus laevis were incubated for 30 min in [3H]arginine and then "chased" for various time periods. By use of this pulse-chase paradigm there were detected 10 trichloroacetic acid (TCA)-precipitable peptides separated on acid-urea polyacrylamide gels and one TCA-soluble peptide separated by high-voltage electrophoresis (pH 4.9) with melanotropic activity. Each of these peptides had a different degree of melanocyte stimulating hormone (MSH) activity as revealed by the Anolis skin bioassay. Three of these TCA-precipitable peptides comigrated with ACTH, beta-lipotrophin, and alpha-MSH on acid-urea gels. Evidence suggesting a precursor-product mode of biosynthesis of the melanotropic peptides is presented. 7 of the 10 TCA-precipitable peptides and the one TCA-soluble peptide with melanotropic activity were released into the medium. The half-time of release of the TCA-precipitable peptides was about 2 h, whereas the half-time of TCA-soluble peptide release was about 30 min. The release of these peptides was inhibited by 5 X 10(-5) M dopamine. Dopamine inhibition of release did not appear to affect the biosynthesis of the melanotropic peptides, but did appear to enhance the degradation of the newly synthesized TCA-soluble peptide in the tissue. White adaptation of the toads greatly decreased the biosynthesis of all of the TCA-precipitable melanotropic peptides.

The ordered secretion of bioactive peptides: oldest or newest first?

The order of secretion of newly synthesized and older bioactive peptides was investigated using primary rat intermediate pituitary melanotropes, which synthesize, store, and secrete peptides derived from pro-ACTH/endorphin (PAE; also POMC). PAE-derived peptides produced by the cells were biosynthetically labeled by incubating the cells with radioactive amino acids at various times preceding the period during which secretion was examined; secreted and cellular peptides were characterized and quantitated by immunoprecipitation, using affinity-purified antibodies to selected regions of PAE, followed by polyacrylamide gel electrophoretic analysis. Release in the absence of secretagogues (basal or constitutive release) was compared to release in the presence of maximally effective levels of 8-bromo-cAMP and BaCl2 (stimulated or regulated release). Both cell types showed short-lived preferential basal release of newly synthesized and not fully mature peptides (less than 2-3 h old). Conversely, the cells showed preferential stimulated secretion of older peptides. A process of maturation occurred, taking 2-4 h, after which the secretion of newly synthesized and older peptides in response to secretagogues was nearly indistinguishable for the smallest product peptides. The data support a model of gradual processing of peptides from precursors into smaller products and maturation from molecules only available for basal release into peptides available for stimulated secretion as well as for basal release. Basal secretion was found to include mature peptides as well as intermediates and precursor molecules. The data do not support the existence of any preferential regulated secretion of newly synthesized peptides.

Plasma immunoreactive beta-melanocyte-stimulating hormone in chronic liver disease and fulminant hepatic failure.

Hyperpigmentation believed to be due to melanin, is a feature of chronic liver disease, especially primary biliary cirrhosis and hemochromatosis. Normal plasma concentrations of immunoreactive beta-melanocyte-stimulating hormone (beta-MSH) have been found in both these conditions; thus elevation of plasma beta-MSH plays no role in the pathogenesis of hepatic pigmentation. Normal levels are also found in hepatocellular failure, which supports the hypothesis that the kidney and not the liver is the site of metabolism of this hormone.

Two distinct pituitary cell lines from mouse intermediate lobe tumors: a cell that produces prolactin-regulating factor and a melanotroph [seecomments].

The intermediate lobe (IL) of the pituitary produces a PRL-regulating factor (PRF). Targeted tumorigenesis, using the POMC promoter ligated to SV40 large T antigen (Tag), generated transgenic mice that develop IL tumors with PRF activity. Our goal was to establish and characterize a PRF-producing cell line. Two cell lines, which differ markedly in size and morphology, were independently developed from IL tumors and designated mIL5 and mIL39. These cells are transformed, as judged by rapid proliferation, low serum requirements, and generation of secondary tumors in nude mice. RT-PCR revealed that mIL39, but not mIL5 cells, express POMC and dopamine D2 receptors, typical of a melanotroph phenotype. Although mIL5 cells originated from an IL tumor, they do not express messenger RNA for SV40 Tag. The bioassay for PRF used GH3 cells stably transfected with the PRL promoter ligated to a luciferase reporter gene (GH3/luc). Coculture of mIL5 with GH3/luc cells induced cell-density dependent increases in PRL gene expression and release, whereas mIL39 cells showed negligible PRF activity. Incubation of GH3/luc cells with conditioned media from mIL5, but not mIL39 cells, stimulated PRL gene expression and release up to 10-fold. Coculture of mIL5 cells with primary rat anterior pituitary cells stimulated PRL, but not GH, release. Fractionation of mIL5 cell extracts by reverse phase HPLC resolved PRF activity into one major and one minor peak. In conclusion, we have developed two novel and distinct cell lines from mouse intermediate lobe tumors. The first reported melanotroph cell line, mIL39, could provide a valuable model for studying dopaminergic regulation of POMC gene expression and release. In contrast, the mIL5 cells do not express POMC, D2 receptors, or SV40 Tag and appear to have been immortalized by a spontaneous mutation(s). These cells produce and secrete a potent PRF and could be used for the purification and biochemical characterization of PRF.

Dopaminergic regulation of the biosynthetic activity of individual melanotropes in the rat pituitary intermediate lobe: a morphometric analysis by light and electron microscopy and in situ hybridization.

The primary cell type of the intermediate lobe (IL) of the rat pituitary is a polyhedral secretory cell with a smooth ovoid nucleus. The results of this study demonstrate, however, that IL melanotropes are a heterogeneous cell population. Melanotropes differed in the tinctorial properties of their cytoplasm; some cells appeared distinctly darker, others lighter, and cells staining in intermediate shades were also found. Electron microscopical morphometry revealed that darkly staining melanotropes have a denser cytosol and contain a greater amount of rough endoplasmic reticulum, mitochondria, and secretory vesicles than light cells. In addition, in situ hybridization studies, using a POMC probe, showed that POMC mRNA was distributed unevenly among melanotropes in a pattern comparable to the distribution of light and dark cells. These studies further demonstrated that dopaminergic drug treatments, which are known to alter the secretion of POMC-related peptides from the IL, produced parallel changes in both the histological staining properties and the amount of POMC mRNA per cell. Haloperidol treatment dramatically increased the number of dark melanotropes and the amount of POMC mRNA in each cell and eliminated the cellular heterogeneity in both staining properties and the distribution of POMC mRNA. After bromocriptine treatment the number of light melanotropes increased, and each cell contained reduced levels of POMC mRNA. These findings indicate that individual melanotropes maintain different levels of biosynthetic activity and that treatments that alter the secretion of POMC peptides affect both the rate of POMC synthesis in individual melanotropes and the cellular heterogeneity of the IL.

Plasticity in the adrenocorticotropin-related peptides produced by primary cultures of neonatal rat pituitary.

Intermediate pituitary lobe cells from newborn rats were maintained in culture to determine the extent to which they continue to exhibit the tissue-specific properties of the newborn and adult intermediate pituitary lobes. At all times examined these cultures contained mostly alpha MSH-sized and corticotropin-like intermediate lobe peptide-sized peptides; the alpha MSH-sized peptides were predominantly diacetyl-ACTH-(1-13)NH2. After 6 days in culture, the intermediate pituitary lobe cells retained the ability to synthesize diacetyl-ACTH-(1-13)NH2. Compared to that of the adult, the newborn anterior pituitary lobe is enriched in high mol wt forms of ACTH-related molecules. Therefore, the ability of newborn anterior pituitary lobe cell cultures to develop the adult processing pattern in culture was investigated. After 6 days in culture, peptides the size of alpha MSH predominated rather than ACTH-(1-39), which is the major form found in the adult. Immunocytochemical studies showed that all cultured newborn corticotropes strongly stained for alpha MSH-related material. The alpha MSH-sized molecules were identified as ACTH-(1-13)NH2 by reverse phase HPLC. In 6-day-old cultures of neonatal anterior pituitary lobes grown in the presence of a synthetic glucocorticoid, dexamethasone, the amount of alpha MSH-sized material was diminished, and instead, precursor forms of the ACTH-related peptides were detected. In biosynthetic labeling experiments, the ratio of newly synthesized ACTH-(1-13)NH2 to ACTH-(1-39) was greatly reduced by treatment of the cells with dexamethasone. The extensive cleavage of ACTH-(1-39) and its regulation by dexamethasone are unique to newborn anterior pituitary lobe corticotropes; such plasticity is not observed in cultures of adult tissue.

Inhibition of voltage-dependent calcium channels by prostaglandin E2 in rat melanotrophs.

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 microM 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 types (N-, P/Q-, L-, and R-types) of high-threshold Ba2+ currents were observed, PGE2 (1 microM) caused significant inhibition of only P/Q- and L-type currents, which were 17.3 and 10.1%, respectively, of the total Ba2+ currents. These results suggest that PGE2 inhibits P/Q- and L-type Ca2+ channels of rat melanotrophs via EP1 and EP3 receptors, which are coupled to pertussis toxin-sensitive G proteins, and produces both voltage-sensitive and -insensitive inhibition of Ca2+ channels.

D-2 dopaminergic agonists and adenosine 3',5'-monophosphate directly regulate the synthesis of alpha-melanocyte-stimulating hormone-like peptides by cultured rat melanotrophs.

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.

Bromocriptine-induced changes in the biochemistry, physiology, and histology of the intermediate lobe of the rat pituitary gland.

Treatment of rats with 2-Br-alpha-ergocryptine (bromocriptine; CB 154) elicits biochemical, physiological, and histological changes in the intermediate lobe (IL) of the rat pituitary gland, suggesting a decrease in activity in the IL. CB 154 treatment decreases 1) the capacity of the IL to synthesize proopiomelanocortin (POMC), 2) the content of mRNA directing the synthesis of POMC, and 3) the capacity of the IL to synthesize the peptides desacetyl alpha MSH, N,O-diacetyl alpha MSH, and alpha MSH. CB 154 treatment also causes a 40% loss of IL protein and an atrophy of the IL. CB 154 treatment has a biphasic effect upon the IL content of alpha MSH-like peptides; the drug first increases and then diminishes the content of these molecules. Control experiments using CB 154-treated IL tissue suggest that these effects of CB 154 are not a toxic effect of CB 154 on the IL. Spiroperidol reverses the effects of CB 154 on POMC synthesis and POMC mRNA content; by itself, spiroperidol increases the IL synthesis of POMC, the IL content of POMC mRNA, and the capacity of the IL to synthesize immunoreactive alpha MSH. Stalk section of rat pituitary gland also results in an increase in the capacity of the IL to synthesize POMC. These results suggest that a D-2 dopamine receptor mediates a tonic inhibition of the function of the IL.

Chemical characterization of adrenocorticotropin and a novel peptide biosynthesized from a pituitary adenoma of a patient with Cushing's disease.

ACTH and another unidentified peptide have been biosynthesized in vitro and purified from a pituitary adenoma of a patient with Cushing's disease. The new peptide had an apparent molecular weightof 4000 daltons and was more acidic than ACTH as analyzed by sodium dodecyl sulfate/urea and acidic (pH 4.5) polyacrylamide gel electrophoreses, respectively. Sequence studies revealed the presence of [35S]methionine at position 3 of the new peptide. High performance liquid chromatographic analysis of [35S]methionine-labeled tryptic fragments of the peptide revealed that this peptide contained gamma MSH- (1-7) within its sequence. Our observations raise the possibility that this peptide contains at its N-terminus gamma MSH, a putative peptide proposed by Nakanishi et al.

Differential glycosylation of N-POMC1-77 regulates the production of gamma 3-MSH by purified pro-opiomelanocortin converting enzyme. A possible mechanism for tissue-specific processing.

The amino terminus of bovine pro-opiomelanocortin (N-POMC1-77) is partially processed in the intermediate lobe of the pituitary to N-POMC1-49 and lys-gamma 3-melanotropin. Two pools of N-POMC1-77 were isolated which were differentially glycosylated at threonine45, while N-POMC1-49 isolated from bovine intermediate lobe extracts existed in a non-glycosylated form. This suggested that differential O-linked glycosylation of N-POMC1-77 may regulate cleavage at the Arg49-Lys50 processing site. We tested this hypothesis by incubating N-POMC1-77 glycoforms with purified proopiomelanocortin converting enzyme. Only non-O-glycosylated N-POMC1-77 and O-glycosylated N-POMC1-77 with truncated oligosaccharide sidechains were sensitive to cleavage and generated predominantly lys-gamma 3-melanotropin, identified by high-performance liquid chromatography. These data provide the first functional evidence to support a role for differential O-linked glycosylation in the regulation of the processing of the N-terminus of bovine POMC.

Identification of POMC processing products in single melanotrope cells by matrix-assisted laser desorption/ionization mass spectrometry.

The use of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) in identifying proopiomelanocortin (POMC) processing products in melanotrope cells of the pituitary intermediate lobe of Xenopus laevis was explored. Mass spectra were obtained with such a high sensitivity of detection that the peptides could be identified in a single melanotrope cell. In addition to known POMC processing products of the Xenopus melanotrope cell, the presence of previously unidentified POMC-derived peptides was demonstrated. Together these POMC processing products accounted for the entire length of the POMC precursor. Furthermore, Xenopus possesses two genes for POMC and the sensitivity and accuracy of the MALDI-MS technique allowed identification of processing products of both the POMCA and POMCB gene. In addition, differences were obtained between the mass spectra of melanotrope cells from Xenopus laevis adapted to different conditions of background illumination. These results show that MALDI-MS is a valuable tool in the study of the expression of peptides in single (neuroendocrine) cells.

Postnatal expression of melanocortin-3 receptor in rat diencephalon and mesencephalon.

In situ hybridization was applied to examine the postnatal expression of melanocortin-3 (MC-3) receptor mRNA in the rat brain. Very weak and limited signals were seen in the hypothalamus on postnatal day 0 (P0) and in the dorsal lateral thalamus on P4. A marked increase was noted in several regions of the diencephalon and mesencephalon on P7. The highest levels were reached on P21, which was the time when an adult-like pattern was established. On P21, intense signals were seen in the ventromedial nucleus and the arcuate nucleus of the tuberal hypothalamus, the habenular nucleus of the epithalamus and the ventral tegmental area. [125I] Nle4, D-Phe7-alpha-MSH showed overlapping, but wider labelling of melanocortin receptors, that followed a similar developmental course. alpha-MSH-like immunoreactivity was seen widely in the forebrain and midbrain from P14. In contrast to the staining of alpha-MSH in neurons and their process, gamma 2-MSH-like immunoreactivity was detected strongly in the blood vessels. The neuronal localization of MC-3 receptor mRNA suggests that this receptor may mediate the neurotropic actions of melanocortin peptides in the developing brain.

Studies on pituitary melanotrophs reveal the novel GABAB antagonist CGP 35-348 to be the first such compound effective on endocrine cells.

One obstacle to understanding the action and physiological significance of the responsiveness of various endocrine cells to gamma-aminobutyric acid (GABA) has been that previously available substances, all active as GABAB antagonists in the nervous system, are ineffective on endocrine cells. The introduction of a potent new member of this class, CGP 35-348, of very different chemical structure, encouraged us to examine its effect on endocrine cells. For this purpose, we studied melanotroph secretion from pituitary neurointermediate lobes. We found that CGP 35-348, in contrast to previously available members of this class, suppressed completely, in rat and toad, secretory responses to baclofen, the classic GABAB agonist. Analysis, in toad, showed CGP 35-348 did not affect responses to GABAA agonists (muscimol; isoguvacine), dopamine, or neuropeptide Y. When tested against GABA, the physiological ligand present in the innervation of melanotrophs (along with dopamine and neuropeptide Y), CGP 35-348 completely suppressed the secretory response, which, in toad, is purely inhibitory and unaffected by bicuculline, the specific GABAA antagonist. In addition, CGP 35-348 unmasked a stimulant effect that bicuculline blocked. In CGP 35-348, we thus have a new tool with which to analyse responses to GABA and their physiological involvement in endocrine cells.