The cloning of a family of genes that encode the melanocortin receptors.

Melanocyte-stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH) regulate pigmentation and adrenal cortical function, respectively. These peptides also have a variety of biological activities in other areas, including the brain, the pituitary, and the immune system. A complete understanding of the biological activities of these hormones requires the isolation and characterization of their corresponding receptors. The murine and human MSH receptors (MSH-Rs) and a human ACTH receptor (ACTH-R) were cloned. These receptors define a subfamily of receptors coupled to guanine nucleotide-binding proteins that may include the cannabinoid receptor.

Localization of the melanocortin-4 receptor (MC4-R) in neuroendocrine and autonomic control circuits in the brain.

POMC, the precursor of ACTH, MSH, and beta-endorphin peptides, is expressed in the pituitary and in two sites in the brain, in the arcuate nucleus of the hypothalamus and the commissural nucleus of the solitary tract of the brain stem. Little is known regarding the functions of melanocortin (ACTH and MSH) peptides in the brain. We report here the detailed neuroanatomical distribution of the MC4-R mRNA in the adult rat brain. The melanocortin 3 receptor (MC3-R), characterized previously, was found to be expressed in arcuate nucleus neurons and in a subset of their presumptive terminal fields but in few regions of the brainstem. The highly conserved MC4-R is much more widely expressed than MC3-R and is pharmacologically distinct. MC4-R mRNA was found in multiple sites in virtually every brain region, including the cortex, thalamus, hypothalamus, brainstem, and spinal cord. Unlike the MC3-R, MC4-R mRNA is found in both parvicellular and magnocellular neurons of the paraventricular nucleus of the hypothalamus, suggesting a role in the central control of pituitary function. MC4-R is also unique in its expression in numerous cortical and brainstem nuclei. Together, MC3-R and/or MC-4R mRNA are found in every nucleus reported to bind MSH in the adult rat brain and define neuronal circuitry known to be involved in the control of diverse neuroendocrine and autonomic functions. The high degree of conservation, distinct pharmacology, and unique neuronal distribution of the MC4 receptor suggest specific and complex roles for the melanocortin peptides in neuroendocrine and autonomic control.

Targeted ablation of pituitary pre-proopiomelanocortin cells by herpes simplex virus-1 thymidine kinase differentially regulates mRNAs encoding the adrenocorticotropin receptor and aldosterone synthase in the mouse adrenal gland.

We have produced and characterized lines of transgenic mice expressing a fusion gene composed of the pituitary expression-specific promoter region of the POMC gene, driving the herpes simplex viral-1 thymidine kinase. Adult mice were treated with the antiherpes agent ganciclovir at 70 mg/kg body weight (ip, twice daily for 10-12 days). Approximately 98% of the pituitary intermediate lobe melanotropes and anterior lobe corticotropes were ablated as determined by immunocytochemistry and RIA specific for the POMC-derived peptides, ACTH, beta-endorophin, and alpha-MSH. The number of lactotropes, somatotropes, thyrotropes, and gonadotropes was not altered compared with controls, indicating that in the adult pituitary, POMC products are not required to maintain the distribution of cell types. As expected, plasma corticosterone levels were substantially decreased after POMC cell ablation. In situ hybridization studies showed that the mouse ACTH receptor was expressed uniformly throughout the adrenal cortex, and RNase protection assays revealed that the ACTH receptor mRNA decreased after pituitary POMC cell ablation. Additionally, RNase protection assays showed that pituitary POMC cell ablation resulted in the decrease of adrenal p450c11 beta transcripts while p450c11AS (aldosterone synthase) mRNA levels remained constant. These data demonstrate differential regulation of steroid pathway-specific enzymes by POMC products. Our results also suggest that the thymidine kinase cell obliteration technique may not be dependent on cell division as a prerequisite for cytotoxicity, thus supporting the idea that targeted molecular ablation using cell- and tissue-specific promoter sequences to drive viral thymidine kinase expression can be refined further to study other nonmitotic cells.

Local tetrodotoxin blocks chronic stress effects on corticotropin-releasing factor and vasopressin messenger ribonucleic acids in hypophysiotropic neurons.

To test the hypothesis that synaptic inputs to the paraventricular nucleus mediate stress-induced increases in corticotropin-releasing peptide expression in the paraventricular nucleus of the hypothalamus (PVH), relative levels of the mRNAs encoding corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) were followed, in situ, in animals subjected to chronic footshock stress and concurrent local administration of tetrodotoxin or vehicle. Consistent with previous findings, a 7-day exposure to chronic footshock resulted in a 2.1-fold increase in CRF mRNA levels in the parvocellular division of the PVH. The footshock paradigm also resulted in at least a 41% increase in AVP transcripts in this same region; this effect was localized predominantly to CRF-immunoreactive neurons. The stressor did not significantly alter AVP mRNA levels in the magnocellular division of the PVH. Tetrodotoxin, administered to the PVH via osmotic minipump, blocked the stress-induced rise in CRF and AVP mRNAs, but had no significant effect on basal levels of these transcripts. The results support the view that maintenance of the enhanced central drive on pituitary-adrenal activity seen in response to chronic stress is mediated via neural inputs to the PVH.

Calcitonin gene-related peptide acts within the central nervous system to inhibit gastric acid secretion.

The central nervous system effect of calcitonin gene-related peptide (CGRP) on gastric acid secretion was studied in conscious freely moving rats. CGRP (220 fmol to 2.2 nmol) injected into the lateral cerebral ventricle or intravenously inhibited gastric acid secretion. Intravenous passive immunization with CGRP antiserum prevented the inhibitory effect of CGRP following intravenous but not intracerebroventricular administration. Adrenalectomy and noradrenergic blockade with bretylium tosylate did not significantly alter the inhibitory action of CGRP given intracerebroventricularly on gastric secretion. These studies indicate that CGRP acts within the central nervous system to potently decrease gastric acid secretion by mechanism(s) not dependent on intact sympathetic nervous function.

Calcitonin gene related peptide inhibits basal, pentagastrin, histamine, and bethanecol stimulated gastric acid secretion.

This study was designed to examine the effect of calcitonin gene related peptide on gastric acid secretion in the rat. Calcitonin gene related peptide (1 pmol-1 nmol/rat) injected intravenously inhibited basal gastric acid secretion in awake, freely moving rats. Calcitonin gene related peptide decreased gastric secretion stimulated by histamine, pentagastrin, or bethanecol in anaesthetised rats. The inhibitory effect was immediate and most pronounced in the first hour and diminished during the second hour. The N- and C-terminal fragments of calcitonin gene related peptide, CGRP1-14 and [Tyr23]CGRP23-37, did not affect gastric acid secretion. [acetamidomethyl-cys2,7]CGRP, the linear cysteine-protected molecule devoid of the disulphide bridge, was not biologically active. After truncal vagotomy or atropine sulphate, calcitonin gene related peptide did not inhibit gastric secretion. These studies indicate that calcitonin gene related peptide administered peripherally inhibits basal and stimulated gastric acid secretion in the rat. Both C- and N-terminal residues as well as the disulphide bridge are necessary for the inhibitory effect on gastric secretion. Inhibition of gastric acid secretion by calcitonin gene related peptide may depend on intact vagal cholinergic fibres.

Central nervous system actions of calcitonin gene-related peptide on gastric acid secretion in the rat.

Calcitonin gene-related peptide (CGRP) and its messenger ribonucleic acid have been identified in brain regions that participate in the central nervous system regulation of gastrointestinal functions. The purpose of this study was to determine the central nervous system effect of CGRP on gastric acid secretion and to delineate the mechanism(s) of its action. Calcitonin gene-related peptide (1 pmol-1 nmol) injected intracerebroventricularly inhibited gastric acid secretion in awake, freely moving, pylorus-ligated rats and the secretory response after intracerebroventricularly administered thyrotropin-releasing hormone. Calcitonin gene-related peptide (rat) and calcitonin (salmon) inhibited gastric secretion similarly. Calcitonin gene-related peptide given intracerebroventricularly inhibited gastric acid secretion stimulated by pentagastrin, histamine, or bethanecol for 2 h in anesthetized rats. Adrenalectomy or noradrenergic blockade with bretylium tosylate did not affect the gastric inhibitory action of CGRP. After subdiaphragmatic truncal vagotomy, CGRP did not exhibit the inhibitory effect on gastric acid secretion stimulated by pentagastrin. These studies indicate that CGRP injected intracerebroventricularly is a potent inhibitor of gastric acid secretion in the rat. Calcitonin gene-related peptide decreases gastric secretion stimulated centrally by thyrotropin-releasing hormone and peripherally by pentagastrin, histamine, or bethanecol. Inhibition of gastric acid secretion by CGRP is not mediated by the sympathetic nervous system but may depend on intact vagus nerves.

Molecular characterization of a nonneuronal human UNC18 homolog.

A cDNA clone encoding a human homolog of the Caenorhabditis elegans unc-18 gene was identified following random sequencing of clones from IL-2-activated human NK cells. This cDNA clone is related to the nonneuronal Munc-18b and so has been called Hunc-18b. The Hunc-18b transcripts were found in most human tissues, with the exception of brain and skeletal muscle, and in cells from all lymphoid lineages. The Hunc-18b gene was localized to human chromosome 19p13.2-p13.3, and the mouse homolog, Munc-18b, was mapped to the proximal region of mouse Chromosome 8.

Identification of a receptor for gamma melanotropin and other proopiomelanocortin peptides in the hypothalamus and limbic system.

Corticotropin (ACTH) and melanotropin (MSH) peptides (melanocortins) are produced not only in the pituitary but also in the brain, with highest concentrations in the arcuate nucleus of the hypothalamus and the commisural nucleus of the solitary tract. We have identified a receptor for MSH and ACTH peptides that is specifically expressed in regions of the hypothalamus and limbic system. This melanocortin receptor (MC3-R) is found in neurons of the arcuate nucleus known to express proopiomelanocortin (POMC) and in a subset of the nuclei to which these neurons send projections. The MC3-R is 43% identical to the MSH receptor present in melanocytes and is strongly coupled to adenylyl cyclase. Unlike the MSH or ACTH receptors, MC3-R is potently activated by gamma-MSH peptides, POMC products that were named for their amino acid homology with alpha- and beta-MSH, but lack melanotropic activity. The primary biological role of the gamma-MSH peptides is not yet understood. The location and properties of this receptor provide a pharmacological basis for the action of POMC peptides produced in the brain and possibly a specific physiological role for gamma-MSH.