Activation of central melanocortin receptors by MT-II increases cavernosal pressure in rabbits by the neuronal release of NO.
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1. Melanotan-II had been reported to cause penile erections in men with erectile dysfunction. In the present study, we investigated the mechanisms by which systemic administration of MT-II increases intracavernosal pressure in anaesthetized rabbits. 2. MT-II (10 microM) had no effect on electrical field stimulation-evoked relaxations of rabbit corpus cavernosal strips in vitro. 3. Intravenous injection of MT-II (66 and 133 microg kg(-1) elicited dose-related increases in cavernosal pressure. SHU 9119 (3 microg kg(-1), i.v.), a non-selective antagonist of MC(3) and MC(4) receptors did not significantly affect either cavernosal pressure or systemic blood pressure but abolished the MT-II-induced increases in cavernosal pressure. SHU 9119 also inhibited the depressor response produced by MT-II. 4. Intracavernosal injection 100 microl of the cocktail containing phentolamine mesylate (1 mg ml(-1)), papaverine (20 mg ml(-1)) and PGE1 (20 microg ml(-1)) increased the cavernosal pressure by about 4 fold. 5. The role of NO-cyclic GMP dependent pathway to MT-II-induced increases in cavernosal pressure was investigated by bilateral transection of the pudendal nerves and by inhibition of NO synthase with L-NAME (20 mg kg(-1), i.v. over 30 min). Ablation of the pudendal nerves or pretreatment with L-NAME abolished the MT-II-induced increases in intracavernosal pressure in anaesthetized rabbits. 6. The data suggest that activation of central melanocortin receptors by MT-II increases cavernosal pressure by the neuronal release of NO. (+info)
Hyperleptinemia in A(y)/a mice upregulates arcuate cocaine- and amphetamine-regulated transcript expression.
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The effects of leptin on cocaine- and amphetamine-regulated transcript (CART) and agouti-related protein (AGRP) expression in the hypothalamic arcuate nucleus of obese A(y)/a mice were investigated. CART mRNA expression was upregulated by 41% and AGRP mRNA downregulated by 78% in hyperleptinemic A(y)/a mice relative to levels in lean a/a mice. The mRNA expression of these neuropeptides in either young nonobese A(y)/a mice or rats treated with SHU-9119, a synthetic melanocortin-4 receptor (MC4R) antagonist, did not differ significantly from that in the corresponding controls. After a 72-h fast, which decreased the concentration of serum leptin, CART and AGRP mRNA expression decreased and increased, respectively, in A(y)/a mice. The expression levels of these neuropeptides in leptin-deficient A(y)/a ob/ob double mutants were comparable to those in a/a ob/ob mice. Leptin thus modulates both CART and AGRP mRNA expression in obese A(y)/a mice, whereas leptin signals are blocked at the MCR4R level. Taken together, the present findings indicate that differential expression of these neuropeptides in A(y)/a and ob/ob mice results in dissimilar progression toward obesity. (+info)
Molecular determinants of human melanocortin-4 receptor responsible for antagonist SHU9119 selective activity.
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The hypothalamic melanocortin-4 receptor (MC4R), a seven transmembrane G-protein-coupled receptor, plays an important role in the regulation of body weight. The synthetic melanocortin analog SHU9119 has been widely used to characterize the physiological role of MC4R in feeding behavior and energy homeostasis. Previous studies indicated that SHU9119 is an agonist at the melanocortin-1 receptor (MC1R) but an antagonist at the MC4R. However, the molecular basis of the interaction between hMC4R and SHU9119 has not been clearly defined. To gain insight into the molecular determinants of hMC4R in the selectivity of SHU9119 chimeras and mutants hMC1R and hMC4R were expressed in cell lines and pharmacologically analyzed. A region of receptor containing the third transmembrane of hMC4R was found to be required for selective SHU9119 antagonism. Further mutagenesis studies of this region of hMC4R demonstrated that the amino acid residue leucine 133 in the third transmembrane was critical for the selective antagonist activity of SHU9119. The single substitution of leucine 133 to methionine did not affect SHU9119 binding to hMC4R. However, this substitution did convert SHU9119 from an antagonist to an agonist. Conversely, exchange of Met(128) in hMC1R to Leu, the homologous residue 133 of hMC4R, displayed a reduction in SHU9119 binding affinity and potency. This report provides the details of the molecular recognition of SHU9119 antagonism at hMC4R and shows that amino acid Leu(133) of hMC4R plays a key role in melanocortin receptor subtype specificity. (+info)
Proteo-chemometrics analysis of MSH peptide binding to melanocortin receptors.
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The published data for six melanocortin peptides binding to wild-type and chimeric melanocortin MC(1)/MC(3) receptors were analysed using the novel proteo-chemometrics modelling approach. The chimeric receptors and the peptides were coded using binary descriptors and used to correlate with the experimental data for affinity or selectivity for peptides binding to receptors. Correlations were achieved using partial least squares projection to latent structures (PLS) and statistically valid models were obtained. The models were further improved by adding cross-terms and applying orthogonal signal correction. The models were validated using external prediction, with half of the data being excluded from the modelling. Interpretation of the results using PLS coefficient plots revealed that the binding pocket for the melanocortins is located between the first, second, third, sixth and seventh transmembrane regions of the melanocortin receptors, in good agreement with previous three-dimensional models for the interactions of melanocortins with melanocortin receptors. Further, analysis of cross-terms between peptide descriptors indicated that the proteo-chemometrics modelling is able to distinguish between differences in the conformational space of the peptides that affect binding affinity and selectivity. (+info)
Identification of domains directing specificity of coupling to G-proteins for the melanocortin MC3 and MC4 receptors.
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The melanocortin receptors, MC3R and MC4R, are G protein-coupled receptors that are involved in regulating energy homeostasis. Using a luciferase reporter gene under the transcriptional control of a cAMP- responsive element (CRE), the coupling efficiency of the MC4R and MC3R to G-proteins was previously shown to be different. MC4R exhibited only 30-50% of the maximum activity induced by MC3R. To assess the role of the different MC3R and MC4R domains in G-protein coupling, several chimeric MC3R/MC4R receptors were constructed. The relative luciferase activities, which were assessed after transfecting the chimeric receptors into HEK 293T cells, showed that the i3 (3rd intracellular) loop domain has an essential role in the differential signaling of MC3R and MC4R. To reveal which amino acid residue was involved in the MC4R-specific signaling in the i3 loop, a series of mutant MC4Rs was constructed. Reporter gene analysis showed that single mutations of Arg(220) to Ala and Thr(232) to either Val or Ala increased the relative luciferase activities, which suggests that these specific amino acids, Arg(220) and Thr(232), in the i3 loop of MC4R play crucial roles in G-protein coupling and the subtype-specific signaling pathways. An examination of the inositol phosphate (IP) levels in the cells transfected with either MC3R or MC4R after being exposed to the melanocortin peptides revealed significant stimulation of IP production by MC3R but no detectable increase in IP production was observed by MC4R. Furthermore, none of the MC4R mutants displayed melanocortin peptide-stimulated IP production. Overall, this study demonstrated that MC3R and MC4R have distinct signaling in either the cAMP- or the inositol phospholipid-mediated pathway with different conformational requirements. (+info)
Activation of central melanocortin pathways by fenfluramine.
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D-fenfluramine (d-FEN) was once widely prescribed and was among the most effective weight loss drugs, but was withdrawn from clinical use because of reports of cardiac complications in a subset of patients. Discerning the neurobiology underlying the anorexic action of d-FEN may facilitate the development of new drugs to prevent and treat obesity. Through a combination of functional neuroanatomy, feeding, and electrophysiology studies in rodents, we show that d-FEN-induced anorexia requires activation of central nervous system melanocortin pathways. These results provide a mechanistic explanation of d-FEN's anorexic actions and indicate that drugs targeting these downstream melanocortin pathways may prove to be effective and more selective anti-obesity treatments. (+info)
The catabolic action of insulin in the brain is mediated by melanocortins.
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Like leptin, the pancreatic hormone insulin is an important adiposity signal to the brain. We report that the hypothalamic melanocortin system is an important target of the actions of insulin to regulate food intake and body weight. Hypothalamic neurons expressing insulin receptors were found to coexpress the melanocortin precursor molecule pro-opiomelanocortin (POMC), and administration of insulin into the third cerebral ventricle of fasted rats increased expression of POMC mRNA. Finally, a subthreshold dose of the melanocortin antagonist SHU-9119 prevented the reduction in food intake caused by third-ventricular insulin administration. These data suggest that the hypothalamic melanocortin system mediates the anorexic effects of central insulin, as well as of leptin. (+info)
Distinct effect of actin cytoskeleton disassembly on exo- and endocytic events in a membrane patch of rat melanotrophs.
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We used the cell-attached mode of patch-clamp technique to measure discrete attofarad steps in membrane capacitance (C(m)), reporting area changes in the plasma membrane due to unitary exocytic and endocytic events. To investigate the role of the actin cytoskeleton in elementary exocytic and endocytic events, neuroendocrine rat melanotrophs were treated with Clostridium spiroforme toxin (CST), which specifically depolymerises F-actin. The average amplitude of exocytic events was not significantly different in control and in CST-treated cells. However, the amplitude of endocytic events was significantly smaller in CST-treated cells as compared to controls. The frequency of exocytic events increased by 2-fold in CST-treated cells relative to controls. In control cells the average frequency of exocytic events (upsilon;(exo)) was lower than the frequency of endocytic events (upsilon;(endo)) with a ratio upsilon;(exo)/upsilon;(endo) < 1. In the toxin treated cells, the predominant process was exocytosis with a ratio (upsilon;(exo)/upsilon;(endo) > 1). To study the coupling between the two processes, the slopes of regression lines relating upsilon;(exo) and upsilon;(endo) in a given patch of membrane were studied. The slopes of regression lines were similar, whereas the line intercepts with the y-axis were significantly different. The increased frequency of unitary exocytic events in CST-treated cells is consistent with the view, that the actin cytoskeleton acts as a barrier for exocytosis. While the disassembly of the actin cytoskeleton diminishes the size of unitary endocytic events, suggesting an important role of the actin cytoskeleton in determining the size of endocytic vesicles, the coupling between exocytosis and endocytosis in a given patch of membrane was independent of the state of the actin cytoskeleton. (+info)