Cellular localization and role of prohormone convertases in the processing of pro-melanin concentrating hormone in mammals.
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Melanin concentrating hormone (MCH) and neuropeptide EI (NEI) are two peptides produced from the same precursor in mammals, by cleavage at the Arg145-Arg146 site and the Lys129-Arg130 site, respectively. We performed co-localization studies to reveal simultaneously the expression of MCH mRNA and proconvertases (PCs) such as PC1/3 or PC2. In the rat hypothalamus, PC2 was present in all MCH neurons, and PC1/3 was present in about 15-20% of these cells. PC1/3 or PC2 was not found in MCH-positive cells in the spleen. In GH4C1 cells co-infected with vaccinia virus (VV):pro-MCH along with VV:furin, PACE4, PC1/3, PC2, PC5/6A, PC5/6B, or PC7, we observed only efficient cleavage at the Arg145-Arg146 site to generate mature MCH. Co-expression of pro-MCH together with PC2 and 7B2 resulted in very weak processing to NEI. Comparison of pro-MCH processing patterns in PC1/3- or PC2-transfected PC12 cells showed that PC2 but not PC1/3 generated NEI. Finally, we analyzed the pattern of pro-MCH processing in PC2 null mice. In the brain of homozygotic mutants, the production of mature NEI was dramatically reduced. In contrast, MCH content was increased in the hypothalamus of PC2 null mice. In the spleen, a single large MCH-containing peptide was identified in both wild type and PC2 null mice. Together, our data suggest that pro-MCH is processed differently in the brain and in peripheral organs of mammals. PC2 is the key enzyme that produces NEI, whereas several PCs may cleave at the Arg145-Arg146 site to generate MCH in neuronal cell types. (+info)
The effect of the orexins on food intake: comparison with neuropeptide Y, melanin-concentrating hormone and galanin.
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Orexin-A and orexin-B (the hypocretins) are recently described neuropeptides suggested to have a physiological role in the regulation of food intake in the rat. We compared the orexigenic effect of the orexins administered intracerebroventricular (ICV) with other known stimulants of food intake, one strong, neuropeptide Y (NPY), and two weaker, melanin-concentrating hormone (MCH) and galanin. Orexin-A consistently stimulated food intake, but orexin-B only on occasions. Both peptides stimulated food intake significantly less than NPY, but to a similar extent to MCH (2 h food intake: NPY 3 nmol, 7.2+/-0.9 g vs saline, 1.5+/-0.2 g, P<0.001, MCH 3 nmol, 3.2+/-0.8 g vs saline, P<0.01, orexin-B 30 nmol, 2. 6+/-0.5 g vs saline, P=0.11) and to galanin (1 h food intake: galanin 3 nmol, 2.0+/-0.4 g vs saline, 0.8+/-0.2 g, P<0.05, orexin-A 3 nmol 2.2+/-0.4 g vs saline, P<0.01; 2 hour food intake: orexin-B 3 nmol, 2.4+/-0.3 g vs saline, 1.3+/-0.2 g, P<0.05). Following ICV orexin-A, hypothalamic c-fos, a maker of neuronal activation, was highly expressed in the paraventricular nucleus (PVN), and the arcuate nucleus (P<0.005 for both). IntraPVN injection of orexin-A stimulated 2 h food intake by one gram (orexin-A 0.03 nmol, 1.6+/-0. 3 g vs saline, 0.5+/-0.3 g, P<0.005). These findings support the suggestion that the orexins stimulate food intake. However, this effect is weak and may cast doubt upon their physiological importance in appetite regulation in the rat. (+info)
Characterization of a cDNA encoding a precursor of Carassius RFamide, structurally related to a mammalian prolactin-releasing peptide.
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We have characterized the cDNA encoding Carassius RFamide (C-RFa), which is structurally related to mammalian prolactin-releasing peptides (PrRPs), from the brain of the crucian carp. The deduced C-RFa precursor has been shown to comprise 117 amino acids, encoding a single C-RFa sequence. A comparative study of amino acid sequences has revealed that several sequences conserved in preproPrRPs are also found in the C-RFa precursor. Furthermore, the abundant presence of the C-RFa mRNA in the telencephalon, optic tectum, medulla oblongata, and proximal half eye ball was demonstrated by Southern blot analysis of RT-PCR products. (+info)
Identification of melanin concentrating hormone (MCH) as the natural ligand for the orphan somatostatin-like receptor 1 (SLC-1).
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To identify possible ligands of the orphan somatostatin-like receptor 1 (SLC-1), rat brain extracts were analyzed by using the functional expression system of Xenopus oocytes injected with cRNAs encoding SLC-1 and G protein-gated inwardly rectifying potassium channels (GIRK). A strong inward current was observed with crude rat brain extracts which upon further purification by cation exchange chromatography and high performance liquid chromatography (HPLC) yielded two peptides with a high agonist activity. Mass spectrometry and partial peptide sequencing revealed that one peptide is identical with the neuropeptide melanin concentrating hormone (MCH), the other represents a truncated version of MCH lacking the three N-terminal amino acid residues. Xenopus oocytes expressing the MCH receptor responded to nM concentrations of synthetic MCH not only by the activation of GIRK-mediated currents but also by the induction of Ca(2+) dependent chloride currents mediated by phospholipase C. This indicates that the MCH receptor can couple either to the G(i)- or G(q)-mediated signal transduction pathway, suggesting that MCH may serve for a number of distinct brain functions including food uptake behavior. (+info)
Prolactin-releasing peptide activation of the prolactin promoter is differentially mediated by extracellular signal-regulated protein kinase and c-Jun N-terminal protein kinase.
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Regulation of the mitogen-activated protein kinase (MAPK) family by prolactin-releasing peptide (PrRP) in both GH3 rat pituitary tumor cells and primary cultures of rat anterior pituitary cells was investigated. PrRP rapidly and transiently activated extracellular signal-regulated protein kinase (ERK) in both types of cells. Both pertussis toxin, which inactivates G(i)/G(o) proteins, and exogenous expression of a peptide derived from the carboxyl terminus of the beta-adrenergic receptor kinase I, which specifically blocks signaling mediated by the betagamma subunits of G proteins, completely blocked the PrRP-induced ERK activation, suggesting the involvement of G(i)/G(o) proteins in the PrRP-induced ERK activation. Down-regulation of cellular protein kinase C did not significantly inhibit the PrRP-induced ERK activation, suggesting that a protein kinase C-independent pathway is mainly involved. PrRP-induced ERK activation was not dependent on either extracellular Ca(2+) or intracellular Ca(2+). However, the ERK cascade was not the only route by which PrRP communicated with the nucleus. JNK was also shown to be significantly activated in response to PrRP. JNK activation in response to PrRP was slower than ERK activation. Moreover, to determine whether a MAPK family cascade regulates rat prolactin (rPRL) promoter activity, we transfected the intact rPRL promoter ligated to the firefly luciferase reporter gene into GH3 cells. PrRP activated the rPRL promoter activity in a time-dependent manner. Co-transfection with a catalytically inactive form of a MAPK construct or a dominant negative JNK, partially but significantly inhibited the induction of the rPRL promoter by PrRP. Furthermore, co-transfection with a dominant negative Ets completely abolished the response of the rPRL promoter to PrRP. These results suggest that PrRP differentially activates ERK and JNK, and both cascades are necessary to elicit rPRL promoter activity in an Ets-dependent mechanism. (+info)
Molecular characterization of the melanin-concentrating hormone/receptor complex: identification of critical residues involved in binding and activation.
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A molecular model of the human melanin-concentrating hormone (MCH) peptide was constructed and docked into a helical, bacteriorhodopsin-based model of the recently identified human MCH receptor. From this hormone-receptor complex, potential sites of agonist-receptor interaction were identified, and site-directed mutagenesis was used to substitute residues predicted to reside within the receptor binding pocket. Substitution of Asp(123)(3.32) in the third transmembrane domain of the receptor resulted in a loss of detectable (125)I-MCH binding and of MCH-stimulated Ca(2+) flux; cell surface expression of the mutant receptor was not affected. Arg(11) and Arg(14) of the MCH ligand were identified as potential sites of interaction with Asp(123)(3.32). [Ala(14)]-MCH was equipotent to native MCH in its ability to bind to and activate the wild-type MCH receptor, whereas [Ala(11)]-MCH displayed a 3000-fold reduction in binding affinity and a complete loss of measurable functional activity. Furthermore, [Lys(11)]-MCH and [D-Arg(11)]-MCH displayed reduced affinity for the receptor. [Lys(11)]-MCH was observed to be a partial agonist, eliciting approximately 67% of the native peptide's activity in a Ca(2+) flux assay, and [D-Arg(11)]-MCH was determined to be a functional antagonist with a K(b) valve of 15.8 microM. These data provide evidence that a basic moiety with specific stereochemical requirements at this site is needed for receptor activation. We conclude that both Asp(123)(3.32) in the MCH receptor and Arg(11) in the MCH peptide are required for the formation of the MCH peptide/receptor complex and propose that they form a direct interaction that is critical for receptor function. (+info)
Melanin-concentrating hormone regulates leptin synthesis and secretion in rat adipocytes.
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Obesity is a common problem in Western society and is associated with significant morbidity and mortality. Energy homeostasis is regulated by a complex system involving both peripheral signals such as leptin and a number of orexigenic and anorectic neuropeptides. Obesity can result from dysregulation of the peripheral and/or central signals. Melanin-concentrating hormone (MCH) is a hypothalamic peptide that is important in the regulation of feeding behavior, primarily via uncharacterized signaling pathways in the central nervous system. Leptin, expressed in adipose tissue, mediates some of its actions through several hypothalamic neuropeptides, notably agouti-related peptide, proopiomelanocortin, and neuropeptide Y. Expression of leptin is regulated by dietary status, insulin, and glucocorticoids. Furthermore, certain neuropeptides may act on adipocytes. However, the potential effect of MCH has not been investigated. We report that MCH stimulates leptin mRNA expression and leptin secretion. MCH stimulated a 2-fold increase in leptin secretion by isolated rat adipocytes after 4 h of treatment. This increase in secreted leptin was preceded by a rapid and transient increase in ob mRNA levels; MCH stimulated a 2.5-fold increase in ob mRNA within 1 h of treatment, followed by a decline to basal levels within 4 h. In addition, we demonstrate that the MCH receptor SLC-1 is expressed in adipocytes, suggesting that fat cells may be targets of MCH or an MCH-like peptide under physiological conditions. Finally, using a radioimmunoassay, MCH/MCH-like peptide was detected in rat plasma. This study establishes a novel in vitro mammalian system for examining MCH signaling pathways. (+info)
The neuroimmune-endocrine axis: pathophysiological implications for the central nervous system cytokines and hypothalamus-pituitary-adrenal hormone dynamics.
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Cytokines are molecules that were initially discovered in the immune system as mediators of communication between various types of immune cells. However, it soon became evident that cytokines exert profound effects on key functions of the central nervous system, such as food intake, fever, neuroendocrine regulation, long-term potentiation, and behavior. In the 80's and 90's our group and others discovered that the genes encoding various cytokines and their receptors are expressed in vascular, glial, and neuronal structures of the adult brain. Most cytokines act through cell surface receptors that have one transmembrane domain and which transduce a signal through the JAK/STAT pathway. Of particular physiological and pathophysiological relevance is the fact that cytokines are potent regulators of hypothalamic neuropeptidergic systems that maintain neuroendocrine homeostasis and which regulate the body's response to stress. The mechanisms by which cytokine signaling affects the function of stress-related neuroendocrine systems are reviewed in this article. (+info)