Endosomal colocalization of melanocortin-3 receptor and beta-arrestins in CAD cells with altered modification of AKT/PKB.

The melanocortin 3-receptor is involved in regulating energy metabolism, body fluid composition and inflammatory responses. Melanocortin receptors function by activating membrane bound adenylate cyclase. However, the literature reports indicate that some G protein coupled receptors (GPCRs) can also activate mitogen activated protein kinase (MAPK) or phosphoinositide 3 kinase (PI3K) signaling pathways consequent to their endocytosis. These studies were undertaken to evaluate the role of these pathways in MC3R signaling in brain-stem neuronal cells. Recruitment of arrestins is implicated in the activation of secondary pathways by GPCRs and our data shows the colocalization of either arrestin B1 or B2 with MC3R in endosomes. An alteration in PKB phosphorylation pattern was observed in MC3R expressing cells independent of agonist stimulation. MC3R transfectants exhibited increased proliferation rates and inhibition of PKB pathway with triciribine abrogated cell proliferation in both vector control and MC3R transfectants. PKB is constitutively active in proliferating CAD cells but could be further activated by culturing the cells in differentiation medium. These studies suggest that the AKT/PKB pathway plays an important role in the proliferation of CAD cells and suggest a link between MC3R and cell growth pathways that may involve the alteration of AKT/PKB signaling pathway.

Prevalence of melanocortin system transcripts in rat salt homeostasis endocrine tissues.

Melanocortin receptors have been implicated in the confounding factors of cardiovascular diseases such as obesity, insulin resistance and salt-sensitive hypertension. The aim of this study was determine how increased dietary salt intake affects the expression profiles of melanocortin system genes in relevant endocrine tissues. Total RNA was isolated from the pituitary and adrenal glands of Wistar Kyoto (WKY) and stroke-prone spontaneously hypertensive rats (SP-SHR) and subjected to real-time PCR analysis. Expression levels of pro-opiomelanocortin (POMC), POMC processing enzymes prohormone convertases 1 and 2 (PC1/PC2), melanocortin 3 receptor (MC3R) and melanocortin 5 receptor (MC5R) were not significantly affected by high dietary salt intake in either WKY or SP-SHR tissues. Consistent with known endocrine relationship between the pituitary and adrenal glands, the expression levels of the ACTH receptor, MC2R, were five orders of magnitude higher in adrenal tissues whereas those of POMC were three orders of magnitude higher in the pituitary. MC3R, PC1 and PC2 transcripts were expressed at similar levels in both tissues while MC5R was expressed at a higher level in the adrenal tissues. These results are therefore inconsistent with an endocrine pathway that involves pituitary derived gamma-MSH modulating adrenal function in response to high dietary salt intake.

Activation and endocytic internalization of melanocortin 3 receptor in neuronal cells.

Melanocortins play a central role in autonomic modulation of metabolism by acting through a family of highly homologous G protein-coupled receptors. Studies with gene knockout mice have implicated neural melanocortin receptors, MC3R and MC4R, in the etiology of obesity, insulin resistance, and salt-sensitive hypertension. In an attempt to better understand the mechanisms of function of these receptors, we expressed MC3R and MC4R in neuronal cells and demonstrated their co-localization to several membrane regions. We now show that in cultured neuronal cells, MC3R localizes to lipid rafts and undergoes endocytic internalization upon activation by gamma-MSH through a protein kinase-sensitive pathway. The appearance of the internalized receptor in lysosomes suggests that it is subsequently degraded. The expression of protein kinase A regulatory subunits and of c-Jun and c-Fos was analyzed by either immunoblotting or real-time PCR. No discernable changes were observed in the expression levels of these protein kinase A and protein kinase C responsive genes. Immunohistochemical studies showed a robust expression of MC3R protein in brain nuclei with relevance to cardiovascular function and fluid homeostasis further supporting the notion that the physiological effects of melanocortins on the cardiovascular system arise from effects on the central nervous system.

Neural melanocortin receptors are differentially expressed and regulated by stress in rat hypothalamic-pituitary-adrenal axis.

Genetic knockout and null mutations of melanocortin system components lead to phenotypes that recapitulate the metabolic syndrome such as obesity, hypertension and insulin resistance. Since stress is known to modify metabolic and cardiovascular function, we hypothesized the involvement of the neural melanocortin system in the stress response. Male rats were subjected to rapid-eye-movement sleep deprivation stress and the levels of proopiomelanocortin (POMC), MC3R, MC4R and MC5R transcripts in the hypothalamic-pituitary-adrenal axis (HPA) determined by real-time PCR. Increased levels of POMC transcripts were observed in the hypothalamus and adrenal gland tissues but there were no significant changes in the expression of the receptors genes. Whereas MC3R and MC5R are expressed in all HPA tissues, MC4R seems to be restricted mainly to the hypothalamus. It is possible that melanocortin receptors function in different aspects of the neuron. In vitro studies showed similar cellular distribution patterns for MC3R and MC4R and sequence analyses revealed strong conservation of the putative G-protein coupled receptors (GPCR) C-terminal membrane localization signal, EX(3-7)II/L motif, in MC3R, MC4R and MC5R. These data suggest that the physiological roles of neural melanocortin receptors, MC3R and MC4R, are likely determined by distinct tissue distribution patterns and suggest a role for hypothalamic and intra-adrenal melanocortin systems in the manifestation of stress related pathologies.

Effects of gamma-2-melanocyte-stimulating hormone on protein kinase C activity and expression in spontaneous hypertensive rats (SHR).

Administration of gamma-2-melanocyte stimulating hormone (gamma-2-MSH) to rats increases blood pressure, heart rate and natriuresis by acting through the nervous system and this response is more pronounced in spontaneous hypertensive rat (SHR). The molecular mechanisms underlying these effects are unknown, however, protein kinase C (PKC) activity is higher in SHR tissues and melanocortins are known to activate the phosphoinositide (PI) signaling pathway. In this study, we tested the hypothesis that gamma-2-MSH potentiation of PKC activation is increased in nerve terminals from SHR brain. Synaptosomes were isolated from SHR and age-matched control Wistar Kyoto (WKY) rats and incubated with gamma-2-MSH. Total particulate-fraction associated PKC activity was determined and the expression of individual isozymes analyzed by immunoblotting. Treatment with gamma-2-MSH resulted in an increase in particulate-associated PKC activity in hindbrain synaptosomes that was more prominent in SHR. The levels of membrane-associated PKC-alpha and beta-isozymes were considerably less than for PKC-gamma in these tissues as determined by immunoblotting. The novel PKC isozymes delta and epsilon were detected in total synaptosomes but not in membrane fractions. These data suggest that PKC-gamma is the major presynaptic PKC isozyme and that PKC may be an important mediator for gamma-2-MSH in neural tissues.

Expression and distribution of protein kinase C isozymes in brain tissue of spontaneous hypertensive rats.

Hypertension activates many endocrine, neuroendocrine and metabolic responses. How hypertension alters these functions remains unknown. Consequently the pathophysiology of hypertension related illnesses are incompletely understood. Protein kinase C (PKC) isoforms play an important role in cellular signal transduction and altered PKC activity has been reported in spontaneous hypertensive rats (SHR). In order to understand the role that PKC plays in hypertension, we hypothesized that PKC activity is significantly expressed in synaptosomal preparations from the brains of SHRs. In addition, the neuroanatomical distribution of this expression was mapped and compared to control animals. The brains were further studied for signs of neuropathology. Total PKC activity was significantly increased in synaptosomal samples isolated from the forebrain, midbrain, and hindbrain of SHR rats. Westem blot analysis identified PKC-alpha, -beta, -gamma, -delta, -epsilon and -zeta in all brain regions. Immunohistochemical analyses indicated that PKC-gamma was localized in cell bodies and processes in many autonomic cardiovascular control regions. These results suggest that PKC may be an important modulator of autonomic blood pressure control.