This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The long-term objectives of this study are to delineate the molecular mechanisms that underlie modification of cardiovascular function during stress. Stress is an important risk factor for cardiovascular diseases, but the underlying mechanisms are not well understood. The REM_SD (REMsleep deprivation) rat model of stress displays profound alterations in central autonomic pathways including cardiovascular homeostasis, feeding behavior, energy balance, and thermoregulation. Since melanocortins have been implicated in these functions, it is hypothesized that a perturbation of neural melanocortin system occurs during stress, leading to changes the responsive signal transduction pathways in central autonomic regions. Therefore, the first aim of this study to further characterize the neural melanocortin signaling system and determine the changes that occur during REM-SD stress. Antibodies will be generated against melanocortin receptors MC3-R, MC4-R and MC- 5R. The antibodies will be used to map the expression of receptor proteins in rat brain and to analyze alterations in expression patterns during REM-SD using immunoblot and immunohistochemical techniques. Proopiomelanocortin (POMC) and melanocortin receptor gene expression will also be determined by Northern blotting and RT-PCR methods. Antibodies will also be used to characterize the melanocortin signaling system by identifying the interacting partners using pull-down assays, colocalization immunohistochemistry and immunoblot analysis. Further, melanocortins are involved in many physiological processes acting both in the CNS and peripheral tissues. Literature reports indicate that in peripheral tissue cell lines, melanocortin receptors activate the cAMP, PKC and JAK/STAT signaling pathways. Neuronal signaling pathways that are modulated by melanocortins in a rodent brain stem cell line and in rats subjected to REM-SD stress will be identified using array technology and immunoblotting techniques. Those transcripts identified will be localized using in-situ hybridization and northern blotting. This study will not only provide important insights into the role and mechanism of neural melanocortin signaling system in autonomic functions, but will also identify molecular markers that play a potential role in stress related cardiovascular disease.
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