ACTH/MSH PEPTIDES & PHORBOL ESTERS ON PKC PHOSPHORYLATION & CA LEVELS IN SHR

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Description (Adapted from Application): Phosphoinositide signaling system and calcium have been reported to play a role in the regulation of blood pressure. In preliminary studies, it has been established by immunoblot and immunohistochemical studies that PKC isozymes alpha, beta, gamma, delta, epsilon, and zeta are expressed in synaptosomes isolated from brain regions of normal and hypertensive rats. It has also been established that gamma-2-MSH alters the expression of PKC isozymes as well as PKC activity in hypertensive rats. It has been reported that ACTH-like peptides derived either from the extracellular space affect the B-50 (GAP-43) phosphoprotein through interaction with the presynaptic membrane of the nerve terminal. Also, the brain is rick in PKC and Ca2+ and it is sensitive to hypertension, which causes a variety of vascular and neuronal cerebral changes. Taken together, it is hypothesized that in hypertension, there is a link between peptide (i.e., gamma-2-MSH, ACTH[4-10], and ACTH] PKC-mediated phosphorylation of specific PKC isozymes, which results in changes in PKC-mediated phosphorylation of specific proteins as well as alterations in the levels of calcium. In order to understand the role of the central nervous system and the phosphoinositol signaling pathway in hypertension, the investigators plan to use: (1) protein phosphorylation, immunoprecipitation, and Western immunoblot assays to identify the specific proteins that are phosphorylated as a result of the activation of PKC-delta, -epsilon, and-gamma in the forebrain, midbrain, and hindbrain regions of normal and hypertensive rats in the presence and absence of gamma-2-MSH and/or ACTH[4-10]; (2) protein phosphorylation immunoprecipitation and Western immunoblot assays to determine whether phorbol esters, in the presence and absence of gamma-2-MSH and ACTH[4-10] interfere with the activation of PKC-delta, -epsilon, and-gamma synaptosomes from the forebrain, midbrain, and hindbrain regions of normal and hypertensive rats; and (3) calcium imaging studies to determine whether phorbol esters increase or decrease intracellular Ca2+ levels in synaptosomes isolated from the forebrain, midbrain, and hindbrain regions of normal and hypertensive rats in the presence and absence of gamma-2-MSH and ACTH[4-10].