Atriopeptin is a potent natriuretic, diuretic and vasodilating peptide which is synthesized and stored in the atrial myocyte as a 126 amino acid precursor prohormone, but secreted from the heart in response to stretch as the 28 amino acid, physiologically active species. The mechanism(s) that regulate its synthesis, storage, secretion and processing are currently not well defined. We have shown that stretching the atrial myocyte with hypotonic solutions results in atriopeptin secretion. The central hypothesis of the proposed research is that mechanical perturbation (i.e. stretch) of the myocyte sarcolemma and other atriopeptin secretagogues activate phospholipase C, which results in the formation of two putative intracellular mediators of atriopeptin secretion, inositol triphosphate and diacylglycerol. To initially test the hypothesis, the intracellular mass of inositol triphosphate will be measured by GC-MS in stimulated atrial myocytes. Furthermore, atriopeptin secretion as a result of the direct application of inositol triphosphate to permeabilized myocytes or by electroporation will be evaluated. The role of diacylglycerol formation and protein kinase C activation on atriopeptin secretion will be assessed by pharmacologically activating protein kinase C by exposing atrial myocytes to a phorbol esther, and l-oleoy1-2-acetylglycerol, a synthetic analogue of diacylglycerol. Once secreted from the atrial myocyte, we hypothesize that the atriopeptin prohormone is processed to atriopeptin 28 by the cardiac mesenchymal cell before release into the circulation. Amino acid sequence analysis of the peptide fragment(s) generated upon exposure of the prohormone to cultured cardiac mesenchymal cells will either substantiate or disprove this hypothesis. By employing techniques used to separate and isolate subcellular organelles, as well as chemically and physically inhibiting cellular internalization of the atriopeptin prohormone, the cellular site(s) of prohormone processing will be determined. The atria is thought to be the major site of atriopeptin synthesis and secretion, however, the hypertrophied ventricle possesses significant synthetic capabilities. In order to characterize the cellular mechanism(s) involved in the induction and secretion of atriopeptin in ventricular tissue, atriopeptin synthesis in cultured ventricular myocytes will be assessed in response to stimuli that induce either cell growth or atriopeptin secretion. Characterization of whether ventricular atriopeptin secretion is the result of regulated and/or constitutive release will be assessed. Hopefully, these experiments will clarify approaches to pharmacologically manipulate atriopeptin synthesis, storage and release in pathophysiological states.
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