The proposed research program focuses on ionic, biochemical and cellular mechanisms of vasoconstrictor agonist (angiotensin II, KC1) and growth factor (platelet-derived growth factor) action on diploid and polyploid vascular smooth muscle cells (VSMC) isolated from different blood vessels (aorta, caudal artery and mesenteric artery) of normotensive, spontaneously hypertensive and DOCA/salt hypertensive rats. Freshly isolated, as well as cultured VSMC will be used in these studies. A detailed analysis of cytosolic calcium and pH will be made using flow cytometry and newly developed multiparameter analytical methods related to fluorescence-ratio imaging microscopy.
The specific aims i nclude: 1) providing a detailed cell-by-cell analysis of resting cytosolic calcium and pH, as well as membrane potential, in diploid/tetraploid VSMC via flow cytometry. In addition, differences in the spatial and regional distribution (nuclear vs. cytosolic) of cellular calcium and pH will be assessed; 2) determining whether diploid/polyploid (tetraploid, octaploid) VSMC from hypertensive animals possess altered signal transduction pathways as compared to normal diploid VSMC. We will also compare and contrast the regional differences in cytosolic calcium and pH following angiotensin II and PDGF stimulation; 3) evaluating the physiological relevance of cell-cell interactions between diploid and polyploid populations of VSMC; and 4) examining the cytogenetic mechanisms which contribute to the development of the polyploid VSMC phenotype. These studies should contribute to our basic understanding of the mechanisms of normal and altered VSMC responsiveness related to the pathogenesis of hypertension, atherosclerosis and arterial vasospasm.
