The overall objective of our research is to understand the mechanisms that regulate ion channels in the cardiovascular system. We made a novel observation that the large conductance Ca2+-activated K+ (BK) channels in smooth muscle cells from small coronary arteries of the Zucker diabetic fatty (ZDF) rats showed altered response to voltage and Ca 2+ activation. ZDF rats are an established model for the study of type 2 diabetes and their coronary vessels showed impaired endothelium-dependent and -independent relaxation. Since BK channels are the major ionic determinant in mediating vasorelaxation and are the target of endothelium-derived hyperpolarizing factors (EDHFs), altered BK channel regulation may contribute to both endothelium-dependent and -independent vascular dysfunction in type 2 diabetes. The following hypotheses will be tested: A) Regulation of vasorelaxation by arachidonic acid metabolites, which are putative EDHFs, is abnormal in type 2 diabetes. B) Abnormal EDHF-mediated vasorelaxation in type 2 diabetes is due to abnormal regulation of vascular BK channels. C) Abnormal BK channel regulation is due to structural modification of the channel. We propose the following specific aims: 1) To determine the arachidonic acid-mediated coronary vasorelaxation in control and ZDF rats, we plan to determine the contribution of the cytochrome P450 and the lipoxygenase pathways to the arachidonic acid effects using pharmacological approaches and microelectrode measurements of membrane potential in isolated coronary vessels. 2) To determine the alterations in the regulation of BK channels in ZDF rats, we will determine the BK channel properties including channel density, I-V relationships, V1/2, Ca 2+ EC50, response to channel activators, and channel kinetics using the whole-cell and single-channel electrophysiological approaches. 3) To determine the molecular mechanisms of the BK channel defects, we will explore three plausible mechanisms, namely activation of protein kinase C, expression of channel variants, and functional uncoupling with the channel beta subunit using biochemical and molecular approaches. This study may help us to better understand the regulation of vascular BK channels and to delineate the role of EDHF in the regulation of coronary physiology in diabetes. Our results may provide important mechanistic insight into the pathogenesis of abnormal vasoreactivity associated with type 2 diabetes and may lead to the development of new approaches in the prevention and treatment of cardiovascular complications in patients with type 2 diabetes.
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