Potassium Channels and Arteriolar Hyperpolarization

Jackson, William

Abstract

Isoproterenol, acting through Beta-adrenergic receptors on arteriolar muscle cells causes hyper polarization. However, the mechanisms underlying this action of isoproterenol on vascular smooth muscle cells remain unclear. Potassium channels have been implicated in the isoproterenol-induced hyperpolarization of a number of smooth muscles, but the channels that mediate this increased membrane potential remain to be identified. Therefore the overall aims of this proposal are to establish the mechanism by which the Beta-adrenergic agonist, isoproterenol, induces hyperpolarization arteriolar muscle cells and to determine the role of K+ channels in this process. To accomplish these aims, we will test three hypotheses using patch-clamp techniques applied to enzymatically isolated rat cremasteric arteriolar muscle cells, and use microelectrodes to measure membrane potential in in situ cremasteric arterioles. First, we will test the hypothesis that isoproterenol increases macroscopic K+ currents in voltage-clamped cells, and causes hyperpolarization of current-clamped cells. We will assess the effects of isoproterenol on whole-cell current-voltage relationships and membrane potential, and perform ion substitution experiments to verify a role for K+ channels. Potassium channel blockers will be used to obtain preliminary identification of the types of K+ currents involved. Second, we will test the hypothesis that isoproterenol activates K+ channel currents in membranes from rat cremasteric arteriolar cells by characterizing single channel K+ currents expressed in inside-out patches, and by assessing the effects of isoproterenol on the cumulative open-state probability (NP0) of K+ channels in cell-attached and outside- out membrane patches. Third, we will test the hypothesis that isoproterenol-induced hyperpolarization of cremasteric arterioles in situ is inhibited by the same K+ channel blockers identified in the in vitro experiments above. These experiments will allow us to identify the K+ channel types that mediate Beta-adrenergic agonist-induced arteriolar muscle hyperpolarization, which represents a first step in understanding the mechanism of action of isoproterenol on arteriolar muscle cells. These experiments also will increase our understanding of the regulation of arteriolar muscle, train the applicant in patch-clamp techniques and vascular smooth muscle electrophysiology and establish a working collaboration between the applicant and the sponsor, all three of which will enhance the applicant's research career and assist in achieving his career goals.