Endothelial cells line the inner surface of blood vessels and form the primary barrier between blood and tissues. The endothelium participates in a number of important vascular functions including vascular transport, control of vascular smooth muscle tone and reactivity, and hemostasis. A large body of experimental work indicates that endothelial function may be regulated by mechanical forces generated by the flow of blood under pressure. The goal of this project is to understand ionic mechanisms underlying excitation and mechano-electrical transduction by the vascular endothelium. Patch-clamp methods will be used to identify and study ion channels in cultured bovine aortic endothelial cells. A primary objective is to analyze the activity of single stretch- activated ion channels in cell-attached membrane patches. Experiments will determine: (1) whether spontaneous opening of a 20 and 40 pS channel occur randomly and independently, (2) the effects of pressure on channel open probability and gating kinetics, (3) the effects of voltage on spontaneous and pressure- activated activity, (4) the involvement of cytoplasmic messengers in the gating of the 20 pS channel, and (5) the permeability of the channels to monovalent and divalent cations. The second set of experiments will analyze the macroscopic membrane properties of the endothelial membrane: the resting potential, response of membrane potential to injected current, and ionic components of whole-cell membrane currents under voltage-clamp to evaluate the hypothesis that mechanotransducing ion channels couple membrane deformation to Ca influx through activation of voltage-gated channels and to identify functionally important ion channels. These studies will provide fundamental information on how the endothelium regulates vascular structure and function and will help elucidate its role in the pathogenesis of hypertension, coronary vasospasm, atherosclerosis, and thrombosis.
