IONIC CHANNELS IN DISSOCIATED SMOOTH MUSCLE CELLS

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The present application is a continuation of ionic channels and their regulation in single, freshly dissociated smooth muscle cells. These ionic channels control the membrane potential, are the targets of neurotransmitter action, underlie the action potential and together with the channels responsible for Ca (2+) release from intracellular stores regulate the cytosolic Ca (2+) concentration. Consequently, these channels play a central role in the control of smooth muscle contraction, a topic of considerable therapeutic importance, for example, in hypertension, vasospasm and uterine and gastrointestinal contractility. Most studies will be carried out on a preparation of isolated cells from the stomach of the toad, Bufo marinus, which have been characterized in considerable detail electrophysiologically, biochemically, and morphologically, and have proven to be excellent predictors of general properties of smooth muscle from a variety of sources. There will be three major areas of study. 1. Neurotransmitter and second messenger regulation of ion channels. Muscarinic, cholinergic regulation of Ca (2+) channels and K+ channels responsible for M-current will be studied to determine the role of cytosolic second messengers such as protein kinase C and "membrane delimited" mechanisms such as G-protein channel interactions. Using this study as a prototype, the mechanisms underlying the actions of a variety of other excitatory and inhibitory agents such as neuropeptides will be examined. 2. Fatty acid of ionic channels. Recently fatty acids have been shown to exert a direct effect on a number of ionic channels in smooth muscle and other cell types. The mechanisms of this fatty acid activation and its physiological role will be examined. Regulation of mechanically gated channels. The smooth muscle cells have a considerable variety of mechanically gated channels, including stretch-activated channels, stretch inactivated channels, channels activated both by stretch and membrane hyperpolarization, and flow- activated channels. The properties of these channels and their regulation will be studied. The studies will emphasize the use of patch-clamp technology to record currents through single ionic channels and also record macroscopic currents under conditions where the cytosol can be altered. In addition, physical techniques such as NMR will be used in studying fatty acid interactions with membrane proteins and lipids. Finally, the Ca (2+) indicator dye fura-2 will be employed to measure cytosolic {Ca (2+)} in single, voltage- clamped cells to determine the effect of various agents on release of Ca (2+) from intracellular stores and the relationship between transmembrane Ca (2+) current and {Ca (2+)}.