The proposed studies will examine alterations in binding or mobilization of Ca ions from high and low affinity sites important for the initiation, maintenance or inhibition of contractility in vascular smooth muscle. Conventional techniques will be employed to measure isometric tension responses, 45Ca uptake and rapid washout, and total ionic (Ca ions, Na ion, K ion) concentrations as well as to obtain vascular smooth muscle microsomal preparations. In intact preparations, emphasis will be placed upon measurement of residual Ca ions retention (La ions-resistant Ca ions) after exposure to isosmotic (80.8 mM) La ions at 0.5 degrees C under conditions optimizing high or low affinity Ca ions binding. Further dissociation and analysis of high and low affinity sites will be attained with Sr ions (which can displace Ca ions from high but not low affinity sites), high K ion (which mobilizes La ions-accessible low affinity Ca ions, and agents such as norepinephrine (which releases La ions-resistant high affinity Ca ions). This information could provide a basis for development of a model including various mechanisms by which Ca ions at different membrane sites is involved in events leading to enhanced or decreased contractile tone. The effects of selected ions, agonists and vasodilators will be described in terms of alterations of Ca ions at or in specific membrane binding sites or channels. Vascular preparations which differ qualitatively in the mechanisms by which Ca ions mobilization occurs will be compared. Vascular smooth muscle microsomal membrane components will be developed, characterized, and employed in a comparative manner. Measurement of 45Ca fluxes and binding in these preparations will permit comparison of effects obtained with stimulatory and inhibitory agents in these systems with corresponding effects in more anatomically complex isolated vascular strips. Thus, insight concerning the cellular and subcellular actions and mechanisms of action of a variety of ions and drugs in different types of vascular smooth muscle will be obtained.
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