Nitric oxide (NO) and compounds that generate NO (e.g., nitroglycerin) play an increasingly important therapeutic role in the perioperative period (e.g., in the treatment of acute pulmonary and systemic hypertension). The mechanisms of action of NO on smooth muscle are not fully understood. Recent evidence suggests that NO relaxes smooth muscle not only by reducing cytosolic calcium (Ca2+) concentration ([Ca2+]i), the focus of most previous work, but also by reducing the amount of force produced for a given [Ca2+]i (i.e., """"""""myofilament Ca2+ sensitivity""""""""). Our preliminary data suggest two categories of mechanisms responsible for these effects on myofilament Ca2+ sensitivity: 1) mechanisms mediated by NO-induced increases in guanosine cyclic 3',5'-monophosphate (cGMP), and 2) mechanisms that are independent of cGMP. The goal of this proposal is to explore novel mechanisms specifically related to cGMP- dependent (AIM A) and cGMP-independent (AIM B) effects of NO on myofilament Ca2+ sensitivity in smooth muscle. Both inhibition of Ca2+- calmodulin activation of the contractile proteins and inhibition of membrane receptor-linked second messenger systems that regulate myofilament Ca2+ sensitivity by cGMP-dependent and cGMP-independent effects of NO will be studied. These studies will be facilitated by the use of a unique permeabilized smooth muscle preparation in which intracellular concentrations of NO and cGMP can be separately manipulated under conditions of constant [Ca2+]i. In this preparation, second messenger systems that regulate myofilament Ca2+ sensitivity remain intact. Preliminary studies support the overall hypothesis that cGMP- dependent effects of NO on myofilament Ca2+ sensitivity are mediated exclusively by inhibition of the second messenger systems that normally function to regulate myofilament Ca2+ sensitivity, whereas cGMP- independent effects of NO are mediated by inhibition of both categories of cellular processes. Biochemical measurements of myosin light chain phosphorylation, and myosin light chain inase, myosin ATPase, and protein kinase C activities will identify the specific mechanisms involved in these actions. Measurements of smooth muscle mechanics will provide insight into effects on actin-myosin crossbridge kinetics. These experiments will reveal whether the fraction of force-generating actin- myosin crossbridges or the mean force per crossbridge is reduced. An understanding of the complex mechanisms producing these effects may provide insights into mechanisms of NO actions in other tissues, as many of the intracellular signal transduction systems found in smooth muscle are common to other cell types. Additionally, elucidation of these mechanisms may suggest strategies for the future development of therapeutic smooth muscle relaxants.
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