Evidence exists that arterial smooth muscle contraction is regulated by phosphorylation of the 20,000 dalton light chain (LC20). The proposed project seeks to extend these studies to venous smooth muscle by using pharmacologically characterized tissues from canine jugular and femoral vein. Isolated tissues, incubated in muscle baths, will be quick frozen during isometric contraction and relaxation and analyzed for LC20 phosphorylation using a 2-dimensional isoelectric-focusing SDS-electrophoresis gel system. Specific questions to be addressed include: 1) Is phosphorylation functionally associated with contractile stimulation and active cross bridge cycling? 2) Is contractile force sustained solely through phosphorylation dependent cross bridge cycling or can phosphorylation decrease to allow force maintenance through a catch-like state as observed in certain arteries? 3) To what extent is the course of phosphorylation and nature of contraction influenced by possible transients in myoplasmic free calcium during contraction? 4) How do different stimuli compare in their effect on the time course of phosphorylation and can this explain the nature of the contractile response? Eventual emphasis will be on vasoactive autocoids released through injury and surgical trauma which may be implicated by way of their contractile effects in the pathogenesis of deep venous thrombosis. Canine jugular and femoral vein are relevant in this respect since they have been used as models for in vivo studies on formation of autocoid-induced lesions which could serve as sites for thrombus development. The long term goals of this project are to delineate the regulatory role of LC20 phosphorylation on the venous side of the vasculature, the extent to which this role may be influenced by the stimulus employed, and the ability of phosphorylation to explain certain vasoactive effects of autocoids.
