In disease states, such as hypertension, the primary defect may be traced to the regulation of the mechanisms for maintenance of tone in smooth muscle. The basic mechanism(s) controlling tone are not well understood. A unique and important characteristic of all smooth muscles is the ability to vary the energy cost of force production and maintenance during the time course of isometric contractions. It has become obvious that there are many similarities among the mechanical and energetic properties of mammalian smooth muscles during the """"""""latch"""""""" state and molluscan muscles during """"""""catch,"""""""" yet the molecular basis of these states and their regulation is not well understood. A unique feature of catch muscle is the operation of a mechanism that appears to control the detachment rate of the crossbridge. Such a mechanism has not yet been identified in mammalian muscle, but would be of great importance as a means of regulating tone and consequently physiological functions, such as blood pressure. The elucidation of such a mechanism in catch thus has the potential to provide important insights into normal and abnormal function of the cardiovascular, pulmonary, digestive, and reproductive systems. The overall goal of this proposal is to define the state of the myosin crossbridge and its kinetics of turnover at rest and during phasic and catch contractions in the anterior byssus retractor muscle (ABRM) of Mytilus edulis, and to identify proteins whose change in phosphorylation state is the molecular basis for the regulation of the transition of the crossbridge into and out of the catch state.
The Specific Aims are to determine: (1) in permeabilized ABRM, the nucleotide and/or inorganic phosphate bound to myosin under different mechanical conditions, including the resting state (crossbridges detached or weakly bound to actin), phasic contraction (crossbridges cycling and maintaining force) and the catch state (crossbridges likely to be attached and maintaining force, but cycling very slowly); (2) the rate of nucleotide and/or phosphate turnover on myosin under different mechanical conditions using single turnover techniques in which radiolabeled nucleotides are generated by photolysis of caged compounds, and thereby gain quantitative information concerning the rate of the myosin ATPase cycle under these very different mechanical conditions. We will also determine whether the rapid relaxation from catch initiated by cAMP is associated with a rapid exchange of the products of ATP hydrolysis bound to myosin, in order to directly address the question of whether myosin changes its state in the transition from catch to rest. (3) during the release of catch, which proteins show changes in levels of phosphorylation on a time course that corresponds to the change in state of myosin and/or relaxation following cAMP formation from photolysis of caged cAMP. These experiments will identify proteins which are candidates for the molecular regulator(s) of catch.
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