Phosphorylation, calcium and smooth muscle contraction

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DESCRIPTION (provided by applicant): It is widely accepted that an increase in [Ca2+] initiates contraction by activation of myosin light chain (MLC) kinase and phosphorylation of the 20 kDa MLC. We have previously suggested that a second parallel, independent pathway exists for the regulation of contraction. In this proposal for renewal of support, we will continue our studies on the mechanism(s) of this pathway by testing the hypothesis that disinhibition of caldesmon by phosphorylation allows constitutively active myosin crossbridges to interact with actin and produce force. Our alternate hypothesis is that caldesmon is important in the transmission of the signal that allows phosphorylated crossbridges to cooperatively turn on unphosphorylated crossbridges. To test these hypotheses, we will utilize an antisense oligonucleotide (ON) approach to produce a caldesmon-deficient vascular tissue. We will use, as our model, intact and skinned strips of swine carotid artery. We will determine the Ca2+ dependence of force, crossbridge cycling rate and attachment, kinase activities, protein content, and protein phosphorylation in control strips containing endogenous caldesmon and in caldesmon-deficient strips. Once we determine how caldesmon regulates smooth muscle, we will determine how caldesmon is regulated. The following specific aims will be pursued in this proposal: 1. To use ONs to inhibit the synthesis of caldesmon and to identify potential regulators of caldesmon using the yeast two-hybrid screen. 2. To test the hypotheses that caldesmon tonically inhibits constitutively active crossbridges or alternatively, that caldesmon regulates the cooperative activation of non-phosphorylated crossbridges by phosphorylated crossbridges. This will be performed by determining if active crossbridge cycling and attachment can be quantified in caldesmon deficient-tissues and to determine the effect of caldesmon removal on the curvilinear force/MLC phosphorylation relationship. And 3. To determine the identity of the physiologically relevant kinase that is responsible for phosphorylation and therefore disinhibition of caldesmon. This will be performed using 32p labeling in conjunction with pharmacological agents and specific anti-phosphocaldesmon antibodies. We will also utilize TOF-mass spectrometry to identify phosphopeptide sequences of caldesmon. We believe the results of this proposal will provide reasonably definitive answers to the questions: Is caldesmon important in contraction? And if so, how does caldesmon regulate smooth muscle and how is caldesmon regulated.