Control of signal transduction and contraction in smooth muscle (SM) are complex processes involving multiple regulatory mechanisms. Resolution of these mechanisms is a major problem in SM physiology. This study is based on a new hypothesis which suggests that phosphorylation of proteins on tyr-residues is an important regulatory mechanism for control of receptor mediated contraction of SM. Our objective is to determine and elucidate physiological relationships between receptor mediated activation of SM, protein tyr-phosphorylation, and contraction. The specific hypothesis is that tyrphosphorylation of GAP (GTPase activating protein) and ERK/MAP (extracellular signal related kinase/mitogen activated protein kinase) are significant regulatory mechanisms which function between receptor- activation and Ca2+-sensitivity for contraction of SM.
The specific aims are to (l) delineate specific signalling mechanisms in the activation- contraction pathway involving the tyr-kinase pp60c-src, GAP, ERK, and substrates for ERK which culminate in regulation of Ca2+-sensitivity, (2) delineate between these mechanisms during receptor-activation and direct activation by Ca2+, (3) determine mechanisms whereby tyr-phosphorylation of ERK, and protein kinase C-mediated activation of ERK, function in control of Ca2+-sensitivity, and (4) utilize specific peptides to elucidate sequential signalling steps in permeabilized SM. Experiments will be performed with vascular and visceral SM, and include (a) quantitation of tyr-phosphorylation of GAP/ERK, (b) changes in complex formation between specific molecules such as pp60c-src and GAP, GAP and ras, and ERK and substrates for BRK (i.e., caldesmon) (c) isometric force, and (d) Ca2+-sensitivity. The new data generated will enhance our understanding of basic signalling pathways involving tyr-phosphorylation which regulate contraction of SM. Defects in this previously unrecognized regulatory mechanism will have pronounced impact on the basic physiology of SM contractility, and diseases such as hypertension. Moreover, this impact may lead to new rational pharmacological interventions in the treatment of hypertension.
