A system for studying basic biochemical and physiologic aspects of the contraction of intact mammalian smooth muscle has been established in this laboratory during the past year. Utilizing fresh strips of bovine trachealis muscle, methods have been developed to correlate the contractile response to certain agonists with the state of myosin phosphorylation. Of particular interest is whether phosphorylation of the 20 kDa-myosin light chain (MLC) at an amino acid residue(s) other than serine 19, known (Pearson, R.B. et al. (1984) FEBS Lett. 168, 108-112) to be preferentially phosphorylated by myosin light chain kinase (MLCK), can be shown to occur and to have a regulatory role in tension generation. Baseline studies have demonstrated the expected single phosphopeptide upon activation of MLCK via the muscarinic cholinergic receptor agonist carbachol. In contrast, upon stimulation with a phorbol ester which preferentially activates intracellular proteins kinase C (PKC), preliminary results indicate the presence of an additional phosphopeptide. Experiments to more precisely characterize this response are in progress.
