Motile force needed for propulsion of material through the colon is produced by the contractile system of smooth muscle cells in the muscularis. The long range goal of this project is to understand how contraction is regulated at the molecular level. The working hypothesis is that the crossbridge cycle is controlled by phosphorylation of three regulatory proteins - the 20 kDa myosin light chains, caldesmon and calponin. A regulatory model is suggested in which one or more intracellular signal pathway controls each regulatory protein. The signalling pathways suggested to be important are activation of myosin light chain kinase, inhibition of myosin phosphatase activities, activation of MAP kinases (p44 and/or p42) by both Ca2+-dependent and Ca2+-independent pathways, activation of CaM kinase II and activation of protein kinase C.
The specific aims are: 1. Investigate regulation of myosin light chain kinase and phosphatase activities by agonists. 2. Establish the signal transduction pathway that controls caldesmon phosphorylation. 3. Establish the signal transduction pathway that controls calponin phosphorylation. Myosin light chain, caldesmon, and calponin phosphorylation will be measured in intact and permeabilized smooth muscle to correlate with force developed in response to methacholine, histamine and neurokinin A. Agonist and inhibitor effects on myosin phosphatases, CaM kinase II, PKC and MAP kinase activities will also be measured to define which kinases participate in contractile protein phosphorylation and contraction. Multiple regulatory proteins controlled by several signalling pathways may be why transduction of the Ca2+ signal to force is not a fixed characteristic of colonic smooth muscle, but varies with the agonist or combination of agonists stimulating the muscle. This may be relevant to normal function of the colon in that multiple stimuli (eg. acetylcholine, neurokinin A and prostaglandins) are likely to be present during periods of enhanced motility. Furthermore, during periods of inflammation such as bacterial infections or chronic inflammatory bowel diseases there are multiple stimuli in the milieu of the smooth muscle cell (eg. neurotransmitters, histamine, leukotrienes and cytokines). Understanding contractile protein regulation will help in understanding the integrated mechanical response of the muscularis to multiple extracellular messengers.
Showing the most recent 10 out of 365 publications
