Contraction of smooth muscle in the walls of blood vessels can decrease flow to a tissue or cause an elevation of pressure. Our long range goal is to determine how contraction of intact vascular smooth muscle is regulated at the biochemical level. In smooth muscle, one of the light chains of myosin (the protein that converts the energy of ATP into work) can be phosphorylated by a specific calcium dependent enzyme, light chain kinase. This is thought to initiate contraction, but our studies on intact hog carotid artery smooth muscle have not shown a simple correlation of light chain phosphorylation with muscle force. Because of this, it has been proposed that phosphorylation is necessary for rapid crossbridge cycling, and that dephosphorylation of attached crossbridges converts them to slowly-cycling """"""""latchbridges"""""""" which maintain force. Continued attachment of latchbridges appears to be regulated by other, unknown, calcium-dependent mechanisms. One such possible mechanism is phosphorylation of caldesmon, a thin filament protein that inhibits actomyosin ATPase in vitro. Phosphorylation of caldesmon by a calcium-dependent kinase removes this inhibition, and one of our objectives is to determine if this is a physioligical regulatory machanism. Progress in understanding light chain phosphorylation as a regulatory mechanism has been hampered because 1) the kinetics of the crossbridge cycle in relation to dephosphorylation rates are not known; and 2) it has not been possible to distinguish between phosphorylation of light chains by two different kinases: myosin light chain kinase, and protein kinase C, the calcium-, phospholipid-dependent kinase. We propose to quantitate light chain phosphorylation by kinase C and to determine the role, if any, of this phosphorylation in regulating contraction of smooth muscle stips. Because this latter enzyme phosphorylates a threonine residue (light chain kinase phosphorylates a serine residue), ultrasensitive HPLC amino acid analysis can be used to differentiate between phosphorylation by the two different kinases. We will also make definitive measurements of light chain phosphorylation and dephosphorylation rates in isolated smooth muscle cells, and will extend our studies on dephosphorylation during rhythmic contractions. We have developed a method to prepare isolated smooth muscle cells from the hog carotid artery by papain digestion, and these cells will be used for the phosphorylation measurements. Note: Smooth muscle tissues will be obtained from slaughterhouses (hog, turkey, chicken)
| Dougherty, T J; Driska, S P (1991) Unusual [Ca2+] dependence of vascular smooth muscle cell shortening velocity. Am J Physiol 260:C449-56 |
