The long-term goal of this proposal, is to investigate the molecular regulation of smooth muscle contractile proteins. Two general hypothesis will be tested. Hypothesis 1: Myosin phosphorylation regulates the number of active crossbridges in vascular and nonvascular smooth muscle. Hypothesis 2: The actin-binding proteins caldesmon and filamin contribute to an actin-linked regulatory system operating in concert with the myosin-linked phosphorylation system.
The specific aims which will be addressed by these studies are: (1) to determine if myosin phosphorylation (ser-19) functions as a simple switch to activate force-generating crossbridges. (2) to determine whether slowly- cycling, latch-bridges exist in chemically skinned vascular and/or nonvascular smooth muscle, (3) to determine if the rate of LC20 phosphate turnover influences the formation of latch-bridges, (4) to determine if the regulation by myosin phosphorylation (ser-19) is mechanistically similar for vascular (both tonic and phasic) and nonvascular smooth muscle, (5) to determine if the presence of a latch-static contributes to the mechanical differences (e.g. different rates of determine isotonic shortening velocity) observed for different smooth-muscle types, (6) to determine if caldesmon/filamin contributes to the regulation smooth muscle contraction, and (7) to investigate the possible involvement of caldsmon/filamin in the regulation of the latch-static in vascular (tonic and phasic) and nonvascular smooth-muscle.
These specific aims will be approached experimentally by utilizing both intact and skinned preparations from porcine caotid, rat protal vein, and rat uterine smooth muscle. The relationship between myosin phosphorylation and isometric force, stiffness, and shortening velocity will be determine for both intact and skinned muscles. Experiments with the skinned muscle preparation involving the addition of purified myosin light chains, caldsmon, filamin, and both enzymatically active and inactive proteolytic fragments of caldesmon and filamin mechanisms. In general, these studies should provide data essential to the understanding of the molecular regulation of vascular and nonvascular smooth muscle contraction. An understanding of the regulatory processes in vascular smooth muscle is of particular importance because of the human morbidity and mortality associated with blood vessel disease and inappropriate vascular smooth muscle contraction.
| Haeberle, J R; Hathaway, D R; Smith, C L (1992) Caldesmon content of mammalian smooth muscles. J Muscle Res Cell Motil 13:81-9 |
| Sutton, T A; Haeberle, J R (1990) Phosphorylation by protein kinase C of the 20,000-dalton light chain of myosin in intact and chemically skinned vascular smooth muscle. J Biol Chem 265:2749-54 |
