The receptor-mediated contraction of vascular smooth muscle (VSM) involves activation of phospholipase C (PLC), protein kinase C (PKC), and perhaps phospholipase D (PLD). Although several isoforms of these enzymes exist in mammalian cells, little is known regarding which isoforms are present or which are involved in force development in VSM. This proposal is based upon the following hypotheses: 1) only certain isoforms of PLC and PKC exist in vascular smooth muscle and only certain isoforms are required for force development in VSM; and 2) PLD is also important during agonist-mediated force development and its activity is controlled by G proteins and/or cytosolic calcium levels. In order to test these hypotheses, the following specific aims will be addressed:
Aim 1) To determine which isoforms of PLC exist in VSM and which isoforms are involved in force development.
Aim 2) To determine the importance of the tyrosine phosphorylation of phospholipase C during smooth muscle contraction.
Aim 3) To determine which isoforms of protein kinase C exist in smooth muscle and which isoforms are important for force development.
Aim 4) To determine the physiological role of phospholipase D activation in smooth muscle by determining if this enzyme covalently links some membrane protein to phosphatidic acid.
Aim 5) To determine if phospholipase C in vascular smooth muscle is subject to regulation by G proteins, cytosolic calcium levels or tyrosine phosphorylation. These studies will be performed using rat tail artery. We will identify the isoforms of PLC and PKC by separating SDS extracts of the tissue by SDS polyacrylamide gel electrophoresis, Western blotting, and immunodetection with monoclonal antibodies. PLC activity will be determined by prelabeling tissue with [3H] inositol (to label inositol lipids) and measuring inositol phosphate levels after agonist-stimulation. PLD activity will be determined by prelabeling tissue with tritiated choline (to label the phosphatidyl choline) and measuring choline release after agonist-stimulation, and also by measuring phosphatidyl ethanol production in agonist-stimulated tissue that has been prelabeled with [3H] myristic acid. These studies are designed to further elucidate the molecular mechanisms underlying signal transduction by phospholipid- derived second messengers in the regulation of vascular smooth muscle contraction.
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