Intracellular mechanisms involved in vascular smooth muscle responses include both initial and sustained components. Initial components are receptor-coupled phospholipase C (PLC) activation, inositol phosphate and diacylglycerol (DAG) formation, intracellular Ca2+ mobilization, and protein kinase C (PKC) activation. Sustained components are less well documented but include DAG formation and PKC activation. Elevated cytosolic Ca2+ leads to contraction by calmodulin-mediated activation of myosin light chain kinase. However, Ca2+-independent contraction also occurs and may be the dominant mechanism involved in intrinsic basal tone, contraction to prostaglandin F(2a), and persistent agonist-induced contraction observed after Ca2+ mobilization has returned to baseline. While PKC activity has been shown to be important in these Ca2+- independent responses, PKC has also been shown to elevate Ca2+ in some vascular preparations. Since activation of PKC contracts vascular smooth muscle, and DAG is its intracellular activator, it is critical to understand mechanisms for DAG formation in smooth muscle. PLC activation leads to rapid-initial DAG formation, but sustained DAG production likely results from phospholipase D (PLD) activation. The objective of this proposal is to investigate mechanisms for agonist activation of PLD and production of DAG in vascular smooth muscle. Our proposal that a tyrosine kinase is involved in endothelin-stimulated PLD in smooth muscle is novel. Our approach will be to investigate the mechanisms involved in DAG formation looking at the role of PLCs and PLD. We will study the effects of vasoactive peptides on PLD by determining the kinetics of activation, substrate specificity and calcium dependence. Vasoactive peptide stimulation of protein tyrosine formation will also be studied, determining kinetics, calcium dependence and proteins involved. The role of a tyrosine kinase will be investigated using both specific tyrosine kinase inhibitors and protein tyrosine phosphatase inhibitors. Agents which alter agonist-activated protein tyrosine formation should alter agonist-stimulated PLD. We will use receptor-mediated activation (peptides), and receptor-independent activation (phorbol esters, thapsigargin, ionophores) to clearly establish the components of the mechanism involved. These studies will be done using vascular smooth muscle cells in culture.
