The long-term objectives of this proposals are to define the mechanism by which anesthetics alter agonist-mediated intracellular signalling pathways and to understand the cellular mechanisms responsible for anesthetic-induced hypotension. As Ca2+ is a primary determinant controlling contraction and myosin light chain (MLC) phosphorylation is recognized biochemical index of contraction, the regulation of anesthetics on agonist-mediated Ca2+ mobilization and MLC phosphorylation will be studied using vascular smooth muscle cells. Agonists and drugs which alter[Ca2+]i and MLC phosphorylation, alter contraction and thereby regulate important vascular parameters including vascular tone and blood pressure. Endothelin will be used as the primary agonist because this peptide produces a slowly-developing, sustained contraction by mobilizing both Ca2+ release and Ca2+ entry through L-type Ca2+ channels and by stimulating formation of Ins(1,4,5)P3 and diacylglycerol (DAG), and protein kinase C (PKC) activation. Propofol, a new intravenous anesthetic, produces vasodilation and hypotension by an as yet unknown mechanism. Preliminary studies have found that propofol inhibits endothelin-stimulated Ins(1.4,5)P3 formation phospholipase D activity, and Ca2+ entry through the L-type channel. We hypothesize that propofol: 1) inhibits the phospholipase C pathway, 2) reduces Ca2+ entry by interfering at sites leading to PKC through the L-type channel by modulating dihydropyridine sites, and 4) alters MLC phosphorylation.
In specific aim #1, experiments are designed to investigate the interaction of propofol with the phospholipase C pathway and the mechanisms involved.
In specific aim #2, the Ca2+ entry pathways altered by propofol will be studied by investigating effects on Ca2+ entry through the L-type channel and the receptor-operated channel.
In specific aim #3, the mechanisms by which propofol regulates Ca2+ entry through the L-type channel will be studied by investigating formation of DAG, the role of phospholipase D in the regulation of PKC activation, and dihydropyridine binding.
In specific aim #4, the regulation of anesthetics and PKC on MLC phosphorylation will be studied. These experiments will use both A10 cells and primary cultures of rat aortic smooth muscle. Methods to be used will include the measurements of: a) Ca2+ mobilization using the fluorescent probe fura-2 and 45Ca2+ fluxes; b) PKC activity by measuring the transfer 32P-ATP to histone III; c) phospholipase D activation by dihydropyridine binding using [3H] PN200-110; e) MLC phosphorylation using a method of SDS-PAGE coupled with immunoblotting. Information from these studies will provide important insights into understanding of the cellular mechanism of anesthetic actions and may lead to development of safer anesthetics.
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