The long-term objectives of this proposal are to determine the mechanism by which anesthetic agents alter agonist-mediated signal pathways and to define the cellular mechanisms responsible for anesthetic-induced hypotension. As Ca2+ is known to be a primary determinant controlling smooth muscle contraction, vascular smooth muscle cells will be used to investigate the role of anesthetics in agonist-mediated Ca2+ mobilization. Agonists and drugs which alter [Ca2+]i, alter contraction and thereby regulate important vascular parameters including blood pressure. Endothelin will be used as the agonist because this peptide produces a slowly-developing, sustained contraction by mobilizing both intracellular Ca2+ release and Ca2+ entry through L-type Ca2+ channels and by involving formation of Ins(1,4,5)P3 and diacylglycerol (DAG), and protein kinase C 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 inositol phosphate formation and Ca2+ entry through the L-type channel. We hypothesize that propofol regulates agonist-induced Ca2+ mobilization and L-type channel activity by interfering at sites leading to and beyond protein kinase C activation in the agonist-activated phospholipase C and/or phospholipase D signal pathways.
In specific aim #1, experiments are designed to investigate the ability of propofol to regulate intracellular Ca2+ release and the mechanisms involved by investigating effects on the Ca2+ release, sarcoplasmic reticulum Ca2+- ATPase activity, phospholipase C activity, and agonist/receptor binding.
In specific aim #2, experiments are designed to determine the Ca2+ entry pathway altered by propofol. Ca2+ entry through either the L-type channel or the receptor-operated channel will be initiated by a receptor-mediated system (endothelin), by phorbol esters bypassing the receptor, by depolarization bypassing intracellular signals, and by emptying of intracellular pools with thapsigargin. The effects of propofol on Ca2+ entry through both channels will be determined.
In specific aim#3, the cellular mechanisms by which propofol regulates Ca2+ entry through the L- type channel will be studied by investigating the formation of DAG, protein kinase C activation, phospholipase D activity, phosphorylation, and dihydropyridine binding. Similar experiments will be performed with volatile anesthetics and the mechanism of action compared with propofol. These experiments will use both A10 cells and primary cultures of smooth muscle isolated from rat aortas. Methods to be used will include measurements of inositol phosphate production, DAG levels, protein kinase C activation, phospholipase D activity, agonist and dihydropyridine receptor binding, as well as Ca2+ mobilization using Ca2+ indicator dye, fura-2, and 45Ca2+.
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| Xuan, Y T; Tang, X L; Banerjee, S et al. (1999) Nuclear factor-kappaB plays an essential role in the late phase of ischemic preconditioning in conscious rabbits. Circ Res 84:1095-109 |
| Xuan, Y T; Glass, P S (1996) Propofol regulation of calcium entry pathways in cultured A10 and rat aortic smooth muscle cells. Br J Pharmacol 117:5-12 |
| Xuan, Y T; Wang, O L; Whorton, A R (1994) Regulation of endothelin-induced Ca2+ mobilization in smooth muscle cells by protein kinase C. Am J Physiol 266:C1560-7 |
