INOSITOL LIPID STIMULATION OF AORTAL CONTRACTION

Mcp Hahnemann University, Philadelphia PA

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Abstract

DESCRIPTION: (Adapted from the application) Force development in vascular smooth muscle appears to require the activation of the enzyme phospholipase C that breaks down the membrane lipid phosphatidyl inositol 4,5-bisphosphate to form inositol 1,4,5-trisphosphate and diacylglycerol. Since the isoforms of phospholipase C that exist in rat tail artery are currently known, the following hypothesis has been formulated: the formation of lipid-derived second messengers by phospholipase C beta-2 and phospholipase A2 play a key role in the production of agonist-stimulated force in vascular smooth muscle. To test this hypothesis, the following specific aims will be addressed: (1) to determine the functional importance of PLC isoforms in vascular smooth muscle by separating PLC isoforms by column chromatography to measure the PLC activity of each isoform; (2) to determine if the isoforms of PLC that exist in pellets contained from rat tail artery exist in plasma membrane or nuclei; (3) to determine whether the isoforms of PLC in rat tail artery undergo phosphorylation during force generation or maintenance; (4) to determine if norepinephrine stimulates PLA2 in rat-tail artery and determine the mechanism by which it is activated; (5) to determine the effects of arachidonate on force and calcium in vascular smooth muscle. These studies will be performed using rat-tail artery. PLC isoforms will be identified by Western blotting. Using specific antibodies, PLC activity will be determined by incubating fractions from tail artery with [3H]PIP2 and determining the [3H]IP3 released. PLA2 activity will be determined by measuring the production of [3H] arachidonic acid in prelabeled tail artery after norepinephrine stimulation. The effects of arachidonate on cystosolic calcium levels will be determined by measuring indo-1 fluorescence. The studies are designed to further elucidate the molecular mechanism underlying signal transduction by phospholipid-derived second messengers in the regulation of vascular smooth muscle contraction.