Activation of phospholipase C by oxyhemoglobin in cultured aortic cells

emoglobm pro&es a contraction in cerebral blood tessels, ar:d it is generally accepted that the release of this compound from e@rocytes in the subarachnoid clot may be r&ponsible for the delayed vasospasm which is frequently observed after subarachnoid hemorrhage (Welhun et al., 1982). Important evidence in this regard is that the release of hemoglobm from clotted blood shows a similar time course to the initiation of the vasospastic response. The mechanism by which hemoglobim produces a contraction is not known, but since neither the response to hemoglobin nor the development of cerebrovascular spasm after subarachnoid hemorrhage, are antagonised well by agents which inhibit voltage-operated calcium channels, it is likely that the hetrqlobii-induced contraction involves release of calcium from intracellular stores. The observation that an intracellular calcium antagonist, HA 1077, may prevent delayed cerebral vasospasm (Shibuya et al., 1988) is consistent with this suggestion. One mechanism by which this process can take place involves the breakdown of inositol phosphobpids to inositol(l,4,5~~sphosphat~ (1~1~4,S)P~), which is a reaction mediated by a Ga-biiding protein (Cockcroft and Gomperts, 1985). Ins(1,4,5)P, is responsible for the release of calcium from stores on the sarcoplasmic reticulum. We have examined this hypothesis in cultured cells from rat aorta (A,r& and correlated the activity of hemoglobin on inositol phospholipid metabolism in this system, with its contractile effects. Ins(1,4,5)P, was measured using the 3H-Ins(l,4,S)P3 assay system (Atnersham) or by HPLC analysis after incubation of the cells with 3H-myo-inositol. The production of Ins(1,4,5)P3 was stimulated by hemoglobin in a timedependent manner, the first elevation beiig observed as early as 15 seconds after exposure to hemo~obin. ~~ sedition was observed after about 1 minute, after which the effect subsided over about ten minutes. There was a good correlation between the elevation of Ins(l,4,5)P3 and the initiation of the contractile response in rings of rat aorta, suggesting that this is the mechanism by which the initial response to hemoglobin is produced, The ~n~action was not well antagonised by nifedipine even at a inundation of 10 FM. Studies of the action of agents which interfere selectively with components of the inositide pathw.ay, and analysis of the time course of the production of further metabolites of Ins(1,4,5)P3 using HPLC, support the conclusion that the hemoglobin-induced contraction of kascular smooth muscle arises, at least in part, from stimulation of the production of Ins(l;Q,S)P*

Co&oft, S. and Gomperts,B.D., 1985, Nature (Land.) 314, 534. Shibuya, M., Suzuki,Y., Talcayasu,M., Asano,T., Kanamori,M., Okada,T. and Hid&a, H., 1988, Acta NeUrochiF.(wien) 90.53. Wetbun,G.R., b-vine,T.W. and Zervas,N.T., 1982, J. Neurosurg. 56,777.

I.-J

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Jamsen, C.M.J., Schiffers, B.M.H. and De Mey, J.G.R. Deporrmenr of Pkrmacalogy,

University of Limburg

P.O. Box dlQ,6200 MD Muawicht, The IVefherlunL

organ culture of isolated renal arteries induces expression of relaxing responses to i~proterenol {De Mey et al. Circ. l&s. 65: 112%1135, 1989). In this study we attempted to identify the biochemical changes that underly this pharmacological alteration. The experiments were performed in segments of carotid (CA) and renal arteries (RA) that hd been isolated from adult Wistar-Kyoto rats and chemically sympathectomized. In freshly isolated CA and RA, ~~p~ote~no~and forskolin stimulated the pr~uction of CAMP. In freshly isolated CA, that had been made to