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Muscarinic receptors and calcium signalling in rabbit isolated pulmonary artery smooth muscle cells

O'Neill, G.T. and Rowan, E.G. and Gurney, A.M. (2002) Muscarinic receptors and calcium signalling in rabbit isolated pulmonary artery smooth muscle cells. British Journal of Pharmacology, 137 (Suppl.). 24P-24P. ISSN 0007-1188

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Abstract

Acetylcholine (ACh) is an endothelium-dependent vasodilator, but when pulmonary artery endothelium is damaged it causes vasoconstriction through a direct action on smooth muscle muscarinic receptors (Dinh-Xuan et al., 1989). In rabbit main pulmonary artery this action involves M2 and M3 receptors The muscarinic agonist, oxotremorine sesquifumarate (Oxo-S), also contracts this preparation, but the effects appear to be mediated solely through M2 receptors This study characterised responses to ACh and Oxo-S in isolated pulmonary artery smooth muscle cells (PASMC) by monitoring changes in the fluorescence of the Ca2+ indicator, fluo-4. Arteries were obtained from male New Zealand rabbits (2-2.5 kg) killed by lethal injection of sodium pentobarbitone (140 mg kg-' i.v.). PASMC were isolated by enzymatic digestion, loaded with 1 jM fluo-4 at 221C for 45 min, washed and bathed in physiological salt solution (PSS). Fluorescence was excited at 488 nm and measured between 503 and 553 nm using a confocal microscope. Calcium signals were quantified as the change in fluorescence (AF) relative to the background fluorescence (F0). Ca2+-free solution was prepared by replacing CaCl2 in the PSS with equimolar MgC12 and adding 5mM EGTA. To study Gi involvement, PASMC were incubated with pertussis toxin (PTX; 5gig ml-') for 3-4 hr at 22°C and compared with control cells treated in the same way, but without exposure to PTX. Experiments were performed 22°C. Results are expressed as mean ± s.e.m.