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

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.