Muscarinic receptor-mediated increase in cytoplasmic free Ca2+ in isolated bovine adrenal medullary cells

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Abstract
The change in cytoplasmic free calcium, [Ca2+]iin isolated bovine adrenal medullary cells during stimulation by acetylcholine (ACh) in Ca2+-free incubation medium was measured using the fluorescent Ca2+ indicator quin2. ACh (1–100 μM) caused an increase in [Ca2+]i by mobilization of Ca2+ from the intracellular pool. Nicotine (10 μM) did not increase [Ca2+]iin the absence of extracellular Ca2+. Pretreatment of the cells with atropine (10 μM) completely inhibited ACh-induced increase in [Ca2+]i, whereas pretreatment with hexamethonium (100 μM) did not. The intracellular Ca2+ antagonist 8-(N,N-diethylamino)octyl-3,4,5trimethoxybenzoate (TMB-8), inhibited ACh-induced increase in [Ca2+]i. The activator of protein kinase C 12-O-tetradecanoylphorbol-13-acetate (TPA), but not its ‘inactive’ analog 4α-phorbol-12,13-didecanoate (PDD), also inhibited ACh-induced increase in [Ca2+]i. These findings suggest that in bovine adrenal medullary cells, stimulation of muscarinic ACh receptor causes an increase in [Ca2+]i by mobilizing Ca2+ from the intracellular pool and that protein kinase C is involved in ‘termination’ or ‘down regulation’ of this response.