FUNCTIONAL MUSCARINIC RECEPTORS IN CULTURED SKELETAL-MUSCLE

We studied the influence of muscarinic and nicotinic stimulation on bo th phosphoinositide metabolism and intracellular calcium levels in rat skeletal muscle primary cultures. Both nicotine and muscarine induced an increase in cytosolic calcium measured by flue 3 fluorescence in c onfocal microscopy. The mass of inositol (1,4,5)trisphosphate measured by radioreceptor assay rose 2- to 3.5-fold upon carbachol, nicotine, or muscarine stimulation. The muscarine effect was mimicked by oxotrem orine-M; pirenzepine prevented the muscarine-induced inositol (1,4,5)t risphosphate increase, whereas 4-diphenylacetoxy-N-methyl piperidine m ethiodide was ineffective. A relatively small (40 fmol/mg protein) hig h-affinity 3-quinuclidinylbenzilate binding to rat myotube microsomes was consistent with the muscarinic effect found. On the other hand, th e effect of nicotine on the mass of inositol (1,4,5)trisphosphate was totally suppressed in sodium-free medium. Expression of M(1) muscarini c receptors coupled to phospholipase C and to internal calcium stores in cultured skeletal muscle is proposed; nicotinic receptors could be acting via ion fluxes and membrane depolarization. (C) 1996 Academic P ress, Inc.