SIGNALING MECHANISMS UNDERLYING MUSCARINIC RECEPTOR-MEDIATED INCREASEIN CONTRACTION RATE IN CULTURED HEART-CELLS

We have investigated the mechanisms by which stimulation of cardiac mu scarinic receptors result in paradoxical stimulatory effects on cardia c function, using cultured neonatal rat ventricular myocytes as a mode l system. Application of low concentrations of carbachol (CCh) (EC50 = 35 nM) produced an atropine-sensitive decrease in spontaneous contrac tion rate, while, in cells pretreated with pertussis toxin, higher con centrations of CCh (EC50 = 26 mu M) elicited an atropine-sensitive inc rease in contraction rate. Oxotremorine, an m(2) muscarinic acetylchol ine receptor (mAChR) agonist, mimicked the negative but not the positi ve chronotropic response to CCh, Reverse transcription followed by pol ymerase chain reaction carried out on mRNA obtained from single cells indicated that ventricular myocytes express mRNA for the m(1), m(2), a nd, possibly, m(4) mAChRs, The presence of m(1) and m(2) mAChR protein on the surface membranes of the cultured ventricular myocytes was con firmed by immunofluorescence, The CCh-induced positive chronotropic re sponse was significantly inhibited by fluorescein-tagged antisense oli gonucleotides directed against the m(1), but not the m(2) and m(4), mA ChR subtypes, The response was also inhibited by antisense oligonucleo tides against G(q)alpha protein. Finally, inhibition of CCh-induced ph osphoinositide hydrolysis with 500 mu M neomycin or 5 mu M U73122 comp letely abolished the CCh-induced positive chronotropic response. These results are consistent with the stimulatory effects of mAChR activati on on the rate of contractions in cultured ventricular myocytes being mediated through the m(1) mAChR coupled through G(q) to phospholipase C-induced phosphoinositide hydrolysis.