Stimulation of muscarinic receptors raises free intracellular Ca2+ concentration in rat ventricular myocytes

Abstract

The effect of carbachol on free intracellular calcium concentration, ([Ca2+](i)) and on intracellular hydrogen concentration (pH(i)) was determined from fluorescence signals obtained from rat ventricular myocytes. Application of carbachol (300 μmol/l) to quin2-loaded myocytes bathed in 2 mmol/l Ca2+-containing solution caused [Ca2+](i) to increase within 7-10 minutes from 182±9 to 212±11 nmol/l (n=4). Carbachol acted via stimulation of muscarinic receptors because atropine (1 μmol/l) either prevented or abolished the increase in [Ca2+](i). Carbachol also produced a positive inotropic effect in rat papillary muscles contracting isometrically at a frequency of 0.5 Hz and enhanced contracture in resting preparations in the presence of high extracellular Ca2+ concentration ([Ca2+]0) (20 mmol/l). The effect of carbachol on [Ca2+](i) was dependent on [Ca2+]0. In the presence of 10 mmol/l [Ca2+]0, the increase in [Ca2+](i) was about two times that elicited by carbachol when bath [Ca2+]0 was 2 mmol/l. Reduction of [Ca2+]0 to 50 μmol/l abolished the carbachol effect but did not prevent caffeine-induced Ca2+ release. The carbachol-induced rise in [Ca2+](i) remained unchanged in the presence of either 10 mmol/l caffeine or 1 μmol/l ryanodine. In the absence of extracellular Na+ concentration ([Na+]0), carbachol no longer produced an increase in [Ca2+](i) of cardiomyocytes and failed to enhance Na+-withdrawal contracture of the rat papillary muscle. In contrast to the effect on [Ca2+](i), carbachol did not produce any change in pH(i) as determined from fluorescence signals obtained from rat ventricular myocytes loaded with 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. The results demonstrate that activation of muscarinic receptors in ventricular myocardium is associated with an increase in [Ca2+](i), which is 1) dependent on [Ca2+]0, 2) not the result of Ca2+ release from caffeine- and ryanodine-sensitive intracellular stores, 3) abolished in [Na+]0-free solution, and 4) not dependent on pH(i). From both these results and recent work showing that carbachol increases intracellular Na+ activity (a(Na)(i)) in papillary muscle of the guinea pig (Korth M, Kuhlkamp V: Muscarinic receptor-mediated increace of intracellular Na+-ion activity and force of contraction. Pflugers Arch 1985;403:266-272), we conclude that the source of Ca2+ that contributes to the carbachol-induced [Ca2+](i) increase is from extracellular medium. Stimulation of muscarinic receptors probably leads first to an increase in a(Na)(i) and then to an increase in [Ca2+](i) via Na+-Ca2+ exchange.