Opioid Effects on Contractility, Ca²⁺-transients and Intracellular pH in Cultured Cardiac Myocytes

Morphine, the opioid-agonist, and the antagonist naloxone and levallorphan exerted direct effects on spontaneously-contracting cultures of cardiac myocytes from neonatal rats. Naloxone and levallorphan induced an increase in the amplitude of systolic cell motion (ASM) and in the size of [Ca²⁺],-transients, measured as indo-1 fluorescence ratio (IFR), whereas morphine caused an increase in IFR with no change in ASM. Both morphine and naloxone caused a transient increase in ⁴⁵Ca²⁺ influx into the cardiomyocytes. Analysis of the relationship between changes in ASM and IFR indicated dual action of the drug: (a) An increase in [Ca²⁺]-transients elicited by morphine and the antagonists, apparently resulting from a transient increase of Ca²⁺ influx. (b) Altered myofibril responsiveness to Ca²⁺; the agonists decreased it, and the antagonists increased it. Intracellular pH_i measurements in cardiomyocytes loaded with the fluorescent indicator BCECF revealed that morphine caused acidosis and the antagonists caused alkalosis. These pH changes were inhibited by pertussis-toxin, protein kinase inhibitor K323a, phorbol-ester and ethylisopropyl-amiloride, indicating pathways mediated by GTP-binding proteins and altered activities of protein kinase C and Na⁺/H⁺ exchanger. Preincubation with pertussis toxin prior to the addition of morphine prevented the decrease in the myofibril responsiveness to Ca²⁺ as well as the decrease in pH_i but did not affect the increase in [Ca²⁺]-transients and the increase in the rate of Ca²⁺ influx. As a result, addition of morphine after preincubation with pertussis toxin caused a positive inotropic effect. Our results indicate that morphine acts by two different pathways distinguishable by their sensitivity to pertussis toxin (1), increases Ca²⁺ influx leading to increased Ca²⁺-transients and (2) decreased intracellular pH leading to reduced myofibril responsiveness to Ca²⁺.