EFFECTS OF PHORBOL ESTER ON CONTRACTION, INTRACELLULAR PH AND INTRACELLULAR CA2+ IN ISOLATED MAMMALIAN VENTRICULAR MYOCYTES

1. We have investigated the actions of certain phorbol esters on the intracellular pH, intracellular Ca2+ and contractility of isolated rat and guinea-pig cardiac myocytes. Intracellular pH was measured using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and intracellular Ca2+ was measured using Fura-2. 2. Application of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (also called phorbol 12-myristate 13-acetate) (TPA) (which activates protein kinase C) to rat cardiac myocytes significantly increased cell shortening by 116 +/- 34% (n = 8) (p < 0.02). The rate of change of cell length during contraction (i.e. + dL/dt) increased from 67.2 +/- 8.7-mu-m/s to 127.7 +/- 14.1-mu-m/s (n = 7). The rate of change of cell length during relaxation (-dL/dt) increased from 55.8 +/- 7.4-mu-m/s to 118.9 +/- 12.1-mu-m/s (n = 7). Time to peak shortening was unchanged. 3. Application of 4-alpha-phorbol 12,13-didecanoate, which does not activate protein kinase C, did not affect rat myocyte contractility. An insignificant decrease in contractility (by 7.5 +/- 7.5%) was observed (n = 5). The positive inotropic effect of TPA may therefore be evoked through an activation of protein kinase C. 4. In rat myocytes we have measured the changes of pH(i) and contractility (cell shortening) during an alkalosis and acidosis induced by exposure to and subsequent removal of NH4Cl both in the presence and absence of TPA. Recovery times from an acid load were significantly (p < 0.05) enhanced by 15.1 +/- 6.9% (n = 13) in the presence of TPA. Recovery times of cell shortening were also more rapid (p < 0.05) by an average of 59.1 +/- 10.6% (n = 5) in the presence of TPA. Recovery times were unchanged in the presence of 4-phorbol 12,13-didecanoate (which does not activate protein kinase C). 5. Since pH(i) recovery of an isolated myocyte from an acid load is partially inhibited by the presence of 1 mM-amiloride and inhibited by removing extracellular Na+ then it is suggested that, like pH(i) regulation in sheep heart Purkinje fibres, pH(i) recovery in rat cardiac ventricular myocytes is mainly through sarcolemmal Na+-H+ exchange. We suggest that in the presence of TPA the Na+-H+ exchange is stimulated. 6. The relationship between pH(i) and cell shortening is non-linear as has been observed by others in whole tissue preparations. The presence of TPA shifts the relationship upwards such that at any one pH(i), cell shortening is greater. 7. Addition of the phorbol ester did not change steady-state pH(i) so the positive inotropic effect cannot simply be due to pH(i) becoming more alkaline. 8. In rat and guinea-pig myocytes TPA increased peak systolic [Ca2+] but did not alter resting (diastolic) [Ca2+]. Fura-2 ratios increased by 21 +/- 4% (n = 5). We conclude that some of the positive inotropic effect produced by phorbol esters which activate protein kinase C is caused by an increase in systolic Ca2+.