Positive and negative inotropic effects of phorbol 12-myristate 13-acetate: Relationship to PKC-dependence and changes in [Ca 2+] i

Abstract

The present study examined the concentration-dependent effects of phorbol 12-myristate 13-acetate (PMA), a PKC-activating phorbol ester, on contractile force and [Ca 2+] i in guinea-pig hearts and isolated cardiac myocytes, respectively. Contractile force was measured using isolated Langendorff-perfused hearts while [Ca 2+] i was measured independently in isolated cardiac myocytes loaded with fura2-AM. Phorbol 12-myristate 13-acetate, as well as another PKC-activating phorbol, phorbol dibutyrate (PDBu), and two non-PKC-activating phorbols, α-phorbol didecanoate (αPDD) and 4α-phorbol, exerted time- and concentration-dependent effects on contractility. A significant positive inotropic response was observed with either PMA (10 −12 m; 5–15 min of perfusion) or PDBu (10 −12 m; 5 min of perfusion). In contrast, 10 −10 m PMA caused a significant negative inotropic effect following 30 min of perfusion while 10 −8 m PMA produced a significant negative inotropic effect which occurred earlier (10 min) and was sustained throughout the 30 min perfusion period. A similar negative inotropic effect was seen with 10 −8 m of either PDBu or αPDD. In addition, 4α-phorbol (10 −8 m) exerted a modest, but significant negative inotropic effect following 25 and 30 min of perfusion. Both concentration-dependent increases and decreases of + dF dt and − dF dt were observed in the presence of PMA. In addition, both PMA and PDBu caused a concentration-dependent increase in coronary perfusion pressure. The positive inotropic responses and coronary perfusion pressure effects elicited by PMA and PDBu were largely prevented by the addition of the PKC inhibitors H7 (6 n m) or HAG (10 n m); however, these drugs were without effect on the negative inotropic response to higher concentrations of both PKC-activating (PMA, PDBu) and non-PKC-activating (αPDD, 4α-phorbol) phorbol compounds. The lowest concentration of either PMA or PDBu (10 −12 m) increased the 340 380 fluorescence ratio of isolated cardiac myocytes loaded with fura2-AM on a time scale similar to that at which the positive inotropic response was seen in the whole heart. However, in contrast to results in the isolated heart, PDBu elicited a greater and sustained increase in the fluorescence ratio measured in isolated cardiac myocytes. The higher concentration of either PMA or PDBu (10 −8 m), resulted in a decrease in the 340 380 ratio. The results of this study demonstrate that the phorbol esters, PMA and PDBu, elicit concentration-dependent positive and negative inotropic responses in isolated perfused hearts which are associated with a parallel increase and decrease in the 340 380 fluorescence ratio in isolated cardiac myocytes, respectively. Therefore, it is likely that both PKC-dependent and PKC-independent actions of phorbol esters on myocardial contractile activity are related to changes in the intracellular Ca 2+ concentration.