Phorbol ester and dioctanoylglycerol stimulate membrane association of protein kinase C and have a negative inotropic effect mediated by changes in cytosolic Ca2+ in adult rat cardiac myocytes

Abstract

We used left ventricular myocytes from adult rats to investigate the effect of 4β-phorbol 12-myristate 13-acetate (PMA) and of sn-1,2-dioctanoylglycerol (DiC-8) on the membrane association of protein kinase C (PKC), cytosolic [Ca2+], (Ca(i)) homeostasis, and the contractile properties of single cardiac cells. Because PKC activity is known to be highly Ca2+ sensitive, the K+ concentration of the bathing medium was raised from 5 to 30 mM in some experiments, a perturbation known to depolarize the cell and increase Ca(i). In cell suspensions both PMA (3 x 10-10 and 3 x 10-7 M) and DiC-8 (10-5 and 10-4 M) increased membrane association of PKC. The effect of PMA (10-7 M) on PKC translocation was enhanced in 30 mM KCl compared with 5 mM KCl. During steady field stimulation at 1 Hz in 1 mM bathing [Ca2+], both PMA (10-7 M) and DiC-8 (10-5 M) decreased twitch amplitude to approximately 60% of control in 5 mM KCl, and the negative inotropic effect of either drug was more pronounced in 30 mM KCl than in 5 mM KCl. In single cardiac myocytes loaded with the Ca2+ indicator indo-1 and bathed in 5 mM KCl, we simultaneously measured cell length and Ca(i). The myofilament responsiveness to Ca2+ was assessed by the relation between contraction amplitude and the peak of the Ca(i) transient. The negative inotropic effect of both PMA and DiC-8 was related to a diminished amplitude of the Ca(i) transient and not to a decreased myofilament responsiveness to Ca2+. In the absence of electrical stimulation, PMA (10-7 M) and DiC-8 (10-5 M) decreased the frequency of contractile waves due to spontaneous Ca2+ release from the sarcoplasmic reticulum, and DiC-8 also decreased resting Ca(i). Thus, activation of PKC, which is thought to occur as part of the response of cardiac muscle to α1-adrenergic stimulation, is associated with a negative inotropic action due to a smaller Ca(i) transient rather than to a decrease in the myofilament responsiveness to Ca2+. These effects on the membrane association of PKC and on contractility are enhanced by cell depolarization achieved by raising [KCl] in the bathing medium.