Role of protein kinase C-epsilon in the development of κ-opioid receptor tolerance to U50,488H in rat ventricular myocytes

1 The role of protein kinase C-epsilon (PKC-epsilon) in the development Of K-opioid receptor (K-OR) tolerance to the effects of trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[I-pyrrolidinyl]cyclohexyl) (U50,488H), the selective agonist of kappa-OR, was determined in rat ventricular myocytes. 2 Incubation of ventricular myocytes with 1 mum U50,488H for 24 h significantly attenuated the inhibitory effects of 30 muM U50,488H on the electrically-induced [Ca2+](i) transient and forskolin-stimulated cyclic AMP accumulation, indicating the development of tolerance to the K-OR agonist. Chronic treatment of ventricular myocytes with U50,488H also induced translocation of PKC-epsilon to the particulate fraction. On the other hand, administration of 30 um U50,488H for 10 min induced translocation of PKC-alpha to the particulate fraction in naive ventricular myocytes, but not in cells pretreated with I muM U50,488H for 24 h. 3 In ventricular myocytes incubated for 24 h with 1 mum U50,488H together with I pm chelerythrine or 1 muM GF109203X, PKC inhibitors, or 0.1 muM epsilonVI-2 peptide, a selective inhibitor of PKC-epsilon, 30 muM U50,488H still produced the inhibitory effect on the electrically-induced [Ca2+](i) transient as it did in naive ventricular myocytes. Chronic treatment of ventricular myocytes with U50,48814 and chelerythrine also attenuated the development of tolerance to acute U50,488H on cyclic AMP accumulation. Cells exposed to chelerythrine, GF109203X, or epsilonVI-2 peptide alone did not show an altered [Ca2+](i) response to U50,488H. 4 These results indicate that activation of PKC-epsilon is a critical step in the development of tolerance in the kappa-OR.