EFFECTS OF PHORBOL ESTER ON GAP-JUNCTIONS OF NEONATAL RAT-HEART CELLS

Myocytes were isolated from the ventricles of neonatal rat hearts and cultured for 1-3 days. Newly formed cell pairs were used to examine the conductance of gap junctions, g(j). Measurements were performed using a dual voltage-clamp method in conjunction with a whole-cell, tight-seal recording. Exposure to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, 100-160 nM) led to a decrease in g(j). Single-channel events recorded immediately before complete uncoupling yielded a single-channel conductance, gamma(j), of 40.5 pS, implying that TPA affects the channel kinetics rather than gamma(j). TPA-induced uncoupling was observed at subphysiological levels of cytosolic Ca2+ (pipette solution = 18 nM), not at physiological levels (pipette solution = 170 nM). The effects of TPA could not be mimicked by 250 muM 1-oleoyl-2-acetyl-glycerol (OAG). Preincubation with TPA (up to 24 h) revealed no changes in g(j) attributable to down-regulation of protein kinase C, PKC. Pretreatment with PKC inhibitors, staurosporine or PKCI, prevented the TPA-dependent decrease in g(j). TPA-dependent uncoupling was not impaired by 4-bromophenacyl bromide, an inhibitor of phospholipase A2, PLA2; conversely, an arachidonic acid-dependent decrease in g(j) was not prevented by PKCI. This suggests that g(j) regulation does not involve an interaction between PLA2 and PKC.