Cross-talk between receptor-mediated phospholipase C-beta and D via protein kinase C as intracellular signal possibly leading to hypertrophy in serum-free cultured cardiomyocytes

Phospholipase C-beta (PLC-beta) signalling via protein kinase C (PKC) has been recognized as a major route by which stimuli such as alpha(1)-adrenergic agonists, endothelin-l (ET-1) and angiotensin IT (Ang LI) induce hypertrophy of myocytes, The goal of this study was to evaluate the role of phospholipase D (PLD) in contributing to the formation of the PI(C activator 1,2-diacylglycerol (1,2-DAG) and to study the mechanism(s) of PLD activation by agonists. Stimulation of serum-free cultured neonatal rat cardiomyocytes with ET-1 (10(-8) M), phenylephrine (PHE, 10(-5) M) or Ang lI (10(-7) M) resulted in a rapid (0-10 min) activation of PLC-beta to an extent (ET-1>PHE>Ang II) that correlated with the magnitude of stimulation of protein synthesis ([H-3]leucine incorporation into protein) measured after 24 h. Phorbol 12-myristate 13-acetate (PMA, 10(-6) M) and ET-1 were equipotent in stimulating protein synthesis. ET-1 and PMA, but not PHE and Ang II stimulated [3H]choline formation from labelled PtdCho after a lag-phase of about 10 min. That this [H-3]choline formation was due to the action of PLD was confirmed by measurement of phosphatidylgroup-transfer from cellular [C-14]palmitoyl-phosphatidylcholine to exogenous ethanol. ET-1 and PHE, to much lesser extent, produced a rapid (0-5 min) translocation of PKC-E immunoreactivity from the cytosol to the membrane fraction, whereas no intracellular redistribution of PKC-alpha, -delta and -xi immunoreactivities was observed, PMA caused translocation of PKC-alpha, PKC-epsilon as well as PKC-delta. Cellular redistribution of PKC activity measured by [P-32]-incorporation into histone III-S was not observed with ET-1 and PHE, but only with PMA stimulation, Down-regulation of PKC isozymes by 24 h pretreatment of cells with PMA or blockade of PKC by chelerythrine (10(-4) M) inhibited ET-1 and PMA stimulated [H-3]choline production, Staurosporine (10(-6) M) had, however, no effect. In conclusion, the results indicate that in serum-free cultured cardiomyocytes, ET-1 initially activates PLC-beta and after a lag-phase PLD, whereas PHE and Ang II activate only PLC-beta. PLC-beta stimulated by ET-1, may cross-tall( with PLD via translocation of PKC-epsilon. These signals are possibly linked to the hypertrophic response. (C) 1997 Academic Press Limited.