Cross-talk Between Receptor-mediated Phospholipase C-βand D via Protein Kinase C as Intracellular Signal Possibly Leading to Hypertrophy in Serum-free Cultured Cardiomyocytes

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Phospholipase C-β(PLC-β) signalling via protein kinase C (PKC) has been recognized as a major route by which stimuli such asα1-adrenergic agonists, endothelin-1 (ET-1) and angiotensin II (Ang II) 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 PKC 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 (10−8m), phenylephrine (PHE, 10−5m) or Ang II (10−7m) resulted in a rapid (0–10 min) activation of PLC-βto an extent (ET-1>PHE>Ang II) that correlated with the magnitude of stimulation of protein synthesis ([3H]leucine incorporation into protein) measured after 24 h. Phorbol 12-myristate 13-acetate (PMA, 10−6m) 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 [3H]choline formation was due to the action of PLD was confirmed by measurement of phosphatidylgroup-transfer from cellular [14C]palmitoyl-phosphatidylcholine to exogenous ethanol. ET-1 and PHE, to much lesser extent, produced a rapid (0–5 min) translocation of PKC-εimmunoreactivity from the cytosol to the membrane fraction, whereas no intracellular redistribution of PKC-α, -δand -ξimmunoreactivities was observed. PMA caused translocation of PKC-α, PKC-εas well as PKC-δ. Cellular redistribution of PKC activity measured by [32P]-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 (10−4m) inhibited ET-1 and PMA stimulated [3H]choline production. Staurosporine (10−6m) had, however, no effect. In conclusion, the results indicate that in serum-free cultured cardiomyocytes, ET-1 initially activates PLC-βand after a lag-phase PLD, whereas PHE and Ang II activate only PLC-β. PLC-βstimulated by ET-1, may cross-talk with PLD via translocation of PKC-ε. These signals are possibly linked to the hypertrophic response.