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 ☆

Abstract Phospholipase C- β (PLC- β ) signalling via protein kinase C (PKC) has been recognized as a major route by which stimuli such as α 1 -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 −8 m ), phenylephrine (PHE, 10 −5 m ) or Ang II (10 −7 m ) resulted in a rapid (0–10 min) activation of PLC- β to an extent (ET-1>PHE>Ang II) that correlated with the magnitude of stimulation of protein synthesis ([ 3 H]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 [ 3 H]choline formation from labelled PtdCho after a lag-phase of about 10 min. That this [ 3 H]choline formation was due to the action of PLD was confirmed by measurement of phosphatidylgroup-transfer from cellular [ 14 C]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- α , - δ and - ξ immunoreactivities was observed. PMA caused translocation of PKC- α , PKC- e as well as PKC- δ . Cellular redistribution of PKC activity measured by [ 32 P]-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 [ 3 H]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- β and after a lag-phase PLD, whereas PHE and Ang II activate only PLC- β . PLC- β stimulated by ET-1, may cross-talk with PLD via translocation of PKC- e . These signals are possibly linked to the hypertrophic response.