title: 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.
abstract: Phospholipase C-beta (PLC-beta) signalling via protein kinase C (PKC) has been recognized as a major route by which stimuli such as alpha1-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-beta to 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(-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 [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- 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 [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(-4)M) inhibited ET-1 and PMA stimulated [3H]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-talk with PLD via translocation of PKC-epsilon. These signals are possibly linked to the hypertrophic response.
authors.affiliation: Department of Biochemistry, Cardiovascular Research Institute COEUR, Faculty of Medicine & Health Sciences, Erasmus University Rotterdam, P.O. Box 1738, Rotterdam, 3000 DR, The Netherlands.
authors.forename: Y E
authors.initials: YE
authors.lastname: Eskildsen-Helmond
authors.forename: K
authors.initials: K
authors.lastname: Bezstarosti
authors.forename: D H
authors.initials: DH
authors.lastname: Dekkers
authors.forename: H A
authors.initials: HA
authors.lastname: van Heugten
authors.forename: J M
authors.initials: JM
authors.lastname: Lamers
mesh_terms: D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D006332:Cardiomegaly / Q000378:metabolism*; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D015536:Down-Regulation; D019332:Endothelin-1 / Q000494:pharmacology; D006657:Histones / Q000378:metabolism; D015151:Immunoblotting; D007527:Isoenzymes / Q000187:drug effects / Q000378:metabolism*; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D010656:Phenylephrine / Q000494:pharmacology; D054799:Phospholipase C beta; D010739:Phospholipase D / Q000187:drug effects / Q000378:metabolism*; D014176:Protein Biosynthesis; D011493:Protein Kinase C / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D011506:Proteins / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010738:Type C Phospholipases / Q000187:drug effects / Q000378:metabolism*
chemical_list: D016895:Culture Media, Serum-Free; D019332:Endothelin-1; D006657:Histones; D007527:Isoenzymes; D011506:Proteins; D000804:Angiotensin II; D010656:Phenylephrine; D011493:Protein Kinase C; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate