title: Platelet-derived growth factor induces cellular growth in cultured chick ventricular myocytes.
abstract: OBJECTIVE
  Platelet-derived growth factor (PDGF) stimulates growth in various types of cells, but little is known about its effect on cardiac myocytes. Therefore, we examined whether PDGF had a direct effect on cardiac myocytes and investigated their intracellular signaling pathways.
  
  
  METHODS
  A primary culture of chick embryonic (Hamburger and Hamilton stage 36) ventricular myocytes was prepared. Cellular growth was estimated by 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide assay and 5-bromo-2'-deoxyuridine incorporation assay. The number of PDGF binding sites was measured by binding assay. Induction of c-fos mRNA was analyzed by Northern blot analysis. The binding activity of activator protein (AP)-1 was examined by electrophoretic mobility shift assay. The activation of mitogen-activated protein kinase (MAPK) and signal transducers and activators of transcription (STATs) was analyzed by Western blot analysis, immunoprecipitation, and immunocytochemistry. Furthermore, intracellular Ca2+ concentration ([Ca2+]i) was measured with indo-1 and L-type Ca(2+)- channel current (ICa) was recorded with the patch clamp technique.
  
  
  RESULTS
  PDGF-AB and -BB, but not PDGF-AA, increased viable cell number (5 ng/ml of PDGF-AA, -AB, -BB: 101 +/- 4%, 115* +/- 4%, 122* +/- 4%, respectively, n = 4, *P < 0.05) and DNA synthesis (104 +/- 11%, 202* +/- 18%, 295* +/- 25%, respectively, n = 4, *P < 0.05). Scatchard analysis demonstrated that the maximal number of PDGF-AA, -AB, -BB binding sites was 5 +/- 1, 63 +/- 12, 126 +/- 24 fmol/10(6) cells, respectively. PDGF-BB provoked induction of c-fos mRNA and increases in binding activity to the AP-1 site. PDGF-BB also induced tyrosine phosphorylation and nuclear translocation of MAPK. The c-fos induction, the increased AP-1 binding activity and the acceleration of DNA synthesis were all attenuated by genistein (100 microM) or MAPK kinase inhibitor (10 or 50 microM PD98059). Interestingly, protein kinase C inhibitor (250 nM calphostin C) attenuated the increases of AP-1 binding activity to some extent, but did not inhibit the c-fos induction at all. The phosphorylation states of STATs were not significantly affected by PDGF-BB. PDGF-BB did not alter [Ca2+]i or ICa.
  
  
  CONCLUSIONS
  We conclude that PDGF can exert direct effects on embryonic cardiac myocytes and induce their growth. MAPK cascade may play an important role in the PDGF-induced embryonic myocardial growth.
authors.affiliation: Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Shimizu
authors.forename: K
authors.initials: K
authors.lastname: Kinugawa
authors.forename: A
authors.initials: A
authors.lastname: Yao
authors.forename: Y
authors.initials: Y
authors.lastname: Sugishita
authors.forename: K
authors.initials: K
authors.lastname: Sugishita
authors.forename: K
authors.initials: K
authors.lastname: Harada
authors.forename: H
authors.initials: H
authors.lastname: Matsui
authors.forename: O
authors.initials: O
authors.lastname: Kohmoto
authors.forename: T
authors.initials: T
authors.lastname: Serizawa
authors.forename: T
authors.initials: T
authors.lastname: Takahashi
mesh_terms: D000818:Animals; D001665:Binding Sites; D015152:Blotting, Northern; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002478:Cells, Cultured; D002642:Chick Embryo; D016762:Genes, fos; D007150:Immunohistochemistry; D009206:Myocardium / Q000166:cytology*; D018408:Patch-Clamp Techniques; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D015398:Signal Transduction / Q000187:drug effects*; D018808:Transcription Factor AP-1 / Q000378:metabolism
chemical_list: D010982:Platelet-Derived Growth Factor; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium