title: Roles of protein kinase C, Ca2+, Pyk2, and c-Src in agonist activation of rat lacrimal gland p42/p44 MAPK.
abstract: OBJECTIVE
  Although p42/p44 mitogen-activated protein kinase (MAPK) negatively modulates protein secretion stimulated by cholinergic and alpha(1D)-adrenergic agonists, it does not play a role in epidermal growth factor (EGF)-stimulated protein secretion. Therefore, this study was conducted to determine the roles that protein kinase C (PKC), intracellular Ca(2+) ([Ca(2+)](i)), and nonreceptor tyrosine kinases Pyk2 and Src play in the activation of agonist- and EGF-stimulated MAPK activation.
  
  
  METHODS
  Lacrimal gland acini were isolated by collagenase digestion and incubated with phorbol 12-myristate 13-acetate (PMA) to activate PKC or ionomycin, a Ca(2+) ionophore. Acini were preincubated with the PKC inhibitors calphostin C or Ro-31-8220, EGTA to chelate Ca(2+), or the c-Src inhibitor PP1 before stimulation with the cholinergic agonist carbachol, the alpha(1D)-adrenergic agonist phenylephrine, or EGF. Activated MAPK, Pyk2, and c-Src amounts were measured by Western blot analysis.
  
  
  RESULTS
  PMA and ionomycin significantly increased the activation of MAPK in a time- and concentration-dependent manner. Inhibition of PKC partially inhibited carbachol-stimulated MAPK activation while completely inhibiting phenylephrine- and EGF-stimulated MAPK activation. Chelation of Ca(2+) also partially inhibited carbachol-stimulated MAPK with no effect on phenylephrine- and EGF-stimulated MAPK activation. Carbachol increased the phosphorylation of Pyk2 on tyrosine 402 and c-src on tyrosine 416 in a time-dependent manner. The c-src inhibitor PP1 inhibited carbachol-stimulated phosphorylation of Pyk2.
  
  
  CONCLUSIONS
  It was concluded that cholinergic agonists use Ca(2+) and PKC to phosphorylate Pyk2 and c-Src, which subsequently stimulate MAPK activity. In contrast, alpha(1D)-adrenergic agonists and EGF do not use Pyk2 and Src but do use PKC to activate MAPK.
authors.affiliation: Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA. hodges@vision.eri.harvard.edu
authors.forename: Robin R
authors.initials: RR
authors.lastname: Hodges
authors.forename: Jose D
authors.initials: JD
authors.lastname: Rios
authors.forename: Joanna
authors.initials: J
authors.lastname: Vrouvlianis
authors.forename: Isao
authors.initials: I
authors.lastname: Ota
authors.forename: Driss
authors.initials: D
authors.lastname: Zoukhri
authors.forename: Darlene A
authors.initials: DA
authors.lastname: Dartt
mesh_terms: D000316:Adrenergic alpha-Agonists / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000502:physiology*; D002217:Carbachol / Q000494:pharmacology; D018679:Cholinergic Agonists / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D051418:Focal Adhesion Kinase 2 / Q000502:physiology; D016391:Genes, src / Q000502:physiology*; D015759:Ionomycin / Q000494:pharmacology; D007765:Lacrimal Apparatus / Q000187:drug effects* / Q000201:enzymology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010656:Phenylephrine / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology; D011494:Protein Kinases / Q000502:physiology*; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors
chemical_list: D000316:Adrenergic alpha-Agonists; D018679:Cholinergic Agonists; D004791:Enzyme Inhibitors; D010656:Phenylephrine; D015759:Ionomycin; D004815:Epidermal Growth Factor; D002217:Carbachol; D011494:Protein Kinases; D051418:Focal Adhesion Kinase 2; D011493:Protein Kinase C; D048049:Extracellular Signal-Regulated MAP Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium