title: Signal transduction pathways used by EGF to stimulate protein secretion in rat lacrimal gland.
abstract: OBJECTIVE
  To determine the effects of epidermal growth factor (EGF) on lacrimal gland secretion of proteins and characterize its signal-transducing components.
  
  
  METHODS
  Both exorbital lacrimal glands were removed from male Sprague-Dawley rats. Dispersed acini were isolated by collagenase digestion in Krebs-Ringer bicarbonate (KRB) buffer at 37 degrees C. Acini were incubated with EGF (10(-7) M), the cholinergic agonist carbachol (10(-4) M), or the alpha(1)-adrenergic agonist phenylephrine (10(-4) M), and peroxidase secretion was measured by a fluorescence assay. To measure intracellular calcium ([Ca(2+)](i)), acini were incubated in fura-2 tetra-acetoxymethyl ester for 60 minutes at 22 degrees C, and fluorescence was measured at 340 and 380 nm with an emission wavelength of 505 nm. Extracellular Ca(2+) was chelated with KRB-BSA without CaCl(2) and with 2 mM EGTA before measurement of peroxidase secretion. Protein kinase C (PKC) was downregulated by incubating acini overnight, with or without the phorbol ester, phorbol 12-myristate 13-acetate (PMA; 10(-6) M), and peroxidase secretion was measured.
  
  
  RESULTS
  EGF-stimulated peroxidase secretion in a concentration-dependent manner with a significant increase at 10(-7) M. EGF-stimulated secretion was inhibited by the EGF receptor (EGFR) inhibitor AG1478, but not by the phosphoinositide-3 kinase inhibitor LY292004 or the mitogen-activated kinase kinase (MEK) inhibitor U0126. EGF increased [Ca(2+)](i), whereas chelation of extracellular Ca(2+) inhibited EGF-induced peroxidase secretion by 90%. Downregulation of PKC also inhibited EGF-stimulated peroxidase secretion.
  
  
  CONCLUSIONS
  EGF stimulates lacrimal gland secretion of protein by activating the EGFR to increase [Ca(2+)](i) and activate PKC.
authors.affiliation: Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114, USA.
authors.forename: Vanja
authors.initials: V
authors.lastname: Tepavcevic
authors.forename: Robin R
authors.initials: RR
authors.lastname: Hodges
authors.forename: Driss
authors.initials: D
authors.lastname: Zoukhri
authors.forename: Darlene A
authors.initials: DA
authors.lastname: Dartt
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005136:Eye Proteins / Q000378:metabolism*; D007765:Lacrimal Apparatus / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D009195:Peroxidase / Q000378:metabolism; D010656:Phenylephrine / Q000494:pharmacology; D051966:Phospholipase C gamma; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011799:Quinazolines; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism; D020032:Tyrphostins / Q000494:pharmacology
chemical_list: D005136:Eye Proteins; D011799:Quinazolines; D020032:Tyrphostins; C101044:RTKI cpd; D010656:Phenylephrine; D004815:Epidermal Growth Factor; D002217:Carbachol; D009195:Peroxidase; D066246:ErbB Receptors; D011493:Protein Kinase C; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium