title: Cholinergic agonists transactivate EGFR and stimulate MAPK to induce goblet cell secretion.
abstract: Conjunctival goblet cells are the primary source of mucins in the mucous layer, the innermost layer of the tear film. Conjunctival goblet cell mucin secretion is under neural control because exogenous addition of parasympathetic agonists stimulates goblet cell secretion. To elucidate the intracellular signal pathways used by cholinergic agonists to stimulate goblet cell mucin secretion, we determined whether p42/p44 mitogen-activated protein kinase (MAPK) is activated during cholinergic agonist-stimulated mucin secretion. Rat conjunctiva was removed, preincubated with or without antagonists, and stimulated with the cholinergic agonist carbachol (10(-4) M). Carbachol statistically significantly stimulated the phosphorylation of MAPK in a time- and concentration-dependent manner. U-0126, an inhibitor of MAPK activation, completely inhibited both the activation of MAPK and goblet cell secretion stimulated by carbachol. The M(1) muscarinic antagonist pirenzepine, the M(2) muscarinic antagonist gallamine, and the M(1)/M(3) muscarinic receptor antagonist N-(3-chloropropyl)-4-piperidinyl diphenylacetate (4-DAMP) also inhibited carbachol-stimulated MAPK activation. Increasing the intracellular Ca(2+) concentration with a Ca(2+) ionophore increased MAPK activation, and chelation of extracellular Ca(2+) inhibited carbachol-stimulated activation. Carbachol also increased tyrosine phosphorylation of Pyk2, p60Src, and the epidermal growth factor receptor (EGFR). The Src inhibitor PP1 and the EGFR inhibitor AG-1478 completely inhibited carbachol-stimulated MAPK activation. AG-1478 also inhibited goblet cell secretion. We conclude that carbachol transactivates the EGFR to activate MAPK, leading to conjunctival goblet cell secretion. In addition, carbachol also activates Pyk2 and p60Src that could play a role in the transactivation of the EGFR.
authors.affiliation: Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114, USA.
authors.forename: Harumi
authors.initials: H
authors.lastname: Kanno
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Horikawa
authors.forename: Robin R
authors.initials: RR
authors.lastname: Hodges
authors.forename: Driss
authors.initials: D
authors.lastname: Zoukhri
authors.forename: Marie A
authors.initials: MA
authors.lastname: Shatos
authors.forename: Jose D
authors.initials: JD
authors.lastname: Rios
authors.forename: Darlene A
authors.initials: DA
authors.lastname: Dartt
mesh_terms: D000818:Animals; D002118:Calcium / Q000502:physiology; D002217:Carbachol / Q000494:pharmacology*; D018679:Cholinergic Agonists / Q000494:pharmacology*; D004789:Enzyme Activation / Q000502:physiology; D066246:ErbB Receptors / Q000187:drug effects* / Q000502:physiology*; D051418:Focal Adhesion Kinase 2; D020397:Goblet Cells / Q000378:metabolism*; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology*; D009093:Mucus / Q000378:metabolism; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism; D009994:Osmolar Concentration; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000502:physiology; D013997:Time Factors; D015533:Transcriptional Activation / Q000502:physiology*
chemical_list: D018679:Cholinergic Agonists; D011976:Receptors, Muscarinic; D002217:Carbachol; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D051418:Focal Adhesion Kinase 2; D015688:Oncogene Protein pp60(v-src); C496054:Ptk2b protein, rat; D020928:Mitogen-Activated Protein Kinases; D002118:Calcium