title: Effect of VIP on intracellular [Ca2+], extracellular regulated kinase 1/2, and secretion in cultured rat conjunctival goblet cells.
abstract: OBJECTIVE
  To determine the intracellular signaling pathways that vasoactive intestinal peptide (VIP) uses to stimulate high molecular weight glycoconjugate secretion from cultured rat conjunctival goblet cells.
  
  
  METHODS
  Goblet cells from rat bulbar and forniceal conjunctiva were grown in organ culture. Presence and localization of VIP receptors (VPAC1 and 2) were determined by RT-PCR, immunofluorescence microscopy and Western blot analysis. Intracellular [Ca(2+)] ([Ca(2+)]i) was measured using fura-2. Extracellular signal-regulated kinase (ERK)-1/2 activity was determined by Western blot analysis. High molecular weight glycoconjugate secretion was measured with an enzyme-linked lectin assay on cultured goblet cells that were serum-starved for 2 hours before stimulation with VIP, VPAC1-, or VPAC2-specific agonists. Inhibitors were added 30 minutes prior to VIP. Activation of epidermal growth factor receptor (EGFR) was measured by immunoprecipitation using an antibody against pTyr followed by Western blot analysis with an antibody against EGFR.
  
  
  RESULTS
  Both VIP receptors were present in rat conjunctiva and cultured goblet cells. VIP- and VPAC-specific agonists increased [Ca(2+)]i and secretion in a concentration-dependent manner. VIP also increased ERK1/2 activity, VIP-stimulated increase in [Ca(2+)]i. Secretion, but not ERK1/2 activity, was inhibited by the protein kinase A inhibitor, H89. VIP-stimulated secretion was inhibited by siRNA for ERK2 but not by siRNA for EGFR. VIP did not increase the phosphorylation of the EGFR.
  
  
  CONCLUSIONS
  In conclusion, in cultured rat conjunctival goblet cells, VPAC1 and 2 receptors are functional. VIP stimulates a cAMP-dependent increase in [Ca(2+)]i and glycoconjugate secretion, but not ERK1/2 activation. VIP does not activate with EGFR.
authors.affiliation: Schepens Eye Research Institute, Massachusetts Eye and Ear and Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA.
authors.forename: Dayu
authors.initials: D
authors.lastname: Li
authors.forename: Jianwei
authors.initials: J
authors.lastname: Jiao
authors.forename: Marie A
authors.initials: MA
authors.lastname: Shatos
authors.forename: Robin R
authors.initials: RR
authors.lastname: Hodges
authors.forename: Darlene A
authors.initials: DA
authors.lastname: Dartt
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology / Q000294:innervation / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D006001:Glycoconjugates / Q000378:metabolism; D020397:Goblet Cells / Q000187:drug effects / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D055271:Mucin 5AC / Q000378:metabolism; D009924:Organ Culture Techniques; D010275:Parasympathetic Nervous System / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D051239:Receptors, Vasoactive Intestinal Peptide, Type II / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism / Q000494:pharmacology
chemical_list: D018076:DNA, Complementary; D006001:Glycoconjugates; C524680:Muc5ac protein, rat; D055271:Mucin 5AC; D051239:Receptors, Vasoactive Intestinal Peptide, Type II; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D014660:Vasoactive Intestinal Peptide; D017868:Cyclic AMP-Dependent Protein Kinases; C535149:Mapk1 protein, rat; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D002118:Calcium