title: Phospholipase D1, but not D2, regulates protein secretion via Rho/ROCK in a Ras/Raf-independent, MEK-dependent manner in rat lacrimal gland.
abstract: OBJECTIVE
  A prior study showed that cholinergic agonists activate phospholipase D (PLD). The purpose of this study was to determine whether cholinergic agonists use the PLD pathway to alter protein secretion and to identify the molecular signaling components of this pathway in rat lacrimal gland acini.
  
  
  METHODS
  Rat lacrimal gland acini were isolated by collagenase digestion. Presence and localization of PLD1 and -2 were determined by immunofluorescence and Western blot experiments. Acini were incubated with adenoviruses overnight or the inhibitors 1-butanol, Y-27632, or C3 exotoxin before stimulation with the cholinergic agonist carbachol (Cch, 10(-4) M) for 5 minutes. Western blot analysis was performed for 20 minutes, and protein secretion was measured spectrophotometrically. Activation of ERK, MEK, Pyk2, Ras, and Raf was determined by Western blot analysis.
  
  
  RESULTS
  1-Butanol increased Cch-stimulated protein secretion and decreased ERK activity. Incubation with catalytically inactive PLD1, but not catalytically inactive mutant PLD2 adenovirus, also increased Cch-stimulated protein secretion and decreased ERK activity. Inhibition of Rho with C3 exotoxin and a dominant negative Rho adenovirus and inhibition of ROCK with Y-27632 inhibited Cch-stimulated PLD1 activity, increased protein secretion, and decreased ERK activity. The association of PLD1 and ROCK increased with Cch stimulation, as determined by immunoprecipitation. PMA-stimulated ERK activity was also inhibited by 1-butanol. 1-Butanol had no effect on Cch-stimulated Pyk2, Ras, and Raf activity, but decreased MEK activity.
  
  
  CONCLUSIONS
  Cholinergic agonists activate PLD1 through Rho and ROCK, which in turn activate MEK and ERK, which attenuate protein secretion in freshly isolated epithelial cells.
authors.affiliation: Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, 20 Staniford Street, Boston, MA 02114, USA. robin.hodges@schepens.harvard.edu
authors.forename: Robin R
authors.initials: RR
authors.lastname: Hodges
authors.forename: Erin
authors.initials: E
authors.lastname: Guilbert
authors.forename: Marie A
authors.initials: MA
authors.lastname: Shatos
authors.forename: Viswanathan
authors.initials: V
authors.lastname: Natarajan
authors.forename: Darlene A
authors.initials: DA
authors.lastname: Dartt
mesh_terms: D020001:1-Butanol / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002217:Carbachol / Q000494:pharmacology; D018679:Cholinergic Agonists / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005136:Eye Proteins / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D047468:Immunoprecipitation; D007765:Lacrimal Apparatus / Q000187:drug effects* / Q000201:enzymology; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D048490:raf Kinases / Q000378:metabolism*; D020663:ras Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000378:metabolism*
chemical_list: D018679:Cholinergic Agonists; D004791:Enzyme Inhibitors; D005136:Eye Proteins; D020663:ras Guanine Nucleotide Exchange Factors; C042434:tear proteins; D020001:1-Butanol; D002217:Carbachol; D048490:raf Kinases; D054460:rho-Associated Kinases; D020928:Mitogen-Activated Protein Kinases; C105606:phospholipase D2; D010739:Phospholipase D; C106801:phospholipase D1