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R. R. Hodges, D. A. Dartt; Cholinergic Agonists Activate Phospholipase D via Rock to Activate P42/p44 MAPK and Attenuate Protein Secretion in Rat Lacrimal Gland. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1909.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the effects of cholinergic agonists on phospholipase D (PLD), the effects of PLD activation, and the signal transduction pathways involved.
Acini were isolated from male rat lacrimal glands by collagenase digestion and incubated with the cholinergic agonist carbachol (Cch, 10-4 M). Immunoprecipitation (IP) experiments were performed after Cch stimulation for 0 - 60 s with a PLD antibody. PLD and p42/p44 MAPK (MAPK) activation were determined after a 5 min Cch stimulation while protein secretion was determined after a 20 min Cch stimulation. Inhibitors were added 15 min prior to stimulation with Cch. Activity assays were performed via western blot analysis using antibodies to phosphorylated (active) or total enzyme. Secretion was determined spectrophotometrically.
As determined by IP experiments, Cch increased the association of PLD with ROCK 1 and ROCK 2 with maximum increase of association of 2.5 ± 0.4 and 2.1 ± 0.7 fold above basal after 15 s. Cch increased PLD activity 2.6 ± 0.5 fold above basal. Cch-stimulated PLD activity was inhibited 83 ± 17% with the ROCK inhibitor Y-27632 (10-5 M). Cholinergic agonist-stimulated MAPK activity was inhibited 84 ± 9% by the PLD inhibitor 1-butanol (3% solution) and 86 ± 8% by Y-27632 (10-4 M). We previously determined that MAPK activation negatively modulates protein secretion. Consistent with this, Cch-stimulated protein secretion was increased 220 ± 95% and 194 ± 23% by 1-butanol and Y-27632, respectively.
We conclude that Cch activates PLD via ROCK to activate MAPK to negatively modulate protein secretion from rat lacrimal gland.
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