Abstract
Abstract: :
Purpose:To determine the effect of activation of MAPK on protein secretion from lacrimal gland. Methods:Lacrimal glands were removed from male rats. Acini were prepared by collagenase digestion and stimulated for varying times (to measure MAPK) or 20 min (to measure secretion) with either the cholinergic agonist carbachol (10-4 M), the α1-adrenergic agonist phenylephrine (10-4 M) or EGF (10-7 M), as a positive control. Either U0126 (10-8 - 10-6 M), an inhibitor of MAPK activation, or AG1478 (10-6 M), an inhibitor of EGF receptor activity, were added 10 min prior to agonists. Acini were either homogenized and samples prepared for Western blot analysis with antibodies against phosphorylated or total MAPK or the supernatant and pellet were analyzed by spectrophotometry for peroxidase, our index of protein secretion. Results:Both carbachol and phenylephrine increased the phosphorylation of MAPK (pMAPK) in a time-dependent manner. Carbachol at 10 min increased the amount of pMAPK a maximum of 5.5 ±1.3 fold over control. Carbachol-induced activation of MAPK in the lacrimal gland was completely inhibited by U0126 at 6 x10-7 and 6 x 10-6 M. Phenylephrine also increased the amount of pMAPK in a time-dependent manner with a maximum of 2.7 ± 0.6 at 5 min, while EGF increased MAPK phosphorylation 4.1 ± 1.5 fold over control after 5 min. As carbachol and phenylephrine are both potent stimulators of lacrimal gland secretion, we tested U0126 on protein secretion. U0126 (6 x 10-7 M) significantly increased carbachol- and phenylephrine-induced secretion 2.5 ± 0.5 and 1.3 ± 0.1 fold, respectively. In contrast, U0126 completely abolished EGF-induced protein secretion. Carbachol-stimulated peroxidase secretion was unchanged after incubation with AG1478 while phenylephrine-induced secretion was significantly increased 4.4 ± 0.9 fold. EGF-stimulated protein secretion was completely inhibited by AG1478. Conclusion:Cholinergic and α1-adrenergic agonists use different pathways to activate MAPK. α1-Adrenergic, but not cholinergic, agonists transactivate the EGF receptor. Activation of MAPK by either mechanism inhibits protein secretion.
Keywords: 452 lacrimal gland • 580 signal transduction • 576 second messengers