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C. Funaki, R. R. Hodges, D. A. Dartt; Inhibition of Ras/Raf/MEK/MAPK Pathway by dbcAMP in Rat Lacrimal Gland. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5301.
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We previously showed that increasing intracellular cyclic AMP (cAMP) inhibited both basal and stimulated p42/p44 mitogen-activated protein kinase (MAPK, ERK1/2) by the agonists carbachol (a cholinergic agonist, Cch), phenylephrine (an alpha1D adrenergic agonist, Ph) and EGF (epidermal growth factor). Activation of this pathway accounts for the potentiation of lacrimal gland protein secretion. In this study we determine which step in the Ras/Raf/MEK/MAPK pathway cyclic AMP inhibits.
Exorbital lacrimal glands were removed from male Sprague-Dawley rats. Acini were isolated by collagenase digestion. Cellular cAMP levels were increased by using the cell permeant cAMP analogs, dibutyryl cAMP (dbcAMP) for 0-30 min. In addition, acini were preincubated with or without dbcAMP (10-3M) for 30 min prior to stimulation for 5 min with Cch (10-4M), Ph (10-4M) or EGF (10-7M). Acini were homogenized and western blot analysis was performed using antibodies specific to phosphorylated (activated) p42/44 MAPK, total p42-MAPK, phosphorylated Raf-1(ser338), total Raf-1, phosphorylated MEK, total MEK, phosphorylated EGF receptor and total EGF receptor. Ras activity was measured by using Ras activation kit.
DbcAMP (10-3M) significantly inhibited basal MEK (at 30 min) and MAPK at all times and agonist (Cch, Ph and EGF)-stimulated MAPK activity. However dbcAMP did not inhibit basal phosphorylation of EGF receptor or Ras and Raf-1 activity at any time point. Agonist (Cch, Ph and EGF)-stimulated Raf-1 and MEK activity was inhibited by dbcAMP, although not significantly.
dbcAMP inhibits basal MAPK activity either directly or upstream of MEK but downstream of Raf-1. In turn, agonist-stimulated MAPK pathway could be inhibited by dbcAMP directly at MAPK and potentially at Ras activation of Raf-1.
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