Characterization of the at α1-adrenoceptor subtype activating extracellular signal-regulated kinase in submandibular gland acinar cells

alpha(1)-Adrenoceptors and extracellular signal-regulated kinases 1 and 2 (ERK1/2) regulate salivary secretion. However, whether alpha(1)-adrenoceptors couple to ERK1/2 activation and the specific alpha(1)-adrenoceptor subtypes involved in salivary glands is unknown. Western blotting of ERK1/2 phosphorylation showed plienylephrine activated ERK1/2 by 2-3-fold in submandibular gland slices and 3-4-fold in submandibular acinar (SMG-C10) cells with an EC50 of 2.7 +/- 2 mu M. ERK1/2 activation was blocked by either prazosin or HEAT, indicating alpha(1)-adrenoceptors stimulate ERK1/2 in native glands and SMG-C10 cells. Inhibition of [I-125]HEAT binding by 5-methylurapidil (selective for alpha(1A) over alpha(1B)/alpha(1D)), but not BMY 73 78 (selective for alpha(1D) over alpha(1A)/alpha(1B)), Was biphasic and best-fit by a two-site binding model with K-iH and K-iL values for 5-methylurapidil of 0.64 +/- 0.3 and 91 +/- 7 nM, respectively, in SMG-C10 membranes. From these binding data, we obtained subtype-selective concentrations of 5-methylurapidil to determine the alpha(1)-adrenoceptor subtype/s activating ERK1/2 in SMG-C10 cells. 5-methylurapidil (20 nM) did not affect phenylephrine- or A-61603.. (alpha(1A)-selective agonist) induced ERK1/2 activation; whereas, 30 mu M chloroethylclonidine (alpha(1B)-selective antagonist) inhibited ERK1/2 activation by phenylephrine, indicating alpha(1B)-adrenoceptors, but not CL I alpha(1A)-adrenoceptors, activate ERK1/2 in submandibular cells. We also examined alpha(1)-adrenoceptor location and dependence on cholesterol-rich microdomains for activating ERK1/2. Sucrose density gradient centrifugation showed 71 +/- 3 % of alpha(1)-adrenoceptor binding sites were in plasma membranes. Cholesterol-disrupting agents filipin and methyl-beta-cyclodextrin inhibited phenylephrine-stimulated ERK1/2. These results show only alpha(1B)-adrenoceptors activate ERK1/2 and suggest subtypespecific ERK1/2 signaling by (alpha(1B)-adrenoceptors may be determined by localization to cholesterol-rich microdomains in submandibular cells. (c) 2007 Elsevier B.V. All rights reserved.