α1-Adrenoceptors and extracellular signal-regulated kinases 1 and 2 (ERK1/2) regulate salivary secretion. However, whether α1-adrenoceptors couple to ERK1/2 activation and the specific α1-adrenoceptor subtypes involved in salivary glands is unknown. Western blotting of ERK1/2 phosphorylation showed phenylephrine 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 μM. ERK1/2 activation was blocked by either prazosin or HEAT, indicating α1-adrenoceptors stimulate ERK1/2 in native glands and SMG-C10 cells. Inhibition of [125I]HEAT binding by 5-methylurapidil (selective for α1A over α1B/α1D), but not BMY 7378 (selective for α1D over α1A/α1B), was biphasic and best-fit by a two-site binding model with KiH and KiL 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 α1-adrenoceptor subtype/s activating ERK1/2 in SMG-C10 cells. 5-methylurapidil (20 nM) did not affect phenylephrine- or A-61603- (α1A-selective agonist) induced ERK1/2 activation; whereas, 30 μM chloroethylclonidine (α1B-selective antagonist) inhibited ERK1/2 activation by phenylephrine, indicating α1B-adrenoceptors, but not α1A-adrenoceptors, activate ERK1/2 in submandibular cells. We also examined α1-adrenoceptor location and dependence on cholesterol-rich microdomains for activating ERK1/2. Sucrose density gradient centrifugation showed 71 ± 3% of α1-adrenoceptor binding sites were in plasma membranes. Cholesterol-disrupting agents filipin and methyl-β-cyclodextrin inhibited phenylephrine-stimulated ERK1/2. These results show only α1B-adrenoceptors activate ERK1/2 and suggest subtype-specific ERK1/2 signaling by α1B-adrenoceptors may be determined by localization to cholesterol-rich microdomains in submandibular cells.
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