Characterization of the alpha1-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 phenylephrine activated ERK1/2 by 2-3-fold in submandibular gland slices and 3-4-fold in submandibular acinar (SMG-C10) cells with an EC(50) of 2.7+/-2 microM. 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 [(125)I]HEAT binding by 5-methylurapidil (selective for alpha(1A) over alpha(1B/)alpha(1D)), but not BMY 7378 (selective for alpha(1D) over alpha(1A/)alpha(1B)), was biphasic and best-fit by a two-site binding model with K(i)(H) and K(i)(L) 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 microM chloroethylclonidine (alpha(1B)-selective antagonist) inhibited ERK1/2 activation by phenylephrine, indicating alpha(1B)-adrenoceptors, but not 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 subtype-specific ERK1/2 signaling by alpha(1B)-adrenoceptors may be determined by localization to cholesterol-rich microdomains in submandibular cells.
Bruchas
Michael R
MR
Department of Pharmacology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178, USA.
Toews
Myron L
ML
Bockman
Charles S
CS
Abel
Peter W
PW
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000317:Adrenergic alpha-Antagonists / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D066298:In Vitro Techniques; D008297:Male; D021962:Membrane Microdomains / Q000201:enzymology / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D018340:Receptors, Adrenergic, alpha-1 / Q000187:drug effects / Q000378:metabolism*; D012463:Saliva / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013363:Submandibular Gland / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*
D000316:Adrenergic alpha-Agonists; D000317:Adrenergic alpha-Antagonists; D018340:Receptors, Adrenergic, alpha-1; D002784:Cholesterol; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3