title: Characterization of the alpha-1 adrenoceptor subtype mediating [3H]-arachidonic acid release and calcium mobilization in Madin-Darby canine kidney cells.
abstract: The authors characterized the alpha-1 adrenoceptor subtype located on Madin-Darby canine kidney (MDCK) cells by measuring norepinephrine-mediated [3H]-arachidonic acid release and Ca++ mobilization in fura 2-loaded cells. In both assays, prazosin and chloroethylclonidine acted as unsurmountable antagonists, whereas 5-methyl-urapidil acted as a competitive antagonist with pA2 estimates of 7.3 (arachidonic acid assay) and 7.7 (Ca++ assay). Competitive antagonism toward arachidonic acid release was also obtained with the following alpha-1 adrenoceptor antagonists (pA2): (+)-niguldipine (7.6), 2-(2,6-dimethoxy-phenoxy-yethyl)aminomethyl-1,4-benzodioxane hydrochloride (WB 4101; 8.3), phentolamine (7.6) and oxymetazoline (6.4). Arachidonic acid release by norepinephrine was abolished in the absence of extracellular Ca++ and was antagonized by 1-[beta-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SK&F 96365) but was insensitive to antagonism by L-type Ca++ channel antagonists and La . Norepinephrine-mediated increases in intracellular Ca++ consisted of two distinct phases: a transient phase followed by a sustained plateau. The transient phase was resistant to EGTA, whereas the plateau phase was abolished by EGTA. Potassium chloride did not evoke a response in either assay. Northern blot analysis demonstrated that MDCK cells express alpha-1B adrenoceptor messenger RNA. It was concluded that typical alpha-1B adrenoceptors mediate responses to norepinephrine in MDCK cells and that these receptors couple to both intracellular Ca++ release and Ca++ influx by a voltage-independent mechanism. This influx pathway is insensitive to L-type Ca++ channel antagonists but is antagonized by the receptor-operated Ca++ channel antagonist SK&F 96365.
authors.affiliation: Institute of Pharmacology, Syntex Discovery Research, Palo Alto, California.
authors.forename: D R
authors.initials: DR
authors.lastname: Blue
authors.forename: D A
authors.initials: DA
authors.lastname: Craig
authors.forename: J T
authors.initials: JT
authors.lastname: Ransom
authors.forename: J A
authors.initials: JA
authors.lastname: Camacho
authors.forename: P A
authors.initials: PA
authors.lastname: Insel
authors.forename: D E
authors.initials: DE
authors.lastname: Clarke
mesh_terms: D058668:Adrenergic alpha-1 Receptor Antagonists; D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D004285:Dogs; D016257:Fura-2; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D018340:Receptors, Adrenergic, alpha-1 / Q000145:classification / Q000235:genetics / Q000502:physiology*
chemical_list: D058668:Adrenergic alpha-1 Receptor Antagonists; D000317:Adrenergic alpha-Antagonists; D012333:RNA, Messenger; D018340:Receptors, Adrenergic, alpha-1; D016718:Arachidonic Acid; D002118:Calcium; D016257:Fura-2