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.