Molecular cloning and functional expression of the guinea pig α1a-adrenoceptor

In the present paper, the cloning and expression of the guinea pig alpha (1A)-adrenoceptor is presented. The nucleotide sequence had an open reading frame of 1401 bp that encoded a 466 aniino-acid protein with an estimated molecular mass of = 51.5 kDa. When the clone was expressed in Cos-1 cells, specific high-affinity binding of [H-3]prazosin and [H-3]tamsulosin was observed. Chloroethylclonidine treatment of membranes slightly decreased the total binding with both radioligands. Binding competition experiments using [H-3]tamsulosin showed the following potency order: (a) for agonists: oxymetazoline >> epinephrine > norepinephrine > methoxamine, and (b) for antagonists: prazosin greater than or equal to 5-methyl-urapidil = benoxathian > phentolamine >> BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione). Photoaffinity labeling using [I-125-aryl]azido-prazosin revealed a major broad band with a molecular mass between 70 and 80 kDa. The receptor was functional, as evidenced by an epinephrine-increased production of [H-3]inositol phosphates that was blocked by prazosin. (C) 2001 Published by Elsevier Science B.V.