Stable expression of recombinant human alpha 2-adrenoceptor subtypes in two mammalian cell lines: characterization with [3H]rauwolscine binding, inhibition of adenylate cyclase and RNase protection assay.
Cloning of the genes encoding distinct subtypes of human alpha 2-adrenergic receptors (alpha 2-AR) allows the separate recombinant expression of each individual subtype in heterologous systems. We report here the transfection, selection and preliminary pharmacological characterization of two mammalian cell lines, adherent Shionogi S115 mouse mammary tumour cells and human B-lymphoblastoid IBW4 cells growing in suspension, expressing the human alpha 2-AR subtypes alpha 2-C4 and alpha 2-C10 at densities of approx. 2 x 10(5) receptors/cell. Transfection of the subtype genes was verified using a specific RNase protection assay. Pharmacological characterization was carried out with [3H]rauwolscine binding, which was inhibited by oxymetazoline and prazosin in a subtype-selective manner. The sensitivity of (-)-noradrenaline binding to the GTP-analogue 5'-guanylylimidodiphosphate suggested that the receptors are coupled to G-proteins. This was verified in S115 cells by efficient inhibition of forskolin-stimulated cAMP production by the alpha 2-AR agonists, (-)-noradrenaline and clonidine. These cell lines thus appear to be suitable for pharmacological studies on receptor function and ligand binding.
Marjamäki
A
A
Department of Pharmacology, University of Turku, Finland.
Ala-Uotila
S
S
Luomala
K
K
Perälä
M
M
Jansson
C
C
Jalkanen
M
M
Regan
J W
JW
Scheinin
M
M
D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D001345:Autoradiography; D002460:Cell Line; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000096:biosynthesis; D019204:GTP-Binding Proteins / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008322:Mammals; D010957:Plasmids; D011224:Prazosin / Q000494:pharmacology; D015347:RNA Probes; D011942:Receptors, Adrenergic, alpha / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism*; D014162:Transfection; D015016:Yohimbine / Q000378:metabolism*
D000067956:Adenylyl Cyclase Inhibitors; D015347:RNA Probes; D011942:Receptors, Adrenergic, alpha; D011994:Recombinant Proteins; D015016:Yohimbine; D000242:Cyclic AMP; D012260:Ribonucleases; D019204:GTP-Binding Proteins; D011224:Prazosin