title: Alpha 2A-adrenoceptors mediate activation of non-selective cation channels via Gi-proteins in human erythroleukaemia (HEL) cells. No evidence for a functional role of imidazoline receptors in modulating calcium.
abstract: Human erythroleukaemia (HEL) cells were investigated to characterize their alpha 2-adrenoceptor and imidazoline receptor sites. Membranes from HEL cells bound [3H]2-(2-methoxy-1, 4-benzodioxan-2yl)-2-imidazoline ([3H]RX821002) in a saturable and specific manner with a KD of 0.64 +/- 0.07 nM and a Bmax of 126 +/- 4 fmol/mg protein. [3H]RX821002 was displaced from HEL membranes by adrenergic drugs with the order of potency being yohimbine approximately oxymetazoline >> prazosin = 2-[2-[4-(o-methoxyphenyl)piperazin-1-yl]ethyl]-4,4-dimethyl- 1,3(2H,4H)-isochinolindione HCl (ARC 239), consistent with this site being an alpha 2A-adrenoceptor. HEL membranes also bound [3H]idazoxan in the presence of adrenaline to block alpha 2-adrenoceptors. This binding was saturable and specific with a KD of 3.5 +/- 1.0 nM and a Bmax of 31 +/- 6 fmol/mg protein. Adrenergic drugs from both the phenylethylamine and imidazoline classes increased high-affinity GTPase activity, an index of activation of regulatory heterotrimeric guanine-nucleotide binding proteins (G-proteins), and produced increases in cytosolic free calcium concentration ([Ca2+]i). The effects of these agonists in both systems were abolished by pertussis toxin pretreatment, and oxymetazoline and clonidine were antagonists. The potency of adrenergic drugs to inhibit 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304)-induced increases in [Ca2+]i was yohimbine approximately oxymetazoline >> ARC 239, consistent with the binding data and an action at alpha 2A-adrenoceptors. No evidence was found for a role of imidazoline receptors in stimulating G-proteins or modulating [Ca2+]i. The adrenergic agonist-induced increases in [Ca2+]i were due to both release of Ca2+ from intracellular stores and entry of extracellular Ca2+. Ca2+ entry was blocked by 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenylethyl)-1H- imidazole hydrochloride (SKF 96365), but not by nitrendipine. Adrenaline also stimulated Mn2+ entry in HEL cells. Taken together, these results suggest that HEL cells have alpha 2A-adrenoceptors that activate non-selective cation channels via pertussis toxin-sensitive G-proteins, i.e. Gi-proteins.
authors.affiliation: Institut für Pharmakologie, Freie Universität Berlin, Germany.
authors.forename: I F
authors.initials: IF
authors.lastname: Musgrave
authors.forename: R
authors.initials: R
authors.lastname: Seifert
mesh_terms: D001667:Binding, Competitive; D002118:Calcium / Q000032:analysis; D002458:Cell Fractionation; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D054755:Imidazoline Receptors; D007473:Ion Channels / Q000378:metabolism*; D007546:Isoquinolines / Q000494:pharmacology; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism*; D010109:Oxymetazoline / Q000494:pharmacology; D037342:Pertussis Toxin; D010879:Piperazines / Q000494:pharmacology; D011942:Receptors, Adrenergic, alpha / Q000187:drug effects* / Q000378:metabolism; D011955:Receptors, Drug / Q000187:drug effects; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D015016:Yohimbine / Q000494:pharmacology
chemical_list: D054755:Imidazoline Receptors; D007473:Ion Channels; D007546:Isoquinolines; D010879:Piperazines; D011942:Receptors, Adrenergic, alpha; D011955:Receptors, Drug; D010566:Virulence Factors, Bordetella; D015016:Yohimbine; D010109:Oxymetazoline; D037342:Pertussis Toxin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C032994:AR-C239; D002118:Calcium