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TA Esbenshade, C Han, TJ Murphy and KP Minneman
Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322.
We compared the alpha 1-adrenergic receptor subtypes in two neuronal cell lines, SK-N-MC (human neuroepithelioma) and NB41A3 (murine neuroblastoma). 125I-BE 2254 labeled alpha 1-adrenergic receptor binding sites in membranes from both cell lines. Pretreatment with the alpha 1B-selective alkylating agent chloroethylclonidine (CEC) completely eliminated these binding sites in NB41A3 cells but caused only a 50% loss in SK-N-MC cells. Displacement with subtype-selective antagonists suggested that NB41A3 cells express only the alpha 1B subtype, whereas SK-N-MC cells express a pharmacologically heterogeneous receptor population, including both alpha 1A and alpha 1B subtypes. Norepinephrine increased [3H] inositol phosphate formation in both cell lines, but with different sensitivities to pertussis toxin and the presence of extracellular Ca2+. CEC pretreatment eliminated this response in NB41A3 cells but caused a maximal 42% reduction in SK- N-MC cells. Use of subtype-selective antagonists showed that the [3H]inositol phosphate response involved only the alpha 1B subtype in NB41A3 cells but a combination of subtypes in SK-N-MC cells. Norepinephrine induced both transient and sustained increases in intracellular Ca2+ concentrations in both cell lines, as measured with fura-2. CEC pretreatment abolished the Ca2+ response in NB41A3 cells but had little effect in SK-N-MC cells. In SK-N-MC cells the Ca2+ response was potently blocked by alpha 1A-selective antagonists. Chelation of extracellular Ca2+ eliminated the sustained component of the Ca2+ signal in both cell lines. Poly(A)+ RNA from NB41A3, DDT1MF-2, BC3H1, and MDCK-D1 cell lines showed one or more prominent transcripts (2.2-4.2 kilobases) that strongly hybridized to the hamster alpha 1B cDNA probe but not to the bovine alpha 1C or rat alpha 1D cDNA probes. Poly(A)+ RNA from SK-N-MC cells showed multiple transcripts (1.3-5.6 kilobases) that hybridized to both hamster alpha 1B and rat alpha 1D but not bovine alpha 1C cDNA probes. We conclude that NB41A3 cells contain exclusively alpha 1B-adrenergic receptors linked to inositol phosphate formation and mobilization of intracellular Ca2+, whereas at least two alpha 1-adrenergic receptor forms, which resemble the alpha 1A and alpha 1B subtypes, coexist in SK-N-MC cells. The CEC-insensitive alpha 1A-like subtype in SK-N-MC cells is capable of increasing inositol phosphate formation and mobilizing intracellular Ca2+.
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