Comparison of alpha 1-adrenergic receptor subtypes and signal transduction in SK-N-MC and NB41A3 neuronal cell lines.
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+.
Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322.
T A
TA
Esbenshade
C
C
Han
T J
TJ
Murphy
K P
KP
Minneman
D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000502:physiology; D003000:Clonidine / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D051379:Mice; D009474:Neurons / Q000378:metabolism / Q000502:physiology*; D009638:Norepinephrine / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D011869:Radioligand Assay; D011942:Receptors, Adrenergic, alpha / Q000145:classification* / Q000187:drug effects / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D014407:Tumor Cells, Cultured
D010716:Phosphatidylinositols; D012333:RNA, Messenger; D011942:Receptors, Adrenergic, alpha; C051731:chlorethylclonidine; D003000:Clonidine; D002118:Calcium; D009638:Norepinephrine