title: Characterization of the calcium response to thyrotropin-releasing hormone (TRH) in cells transfected with TRH receptor complementary DNA: importance of voltage-sensitive calcium channels.
abstract: TRH stimulates a biphasic increase in intracellular free calcium ion, [Ca2+]i. Cells stably transfected with TRH receptor cDNA were used to compare the response in lines with and without L type voltage-gated calcium channels. Rat pituitary GH-Y cells that do not normally express TRH receptors, rat glial C6 cells, and human epithelial Hela cells were transfected with mouse TRH receptor cDNA. All lines bound similar amounts of [3H][N3-Me-His2]TRH with identical affinities (dissociation constant = 1.5 nM). Both pituitary lines expressed L type voltage-gated calcium channels; depolarization with high K+ increased 45Ca2+ uptake 20- to 25-fold and [Ca2+]i 12- to 14-fold. C6 and Hela cells, in contrast, appeared to have no L channel activity. GH4C1 cells responded to TRH with a calcium spike (6-fold) followed by a sustained second phase. When TRH was added after 100 nM nimodipine, an L channel blocker, the initial calcium burst was unaffected but the second phase was abolished. GH-Y cells transfected with TRH receptor cDNA responded to TRH with a 6-fold [Ca2+]i spike followed by a plateau phase (>8 min) in which [Ca2+]i remained elevated or increased. Nimodipine did not alter the peak TRH response or resting [Ca2+]i but reduced the sustained phase, which was eliminated by chelation of extracellular Ca2+. In the transfected glial C6 and Hela cells without calcium channels, TRH evoked transient, monophasic 7- to 9-fold increases in [Ca2+]i, and [Ca2+]i returned to resting levels within 3 min. Thapsigargin stimulated a gradual, large increase in [Ca2+]i in transfected C6 cells, and subsequent addition of TRH caused no further rise. Removal of extracellular Ca2+ from transfected C6 cells shortened the [Ca2+]i responses to TRH, to endothelin 1, and to thapsigargin. The TRH responses were pertussis toxin-insensitive. In summary, TRH can generate a calcium spike in pituitary, C6, and Hela cells transfected with TRH receptor cDNA, but the plateau phase of the [Ca2+]i response is not observed when the receptor is expressed in a cell line without L channel activity.
mesh_terms: D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D004247:DNA / Q000235:genetics; D016232:Endothelins / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D051379:Mice; D009457:Neuroglia / Q000166:cytology / Q000187:drug effects; D009553:Nimodipine / Q000494:pharmacology; D010911:Pituitary Neoplasms / Q000473:pathology; D051381:Rats; D017981:Receptors, Neurotransmitter / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D018025:Receptors, Thyrotropin-Releasing Hormone; D011994:Recombinant Proteins; D013268:Stimulation, Chemical; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology*; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D015220:Calcium Channels; D016232:Endothelins; D017981:Receptors, Neurotransmitter; D018025:Receptors, Thyrotropin-Releasing Hormone; D011994:Recombinant Proteins; D013729:Terpenes; D009553:Nimodipine; D013973:Thyrotropin-Releasing Hormone; D019284:Thapsigargin; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D004247:DNA; D002118:Calcium