title: Increased voltage-dependent calcium influx produced by alpha 1B-adrenergic receptor activation in rat medullary thyroid carcinoma 6-23 cells.
abstract: We characterized norepinephrine (NE)-activated Ca2+ influx in the rat medullary thyroid carcinoma (rMTC) 6-23 cell line using fura-2. NE caused a sustained increase in the intracellular Ca2+ concentration ([Ca2+]i), which was completely reversed by addition of nifedipine or removal of extracellular Ca2+. Bay K8644, KCl-induced depolarization, and ATP also increased [Ca2+]i in rMTC 6-23 cells, effects that were also reversed by nifedipine. Release of intracellular Ca2+ by thapsigargin was not blocked by nifedipine, and NE caused nifedipine-sensitive increases in [Ca2+]i even in the presence of thapsigargin. NE-stimulated increases in [Ca2+]i were mimicked by the alpha 1-adrenergic receptor (AR) agonist phenylephrine but not by the beta-AR agonist isoproterenol. The response to NE was blocked by the alpha-AR antagonist phentolamine and by pretreatment with the alpha 1B-selective alkylating agent chloroethylclonidine (CEC) but was not blocked by alpha 1A-selective concentrations of the subtype-selective antagonist 5-methylurapidil. alpha 1-AR binding sites labeled by 125I-BE 2254 in membranes from this cell line were highly sensitive to inactivation by CEC (> 80%), and competition with subtype-selective antagonists suggested the presence of a homogeneous population of alpha 1B-ARs. NE, epinephrine, and phenylephrine, but not KCl, ATP, or isoproterenol, caused large increases in [3H]inositol phosphate (InsP) formation in these cells. This [3H]InsP response was greatly reduced by CEC pretreatment, and competitive antagonists blocked this response with an alpha 1B-like pharmacology. Northern blots of poly(A)+ RNA from rMTC 6-23 cells showed single transcripts hybridizing to the hamster alpha 1B-AR (2.2-kilo-base) and less prominently to the rat alpha 1D-AR (4.0-kilobase) cDNAs but no detectable hybridization to the bovine alpha 1C-AR cDNA. The phospholipase C inhibitor U-73122 reduced the [3H] InsP response to NE in a concentration-dependent manner but had little or no effect on the NE-induced increases in [Ca2+]i. Phorbol myristate acetate also increased [Ca2+]i in rMTC 6-23 cells, although this response was not blocked by nifedipine. We conclude that activation of alpha 1B-like ARs (including possibly both alpha 1B- and alpha 1D-ARs) increases voltage-dependent Ca2+ influx in rat rMTC 6-23 cells. This effect appears to be independent of release of intracellular Ca2+, activation of phospholipase C, and/or activation of protein kinase C. This cell line should be very useful in defining the mechanisms underlying the known effects of alpha 1-ARs on voltage-gated Ca2+ influx, which plays an important functional role in vascular smooth muscle.
authors.affiliation: Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322.
authors.forename: T A
authors.initials: TA
authors.lastname: Esbenshade
authors.forename: T L
authors.initials: TL
authors.lastname: Theroux
authors.forename: K P
authors.initials: KP
authors.lastname: Minneman
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D018276:Carcinoma, Medullary / Q000378:metabolism*; D002460:Cell Line; D004963:Estrenes / Q000494:pharmacology; D066298:In Vitro Techniques; D007295:Inositol Phosphates / Q000378:metabolism; D008564:Membrane Potentials; D009638:Norepinephrine / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D051381:Rats; D011942:Receptors, Adrenergic, alpha / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism; D013961:Thyroid Gland / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D004963:Estrenes; D007295:Inositol Phosphates; D011760:Pyrrolidinones; D011942:Receptors, Adrenergic, alpha; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C074854:U 73343; D010738:Type C Phospholipases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D009638:Norepinephrine