Muscarinic receptor subtypes and calcium signaling in Fischer rat thyroid cells

Abstract

1 A specific and saturable binding site for [ 3H]N-methyl-scopolamine ([ 3H]NMS) was observed in plasma membrane of Fischer rat thyroid (FRT) cells with an equilibrium dissociation constant ( K d ) of 0.11 ± 0.02 nM and a concentration of receptor sites ( B max) of 14.1 ± 3.9 fmol/mg protein. Pharmacological characterization of this binding site using pirenzepine, himbacine, (11(2-diethyl-amino)methyl)-1-piperidinylacetyl-5-11-dihydro-6 H-pyrido(14) benzodiazepine (AF-DX 116), dicyclomine, 4-diphenylacetoxy- N-methylpiperidine methiodide (4-DAMP), and hexahydro-sila-difenidol (HHSD) showed clear differences, in terms of affinities, between these muscarinic receptor antagonists. The order of potency for inhibiting [ 3H]NMS binding was HHSD = dicyclomine > 4-DAMP > pirenzepine = himbacine > AF-DX 116. These findings suggest that the muscarinic receptors found in FRT cells belong to the M 3 subtype. Stimulation of FRT cells with carbachol produced a biphasic and dose-dependent increase in the intracellular calcium concentration ([Ca 2+] i), which was blocked in pretreated cells with atropine and almost abolished by a low concentration of 4-DAMP and HHSD. Removal of extracellular Ca 2+ from the incubation medium reduced the initial transient peak and completely abolished the plateau phase, while the transient phase was markedly reduced by the phospholipase C inhibitor U73122. These data indicate that [Ca 2+] i results from both Ca 2+ influx across Ca 2+ channels and mobilization of Ca 2+ from intracellular Ca 2+ stores. The present data showed the presence of the M 3 muscarinic acetylcholine receptor subtype in plasma membrane of FRT cells, which may influence cellular function via modulation of [Ca 2+] i.