M1 muscarinic receptors mediate intracellular calcium release in NB-OK1 human neuroblastoma cells.

Abstract

Muscarine acetylcholine receptors were characterized in NB-OK1 cells using radioligand (3H-NMS) binding experiments and second messenger (calcium and phosphatidylinositol (PI) turnover) studies. In radioligand binding experiments the displacement curves of pirenzepine (KI = 1.3 x 10(-8) M), AF-DX 116 (KI = 8.2 x 10(-7) M), methoctramine (KI = 8.4 x 10(-8) M) and parafluorohexahydrosiladifenidol (pF-HHSiD) (KI = 1.8 x 10(-8) M) were monophasic and indicated the presence of M1 muscarinic receptors. Schild analysis with the muscarinic antagonists pirenzepine, AF-DX 116, methoctramine and pF-HHSiD yielded pA2 values of 8.40 +/- 0.13, 6.48 +/- 0.09, 7.61 +/- 0.12 and 7.22 +/- 0.08 in the calcium experiments and pA2 values of 8.13 +/- 0.30 and 6.26 +/- 0.26, 7.65 +/- 0.16 and 7.46 +/- 0.11, respectively, in the PI turnover experiments. These results indicate that both the carbachol-induced increase in intracellular calcium and the increase in PI turnover are mediated by M1 muscarinic receptors. In calcium free buffer, stimulation with carbachol induced similar responses to those seen under control conditions. From functional and radioligand binding experiments we conclude that the muscarinic receptor expressed in NB-OK1 cells is the M1 subtype. In addition, the M1 receptor-induced calcium response is related to PI turnover and is independent on extracellular calcium.