Bibliothek

Notizen: The regulation of intracellular calcium by cholinergic agonists was investigated in the human neuroblastoma SH-SY5Y, loaded with fura-2. The resting free Ca2+ concentration in this cell line was 199 ± 14 nM (mean ± SEM, n = 19). At 1 mM extracellular Ca2+, high concentrations of carbachol and acetylcholine evoked a biphasic change in intracellular Ca2+ concentration, consisting of a transient initial peak followed by a decline to a plateau that was significantly higher than the basal level. Carbachol (0.5 mM) and acetylcholine (10 μM) caused a maximal increase in the intracellular Ca2+ concentration, reaching a peak of 465 ± 52 (mean ± SEM, n = 12) and 422 ± 48 nM (mean ± SEM, n = 7), respectively, in 〈4 s. This initial calcium transient declined to a plateau of 268 ± 36 and 240 ± 27 nM for carbachol and acetylcholine, respectively, in ∼40 s. The plateau persisted until the agonist was displaced by the addition of antagonist. Atropine, hexahydrosiladifenidol (HHSD), pirenzepine, and methoctramine inhibited the carbachol-evoked initial calcium transient with Ki values of 0.85 ± 0.05, 8.3 ± 1.6, 411 ± 36, and 240 ± 46 nM (mean ± SEM, n = 3), respectively, and the acetylcholine-induced initial calcium transient with Ki values of 0.48 ± 0.18, 13.5 ± 8.5, 192 ± 32, and 414 ± 25 nM (mean ± SEM of two experiments), respectively, results suggesting that an M3 muscarinic receptor was predominantly mediating these effects. Furthermore, atropine, HHSD, and pirenzepine inhibited the plateau phase of the carbachol- evoked change in intracellular Ca2+ concentration with Ki values of 0.25, 1, and 861 nM, respectively, which are consistent with an M3 receptor being coupled to this effect. The plateau was abolished in the presence of EGTA, thereby converting the biphasic signal into a monophasic response. Under these conditions, the initial calcium transient or its pharmacology was not altered. However, if the cells were washed in calcium-free buffer for 3 min, the initial peak was decreased by∼30%. The subsequent readdition of calcium caused a further increase in fura-2 fluorescence. The influx of calcium and hence the plateau were also blocked by nickel but were insensitive to verapamil. We may conclude, therefore, on pharmacological grounds, that an M3 muscarinic receptor is coupled both to the peak response, presumably due to inositol trisphosphate mobilization of intracellular calcium, and to the plateau due to an influx of extracellular calcium.