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Notes: 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.

Notes: Abstract: The human neuroblastoma clone SH-SY5Y expresses potassium-, carbachol-, and calcium ionophore A23187-evoked, calcium-dependent release of [3H]noradrenaline. Release in response to carbachol and potassium was greater than additive. Atropine (Ki= 0.33 nM), hexahydrosiladifenidol (Ki= 18 nM), and pirenzepine (Ki= 1,183 nM) completely inhibited the carbachol-evoked noradrenaline release, an order of potency suggesting that an M3 receptor was linked to release. In contrast, noradrenaline release was only partially inhibited by the M2-selective antagonists meth-octramine (10-4M) and AFDX-116 (10-4M), by ∼14 and 46%, respectively. The nicotinic antagonist d-tubocurarine (10-4M) resulted in a partial inhibition of release, a finding suggesting that a nicotinic receptor may also be involved. SH-SY5Y provides a suitable cell line in which to study the biochemical mechanisms underlying the cholinergic receptor regulation of noradrenaline release.

Notes: The present study investigates the roles of protein kinase C (PKC) and A (PKA) activities in NMDA-mediated Ca2+ entry in primary cultures of mouse striatal neurons. Inhibitors of protein kinases, such as sphingosine, RO 31 – 8220 and staurosporine inhibited the NMDA- but also the KCI-induced rise in cytosolic Ca2+. However, the PKA antagonist Rp-adenosine-3′,5′monophosphothioate (Rp-cAMPS) did not alter the NMDA +d-serine response, whereas it completely suppressed the KCI response. The NMDA +d-serine-evoked rise in cytosolic Ca2+, observed in the absence of external Mg2+, was potentiated by the PKC activator phorbol 12-myristate 13-acetate (PMA) only when submaximal effective concentrations of this agonist and co-agonist were used. In addition, the PKC activator did not alter the NMDA +d-serine-evoked response in the presence of varying concentrations of Mg2+. Confirming the dependence on PKC activity, desensitization of PKC resulting from long-term PMA treatment led to an impairment of the NMDA response, leaving the KCI-induced response intact. We therefore propose that PKC not only potentiates but is also required for the NMDA-evoked elevation in cytosolic Ca2+ in mouse striatal neurons.