Muscarinic (Mi) receptor‐mediated inhibition of K+‐evoked [3H]‐noradrenaline release from human neuroblastoma (SH‐SY5Y) cells via inhibition of L‐ and N‐type Ca2+ channels

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Abstract
1 Human neuroblastoma (SH-SY5Y) cells were preincubated with [3H]-noradrenaline ([3H]-NA) in the presence of 0.2 mm pargyline to examine the modulation of K+-evoked [3H]-NA release by muscarinic agonists. 2 Release of [3H]-NA evoked by 4 min exposure to 100 mm K+ could be partially inhibited by 5 μm nifedipine and partially inhibited by 100 nm ω-conotoxin GVIA (ω-CgTx). When nifedipine and (ω-CgTx were added together, evoked release was inhibited by approximately 93%. 3 K+-evoked [3H]-NA release was inhibited by > 90% by pretreatment of cells for 2 min with muscarine, carbachol or oxotremorine methiodide (each at 300 μm). For muscarine, inhibition of evoked release was both time- and concentration-dependent and was reversible. Muscarine also inhibited [3H]-NA release evoked by veratridine (28 μm) and replacement of extracellular Ca2+ with Ba2+, but not that evoked by the Ca2+ ionophore, A23187 (19 μm). 4 Residual K+-evoked [3H]-NA release measured in the presence of either nifedipine (5 μm) or ω-CgTx (100 nm) was inhibited by muscarine with a similar potency as release evoked in the absence of either Ca2+ channel blocker. Pretreatment of cells for 16–24 h with pertussis toxin (200 ng ml−1) did not affect K+-evoked release per se or the ability of muscarine to inhibit such release. 5 Muscarinic inhibition of K+-evoked [3H]-NA release was potently antagonized by pirenzepine (pA2 8.14) and by hexahydrosiladiphenidol (pA2 9.03), suggesting the involvement of an M1 receptor. 6 Our results demonstrate that 100 mm K+-evoked release of [3H]-NA from the human neuroblastoma is mediated by activation of both L- and N-type Ca2+ channels. Activation of muscarinic M1 receptors can inhibit release via a pertussis toxin-insensitive mechanism which involves non-selective inhibition of L- and N-type Ca2+ channels.

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