MUSCARINIC (M(1)) RECEPTOR-MEDIATED INHIBITION OF K+-EVOKED [H-3] NORADRENALINE RELEASE FROM HUMAN NEUROBLASTOMA (SH-SY5Y) CELLS VIA INHIBITION OF L-TYPE AND N-TYPE CA2+ CHANNELS

1 Human neuroblastoma (SH-SY5Y) cells were preincubated with [H-3]-noradrenaline ([H-3]-NA) in the presence of 0.2 mM pargyline to examine the modulation of K+-evoked [H-3]-NA release by muscarinic agonists. 2 Release of [H-3]-NA evoked by 4 min exposure to 100 mM K+ could be partially inhibited by 5 mu M nifedipine and partially inhibited by 100 nM omega-conotoxin GVIA (omega-CgTx). When nifedipine and omega-CgTx were added together, evoked release was inhibited by approximately 93%. 3 K+-evoked [H-3]-NA release was inhibited by > 90% by pretreatment of cells for 2 min with muscarine, carbachol or oxotremorine methiodide (each at 300 mu M). For muscarine, inhibition of evoked release was both time- and concentration-dependent and was reversible. Muscarine also inhibited [H-3]-NA release evoked by veratridine (28 mu M) and replacement of extracellular Ca2+ with Ba2+, but not that evoked by the Ca2+ ionophore, A23187 (19 mu M). 4 Residual K+-evoked [H-3]-NA release measured in the presence of either nifedipine (5 mu M) or omega-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 [H-3]-NA release was potently antagonized by pirenzepine (pA(2) 8.14) and by hexahydrosiladiphenidol (pA(2) 9.03), suggesting the involvement of an M(1) 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 M(1) receptors can inhibit release via a pertussis toxin-insensitive mechanism which involves non-selective inhibition of L- and N-type Ca2+ channels.