Muscarinic (M1) receptor-mediated inhibition of K(+)-evoked [3H]-noradrenaline release from human neuroblastoma (SH-SY5Y) cells via inhibition of L- and N-type Ca2+ channels.
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 microM 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 [3H]-NA release was inhibited by > 90% by pretreatment of cells for 2 min with muscarine, carbachol or oxotremorine methiodide (each at 300 microM). 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 microM) and replacement of extracellular Ca2+ with Ba2+, but not that evoked by the Ca2+ ionophore, A23187 (19 microM). 4. Residual K(+)-evoked [3H]-NA release measured in the presence of either nifedipine (5 microM) 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 [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 Ml receptors can inhibit release via a pertussis toxin-insensitive mechanism which involves non-selective inhibition of L- and N-type Ca2+ channels.
Department of Pharmacology, University of Leeds.
R L
RL
McDonald
P F
PF
Vaughan
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Peers
D002121:Calcium Channel Blockers / Q000494:pharmacology*; D006801:Humans; D018721:Muscarinic Agonists / Q000494:pharmacology; D018727:Muscarinic Antagonists / Q000494:pharmacology; D009447:Neuroblastoma / Q000378:metabolism*; D009543:Nifedipine / Q000494:pharmacology; D009638:Norepinephrine / Q000378:metabolism*; D010455:Peptides / Q000494:pharmacology; D037342:Pertussis Toxin; D011188:Potassium / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D011976:Receptors, Muscarinic / Q000502:physiology*; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D020866:omega-Conotoxin GVIA
D002121:Calcium Channel Blockers; D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D010455:Peptides; D011976:Receptors, Muscarinic; D010566:Virulence Factors, Bordetella; D020866:omega-Conotoxin GVIA; D037342:Pertussis Toxin; D009543:Nifedipine; D011188:Potassium; D009638:Norepinephrine