Muscarine inhibits omega-conotoxin-sensitive calcium channels in a voltage- and time-dependent mode in the human neuroblastoma cell line SH-SY5Y.
1. Calcium channel modulation by muscarine was investigated in cultured human neuroblastoma SH-SY5Y cells using the whole cell variant of the patch-clamp technique. 2. In SH-SY5Y cells, omega-conotoxin (omega-CgTx)-sensitive, high-voltage-activated Ca2+ current density gradually increased from approximately 1 microA/cm2 in undifferentiated cells to 4 microA/cm2 after approximately 20 days of application of the differentiating agent retinoic acid. 3. In differentiated SH-SY5Y cells, muscarine reversibly decreased high-voltage-activated omega-CgTx-sensitive Ba2+ currents in a concentration-dependent way. Maximum inhibition (approximately 65%) measured at 0 mV was obtained with 30 microM muscarine and the IC50 was 1 microM. 4. Current inhibition obtained with 30 microM muscarine was suppressed by the specific M2 and M3 antagonists AFDX-116 and 4-diphenylacetoxy-N-methyl-piperidine methiodide (0.3 microM; 87% suppression), but not by the M1 antagonist pirenzepine. 5. Muscarine-induced current suppression was prevented by pretreatment of the cells with pertussis toxin and mimicked by intracellular application of guanosine 5'-[gamma-thio]triphosphate. 6. In several cells, muscarinic inhibition was characterized by a clear slowdown of Ba2+ current activation at low test potentials. Both inhibition and slowdown of activation were attenuated at more positive potentials and could be partially relieved by strong conditioning depolarizations. 7. These results indicate that muscarinic inhibition of omega-CgTx-sensitive Ca2+ channel current occurs through activation of specific muscarinic receptors and the modulatory mechanism operates through activation of a guanosine 5'-triphosphate-binding protein sensitive to pertussis toxin. Our results suggest that a blocking molecule interacts in a voltage-dependent manner with the Ca2+ channel without involvement of intracellular Ca2+ mobilization or activation of protein kinase C or cyclic nucleotide protein kinases. A simple model describing the reactions involved is proposed.
Toselli
M
M
Istituto di Fisiologia Generale, Universitá di Pavia, Italy.
Perin
P
P
Taglietti
V
V
D002121:Calcium Channel Blockers / Q000494:pharmacology*; D015220:Calcium Channels / Q000187:drug effects* / Q000502:physiology; D004594:Electrophysiology; D006801:Humans; D007700:Kinetics; D009116:Muscarine / Q000494:pharmacology*; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D010455:Peptides / Q000494:pharmacology*; D013997:Time Factors; D014407:Tumor Cells, Cultured; D020866:omega-Conotoxin GVIA
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D010455:Peptides; D009116:Muscarine; D020866:omega-Conotoxin GVIA