Muscarinic depolarization of SH-SY5Y human neuroblastoma cells as determined using oxonol V.
Membrane potential was measured in the suspension of SH-SY5Y cells using the anionic potentiometric probe, oxonol V. The relation of fluorescence to membrane potential was assessed by increasing the external [K+] in the presence of the K+ ionophore valinomycin. The response was linear in the range of 5 to 30 mM K+ (membrane potential change of approximately 40 mV). Muscarine increased the fluorescence indicating a depolarization. The competitive inhibitory constant (112 nM) of the muscarinic antagonist pirenzepine (5,11-dihydro-11-([4-methyl-1-piperazinyl]acetyl)-6H-pyrido[2,3-b] (1,4)benzodiazepin-6-one-dihydrochloride) suggests that Hm1 receptors are not involved. The protein kinase C inhibitor, GF 109203X (3-[1-(3-demethylaminopropyl)-indol-3-yl]-3-(indol-3-yl)-maleimide ), and a reduction of extracellular Na+ both produced an additive partial inhibition. The results suggest that muscarinic receptors depolarize these cells by separate Na(+)-dependent and -independent mechanisms, the Na(+)-independent mechanism being protein kinase C-dependent.
Kukkonen
J P
JP
Department of Biochemistry and Pharmacy, Abo Akademi University BioCity, Turku, Finland. jkukkone@aton.abo.fi
Hautala
R
R
Akerman
K E
KE
D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004594:Electrophysiology / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology; D007555:Isoxazoles; D008564:Membrane Potentials / Q000502:physiology; D009116:Muscarine / Q000494:pharmacology; D018721:Muscarinic Agonists / Q000494:pharmacology; D009447:Neuroblastoma; D011976:Receptors, Muscarinic / Q000502:physiology*; D013779:Tetrodotoxin / Q000494:pharmacology; D013997:Time Factors; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000502:physiology
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007555:Isoxazoles; D018721:Muscarinic Agonists; D011976:Receptors, Muscarinic; D013779:Tetrodotoxin; D004533:Egtazic Acid; C013633:oxonol V; D009116:Muscarine; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid