State-dependent inhibition of Na+ currents by the neuroprotective agent 619C89 in rat hippocampal neurons and in a mammalian cell line expressing rat brain type IIA Na+ channels.
The compound 619C89 [4-amino-2-(4-methyl-1-piperazinyl)-5-(2,3,5-tricholorophenyl)-pyr imidine] is an effective neuroprotective agent in in vivo models of cerebral ischaemia. It has been suggested to act by inhibiting voltage-gated Na+ channels. To test this hypothesis, the action of 619C89 on recombinant rat brain type IIA Na+ channels expressed in Chinese hamster ovary cells and on native Na+ channels in acutely dissociated rat hippocampal neurons has been studied using whole-cell voltage-clamp recording techniques. In the cell line expressing type IIA Na+ channels, 619C89 caused a reversible inhibition of Na+ currents in a concentration- and voltage-dependent manner. A half-maximal inhibitory concentration (IC50) of approximately 50 microM was obtained at a holding potential of -90 mV whereas, with a conditioning prepulse to -60 mV for 30 s, the IC50 was reduced to 8 microM. Furthermore, the inhibition was markedly enhanced by a use-dependent action, which was dependent not only on the frequency of stimulation, but also on the duration (3.5-40 ms) of the pulses. Trains (10-50 Hz) of up to 60 depolarizing pulses of 0.7 ms duration did not evoke any use-dependent inhibition in the presence of 619C89, suggesting that this compound is not an open channel blocker. The voltage- and use-dependent inhibition by 619C89 was also observed on native Na+ channels in hippocampal neurons. 619C89 (10 microM) produced a small hyperpolarizing shift in the fast inactivation curve and a substantial (13 mV) hyperpolarizing shift in slow inactivation. The compound dramatically delayed the recovery from inactivation without affecting the development of inactivation. Moreover, 619C89 has no effect on the shape of the current-voltage relationship or on the voltage activation curve. These data indicate that 619C89 interacts selectively with the inactivated state of the Na+ channel with an estimated affinity of 3 microM. This primary action of 619C89 may underlie its neuroprotective effects.
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Neuroscience Research Group, Wellcome Research Laboratories, Beckenham, Kent, U.K.
Garthwaite
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D000818:Animals; D001921:Brain / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004553:Electric Conductivity; D004594:Electrophysiology; D005260:Female; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D018696:Neuroprotective Agents / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D010879:Piperazines / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D012964:Sodium / Q000502:physiology*
D018696:Neuroprotective Agents; D010879:Piperazines; D011743:Pyrimidines; D012964:Sodium; C081980:sipatrigine