R G Vickery, S M Amagasu, R Chang, N Mai, E Kaufman, J Martin, J Hembrador, M D O'Keefe, C Gee, D Marquess, J A M Smith
{"title":"利用膜电位敏感染料和FLIPR比较大鼠Na(V)1.8与大鼠Na(V)1.2a和人Na(V)1.5电压门控钠通道亚型的药理学性质。","authors":"R G Vickery, S M Amagasu, R Chang, N Mai, E Kaufman, J Martin, J Hembrador, M D O'Keefe, C Gee, D Marquess, J A M Smith","doi":"","DOIUrl":null,"url":null,"abstract":"<p><p>A novel, membrane potential sensitive dye and a fluorescence imaging plate reader (FLIPR) have been used to characterize the pharmacological properties of rat Na(v)1.8 voltage-gated sodium channels (VGSC) in parallel with rat Na(v)1.2a and human Na(v)1.5 VGSC subtypes, respectively. The sensitivity of recombinant Na(v)1.2a-CHO, Na(v)1.5-293-EBNA, and Na(v)1.8-F-11 cells to VGSC activators was subtype dependent. Veratridine evoked depolarization of Na(v)1.2a-CHO and Na(v)1.5-293-EBNA cells with pEC(50) values of 4.78 +/- 0.13 and 4.84 +/- 0.12, respectively (n = 3), but had negligible effect on Na(v)1.8-F-11 cells (pEC(50) < 4.5). Type I pyrethroids were without significant effect at all subtypes. In contrast, the type II pyrethroids deltamethrin and fenvalerate evoked direct depolarization of Na(v)1.8-F-11 and Na(v)1.5-293-EBNA cells. Deltamethrin potentiated the veratridine-evoked response in Na(v)1.8-F-11 cells by > or =20-fold, in contrast to a <or =3-fold potentiation of the response in Na(v)1.2a, and Na(v)1.5 cells. Tetrodotoxin (TTX) inhibited VGSC activator-evoked depolarization of Na(v)1.8-F-11 cells with a biphasic concentration-response curve. The calculated pIC(50) values were 8.05 +/- 0.25 (n = 4) and 4.32 +/- 0.21 (n = 4), corresponding to TTX inhibition of endogenous TTX-sensitive (TTX-S), and recombinant Na(v)1.8 TTX-resistant (TTX-R) VGSCs, respectively. With the exception of TTX, the potencies of a number of ion channel blockers for the Na(v)1.8, Na(v)1.2a, and Na(v)1.5 VGSC subtypes were similar. In summary, these high-throughput FLIPR assays represent a valuable tool for the determination of the relative potencies of compounds at different VGSC subtypes and may prove useful for the identification of novel subtype-selective inhibitors.</p>","PeriodicalId":20928,"journal":{"name":"Receptors & channels","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2004-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Comparison of the pharmacological properties of rat Na(V)1.8 with rat Na(V)1.2a and human Na(V)1.5 voltage-gated sodium channel subtypes using a membrane potential sensitive dye and FLIPR.\",\"authors\":\"R G Vickery, S M Amagasu, R Chang, N Mai, E Kaufman, J Martin, J Hembrador, M D O'Keefe, C Gee, D Marquess, J A M Smith\",\"doi\":\"\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>A novel, membrane potential sensitive dye and a fluorescence imaging plate reader (FLIPR) have been used to characterize the pharmacological properties of rat Na(v)1.8 voltage-gated sodium channels (VGSC) in parallel with rat Na(v)1.2a and human Na(v)1.5 VGSC subtypes, respectively. The sensitivity of recombinant Na(v)1.2a-CHO, Na(v)1.5-293-EBNA, and Na(v)1.8-F-11 cells to VGSC activators was subtype dependent. Veratridine evoked depolarization of Na(v)1.2a-CHO and Na(v)1.5-293-EBNA cells with pEC(50) values of 4.78 +/- 0.13 and 4.84 +/- 0.12, respectively (n = 3), but had negligible effect on Na(v)1.8-F-11 cells (pEC(50) < 4.5). Type I pyrethroids were without significant effect at all subtypes. In contrast, the type II pyrethroids deltamethrin and fenvalerate evoked direct depolarization of Na(v)1.8-F-11 and Na(v)1.5-293-EBNA cells. Deltamethrin potentiated the veratridine-evoked response in Na(v)1.8-F-11 cells by > or =20-fold, in contrast to a <or =3-fold potentiation of the response in Na(v)1.2a, and Na(v)1.5 cells. Tetrodotoxin (TTX) inhibited VGSC activator-evoked depolarization of Na(v)1.8-F-11 cells with a biphasic concentration-response curve. The calculated pIC(50) values were 8.05 +/- 0.25 (n = 4) and 4.32 +/- 0.21 (n = 4), corresponding to TTX inhibition of endogenous TTX-sensitive (TTX-S), and recombinant Na(v)1.8 TTX-resistant (TTX-R) VGSCs, respectively. With the exception of TTX, the potencies of a number of ion channel blockers for the Na(v)1.8, Na(v)1.2a, and Na(v)1.5 VGSC subtypes were similar. In summary, these high-throughput FLIPR assays represent a valuable tool for the determination of the relative potencies of compounds at different VGSC subtypes and may prove useful for the identification of novel subtype-selective inhibitors.</p>\",\"PeriodicalId\":20928,\"journal\":{\"name\":\"Receptors & channels\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2004-01-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Receptors & channels\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Receptors & channels","FirstCategoryId":"1085","ListUrlMain":"","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
Comparison of the pharmacological properties of rat Na(V)1.8 with rat Na(V)1.2a and human Na(V)1.5 voltage-gated sodium channel subtypes using a membrane potential sensitive dye and FLIPR.
A novel, membrane potential sensitive dye and a fluorescence imaging plate reader (FLIPR) have been used to characterize the pharmacological properties of rat Na(v)1.8 voltage-gated sodium channels (VGSC) in parallel with rat Na(v)1.2a and human Na(v)1.5 VGSC subtypes, respectively. The sensitivity of recombinant Na(v)1.2a-CHO, Na(v)1.5-293-EBNA, and Na(v)1.8-F-11 cells to VGSC activators was subtype dependent. Veratridine evoked depolarization of Na(v)1.2a-CHO and Na(v)1.5-293-EBNA cells with pEC(50) values of 4.78 +/- 0.13 and 4.84 +/- 0.12, respectively (n = 3), but had negligible effect on Na(v)1.8-F-11 cells (pEC(50) < 4.5). Type I pyrethroids were without significant effect at all subtypes. In contrast, the type II pyrethroids deltamethrin and fenvalerate evoked direct depolarization of Na(v)1.8-F-11 and Na(v)1.5-293-EBNA cells. Deltamethrin potentiated the veratridine-evoked response in Na(v)1.8-F-11 cells by > or =20-fold, in contrast to a
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