Binding of [3H]saxitoxin to the voltage-dependent Na channels and inhibition of 22Na influx in bovine adrenal medullary cells

Wada, Akihiko; Arita, Masahide; Kobayashi, Hideyuki; Izumi, Fujio

doi:10.1016/0306-4522(87)90293-4

Cited by 18 publications

(24 citation statements)

References 20 publications

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“…Riluzole did not reduce 22 Na influx caused by either α-scorpion venom, β-scorpion venom, or PbTx-3. α-scorpion venom, and β-scorpion venom, as well as PbTx-3 potentiated 22 Na influx caused by veratridine, a toxin acting at site 2 of S6I (Trainer et al 1996), by 2.8-, 2.0-, and 4.8-fold, respectively, as reported previously (Wada et al 1987(Wada et al , 1992. Veratridine-induced 22 Na influx was potentiated by either venom/toxin even in the presence of riluzole, although absolute values of 22 Na influx remained decreased in riluzole-treated cells, as compared to nontreated cells.…”

Section: Effects Of Riluzole Verapamil and Nicardipine On Veratridisupporting

confidence: 68%

Selective inhibition by riluzole of voltage-dependent sodium channels and catecholamine secretion in adrenal chromaffin cells

Yokoo

Shiraishi

Kobayashi

et al. 1998

Naunyn-Schmiedeberg's Arch Pharmacol

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We examined the effects of riluzole, a neuroprotective drug, on voltage-dependent Na channels, nicotinic receptors, and voltage-dependent Ca channels, as well as catecholamine secretion, in comparison with those of verapamil and nicardipine, in primary cultures of bovine adrenal chromaffin cells. Riluzole inhibited veratridine-induced 22Na influx via voltage-dependent Na channels even in the presence of ouabain, an inhibitor of Na,K-ATPase. Blockade of Na channels by riluzole was concentration-dependent with an IC50 of 5.3 microM. It was associated with a similar concentration-related reduction of veratridine-induced 45Ca influx via voltage-dependent Ca channels, and of catecholamine secretion. Riluzole had no effect on 45Ca influx caused by high K, which directly activates voltage-dependent Ca channels, and on nicotine-induced 22Na influx, which passes through the nicotinic receptors. Verapamil and nicardipine attenuated 22Na influx caused by veratridine or nicotine at the same concentrations as they suppressed high K-induced 45Ca influx. The inhibitory effect of riluzole on veratridine-induced 22Na influx disappeared at high concentrations of veratridine. A potentiation of veratridine (site 2 toxin)-induced 22Na influx caused by alpha-scorpion venom (site 3 toxin), beta-scorpion venom (site 4 toxin), or brevetoxin PbTx-3 (site 5 toxin), occurred in the presence of riluzole in the same manner as in control cells. These results suggest that riluzole binds to the veratridine site in voltage-dependent Na channels. It does not impair the cooperative interaction between the functional peptide segments of Na channels, but selectively inhibits gating of Na channels, thereby reducing Ca influx via Ca channels and catecholamine secretion. In contrast, verapamil and nicardipine suppress Na influx both Na channels and nicotinic receptors.

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Section: Effects Of Riluzole Verapamil and Nicardipine On Veratridisupporting

confidence: 68%

Selective inhibition by riluzole of voltage-dependent sodium channels and catecholamine secretion in adrenal chromaffin cells

Yokoo

Shiraishi

Kobayashi

et al. 1998

Naunyn-Schmiedeberg's Arch Pharmacol

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“…In cultured bovine adrenal chromaffin cells, veratridine causes a persistent influx of 22 Na + for at least 5 min, which passes through TTX/STX‐sensitive Na + channels ( Wada et al ., 1985a , 1985b ; 1987 ). As shown in Figure 1a, veratridine (100 μ M ) increased Na + influx by 182.9±12.9 nmol over the basal Na + influx (18.9±1.7 nmol) ( n =5) per 4×10 6 cells per 5 min.…”

Section: Resultsmentioning

confidence: 99%

“…Cells were incubated with 1–25 n M [ 3 H]‐STX in 1 ml KRP buffer at 4°C for 15 min in the absence (total binding) and presence (nonspecific binding) of 1 μ M TTX, then washed, solubilized, and counted for radioactivity; specific binding was calculated total binding minus nonspecific binding ( Wada et al ., 1987 ). A mere addition of NS‐7 to the binding assay medium per se did not alter [ 3 H]‐STX binding, as reported previously ( Shimidzu et al ., 1997 ).…”

Section: Methodsmentioning

confidence: 99%

Short‐ and long‐term differential effects of neuroprotective drug NS‐7 on voltage‐dependent sodium channels in adrenal chromaffin cells

Yokoo

Shiraishi

Kobayashi

et al. 2000

British J Pharmacology

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1 In cultured bovine adrenal chroman cells, NS-7 [4-(4-¯uorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride], a newly-synthesized neuroprotective drug, inhibited veratridine-induced 22 Na + in¯ux via voltage-dependent Na + channels (IC 50 =11.4 mM). The inhibition by NS-7 occurred in the presence of ouabain, an inhibitor of Na + ,K + ATPase, but disappeared at higher concentration of veratridine, and upon the washout of NS-7. 2 NS-7 attenuated veratridine-induced 45 Ca2+ in¯ux via voltage-dependent Ca 2+ channels (IC 50 =20.0 mM) and catecholamine secretion (IC 50 =25.8 mM). 3 Chronic (512 h) treatment of cells with NS-7 increased cell surface [ 3 H]-STX binding by 86% (EC 50 =10.5 mM; t 1/2 =27 h), but did not alter the K D value; it was prevented by cycloheximide, an inhibitor of protein synthesis, or brefeldin A, an inhibitor of vesicular transport from the trans-Golgi network, but was not associated with increased levels of Na + channel a-and b 1 -subunit mRNAs. 4 In cells subjected to chronic NS-7 treatment, 22 Na + in¯ux caused by veratridine (site 2 toxin), ascorpion venom (site 3 toxin) or b-scorpion venom (site 4 toxin) was suppressed even after the extensive washout of NS-7, and veratridine-induced 22 Na + in¯ux remained depressed even at higher concentration of veratridine; however, either a-or b-scorpion venom, or Ptychodiscus brevis toxin-3 (site 5 toxin) enhanced veratridine-induced 22 Na + in¯ux as in nontreated cells. 5 These results suggest that in the acute treatment, NS-7 binds to the site 2 and reversibly inhibits Na + channels, thereby reducing Ca 2+ channel gating and catecholamine secretion. Chronic treatment with NS-7 up-regulates cell surface Na + channels via translational and externalization events, but persistently inhibits Na + channel gating without impairing the cooperative interaction between the functional domains of Na + channels.

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“…Klugbauer et al (1995) documented that the TTX/STX‐sensitive human neuroendocrine‐type Na channel α‐subunit (hNE‐Na) was widely distributed only among a variety of the neural crest‐derived cells, including bovine adrenal medulla. In cultured bovine adrenal chromaffin cells, our previous studies showed that the Na channel α‐subunit is structurally different from the brain II/II A Na channel α‐subunit (Yamamoto et al, 1996); the Na channels are sensitive to TTX/STX (Wada et al, 1985, 1987) but modulated by conotoxin GIIIA (Wada et al, 1990) and brevetoxin (Wada et al, 1992; Yuhi et al, 1994) in a manner different from those of Na channels in brain, sympathetic neuron, and cardiac and skeletal muscles. In cultured bovine adrenal chromaffin cells, we have shown that antiepileptic valproic acid raised α‐ and β 1 ‐subunit mRNA levels as well as cell surface [ 3 H]STX binding (Yamamoto et al, 1997), whereas insulin (Yamamoto et al, 1996) and PKA (Yuhi et al, 1996) elevated [ 3 H]STX binding without altering α‐ and β 1 ‐subunit mRNA levels (Wada et al, 1998).…”

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confidence: 99%

Protein Kinase C‐α and ‐ε Down‐Regulate Cell Surface Sodium Channels via Differential Mechanisms in Adrenal Chromaffin Cells

Yanagita

Kobayashi²,

Yamamoto³

et al. 2000

Journal of Neurochemistry

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Abstract:In cultured bovine adrenal chromaffin cells, our [ 3 H]saxitoxin ([ 3 H]STX) binding, immunoblot, and northern blot analyses specified protein kinase C (PKC) isoform-specific posttranscriptional and posttranslational mechanisms that direct down-regulation of cell surface Na channels. Immunoblot analysis showed that among 11 PKC isoforms, adrenal chromaffin cells contained only conventional (c)PKC-␣, novel (n)PKC-⑀, and atypical (a)PKC-. Treatment of adrenal chromaffin cells with 100 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) or 100 nM phorbol 12,13-dibutyrate (PDBu) caused a rapid (Ͻ15 min) and sustained (Ͼ15 h) translocation of PKC-␣ and -⑀ (but not -) from cytosol to membranes, whereas a biologically inactive 4␣-TPA had no effect. Thymeleatoxin (TMX), an activator of cPKC, produced similar membrane association of only PKC-␣ at 100 nM, with the potency of TMX being comparable with those of TPA and PDBu. Treatment with either 100 nM TPA or 100 nM TMX reduced cell surface [ 3 H]STX binding to a comparable extent at 3, 6, and 12 h, whereas TPA lowered the binding to a greater extent than TMX at 15, 18, and 24 h; at 15 h, Gö 6976, a specific inhibitor of cPKC, completely blocked TMX-induced decrease of [ 3 H]STX binding while preventing by merely 57% TPA-induced decrease of [ 3 H]STX binding. Treatment with 100 nM TPA lowered the Na channel ␣-subunit mRNA level between 3 and 12 h, with its maximum 52% fall at 6 h, and it was accompanied by a subsequent 61% rise of the ␤ 1 -subunit mRNA level at 24 h. Gö 6976 failed to prevent TPA-induced reduction of the ␣-subunit mRNA level; TMX did not change the ␣-and ␤ 1 -subunit mRNA levels throughout the 24-h treatment. Brefeldin A, an inhibitor of vesicular exit from the trans-Golgi network, augmented TPA-and TMX-induced decrease of [ 3 H]STX binding at 1 and 3 h. Our previous and present studies suggest that PKC down-regulates cell surface Na channels without altering the allosteric gating of Na channels via PKC isoform-specific mechanisms; cPKC-␣ promotes Na channel internalization, whereas nPKC-⑀ decreases the ␣-subunit mRNA level by shortening the half-life of ␣-subunit mRNA without changing its gene transcription.

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Binding of [3H]saxitoxin to the voltage-dependent Na channels and inhibition of 22Na influx in bovine adrenal medullary cells

Cited by 18 publications

References 20 publications

Selective inhibition by riluzole of voltage-dependent sodium channels and catecholamine secretion in adrenal chromaffin cells

Selective inhibition by riluzole of voltage-dependent sodium channels and catecholamine secretion in adrenal chromaffin cells

Short‐ and long‐term differential effects of neuroprotective drug NS‐7 on voltage‐dependent sodium channels in adrenal chromaffin cells

Protein Kinase C‐α and ‐ε Down‐Regulate Cell Surface Sodium Channels via Differential Mechanisms in Adrenal Chromaffin Cells

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