Gleitz J, Beile A, Peters T
Institute of Naturheilkunde, University Clinics Ulm, Germany.
Neuropharmacology. 1996 Feb;35(2):179-86. doi: 10.1016/0028-3908(95)00163-8.
The action of (+/-)-kavain on the veratridine, monensin and KCl-depolarization evoked increase in free cytosolic Ca2+ concentration ([Ca2+]i), and its influence on the release of endogenous glutamate from rat cerebrocortical synaptosomes were investigated. [Ca2+]i was fluorimetrically determined employing FURA as the Ca2+ sensitive fluorophore, and glutamate was detected by a continuous enzyme-linked fluorimetric assay. The incubation of synaptosomes in the presence of (+/-)-kavain up to a concentration of 500 mumol/l affected neither basal [Ca2+]i nor spontaneous release of glutamate, but dose-dependently reduced both veratridine-elevated [Ca2+]i (IC50 = 63.2 mumol/l) and glutamate-release (IC500 = 116.4 mumol/l). The inhibition of these parameters, attained with 500 mumol/l(+/-)-kavain, could be overcome by inducing an artificial Na+ influx, using monensin as a Na+ ionophore, An application of (+/-)-kavain after veratridine caused a decrease in veratridine-elevated [Ca2+]i, which was similar to the action of tetrodotoxin (TTX) with regard to time course, half-life of [Ca2+]i decline and the final steady state level of [Ca2+]i. Concomitantly, veratridine-induced glutamate-release was blocked. The results indicate that specific inhibition of voltage-dependent Na+ channels is a primary target of (+/-)-kavain, thus preventing a [Na+]i provoked increase in [Ca2+]i and glutamate-release. However, pathways related to the elevation of [Ca2+]i by [Na+]i itself, and the processes involved in normalization of elevated [Ca2+]i and glutamate-release downstream to enhanced [Ca2+]i, seems to be unaffected by (+/-)-kavain. Using KCl-depolarized synaptosomes, 400 mumol/l (+/-)-kavain reduced, in analogy to Aga-GI toxin, KCl-evoked [Ca2+]i and diminished the part of glutamate-exocytosis which is related to external Ca2+ to about 75% of control. At a concentration of 150 mumol/l, which is above the IC50 value necessary to block voltage-dependent Na+ channels, (+/-)-kavain affected neither basal nor the KCl-induced increase in [Ca2+]i. These results might suggest that (+/-)-kavain at concentrations sufficient to block Na+ channels completely. moderately inhibits the non-inactivating Ca2+ channels located on mammalian presynaptic nerve endings.
研究了（±）-卡瓦因对藜芦碱、莫能菌素和氯化钾去极化引起的游离胞质钙浓度（[Ca2+]i）升高的作用，以及它对大鼠大脑皮质突触体中内源性谷氨酸释放的影响。采用FURA作为Ca2+敏感荧光团，通过荧光法测定[Ca2+]i，并通过连续酶联荧光测定法检测谷氨酸。在浓度高达500μmol/L的（±）-卡瓦因存在下孵育突触体，既不影响基础[Ca2+]i，也不影响谷氨酸的自发释放，但剂量依赖性地降低了藜芦碱升高的[Ca2+]i（IC50 = 63.2μmol/L）和谷氨酸释放（IC500 = 116.4μmol/L）。用莫能菌素作为Na+离子载体诱导人工Na+内流，可以克服500μmol/L（±）-卡瓦因对这些参数的抑制作用。在藜芦碱作用后应用（±）-卡瓦因，可使藜芦碱升高的[Ca2+]i降低，在[Ca2+]i下降的时间进程、半衰期以及[Ca2+]i的最终稳态水平方面，这与河豚毒素（TTX）的作用相似。同时，藜芦碱诱导的谷氨酸释放被阻断。结果表明，特异性抑制电压依赖性Na+通道是（±）-卡瓦因的主要靶点，从而防止[Na+]i引起的[Ca2+]i升高和谷氨酸释放。然而，与[Na+]i本身引起的[Ca2+]i升高相关的途径，以及在[Ca2+]i升高下游使升高的[Ca2+]i和谷氨酸释放恢复正常的过程，似乎不受（±）-卡瓦因的影响。使用氯化钾去极化的突触体，400μmol/L的（±）-卡瓦因与Aga-GI毒素类似，可降低氯化钾引起的[Ca2+]i，并使与细胞外Ca2+相关的谷氨酸胞吐作用部分减少至对照的约75%。在150μmol/L的浓度下，该浓度高于阻断电压依赖性Na+通道所需的IC50值，（±）-卡瓦因既不影响基础[Ca2+]i，也不影响氯化钾诱导的[Ca2+]i升高。这些结果可能表明，（±）-卡瓦因在足以完全阻断Na+通道的浓度下，适度抑制位于哺乳动物突触前神经末梢的非失活Ca2+通道。
