Taglialatela M, Secondo A, Fresi A, Rosati B, Pannaccione A, Castaldo P, Giorgio G, Wanke E, Annunziato L
Unit of Pharmacology, Department of Neuroscience, University of Naples Federico II, School of Medicine, Via S. Pansini 5, I-80131, Naples, Italy.
Biochem Pharmacol. 2001 Nov 1;62(9):1229-38. doi: 10.1016/s0006-2952(01)00754-7.
In the present study, the effect of the blockade of membrane calcium channels activated by intracellular Ca(2+) store depletion on basal and depolarization-induced [3H]norepinephrine ([3H]NE) release from SH-SY5Y human neuroblastoma cells was examined. The second-generation H(1) receptor blockers astemizole, terfenadine, and loratadine, as well as the first-generation compound hydroxyzine, inhibited [3H]NE release induced by high extracellular K(+) concentration (K(+)) depolarization in a concentration-dependent manner (the IC(50)s were 2.3, 1.7, 4.8, and 9.4 microM, respectively). In contrast, the more hydrophilic second-generation H(1) receptor blocker cetirizine was completely ineffective (0.1-30 microM). The inhibition of high K(+)-induced [3H]NE release by H(1) receptor blockers seems to be related to their ability to inhibit Ca(2+) channels activated by Ca(i)(2+) store depletion (SOCs). In fact, astemizole, terfenadine, loratadine, and hydroxyzine, but not cetirizine, displayed a dose-dependent inhibitory action on the increase in intracellular Ca(2+) concentrations (Ca(2+)) obtained with extracellular Ca(2+) reintroduction after Ca(i)(2+) store depletion with thapsigargin (1 microM), an inhibitor of the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) pump. The rank order of potency for SOC inhibition by these compounds closely correlated with their inhibitory properties on depolarization-induced [3H]NE release from SH-SY5Y human neuroblastoma cells. Nimodipine (1 microM) plus omega-conotoxin (100 nM) did not interfere with the present model for SOC activation. In addition, the inhibition of depolarization-induced [3H]NE release does not seem to be attributable to the blockade of the K(+) currents carried by the K(+) channels encoded by the human Ether-a-Gogo Related Gene (I(HERG)) by these antihistamines. In fact, whole-cell voltage-clamp experiments revealed that the IC(50) for astemizole-induced hERG blockade is about 300-fold lower than that for the inhibition of high K(+)-induced [3H]NE release. Furthermore, current-clamp experiments in SH-SY5Y cells showed that concentrations of astemizole (3 microM) which were effective in preventing depolarization-induced [3H]NE release were unable to interfere with the cell membrane potential under depolarizing conditions (100 mM K(+)), suggesting that hERG K(+) channels do not contribute to membrane potential control during exposure to elevated K(+). Collectively, the results of the present study suggest that, in SH-SY5Y human neuroblastoma cells, the inhibition of SOCs by some second-generation antihistamines can prevent depolarization-induced neurotransmitter release.
在本研究中，检测了细胞内Ca(2+)储存耗竭激活的膜钙通道阻断对SH-SY5Y人神经母细胞瘤细胞基础及去极化诱导的[3H]去甲肾上腺素([3H]NE)释放的影响。第二代H(1)受体阻滞剂阿司咪唑、特非那定和氯雷他定，以及第一代化合物羟嗪，以浓度依赖性方式抑制高细胞外钾(K(+))浓度去极化诱导的[3H]NE释放(IC(50)分别为2.3、1.7、4.8和9.4 microM)。相比之下，亲水性更强的第二代H(1)受体阻滞剂西替利嗪则完全无效(0.1 - 30 microM)。H(1)受体阻滞剂对高K(+)诱导的[3H]NE释放的抑制作用似乎与其抑制由Ca(i)(2+)储存耗竭激活的钙通道(SOCs)的能力有关。事实上，阿司咪唑、特非那定、氯雷他定和羟嗪，但不包括西替利嗪，对在用毒胡萝卜素(1 microM，一种肌浆网 - 内质网钙ATP酶(SERCA)泵抑制剂)耗竭Ca(i)(2+)储存后再引入细胞外Ca(2+)时细胞内Ca(2+)浓度(Ca(2+))的升高表现出剂量依赖性抑制作用。这些化合物对SOC抑制的效力顺序与其对SH-SY5Y人神经母细胞瘤细胞去极化诱导的[3H]NE释放的抑制特性密切相关。尼莫地平(1 microM)加ω-芋螺毒素(100 nM)不干扰本研究中SOC激活模型。此外，这些抗组胺药对去极化诱导的[3H]NE释放的抑制作用似乎并非归因于对人Ether-a-Gogo相关基因编码的钾通道所携带的钾电流(I(HERG))的阻断。事实上，全细胞膜片钳实验表明，阿司咪唑诱导的hERG阻断的IC(50)比对高钾诱导的[3H]NE释放抑制的IC(50)低约300倍。此外，SH-SY5Y细胞中的电流钳实验表明，有效防止去极化诱导的[3H]NE释放的阿司咪唑浓度(3 microM)在去极化条件下(100 mM K(+))无法干扰细胞膜电位，这表明在暴露于升高的K(+)期间，hERG钾通道对膜电位控制没有贡献。总体而言，本研究结果表明，在SH-SY5Y人神经母细胞瘤细胞中，一些第二代抗组胺药对SOCs的抑制可防止去极化诱导的神经递质释放。
