Groschner K, Graier W F, Kukovetz W R
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Austria.
Circ Res. 1994 Aug;75(2):304-14. doi: 10.1161/01.res.75.2.304.
The nature of the membrane currents mediating agonist-induced Ca2+ entry and enhanced nitric oxide (NO) production in endothelial cells is still unclear. Using both perforated-patch and conventional whole-cell clamp technique, we have studied the membrane response associated with histamine stimulation of human vascular endothelial cells. In perforated-patch experiments, the initial histamine (10 mumol/L)-induced current reversed close to the K+ equilibrium potential and was blocked by tetrabutylammonium ions (TBA, 10 mmol/L). In addition, a TBA-insensitive current that developed slowly in the presence of histamine was recorded. This delayed histamine-induced current reversed close to neutral potential and was inhibited by SK&F 96365 (25 mumol/L), a putative blocker of receptor-operated Ca2+ channels. Similar histamine effects were observed in conventional whole-cell experiments using pipette solutions with low Ca(2+)-buffering capacity. Strong buffering of intracellular free Ca2+ suppressed the initial, but not the delayed, current response. The delayed component of histamine-induced current was substantially inhibited by the Cl- channel blocker N-phenylanthranilic acid (NPA, 100 mumol/L), and an eightfold change in the Cl- gradient shifted the reversal potential of this current by 30 mV. In Cl(-)-free solutions, histamine induced an SK&F 96365-sensitive NPA-resistant current, which, according to reversal potential measurements in 20 mmol/L extracellular Ca2+, corresponded to a cation conductance with 13- to 25-fold selectivity for Ca2+ over K+. Both SK&F 96365 and TBA strongly suppressed histamine-induced rises in intracellular free Ca2+ and cellular cGMP levels, whereas NPA did not. Our results provide the first demonstration that three distinct ionic conductances contribute to the histamine-induced membrane response of endothelial cells. It is suggested that histamine induces a Cl- conductance that is apparently not involved in Ca2+ homeostasis and regulation of NO biosynthesis, while, in parallel, joint activation of a rapidly induced K+ permeability and a slowly developing cation permeability mediate Ca2+ entry and stimulation of endothelial NO production.
介导内皮细胞中激动剂诱导的Ca2+内流和一氧化氮（NO）生成增强的膜电流性质仍不清楚。我们使用穿孔膜片钳和传统全细胞膜片钳技术，研究了组胺刺激人血管内皮细胞相关的膜反应。在穿孔膜片钳实验中，初始组胺（10 μmol/L）诱导的电流在接近K+平衡电位处反转，并被四丁基铵离子（TBA，10 mmol/L）阻断。此外，记录到一种在组胺存在下缓慢形成的对TBA不敏感的电流。这种延迟的组胺诱导电流在接近中性电位处反转，并被SK&F 96365（25 μmol/L）抑制，SK&F 96365是一种推测的受体操纵性Ca2+通道阻滞剂。在使用低Ca2+缓冲能力移液管溶液的传统全细胞实验中观察到类似的组胺效应。细胞内游离Ca2+的强缓冲作用抑制了初始电流反应，但未抑制延迟电流反应。组胺诱导电流的延迟成分被Cl-通道阻滞剂N-苯基邻氨基苯甲酸（NPA，100 μmol/L）显著抑制，Cl-梯度的八倍变化使该电流的反转电位移动了30 mV。在无Cl-溶液中，组胺诱导一种对SK&F 96365敏感、对NPA耐药的电流，根据在20 mmol/L细胞外Ca2+中的反转电位测量，该电流对应于一种对Ca2+的选择性比对K+高13至25倍的阳离子电导。SK&F 96365和TBA都强烈抑制组胺诱导的细胞内游离Ca2+升高和细胞cGMP水平，而NPA则没有。我们的结果首次证明三种不同的离子电导参与了组胺诱导的内皮细胞膜反应。提示组胺诱导一种Cl-电导，该电导显然不参与Ca2+稳态和NO生物合成的调节，同时，快速诱导的K+通透性和缓慢形成的阳离子通透性的联合激活介导Ca2+内流并刺激内皮细胞NO生成。
