Albillos A, García A G, Olivera B, Gandía L
Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid, Arzobispo Morcillo, 4; E-28029 Madrid, Spain.
Pflugers Arch. 1996 Oct;432(6):1030-8. doi: 10.1007/s004240050231.
This study was undertaken to reassess the set of voltage-dependent Ca2+ channel subtypes expressed by bovine adrenal chromaffin cells maintained in primary cultures. Previous views on the pharmacology of such channels had to be revised in the light of the novel data which arose from the use in this study of low and high micromolar concentrations of omega-agatoxin IVA, and low (2 mM) and high (10 mM) concentrations of the charge carrier Ba2+. Whole-cell Ba2+ currents (IBa) through Ca2+ channels were elicited in voltage-clamped chromaffin cells, with a holding potential of -80 mV and depolarising pulses to 0 mV. Mean peak IBa was 425 pA in 2 mM Ba2+ (59 cells) and 787 pA in 10 mM Ba2+ (42 cells). In 2 mM Ba2+, omega-conotoxin MVIIC (3 microM) inhibited IBa by 79%; in 10 mM Ba2+, the blockade developed much more slowly and reached only 44%. A low concentration of omega-agatoxin IVA (20 nM) inhibited IBa by 9%; 2 microM inhibited IBa by 60%. This blockade was similar in low and high Ba2+ concentrations. After giving furnidipine (3 microM) and omega-conotoxin GVIA (1 microM), 2 microM omega-agatoxin IVA inhibited the remaining current (about 40-45%); this blockade was independent of the Ba2+ concentration. The current could be fully blocked by the cocktail furnidipine/omega-conotoxin GVIA/high omega-agatoxin IVA, both in low and high Ba2+ concentrations. The large Q-type channel component of IBa is blocked by micromolar concentrations of omega-agatoxin IVA and omega-conotoxin MVIIC. While solutions with a high Ba2+ concentration strongly delayed the development of blockade by omega-conotoxin MVIIC, the blockade by high concentrations of omega-agatoxin IVA was equally effective in solutions with a low or a high Ba2+ concentration. Hence, the use of appropriate Ba2+ and toxin concentrations in this study reveals that P-type Ca2+ channels are poorly expressed in bovine chromaffin cells; in contrast, a robust component of the current depends on Q-type Ca2+ channels. An R-type residual current is not present in these cells.
本研究旨在重新评估原代培养的牛肾上腺嗜铬细胞所表达的电压依赖性Ca2+通道亚型。鉴于本研究中使用低和高微摩尔浓度的ω-芋螺毒素IVA以及低（2 mM）和高（10 mM）浓度的载流子Ba2+所产生的新数据，此前关于此类通道药理学的观点必须加以修正。在电压钳制的嗜铬细胞中，通过Ca2+通道引发全细胞Ba2+电流（IBa），保持电位为 -80 mV，去极化脉冲至0 mV。在2 mM Ba2+（59个细胞）中，平均峰值IBa为425 pA，在10 mM Ba2+（42个细胞）中为787 pA。在2 mM Ba2+中，ω-芋螺毒素MVIIC（3 μM）使IBa抑制79%；在10 mM Ba2+中，阻断发展得慢得多，仅达到44%。低浓度的ω-芋螺毒素IVA（20 nM）使IBa抑制9%；2 μM使IBa抑制60%。这种阻断在低和高Ba2+浓度下相似。给予法尼地平（3 μM）和ω-芋螺毒素GVIA（1 μM）后，2 μM ω-芋螺毒素IVA抑制剩余电流（约40 - 45%）；这种阻断与Ba2+浓度无关。无论在低还是高Ba2+浓度下，电流都可被法尼地平/ω-芋螺毒素GVIA/高浓度ω-芋螺毒素IVA的混合物完全阻断。IBa的大Q型通道成分被微摩尔浓度的ω-芋螺毒素IVA和ω-芋螺毒素MVIIC阻断。虽然高Ba2+浓度的溶液强烈延迟了ω-芋螺毒素MVIIC的阻断发展，但高浓度ω-芋螺毒素IVA在低或高Ba2+浓度的溶液中阻断效果相同。因此，本研究中使用适当的Ba2+和毒素浓度表明，P型Ca2+通道在牛嗜铬细胞中表达不佳；相反，电流的一个强大成分依赖于Q型Ca2+通道。这些细胞中不存在R型残余电流。
