Instituto Teófilo Hernando, Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid, Av. Arzobispo Morcillo 4, E-28029 Madrid, Spain.
Am J Physiol Cell Physiol. 2011 Mar;300(3):C610-23. doi: 10.1152/ajpcell.00380.2010. Epub 2010 Dec 15.
Modulation of high-threshold voltage-dependent calcium channels by neurotransmitters has been the subject of numerous studies in cultures of neurons and chromaffin cells. However, no studies on such modulation exist in chromaffin cells in their natural environment, the intact adrenal medullary tissue. Here we performed such a study in voltage-clamped chromaffin cells of freshly prepared mouse adrenal slices under the whole cell configuration of the patch-clamp technique. The subcomponents of the whole cell inward Ca(2+) current (I(Ca)) accounted for 49% for L-, 28% for N-, and 36% for P/Q-type channels. T-type Ca(2+) channels or residual R-type Ca(2+) currents were not seen. However, under the perforated-patch configuration, 20% of I(Ca) accounted for a toxin-resistant R-type Ca(2+) current. Exogenously applied ATP and methionine-enkephalin (Met-enk) inhibited I(Ca) by 33%. Stop-flow and Ca(2+) replacement by Ba(2+), which favored the release of endogenous ATP and opioids, also inhibited I(Ca), with no changes in activation or inactivation kinetics. This inhibition was partially voltage independent and insensitive to prepulse facilitation. Furthermore, in about half of the cells, suramin and naloxone augmented I(Ca) in the absence of exogenous application of ATP/Met-enk. No additional modulation of I(Ca) was obtained after bath application of exogenous ATP and opioids to these already inhibited cells. Augmentation of I(Ca) was also seen upon intracellular dialysis of guanosine 5'-[β-thio]diphosphate (GDPβS), indicating the existence in the intact slice of a tonic inhibition of I(Ca) in resting conditions. These results suggest that in the intact adrenal tissue a tonic inhibition of I(Ca) exists, mediated by purinergic and opiate receptors.
神经递质对高阈值电压依赖性钙通道的调制一直是神经元和嗜铬细胞培养物中众多研究的主题。然而，在完整的肾上腺髓质组织中，即在嗜铬细胞的自然环境中，没有关于这种调制的研究。在这里，我们在新鲜制备的小鼠肾上腺切片的电压钳制嗜铬细胞中进行了这样的研究，使用全细胞膜片钳技术的全细胞构型。全细胞内向 Ca(2+)电流（I(Ca)）的亚成分占 L 型的 49%，N 型的 28%，和 P/Q 型通道的 36%。未观察到 T 型 Ca(2+)通道或残留的 R 型 Ca(2+)电流。然而，在穿孔膜片钳构型下，20%的 I(Ca)是一种对毒素有抗性的 R 型 Ca(2+)电流。外源性应用 ATP 和甲硫氨酸脑啡肽（Met-enk）抑制 I(Ca)达 33%。停止流动和 Ca(2+)被 Ba(2+)取代，有利于内源性 ATP 和阿片样物质的释放，也抑制了 I(Ca)，但不改变激活或失活动力学。这种抑制部分是电压非依赖性的，并且对预脉冲易化不敏感。此外，在大约一半的细胞中，苏拉明和纳洛酮在没有外源性应用 ATP/Met-enk 的情况下增强了 I(Ca)。将外源性 ATP 和阿片样物质应用于这些已经被抑制的细胞后，对 I(Ca)没有获得额外的调制。在这些已经被抑制的细胞中，通过细胞内透性酶二磷酸鸟苷 5'-[β-硫]（GDPβS）灌流也观察到 I(Ca)的增强，表明在完整的切片中存在静息状态下对 I(Ca)的紧张性抑制。这些结果表明，在完整的肾上腺组织中，存在由嘌呤能和阿片受体介导的 I(Ca)紧张性抑制。
