Institute for Veterinary Physiology and Biochemistry, Justus-Liebig-University Giessen, Giessen, Germany.
Department of Cell Biology, Physiology and Immunology, Veterinary Faculty, Universitat Autonoma de Barcelona, Barcelona, Spain.
Br J Pharmacol. 2019 May;176(9):1328-1340. doi: 10.1111/bph.14638. Epub 2019 Apr 9.
ACh exerts its actions via nicotinic (nAChR) and muscarinic receptors. In the peripheral nervous system, ionotropic nAChR mediate responses in excitable cells. However, recent studies demonstrate the expression of nAChR in the colonic epithelium, which are coupled to an induction of Cl secretion via activation of the Na -K -pump.
In order to find out whether these epithelial nAChR function as ionotropic receptors, intracellular microelectrode and imaging experiments were performed in isolated crypts from rat colon. Apically permeabilized epithelia were used to measure pump current across the basolateral membrane.
Imaging experiments with the Na -sensitive dye SBFI revealed that nicotine induced a decrease in the cytosolic Na concentration concomitant with a fall in the cytosolic Ca concentration in about 50% of the cells. as shown in fura-2 experiments. Nicotine hyperpolarized the membrane by 6.4 ± 2.1 mV. These observations contradict the assumption that epithelial nAChR function as ligand-gated non-selective cation channels. The decrease in the cytosolic Na concentration was strongly delayed, when the Na -K -pump was inhibited by scilliroside. Ussing chamber experiments revealed a strong dependence of the nicotine-induced pump current on the presence of Ca , and chelation of cytosolic Ca with BAPTA prevented the fall in the cytosolic Na concentration in SBFI-loaded crypts. Inhibition of PKC with GF 109203X or Goe 6983 significantly reduced the nicotine-induced pump current.
These results suggest that epithelial nAChR activate the Na -K -pump via a PKC dependent on a sufficient cytosolic Ca concentration.
乙酰胆碱（ACh）通过烟碱型（nAChR）和毒蕈碱型受体发挥作用。在外周神经系统中，离子型 nAChR 介导可兴奋细胞的反应。然而，最近的研究表明 nAChR 在结肠上皮细胞中表达，通过激活 Na+-K+-泵诱导 Cl-分泌。
为了确定这些上皮 nAChR 是否作为离子型受体发挥作用，我们在大鼠结肠分离的隐窝中进行了细胞内微电极和成像实验。用顶端通透的上皮来测量穿过基底外侧膜的泵电流。
用 Na+敏感染料 SBFI 进行的成像实验表明，尼古丁诱导细胞内 Na+浓度下降，同时伴随胞浆 Ca2+浓度下降，约 50%的细胞中发生这种情况，正如 fura-2 实验所示。尼古丁使膜超极化 6.4±2.1 mV。这些观察结果与上皮 nAChR 作为配体门控非选择性阳离子通道的假设相矛盾。当用 scilliroside 抑制 Na+-K+-泵时，细胞内 Na+浓度的下降被强烈延迟。Ussing 室实验表明，尼古丁诱导的泵电流强烈依赖于 Ca2+的存在，并且用 BAPTA 螯合胞浆 Ca2+可防止 SBFI 加载隐窝中细胞内 Na+浓度下降。用 GF 109203X 或 Goe 6983 抑制 PKC 显著减少了尼古丁诱导的泵电流。
这些结果表明，上皮 nAChR 通过 PKC 依赖性、依赖于足够胞浆 Ca2+浓度的机制激活 Na+-K+-泵。
