Zeiske Wolfgang, Meyer Heiko, Wieczorek Helmut
Department of Biology/Chemistry, Division of Animal Physiology, University of Osnabrück, 49069 Osnabrück, Germany.
J Exp Biol. 2002 Feb;205(Pt 4):463-74. doi: 10.1242/jeb.205.4.463.
In lepidopteran larvae, three transport mechanisms are involved in the active and electrogenic K(+) secretion that occurs in the epithelial goblet cells of the midgut. These consist of (i) basolateral K(+) channels, allowing K(+) entry from the haemolymph into the cytosol, (ii) apical electrogenic K(+)/2H(+) antiporters, which are responsible for secondary active extrusion of K(+) from the cell into the gut lumen via the goblet cavity and (iii) apical V-ATPase-type proton pumps. The latter energize apical K(+) exit by building up a large, cavity-positive electrical potential that drives the antiporters. Net K(+) secretion (I(K)) can be measured as short-circuit current (I(sc)) across the in vitro midgut mounted in an Ussing chamber. We investigated the influence of protons on the transepithelial I(K) and the partial reactions of the basolateral K(+) permeability (P(K)) and the apical, lumped 'K(+) pump' current (I(P)) at various extra- and intracellular pH values. In particular, we wanted to know whether increased cellular acidity could counteract the reversible dissociation of the V-ATPase into its V(1) and V(o) parts, as occurs in yeast after glucose deprivation and in the midgut of Manduca sexta during starvation or moulting, thus possibly enhancing K(+) transport. When intact epithelia were perfused with high-[K(+)] (32 mmol l(-1)) salines with different pH values, I(K) was reversibly reduced when pH values fell below 6 on either side of the epithelium. Attempts to modify the intracellular pH by pulsing with NH(4)(+) or propionate showed that intracellular acidification caused a reduction in I(K) similar to that obtained in response to application of external protons. Treatment with azide, a well-known inhibitor of the mitochondrial ATP synthase, had the same effect as pulsing with ammonium or propionate with, however, much faster kinetics and higher reversibility. Breakdown of the basolateral or apical barrier using the antibiotic nystatin allowed the intracellular pH to be clamped to that of the saline facing the nystatin-treated epithelial border. Cell acidification achieved by this manipulation led to a reduction in both apical I(P) and basolateral P(K). The transepithelial I(K) showed an approximately half-maximal reduction at external pH values close to 5 in intact tissues, and a similar reduction in I(P) and P(K) values was seen at an intracellular pH of 5 in nystatin-permeabilised epithelia. Thus, the hypothesized V(1)V(o) stabilization by cell acidity is not reflected in the pH-sensitivity of I(P). Moreover, all components that transport K(+) are synchronously inhibited below pH 6. The significance of our findings for the midgut in vivo is discussed.
在鳞翅目幼虫中，中肠上皮杯状细胞中发生的主动且生电的钾离子（K⁺）分泌涉及三种转运机制。这些机制包括：（i）基底外侧的K⁺通道，允许K⁺从血淋巴进入细胞质；（ii）顶端生电的K⁺/2H⁺反向转运体，负责通过杯状腔将K⁺从细胞中二次主动转运到肠腔；（iii）顶端V-ATP酶型质子泵。后者通过建立一个大的、腔侧为正的电势来驱动反向转运体，从而为顶端K⁺的排出提供能量。净K⁺分泌（I(K)）可以通过安装在尤斯灌流小室中的体外中肠的短路电流（I(sc)）来测量。我们研究了在不同的细胞外和细胞内pH值下，质子对跨上皮I(K)以及基底外侧K⁺通透性（P(K)）的部分反应和顶端总的“K⁺泵”电流（I(P)）的影响。特别地，我们想知道细胞酸度的增加是否能抵消V-ATP酶可逆解离成其V₁和V₀部分的过程，就像葡萄糖剥夺后酵母中以及饥饿或蜕皮期间烟草天蛾中肠中发生的那样，从而可能增强K⁺的转运。当用不同pH值的高[K⁺]（32 mmol l⁻¹）盐溶液灌注完整上皮时，当上皮两侧的pH值降至6以下时，I(K)可逆性降低。尝试通过用NH₄⁺或丙酸盐脉冲来改变细胞内pH值表明，细胞内酸化导致I(K)降低，类似于施加外部质子时的情况。用叠氮化物（一种著名的线粒体ATP合酶抑制剂）处理产生了与用铵或丙酸盐脉冲相同的效果，然而，其动力学更快且可逆性更高。使用抗生素制霉菌素破坏基底外侧或顶端屏障可使细胞内pH值钳制在面对制霉菌素处理的上皮边界的盐溶液的pH值。通过这种操作实现的细胞酸化导致顶端I(P)和基底外侧P(K)均降低。在完整组织中，当外部pH值接近5时，跨上皮I(K)显示出约一半的最大降低，在制霉菌素通透的上皮中，当细胞内pH值为5时，I(P)和P(K)值也有类似的降低。因此，假设的细胞酸度对V₁V₀的稳定作用并未体现在I(P)的pH敏感性上。此外，所有转运K⁺的成分在pH低于6时均被同步抑制。本文讨论了我们的研究结果对体内中肠的意义。
