Carbon-dioxide-induced exocytotic insertion of H+ pumps in turtle-bladder luminal membrane: role of cell pH and calcium

The contents of endocytic vesicles and other intracellular organelles (such as Golgi and microsomes) are acidified by an electrogenic proton-translocating ATPase that is remarkably similar to that found in urinary epithelia. We recently found that the number of H+ ATPases in the apical plasma membrane of these epithelia is regulated by exocytotic insertion of endocytic vesicles whose membranes contain this H+ pump. Carbon dioxide, a major stimulus for urinary acidification, causes rapid fusion of these vesicles with the luminal membrane, thereby inserting these pumps there and increasing the rate of net transepithelial H+ secretion; CO2 also inhibits endocytic retrieval of the pumps from the luminal membrane. Such reciprocal regulation of endocytosis and exocytosis by a physiological modulator makes this system particularly attractive for studying the cellular events regulating membrane fusion. Here we present evidence that CO2 induces exocytosis by a cascade of events, the first step of which is cytoplasmic acidification. Cell acidification then increases calcium activity, which causes the fusion event.     

pH对油酸水解废水酸化发酵过程的影响

pH对油酸水解废水酸化发酵影响的研究表明,pH不仅对酸化速率有很大影响,而且也会影响酸化产物的构成;不同pH值下酸化的主要产物是乙酸,但酸化的最佳pH值为6.5,此pH条件下VFA的产量最高可达12.53 g/L;在pH>7时,明显有丙酸生成。     

Arginine vasopressin enhances pHi regulation in the presence of HCO3- by stimulating three acid-base transport systems

Growth factors raise intracellular pH (pHi) by stimulating Na+/H+ exchange in the absence of HCO3-. In mutant cells that lack the Na+/H+ exchange activity, this alkalinization does not occur, and the cells do not proliferate without artificial elevation of pHi. It has therefore been widely suggested that an early pHi increase is a necessary signal for mitogenesis. In the presence of HCO3- however, growth factors fail to raise pHi in A431 cells, renal mesangial cells and 3T3 fibroblasts. In mesangial cells, arginine vasopressin (AVP) raises pHi in the absence of HCO3-, but lowers it when HCO3- is present; growth is stimulated under both conditions. We report here that, in the presence of HCO3-, AVP stimulates two potent HCO3- transporters, as well as the Na+/H+ exchanger. These are the Na+-dependent and Na+-independent Cl-/HCO3- exchangers. Our results indicate that AVP causes acidification in the presence of HCO3- because, at the resting pHi, it stimulates Na+-independent Cl-/HCO3- exchange (which lowers pHi) more than it stimulates the sum of Na+/H+ exchange and Na+-dependent Cl-/HCO3- exchange (both of which raise pHi). The stimulation of three acid-base transporters by the growth factor AVP greatly enhances the ability of the cell to regulate pHi.     

Viscous fingering of HCl through gastric mucin.

The HCl in the mammalian stomach is concentrated enough to digest the stomach itself, yet the gastric epithelium remains undamaged. One protective factor is gastric mucus, which forms a protective layer over the surface epithelium and acts as a diffusion barrier Bicarbonate ions secreted by the gastric epithelium are trapped in the mucus gel, establishing a gradient from pH 1-2 at the lumen to pH 6-7 at the cell surface. How does HCl, secreted at the base of gastric glands by parietal cells, traverse the mucus layer without acidifying it? Here we demonstrate that injection of HCl through solutions of pig gastric mucin produces viscous fingering patterns dependent on pH, mucin concentration and acid flow rate. Above pH 4, discrete fingers are observed, whereas below pH 4, HCl neither penetrates the mucin solution nor forms fingers. Our in vitro results suggest that HCl secreted by the gastric gland can penetrate the mucus gel layer (pH 5-7) through narrow fingers, whereas HCl in the lumen (pH 2) is prevented from diffusing back to the epithelium by the high viscosity of gastric mucus gel on the luminal side.     

上皮细胞膜离子转运的短路电流测定

短路电流技术是研究上皮组织膜离子转运的一种方法。当膜两侧浸浴液具有相同的成分时,消除跨膜电位差所需要的电流便等于被非共轭力所驱动的离子电流的代数和;此电流即为短路电流。本文目的是描述短路电流技术的细节和设计改进的上皮细胞膜灌流装置。并通过氨氯吡咪和去甲肾上腺素影响上皮细胞膜短路电流的实验,介绍测定短路电流的一般方法。实验表明氨氯吡咪加入到粘膜侧引起短路电流下降,膜电阻增加(电导减小),去甲肾上腺素加到浆膜侧则引起短路电流增加,膜电阻减小(电导增加)。     

Yeast plasma membrane ATPase is essential for growth and has homology with (Na+ + K+), K+- and Ca2+-ATPases

The plasma membrane ATPase of plants and fungi is a hydrogen ion pump. The proton gradient generated by the enzyme drives the active transport of nutrients by H+-symport. In addition, the external acidification in plants and the internal alkalinization in fungi, both resulting from activation of the H+ pump, have been proposed to mediate growth responses. This ATPase has a relative molecular mass (Mr) similar to those of the Na+-, K+- and Ca2+-ATPases of animal cells and, like these proteins, forms an aspartylphosphate intermediate. We have cloned, mapped and sequenced the gene encoding the yeast plasma membrane ATPase (PMA1) and report here that it maps to chromosome VII adjacent to LEU1. The strong homology between the amino-acid sequence encoded by PMA1 and those of (Na+ + K+), Na+-, K+- and Ca2+- ATPases is consistent with the notion that the family of cation pumps which form a phosphorylated intermediate evolved from a common ancestral ATPase. The function of the PMA1 gene is essential because a null mutation is lethal in haploid cells.     

Chloride/proton antiporter activity of mammalian CLC proteins ClC-4 and ClC-5

ClC-4 and ClC-5 are members of the CLC gene family, with ClC-5 mutated in Dent's disease, a nephropathy associated with low-molecular-mass proteinuria and eventual renal failure. ClC-5 has been proposed to be an electrically shunting Cl- channel in early endosomes, facilitating intraluminal acidification. Motivated by the discovery that certain bacterial CLC proteins are secondary active Cl-/H+ antiporters, we hypothesized that mammalian CLC proteins might not be classical Cl- ion channels but might exhibit Cl(-)-coupled proton transport activity. Here we report that ClC-4 and ClC-5 carry a substantial amount of protons across the plasma membrane when activated by positive voltages, as revealed by measurements of pH close to the cell surface. Both proteins are able to extrude protons against their electrochemical gradient, demonstrating secondary active transport. H+, but not Cl-, transport was abolished when a pore glutamate was mutated to alanine (E211A). ClC-0, ClC-2 and ClC-Ka proteins showed no significant proton transport. The muscle channel ClC-1 exhibited a small H+ transport that might be physiologically relevant. For ClC-5, we estimated that Cl- and H+ transport contribute about equally to the total charge movement, raising the possibility that the coupled Cl-/H+ transport of ClC-4 and ClC-5 is of significant magnitude in vivo.     

An H+-ATPase in opposite plasma membrane domains in kidney epithelial cell subpopulations

Vectorial solute transport by epithelia requires the polarized insertion of transport proteins into apical or basolateral plasmalemmal domains. In the specialized intercalated cells of the kidney collecting duct, the selective placement of an apical plasma membrane proton-pumping ATPase (H+-ATPase) and of a basolateral membrane anion-exchange protein results in transepithelial proton secretion. It is currently believed that amino-acid sequences of membrane proteins contain critical signalling regions involved in sorting these proteins to specific membrane domains. Recently, it was proposed that intercalated cells can reverse their direction of proton secretion under different acid-base conditions by redirecting proton pumps from apical to basolateral membranes, and anion exchangers from basolateral to apical membranes. But others have found that antibodies raised against the red cell anion-exchange protein (Band 3) only labelled intercalated cells at the basolateral plasma membrane, providing evidence against the model of polarity reversal. In this report, we have examined directly the distribution of proton pumps in kidney intercalated cells using specific polyclonal antibodies against subunits of a bovine kidney medullary H+-ATPase. We find that some cortical collecting duct intercalated cells have apical plasma membrane proton pumps, whereas others have basolateral pumps. This is the first direct demonstration of neighbouring epithelial cells maintaining opposite polarities of a transport protein. Thus, either subtle structural differences exist between proton pumps located at opposite poles of the cell, or factors other than protein sequence determine the polarity of H+-ATPase insertion.     

聚吡略/L-半胱胺酸/二硫醇混合自组装膜修饰电极制备的电化学研究

在不同比较L－半胺胺酸／二硫混合溶液中制备了混合自组装膜修饰电极。经过L－半胱胺酸的还原脱附后，利用循环伏安技术研究了吡咯在L－半胱胺酸／二硫醇自组装膜修饰电极表面聚合的过程和影响聚合的几个因素。实验结果表明，下面的几个因素影响着吡咯在自组装膜上的聚合：L－半胱胺酸在混合膜中的比例、支持电解质pH的大小以及聚合单体分子的大小。     

聚吡咯/L-半胱胺酸/二硫醇混合自组装膜修饰电极制备的电化学研究(英文)

在不同比例 L-半胱胺酸 /二硫醇混合溶液中制备了混合自组装膜修饰电极。经过 L-半胱胺酸的还原脱附后 ,利用循环伏安技术研究了吡咯在 L -半胱胺酸 /二硫醇自组装膜修饰电极表面聚合的过程和影响聚合的几个因素。实验结果表明 ,下面的几个因素影响着吡咯在自组装膜上的聚合 :L-半胱胺酸在混合膜中的比例、支持电解质 p H的大小以及聚合单体分子的大小。     

pH值对水稻土Cu^2＋静电吸附与专性吸附的影响

采用平衡液吸附法和CaCl2与HCl溶液的连续解吸法研究了太湖地区水稻土（黄泥土）在pH值为2～6范围内pH值对Cu^2＋的静电吸附和专性吸附的影响以及吸附过程土壤溶液pH值的变化.研究结果表明,吸附量随pH值升高而增加.低pH值条件下利于静电吸附,吸附过程H＋减少使平衡液pH值上升;高pH值条件下主要是专性吸附所控制,释放H＋使平衡液pH值下降.所以土壤酸化可以增加重金属的生物有效性,降低土壤对重金属的缓冲能力.     

Digital image processing of intracellular pH in gastric oxyntic and chief cells

Cytosolic pH (pHi) is a critically regulated determinant of intracellular function. Several mechanisms for pHi regulation in different tissues have been found, such as direct proton pumping, Na/H exchange, Cl/HCO3 exchange, NaHCO3 cotransport, and Na/H/Cl/HCO3 obligatorily linked. All these studies have used either single cells or cell populations assumed to be behaving homogeneously. Most tissues consist of more than one cell type, so it would be desirable to examine pHi regulation simultaneously in many identified individual cells, particularly in epithelia where disaggregation and purification of isolated cells destroys the normal distinction between luminal and serosal environments. We have used a pH-sensitive fluorescent dye, BCECF (2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein) and digital image processing to study pHi regulation simultaneously in the oxyntic cells (OC) and chief cells (CC) of gastric glands isolated from rabbit stomach. CCs become markedly more acidic upon removal of external Na (Nao), but pHi is restored rapidly on return to normal Nao, with or without Cl. Oxyntic cell pHi is much less affected by Nao. Conversely, OCs become strongly more alkaline on removal of external Cl (Clo), pHi being restored when Clo is replaced with or without Na, whereas CCs are relatively insensitive to Clo. Therefore, Na/H exchange is dominant over Cl/HCO3 exchange in CCs, but in the neighbouring OCs, Cl/HCO3 outweighs the Na/H mechanism, a heterogeneity that correlates with the functions of the two cell types.     

Increased pH and tumorigenicity of fibroblasts expressing a yeast proton pump

A common early response of eukaryotic cells to stimuli which activate their proliferation is an increase in intracellular pH (ref. 1). In animal cells this is caused by the activation of an Na+/H+ exchange system; in fungi and plants an H+-pumping ATPase is involved. The critical question is whether this intracellular alkalinization is merely coincident with the activation of cell proliferation or whether it is a regulatory signal. To increase intracellular pH bypassing the usual physiological stimuli (growth factors, hormones etc.) alkaline media or ammonia have been used in the past. Both approaches suffer from long-term toxicity effects and cannot be used in tumorigenic assays with whole organisms. We introduce here a more specific approach which involves expressing the gene for the yeast plasma membrane H+-ATPase in fibroblasts. The resulting cells have an elevated intracellular pH and acquire tumorigenic properties, suggesting that the yeast ATPase gene behaves as an oncogene in mammalian cells. These experiments support a crucial role of intracellular pH in the growth control of animal cells.     

Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells

On activation, T cells undergo distinct developmental pathways, attaining specialized properties and effector functions. T-helper (T(H)) cells are traditionally thought to differentiate into T(H)1 and T(H)2 cell subsets. T(H)1 cells are necessary to clear intracellular pathogens and T(H)2 cells are important for clearing extracellular organisms. Recently, a subset of interleukin (IL)-17-producing T (T(H)17) cells distinct from T(H)1 or T(H)2 cells has been described and shown to have a crucial role in the induction of autoimmune tissue injury. In contrast, CD4+CD25+Foxp3+ regulatory T (T(reg)) cells inhibit autoimmunity and protect against tissue injury. Transforming growth factor-beta (TGF-beta) is a critical differentiation factor for the generation of T(reg) cells. Here we show, using mice with a reporter introduced into the endogenous Foxp3 locus, that IL-6, an acute phase protein induced during inflammation, completely inhibits the generation of Foxp3+ T(reg) cells induced by TGF-beta. We also demonstrate that IL-23 is not the differentiation factor for the generation of T(H)17 cells. Instead, IL-6 and TGF-beta together induce the differentiation of pathogenic T(H)17 cells from naive T cells. Our data demonstrate a dichotomy in the generation of pathogenic (T(H)17) T cells that induce autoimmunity and regulatory (Foxp3+) T cells that inhibit autoimmune tissue injury.     

Cell membranes impermeable to NH3

Classically, there is a direct correlation between the lipophilic nature of a molecule and its rate of permeation across a biological membrane, so cell membranes should be more permeable to small, neutral molecules than they are to charged molecular species of similar size. Consequently, the distribution of NH+4 in biological systems is generally believed to be due to the rapid diffusion and equilibration of lipophilic NH3 across cell membranes and the accumulation of NH+4 to be governed by pH differences between compartments. Here we report that renal tubule cells from the medullary thick ascending limb of Henle have an apical membrane which is not only virtually impermeable to NH3, but is also highly permeable to NH+4. These remarkable properties have been incorporated into a model which explains how this renal epithelium can mediate vectorial movement of NH+4 between compartments of equal pH.     

罗丹明6G与罗丹明6G酰肼测汞的对比研究

以罗丹明6G(R6G)为原料合成了罗丹明6G酰肼(R6GH),并通过荧光法考察了R6G和R6GH酰肼选择性识别金属离子、适合的缓冲溶液体系、最佳缓冲溶液pH及浓度、R6G和R6GH的浓度、Hg~(2+)的浓度以及阳离子的干扰等七个因素的影响。实验确定了R6G测定Hg~(2+)的最佳条件为:0.01mol/L醋酸缓冲体系(pH=4.60),R6GH测定Hg~(2+)的最佳条件为:0.005 mol/L醋酸缓冲体系(pH=4.60)。通过14种阳离子的干扰实验发现,阳离子对R6G测定Hg~(2+)的影响大于对R6GH测定Hg~(2+)的影响。实验结果表明:R6GH比R6G能更好的识别Hg~(2+),最低检出限为5.0×10~(-12) mol/L。     

胞外ATP对VK2/E6E7细胞胞内pH的影响

 利用激光扫描共聚焦显微镜（Confocal microscope）采用pH依赖的荧光探针Snarf-4F测量细胞胞内pH(pHi)方法,发现胞外ATP能剂量依赖（10~1 000 μmol/L）地降低人阴道上皮细胞株VK2/E6E7细胞pHi。当胞外加入200 μmol/L ATP时,能快速地使VK2/E6E7细胞pHi降低,此降低的pHi在洗脱ATP后可迅速恢复。这些结果表明胞外ATP能引起VK2/E6E7细胞胞内酸化。     

The Cl-/H+ antiporter ClC-7 is the primary chloride permeation pathway in lysosomes

Lysosomes are the stomachs of the cell-terminal organelles on the endocytic pathway where internalized macromolecules are degraded. Containing a wide range of hydrolytic enzymes, lysosomes depend on maintaining acidic luminal pH values for efficient function. Although acidification is mediated by a V-type proton ATPase, a parallel anion pathway is essential to allow bulk proton transport. The molecular identity of this anion transporter remains unknown. Recent results of knockout experiments raise the possibility that ClC-7, a member of the CLC family of anion channels and transporters, is a contributor to this pathway in an osteoclast lysosome-like compartment, with loss of ClC-7 function causing osteopetrosis. Several mammalian members of the CLC family have been characterized in detail; some (including ClC-0, ClC-1 and ClC-2) function as Cl--conducting ion channels, whereas others act as Cl-/H+antiporters (ClC-4 and ClC-5). However, previous attempts at heterologous expression of ClC-7 have failed to yield evidence of functional protein, so it is unclear whether ClC-7 has an important function in lysosomal biology, and also whether this protein functions as a Cl- channel, a Cl-/H+ antiporter, or as something else entirely. Here we directly demonstrate an anion transport pathway in lysosomes that has the defining characteristics of a CLC Cl-/H+ antiporter and show that this transporter is the predominant route for Cl- through the lysosomal membrane. Furthermore, knockdown of ClC-7 expression by short interfering RNA can essentially ablate this lysosomal Cl-/H+ antiport activity and can strongly diminish the ability of lysosomes to acidify in vivo, demonstrating that ClC-7 is a Cl-/H+ antiporter, that it constitutes the major Cl- permeability of lysosomes, and that it is important in lysosomal acidification.     

Defective acidification of intracellular organelles in cystic fibrosis.

The phenotype of cystic fibrosis (CF) includes abnormalities in transepithelial transport of Cl- (refs 1-5), decreased sialylation and increased sulphation and fucosylation of glycoproteins, and lung colonization with Pseudomonas. It is not apparent how these abnormalities are interrelated, nor how they result from loss of function of the CF gene-encoded transmembrane regulator (CFTR). We have previously shown that that the pH of a secretory granule is regulated by the vesicular conductance for Cl- (ref. 11). Here we find defective acidification in CF cells of the trans-Golgi/trans-Golgi network, of prelysosomes and of endosomes as a result of diminished Cl- conductance. Sialytation of proteins and lipids is reduced and ligand traffic altered. These abnormalities can result from defective acidification because vacuolar pH regulates glycoprotein processing and ligand transport. The CF phenotype is similar to that of alkalinized cells and acidification-defective mutatants.