Plasma membrane proton-ATPase of a turtle bladder epithelial cell line

Urinary acidification by the turtle bladder is mediated by a proton ATPase located in the apical membrane. The present study describes a proton ATPase in the plasma membrane of a cell line of turtle bladder epithelial cells. In the presence of ouabain to inhibit Na+,K+-ATPase and in the absence of Ca2+ to inhibit Ca2+-ATPase, we measured ATPase activity of the plasma membranes of the cultured cells. This ATPase was resistant to oligomycin but sensitive to dicyclohexylcarbodiimide, N-ethylmaleimide, and vanadate. In the presence of ATP, the ATPase was capable of acidification as assessed by quenching of acridine orange. Acidification could not be elicited by other nucleotides (GTP, UTP). Acidification was inhibited by dicyclohexylcarbodiimide, N-ethylmaleimide, and vanadate but was not affected by replacement of Na+ by K+. The acidification response was dependent on the presence of chloride, abolished in the presence of gluconate, and inhibited partially by nitrate. Experiments utilizing the voltage-sensitive dye 3,3'-dipropylthiodicarbocyanine iodide showed that the proton ATPase was electrogenic and capable of responding to a favorable electric gradient. In summary, the turtle bladder epithelial cell line has a plasma membrane proton ATPase which is similar to the proton ATPase of turtle bladder epithelium and thus should allow purification and characterization of this enzyme.     

Structure-function relationships in H+-secreting epithelia

Stimulation or inhibition of H+ secretion has been associated with characteristic ultrastructural changes in various epithelial cells, including the parietal cell of the gastric mucosa, the carbonic anhydrase (CA)-rich cell of the turtle urinary bladder, and the intercalated (I) cell of the mammalian collecting duct. An electroneutral potassium-activated H+-ATPase is responsible for H+ secretion in the stomach, whereas acidification in the turtle bladder and the mammalian collecting duct is mediated by an electrogenic H+-translocating ATPase. Despite these differences, the parietal cell, the CA-rich cell, and the I cell have several morphological features in common. They are rich in mitochondria, contain numerous tubulovesicular membrane structures in the apical region of the cell, and possess a variable number of microprojections on the luminal surface. After stimulation of H+ secretion there is a significant increase in the surface area of the apical membrane concomitant with a decrease in the tubulovesicular membrane compartment in these cells, as revealed by morphometric analysis. These findings suggest that membrane (possibly containing an H+ pump) is being transferred from the tubulovesicular compartment to the apical plasma membrane on stimulation of H+ secretion. A hypothesis of membrane recycling is proposed to account for the observed morphological changes.     

Structure of the novel membrane-coating material in proton-secreting epithelial cells and identification as an H+ATPase

Specialized proton-secreting cells known collectively as mitochondria-rich cells are found in a variety of transporting epithelia, including the kidney collecting duct (intercalated cells) and toad and turtle urinary bladders. These cells contain a population of characteristic tubulovesicles that are believed to be involved in the shuttling of proton pumps (H+ATPase) to and from the plasma membrane. These transporting vesicles have a dense, studlike material coating the cytoplasmic face of their limiting membranes and similar studs are also found beneath parts of the plasma membrane. We have recently shown that this membrane coat does not contain clathrin. The present study was performed to determine the structure of this coat in rapidly frozen and freeze-dried tissue, and to determine whether the coat contains a major membrane protein transported by these vesicles, a proton pumping H+ATPase. The structure of the coat was examined in proton-secreting, mitochondria-rich cells from toad urinary bladder epithelium by rapidly freezing portions of apical membrane and associated cytoplasm that were sheared away from the remainder of the cell using polylysine-coated coverslips. Regions of the underside of these apical membranes as large as 0.2 micron2 were decorated by studlike projections that were arranged into regular hexagonal arrays. Individual studs had a diameter of 9.5 nm and appeared to be composed of multiple subunits arranged around a central depression, possibly representing a channel. The studs had a density of approximately 16,800 per micron2 of membrane. Similar arrays of studs were also found on vesicles trapped in the residual band of cytoplasm that remained attached to the underside of the plasma membrane, but none were seen in adjacent granular cells. To determine whether these arrays of studs contained H+ATPase molecules, we examined a preparation of affinity-purified bovine medullary H+ATPase, using the same technique, after incorporation of the protein eluted from a monoclonal antibody affinity column into phospholipid liposomes. The affinity-purified protein was shown to be capable of ATP-dependent acidification. In such preparations, large paracrystalline arrays of studs identical in appearance to those seen in situ were found. The dimensions of the studs as well as the number per square micrometer of membrane were identical to those of toad bladder mitochondria-rich cells: 9.5 nm in diameter, 16,770 per micron2 of membrane.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of cell pH by Ca+2-mediated exocytotic insertion of H+- ATPases

Exposure to CO2 acidifies the cytosol of mitochondria-rich cells in turtle bladder epithelium. The result of the decrease in pH in these, the acid-secreting cells of the epithelium, is a transient increase in cell calcium, which causes exocytosis of vesicles containing proton-translocating ATPase. Because mitochondria-rich cells have rapid luminal membrane turnover, we were able to identify single mitochondria-rich cells by their endocytosis of rhodamine-tagged albumin. Using fluorescence emission of 5,6-carboxyfluorescein at two excitation wavelengths, we measured cell pH in these identified mitochondria-rich cells and found that although the cell pH fell, it recovered within 5 min despite continuous exposure to CO2. This pH recovery also occurred at the same rate in Na+-free media. However, pH recovery did not occur when luminal pH was 5.5, a condition under which the H+-pump does not function, suggesting that recovery of cell pH is due to the luminally located H+ ATPase. Chelation of extracellular calcium by EGTA prevented the CO2-induced rise in cell calcium measured with the intracellular fluorescent dyes Quin 2 or Fura 2 and also prevented recovery of cell pH. When the change in cell calcium was buffered by loading the cells with high concentrations of Quin 2, the CO2-induced decrease in pH did not return back to basal levels. We had found previously that buffering intracellular calcium transients prevented CO2-stimulated exocytosis. Further, we show here that the increased H+ current in voltage-clamped turtle bladders, which is directly proportional to the number of H+-pump-containing vesicles that fuse with the luminal membrane, was significantly reduced in calcium-depleted bladders. These results suggest that pH regulation in these acid-secreting cells occurs by calcium-dependent exocytosis of vesicles containing proton pumps, whose subsequent turnover restores the cell pH to its initial levels.     

Effect of calcium on intracellular pH

The effect of intracellular calcium on intracellular pH in the turtle urinary bladder was examined with phosphorus nuclear magnetic resonance. The turtle urinary bladder is capable of acidification in vitro and urinary acidification by this membrane is inhibited by an increase in intracellular calcium. Since calcium is capable of altering intracellular pH, it remains unclear whether the inhibition of urinary acidification is the result of an increase in intracellular pH. In the present study, intracellular calcium was increased by the cholinergic agent, carbachol, the ionophore A23187 and replacement of extracellular Na by sucrose. All agents decreased intracellular pH in the turtle bladder, thus suggesting that inhibition of urinary acidification by these agents is not due to an increase in intracellular pH.     

ATP-dependent H+ transport by the turtle bladder: NBD-C1 preferentially inhibits the vanadate-insensitive component in isolated membranes

We investigated the inhibitory effects of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) on ATP-dependent H+ accumulation by membrane vesicles prepared from the turtle urinary bladder epithelium. NBD-Cl at 30 microM was found to completely inhibit the vanadate-insensitive component of H+ transport, with half-maximal inhibition occurring at 4.2 to 5.4 microM. In contrast, the vanadate-inhibitable component was unaffected by 30 microM NBD-Cl. At high concentrations (300 microM), both components were fully inhibited. The results confirm the presence of two distinct H+ transport processes in turtle bladder membranes and identify selective inhibitors, NBD-Cl and vanadate, for each process.     

Nonlysosomal vesicles (acidosomes) are involved in phagosome acidification in Paramecium

Although acidification of phagocytic vacuoles has received a broadened interest with the development of pH-sensitive fluorescent probes to follow the pH changes of vacuoles and acidic vesicles in living cells, the mechanism responsible for the acidification of such vacuoles still remains in doubt. In previous studies of the digestive vacuole system in the ciliate Paramecium caudatum we observed and described a unique population of apparently nonlysosomal vesicles that quickly fused with the newly released vacuole before the vacuole became acid and before lysosomes fused with the vacuole. In this paper we report the following: (a) these vesicles, named acidosomes, are devoid of acid phosphatase; (b) these vesicles accumulate neutral red as well as acridine orange, two observations that demonstrate their acid content; (c) cytochalasin B given 15 s after exposure of the cells to indicator dye-stained yeast will inhibit the acidification of yeast-containing vacuoles; and that (d) we observed using electron microscopy, that fusion of acidosomes with the vacuole is inhibited by cytochalasin B. We conclude that the mechanism for acidification of phagocytic vacuoles in Paramecium resides, at least partially if not entirely, in the acidosomes.     

Intracellular phosphate pools show compartmentalization of intracellular pH in turtle urinary bladder

The urinary bladder of the fresh water turtle is capable of acidification and Na transport, in vitro, and it has been extensively used as a model of distal nephron of the kidney. In the course of measuring intracellular pH of stripped turtle bladder mucosa with phosphorus nuclear magnetic resonance, we observed the consistent presence of two inorganic phosphorus resonances under aerobic conditions, indicating the existence of a pH gradient possibly between cytosol and mitochondrion. This pH gradient was collapsed by addition of N₂ and could be restored by reintroduction of oxygen. These observations demonstrate the existence of a spontaneous pH gradient between cytosol and mitochondria of turtle bladder epithelial cells.     

H+/ATP stoichiometry of proton pump of turtle urinary bladder

Urinary acidification in the turtle urinary bladder is due to a reversible proton-translocating ATPase. To estimate the H+/ATP stoichiometry of this pump, we measured the delta G'ATP in the epithelial cells and the maximum e.m.f. generated by the pump. The latter is the maximal transepithelial electrochemical gradient for protons placed across the epithelium that is needed to nullify the rate of transport and averaged 179 +/- 7 mV. The delta G'ATP averaged 50.1 kJ/mol. The H+/ATP stoichiometry of these bladders was 2.92 +/- 0.1. In other experiments, the bladders were poisoned by iodoacetate and cyanide and a variable transepithelial electrochemical gradient for protons was placed across them. It was noted that ATP synthesis occurred at a transepithelial electrochemical gradient for protons greater than 120 mV. The delta G'ATP in other bladders treated identically averaged 40.0 kJ/mol, giving a H+/ATP stoichiometry of 3.4 +/- 0.1. We conclude that the H+/ATP stoichiometry of the proton pump of turtle urinary bladder is approximately 3.     

ATPase activity and ATP-dependent proton translocation in plasma membrane vesicles of turtle bladder epithelial cells

ATP-induced quenching of fluorescence of acridine orange (a pH probe) or Oxonol V (a potential difference probe) is evoked in turtle bladder membrane vesicles in suspending media of appropriate ionic composition and is insensitive to oligomycin, valinomycin, and ouabain. These effects are ascribed to a membrane-bound, ouabain-resistant ATPase which mediates an active electrogenic proton transport.     

Physiological role for vanadate-inhibitable active H+ transport: a new model for distal urinary acidification

We have shown elsewhere that membranes isolated from the turtle bladder epithelium have both vanadate-resistant (VR) and vanadate-sensitive (VS) components of ATP-dependent H+ transport. VR appears to be due to a vacuolar-type H+ uniport while VS has properties of an H/K exchanger. In the present study we used bladders from chronically alkalotic turtles, which do not acidify urine, and found them to yield membranes which retain VR but lack VS transport. Thus VS occurs only in membranes from bladders secreting acid, implying that VS H+ transport (1) plays a physiological role in the secretion and (2) is a functional marker for the apical membrane of the acid-secreting cells. These results suggest a new model for distal urinary acidification.     

Electrolyte transport across the basolateral membrane of the parietal cells

The ion-transport properties of the basal lateral membranes of intact isolated parietal cells were studied at the cellular and subcellular level. The presence of an amiloride-sensitive Na+:H+ exchange was demonstrated in cells by proton gradient-driven Na+ uptake and by changes in cell pH as monitored by dimethylcarboxylfluorescein fluorescence both in a fluorimeter and on single isolated cells using a fluorescence microscope and an attached intensified photodiode array spectrophotometer. The presence of the Na+:H+ antiport in vesicles was shown both by intravesicular acidification monitored by acridine orange fluorescent quenching and by proton gradient-dependent Na+ uptake. The presence of Cl-:HCO-3 exchange was determined in intact cells by monitoring changes in cell pH due to Cl- uptake and was shown to be 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid- and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid-sensitive. In vesicles, Cl-:HCO-3 exchange was demonstrated by Cl- flux measurement. The apparent affinities for both Cl- and HCO-3 on either side of the membrane were determined to be Km Cli = 20 mM, Km Clout = 17.5 mM, Km HCO-3in = 2.5 mM, and Km HCO-3out = 7.5 mM. A K+ conductance in cells and vesicles was demonstrated by monitoring K+ gradient-dependent 86Rb uptake. No evidence was found for the presence of a Cl- conductance in either cells or vesicles but a H+ conductance was found to be present in vesicles but not in intact cells. In the latter, by determining the effect of either Na+ or Cl- gradients on cell pH and by flux calculations it was concluded that the Cl-:HCO-3 exchange was the major passive flux mechanism for pH regulation in this cell type.     

Human Fucci Pancreatic Beta Cell Lines: New Tools to Study Beta Cell Cycle and Terminal Differentiation

Regulation of cell cycle in beta cells is poorly understood, especially in humans. We exploited here the recently described human pancreatic beta cell line EndoC-βH2 to set up experimental systems for cell cycle studies. We derived 2 populations from EndoC-βH2 cells that stably harbor the 2 genes encoding the Fucci fluorescent indicators of cell cycle, either from two vectors, or from a unique bicistronic vector. In proliferating non-synchronized cells, the 2 Fucci indicators revealed cells in the expected phases of cell cycle, with orange and green cells being in G1 and S/G2/M cells, respectively, and allowed the sorting of cells in different substeps of G1. The Fucci indicators also faithfully red out alterations in human beta cell proliferative activity since a mitogen-rich medium decreased the proportion of orange cells and inflated the green population, while reciprocal changes were observed when cells were induced to cease proliferation and increased expression of some beta cell genes. In the last situation, acquisition of a more differentiated beta cell phenotype correlates with an increased intensity in orange fluorescence. Hence Fucci beta cell lines provide new tools to address important questions regarding human beta cell cycle and differentiation.     

Non-apoptotic concentrations of prodigiosin (H+/Cl- symporter) inhibit the acidification of lysosomes and induce cell cycle blockage in colon cancer cells

Prodigiosin (PG) is a bacterial red pigment with interesting immunosuppressive and apoptotic properties that have been partly attributed to its ability to uncouple V-ATPase through the promotion of the H+/Cl- symporter. In the present study, we investigate the effect of non-apoptotic concentrations of PG on the lysosomal-pH and on cell cycle progression in colon cancer cells. Lysosomal-pH was tested in DLD-1 cells using acridine orange vital staining. Orange granules, indicative of acidified lysosomes, decreased significantly in cells treated with 25 nM of PG for 1/2 h, and disappeared completely at 100 nM. This suggests that PG can induce lysosomal alkalinization without any apparent cytotoxic effect. Cell cycle progression was analysed in HT29 cells and we found that PG induces a blockage in the G1 phase. This blockage correlates with p21(WAF1/CIP1) induction, and it can be triggered either dependently or independently of p53. In conclusion, the reversible increase in lysosomal-pH and cytosol acidification induced by non-apoptotic concentrations of PG in colon cancer cells, suggests that the apoptotic process induced by PG can not be solely explained by changes in intracellular pH. The effect of intracellular acidification on cell cycle arrest must be analysed more exactly.     

A thermosensitive lesion in a Chinese hamster cell mutant causing differential effects on the acidification of endosomes and lysosomes

We previously described the isolation and preliminary characterization of a Chinese hamster ovary cell mutant, termed G.7.1, that carried a temperature-sensitive, conditional-lethal lesion affecting the acidification of vesicles in crude cellular extracts (Marnell, M. H., Mathis, L. S., Stookey, M., Shia, S.-P., Stone, D. K., and Draper, R. K. (1984) J. Cell Biol. 99, 1907-1916). In the present report, we have separated lysosomal vesicles from more buoyant nonlysosomal vesicles by centrifuging cell extracts with Percoll and correlated the acidification defect with nonlysosomal vesicles, including endosomes, but not with secondary lysosomes. Moreover, the acidification of nonlysosomal vesicles prepared from mutant cells grown at the permissive temperature was more sensitive to thermal inactivation than similar vesicles from parental cells, implying that a heat-sensitive component is a normal resident of nonlysosomal vesicles in the mutant. This heat-sensitive component is apparently not associated with lysosomes, or if it is, it does not inhibit lysosomal acidification at the nonpermissive temperature. We also found that the transferrin-mediated uptake of iron is inhibited by 50% in the mutant cells at the nonpermissive temperature and that the inhibition cannot be accounted for by reduced binding or internalization of transferrin.     

Chloride-induced increment in short-circuiting current of the turtle bladder. Effects of in-vivo acid-base state

Evidence for the participation of conductive and non-conductive (exchange) transmembrane anion pathways in the luminal acidification, alkalinization, and chloride-reabsorptive functions of the turtle bladder is provided from the pattern of Cl- -induced changes in transepithelial electrical parameters of isolated urinary bladders from three groups of donor turtles: control or post-absorptive turtles (those killed 5 days after feeding); acidotic turtles (NH4Cl-loaded); and alkalotic turtles (NaHCO3-loaded). The predominance of each of the three aforementioned transport functions as well as the response to Cl- -addition is altered by the in-vivo electrolyte balance of the turtle. In post-absorptive bladders, which are poised for acidification and Cl- reabsorption, the mucosal and serosal addition of Cl- to Na+-free, (HCO3- + CO2)-containing media increases the negative short-circuiting current (Isc). In acidotic bladders, which are poised for acidification but not Cl- reabsorption, mucosal Cl- addition has no effect on this Isc whereas serosal Cl- addition increases the negative Isc in a manner identical to that observed in the post-absorptive bladders. Alkalotic bladders do not possess an acidification function but instead are poised for Cl- reabsorption and cAMP-dependent electrogenic alkali secretion (positive Isc). In these bladders, serosal Cl- addition is without effect while mucosal Cl- addition produces transient changes in this positive Isc. It is found that these results can be replicated by a model of the turtle bladder in which transmembrane Cl- and HCO3- conductive and exchange paths mediate transepithelial acidification, alkalinization and Cl- reabsorption.     

Rapid intracellular acidification and cell death by H2O2 and alloxan in pancreatic beta cells

Pancreatic beta-cell death induced by oxidative stress plays an important role in the pathogenesis of diabetes mellitus. We studied the relation between rapid intracellular acidification and cell death of pancreatic beta-cell line NIT-1 cells exposed to H2O2 or alloxan. Intracellular pH was measured by a pH-sensitive dye, and cell damage by double staining with Annexin-V and propidium iodide using flow cytometry. H2O2 and alloxan caused a rapid fall in intracellular pH and suppressed Na+/H+ exchanger activity in the NH4Cl prepulse method. H2O2 induced necrotic cell death, which shifted to apoptotic cell death when initial acidification was prevented by pH clamping to 7.4 using nigericin (unclamped cells vs clamped cells, necrosis 43.8 +/- 5.8% vs 21.1 +/- 10.6%, P < 0.05; apoptosis 8.0 +/- 1.9% vs 44.5 +/- 5.0%, P < 0.01). pH-clamped cells showed enhanced caspase 3 activity and proapoptotic Bax expression. On the other hand, NIT-1 cells were resistant to alloxan toxicity, but treatment with alloxan and nigericin strikingly enhanced the cytotoxicity. Antioxidants partly prevented cell death, although intracellular pH remained similarly acidic. The rapid intracellular acidification was not the cause of cell death but a significant determinant of the mode of death of H2O2 -treated beta cells, whereas no link between cell death and acidification was demonstrated in alloxan toxicity.     

Effect of furosemide on ion transport in the turtle bladder: evidence for direct inhibition of active acid-base transport

The diuretic furosemide inhibits acid-base transport in the short-circuited turtle bladder. It inhibits luminal acidification when present in either mucosal or serosal bathing fluids, but decreases alkalinization only from the serosal side of the tissue. The inhibition of both acid-base transport processes is independent of ambient Cl-; and the disulfonic stilbene, SITS, an inhibitor of Cl--HCO3- exchange, fails to prevent the furosemide-elicited inhibition of alkalinization. These results preclude an absolute requirement of a furosemide-sensitive Cl--HCO3- exchange by these transport processes. The drug also interferes with the CO2-induced stimulation of acidification and alkalinization. The inhibition of the residual acidification in acetazolamide-treated, acidotic bladders, however, suggests an action at sites other than cytosolic carbonic anhydrase. Although active Na+ and Cl- reabsorption and tissue oxygen uptake are also decreased by furosemide, the rate of oxygen consumption uncoupled by 2,4-dinitrophenol is not diminished, indicating a primary inhibition of the various ion transport processes, not of metabolism. It is proposed that inhibition of transepithelial acid-base transport by furosemide in the turtle bladder includes inhibition of the acid-base pumps.     

Vanadate inhibition of ATP-dependent H+ transport in membrane vesicles from turtle bladder epithelial cells

The ATP-dependent proton transport into vesicles of a mixed membrane fraction obtained from turtle bladder epithelial cells consists of at least two kinetically defined moieties: one, which is maximally inhibited by 25% with nanomolar levels of vanadate, but not inhibited at all with equimolar levels of N-ethylmaleimide, and another, which is maximally inhibited by 70% with micromolar levels of N-ethylmaleimide and by 25% with equimolar levels of vanadate. In contrast to the transport function, the associated enzymatic function (the ouabain-resistant ATPase activity) in these membranes, not inhibited by nanomolar levels of vanadate or N-ethylmaleimide, is maximally inhibited by 40% with micromolar levels of vanadate and by 13% with equimolar levels of N-ethylmaleimide. Independent of these kinetic differences between the enzyme and the transport functions, membranes containing the N-ethylmaleimide-sensitive proton transport function are electrophoretically separable from those containing the vanadate-sensitive transport function. For example, the kinetically defined, vanadate-sensitive proton transport function is recovered exclusively and kinetically identified in one of four electrophoretic membrane fractions, EF-II; while the N-ethylmaleimide-sensitive function is recovered in EF-III as well as in EF-II. Membranes of EF-IV, maximally enriched in ouabain-resistant ATPase activity, possess no proton transport function at all, even in the absence of N-ethylmaleimide or vanadate. Additional data under in vivo as well as under in vitro conditions are required to prove that the vanadate-sensitive proton transport in these vesicles is an in vitro manifestation of the mechanism responsible for generating the vanadate-sensitive luminal acidification process under in vivo conditions in the intact turtle bladder.     

A cell-free system for regulated exocytosis in PC12 cells

We have developed a cell-free system for regulated exocytosis in the PC12 neuroendocrine cell line. Secretory vesicles were preloaded with acridine orange in intact cells, and the cells were sonicated to produce flat, carrier-supported plasma membrane patches with attached vesicles. Exocytosis resulted in the release of acridine orange which was visible as a disappearance of labeled vesicles and, under optimal conditions, produced light flashes by fluorescence dequenching. Exocytosis in vitro requires cytosol and Ca(2+) at concentrations in the micromolar range, and is sensitive to Tetanus toxin. Imaging of membrane patches at diffraction- limited resolution revealed that 42% of docked granules were released in a Ca(2+)-dependent manner during 1 min of stimulation. Electron microscopy of membrane patches confirmed the presence of dense-core vesicles. Imaging of membrane patches by atomic force microscopy revealed the presence of numerous particles attached to the membrane patches which decreased in number upon stimulation. Thus, exocytotic membrane fusion of single vesicles can be monitored with high temporal and spatial resolution, while providing access to the site of exocytosis for biochemical and molecular tools.