Relationship Between Effect of Ethanol on Proton Flux Across Plasma Membrane and Ethanol Tolerance, inPichia stipitis

Pichia stipitisefficiently converts glucose or xylose into ethanol but is inhibited by ethanol concentrations exceeding 30 g/L. InSaccharomyces cerevisiae, ethanol has been shown to alter the movement of protons into and out of the cell. InP. stipitisthe passive entry of protons into either glucose- or xylose-grown cells is unaffected at physiological ethanol concentrations. In contrast, active proton extrusion is affected differentially by ethanol, depending on the carbon source catabolized. In fact, in glucose-grown cells, the H⁺-extrusion rate is reduced by low ethanol concentrations, whereas, in xylose-grown cells, the H⁺-extrusion rate is reduced only at non-physiological ethanol concentrations. Thus, the ethanol inhibitory effect on growth and ethanol production, in glucose-grown cells, is probably caused by a reduction in H⁺-extrusion. Comparison of the rates of H⁺-flux with the relatedin vitroH⁺-ATPase activity suggests a new mechanism for the regulation of the proton pumping plasma membrane ATPase (EC 3.6.1.3) ofP. stipitis, by both glucose and ethanol. Glucose activates both the ATP hydrolysis and the proton-pumping activities of the H⁺-ATPase, whereas ethanol causes an uncoupling between the ATP hydrolysis and the proton-pumping activities. This uncoupling may well be the cause of ethanol induced growth inhibition of glucose grownP. stipitiscells.     

Glucose- and K+-induced acidification in different yeast species

The process of acidification of the external medium after addition of glucose and subsequently of KCl to a suspension of yeast cells varies substantially from species to species. After glucose it is most pronounced inSaccharomyces cerevisiae andSchizosaccharomyces pombe but is very much lower inLodderomyces elongisporus, Dipodascus magnusii andRhodotorula gracilis. Both the buffering capacity and the varied effects of vanadate, suloctidil and erythrosin B indicate that the acidification is by about one-half due to the activity of plasma membrane H⁺-ATPase and by about one-half to the extrusion of acidic metabolites from cells. This is supported by the finding that a respiratory quotient greater than one (in various strains ofS. cerevisiae and inS. pombe) is indicative of a greater buffering capacity and overall acidification of the medium. Taking into account the virtually negligible buffering capacity of the medium in the pH range where the effect of K⁺ is observed, the effect of K⁺ is generally of a similar magnitude as that of adding glucose. It is clearly dependent on (anaerobic) production of metabolic energy, quite distinct from the dependence of the H⁺-ATPase-caused acidification.     

Effects of salt-adaptation and salt-stress on extracellular acidification and microsome phosphohydrolase activities in tomato cell suspensions

The effects of NaCl-adaptation and NaCl-stress on in vivo H⁺ extrusion and microsomal vanadate- and bafilomycin-sensitive ATPase and PPase activities were studied in tomato cell suspensions. Acidification of the external medium by 50 mM NaCl-adapted and non-adapted (control) tomato cells was similar. Extracellular acidification by both types of cells during the first hour of incubation with 2 μM fusicoccin (FC) in the presence of 100 mM NaCl was lightly increased while in the presence of 100 mM KCl it was increased by 3 (control)- and 6.5 (adapted)-fold. Extracellular alkalinization after 2 h of cell incubation in 100 mM NaCl indicated the possibility that a Na⁺/H⁺ exchange activity could be operating in both types of cells. Moreover, acidification induced by adding 100 mM NaCl + FC to non-adapted cells was relatively less affected by vanadate than that induced by 5 mM KCl + FC, which suggested that salt stress could induce some component other than H⁺ extrusion by H⁺-ATPase. In addition, no differences were observed in microsomal vanadate-sensitive ATPase activity among control, NaCl-adapted and NaCl-stressed cells, while K⁺-stimulated H⁺-PPase and bafilomycin-sensitive H⁺-ATPase activities were higher in microsomes from NaCl-adapted than in those from control cells. Likewise, the stimulation of in vivo H⁺ extrusion in NaCl adapted cells under NaCl or KCl stress in the presence of FC occurred with an inhibition of H⁺-PPase and bafilomycin-sensitive H⁺-ATPase activities and without changes in the vanadate-sensitive H⁺-ATPase activity. These results suggest that the stimulation of tonoplast proton pumps in NaCl-adapted cells, without changes in plasmalemma H⁺-ATPase, could serve to energize Na⁺ efflux across the plasmalemma and Na⁺ fluxes into vacuoles catalyzed by the Na⁺/H⁺ antiports. This revised version was published online in June 2006 with corrections to the Cover Date.     

灵武长枣果实质膜和液泡膜H~+-ATPase和H~+-PPase活性与果实糖分积累

以不同发育时期灵武长枣(Ziziphus jujuba cv.Lingwuchangzao)的果实为材料,通过测定与分析果肉组织中细胞质膜、液泡膜H+-ATPase和H+-PPase活性、果实糖分含量变化,研究了灵武长枣果实质膜、液泡膜H+-ATPase和H+-PPase活性与糖积累特性的关系。结果表明:(1)果实第二次快速生长期之前主要积累葡萄糖和果糖,之后果实迅速积累蔗糖,葡萄糖和果糖含量则逐渐下降,成熟期果实主要积累蔗糖。(2)在果实发育的缓慢生长期S1,质膜H+-ATPase活性最低;第一次快速生长期,质膜H+-ATPase活性最高;缓慢生长期S2,其活性降低;第二次快速生长期,质膜H+-ATPase活性升至次高;完熟期,质膜H+-ATPase活性下降幅度较大。(3)在果实发育过程中,液泡膜H+-ATPase和H+-PPase活性的变化趋势相似。缓慢生长期S1,液泡膜H+-ATPase和H+-PPase活性较低;从缓慢生长期S1至第一次快速生长期缓慢下降至最低;从第一次快速生长期开始,液泡膜H+-ATPase和H+-PPase活性呈现为逐渐增高的变化趋势;除第二次快速生长期以外,液泡膜H+-PPase活性始终高于H+-ATPase。由此推测,质膜H+-ATPase和液泡膜H+-ATPase、H+-PPase对灵武长枣果实糖分的跨膜次级转运起到重要的调控作用。     

Cytochemical demonstration of NPPase activity for detecting proton-translocating ATPase of Golgi complex in rat pancreatic acinar cells

Summary An attempt at cytochemical demonstration of acidification proton-translocating ATPase (H⁺-ATPase) of Golgi complex in rat pancreatic acinar cells has been made by using p-nitrophenylphosphatase (NPPase) cytochemistry which is used for detecting of Na⁺-K⁺-ATPase (Mayahara et al. 1980) and gastric H⁺-K⁺-ATPase (Fujimoto et al. 1986). K⁺-independent NPPase activity was observed on the membrane of the trans cisternae of Golgi complex, but not inside of cisternae. The localization of NPPase activity is different from that of acid phosphatase activity where reaction products were seen on the inside of the trans Golgi cisternae. Since this activity was insensitive to vanadate, ouabain and independent of potassium ions, it was distinct from plasma membranous ATPases such as Na⁺-K⁺-ATPase and Ca²⁺-ATPase. The K⁺-independent NPPase activity was diminished by the inhibitors of H⁺-ATPase such as N-ethylmaleimide (NEM) and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). The NPPase reaction products were also seen on the membranes of other acidic organelles, i.e., lysosomes, endosomes, autophagosomes and coated vesicles. These results suggest that NPPase activity on the membrane of the Golgi complex and other acidic organelles corresponds with H⁺-ATPase which plays a role in acidification.     

Glucose-induced activation of H+-ATPase in Dunaliella salina and its role in hygromycin resistance

Dunaliella salina, a eukaryotic microalga, is known for its highly halophilic nature. The high level of salts in growth medium for this alga has made its genetic transformation a comparatively difficult procedure, particularly during the selection stage. The high salt content decreases the efficiency of most antibiotics which are being used as selection markers. Studies pertaining to the interrelationship between salt concentration and antibiotic sensitivity are scarce in Dunaliella. During our previous experiment at genetic transformation of Dunaliella, an inverse relationship between the amount of antibiotic hygromycin and sodium chloride in the medium was revealed. A possible link between plasma membrane activity and the hygromycin sensitivity was investigated in the present study by modulating plasma membrane H⁺-ATPase activity using glucose. Glucose-induced activation of H⁺-ATPase, reduced the tolerance of D. salina to the antibiotic hygromycin. Hygromycin concentration required for selection during genetic transformation of Dunaliella was lowered from 100 to 25 mg L^?1 in the presence of 10 mM glucose. Conversely, the inhibitors of the plasma membrane H⁺-ATPase, orthovanadate and diethylstilbestrol were found to inhibit the glucose activation at concentrations of 10 and 15 μM, respectively. The activation of H⁺-ATPase by glucose was further confirmed through H⁺-ATPase assay and medium acidification experiments. The results indicated that the sensitivity of Dunaliella to antibiotic is related to H⁺-ATPase and the possible involvement of pH gradient, created through H⁺-ATPase activation during drug transport.     

Modulation of H,K-ATPase activity by the molecular chaperone ERp57 highly expressed in gastric parietal cells

ERp57 is a ubiquitous ER chaperone that has disulfide isomerase activity. Here, we found that both ERp57 and gastric H⁺,K⁺-ATPase are expressed in a sample derived from the apical canalicular membranes of parietal cells. Overexpression of ERp57 in HEK293 cells stably expressing H⁺,K⁺-ATPase significantly increased the ATPase activity without changing the expression level of H⁺,K⁺-ATPase. Interestingly, overexpression of a catalytically inactive mutant of ERp57 (C57S/C60S/C406S/C409S) in the cells also increased H⁺,K⁺-ATPase activity. In contrast, knockdown of endogenous ERp57 in H⁺,K⁺-ATPase-expressing cells significantly decreased ATPase activity without changing the expression level of H⁺,K⁺-ATPase. Overexpression and knockdown of ERp57 had no significant effect on the expression and function of Na⁺,K⁺-ATPase. These results suggest that ERp57 positively regulates H⁺,K⁺-ATPase activity apart from its chaperoning function.     

Glucose activates H(+)-ATPase in kidney epithelial cells

The vacuolar H⁺-ATPase (V-ATPase) acidifies compartments of the vacuolar system of eukaryotic cells. In renal epithelial cells, it resides on the plasma membrane and is essential for bicarbonate transport and acid-base homeostasis. The factors that regulate the H⁺-ATPase remain largely unknown. The present study examines the effect of glucose on H⁺-ATPase activity in the pig kidney epithelial cell line LLC-PK₁. Cellular pH was measured by performing ratiometric fluorescence microscopy using the pH-sensitive indicator BCECF-AM. Intracellular acidification was induced with NH₃/NH₄⁺ prepulse, and rates of intracellular pH (pH_i) recovery (after in situ calibration) were determined by the slopes of linear regression lines during the first 3 min of recovery. The solutions contained 1 µM ethylisopropylamiloride and were K⁺ free to eliminate Na⁺/H⁺ exchange and H⁺-K⁺-ATPase activity. After NH₃/NH₄⁺-induced acidification, LLC-PK₁ cells had a significant pH_i recovery rate that was inhibited entirely by 100 nM of the V-ATPase inhibitor concanamycin A. Acute removal of glucose from medium markedly reduced V-ATPase-dependent pH_i recovery activity. Readdition of glucose induced concentration-dependent reactivation of V-ATPase pH_i recovery activity within 2 min. Glucose replacement produced no significant change in cell ATP or ADP content. H⁺-ATPase activity was completely inhibited by the glycolytic inhibitor 2-deoxy-D-glucose (20 mM) but only partially inhibited by the mitochondrial electron transport inhibitor antimycin A (20 µM). The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (500 nM) abolished glucose activation of V-ATPase, and activity was restored after wortmannin removal. Glucose activates V-ATPase activity in kidney epithelial cells through the glycolytic pathway by a signaling pathway that requires PI3K activity. These findings represent an entirely new physiological effect of glucose, linking it to cellular proton secretion and vacuolar acidification. proton secretion; glycolysis; intracellular pH; concanamycin A     

Activation of H-ATPase by glucose in Saccharomyces cerevisiae involves a membrane serine protease

Background

Glucose induces H⁺-ATPase activation in Saccharomyces cerevisiae. Our previous study showed that (i) S. cerevisiae plasma membrane H⁺-ATPase forms a complex with acetylated tubulin (AcTub), resulting in inhibition of the enzyme activity; (ii) exogenous glucose addition results in the dissociation of the complex and recovery of the enzyme activity.

Methods

We used classic biochemical and molecular biology tools in order to identify the key components in the mechanism that leads to H⁺-ATPase activation after glucose treatment.

Results

We demonstrate that glucose-induced dissociation of the complex is due to pH-dependent activation of a protease that hydrolyzes membrane tubulin. Biochemical analysis identified a serine protease with a kDa of 35–40 and an isoelectric point between 8 and 9. Analysis of several knockout yeast strains led to the detection of Lpx1p as the serine protease responsible of tubulin proteolysis. When lpx1Δ cells were treated with glucose, tubulin was not degraded, the AcTub/H⁺-ATPase complex did not undergo dissociation, and H⁺-ATPase activation was significantly delayed.

Conclusion

Our findings indicate that the mechanism of H⁺-ATPase activation by glucose involves a decrease in the cytosolic pH and consequent activation of a serine protease that hydrolyzes AcTub, accelerating the process of the AcTub/H⁺-ATPase complex dissociation and the activation of the enzyme.

General significance

Our data sheds light into the mechanism by which acetylated tubulin dissociates from the yeast H⁺-ATPase, identifying a degradative step that remained unknown. This finding also proposes an indirect way to pharmacologically regulate yeast H⁺-ATPase activity and open the question about mechanistic similarities with other higher eukaryotes.     

Experimental determination of control by the H+-ATPase inEscherichia coli

Strains carrying deletions in theatp genes, encoding the H⁺-ATPase, were unable to grow on nonfermentable substrates such as succinate, whereas with glucose as the substrate the growth rate of anatp deletion mutant was surprisingly high (some 75–80% of wild-type growth rate). The rate of glucose and oxygen consumption of these mutants was increased compared to the wild-type rates. In order to analyze the importance of the H⁺-ATPase at its physiological level, the cellular concentration of H⁺-ATPase was modulated around the wild-type level, using genetically manipulated strains. The control coefficient by the H⁺-ATPase with respect to growth rate and catabolic fluxes was measured. Control on growth rate was absent at the wild-type concentration of H⁺-ATPase, independent of whether the substrate for growth was glucose or succinate. Control by the H⁺-ATPase on the catabolic fluxes, including respiration, was negative at the wild-type H⁺-ATPase level. Moreover, the turnover number of the individual H⁺-ATPase enzymes increased as the H⁺-ATPase concentration was lowered. The negative control by the H⁺-ATPase on catabolism may thus be involved in a homeostatic control of ATP synthesis and, to some extent, explain the zero control by the H⁺-ATPase onE. coli growth rate.     

KCNQ1 loss-of-function mutation impairs gastric acid secretion in mice

The KCNQ1 channel is abundantly expressed in the gastric parietal cells. Although the functional coupling of KCNQ1 with the H⁺/K⁺-ATPase has already been confirmed on the basis of pharmacological kinetics, the effect of a KCNQ1 loss-of-function mutation on gastric acidification remains unclear. In this study, parietal cells and gastric glands from both C57BL/6 J mice (normal control) and J343 mice (mice with a KCNQ1 loss-of-function mutation) were isolated to study the effects of KCNQ1 on gastric acidification. We found that the mutation limited intracellular acidification of parietal cells and H⁺ secretion of the stomach in response to histamine. Thus, a KCNQ1 loss-of-function mutation may impair gastric acid secretion.     

Ethanol-Induced Activation of ATP-Dependent Proton Extrusion in Elodea densa Leaves

In Elodea densa leaves, ethanol up to 0.17 m stimulates H⁺ extrusion activity. This effect is strictly dependent on the presence of K⁺ in the medium and is suppressed by the presence of the plasmalemma H⁺-ATPase inhibitor vanadate. Stimulation of H⁺ extrusion is associated with (a) a decrease in cellular ATP level, (b) a marked hyperpolarization of transmembrane electrical potential, and (c) an increase in net K⁺ influx. These results suggest that ethanol-induced H⁺ extrusion is mediated by an activation of the plasma membrane ATP-dependent, electrogenic proton pump. This stimulating effect is associated with an increase of cell sap pH and of the capacity to take up the weak acid 5,5-dimethyloxazolidine-2,4-dione, which is interpretable as due to an increase of cytosolic pH. This indicates that the stimulation of H⁺ extrusion by ethanol does not depend on a cytosolic acidification by products of ethanol metabolism. The similarity of the effects of ethanol and those of photosynthesis on proton pump activity in E. densa leaves suggests that a common metabolic situation is responsible for the activation of the ATP-dependent H⁺-extruding mechanism.     

Elucidation of antimicrobial activity and mechanism of action by N-substituted carbazole derivatives

Compounds belonging to a carbazole series have been identified as potent fungal plasma membrane proton adenosine triphophatase (H⁺-ATPase) inhibitors with a broad spectrum of antifungal activity. The carbazole compounds inhibit the adenosine triphosphate (ATP) hydrolysis activity of the essential fungal H⁺-ATPase, thereby functionally inhibiting the extrusion of protons and extracellular acidification, processes that are responsible for maintaining high plasma membrane potential. The compound class binds to and inhibits the H⁺-ATPase within minutes, leading to fungal death after 1–3 h of compound exposure in vitro. The tested compounds are not selective for the fungal H⁺-ATPase, exhibiting an overlap of inhibitory activity with the mammalian protein family of P-type ATPases; the sarco(endo)plasmic reticulum calcium ATPase (Ca²⁺-ATPase) and the sodium potassium ATPase (Na⁺,K⁺-ATPase). The ion transport in the P-type ATPases is energized by the conversion of ATP to adenosine diphosphate (ADP) and phosphate and a general inhibitory mechanism mediated by the carbazole derivative could therefore be blocking of the active site. However, biochemical studies show that increased concentrations of ATP do not change the inhibitory activity of the carbazoles suggesting they act as allosteric inhibitors. Furthermore decreased levels of intracellular ATP would suggest that the compounds inhibit the H⁺-ATPase indirectly, but Candida albicans cells exposed to potent H⁺-ATPase-inhibitory carbazoles result in increased levels of intracellular ATP, indicating direct inhibition of H⁺-ATPase.     

Differential activation of H+-ATPase genes by an arbuscular mycorrhizal fungus in root cells of transgenic tobacco

In arbuscular mycorrhizas, H⁺-ATPase is active in the plant membrane around arbuscules but absent from plant mutants defective in arbuscule development (Gianinazzi-Pearson et al. 1995, Can J Bot 73: S526–S532). The proton-pumping H⁺-ATPase is encoded by a family of genes in plants. Immunocytochemical studies and promoter-gusA fusion assays were performed in transgenic tobacco (Nicotiana tabacum L.) to determine whether the periarbuscular enzyme activity results from de-novo activation of plant genes by an arbuscular mycorrhizal fungus. The H⁺-ATPase protein was localized in the plant membrane around arbuscule hyphae. The enzyme was absent from non-colonized cortical cells. Regulation of seven H⁺-ATPase genes (pma) was compared in non-mycorrhizal and mycorrhizal roots by histochemical detection of β-glucuronidase (GUS) activity. Two genes (pma2, pma4) were induced in arbuscule-containing cells of mycorrhizal roots but not in non-mycorrhizal cortical tissues or senescent mycorrhiza. It is concluded that de-novo H⁺-ATPase activity in the periarbuscular membrane results from selective induction of two H⁺-ATPase genes, which can have diverse roles in plant-fungal interactions at the symbiotic interface. Received: 23 October 1999 / Accepted: 7 February 2000     

Effect of Reduced H+-ATPase Activity on Acid Tolerance in Streptococcus bovis Mutants

In order to confirm that H⁺-ATPase plays an important role in the acid tolerance ofStreptococcus bovis , two mutants with low activities of H⁺-ATPase were isolated by use of ethyl methanesulfonate and neomycin resistance. The activity of H⁺-ATPase per cellular nitrogen was related to the lowest culture pH permitting growth. A mutant with little H⁺-ATPase activity (Mutant 2) was unable to grow below pH 5.5, which suggests that the intracellular pH should be maintained above 5.5 in S. bovis. Since lactate dehydrogenase activity, which is important for acid tolerance, was similar in parent and mutant strains, H⁺-ATPase activity is likely to affect acid tolerance. The amount of H⁺-ATPase protein as determined by Western-blot analysis with polyclonal antibody, was similar in Mutant 2 and its parent, indicating that H⁺-ATPase activity per enzyme protein is reduced by mutation. Probably, H⁺-ATPase synthesis was not changed by mutation. The gene encoding H⁺-ATPase of Mutant 2 had mutations at positions close to the ATP-binding motif A sequence in the β-subunit, which probably explains the reduced activity of H⁺-ATPase in this mutant. These results strongly support the assumption that H⁺-ATPase has a key role in the acid tolerance of S. bovis.     

Colocalization of the (Pro)renin Receptor/Atp6ap2 with H+-ATPases in Mouse Kidney but Prorenin Does Not Acutely Regulate Intercalated Cell H+-ATPase Activity

The (Pro)renin receptor (P)RR/Atp6ap2 is a cell surface protein capable of binding and non-proteolytically activate prorenin. Additionally, (P)RR is associated with H⁺-ATPases and alternative functions in H⁺-ATPase regulation as well as in Wnt signalling have been reported. Kidneys express very high levels of H⁺-ATPases which are involved in multiple functions such as endocytosis, membrane protein recycling as well as urinary acidification, bicarbonate reabsorption, and salt absorption. Here, we wanted to localize the (P)RR/Atp6ap2 along the murine nephron, exmaine whether the (P)RR/Atp6ap2 is coregulated with other H⁺-ATPase subunits, and whether acute stimulation of the (P)RR/Atp6ap2 with prorenin regulates H⁺-ATPase activity in intercalated cells in freshly isolated collecting ducts. We localized (P)PR/Atp6ap2 along the murine nephron by qPCR and immunohistochemistry. (P)RR/Atp6ap2 mRNA was detected in all nephron segments with highest levels in the collecting system coinciding with H⁺-ATPases. Further experiments demonstrated expression at the brush border membrane of proximal tubules and in all types of intercalated cells colocalizing with H⁺-ATPases. In mice treated with NH₄Cl, NaHCO₃, KHCO₃, NaCl, or the mineralocorticoid DOCA for 7 days, (P)RR/Atp6ap2 and H⁺-ATPase subunits were regulated but not co-regulated at protein and mRNA levels. Immunolocalization in kidneys from control, NH₄Cl or NaHCO₃ treated mice demonstrated always colocalization of PRR/Atp6ap2 with H⁺-ATPase subunits at the brush border membrane of proximal tubules, the apical pole of type A intercalated cells, and at basolateral and/or apical membranes of non-type A intercalated cells. Microperfusion of isolated cortical collecting ducts and luminal application of prorenin did not acutely stimulate H⁺-ATPase activity. However, incubation of isolated collecting ducts with prorenin non-significantly increased ERK1/2 phosphorylation. Our results suggest that the PRR/Atp6ap2 may form a complex with H⁺-ATPases in proximal tubule and intercalated cells but that prorenin has no acute effect on H⁺-ATPase activity in intercalated cells.     

Plasma-membrane H+-ATPases are expressed in pitchers of the carnivorous plant Nepenthes alata Blanco

Nepenthes is a unique genus of carnivorous plants that can capture insects in trapping organs called pitchers and digest them in pitcher fluid. The pitcher fluid includes digestive enzymes and is strongly acidic. We found that the fluid pH decreased when prey accumulates in the pitcher fluid of Nepenthes alata. The pH decrease may be important for prey digestion and the absorption of prey-derived nutrients. To identify the proton pump involved in the acidification of pitcher fluid, plant proton-pump homologs were cloned and their expressions were examined. In the lower part of pitchers with natural prey, expression of one putative plasma-membrane (PM) H⁺-ATPase gene, NaPHA3, was considerably higher than that of the putative vacuolar H⁺-ATPase (subunit A) gene, NaVHA1, or the putative vacuolar H⁺-pyrophosphatase gene, NaVHP1. Expression of one PM H⁺-ATPase gene, NaPHA1, was detected in the head cells of digestive glands in the lower part of pitchers, where proton extrusion may occur. Involvement of the PM H⁺-ATPase in the acidification of pitcher fluid was also supported by experiments with proton-pump modulators; vanadate inhibited proton extrusion from the inner surface of pitchers, whereas bafilomycin A₁ did not, and fusicoccin induced proton extrusion. These results strongly suggest that the PM H⁺-ATPase is responsible for acidification of the pitcher fluid of Nepenthes. Received: 8 June 2000 / Accepted: 8 August 2000     

Dicyclohexylcarbodiimide and vanadate sensitive ATPase of lung lamellar bodies

Lung surfactant is synthesized in lung epithelial type II cells and stored in the lamellar bodies prior to its secretion onto the alveolar surface. The lamellar bodies, like other secretory organelles, maintain an ATP-dependent pH gradient that is sensitive to inhibitors of H⁺-ATPase. This report shows that the ATPase activity of lamellar bodies is enriched in a fraction prepared from lamellar bodies that were disrupted after isolation. The apparent V_max for this enzyme was 150 nmol ATP hydrolyzed per min per mg protein and apparent K_m for ATP was approximately 50 μM. The enzyme activity was sensitive to N-ethylmaleimide (NEM), dicyclohexylcarbodiimide (DCCD) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1) (all inhibitors of vacuolar-type H⁺-ATPase) and vanadate (inhibitor of phosphoenzyme-type ATPase). Besides, the activity could also be inhibited with diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and Ca²⁺. Two proteins (of approximately 45 kDa and 17 kDa) of this fraction showed acid-stable phosphorylation with ATP. The labeling of proteins with ATP (-γ-³²P) could be chased with unlabelled ATP, suggesting that phosphorylation and dephosphorylation of these proteins is associated with the ATPase activity. Our results on inhibition characteristics of the enzyme activity suggest that besides a vacuolar type H⁺-ATPase, the lamellar bodies also contain a phosphoenzyme type ATPase that is sensitive to inhibitors of vacuolar type H⁺-ATPase.     

Evidence for activation of an active electrogenic proton pump in Ehrlich ascites tumor cells during glycolysis

Summary The addition of glucose to a suspension of Ehrlich ascites tumor cells results in rapid acidification of the extracellular medium due to lactic acid production. The nature of the H⁺ efflux mechanism has been studied by measuring the time course of the acidification, the rate of proton efflux, the direction and relative magnitude of the H⁺ concentration gradient, and the voltage across the membrane. Using the pH-sensitive dye acridine orange, we have established that after addition of 10mm glucose an outward-directed H⁺ concentration gradient develops. As the rate of glycolysis slows, the continued extrusion of H⁺ reverses the direction of the H⁺ concentration gradient. Changes in absorbance of the voltagesensitive dye diethyloxadicarbocyanine iodide (DOCC), and changes in the distribution of the lipid permeant cation tetraphenyl phosphonium, showed a dramatic and persistent hyperpolarization of the membrane voltage after glucose addition. The hyperpolarization was prevented by the protonophore tetrachlorosalicylanalide (TCS) and by valinomycin, but not by the neutral-exchange ionophore nigericin. Inhibitors of lactate efflux were found to reduce the rate of acidification after glucose addition but they had no effect on the magnitude of the resulting hyperpolarization. On the basis of these and other data we suggest that an active electrogenic pump mechanism for H⁺ efflux may be activated by glucose and that this mechanism operates independently of the lactate carrier system.     

Physiological and molecular analysis of the interactive effects of feeding and high environmental ammonia on branchial ammonia excretion and Na+ uptake in freshwater rainbow trout

Recently, a “Na⁺/NH₄ ⁺ exchange complex” model has been proposed for ammonia excretion in freshwater fish. The model suggests that ammonia transport occurs via Rhesus (Rh) glycoproteins and is facilitated by gill boundary layer acidification attributable to the hydration of CO₂ and H⁺ efflux by Na⁺/H⁺ exchanger (NHE-2) and H⁺-ATPase. The latter two mechanisms of boundary layer acidification would occur in conjunction with Na⁺ influx (through a Na⁺ channel energized by H⁺-ATPase and directly via NHE-2). Here, we show that natural ammonia loading via feeding increases branchial mRNA expression of Rh genes, NHE-2, and H⁺-ATPase, as well as H⁺-ATPase activity in juvenile trout, similar to previous findings with ammonium salt infusions and high environmental ammonia (HEA) exposure. The associated increase in ammonia excretion occurs in conjunction with a fourfold increase in Na⁺ influx after a meal. When exposed to HEA (1.5 mmol/l NH₄HCO₃ at pH 8.0), both unfed and fed trout showed differential increases in mRNA expression of Rhcg2, NHE-2, and H⁺-ATPase, but H⁺-ATPase activity remained at control levels. Unfed fish exposed to HEA displayed a characteristic reversal of ammonia excretion, initially uptaking ammonia, whereas fed fish (4 h after the meal) did not show this reversal, being able to immediately excrete ammonia against the gradient imposed by HEA. Exposure to HEA also led to a depression of Na⁺ influx, demonstrating that ammonia excretion can be uncoupled from Na⁺ influx. We suggest that the efflux of H⁺, rather than Na⁺ influx itself, is critical to the facilitation of ammonia excretion.