Determinants of the phagosomal pH in macrophages. In situ assessment of vacuolar H(+)-ATPase activity, counterion conductance, and H+ "leak".

We studied the factors that determine the intraphagosomal pH (pHp) in elicited murine peritoneal macrophages. pHp was measured in situ by recording the fluorescence of covalently fluoresceinated Staphylococcus aureus ingested by the macrophages. Following spontaneous acidification of the phagosomes, passive (leak) H+ permeability was determined measuring the rate of change of pHp upon complete inhibition of the H+ pump with bafilomycin A1. A significant, but comparatively low passive H+ permeability was detected. The existence of a passive H+ leak implies that continuous energy expenditure is required for the maintenance of an acidic pHp. In combination with ionophores, bafilomycin was also used to estimate the counterion permeability. The counterion conductance was found to be severalfold higher than the H+ leak. Ion substitution experiments in electropermeabilized cells and the inhibitory effects of quinine and 5-nitro-2-(3-phenylpropylamino)benzoic acid suggest that both monovalent anions and cations permeate the phagosomal membrane. The activity of the H+ pump was measured at various pHp levels. In the steady state, the rate of H+ pumping was considerably lower than counterion permeation. These findings suggest that the phagosomal membrane potential is insignificant. Consistent with this notion, increasing phagosomal conductance with ionophores failed to accelerate the rate of H+ pumping. Thus, the transmembrane delta pH is the predominant component of the proton-motive force across the phagosomal membrane in the steady state. The rate of H+ pumping was found to decrease steeply as the phagosomal lumen became acidified. Therefore, the pH sensitivity of the H+ pump, which possibly reflects a kinetic or allosteric effect, is the primary determinant of pHp.     

Bafilomycin A1 inhibits lysosomal,phagosomal, and plasma membrane H+-ATPase and induces lysosomal enzyme secretion in macrophages

Bafilomycin A₁, a specific inhibitor of H⁺-ATPases of the vacuolar type, was in the present study shown, at similar concentrations, to induce secretion of lysosomal enzyme and to elevate lysosomal pH in mouse macrophages. These results lend support to the previous suggestion of a triggering role for an increase in lysosomal pH and a permissive role for cytosolic pH in the exocytosis of macrophage lysosomal enzyme. Vacuolar H⁺-ATPases are present in the macrophage plasma membrane as well as in intracellular membranes, for example, those of the lysosomal and phagosomal compartments. Phagosomal acidification was shown to be achieved in part by a mechanism with a similar sensitivity to bafilomycin A₁ as lysosomal H⁺ transport and in part by an early, bafilomycin A₁-insensitive mechanism. We found a lesser sensitivity towards bafilomycin A₁ of the lysosomal and phagosomal H⁺-ATPase than that localized in the plasma membrane, indicating differences among H⁺-ATPases at the subcellular level. Also, by attempts to mobilize lysosomal H⁺-ATPase to the plasma membrane, support was obtained for the notion that subcellular H⁺-ATPase populations differ and thus possibly could be differentially regulated. © 1995 Wiley-Liss, Inc.     

Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells.

Bafilomycin A1 is known as a strong inhibitor of the vacuolar type H(+)-ATPase in vitro, whereas other type ATPases, e.g. F1,F0-ATPase, are not affected by this antibiotic (Bowman, E.M., Siebers, A., and Altendorf, K. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7972-7976). Effects of this inhibitor on lysosomes of living cultured cells were tested. The acidification of lysosomes revealed by the incubation with acridine orange was completely inhibited when BNL CL.2 and A431 cells were treated with 0.1-1 microM bafilomycin A1. The effect was revealed by washing the cells. Both studies using 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine and fluorescein isothiocyanate-dextran showed that the intralysomal pH of A431 cells increased from about 5.1-5.5 to about 6.3 in the presence of 1 microM bafilomycin A1. The pH increased gradually in about 50 min. In the presence of 1 microM bafilomycin A1, 125I-labeled epidermal growth factor (EGF) bound to the cell surface at 4 degrees C was internalized normally into the cells at 37 degrees C but was not degraded at all, in marked contrast to the rapid degradation of 125I-EGF in the control cells without the drug. Immunogold electron microscopy showed that EGF was transported into lysosomes irrespective of the addition of bafilomycin A1. These results suggest that the vacuolar type H(+)-ATPase plays a pivotal role in acidification and protein degradation in the lysosomes in vivo.     

Measurement of phagosomal pH of normal and CGD-like human neutrophils by dual fluorescence flow cytometry

BACKGROUND: Phagosomal pH is thought to play an important role in the antimicrobial activity of polymorphonuclear leukocytes (PMNs). In this study, we set up a method for a rapid and accurate measurement of phagosomal pH in PMNs with the use of Candida albicans doubly labeled with a pH-insensitive and a pH-sensitive probe and flow cytometry. METHODS: Heat-killed, serum-opsonized C. albicans were doubly labeled with fluorescein, a pH-sensitive probe, and rhodamine, a pH-insensitive probe, and incubated with human PMNs. Flow cytometric readings of PMN-associated Candida were then taken, and the intraphagosomal pH was calculated on the basis of the ratio of fluorescein:rhodamine fluorescence by using a calibration curve obtained after equilibration of phagosomal pH with different external pH values after addition of digitonin. RESULTS: A rapid rise in phagosomal pH, which reached pH 7.8, was observed 2 min after initiation of phagocytosis and progressively declined to pH 6.9 after 15 min. Such a rise was not observed in PMNs with defective microbicidal activity (deficient in nicotinamide adenine dinucleotide phosphate oxidase), where phagosomal pH dropped to pH 6.6, 2 min after phagocytosis. The abnormal initial acidification in PMNs deficient in nicotinamide adenine dinucleotide phosphate oxidase was prevented by using lysosomotropic weak bases or the vacuolar-type H(+) pump inhibitor concanamycin A. CONCLUSIONS: Phagosomal pH of PMNs can be easily and accurately measured by dual fluorescence flow cytometry. The method can be applied to assess phagosomal pH in PMNs with defective microbicidal activity and to monitor the outcome of pharmacologic interventions aimed at correcting its abnormalities.     

The effect of Mg2+, nucleotides and ATPase inhibitors on the uptake of [3H]-cGMP to inside-out vesicles from human erythrocytes.

An ATP-dependent transport system is responsible for the cellular extrusion of cGMP. The objective of the present study was to determine the effect of Mg2+, ATP and other nucleotides (2'-dATP, GTP and ADP), exogenous ATPase modulators (such as metavanadate, ouabain, EGTA, NEM, bafilomycin A1 and oligomycin A) on the cGMP transport. The uptake of [3H]-cGMP (1 microM) at 37 degrees C was studied in inside-out vesicles from human erythrocytes. Magnesium caused a maximal activation between 5 and 10 mM and the optimal ATP concentration was 1.25 mM with K50-values of 0.3-0.5 mM. Among other nucleotides tested, 2'-dATP (K50 of 0.7 mM) was nearly as effective as ATP, whereas cGMP accumulated slowly in the presence of GTP. ADP and metavanadate (P-type ATPase inhibitor) showed to be competitive inhibitors with Ki values of 0.15 mM and 10 microns, respectively. NEM (a sulphydryl agent) reduced the ATP-dependent uptake in a concentration-dependent manner with a Ki value of 10 microM. Ouabain (Na+/K(+)-ATPase inhibitor) had no effect. Bafilomycin A1 (V-type ATPase inhibitor) and oligomycin (F-type ATPase inhibitor) were the most potent inhibitors with Ki values of 0.7 and 1.8 microM, respectively. The present study suggests that the cellular cGMP extrusion is energized by an ATPase with a unique inhibitor profile, which clearly differentiates it from the other major classes of membrane-bound ATPases.     

The dual mechanism of the antifungal effect of new lysosomotropic agents on the Saccharomyces cerevisiae RXII strain

Quinacrine was used to visualize the intracellular pH changes in the yeast strain Saccharomyces cerevisiae RXII occurring after exposure to four recently-synthesized lysosomotropic drugs: DM-11, PY-11, PYG-12s and DMAL-12s. The cells took up quinacrine, mostly accumulating it in their vacuoles. DM-11 and PY-11 gave rise to diffuse quinacrine fluorescence throughout the cells, with the vacuoles staining to a somewhat greater extent than the cytosol. This quinacrine-detected overall acidification of the cell interior is very probably caused by blocking of plasma membrane H(+)-ATPase. PYG-12s gave rise to a strong vacuolar accumulation of the dye. Like the vacuolar ATPase inhibitor bafilomycin A(1), DMAL-12s strongly lowered the intensity of quinacrine fluorescence. Owing to its low pK(a), it can penetrate rapidly into the cells and may inhibit vacuolar H(+)-ATPase and prevent quinacrine-detectable vacuolar acidification without causing strong cell acidification. Since these drugs were found to penetrate into the cells, their lack of effect may reflect a higher resistance of both plasma membrane H(+)-ATPase and vacuolar ATPase to the drugs. Our data indicate that the lysosomotropic drugs under study have a dual action. On entering the cell, they cause intracellular acidification, very probably by inhibiting plasma membrane H(+)-ATPase and curtailing active proton pumping from the cells. Furthermore, they interfere with the function of V-type ATPase, causing vacuolar alkalinization and eventually cell death.     

Activation of the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae by addition of hydrogen peroxide.

Addition of hydrogen peroxide (greater than 10 mM) to aerated derepressed cells of S. cerevisiae in the absence of substrate caused a boost of endogenous respiration and both intra- and extracellular acidification, without any significant change in cellular ATP level. Furthermore, a hyperpolarization of the plasma membrane was indicated by an enhanced accumulation of tetraphenylphosphonium in the cells. The extracellular pH attained was as low as 3.5. The acidification could be suspended by the H(+)-ATPase inhibitors diethylstilbestrol and dicyclohexylcarbodiimide and was, in general, associated with an opposite flux of K+. K+ also stimulated the H(+)-ATPase activity in the purified plasma membrane fraction. These results are consistent with the plasma membrane H(+)-ATPase being involved in the H+ extrusion induced by H2O2 in the absence of substrate. Extended exposure of cells to H2O2 led eventually to an arrest of both respiration and ion fluxes that could be again lifted by depolarizing the plasma membrane. Along with differences in the cellular NADH/NAD+ ratio and in the participation of organic acids, this makes the H2O2-induced acidification distinct from that induced by glucose.     

H+ extrusion by an apical vacuolar-type H+-ATPase in rat renal proximal tubules

The activity of Na+/H(+)-exchange and H(+)-ATPase was measured in the absence of CO2/HCO3 by microfluorometry at the single cell level in rat proximal tubules (superficial S1/S2 segments) loaded with BCECF [2'7'-bis(carboxyethyl)5-6-carboxyfluorescein- acetoxymethylester]. Intracellular pH (pHi) was lowered by a NH4Cl-prepulse technique. In the absence of Na+ in the superfusion solutions, pHi recovered from the acid load by a mechanism inhibited by 0.1 microM bafilomycin A1, a specific inhibitor of a vacuolar-type H(+)-ATPase. Readdition of Na+ in the presence of bafilomycin A1 produced an immediate recovery of pHi by a mechanism sensitive to the addition of 10 microM EIPA (ethylisopropylamiloride), a specific inhibitor of Na+/H+ exchange. The transport rate of the H(+)-ATPase is about 40% of Na+/H(+)-exchange activity at a similar pHi (0.218 +/- 0.028 vs. 0.507 +/- 0.056 pH unit/min. Pre-exposure of the tubules to 30 mM fructose, 0.5 mM iodoacetate and 1 mM KCN (to deplete intracellular ATP) prevented a pHi recovery in Na(+)-free media; readdition of Na+ led to an immediate pHi recovery. Tubules pre-exposed to Cl(-)-free media for 2 hr also reduced the rate of Na(+)-independent pHi recovery. In free-flow electrophoretic separations of brush border membranes and basolateral membranes, a bafilomycin A1-sensitive ATPase activity was found to be associated with the brush border membrane fraction; half maximal inhibition is at 6 x 10(-10) M bafilomycin A1.(ABSTRACT TRUNCATED AT 250 WORDS)     

pH regulation in the intracellular malaria parasite, Plasmodium falciparum. H(+) extrusion via a V-type H(+)-ATPase

The mechanism by which the intra-erythrocytic form of the human malaria parasite, Plasmodium falciparum, extrudes H(+) ions and thereby regulates its cytosolic pH (pH(i)), was investigated using saponin-permeabilized parasitized erythrocytes. The parasite was able both to maintain its resting pH(i) and to recover from an imposed intracellular acidification in the absence of extracellular Na(+), thus ruling out the involvement of a Na(+)/H(+) exchanger in both processes. Both phenomena were ATP-dependent. Amiloride and the related compound ethylisopropylamiloride caused a substantial reduction in the resting pH(i) of the parasite, whereas EMD 96785, a potent and allegedly selective inhibitor of Na(+)/H(+) exchange, had relatively little effect. The resting pH(i) of the parasite was also reduced by the sulfhydryl reagent N-ethylmaleimide, by the carboxyl group blocker N,N'-dicyclohexylcarbodiimide, and by bafilomycin A(1), a potent inhibitor of V-type H(+)-ATPases. Bafilomycin A(1) blocked pH(i) recovery in parasites subjected to an intracellular acidification and reduced the rate of acidification of a weakly buffered solution by parasites under resting conditions. The data are consistent with the hypothesis that the malaria parasite, like other parasitic protozoa, has in its plasma membrane a V-type H(+)-ATPase, which serves as the major route for the efflux of H(+) ions.     

Degradation of mucopolysaccharide in intact isolated lysosomes

The function of isolated lysosomes was studied by measuring mucopolysaccharide degradation. Cultured human diploid skin fibroblasts were grown in medium containing H235SO4 to label endogenous mucopolysaccharide. Lysosome containing preparations at various stages of purity were isolated from disrupted cells. These preparations degraded mucopolysaccharide as indicated by the release of radioactive sulfate. Degradation was temperature-dependent, required intact lysosomes, and was optimal when incubation was carried out at neutral pH in a buffer of low ionic strength. Lysosomes from Hurler fibroblasts were unable to carry out the degradative process. ATP at 0.5 mM was found to stimulate both the rate and the extent of mucopolysaccharide degradation; GTP, UTP, and CTP had similar effects, whereas the noncleavable ATP analog adenosine 5'-(beta gamma-imido)triphosphate gave no stimulation. The ATP stimulation was inhibited by nigericin. ATP also stimulated chloroquine accumulation in lysosomes, the magnitude of which was used to measure the change in intralysosomal pH. The presence of ATP was associated with acidification of lysosome pH by 0.23 units. Acetyl coenzyme A was also found to stimulate lysosome function. This reagent, however, had no effect on chloroquine accumulation and thus appears to stimulate mucopolysaccharide degradation by a mechanism different than that caused by ATP.     

Polyamines inhibit phospholipase C-catalysed polyphosphoinositide hydrolysis. Studies with permeabilized GH3 cells.

[3H]Inositol-labelled GH3 rat anterior pituitary tumour cells were permeabilized with digitonin and were incubated at 37 degrees C in the presence of ATP and Mg2+. [3H]Polyphosphoinositide breakdown and [3H]inositol phosphate production were stimulated by hydrolysis-resistant GTP analogues and by Ca2+. Of the nucleotides tested, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) was the most effective stimulus. Activation by GTP gamma S appeared to be mediated by a guanine nucleotide-binding (G) protein as GTP gamma S-stimulated [3H]inositol phosphate production was inhibited by other nucleotides with a potency order of GTP = GDP = guanosine 5'-[beta-thio]diphosphate greater than ITP greater than GMP greater than UTP = CTP = adenosine 5'-[gamma-thio]triphosphate. The stimulatory effects of 10 microM-GTP gamma S on [3H]inositol phosphate levels were reversed by spermine and spermidine with IC50 values of approx. 0.25 and 2 mM respectively. Putrescine was inhibitory only at higher concentrations. Similarly, GTP gamma S-induced decreases in [3H]polyphosphoinositide levels were reversed by 2.5 mM-spermine. The inhibitory effects of spermine were not overcome by supramaximal concentrations of GTP gamma S. In contrast, [3H]inositol phosphate production stimulated by addition of 0.3-0.6 mM-Ca2+ to incubation media was only partially inhibited by spermine (5 mM), and spermine was not inhibitory when added Ca2+ was increased to 1 mM. These data show that polyamines, particularly spermine, inhibit phospholipase C-catalysed polyphosphoinositide hydrolysis with a marked selectivity towards the stimulatory effects of GTP gamma S.     

Acidification of endocytic vesicles by an ATP-dependent proton pump

One of the early events in the pathway of receptor-mediated endocytosis is the acidification of the newly formed endocytic vesicle. To examine the mechanism of acidification, we used fluorescein-labeled alpha 2- macroglobulin (F-alpha 2M) as a probe for endocytic vesicle pH. Changes in pH were determined from the change in fluorescein fluorescence at 490-nm excitation as measured with a microscope spectrofluorometer. After endocytosis of F-alpha 2M, mouse fibroblast cells were permeabilized by brief exposure to the detergent digitonin. Treatment with the ionophore monensin or the protonophore carbonyl cyanide p- trifluoromethoxyphenylhydrazone (FCCP) caused a rapid increase in the pH of the endocytic vesicle. Upon removal of the ionophore, the endocytic vesicle rapidly acidified only when MgATP or MgGTP was added. Neither ADP nor the nonhydrolyzable analog, adenosine 5'-(beta, gamma- imido)triphosphate (AMP-PNP) could support acidification. The ATP- dependent acidification did not require a specific cation or anion in the external media. Acidification was insensitive to vanadate and amiloride but was inhibited by Zn2+ and the anion transport inhibitor diisothiocyanostilbene disulfonic acid (DIDS). We also examined the acidification of lysosomes with the permeabilized cell system, using fluorescein isothiocyanate dextran as probe. DIDS inhibited the ATP- dependent reacidification of lysosomes, although at a lower concentration than that for inhibition of endocytic vesicle reacidification. These results demonstrate that endocytic vesicles contain an ATP-dependent acidification mechanism that shares similar characteristics with the previously described lysosomal proton pump.     

Chromium(III)ATP inactivating (Na+ + K+)-ATPase supports Na+-Na+ and Rb+-Rb+ exchanges in everted red blood cells but not Na+,K+ transport

The chromium(III) complex of ATP, an MgATP complex analogue, inactivates (Na+ + K+)-ATPase by forming a stable chromo-phosphointermediate. The rate constant k2 of inactivation at 37 degrees C of the beta, gamma-bidentate of CrATP is enhanced by Na+ (K0.5 = 1.08 mM), imidazole (K0.5 = 15 mM) and Mg2+ (K0.5 = 0.7 mM). These cations did not affect the dissociation constant of the enzyme-chromium-ATP complex. The inactive chromophosphoenzyme is reactivated slowly by high concentrations of Na+ at 37 degrees C. The half-maximal effect on the reactivation was reached at 40 mM NaCl, when the maximally observable reactivation was studied. However, 126 mM NaCl was necessary to see the half-maximal effect on the apparent reactivation velocity constant. K+ ions hindered the reactivation with a Ki of 70 microM. Formation of the chromophosphoenzyme led to a reduction of the Rb+ binding sites and of the capacity to occlude Rb+. The beta, gamma-bidentate of chromium(III)ATP (Kd = 8 microM) had a higher than the alpha, beta, gamma-tridentate of chromium(III)ATP (Kd = 44 microM) or the cobalt tetramine complex of ATP (Kd = 500 microM). The beta, gamma-bidentate of the chromium(III) complex of adenosine 5'-[beta, gamma-methylene]triphosphate also inactivated (Na+ + K+)ATPase. Although CrATP could not support Na+, K+ exchange in everted vesicles prepared from human red blood cells, it supported the Na+-Na+ and Rb+-Rb+ exchange. It is concluded that CrATP opens up Na+ and K+ channels by forming a relatively stable modified enzyme-CrATP complex. This stable complex is also formed in the presence of the chromium complex of adenosine 5'-[beta, gamma-methylene]triphosphate. Because the beta, gamma-bidentate of chromium ATP is recognized better than the alpha, beta, gamma-tridentate, it is concluded that the triphosphate site recognizes MgATP with a straight polyphosphate chain and that the Mg2+ resides between the beta- and the gamma-phosphorus. The enhancement of inactivation by Mg2+ and Na+ may be caused by conformational changes at the triphosphate site.     

Influence of ANG II on cytoplasmic sodium in cultured rabbit nonpigmented ciliary epithelium

Angiotensin (ANG) II receptors have been reported in the nonpigmented ciliary epithelium (NPE) of the eye. In cultured NPE, we found ANG II caused a dose-dependent rise of cytoplasmic sodium. The sodium increase was inhibited by the AT(1)-AT(2) receptor antagonist saralasin (IC(50) = 3.7 nM) and the AT(1) antagonist losartan (IC(50) = 0.6 nM) but not by the AT(2) antagonist PD-123319. ANG II also caused a dose-dependent increase in the rate of ouabain-sensitive (86)Rb uptake. The ANG II-induced cell sodium increase and (86)Rb uptake increase were reduced by dimethylamiloride (DMA; 10 microM). On the basis of this finding, we propose that Na(+)/H(+) exchange is stimulated by ANG II. Simultaneously, ANG II appears to inhibit H(+)-ATPase-mediated proton export. Thus Ang II (10 nM) did not alter the baseline cytoplasmic pH (pH(i)) but reduced pH(i) in cells that were also exposed to 10 microM DMA. Consistent with the notion of H(+)-ATPase inhibition in ANG II-treated NPE, bafilomycin A(1) (100 nM) (BAF) and ANG II were both observed to suppress the pH(i) increase that occurs upon exposure to a mixture of epinephrine (1 microM) and acetylcholine (10 microM) and the pH(i) increase elicited by depolarization. In ATP hydrolysis measurements, H(+)-ATPase activity (bafilomycin A(1)-sensitive ATP hydrolysis) was reduced significantly in cells that had been pretreated 10 min with 10 nM ANG II. In summary, these studies suggest that ANG II causes H(+)-ATPase inhibition and an increase of cell sodium due to activation of Na(+)/H(+) exchange.     

Involvement of a non-proton pump factor (possibly Donnan-type equilibrium) in maintenance of an acidic pH in lysosomes.

Change of the internal pH of isolated lysosomes was measured with fluorescein isothiocyanate-dextran. In buffer of pH 7.0, isolated lysosomes had an acidic pH of about 5.5, which decreased to pH 5.2 on addition of ATP. Addition of bafilomycin inhibited the acidification by H(+)-ATPase and resulted in an increase of the internal pH to 5.5 due to passive diffusion of protons across the lysosomal membrane. However, no further alkalization was observed. The acidic pH (pH 5.5) of isolated lysosomes could be maintained for at least 48 h in the absence of ATP, but increased gradually to pH 5.9-6.4 upon incubation with monovalent cations (K+ or Na+), amines, or ionophores. These results suggest that a non-proton pump factor (possibly Donnan equilibrium) is involved in maintaining the acidic pH of isolated lysosomes.     

Vacuolar H(+)-ATPase localized in plasma membranes of malaria parasite cells, Plasmodium falciparum, is involved in regional acidification of parasitized erythrocytes

Recent biochemical studies involving 2',7'-bis-(2-carboxyethyl)-5, 6-carboxylfluorescein (BCECF)-labeled saponin-permeabilized and parasitized erythrocytes indicated that malaria parasite cells maintain the resting cytoplasmic pH at about 7.3, and treatment with vacuolar proton-pump inhibitors reduces the resting pH to 6.7, suggesting proton extrusion from the parasite cells via vacuolar H(+)-ATPase (Saliba, K. J., and Kirk, K. (1999) J. Biol. Chem. 274, 33213-33219). In the present study, we investigated the localization of vacuolar H(+)-ATPase in Plasmodium falciparum cells infecting erythrocytes. Antibodies against vacuolar H(+)-ATPase subunit A and B specifically immunostained the infecting parasite cells and recognized a single 67- and 55-kDa polypeptide, respectively. Immunoelectron microscopy indicated that the immunological counterpart of V-ATPase subunits A and B is localized at the plasma membrane, small clear vesicles, and food vacuoles, a lower extent being detected at the parasitophorus vacuolar membrane of the parasite cells. We measured the cytoplasmic pH of both infected erythrocytes and invading malaria parasite cells by microfluorimetry using BCECF fluorescence. It was found that a restricted area of the erythrocyte cytoplasm near a parasite cell is slightly acidic, being about pH 6.9. The pH increased to pH 7.3 upon the addition of either concanamycin B or bafilomycin A(1), specific inhibitors of vacuolar H(+)-ATPase. Simultaneously, the cytoplasmic pH of the infecting parasite cell decreased from pH 7.3 to 7.1. Neither vanadate at 0.5 mm, an inhibitor of P-type H(+)-ATPase, nor ethylisopropylamiloride at 0.2 mm, an inhibitor of Na(+)/H(+)-exchanger, affected the cytoplasmic pH of erythrocytes or infecting parasite cells. These results constitute direct evidence that plasma membrane vacuolar H(+)-ATPase is responsible for active extrusion of protons from the parasite cells.     

The HPV-16 E5 oncogene and bafilomycin A(1) influence cell motility

It is known that the proper function of the vacuolar H(+)-ATPase is inhibited by bafilomycin A(1). In transfected cells the E5 protein interacts with the 16 kDa subunit of the vacuolar H(+)-ATPase. Thereby the pH gradient in endocytic structures is impaired. The present study demonstrates for the first time that the inhibition of the vacuolar H(+)-ATPase in NIH3T3 cells with bafilomycin A(1) or by transfection of cells with the HPV-16 E5 oncogene leads to a changed morphology and a reduced motility as shown by computer-assisted video recordings and image analysis. Bafilomycin A(1) potentiates the effect of the E5 protein on cell motility and this cooperative effect indicates that the E5 protein and bafilomycin A(1) either target the vacuolar H(+)-ATPase differently or that the E5 protein has additional targets in transfected cells. Our data therefore show that proper function of the vacuolar H(+)-ATPase is needed for normal cell locomotion.     

Effect of ATP analogs of DNA synthesis in isolated nuclei

Optimal synthesis of DNA in Ehrlich ascites cell nuclei is shown to be dependent upon the presence of both ATP and ADP. ATP can be replaced only by dATP. An ATP regenerating system is less effective than ATP alone or ATP in combination with ADP. ATP does not stimulate DNA synthesis primarily by maintenance of deoxyribonucleotide triphosphate levels. When the inhibition of DNA synthesis by high ATP levels is taken into account, the ATP analogs adenosine 5'-(alpha,beta-methylene)triphosphate, adenosine 5'-(beta, gamma-methylene)-triphosphate, and adenosine 5'-(beta, gamma-imino)triphosphate can neither substitute for ATP nor inhibit the ATP stimulation of DNA synthesis. Adenosine 5'-(3-thio)triphosphate, however, is a competitive inhibitor of DNA synthesis.     

Requirement for vacuolar H+ -ATPase activity and Ca2+ gradient during entry of rotavirus into MA104 cells

The mechanism by which rotavirus and other nonenveloped viruses enter the cell is still not clear. We have proposed an endocytosis model where the critical step for virus uncoating and membrane permeabilization is the decrease in Ca(2+) concentration in the endosome. In this paper, we monitored rotavirus entry by measuring alpha-sarcin-rotavirus coentry and infectivity in MA104 cells. The participation of endocytosis, acidification, and endosomal Ca(2+) concentration on virus entry was studied by inhibiting the endosomal H(+)-ATPase with bafilomycin A1 and/or increasing the extracellular calcium reservoir by addition of 10 mM CaEGTA. Rotavirus-alpha-sarcin coentry was inhibited by bafilomycin A1 and by addition of 10 mM CaEGTA. These effects were additive. These substances induced a significant inhibition of infectivity without affecting virus binding and postentry steps. These results are compatible with the interpretation that bafilomycin A1 and CaEGTA block rotavirus penetration from the endosome into the cytoplasm and support our hypothesis of a Ca(2+)-dependent endocytosis model.