Proton Extrusion Activity Research Articles

Acid Microenvironment in Bone Sarcomas.

Simple SummaryAlthough rare, malignant bone sarcomas have devastating clinical implications for the health and survival of young adults and children. To date, efforts to identify the molecular drivers and targets have focused on cancer cells or on the interplay between cancer cells and stromal cells in the tumour microenvironment. On the contrary, in the current literature, the role of the chemical-physical conditions of the tumour microenvironment that may be implicated in sarcoma aggressiveness and progression are poorly reported and discussed. Among these, extracellular acidosis is a well-recognized hallmark of bone sarcomas and promotes cancer growth and dissemination but data presented on this topic are fragmented. Hence, we intended to provide a general and comprehensive overview of the causes and implications of acidosis in bone sarcoma.In bone sarcomas, extracellular proton accumulation is an intrinsic driver of malignancy. Extracellular acidosis increases stemness, invasion, angiogenesis, metastasis, and resistance to therapy of cancer cells. It reprograms tumour-associated stroma into a protumour phenotype through the release of inflammatory cytokines. It affects bone homeostasis, as extracellular proton accumulation is perceived by acid-sensing ion channels located at the cell membrane of normal bone cells. In bone, acidosis results from the altered glycolytic metabolism of bone cancer cells and the resorption activity of tumour-induced osteoclasts that share the same ecosystem. Proton extrusion activity is mediated by extruders and transporters located at the cell membrane of normal and transformed cells, including vacuolar ATPase and carbonic anhydrase IX, or by the release of highly acidic lysosomes by exocytosis. To date, a number of investigations have focused on the effects of acidosis and its inhibition in bone sarcomas, including studies evaluating the use of photodynamic therapy. In this review, we will discuss the current status of all findings on extracellular acidosis in bone sarcomas, with a specific focus on the characteristics of the bone microenvironment and the acid-targeting therapeutic approaches that are currently being evaluated.

Dissecting iron deficiency‐induced proton extrusion in Arabidopsis roots

Here, we have analysed the H(+)-ATPase-mediated extrusion of protons across the plasma membrane (PM) of rhizodermic cells, a process that is inducible by iron (Fe) deficiency and thought to serve in the mobilization of sparingly soluble Fe sources. The induction and function of Fe-responsive PM H(+)-ATPases in Arabidopsis roots was investigated by gene expression analysis and by using mutants defective in the expression or function of one of the isogenes. In addition, the expression of the most responsive isogenes was investigated in natural Arabidopsis accessions that have been selected for their in vivo proton extrusion activity. Our data suggest that the rhizosphere acidification in response to Fe deficiency is chiefly mediated by AHA2, while AHA1 functions as a housekeeping isoform. The aha7 knock-out mutant plants showed a reduced frequency of root hairs, suggesting an involvement of AHA7 in the differentiation of rhizodermic cells. Acidification capacity varied among Arabidopsis accessions and was associated with a high induction of AHA2 and IRT1, a high relative growth rate and a shoot-root ratio that was unaffected by the external Fe supply. An effective regulation of the Fe-responsive genes and a stable shoot-root ratio may represent important characteristics for the Fe uptake efficiency.

Heterologously expressed protein phosphatase calcineurin downregulates plant plasma membrane H +-ATPase activity at the post-translational level

To investigate the effects of calcineurin expression on cellular ion homeostasis in plants, we have obtained a transgenic cell culture of tomato, expressing constitutively activated yeast calcineurin. Transgenic cells exhibited reduced growth rates and proton extrusion activity in vivo. We show that reduction of plasma membrane H +-ATPase activity by expression of calcineurin is the basis for the observed phenotypes. Transgenic calli and cell suspensions displayed also increased salt tolerance and contained slightly higher Ca 2+ and K + levels. This demonstrates that calcineurin can modulate ion homeostasis in plants as it does in yeast by affecting the activity of primary ion transporters.

Proton pumping by tomato roots. Effect of Fe deficiency and hormones on the activity and distribution of plasma membrane H+‐ATPase in rhizodermal cells

ABSTRACTThe immunocytochemical localization of the plasma membrane H+‐ATPase in epidermal cells of tomato roots was studied using a monoclonal antibody raised against purified maize P‐type H+‐ATPase. Plants subjected to iron starvation exhibited increased proton extrusion that was confined to the root elongation zones. Immunogold labelling of the H+‐ATPase on the plasma membrane was considerably higher in rhizodermal cells within zones with intense proton extrusion than in non‐acidifying areas of the roots. Transfer cells were formed in rhizodermal cells of Fe‐deficient plants. Quantitative determination of immunolabelling revealed that the density of PM H+‐ATPase in transfer cells was about twice that of ordinary epidermal cells. In transfer cells, H+‐ATPase was most abundant on the plasma membrane lining the labyrinthine invaginations of the peripheral cell wall. While the number of immunologically detectable ATPase molecules in transfer cells was not spatially correlated with proton extrusion activity, the frequency of transfer cells was considerably higher in acidifying root areas relative to non‐active segments. Split‐root experiments indicated that both the steady‐state level of plasma membrane H+‐ATPase and proton extrusion activity are systemically regulated, indicating inter‐organ regulation of rhizosphere acidification. Exogenous application of the auxin analog 2,4‐dichlorophenoxyacetic acid and the ethylene precursor 1‐aminocyclopropane‐1‐carboxlic acid caused the formation of transfer cells at a frequency similar to that observed in Fe‐deficient roots. However, the number of proton pumps was not affected by the hormone treatment, suggesting that both responses are regulated independently. It is concluded that transfer cells in the rhizodermis may be important but not crucial for rhizosphere acidification.

Quantification of presumptive Na+/H+ antiporters of the erythrocytes of trout and eel

The presumptive Na(+)/H(+) exchange sites of trout and eel erythrocytes were quantified using amiloride-displaceable 5-(N-methyl-N-[(3)H]isobutyl)-amiloride ((3)H-MIA) equilibrium binding to further evaluate the mechanisms of i) hypoxia-mediated modifications in the trout erythrocyte β-adrenergic signal transduction system and ii) the marked differences in the catecholamine responsiveness of this system between the trout and eel. MIA was a more potent inhibitor of both trout apparent erythrocyte proton extrusion (IC50 = 20.1 ± 1.1 μmol l(-1), N = 6) activity (as evaluated by measuring plasma pH changes after addition of catecholamine in vitro) and specific (3)H-MIA binding (IC50 = 257 ± 8.2 nmol l(-1), N = 3) than amiloride, which possessed a proton extrusion IC50 of 26.1 ± 1.6 μmol l(-1) (N = 6) and a binding IC50 of 891 ± 113 nmol l(-1) (N = 3). The specific Na(+) channel blocker phenamil was without effect on adrenergic proton extrusion activity or specific (3)H-MIA binding. Trout erythrocytes suspended in Na(+)-free saline and maintained under normoxic conditions possessed 37,675 ± 6,678 (N = 6) amiloride-displaceable (3)H-MIA binding sites per cell (Bmax, presumptive Na(+)/H(+) antiporters) with an apparent dissociation constant (KD) of 244 ± 29 nmol l(-1) (N = 6). Acute hypoxia (PO2 = 1.2 kPa; 30 min) did not affect the KD, yet resulted in a 65% increase in the number of presumptive Na(+)/H(+) antiporters. Normoxic eel erythrocytes, similarly suspended in Na(+)-free saline, possessed only 17,133 ± 3,716 presumptive Na(+)/H(+) antiporters (N = 6), 45% of that of trout erythrocytes, with a similar KD (246 ± 41 nmol l(-1), N = 6). These findings suggest that inter- and intra-specific differences in the responsiveness of the teleost erythrocyte β-adrenergic signal transduction system can be explained, in part, by differences in the numbers of Na(+)/H(+) exchange sites.

Studies of the Physiological and Genetic Basis of Acid Tolerance in Rhizobium leguminosarum biovar trifolii

Acid-tolerant Rhizobium leguminosarum biovar trifolii ANU1173 was able to grow on laboratory media at a pH as low as 4.5. Transposon Tn5 mutagenesis was used to isolate mutants of strain ANU1173, which were unable to grow on media at a pH of less than 4.8. The acid-tolerant strain ANU1173 maintained a near-neutral intracellular pH when the external pH was as low as 4.5. In contrast, the acid-sensitive mutants AS25 and AS28 derived from ANU1173 had an acidic intracellular pH when the external pH was less than 5.5. The acid-sensitive R. leguminosarum biovar trifolii ANU794, which was comparatively more sensitive to low pH than mutants AS25 and AS28, showed a more acidic internal pH than the two mutants when the three strains were exposed to medium buffered at a pH of less than 5.5. The two acid-sensitive mutants had an increased membrane permeability to protons but did not change their proton extrusion activities. However, the acid-sensitive strain ANU794 exhibited both a higher membrane permeability to protons and a lower proton extrusion activity compared with the acid-tolerant strain ANU1173. DNA hybridization analysis showed that mutants AS25 and AS28 carried a single copy of Tn5 located in 13.7-kb (AS25) and 10.0-kb (AS28) EcoRI DNA fragments. The wild-type DNA sequences spanning the mutation sites of mutants AS25 and AS28 were cloned from genomic DNA of strain ANU1173. Transfer of these wild-type DNA sequences into corresponding Tn5-induced acid-sensitive mutants, respectively, restored the mutants to their acid tolerance phenotypes. Mapping studies showed that the AS25 locus was mapped to a 5.6-kb EcoRI-BamHI megaplasmid DNA fragment, whilst the AS28 locus was located in an 8.7-kb BglII chromosomal DNA fragment.

Involvement of Genes on a Megaplasmid in the Acid-Tolerant Phenotype of Rhizobium leguminosarum Biovar Trifolii

The acid-tolerant Rhizobium leguminosarum biovar trifolii strain ANU1173 exhibited several new phenotypes when cured of its symbiotic (Sym) plasmid and the second largest megaplasmid. Strain P22, which has lost these two plasmids, had reduced exopolysaccharide production and cell mobility on TY medium. The parent strain ANU1173 was able to grow easily in laboratory media at pH 4.5, whereas the derivative strain P22 was unable to grow in media at a pH of <4.7. The intracellular pH of strain ANU1173 was 6.8 when the external pH was 4.5. In contrast, strain P22 had an acidic intracellular pH of <6.4 when the external pH was <5.5. Strain P22 had a dramatically increased membrane permeability to protons and decreased proton extrusion activity. Analysis with sodium dodecyl sulfate-polyacrylamide gels showed that strain P22 lacked a slow-migrating lipopolysaccharide (LPS) banding group which was present in the parent strain. Mobilization of the second largest megaplasmid of strain ANU1173 back into strain P22 restored the altered LPS structure and physiological characteristics of strain P22. Mobilization of the Sym plasmid of strain ANU1173 into strain P22 showed that the second largest megaplasmid of strain ANU1173 was required for the establishment of nitrogen-fixing nodules on Trifolium repens and Trifolium subterraneum. Furthermore, an examination of a large number of specific exopolysaccharide- or LPS-deficient Rhizobium mutants did not show a positive correlation between exopolysaccharide or LPS synthesis and acid tolerance.

Alteration of distribution of regions with high net proton extrusion in tomato roots infected with Meloidogyne incognita

Regions of net proton extrusion activity were localized by use of the fluorescent dye, fluorescein (pK a ≈ 4·9), which can enter plant cells with a localized low apoplastic pH. After incubation in a fluorescein solution for 16 h, the root systems were surface screened with UV-irradiation. Uninfected root systems fluoresced brightly at apical root regions (2–3 cm from the root tip), whereas root systems infected with M. incognita showed only slight fluorescence. In the latter case fluorescence was restricted to uninfected root regions distal to infection sites and young growing galls near the root apex. Older galls displayed a sharp transition zone between the active and the less active proton-extruding root regions. The nematode-induced giant cells accumulated fluorescein from the apoplast. Dissipation of the proton-motive force by carbonylcyanide m-chlorophenylhydrazone strongly suppressed dye uptake by the apical root regions and giant cells. It is concluded that net proton extrusion activity of infected apical root regions is decreased compared to similar uninfected ones. This decreased proton extrusion may have major consequences for the uptake and mobilization of minerals. Giant cells have an increased net proton extrusion activity compared to other cell types at the infection site, suggesting that proton extrusion might be a driving force for solute uptake from the apoplast.

Determining Apoplastic pH Differences in Pea Roots by Use of the Fluorescent Dye Fluorescein

The uptake of the fluorescent dye fluorescein (uranin) was used to determine apoplastic pH differences in pea roots. Fluorescein, an acidic dye (pKa≊4·9), is able to pass cell membranes in its undissociated form. The uptake was studied in roots of young and older pea seedlings growing in solution or in sand, respectively. Following 16 h of incubation in an unbuffered 0·01% (w/v) fluorescein solution with a pH of 7·9, the dye only accumulated in cells of the root apex, as was visible under ultraviolet irradiation. Dye accumulation was prevented when the proton buffering strength of the incubation medium was increased. Under more acidic conditions (pH 5·0) it also accumulated in the root base. Fluorescein uptake was strongly suppressed by CCCP, reduced by DNP and stimulated by KCl. The results indicate that net proton extrusion activity is the main mechanism for generating a low apoplastic pH. Cross-sections of roots of seedlings, incubated in the unbuffered fluorescein-containing medium, showed discriminative dye uptake by the different root tissues and cell types, suggesting that net proton extrusion activity may vary with the cell type and its location in the root. The method can be used to localize root regions, tissues, and cells inside the root, with a relatively low apoplastic pH.

Expression of Na(+)-H+ exchange and ATP-dependent proton extrusion in growing rat IMCD cells

As the last step of urinary acidification, the inner medullary collecting duct (IMCD) is thought to secrete protons into the tubular lumens by means of a H(+)-translocating adenosinetriphosphatase (H(+)-ATPase). However, recent studies have also shown the existence of Na(+)-H+ exchange activity in IMCD cells. Although the physiological function of the antiporter in IMCD cells is unknown, activation of Na(+)-H+ exchange in other cell-culture systems has been suggested to be closely associated with the process of cell growth. Thus presence of Na(+)-H+ exchange may relate to the growth phase of these cells. To examine intracellular pH (pHi) regulation in growing IMCD cells, we studied proton transport by Na(+)-dependent and Na(+)-independent mechanisms by microfluorimetry using the pHi-sensitive dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF/AM). Actively growing cells, defined by [3H]thymidine incorporations, demonstrated an amiloride-sensitive Na(+)-dependent pHi recovery after an acid load. No pHi recovery was evident in the absence of Na+, indicating the importance of Na(+)-H+ exchange for pHi recovery. However, when evaluated in quiescent cells, Na(+)-dependent pHi recovery appeared to be diminished. Instead, a Na(+)-independent pHi recovery which was inhibitable by ATP depletion and by 1 mM N-ethylmaleimide was present, suggesting function of a H(+)-ATPase. These findings indicate that Na(+)-dependent proton extrusion activity (Na(+)-H+ exchange) but not Na(+)-independent proton extrusion activity is expressed during the rapid growth phase of IMCD cells, whereas the more quiescent cells express Na(+)-independent ATP-dependent proton extrusion activity and a possibly less active Na(+)-H+ exchanger.

Proton pump activity in bundle sheath tissues of broad‐leaved trees in relation to leaf age

The fluorescent probe sulphorhodamine G (SR) has been previously used as an indicator of low extra‐cellular pH and, by inference, of proton extrusion activity in living leaves. In legumes the SR uptake and proton extrusion was characteristic of the extended bundle sheath system (EBS) or paraveinal mesophyll, composed of bundle sheath cells and the related network of bridging cells between veins. This system has been identified as a site of temporary storage of amino carbon in soybean. A tree species. Populus deltoides Bartr. ex Marsh, was known both to have the EBS system in its leaves and to carry organic nitrogen in its xylem sap. It is now shown that P. deltoides also accumulates the SR probe in the EBS system. This association has been explored in 8 other broad‐leaved tree species. Seven of the 8 species have EBS systems and accumulate SR in them in early summer. The 8th species, Tilia americana L. has no EBS system and shows weak SR accumulation. The capacity to accumulate SR (and by inference to scavenge solutes from the transpiration stream) disappeared in all species at various stages in late summer. In two species, in addition, SR accumulation is interrupted for several weeks during fruit growth. It is proposed that EBS systems will be found in many dicotyledonous leaves, and will be found to scavenge solutes, especially organic nitrogen, from the xylem sap.

A possible relationship between stalk necrosis and membrane transport in grapevine cultivars

The higher susceptibility to stalk necrosis of the grape ( Vitis vinifera L.) cultivar ‘Bonarda’ (clusters with few berries) than the cultivar ‘Barbera’ (clusters with many berries) was correlated to higher K + Ca 2+ ratios in the tissue and to a different functioning of the transport mechanism. At the middle of the meristematic growth phase of the berries, segments excised from grapevine clusters of the more susceptible cultivars showed lower proton extrusion activity, lower K + and Ca 2+ leakage and similar uptake capacity both for K + and for Ca 2+. These results are discussed in regard to the onset of stalk necrosis in grapevine clusters.

Osmotically Induced Proton Extrusion from Carrot Cells in Suspension Culture

Addition of 200 mm of a polyol to anthocyanin containing carrot (Daucus carota L.) cells in suspension culture decreased turgor pressure to zero and induced hyperpolarization of the membrane potential and acidification of the medium due to H(+) extrusion. These changes were shown to be slightly affected by vanadate. In parallel, a decrease in intracellular ATP and total adenylate concentrations were observed. However, when the osmoticum was NaCl acidification of the medium occurred in the absence of considerable changes in intracellular ATP concentration. These results are interpreted as indicating that a drop of turgor, by addition of a polyol, triggers a proton extrusion activity which is only slightly inhibited by vanadate but apparently ATP utilizing. The observed decrease in ATP level occurs without a change in respiration rate and is accompanied by a drop in total adenylate pool. However when NaCl is the osmoticum it is assumed that Delta(muH+) is enhanced through a Na(+)/H(+) antiporter. The difference between the two types of osmotica as related to their ability to penetrate through the cellular membrane is discussed.

The Effect of Fusicoccin and Monovalent Cations on the Viability of Wheat Seeds

Fusicoccin (FC), K+ and Na+ restore the viability of aged wheat seeds and isolated wheat embryos. We studied the effect of FC and K+ on the evolution of the proton extrusion activity and net K+ uptake during the early phases of germination of wheat embryos isolated from unaged and aged seeds. Our data show that in this material, germination is also characterized by the activation of the proton-extruding activity and by the leakage and subsequent re-absorption of K+. The percentage of germination appeared to be correlated with the amount of proton extrusion and K+ net uptake. In aged embryos the positive effects of FC and K+ on germination were correlated with a marked increase in both proton extrusion and K+ net uptake. These findings suggest that: (a) in wheat seeds, germination is also correlated to the activation of a mechanism linked to H+/K+ exchange; (b) the damage of this mechanism is, under our conditions, responsible for the loss of viability.