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PubMed2004

Groundwater acidification and the mobilization of trace metals in a sandy aquifer.

The acidification of groundwater due to acid rain impact and the mobilization of the trace metals Ni, Be, Cd and Co was studied in a noncalcareous sandy aquifer. The groundwater is acidified down to pH 4.4 in the upper 3-4 m of the saturated zone. There is a sharp acidification front and below that the pH increases to 5.2-6.5. The acid zone groundwater contains an Al concentration of approximately 0.2 mM. These observations could be explained by a reactive transport model for downward groundwater movement based on ion exchange and equilibrium with Al(OH)3. At the acidification front, the Al3+ in groundwater exchanges for sorbed Ca2+ and Mg2+ and the coupled dissolution of Al(OH)3 causes the pH to increase. The downward migration rate of the acidification front is 3.5-5.0 cm/yr. Trace metals (Ni, Be, Cd and Co) are found to accumulate near the acidification front. Downward moving, low pH, and trace metal containing groundwater passes the acidification front, and the trace metals adsorb as the pH increases. The acidification front moves downward at a slower rate, and in this process the heavy metals are desorbed. Accordingly, the acidification front functions as a geochemical trap where trace metals accumulate, and their amount will increase with time. Different surface complexation models were explored to explain the behavior of Ni. Neither a simple iron oxide surface complexation model nor ion exchange could explain the field observations of the Ni distribution. The sediment appeared, even at low pH, to have a much stronger affinity toward Ni than predicted by the iron oxide model. The discrepancy can be accounted for in the model by increasing the Ni binding strength constant in combination with an increased number of reactive sites.

Acid Rain↗

PubMed2004

Stratum corneum acidification in neonatal skin: secretory phospholipase A2 and the sodium/hydrogen antiporter-1 acidify neonatal rat stratum corneum.

At birth, human stratum corneum (SC) displays a near-neutral surface pH, which declines over several days to weeks to months to an acidic pH, comparable to that of adults. Recent studies suggest that an acidic pH is required for normal permeability barrier homeostasis and SC integrity/cohesion. We assessed here the basis for postnatal acidification in the neonatal rat, where SC pH, as measured with a flat surface electrode, declines progressively from near-neutral levels (pH 6.63) on postnatal days 0 to 1 to adult levels (pH 5.9) or even below over the subsequent 7 to 8 d. The postnatal decline in SC pH was paralleled by a progressive activation of a pH-dependent hydrolytic enzyme, beta-glucocerebrosidase. Because SC acidification could not be linked to commonly implicated exogenous factors, such as bacterial colonization, or the deposition of sebaceous gland products. We next assessed whether changes in one or more of three endogenous mechanisms demonstrate postnatal activity changes that contribute to the progressive development of an acidic SC pH. Although the histidine-to-urocanic acid pathway has been implicated in acidification of the adult SC, surface pH is completely normal in histidase-deficient (his/his, Peruvian) mice, ruling out a requirement for this mechanism. In contrast, when sodium/hydrogen antiporter-1 (NHE1), which predominantly acidifies membrane domains at the stratum granulosum-SC interface, is inhibited, postnatal acidification of the SC is partially blocked. Likewise, SC secretory phospholipase A2 (sPLA2) activity, measured with a fluorometric assay, is low at birth, but increases progressively (by 66%) over the first 5 d after birth, and inhibition of sPLA2 between days 0 to 1 and days 5 to 6 delays postnatal SC acidification. Together, these results describe a neonatal model, in which the development of an acidic surface pH can be ascribed, in part, to progressive SC acidification by two endogenous mechanisms, namely, sPLA2 and NHE1, which are known to be important for acidification of adult rodent SC. Conversely, the impaired acidification of neonatal SC, which has important functional and clinical consequences, can be explained by the relatively low activities of one or both of these mechanisms at birth.

Acids↗

PubMed1996

Events in apoptosis. Acidification is downstream of protease activation and BCL-2 protection.

Cytoplasmic acidification is now recognized as a feature of apoptosis in a variety of systems. However, its relation to other events in the process of apoptosis is not yet characterized. In this work, we examined the effect of BCL-2 overexpression on acidification mediated by cycloheximide treatment or Fas ligation in Jurkat T-lymphoblasts. We find that BCL-2 overexpression attenuates cytoplasmic acidification and apoptosis detected by annexin V labeling. Acidification and phosphatidylserine externalization were found to occur concurrently. We also examined the requirement for protease activation for cytoplasmic acidification to occur and found that inhibition of interleukin-1beta converting enzyme/CED-3 family proteases (using carbobenzoxy-Val-Ala-Asp-fluoromethylketone, an inhibitor of these proteases) prevents acidification and apoptosis mediated by Fas ligation. These studies suggest that BCL-2 acts at a point upstream of acidification and that protease activation is also upstream of acidification.

Amino Acid Chloromethyl Ketones↗

PubMed1992

Second messengers regulate endosomal acidification in Swiss 3T3 fibroblasts.

Acidification of the endosomal pathway is important for ligand and receptor sorting, toxin activation, and protein degradation by lysosomal acid hydrolases. Fluorescent probes and imaging methods were developed to measure pH to better than 0.2 U accuracy in individual endocytic vesicles in Swiss 3T3 fibroblasts. Endosomes were pulse labeled with transferrin (Tf), alpha 2-macroglobulin (alpha 2M), or dextran, each conjugated with tetramethylrhodamine and carboxyfluorescein (for pH 5-8) or dichlorocarboxyfluorescein (for pH 4-6); pH in individual labeled vesicles was measured by ratio imaging using a cooled CCD camera and novel image analysis software. Tf-labeled endosomes acidified to pH 6.2 +/- 0.1 with a t1/2 of 4 min at 37 degrees C, and remained small and near the cell periphery. Dextran- and alpha 2M-labeled endosomes acidified to pH 4.7 +/- 0.2, becoming larger and moving toward the nucleus over 30 min; approximately 15% of alpha 2M-labeled endosomes were strongly acidic (pH less than 5.5) at only 1 min after labeling. Replacement of external Cl by NO3 or isethionate strongly and reversibly inhibited acidification. Addition of ouabain (1 mM) at the time of labeling strongly enhanced acidification in the first 5 min; Tf-labeled endosomes acidified to pH 5.3 without a change in morphology. Activation of phospholipase C by vasopressin (50 nM) enhanced acidification of early endosomes; activation of protein kinase C by PMA (100 nM) enhanced acidification strongly, whereas elevation of intracellular Ca by A23187 (1 microM) had no effect on acidification. Activation of protein kinase A by CPT-cAMP (0.5 mM) or forskolin (50 microM) inhibited acidification. Lysosomal pH was not affected by ouabain or the protein kinase activators. These results establish a methodology for quantitative measurement of pH in individual endocytic vesicles, and demonstrate that acidification of endosomes labeled with Tf and alpha 2M (receptor-mediated endocytosis) and dextran (fluid-phase endocytosis) is sensitive to intracellular anion composition, Na/K pump inhibition, and multiple intracellular second messengers.

3T3 Cells↗

PubMed1980

Luminal acidification by the perfused rat cauda epididymidis.

1. Acid secretion by the rat cauda epididymidis was studied by microperfusion of the epididymal duct and by measuring the pH of the perfusate at a constant pCO(2) using a micro pH sensitive electrode. The rate of acidification was expressed as the rate of fall of intraluminal bicarbonate per cm duct per min.2. When the cauda epididymal duct was perfused with normal bicarbonate solution, the luminal bicarbonate concentration fell at a rate of 0.59 +/- 0.39 n-equiv cm(-1) min(-1) (mean +/- s.e.,n = 22).3. The rate of luminal acidification was independent of the perfusion rate but was dependent on the concentration of bicarbonate in the perfusion fluid. The rate of fall of luminal bicarbonate increased with increasing bicarbonate concentration and showed saturation at an intraluminal bicarbonate concentration of 25 m-mole/l.4. Acidification was abolished in the absence of intraluminal sodium ions. This may suggest a linked sodium reabsorption and hydrogen ion secretion.5. Acidification of the luminal fluid was studied under different acid-base conditions. In animals undergoing metabolic acidosis, the rate of acidification was enhanced, and conversely in animals undergoing metabolic alkalosis, the rate was depressed.6. Intravenous infusion of acetazolamide into rats at a dose rate of 20 mg/kg.hr markedly inhibited the acidification process. This effect was still observed in animals undergoing metabolic acidosis. Acetazolamide (10(-5)m) applied luminally was found to have no effect but higher concentration (10(-4)m) was found to inhibit acidification by 50%.7. The role of acidification of the epididymal fluid in sperm maturation was discussed.

Acetazolamide↗

PubMed1993

Acidification of rat liver lysosomes: quantitation and comparison with endosomes.

Both lysosomes and endosomes are acidified by an electrogenic proton pump, although studies in intact cells indicate that the steady-state internal pH (pHi) of lysosomes is more acid than that of endosomes. We undertook the present study to examine in detail the acidification mechanism of purified rat liver secondary lysosomes and to compare it with that of a population of early endosomes. Both endosomes and lysosomes exhibited ATP-dependent acidification, but proton influx rates were 2.4- to 2.7-fold greater for endosomes than for lysosomes because of differences in both buffering capacity and acidification rates, suggesting that endosomes exhibited greater numbers or rates of proton pumps. Lysosomes, however, exhibited a more acidic steady-state pHi due in part to a slower proton leak rate. Changes in medium Cl- increased acidification rates of endosomes more than lysosomes, and the lysosome ATP-dependent interior-positive membrane potential was only partially eliminated by high-Cl- medium. Permeability studies suggested that lysosomes were less permeable to Na+, Li+, and Cl- and more permeable to K+ and PO4(2-) than endosomes. Na-K-adenosine-triphosphatase did not appear to regulate acidification of either vesicle type. Endosome and lysosome acidification displayed similar inhibition profiles to N-ethylmaleimide, dicyclohexyl-carbodiimide, and vanadate, although lysosomes were somewhat more sensitive [concentration producing 50% maximal inhibition (IC50) 1 nM] to bafilomycin A1 than endosomes (IC50 7.6 nM). Oligomycin (1.5-3 microM) stimulated lysosome acidification due to shunting of membrane potential. Overall, acidification of endosomes and lysosomes was qualitatively similar but quantitatively somewhat different, possibly related to differences in the density or rate of proton pumps as well as vesicle permeability to protons, anions, and other cations.

Acids↗

PubMed1997

Intracellular acidification inhibits opposum kidney cell phosphate uptake.

The aim of our study is to examine the effect of intracellular pH (pHi) on inorganic phosphate (Pi) uptake by a proximal tubular cell line, the opposum kidney (OK) cells. The OK cell pHi (7.48 +/- 0.02; n = 12) was altered to levels between 6.5 and 8.5 by the high-K+ nigericin method, and cell uptakes were measured at 7.5 extracellular pH. It was found that pHi acidification suppressed Pi uptake with a decrease in maximal reaction rate, whereas alkalinization had no significant effect. Other Na(+)-dependent transport systems for glucose and amino acid were not affected by these pHi changes. The inhibition of cell Pi uptake by pHi acidification was not prevented by protein synthesis inhibitors (actinomycin D or cycloheximide) or by Na+/H+ exchange inhibitor [5-(N-ethyl-N-isopropyl)-amiloride]. pHi acidification caused a significant decrease in cellular adenosine 3',5'-cyclic monophosphate (cAMP) content, and cAMP-dependent protein kinase inhibitor (H-89) also did not prevent inhibition of cell Pi uptake by pHi acidification. However, pHi acidification stimulated protein kinase C (PKC) activity and inhibition of PKC by PKC inhibitors (bisindolylmaleimide, calphostin C, or staurosporine) or prolonged exposure to phorbol ester abrogated the inhibitory effect of pHi acidification on cell Pi uptake. In summary, these studies showed that pHi acidification inhibits Pi uptake in OK cells, probably through PKC activation. These effects of pHi acidification may thus contribute to increase acid excretion in systemic acidosis.

Acids↗

PubMed1992

Dopamine D2 receptor stimulation of Na+/H+ exchange assessed by quantification of extracellular acidification.

A microphysiometer was used to quantify the rate of extracellular acidification by C6 glioma cells and L fibroblasts expressing recombinant dopamine D2 receptors. The dopamine D2 receptor agonist, quinpirole, accelerated the rate of acidification of the medium by C6 cells expressing either the short or long form of D2 receptors, D2(415) and D2(444), but not by wild-type cells that were not transfected with a D2 receptor cDNA. The rate of acidification increased with increasing concentrations of quinpirole up to 100 nM. Inhibition of the response by the dopamine D2 antagonist, spiperone, provided additional evidence that the enhanced extracellular acidification resulted from stimulation of D2 receptors. To test the hypothesis that D2 receptor-stimulated extracellular acidification was due to transport of protons by a Na+/H+ antiporter and reflected intracellular alkalinization, the effect of two inhibitors of Na+/H+ exchange, amiloride and methyl-isobutyl-amiloride, was determined. Both compounds inhibited quinpirole-induced extracellular acidification at concentrations that did not alter D2 receptor-mediated inhibition of adenylylcyclase or radioligand binding to D2 receptors. In addition, quinpirole-induced extracellular acidification was greatly inhibited by removal of sodium from the extracellular medium, confirming the participation of Na+/H+ exchange in the extrusion of acid. Quinpirole (100 nM) also increased the rate of extracellular acidification by L cells expressing D2(415), LZR1 cells. Treatment with pertussis toxin (100 ng/ml for 18 h) had no effect on the quinpirole-induced acid extrusion by C6D2(415) and LZR1 cells, although the same pertussis toxin treatment regimen completely prevented inhibition of adenylylcyclase. We conclude that recombinant D2 receptors accelerate Na+/H+ exchange in C6 cells and L fibroblasts by a pathway that does not involve inhibition of adenylylcyclase or pertussis toxin-sensitive G proteins.

Amiloride↗

PubMed2001

Response of alkalinization or acidification by phytohemagglutinin is dependent on the activity of protein kinase C in human peripheral T Cells.

The increase of intracellular free calcium concentration ([Ca(2+)](i)) and protein kinase C (PKC) activity are two major early mitogenic signals to initiate proliferation of human T cells. However, a rapid change in intracellular pH (pH(i)), acidification or alkalinization during the activation, is also associated after these two signals. The aim of this study was to define whether the change in pH(i) is affected by calcium and protein kinase C (PKC), in phytohemagglutinin (PHA)-stimulated T cells. T cells were isolated from human peripheral blood. The [Ca(2+)](i) and the pH(i) were measured using, respectively, the fluorescent dyes, Fura-2, and BCECF. In addition, down-regulation of PKC activity by PMA (1 microM, 18 h) was confirmed in these cells using a protein kinase assay. The results indicated that, (1) alkalinization was induced by PHA or PMA in T cells; the results of alkalinization was PKC-dependent and Ca(2+)-independent, (2) in PKC down-regulated T cells, PHA induced acidification; this effect was enhanced by pre-treating the cells with the Na(+)/H(+) exchange inhibitor, 5-(N,N-dimethyl)-amiloride, (DMA, 10 microM, 20 min), (3) the acidification was dependent on the Ca(2+) influx and blocked by removal of extracellular calcium or the addition of the inorganic channel blocker, Ni(2+), and (4) Thapsigargin (TG), a Ca(2+)-ATPase inhibitor, confirmed that acidification by the Ca(2+) influx occurred in T cells in which PKC was not down-regulated. These findings indicate two mechanisms, alkalinization by PKC and acidification by Ca(2+) influx, exist in regulating pH(i) in T cells. This is the first report that PHA stimulates the acidification by Ca(2+) influx but not alkalinization in T cells after down-regulation of PKC. In conclusion, the activity of PKC in T cells determines the response in alkalinization or acidification by PHA.

Acid-Base Equilibrium↗

PubMed1993

GM-CSF triggers a rapid, glucose dependent extracellular acidification by TF-1 cells: evidence for sodium/proton antiporter and PKC mediated activation of acid production.

The extracellular acidification rate of the human bone marrow cell line, TF-1, increases rapidly in response to a bolus of recombinant granulocyte-macrophage colony stimulating factor (GM-CSF). Extracellular acidification rates were measured using a silicon microphysiometer. This instrument contains micro-flow chambers equipped with potentiometric sensors to monitor pH. The cells are immobilized in a fibrin clot sandwiched between two porous polycarbonate membranes. The membranes are part of a disposable plastic "cell capsule" that fits into the microphysiometer flow chamber. The GM-CSF activated acidification burst is dose dependent and can be neutralized by pretreating the cytokine with anti-GM-CSF antibody. The acidification burst can be resolved kinetically into at least two components. A rapid component of the burst is due to activation of the sodium/proton antiporter as evidenced by its elimination in sodium-free medium and in the presence of amiloride. A slower component of the GM-CSF response is a consequence of increased glycolytic metabolism as demonstrated by its dependence on D-glucose as a medium nutrient. Okadaic acid (a phospho-serine/threonine phosphatase inhibitor), phorbol 12-myristate 13-acetate (PMA, a protein kinase C (PKC) activator), and ionomycin (a calcium ionophore) all produce metabolic bursts in TF-1 cells similar to the GM-CSF response. Pretreatment of TF-1 cells with PMA for 18 h resulted in loss of the GM-CSF acidification response. Although this treatment is reported to destroy protein kinase activity, we demonstrate here that it also down-regulates expression of high-affinity GM-CSF receptors on the surface of TF-1 cells. In addition, GM-CSF driven TF-1 cell proliferation was decreased after the 18 h PMA treatment. Short-term treatment with PMA (1-2 h) again resulted in loss of the GM-CSF acidification response, but without a decrease in expression of high-affinity GM-CSF receptors. Evidence for involvement of PKC in GM-CSF signal transduction was obtained using calphostin C, a specific inhibitor of PKC, which inhibited the GM-CSF metabolic burst at a subtoxic concentration. Genistein and herbimycin A, tyrosine kinase inhibitors, both inhibited the GM-CSF response of TF-1 cells, but only at levels high enough to also inhibit stimulation by PMA. These results indicate that GM-CSF activated extracellular acidification of TF-1 cells is caused by increases in sodium/proton antiporter activity and glycolysis, through protein kinase signalling pathways which can be both activated and down-regulated by PMA.

Benzoquinones↗

PubMed1994

Mucosal acidification and an acid microclimate in the hen colon in vitro.

Experiments were performed on isolated, stripped colonic epithelia of low-salt-adapted hens (Gallus domesticus) in order to characterize acid secretion by this tissue. With symmetric, weak buffer solutions, colonic epithelia acidified both mucosal and serosal sides. Titration measurements of the mucosal acidification rate (pH-stat technique) averaged 1.63 +/- 0.25 microEq.cm-2.h-1. Mucosal acidification was also evident in colons from high-salt-adapted birds and in low-salt-adapted coprodeum, but was completely abolished in the high-salt coprodeum. Mucosal acidification by low-salt-adapted colonic epithelium was unaffected by sodium replacement, mucosal amiloride (10(-3) mol.l-1), and serosal ouabain (5 x 10(-4) mol.l-1), although all three treatments significantly reduced or reversed the short-circuit current. Acetazolamide (10(-3) mol.l-1, serosal) reduced mucosal acidification by 15% and simultaneously increased short-circuit current by a similar amount. Colonic epithelia incubated in glucose-free solutions had significantly lower acidification rates (0.59 +/- 0.13 microEq.cm-2.h-1, P < 0.002 versus controls) and addition of glucose (15 mmol.l-1), but not galactose, partially restored acidification to control levels. Anoxia (N2 gassing) completely inhibited short-circuit current, but reduced acidification by only 30%. A surface microclimate pH, nearly 2 pH units more acidic than the bath pH of 7.1-7.4, was measured in low-salt-adapted colon and coprodeum. The acid microclimate of both tissues was partially attenuated by adaptation to a high-salt diet. Colonic microclimate pH was dependent on the presence of glucose and sensitive to the bath pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Acids↗

PubMed2005

Acidification of forested podzols in North Belgium during the period 1950-2000.

Acidification of forest soils in Europe and North America has been an important concern over the last decades. The last area-covering survey of forest soil acidification in Flanders (North Belgium) goes back to 1985 [Ronse A, De Temmerman L, Guns M, De Borger R. Evolution of acidity, organic matter content, and CEC in uncultivated soils of North Belgium during the past 25 years. Soil Sci; 146, (1988), 453-460] and highlighted a significant acidification of the upper layer (0.3-0.4 m) of forested podzols during the period 1950-1985. The present study aimed to assess (1) to what extent further acidification of forested podzols occurred during the period 1985-2000 at different depths and (2) whether the average annual acidification rate accelerated or slowed down between 1985 and 2000 compared to the period 1950-1985. Average soil pH-KCl values of podzols in northern Belgium dropped during the period 1985-2000. This decline extends to a depth of about 50 cm but was most pronounced and significant in the A horizon. In the A(0), A(1) and A(2) horizons, average pH dropped with 0.2, 0.3 and 0.1 units, and in the B(ir) and C horizons with 0.1 units. No change in average pH value occurred in the B(h) horizon. Average annual acidification rate of the A(1) horizon was significantly higher in the period 1985-2000 than in the period 1950-1985. Changes in pH occurred in the entire soil profile during the period 1950/67-1985 likely because sulphate was the major form of acid deposition before 1985. After 1985, acid sulphur deposition decreased with more than 50% in North Belgium. In contrast, ammonium deposition almost doubled between 1950 and 1980, which may explain why soil acidification between 1985 and 2000 has been restricted to the upper soil horizons.

Acid Rain↗

PubMed1983

Uricemia and urinary acidification in renal calcium stone disease.

Serum urate concentrations were determined in 487 consecutive patients with stone disease and in 89 controls. Male controls had higher concentrations than female controls but men and women with stones had lower concentrations than controls. Renal acidification defects, incomplete and complete forms, were found in 25 per cent of the patients with stones, with the highest frequency in patients with hyperuricemia. Patients with proximal renal acidification defects had a higher serum urate concentration than patients with normal acidification of the urine. The urinary excretion of urate was higher in hyperuricemic than in normouricemic patients but lower in patients with hyperuricemia and renal acidification defects than in those with hyperuricemia and normal acidification of the urine. This finding indicates that underexcretion rather than overproduction contributes to the higher serum urate concentration in patients with renal acidification defects. Stone patients with high serum urate levels, especially in combination with renal acidification defects, had more severe stone disease and underwent more operations than other patients with stones. Thus, hyperuricemia does not appear to be a common feature in patients with calcium stone disease, although it is associated with formation of large stones for unknown reasons.

Adult↗

PubMed2003

Effects of glial toxins on extracellular acidification in the hippocampal CA1 region in vivo.

In the pyramidal cell layer of the CA1 region of the hippocampus in the urethane-anesthetized adult rat, there is an initial alkalinization followed by an acidification in response to synchronized seizure activity induced by stimulus trains. In this study, the role of astrocytes in these extracellular pH changes during neuronal activity was examined using local injection of two relatively selective glial toxins (fluorocitrate (FC) and fluoroacetate (FA)) into the CA1 cell layer. Both glial toxins reduced the peak level of acidification reached after 20 Hz stimulus trains to the contralateral CA3 region, without changing the lengthening of the afterdischarge, when compared to animals that had received a local injection of vehicle. After administration of either glial toxin, the peak level of acidification still correlated with the total discharge duration, but the levels of acidification were consistently lower than in control animals. Administration of either glial toxin had no effect on the peak alkalinization during the stimulus train, or on the rate of recovery from peak level of acidification. Injection of either vehicle, FA, or FC had no effect on the amplitude or frequency of the neuronal discharge during the afterdischarge. The results suggest that, in normal conditions, astrocytes contribute to the acidification of the extracellular space that occurs in response to intense neuronal activity. This acidification may contribute to feedback regulation of neuronal excitability.

Animals↗

PubMed1989

A possible role for Na+,K+-ATPase in regulating ATP-dependent endosome acidification.

Endosomes maintain a slightly acidic internal pH, which is directly responsible for their ability to ensure proper sorting of incoming receptors and ligands during endocytosis. At least two distinct subpopulations of endosomes can be distinguished, designated "early" and "late" on the basis of their kinetics of labeling with endocytic tracers. The subpopulations differ not only in their functions (rapid receptor recycling and transport to lysosomes, respectively) but also in their capacities for acidification in intact cells and in vitro. To investigate the possible basis for pH regulation in endosomes, we have studied the transport properties and ion permeabilities of early and late endosomes isolated from Chinese hamster ovary cells. Using endosomes selectively labeled with pH-sensitive endocytic tracers, we found that ATP-dependent acidification is electrogenic, being accompanied by the generation of an interior-positive membrane potential which opposes further acidification. While membrane potential and, consequently, acidification was controlled by the influx of permeant anions and efflux of protons and alkali cations, acidification was further modulated in Na+ and K+-containing buffers by the ouabain- and vanadate-sensitive Na+,K+-ATPase, which appears to be a functional component of the endosomal membrane. The data suggest that electrogenic Na+ transport due to Na+,K+-ATPase activity contributes to the interior-positive membrane potential, thereby reducing ATP-dependent H+ transport. Importantly, inhibition of acidification by Na+,K+-ATPase activity was found only in early endosomes, consistent with their limited acidification capacity relative to late endosomes and lysosomes.

Animals↗

PubMed1996

Multiphasic action of glucose and alpha-ketoisocaproic acid on the cytosolic pH of pancreatic beta-cells. Evidence for an acidification pathway linked to the stimulation of Ca2+ influx.

Glucose stimulation raises the pHi of pancreatic beta-cells, but the underlying mechanisms are not well understood. We have now investigated the acute effects of metabolizable (glucose and the mitochondrial substrate alpha-ketoisocaproic acid, KIC) and nonmetabolizable (high K+ and the K-ATP channel blocker tolbutamide) insulin secretagogues on the pHi of pancreatic beta-cells isolated from normal mice, as assessed by BCECF fluorescence from single cells or islets in the presence of external bicarbonate. The typical acute effect of glucose (22-30 mM) on the pHi was a fast alkalinization of approximately 0.11 unit, followed by a slower acidification. The relative expression of the alkalinizing and acidifying components was variable, with some cells and islets displaying a predominant alkalinization, others a predominant acidification, and others yet a mixed combination of the two. The initial alkalinization preceded the [Ca2+]i rise associated with the activation of voltage-sensitive Ca2+ channels. There was a significant overlap between the glucose-evoked [Ca2+]i rise and the development of the secondary acidification. Depolarization with 30 mM K+ and tolbutamide evoked pronounced [Ca2+]i rises and concomitant cytosolic acidifications. Blocking glucose-induced Ca2+ influx (with 0 Ca2+, nifedipine, or the K-ATP channel agonist diazoxide) suppressed the secondary acidification while having variable effects (potentiation or slight attenuation) on the initial alkalinization. KIC exerted glucose-like effects on the pHi and [Ca2+]i, but the amplitude of the initial alkalinization was about twice as large for KIC relative to glucose. It is concluded that the acute effect of glucose on the pHi of pancreatic beta-cells is biphasic. While the initial cytosolic alkalinization is an immediate consequence of the activation of H+-consuming metabolic steps in the mitochondria, the secondary acidification appears to originate from enhanced Ca2+ turnover in the cytoplasm. The degree of coupling between glucose metabolism and Ca2+ influx as well as the relative efficacies of these processes determines whether the acute pHi response of a beta-cell (or of a tightly coupled multicellular system such as an islet of Langerhans) is predominantly an alkalinization, an acidification, or a mixed proportion of the two.

Animals↗

PubMed1998

Different sources of acidity in glucose-elicited extracellular acidification in the yeast Saccharomyces cerevisiae.

Three wild-type strains of Saccharomyces cerevisiae, viz. K, Y55 and sigma 1278b, two mutants lacking one or both of the putative K+ transporters, trk1 delta and trk1 delta trk2 delta, and a mutant in the plasma membrane H(+)-ATPase, viz. pma1-105, were compared in their extracellular acidification following addition of glucose and subsequent addition of KCl; in ATPase activity in purified plasma membranes; and in respiration on glucose. The glucose-induced acidification was the greater the higher the respiratory quotient, i.e. the higher the anaerobic metabolism. A markedly lower acidification was found in the ATPase-deficient pma1-105 strain but also in the TRK-deficient double mutant. The acidification pattern after addition of KCl corresponds to expectations in the TRK mutants; however, a similarly decreased acid production was found in the ATPase-deficient mutant pma1-105. The highest rate of ATP hydrolysis in vitro was found with the trk1 delta trk2 delta mutant where glucose-, as well as KCl-induced acidification were lowest. Likewise, the pma1-105 mutant with extremely low acidification showed only a minutely lower ATP hydrolysis than did its parent Y55 strain. Apparently, several different sources of acidity are involved in the glucose-induced acidification (including extrusion of organic acids); in fact, contrary to the general belief, the H(+)-ATPase may play a minor role in this process in some strains.

Acids↗

PubMed1992

Proteolysis of the major yolk glycoproteins is regulated by acidification of the yolk platelets in sea urchin embryos.

The precise function of the yolk platelets of sea urchin embryos during early development is unknown. We have shown previously that the chemical composition of the yolk platelets remains unchanged in terms of phospholipid, triglyceride, hexose, sialic acid, RNA, and total protein content after fertilization and early development. However, the platelet is not entirely static because the major 160-kD yolk glycoprotein YP-160 undergoes limited, step-wise proteolytic cleavage during early development. Based on previous studies by us and others, it has been postulated that yolk platelets become acidified during development, leading to the activation of a cathepsin B-like yolk proteinase that is believed to be responsible for the degradation of the major yolk glycoprotein. To investigate this possibility, we studied the effect of addition of chloroquine, which prevents acidification of lysosomes. Consistent with the postulated requirement for acidification, it was found that chloroquine blocked YP-160 breakdown but had no effect on embryonic development. To directly test the possibility that acidification of the yolk platelets over the course of development temporally correlated with YP-160 proteolysis, we added 3-(2,4-dinitroanilo)-3-amino-N-methyldipropylamine (DAMP) to eggs or embryos. This compound localizes to acidic organelles and can be detected in these organelles by EM. The results of these studies revealed that yolk platelets did, in fact, become transiently acidified during development. This acidification occurred at the same time as yolk protein proteolysis, i.e., at 6 h after fertilization (64-cell stage) in Strongylocentrotus purpuratus and at 48 h after fertilization (late gastrula) in L. pictus. Furthermore, the pH value at the point of maximal acidification of the yolk platelets in vivo was equal to the pH optimum of the enzyme measured in vitro, indicating that this acidification is sufficient to activate the enzyme. For both S. purpuratus and Lytechinus pictus, the observed decrease in the pH was approximately 0.8 U, from 7.0 to 6.2. The trypsin inhibitor benzamidine was found to inhibit the yolk proteinase in vivo. By virtue of the fact that this inhibitor was reversible we established that the activity of the yolk proteinase is developmentally regulated even though the enzyme is present throughout the course of development. These findings indicate that acidification of yolk platelets is a developmentally regulated process that is a prerequisite to initiation of the catabolism of the major yolk glycoprotein.

Animals↗