Modulation of acid extrusion and acid loading in neurons and astrocytes by RPTPγ and RPTPζ

Changes of the intracellular pH of rat diaphragm muscle were monitored at 30-min intervals with the weak acid DMO (5,5-dimethyl-2,4-oxazolidinedione). Transferring the muscle from a CO2-containing to a CO2-free solution caused intracellular pH (pHi) to rise by an average of 0.18 during the first 30 min and then to level off at a slightly lower value over the next 60-90 min. Transferring the muscle from a CO2-free to a CO2-containing solution caused pHi to fall by 0.18 during the first 30 min and then to recover by 0.05 over the next 90 min. Subsequent return to the CO2-free solution caused pHi to overshoot the control value by 0.10. Both the recovery and the overshoot can be accounted for by an acid-extruding pump. Intracellular acid loading with 118 mM DMO similarly caused pHi to fall initially, to recover slowly during the acid loading, and then to overshoot the control pHi on removal of the acid load. In the absence of HCO3-/CO2, acid extrusion was reduced by about a fifth. SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid) had no effect. The absence of either Na+ or Cl- from HCO3-/CO2- free solution reduced acid extrusion by about a half.

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Regulation of intracellular pH

AJP Advances in Physiology Education ◽

10.1152/advan.00045.2004 ◽

2004 ◽

Vol 28 (4) ◽

pp. 160-179 ◽

Cited By ~ 198

Author(s):

Walter F. Boron

Keyword(s):

Steady State ◽

Intracellular Ph ◽

Temperature Control System ◽

Air Conditioner ◽

Environmental Sensors ◽

Animal Cells ◽

Buffering Power ◽

A Cell ◽

Acid Extrusion ◽

Acid Loading

The approach that most animal cells employ to regulate intracellular pH (pHi) is not too different conceptually from the way a sophisticated system might regulate the temperature of a house. Just as the heat capacity (C) of a house minimizes sudden temperature (T) shifts caused by acute cold and heat loads, the buffering power (β) of a cell minimizes sudden pHi shifts caused by acute acid and alkali loads. However, increasing C (or β) only minimizes T (or pHi) changes; it does not eliminate the changes, return T (or pHi) to normal, or shift steady-state T (or pHi). Whereas a house may have a furnace to raise T, a cell generally has more than one acid-extruding transporter (which exports acid and/or imports alkali) to raise pHi. Whereas an air conditioner lowers T, a cell generally has more than one acid-loading transporter to lower pHi. Just as a house might respond to graded decreases (or increases) in T by producing graded increases in heat (or cold) output, cells respond to graded decreases (or increases) in pHi with graded increases (or decreases) in acid-extrusion (or acid-loading) rate. Steady-state T (or pHi) can change only in response to a change in chronic cold (or acid) loading or chronic heat (or alkali) loading as produced, for example, by a change in environmental T (or pH) or a change in the kinetics of the furnace (or acid extrudes) or air conditioner (or acid loaders). Finally, just as a temperature-control system might benefit from environmental sensors that provide clues about cold and heat loading, at least some cells seem to have extracellular CO2 or extracellular HCO3− sensors that modulate acid-base transport.

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Phorbol ester-induced changes of cytoplasmic pH in neutrophils: role of exocytosis in Na+-H+ exchange

AJP Cell Physiology ◽

10.1152/ajpcell.1985.248.3.c379 ◽

1985 ◽

Vol 248 (3) ◽

pp. C379-C386 ◽

Cited By ~ 46

Author(s):

S. Grinstein ◽

B. Elder ◽

W. Furuya

Keyword(s):

Phorbol Ester ◽

Secretory Granules ◽

Cytoplasmic Ph ◽

Ph Homeostasis ◽

Intact Cells ◽

Acid Load ◽

Induced Changes ◽

Acid Extrusion ◽

Acid Loading

Cytoplasmic pH homeostasis was studied in intact and granule-free porcine neutrophils following activation with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). In intact cells, TPA activated at least two separate processes: a Na+-independent and amiloride-insensitive acidification, and a compensatory acid extrusion. The latter is Na+ dependent and blocked by amiloride and is likely to represent Na+-H+ exchange. Enucleated and degranulated neutrophils (cytoplasts) were prepared by sedimentation of cytochalasin B-treated neutrophils through a discontinuous density gradient. Cytoplasts responded to an artificially imposed acid load with activation of an amiloride-sensitive Na+-H+ exchange. TPA also activated both acid production and Na+-dependent acid extrusion in cytoplasts. The magnitude of the responses was comparable in intact neutrophils and in cytoplasts. These data suggest that 1) the nucleus and the secretory granules are not involved in the acidifying response to TPA, and 2) exocytosis of secretory vesicles is not required for activation of Na+-H+ exchange during acid loading or following treatment with the phorbol ester TPA.

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Effect of basolateral CO2/HCO3- on intracellular pH regulation in the rabbit S3 proximal tubule.

The Journal of General Physiology ◽

10.1085/jgp.102.6.1171 ◽

1993 ◽

Vol 102 (6) ◽

pp. 1171-1205 ◽

Cited By ~ 16

Author(s):

N L Nakhoul ◽

L K Chen ◽

W F Boron

Keyword(s):

Steady State ◽

Proximal Tubule ◽

Intracellular Ph ◽

Ph Regulation ◽

Uptake Mechanism ◽

Luminal Membrane ◽

S3 Segment ◽

Acid Extrusion ◽

Acid Loading

We used the absorbance spectrum of the pH-sensitive dye dimethylcarboxyfluorescein to monitor intracellular pH (pHi) in the isolated perfused S3 segment of the rabbit proximal tubule, and examined the effect on pHi of switching from a HEPES to a CO2/HCO3- buffer in the lumen and/or the bath (i.e., basolateral solution). Solutions were titrated to pH 7.40 at 37 degrees C. With 10 mM acetate present bilaterally (lumen and bath), this causing steady-state pHi to be rather high (approximately 7.45), bilaterally switching the buffer from 32 mM HEPES to 5% CO2/25 mM HCO3- caused a sustained fall in pHi of approximately 0.26. However, with acetate absent bilaterally, this causing steady-state pHi to be substantially lower (approximately 6.9), bilaterally switching to CO2/HCO3- caused a transient pHi fall (due to the influx of CO2), followed by a sustained rise to a level approximately 0.18 higher than the initial one. The remainder of the experiments was devoted to examining this alkalinization in the absence of acetate. Switching to CO2/HCO3- only in the lumen caused a sustained pHi fall of approximately 0.15, whereas switching to CO2/HCO3- only in the bath caused a transient fall followed by a sustained pHi increase to approximately 0.26 above the initial value. This basolateral CO2/HCO3(-)-induced alkalinization was not inhibited by 50 microM DIDS applied shortly after CO2/HCO3- washout, but was slowed approximately 73% by DIDS applied more than 30 min after CO2/HCO3- washout. The rate was unaffected by 100 microM bilateral acetazolamide, although this drug greatly reduced CO2-induced pHi transients. The alkalinization was not blocked by bilateral removal of Na+ per se, but was abolished at pHi values below approximately 6.5. The alkalinization was also unaffected by short-term bilateral removal of Cl- or SO4=. Basolateral CO2/HCO3- elicited the usual pHi increase even when all solutes were replaced, short or long-term (> 45 min), by N-methyl-D-glucammonium/glucuronate (NMDG+/Glr-). Luminal CO2/HCO3- did not elicit a pHi increase in NMDG+/Glr-. Although the sustained pHi increase elicited by basolateral CO2/HCO3- could be due to a basolateral HCO3- uptake mechanism, net reabsorption of HCO3- by the S3 segment, as well as our ACZ data, suggest instead that basolateral CO2/HCO3- elicits the sustained pHi increase either by inhibiting an acid-loading process or stimulating acid extrusion across the luminal membrane (e.g., via an H+ pump).

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pH regulation in the vertebrate central nervous system: microelectrode studies in the brain stem of the lamprey

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y87-156 ◽

1987 ◽

Vol 65 (5) ◽

pp. 986-993 ◽

Cited By ~ 13

Author(s):

Mitchell Chesler

Keyword(s):

Brain Stem ◽

Ph Regulation ◽

Disulfonic Acid ◽

Small Rise ◽

Free Media ◽

Acid Extrusion ◽

Acid Loading ◽

The Brain ◽

Vertebrate Central Nervous System ◽

Dependent Mechanism

Studies of intracellular pH (pHi) in nervous tissue are summarized and recent investigation of intracellular and extracellular pH (pHo) in the isolated brain stem of the lamprey is reviewed. In the lamprey, pHi regulation was studied in single reticulospinal neurons using double-barrel ion-selective microelectrodes (ISMs). In nominally [Formula: see text]-free HEPES-buffered media, acute acid loading was followed by a spontaneous recovery of pHi requiring 10–20 min and was associated with a prolonged rise in intracellular Na+. The recovery of pHi was blocked by 1–2 mM amiloride. Amiloride also caused a small rise in pHo. Substitution of external Na+ caused a slow intracellular acidification and extracellular alkalinization. Return of external Na+ reversed these effects. Transition from HEPES to [Formula: see text]-buffered media increased the rate of acid extrusion during recovery of pHi. Recovery in [Formula: see text]-buffered media was inhibited by 4,4′-diisothio-cyanostilbene-2,2′-disulfonic acid and was slowed after exposure to Cl−-free media. Following inhibition of acid extrusion by amiloride, transition to [Formula: see text] media restored pHi recovery. These data indicate that lamprey neurons recover from acute acid loads by both Na+–H+ exchange and an independent [Formula: see text]-dependent mechanism. Evidence for [Formula: see text]-dependent acid extrusion in other vertebrate cells and the protocols of pHi studies using ISMs are discussed.

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pH regulation in single glomerular mesangial cells. I. Acid extrusion in absence and presence of HCO3-

AJP Cell Physiology ◽

10.1152/ajpcell.1988.255.6.c844 ◽

1988 ◽

Vol 255 (6) ◽

pp. C844-C856 ◽

Cited By ~ 286

Author(s):

G. Boyarsky ◽

M. B. Ganz ◽

R. B. Sterzel ◽

W. F. Boron

Keyword(s):

Steady State ◽

Loading Rate ◽

Mesangial Cells ◽

Ph Regulation ◽

Disulfonic Acid ◽

Glomerular Mesangial Cells ◽

Fluorescence Excitation ◽

Dependent Transport ◽

Acid Extrusion ◽

Acid Loading

We have developed a technique to measure the fluorescence of a pH-sensitive dye (2,7-biscarboxyethyl-5(6)-carboxyfluorescein) in single glomerular mesangial cells in culture. The intracellular fluorescence excitation ratio of the dye was calibrated using the nigericin-high-K+ approach. In the absence of CO2-HCO3-, mesangial cells that are acid loaded by an NH+4 prepulse exhibit a spontaneous intracellular pH (pHi) recovery that is blocked either by ethylisopropylamiloride (EIPA) or removal of external Na+. This pHi recovery most probably reflects the activity of a Na+-H+ exchanger. When the cells are switched from a N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution to one containing CO2-HCO3-, there is an abrupt acidification due to CO2 entry, which is followed by a spontaneous recovery of pHi to a steady-state value higher than that prevailing in HEPES. Both the rate of recovery and the higher steady-state pHi imply that the application of CO2-HCO3- introduces an increase in net acid extrusion from the cell. One third of total net acid extrusion in CO2-HCO3- is EIPA sensitive and most likely is mediated by the Na+-H+ exchanger. The remaining two thirds of acid extrusion could be caused by a decrease in the background acid-loading rate and/or the introduction of a new, HCO3- -dependent acid-extrusion mechanism. The HCO3- -induced alkalinization cannot be accounted for by a HCO3- -induced reduction in the acid-loading rate. The latter can be estimated by applying EIPA in the absence of HCO3- and observing the rate of pHi decline. We found that this acid-loading rate is only about one fifth as great as the total net acid extrusion rate in the presence of HCO3-. Indeed, two thirds of net acid extrusion in HCO3- is blocked by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), an inhibitor of HCO3- -dependent transport. Furthermore, the effects of EIPA and SITS were additive. Thus, in the presence of CO2-HCO3-, a SITS-sensitive-HCO3- -dependent transporter is the dominant mechanism of acid extrusion. This mechanism also accounts for the increase in steady-state pHi on addition of CO2-HCO3-.

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Functional expression of the cDNA encoded by the human ATP1AL1 gene

AJP Renal Physiology ◽

10.1152/ajprenal.1996.271.3.f539 ◽

1996 ◽

Vol 271 (3) ◽

pp. F539-F551 ◽

Cited By ~ 13

Author(s):

A. V. Grishin ◽

M. O. Bevensee ◽

N. N. Modyanov ◽

V. Rajendran ◽

W. F. Boron ◽

...

Keyword(s):

Polyclonal Antibodies ◽

Ph Dependence ◽

Functional Expression ◽

Beta Subunit ◽

Hek 293 ◽

Cos Cells ◽

293 Cells ◽

Uptake Activity ◽

Acid Extrusion ◽

Acid Loading

The human ATP1AL1 gene encodes a protein expressed in brain, kidney, and skin and that is highly homologous to the recently cloned nongastric isoforms of H-K-adenosinetriphosphatase H-K-ATPase). We have generated polyclonal antibodies against the protein encoded by ATP1AL1 and used them to monitor the protein's expression and distribution in transfection studies. The protein was retained in the endplasmic reticulum when it was transiently expressed alone in COS cells. In COS cells cotransfected with ATP1AL1 plus gastric H-K-ATPase beta-subunit cDNAs (ATP1AL1-gH-K beta), both proteins reached the surface. Stably transfected lines of HEK 293 cells expressing both of these proteins demonstrate a 86Rb+ uptake activity sensitive to both 2-methyl,8-(phenylmeoxy)imidazo(1,2-a)pyridine 3-acetonitrile (SCH-28080) and ouabain (inhibitory constants of approximately 131 and 42 microM, respectively). Outward proton fluxes were measured in the same cells as the spontaneous intracellular pH (pHi) recovery in Cells loaded with a pH-sensitive dye [2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein] and subjected to acid loading through an NH4Cl pulse. The cells expressing both the ATP1AL1-encoded protein and the gastric H-K-ATPase beta-subunit possess a net acid extrusion activity that can be inhibited by 1 mM ouabain. Comparison of the 86Rb+ influx and proton efflux, however, does not support equal H+/Rb+ exchange mediated by this pump under the conditions of pHi-monitoring experiments. Moreover, whereas the acid extrusion activity mediated by the pump shows a marked pH dependence, the 86Rb+ uptake activity present in the cells expressing the ATP1AL1-gH-K beta complex cannot be stimulated by acute lowering of pHi. These data suggest that the ATP1AL1-encoded protein is the catalytic alpha-subunit of a human K(+)-dependent ATPase. The possible implications of the discrepancy between 86Rb+ uptake and pHi monitoring data are discussed.

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Monovalent ions control proliferation of Ehrlich Lettre ascites cells

AJP Cell Physiology ◽

10.1152/ajpcell.00445.2009 ◽

2010 ◽

Vol 299 (3) ◽

pp. C714-C725 ◽

Cited By ~ 18

Author(s):

Thomas Kjær Klausen ◽

Sarah Preisler ◽

Stine Falsig Pedersen ◽

Else Kay Hoffmann

Keyword(s):

Intracellular Ph ◽

S Phase ◽

Fine Tuning ◽

Serum Starvation ◽

Western Blots ◽

Monovalent Ions ◽

Intracellular Ca ◽

Acid Extrusion ◽

Acid Loading

Channels and transporters of monovalent ions are increasingly suggested as putative anticarcinogenic targets. However, the mechanisms involved in modulation of proliferation by monovalent ions are poorly understood. Here, we investigated the role of K+, Na+, and Cl− ions for the proliferation of Ehrlich Lettre ascites (ELA) cells. We measured the intracellular concentration of each ion in G0, G1, and S phases of the cell cycle following synchronization by serum starvation and release. We show that intracellular concentrations and content of Na+ and Cl− were reduced in the G0–G1 phase transition, followed by an increased content of both ions in S phase concomitant with water uptake. The effect of substituting extracellular monovalent ions was investigated by bromodeoxyuridine incorporation and showed marked reduction after Na+ and Cl− substitution. In spectrofluorometric measurements with the pH-sensitive dye BCECF, substitution of Na+ was observed to upregulate the activity of the Na+/H+ exchanger NHE1 as well as of Na+-independent acid extrusion mechanisms, facilitating intracellular pH (pHi) recovery after acid loading and increasing pHi. Results using the potential sensitive dye DiBaC4( 3 ) showed a reduced Cl− conductance in S compared with G1 followed by transmembrane potential ( Em) hyperpolarization in S. Cl− substitution by impermeable anions strongly inhibited proliferation and increased free, intracellular Ca2+ ([Ca2+]i), whereas a more permeable anion had little effect. Western blots showed reduced chloride intracellular channel CLIC1 and chloride channel ClC-2 expression in the plasma membrane in S compared with G1. Our results suggest that Na+ regulates ELA cell proliferation by regulating intracellular pH while Cl− may regulate proliferation by fine-tuning of Em in S phase and altered Ca2+ signaling.

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Intracellular pH recovery during respiratory acidosis in perfused hearts

AJP Cell Physiology ◽

10.1152/ajpcell.1994.266.2.c489 ◽

1994 ◽

Vol 266 (2) ◽

pp. C489-C497 ◽

Cited By ~ 24

Author(s):

J. I. Vandenberg ◽

J. C. Metcalfe ◽

A. A. Grace

Keyword(s):

Intracellular Ph ◽

Cardiac Tissue ◽

Respiratory Acidosis ◽

Ph Indicator ◽

Ethanesulfonic Acid ◽

Intracellular Buffering Capacity ◽

Acid Extrusion ◽

Acid Loading ◽

Perfused Hearts ◽

Ferret Heart

Na(+)-H+ exchange and Na(+)-dependent HCO3- influx both contribute to recovery of intracellular pH (pHi) after an acidosis induced by using the NH4Cl prepulse technique in mammalian and avian cardiac tissue. We have investigated the relative contributions of these mechanisms to pHi recovery during respiratory acidosis in the Langendorff-perfused ferret heart with and without correction of extracellular pH (pHo). pHi was measured from the chemical shift of the exogenous 31P nuclear magnetic resonance pH indicator 2-deoxy-D-glucose 6-phosphate. Intrinsic intracellular buffering capacity, calculated from the change in intracellular HCO3- concentration after a change in CO2, was reduced from approximately 33 (no inhibitors of acid extrusion present) to 19 +/- 5 mM when H+ extrusion during the acid loading phase was inhibited. During respiratory acidosis (pHo approximately 6.95), the proton efflux rate (JH) calculated at pHi 6.85 was 0.30 +/- 0.04 mmol.l-1.min-1 (n = 9). When pHo was corrected by increasing external HCO3- concentration to 60 mM during respiratory acidosis (pHo approximately 7.33), JH was 1.11 +/- 0.11 mmol.l-1.min-1 (n = 7), and when pHo was partially corrected by the addition of 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid to the perfusion solution (pHo approximately 7.1), JH was 0.64 +/- 0.08 mmol.l-1.min-1 (n = 6). In all three groups Na(+)-H+ exchange and HCO3- influx each contributed approximately 50% to acid-equivalent efflux.(ABSTRACT TRUNCATED AT 250 WORDS)

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Shrinkage-induced activation of Na+/H+exchange in rat renal mesangial cells

AJP Cell Physiology ◽

10.1152/ajpcell.1999.276.3.c674 ◽

1999 ◽

Vol 276 (3) ◽

pp. C674-C683 ◽

Cited By ~ 18

Author(s):

Mark O. Bevensee ◽

Esther Bashi ◽

Wolf-Rüdiger Schlue ◽

Gregory Boyarsky ◽

Walter F. Boron

Keyword(s):

Mesangial Cells ◽

Rat Kidney ◽

Single Cells ◽

Cell Populations ◽

Cell Shrinkage ◽

Total Acid ◽

Buffering Power ◽

Hyperosmolar Solutions ◽

Acid Extrusion ◽

Acid Loading

Using the pH-sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), we examined the effect of hyperosmolar solutions, which presumably caused cell shrinkage, on intracellular pH (pHi) regulation in mesangial cells (single cells or populations) cultured from the rat kidney. The calibration of BCECF is identical in shrunken and unshrunken mesangial cells if the extracellular K+concentration ([K+]) is adjusted to match the predicted intracellular [K+]. For pHi values between ∼6.7 and ∼7.4, the intrinsic buffering power in shrunken cells (600 mosmol/kgH2O) is threefold larger than in unshrunken cells (∼300 mosmol/kgH2O). In the nominal absence of CO2/[Formula: see text], exposing cell populations to a HEPES-buffered solution supplemented with ∼300 mM mannitol (600 mosmol/kgH2O) causes steady-state pHi to increase by ∼0.4. The pHi increase is due to activation of Na+/H+exchange because, in single cells, it is blocked in the absence of external Na+ or in the presence of 50 μM ethylisopropylamiloride (EIPA). Preincubating cells in a Cl−-free solution for at least 14 min inhibits the shrinkage-induced pHi increase by 80%. We calculated the pHi dependence of the Na+/H+exchange rate in cell populations under normosmolar and hyperosmolar conditions by summing 1) the pHi dependence of the total acid-extrusion rate and 2) the pHi dependence of the EIPA-insensitive acid-loading rate. Shrinkage alkali shifts the pHi dependence of Na+/H+exchange by ∼0.7 pH units.

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