pH regulation in hepatoma cells: roles for Na-H exchange, Cl-HCO3 exchange, and Na-HCO3 cotransport

Regulation of intracellular pH (pHi) was studied in Fu5, a rat hepatoma cell line that maintains a variety of differentiated functions. Microspectrofluorimetry of the pH-sensitive dye 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) was used to measure pHi in 10-15 cells growing on cover glasses that were mounted in a flow-through chamber on the stage of a microscope. In N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solutions, pHi was 7.14, and intrinsic buffer capacity was inversely related to pHi. Amiloride (0.1 mM) caused pHi to decrease by 0.33 pH units in 4 min. Recovery from an acid load (using either NH4 prepulse technique or Na-free solutions) was completely blocked by amiloride. In HCO3-CO2-buffered solutions, pHi was 7.15, and buffer capacity was relatively insensitive to pHi between pHi of 6.6 and 7.2. Amiloride caused pHi to decrease by only 0.09 units. Recovery from an acid load was Na dependent, occurred in Cl-free solutions, and was totally blocked by the combination of amiloride plus 0.5 mM dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (H2DIDS); recovery occurred when either amiloride or H2DIDS was removed. Removal of external Cl caused a rapid, H2DIDS-blockable alkalinization that was faster in HCO3-CO2 than in HEPES. The apparent Km for Clout for relaxation of Cl-free alkalinization was 4.5 mM. Rate of HCO3 transport during Cl-free treatment increased at alkaline resting pHi. It is concluded that Fu5 cells have two Na-dependent base-loading mechanisms and an acid-loading Cl-HCO3 exchanger. In solutions containing HCO3-CO2, the Na-H exchanger accounts for approximately 40% of recovery from an acid load, and a Na-HCO3 cotransporter accounts for the remainder. Recovery from an alkaline load appears to occur through the activity of the Cl-HCO3 exchanger.

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Intracellular pH transients in rat diaphragm muscle measured with DMO

AJP Cell Physiology ◽

10.1152/ajpcell.1978.235.1.c49 ◽

1978 ◽

Vol 235 (1) ◽

pp. C49-C54 ◽

Cited By ~ 16

Author(s):

A. Roos ◽

W. F. Boron

Keyword(s):

Intracellular Ph ◽

Weak Acid ◽

Diaphragm Muscle ◽

Disulfonic Acid ◽

Free Solution ◽

Rat Diaphragm ◽

Control Value ◽

Acid Load ◽

Acid Extrusion ◽

Acid Loading

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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pH regulatory transport systems in a smooth muscle-like cell line

AJP Cell Physiology ◽

10.1152/ajpcell.1990.258.3.c470 ◽

1990 ◽

Vol 258 (3) ◽

pp. C470-C479 ◽

Cited By ~ 22

Author(s):

R. W. Putnam

Keyword(s):

Prolonged Exposure ◽

Ph Regulation ◽

Transport Systems ◽

Disulfonic Acid ◽

High K ◽

Recovery From Acidification ◽

Ethanesulfonic Acid ◽

Membrane Transport Systems ◽

External Ph ◽

External K

The membrane transport systems responsible for pH regulation in BC3H-1 cells were studied using the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). In nominally CO2-free Na N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer (NHB) recovery from acidification after an NH4Cl pulse was reversibly inhibited by 1 mM amiloride or by Na-free solutions. On exposure to 5% CO2-HCO3 (external pH constant at 7.4), BC3H-1 cells alkalinized by approximately 0.3-0.4 pH unit. This CO2-induced alkalinization was unaffected by 1 mM amiloride, markedly reduced by 0.5 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), and inhibited by Na-free solutions. On readdition of Na, cells rapidly alkalinized, even in the presence of 1 mM amiloride. Exposure to Cl-free CO2-HCO3 solutions caused a rapid alkalinization of nearly 1 pH unit that was abolished by SITS, largely independent of Na, unaffected by amiloride, and unchanged by membrane depolarization in high external K solutions. CO2-induced alkalinization was slowed by approximately 75% after prolonged exposure of cells to Cl-free NHB, but a distinct recovery from acidification remained in these Cl-depleted cells. This recovery was Na-dependent, SITS-inhibitable, and unaffected by depolarization in high-K solutions. In the presence of CO2, the acidification seen in response to NH4Cl-induced alkalinization was reduced 50% by 0.5 mM SITS. These data suggest that the regulation of pH in BC3H-1 cells is mediated by at least three transport systems: 1) Na-H exchange; 2) Cl-HCO3 exchange; and 3) electroneutral (Na + HCO3)-Cl exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

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Characterization of ion transport mechanisms regulating intracellular pH in hepatic stellate cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1997.273.1.g39 ◽

1997 ◽

Vol 273 (1) ◽

pp. G39-G48 ◽

Cited By ~ 5

Author(s):

A. Di Sario ◽

G. S. Baroni ◽

E. Bendia ◽

L. D'Ambrosio ◽

F. Ridolfi ◽

...

Keyword(s):

Intracellular Ph ◽

Hepatic Stellate Cells ◽

Stellate Cells ◽

Platelet Derived Growth Factor ◽

Disulfonic Acid ◽

Transport Mechanisms ◽

Independent Manner ◽

Acid Load ◽

Acid Loading

The aim of this study was to evaluate intracellular pH (pHi) regulation in nonactivated and activated rat hepatic stellate cells (HSC). The fluorescent pHi indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein was used to measure pHi in the presence and absence of HCO3-. In the absence of HCO3-, baseline pHi was significantly higher (P < 0.001) in activated than in nonactivated HSC (7.1 +/- 0.1 vs. 6.9 +/- 0.2) and decreased, in both groups, after amiloride administration and after Na+ removal. After an acid-loading maneuver, pHi recovery was significantly higher (P < 0.03) in activated than in nonactivated HSC (H+ flux = 11.0 +/- 3.8 vs. 7.7 +/- 2.9 mM/min at pHi 6.6) and was inhibited by amiloride and Na+ removal. In the presence of HCO3-, baseline pHi was higher in both groups and decreased after amiloride administration. Amiloride and Na+ removal inhibited pHi recovery after an intracellular acid load by 77 and 93%, respectively, in nonactivated and by 82 and 92%, respectively, in activated HSC, whereas 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibited pHi recovery by only 27%. Acute Cl- removal increased pHi by 0.07 +/- 0.01 pH unit/min in the absence but not in the presence of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid in nonactivated and activated HSC in an Na(+)-independent manner. In activated HSC, 24 h of incubation with 25 ng/ml platelet-derived growth factor (PDGF)-BB (in 0.5% serum) did not modify baseline pHi (7.07 +/- 0.1 vs. 7.08 +/- 0.1 in HSC cultured in 0.5% serum only) but significantly (P < 0.02) increased, with respect to controls, pHi recovery after an acute acid load. Incubation with PDGF for 24 h induced a fivefold increase in HSC proliferation expressed as percentage of bromodeoxyuridine-positive cells (30.8 +/- 6.7 vs. 6.1 +/- 1.9% in controls). When amiloride (0.1 mM) was present, PDGF-induced HSC proliferation was significantly inhibited (8.1 +/- 0.4%, P < 0.001). Our results show that 1) the Na+/H+ exchanger is the main pHi regulator in rat HSC, 2) activation of HSC is associated with an increase in pHi and in the activity of the Na+/H+ exchanger, 3) PDGF increases the activity of this exchanger, and 4) amiloride is able to inhibit HSC proliferation induced by PDGF.

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Mechanisms of pHi control and relationships between tension and pHi in human subcutaneous small arteries

AJP Cell Physiology ◽

10.1152/ajpcell.1995.268.3.c580 ◽

1995 ◽

Vol 268 (3) ◽

pp. C580-C589 ◽

Cited By ~ 9

Author(s):

P. Carr ◽

W. McKinnon ◽

L. Poston

Keyword(s):

Isometric Tension ◽

Disulfonic Acid ◽

Physiological Salt Solution ◽

Ang Ii ◽

Tension Development ◽

Small Arteries ◽

Transport Inhibitor ◽

Acid Load ◽

Ethanesulfonic Acid

Intracellular pH (pHi) control and relationships between pHi and tension have been investigated in human subcutaneous small arteries. Isometric tension and pHi (using 2',7'-bis(carboxyethyl)- 5(6)-carboxyfluorescein) were estimated simultaneously. pHi recovery from an acute acid load was dependent on external Na+ and partially inhibited by the absence of HCO3(-) [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution] or by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). In an HCO3(-)-buffered physiological salt solution (PSS), pHi recovery was partially blocked by hexamethylene amiloride (HMA), an inhibitor of Na+/H+ exchange, and completely blocked by DIDS and HMA together. Intracellular Cl- depletion of arteries did not affect the rate of pHi recovery in PSS from an acid load. pHi recovery from acute alkalosis was unaffected by external Na+ removal, reduced in HEPES buffer, and abolished by removal of external Cl-. These data suggest that human small arteries maintain pHi by Na+/H+ exchange and Na(+)-dependent HCO3(-) exchange in response to an acid load, and Na(+)-independent Cl-/HCO3(-) exchange to counteract intracellular alkalosis. Norepinephrine (NE)-, endothelin-1 (ET-1)-, arginine vasopressin (AVP)-, and K(+)-induced tension did not alter pHi in PSS, but there was a small fall with angiotensin II (ANG II). In HEPES, stimulation with K+, NE, ANG II, or AVP led to a fall in pHi, but this did not occur with ET-1. It is therefore unlikely in vivo that an increase in pHi in these arteries would be involved in either tension development or growth induced by these agonists.

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Intracellular pH regulation in IEC-6 cells, a cryptlike intestinal cell line

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1989.257.5.g732 ◽

1989 ◽

Vol 257 (5) ◽

pp. G732-G740 ◽

Cited By ~ 1

Author(s):

E. Wenzl ◽

M. D. Sjaastad ◽

W. H. Weintraub ◽

T. E. Machen

Keyword(s):

Cell Line ◽

Intracellular Ph ◽

Ph Regulation ◽

Control Level ◽

Intestinal Cell ◽

Disulfonic Acid ◽

Intestinal Crypt ◽

Intestinal Cell Line ◽

Ethanesulfonic Acid ◽

Intestinal Crypt Cell

Regulation of intracellular pH, pHi, was studied using microspectrofluorimetry of the pH-sensitive, fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein in the rat intestinal crypt cell line, IEC-6. In N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solutions with pHi 7.25, treatment with a pulse of NH4Cl caused cells to acidify and then recover to control level. Because recovery was Na dependent, blocked by 1 mM amiloride, and unaffected by the presence and absence of Cl, it was likely because of a Na+-H+ exchanger. Cells were also acid loaded by changing from HEPES to HCO3-CO2-buffered solutions. pHi again recovered, but 1 mM amiloride reduced the rate of H+ efflux by only 47%. This HCO3-dependent, amiloride-insensitive H efflux required Na+ but not Cl- and was completely blocked by 200 microM [H2] 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). We conclude that a Na+-HCO3- cotransporter was operative. Cl-free solutions caused pHi to increase from 7.19 to 7.41; this effect required the presence of exogenous HCO3-CO2 but not Na and was blocked by 200 microM [H2]DIDS. A Cl- -HCO3- exchanger is the most likely explanation for these data. All the pHi regulatory mechanisms are operative in NaCl-HCO3-CO2-buffered solutions. The Na+-H+ and Na+-HCO3- mechanisms are acid extruders, whereas the Cl- -HCO3- exchanger is an acid loader. These transporters may be important for generating HCO3 secretion by intestinal crypt cells.

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Intracellular pH dependence of buffer capacity and anion exchange in the parietal cell

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1989.257.5.g741 ◽

1989 ◽

Vol 257 (5) ◽

pp. G741-G747 ◽

Cited By ~ 2

Author(s):

E. Wenzl ◽

T. E. Machen

Keyword(s):

Anion Exchange ◽

Intracellular Ph ◽

Anion Exchanger ◽

Buffer Capacity ◽

Ph Dependence ◽

Disulfonic Acid ◽

Rates Of Change ◽

Ethanesulfonic Acid ◽

Increased Activity ◽

Stimulation Of

When parietal cells (PC) are stimulated with histamine, the anion exchanger rate increases three to five times to compensate for alkaline loading induced by H+-K+ adenosinetriphosphatase (ATPase) and to provide Cl for acid secretion. It has been hypothesized that this increased activity is caused by the increase in intracellular pH (pHi) that often occurs in stimulated PC (from 7.1 to a maximum of 7.3). The dependence of the anion exchanger on pHi was studied using microspectrofluorimetry of the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solutions were used because the anion exchanger can transport OH- (or HCO3) in exchange for Cl- even with [HCO3]o = 200 microM. It was found that when solutions were changed either from NaCl to Cl- free or Cl- free to NaCl, rates of change of pHi (delta pH/delta t) were strongly dependent on pHi: nearly 0 at pHi 6.6 and 1.25 pH/min at pHi 8.0. To convert these pHi changes into anion flux rates, the intrinsic buffer capacity (beta i) was determined over the same pHi range by making small changes of [NH4]o to determine the resulting changes of [NH4]i and pHi (i.e., beta i = delta[NH4]i/delta pHi) in PC that had been pretreated with 1 mM amiloride and 200 microM [H2]4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) [to block Na+-H+ and Cl- -OH-(HCO3-) exchange]. beta i was also strongly dependent on pHi: at pHi 6.5 beta i = 48 mM/pH, and this decreased as pHi increased; at pHi 7.75 beta i = 8 mM/pH. The derived anion fluxes (i.e., JOH = beta i x delta pH/delta t) were roughly linearly related to pHi between 6.6 (JOH near 0) and 8.1 (JOH = 13 mM/min). Between pHi 7.1 and 7.3, the range normally observed during stimulation of PC, rates of anion exchange increased by 75%. This pHi sensitivity cannot explain the 300-500% increase in anion exchanger activity observed during secretagogue stimulation of PC.

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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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Effects of sodium and proton pump activity on respiratory burst and pH regulation of rat alveolar macrophages

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1993.264.5.l523 ◽

1993 ◽

Vol 264 (5) ◽

pp. L523-L532 ◽

Cited By ~ 6

Author(s):

J. K. Murphy ◽

H. J. Forman

Keyword(s):

Proton Pump ◽

Respiratory Burst ◽

Ph Regulation ◽

Atpase Inhibitor ◽

Atpase Inhibitors ◽

Pump Activity ◽

Acid Load ◽

Na Effect ◽

Acid Loading ◽

Phi Regulation

At pH 7.4, extracellular Na+ removal inhibited the rat alveolar macrophage respiratory burst (RB) stimulated by phorbol 12-myristate 13-acetate (PMA) or zymosan-activated serum (ZAS). At pH 6.8, the RB was lower and decreased the Na+ effect. Amiloride inhibited the ZAS RB independently of effects on Na(+)-H+ exchange, but did not affect PMA stimulation. NBD-Cl, an H(+)-ATPase inhibitor, significantly inhibited the PMA or ZAS RB. Na+ removal caused sustained elevation of intracellular free [Ca2+], which previous studies suggested inhibits the RB. Intracellular pH (pHi) was lower at pHo 6.8 compared with pHo 7.4, but not altered by Na+ removal. PMA stimulation resulted in acidification corresponding with onset of superoxide production. At pHo 7.4, recovery to baseline pHi occurred that was not inhibited by amiloride or Na+ removal. In contrast, amiloride slowed pHi recovery after an exogenous acid load. Addition of H(+)-ATPase inhibitors, NBD-Cl or bafilomycin, following PMA stimulation or acid loading, inhibited pHi restoration. These studies suggest that pHi regulation following stimulation was mainly through a proton pump, whereas Na(+)-H+ exchange occurred only after greater acid loading. Nevertheless, Na+ and pH interacted to modulate the RB independent of Na(+)-H+ exchange.

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