Regulation of intracellular pH in J774 murine macrophage cells: H+ extrusion processes

1995 ◽  
Vol 268 (1) ◽  
pp. C210-C217 ◽  
Author(s):  
L. C. McKinney ◽  
A. Moran
Keyword(s):  
Intracellular Ph ◽  
Initial Rate ◽  
Adherent Cells ◽  
Murine Macrophage ◽  
Bafilomycin A1 ◽  
Macrophage Cell ◽  
Recovery From Acidification ◽  
J774 Cells ◽  
Acid Load ◽  
Rate Of Recovery

Mechanisms of intracellular pH (pHi) regulation were characterized in the murine macrophage cell line J774.1, using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein to measure pHi. Under nominally HCO3(-)-free conditions, resting pHi of nonadherent J774.1 cells was 7.53 +/- 0.02 (n = 86), and of adherent cells was 7.59 +/- 0.02 (n = 97). In the presence of HCO3-/CO2, pHi values were reduced to 7.41 +/- 0.02 (n = 12) and 7.40 +/- 0.01 (n = 28), respectively. Amiloride, an inhibitor of Na+/H+ exchange, did not affect resting pHi. Inhibitors of a vacuolar type H(+)-ATPase [bafilomycin A1, N-ethylmaleimide (NEM), 7-chloro-4-nitrobenz-2-oxa-1,3-diazide (NBD), and p-chloromercuriphenylsulfonic acid (pCMBS)] reduced pHi by at least 0.2 pH units. Inhibitors of other classes of H(+)-ATPases (oligomycin, azide, vanadate, and ouabain) were without effect. Inhibition of H+ efflux, measured by the change in extracellular pH of a weakly buffered cell suspension, followed the same pharmacological profile, indicating that the reduction of pHi was due to inhibition of H+ extrusion. Mechanisms of recovery from an imposed intracellular acid load were also investigated. In NaCl-Hanks' solution, pHi recovered exponentially to normal within 2 min. The initial rate of recovery was inhibited > 90% by amiloride or by replacement of extracellular Na+ concentration by N-methyl-glucamine. Inhibitors of the vacuolar H(+)-ATPase also inhibited recovery. NEM and NBD nonspecifically inhibited all recovery. Bafilomycin A1 and pCMBS did not inhibit the initial amiloride-sensitive portion of recovery, but they did inhibit a late component of recovery when pHi was above 7.0. We conclude that the Na+/H+ exchanger is primarily responsible for recovery from an acid load but does not regulate resting pHi. Conversely, a vacuolar H(+)-ATPase regulates the resting pHi of J774 cells but contributes little to recovery from acidification.

Intracellular pH homeostasis in cultured human placental syncytiotrophoblast cells: recovery from acidification

2005 ◽  
Vol 288 (4) ◽  
pp. C891-C898 ◽  
Author(s):  
Elizabeth A. Cowley ◽  
Mary C. Sellers ◽  
Nicholas P. Illsley
Keyword(s):  
Intracellular Ph ◽  
Driving Force ◽  
Progressive Decrease ◽  
Ph Homeostasis ◽  
Ion Substitution ◽  
Recovery From Acidification ◽  
Acid Load ◽  
Acidification Recovery ◽  
Intracellular Acid

Resting or basal intracellular pH (pHi) measured in cultured human syncytiotrophoblast cells was 7.26 ± 0.04 (without HCO3−) or 7.24 ± 0.03 (with HCO3−). Ion substitution and inhibitor experiments were performed to determine whether common H+-transporting species were operating to maintain basal pHi. Removal of extracellular Na+ or Cl− or addition of amiloride or dihydro-4,4′-diisothiocyanatostilbene-2,2′-disulfonate (H2DIDS) had no effect. Acidification with the K+/H+ exchanger nigericin reduced pHi to 6.25 ± 0.15 (without HCO3−) or 6.53 ± 0.10 (with HCO3−). In the presence of extracellular Na+, recovery to basal pHi was prompt and occurred at similar rates in the absence and presence of HCO3−. Ion substitution and inhibition experiments were also used to identify the species mediating the return to basal pHi after acidification. Recovery was inhibited by removal of Na+ or addition of amiloride, whereas removal of Cl− and addition of H2DIDS were ineffective. Addition of the Na+/H+ exchanger monensin to cells that had returned to basal pHi elicited a further increase in pHi to 7.48 ± 0.07. Analysis of recovery data showed that there was a progressive decrease in ΔpH per minute as pHi approached the basal level, despite the continued presence of a driving force for H+ extrusion. These data show that in cultured syncytial cells, in the absence of perturbation, basal pHi is preserved despite the absence of active, mediated pH maintenance. They also demonstrate that an Na+/H+ antiporter acts to defend the cells against acidification and that it is the sole transporter necessary for recovery from an intracellular acid load.

Intracellular pH regulation in the rabbit cortical collecting duct A-type intercalated cell

1997 ◽  
Vol 273 (3) ◽  
pp. F340-F347 ◽  
Author(s):  
A. E. Milton ◽  
I. D. Weiner
Keyword(s):  
Intracellular Ph ◽  
Ph Regulation ◽  
Collecting Duct ◽  
Cortical Collecting Duct ◽  
Bafilomycin A1 ◽  
Proton Secretion ◽  
Acid Load ◽  
A Cell ◽  
Phi Regulation ◽  
Intracellular Acid

The A cell may possess multiple H+ transporters, including H(+)-adenosinetriphosphatase (H(+)-ATPase) and H(+)-K(+)-ATPase. The current study examines the relative roles of proton transporters in the A cell by observing their contribution to both basal intracellular pH (pHi) regulation and pHi recovery from an intracellular acid load. CCD were studied using in vitro microperfusion, and pHi was measured in the individual A cell using the fluorescent, pH-sensitive dye, 2',7'-bis(carboxyethyl)-5(6)-carboxy-fluorescein (BCECF). Inhibiting H(+)-ATPase with luminal bafilomycin A1 decreased basal pHi, whereas inhibiting apical H(+)-K(+)-ATPase with either luminal Sch-28080 or luminal potassium removal did not. The predominant mechanism of pHi, recovery from an intracellular acid load was peritubular sodium dependent and peritubular ethylisopropylamiloride (EIPA) sensitive, identifying basolateral Na+/H+ exchange activity. In the absence of peritubular sodium, pHi recovery was inhibited by luminal bafilomycin A1 but not by luminal Sch-28080 addition or by luminal potassium removal. However, when Na+/H+ exchange was inhibited with EIPA, both bafilomycin A1 sensitive and potassium dependent, Sch-28080-sensitive components of pHi recovery were present. Quantitatively, the rate of H(+)-ATPase proton secretion was greater than the rate of H(+)-K(+)-ATPase proton secretion. We conclude that basolateral Na+/H+ exchange is the predominant mechanism of A cell pHi recovery from an intracellular acid load. An apical H(+)-ATPase is the primary apical transporter contributing to A cell pHi regulation. An apical H(+)-K(+)-ATPase, while present, plays a more limited role under the conditions tested.

Glucocorticoids stimulate Na+/H+ antiporter in OKP cells

1993 ◽  
Vol 264 (6) ◽  
pp. F1027-F1031 ◽  
Author(s):  
M. Baum ◽  
A. Cano ◽  
R. J. Alpern
Keyword(s):  
Proximal Tubule ◽  
Intracellular Ph ◽  
Direct Effect ◽  
Initial Rate ◽  
Time Dependent ◽  
Affinity Constant ◽  
Maximal Velocity ◽  
Hemodynamic Changes ◽  
Acid Load ◽  
Stimulation Of

Previous studies have demonstrated that systemic administration of glucocorticoids stimulates proximal tubule acidification in part by increasing Na+/H+ antiporter activity; however, these studies could not exclude the possibility that changes in Na+/H+ antiporter activity were secondary to glucocorticoid-induced hemodynamic changes. The present study examined the effect of dexamethasone on Na+/H+ antiporter activity in quiescent OKP cells. Na+/H+ antiporter activity was assayed as the initial rate of Na(+)-dependent pH recovery from an acid load. Intracellular pH was measured using the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Dexamethasone produced a dose- and time-dependent stimulation of Na+/H+ antiporter activity in OKP cells. Dexamethasone produced a 24% stimulation in Na+/H+ antiporter activity at 10(-9) M and an approximately 40% stimulation of Na+/H+ antiporter activity at both 10(-8) and 10(-6) M. The effect of 10(-6) M dexamethasone was seen within 4 h of incubation and was due to an increase in maximal velocity (Vmax, 3.03 vs. 1.79 pH units/min) with no change in the affinity constant for sodium (KNa, 47.2 vs. 42.0 mM). The stimulatory effect of dexamethasone on Na+/H+ antiporter activity was blocked by cycloheximide and was not observed with 10(-8) M aldosterone. These data demonstrate a direct effect of glucocorticoids to stimulate Na+/H+ antiporter activity in OKP cells.

Regulation of intracellular pH in crypt cells from rabbit distal colon

1994 ◽  
Vol 267 (3) ◽  
pp. G409-G415 ◽  
Author(s):  
S. L. Abrahamse ◽  
A. Vis ◽  
R. J. Bindels ◽  
C. H. van Os
Keyword(s):  
Intracellular Ph ◽  
Distal Colon ◽  
Buffering Capacity ◽  
Colonic Crypt ◽  
Bafilomycin A1 ◽  
Acid Load ◽  
Intracellular Buffering Capacity ◽  
Crypt Cells ◽  
Exchange Activity ◽  
In Cells

H+ secretory mechanisms and intrinsic intracellular buffering capacity were studied in crypt cells from rabbit distal colon. To this end crypts of Lieberkuhn were isolated by microdissection, and intracellular pH (pHi) was measured using digital imaging fluorescence microscopy and the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)- 5(6)-carboxyfluorescein. In the absence of HCO(3-)-CO2 and presence of Na+, resting pHi was 7.51 +/- 0.04 (n = 237/23, cells/crypts). However, 6 min after superfusion with a solution containing zero Na+, 1 x 10(5) M Sch-28080 and 5 x 10(-8) M bafilomycin A1, pHi in cells at the bottom of the crypts was significantly reduced, whereas pHi in cells at the top of the crypts remained unchanged. The intrinsic buffering capacity of cells from the middle to the top portion of crypts was significantly higher in the pHi range 7.2-7.6 than of cells at the bottom of the crypt. H+ secretion after an NH(4+)-NH3 pulse amounted to 245 +/- 53 microM/s (n = 73/7) at pHi 7.1 and was largely Na+ dependent and ethylisopropylamiloride sensitive. The Na(+)-independent recovery of pHi after an acid load was insensitive to Sch-28080 and bafilomycin A1. In conclusion, pHi in colonic crypt cells is regulated through Na+/H+ exchange activity in the absence of HCO3-. In addition, intracellular buffering capacity varied with the position along the crypt axis, whereas Na+/H+ exchange activity and pHi did not.

Identification of exchange mechanisms for the regulation of intracellular pH in rat thyroid FRTL-5 cells

1992 ◽  
Vol 9 (3) ◽  
pp. 301-308 ◽  
Author(s):  
A. M. Wood ◽  
G. Warhurst ◽  
S. P. Bidey ◽  
J. Soden ◽  
R. Taylor ◽  
...  
Keyword(s):  
Intracellular Ph ◽  
Similar Rate ◽  
Thyroid Cell ◽  
Cell Monolayers ◽  
Ionic Substitution ◽  
Acid Load ◽  
Rate Of Recovery ◽  
Disulphonic Acid ◽  
Rat Thyroid ◽  
Free Media

ABSTRACT pH is maintained in cells by plasma membrane exchange mechanisms. In the absence of HCO3− ions, FRTL-5 cells regulate intracellular pH (pHi) by an Na+/H+ antiport but HCO3−-dependent exchangers cannot operate. We have investigated pHi regulation (by microfluorimetry and the pH sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6′)-carboxy-fluorescein) in small groups (five to six cells) of FRTL-5 thyroid cell monolayers held in kREBS—Ringer buffer (pH 7·4) with or without HCO3− ions. The exchangers were investigated with inhibitors (amiloride or its derivative dimethylamiloride for the Na+/H+ antiporter and the stilbene derivative disodium 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) for HCO3 −-dependent mechanisms), ionic substitution and by NH4+/NH3 (10mm) acid loading. Basal pHi was lower in the presence (7·3±0·058, mean±s.d., n= 14) than in the absence (7·59±0·078, n=10) of HCO3 ions. In HCO3 −-free media, cells recovered from acid load by 0·34±0·04 pH units in the first 2 min and finally reached a pHi of 7·35±0·06. This recovery was Na+-dependent and blocked by dimethylamiloride during the 15 min following intracellular acidification. In HCO3−-containing media, cells recovered from an acid load at a similar rate, but reached 99 ± 10% (n = 9) of the baseline pH; this recovery was also dependent on Na+ ions. Moreover, although dimethylamiloride and DIDS reduced the rate of recovery to 0·06±0·02 and 0·18±0·04 pH units respectively during the 2-min period, the cells returned to the basal pHi within 15 min. Removal of Na+ from HCO3−-containing media acidified the cells (ΔpH=–0·82±0·05, n=10) within 40 min; this acidification was partially blocked by either amiloride or DIDS. Removal of Cl− alkalinized the cells (ΔpH=+0·51 ± 0·06, n=10) after 40 min, and this alkalinization was totally prevented by DIDS. Furthermore, in the absence of Na+ and presence of amiloride, alkalinization was still seen on the removal of Cl−, albeit at a diminished rate (i.e. ΔpH = +0·25±0·05, n=8) after 40 min. In conclusion, FRTL-5 cells maintain pHi by two Na+-dependent exchangers, one sensitive to amiloride, the other to DIDS, and a Na+-independent, Cl−/HCO3− mechanism.

scholarly journals l-arginine transport is increased in macrophages generating nitric oxide

1992 ◽  
Vol 284 (1) ◽  
pp. 15-18 ◽  
Author(s):  
R G Bogle ◽  
A R Baydoun ◽  
J D Pearson ◽  
S Moncada ◽  
G E Mann
Keyword(s):  
Nitric Oxide ◽  
Time Dependent ◽  
Murine Macrophage ◽  
Macrophage Cell Line ◽  
Macrophage Cell ◽  
Arginine Transport ◽  
Nitrite Production ◽  
J774 Cells ◽  
Elevated Rate ◽  
Stimulation Of

Transport of L-arginine and nitrite production were examined in the murine macrophage cell line J774. Bacterial lipopolysaccharide (LPS) induced a dose- and time-dependent stimulation of nitrite production, which was further increased in the presence of interferon-gamma. Nitrite synthesis was absolutely dependent on extracellular L-arginine and inhibited in the presence of L-lysine or L-ornithine. In unactivated J774 cells L-arginine transport was saturable, with an apparent Km of 0.14 +/- 0.04 mM and Vmax. of 15 +/- 2 nmol/h per 10(6) cells. LPS (1 microgram/ml) induced a time-dependent stimulation of L-arginine transport, and after 24 h the Vmax. increased to 34 +/- 2 nmol/h per 10(6) cells. These findings indicate that activation of J774 cells with LPS produces an increase in both L-arginine transport and nitrite synthesis. The elevated rate of L-arginine transport in activated J774 cells may provide a mechanism for sustained substrate supply during enhanced utilization of L-arginine for the generation of NO.

Influence of surface pH on intracellular pH regulation in cardiac and skeletal muscle

1986 ◽  
Vol 250 (5) ◽  
pp. C748-C760 ◽  
Author(s):  
B. Vanheel ◽  
A. de Hemptinne ◽  
I. Leusen
Keyword(s):  
Intracellular Ph ◽  
Soleus Muscle ◽  
Ph Regulation ◽  
Purkinje Fiber ◽  
Intracellular Acidification ◽  
Surface Ph ◽  
Acid Load ◽  
Rat Soleus Muscle ◽  
Rate Of Recovery ◽  
Intracellular Acid

The influence of the surface pH (pHs) on the intracellular pH (pHi) and the recovery of pHi after an imposed intracellular acid load was investigated in isolated sheep cardiac Purkinje fiber, rabbit papillary muscle, and mouse and rat soleus muscle. pHs and pHi, respectively, were continuously measured by use of single- and double-barreled pH-sensitive glass microelectrodes. Surface acidosis, usually obtained by superfusion with solutions of acid pH, was also produced with low buffered (5 mM N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid) solutions at control pH. The pHs decrease (delta pHs) induced by low buffering was smallest (-0.08 pH unit) in Purkinje fiber and largest (-0.31 pH unit) in rat soleus muscle, which already had a more acid surface in control conditions. delta pHs was somewhat dependent on the superfusion rate. Higher superfusion rates decreased but did not abolish delta pHs. Surface acidosis was associated with a small intracellular acidification. Intracellular acid loads were produced by adding and subsequently withdrawing 20 meq/l NH4+ from the superfusate. In all preparations, the rate of recovery of pHi after NH4+ withdrawal was notably decreased at acidified pHs. This effect was amiloride sensitive. It is concluded that, in superfused multi-cellular preparations, pHs and therefore the buffer concentration of a superfusate can considerably influence steady-state pHi and pHi recovery from an imposed intracellular acid load.

Na+/H+ exchange in mosquito Malpighian tubules

2000 ◽  
Vol 279 (6) ◽  
pp. R1996-R2003 ◽  
Author(s):  
D. H. Petzel
Keyword(s):  
Steady State ◽  
Intracellular Ph ◽  
Specific Inhibitor ◽  
Fluid Secretion ◽  
Malpighian Tubules ◽  
Pulse Technique ◽  
Acid Load ◽  
Rate Of Recovery ◽  
Stimulation Of

Fluid secretion and intracellular pH were measured in isolated mosquito Malpighian tubules to determine the presence of Na+/H+ exchange. Rates of fluid secretion by individual Malpighian tubules in vitro were inhibited by 78% of control in the presence of 100 μM 5-( N-ethyl- n-isopropyl)-amiloride (EIPA), a specific inhibitor of Na+/H+ exchange. Steady-state intracellular pH was measured microfluorometrically by using 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in individual Malpighian tubules. Bathing the Malpighian tubules in 0 mM extracellular Na+ or in the presence of 100 μM EIPA reduced the steady-state intracellular pH by 0.5 pH units. Stimulation of the Na+/H+ exchanger by using the NH4Cl pulse technique resulted in a rate of recovery from the NH4Cl-induced acute acid load of 8.7 ± 1.0 × 10−3 pH/s. The rates of recovery of intracellular pH after the acute acid load in the absence of extracellular Na+ or in the presence of 100 μM EIPA were 0.7 ± 0.6 and −0.3 ± 0.3 × 10−3 pH/s, respectively. These results indicate that mosquito Malpighian tubules possess a Na+/H+ exchanger.

scholarly journals The role of a H+-ATPase in the regulation of cytoplasmic pH in Trypanosoma cruzi epimastigotes

1996 ◽  
Vol 318 (1) ◽  
pp. 103-109 ◽  
Author(s):  
Nicole VANDERHEYDEN ◽  
Gustavo BENAIM ◽  
Roberto DOCAMPO
Keyword(s):  
Trypanosoma Cruzi ◽  
Cytoplasmic Ph ◽  
Bafilomycin A1 ◽  
Intact Cells ◽  
Low Concentrations ◽  
Acid Load ◽  
Rate Of Recovery ◽  
Acid Loading ◽  
Cellular Acidification

Cytoplasmic pH (pHi) regulation was studied in Trypanosoma cruzi epimastigotes using fluorescent probes. Steady-state pHi was maintained even in the absence of extracellular Na+ or K+, but was significantly decreased in the absence of Cl-. Acid-loaded epimastigotes regained normal pHi by a process that was ATP-dependent and sensitive to N-ethylmaleimide, dicyclohexylcarbodi-imide and diethylstilboestrol, suggesting involvement of a H+-pumping ATPase. Recovery from an acid load was independent of extracellular Na+ or K+ and insensitive to omeprazole, vanadate and low concentrations of bafilomycin A1. Using the fluorescent probe bisoxonol to measure the membrane potential of intact cells, acid loading of epimastigotes was shown to result in a dicyclohexylcarbodi-imide-sensitive hyperpolarization, which suggests electrogenic pumping of protons across the plasma membrane. Addition of glucose, but not of 6-deoxyglucose, produced a transient cellular acidification of possible metabolic origin, and increased the rate of recovery from an acid load. Taken together, these results are consistent with an important role of a H+-ATPase in the regulation of pHi homoeostasis in T. cruzi.

Ionic requirements for intracellular pH regulation in rainbow trout hepatocytes

1986 ◽  
Vol 250 (1) ◽  
pp. R24-R29 ◽  
Author(s):  
P. J. Walsh
Keyword(s):  
Rainbow Trout ◽  
Intracellular Ph ◽  
Ph Regulation ◽  
Disulfonic Acid ◽  
Salmo Gairdneri ◽  
Exchange System ◽  
Exchange Processes ◽  
Acid Load ◽  
Rate Of Recovery ◽  
22Na Uptake

The ionic requirements for pH regulation in isolated rainbow trout (Salmo gairdneri) hepatocytes were determined by manipulation of intracellular pH (pHi; measured by the dimethyloxazolidinedione distribution technique) by NH4Cl prepulse and changes in external [CO2] in the presence and absence of various drugs and external ions. The presence of a Na+/H+(NH+4) exchange system is supported by the following results: 1) the rate of recovery from an acid load is decreased by amiloride (0.5 mM) or reduction of external [Na+]; 2) the rate of 22Na uptake is increased during recovery from an acid load, and this increase in amiloride sensitive. The presence of a Cl-/HCO3- exchange system is supported by the observations that 1) pHi is increased, and 2) rates of recovery of pHi from acid loading are enhanced, by exposure to 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (0.5 mM) and reductions in external [Cl-]. Further studies are required to determine the role of these exchange processes during physiological pHi perturbations.

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