scholarly journals Intracellular pH regulation in the S3 segment of the rabbit proximal tubule in HCO3- -free solutions.

1988 ◽  
Vol 92 (3) ◽  
pp. 369-393 ◽  
Author(s):  
N L Nakhoul ◽  
A G Lopes ◽  
J R Chaillet ◽  
W F Boron
Keyword(s):  
Proximal Tubule ◽  
Intracellular Ph ◽  
Ph Regulation ◽  
Transport Systems ◽  
Total Acid ◽  
Luminal Membrane ◽  
Acid Load ◽  
S3 Segment ◽  
Acid Extrusion ◽  
Dependent Mechanism

We used the absorbance spectrum of 4',5'-dimethyl-5-(and 6) carboxyfluorescein to measure intracellular pH (pHi) in the isolated, perfused S3 segment of the rabbit proximal tubule. Experiments were conducted in HCO3- -free solutions. pHi recovered from an acid load imposed by an NH4+ prepulse, indicating the presence of one or more active acid-extrusion mechanisms. Removal of Na+ from bath and lumen caused pHi to decrease by approximately 0.6, whereas Na+ readdition caused complete pHi recovery. Removal of Na+ from the bath caused only a slow pHi decrease that was enhanced about fourfold when Na+ was subsequently removed from the lumen also. Similarly, the pHi recovery produced by the readdition of Na+ to the bath and lumen was about ninefold faster than when Na+ was returned to the bath only. Amiloride (1-2 mM) inhibited the pHi recovery that was elicited by returning 15 or 29 mM Na+ to lumen by only approximately 30%. However, in the absence of external acetate (Ac-), 1 mM amiloride inhibited approximately 66% of the pHi recovery induced by the readdition of 29 mM Na+ to the lumen only. The removal of external Ac- reduced the pHi recovery rate from an NH4+-induced acid load by approximately 47%, and that elicited by Na+ readdition, by approximately 67%. Finally, when bilateral removal of Na+ was maintained for several minutes, pHi recovered from the initial acidification, slowly at first, and then more rapidly, eventually reaching a pHi approximately 0.1 higher than the initial one. This Na+-independent pHi recovery was not significantly affected by lowering [HEPES]o from 32 to 3 mM or by adding N'N'-dicyclohexylcarbodiimide (10(-4) M) to the lumen, but it was reduced approximately 57% by iodoacetate (0.5 mM) plus cyanide (1 mM). We conclude that in the nominal absence of HCO3-, three transport systems contribute to acid extrusion by S3 cells: (a) a Na+-independent mechanism, possibly an H+ pump; (b) a Na-H exchanger, confined primarily to the luminal membrane; and (c) an Ac- and luminal Na+-dependent mechanism. The contribution of these three mechanisms to total acid extrusion, assessed by the rapid readdition of Na+, was approximately 13, approximately 30, and approximately 57%, respectively.

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

1993 ◽  
Vol 102 (6) ◽  
pp. 1171-1205 ◽  
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).

Intracellular pH regulation in rabbit S3 proximal tubule: basolateral Cl-HCO3 exchange and Na-HCO3 cotransport

1990 ◽  
Vol 258 (2) ◽  
pp. F371-F381 ◽  
Author(s):  
N. L. Nakhoul ◽  
L. K. Chen ◽  
W. F. Boron
Keyword(s):  
Proximal Tubule ◽  
Intracellular Ph ◽  
Initial Rate ◽  
Ph Regulation ◽  
Basolateral Membrane ◽  
Intracellular Ph Regulation ◽  
S3 Segment ◽  
Absorbance Spectra ◽  
Rule Out

We studied the role of basolateral HCO3- transport in the regulation of intracellular pH (pHi) in the isolated perfused S3 segment of the rabbit proximal tubule. pHi was calculated from absorbance spectra of the pH-sensitive dye dimethylcarboxyfluorescein. Solutions were normally buffered to pH 7.4 at 37 degrees C with 25 mM HCO3- 5% CO2. pHi fell by approximately 0.17 when luminal [HCO3-] was lowered to 5 mM at fixed PCO2 (i.e., reducing pH to 6.8) but by approximately 0.42 when [HCO3-] in the bath (i.e., basolateral solution) was lowered to 5 mM. The pHi decrease elicited by reducing bath [HCO3-] was substantially reduced by removal of Cl- or Na+, suggesting that components of basolateral HCO3- transport are Cl- and/or Na+ dependent. We tested for the presence of basolateral Cl-HCO3 exchange by removing bath Cl-. This caused pHi to increase by approximately 0.23, with an initial rate of approximately 100 X 10(-4) pH/s. Although the initial rate of this pHi increase was not reduced by removing Na+ bilaterally, it was substantially lowered by the nominal removal of HCO3- from bath and lumen or by the addition of 0.1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) to the bath. The results thus suggest that a Na-independent Cl-HCO3 exchanger is present at the basolateral membrane. We tested for the presence of basolateral Na-HCO3 cotransport by removing bath Na+. This caused pHi to fall reversibly by approximately 0.26 with initial rates of pHi decline and recovery being approximately 30 and approximately 41 X 10(-4) pH/s, respectively. Although the bilateral removal of Cl- had no effect on these rates, the nominal removal of HCO3- or the presence of DIDS substantially slowed the pHi changes. Thus, in addition to a Cl-HCO3 exchanger, the basolateral membrane of the S3 proximal tubule also appears to possess a Na-HCO3 cotransport mechanism. The data do not rule out the possibility of other basolateral HCO3- transporters.

Acid extrusion in S3 segment of rabbit proximal tubule. I. Effect of bilateral CO2/HCO3-

1995 ◽  
Vol 268 (2) ◽  
pp. F179-F192 ◽  
Author(s):  
L. K. Chen ◽  
W. F. Boron
Keyword(s):  
Steady State ◽  
Proximal Tubule ◽  
Initial Rate ◽  
Iodoacetic Acid ◽  
Buffering Power ◽  
Acid Load ◽  
S3 Segment ◽  
Series Of Experiments ◽  
Acid Extrusion ◽  
Stimulation Of

Monitoring the absorbance spectra of the pH-sensitive dye dimethylcarboxyfluorescein, we studied intracellular pH (pHi) regulation in the isolated perfused S3 segment of rabbit proximal tubule. To explain a previous observation, that steady-state pHi is higher in the presence than in the absence of CO2/HCO3- (N. L. Nakhoul, L. K. Chen, and W. F. Boron. J. Gen. Physiol. 102: 1171-1205, 1993), we examined the effect of bilateral (i.e., luminal and basolateral) CO2/HCO3- on the acid extrusion processes responsible for recovery of pHi from acid loads. To compute fluxes from rates of pHi change, we determined the pHi dependence of intrinsic intracellular buffering power, which was approximately 50 mM/pH at pHi 6.5 and fell linearly to approximately 20 mM at pHi 7.4. In one series of experiments, we monitored the rate of pHi recovery from an acid load imposed by an NH4+/NH3 prepulse. Over a broad range of pHi values, total net acid extrusion was approximately four times higher in bilateral presence of CO2/HCO3- than in its absence. In a second group of experiments, which were designed to determine the effect of CO2/HCO3- on luminal Na+/H+ exchange, we monitored the rate of pHi recovery elicited by adding Na+ back to only the lumen, after first removing Na+ bilaterally. Initial rate of luminal Na(+)-dependent net acid extrusion in presence of CO2/HCO3- was approximately 229 microM/s (pHi 6.92), approximately 1.8 times higher than the flux of approximately 127 microM/s (P < 0.005) obtained in absence of CO2/HCO3- (pHi 6.66). CO2/HCO3- alkali-shifted the flux vs. pHi relationship by 0.3-0.4 pH units. In a final series of experiments, we examined the effect of CO2/HCO3- on the Na(+)-independent alkalinization that follows the rapid, initial acidification elicited by bilateral Na+ removal. In the presence of CO2/HCO3-, lag time for initiation of the Na(+)-independent alkalinization was only approximately 36 vs. approximately 211 s (P < 0.002) in absence of CO2/HCO3-. Also, Na(+)-independent net acid extrusion rate was approximately two to three times higher in presence than in absence of CO2/HCO3- at comparable pHi. This Na(+)-independent acid extrusion was insensitive to N-ethylmaleimide (2 mM), but was inhibited approximately 94% by efforts to deplete intracellular ATP (i.e., removal of glucose and amino acids, plus addition of 2 mM cyanide and 10 mM iodoacetic acid). Stimulation of luminal Na+/H+ exchange and Na(+)-independent acid extrusion appears to be the major, if not the entire, explanation for the higher steady-state pHi caused by bilateral addition of CO2/HCO3-.

scholarly journals Acetate transport in the S3 segment of the rabbit proximal tubule and its effect on intracellular pH.

1988 ◽  
Vol 92 (3) ◽  
pp. 395-412 ◽  
Author(s):  
N L Nakhoul ◽  
W F Boron
Keyword(s):  
Acetic Acid ◽  
Proximal Tubule ◽  
Intracellular Ph ◽  
Acid Transport ◽  
Subsequent Removal ◽  
Luminal Membrane ◽  
A Value ◽  
All Solutions ◽  
S3 Segment ◽  
The One

We monitored intracellular pH (pHi) in isolated perfused S3 segments of the rabbit proximal tubule, and studied the effect of acetate (Ac-) transport on pHi. pHi was calculated from the absorbance spectrum of 4',5'-dimethyl-5-(and 6) carboxyfluorescein trapped intracellularly. All solutions were nominally HCO3(-)-free. Removal of 10 mM Ac- from bath and lumen caused pHi to rapidly rise by approximately 0.2, and then to decline more slowly to a value approximately 0.35 below the initial one. Removal of only luminal Ac- caused pHi changes very similar to those resulting from bilateral removal of Ac-. When Ac- was removed from bath only, pHi rose rapidly at first, and then continued to rise more slowly. Readdition of Ac- to bath caused pHi to rapidly fall to a value slightly higher than the one prevailing before the removal of Ac- from the bath. In experiments in which Ac- was first removed from both bath and lumen, readdition of 10 mM Ac- to only lumen caused a rapid but small acidification, followed by a slower alkalinization that brought the pHi near the value that prevailed before the bilateral removal of Ac-. The alkalinizing effects caused by the readdition of 10 or 0.5 mM Ac- were indistinguishable. When Ac- was returned to only lumen in the absence of luminal Na+, there was a small and rapid pHi decrease, but no pHi recovery. Removal of Na+ from bath did not affect the pHi transients caused by the addition of Ac- to lumen. In experiments in which Ac- was first removed bilaterally, readdition of Ac- to only bath caused a large and sustained drop in pHi, whereas the subsequent removal of Ac- from the bath caused a slight alkalinization. These pHi changes caused by readdition or removal of Ac- from baths were unaffected by the absence of external Na+. We conclude that there is a Na+/Ac- cotransporter at the luminal membrane, and pathways for acetic acid transport at both luminal and basolateral membranes. The net effect of Ac- transport on pHi is to alkalinize the cell as a result of the luminal entry of Na+/Ac-, which is followed by the luminal and basolateral exit of acetic acid.

scholarly journals Intracellular pH Regulation in Cultured Astrocytes from Rat Hippocampus

1997 ◽  
Vol 110 (4) ◽  
pp. 453-465 ◽  
Author(s):  
Mark O. Bevensee ◽  
Regina A. Weed ◽  
Walter F. Boron
Keyword(s):  
Intracellular Ph ◽  
Ph Regulation ◽  
Disulfonic Acid ◽  
Bath Solution ◽  
Major Cation ◽  
Cultured Astrocytes ◽  
Buffering Power ◽  
Acid Load ◽  
Acid Extrusion ◽  
Best Fit

We studied the regulation of intracellular pH (pHi) in single cultured astrocytes passaged once from the hippocampus of the rat, using the dye 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) to monitor pHi. Intrinsic buffering power (βI) was 10.5 mM (pH unit)−1 at pHi 7.0, and decreased linearly with pHi; the best-fit line to the data had a slope of −10.0 mM (pH unit)−2. In the absence of HCO3−, pHi recovery from an acid load was mediated predominantly by a Na-H exchanger because the recovery was inhibited 88% by amiloride and 79% by ethylisopropylamiloride (EIPA) at pHi 6.05. The ethylisopropylamiloride-sensitive component of acid extrusion fell linearly with pHi. Acid extrusion was inhibited 68% (pHi 6.23) by substituting Li+ for Na+ in the bath solution. Switching from a CO2/HCO3−-free to a CO2/HCO3−-containing bath solution caused mean steady state pHi to increase from 6.82 to 6.90, due to a Na+-driven HCO3− transporter. The HCO3−-induced pHi increase was unaffected by amiloride, but was inhibited 75% (pHi 6.85) by 400 μM 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), and 65% (pHi 6.55–6.75) by pretreating astrocytes for up to ∼6.3 h with 400 μM 4-acetamide-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS). The CO2/HCO3−-induced pHi increase was blocked when external Na+ was replaced with N-methyl-d-glucammonium (NMDG+). In the presence of HCO3−, the Na+-driven HCO3− transporter contributed to the pHi recovery from an acid load. For example, HCO3− shifted the plot of acid-extrusion rate vs. pHi by 0.15–0.3 pH units in the alkaline direction. Also, with Na-H exchange inhibited by amiloride, HCO3− increased acid extrusion 3.8-fold (pHi 6.20). When astrocytes were acid loaded in amiloride, with Li+ as the major cation, HCO3− failed to elicit a substantial increase in pHi. Thus, Li+ does not appear to substitute well for Na+ on the HCO3− transporter. We conclude that an amiloride-sensitive Na-H exchanger and a Na+-driven HCO3− transporter are the predominant acid extruders in astrocytes.

Acid extrusion in S3 segment of rabbit proximal tubule. II. Effect of basolateral CO2/HCO3-

1995 ◽  
Vol 268 (2) ◽  
pp. F193-F203 ◽  
Author(s):  
L. K. Chen ◽  
W. F. Boron
Keyword(s):  
Proximal Tubule ◽  
Recovery Rate ◽  
Lag Time ◽  
The Other ◽  
Total Rate ◽  
Other Hand ◽  
Acid Load ◽  
S3 Segment ◽  
Acid Extrusion ◽  
Absorbance Spectra

Monitoring intracellular absorbance spectra of the pH-sensitive dye dimethylcarboxyfluorescein we studied intracellular pH (pHi) regulation in the isolated perfused S3 segment of the rabbit proximal tubule. In the preceding study [Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F179-F192, 1995.], we demonstrated that simultaneously adding CO2/HCO3- to both lumen and bath stimulated two acid-extruding mechanisms, one dependent on luminal Na+ (a Na+/H+ exchanger) and one independent of Na+ (presumably a H+ pump). Here, we examine the effects of adding CO2/HCO3- to the lumen only or to the bath only. Over a broad pHi range, the total rate of pHi recovery from an acid load in the presence of Na+ was not increased by luminal CO2/HCO3-. On the other hand, basolateral CO2/HCO3- increased the pHi recovery rate to an even greater extent than had bilateral CO2/HCO3-. Regarding the Na(+)-independent pHi recovery mechanism, we found that luminal CO2/HCO3- failed to increase the pHi recovery rate compared with controls also studied in the absence of Na+. Neither did luminal CO2/HCO3- significantly affect the lag time between the maximal acid load and the initiation of the Na(+)-independent pHi recovery (approximately 147 vs. approximately 212 s in controls). On the other hand, adding CO2/HCO3- to only the bath substantially increased the rate of Na(+)-independent pHi recovery, which generally was greater than that observed with bilateral CO2/HCO3-. CO2/HCO3- only in the bath also reduced the lag time to approximately 51 s, which is not significantly different from the value of approximately 36 s observed with bilateral CO2/HCO3-. Our data are consistent with the hypothesis that basolateral (but not luminal) CO2/HCO3- stimulates both a luminal Na+/H+ exchanger and a luminal H+ pump.

Intracellular pH transients in rat diaphragm muscle measured with DMO

1978 ◽  
Vol 235 (1) ◽  
pp. C49-C54 ◽  
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.

pH regulation in ileum: Na(+)-H+ and Cl(-)-HCO3- exchange in isolated crypt and villus cells

1991 ◽  
Vol 260 (3) ◽  
pp. G440-G449 ◽  
Author(s):  
U. Sundaram ◽  
R. G. Knickelbein ◽  
J. W. Dobbins
Keyword(s):  
Intracellular Ph ◽  
Ph Regulation ◽  
Second Messengers ◽  
Cell Types ◽  
Current Evidence ◽  
Leucine Incorporation ◽  
Trypan Blue Exclusion ◽  
Morphological Criteria ◽  
Acid Load ◽  
Separation Cell

Current evidence suggests that intestinal crypt and villus cells have different functions in electrolyte transport. To study the regulation of transporters, we isolated and separated these two cell types. This was accomplished by sequential collection of enterocytes from rabbit ileal loops incubated with buffered solutions of calcium chelators. Alkaline phosphatase and thymidine kinase activity, sodium-glucose cotransport, and morphological criteria were used to determine cell separation. Cell viability was evaluated with trypan blue exclusion, leucine incorporation into protein, and morphological features. The role of Na(+)-H+ and Cl(-)-HCO3- exchange in the regulation of intracellular pH was analyzed using an intracellular pH sensitive dye, BCECF. Removal of external Na+ or the addition of amiloride resulted in acidification of both crypt and villus cells. Removal of Cl- or the addition of DIDS resulted in alkalinization of both cell types. The cells could be acidified with NH4Cl, and recovery from this acid load was dependent on Na+ and inhibited by amiloride. Similarly, the cells could be alkalinized with propionate and recovery was Cl- dependent and DIDS sensitive. These data are consistent with the presence of Na(+)-H+ and Cl(-)-HCO3- exchange in both crypt and villus cells. Both exchanges appear to be involved in the regulation of basal pH as well as in recovery from alterations in intracellular pH. Having demonstrated the presence of Na(+)-H+ and Cl(-)-HCO3- exchange activity in both crypt and villus cells, we can now use these cells to determine the regulation of these exchangers by intracellular second messengers.

Hyperosmotic urea activates basolateral NHE in proximal tubule from P-gp null and wild-type mice

2002 ◽  
Vol 283 (4) ◽  
pp. F771-F783 ◽  
Author(s):  
Yukio Miyata ◽  
Yasushi Asano ◽  
Shigeaki Muto
Keyword(s):  
Tyrosine Kinase ◽  
Proximal Tubule ◽  
Entire Range ◽  
Kinase Inhibitor ◽  
Urea Solution ◽  
Wild Type ◽  
Pkc Inhibitor ◽  
Mannitol Solution ◽  
Acid Load ◽  
Acid Extrusion

Using the pH-sensitive fluorescent dye BCECF, we compared the effects of hyperosmotic urea on basolateral Na+/H+ exchange (NHE) with those of hyperosmotic mannitol in isolated nonperfused proximal tubule S2 segments from mice lacking both the mdr1a and mdr1b genes (KO) and wild-type (WT) mice. All the experiments were performed in CO2/HCO[Formula: see text]-free HEPES solutions. Osmolality of the peritubular solution was raised from 300 to 500 mosmol/kgH2O by adding mannitol or urea. NHE activity was assessed by the Na+-dependent acid extrusion rate ( J H) after an acid load with NH4Cl prepulse. In WT mice, hyperosmotic mannitol had no effect on J H at over the entire range of intracellular pH (pHi) studied (6.20–6.90), whereas in KO mice it increased J H at a pHi range of 6.20–6.45. In contrast, in both WT and KO mice, hyperosmotic urea increased J H at a pHi range of 6.20–6.90. In KO mice, J H in a hyperosmotic urea solution were similar to those in a hyperosmotic mannitol solution at a pHi range of 6.20–6.40 but were greater than in a hyperosmotic mannitol solution at a pHi range of 6.45–6.90. In WT mice, hyperosmotic urea caused an increase in V max without changing K mfor peritubular Na+. Staurosporine (the PKC inhibitor) inhibited hyperosmotic mannitol-induced NHE activation in KO mice, whereas it had no effect on hyperosmotic urea-induced NHE activation in WT or KO mice. Genistein (the tyrosine kinase inhibitor) inhibited hyperosmotic urea-induced NHE activation in WT and KO mice, whereas it caused no effect on hyperosmotic mannitol-induced NHE activation in KO mice. We conclude that hyperosmotic urea activates basolateral NHE via tyrosine kinase in tubules from both WT and KO mice, whereas hyperosmotic mannitol activates it via PKC only in tubules from KO mice.

Control of intracellular pH during regulatory volume decrease in HL-60 cells

1994 ◽  
Vol 267 (4) ◽  
pp. C1057-C1066 ◽  
Author(s):  
K. R. Hallows ◽  
D. Restrepo ◽  
P. A. Knauf
Keyword(s):  
Intracellular Ph ◽  
Ph Regulation ◽  
Regulatory Volume Decrease ◽  
Transport Systems ◽  
Disulfonic Acid ◽  
Volume Dependence ◽  
Supportive Role ◽  
22Na Uptake ◽  
Volume Decrease ◽  
Stimulation Of

Intracellular pH (pHi) homeostasis was investigated in human promyelocytic leukemic HL-60 cells as they undergo regulatory volume decrease (RVD) in hypotonic media to determine how well pHi is regulated and which transport systems are involved. Cells suspended in hypotonic (50-60% of isotonic) media undergo a small (< 0.2 pH units), but significant (P < 0.05), intracellular acidification within 5 min. However, after 30 min of RVD, pHi is not significantly different from the initial pHi in 20 mM HCO3- medium and is significantly higher in HCO3(-)-free medium. Experiments performed in media with or without 150 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and HCO3- demonstrate that the anion exchanger (AE) mediates a net Cl- influx, with compensating HCO3- efflux, during RVD. To determine which transport systems are involved in counteracting this tendency toward acidification, we measured transport rates and examined the effect of transport system inhibitors on pHi. We found that inhibition of Na+/H+ exchange (NHE) with 12.5 microM ethylisoproplamiloride (EIPA) causes pHi to fall significantly by the end of 30 min of RVD. As assessed by EIPA-sensitive 22Na+ uptake measurements, NHE, largely dormant under resting isotonic conditions, becomes significantly activated by the end of 30 min of RVD, despite recovery of pHi and cell volume to near-normal levels. Thus a shift in the normal pHi dependence and/or volume dependence of NHE activity must occur during RVD under hypotonic conditions. In contrast, H(+)-monocarboxylate cotransport appears to play only a supportive role in pH regulation during RVD, as indicated by lack of stimulation of [14C]lactate efflux during RVD.

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