scholarly journals Intracellular pH regulation in human Sertoli cells: role of membrane transporters

Reproduction ◽  
2009 ◽  
Vol 137 (2) ◽  
pp. 353-359 ◽  
Author(s):  
P F Oliveira ◽  
M Sousa ◽  
A Barros ◽  
T Moura ◽  
A Rebelo da Costa
Keyword(s):  
Intracellular Ph ◽  
Sertoli Cells ◽  
Ph Regulation ◽  
Primary Cultures ◽  
Disulfonic Acid ◽  
Cell Physiology ◽  
Experimental Conditions ◽  
Concanamycin A ◽  
Wide Range ◽  
Phi Regulation

Sertoli cells are responsible for regulating a wide range of processes that lead to the differentiation of male germ cells into spermatozoa. Intracellular pH (pHi) is an important parameter in cell physiology regulating namely cell metabolism and differentiation. However, pHi regulation mechanisms in Sertoli cells have not yet been systematically elucidated. In this work, pHi was determined in primary cultures of human Sertoli cells. Sertoli cells were exposed to weak acids, which caused a rapid acidification of the intracellular milieu. pHi then recovered by a mechanism that was shown to be particularly sensitive to the presence of the inhibitor DIDS (4,4′-diisothiocyanostilbene disulfonic acid). In the presence of amiloride and PSA (picrylsulfonic acid), pHi recovery was also significantly affected. These results indicate that, in the experimental conditions used, pHi is regulated by the action of an Na+-driven HCO3−/Cl−exchanger and an Na+/HCO3−co-transporter and also by the action of the Na+/H+exchanger. On the other hand, pHi recovery was only slightly affected by concanamycin A, suggesting that V-Type ATPases do not have a relevant action on pHi regulation in human Sertoli cells, and was independent of the presence of bumetanide, suggesting that the inhibition of the Na+/K+/Cl−co-transporter does not affect pHi recovery, not even indirectly via the shift of ionic gradients. Finally, pHi was shown to be sensitive to the removal of external Cl−, but not of Na+or K+, evidencing the presence of a membrane Cl−-dependent base extruder, namely the Na+-independent HCO3−/Cl−exchanger, and its role on pHi maintenance on these cells.

scholarly journals The Boron & De Weer Model of Intracellular pH Regulation

2020 ◽  
Author(s):  
Rossana Occhipinti ◽  
Soroush Safaei ◽  
Peter J. Hunter ◽  
Walter F. Boron
Keyword(s):  
Cell Membrane ◽  
Intracellular Ph ◽  
Ph Regulation ◽  
Quantitative Model ◽  
Historical Background ◽  
Acid Base ◽  
Experimental Conditions ◽  
Subsequent Work ◽  
Energy Dependent ◽  
Parameter Values

The classic Boron & De Weer (1976) paper provided the first evidence of active regulation of pH} in cells by an energy-dependent acid-base transporter. These authors also developed a quantitative model --- comprising passive fluxes of acid-base equivalents across the cell membrane, intracellular reactions, and an active transport mechanism in the cell membrane (modelled as a proton pump) --- to help interpret their measurements of intracellular pH under perturbations of both extracellular CO2/HCO3- and extracellular NH3/NH4+. This Physiome paper seeks to make that model, and the experimental conditions under which it was developed, available in a reproducible and well-documented form, along with a software implementation that makes the model easy to use and understand. We have also taken the opportunity to update some of the units used in the original paper, and to provide a few parameter values that were missing in the original paper. Finally, we provide an historical background to the Boron & De Weer (1976) proposal for active pH regulation and a commentary on subsequent work that has enriched our understanding of this most basic aspect of cellular physiology.

K(+)-induced alkalinization in mouse cerebral astrocytes mediated by reversal of electrogenic Na(+)-HCO3- cotransport

1994 ◽  
Vol 267 (6) ◽  
pp. C1633-C1640 ◽  
Author(s):  
N. Brookes ◽  
R. J. Turner
Keyword(s):  
Intracellular Ph ◽  
Primary Cultures ◽  
Atmospheric Co2 ◽  
Disulfonic Acid ◽  
Free Solution ◽  
High Affinity ◽  
Alkaline Shift ◽  
Net Flux ◽  
K Concentration ◽  
The Relationship

Raising extracellular K+ concentration ([K+]o) induces an alkaline shift of intracellular pH (pHi) in astrocytes. The mechanism of this effect was examined using the fluorescent pHi indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein in primary cultures of mouse cerebral astrocytes. Raising [K+]o from 3 to 12 mM increased pHi by 0.28 pH units in 26 mM HCO(3-)-buffered solution. In nominally HCO(3-)-free solution (containing approximately 95 microM HCO3-), the alkalinization fell to 0.21 pH units and further to 0.08 pH units on removal of atmospheric CO2, suggesting a process with high affinity for HCO3-. This effect was Na+ dependent, Cl- independent, and inhibited by 0.5 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, indicating the involvement of Na(+)-HCO3- cotransport. The relationship between pHi and log[K+]o was found to be linear and to predict a stoichiometry of at least two HCO3- transported with each Na+. After removal of exogenous CO2/HCO3-, the direction of changes in pHi elicited by adding 1 mM HCO3- showed that net flux of HCO3- via the Na(+)-HCO3- cotransporter was outward at rest and was reversed by depolarization.

Na-H exchange regulates intracellular pH in isolated rat hepatocyte couplets

1987 ◽  
Vol 252 (1) ◽  
pp. G109-G113
Author(s):  
R. M. Henderson ◽  
J. Graf ◽  
J. L. Boyer
Keyword(s):  
Carbon Dioxide ◽  
Intracellular Ph ◽  
Ph Regulation ◽  
Intracellular Acidification ◽  
Rat Hepatocyte ◽  
Buffering Power ◽  
Recovery From Acidification ◽  
Regulation Mechanisms ◽  
Phi Regulation ◽  
Isolated Rat

Intracellular pH (pHi) was measured directly in isolated rat hepatocyte couplets using pH sensitive microelectrodes. The hepatocytes were maintained in a minimal salt buffer without added hormones or serum. Values of pHi (6.99 +/- 0.12, mean +/- SE) were close to their Nernst equilibria. After intracellular acidification with ammonium chloride, pH regulation was inhibited with 1 mM amiloride or by omission of external sodium, consistent with a Na-H exchange mechanism. Mean intracellular buffering power, in the nominal absence of carbon dioxide, was 34.1 +/- 11.4 mM. In the presence of external bicarbonate, amiloride or omission of sodium slowed, but did not completely inhibit recovery from acidification, indicating that additional pHi regulation mechanisms may operate in this preparation. These studies provide a direct measurement of pHi in hepatocyte couplets and indicate that Na-H exchange, together with a bicarbonate dependent system are important mechanisms for pHi regulation in this preparation.

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.

Na(+)-H+ antiporter and Na(+)-(HCO3-)n symporter regulate intracellular pH in mouse medullary thick limbs of Henle

1990 ◽  
Vol 258 (3) ◽  
pp. F445-F456 ◽  
Author(s):  
D. Kikeri ◽  
S. Azar ◽  
A. Sun ◽  
M. L. Zeidel ◽  
S. C. Hebert
Keyword(s):  
Steady State ◽  
Intracellular Ph ◽  
Dominant Role ◽  
Disulfonic Acid ◽  
Outer Medulla ◽  
Ethanesulfonic Acid ◽  
Antibody Labeling ◽  
Intracellular Alkalinization ◽  
Phi Regulation

To determine mechanisms of intracellular pH (pHi) regulation in mouse medullary thick limbs (MTAL), pHi was measured in MTAL suspensions and in the isolated perfused MTAL by use of 2',7'-bis(carboxyethyl)-5(6)carboxyfluorescein (BCECF). A method to obtain MTAL suspensions from the mouse outer medulla is reported. Characterization of suspensions with microscopy, anti-Tamm-Horsfall antibody labeling, measurement of O2 consumption, and adenosine 3',5'-cyclic monophosphate responses to antidiuretic hormone indicated that these suspensions were highly purified for viable MTAL tubules. The resting pHi was 7.41 +/- 0.02 (means +/- SE) in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered media and 7.23 +/- 0.02 in CO2- HCO3(-)-buffered media, both at extracellular pH 7.4. MTAL tubules exhibited rapid pHi recovery from intracellular acidification. Recovery of pHi was dependent on luminal Na+ (apparent Km = 13.2 +/- 3.2 mM) and was inhibited by amiloride (apparent Ki = 10.6 microM), consistent with the activity of an apical Na(+)-H+ antiporter. Antiporter activity was enhanced by acidification and was diminished at the resting pHi. Recovery from intracellular alkalinization (rapid withdrawal of CO2- HCO3-) was sensitive to the stilbene anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, Cl(-)-insensitive, and Na(+)-sensitive, consistent with the activity of a Na(+)-(HCO3-)n symporter. Both transporters were significantly involved in steady-state pHi regulation in the presence of CO2- HCO3-. In contrast, the Na(+)-H+ antiporter played the dominant role in steady-state pHi regulation in the absence of CO2- HCO3-.

Intracellular pH regulation in cultured renal proximal tubule cells in different stages of maturation

1992 ◽  
Vol 263 (4) ◽  
pp. F716-F721 ◽  
Author(s):  
H. Ekblad ◽  
A. Aperia ◽  
S. H. Larsson
Keyword(s):  
Proximal Tubule ◽  
Intracellular Ph ◽  
Recovery Rate ◽  
Ph Regulation ◽  
Renal Proximal Tubule ◽  
Disulfonic Acid ◽  
Intracellular Acidosis ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells

This study examines the ontogeny of cellular pH regulation in renal proximal tubule cells (RPTC). RPTC from 8- to 40-day-old Sprague-Dawley rats (RPTC-8 to RPTC-40) were studied after 48 h of primary culture. Intracellular pH (pHi) was measured by quantitative fluorescence microscopy using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Recordings were made under basal conditions and after imposing a cytoplasmic alkalosis and acidosis using 15 mM NH4+ salt. The net recovery rate (dpHi/dt) from intracellular acidosis increases significantly between 10 and 12 days of age from 0.39 +/- 0.04 to 0.54 +/- 0.06 pH units/min (P < 0.05, n = 10 vs. 6). This increase can be completely accounted for by an increase in the rate of amiloride (100 microM)-inhibitable Na(+)-H+ exchange (0.29 +/- 0.04 vs. 0.42 +/- 0.05 pH units/min, P < 0.05, n = 6 vs. 6). The rate of Na(+)-H+ exchange increases similarly in RPTC-10 and RPTC-40 when the transmembrane Na+ gradient is increased by Na+ depleting the cells (48 and 49%, respectively). The amiloride-insensitive recovery is Na+ independent and insensitive to 4-acetamido-4'-isothiocyanostilbene-2-2'-disulfonic acid (SITS, 500 microM) (range 0.08-0.14 pH units/min). The net recovery rate from intracellular alkalosis is significantly lower in RPTC-10 than in RPTC-40 (0.16 +/- 0.02 vs. 0.28 +/- 0.02 pH units/min, P < 0.01, n = 4 vs. 5). SITS (500 microM) inhibits the recovery by 27 +/- 8 and 26 +/- 9%, respectively, whereas amiloride has no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Na(+)-HCO3- symport modulates intracellular pH in alveolar epithelial cells

1991 ◽  
Vol 260 (6) ◽  
pp. L555-L561 ◽  
Author(s):  
R. L. Lubman ◽  
E. D. Crandall
Keyword(s):  
Intracellular Ph ◽  
Initial Rate ◽  
Alveolar Epithelium ◽  
Alveolar Epithelial Cells ◽  
Transepithelial Transport ◽  
Disulfonic Acid ◽  
Experimental Conditions ◽  
Alveolar Epithelial ◽  
Ethanesulfonic Acid ◽  
Acid Extrusion

We investigated Na(+)-HCO3- cotransport as a mechanism for regulation of intracellular pH (pHi) in rat alveolar pneumocytes grown in primary culture. pHi was monitored using the fluorescent pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Cells incubated in 6 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) medium at pH 7.4 were subjected to rapid acidification by CO2 pulse. pHi recovered in the presence of Na+ with an initial rate (dpHi/dt) of 0.15 min-1, which was reduced by 67% when Na+ was replaced by choline, unaffected by substitution of gluconate for Cl-, reduced 40% in the presence of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 500 microM), and unchanged by amiloride (1 mM). In parallel experiments, cells were incubated at pH 7.4 with 20 mM HCO3- and pHi acutely lowered by NH3 prepulse. dpHi/dt in these experiments was 0.14 min-1 in the presence of Na+ and HCO3-, and reduced 79% under Na(+)-free conditions. These data indicate the presence of a Na(+)-dependent, Cl(-)-independent, DIDS-sensitive and amiloride-insensitive mechanism of recovery from acute intracellular acidification in alveolar pneumocytes, most consistent with Na(+)-HCO3- cotransport (symport) effecting acid extrusion under these experimental conditions. This ion transport mechanism may contribute to regulation of pHi in alveolar pneumocytes, transepithelial transport of acid-base equivalents across the alveolar epithelium, and modulation of pH of alveolar fluid in adult mammalian lungs.

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

1989 ◽  
Vol 257 (5) ◽  
pp. G732-G740 ◽  
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.

Relationship between intra- and extracellular pH in primary cultures of rat astrocytes

1994 ◽  
Vol 267 (2) ◽  
pp. C581-C589 ◽  
Author(s):  
P. Mellergard ◽  
Y. Ou-Yang ◽  
B. K. Siesjo
Keyword(s):  
Kynurenic Acid ◽  
Prolonged Exposure ◽  
Primary Cultures ◽  
Low Ph ◽  
Extracellular Ph ◽  
Neonatal Rat ◽  
Disulfonic Acid ◽  
Rat Cortex ◽  
Rat Astrocytes ◽  
Phi Regulation

We studied the influence of extracellular pH (pHe) on the mechanisms regulating intracellular pH (pHi) in astrocytes cultured from neonatal rat cortex, using single cell microspectrofluorometry and the pH-sensitive fluorophore 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. When pHe was maintained at control values of 7.35 during acid transients caused by an increased CO2 tension, pHi was rapidly regulated back to normal. However, at pHe 6.9 or below, there was no recovery of pHi. Steady-state pHi was also strongly dependent on pHe (pHi = 1.14 + 0.80 pHe). The pHi recovery after normalization of pHe was very rapid, indicating that a prolonged exposure to a low pH stimulates pH-regulating mechanisms, and was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and amiloride or the removal of Na+. Recovery was also slowed down by Cd2+. The pHe-dependent acidification was not critically influenced by DIDS or amiloride and was not inhibited by tetrodotoxin, tetraethylammonium, Ba2+,2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)-quinoxaline, kynurenic acid, 5-hydroxysaclofen, bicuculline, Cd2+, or albumin. The results emphasize the importance of pHe for pHi regulation and suggest that in astrocytes pHi is not regulated back to normal levels until pHe is normalized.

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