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Frank B. Jensen - One of the best experts on this subject based on the ideXlab platform.
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Red blood Cell pH, the Bohr effect, and other oxygenation‐linked pHenomena in blood O2 and CO2 transport
Acta Physiologica Scandinavica, 2004Co-Authors: Frank B. JensenAbstract:The discovery of the S-shaped O 2 equilibrium curve and the Bohr effect in 1904 stimulated a fertile and continued research into respiratory functions of blood and allosteric mechanisms in haemoglobin (Hb). The Bohr effect (influence of pH/CO 2 on Hb O 2 affinity) and the reciprocal Haldane effect (influence of HbO 2 saturation on H + /CO 2 binding) originate in the Hb oxydeoxy conformational change and allosteric interactions between O 2 and H + / CO 2 binding sites. In steady state, H + is passively distributed across the vertebrate red blood Cell (RBC) membrane, and intraCellular pH (pH i ) changes are related to changes in extraCellular pH, Hb-O 2 saturation and RBC organic pHospHate content. As the Hb molecule shifts between the oxy and deoxy conformation in arterial-venous gas transport, it delivers O 2 and takes up CO 2 and H + in tissue capillaries (elegantly aided by the Bohr effect). Concomitantly, the RBC may sense local O 2 demand via the degree of Hb deoxygenation and release vasodilatory agents to match local blood flow with requirements. Three recent hypotheses suggest (1) release of NO from S-nitroso-Hb upon deoxygenation, (2) reduction of nitrite to vasoactive NO by deoxy haems, and (3) release of ATP. Inside RBCs, carbonic anhydrase (CA) provides fast hydration of metabolic CO 2 and ensures that the Bohr shift occurs during capillary transit. The formed H + is bound to Hb (Haldane effect) while HCO - 3 is shifted to plasma via the anion exchanger (AE1). The magnitude of the oxylabile H + binding shows characteristic differences among vertebrates. Alternative strategies for CO 2 transport include direct HCO - 3 binding to deoxyHb in crocodilians, and high intraCellular free [HCO - 3 ] (due to high pH i ) in lampreys. At the RBC membrane, CA, AE1 and other proteins may associate into what appears to be an integrated gas exchange metabolon. Oxygenation-linked binding of Hb to the membrane may regulate glycolysis in mammals and perhaps also oxygen-sensitive ion transport involved in RBC volume and pH i regulation. Blood O 2 transport shows several adaptive changes during exposure to environmental hypoxia. The Bohr effect is involved via the respiratory alkalosis induced by hyperventilation, and also via the pH i change that results from modulation of RBC organic pHospHate content. In teleost fish, β-adrenergic activation of Na + /H + exchange rapidly elevates pH i and O 2 affinity, particularly under low O 2 conditions.
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red blood Cell pH the bohr effect and other oxygenation linked pHenomena in blood o2 and co2 transport
Acta Physiologica Scandinavica, 2004Co-Authors: Frank B. JensenAbstract:The discovery of the S-shaped O 2 equilibrium curve and the Bohr effect in 1904 stimulated a fertile and continued research into respiratory functions of blood and allosteric mechanisms in haemoglobin (Hb). The Bohr effect (influence of pH/CO 2 on Hb O 2 affinity) and the reciprocal Haldane effect (influence of HbO 2 saturation on H + /CO 2 binding) originate in the Hb oxydeoxy conformational change and allosteric interactions between O 2 and H + / CO 2 binding sites. In steady state, H + is passively distributed across the vertebrate red blood Cell (RBC) membrane, and intraCellular pH (pH i ) changes are related to changes in extraCellular pH, Hb-O 2 saturation and RBC organic pHospHate content. As the Hb molecule shifts between the oxy and deoxy conformation in arterial-venous gas transport, it delivers O 2 and takes up CO 2 and H + in tissue capillaries (elegantly aided by the Bohr effect). Concomitantly, the RBC may sense local O 2 demand via the degree of Hb deoxygenation and release vasodilatory agents to match local blood flow with requirements. Three recent hypotheses suggest (1) release of NO from S-nitroso-Hb upon deoxygenation, (2) reduction of nitrite to vasoactive NO by deoxy haems, and (3) release of ATP. Inside RBCs, carbonic anhydrase (CA) provides fast hydration of metabolic CO 2 and ensures that the Bohr shift occurs during capillary transit. The formed H + is bound to Hb (Haldane effect) while HCO - 3 is shifted to plasma via the anion exchanger (AE1). The magnitude of the oxylabile H + binding shows characteristic differences among vertebrates. Alternative strategies for CO 2 transport include direct HCO - 3 binding to deoxyHb in crocodilians, and high intraCellular free [HCO - 3 ] (due to high pH i ) in lampreys. At the RBC membrane, CA, AE1 and other proteins may associate into what appears to be an integrated gas exchange metabolon. Oxygenation-linked binding of Hb to the membrane may regulate glycolysis in mammals and perhaps also oxygen-sensitive ion transport involved in RBC volume and pH i regulation. Blood O 2 transport shows several adaptive changes during exposure to environmental hypoxia. The Bohr effect is involved via the respiratory alkalosis induced by hyperventilation, and also via the pH i change that results from modulation of RBC organic pHospHate content. In teleost fish, β-adrenergic activation of Na + /H + exchange rapidly elevates pH i and O 2 affinity, particularly under low O 2 conditions.
Lawrence G. Palmer - One of the best experts on this subject based on the ideXlab platform.
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Regulation of principal Cell pH by Na/H exchange in rabbit cortical collecting tubule
The Journal of Membrane Biology, 1992Co-Authors: Randi B. Silver, Gustavo Frindt, Lawrence G. PalmerAbstract:Changes in intraCellular pH (pH_ i ) were measured using the pH indicator, BCECF, in principal Cells from split opened cortical collecting tubules (CCTs) derived from rabbits maintained on a normal diet. This monolayer preparation has the advantage of allowing us to visualize the morpHological differences in the two major Cell types in this nepHron segment under transmitted light. The visual identification of the Cell types was verified using emission measurements taken from single principal and intercalated Cells in the opened tubule which had been exposed to fluorescein isothiocyanate (FITC)-labeled peanut lectin. We confirmed the existence of an amiloride-sensitive Na/H exchange process activated during intraCellular acidosis in principal Cells. In addition, the exchanger was active under basal conditions and over a wide range of pH_ i . Because the exchanger was active under basal conditions we tested the hypothesis that changes in intraCellular Na (Na_ i ) would alter pH_ i in a predictable way. Maneuvers designed to alter Na_ i were without significant effects within a 10-min time frame. Specifically, addition of 100 μ m ouabain to increase Na_ i or exposure of the tubules to 10^−5 m amiloride to decrease luminal Na entry and reduce Na_ i did not have an effect on pH_ i . In some experiments we did observe however, after a 30-min exposure to ouabain, a small decrease in pH_ i . These results suggest that Na/H exchange is a major regulator of pH_ i in principal Cells. However, regulation of Na transport by changes in pH_ i in principal Cells of rabbit CCT via the activity of a Na/H exchanger do not seem to contribute to the feedback control of Na transport.
Wray H. Huestis - One of the best experts on this subject based on the ideXlab platform.
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Membrane potential and human erythrocyte shape
Biophysical Journal, 1997Co-Authors: Margaret M. Gedde, Wray H. HuestisAbstract:Altered external pH transforms human erythrocytes from discocytes to stomatocytes (low pH) or echinocytes (high pH). The process is fast and reversible at room temperature, so it seems to involve shifts in weak inter- or intramolecular bonds. This shape change has been reported to depend on changes in membrane potential, but control experiments excluding roles for other simultaneously varying Cell properties (Cell pH, Cell water, and Cell chloride concentration) were not reported. The present study examined the effect of independent variation of membrane potential on red Cell shape. Red Cells were equilibrated in a set of solutions with graduated chloride concentrations, producing in them a wide range of membrane potentials at normal Cell pH and Cell water. By using assays that were rapid and accurate, Cell pH, Cell water, Cell chloride, and membrane potential were measured in each sample. Cells remained discoid over the entire range of membrane potentials examined (-45 to +45 mV). It was concluded that membrane potential has no independent effect on red Cell shape and does not mediate the membrane curvature changes known to occur in red Cells equilibrated at altered pH.
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Shape response of human erythrocytes to altered Cell pH
Blood, 1995Co-Authors: Margaret M. Gedde, Eungyeong Yang, Wray H. HuestisAbstract:Alteration of red blood Cell (RBC) pH produces stomatocytosis (at low pH) and echinocytosis (at high pH). Cell shrinkage potentiates high pH echinocytosis, but shrinkage alone does not cause echinocytosis. Mechanisms for these shape changes have not been described. In this study, measured dependence of RBC shape on Cell pH was nonlinear, with a broad pH range in which normal discoid shape was maintained. Transbilayer distribution of pHospHatidylcholine and pHospHatidylserine, measured by back-extraction of radiolabeled lipid, was the same in control and altered pH Cells. Possible roles of pH- titratable inner monolayer pHospHolipids were examined by assessing pH- dependent shape in Cells in which their levels had been perturbed. In metabolically depleted Cells and calcium-treated Cells, which have altered levels of pHospHatidic acid, pHospHatidylinositol-4-pHospHate, and/or pHospHatidylinositol-4,5-bispHospHate, low Cell pH was stomatocytogenic and high Cell pH was echinocytogenic, as in control Cells. Thus, neither change in membrane lipid asymmetry nor normal levels of the pH-titratable inner monolayer lipids is necessary for Cell pH-mediated shape change.
Jared J. Grantham - One of the best experts on this subject based on the ideXlab platform.
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Effect of Cellular acidosis on Cell volume in S2 segments of renal proximal tubules.
The American journal of physiology, 1990Co-Authors: Lawrence P. Sullivan, Darren P. Wallace, R. L. Clancy, Jared J. GranthamAbstract:The presence of pH-sensitive transport mechanisms in the basolateral membrane of proximal tubular Cells suggests that Cell volume and its regulation may be sensitive to changes in Cell pH. We have measured the response of Cell pH and Cell volume to changes in the acid-base composition of solutions bathing isolated, lumen-collapsed, proximal S2 tubular segments taken from the rabbit kidney. Cell pH was determined by measurement of the fluorescence emission of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Cell volume was calculated from measurements of tubular diameter. An increase in CO2 from 5 to 15% reduced Cell pH 0.30 units and raised Cell bicarbonate concentration ([HCO3]) 10 mM. Cell volume rose to 108.6% of control in 4 min. A decrease in bath [HCO3] from 25 to 5 mM reduced Cell pH 0.41 units and Cell [HCO3] by 15 mM. Cell volume gradually increased to 105.7% at 8 min. The rate of the regulatory volume decrease after Cell swelling on exposure to a 160 mosM solution was determined in the presence of 5 and 15% CO2. The latter reduced the maximum fractional rate of recovery of volume from 0.18 to 0.11 min-1 but did not affect the extent of regulation. We conclude that acidosis causes Cell swelling and reduces the rate of volume regulation in response to hypotonic media.
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Effect of Cellular acidosis on Cell volume in S2 segments of renal proximal tubules
American Journal of Physiology-renal Physiology, 1990Co-Authors: Lawrence P. Sullivan, Darren P. Wallace, R. L. Clancy, Jared J. GranthamAbstract:The presence of pH-sensitive transport mechanisms in the basolateral membrane of proximal tubular Cells suggests that Cell volume and its regulation may be sensitive to changes in Cell pH. We have measured the response of Cell pH and Cell volume to changes in the acid-base composition of solutions bathing isolated, lumen-collapsed, proximal S2 tubular segments taken from the rabbit kidney. Cell pH was determined by measurement of the fluorescence emission of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Cell volume was calculated from measurements of tubular diameter. An increase in CO2 from 5 to 15% reduced Cell pH 0.30 units and raised Cell bicarbonate concentration ([HCO3]) 10 mM. Cell volume rose to 108.6% of control in 4 min. A decrease in bath [HCO3] from 25 to 5 mM reduced Cell pH 0.41 units and Cell [HCO3] by 15 mM. Cell volume gradually increased to 105.7% at 8 min. The rate of the regulatory volume decrease after Cell swelling on exposure to a 160 mosM solution was determined in the presence ...
Gerhard Malnic - One of the best experts on this subject based on the ideXlab platform.
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NHE1, NHE2, and NHE4 contribute to regulation of Cell pH in T84 colon cancer Cells
Pflügers Archiv: European Journal of Physiology, 2007Co-Authors: Ana Rosa Beltrán, M. A. Ramírez, Luciene Regina Carraro-lacroix, Yumi Hiraki, Nancy Amaral Rebouças, Gerhard MalnicAbstract:The isoforms of the Na+/H+ exchanger present in T84 human colon Cells were identified by functional and molecular methods. Cell pH was measured by fluorescence microscopy using the probe BCECF. Based on the pH recovery after an ammonium pulse and determination of buffering capacity of these Cells, the rate of H+ extrusion (JH) was 3.68 mM/min. After the use of the amiloride derivative HOE-694 at 25 μM, which inhibits the isoforms NHE1 and NHE2, there remained 43% of the above transport rate, the nature of which was investigated. Evidence of the presence of NHE1, NHE2, and NHE4 was obtained by reverse transcriptase polymerase chain reaction (RT-PCR) (mRNA) and Western blot. There was no decrease of JH by the NHE3 inhibitor S3226 (1 μM) and no evidence of this isoform by RT-PCR was found. The following functional evidence for the presence of NHE4 was obtained: 25 μM EIPA abolished JH entirely, but NHE4 was not inhibited at 10 μM; substitution of Na by K increased the remainder, a property of NHE4; hypertonicity also increased this fraction of JH. Cl−-dependent NHE was not detected: in 0 Cl− solutions JH was increased and not reduced. In 0 Cl− Cell volume decreased significantly, which was abolished by the Cl− channel blocker NPPB, indicating that the 0 Cl− effect was because of reduction of Cell volume. In conclusion, T84 human colon Cells contain three isoforms of the Na+/H+ exchanger, NHE1, NHE2, and NHE4, but not the Cl-dependent NHE.
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Control of Cell pH in the T84 colon Cell line.
The Journal of Membrane Biology, 2000Co-Authors: M. A. Ramírez, Roxana Toriano, Mario Parisi, Gerhard MalnicAbstract:Cell pH regulation was investigated in the T84 Cell line derived from epithelial colon cancer. Cell pH was measured by ratiometric fluorescence microscopy using the fluorescent probe BCECF. Basal pH was 7.17 ± 0.023 (n= 48) in HEPES Ringer. After acidification by an ammonium pulse, Cell pH recovered toward normal at a rate of 0.13 ± 0.011 pH units/min in the presence of Na+, but in the absence of this ion or after treatment with 0.1 mm hexamethylene amiloride (HMA) no significant recovery was observed, indicating absence of Na+ independent H+ transport mechanisms in HEPES Ringer. In CO2/HCO− 3 Ringer, basal Cell pH was 7.21 ± 0.020 (n= 35). Changing to HEPES Ringer, a marked alkalinization was observed due to loss of CO2, followed by return to the initial pH at a rate of −0.14 ± 0.012 (n= 8) pH/min; this return was retarded or abolished in the absence of Cl− or after addition of 0.2 mm DIDS, suggesting extrusion of bicarbonate by Cl−/HCO− 3 exchange. This exchange was not Na+ dependent. When Na+ was added to Cells incubated in 0 Na+ Ringer while blocking Na+/H+ exchange by HMA, Cell alkalinization by 0.19 ± 0.04 (n= 11) pH units was observed, suggesting the presence of Na+/HCO− 3 cotransport carrying HCO− 3 into these Cells, which was abolished by DIDS. These experiments, thus, show that Na+/H+ and Cl−/HCO− 3 exchange and Na+/HCO− 3 cotransport participate in Cell pH regulation in T84 Cells.
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Permeation of NH3/NH4+ and Cell pH in colonic crypts of the rat.
Pflügers Archiv: European Journal of Physiology, 1999Co-Authors: M. A. Ramírez, R. Fernández, Gerhard MalnicAbstract:Colon Cells are subjected to high concentrations of NH3 and NH4 +, and a sizeable portion of this buffer is absorbed. The flux of these components into Cells causes opposite effects on their pH; this effect is largely used to induce an acid load and to observe the mechanism of acid extrusion from Cells. We studied Cells of microdissected colon crypts loaded with BCECF and superfused with NH4Cl-containing Krebs-Ringer solution. We found a marked transient reduction in pH measured by ratiometric fluorescence microscopy, from a control value of 7.51±0.041 to 7.15±0.041 (n=21), instead of the initial alkalinization found in most Cells. This pH was reached at a rate of 0.95±0.07 pH units/min. Addition of 1 mmol/l furosemide, a blocker of Na+,K+,2Cl– cotransport, to the ammonium solution inverted this acidification toward alkalinization (pH 7.89±0.041, n=5), and superfusion with furosemide plus 0.1 mmol/l hexamethylene amiloride, a specific blocker of Na+/H+ exchange, increased this initial alkalinization further to 8.10±0.117 (n=7). When Krebs-Ringer with 0 Cl– containing (NH4)2SO4 instead of NH4Cl was superfused, the acid transient was also reverted to alkalinization; however, a higher degree of alkalinization was observed either when 1 mmol/l furosemide was added to the superfusing sulfate solution (when a pH of 7.78±0.010 was reached), or when ammonium gluconate was used instead of ammonium sulfate. The addition of Ba2+ to the superfusion solution did not alter the initial acidification. These data indicate that in colon crypt Cells, basolateral membrane transporters, in particular the Na+,K+,2Cl– cotransporter and the Na+/H+ exchanger (but not Ba2+-sensitive K+ channels), mediate the predominant influx of NH4 + ions leading to the initial transient acidification.
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H+ ATPase and Cl− Interaction in Regulation of MDCK Cell pH
The Journal of Membrane Biology, 1998Co-Authors: R. Fernández, Gerhard MalnicAbstract:MDCK Cells display several acid-base transport systems found in intercalated Cells, such as Na+-H+ exchange, H+–K+ ATPase and Cl−/HCO−3 exchange. In this work we studied the functional activity of a vacuolar H+-ATPase in MDCK Cells and its chloride dependence. We measured intraCellular pH (pHi) in monolayers grown on glass cover slips utilizing the pH sensitive probe BCECF. To analyze the functional activity of the H+ transporters we observed the intraCellular alkalinization in response to an acute acid load due to a 20 mm NH+4 pulse, and calculated the initial rate of pHi recovery (dpHi/dt). The Cells have a basal pHi of 7.17 ± 0.01 (n= 23) and control dpHi/dt of 0.121 ± 0.006 (n= 23) pHi units/min. This pHi recovery rate is markedly decreased when Na+ was removed, to 0.069 ± 0.004 (n= 16). It was further reduced to 0.042 ± 0.005 (n= 12) when concanamycin 4.6 × 10−8m (a specific inhibitor of the vacuolar H+-ATPase) was added to the zero Na+ solution. When using a solution with zero Na+, low K+ (0.5 mm) plus concanamycin, pHi recovery fell again, significantly, to 0.023 ± 0.006 (n= 14) as expected in the presence of a H+–K+-ATPase. This result was confirmed by the use of 5 × 10−5m Schering 28080. The Na+ independent pHi recovery was significantly reduced from 0.069 ± 0.004 to 0.042 ± 0.004 (n= 12) when NPPB 10−5m (a specific blocker of Cl− channels in renal tubules) was utilized. When the Cells were preincubated in 0 Cl−/normal Na+ solution for 8 min. before the ammonium pulse, the pHi recovery fell from 0.069 ± 0.004 to 0.041 ± 0.007 (n= 12) in a Na+ and Cl− free solution. From these results we conclude that: (i) MDCK Cells have two Na+-independent mechanisms of pHi recovery, a concanamycin sensitive H+-ATPase and a K+ dependent, Schering 28080 sensitive H+–K+ ATPase; and, (ii) pHi recovery in Na+-free medium depends on the presence of a chloride current which can be blocked by NPPB and impaired by preincubation in Cl−–free medium. This finding supports a role for chloride in the function of the H+ ATPase, which might be electrical shunting or a biochemical interaction.
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MECHANISMS AND REGULATION OF H+ TRANSPORT IN DISTAL TUBULE EPITHELIAL CellS
Wiener Klinische Wochenschrift, 1997Co-Authors: Gerhard Malnic, R. Fernández, A.c. Cassola, Maria Luiza M. Barreto-chaves, De Souza Mo, Aires Mde MAbstract:The mechanism of acidification in the cortical distal tubule of mammalian kidney was analysed by "in vivo" microperfusion and using MDCK Cells in culture, by electropHysiological and by Cell pH microfluorescence techniques. An electrogenic effect of the vacuolar H(+)-ATPase, which has been localized to the intercalated Cells of the cortical distal tubule (connecting segment and initial collecting duct) was only observed after blocking Cl- channels by NPPB. In MDCK Cells, the recovery of Cell pH after an acid pulse in Na(+)-free medium was also depressed by NPPB, indicating that Cl- ions have an important role in the function of H+ ATPase. The regulation by hormonal agents of distal H+ transport due to Na+/H+ exchange and to vacuolar H+ ATPase, was also studied by microperfusion and Cell pH techniques. Angiotensin and vasopressin at picomolar concentrations stimulated both transport mechanisms in late distal tubule, and only Na+/H+ exchange in the early segment. In MDCK Cells, Cell pH recovery in the presence of Na+ was stimulated by picomolar concentrations of angiotensin and vasopressin, and inhibited by micromolar levels, both effects being reverted by micromolar ANP. Studies with specific antagonists suggest that the luminal effect of angiotensin is mediated by AT1 receptors, and of vasopressin by V1 receptors. There is evidence that Cell Ca2+ may have an important regulatory role in the action of these hormones.