scholarly journals Ion exchange mechanisms on the erythrocyte membrane of the aquatic salamander, Amphiuma tridactylum

1987 ◽  
Vol 133 (1) ◽  
pp. 329-338
Author(s):  
B. L. Tufts ◽  
M. Nikinmaa ◽  
J. F. Steffensen ◽  
D. J. Randall
Keyword(s):  
Steady State ◽  
Ion Exchange ◽  
Water Content ◽  
Intracellular Ph ◽  
Erythrocyte Membrane ◽  
Incubation Medium ◽  
Cell Water ◽  
Pharmacological Agents ◽  
Intracellular Alkalinization

The effects of different pharmacological agents and incubation media on the intracellular pH and water content of Amphiuma erythrocytes were investigated in vitro. Adrenaline had no significant effect on the intracellular pH or cell water content. DIDS caused an intracellular alkalinization that could be abolished by amiloride, ouabain or removal of sodium from the incubation medium. In addition, amiloride and DIDS both caused a decrease in cell water content. The data indicate that sodium/proton and chloride/bicarbonate exchangers are present on the membrane of Amphiuma erythrocytes and these exchangers are active under steady-state conditions.

scholarly journals Effects of ammonium on intracellular pH in rat medullary thick ascending limb: mechanisms of apical membrane NH4+ transport.

1994 ◽  
Vol 103 (5) ◽  
pp. 917-936 ◽  
Author(s):  
B A Watts ◽  
D W Good
Keyword(s):  
Steady State ◽  
Intracellular Ph ◽  
Apical Membrane ◽  
Ammonium Transport ◽  
Thick Ascending Limb ◽  
Transport Pathway ◽  
Critical Determinant ◽  
Medullary Thick Ascending Limb ◽  
Intracellular Alkalinization

The renal medullary thick ascending limb (MTAL) actively reabsorbs ammonium ions. To examine the effects of NH4+ transport on intracellular pH (pHi) and the mechanisms of apical membrane NH4+ transport, MTALs from rats were isolated and perfused in vitro with 25 mM HCO3(-)-buffered solutions (pH 7.4). pHi was monitored using the fluorescent dye BCECF. In the absence of NH4+, the mean pHi was 7.16. Luminal addition of 20 mM NH4+ caused a rapid intracellular acidification (dpHi/dt = 11.1 U/min) and reduced the steady state pHi to 6.67 (delta pHi = 0.5 U), indicating that apical NH4+ entry was more rapid than entry of NH3. Luminal furosemide (10(-4) M) reduced the initial rate of cell acidification by 70% and the fall in steady state pHi by 35%. The residual acidification observed with furosemide was inhibited by luminal barium (12 mM), indicating that apical NH4+ entry occurred via both furosemide (Na(+)-NH4(+)-2Cl- cotransport) and barium-sensitive pathways. The role of these pathways in NH4+ absorption was assessed under symmetric ammonium conditions. With 4 mM NH4+ in perfusate and bath, mean steady state pHi was 6.61 and net ammonium absorption was 12 pmol/min/mm. Addition of furosemide to the lumen abolished net ammonium absorption and caused pHi to increase abruptly (dpHi/dt = 0.8 U/min) to 7.0. Increasing luminal [K+] from 4 to 25 mM caused a similar, rapid cell alkalinization. The pronounced cell alkalinization observed with furosemide or increasing [K+] was not observed in the absence of NH4+. In symmetric 4 mM NH4+ solutions, addition of barium to the lumen caused a slow intracellular alkalinization and reduced net ammonium absorption only by 14%. Conclusions: (a) ammonium transport is a critical determinant of pHi in the MTAL, with NH4+ absorption markedly acidifying the cells and maneuvers that inhibit apical NH4+ uptake (furosemide or elevation of luminal [K+]) causing intracellular alkalinization; (b) most or all of transcellular ammonium absorption is mediated by apical membrane Na(+)-NH4(+)-2Cl- cotransport; (c) NH4+ also permeates a barium-sensitive apical membrane transport pathway (presumably apical membrane K+ channels) but this pathway does not contribute significantly to ammonium absorption under physiologic (symmetric ammonium) conditions.

Adaptation to hypercapnia vs. intracellular pH in cat carotid body: responses in vitro

1996 ◽  
Vol 80 (4) ◽  
pp. 1090-1099 ◽  
Author(s):  
S. Lahiri ◽  
R. Iturriaga ◽  
A. Mokashi ◽  
F. Botre ◽  
D. Chugh ◽  
...  
Keyword(s):  
Steady State ◽  
Intracellular Ph ◽  
Baseline Level ◽  
Discharge Rate ◽  
Initial Response ◽  
Extracellular Ph ◽  
Carotid Bodies ◽  
Initial Peak ◽  
Initial Change

The hypotheses that the chemosensory discharge rate parallels the intracellular pH (pHi) during hypercapnia and that the initial change in pHi (delta pHi) is always more than the stead-state delta pHi were studied by using cat carotid bodies in vitro at 36.5 degrees C in the absence and presence of methazolamide (30-100 mg/l). Incremental acidic hypercapnia was followed by an incremental initial peak response and a greater adaptation. A given acidic hypercapnia elicited a rapid initial response followed by a slower adaptation; isohydric hypercapnia produced an equally rapid initial response but of smaller magnitude that returned to near-baseline level; alkaline hypercapnia induced a similar rapid initial response but one of still smaller magnitude that decreased rapidly to below the baseline. Methazolamide eliminated the initial overshoot, which also suggested involvement of the initial rapid pHi in the overshoot. These results show that the initial delta pHi is always greater than the steady-state delta pHi and during hypercapnia. Also, the steady-state chemoreceptor activity varied linearly with the extracellular pH, indicating a linear relationship between extracellular pH and pHi.

An increase in intracellular pH during neural induction in Xenopus

Development ◽  
1994 ◽  
Vol 120 (2) ◽  
pp. 433-442 ◽  
Author(s):  
A.K. Sater ◽  
J.M. Alderton ◽  
R.A. Steinhardt
Keyword(s):  
Intracellular Ph ◽  
Marginal Zone ◽  
Phorbol Esters ◽  
Regulation Of Gene Expression ◽  
Homeobox Gene ◽  
Neural Induction ◽  
Ph Indicator ◽  
Pharmacological Agents ◽  
Dorsal Ectoderm ◽  
Intracellular Alkalinization

In this paper, we show that an intracellular alkalinization of the dorsal ectoderm cells is among the earliest responses to neural induction in Xenopus. Planar explants of the dorsal marginal zone were prepared from embryos that had been microinjected during cleavage stages with the fluorescent pH indicator bis-carboxyethyl-carboxyfluorescein-dextran (BCECF-dextran), and intracellular pH (pHi) was monitored continuously by emission ratio microfluorimetry. During stage 10.5, the dorsal ectoderm cells undergo a sustained intracellular alkalinization of approximately 0.1 pH units in response to neural induction; in the absence of the inductive signal, the pH of the dorsal ectoderm cells decreases slightly. Ectoderm cells within planar explants of the ventral marginal zone show little change in pH during a similar period. This increase in intracellular pH is inhibited by 4, 4′-dihydrodiisothiocyanatostilbene-2, 2′-disulfonate (H2DIDS) or a low Na+/high Cl- medium, treatments that presumably affect anion transport. Under these conditions, expression of the anterior neural-specific homeobox gene engrailed is not detected, while the notochord-specific epitope recognized by the Tor-70 antibody is expressed in the presence of H2DIDS. This characteristic alkalinization is not evoked by pharmacological agents that reportedly alter ectodermal developmental pathways in Xenopus embryos, such as NH4Cl, phorbol esters, or cAMP-dependent protein kinase agonists. Our results suggest that an ionic regulatory event may participate in the regulation of gene expression in response to neural induction.

Bicarbonate Therapy and Intracellular Acidosis

1997 ◽  
Vol 93 (6) ◽  
pp. 593-598 ◽  
Author(s):  
D. J. A. Goldsmith ◽  
L. G. Forni ◽  
P. J. Hilton
Keyword(s):  
Carbon Dioxide ◽  
Metabolic Acidosis ◽  
Sodium Bicarbonate ◽  
Intracellular Ph ◽  
Extracellular Fluid ◽  
Intracellular Acidification ◽  
Intracellular Acidosis ◽  
Bicarbonate Therapy ◽  
Intracellular Alkalinization

1. The correction of metabolic acidosis with sodium bicarbonate remains controversial. Experiments in vitro have suggested possible deleterious effects after alkalinization of the extracellular fluid. Disequilibrium of carbon dioxide and bicarbonate across cell membranes after alkali administration, leading to the phenomenon of ‘paradoxical’ intracellular acidosis, has been held responsible for some of these adverse effects. 2. Changes in intracellular pH in suspensions of leucocytes from healthy volunteers were monitored using a fluorescent intracellular dye. The effect in vitro of increasing extracellular pH with sodium bicarbonate was studied at different sodium bicarbonate concentrations. Lactic acid and propionic acid were added to the extracellular buffer to mimic conditions of metabolic acidosis. 3. The addition of a large bolus of sodium bicarbonate caused intracellular acidification as has been observed previously. The extent of the intracellular acidosis was dependent on several factors, being most evident at higher starting intracellular pH. When sodium bicarbonate was added as a series of small boluses the reduction in intracellular pH was small. Under conditions of initial acidosis this was rapidly followed by intracellular alkalinization. 4. Although intracellular acidification occurs after addition of sodium bicarbonate to a suspension of human leucocytes in vitro, the effect is minimal when the conditions approximate those seen in clinical practice. We suggest that the observed small and transient lowering of intracellular pH is insufficient grounds in itself to abandon the use of sodium bicarbonate in human acidosis.

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 in the OK cell. I. Identification of H+ conductance and observations on buffering capacity

1991 ◽  
Vol 261 (6) ◽  
pp. C1143-C1153 ◽  
Author(s):  
M. Graber ◽  
J. DiPaola ◽  
F. L. Hsiang ◽  
C. Barry ◽  
E. Pastoriza
Keyword(s):  
Steady State ◽  
Intracellular Ph ◽  
Large Cell ◽  
Buffering Capacity ◽  
Disulfonic Acid ◽  
Transport Pathways ◽  
Opossum Kidney ◽  
Direct Titration ◽  
Cell Ph

The regulation of intracellular pH (pHi) in the opossum kidney (OK) cell line was studied in vitro using the pH-sensitive excitation ratio of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Recovery from an NH4Cl acid load disclosed a Na-dependent component blocked by amiloride and a smaller Na-independent component. The Na-independent recovery rate was proportional to the H+ gradient from cell to buffer and was zero in the absence of an electrochemical gradient. The Na-independent recovery was not affected by N-ethylmaleimide, dicyclohexylcarbodiimide, HCO3, phloretin, or ZnCl2 but was accelerated in depolarized cells and by membrane-fluidizing drugs and was inhibited by glutaraldehyde. The apparent cellular buffering capacity changed in proportion to this H+ conductance. Consistent with an electrogenic H+ leak, steady-state cell pH alkalinized with depolarization and acidified with hyperpolarization. Removal of buffer Na+ produced a profound acidification, as did amiloride. In 0-Na+ buffers, extremely large cell-to-buffer H+ gradients were present and proportional to buffer pH. 4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid had no effect on steady-state pHi. Measurements of intracellular buffering capacity were derived from the change of cell pH induced by withdrawing NH4Cl. This buffering capacity was increased threefold in Na-free buffers, whereas the value measured by direct titration of cell lysate was the same or less than that of control cells. The NH4Cl-derived buffering capacity varied in direct proportion to the magnitude of the H+ leak. Drugs that changed H+ permeability produced the apparent changes of the measured buffering capacity within a few minutes. We conclude that, in HCO3-free buffer, the OK cell uses two membrane acid-base transport pathways: a Na-H antiporter active at physiological pH and a substantial passive H+ conductance. The results also reveal that the NH4Cl-derived buffering capacity is subject to artifacts, possibly due to a finite leak of ionic NH4+.

scholarly journals THE IN VITRO SYNTHESIS OF GLYCINE BY RUMEN PAPILLAE

1980 ◽  
Vol 60 (1) ◽  
pp. 43-51 ◽  
Author(s):  
R. J. BOILA ◽  
L. P. MILLIGAN
Keyword(s):  
Amino Acids ◽  
Ion Exchange ◽  
Chromatographic Separation ◽  
Incubation Medium ◽  
Amino Group ◽  
In Vitro Synthesis ◽  
Glycine Release ◽  
Liquid Chromatographic Separation ◽  
Liquid Chromatographic

Following aerobic in vitro incubation of papillae from the ventral sac of the bovine rumen, amino acids released into the incubation medium were isolated using a rapid ion exchange procedure and measured quantitatively by gas-liquid chromatographic separation of the isobutyl-N(0)-heptafluorobutyryl esters. Of the potential precursors of the carbon skeleton of glycine that were added as substrates, only glyoxylate and serine (0.5–2 mM) resulted in enhanced glycine release while 10 mM glycolaldehyde, glycollate, glyoxal or oxalate or 1 mM monoethanolamine or hydroxyproline did not specifically influence glycine release. Each increase in glyoxylate concentration through 5, 10 or 20 mM caused an increased glycine release. In the presence of formate, inclusion of glycine as the added substrate increased serine release. Oxalate caused a non-specific release of amino acids. In the presence of glyoxylate, alanine and glutamate (1 mM) were effective precursors of the amino group of glycine.

scholarly journals Parallel decrease of erythrocyte membrane deformability and spectrin solubility at low pH

Blood ◽  
1979 ◽  
Vol 53 (1) ◽  
pp. 15-18 ◽  
Author(s):  
BD Smith ◽  
PL La Celle
Keyword(s):  
Intracellular Ph ◽  
Erythrocyte Membrane ◽  
Low Ph ◽  
Erythrocyte Deformability ◽  
Pronounced Change ◽  
Membrane Elasticity ◽  
Ph Values ◽  
Cellular Deformability ◽  
Elastic Characteristics

Abstract Reduction of pH over the range 6.0–4.5 results in a decrease of erythrocyte deformability in parallel with the induced progressive sphericity of cells. At low deformation rates employed, increase of hemoglobin viscosity was not significant. A decrease of membrane elasticity was detected in cells when sphering, the major determinant of cellular deformability, was prevented at pH 5.0 by hyperosmotic medium. The pronounced change of deformability and the reduced elasticity occurred at calculated intracellular pH values at which solubility in vitro of extracted erythrocyte spectrin is also markedly reduced. The parallel decrease of deformability and spectrin solubility supports the hypothesis that spectrin aggregation may contribute to regulation of erythrocyte deformability through effects on shape and membrane elastic characteristics.

scholarly journals Regulation of Intracellular pH in Single Rat Cortical Neurons in vitro: A Microspectrofluorometric Study

1993 ◽  
Vol 13 (5) ◽  
pp. 827-840 ◽  
Author(s):  
Yibing Ou-Yang ◽  
Pekka Mellergård ◽  
Bo K. Siesjö
Keyword(s):  
Steady State ◽  
Intracellular Ph ◽  
Cortical Neurons ◽  
Ph Sensitive ◽  
Disulfonic Acid ◽  
Extrusion Rate ◽  
Rat Cortical Neurons ◽  
Acid Extrusion

Intracellular pH (pHi) and the mechanisms of pHi regulation in cultured rat cortical neurons were studied with microspectrofluorometry and the pH-sensitive fluorophore 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein. Steady-state pHi was 7.00 ± 0.17 (mean ± SD) and 7.09 ± 0.14 in nominally HCO3− -free and HCO3−-containing solutions, respectively, and was dependent on extracellular Na+ and Cl−. Following an acid transient, induced by an NH1 prepulse or an increase in CO2 tension, pHi decreased and then rapidly returned to baseline, with an average net acid extrusion rate of 2.6 and 2.8 mmol/L/min, in nominally HCO3− -free and HCO3− -containing solutions, respectively. The recovery was completely blocked by removal of extracellular Na+ and was partially inhibited by amiloride or 5- N-methyl- N-isobutylamiloride. In most cells pHi recovery was completely blocked in the presence of harmaline. The recovery of pHi was not influenced by addition of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) or removal of Cl−. The rapid regulation of pHi seen following a transient alkalinization was not inhibited by amiloride or by removal of extracellular Na+, but was partially inhibited by DIDS and by removal of extracellular Cl−. The results are compatible with the presence of at least two different pHi-regulating mechanisms: an acid-extruding Na+/H+ antiporter, possibly consisting of different subtypes, and a passive Cl−/HCO3− exchanger, mediating loss of HCO3− from the cell.

Effect of PCO2 on intracellular pH in in vitro frog gastric mucosa

1989 ◽  
Vol 256 (1) ◽  
pp. G206-G213 ◽  
Author(s):  
K. J. Carter ◽  
I. Saario ◽  
U. Seidler ◽  
W. Silen
Keyword(s):  
Steady State ◽  
Gastric Mucosa ◽  
Intracellular Ph ◽  
Ringer Solution ◽  
Partial Recovery ◽  
Muscularis Mucosa ◽  
Fundic Mucosa ◽  
Ethanesulfonic Acid ◽  
Oxynticopeptic Cells

Steady-state intracellular pH (pHi) in 0, 5, and 10% CO2-buffered Ringer solution in sheets of in vitro frog gastric antral or fundic mucosa has been measured using the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). In tissues perfused with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-100% O2 buffer [extracellular pH (pHo) = 7.14], steady-state pHi in antral surface cells was 7.08 +/- 0.06 (n = 8), in fundic oxynticopeptic cells 6.91 +/- 0.03 (n = 13), in the muscularis mucosa 7.58 +/- 0.06 (n = 4). In mucosae perfused with 17.8 mM HCO3- -95% O2-5% CO2 buffer (pHo = 7.14), steady-state pHi in antral surface cells was 6.97 +/- 0.02 (n = 22), in fundic oxynticopeptic cells 7.00 +/- 0.04 (n = 18), and in fundic muscularis mucosa 7.39 +/- 0.05 (n = 8). In fundic oxynticopeptic cells perfused with 35.6 mM HCO3- -90% O2-10% CO2 (pHo = 7.14) steady-state pHi was 6.77 +/- 0.07 (n = 4). In tissues equilibrated initially with 100% O2 and changed to 5% CO2, antral surface cells acidified by 0.21 pH units and fundic oxynticopeptic cells by 0.10 pH units, with restoration of pHi to resting levels within 30 and 10 min, respectively. Exposure of tissues initially equilibrated with 5% CO2 to 100% O2 alkalinized antral surface cells by 0.22 pH units and fundic oxynticopeptic cells by 0.23 pH units, with only partial recovery of pHi by 30 min. These data suggest that steady-state pHi is equivalent in surface and oxynticopeptic cells and is lower than in the muscularis mucosa.(ABSTRACT TRUNCATED AT 250 WORDS)

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