scholarly journals Adrenalin-Induced Na+/H+ Exchange in' Trout Erythrocytes and its Effects Upon Oxygen-Carrying Capacity

1985 ◽  
Vol 118 (1) ◽  
pp. 229-246 ◽  
Author(s):  
A. R. COSSINS ◽  
P. A. RICHARDSON
Keyword(s):  
Steady State ◽  
Intracellular Ph ◽  
Carrying Capacity ◽  
Extracellular Ph ◽  
Extracellular Medium ◽  
Root Effect ◽  
Fish Blood ◽  
Wide Range ◽  
Oxygen Carrying Capacity ◽  
Normal Relationship

Addition of adrenalin (10−4moll−1) to trout erythrocytes in an unbuffered saline resulted in a rapid acidification of the extracellular medium. This process was inhibited by amiloride (K½10−4moll−1) and by the removal of extracellular Na+. The rate of acidification was a saturable function of extracellular Na+ concentration. When extracellular pH was maintained constant by continual titration with KOH, adrenalin induced a transient burst of H+ efflux. During this period the loss of cellular H+ equivalents was approximately equal to the net gain of Na+, providing evidence for a Na+/H+ exchange with a stoichiometry of 1. The steady state following stimulation with adrenalin could be disturbed by changes in extracellular pH. After the addition of adrenalin, intracellular pH (pHi) was increased by 0.2-0.3 units but did not exceed extracellular pH, as required if the Na+ and H+ concentration ratios came into equilibrium. The increase in pHi in stimulated compared with control cells was maintained approximately constant over a wide range of pHo, suggesting that pH equilibration by the Jacob-Stewart cycle was operating normally and that the activation of Na+/H+ exchange provides an offset to the normal relationship between pHi and pHo. The steady state results from a balance of an increase Na+/H+ and CI−/HCO3− exchange with an increased rate of Na+ pumping and next KCl efflux. In a buffered saline, adrenalin caused a 22–46% increase in the oxygen-carrying capacity of trout erythrocytes. It is suggested that this was due to a Root effect of trout haemoglobin caused by cellular alkalinization when the Na+/H+ exchange mechanism was activiated. This observation suggests that many published values for oxygencarrying capacity of fish blood require re-evaluation.

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.

scholarly journals Hydrogen Sulfide Is a Regulator of Hemoglobin Oxygen-Carrying Capacity via Controlling 2,3-BPG Production in Erythrocytes

2021 ◽  
Vol 2021 ◽  
pp. 1-16
Author(s):  
Gang Wang ◽  
Yan Huang ◽  
Ningning Zhang ◽  
Wenhu Liu ◽  
Changnan Wang ◽  
...  
Keyword(s):  
Hydrogen Sulfide ◽  
Binding Affinity ◽  
Carrying Capacity ◽  
Inhibitory Effect ◽  
Peripheral Tissues ◽  
Metabolomic Profiling ◽  
Wide Range ◽  
Mammalian Tissues ◽  
Oxygen Carrying Capacity ◽  
O2 Binding

Hydrogen sulfide (H2S) is naturally synthesized in a wide range of mammalian tissues. Whether H2S is involved in the regulation of erythrocyte functions remains unknown. Using mice with a genetic deficiency in a H2S natural synthesis enzyme cystathionine-γ-lyase (CSE) and high-throughput metabolomic profiling, we found that levels of erythrocyte 2,3-bisphosphoglycerate (2,3-BPG), an erythroid-specific metabolite negatively regulating hemoglobin- (Hb-) oxygen (O2) binding affinity, were increased in CSE knockout (Cse-/-) mice under normoxia. Consistently, the 50% oxygen saturation (P50) value was increased in erythrocytes of Cse-/- mice. These effects were reversed by treatment with H2S donor GYY4137. In the models of cultured mouse and human erythrocytes, we found that H2S directly acts on erythrocytes to decrease 2,3-BPG production, thereby enhancing Hb-O2 binding affinity. Mouse genetic studies showed that H2S produced by peripheral tissues has a tonic inhibitory effect on 2,3-BPG production and consequently maintains Hb-O2 binding affinity in erythrocytes. We further revealed that H2S promotes Hb release from the membrane to the cytosol and consequently enhances bisphosphoglycerate mutase (BPGM) anchoring to the membrane. These processes might be associated with S-sulfhydration of Hb. Moreover, hypoxia decreased the circulatory H2S level and increased the erythrocyte 2,3-BPG content in mice, which could be reversed by GYY4137 treatment. Altogether, our study revealed a novel signaling pathway that regulates oxygen-carrying capacity in erythrocytes and highlights a previously unrecognized role of H2S in erythrocyte 2,3-BPG production.

Modulation of agonist-activated calcium influx by extracellular pH in rat pancreatic acini

1989 ◽  
Vol 257 (6) ◽  
pp. G917-G924 ◽  
Author(s):  
S. Muallem ◽  
S. J. Pandol ◽  
T. G. Beeker
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Intracellular Ph ◽  
Calcium Influx ◽  
Extracellular Ph ◽  
Transport Pathways ◽  
Pancreatic Acini ◽  
Hormone Stimulation ◽  
Entry Pathway ◽  
Agonist Stimulation

The biochemical and Ca2+ transport pathways involved in generating the hormone-evoked Ca2+ signal are reported to be influenced by pH. The present study was designed to determine the effect of extracellular pH (pHo) and intracellular pH (pHi) on hormone-stimulated Ca2+ transport. We used rat pancreatic acini and measured free cytosolic Ca2+ concentration ([Ca2+]i) with fura-2, pHi with 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and Ca2+ fluxes with 45Ca2+. In the presence of external Ca2+, increasing pHo increased steady-state [Ca2+]i during sustained agonist stimulation; in the absence of external Ca2+, this increase in [Ca2+]i did not occur. The addition of an antagonist or blocking plasma membrane Ca2+ influx with La3+ in stimulated cells suspended at pHo 8.2 resulted in a reduction in [Ca2+]i. Increasing pHo increased the rate and extent of 45Ca2+ uptake into stimulated cells and the rate and extent of Ca2+ reloading of intracellular stores. The increased Ca2+ content of the intracellular stores with increased pHo indicated that at physiological pHo and pHi the agonist-mobilizable internal stores are not saturated with Ca2+. Changes in pHo affected pHi. However, changes in pHi at constant pHo had no effect on hormone-evoked [Ca2+]i increase, reduction in [Ca2+]i after hormone stimulation, or reloading of intracellular stores. We conclude that the hormone-activated plasma membrane Ca2+ entry pathway responsible for Ca2+ reloading is directly modulated by external H+.

Coupled Na+-H+ exchange in isolated acinar cells from rat exocrine pancreas

1985 ◽  
Vol 249 (1) ◽  
pp. G125-G136 ◽  
Author(s):  
W. Hellmessen ◽  
A. L. Christian ◽  
H. Fasold ◽  
I. Schulz
Keyword(s):  
Intracellular Ph ◽  
Fluorescence Intensity ◽  
Exocrine Pancreas ◽  
Acinar Cells ◽  
Extracellular Ph ◽  
Pancreatic Acinar Cells ◽  
Intracellular Acidification ◽  
Extracellular Medium ◽  
Rat Exocrine Pancreas ◽  
Intracellular Fluorescence

Isolated acinar cells from the rat exocrine pancreas were loaded with 6-carboxyfluorescein diacetate (CFDA), and the intracellular pH (pHi) was estimated from the pH-dependent fluorescence intensity of trapped 6-carboxyfluorescein liberated from CFDA by intracellular esterases. The intracellular fluorescence intensity was calibrated by equilibrating the internal and external pH with nigericin in K+ buffers. In the absence of Na+ (130 mmol/l K+) a pHi of 6.86 +/- 0.04 was found; in its presence (130 mmol/l Na+) a pHi of 7.17. Acute addition of Na+ increased intracellular pH with increasing Na+ concentrations, reaching a maximum at 150 mmol/l with an apparent Km of approximately 40 mmol/l. Of the different cations tested on pHi, such as Li+, K+, Rb+, and Cs+, only Li+ showed an effect on pHi similar to that of Na+. Amiloride dose dependently inhibited both Na+- and Li+-induced alkalinization (apparent Km approximately 10(-5) mol/l). In the presence of ouabain pHi was decreased by 0.2 pH units. Intracellular acidification induced by permeable buffers such as acetic acid-acetate or CO2-HCO3- was dissipated more rapidly in the presence of Na+ compared with K+ or with Na+ and amiloride in the medium. In Li+-preincubated cells intracellular acidification was higher in the absence of Li+ in the extracellular medium than in its presence. This Li+ gradient-induced acidification was dependent on the extracellular pH, was highest at an extracellular pH of 7.05, and decreased with increasing pH to 7.5. The results allow the conclusion that a coupled Na+-H+ exchange is present in pancreatic acinar cells and that the intracellular pH rather than the extracellular Na+ concentration regulates this transport mechanism.

scholarly journals A pyruvate–proton symport and an H+-ATPase regulate the intracellular pH of Trypanosoma brucei at different stages of its life cycle

2000 ◽  
Vol 346 (1) ◽  
pp. 53-62 ◽  
Author(s):  
Nicole VANDERHEYDEN ◽  
Julian WONG ◽  
Roberto DOCAMPO
Keyword(s):  
Steady State ◽  
Trypanosoma Brucei ◽  
Intracellular Ph ◽  
Fluorescent Dyes ◽  
Minor Role ◽  
Proton Efflux ◽  
Acetoxymethyl Ester ◽  
Wide Range ◽  
Atpase Inhibitors ◽  
Recovery From Acidification

Regulation of intracellular pH (pHi) and H+ efflux were investigated in Trypanosoma brucei bloodstream and procyclic trypomastigotes using the fluorescent dyes 2ʹ,7ʹ-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) acetoxymethyl ester and free BCECF respectively. pHi in bloodstream and procyclic trypomastigotes was 7.47±0.06 and 7.53±0.07 respectively. Differences in the mechanisms for the regulation of pHi were noted between bloodstream and procyclic forms. Procyclic trypomastigotes maintained their pHi at neutral over a wide range of external pH values from 6 to 8, and in the absence of K+ or Na+. The H+-ATPase inhibitors N,Nʹ-dicyclohexylcarbodi-imide (DCCD), diethylstilboestrol and N-ethylmaleimide substantially decreased the steady-state pHi and inhibited its recovery from acidification. The rate of H+ efflux in these forms was determined to be 62±6.5 nmol/min per mg of protein, and was substantially decreased by H+-ATPase inhibitors. The data support the presence of an H+-ATPase as the major regulator of pHi in procyclic trypomastigotes. In contrast, bloodstream trypomastigotes were unable to maintain a neutral pH under acidic conditions, and their steady-state pHi and recovery from acidification were unaffected by H+-ATPase inhibitors, except for DCCD (100 μM). Their steady-state pHi was markedly decreased in glucose-free buffer or by ≥ 10 mM pyruvate, whereas procyclic trypomastigotes were unaffected by similar treatments. The rate of H+ efflux in bloodstream trypomastigotes was 534±38 nmol/min per mg of protein, and was decreased in the absence of glucose and by the addition of pyruvate or DCCD. Pyruvate efflux in these forms was calculated to be 499±34 nmol/min per mg of protein, and was significantly inhibited by DCCD, 4,4ʹ-di-isothiocyanatodihydrostilbene-2,2ʹ-disulphonic acid and α-cyanohydroxycinnamic acid. The pyruvate analogues β-hydroxypyruvate, 3-bromopyruvate, 3-oxoglutarate, oxaloacetate, 3-oxoisovalerate and 3-oxoisohexanoate significantly decreased pHi, as well as proton and pyruvate efflux, whereas lactate had only a small effect, and no effect was observed with citrate or fumarate. The inhibition by pyruvate analogues of pyruvate efflux, proton efflux and acidification of pHi supports the hypothesis that pyruvate efflux is accompanied by proton efflux and that this is the major pHi control mechanism in bloodstream forms. Inhibition by H+-ATPase inhibitors of residual H+ efflux in the absence of glucose or in the presence of high extracellular pyruvate indicates a minor role for H+-ATPase(s) in control of pHi in bloodstream forms.

Determinants of intracellular pH in gas gland cells of the swimbladder of the European eel Anguilla anguilla

2002 ◽  
Vol 205 (8) ◽  
pp. 1069-1075 ◽  
Author(s):  
E. Sötz ◽  
H. Niederstätter ◽  
B. Pelster
Keyword(s):  
Steady State ◽  
Anion Exchange ◽  
Intracellular Ph ◽  
Specific Inhibitor ◽  
Anguilla Anguilla ◽  
Extracellular Ph ◽  
European Eel ◽  
Pulse Technique ◽  
Gland Cells ◽  
Gas Gland

SUMMARY Gas gland cells of the European eel (Anguilla anguilla) were cultured on collagen-coated coverslips, and intracellular pH was measured using the pH-sensitive fluorescent probe 2′,7′-bis-(2-carboxypropyl)-5-(6)-carboxyfluorescein (BCPCF). The contributions of various proton-translocating mechanisms to homeostasis of intracellular pH (pHi) were assessed by adding specific inhibitors of the various proton-translocating mechanisms at a constant extracellular pH (pHe)of 7.4 and after artificial acidification of the cells using the ammonium pulse technique. The greatest decrease in pHi was observed after addition of 5-(N-ethyl-N-isobutyl)-amiloride (MIA), an inhibitor of Na+/H+ exchange. Na+/H+ exchange was active under steady-state conditions at an extracellular pH of 7.4, and activity increased after intracellular acidification. Incubation of gas gland cells with 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid(DIDS), an inhibitor of anion exchange, also caused a decrease in pHi, but this decrease was not as pronounced as in the presence of MIA. Furthermore, at low pHi, the effect of DIDS was further reduced, suggesting that bicarbonate-exchanging mechanisms are involved in maintaining a steady-state pHi but that their importance is reduced at low pH. Bafilomycin A1,a specific inhibitor of the V-ATPase, had no effect on steady-state pHi. However, recovery of intracellular pH after an artificial acid load was significantly impaired in the presence of bafilomycin. Our results suggest that Na+/H+ exchange and anion exchange are important for the regulation of pHi at alkaline values of pHe. When pHi is low, a situation probably often encountered by gas gland cells during gas secretion,Na+/H+ exchange continues to play an important role in acid secretion and a V-ATPase appears to contribute to proton secretion.

Simulation of C1 fluxes in cyanobacteria: comparison between the carboxysome hypothesis and the equilibrium proposal

1997 ◽  
Vol 75 (12) ◽  
pp. 2117-2130 ◽  
Author(s):  
Christophe Salon ◽  
David Thomas Canvin
Keyword(s):  
Mathematical Model ◽  
Steady State ◽  
Kinetic Constants ◽  
Experimental Conditions ◽  
Extracellular Medium ◽  
Blue Green Algae ◽  
Permeability Coefficients ◽  
Inorganic Species ◽  
Wide Range ◽  
Entire Cell

Inorganic carbon fluxes were simulated by a mathematical model using an equilibrium hypothesis for a wide range of conditions in a closed system composed of air-grown cells of Synechococcus UTEX 625 in a reaction vessel connected to a mass spectrometer. The metabolic scheme took into account the input fluxes of CO2 and HCO3− transport into the cells, the output fluxes of CO2 and HCO3− efflux, the diversion of Q toward the formation of the internal C2 pool, and photosynthetic CO2 fixation. The equations expressed the variation in concentration of each inorganic species outside and inside the cell as a function of time. The input fluxes were previously characterized by their kinetic constants (K1/2 and Vm) both during initial uptake occurring upon illumination of the cells and under steady-state photosynthesis conditions. The efflux rates of the various Ci species from the cells were investigated under a wide variety of experimental conditions. Using these efflux rates, the permeability coefficients of the cell for CO2 and HCO3− were calculated previously. Using the kinetic constants for CO2 and HCO3− transport, the permeability coefficients of the cell for CO2 and HCO3− and the geometrical characteristics of the cells, the model simulated precisely the [HCO3−]/[CO2] ratio and the [CO2] and [O2] changes in the extracellular medium as well as the rate of filling of the internal Ci pool under various conditions. Accurate fitting of experimental data with calculated values were possible only when the intracellular Ci species were assumed to be in equilibrium throughout the entire cell volume. Results are discussed and compared with those given by previous hypotheses. Key words: Synechococcus UTEX 625, blue green algae, cyanobacteria, mathematical model, active CO2 transport, active HCO3− transport, steady state, photosynthesis, Ci concentrating mechanism.

The Generation of Hyperbaric Oxygen Tensions in Fish

Physiology ◽  
2001 ◽  
Vol 16 (6) ◽  
pp. 287-291 ◽  
Author(s):  
Bernd Pelster
Keyword(s):  
Hyperbaric Oxygen ◽  
Carrying Capacity ◽  
Oxygen Supply ◽  
Low Ph ◽  
Swim Bladder ◽  
Root Effect ◽  
Metabolic Adaptations ◽  
Oxygen Tensions ◽  
Gas Gland ◽  
Oxygen Carrying Capacity

Surprising inventiveness in the molecular interactions in fish hemoglobins that express the Root effect (decreased oxygen-carrying capacity at low pH) and in metabolic adaptations in swim bladder gas gland cells and retinal tissues causes local acidification of blood and generates hyperbaric oxygen tensions that drive oxygen into the swim bladder (regulating buoyancy) and ensures the oxygen supply to the avascularized retinae.

The functional significance of adrenergic pH regulation in fish erythrocytes

1989 ◽  
Vol 67 (1) ◽  
pp. 235-238 ◽  
Author(s):  
Bruce L. Tufts ◽  
D. J. Randall
Keyword(s):  
Carrying Capacity ◽  
Functional Significance ◽  
Oncorhynchus Tshawytscha ◽  
Ph Regulation ◽  
Ph Effect ◽  
Blood Oxygen ◽  
Adrenergic Stimulation ◽  
Root Effect ◽  
Oxygen Carrying Capacity

Experiments were conducted in vitro to determine the presence or absence of an adrenergic pH effect in the erythrocytes of Squalus suckleyi, Raja binoculata, and Oncorhynchus tshawytscha. Adrenergic stimulation significantly increased the erythrocyte pH of Oncorhynchus tshawytscha, but did not significantly affect the erythrocyte pH of Squalus suckleyi or Raja binoculata. The results support the view that the functional significance of adrenergic pH regulation in fish erythrocytes is to offset the reduction in blood oxygen carrying capacity caused by the Root effect.

Spectroscopic Studies of Asparaginyl-tRNA Synthetase from Entamoeba histolytica

2019 ◽  
Vol 26 (6) ◽  
pp. 435-448
Author(s):  
Priyanka Biswas ◽  
Dillip K. Sahu ◽  
Kalyanasis Sahu ◽  
Rajat Banerjee
Keyword(s):  
Circular Dichroism ◽  
Steady State ◽  
Entamoeba Histolytica ◽  
Protein Concentration ◽  
Trna Synthetase ◽  
Solvent Exposure ◽  
Steady State Fluorescence ◽  
State Process ◽  
Wide Range ◽  
Circular Dichroïsm

Background: Aminoacyl-tRNA synthetases play an important role in catalyzing the first step in protein synthesis by attaching the appropriate amino acid to its cognate tRNA which then transported to the growing polypeptide chain. Asparaginyl-tRNA Synthetase (AsnRS) from Brugia malayi, Leishmania major, Thermus thermophilus, Trypanosoma brucei have been shown to play an important role in survival and pathogenesis. Entamoeba histolytica (Ehis) is an anaerobic eukaryotic pathogen that infects the large intestines of humans. It is a major cause of dysentery and has the potential to cause life-threatening abscesses in the liver and other organs making it the second leading cause of parasitic death after malaria. Ehis-AsnRS has not been studied in detail, except the crystal structure determined at 3 Å resolution showing that it is primarily α-helical and dimeric. It is a homodimer, with each 52 kDa monomer consisting of 451 amino acids. It has a relatively short N-terminal as compared to its human and yeast counterparts. Objective: Our study focusses to understand certain structural characteristics of Ehis-AsnRS using biophysical tools to decipher the thermodynamics of unfolding and its binding properties. Methods: Ehis-AsnRS was cloned and expressed in E. coli BL21DE3 cells. Protein purification was performed using Ni-NTA affinity chromatography, following which the protein was used for biophysical studies. Various techniques such as steady-state fluorescence, quenching, circular dichroism, differential scanning fluorimetry, isothermal calorimetry and fluorescence lifetime studies were employed for the conformational characterization of Ehis-AsnRS. Protein concentration for far-UV and near-UV circular dichroism experiments was 8 µM and 20 µM respectively, while 4 µM protein was used for the rest of the experiments. Results: The present study revealed that Ehis-AsnRS undergoes unfolding when subjected to increasing concentration of GdnHCl and the process is reversible. With increasing temperature, it retains its structural compactness up to 45ºC before it unfolds. Steady-state fluorescence, circular dichroism and hydrophobic dye binding experiments cumulatively suggest that Ehis-AsnRS undergoes a two-state transition during unfolding. Shifting of the transition mid-point with increasing protein concentration further illustrate that dissociation and unfolding processes are coupled indicating the absence of any detectable folded monomer. Conclusion: This article indicates that GdnHCl induced denaturation of Ehis-AsnRS is a two – state process and does not involve any intermediate; unfolding occurs directly from native dimer to unfolded monomer. The solvent exposure of the tryptophan residues is biphasic, indicating selective quenching. Ehis-AsnRS also exhibits a structural as well as functional stability over a wide range of pH.

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