Metabolic Acidosis is a Potent Stimulus for Cellular Inorganic Phosphate Generation in Uraemia

1995 ◽  
Vol 88 (4) ◽  
pp. 405-412 ◽  
Author(s):  
Alan Bevington ◽  
Dennis Brough ◽  
Frease E. Baker ◽  
Jane Hattersley ◽  
John Walls
Keyword(s):  
Steady State ◽  
Metabolic Acidosis ◽  
Inorganic Phosphate ◽  
Lactate Production ◽  
Extracellular Fluid ◽  
Low Ph ◽  
Organic Phosphate ◽  
Glycolytic Flux ◽  
Before And After

1. During metabolic acidosis, significant fluxes of inorganic phosphate (Pi) may occur from cellular to extracellular fluid. In this study Pi was measured in erythrocytes of uraemic patients before and after haemodialysis and was related to their plasma pH (acidosis), plasma Pi (hyperphosphataemia) and cellular organic phosphate concentrations. 2. Before dialysis, the ratio of cellular to extracellular Pi concentration correlated inversely with plasma pH, increasing 2.5-fold as pH fell from 7.4 to 7.2. 3. An increase in cellular Pi similar to that seen in the patients was observed within 90 min of adding acid to normal erythrocytes in vitro. 4. The total Pi content of the cell suspension increased 25% on decreasing plasma pH from 7.4 to 7.2, largely as a result of generation of Pi from 2,3-bisphosphoglycerate in the cells. This was accompanied by net efflux of Pi into plasma. 5. In addition, the increase in the steady-state cellular Pi concentration on adding a constant extracellular Pi load was 50% greater at pH 7.2 than at 7.4, implying that alterations in the regulation of the transmembrane Pi gradient also contribute to the rise in cellular Pi observed at low pH. 6. At normal plasma Pi concentration (1 mM), glycolytic flux (lactate production) was inhibited by 20% when pH was lowered from 7.4 to 7.2. However, this inhibition was blocked when cellular Pi was increased by adding Pi to the plasma in vitro. 7. Metabolic acidosis is therefore a potent stimulus for Pi generation in erythrocytes, and this Pi may serve to stimulate glycolysis which is normally inhibited by low pH.

Adaptation of the outer medullary collecting duct to metabolic acidosis in vitro

1998 ◽  
Vol 275 (6) ◽  
pp. F982-F990 ◽  
Author(s):  
Shuichi Tsuruoka ◽  
George J. Schwartz
Keyword(s):  
Protein Synthesis ◽  
Metabolic Acidosis ◽  
Collecting Duct ◽  
Low Ph ◽  
Acetoxymethyl Ester ◽  
Calmodulin Antagonist ◽  
Cell Calcium ◽  
Signal Transduction Inhibitors

Metabolic acidosis in vivo, as well as in vitro (1 h at pH 6.8 followed by 2 h at pH 7.4) stimulates H+-ATPase-dependent H+ secretion in outer medullary collecting ducts from the inner stripe (OMCDi) (S. Tsuruoka and G. J. Schwartz. J. Clin. Invest. 99: 1420–1431, 1997). Another group has shown that the adaptation to metabolic acidosis in vivo is mediated by an apical polarization of H+ pumps without an increase in total H+pump mRNA or protein (B. Bastani, H. Purcell, P. Hemken, D. Trigg, and S. Gluck. J. Clin. Invest. 88: 126–136, 1991). To further address the mechanism of adaptation, we measured net [Formula: see text] absorption before and after applying protein/RNA synthesis and signal transduction inhibitors during the 1 h of low pH and a cytoskeletal inhibitor during the entire 3-h incubation. Net [Formula: see text]transport, measured by microcalorimetry, increased ∼33% after in vitro acidosis. This increase was prevented by application during the first hour of anisomycin (10 μM) or actinomycin D (4 μM), but not by anisomycin applied during the 2-h incubation at pH 7.4. Similar results were obtained with the cell calcium chelator, 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM, 20 μM), the calmodulin antagonist, calmidazolium (30 nM), the endoplasmic reticulum Ca-ATPase inhibitor, thapsigargin (100 nM), and the protein kinase C (PKC) inhibitor, staurosporine (100 nM), applied during the 1 h at pH 6.8, but not with BAPTA-AM or thapsigargin used during the 2-h incubation at pH 7.4. Colchicine (10 μM) applied during the entire 3-h incubation also prevented this adaptive increase in H+ secretion, whereas lumicolchicine (10 μM, the inactive congener) did not. Colchicine also reversibly prevented any adaptive increases in transepithelial positive voltage. Thus the adaptation to acidosis in vitro required RNA and protein synthesis, changes in intracellular calcium and PKC activity, and intact microtubules. Time was required for the adaptation to occur, as the increase in [Formula: see text]transport was small after <3-h incubation. Protein synthesis and changes in cell calcium were critical during the initial period of low pH but not once the acid stimulus had been removed. Exocytosis of H+ pumps appears to occur continually during the entire 3-h incubation. These data would suggest that the synthesis and regulation of proteins involved in shuttling H+ pumps in cytoplasmic vesicles to the apical membrane via exocytosis are important for the OMCDi to adapt to low pH in vitro and probably to metabolic acidosis in vivo.

THE EFFECTS OF LOW PH ON CORNEAL LACTATE PRODUCTION IN VITRO

1995 ◽  
Vol 72 (SUPPLEMENT) ◽  
pp. 229
Author(s):  
Susan Lin ◽  
Arlene Sarroca ◽  
Joseph A. Bonanno
Keyword(s):  
Lactate Production ◽  
Low Ph

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.

Response of renal NH3 production to chronic respiratory acidosis

1984 ◽  
Vol 247 (6) ◽  
pp. F896-F903 ◽  
Author(s):  
F. Rodriguez-Nichols ◽  
E. Laughrey ◽  
R. L. Tannen
Keyword(s):  
Metabolic Acidosis ◽  
Respiratory Acidosis ◽  
Low Ph ◽  
Chronic Metabolic Acidosis ◽  
Conscious Rats ◽  
Ambient Co2 ◽  
Normal Controls ◽  
Isolated Kidneys

Although chronic metabolic acidosis results in an adaptive increase in the renal capacity to produce NH3, the response to a low pH produced by chronic respiratory acidosis is unknown. Rats were placed in a specially constructed chamber with an ambient CO2 of 10% for 3 days, which increased their PCO2 to 76 +/- 4 mmHg. NH3 production was determined in vitro using both isolated kidneys perfused with 0.5 mM glutamine and cortical tubules incubated with 1 mM glutamine. Conscious rats with chronic respiratory and chronic metabolic acidosis had similar arterial pHs (7.29 +/- 0.01 and 7.31 +/- 0.01), which were significantly lower than controls (7.41 +/- 0.04). NH3 production by kidneys from rats with chronic respiratory acidosis perfused at pH 7.4 did not differ significantly from normal controls (1.13 +/- 0.13 vs. 1.07 +/- 0.17 mumol X min-1 X g-1). By contrast, kidneys from rats with chronic metabolic acidosis produced significantly more NH3 than both these groups (2.73 +/- 0.29 mumol X min-1 X g-1). Cortical tubules from rats with chronic respiratory acidosis also showed no evidence of adaptation in both NH3 (8.8 +/- 0.8 vs. 11.6 +/- 0.8 mumol X min-1 X g-1) and glucose (1.38 +/- 0.08 vs. 1.41 +/- 0.13 mumol X min-1 X g-1) production in comparison with controls, whereas chronic metabolic acidosis stimulated both ammoniagenesis and gluconeogenesis twofold or more. Thus a low systemic pH does not account for the adaptation in the capacity of the kidney to produce either ammonia or glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals The In Vitro Restoration of Red Cell 2,3-Diphosphoglycerate Levels in Banked Blood

Blood ◽  
1971 ◽  
Vol 37 (1) ◽  
pp. 52-58 ◽  
Author(s):  
FRANK A. OSKI ◽  
SUSAN F. TRAVIS ◽  
LEONARD D. MILLER ◽  
MARIA DELIVORIA-PAPADOPOULOS ◽  
ELIZABETH CANNON
Keyword(s):  
Oxygen Tension ◽  
Inorganic Phosphate ◽  
Final Concentration ◽  
Organic Phosphate ◽  
Red Cell ◽  
Fresh Blood ◽  
Stored Blood ◽  
2,3 Dpg

Abstract The demonstration that red cell levels of 2,3-DPG play a central role in determining the affinity of hemoglobin for oxygen has resulted in a renewed interest in methods for maintaining or restoring the level of this organic phosphate in stored blood. The effects of addition of inorganic phosphate, inosine and pyruvate, individually or in various combinations, all in a final concentration of 10 mM were evaluated 1 and 4 hours after supplementation of ACD-stored blood, 21 to 28 days old. In 14 studies the initial 2,3-DPG level averaged 176 mµmoles/ ml. RBC. In normal fresh blood the 2,3-DPG was 4200 ± 400 mµmoles/ml. RBC. Inosine addition raised the 2,3-DPG to 1395, inosine and phosphate to 1528, inosine and pyruvate to 3363, while the combination of inosine, pyruvate and phosphate increased the level to 6637 mµmoles/ml. RBC. After 2-3 hours of incubation most of the 2,3-DPG restoration had occured. The maximum effects did not require prior pH correction of the blood. Although inosine in a final concentration of 10 mM was required for optimum effects, the phosphate and pyruvate concentrations could be reduced to 4 mM. In the presence of inosine and phosphate alone the red cell accumulated large quantities of triose phosphates, fructose diphosphate and glucose-6-phosphate. These levels were reduced by the addition of pyruvate. Pyruvate addition appears necessary to provide sufficient NAD for maximum 2,3-DPG synthesis. Associated with 2,3-DPG restoration of the stored blood there was a rise in the P50 (the oxygen tension at which hemoglobin is 50% saturated) from a mean of 16.7 to 31.6 mm. Hg.

Exogenous protein affects developmental competence and metabolic activity of bovine pre-implantation embryos in vitro

1998 ◽  
Vol 10 (4) ◽  
pp. 327 ◽  
Author(s):  
J. Eckert ◽  
P. A. Pugh ◽  
J. G. Thompson ◽  
H. Niemann ◽  
H. R. Tervit
Keyword(s):  
Metabolic Activity ◽  
Production Systems ◽  
Lactate Production ◽  
Developmental Competence ◽  
Morphological Development ◽  
Bovine Embryo ◽  
In Vitro Production ◽  
Exogenous Protein ◽  
Before And After

The role of exogenous protein during bovine pre-implantation embryo development in two in vitro production systems was investigated. Morphological development, survival after vitrification and metabolic activity before and after vitrification were recorded in blastocysts generated in vitro in synthetic oviduct fluid (SOF) medium in the presence of either bovine serum albumin (BSA) or polyvinyl-alcohol (PVA). Metabolic activity was determined by measuring oxygen consumption, glucose and pyruvate uptake as well as lactate production. Development to blastocysts and survival after vitrification were reduced significantly in medium lacking protein. Of the metabolic parameters measured, only pyruvate uptake was increased significantly in embryos cultured in medium supplemented with PVA. Whereas in BSA-supplemented medium pyruvate uptake was correlated with lactate production, in PVA-supplemented medium glucose uptake was correlated with lactate production. Lactate production increased significantly after vitrification as compared with fresh embryos. Thus, exogenously added protein significantly alters oxidative metabolism. In medium lacking protein, the additional pyruvate may be used for the maintenance of intracellular amino acid pools. Vitrification appears to alter glycolytic metabolic profiles indicating a stress-response. In conclusion, the perturbed metabolism corresponding to reduced developmental capacity of embryos produced under protein-free conditions emphasizes the ambiguity between maximum develop-ment, technical and hygienic requirements and physiological demands of the early bovine embryo in vitro. The use of well-defined recombinant proteins might assist in closing this gap.

scholarly journals Pulmonary artery smooth muscle cell hyperproliferation and metabolic shift triggered by pulmonary overcirculation

2016 ◽  
Vol 311 (4) ◽  
pp. H944-H957 ◽  
Author(s):  
Jason Boehme ◽  
Xutong Sun ◽  
Kathryn V. Tormos ◽  
Wenhui Gong ◽  
Manuela Kellner ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Smooth Muscle Cell ◽  
Muscle Cell ◽  
Lactate Production ◽  
Pentose Phosphate ◽  
Metabolic Reprogramming ◽  
Glycolytic Flux ◽  
Pulmonary Artery Smooth Muscle

Vascular cell hyperproliferation and metabolic reprogramming contribute to the pathophysiology of pulmonary arterial hypertension (PAH). An important cause of PAH in children with congenital heart disease (CHD) is increased pulmonary blood flow (PBF). To better characterize this disease course we studied early changes in pulmonary artery smooth muscle cell (PASMC) proliferation and metabolism using a unique ovine model of pulmonary overcirculation. Consistent with PAH in adults, PASMCs derived from 4-wk-old lambs exposed to increased PBF (shunt) exhibited increased rates of proliferation. While shunt PASMCs also exhibited significant decreases in mitochondrial oxygen consumption, membrane potential, and tricarboxylic acid (TCA) cycle function, suggesting a switch to Warburg metabolism as observed in advanced PAH in adults, they unexpectedly demonstrated decreased glycolytic lactate production, likely due to enhanced flux through the pentose phosphate pathway (PPP). This may be a response to the marked increase in NADPH oxidase (Nox) activity and decreased NADPH/NADP+ ratios observed in shunt PASMCs. Consistent with these findings, pharmacological inhibition of Nox activity preferentially slowed the growth of shunt PASMCs in vitro. Our results therefore indicate that PASMC hyperproliferation is observed early in the setting of pulmonary overcirculation and is accompanied by a unique metabolic profile that is independent of HIF-1α, PDHK1, or increased glycolytic flux. Our results also suggest that Nox inhibition may help prevent pulmonary overcirculation-induced PAH in children born with CHD.

Ex vivo zidovudine (AZT) treatment of CD34+ bone marrow progenitors causes decreased steady state mitochondrial DNA (mtDNA) and increased lactate production

2004 ◽  
Vol 23 (4) ◽  
pp. 173-185 ◽  
Author(s):  
L D Lewis ◽  
S Amin ◽  
C I Civin ◽  
P S Lietman
Keyword(s):  
Bone Marrow ◽  
Mitochondrial Dna ◽  
Steady State ◽  
Nuclear Dna ◽  
Ex Vivo ◽  
Lactate Production ◽  
Nuclear Dna Content ◽  
Content Ratio ◽  
The Mean

Haematopoietic suppression is one of the dose-limiting side effects of chronic zidovudine (AZT) therapy. We tested the hypothesis that AZT would reduce mitochondrial DNA (mtDNA) content in haematopoietic progenitors causing impaired haematopoiesis and mitochondrial dysfunction. We studied the effects of AZT 0 / 50 M in vitro, on normal human CD34 / haematopoietic progenitor cells cultured ex vivo for up to 12 days. The mean AZT IC50 for granulocyte (phenotype CD15 / /CD14 /) and erythroid (phenotype glycophorin / /CD45 /) cell proliferation was 2.5 M (SD9 / 0.7) and 0.023 M (SD9 / 0.005), respectively. In myeloid-rich cell cultures, the mean lactate content of the media, compared to untreated controls, increased by 86% (SD9 / 23) at 10 M AZT and in erythroid-rich cultures it increased by 134% (SD9 / 24) in the presence of 0.5 M AZT. In myeloid-rich cultures the AZT IC50 for the reduction in the mitochondrial/nuclear DNA content ratio was 5.6 M, whereas in erythroid rich cultures this AZT IC50 was B / 0.0005 M. AZT produced concentration-dependent inhibition of CD34 / progenitor proliferation into both myeloid and erythroid lineages; erythropoiesis was more sensitive than myelopoiesis. Concurrently, AZT reduced steady state mtDNA content, while increasing lactate production. These findings support the hypothesis that mtDNA is one of the intracellular targets involved in the pathogenesis of AZT-associated bone marrow progenitor cell toxicity.

scholarly journals The regulatory principles of glycolysis in erythrocytes in vivo and in vitro. A minimal comprehensive model describing steady states, quasi-steady states and time-dependent processes

1976 ◽  
Vol 154 (2) ◽  
pp. 449-469 ◽  
Author(s):  
T A. Rapoport ◽  
R Heinrich ◽  
S M. Rapoport
Keyword(s):  
Steady State ◽  
Atp Synthesis ◽  
Steady States ◽  
Time Dependent ◽  
Glycolytic Flux ◽  
Stable Steady State ◽  
Time Interval ◽  
Fructose 1,6 Bisphosphate

A simple mathematical model for glycolysis in erythrocytes is presented which takes into account ATP synthesis and consumption. The system is described by four ordinary differential equations. Conditions in vivo are described by a stable steady state. The model predicts correctly the metabolite concentrations found in vivo. The parameters involved are in agreement with data on the separate steps. The metabolite changes found in pyruvate kinase-deficient erythrocytes and the species variations among erythrocytes from different animals are described satisfactorily. The roles of the enzymes in the control of metabolites and glycolytic flux are expressed in the form of a control matrix and control strengths [R. Heinrich & T.A. Rapoport (1974) Eur. J. Biochem. 42, 89-95] respectively. Erythrocytes from various species are shown to be adapted to a maximal ATP-consumption rate. The calculated eigenvalues reveal the pronounced time-hierarchy of the glycolytic reactions. Owing to the slowness of the 2,3-bisphospho-glycerate phosphatase reaction, quasi-steady states occur during the time-interval of about 0.5-2h incubation, which are defined by perturbed 2,3-bisphosphoglycerate concentrations. The theoretical predictions agree with experimental data. In the quasi-steady state the flux control is exerted almost entirely by the hexokinase-phosphofructokinase system. The model describes satisfactorily the time-dependent changes after addition of glucose to starved erythrocytes. The theoretical consequences are discussed of the conditions in vitro with lactate accumulation and the existence of a time-independent conservation quantity for the oxidized metabolites. Even in this closed system quasi-steady states occur which are characterized by approximately constant concentrations of all glycolytic metabolites except for the accumulation of lactate, fructose 1,6-bisphosphate and triose phosphate.

Incorporation of radioactive phosphate into organic phosphates of tocopherol-deficient and control rabbit muscle

1960 ◽  
Vol 199 (2) ◽  
pp. 295-298 ◽  
Author(s):  
Y. F. Masters ◽  
P. C. Johnson ◽  
W. H. Mosley ◽  
P. B. McCay ◽  
R. Caputto
Keyword(s):  
Vitamin E ◽  
Inorganic Phosphate ◽  
Specific Activity ◽  
Extracellular Fluid ◽  
Organic Phosphate ◽  
Rabbit Muscle ◽  
Dystrophic Muscle ◽  
Control Rabbit ◽  
Activity Ratios ◽  
And Control

Control and vitamin E-deficient rabbits were compared in their ability to incorporate intravenously injected radioactive phosphate into muscle intracellular organic phosphate. Vitamin E-deficient dystrophic muscle received inorganic phosphate from the extracellular fluid at a higher rate than did the control. Specific activity ratios showed that a smaller portion of this phosphate was converted into organic phosphate, making it appear that dystrophic muscle is less efficient than the control. Evidence is presented suggesting that at least two inorganic phosphate pools may exist in muscle cells.

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