Biomedical subjects

P Canioni

Publications and source records attributed to P Canioni.

At least 19 recordsLinked to original sources

PubMed1997

NMR studies of inorganic phosphate compartmentation in the isolated rat liver during acidic perfusion.

Mitochondrial inorganic phosphate has been shown to be undetectable by 31P NMR in the isolated rat liver perfused under physiological conditions. Cold perfusion (4 degrees C) with valinomycin (K+ ionophore) induced the appearance of an additional resonance assigned to P(i) from mitochondrial compartment (P(i,mito)) (Thiaudière et al., 1993, FEBS Lett. 330, 232-235). Here we have demonstrated that P(i,mito) can be detected by NMR under normothermic conditions (37 degrees C) in acidic (pH 6.5, bicarbonate-free) perfused liver using 50 nM valinomycin or 10 microM N,N'-dicyclohexylcarbodiimide (DCCD, a mitochondrial H+-ATP synthase inhibitor). These conditions resulted in a significant increase in mitochondrial P(i) content. In the presence of valimomycin, pH values of 7.00+/-0.07 and 6.60+/-0.10 (n = 7) for mitochondria and cytosol, respectively, were determined from the chemical shift values of P(i) resonances. Electron microscopy demonstrated a large matrix swelling under valinomycin perfusion, explaining the increased level of mitochondrial P(i). The amount of mitochondrial P(i) measured by NMR increased linearly with the cellular ATP depletion, suggesting a mitochondrial influx of P(i) from the cytosolic compartment with valinomycin perfusion. Moreover, the level of matrix P(i) was dependent on the cytosolic pH value, the resonance being not detectable at physiological cytosolic pH. During mitochondrial swelling, P(i) influx was likely to be associated with proton influx, owing to the stability of transmembrane pH gradient and matrix proton concentration.

Adenosine Triphosphate↗

PubMed1996

Mathematical modelling of the citric acid cycle for the analysis of glutamine isotopomers from cerebellar astrocytes incubated with [1(-13)C]glucose.

A mathematical model of the citric acid cycle devoted to the analysis of 13C-NMR data was developed for determining the relative flux of molecules through the anaplerotic versus oxidative pathways and the relative pyruvate carboxylase versus pyruvate dehydrogenase activities. Different variants of the model were considered depending on the reversibility of the conversion of fumarate into malate and oxaloacetate. The model also included the possibility of orientation-conserved transfer of the four-carbon citric acid cycle intermediates, leading to conversion of succinyl-CoA C1 into either malate C1 or C4. It was used to analyse NMR data from glutamine isotopomers produced by cerebellar astrocytes incubated with [1-13C]glucose. Partial cycling (39%) between oxaloacetate and fumarate was evident from the analysis. Application of the model to glutamate isotopomers from granule cells incubated with [1-13C]glucose [Martin, M.. Portais, J.C.. Labouesse. J., Canioni. P, & Merle, M. (1993) Eur. J. Biochem. 217, 617-625] indicated that total cycling of oxaloacetate into fumarate was, in this case, required to get the best fit. The results emphasized some important differences in carbon metabolism between cerebellar astrocytes and granule cells concerning the sources of carbon fuelling the citric acid cycle and the carbon fluxes on different pathways.

Acetates↗

PubMed1996

Top-down control analysis of temperature effect on oxidative phosphorylation.

The effects of temperature on the control of respiration rate, phosphorylation rate, proton leakage rate, the protonmotive force and the effective ATP/O ratio were determined in isolated rat liver mitochondria over a range of respiratory conditions by applying top-down elasticity and control analyses. Simultaneous measurements of membrane potential, oxidation and phosphorylation rates were performed under various ATP turnover rates, ranging from state 4 to state 3. Although the activities of the three subsystems decreased with temperature (over 30-fold between 37 and 4 degrees C), the effective ATP/O ratio exhibited a maximum at 25 degrees C, far below the physiological value. Top-down elasticity analysis revealed that maximal membrane potential was maintained over the range of temperature studied, and that the proton leakage rate was considerably reduced at 4 degrees C. These results definitely rule out a possible uncoupling of mitochondria at low temperature. At 4 degrees C, the decrease in ATP/O ratio is explained by the relative decrease in phosphorylation processes revealed by the decrease in depolarization after ADP addition [Diolez and Moreau (1985) Biochim. Biophys. Acta 806, 56-63]. The change in depolarization between 37 and 25 degrees C was too small to explain the decrease in ATP/O ratio. This result is best explained by the changes in the elasticity of proton leakage to membrane potential between 37 and 25 degrees C, leading to a higher leak rate at 37 degrees C for the same value of membrane potential. Top-down control analysis showed that despite the important changes in activities of the three subsystems between 37 and 25 degrees C, the patterns of the control distribution are very similar. However, a different pattern was obtained at 4 degrees C under all phosphorylating conditions. Surprisingly, control by the proton leakage subsystem was almost unchanged, although both control patterns by substrate oxidation and phosphorylation subsystems were affected at 4 degrees C. In comparison with results for 25 and 37 degrees C, at 4 degrees C there was evidence for increased control by the phosphorylation subsystem over both fluxes of oxidation and phosphorylation as well as on the ATP/O ratio when the system is close to state 3. However, the pattern of control coefficients as a function of mitochondrial activity also showed enhanced control exerted by the substrate oxidation subsystem under all intermediate conditions. These results suggest that passive membrane permeability to protons is not involved in the effect of temperature on the control of oxidative phosphorylation.

Adenosine Triphosphate↗

PubMed1996

Temperature dependence of NMR relaxation times of nucleoside triphosphates and inorganic phosphate in the isolated perfused rat liver. Effect on Pi compartmentation.

The effect of temperature on 31P NMR spectra from isolated perfused rat livers was studied at 9.4 T. Relaxation times (T1 and T2) of uncleoside triphosphates (NTP) and inorganic phosphate (Pi) were determined at 37, 25, 15, and 4 degrees C. Under hypothermic conditions, an unexpected apparent line sharpening in the Pi spectral region and a clear emergence of an additional Pi resonance were observed. This additional signal was assigned to mitochondrial Pi. T1 values obtained for cytosolic and mitochondrial Pi at 4 degrees C were 1.14 +/- 0.24 s (n = 5) and 0.71 +/- 0.18.s (n = 5), respectively. No significant mitochondrial contribution to the Pi resonance was observed at 37 degrees C. Quantification of Pi and NTP liver contents at 37 and 4 degrees C was performed by comparing the perfused liver spectrum and the corresponding perchloric acid extract spectrum. Under experimental conditions of low external Pi (0.12 mM), it was concluded that intracellular Pi was completely NMR-visible at 4 and 37 degrees C. The observation of the mitochondrial Pi signal at 4 degrees C was well explained by an increase in the Pi level within the matrix, in response to the mitochondrial swelling induced by hypothermia, as observed by electron microscopy. T2 values for the cytosolic Pi at 37 and 4 degrees C were 17 +/- 4 ms (n = 8) and 22 +/- 4 ms (n = 10), respectively. Comparison with measured linewidths indicated that line broadening for the main phosphorylated metabolites--including matrix Pi--was the result of B0 field inhomogeneity. The additional broadening of the cytosolic Pi resonance at 4 and 37 degrees C was attributed to pH heterogeneity within the liver.

Animals↗

PubMed1996

Glucose and glutamine metabolism in C6 glioma cells studied by carbon 13 NMR.

The question as to whether glutamine and glucose are both required for optimal growth of glioma cells is studied through the role of these substrates on the metabolism of the cells. C6 rat glioma cells grow only very slowly when glutamine is omitted from the culture medium. The rates of glucose consumption and lactate production on confluent cells in glutamine-free medium were 0.88 +/- 0.09 and 1.06 +/- 0.25 mumol/h/mg protein, respectively. In the presence of 4 mM glutamine, glucose utilization increase to 60% leading to a 45% increase of lactate production. We have studied the kinetics of enrichment of intracellular glutamate at C2, C3 and C4 positions on cells incubated with 5 mM 99% enriched [1-(13)C]glucose in the presence or the absence of glutamine in the incubation medium. The specific enrichments at metabolic steady state of all carbon positions were the same under both conditions, but we observed a significantly reduced rate of 13C incorporation in the presence of glutamine, showing an isotopic dilution of tricarboxylic acid cycle intermediates and indicating the use of this amino acid as an anaplerotic substrate. The fact that no dilution occurred at the level of pyruvate suggests strongly the lack of glutaminolysis in these cells. The main conclusion from this work is that glutamine metabolism in C6 cells appears complementary to that of glucose as far as energy production and carbon sources for the growing of the cells are concerned: glutamine is mainly utilized for anaplerosis as carbon donor to replenish the tricarboxylic acid cycle; it is not a substrate for energy metabolism. In contrast, glucose is poorly anaplerotic and is essentially used as energetic fuel by the C6 cells.

Amino Acids↗

PubMed1996

31P magnetic resonance spectroscopy of human liver in elderly patients: changes according to nutritional status and inflammatory state.

Magnetic resonance spectroscopy (MRS) was used to determine the phosphorylated metabolite content in the liver of elderly patients in various nutritional states: normal, with protein deprivation, and with acute inflammatory syndrome. 31P-MRS investigations were performed at 1.5 T, and localized liver spectra were recorded using a two-dimensional chemical shift imaging sequence. Comparison to control spectra recorded on 10 healthy volunteers (age, 30.5 +/- 2.1 years) showed that the aging process does not significantly modify 31P-MRS liver spectra. Patients with protein deprivation exhibited a higher value than controls for the phosphomonoesters/nucleoside triphosphates (PME/NTP) ratio (P < .05). This increase was not due to the decrease of NTP, since the ratio of inorganic phosphate to NTP (Pi/NTP) remained constant. A decrease in the phosphodiesters to NTP (PDE/NTP) ratio (P < .04) contributed to the observed increase in the PME/PDE ratio (P < .01). In contrast, no significant difference in 31P-MRS spectra was found between elderly patients with hypoalbuminemia associated with inflammatory syndrome and the control group. We conclude that elderly patients with protein deprivation displayed changes in the level of phosphorylated metabolites in the liver that were not observed in the case of inflammatory syndrome despite lower serum albumin (Alb) concentrations.

Aged↗

PubMed1996

[1-13C]glucose metabolism in brain cells: isotopomer analysis of glutamine from cerebellar astrocytes and glutamate from granule cells.

We analyzed the glutamine isotopomers released into the extracellular medium by cerebellar astrocytes incubated with [1-13C]glucose. We developed a mathematical model of the tricarboxylic acid (TCA) cycle to determine the relative flux of molecules through the anaplerotic versus oxidative pathways and the relative pyruvate carboxylase versus pyruvate dehydrogenase activities. As glutamine C2 and C3 exhibited unequivalent enrichments, we examined the possibility of: (1) both the entry of the label into the TCA cycle from pyruvate and the conversion of the oxaloacetate into citrate before equilibration with fumarate, and (2) the occurrence of an orientation-conserved transfer of the symmetrical 4-carbon intermediates. The best fit required partial cycling between oxaloacetate and fumarate, whereas the occurrence of any orientation-conserved transfer was rejected. On the other hand, the analysis of glutamate isotopomers from perchloric acid extracts of granule cells incubated with [1-13C]glucose indicated that total cycling of oxaloacetate into fumarate occurred in these cells.

Animals↗

PubMed1995

Comparative analysis of 13C-enriched metabolites released in the medium of cerebellar and cortical astrocytes incubated with [1-13C]glucose.

Rat cerebellar and cortical astrocytes cultured for 15 or 35 days were incubated with [1-13C]glucose in the presence or absence of 4 mM exogenous glutamine and the release of 13C-enriched metabolites into cell media was studied by 13C-NMR spectroscopy. In the presence of exogenous glutamine, both cerebellar and cortical astrocytes consumed the amino acid. In contrast, a net production of glutamine occurred in the absence of the amino acid. Simultaneously, a release of 13C-enriched glutamine into cell media was observed and was higher in the presence than in the absence of exogenous glutamine. This demonstrated the occurrence of an isotopic-exchange process which may involve a futile cycle at the level of glutamine synthetase and glutaminase activities. The 13C-enrichment ratio between glutamine carbons C2 and C3 was close to 1 in the presence of exogenous glutamine whereas it was higher than 1 in its absence, indicating that pyruvate carboxylase was more active in the absence of glutamine. In addition to glutamine, alanine was synthesized and exported into the medium of both cerebellar and cortical astrocytes. In contrast, citrate was specifically produced by cortical astrocytes. Slight increases in alanine and glutamine productions were observed for cortical astrocyte cultures between 15 and 35 days, whereas the amino acid production by cerebellar astrocytes increased several-fold after 35 days compared with that at 15 days of culture.

Alanine↗

PubMed1995

Quantification of compartmented metabolic fluxes in maize root tips using isotope distribution from 13C- or 14C-labeled glucose.

Metabolic pathways of the intermediate metabolism of maize root tips were identified and quantified after labeling to isotopic and metabolic steady state using glucose labeled on carbon-1, -2, or -6 with 14C or 13C. The specific radioactivity of amino acids and the 13C-specific enrichment of specific carbons of free glucose, sucrose, alanine and glutamate were measured and used to calculate metabolic fluxes. The non-triose pathways, including synthesis of polysaccharides, accumulation of free hexoses, and to a lesser extent starch synthesis, were found to consume 75% of the glucose entering the root tips. The cycle of synthesis and hydrolysis of sucrose was found to consume about 70% of the ATP produced by respiration. The comparison of the specific radioactivities of amino acids and phospholipid glycerol phosphate after labeling with [1-(14)C] or [6-(14)C]glucose revealed the operation of the pentose phosphate pathway. The transfer of label from [2-(14)C]glucose to carbon-1 of starch glucosyl units confirmed the operation of this pathway and indicated that it is located in plastids. It was found to consume 32% of the hexose phosphates entering the triose pathways. The remaining 68% were consumed by glycolysis. The determination of the specific enrichment of carbohydrate carbons -1 and -6 after labeling with [1-(13)C]glucose indicated that both the conversion of triose phosphates back to hexose phosphates and the transaldolase exchange contributed to this randomization. Of the triose phosphates produced by glycolysis and the pentose phosphate pathway, about 60% were found to be recycled to hexose phosphates, and 28% were directed to the tricarboxylic acid cycle. Of this 28%, two-thirds were found to be directed through the pyruvate kinase branch and one-third through the phosphoenolpyruvate branch. The latter essentially has an anaplerotic function since little malate was found to be converted to pyruvate (malic enzyme reaction).

Biological Transport↗

PubMed1994

Postnatal decrease of acetate concentration in rat cerebellum.

We examined the evolution of 1H-NMR detectable metabolites in rat cerebellum from the postnatal day 1-25, a period associated with intense metabolic changes as well as physiological and morphological modifications. The unexpected result reported here is the existence of a high concentration of acetate in neonatal rat cerebellum, progressively decreasing with age and maturation. In contrast, the cerebellum content of various metabolites such as N-acetyl-L-aspartate (NAA), glutamate, and aspartate increases from the first day onward.

Acetates↗

PubMed1993

[1-13C]glucose metabolism in rat cerebellar granule cells and astrocytes in primary culture. Evaluation of flux parameters by 13C- and 1H-NMR spectroscopy.

The metabolism of [1-13C]glucose in rat cerebellum astrocytes and granule cells was investigated using 13C- and 1H-NMR spectroscopy. Near homogeneous primary cultures of each cell type were incubated with [1-13C]glucose, under the same conditions. Analysing the relative 13C enrichments of metabolites in spectra of cell perchloric acid extracts, on the one hand, the 13C-1H spin-coupling patterns in 1H-NMR spectra of cell medium lactate and the 13C-13C spin-coupling patterns in 13C-NMR spectra of purified cell glutamate, on the other hand, showed significant differences, between the two cell types, in the activity of various metabolic ways. First, the carbon flux through the oxidative branch of the hexose monophosphate shunt, which leads to unenriched lactate, was found higher in granule cells than in astrocytes. Second, although the specific 13C enrichment of lactate was higher in astrocytes than in granule cells, the fraction of 13C-enriched acetyl-CoA entering the citric acid cycle was more than twice as high in granule cells as in astrocytes. Lactate C3 and acetyl-CoA C2 enrichments were very similar in granule cells, whereas acetyl-CoA C2 enrichment was 60% lower than that of lactate C3 in astrocytes. These results can be explained by the fact that granule cells used almost exclusively the exogenous glucose to fuel the citric acid cycle, whereas astrocytes used concomitantly glucose and other carbon sources. Last, in the case of granule cells, glutamate C2 and C3 enrichments were equivalent; the carbon flux through the pyruvate carboxylase route was evaluated to be around 15% of the carbon flux through the citrate synthetase route. In astrocytes, glutamate C2 enrichment was higher than that of C3, which could be explained by a pyruvate carboxylase activity much more active in these cells than in granule cells.

Animals↗

PubMed1993

Metabolic flux determination in C6 glioma cells using carbon-13 distribution upon [1-13C]glucose incubation.

A mathematical model of mammalian cell intermediary metabolism is presented. It describes the distribution of the carbon-13 isotope (13C) at the different carbon positions of metabolites in cells fed with 13C-enriched substrates. The model allows the determination of fluxes through different metabolic pathways from 13C- and 1H-NMR spectroscopy and mass spectrometry data. The considered metabolic network includes glycolysis, gluconeogenesis, the citric acid cycle and a number of reactions corresponding to protein or fatty acid metabolism. The model was used for calculating metabolic fluxes in a rat tumor cell line, the C6 glioma, incubated with [1-13C]glucose. After evolution to metabolic and isotopic steady states, the intracellular metabolites were extracted with perchloric acid. The specific enrichments of glutamate, aspartate and alanine carbons were determined from 13C-, 1H-NMR spectroscopy, or mass spectrometry data. Taking into account the rate of glucose consumption and of lactate formation, determined from the evolution of glucose and lactate contents in the cell medium, and knowing the activity of the hexose monophosphate shunt, it was possible to estimate the absolute values of all the considered fluxes. From the analysis the following results were obtained. (a) Glucose accounts for about 78% of the pyruvate and 57% of the CoASAc. (b) A metabolic channelling occurs at the citric acid cycle level; it favours the conversion of carbons 2, 3, 4, and 5 of 2-oxoglutarate into carbons 1, 2, 3, and 4 of oxaloacetate, respectively. The percentage of channelled metabolites amounts to 39%. (c) The pyruvate carboxylase activity and the efflux from the citric acid cycle are estimated to be very low, suggesting a lack of glutamine production in C6 cells. The results emphasize different metabolic characteristics of C6 cells when compared to astrocytes, their normal counterpart.

Alanine↗

PubMed1993

Compartmentation of inorganic phosphate in perfused rat liver. Can cytosol be distinguished from mitochondria by 31P NMR?

Compartmentation of inorganic phosphate was studied in intact perfused rat liver at 4 degrees C by 31P NMR. It was shown that decreases in cytosolic pH or cytosolic Pi concentration induced the appearance of an additional Pi resonance at low field which was assigned to Pi from an alkaline compartment. Valinomycin (K+ ionophore) induced a further splitting of the lines whereas nigericin (K+/H+ antiport) or potassium cyanide (inhibitor of cytochrome oxidase) had opposite effects. As valinomycin acts mainly on the cytosolic/mitochondrial K+ gradient and KCN on the mitochondrial respiratory chain, it was deduced that the alkaline compartment as revealed by the second Pi resonance was probably mitochondria. Single Pi lines observed on perchloric extracts of livers exhibiting two resonances during cold perfusion confirmed that the split peaks in the intact liver indeed arose from the same molecular species.

Animals↗

PubMed1993

Glutathione, but not glutamine, is detected in 13C-NMR spectra of perchloric acid extracts from C6 glioma cells.

Glutamine, which is expected to be produced by C6 glioma cells, is not detected in both amino-acid analyses and 13C-NMR spectra of perchloric acid extracts of cells incubated for 4 h with [1-13C]glucose in the absence of extracellular glutamine. However, the resonances of a glutamate-linked product are observed in these spectra. The analysis of the pH dependence of chemical shifts from various glutamate-derived compounds shows that the observed resonances came from glutathione. Glutamine and glutathione signals are in close proximity on the frequency scale, leading to possible misinterpretation of the spectra.

Brain Neoplasms↗

PubMed1993

Interactions between glucose metabolism and oxidative phosphorylations on respiratory-competent Saccharomyces cerevisiae cells.

The purpose of this work was to analyze the interactions between oxidative phosphorylations and glucose metabolism on yeast cells aerobically grown on lactate as carbon source and incubated in a resting cell medium. On such respiratory-competent yeast cells, four different metabolic steady states have particularly been studied: (a) glucose feeding under anaerobiosis, (b) ethanol supply under aerobiosis, (c) glucose supply under aerobiosis and (d) glucose plus ethanol under aerobiosis. For each condition, we measured: (a) the cellular ATP/ADP ratio and NADH content sustained under these conditions, (b) the glucose consumption rate (glucose conditions) and the respiratory rate (aerobic conditions). Under aerobic conditions, when ethanol is used as substrate, the ATP/ADP ratio and NADH level are very high as compared with glucose feeding. However, the rate of oxygen consumption is similar under both conditions. The main observation is a large increase in the respiratory rate when both glucose and ethanol are added. This increase corresponds to an ATP/ADP ratio and a NADH level lower than those observed with ethanol but higher than those with glucose. Therefore the response of the respiratory rate to the ATP/ADP ratio depends on the redox potential. We studied the way in which the ATP-consuming activity was increased under glucose+ethanol conditions. By NMR experiments, it appears that neither the futile cycle at the level of the phosphofructo-1-kinase/fructo-1,6-bisphosphatase couple nor the synthesis of carbohydrate stores could account for the increase in oxidative phosphorylation. However, it is shown that, in the presence of glucose+ethanol, ATP consumption is strongly stimulated. It is hypothesized that this consumption is essentially due to the combination of the well-known plasma membrane proton-ATPase activation by glucose and the high phosphate potential due to oxidative ethanol metabolism. While it is well documented that oxidative phosphorylations inhibit the glycolytic flux, i.e. the Pasteur effect, we clearly show in this work that the glycolytic pathway limits the ability of mitochondria to maintain a cellular phosphate potential.

Adenosine Diphosphate↗

PubMed1993

N-acetyl-L-aspartate and acetate 1H NMR signal overlapping under mild acidic pH conditions.

The pH dependence of methyl proton chemical shifts of acetate, acetoacetate, N-acetyl-L-aspartate (NAA), and N-acetyl-L-aspartyl-L-glutamate (NAAG) were studied from pH 3 to pH 9. Only slight shifts of acetoacetate, NAA, and NAAG methyl signals were observed, whereas the acetate signal was largely shifted as a result of the titration of its acidic function. At pH 4.7, acetate and NAA methyl signals overlapped, whereas at more acidic pH, the acetate signal appeared downfield when compared to that of NAA. Results are discussed in terms of spectra misinterpretation risks linked to uncontrolled sample pH, on the one hand, and in terms of pH control and contamination by exogenous acetate during perchloric acid cell extract preparation, on the other.

Acetates↗

PubMed1993

31P NMR studies of rat liver cold preservation with histidine-buffered lactobionate solution.

The efficiency of a preservation medium, histidine-buffered lactobionate solution (HBLS), was determined by measuring post-ischemic recoveries of ATP and intracellular pH under Krebs-Henseleit buffer (KHB) perfusion. We used NMR spectroscopy to study the effect of 24-h cold ischemia, followed by 4 degrees C then 37 degrees C reperfusion on the isolated rat liver. Three media were compared: University of Wisconsin solution (UW-lactobionate); Bretschneider's solution (HTK); HBLS and HBLS supplemented with 2 mM Gly and 2 mM Cys (HBLSg2) or with 10 mM Gly and 2 mM Cys (HBLSg10). All values were compared to control values measured during pre-ischemic cold perfusion with KHB (ATP = 8.60 +/- 0.6 mumol/g of dry weigh and pH(in) = 7.41 +/- 0.05). The main result from 31P NMR data concerned ATP recovery during cold reperfusion, which was significantly higher in the HBLS group (112 +/- 10%) as compared to the UW and HTK groups (around 66%). The presence of glycine decreased ATP recovery (88 +/- 8% in HBLSg2, 79 +/- 15% in HBLSg10). Higher values of recovered pHin were observed in livers stored in histidine buffered solutions (around 7.30) as compared to UW (around 7.20); histidine was by 13C NMR proved to accumulate in the liver cells, thus ensuring a good buffering capacity. The thermal transition induced a decrease in both ATP level and pHin in all groups. This might be the result of a stimulation of the carbohydrate metabolism (as demonstrated by 13C NMR) especially when glycine was present in the storage solution.

Adenosine↗

PubMed1993

pH regulation in perfused rat liver: respective role of Na(+)-H+ exchanger and Na(+)-HCO3- cotransport.

Na(+)-H+ antiport and Na(+)-HCO3- symport are involved in intracellular pH (pHi) homeostasis in cultured hepatocytes. We have studied the occurrence of these transport systems in the intact rat liver by 31P nuclear magnetic resonance. Livers perfused with a Krebs medium (25 mM HCO3-, pH 7.4, 37 degrees C) displayed a cytosolic pH 7.18 +/- 0.05 (n = 32). In response to an acid load (35 mM isobutyric acid), pHi remained constant. The same result was obtained in the presence of 1 mM amiloride (with or without acid load), indicating that the amiloride-sensitive Na(+)-H+ exchanger is inactive at external physiological pH (pHe). Under systemic acidosis (6.5-7.0 pHe range), during the acid load, pHi decreased with increased external proton concentrations and became amiloride sensitive. The pHi set point for the activation of the Na(+)-H+ exchange is 7.0. In the absence of HCO3-, livers showed a constant acidic shift of pHi (0.2 pH unit) in the 6.5-7.5 pHe range. Perfusion with 1 mM stilbene derivative (4-acetamido-4'-isothiocyanostilbene-2-2' disulfonic acid) in the presence of HCO3- and at pHe 7.4 induced a dramatic pHi fall (delta pH 0.15), further accentuated during an acid load (delta pH 0.25). Our results suggest that 1) the symport is always involved in pH homeostasis over a large range of pH variation (6.5-7.5) and 2) the Na(+)-H+ exchanger is activated under systemic acidosis as soon as pHi reaches the set point value.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗