Cytosolic pH regulation in perfused rat liver: role of intracellular bicarbonate production

The contribution of metabolic bicarbonate to cytosolic pH (pH_cyto) regulation was studied on isolated perfused rat liver using phosphorus-31 NMR spectroscopy. Removal of external HCO^?₃ decreased proton efflux from 18.6±5.0 to 1.64±0.29 μmol/min per g liver wet weight (w.w.) and pH_cyto from 7.17±0.06 to 6.87±0.06. In the nominal absence of bicarbonate, inhibition of carbonic anhydrase by acetazolamide induced a further decrease of proton efflux of 0.69±0.26 μmol/min per g liver w.w. reflecting a reduction in metabolic CO₂ hydration, and hence a decrease of H⁺ and HCO^?₃ supplies. Even though 27% of the proton efflux was amiloride-sensitive under bicarbonate-free conditions, amiloride did not change pH_cyto, revealing the contribution of additional regulatory processes. Indeed, pH regulation was affected by the combined use of 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS) and amiloride since pH_cyto decreased by 0.16±0.05 and proton efflux by 0.60±0.14 μmol/min per g liver w.w. The data suggest that amiloride-sensitive or SITS-sensitive transport activities could achieve, by themselves, pH_cyto regulation. The involvement of two mechanisms, most likely Na⁺/H⁺ antiport and Na⁺:HCO^?₃ symport, was confirmed in the whole organ under intracellular and extracellular acidosis. The evidence of Na-dependent transport of HCO^?₃ in the absence of exogenous bicarbonate implies that the amount of metabolic bicarbonate is sufficient to effectively participate to pH_cyto regulation.     

Development of thermotolerance and changes in intracellular pH in CHO cells heated at 45.0 degrees C at pH 6.6

Chinese hamster ovary (CHO) cells were given short heat pulses (5 to 20 min) at 45.0 degrees C and incubated at 37 degrees C for up to 20 h under either pH 7.3 or 6.6 conditions. Thermotolerance developed under both pH conditions, but at a slower rate in the pH 6.6 medium. Intracellular pH (pHi) was measured with the dye, 1,4-diacetoxy-2,3-dicyanobenzene, combined with flow cytometry. Time-dependent changes in the intracellular pH occurred under either pH condition. CHO cells incubated under normal pH conditions had a transient increase in the pHi. This pHi elevation was followed by a rapid intracellular acidification of approximately 0.15 to 0.25 pH units. The timing of both the increases and decreases in the pHi was dependent on the magnitude of the initial heat dose. With heat doses less than or equal to 10 min, the pHi returned to normal unheated levels after the acidification phase. Although cells incubated under low pH (6.6) conditions showed similar pHi alterations, differences in the kinetics were measured. The intracellular pH increased immediately after heating. In addition, when intracellular acidification occurred, the rate of acidification was significantly reduced. With heat doses longer than 5 min under the low pH conditions, the pHi did not return to normal unheated levels.     

Studies on rat liver mitochondria: 4. Enzyme activities in mitochondria preserved at 0-4 degrees C

Rat liver mitochondria, stored with the energy-linked functions preserved or in aging conditions, were used to assay the activity of various enzymes during five days. The preservation of energy-linked functions was monitored by the respiratory control coefficient. ATPase, cytochrome oxidase and NADH dehydrogenase showed increased activity when the energy-linked functions were preserved. In aging conditions, cytochrome oxidase, NADH dehydrogenase and ATPase showed decreased activity. The ATPase activity increased only when mitochondria were stored in the presence of inhibitors of the electron transport chain. The activity of NADH oxidase did not change, and succinate oxidase and succinate dehydrogenase showed a small decrease in their activity. The enzymes of the matrix, alpha-ketoglutarate dehydrogenase, malate dehydrogenase and aspartate aminotransferase showed little decrease in activity under either of the conditions of storage. The total protein content decreased slightly under both conditions of storage. These results show that the activity of the enzymes analysed was maintained at reasonable levels, when the energy-linked functions of isolated mitochondria were preserved.     

Dynamics of the changes in homogenate respiratory activity after rat whole liver storage at 4 degrees C

Homogenate respiratory activity was studied after different storage terms of the whole rat liver at 4 degrees C in sucrose-based solution and following normothermic reperfusion. Preservation of homogenate respiratory activity in all metabolic states after normothermic reperfusion of the control liver (60 min, 4 degrees C) is shown. Further storage (6 and 24 hrs) of isolated liver under the mentioned above conditions strengthens the substrate respiration of homogenate both after storage and after normothermic reperfusion. At the same time oxidative phosphorylation does not practically change. No change was noted in respiratory activity in the states 3, 4ADP and 3DNP after 24 hrs of liver storage in respect of a previous term. Following normothermic liver reperfusion contributes to a statistically true reduction of mentioned parameters of respiration, that correlates with a decrease in the degree of respiration and phosphorylation coupled of the studied system.     

Effects of procaine on the intracellular pH of Chinese hamster ovary cells heated at 42.0 or 45.0 degrees C

The local anesthetic procaine greatly sensitizes cells to hyperthermia. Though it is generally accepted that procaine is a membrane-active agent that increases membrane fluidity in cells, the mechanism by which it potentiates heat killing is unknown. In this paper we report changes in intracellular pH (pHi) of Chinese hamster ovary (CHO) cells heated at 42.0 or 45.0 degrees C in the presence of procaine. The pHi was measured with flow cytometry using the dye 1,4-diacetoxy-2,3-dicyanobenzene (ADB). Studies were carried out using cells grown at normal pH (7.3) or cells placed in low-pH (6.6) medium 4 h prior to and during heating (acute low-pH treatment). Low-pH-adapted cells (PHV2), which were obtained previously by continuous culture in pH 6.6 medium, were also used. Normal cells heated in the presence of procaine at pH 7.3 underwent a large decrease in pHi compared to cells heated without procaine. Procaine had little additional effect on the intracellular pH of cells in medium with a pH of 6.6 for 4 h before and during 30 min of heating. PHV2 cells exposed to chronic low-pH conditions were resistant to acidification when heated with or without procaine. The surviving fraction of cells heated with procaine was significantly lower under all pH conditions than that of cells heated without procaine. Cells heated at 42.0 degrees C with procaine also became greatly acidified and their survival was reduced. These data suggest that the reduction in pHi caused by procaine may be part of the mechanism of heat sensitization, but cannot account for it entirely. Furthermore, the degree of procaine sensitization and intracellular acidification is dependent on the extracellular pH, with a larger effect occurring at pH 7.3 than at pH 6.6.     

Influence of vanadate on glycolysis,intracellular sodium,and pH in perfused rat hearts

Vanadium compounds have been shown to cause a variety of biological and metabolic effects including inhibition of certain enzymes, alteration of contractile function, and as an insulin like regulator of glucose metabolism. However, the influence of vanadium on metabolic and ionic changes in hearts remains to be understood. In this study we have examined the influence of vanadate on glucose metabolism and sodium transport in isolated perfused rat hearts. Hearts were perfused with 10 mM glucose and varying vanadate concentrations (0.7100 M) while changes in high energy phosphates (ATP and phosphocreatine (PCr)), intracellular pH, and intracellular sodium were monitored using 31P and 23Na NMR spectroscopy. Tissue lactate, glycogen, and (Na+, K+)-ATPase activity were also measured using biochemical assays. Under baseline conditions, vanadate increased tissue glycogen levels two fold and reduced (Na+, K+)-ATPase activity. Significant decreases in ATP and PCr were observed in the presence of vanadate, with little change in intracellular pH. These changes under baseline conditions were less severe when the hearts were perfused with glucose, palmitate and b-hydroxybutyrate. During ischemia vanadate did not limit the rise in intracellular sodium, but slowed sodium recovery on reperfusion. The presence of vanadate during ischemia resulted in attenuation of acidosis, and reduced lactate accumulation. Reperfusion in the presence of vanadate resulted in a slower ATP recovery, while intracellular pH and PCr recovery was not affected. These results indicate that vanadate alters glucose utilization and (Na+, K+)-ATPase activity and thereby influences the response of the myocardium to an ischemic insult.     

Inhibition of intracellular protein degradation by ethanol in perfused rat liver.

Ethanol (50 mM) inhibited proteolysis in the perfused rat liver during stringent amino acid deprivation and also in the presence of normal and 10 times normal concentrations of plasma amino acids. The concentration-response curve of ethanol reached a plateau after 5 mM in both the presence and the absence of normal plasma amino acids, suggesting inhibition by oxidation products of ethanol. Intracellular glutamine, tyrosine and proline increased in concentration with ethanol, but the increases were too small to explain the observed inhibition of proteolysis. The uptake of 125I-asialofetuin was slightly decreased and the output of ammonia increased in the presence of ethanol. These, together with a significant suppression of basal proteolysis in the presence of amino acids, suggest that lysosomal function was directly affected. Electron-microscopic examination of lysosomal components showed that the aggregate volume of autophagosomes (initial vacuoles) were significantly smaller in livers perfused with ethanol than in controls. However, the equivalent volume of autolysosomes (degradative vacuoles) was the same in both groups. According to these results, ethanol inhibits protein degradation in the liver by two discrete mechanisms: one decreasing the formation of autophagic vacuoles and the other involving lysosomotropic inhibition, possibly via ammonia.     

The effect of urea synthesis on extracellular pH in isolated perfused rat liver.

In a non-recirculating system of isolated liver perfusion, stimulation of urea synthesis by NH4Cl is followed by a decrease of effluent pH by up to 0.2 pH unit. This effect is not observed when urea synthesis is inhibited by amino-oxyacetate or norvaline. When the urea formed by the liver is immediately hydrolysed with urease before the effluent perfusate reaches the pH electrode, the urea-synthesis-induced acidification is no longer observed. This indicates that accompanying alterations in hepatic metabolism after stimulation of urea synthesis, such as increased energy provision and consumption, are not responsible for the extracellular acidification, but that the effect is due to the formation of urea itself. The acidification of the extracellular space after stimulation of urea synthesis by NH4Cl is quantitatively explained by the consumption of 2 mol of HCO3-/mol of urea formed: 1 mol being incorporated into urea, the other being protonated to yield CO2 and H2O. The data match the theoretically predicted HCO3- consumption during ureogenesis and underline the role of hepatic urea synthesis for disposal of HCO3- by converting it into the excretable products CO2 and urea.     

The effect of purification of commercial bovine serum albumin on the performance of isolated rat livers perfused at 5 degrees C

Commercial bovine serum albumin was purified by gel filtration, in an attempt to extend the period during which isolated rat livers could be maintained in a viable condition while in a state of hypothermia, and to remove some of the variability of data obtained from livers perfused with different batches of BSA. A minor component, accounting for about 5% of the total protein, was removed and the remainder used to support isolated perfused rat livers.Perfusion of rat livers with purified albumin at a concentration of 62 g/liter achieved two results: one, an improvement in the biochemical performance of rat livers perfused at 35 °C; and two, an increase in the period of hypothermic perfusion from 12 to 24 hr during which the liver would remain viable, as indicated by the biochemical tests performed on rewarming to 35 °C.     

Chloroethene biodegradation in sediments at 4 degrees C

Microbial reductive dechlorination of [1,2-14C]trichloroethene to [14C]cis-dichloroethene and [14C]vinyl chloride was observed at 4 degrees C in anoxic microcosms prepared with cold temperature-adapted aquifer and river sediments from Alaska. Microbial anaerobic oxidation of [1,2-14C]cis-dichloroethene and [1,2-14C]vinyl chloride to 14CO2 also was observed under these conditions.     

Change in intracellular pH of rat liver during azo-dye carcinogenesis.

The change in intracellular pH of rat liver during 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) feeding was examined, contrasting with that during 2-methyl-4- dimethylaminoazobenzene (2-Me-DAB) feeding. Intracellular pH of liver was measured by the DMO method.The intracellular pH decreased markedly until the 5th week after the beginning of 3'-Me-DAB feeding, and then somewhat recovered. After 11 weeks, however, it decreased rapidly again with a lower point in the 15th week. When rats were returned to a basal diet after the dye had been fed for various periods, the pH value returned to the normal range. No significant change in rat liver pH was found during 2-Me-DAB feeding. Although it is not obvious what causes the decrease in intracellular pH of rat liver fed on the 3'-Me-DAB diet, or what role it plays in hepatocarcinogenesis, this alteration in cellular environment seems to be associated with biochemical changes accompanied by carcinogenesis.     

Studies on rat liver mitochondria. 6. The effect of contaminating particles in mitochondria stored at 0-4 degrees C

Rat liver mitochondria were stored at 0-4 degrees C for several days using an appropriate medium and energy source. The elimination of the majority of microsomes and lysosomes, that normally contaminate isolated mitochondria, had a positive effect in preservation of respiratory control, P:O ratio, and monoamine oxidase activity during long term storage.     

31P-NMR study of high-energy phosphorylated compounds metabolism and intracellular pH in the perfused rat heart

Using 31P-NMR and haemodynamical measurements, this work assesses different aspects of myocardial preservation improvement during a global ischaemia, based on a simultaneous and correlated study of high-energy phosphorylated compounds, intracellular pH and left ventricular function. Isolated perfused working rat hearts were subjected to 2 or 3 h of hypothermic ischaemia followed by 30 or 45 min of reperfusion. A study of the influence of pH and buffer used in cardioplegic solutions has demonstrated a better preservation of high-energy phosphates and an improved functional recovery when using a pH 7.0, glutamate - containing solution. Protection provided by cardioplegia can be enhanced by the appropriate use of a fluorocarbon-oxygenated cardioplegic reperfusate. The use of nifedipine, a calcium antagonist, in the cardioplegic solutions, does not provide any additional protection under hypothermic conditions.     

Pathophysiological consequences of enhanced intracellular superoxide formation in isolated perfused rat liver.

The potential toxicity of enhanced intracellular reactive oxygen formation was investigated in isolated perfused livers of male Fischer rats. The presence of the redox-cycling agent diquat in the perfusate (200 microM) increased the basal efflux of glutathione disulfide (GSSG) into bile (2.65 +/- 0.26 nmol GSH-equivalents/min per g liver wt.) and perfusate (0.55 +/- 0.15 nmol/min per g) approximately 10-fold. Since no evidence was found for degradation of GSSG in the biliary tract of these animals, it could be estimated that diquat induced a constant O2- generation of approximately 1000 nmol/min per g liver wt for 1 h. Thus, reactive oxygen formation under these conditions was 1-2 orders of magnitude higher than under various pathophysiological conditions. Only minor liver injury (release of lactate dehydrogenase activity) was observed. To increase the susceptibility of the liver to the oxidant stress, animals were pretreated in vivo with 200 mg/kg body wt. phorone, which caused a 90% depletion of the hepatic glutathione content, 100 mg/kg ferrous sulfate, a combination of phorone and ferrous sulfate, or 40 mg/kg BCNU, which caused a 60% inhibition of hepatic GSSG reductase. Only the combined treatment of phorone + ferrous sulfate or BCNU caused a significant increase of the diquat-induced liver injury. Our results demonstrated an extremely high resistance of the liver against intracellular reactive oxygen formation (even with impaired detoxification systems) and can serve as reference for the evaluation of potential contributions of reactive oxygen to liver injury in various disease states.     

13C NMR studies of glycogen turnover in the perfused rat liver

To assess whether hepatic glycogen is actively turning over under conditions which promote net glycogen synthesis we perfused livers from 24-h fasted rats with 20 mM D-[1-13C]glucose, 10 mM L-[3-13C]alanine, 10 mM L-[3-13C]lactate, and 1 microM insulin for 90 min followed by a 75-min "chase" period with perfusate of the same composition containing either 13C-enriched or unlabeled substrates. The peak height of the C-1 resonance of the glucosyl subunits in glycogen was monitored, in real time, using 13C NMR techniques. During the initial 90 min the peak height of the C-1 resonance of glycogen increased at almost a constant rate reflecting a near linear increase in net glycogen synthesis, which persisted for a further 75 min if 13C-enriched substrates were present during the "chase" period. However, when the perfusate was switched to the unenriched substrates, the peak height of the C-1 resonance of glycogen declined in a nearly linear manner reflecting active glycogenolysis during a time of net glycogen synthesis. By comparing the slopes of the curve describing the time course of the net [1-13C] glucose incorporation into glycogen with the rate of net loss of 13C label from the C-1 resonance of glycogen during the "chase" period we estimated the relative rate of glycogen breakdown to be 60% of the net glycogen synthetic rate. Whether this same phenomenon occurs to such an appreciable extent in vivo remains to be determined.