Intracellular Acid-base Balance Research Articles

Ion Interference Therapy of Tumors Based on Inorganic Nanoparticles.

As an essential substance for cell life activities, ions play an important role in controlling cell osmotic pressure balance, intracellular acid–base balance, signal transmission, biocatalysis and so on. The imbalance of ion homeostasis in cells will seriously affect the activities of cells, cause irreversible damage to cells or induce cell death. Therefore, artificially interfering with the ion homeostasis in tumor cells has become a new means to inhibit the proliferation of tumor cells. This treatment is called ion interference therapy (IIT). Although some molecular carriers of ions have been developed for intracellular ion delivery, inorganic nanoparticles are widely used in ion interference therapy because of their higher ion delivery ability and higher biocompatibility compared with molecular carriers. This article reviewed the recent development of IIT based on inorganic nanoparticles and summarized the advantages and disadvantages of this treatment and the challenges of future development, hoping to provide a reference for future research.

2179-P: Overexpression Screening Identifies Mitochondrial Carbonic Anhydrase 5b as an Important Regulator of Glucose-Stimulated Insulin Secretion in Beta Cells

Despite extensive research efforts, a precise picture of how glucose itself and its metabolites initiates insulin secretion in a concentration-dependent manner has unveiled. To identify candidate genes important for glucose-stimulated insulin secretion (GSIS), we have conducted screening of differentially expressed genes identified by microarray analyses between highly glucose-responsive and less responsive MIN6 subclones. For this purpose, a platform containing zeocin resistant gene flanked with a wild type and mutated flippase recognition target sites was integrated in the genome of MIN6 cells expressing Tet3G. The modified MIN6 cells have been used to generate stable transfectants expressing mRNA or shRNA of genes of interest by means of the recombinease-mediated cassette exchange method. Using this system, more than ten stable MIN6 cell transformants can be gnenerated at a time. By creating approximately 100 overexpressing transfectants, we have identified mitochondrial carbonic anhydrase 5b (Car5b) as an important regulator of GSIS. Car5b is a one of 15 members of carbonic anhydrases, which catalyze the interconversion of carbon dioxide and water to bicarbonate and protons, and modulate various intracellular processes, such as urea formation, HCO3- supply for carboxylation and regulation of intracellular acid-base balance. Car5b overexpression in MIN6 cells increased 14CO2 formation from H14CO3- (37 ± 14% increases, p < 0.05) in isolated mitochondria and resulted in 32 ± 12% increases (P < 0.05) in GSIS at 20 mM, while shCar5b expression reduced Car5b protein levels to 31.4 ± 4.4% (p < 0.05), 14CO2 formation to 68.7 ± 8.1% (p < 0.05), and GSIS at 20 mM to 77 ± 5% (p < 0.05). In addition, we found that Car5b expressions in ob/ob mice were 3.7 ± 0.4-fold higher (p < 0.01) and tended to be higher (p = 0.073) in HFD-mice. These data indicated that mitochondrial carbonic anhydrase Car5b plays a role in GSIS in pancreatic β-cells. Disclosure M. Kosuda: None. S. Yamaguchi: None. A. Nagasawa: None. M. Yamana: None. M. Koike: None. H. Nishioka: None. H. Ishihara: Research Support; Self; Daiichi Sankyo Company, Limited, Lilly Diabetes. Speaker's Bureau; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Kyowa Hakko Kirin Co., Ltd., Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi, Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited.

Acid-resistant genes in Mycobacterium tuberculosis and the underlying regulatory network

The molecular mechanisms of pathogen persistence within host cells are emerging hotspots, and one of the causes of its persistence is the acid resistance of bacteria. Currently, tuberculosis remains a serious threat to global public health and it is caused by Mycobacterium tuberculosis. In particular, acid resistance of M. tuberculosis and its persistence within macrophages contribute significantly to tuberculosis. Investigations have uncovered three major mechanisms underlying its acid resistance: the control of proton entry, metabolic regulation of intracellular acid-base balance and regulation of the two-component signaling system. In this review, we summarize the overall regulation network of M. tuberculosis in the acidic environment, aiming at providing a new overall idea for treating M. tuberculosis persistence and exploring new targets for tuberculosis control.

Anion exchanger inhibitor DIDS induces human poorly-differentiated malignant hepatocellular carcinoma HA22T cell apoptosis

Anion exchangers (AEs) of the Cl(-)/HCO3(-) exchanger family contribute to the regulation of intracellular acid-base balance. Recently, we found that anion exchanger 2 (AE2) was significantly expressed in human hepatocellular carcinoma (HCC) and in poorly-differentiated human HCC HA22T/VGH cells. In the present study, we further explored the pharmacological function of AE in four human HCC cell lines (SK-Hep-1, HA22T/VGH, HepG2, and Hep3B) following the treatment of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an AEs specific inhibitor. After administrations with 400-1000 microM of DIDS, cell proliferation was greatly inhibited in a dose-dependent manner from 47.5 to 65.0% in higher malignant HA22T/VGH cells, but not in other cell lines. The results of 4,6-diamidino-2-phenylindole (DAPI) staining, DNA fragmentation and flow cytometric analysis further revealed that cell apoptosis induced by DIDS was also observed in HA22T/VGH cells. Therefore, these findings suggested that AE may be involved, in part, in the proliferation and survival of HA22T cells and could be a new potential therapeutic target against specific human HCC.

Extra- and intracellular acid-base balance and ionic regulation in cod ( Gadus morhua ) during combined and isolated exposures to hypercapnia and copper

Cod (Gadus morhua) were exposed to hypercapnia (water Pco2 = 7.5 mmHg), elevated copper level (0.4 ppm) or a combination of both in order to study extra- and intracellular acid-base regulation and the influence hereupon of copper. During pure hypercapnia, the extracellular respiratory acidosis was completely compensated within 12 to 24 h via a chloride-mediated increase in extracellular [HCO3 −]. Exposure to copper in normocapnic seawater caused a large and progressive increase in plasma [Na+] and [Cl−] and a metabolic acidosis. Exposure to copper in hypercapnic seawater was associated with smaller elevations of plasma [Na+] and [Cl−] than in normocapnic seawater, showing that hypercapnia had a protective effect on the copper-induced osmoregulatory disturbances. The compensation of the hypercapnic acidosis was, however, slow and incomplete in fish exposed to both copper and hypercapnia. Extracellular pH remained depressed by 0.3 pH units after 72 h. The data reveal that acid-base regulation was immediately and persistently inhibited by copper. The limited acid-base regulation during combined copper and hypercapnia exposure was chloride-mediated as during hypercapnia alone. Intracellular pH recovery was complete and very rapid in ventricular and skeletal muscle tissues during environmental hypercapnia, whereas acid-base compensation in liver tissue was slower, the kinetics being similar to that in the extracellular compartment. Intracellular pH compensation was significantly slowed down by copper. Copper concentration increased drastically in gill tissue already at 3 h, while copper concentrations in liver, muscle and plasma were significantly elevated only after 48 h, with liver showing the largest elevation.

Propionate-initiated changes in intracellular pH in rabbit colonocytes

Background/Aims: Because regulation of intracellular pH (pHi) is critical to basic cell functions, most celts have evolved mechanisms to closely regulate intracellular acid-base balance. Short-chain fatty acids (SCFAs), the predominant luminal anion in the colon, acidify the cell interior in several cell systems, but their effect on their “natural target,” the colonocytes, has not been examined thoroughly. Methods: We monitored the pHi response to a model SCFA, propionate, in isolated cells and epithelial sheets from rabbit proximal colon loaded with the pH-sensitive dye 2′,7′-bis-(2-carboxyethyl)-5-(and -6) carboxyfluorescein.Results: SCFAs induced a characteristic pHi response curve in colonocytes: an immediate acidification and a recovery phase returning to baseline in 100–200 seconds. Acidification was altered by increasing concentrations of SCFAs, by increasing SCFA chain length, extracellular osmolarity, and intracellular pH, and finally, Na+ removal. The recovery phase was slowed by amiloride and 4-α-OH cinnamate, an inhibitor of proton-monocar-boxylate cotransport. Conclusions: Physiological concentrations of SCFAs have profound effects on intracellular pH. Simple diffusion of the SCFA may not explain the complexities of propionate-induced protonated acidification; the pH recovery phase may involve multiple processes including Na+-H+ exchange and H+-SCFA cotransport. Luminal constituents such as SCFAs may have significant effects on the intracellular pH and function of colonocytes.

Interaction of Respiration, Ion Regulation, and Acid-Base Balance in the Everyday Life of Aquatic Crustaceans

Extracellular and intracellular acid-base balance is necessary for the maintenance of normal metabolic processes. The primary source of acid is metabolically produced CO2, and the CO2/HCO3− system is the most significant buffer. The regulation of acid-base balance is complex, involving the interaction between respiratory gas exchange and ion transport. In aquatic crustaceans respiration is governed by the need to extract oxygen from water, an O2-poor medium; thus, acid-base balance is maintained primarily through ion transport mechanisms. These mechanisms include Na+/H+ and Cl−/HCO3− exchange processes that are sensitive to the extracellular acid-base status of the animal. In marine crabs, ion regulation and acid-base balance are accomplished by the posterior gills, while in freshwater species all gills and the antennal gland perform these functions. Intracellular acid-base balance appears to be maintained primarily by ion transport across the cell membrane. Hemolymph pH varies inversely with acclimation temperature and salinity. In both cases Pco2 remains nearly constant, and the pH change is a result of changes in hemolymph HCO3− concentrations brought about by ion exchange mechanisms. Environmental hypercapnia or hyperoxia induces a repiratory acidosis characterized by increased Pco2, low pH, and elevated HCO3−; this is partially compensated for by ion exchange processes that bring about a further increase in hemolymph HCO3−. Exercise causes a mixed respiratory and metabolic acidosis with compensation via H+ ion excretion and hyperventilation.

Lactate compartmentation in hippocampal slices: Evidence for a transporter

Lactic acid accumulation has been implicated in the evolution of brain damage after ischemia. Since compartmentation of lactate may play a role in acid-base balance, lactate release from gerbil hippocampal slices was examined during a number of metabolic stresses including elevated [K+]e, ischemia, anoxia, and aglycemia. Slices were preincubated for 1 hr in artificial cerebrospinal fluid (ACSF) equilibrated with 95% O2/5% CO2 (pH 7.4 at 37 degrees C) and then transferred to tubes containing 300 microliters of test medium. The rate of lactate release in control slices was 9.64 nmol/min/mg protein and increased 2.6- and 3.2-fold in the presence of 60 mM potassium and anoxia, whereas the rate of lactate release was decreased by 50 and 25% during ischemia and aglycemia. Lactate release was temperature dependent and was only minimally influenced by removing Ca2+ or by adding 5 mM d-lactate to the ACSF. In contrast, pyruvate inhibited lactate release with an apparent Ki of 2.4 mM. The results suggest that lactate can be released from cells via a saturable and stereospecific lactate transporter with an apparent Km of 10.7 mM and Vmax of 43.7 nmol/mg protein/min. Such a relatively high-capacity transporter system can rapidly equilibrate brain lactate but is probably not involved in regulating intracellular acid-base balance.

Intracellular pH in isolated Necturus antral mucosa in simulated ulcerogenic conditions

Intracellular pH (pHi) was measured with protonsensitive liquid sensor microelectrodes in isolated Necturus antral mucosa, paying special attention to arranging experimental conditions to simulate conditions frequently associated with in vivo “stress ulceration.” Intracellular pH in mucosas perfused under standard conditions (Ringer's solution containing HCO3staggered−/CO2) was 7.22 + 0.02 (n = 27). Removal of Na+ and HCO3staggered− or addition of amiloride or 4-acetamido-4-isothiosyanostillbene-2,2disulfonic acid (blockers of Na+/H+ and Cl−/HCO3staggered− exchangers) had no influence on steady-state pHi, suggesting that these ion exchangers do not significantly contribute to the maintenance of pHi in the presence of normal external pH. Acidification of mucosal (luminal) perfusate to pH 3 (mimicking the presence of gastric acid) had no influence on pHi, but mucosal pH 2 (10 mM HCl) acidified pHi to 6.93 ± 0.07. Acidification of serosal (nutrient) perfusate to pH 6 (mimicking intramucosal acidosis caused by back-diffusion of luminal H+) acidified pHi to 6.72 ± 0.10. Removal of Na+ from and addition of amiloride to the serosal perfusate during exposure to serosal pH 6.0 induced further acidification of pHi, suggesting that in this acidotic situation (with very low ambient HCO3staggered− concentration) a Na+/H+ exchanger does contribute to the maintenance of steady-state pHi. Increased Pco2 (10% vol/vol in the gas) in a slightly acidic milieu (mimicking mucosal ischemia) likewise acidified pHi to 6.73 ± 0.05. A combination of mucosal acid (pH 3), high Pco2 (10% CO2), and low serosal pH (pH 6) (mimicking conditions that prevail, for example, during hemorrhagic shock) acidified pHi and ultimately resulted in cell death. These derangements of intracellular acid-base balance may have pathogenetic importance also in in vivo stress ulceration.

Experimental studies on therapy of metabolic alkalosis during the beginning of uremia. Influences of ammonium chloride on the intra- and extracellular acid-base status of the rat.

Metabolic alkalosis during renal failure or uremia presents a difficult problem for the clinician. In this study the effects of ammonium chloride, an agent clinically used for the correction of severe metabolic alkalosis, were studied on the extra- and intracellular acid-base balance of nephrectomized rats. While the extracellular acid-base status was determined from blood gas measurements, intracellular pH was calculated from the distribution of 5,5-dimethyl-2,4-oxazolidinedione. It was found that the administration of NH4Cl leads to a significant increase of intracellular pH though extracellular plasma pH decreases, and that ammonium chloride causes only an insignificant reduction of the intracellular bicarbonate concentration. The observed intracellular pH increase may have adverse consequences for patients and raises objections to the further use of ammonium chloride in the treatment of metabolic alkalosis, especially during renal failure or uremia.

TANSLEY REVIEW No. 2: REGULATION OF PH AND GENERATION OF OSMOLARITY IN VASCULAR PLANTS: A COST-BENEFIT ANALYSIS IN RELATION TO EFFICIENCY OF USE OF ENERGY, NITROGEN AND WATER.

The benefits which this paper addresses are those of maintaining the intracellular acid-base balance during growth, and of generating osmolarity related to regulation of turgor in environments of low water potential. These benefits may incur costs in terms of the quantity of potentially growth-limiting resources (photons, water, nitrogen) which are needed to produce unit quantity of 'baseline' plant biomass. The direction (excess H+ or excess OH- ) and magnitude of acid-base perturbation during growth depends on the nature of the N-source (NH4 + , N2 or NO3 - ), so that the costing of pH homoiostasis involves consideration of the costs of overall N-assimilation for comparison with the other costs of growth of a terrestrial C3 plant. Photon costs for the various biochemical and transport processes involved in overall growth, N-assimilation, pH regulation and osmolarity generation are computed using known stoichiometries of coupled reactions. Water costs are deduced from the C-requirements for the various processes (including C lost in associated decarboxylations) by assuming a constant value of water lost in transpiration per unit net C fixed in an illuminated shoot. Nitrogen costs are deduced from the N-content of the plants or compounds under consideration. The computed costs for N-assimilation and the generation of osmolarity are referred to the costs of 'baseline' plant synthesis using the cheapest mechanisms (NH4 + as source for N-assimilation; inorganic ions as the basis for osmolarity generation) so that the increment of cost related to assimilation of N2 or NO3 - , or of osmolarity generation using an organic compatible solute, can be presented. Photon costs of growth with N2 fixation and the processes associated with regulation of pH are (granted the assumptions made as to stoichiometries and plant composition) 9 % higher than are those of growth with NH4 + as N˜ source. The predicted cost of growth with NO3 - as N source depends on the location of NO3 - reduction and the mechanism of OH- disposal, and ranges from 5 to 12% more than that for growth with NH4 + as N source. H2 O (transpiration) costs follow a similar pattern, with growth on N2 as N source costing 12% more, and growth on NO3 - costing to 1-2 to 167 % more, than growth with NH4 + as N source. The extra costs in photons of using compatible solutes (sorbitol, proline or glycine betaine) to generate an osmolarity of 500 osmol m-3 in all of the non-apoplastic water of the plant add 21·5 to 26·1 % to the total costs of growth, while use of compatible solutes to generate osmolarity in 'N' phases (i.e. cytosol, plastid stroma, mitochondrial matrix) alone would add 5·2 to 6·2% The costs of growth in terms of transpirational water are increased 7·9 to 98 % by the use of compatible solutes for osmolarity generation in the 'N' phases only. The increments for the N-containing solutes are higher when NO3 - is the N-source rather than NH4 + . The N-cost of growth with N-containing compatible solutes generating 500 osmol m-3 in 'N' phases increases the N cost of growth by 33%. These predicted costs are under-estimates of 'real' costs which take into account the occurrence of alternate oxidase activity under some growth conditions and the production of additional organic acid anions with N2 as opposed to NH4 + as N source. Nevertheless, the predicted minimum costs of attaining the benefits of pH regulation and of turgor generation are of use in suggesting where selectively significant (i.e. low requirement for a scarce resource) alternative mechanisms may occur. Examples include a possible photon saving by using NH4 + rather than N2 or NO3 - where all three are available; a possible water saving by use of photoreduction of NO3 - in leaves in arid environments; and a possible N saving by use of non-N-containing compatible solutes (polyols) in environments of low water potential. Proof of these suggestions involves comparisons of inclusive fitness of genotypes possessing the trait under consideration with that of genotypes lacking the trait. CONTENTS Summary 26 I. Introduction 27 II. pH Regulation and Osmolarity Generation 27 III. Photon Costs of Various Syntheses Related to pH Regulation and Osmolarity Generation 31 IV. Conclusions on Energy Costs of pH Regulation During Nitrogen Assimilation and Growth 56 V. Conclusions on Energy Costs of Osmolarity Generation 60 VI. Water Costs of pH Regulation and Nitrogen Assimilation 61 VII. Water Costs of Osmolarity Generation 67 VIII. Nitrogen Costs of Osmolarity Generation 69 IX. Conclusions 70 Acknowledgements 72 References 73.

The effect of bicarbonate concentration on fatigue and recovery in isolated rat diaphragm muscle.

Diaphragm strips from young rats (45--60 g) about 0.3 mm thick were fatigued by tetanic stimulation at a train repetition rate of 2 HZ for 3 min. The isometric tension developed was measured during fatigue and recovery in solutions containing 25, 10, or 2 mM bicarbonate at both 37 and 30 degrees C. Tension fell during fatigue to between 20 and 30% of the initial value and this was not significantly influenced by external bicarbonate concentration or temperature over the range considered. Recovery of tension was complete and rapid (t1/2 < 1 min) in 25 mM bicarbonate at both temperatures. In 10 and 2 mM bicarbonate recovery was slowed (t1/2 3.5 and 7 min, respectively, at 30 degrees C, 1.6 and 4.5 min at 37 degrees C) and incomplete (85 and 72% at 30 degrees C, 82 and 61% at 37 degrees C). Muscle creatine phosphate fell during fatigue but was completely restored within 4 min at 30 degrees C in either 2 or 25 mM HCO3. Lactate increased less in muscles fatigued in 2 mM HCO3 and fell at a slower rate during recovery. The results seem to exclude intracellular creatine phosphate concentration as a major determinant in recovery. The evidence suggests that external bicarbonate can affect the recovery of tension following fatigue by altering intracellular acid-base balance.

Sodium bicarbonate, sodium lactate and Tris in the treatment of metabolic acidosis: Effects on intracellular pH and electrolyte content of voluntary muscle

Acute metabolic acidosis was produced in bilaterally nephrectomized rats by the intravenous infusion of 5 mM/Kg. HCl (as 0.3 N HCl) for 1 to 2 hours. This was followed by infusion of either 0.9 per cent NaCl, 0.3 M NaHCO 3, 0.3 M Na lactate or 0.3 M Tris for 1 2 to 1 hour. The amount given was 5 mM/Kg. except for the NaCl, in which the amount given was 2.5 mM/Kg. The effects on extracellular and intracellular acid-base balance and on the electrolyte content of plasma and voluntary muscle were studied. Intracellular pH (pHi) of voluntary muscle was obtained from the distribution of the weak acid 5,5-dimethyl-2,4-oxazolidinedione (DMO) between muscle and plasma. The HCl NaCl group showed acidemia (pH 7.20), but the pHi (6.97) was not significantly different from that of rats given only NaCl. NaHCO 3 and Na lactate both restored extracellular pH to normal levels without causing any significant change in pHi. In the HCl Tris group, extracellular pH (7.30) was not restored to normal. Tris caused respiratory depression and, in addition, raised slightly the plasma potassium. Under the conditions of this experiment, NaHCO 3, and Na lactate were equally effective in the treatment of acute metabolic acidosis. Equal amounts of Tris were less successful.