Anaerobiosis and acid-base status in marine invertebrates: a theoretical analysis of proton generation by anaerobic metabolism

Summary In animals, various organic acids are accumulated during hypoxia or anoxia as products of anaerobic energy metabolism. The diversity of such acids is largest in marine invertebrates where succinate, propionate, acetate, lactate, alanine, octopine, strombine, and alanopine, are produced mainly from glycogen and aspartate. The effect of these substances on the acid-base status was assessed by a theoretical analysis of the respective metabolic pathways. This resulted in a general rule which was applied to evaluate the proton balance of the reactions in energy metabolism: net changes in the number of carboxyl groups or changes in the degree of dissociation of other groups (e.g. phosphate or ammonia) determine the net amount of H⁺ ions released or bound by the substrates and the metabolic end products.For marine invertebrates the results of the analysis can be summarized as follows: In glycogenolysis one mol of protons per mol of end products is released during cytosolic glycolysis, independent of the type of metabolic end product (lactate, octopine, alanopine, strombine, or alanine). The same applies for mitochondrial production of propionate and acetate, whereas formation of succinate results in dissociation of two mol H⁺ per mol. Fermentation of aspartate, however, diminishes the amount of protons which is produced in the succinate-propionate pathway. Net metabolisation of Mg ATP^2– yields extra protons, whereas the cleavage of phosphagens (e.g. creatine phosphate, arginine phosphate) consumes protons.Additionally the break-down of energy-rich phosphates to inorganic phosphate has to be taken into account because of the shift of the intracellular buffer curve caused by changes of the respective effective pK values.     

Influence of extracellular pH on intracellular pH and cell energy status: relationship to hyperthermic sensitivity

The effect of variable extracellular pH on intracellular pH, cell energy status, and thermal sensitivity was evaluated in CHO cells over the extracellular pH range of 6.0 to 8.6. Extracellular pH was adjusted with either lactic acid, HCl, or NaOH. Regardless of the method of pH adjustment, the results obtained were similar. The relationship between extracellular and intracellular pH was dependent upon the pH range examined. Intracellular pH was relatively resistant to a change in extracellular pH over the pHe range of 6.8 to 7.8 (i.e., delta pHi congruent to delta pHe X 0.33). Above and below this range, delta pHi congruent to delta pHe X 1.08 or X 0.76, respectively. Cellular survival after a 30-min heat treatment at 44 degrees C remained constant over the extracellular pH range of 7.0 to 8.4, but varied substantially over a similar intracellular pH range. The cellular concentration of the high energy phosphate reservoir, phosphocreatine, decreased with decreasing pH. However, the cellular concentrations of ATP, ADP, and AMP remained constant over the entire pH range examined. It is concluded that increased thermal sensitivity resulting from a change in extracellular pH is not due to cellular energy depletion. Furthermore, intracellular pH is a more accurate indicator of thermal sensitivity than is extracellular pH.     

Effects of acute hypobaric hypoxia on acid-base status of fowl blood

Arterial blood Po/, Pco2, lactate levels and Cl- ion concentration as well as pH were measured on the time course in chickens (Gallus domesticus) as they settled in normoxic conditions and during exposure to acute hypobaric hypoxia (Pb = 450 Torr). Hypoxia provoked at first a CO2 increased output from blood and a brief stage of deep metabolic acidosis during which lactate levels suddenly increased. This acidosis was then compensated producing a return to the initial pH and a decrease in [HCO3-] + [CO3(2-)] after 60 min. Subsequently respiratory alkalosis associated with an increase in [HCO-3] + [CO3(2-)], a decrease in cl- ion concentration and a small decrease in lactate levels were observed. Prolonged exposure to hypoxia (16 h) resulted in a new return to the initial pH, a decrease in concentration of [HCO3-] + [CO3(2-)] and a high lactate level. The hematocrit value, the Hb concentration, and the plasma Na+, K+, Ca++ and Mg++ ion concentration did not change significantly.     

Temperature-induced changes in blood acid-base status: pH and PCO2 in a binary buffer.

Equations for proton equilibria of a single-phase binary buffer system have been applied to temperature-induced changes in pH and PCO2 of separated dog plasma at constant carbon dioxide content. Predicted behaviour, measured as deltapH/deltaT and deltalog PCO2 /deltaT, and pH and PCO2 as a function of temperature (range 8-45 degrees C), are in reasonable agreement with theory. Theory predicts and data confirm that deltapH/delta T and deltalog PCO2/deltaT functions of temperature; no single "temperature correction factor" is applicable. Comparison of whole blood with binary buffer equations also shows acceptable agreement between theory and experiment. Blood and separated plasma show similar responses in deltapH/deltaT and deltalog PCO2/delta T when compared over identical temperature intervals. For blood or plasma with initial pH (AT 37.5 DEGREES C) values in the range 7.53-7.45 deltapH/delta T (u/ degrees C) values are -0.0139 (37.5-27.5 degrees C) and -0.0192 (19-7 degrees C); comparable deltalog PCO2/deltaT values are 0.0195 (37.5-27.5 degrees C) and 0.0240 (19-7 degrees C). The charge state of protein components in this system remains nearly constant as temperature varies.     

Effects of environmental water salinity on blood acid-base status in juvenile turbot (Scophthalmus maximus L.)

The time courses of extracellular ionic and acid-base adjustments were studied in juvenile turbot (Scophthalmus maximus) following a decrease of water salinity, either abruptly from 32 to 10%. or after a first step (4 weeks) in 19%. salinity followed by a direct transfer to 10%. brackish water (BW). Net exchanges of acid-base equivalents with the external water were also determined after transfer from 32%. SW to 10%. BW. Direct transfer from seawater (SW) to 10%. BW induced a transient decrease in plasma osmolarity, plasma sodium and chloride concentrations, associated with a marked and transient metabolic alkalosis in the blood. A significant net outflux of acidic equivalents was also measured only during the first day in BW. Four weeks preadaptation in 19%. BW reduced the intensity of the osmotic disturbances elicited by a subsequent abrupt transfer to 10%. BW. These ionic readjustments were also coupled with minimal acid-base changes, of lesser magnitude than those described after directly from SW to 10%. BW.     

Stimulus-secretion coupling of arginine-induced insulin release: significance of changes in extracellular and intracellular pH.

The possible relevance of changes in extracellular and/or intracellular pH to the insulinotropic action of L-arginine and L-homoarginine was investigated in rat pancreatic islets. A rise in extracellular pH from 7.0 to 7.4 and 7.8 augmented the secretory response to these cationic amino acids whilst failing to affect the uptake of L-arginine by islet cells and whilst decreasing the release of insulin evoked by D-glucose. Under these conditions, a qualified dissociation was also observed between secretory data and 45Ca net uptake. Moreover, at high extracellular pH, the homoarginine-induced increase in 86Rb outflow from prelabelled islets rapidly faded out, despite sustained stimulation of insulin release. The cationic amino acids failed to affect the intracellular pH of islet cells, whether in the absence or presence of D-glucose and whether at normal or abnormal extracellular pH. These findings argue against the view that the secretory response to L-arginine would be related to either a change in cytosolic pH or the accumulation of this positively charged amino acid in the beta-cell. Nevertheless, they suggest that the yet unidentified target for L-arginine and its non-metabolized analogue in islet cells displays pH-dependency with optimal responsiveness at alkaline pH.     

环境污染对几类水生无脊椎动物内分泌功能扰乱的研究现状

近年来，在环境毒理学这门边缘学科中又诞生了一个新的领域，即环境污染对内分泌功能的扰乱。研究发现，许多人工合成的杀虫剂和工业化合物能够扰乱脊椎动物的内分泌功能，这些化合物也存在于水环境中。近年来，这些环境有机污染物是否对水生无脊椎动物的内分泌功能同样具有扰乱作用成了环境内分泌学这个新领域的热点之一。由于近年来的研究侧重于腔肠动物、轮虫、软体动物、甲壳动物及棘皮动物，因此，本文主要介绍有关环境污染物对这几类水生无脊椎动物内分泌功能扰乱的研究进展。另外，对环境污染对水生无脊椎动物内分泌扰乱这个研究热点的现状以及今后的发展方向进行了评述。在从事环境污染对无脊椎动物内分泌功能影响的研究时，研究者必须意识到无脊椎动物和脊椎动物在内分泌机制上的差异，不可随意地在这两大类动物类群之间互相引伸研究结果。     

Relationships between extracellular pH, intracellular pH, and gene expression in Dictyostelium discoideum

A variety of studies have shown that differentiation of Dictyostelium discoideum amoebae in the presence of cAMP is strongly influenced by extracellular pH and various other treatments thought to act by modifying intracellular pH. Thus conditions expected to lower intracellular pH markedly enhance stalk cell formation, while treatments with the opposite effect favor spores. To directly test the idea that intracellular pH is a cell-type-specific messenger in Dictyostelium, we have measured intracellular pH in cells exposed to either low extracellular pH plus weak acid or high extracellular pH plus weak base using 31P nuclear magnetic resonance (NMR). Our results show that there is no significant difference in intracellular pH (cytosolic or mitochondrial) between pH conditions which strongly promote either stalk cell or spore formation, respectively. We have also examined the effects of external pH on the expression of various cell-type-specific markers, particularly mRNAs. Some mRNAs, such as those of the prestalk II (PL1 and 2H6) and prespore II (D19, 2H3) categories, are strongly regulated by external pH in a manner consistent with their cell-type specificity during normal development. Other markers such as mRNAs D14 (prestalk I), D18 (prespore I), 10C3 (common), or the enzyme UDP-galactose polysaccharide transferase are regulated only weakly or not at all by external pH. In sum, our results show that modulation of phenotype by extracellular pH in cell monolayers incubated with cAMP does not precisely mimic the regulation of stalk and spore pathways during normal development and that this phenotypic regulation by extracellular pH does not involve changes in intracellular pH.     

Regulation of intracellular pH in cancer cell lines under normoxia and hypoxia

Acid‐extrusion by active transport is important in metabolically active cancer cells, where it removes excess intracellular acid and sets the intracellular resting pH. Hypoxia is a major trigger of adaptive responses in cancer, but its effect on acid‐extrusion remains unclear. We studied pH‐regulation under normoxia and hypoxia in eight cancer cell‐lines (HCT116, RT112, MDA‐MB‐468, MCF10A, HT29, HT1080, MiaPaca2, HeLa) using the pH‐sensitive fluorophore, cSNARF‐1. Hypoxia responses were triggered by pre‐incubation in low O₂ or with the 2‐oxoglutarate‐dependent dioxygenase inhibitor dimethyloxalylglycine (DMOG). By selective pharmacological inhibition or transport‐substrate removal, acid‐extrusion flux was dissected into components due to Na⁺/H⁺ exchange (NHE) and Na⁺‐dependent HCO transport. In half of the cell‐lines (HCT116, RT112, MDA‐MB‐468, MCF10A), acid‐extrusion on NHE was the dominant flux during an acid load, and in all of these, bar one (MDA‐MB‐468), NHE‐flux was reduced following hypoxic incubation. Further studies in HCT116 cells showed that <4‐h hypoxic incubation reduced NHE‐flux reversibly with a time‐constant of 1–2 h. This was not associated with a change in expression of NHE1, the principal NHE isoform. Following 48‐h hypoxia, inhibition of NHE‐flux persisted but became only slowly reversible and associated with reduced expression of the glycosylated form of NHE1. Acid‐extrusion by Na⁺‐dependent HCO transport was hypoxia‐insensitive and comparable in all cell lines. This constitutive and stable element of pH‐regulation was found to be important for setting and stabilizing resting pH at a mildly alkaline level (conducive for growth), irrespective of oxygenation status. In contrast, the more variable flux on NHE underlies cell‐specific differences in their dynamic response to larger acid loads. J. Cell. Physiol. 228: 743–752, 2013. © 2012 Wiley Periodicals, Inc.     

Antiinfluenza virus effect of extracts from marine algae and invertebrates

Sixty products, derived from marine organisms, typical of the Bulgarian Black Sea coast, were examined for inhibitory activity on the reproduction of influenza viruses in tissue cultures. The antiviral effect was investigated by the reduction of virus infectivity. Using representative strains of influenza virus it was shown that apparently the inhibitory effect was strain-specific. The most effective products were further studied in fertile hen's eggs and in experimental influenza infection in white mice.     

Evolution of discriminatory aggression in marine invertebrates.

Many species of sessile marine organisms show allotype-conditional aggression towards conspecifics. However, a recent theoretical analysis (Grosberg & Quinn, 1989, Evolution 43, 504-515.) was unable to find conditions permitting a discriminatory ESS against unconditionally aggressive or non-aggressive strategies. This study shows that discrimination can be an ESS if animals interact with clonemates more frequently than randomly as occurs when animals reproduce by budding of fission. This agrees well with the observation that clonal sea anemones are usually discriminators and solitary species usually non-aggressive to conspecifics (Francis, 1988, Biol. Bull. 174, 241-253.) In addition, discrimination can be an ESS if discriminators retaliate against unconditionally aggressive conspecifics of the same allotype, or if the payoff to two sharers of a resource is greater than the payoff to both when sharing does not occur.     

Stimulation of Solute Loss from Radicles of Gossypium hirsutum L. by Chilling, Anaerobiosis, and Low pH

The loss of organic substances from cotton (Gossypium hirsutum L.) radicles is enhanced by chilling, low pH, or anaerobic conditions. Solute loss returns to a low level when the stimulus is removed, indicating no permanent injury to membranes. Loss of solute induced by chilling or anaerobiosis is reversed or prevented by calcium or magnesium. These cations did not reduce the solute loss resulting from low pH. The site of loss appears to be localized at or near the root tip. If the seedling cotyledons are removed, the loss is greatly reduced, indicating the need for a reserve pool of organic substances.     

Effects of prolonged acclimation to cold on the extra--and intracellular acid-base status in the land snail Helix lucorum (L.)

The aim of this study was to examine the effect of prolonged acclimation to cold on the acid-base status of extra- and intracellular fluids in the land snail Helix lucorum. For this purpose, acid-base parameters in the hemolymph and tissues were determined. In addition, the buffer values of hemolymph and tissues were determined in order to examine whether they change in the snails during acclimation to cold. According to the results presented, there is an inverse pH-temperature relationship in the hemolymph within the first day of acclimation, which is consistent with alphastat regulation. The Pco2 decreased, and pH in the hemolymph (pH(e)) increased by 0.32 U within the first day of acclimation to cold, which corresponds to a change of 0.013 U degrees C(-1). After the first day of acclimation, Pco2 increased in the hemolymph, resulting in a significant drop in pH(e) by 90 d of acclimation to cold. Acclimation of snails to low temperatures did not change the buffer value of the hemolymph. Also, intracellular pH (pH(i)) and intracellular buffer values remained stable during acclimation to cold for prolonged periods. The latter results in conjunction with those obtained by the in vitro determination of the passive component of intracellular fluids indicate an active regulation of pH(i) in H. lucorum during acclimation to cold.     

Activation and damage of endothelial cells upon hypoxia/reoxygenation. Effect of extracellular pH

Disturbances of blood flow upon vascular occlusions and spasms result in hypoxia and acidosis, while its subsequent restoration leads to reoxygenation and pH normalization (re-alkalization) in ischemic sites of the vascular bed. The effect of hypoxia/reoxygenation on activation and stimulation of apoptosis in cultured human endothelial cells was studied. The cells were subjected to hypoxia (2% O₂, 5% CO₂, 93% N₂) for 24 h followed by reoxygenation (21% O₂, 5% CO₂, 74% N₂) for 5 h. Reoxygenation was carried out at different pH-6.4 (preservation of acidosis after hypoxia), 7.0, and 7.4 (partial and complete re-alkalization, respectively). Hypoxia only slightly (by ～30%) increased the cell adhesion molecule ICAM-1 content on the cell surface, whereas reoxygenation more than doubled its expression. The reoxygenation effect depended on the medium acidity, and ICAM-1 increase was more pronounced at pH 7.0 compared to that at pH 6.4 and 7.4. Neither hypoxia nor reoxygenation induced expression of two other cell adhesion molecules, VCAM and E-selectin. Incubation of cells under hypoxic conditions but not reoxygenation stimulated secretion of von Willebrand factor and increased its concentration in the culture medium by more than 4 times. The percentage of cells containing apoptosis marker, activated caspase-3, was increased by approximately 1.5 times upon hypoxia as well as hypoxia/reoxygenation. Maximal values were achieved when reoxygenation was performed at pH 7.0. These data show that hypoxia/reoxygenation stimulate pro-inflammatory activation (ICAM-1 expression) and apoptosis (caspase-3 activation) of endothelial cells, and the extracellular pH influences both processes.     

Temperature effects on intra- and extracellular acid-base status in the American locust,Schistocerca nitens

Summary This study examined the effect of temperature on hemolymph and intracellular acid-base status in the locust,Schistocerca nitens. Hemolymph pH decreased with temperature by 0.017 pH units·°C^–1 above 25°C, but was stable at lower temperatures. Average intracellular pH (pH_i, calculated from CO₂ distribution) decreased by approximately 0.018 pH units·°C^–1, in accordance with predictions for preservation of relative alkalinity and protein charge state. DMO was found to be unsuitable for use as an in vivo pH_i marker in locusts due to rapid metabolism and excretion of this compound. Hemolymph pH regulation when temperature changed was accomplished by a combination of variation ofP _CO2 and [HCO ₃ ^– ], with changes in [HCO ₃ ^– ] predominating. Digestive tracts contained a large portion of total body water (over 30%), and total body CO₂ (over 40%). Variation in [HCO ₃ ^– ] may dominate pH regulation in locusts because of the relatively large size of the digestive tract and its powerful acid-base transporting capacities.Abbreviations C _car carcass total CO₂ - C _hem hemolymph total CO₂ - C _in intracellular total CO₂ - C _tot total CO₂ - DMO 5,5 dimethyl-2,4-oxazolidinedione - ECW extracellular water mass - ECWF fraction of total body water which is extracellular - ICW intracellular water mass - ICWF fraction of total body water which is intracellular - PCA perchloric acid - S dissolved CO₂ - TBW total body water - TV tracheal volume     

Effect of pH and nonionic surfactant on profile of intracellular and extracellular Monascus pigments

The uptake of nutrients and export of intracellular products are essential issues of microbial fermentation. Two-stage of Monascus fermentation using monosodium glutamate (MSG) as the sole nitrogen source, i.e., the first stage microbial fermentation in an aqueous solution and the second stage perstractive fermentation in a nonionic surfactant micelle aqueous solution, was carried out under different initial pH conditions. The results revealed that extracellular pH influences on the uptake of MSG and the export of water-soluble red pigment derivates into its extracellular broth. On the other hand, the export of intracellular hydrophobic pigments is affected by perstractive fermentation in nonionic surfactant micelle aqueous solution. All of those factors exhibit strong effect on the profile of Monascus pigments. This information is key to controlling profile of extracellular Monascus pigments by perstractive fermentation in nonionic surfactant micelle aqueous solution.