Evaluation of isotope discrimination in C-based metabolic flux analysis

In a ¹³C experiment for metabolic flux analysis (¹³C MFA), we examined isotope discrimination by measuring the labeling of glucose, amino acids, and hexose monophosphates via mass spectrometry. When Escherichia coli grew in a mix of 20% fully labeled and 80% naturally labeled glucose medium, the cell metabolism favored light isotopes and the measured isotopic ratios (δ¹³C) were in the range of −35 to −92. Glucose transporters might play an important role in such isotopic fractionation. Flux analysis showed that both isotopic discrimination and isotopic impurities in labeled substrates could affect the solution of ¹³C MFA.     

Overexpression of SeNHX1 improves both salt tolerance and disease resistance in tobacco

Recently, we found NHX1, the gene encoding a Na⁺/H⁺ exchanger, participated in plant disease defense. Although NHX1 has been confirmed to be involved in plant salt tolerance, whether the NHX1 transgenic plants exhibit both salt tolerance and disease resistance has not been investigated. The T1 progenies of Nicotiana tabacum L. lines expressing SeNHX1 (from Salicornia europaea) were generated for the present study. Compared with PBI-type control plants, SeNHX1 transgenic tobaccos exhibited more biomass, longer root length, and higher K⁺/Na⁺ ratio at post germination or seedling stage under NaCl treatment, indicating enhanced salt tolerance. The vacuolar H⁺ efflux in SeNHX1 transgenic tobacco was increased after treatment of NaCl with different concentration. Meanwhile, the SeNHX1 transgenic tobaccos showed smaller wilted spot area, less H₂O₂ accumulation in leaves after infection of Phytophthora parasitica var. nicotianae. Further investigation demonstrated a larger NAD(P)(H) pool in SeNHX1 transgenic tobacco. These evidences revealed that overexpression of SeNHX1 intensified the compartmentation of Na⁺ into vacuole under salt stress and improved the ability of eliminating ROS after pathogen attack, which then enhanced salt tolerance and disease resistance simultaneously in tobacco. Our findings indicate NHX1 has potential value in creating crops with both improved salt tolerance and disease resistance.     

Cloning of a novel human NHEDC1 (Na+/H+ exchanger like domain containing 1) gene expressed specifically in testis

The Na⁺/H⁺ exchangers (NHEs) catalyze the transport of Na⁺ in exchange for H⁺ across membranes in organisms and are required for numerous physiological processes. Here we report the cloning and characterization of a novel human NHEDC1 (Na⁺/H⁺ exchanger like domain containing 1) gene, which was mapped to human chromosome 4p24. This cDNA is 1859 bp in length, encoding a putative protein of 515 amino acids. The NHEDC1 proteins are highly conserved in mammals including human, mouse, rat, and Macaca fascicularis. One remarkable characteristic of human NHEDC1 gene is that it is exclusively expressed in the testis by RT-PCR analysis. Western blot analysis showed that the molecular weight of NHEDC1 is about 56 KDa. Guangming Ye and Cong Chen contributed equally to this work.     

Mechanisms of liquid flux across pulmonary alveolar epithelial cell monolayers

Summary Active transport of sodium by pulmonary alveolar epithelial cells (AEC) is believed to be an important component of edema clearance in the normal and injured lung. Data supporting this premise have come from measurements of sodium movement across AEC monolayers or from perfused lung model systems. However, direct measurement of fluid flux across AEC monolayers has not been reported. In the present work, AEC were studied with an experimental system for the measurement of fluid flux (Jv) across functionally intact cell monolayers. Primary adult rat type II alveolar epithelial cells were cultured on 0.8 μm nuleopore filters previously coated with gelatin and fibronectin. Intact monolayers were verified by high electrical resistance (> 1000 Θ) at 4–5 d of primary culture. At the same time interval, transmission electron microscopy revealed cells with type I cell-like morphology throughout the monolayer. These were characterized by both adherens and tight junctional attachments. Fluid flux across the monolayers was measured volumetrically over a period of 2 h in the presence of HEPES-buffered DMEM containing 3% fatty acid-free bovine serum albumin. Flux (Jv) was inhibited 39% by 1 × 10⁻⁴ M ouabain (P < 0.01) and 27% by 5 × 10⁻⁴ M amiloride (P < 0.05). These data support the concept that AEC Na⁺/K⁺-ATPase and Na⁺ transport systems are important determinants of AEC transepithelial fluid movement in vitro.     

短期NaCl胁迫对不同小麦品种幼苗K+吸收和Na+、K+积累的影响

为研究盐胁迫下小麦幼苗生长及Na⁺、K⁺的吸收和积累规律,以中国春、洲元9369和长武134等3种耐盐性不同小麦品种为材料,采用非损伤微测技术检测盐胁迫2 d后的根系K⁺离子流变化,并对植株体内的Na⁺、K⁺含量进行测定。结果表明:短期(2d)盐胁迫对小麦生长有抑制作用,且对根系的抑制大于地上部,耐盐品种下降幅度小于盐敏感品种。盐胁迫下,小麦根际的 K⁺大量外流,盐敏感品种中国春K⁺流速显著高于耐盐品种长武134,最高可达15倍。小麦幼苗地上部分和根系均表现为Na⁺积累增加,K⁺积累减少,Na⁺/K⁺比随盐浓度增加而上升。中国春限Na⁺能力显著低于长武134,Na⁺/K⁺则显著高于长武134。综上所述,盐胁迫下造成小麦组织器官中Na⁺/K⁺比上升的主要原因是根系K⁺大量外流和Na⁺的过量积累,耐盐性不同的小麦品种间差异显著,并认为根系对K⁺的保有能力可能是作物耐盐性评价的一个重要指标。     

Increased platelet sodium-proton exchange rates in insulin-dependent (type 1) diabetic patients with nephropathy and hypertension

In order to assess the potential role of the plasma membrane sodium-proton (Na^–/H⁺) exchanger in the pathogenesis of diabetic nephropathy, we investigated 32 insulin dependent (type 1) diabetic patients and 21 control subjects. We tested the Na⁺/H⁺ exchange as the rate of amiloride sensitive and sodium dependent volume gain of platelets suspended in sodium propionate. Patients with diabetic nephropathy had significantly increased rates of Na⁺/H⁺ exchange (0.31 ± 0.06 s^–1 × 10–2) when compared to those without nephropathy (0.24 ± 0.07, p < 0.05) or to a control group (0.23 ± 05, p < 0.05). Nine patients who were classified as hypertensive had a highly significant increase in the Na⁺/H⁺ exchange rates when compared to 23 non-hypertensive diabetic patients: 0.33 ± 0.04 versus 0.24 ± 0.06 (p < 0.001). There was no significant correlation between the Na⁺/H⁺ exchange rates and age, diabetes duration, glycated hemoglobin or fructosamine levels on the day of the test. In summary, the data presented here demonstrate an increase in the Na⁺/H⁺ exchange rate in insulin-dependent diabetic patients with nephropathy and hypertension     

Subcellular localization of H+-ATPase from pumpkin hypocotyls (Cucurbita maxima L.) by membrane fractionation

A new method of preparing sealed vesicles from membrane fractions of pumpkin hypocotyls in ethanolamine-containing buffers was used to investigate the subcellular localization of H⁺-ATPase measured as nigericin-stimulated ATPase. In a fluorescence-quench assay, the H⁺ pump was directly demonstrated. The H⁺ pump was substrate-specific for Mg·ATP and 0.1 mM diethylstilbestrol completely prevented the development of a pH. The presence of unsupecific phosphatase hampered the detection of nigericin-stimulated ATPase. Unspecific phosphatases could be demonstrated by comparing the broad substrate specificity of the hydrolytic activities of the fractions with the clear preference for Mg·ATP as the substrate for the proton pump. Inhibitor studies showed that neither orthovanadate nor molybdate are absolutely specific for ATPase or acid phosphatase, respectively. Diethylstilbestrol seemed to be a specific inhibitor of ATPase activity in fractions containing nigericin-stimulated ATPase, but it stimulated acid phosphatase which tended to obscure its effect on ATPase activity. Nigericin-stimulated ATPase had its optimum at pH 6.0 and the nigericin effect was K⁺-dependent. The combination of valinomycin and carbonylcyanide m-chlorophenylhydrazone had a similar effect to nigericin, but singly these ionophores were much less stimulatory. After prolonged centrifugation on linear sucrose gradients, nigericin-stimulated ATPase correlated in dense fractions with plasma membrane markers but a part of it remained at the interphase. This lessdense part of the nigericin-stimulated ATPase could be derived from tonoplast vesicles because -mannosidase, an enzyme of the vacuolar sap, remained in the upper part of the gradient. Nigericinstimulated ATPase did not correlate with the mitochondrial marker, cytochrome c oxidase, whereas azide inhibition of ATPase activity did.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DES dethyltilbestrol     

Phytohormone effects on hydrolytic activity of phosphohydrolases in red beet (Beta vulgaris L.) tonoplasts

The effects of indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellic acid (GA₃) and kinetin on the hydrolytic activity of proton pumps (adenosine triphosphatase, H⁺-ATPase, pyrophosphatase, H⁺-PPase) of tonoplasts isolated from stored red beet (Beta vulgaris L. cv. Bordo) roots were studied. Results suggest that the phytohormones can regulate the hydrolytic activities of H⁺-ATPase and H⁺-PPase of the vacuolar membrane. Each of the proton pumps of the tonoplast has its own regulators in spite of similar localization and functions. IAA and kinetin seem to be regulators of the hydrolytic activity for H⁺-PPase whereas for H⁺-ATPase it may be GA₃. Stimulation of enzyme activity by all hormones occurred at concentrations of 10^–6 to 10^–7 M.Abbreviations IAA indole-3-acetic acid - ABA abscisic acid - GA₃ gibberellic acid - H⁺-ATPase adenosine triphosphatase - H⁺-PPase pyrophosphatase - ATP adenosine triphosphate - Tris Tris (hydroxymethyl)-aminomethane - MES (2[N-Morpholino]) ethane sulfonic acid - EDTA ethylene diamine tetraacetic acid - P_i inorganic phosphate     

Proton pumps of the vacuolar membrane in growing plant cells

Plant growth results from the division, enlargement and specialization of cells. The two processes of the enlargement and the differentiation of cells are not spatially separated in plant tissue. We focus our attention here on the enlargement and elongation of cells. In most cases, growing plant cells contain a large central vacuole. The acid growth theory is based on the space-filling function of the large vacuole. The active transport systems in the vacuolar membrane are essential for maintenance of high osmotic pressure and for the expansion of the vacuole. The secondary active transport systems of the vacuole for sugars and ions are driven by the proton-motive force which is generated by the vacuolar H⁺-ATPase and H⁺-translocating inorganic pyrophosphatase. In this review, the relationship between cell elongation and these enzymes of the vacuolar membrane is emphasized.     

Characterization of inositol 1,4,5-trisphosphate-sensitive (IsCaP) and-insensitive (IisCaP) nonmitochondrial Ca2+ pools in rat pancreatic acinar cells

Summary We have measured Ca²⁺ uptake and Ca²⁺ release in isolated permeabilized pancreatic acinar cells and in isolated membrane vesicles of endoplasmic reticulum prepared from these cells. Ca²⁺ uptake into cells was monitored with a Ca²⁺ electrode, whereas Ca²⁺ uptake into membrane vesicles was measured with⁴⁵Ca²⁺. Using inhibitors of known action, such as the H⁺ ATPase inhibitors NBD-Cl and NEM, the Ca²⁺ ATPase inhibitor vanadate as well as the second messenger inositol 1,4,5-trisphosphate (IP₃) and its analog inositol 1,4,5-trisphosphorothioate (IPS₃), we could functionally differentiate two non-mitochondrial Ca²⁺ pools. Ca²⁺ uptake into the IP₃-sensitive Ca²⁺ pool (IsCaP) occurs by a MgATP-dependent Ca²⁺ uptake mechanism that exchanges Ca²⁺ for H⁺ ions. In the absence of ATP Ca²⁺ uptake can occur to some extent at the expense of an H⁺ gradient that is established by a vacuolar-type MgATP-dependent H⁺ pump present in the same organelle. The other Ca²⁺ pool takes up Ca²⁺ by a vanadate-sensitive Ca²⁺ ATPase and is insensitive to IP₃ (IisCaP). The IsCaP is filled at higher Ca²⁺ concentrations (10^–6 mol/liter) which may occur during stimulation. The low steady-state [Ca²⁺] of 10^–7 mol/liter is adjusted by the IisCaP.It is speculated that both Ca²⁺ pools can communicate with each other, the possible mechanism of which, however, is at present unknown.     

Maize tonoplast PP(i)-dependent H(+)/Ca(2+) exchange: two K(s) for Ca(2+) and inhibition by thapsigargin

Maize root tonoplasts are able to accumulate Ca(2+) using the energy derived from the H(+) gradient formed during PP(i) hydrolysis. Oxalate increases 6- to 10-fold the amount of Ca(2+) accumulated by tonoplast. Two apparently different K(s) values for Ca(2+) with values of 0.36 and 4.70 microM were detected when oxalate was included in the medium and the free Ca(2+) concentration in the medium was buffered with the use of EGTA. Binding of Ca(2+) to the outer surface of tonoplasts inhibits the outflow of Ca(2+) previously accumulated by the tonoplast, half-maximal inhibition being observed in presence of 1 microM Ca(2+). Thapsigargin, a specific inhibitor of Ca(2+)-ATPase, inhibits the Ca(2+) uptake driven by H(+) gradient but does not inhibit the hydrolysis of PP(i) nor the formation of a H(+) gradient.     

Activation of Na/H exchanger 1 by neurotensin signaling in pancreatic cancer cell lines

Acidosis commonly observed in solid tumors like pancreatic cancer promotes genetic instability and selection of a more malignant phenotype of cancer cells. Overexpression or activation of integral membrane proteins mediating H⁺ efflux may contribute to extracellular acidification. Neurotensin (NT) induces intracellular alkalinization and stimulates interleukin-8 production in pancreatic cancer cells and, as demonstrated here, the stable NT analog Lys⁸-ψ-Lys⁹NT(8-13) enhances the amiloride-sensitive, Na⁺-dependent transmembrane H⁺ flux by a factor of 2.05 ± 0.28 and 2.69 ± 0.07 in BxPC-3 and PANC-1 pancreatic cancer cells, respectively, by phosphorylation of the Na⁺/H⁺ exchanger 1 (NHE1). Human genome-wide gene expression analysis was performed to detect effects of Lys⁸-ψ-Lys⁹NT(8-13) on BxPC-3 cells. Results indicated upregulation of genes involved in regulation of NHE1, hypoxic response and glycolysis in response to Lys⁸-ψ-Lys⁹NT(8-13) even under normoxic conditions. Therefore, our findings suggest that growth factors like NT may be implicated in the early progression of pancreatic cancer by localized acidification and induction of an aerobic glycolytic phenotype with higher metastatic potential in small cell aggregates.     

Regulation of intestinal hPepT1 (SLC15A1) activity by phosphodiesterase inhibitors is via inhibition of NHE3 (SLC9A3)

The H⁺-coupled transporter hPepT1 (SLC15A1) mediates the transport of di/tripeptides and many orally-active drugs across the brush-border membrane of the small intestinal epithelium. Incubation of Caco-2 cell monolayers (15 min) with the dietary phosphodiesterase inhibitors caffeine and theophylline inhibited Gly-Sar uptake across the apical membrane. Pentoxifylline, a phosphodiesterase inhibitor given orally to treat intermittent claudication, also decreased Gly-Sar uptake through a reduction in capacity (V_max) without any effect on affinity (K_m). The reduction in dipeptide transport was dependent upon both extracellular Na⁺ and apical pH but was not observed in the presence of the selective Na⁺/H⁺ exchanger NHE3 (SLC9A3) inhibitor S1611. Measurement of intracellular pH confirmed that caffeine was not directly inhibiting hPepT1 but rather having an indirect effect through inhibition of NHE3 activity. NHE3 maintains the H⁺-electrochemical gradient which, in turn, acts as the driving force for H⁺-coupled solute transport. Uptake of β-alanine, a substrate for the H⁺-coupled amino acid transporter hPAT1 (SLC36A1), was also inhibited by caffeine. The regulation of NHE3 by non-nutrient components of diet or orally-delivered drugs may alter the function of any solute carrier dependent upon the H⁺-electrochemical gradient and may, therefore, be a site for both nutrient-drug and drug-drug interactions in the small intestine.     

pH-stat experiments in proximal renal tubules

Summary The pH-stat technique has been used to measure H⁺ fluxes in gastric mucosa and urinary bladder in vitro while keeping mucosal pH constant. We now report application of this method in renal tubules. We perfused proximal tubules with double-barreled micropipettes, blocked luminal fluid columns with oil and used a double-barreled Sb/reference microelectrode to measure pH, and Sb or 1n HC1-filled microelectrodes to inject OH^– or H⁺ ions into the tubule lumen. By varying current injection, pH was kept constant at adjustable levels by an electronic clamping circuit. We could thus obtain ratios of current (nA) to pH change (apparent H⁺-ion conductance). These ratios were reduced after luminal 10^–4 m acetazolamide, during injection of OH^–, but they increased during injection of H⁺. The point-like injection source causes pH to fall off with distance from the injecting electrode tip even in oil-blocked segments. Therefore, a method analogous to cable analysis was used to obtain H⁺ fluxes per cm² epithelium. The relation betweenJ _H ⁺ and pH gradient showed saturation kinetics of H fluxes, both during OH^– and H⁺ injection. This kinetic behavior is compatible with inhibition ofJ _H by luminal H⁺. It is also compatible with dependence on Na⁺ and H⁺ gradients of a saturable Na/H exchanger. H⁺-ion back-flux into the tubule lumen also showed saturation kinetics. This suggests that H⁺ flow is mediated by a membrane component, most likely the Na⁺–H⁺ exchanger.     

Histamine treatment induces rearrangements of orthogonal arrays of particles (OAPs) in human AQP4-expressing gastric cells

To test the involvement of the water channel aquaporin (AQP)-4 in gastric acid physiology, the human gastric cell line (HGT)-1 was stably transfected with rat AQP4. AQP4 was immunolocalized to the basolateral membrane of transfected HGT-1 cells, like in native parietal cells. Expression of AQP4 in transfected cells increased the osmotic water permeability coefficient (Pf) from 2.02 +/- 0.3 x 10-4 to 16.37 +/- 0.5 x 10-4 cm/s at 20 degrees C. Freeze-fracture EM showed distinct orthogonal arrays of particles (OAPs), the morphological signature of AQP4, on the plasma membrane of AQP4-expressing cells. Quantitative morphometry showed that the density of OAPs was 2.5 +/- 0.3% under basal condition and decreased by 50% to 1.2 +/- 0.3% after 20 min of histamine stimulation, mainly due to a significant decrease of the OAPs number. Concomitantly, Pf decreased by approximately 35% in 20-min histamine-stimulated cells. Both Pf and OAPs density were not modified after 10 min of histamine exposure, time at which the maximal hormonal response is observed. Cell surface biotinylation experiments confirmed that AQP4 is internalized after 20 min of histamine exposure, which may account for the downregulation of water transport. This is the first evidence for short term rearrangement of OAPs in an established AQP4-expressing cell line.     

Evaluation of ion gradient-dependent H+ transport systems in isolated enterocytes from the chick

Summary The experiments reported here evaluate the capability of isolated intestinal epithelial cells to accomplish net H⁺ transport in response to imposed ion gradients. In most cases, the membrane potential was kept constant by means of a K⁺ plus valinomycin voltage clamp in order to prevent electrical coupling of ion fluxes. Net H⁺ flux across the cellular membrane was examined at pH 6.0 (the physiological lumenal pH) and at pH 7.4 using methylamine distribution or recordings of changes in media pH. Results from both techniques suggest that the cells have an Na⁺/H⁺ exchange system in the plasma membrane that is capable of rapid and sustained changes in intracellular pH in response to an imposed Na⁺ gradient. The kinetics of the Na⁺/H⁺ exchange reaction at pH 6.0 [K _t for Na⁺=57mm,V _max=42 mmol H⁺/liter 3OMG (3-O-methylglucose) space/min] are dramatically different from those at pH 7.4 (K _t for Na⁺=15mm,V _max=1.7 mmol H⁺/liter 3OMG space/min). Experiments involving imposed K⁺ gradients suggest that these cells have negligible K⁺/H⁺ exchange capability. They exhibit limited but measurable H⁺ conductance. Anion exchange for base equivalents was not detected in experiments performed in media nominally free of bicarbonate.     

Effect ofin vivo andin vitro application of the cytokinin N6 -(m-hydroxybenzyl)adenosine on respiration and membrane transport processes in sugar beet

Sugar beet grown in pots was sprayed with N⁶-(m-hydroxybenzyl)adenosine, (mOH)- [9R]BAP, one of the synthetic cytokinins. Root tissue was then examined for respiration and for H⁺-adenosinetriphosphatase activity and both leaf and root tissue served as the object for 6-deoxy-D-glucose and 2-aminoisobutyric acid uptake estimations. Treatment with (mOH)[9R]BAP depressed the uptake of oxygen by the roots of both young and old plants by 17 – 30 % while addition of (mOH)[9R]BAP to the respiring slices decreased it by 10 – 23 %. Uptake of 6-deoxy-D-glucose was mostly diminished byin vivo spraying with the cytokinin (by up to 12 % in leaves and by up to 60 % in roots), as well as by adding it to the experimental vessel (insignificantly in the leaves but by up to 80 % in the roots). The H⁺-ATPase activity was stimulated bothin vivo andin vitro appreciably in young plants but not at all in plants at the end of their vegetation period. Acknowledgement: The work described here was supported by grant No. 501/94/0413 of the Grant Agency of the Czech Republic     

The in vivo nitrogen isotope discrimination among organic plant compounds

The bulk delta 15 N-value of plant (leaf) biomass is determined by that of the inorganic primary nitrogen sources NO(3)(-), NH(4)(+) and N(2), and by isotope discriminations on their uptake or assimilation. NH(4)(+) from these is transferred into "organic N" mainly by the glutamine synthetase reaction. The involved kinetic nitrogen isotope effect does not become manifest, because the turnover is quantitative. From the product glutamine any further conversion proceeds in a "closed system", where kinetic isotope effects become only efficient in connection with metabolic branching. The central and most important corresponding process is the GOGAT-reaction, involved in the de novo nitrogen binding and in recycling processes like the phenylpropanoid biosynthesis and photorespiration. The reaction yields relatively 15N-depleted glutamate and remaining glutamine, source of 15N-enriched amide-N in heteroaromatic compounds. Glutamate provides nitrogen for all amino acids and some other compounds with different 15N-abundances. An isotope equilibration is not connected to transamination; the relative delta 15 N-value of individual amino acids is determined by their metabolic tasks. Relative to the bulk delta 15 N-value of the plant cell, proteins are generally 15N-enriched, secondary products like chlorophyll, lipids, amino sugars and alkaloids are depleted in 15N. Global delta 15 N-values and 15N-patterns of compounds with several N-atoms can be calculated from those of their precursors and isotope discriminations in their biosyntheses.     

Catecholamine- and volume-dependent ion fluxes in carp (Cyprinus carpio) red blood cells

In carp erythrocytes, noradrenaline (10^-6 mol·l^-1) induces a 30- to 40-fold activation of Na⁺/H⁺ exchange (the ethylisopropylamiloride-inhibited component of the ²²Na influx) and a fourfold stimulation of the Na⁺, K⁺ pump (ouabain-inhibited component of ⁸⁶Rb influx). In both cases the effect of noradrenaline is blocked by propranolol but not phentolamine and is imitated by forskolin. An activator of protein kinase C (-phorbol 12-myristate, 13-acetate) increases Na⁺/H⁺ exchange by 10 times and decreases the Na⁺, K⁺ pump activity by 20–30 percent. In the presence of ethylisopropylamiloride the increment of the Na⁺, K⁺ pump activity induced by noradrenaline is reduced by 35–45 percent, indicating the existence of a Na⁺/H⁺ exchange-independent mechanism of the Na⁺, K⁺ pump regulation by -adrenergic catecholamines. Hypertonic shrinkage of carp erythrocytes results in a 40- to 80-fold activation of Na⁺/H⁺ exchange, whereas hypotonic swelling induces an increase in the rate of ⁸⁶Rb⁺ efflux which is inhibited by furosemide by about 30–40 percent. The rate of pH₀ recovery in response to acidification or alkalinization in rat erythrocytes is approximately 15 times as fast as in carp erythrocytes. Unlike in rat erythrocytes, valinomycin does not cause an alkalinization of incubation medium in carp erythrocytes indicating the absence of conductive pathway in the operation of anion transporter protein. A scheme is suggested which describes the interrelation of Na⁺/H⁺ exchange, Na⁺, K⁺ pump and a non-identified system providing for K⁺ efflux in cell swelling, regulation of cell volume and cytoplasmic pH in fish erythrocytes under conditions of deep hypoxia and high activity.Abbreviations cAMP cyclic adenosine monophosphate - CCCP carbonylcyamide m-chlorophenylhydrazone - DMSO dimethylsulphoxide - EIPA ethylisopropylamiloride - NA noradrenaline - PMA -phorbol 12-myristate, 13-acetate - RVD regulatory volume decrease - RVI regulatory volume increase