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     

Mechanisms underlying turgor regulation in the estuarine alga Vaucheria erythrospora (Xanthophyceae) exposed to hyperosmotic shock

Aquatic organisms are often exposed to dramatic changes in salinity in the environment. Despite decades of research, many questions related to molecular and physiological mechanisms mediating sensing and adaptation to salinity stress remain unanswered. Here, responses of Vaucheria erythrospora, a turgor‐regulating xanthophycean alga from an estuarine habitat, have been investigated. The role of ion uptake in turgor regulation was studied using a single cell pressure probe, microelectrode ion flux estimation (MIFE) technique and membrane potential (E_m) measurements. Turgor recovery was inhibited by Gd³⁺, tetraethylammonium chloride (TEA), verapamil and orthovanadate. A NaCl‐induced shock rapidly depolarized the plasma membrane while an isotonic sorbitol treatment hyperpolarized it. Turgor recovery was critically dependent on the presence of Na⁺ but not K⁺ and Cl^? in the incubation media. Na⁺ uptake was strongly decreased by amiloride and changes in net Na⁺ and H⁺ fluxes were oppositely directed. This suggests active uptake of Na⁺ in V. erythrospora mediated by an antiport Na⁺/H⁺ system, functioning in the direction opposite to that of the SOS1 exchanger in higher plants. The alga also retains K⁺ efficiently when exposed to high NaCl concentrations. Overall, this study provides insights into mechanisms enabling V. erythrospora to regulate turgor via ion movements during hyperosmotic stress.     

Intrasynaptosomal Free Mg2+ Concentration Measured with the Fluorescent Indicator Mag-Fura-2: Modulation by Na+ Gradient and by Extrasynaptosomal ATP

Abstract: Synaptosomes can be loaded with mag-fura-2 without significant perturbation of their ATP content by incubation for 10 min at 37°C with 10 µM mag-fura-2 acetoxymethyl ester in Hanks'-HEPES buffer (pH 7.45). The intrasynaptosomal free Mg²⁺ concentration ([Mg²⁺]_i) was found to be dependent on external Mg²⁺ concentration, increasing from 0.8 to 1.25 mM when the concentration of Mg²⁺ in the incubation medium increased from 1 to 8 mM. Dissipation of the Na⁺ gradient across the plasma membrane of synaptosomes by treatment with the Na⁺ ionophore monensin (0.2 mM) or with veratridine (0.2 mM) and ouabain (0.6 mM) produced a moderate increase of [Mg²⁺]_i, from 1.0 to 1.2–1.3 mM in an incubation medium containing 5 mM Mg²⁺. Plasma membrane depolarization by incubation of synaptosomes in a medium containing 68 mM KCl and 68 mM NaCl had no effect on [Mg²⁺]_i. Reversal of the Na⁺ gradient by incubation of synaptosomes in a medium in which external Na⁺ was replaced by choline increased [Mg²⁺]_i up to 1.6 and 2.2 mM for extrasynaptosomal Mg²⁺ concentrations of 1 and 8 mM, respectively. We conclude that a Na⁺/Mg²⁺ exchange operates in the plasma membrane of synaptosomes. In the presence of Mg²⁺ in the incubation medium, extrasynaptosomal ATP, but not ADP or adenosine, increased [Mg²⁺]_i from 1.1 ± 0.1 up to 1.6 ± 0.1 mM. The nonhydrolyzable ATP analogue adenosine 5′-(βγ-imido)triphosphate antagonized the effect of ATP, but had no effect by itself on [Mg²⁺]_i. It is concluded that Mg²⁺ transport across the plasma membrane of synaptosomes is modulated by the activity of an ecto-ATPase or an ecto-protein kinase.     

Na+/H+ antiport activity in tonoplast vesicles isolated from sunflower roots induced by NaCl stress

Na⁺ transport across the tonoplast and its accumulation in the vacuoles is of crucial importance for plant adaptation to salinity. Mild and severe salt stress increased both ATP- and PP_i-dependent H⁺ transport in tonoplast vesicles from sunflower seedling roots, suggesting the possibility that a Na⁺/H⁺ antiport system could be operating in such vesicles under salt conditions (E. Ballesteros et al. 1996. Physiol. Plant. 97: 259–268). During a mild salt stress, Na⁺ was mainly accumulated in the roots. Under a more severe salt treatment, Na⁺ was equally distributed in shoots and roots. In contrast to what was observed with Na⁺, all the salt treatments reduced the shoot K⁺ content. Dissipation by Na⁺ of the H⁺ gradient generated by the tonoplast H⁺-ATPase, monitored as fluorescence quenching of acridine orange, was used to measure Na⁺/H⁺ exchange across tonoplast-enriched vesicles isolated by sucrose gradient centrifugation from sunflower (Helianthus annuus L.) roots treated for 3 days with different NaCl regimes. Salt treatments induced a Na⁺/H⁺ exchange activity, which displayed saturation kinetics for Na⁺ added to the assay medium. This activity was partially inhibited by 125 μM amiloride, a competitive inhibitor of Na⁺/H⁺ antiports. No Na⁺/H⁺ exchange was detected in vesicles from control roots. The activity was specific for Na⁺. since K⁺ added to the assay medium slightly dissipated H⁺ gradients and displayed non-saturating kinetics for all salt treatments. Apparent K_m for Na⁺/H⁺ exchange in tonoplast vesicles from 150 mM NaCl-treated roots was lower than that of 75 mM NaCl-treated roots, V_max remaining unchanged. The results suggest that the existence of a specific Na⁺/H⁺ exchange activity in tonoplast-enriched vesicle fractions, induced by salt stress, could represent an adaptative response in sunflower plants, moderately tolerant to salinity.     

PURIFICATION AND CHARACTERIZATION OF PYRUVATE KINASE FROM THE GREEN ALGA CHLAMYDOMONAS REINHARDTII1

A single form of pyruvate kinase was isolated from the green alga Chlamydomonas reinhardtii Dang. (Chlorophyta) and partially purified over twentyfold, yielding a final specific activity of 2.68 μmol pyruvate produced-min^-1.mg^-1 protein. Studies of its physical characteristics reveal that the pyruvate kinase is heat stable, is partially inactivated by sulfhydryl reagent N-ethylmaleimide, and has a pH optimum at 6.8 and a native molecular mass of 224 kDa. Immunological precipitation and western blotting, using antibodies raised against Selenastrum minutum Naeg. (Chlorophyta) cytosolic pyruvate kinase, reveal that C. reinhardtii pyruvate kinase possesses a subunit molecular mass of 57 kDa, indicating a homo-tetrameric structure. This enzyme exhibits an absolute requirement for a divalent cation that can be fulfilled, by Mg²⁺. The monovalent cation K⁺ acts as a strong activator. The K_m values for phosphoenolpyruvate and adenosine diphosphate (ADP) are 0.16 mM and 0.18 mM, respectively. The enzyme is capable of using other nucleotides with V_max for UDP, GDP, IDP, and CDP of 70%, 55%, 53%, and 25% of that with ADP, respectively. Dihydroxyacetone phosphate, ribulose 1,5-bisphosphate, adenosine monophosphate (AMP), ribose-5-phosphate, and glyceraldehyde-3-phosphate are activators, whereas glutamate, orthophosphate, adenosine triphosphate (ATP), citrate, isocitrate, malate, oxalate, phosphoglycolate, and 2,3-diphosphoglycerate are potent inhibitors of this enzyme. Dihydroxyacetone phosphate can reverse the inhibition by glutamate and phosphate. These properties are discussed in light of pyruvate kinase regulation during anabolic and catabolic respiration. Substrate interaction and product inhibition studies indicate that ADP is the first substrate bound to the enzyme and pyruvate is the last product released (Ordered Bi Bi mechanism).     

Hormone-induced changes in pyruvate kinase activity in isolated hepatocytes II. Relation to the hormonal regulation of gluconeogenesis gluconeogenesis

1. 1. Incubation of isolated hepatocytes with glucagon (10⁻⁶ M) or dibutyryl cyclic AMP (0.1 mM) causes a decrease in pyruvate kinase activity of 50%, measured at suboptimal substrate (phosphoenolpyruvate) concentrations and 1 mM Mg_free²⁺. The magnitude of the decrease in activity is not influenced by the applied extracellular concentrations of lactate (1 and 5 mM), glucose (5 and 30 mM) or fructose (10 and 25 mM). With all three substrates comparable inhibition percentages are induced by glucagon or dibutyryl cyclic AMP.

2. 2. The extent of inhibition of pyruvate kinase induced by incubation of hepatocytes with glucagon or dibutytyl cyclic AMP is not influenced by the extracellular Ca²⁺ concentration nor by the presence of 2 mM EGTA. The reactivation of pyruvate kinase seems to be inhibited by a high concentration of extracellular Ca²⁺ (2.6 mM) as compared to a low concentration of extracellular Ca²⁺ (0.26 mM).

3. 3. Incubation of hepatocytes in a Na⁺-free, high K⁺-concentration medium does not influence the magnitude of the pyruvate kinase inhibition induced by dibutyryl cyclic AMP. However, the reactivation reaction is stimulated under these incubation conditions.

4. 4. Incubation of hepatocytes with dibutyryl cyclic GMP (0.1 mM) leads to a 25% decrease in pyruvate kinase activity. The magnitude of the inhibition by dibutyryl cyclic (GMP) is not influenced by the presence of pyruvate (1 mM) or glucose (5 mM and 30 mM).

5. 5. The relative insensitivity of the pyruvate kinase inhibition induced by glucagon, dibutyryl cyclic AMP and dibutyryl cyclic GMP to the extracellular environment leads to the conclusion that the hormonal regulation of pyruvate kinase is not the only site of hormonal regulation of glycolysis and gluconeogenesis. It is concluded that hormonal regulation of pyruvate kinase activity is exerted by changes in the degree of (de)phosphorylation of the enzyme reflecting acute hormonal control as well as by changes in the concentration of the allosteric activator fructose 1,6-diphosphate. The latter depends at least in part on the hormonal control of the phosphofructokinase-fructose-1,6-phosphatase cycle.

Role of calcium and calmodulin in the regulation of the rabbit ileal brush-border membrane Na+/H+ antiporter

Summary In rabbit ileum, Ca²⁺/calmodulin (CaM) appears to be involved in physiologically inhibiting the linked NaCl absorptive process, since inhibitors of Ca²⁺/CaM stimulate linked Na⁺ and Cl^– absorption. The role of Ca²⁺/CaM-dependent phosphorylation in regulation of the brush-border Na⁺/H⁺ antiporter, which is believed to be part of the neutral linked NaCl absorptive process, was studied using purified brush-border membrane vesicles, which contain both the Na⁺/H⁺ antiporter and Ca²⁺/CaM-dependent protein kinase(s) and its phosphoprotein substrates. Rabbit ileal villus cell brush-border membrane vesicles were prepared by Mg precipitation and depleted of ATP. Using a freezethaw technique, the ATP-depleted vesicles were loaded with Ca²⁺, CaM, ATP and an ATP-regenerating system consisting of creatine kinase and creatine phosphate. The combination of Ca²⁺/CaM and ATP inhibited Na⁺/H⁺ exchange by 45±13%. This effect was specific since Ca²⁺/CaM and ATP did not alter diffusive Na⁺ uptake, Na⁺-dependent glucose entry, or Na⁺ or glucose equilibrium volumes. The inhibition of the Na⁺/H⁺ exchanger by Ca²⁺/CaM/ATP was due to an effect on theV _max and not on theK _m for Na⁺. In the presence of CaM and ATP, Ca²⁺ caused a concentration-dependent inhibition of Na⁺ uptake, with an effect 50% of maximum occurring at 120nm. This Ca²⁺ concentration dependence was similar to the Ca²⁺ concentration dependence of Ca²⁺/CaM-dependent phosphorylation of specific proteins in the vesicles. The Ca²⁺/CaM/ATP-inhibition of Na⁺/H⁺ exchange was reversed by W₁₃, a Ca²⁺/CaM antagonist, but not by a hydrophobic control, W₁₂, or by H-7, a protein kinase C antagonist. we conclude that Ca²⁺, acting through CaM, regulates ileal brush-border Na⁺/H⁺ exchange, and that this may be involved in the regulation of neutral linked NaCl absorption.     

Adaptation to phosphate deprivation in osteoblast-like cells

The rat osteosarcoma cell line UMR-106–01 has an osteoblast-like phenotype. When grown in monolyer culture these cells transport inroganic phosphate and L-alanine via Na⁺-dependent transport systems. Exposure of these cells to a low phosphate medium for 4 h produced a 60–70 per cent increase in Na⁺-dependent phosphate uptake compared to control cells maintained in medium with a normal phosphate concentration. In contrast, Na⁺-dependent alanine uptake and Na⁺-independent phosphate uptake were not changed during phosphate deprivation. The increased phosphate uptake was due, in part, to an increased V_max and was blocked completely by pretreatment with cycloheximide (70 μM). In these cells recovery of intracellular pH after acidification with NH₄Cl is due primarily to the Na⁺/H⁺ exchange system. The rate of this recovery process, monitored with a pH sensitive indicator (BCECF), was decreased by more than 50 per cent in phosphate-deprived cells compared to controls indicating that Na⁺/H⁺ exchange was inhibited during phosphate deprivation.     

Role of Na+-Ca2+ Exchanger in Agonist-Induced Ca2+ Signaling in Cultured Rat Astrocytes

Abstract: We have previously demonstrated that activation of the Na⁺-Ca²⁺ exchanger in the reverse mode causes Ca²⁺ influx in astrocytes. In addition, we showed that the exchange activity was stimulated by nitric oxide (NO)/cyclic GMP and inhibited by ascorbic acid. The present study demonstrates that the Na⁺-Ca²⁺ exchanger is involved in agonist-induced Ca²⁺ signaling in cultured rat astrocytes. The astrocytic intracellular Ca²⁺ concentration ([Ca²⁺]_i) was increased by l -glutamate, noradrenaline (NA), and ATP, and the increases were all attenuated by the NO generator sodium nitroprusside (SNP). SNP also reduced the ionomycin-induced increase in [Ca²⁺]_i. The Na-induced Ca²⁺ signal was also attenuated by S-nitroso-l -cysteine and 8-bromo cyclic GMP, whereas it was enhanced by 3,4-dichlorobenzamil, an inhibitor of the Na⁺-Ca²⁺ exchanger. Treatment of astrocytes with antisense, but not sense, deoxynucleotides to the sequence encoding the Na⁺-Ca²⁺ exchanger enhanced the ionomycin-induced increase in [Ca²⁺]_i and blocked the effects of SNP and 8-bromo cyclic GMP in reducing the NA-induced Ca²⁺ signal. Furthermore, the ionomycin-induced Ca²⁺ signal was enhanced by removal of extracellular Na⁺ and pretreatment with ascorbic acid. These findings indicate that the Na⁺-Ca²⁺ exchanger is a target for NO modulation of elevated [Ca²⁺]_i and that the exchanger plays a role in Ca²⁺ efflux when [Ca²⁺]_i is raised above basal levels in astrocytes.     

Regulation of Intestinal Epithelial Brush Border Na+/H+ Exchanger Isoforms, NHE2 and NHE3, in C2bbe Cells

Until recently, studies to characterize the intestinal epithelial Na⁺/H⁺ exchangers had to be done in nonepithelial, mutated fibroblasts. In these cells, detection of any Na⁺/H⁺ exchange activity requires prior acid loading. Furthermore, most of these experiments used intracellular pH changes to measure NHE activity. Because changes in pH _i only approximate Na⁺/H⁺ exchange activity, and may be confounded by alterations in buffering capacity and/or non-NHE contributions to pH regulation, we have used ²²[Na] unidirectional apical to cell uptake to measure activities specific to NHE2 or NHE3. Furthermore, we performed these measurements under basal, nonacid-stimulated conditions to avoid bias from this nonphysiological experimental precondition. Both brush border NHEs, when expressed in the well-differentiated, intestinal villuslike Caco-2 subclone, C2bbe (C2), localize to the C2 apical domain and are regulated by second messengers in the same way they are regulated in vivo. Increases in intracellular calcium and cAMP inhibit both isoforms, while phorbol ester affects only NHE3. NHE2 inhibition by cAMP and Ca⁺⁺ involves changes to both K _Na and V _max . In contrast, the same two second messengers inhibit NHE3 by a decrease in V _max exclusively. Phorbol ester activation of protein kinase C alters both V _max and K _Na of NHE3, suggesting a multilevel regulatory mechanism. We conclude that NHE2 and NHE3, in epithelial cells, are basally active and are differentially regulated by signal transduction pathways. Received: 28 January 1999/Revised: 18 May 1999     

Ion transport across leech integument

The dorsal skin of the leech Hirudo medicinalis was used for electrophysiological measurements performed in Ussing chambers. The leech skin is a tight epithelium (transepithelial resistance = 10.5±0.5 k· cm^-2) with an initial short-circuit current of 29.0±2.9 A·cm^-2. Removal of Na⁺ from the apical bath medium reduced short-circuit current about 55%. Ouabain (50mol·l^-1) added to the basolateral solution, depressed the short-circuit current completely. The Na⁺ current saturated at a concentration of 90 mmol Na⁺·l^-1 in the apical solution (K _M=11.2±1.8 mmol·l^-1). Amiloride (100 mol·l^-1) on the apical side inhibited ca. 40% of the Na⁺ current and indicated the presence of Na⁺ channels. The dependence of Na⁺ current on the amiloride concentration followed Michaclis-Menten kinetics (K _i=2.9±0.4 mol·l^-1). The amiloride analogue benzamil had a higher affinity to the Na⁺ channel (K _i=0.7±0.2 mol·l^-1). Thus, Na⁺ channels in leech integument are less sensitive to amiloride than channels known from vertebrate epithelia. With 20 mmol Na⁺·l^-1 in the mucosal solution the tissue showed an optimum amiloride-inhibitable current, and the amiloride-sensitive current under this condition was 86.8±2.3% of total short-circuit current. Higher Na⁺ concentrations lead to a decrease in amiloride-blockade short-circuit current. Sitmulation of the tissue with cyclic adenosine monophosphate (100 mol·l^-1) and isobutylmethylxanthine (1 mmol·l^-1) nearly doubled short-circuit current and increased amiloride-sensitive Na⁺ currents by 50%. By current fluctuation analysis we estimated single Na⁺ channel current (2.7±0.9 pA) and Na⁺ channel density (3.6±0.6 channels·m^-2) under control conditions. After cyclic adenosine monophosphate stimulation Na⁺ channel density increased to 5.4±1.1 channels·m^-2, whereas single Na⁺ channel current showed no significant change (1.9±0.2 pA). These data present a detailed investigation of an invertebrate epithelial Na⁺ channel, and show the similarities and differences to vertebrate Na⁺ channels. Whereas the channel properties are different from the classical vertebrate Na⁺ channel, the regulation by cyclic adenosine monophosphate seems similar. Stimulation of Na⁺ uptake by cyclic adenosine monophosphate is mediated by an increasing number of Na⁺ channels.Abbreviations slope of the background noise component - ADH antidiuretic hormone - cAMP cyclic adenosine monophosphate - f frequency - f _c coner frequency of the Lorentzian noise component - Hepes N-hydroxyethylpiperazine-N-ethanesulphonic acid - BMX isobutyl-methylxanthine - i _Na single Na⁺ channel current - I _Na max, maximal inhibitable Na⁺ current - I _SC short circuit current - K _i half maximal blocker concentration - K _M Michaelis constandard error of the mean - S _(f) power density of the Lorentzian noise component - S ₀ plateau value of the Lorentzian noise component - TMA tetramethylammonium - Trizma TRIS-hydroxymethyl-amino-methane - V _max maximal reaction velocity - V _T transepithelial potential - K half maximal blocker concentration     

Endothelin 1 Stimulates Na+,K+-ATPase and Na+-K+-Cl− Cotransport Through ETA Receptors and Protein Kinase C-Dependent Pathway in Cerebral Capillary Endothelium

Abstract: The effect of endothelins (ET-1 and ET-3) on ⁸⁶Rb⁺ uptake as a measure of K⁺ uptake was investigated in cultured rat brain capillary endothelium. ET-1 or ET-3 dose-dependently enhanced K⁺ uptake (EC₅₀ = 0.60 ± 0.15 and 21.5 ± 4.1 nM, respectively), which was inhibited by the selective ET_A receptor antagonist BQ 123 (cyclo-d -Trp-d -Asp-Pro-d -Val-Leu). Neither the selective ET_B agonists IRL 1620 [N-succinyl-(Glu⁹,-Ala^11,15)-ET-1] and sarafotoxin S6c, nor the ET_B receptor antagonist IRL 1038 [(Cys¹¹,Cys¹⁵)-ET-1] had any effect on K⁺ uptake. Ouabain (inhibitor of Na⁺,K⁺-ATPase) and bumetanide (inhibitor of Na⁺-K⁺-Cl^? cotransport) reduced (up to 40% and up to 70%, respectively) the ET-1-stimulated K⁺ uptake. Complete inhibition was seen with both agents. Phorbol 12-myristate 13-acetate (PMA), activator of protein kinase C (PKC), stimulated Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport. ET-1-but not PMA-stimulated K⁺ uptake was inhibited by 5-(N-ethyl-N-isopropyl)amiloride (inhibitor of Na⁺/H⁺ exchange system), suggesting a linkage of Na⁺/H⁺ exchange with ET-1-stimulated Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport activity that is not mediated by PKC.     

Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange activity in the alkaliphile halotolerant cyanobacterium <Emphasis Type="Italic">Aphanothece halophytica</Emphasis>

The activity of Na⁺/H⁺ exchanger to remove toxic Na⁺ is important for growth of organisms under high salinity. In this study, the halotolerant cyanobacterium Aphanothece halophytica was shown to possess Na⁺/H⁺ exchange activity since exogenously added Na⁺ could dissipate a pre-formed pH gradient, and decrease extracellular pH. Kinetic analysis yielded apparent K _m (Na⁺) and V _max of 20.7 ± 3.1 mM and 3,333 ± 370 nmol H⁺ min⁻¹ mg⁻¹, respectively. For cells grown under salt-stress condition, the apparent K _m (Na⁺) and V _max was 18.3 ± 3.5 mM and 3,703 ± 350 nmol H⁺ min⁻¹ mg⁻¹, respectively. Three cations with decreasing efficiency namely Li⁺, Ca²⁺, and K⁺ were also able to dissipate pH gradient. Only marginal exchange activity was observed for Mg²⁺. The exchange activity was strongly inhibited by Na⁺-gradient dissipators, monensin, and sodium ionophore as well as by CCCP, a protonophore. A. halophytica showed high Na⁺/H⁺ exchange activity at neutral and alkaline pH up to pH 10. Cells grown at pH 7.6 under high salinity exhibited higher Na⁺/H⁺ exchange activity than those grown under low salinity during 15 days of growth suggesting a role of Na⁺/H⁺ exchanger for salt tolerance in A. halophytica. Cells grown at alkaline pH of 9.0 also exhibited a progressive increase of Na⁺/H⁺ exchange activity during 15 days of growth.     

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.     

Activation of Na+ /H+ exchange on rat preadipocyte plasma membrane and its role in cell proliferation and differentiation

Anin vitro cultured rat perirenal preadipocyte (PA) was established as a model system to investigate the role of the intracellular pH (pHi) and of the Na⁺ /H⁺ exchanger during PA proliferation and differentiation. pH sensitive probe, 2′,7′-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein(BCECF), was employed to measure the pHi of PA and to determine the Na⁺/H⁺ exchange activity. The results showed that there was Na⁺/H⁺ exchange activity in the plasma membrane of PA, FCS stimulated DNA synthesis measured by³H-TdR incorporation, and the activation of Na⁺ /H⁺ exchanger resulted in pHi increase (nearly 0.2 pH unit) within 2 min. Ethyl-isopropyl-amiloride (EIPA), a specific Na⁺/H⁺ exchange inhibitor, inhibited Na⁺/H⁺ exchange activity and DNA synthesis. In the absence of serum insulin did not stimulate DNA synthesis but did induce PA differentiation characterized by the appearance of adiposome in the cell and the enhancement of glyeerol-3-phosphate dehydrogenase (G₃PDHase) activity. Meantime, insulin was also found to stimulate Na⁺/H⁺ exchange activity and pHi increase. EIPA inhibited Na⁺/H⁺ exchanger activation induced by insulin and also partially inhibited the enhancement of G₃PDHase activity. These results demonstrated that the activation of Na⁺ /H⁺ exchange and the resulting pHi increase are the early events related to both proliferation and differentiation of PA.     

Kinetic properties of sodium transport pathways in the river lamprey <Emphasis Type="Italic">Lampetra fluviatilis</Emphasis> erythrocytes

To activate Na⁺/H⁺ exchange, intracellular pH (pH_i) of erythrocytes of the river lamprey Lampetra fluviatilis were changed from 6 and 8 using nigericin. The Na⁺/H⁺ exchanger activity was estimated from the values of amiloride-sensitive components of Na⁺ (²²Na) inflow or of H⁺ outflow from erythrocytes. Kinetic parameters of the carrier functioning were determined by using Hill equation. Dependence of Na⁺ and H⁺ transport on pH_i value is described by hyperbolic function with the Hill coefficient value (n) close to 1. Maximal rate of ion transport was within the limits of 9–10 mmol/l cells/min, and the H⁺ concentration producing the exchanger 50% activation amounted to 0.6–1.0 μM. Stimulation of H⁺ outcome from acidified erythrocytes (pH_i 5.9) with increase of H⁺ concentration in the incubation medium is described by Hill equation with n value of 1.6. Concentration Na⁺ for the semimaximal stimulation of H⁺ outcome amounted to 10 mM. The obtained results indicate the presence in lamprey erythrocytes of only binding site for H⁺ from the cytoplasm side and the presence of positive cooperativity in Na⁺-binding from the extracellular side of the Na⁺/H⁺ exchanger. Na⁺ efflux from cells in the Na⁺-free medium did not change at a 10-fold increase of H⁺ concentration in the incubation medium. The presented data indicate differences of kinetic properties of the lamprey erythrocyte Na⁺/H⁺ exchanger and of this carrier isoforms in mammalian cells. In intact erythrocytes the dependence of the amiloride-sensitive Na⁺ inflow on its concentration in the medium is described by Hill equitation with n 1.6. The Na⁺ concentration producing the 50% transport activation amounted to 39 mM and was essentially higher as compared with that in acidified erythrocytes. These data confirm conception of the presence of two amiloride-sensitive pathways of Na+ transport in lamprey erythrocytes.     

Interactions of external and internal H+ and Na+ with Na+/Na+ and Na+/H+ exchange of rabbit red cells: Evidence for a common pathway

Summary We have studied the kinetic properties of rabbit red cell (RRBC) Na⁺/Na⁺ and Na⁺/H⁺ exchanges (EXC) in order to define whether or not both transport functions are conducted by the same molecule. The strategy has been to determine the interactions of Na⁺ and H⁺ at the internal (i) and external (o) sites for both exchanges modes. RRBC containing varying Na _i and H _l were prepared by nystatin and DIDS treatment of acid-loaded cells. Na⁺/Na⁺ EXC was measured as Na _o -stimulated Na⁺ efflux and Na⁺/H⁺ EXC as Na _o -stimulated H⁺ efflux and pH _o -stimulated Na⁺ influx into acid-loaded cells.The activation of Na⁺/Na⁺ EXC by Na _o at pH _i 7.4 did not follow simple hyperbolic kinetics. Testing of different kinetic models to obtain the best fit for the experimental data indicated the presence of high (K _m 2.2 mM) and low affinity (K _m 108 mM) sites for a single- or two-carrier system. The activation of Na⁺/H⁺ EXC by Na _o (pH _i 6.6, Na _i <1 mM) also showed high (K _m 11 mM) and low (K _m 248 mM) affinity sites. External H⁺ competitively inhibited Na⁺/Na⁺ EXC at the low affinity Na _o site (K _H 52 nM) while internally H⁺ were competitive inhibitors (pK 6.7) at low Na _i and allosteric activators (pK 7.0) at high Na _i .Na⁺/H₊ EXC was also inhibited by acid pH _o and allosterically activated by H _i (pK 6.4). We also established the presence of a Na _i regulatory site which activates Na⁺/H⁺ and Na⁺/Na⁺ EXC modifying the affinity for Na _o of both pathways. At low Na _i , Na⁺/Na⁺ EXC was inhibited by acid pH _i and Na⁺/H⁺ stimulated but at high Na _i , Na⁺/Na⁺ EXC was stimulated and Na⁺/H⁺ inhibited being the sum of both pathways kept constant. Both exchange modes were activated by two classes of Na _o sites,cis-inhibited by external H _o , allosterically modified by the binding of H⁺ to a H _i regulatory site and regulated by Na _i . These findings are consistent with Na⁺/Na⁺ EXC being a mode of operation of the Na⁺/H⁺ exchanger.Na⁺/H⁺ EXC was partially inhibited (80–100%) by dimethyl-amiloride (DMA) but basal or pH _i -stimulated Na⁺/Na⁺ EXC (pH _i 6.5, Na _i 80 mM) was completely insensitive indicating that Na⁺/Na⁺ EXC is an amiloride-insensitive component of Na⁺/H⁺ EXC. However, Na⁺ and H⁺ efflux into Na-free media were stimulated by cell acidification and also partially (10 to 40%) inhibited by DMA: this also indicates that the Na⁺/H⁺ EXC might operate in reverse or uncoupled modes in the absence of Na⁺/Na⁺ EXC.In summary, the observed kinetic properties can be explained by a model of Na⁺/H⁺ EXC with several conformational states, H _i and Na _i regulatory sites and loaded/unloaded internal and external transport sites at which Na⁺ and H⁺ can compete. The occupancy of the H⁺ regulatory site induces a conformational change and the occupancy of the Na _i regulatory site modulates the flow through both pathways so that it will conduct Na⁺/H⁺ and/or Na⁺/Na⁺ EXC depending on the ratio of internal Na⁺:H⁺.     

Involvement of na in active uptake of pyruvate in mesophyll chloroplasts of some c(4) plants : na/pyruvate cotransport

An artificial Na⁺ gradient across the envelope (Na⁺ jump) enhanced pyruvate uptake in the dark into mesophyll chloroplasts of a C₄ plant, Panicum miliaceum (NAD-malic enzyme type) (J Ohnishi, R Kanai [1987] FEBS Lett 219:347). In the present study, ²²Na⁺ and pyruvate uptake were examined in mesophyll chloroplasts of several species of C₄ plants. Enhancement of pyruvate uptake by a Na⁺ jump in the dark was also seen in mesophyll chloroplasts of Urochloa panicoides and Panicum maximum (phosphoenolpyruvate carboxykinase types) but not in Zea mays or Sorghum bicolor (NADP-malic enzyme types). In mesophyll chloroplasts of P. miliaceum and P. maximum, pyruvate in turn enhanced Na⁺ uptake in the dark when added together with Na⁺. When flux of endogenous Na⁺ was measured in these mesophyll chloroplasts preincubated with ²²Na⁺, pyruvate addition induced Na⁺ influx, and the extent of the pyruvate-induced Na⁺ influx positively correlated with that of pyruvate uptake. A Na⁺/H⁺ exchange ionophore, monensin, nullified all the above mutual effects of Na⁺ and pyruvate in mesophyll chloroplasts of P. miliaceum, while it accelerated Na⁺ uptake and increased equilibrium level of chloroplast ²²Na⁺. Measurements of initial uptake rates of pyruvate and Na⁺ gave a stoichiometry close to 1:1. These results point to Na⁺/pyruvate cotransport into mesophyll chloroplasts of some C₄ plants.     

Regulation of pyruvate kinase from Propionibacterium shermanii

Pyruvate kinase from Propionibacterium shermanii was shown to be activated by glucose-6-phosphate (G-6-P) at non-saturating phosphoenol pyruvate (PEP) concentrations but other glycolytic and hexose monophosphate pathway intermediates and AMP were without effect. Half-maximal activation was obtained at 1 mM G-6-P. The presence of G-6-P decreased both the PEP_0.5V and ADP_0.5V values and the slope of the Hill plots for both substrates. The enzyme was strongly inhibited by ATP and inorganic phosphate (P_i) at all PEP concentrations. At non-saturating (0.5 mM) PEP, half-maximal inhibition was obtained at 1.8 mM ATP or 1.4 mM P_i. The inhibition by both P_i and ATP was largely overcome by 4 mM G-6-P. The specific activity of pyruvate kinase was considerably higher in lactate-, glucose- and glycerol-grown cultures than that of the enzyme catalysing the reverse reaction, pyruvate, phosphate dikinase. It is suggested that the activity of pyruvate kinase in vivo is determined by the balance between activators and inhibitors such that it is inhibited during gluconeogenesis while, during glycolysis, the inhibition is relieved by G-6-P.Abbreviations PEP phosphoenolpyruvate - G-6-P glucose-6-phosphate - P_i inorganic phosphate     

The Temperature Dependence of Intracellular pH in Isolated Frog Skeletal Muscle: Lessons Concerning the Na+-H+ Exchanger

We used ³¹P NMR to investigate the temperature-dependence of intracellular pH (pH _i ) in isolated frog skeletal muscles. We found that ln[H⁺ _i ] is a linear function of 1/T _abs paralleling those of neutral water (i.e., H⁺= OH⁻) and of a solution containing the fixed pH buffers of frog muscle cytosol. This classical van't Hoff relationship was unaffected by inhibition of glycolysis and was not dependent upon the pH or [Na⁺] in the bathing solution. Insulin stimulation of Na⁺-H⁺ exchange shifted the intercept in the alkaline direction but had no effect on the slope. Acid loading followed by washout resulted in an amiloride-sensitive return to the (temperature dependent) basal pH _i . These results show that the temperature dependence of activation of Na⁺-H⁺ exchange is similar to that of the intracellular buffers, and suggest that constancy of [H⁺]/[OH⁻] with changing temperature is achieved in the short term by intracellular buffering and in the long term by the set-point of the Na⁺-H⁺ exchanger. Proton activation of the exchanger has an apparent standard enthalpy change (ΔH°) under both control and insulin-stimulated conditions that is similar to the ΔH° of the intracellular buffers and approximately half of the ΔH° for the dissociation of water. Thus, the temperature-dependent component of the standard free-energy change (ΔF°) is unaffected by insulin stimulation, suggesting that changes in Arrhenius activation energy (E _a ) may not be a part of the mechanism of hormone stimulation. Received: 12 February 1997/Revised: 1 October 1997