Characterization of Na(+)-dependent Mg2+ efflux from Mg2(+)-loaded rat erythrocytes

Na(+)-dependent Mg2+ efflux from Mg2(+)-loaded rat erythrocytes was determined from the increase of extracellular Mg2+ concentration or decrease of intracellular Mg2+ content, as measured by means of atomic absorption spectrophotometry. Mg2+ efflux was specifically combined with the uptake of Na+ at a stoichiometric ratio of 2Na+:1Mg2+, indicating electroneutral Na+/Mg2+ antiport. Na+/Mg2+ antiport depended on intracellular ATP and was inhibited by amiloride and quinidine, but was insensitive to strophanthin. Net Mg2+ efflux was only occurring at increased concentration of intracellular Mg2+ ([Mg2+]i), and stopped when the physiological Mg2+ content was reached. Intracellular Mg2+ acted cooperatively with a Hill coefficient of 2.4, which may indicate gating of Na+/Mg2+ antiport at increased [Mg2+]i. At increased intracellular Na+ concentration, Na+ competed with intracellular Mg2+ for Mg2+ efflux and Na+ could leave the rat erythrocyte via this transport system. Na+/Mg2+ antiport was working asymmetrically with respect to extra- and intracellular Na+ and Mg2+, and did not perform net Mg2+ uptake.     

Regulation of Na+/Mg2+ antiport in rat erythrocytes

In rat erythrocytes, the regulation of Na+/Mg2+ antiport by protein kinases (PKs), protein phosphatases (PPs), intracellular Mg2+, ATP and Cl- was investigated. In untreated erythrocytes, Na+/Mg2+ antiport was slightly inhibited by the PK inhibitor staurosporine, slightly stimulated by the PP inhibitor calyculin A and strongly stimulated by vanadate. PMA stimulated Na+/Mg2+ antiport. This effect was completely inhibited by staurosporine and partially inhibited by the PKC inhibitors Ro-31-8425 and BIM I. Participation of other PKs such as PKA, the MAPK cascade, PTK, CK I, CK II, CAM II-K, PI 3-K, and MLCK was excluded by use of inhibitors. Na+/Mg2+ antiport in rat erythrocytes can thus be stimulated by PKCalpha. In non-Mg2+ -loaded erythrocytes, ATP depletion reduced Mg2+ efflux and PMA stimulation in NaCl medium. A drastic activation of Na+/Mg2+ antiport was induced by Mg2+ loading which was not further stimulated by PMA. Staurosporine, Ro-31-8425, BIM I and calyculin A did not inhibit Na+/Mg2+ antiport of Mg2+ -loaded cells. Obviously, at high [Mg2+]i Na+/Mg2+ antiport is maximally stimulated. PKCalpha or PPs are not involved in stimulation by intracellular Mg2+. ATP depletion of Mg2+ -loaded erythrocytes reduced Mg2+ efflux and the affinity of Mg2+ binding sites of the Na+/Mg2+ antiporter to Mg2+. In non-Mg2+ -loaded erythrocytes Na+/Mg2+ antiport essentially depends on Cl-. Mg2+ -loaded erythrocytes were less sensitive to the activation of Na+/Mg2+ antiport by [Cl-]i.     

Na+-independent Mg2+ efflux from Mg2+-loaded human erythrocytes

Net Mg2+ efflux from Mg2+-loaded human erythrocytes was maximal after reincubation in sucrose. Net Mg2+ efflux was not inhibited by furosemide or bumetanide and, therefore, was not performed by the (Na,K,Cl)- or (K,Cl)-cotransport system. A component of net Mg2+ efflux was inhibited by extracellular NaC1, KCl, LiCl, choline Cl and SITS, in analogy to the inhibition of net Cl- and SITS. Therefore, it was concluded that net Mg2+ efflux is dependent on net Cl- efflux for charge compensation. Cl- -dependent net Mg2+ efflux was inhibited by amiloride. Only 10% of the maximal net Mg2+ efflux may depend on extracellular Na+.     

Stimulation of Na+/Mg2+ antiport in rat erythrocytes by intracellular Cl-

Mg(2+) efflux from rat erythrocytes was measured in NaCl, NaNO(3), NaSCN and Na gluconate medium. Substitution of extracellular and intracellular Cl(-) with the permeant anions NO(3)(-) and SCN(-) reduced Mg(2+) efflux via Na(+)/Mg(2+) antiport. After substitution of extracellular Cl(-) with the non-permeant anion gluconate, Mg(2+) efflux was not significantly reduced. In Na gluconate medium, an influence of the changed membrane potential and intracellular pH on Mg(2+) efflux could be excluded. The results indicate the existence of Cl(-)-independent Na(+)/Mg(2+) antiport and of Na(+)/Mg(2+) antiport stimulated by intracellular Cl(-). Intracellular Cl(-), as determined by means of (36)Cl(-), was found to stimulate Na(+)/Mg(2+) antiport through a cooperative effect according to a sigmoidal kinetics. The Hill coefficient for intracellular Cl(-) amounted to 1.4-1.8, indicating that two intracellular Cl(-) may be simultaneously active. With respect to specificity, Cl(-) was most effective, followed by Br(-), J(-), and F(-). Stimulation of Na(+)/Mg(2+) antiport by intracellular Cl(-) together with intracellular Mg(2+) may play a role during deoxygenation of erythrocytes and in essential hypertension.     

Characterization of Na+/Mg2+ antiport by simultaneous 28Mg2+ influx

During net Mg2+ efflux from Mg2+-preloaded chicken erythrocytes, which occurs via Na+/Mg2+ antiport, 28Mg2+ is taken up intracellularly. Km of 28Mg2+ influx amounted to 1 mM. In Na+-free medium Vmax of 28Mg2+ influx was increased and Km was reduced to 0.2 mM. 28Mg2+ influx was noncompetitively inhibited by amiloride as was found for Na+/Mg2+ antiport. The results indicate that, extracellularly, Mg2+ can compete with Na+ for common binding sites of the Na+/Mg2+ antiporter, resulting in 28Mg2+-24Mg2+ exchange. The rate of Mg2+ exchange depends on extracellular Na+ and on the rate of net Mg2+ efflux.     

Mg2+ efflux is accomplished by an amiloride-sensitive Na+/Mg2+ antiport

Mg2+ efflux from Mg2+-preloaded chicken erythrocytes is caused by an electroneutral Na+/Mg2+ antiport. It depends specifically on extracellular Na+, according to Michaelis-Menten kinetics (Km = 25 mM), and is reversibly noncompetitively inhibited by amiloride (Ki = 0.59 mM). In contrast to Na+/H+ antiport, Li+, Ca2+ and N-ethylmaleimide do not interfere with Na+/Mg2+ antiport. The Na+/Mg2+ antiport is driven by the intracellular/extracellular Mg2+ gradient.     

Activation of Na+/Mg2+ antiport in thymocytes by cAMP.

Mg2+ efflux from Mg(2+)-loaded rat thymocytes was stimulated by 0.1 mM dibutyryl cAMP (db cAMP). The activation of Mg2+ efflux by db cAMP was more expressed at lower Mg(2+)-loading. cAMP induced only a very small increase in the concentration of intracellular free Mg2+ which cannot explain the activation of Na+/Mg2+ antiport. From these results it was concluded that cAMP increases the affinity of the Na+/Mg2+ antiporter for intracellular Mg2+, probably by phosphorylation.     

A one-to-one Mg2+:Mn2+ exchange in rat erythrocytes

Mg2+ efflux in rat erythrocytes was stimulated by increases in external Na+ concentration following a Michaelian-like function with an apparent dissociation constant (KNa) of 11 +/- 3 mM (mean +/- S.D. of three experiments) and a variable maximal rate ranging from 150 to 1200 mumol (liter (1) cells X h)-1. Na+-stimulated Mg2+ efflux was inhibited by quinidine and by ATP depletion. In the absence of external Na+, Mg2+ efflux was stimulated by increases in external Mn2+ concentration following a Michaelian-like function with an apparent dissociation constant (KMn) of 35 +/- 15 microM (mean +/- S.D. of four experiments) and a variable maximal rate ranging from 350 to 1400 mumol (1 cells X h)-1. Mn2+-stimulated Mg2+ efflux was inhibited by quinidine, by ATP depletion, and by increasing the external Na+ concentration. Quinidine-sensitive (or ATP-dependent) Mg2+ efflux exhibited very similar values when compared with quinidine-sensitive (or ATP-dependent) Mn2+ influx. Mn2+ efflux in rat erythrocytes (loaded with total internal Mn2+ contents of 230-450 mumol/l cells) was stimulated by increases in external Na+ concentration and inhibited by quinidine. In the absence of external Na+, Mn2+ efflux was stimulated by increases in external Mg2+ concentration following a Michaelian-like function with an apparent dissociation constant (KMg) of about 35 +/- 5 microM (mean +/- range of two experiments) and a maximal rate of about 60-100 mumol (1 cells X h)-1. In conclusion, the Na+-stimulated Mg2+ carrier of rat erythrocytes may catalyze a one-to-one and reversible Mn2+:Mg2+ exchange in the absence of external Na+.     

Regulated release of Ca2+ from respiring mitochondria by Ca2+/2H+ antiport.

Simultaneous measurements of oxygen consumption and transmembrane transport of Ca2+, H+, and phosphate show that the efflux of Ca2+ from respiring tightly coupled rat liver mitochondria takes place by an electroneutral Ca2+/2H+ antiport process that is ruthenium red-insensitive and that is regulated by the oxidation-reduction state of the mitochondrial pyridine nucleotides. When mitochondrial pyridine nucleotides are kept in a reduced steady state, the efflux of Ca2+ is inhibited; when they are in an oxidized state, Ca2+ efflux is activated. These processes were demonstrated by allowing phosphate-depleted mitochondria respiring on succinate in the presence of rotenone to take up Ca2+ from the medium. Upon subsequent addition of ruthenium red to block Ca2+ transport via the electrophoretic influx pathway, and acetoacetate, to bring mitochondrial pyridine nucleotides into the oxidized state, Ca2+ efflux and H+ influx ensued. The observed H+ influx/Ca2+ efflux ratio was close to the value 2.0 predicted for the operation of an electrically neutral Ca2+/2H+ antiport process.     

Na+/Na+ exchange and Na+/H+ antiport in rabbit erythrocytes: Two distinct transport systems

Summary Rabbit erythrocytes are well known for possessing highly active Na⁺/Na⁺ and Na⁺/H⁺ countertransport systems. Since these two transport systems share many similar properties, the possibility exists that they represent different transport modes of a single transport molecule. Therefore, we evaluated this hypothesis by measuring Na⁺ transport through these exchangers in acid-loaded cells. In addition, selective inhibitors of these transport systems such as ethylisopropyl-amiloride (EIPA) and N-ethylmaleimide (NEM) were used. Na⁺/Na⁺ exchange activity, determined as the Na _o ⁺ -dependent²²Na efflux or Na _i ⁺ -induced²²Na entry was completely abolished by NEM. This inhibitor, however, did not affect the H _i ⁺ -induced Na⁺ entry sensitive to amiloride (Na⁺/H⁺ exchange activity). Similarly, EIPA, a strong inhibitor of the Na⁺/H⁺ exchanger, did not inhibit Na⁺/Na^– countertransport, suggesting the independent nature of both transport systems. The possibility that the NEM-sensitive Na⁺/Na⁺ exchanger could be involved in Na⁺/H⁺ countertransport was suggested by studies in which the net Na⁺ transport sensitive to NEM was determined. As expected, net Na⁺ transport through this transport system was zero at different [Na⁺] _i /[Na⁺] _o ratios when intracellular pH was 7.2. However, at pH _i =6.1, net Na⁺ influx occurred when [Na⁺] _i was lower than 39mm. Valinomycin, which at low [K⁺] _o was lower than 39mm. Valinomycin, which at low [K⁺] _o clamps the membrane potential close to the K⁺ equilibrium potential, did not affect the net NEM-sensitive Na⁺ entry but markedly stimulated, the EIPA-and NEM-resistant Na⁺ uptake. This suggest that the net Na⁺ entry through the NEM-sensitive pathway at low pH _i , is mediated by an electroneutral process possibly involving Na⁺/H⁺ exchange. In contrast, the EIPA-sensitive Na⁺/H⁺ exchanger is not involved in Na⁺/Na⁺ countertransport, because Na⁺ transport through this mechanism is not affected by an increase in cell Na^– from 0.4 to 39mm. Altogether, these findings indicate that both transport systems: the Na⁺/Na⁺ and Na⁺/H⁺ exchangers, are mediated by distinct transport proteins.     

K+/H+ antiport in mitochondria

Mitochondria contain a latent K⁺/H⁺ antiporter that is activated by Mg²⁺-depletion and shows optimal activity in alkaline, hypotonic suspending media. This K⁺/H⁺ antiport activity appears responsible for a respiration-dependent extrusion of endogenous K⁺, for passive swelling in K⁺ acetate and other media, for a passive exchange of matrix⁴²K⁺ against external K⁺, Na⁺, or Li⁺, and for the respiration-dependent ion extrusion and osmotic contraction of mitochondria swollen passively in K⁺ nitrate. K⁺/H⁺ antiport is inhibited by quinine and by dicyclohexylcarbodiimide when this reagent is reacted with Mg²⁺-depleted mitochondria. There is good suggestive evidence that the K⁺/H⁺ antiport may serve as the endogenous K⁺-extruding device of the mitochondrion. There is also considerable experimental support for the concept that the K⁺/H⁺ antiport is regulated to prevent futile influx-efflux cycling of K⁺. However, it is not yet clear whether such regulation depends on matrix free Mg²⁺, on membrane conformational changes, or other as yet unknown factors.     

Regulation of Na/Mg antiport in rat erythrocytes

In rat erythrocytes, the regulation of Na⁺/Mg²⁺ antiport by protein kinases (PKs), protein phosphatases (PPs), intracellular Mg²⁺, ATP and Cl⁻ was investigated. In untreated erythrocytes, Na⁺/Mg²⁺ antiport was slightly inhibited by the PK inhibitor staurosporine, slightly stimulated by the PP inhibitor calyculin A and strongly stimulated by vanadate. PMA stimulated Na⁺/Mg²⁺ antiport. This effect was completely inhibited by staurosporine and partially inhibited by the PKC inhibitors Ro-31-8425 and BIM I. Participation of other PKs such as PKA, the MAPK cascade, PTK, CK I, CK II, CAM II-K, PI 3-K, and MLCK was excluded by use of inhibitors. Na⁺/Mg²⁺ antiport in rat erythrocytes can thus be stimulated by PKCα.In non-Mg²⁺-loaded erythrocytes, ATP depletion reduced Mg²⁺ efflux and PMA stimulation in NaCl medium. A drastic activation of Na⁺/Mg²⁺ antiport was induced by Mg²⁺ loading which was not further stimulated by PMA. Staurosporine, Ro-31-8425, BIM I and calyculin A did not inhibit Na⁺/Mg²⁺ antiport of Mg²⁺-loaded cells. Obviously, at high [Mg²⁺]_i Na⁺/Mg²⁺ antiport is maximally stimulated. PKC_α or PPs are not involved in stimulation by intracellular Mg²⁺. ATP depletion of Mg²⁺-loaded erythrocytes reduced Mg²⁺ efflux and the affinity of Mg²⁺ binding sites of the Na⁺/Mg²⁺ antiporter to Mg²⁺. In non-Mg²⁺-loaded erythrocytes Na⁺/Mg²⁺ antiport essentially depends on Cl⁻. Mg²⁺-loaded erythrocytes were less sensitive to the activation of Na⁺/Mg²⁺ antiport by [Cl⁻]_i.     

Mn2+/Mg2+-dependent pyruvate kinase from a d-lactic acid-producing bacterium Sporolactobacillus inulinus: characterization of a novel Mn2+-mediated allosterically regulated enzyme

Sporolactobacillus inulinus has attracted scientific and commercial interest due to its high efficiency in d-lactic acid production. Pyruvate kinase (PYK) is one of the key regulatory points in glycolysis, and well-activated PYK can improve d-lactic acid production. A novel Mn²⁺/Mg²⁺-dependent PYK from S. inulinus was expressed in Escherichia coli and purified to homogeneity. Kinetic characterization demonstrated that the S. inulinus PYK had drastically higher activity and affinity toward substrates in the presence of Mn²⁺ compared to those of the common PYK cofactor Mg²⁺, and the circular dichroism spectra of the S. inulinus PYK suggested a Mn²⁺-mediated allosteric activation. The S. inulinus PYK was also allosterically regulated by ribose-5-phosphate or AMP activation and inorganic phosphate or ATP inhibition. The inhibition could be marked reduced or fully eliminated in the presence of activators. The result of fermentations by S. inulinus Y2-8 showed that the extracellular-added MnSO₄ and KH₂PO₄ significantly affected glycolysis flux and d-lactic acid production, which is consistent with the allosteric regulation of Mn²⁺ and inorganic phosphate on PYK. The sophisticated regulatory role of PYK would establish the foundation of substantial disturbance or restructuring of cellular metabolism for improving the S. inulinus d-lactic acid production.     

K+/H+ antiport in heart mitochondria

Heart mitochondria depleted of endogenous divalent cations by treatment with A23187 and EDTA swell in (a) K+ acetate or (b) K+ nitrate when an uncoupler is present. These mitochondria also exchange matrix 42K+ with external K+, Na+, or Li+ in a reaction that does not require respiration and is insensitive to uncouplers. Untreated control mitochondria do not swell in either medium nor do they show the passive cation exchange. Both the swelling and the exchange reactions are inhibited by Mg2+ and by quinine and other lipophilic amines. Swelling and exchange are both strongly activated at alkaline pH, and the exchange reaction is also increased markedly by hypotonic conditions. All of these properties correspond to those reported for a respiration-dependent extrusion of K+ from Mg2+-depleted mitochondria, a reaction attributed to a latent Mg2+- and H+-sensitive K+/H+ antiport. The swelling reactions are strongly inhibited by dicyclohexylcarbodiimide reacted under hypotonic conditions, but the exchange reaction is not sensitive to this reagent. Heart mitochondria depleted of Mg2+ show marked increases in their permeability to H+, to anions, and possibly to cations, and the permeability to each of these components is further increased at alkaline pH. This generalized increase in membrane permeability makes it likely that K+/H+ antiport is not the only pathway available for K+ movement in these mitochondria. It is concluded that the swelling, 42K+ exchange, and K+ extrusion data are all consistent with the presence of the putative K+/H+ antiport but that definitive evidence for the participation of such a component in these reactions is still lacking.     

Evidence for Na+/H+ antiport inMethanospirillum Hungatei

The growth and methane formation ofMethanospirillum hungatei were inhibited by an inhibitor of Na⁺/H⁺ antiport amiloride. After addition of NaCl or LiCl, when the cells had a lower intracellular pH and were deenergized, they extruded protons into the external medium. The acidification of the external medium was stimulated by protonophores and inhibited by amiloride. These findings suggest the existence of an Na⁺/H⁺ antiport in the cytoplasmic membrane ofM. hungatei and its role in the energetics of methanogenic bacteria.     

Stimulation of Na+/H+ antiport and pyruvate kinase activities by high glucose concentration in human erythrocytes

This study aims to demonstrate the effect of high glucose concentrations on NHE-1 and PK activities and investigate the implicated signal transduction pathways. Erythrocytes drawn from healthy volunteers were incubated in the presence of 5 or 50 mM of glucose, fructose, galactose or mannitol. When appropriate, specific inhibitors of NHE-1, PKC or p42/44 MAPK were used. Erythrocyte NHE-1 activity has been estimated by fluorometrical determination of the intracellular pH and quantification of sodium uptake using 22Na. Pyruvate kinase activity was measured by a NADH-lactate dehydrogenase enzymatic assay. p42/44 MAPK activity was assessed with a specific enzyme linked immunosorbent assay (ELISA). Increased concentrations of glucose but not galactose, fructose or mannitol enhanced erythrocyte NHE-1, PK and p42/44 MAPK activity. Inhibition of PKC, counteracted these effects of glucose. Similarly, inhibition of NHE 1 abolished the effect of high glucose on PK and p42/44 MAPK as well. Finally, inhibition of p42/44 MAPK also hindered the effect of glucose on NHE-1 and PK activities. The data of the present study indicate an acute effect of glucose on signal transduction pathways in human erythrocytes. This pathway involves NHE-1, PKC, and p42/44 MAPK. A positive feedback between NHE 1 and p42/44 MAPK is suggested.     

Regulation of the mitochondrial Na+/Ca2+ antiport by matrix pH

The effect of matrix pH (pHi) on the activity of the mitochondrial Na+/Ca2+ antiport has been studied using the fluorescence of SNARF-1 to monitor pHi and Na(+)-dependent efflux of accumulated Ca2+ to follow antiport activity. Heart mitochondria respiring in a KCl medium maintain a large delta pH (interior alkaline) and show optimal Na+/Ca2+ antiport only when the pH of the medium (pH0) is acid. Addition of nigericin to these mitochondria decreases delta pH and increases the membrane potential (delta psi). Nigericin strongly activates Na+/Ca2+ antiport at values of pH0 near 7.4 but inhibits antiport activity at acid pH0. When pHi is evaluated in these protocols, a sharp optimum in Na+/Ca2+ antiport activity is seen near pHi 7.6 in the presence or absence of nigericin. Activity falls off rapidly at more alkaline values of pHi. The effects of nigericin on Na+/Ca2+ antiport are duplicated by 20 mM acetate and by 3 mM phosphate. In each case the optimum rate of Na+/Ca2+ antiport is obtained at pHi 7.5 to 7.6 and changes in antiport activity do not correlate with changes in components of the driving force of the reaction (i.e., delta psi, delta pH, or the steady-state Na+ gradient). It is concluded that the Na+/Ca2+ antiport of heart mitochondria is very sensitive to matrix [H+] and that changes in pHi may contribute to the regulation of matrix Ca2+ levels.