Coupled Na+/H+ exchange in rat parotid basolateral membrane vesicles

Summary pH gradient-dependent sodium transport in highly purified rat parotid basolateral membrane vesicles was studied under voltage-clamped conditions. In the presence of an outwardly directed H⁺ gradient (pH_in=6.0, pH_out=8.0)²²Na uptake was approximately ten times greater than uptake measured at pH equilibrium (pH_in=pH_out=6.0). More than 90% of this sodium flux was inhibited by the potassium-sparing diuretic drug amiloride (K ₁ =1.6 m) while the transport inhibitors furosemide (1mm), bumetanide (1mm) SITS (0.5mm) and DIDS (0.1mm) were without effect. This transport activity copurified with the basolateral membrane marker K⁺-stimulatedp-nitrophenyl phosphatase. In addition²²Na uptake into the vesicles could be driven against a concentration gradient by an outwardly directed H⁺ gradient. pH gradient-dependent sodium flux exhibited a simple Michaelis-Menten-type dependence on sodium concentration cosistent with the existence of a single transport system withK _M =8.0mm at 23°C. A component of pH gradient-dependent, amiloride-sensitive sodium flux was also observed in rabbit parotid basolateral membrane vesicles. These results provide strong evidence for the existence of a Na⁺/H⁺ antiport in rat and rabbit parotid acinar basolateral membranes and extend earlier less direct studies which suggested that such a transporter was present in salivary acinar cells and might play a significant role in salivary fluid secretion.     

Bicarbonate uptake by basolateral membrane vesicles from rat jejunum

Basolateral membranes from rat jejunal enterocytes have been obtained by self-orienting Percoll-gradient centrifugation. Bicarbonate and L-glucose uptake into osmotically active basolateral membrane vesicles has been studied by a rapid filtration technique. In closed vessels and at pH 8.2 the uptake kinetics of both [14C]bicarbonate and L[3H]glucose have been followed for 30 min at 18 degrees C. Bicarbonate uptake seems to be fast and in efflux experiments SITS and DIDS effect is negligible. This work demonstrates that it is possible to determine bicarbonate flux across basolateral membrane vesicles at pH and temperature values close to usual experimental conditions.     

Proton-lactate co transport in basolateral membrane vesicles from rat jejunum

Proton-coupled lactate transport across the basolateral membrane of rat jejunal enterocyte was studied using well purified membrane vesicles. L-lactate uptake is stimulated by an inwardly directed H⁺ gradient; the effect of the pH difference is drastically reduced by FCCP and by pCMBS; unlabelled L-lactate causes a strong inhibition, whilst furosemide is uneffective. The H⁺ gradient-dependent stimulation of L-lactate uptake is significantly inhibited also by SCN^–: this finding could explain results recently reported in the literature in which H⁺-lactate symport was not evidenced in basolateral membranes from rat jejunum.     

Ouabain-insensitive Na-ATPase activity in the basolateral membrane from rat jejunum

1. In the basolateral membrane preparation of the rat enterocyte (jejunal tract) there is not only the well-known (Na,K)-ATPase activity, but also a ouabain-insensitive Na-ATPase. 2. The Na-ATPase is not activated by anions or other monovalent cations. As a substrate, ATP cannot be replaced by other nucleotides. 3. The Na-ATPase is insensitive to ouabain and bumetanide, inhibited partially by furosemide and totally by ethacrynate. 4. The activation of Na-ATPase at different Na concentrations shows an hyperbolic curve (Km = 15.7 +/- 2.3 mM and Vmax = 204 +/- 19 nmoles Pi/mg protein per min) different from the sigmoidal curve (Km = 9.8 +/- 1.2 mM and Vmax = 640 +/- 15 nmoles Pi/mg protein per min) shown by (Na,K)-ATPase. 5. These results are compared with the corresponding ones found in other animals and tissues in which the Na-ATPase was found. 6. The Na-ATPase activity can be interpreted as the enzymatic correspondent of a ouabain-insensitive Na pump, present in the basolateral membrane of the enterocyte different in behaviour with respect to the known Na pump.     

Na+/H+ exchange mechanism in the basolateral membrane of the rat enterocyte

Basolateral membrane vesicles from rat jejunal enterocytes, especially purified of brush-border contamination, were used for Na+ uptake. The basolateral membrane vesicles are osmotically active and under our experimental conditions Na+ binding is much lower than transport. An outwardly directed proton gradient stimulates Na+ uptake at both 5 microM and 5 mM concentrations. The proton gradient effect can be inhibited completely by 2 mM amiloride and partially by either FCCP or NH4Cl (NH3 diffusion). Membrane potential effects can be excluded by having valinomycin plus K+ on both sides of the vesicles. These results suggest that there is an Na+/H+ exchanger in the basolateral membrane of rat enterocytes.     

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.     

Interaction between Na ions and the Cl/HCO3 exchanger in basolateral membranes from rat jejunum

The jejunal basolateral Cl/HCO₃ exchanger is modulated by two Na-dependent regulatory sites located on the inner and outer membrane surfaces. The aim of this work was to focus on the interaction between the anion exchanger and intracellular or extracellular sodium. Uptake studies, performed using basolateral membrane vesicles, provided kinetic parameters as a function of outside or inside Na concentration. The intracellular Na-sensitive modifier site seems to be primarily involved in the modulation of the Cl/HCO₃ exchanger.     

Kinetic characterization of Na+/H+ antiport of Plasmodium falciparum membrane

Intraerythrocytic malaria parasites produce vast amounts of lactic acid through glycolysis. While the egress of lactate is very rapid, the mode of extrusion of H⁺ is not known. The possible involvement of a Na⁺/H⁺ antiport in the extrusion of protons across the plasma membrane of Plasmodium falciparum has been investigated by using the fluorescent pH probe 6-carboxyfluorescein. The resting cytosolic pH was 7.27 ± 0.1 in ring stage parasites and 7.31 ± 0.12 in trophozoites. Spontaneous acidification of parasite cytosol was observed in Na⁺-medium and realkalinization occurred upon addition of Na⁺ to the medium in a concentration-dependent manner, with no apparent saturation. The rate of H⁺-at the ring stage was higher than that at the trophozoite stage due to the larger surface/volume ratio of the young parasite stage. Na⁺-H⁺-was: 1) inhibited by the Na⁺/H⁺ inhibitors amiloride and 5-(N-ethyl-isopropyl) amiloride (EIPA), though at relatively high concentrations; 2) augmented with rising pH₆ (pH_i = 6.2 [Na⁺]_o = 30 mM); and 3) decreased with increasing pH_i (pH_o = 7.4; [Na⁺]_o = 30 mM). The pH_i and the pH_o dependencies of H⁺-were almost identical at all parasite stages. Only at pH_i > 7.6 efflux was totally obliterated. The target of this inhibitory effect is probably other than the antiport. Results indicate that H⁺-is mediated by a Na⁺/H⁺ antiport which is regulated by host and parasite pH and by the host cytosol sodium concentration. The proton transport capacity of the antiport can easily cope with all the protons of lactic acid produced by parasite's glycolysis. © 1993 Wiley-Liss, Inc.     

Characterization of apical membrane Cl-dependent Na/H exchange in crypt cells of rat distal colon

A novel Cl-dependent Na/H exchange (Cl-NHE) has been identified in apical membranes of crypt cells of rat distal colon. The presence of Cl is required for both outward proton gradient-driven Na uptake in apical membrane vesicles (AMV) and Na-dependent intracellular pH recovery from an acid load in the crypt gland. The present study establishes that Cl-dependent outward proton gradient-driven (22)Na uptake 1) is saturated with increasing extravesicular Na concentration with a Michaelis constant (K(m)) for Na of approximately 24.2 mM; 2) is saturated with increasing outward H concentration gradient with a hyperbolic curve and a K(m) for H of approximately 1.5 microM; 3) is inhibited by the Na/H exchange (NHE) inhibitors amiloride, ethylisopropylamiloride, and HOE-694 with an inhibitory constant (K(i)) of approximately 480.2, 1.1, and 9.5 microM, respectively; 4) is inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, an anion exchange inhibitor at low concentration and a Cl channel blocker at high dose, and by 5-nitro-2(3-phenylpropylamino)benzoic acid, a Cl channel blocker, with a K(i) of approximately 280.6 and 18.3 microM, respectively; and 5) substantially stimulated Cl-NHE activity by dietary Na depletion, which increases plasma aldosterone and inhibits NHE in surface cell AMV. These properties of Cl-NHE are distinct from those of NHE1, NHE2, and NHE3 isoforms that are present in colonic epithelial cells; thus these results suggest that the colonic crypt cell Cl-NHE is a novel NHE isoform.     

The sinusoidal domain of the plasma membrane of rat hepatocytes contains an amiloride-sensitive Na+/H+ antiport

ATP-dependent trapping of [14C]methylamine was demonstrated in vesicles selectively derived from the sinusoidal plasma membrane of rat hepatocytes; activity was lacking in vesicles prepared from the canalicular domain of the plasma membrane of rat hepatocytes. The proton movement was inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, strophanthidin, vanadate, amiloride, and absence of sodium. 22Na efflux from sinusoidal membrane vesicles increased inversely to extravesicular pH. The results indicate that the sinusoidal plasma membrane of rat hepatocytes contains a Na+/H+ antiport.     

ATP-driven Ca2+ pump in the basolateral membrane of rat kidney cortex catalyzes an electroneutral Ca2+/H+ antiport

An ATP-driven Ca2+ pump in the basolateral membrane of rat kidney cortex pumps Ca2+ out of the cell at the expense of MgATP (Km = 0.191 mM). This pump has a high affinity for free Ca2+ (26 nM). Vanadate, lanthanum, N-ethylmaleimide and calmodulin inhibitor R24571 inhibited this pump activity. Dimethyl[2-14C]oxazolidine-2,4-dione [( 14C]DMO) was entrapped in the vesicles in association with the ATP-driven Ca2+ influx. The ATP-driven Ca2+ influx was stimulated by the intravesicular acid pH and an upper convex Lineweaver-Burk reciprocal plot suggested two possible kinetics; one is that this Ca2+ pump is an allosteric enzyme with more than 1.72 H+ binding sites and another is the presence of two Ca2+ pumps with different affinities for H+. Valinomycin study indicated that the ATP-dependent Ca2+ transport by the BLMV was electroneutral and voltage independent. These results strongly suggest that the ATP-driven Ca2+ pump in the renal basolateral membrane catalyzes an electroneutral Ca2+/H+ antiport.     

Lactoferrin activates plasma membrane oxidase and Na+/H+ antiport activity.

Lactoferrin is a growth stimulant. The basis for this effect is not clear since it is not thought to be involved in iron uptake through endocytosis. Ferric lactoferrin supports external ferrous chelate formation by K562 and HeLa cells, and ferric lactoferrin stimulates the reduction of external ferric iron by cells. Ferric lactoferrin also stimulates NADH oxidase activity in isolated rat liver plasma membranes and stimulates amiloride sensitive proton release from K562 cells. The evidence that ferric lactoferrin can participate in oxidoreduction reactions at the plasma membrane leading to activation of Na+/H+ exchange provides an alternative explanation for the proliferative effect.     

The Na+:H+ antiport in cancer

Several carriers mediate ionic fluxes across the plasma membrane in a variety of mammalian cell types. Intracellular proton concentration is regulated by virtue of the operation of at least two distinct systems: a stilbene-sensitive, Na+- dependent HCO3-/Cl- exchange system, and an amiloride-sensitive Na+/H+ antiporter. The contribution of these two transporters to the modulation of intracellular pH in response to either extracellular pH variations or cell stimulation by growth factors and tumor promoters has been studied in several cell lines, including fibroblast mutants lacking Na+/H+ antiport activity. The attainment of a permissive intracellular pH value is critical to the development of the mitogenic response elicited by growth factors. Kinetic studies have revealed particular features of the Na+/H+ antiporter that explain its function in the early sequence of biochemical events leading to DNA replication. The detailed investigation of the mechanisms by which protons and other ions might regulate cell proliferation has important implications for the understanding of the role of pH microenvironment in carcinogenesis, tumor development and chemotherapy.     

Possible role of the membrane Na+/H+ antiport in ornithine decarboxylase induction by L-asparagine

Amino acids of transport systems A and N play certain important role in cell activation. For example, the presence of these amino acids is essential in the induction of ornithine decarboxylase by growth factors and hormones. At mM concentrations, each of these amino acids, particularly L-asparagine, can also induce the enzyme without being further metabolized or incorporated into proteins. We have reported that the addition of 10 mM L-asparagine to quiescent Reuber's H-35 rat hepatoma cells caused an immediate and transient increase in intracellular pH. Here we report that concomitant with the intracellular alkalinization was an increase in H+ extrusion which was amiloride-sensitive and Na+-dependent. The induction of ornithine decarboxylase by L-asparagine was also amiloride-sensitive.     

Ouabain-insensitive active sodium transport in rat jejunum: Evidence from ATPase activities,Na uptake by basolateral membrane vesicles and in vitro transintestinal transport

The basolateral membrane of the jejunal enterocyte of the rat was separated by self-orienting Percoll-gradient centrifugation and further purified from brush border contamination. Pellets were analysed for Mg-, Na- and (Na, K)-ATPase activities. The uptake of 0·02 M NaCl was also followed by the rapid micro-filtration technique. Transintestinal transport of fluid and electrolytes, and cell water, Na and K were determined in the in vitro everted and incubated jejunum. There is ouabain-insensitive Na-ATPase in addition to the well-known (Na, K)-ATPase in the basolateral membrane. These are differently inhibited by furosemide and ethacrynate. Na uptake by osmotically active basolateral membrane vesicles is enhanced by ATP and a further enhancement is obtained if there is intravesicular K. The ATP effect is inhibited differently by strophanthidin, furosemide and ethacrynate. In the everted sac preparation, transintestinal transport of Na and fluid still occurs when the Na/K pump is totally inhibited by ouabain. These experimental results suggest that there is also a ouabain-insensitive Na pump, different from the Na/K pump, in the basolateral membrane.     

Na/H and k/h antiport in root membrane vesicles isolated from the halophyte atriplex and the glycophyte cotton

Proton fluxes have been followed into and out of membrane vesicles isolated from the roots of the halophyte Atriplex nummularia and the glycophyte Gossypium hirsutum, with the aid of the ΔpH probe [¹⁴C]methylamine. Evidence is presented for the operation of Na⁺/H⁺ and K⁺/H⁺ antiporters in the membranes of both plants. Cation supply after a pH gradient has been set up across the vesicle membrane (either as a result of providing ATP to the H⁺-ATPase or by imposing an artificial pH gradient) brings about dissipation of the ΔpH, but does not depolarize the membrane potential as observed in similar experiments, but in the absence of Cl⁻, using the ΔΨ probe SCN⁻. Cation/H⁺ exchange is thus indicated. This exchange is not due to nonspecific electric coupling, nor to competition for anionic adsorption sites on the membrane, nor to inhibition of the H⁺-ATPase; coupling of the opposed cation and H⁺ fluxes by a membrane component is the most likely explanation. Saturation kinetics have been observed for both Na⁺/H⁺ and K⁺/H⁺ antiport in Atriplex. Moreover, additive effects are obtained when Na⁺ is supplied together with saturating concentrations of K⁺, and vice versa, suggesting that separate antiporters for Na⁺ and for K⁺ may be operating. In the case of both Atriplex and Gossypium evidence was obtained suggesting the presence of antiporters in both plasmalemma and tonoplast.     

Characterization of Cl-HCO3 exchange in basolateral membrane of rat distal colon

Sodium-independent Cl movement (i.e., Cl-anion exchange) has not previously been identified in the basolateral membranes of rat colonic epithelial cells. The present study demonstrates Cl-HCO₃ exchange as the mechanism for ³⁶Cl uptake in basolateral membrane vesicles (BLMV) prepared in the presence of a protease inhibitor cocktail from rat distal colon. Studies of ³⁶Cl uptake performed with BLMV prepared with different types of protease inhibitors indicate that preventing the cleavage of the COOH-terminal end of AE2 protein by serine-type proteases was responsible for the demonstration of Cl-HCO₃ exchange. In the absence of voltage clamping, both outward OH gradient (pH_out/pH_in: 7.5/5.5) and outward HCO₃ gradient stimulated transient ³⁶Cl uptake accumulation. However, voltage clamping with K-ionophore, valinomycin, almost completely (87%) inhibited the OH gradient-driven ³⁶Cl uptake, whereas HCO₃ gradient-driven ³⁶Cl uptake was only partially inhibited (38%). Both electroneutral HCO₃ and OH gradient-driven ³⁶Cl uptake were 1) completely inhibited by DIDS, an anion exchange inhibitor, with a half-maximal inhibitory constant (K_i) of 26.9 and 30.6 µM, respectively, 2) not inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid(NPPB), a Cl channel blocker, 3) saturated by increasing extravesicular Cl concentration with a K_m for Cl of 12.6 and 14.2 mM, respectively, and 4) present in both surface and crypt cells. Intracellular pH (pH_i) was also determined with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetomethylester (BCECF-AM) in an isolated superfused crypt preparation. Removal of Cl resulted in a DIDS-inhibitable increase in pH_i both in HCO₃-buffered and in the nominally HCO₃-free buffered solutions (0.28 ± 0.02 and 0.11 ± 0.02 pH units, respectively). We conclude that a carrier-mediated electroneutral Cl-HCO₃ exchange is present in basolateral membranes and that, in the absence of HCO₃, Cl-HCO₃ exchange can function as a Cl-OH exchange and regulate pH_i across basolateral membranes of rat distal colon. crypt glands; superfusion; intracellular pH; membrane vesicles; ³⁶Cl uptake; Cl-anion exchange     

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.