High affinity binding of amiloride analogs at an internal site in renal microvillus membrane vesicles.

Amiloride analogs with hydrophobic substitutions on the 5-amino nitrogen atom are relatively high affinity inhibitors of the plasma membrane Na(+)-H+ exchanger. We demonstrated that a high affinity-binding site for [3H]5-(N-methyl-N-isobutyl)amiloride ([3H]MIA) (Kd = 6.3 nM, Bmax = 1.2 pmol/mg of protein) is present in microvillus membrane vesicles but not in basolateral membrane vesicles isolated from rabbit renal cortex, in accord with the known membrane localization of the Na(+)-H+ exchanger in this tissue. The rank order potency for inhibition of microvillus membrane [3H]MIA binding by amiloride analogs was: MIA (I50 approximately 10 nM) greater than amiloride (I50 approximately 200 nM) greater than benzamil (I50 approximately 1200 nM). This correlated with a qualitatively similar rank order potency for inhibition of Na(+)-H+ exchange: MIA (I50 approximately 4 microM) greater than amiloride (I50 approximately 15 microM) greater than benzamil (I50 approximately 100 microM), but did not correlate with the rank order potency for inhibition of the organic cation-H+ exchanger in microvillus membrane vesicles: MIA approximately benzamil (I50 approximately 0.5 microM) greater than amiloride (I50 approximately 10 microM). However, tetraphenylammonium, an inhibitor of organic cation-H+ exchange, inhibited the rate of [3H]MIA binding without an effect on equilibrium [3H]MIA binding; the dissociation of bound [3H]MIA was inhibited by preloading the membrane vesicles with tetraphenylammonium. These findings indicated that high affinity [3H]MIA binding to renal microvillus membrane vesicles takes place at an internal site to which access is rate-limited by the tetraphenylammonium-sensitive organic cation transporter. Equilibrium [3H]MIA binding was inhibited by H+ but was unaffected by concentrations of Na+ or Li+ that saturate the external transport site of the Na(+)-H+ exchanger. Binding of MIA to its high affinity binding site had no effect on the rate of Na(+)-H+ exchange. This study suggests that the renal Na(+)-H+ exchanger has a high affinity internal binding site for amiloride analogs that is distinct from the external amiloride inhibitory site.     

A dextran-bound amiloride derivative is a selective inhibitor of Na+/H+ antiport. Application for studying the role of the antiporter in cellular proliferation in human fibroblasts

Inhibitors of Na+/H+ exchange from the amiloride series are known to accumulate within the cell and cause an inhibition of a variety of cellular functions. In order to render the amiloride molecule impermeable to cells, we have synthesized a potent amiloride analog, 5-N-(3-aminophenyl)amiloride (compound A35, Ki = 60 nM). The isothiocyanate derivative of A35 (A35-NCS) was coupled to soluble dextrans of 15-20 kDa that have been derivatized with diaminoalkane spacer groups. Dextran-bound amiloride derivatives showed good inhibition of Na+/H+ exchange in human foreskin fibroblasts and A431 cells. Among several spacer groups tested, dextran derivatized with ethylenediamine showed the highest inhibitory activity. The intrinsic inhibitory potency of this polymer increased with increasing degree of substitution with A35, approaching that of free A35 with substitution of approximately 3 mol of A35 per mole of dextran. Coupling to dextran largely diminished side effects of the amiloride derivative on cells such as the inhibition of protein synthesis. A35-dextran was an effective inhibitor of serum-induced reinitiation of DNA synthesis in human foreskin fibroblasts in a bicarbonate-free medium, pH 7.1, but had little effect when either the pH of the medium was more alkaline or when the medium contained a bicarbonate buffer. These findings suggest that the selective inhibition of Na+/H+ antiport by A35-dextran prevents the reinitiation of DNA synthesis when the external conditions are such that the antiporter activity is required for the establishment of a permissive intracellular pH. Polymer-bound amiloride analogs should be useful as selective inhibitors in studies of the physiological role of the Na+/H+ antiporter, as well as for affinity purification of the antiporter.     

Relationship among activation of the Na+/H+ antiporter, ornithine decarboxylase induction, and DNA synthesis

The relationship among activation of the Na+/H+ antiporter, ornithine decarboxylase, and DNA synthesis was examined with bovine small lymphocytes stimulated by concanavalin A (Con A). The Na+/H+ antiport activity was activated immediately after addition of concanavalin A; the maximum was reached 1 h after Con A addition and the activation continued at least 6 h. With increasing concanavalin A concentrations, the activities of the Na+/H+ antiporter, ornithine decarboxylase, and DNA synthesis increased in a parallel manner. In the presence of HCO3- in the medium, the internal alkalinization of lymphocytes was not induced by Con A. Ornithine decarboxylase and DNA synthetic activities were not inhibited by 5-(N-ethyl-N-isopropyl) amiloride (EIPA), a specific inhibitor of the Na+/H+ antiporter. In contrast, in the absence of HCO3- in the medium, the internal pH was alkalinized approximately 0.06 pH units by Con A. EIPA did inhibit the alkalinization of the internal pH or DNA synthesis significantly. Ornithine decarboxylase activity was not inhibited by EIPA. These results indicate that the activation of a Na+/H+ antiporter is not a trigger for cell proliferation, but its activation is important probably through the maintenance of the internal pH optimum, especially in HCO3(-)-free medium.     

Na+/H+ exchange modulates the production of leukotriene B4 by human neutrophils.

Human neutrophils produce various compounds of the 5-lipoxygenase pathway, including (5S)-hydroxyeicosatetraenoic acid, leukotriene B4, its 6-trans isomers and omega-oxidation metabolites of LTB4, when the cells are stimulated with the Ca2+ ionophore A23187. The elevation in the extracellular pH (pHo) facilitated the cytoplasmic alkalinization induced by the ionophore as determined fluorometrically using 2',7'-bis(carboxyethyl)carboxyfluorescein and enhanced the production of all the 5-lipoxygenase metabolites. The production decreased when the alkalinization was blocked by the decrease in the pHo, the removal of the extracellular Na+ or the addition of specific inhibitors of the Na+/H+ exchange, such as 5-(NN-hexamethylene)amiloride, 5-(N-methyl-N-isobutyl)amiloride and 5-(N-ethyl-N-isopropyl)amiloride. The alkalinization of the cytoplasm with methylamine completely restored the production suppressed by the removal of Na+ from the medium. These findings suggest that the change in the cytoplasmic pH (pHi) mediated by the Na+/H+ exchange regulates the production of the lipoxygenase metabolites. The site of the metabolism controlled by the pHi change seemed to be the 5-lipoxygenase, because the production of all the metabolites decreased in parallel and the release of [3H]arachidonic acid from the neutrophils in response to the ionophore was not affected by the pHi change. Furthermore, the production of the 5-lipoxygenase metabolites stimulated by A23187 with or without exogenous arachidonic acid showed a similar pHo-dependence and the production induced by N-formylmethionyl-leucylphenylalanine (chemotactic peptide) with exogenous arachidonic acid also decreased when the cytoplasmic alkalinization was inhibited.     

Regulation of porcine brain alpha 2-adrenergic receptors by Na+,H+ and inhibitors of Na+/H+ exchange

Previous reports from this laboratory have demonstrated that alpha 2-adrenergic receptors accelerate Na+/H+ exchange in NG108-15 neuroblastoma X glioma cells and evoke platelet secretion via a pathway involving Na+/H+ exchange. The present studies were designed to examine whether agents that interact with Na+/H+ antiporters also might influence alpha 2-adrenergic receptor-ligand interactions. We observed that Na+ decreases receptor affinity for the agonists epinephrine, norepinephrine, and UK14304 and slightly increases receptor affinity for the antagonists yohimbine and idazoxan in digitonin-solubilized preparations from porcine brain cortex. Increases in [H+] also decrease receptor affinity for agonists and cause either a slight increase or no change in receptor affinity for antagonists. Amiloride analogs accelerate the rate of [3H] yohimbine dissociation from digitonin-solubilized receptors with a relative effectiveness that parallels their ability to block Na+/H+ exchange in other systems. Interestingly, these modulatory effects of Na+,H+ and 5-amino-substituted analogs of amiloride are retained in homogeneous preparations of the alpha 2-adrenergic receptor, suggesting that the allosteric-binding sites for these agents are on the receptor-binding protein itself.     

Na+/H+ exchanger activity in the pig kidney epithelial cell line, LLC-PK1: inhibition by amiloride and its derivatives

Rapidly growing pig-kidney-derived epithelial cells, LLC-PK1, lack detectable amiloride-sensitive Na+/H+ exchange activity when assayed directly. A large 22Na uptake is induced when the cells are acid-loaded prior to assay by incubation with buffer containing ammonium chloride or nigericin. The acid-stimulated sodium uptake is sensitive to amiloride, with half-maximal inhibition at 3.5-4.5 microM in buffer containing 15 mM sodium ion. There is simple competitive interaction between amiloride and sodium ion when the amiloride concentration is below 25 microM and the sodium ion concentration is above 20 mM. Derivatives of amiloride which carry substituents on the 5-amino group are 35- to 175-fold more inhibitory than amiloride itself.     

Species-specific Mn2+/Mg2+ antiport from Mg2(+)-loaded erythrocytes.

Mg2(+)-loaded rat erythrocytes performed Mn2+/Mg2+ antiport, which was nonspecifically stimulated by anions and cations. Mn2+/Mg2+ antiport was shown to operate via the Na+/Mg2+ antiporter because extracellular Na+ and Mn2+ inhibited the intracellular uptake of each other's ions competitively. Furthermore, Mn2+/Mg2+ antiport and Na+/Mg2+ antiport were identically inhibited by various amiloride derivatives. Na+/Mg2+ antiport of chicken and human erythrocytes cannot perform Mn2+/Mg2+ antiport although chicken erythrocytes took up more Mn2+ than rat erythrocytes.     

Regulation of human neutrophil chemotaxis by intracellular pH

The relationship of N-formyl-methionyl-leucyl-phenylalanine-stimulated Na+/H+ exchange to the chemotactic responsiveness of human neutrophils was investigated. The pHi changes, measured from the equilibrium distribution of 5,5-dimethyloxazolidine-2,4-dione, were correlated with the migratory behavior of the cells as assessed by the leading front method. Exposure of cells to 10 nM FMLP caused activation of Na+/H+ exchange, leading to a rise in pHi from approximately 7.25 to approximately 7.75. This intracellular alkalinization was inhibited by amiloride and by three more potent analogues. All four compounds reduced the chemotactic response to FMLP with apparent Ki values similar to those for inhibition of the pHi transients, thereby suggesting that the blocking effect of the drugs on directed cell migration was related to inhibition of Na+/H+ exchange. The effect was specific for stimulated cell locomotion: FMLP-induced chemotaxis and chemokinesis were inhibited in parallel, whereas random motility was unimpaired. The relationship of pHi to function was also studied as the pHi of FMLP-activated cells was varied between 6.8 and 8.6 by altering the chemical gradients for Na+ and H+ across the cell membrane. There was a direct, positive correlation between the pHi value attained following FMLP-stimulation and the locomotor response to a chemotactic gradient. These results indicate that the motile functions of human neutrophils can be regulated by their pHi.     

Taurine indirectly increases [Ca]i by inducing Ca2+ influx through the Na+-Ca2+ exchanger

Recent studies in heart cells have shown taurine to induce a sustained increase of both intracellular Ca²⁺ and Na⁺. These results led us to believe that the increase in Na⁺ by taurine could be due to Na⁺ entry through the taurine-Na⁺ cotransporter which in turn favours transarcolemmal Ca²⁺ influx through Na⁺-Ca²⁺ exchange. Therefore, we investigated the effect of -alanine, a blocker of the taurine-Na⁺ cotransporter and low concentrations of CBDMB (a pyrazine derivative, 5-(N-4chlorobenzyl)-2,4-dimethylbenzamil), a Na⁺-Ca²⁺ exchanger blocker on taurine-induced [Ca]_i increase in embryonic chick heart cells. Using Fura-2 Ca²⁺ imaging and Fluo-3 Ca²⁺ confocal microscopy techniques, taurine (20 mM) as expected, induced a sustained increase in [Ca]_i at both the cytosolic and the nuclear levels. Preexposure to 500 M of the blocker of the taurine-Na⁺ cotransporter, -alanine, prevented the amino acid-induced increase of total [Ca]_i. On the other hand, application of -alanine did not reverse the action of taurine on total [Ca]_i. However, low concentrations of the Na⁺-Ca²⁺ exchanger blocker, CBDMB, reversed the taurine-induced sustained increase of cytosolic and nuclear free calcium (in presence or absence of -alanine). Thus, the effect of taurine on [Ca]_i in heart cells appears to be due to Na⁺ entry through the taurine-Na⁺ cotransporter which in turn favours transarcolemmal Ca²⁺ influx through the Na⁺-Ca²⁺ exchanger.