Kinetic properties of the Na+/H+ antiport of heart mitochondria

The fluorescence of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) has been used to follow the Na+/H+ antiport activity of isolated heart mitochondria as a Na+-dependent extrusion of matrix H+. The antiport activity measured in this way shows a hyperbolic dependence on external Na+ or Li+ concentration when the external pH (pHo) is 7.2 or higher. The apparent Km for Na+ decreases with increasing pHo to a limit of 4.6 mM. The Ki for external H+ as a competitive inhibitor of Na+/H+ antiport averages 3.0 nM (pHo 8.6). The Vmax at 24 degrees C is 160 ng ion of H+ min-1 (mg of protein)-1 and does not vary with pHo. Li+ reacts with the antiporter with higher affinity, but much lower Vmax, and is a competitive inhibitor of Na+/H+ antiport. The rate of Na+/H+ antiport is optimal when the pHi is near 7.2. When pHo is maintained constant, Na+-dependent extrusion of matrix H+ shows a hyperbolic dependence on [H+]i with an apparent Km corresponding to a pHi of 6.8. The Na+/H+ antiport is inhibited by benzamil and by 5-N-substituted amiloride analogues with I50 values in the range from 50 to 100 microM. The pH profile for this inhibition seems consistent with the availability of a matrix binding site for the amiloride analogues. The mitochondrial Na+/H+ antiport resembles the antiport found in the plasma membrane of mammalian cells in that Na+, Li+, and external H+ appear to compete for a common external binding site and both exchanges are inhibited by amiloride analogues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the mitochondrial Na+-H+ exchange. The effect of amiloride analogues

The kinetic properties and inhibitor sensitivity of the Na+-H+ exchange activity present in the inner membrane of rat heart and liver mitochondria were studied. (1) Na+-induced H+ efflux from mitochondria followed Michaelis-Menten kinetics. In heart mitochondria, the Km for Na+ was 24 +/- 4 mM and the Vmax was 4.5 +/- 1.4 nmol H+/mg protein per s (n = 6). Basically similar values were obtained in liver mitochondria (Km = 31 +/- 2 mM, Vmax = 5.3 +/- 0.2 nmol H+/mg protein per s, n = 4). (2) Li+ proved to be a substrate (Km = 5.9 mM, Vmax = 2.3 nmol H+/mg protein per s) and a potent competitive inhibitor with respect to Na+ (Ki approximately 0.7 mM). (3) External H+ inhibited the mitochondrial Na+-H+ exchange competitively. (4) Two benzamil derivatives of amiloride, 5-(N-4-chlorobenzyl)-N-(2',4'-dimethyl)benzamil and 3',5'-bis(trifluoromethyl)benzamil were effective inhibitors of the mitochondrial Na+-H+ exchange (50% inhibition was attained by approx. 60 microM in the presence of 15 mM Na+). (5) Three 5-amino analogues of amiloride, which are very strong Na+-H+ exchange blockers on the plasma membrane, exerted only weak inhibitory activity on the mitochondrial Na+-H+ exchange. (6) The results indicate that the mitochondrial and the plasma membrane antiporters represent distinct molecular entities.     

Interaction of external H+ with the Na+-H+ exchanger in renal microvillus membrane vesicles

We examined the effects of external H+ on the kinetics of Na+-H+ exchange in microvillus membrane vesicles isolated from the rabbit renal cortex. The initial rate of Na+ influx into vesicles with internal pH 6.0 was optimal at external pH 8.5 and was progressively inhibited as external pH was reduced to 6.0. A plot of 1/V versus [H+]o was linear and yielded apparent KH = 35 nM (apparent pK 7.5). In vesicles with internal pH 6.0 studied at external pH 7.5 or 6.6, apparent KNa was 13 or 54 mM, Ki for inhibition of Na+ influx by external Li+ was 1.2 or 5.2 mM, Ki for inhibition by external NH4+ was 11 or 50 mM, and Ki for inhibition by external amiloride was 7 or 25 microM, respectively. These findings were consistent with competition between each cation and H+ at a site with apparent pK 7.3-7.5. Lastly, stimulation of 22Na efflux by external Na+ (i.e. Na+-Na+ exchange) was inhibited as external pH was reduced from 7.5 to 6.0, also consistent with competition between external H+ and external Na+. Thus, in contrast with internal H+, which interacts at both transport and activator sites, external H+ interacts with the renal microvillus membrane Na+-H+ exchanger at a single site, namely the external transport site, where H+, Na+, Li+, NH4+, and amiloride all compete for binding.     

Interrelationships among quinidine, amiloride, and lithium as inhibitors of the renal Na+-H+ exchanger

We examined the effects of quinidine, amiloride and Li+ on the kinetics of Na+-H+ exchange in microvillus membrane vesicles isolated from the rabbit renal cortex. Quinidine reversibly inhibited the initial rate of Na+-H+ exchange (I50 200 microM). The plot of 1/V versus [quinidine] was curvilinear, with Hill coefficient greater than 1.0, indicating that the drug interacts at two or more inhibitory sites or at a single site on at least two different conformations of the transporter. Quinidine decreased the Vmax for Na+-H+ exchange and increased the Km for Na+, indicating a mixed-type mechanism of inhibition. In contrast, plots of 1/V versus [amiloride] and 1/V versus [Li+] were linear, indicating single inhibitory sites; amiloride and Li+ each increased the Km for Na+ with no effect on Vmax, indicating a competitive mechanism of inhibition. Addition of Li+ increased the intercept with no change in slope of the 1/V versus [amiloride] plot, indicating that Li+ and amiloride are mutually exclusive inhibitors of Na+-H+ exchange. Addition of quinidine increased the slopes of the plots of 1/V versus [amiloride] and 1/V versus [Li+], indicating that the binding of quinidine is not mutually exclusive with the binding of amiloride and Li+. Results from this and previous studies are consistent with the concept that the inhibitor amiloride and the transportable substrates Na+, H+, Li+, and NH+4 all mutually compete for binding to a single site, the external transport site of the renal Na+-H+ exchanger. However, our findings indicate that quinidine interacts with the Na+-H+ exchanger on at least one additional site that is not shared by Na+, Li+, or amiloride.     

Inhibition of cardiac sarcolemmal Na+/H+ antiporter by opioids.

In our routine screening of chemicals that would inhibit cardiac sarcolemmal Na+/H+ antiporter, we discovered that some of the opioids produced inhibition of cardiac sarcolemmal Na+/H+ antiporter in micromolar concentrations. Using U-50,488H, a selective kappa-opioid agonist, we characterized the nature of interaction between opioids and the Na+/H+ antiporter. The inhibitory effect of U-50,488H on Na+/H+ antiporter was immediate and reversible, and was not mediated through the interaction with the opioid receptors but due to the direct interaction of U-50,488H with the Na+/H+ antiporter. The kinetic data show that in the presence of U-50,488H the Km for Na+ was increased from 2.5 +/- 0.2 to 5.0 +/- 0.3 mM, while the Vmax (52.0 +/- 5.0 nmol.mg-1.min-1) remained the same. These results suggest that U-50,488H and Na+ compete for the same site on the antiporter. When testing the effect of U-50,488H on other transport systems of cardiac sarcolemma, we found that U-50,488H also inhibited Na+/Ca2+ antiporter and Na+/K+ pump but at much higher concentrations suggesting that U-50,488H shows some degree of selectivity for cardiac sarcolemmal Na+/H+ antiporter. When we compared the inhibitory potency of U-50,488H with amiloride and its analog, namely 5-(N,N-hexamethylene)amiloride, we found that U-50,488H (IC50 = 100 +/- 15 microM) was threefold more potent than amiloride (IC50 = 300 +/- 20 microM) but it was three-fold less potent than the amiloride analog (IC50 = 30 +/- 10 microM) in inhibiting cardiac sarcolemmal Na+/H+ antiporter. These results show that although U-50,488H is more potent than amiloride, the inhibitory characteristics of U-50,488H on cardiac sarcolemmal Na+/H+ antiporter are similar to amiloride.     

Biochemical characterization of the amiloride-sensitive Na+/H+ antiport in Chinese hamster lung fibroblasts

Net H+ fluxes across the plasma membrane of Chinese hamster lung fibroblasts (CC139) were monitored by pH-stat titration. Na+-depleted cells release H+ upon addition of Na+. Conversely Na+- or Li+-loaded cells take up H+ from the medium when shifted to a Na+,Li+-free medium. This reversible Na+ (or Li+)-dependent H+ flux is inhibited by amiloride and does not occur in digitonin-permeabilized cells. A similar Na+/H+ exchanger was identified in vascular smooth muscle cells, corneal and aortic endothelial cells, lens epithelial cells of bovine origin, and human platelets. Kinetic studies carried out with CC139 cells indicate the following properties: 1) half-saturation of the system is observed at pH = 7.8, in the absence of Na+; 2) external Na+ stimulates H+ release and inhibits H+ uptake in a competitive manner (Ki = 2-3 mM); 3) amiloride is a competitive inhibitor for Na+ (Ki congruent to 1 microM) and a noncompetitive inhibitor for H+; 4) a coupling ratio of 1.3 +/- 0.3 for the H+/Li+ exchange suggests a stoichiometry of 1:1. We conclude that CC139 cells possess in their plasma membrane a reversible, electroneutral, and amiloride-sensitive Na+/H+ antiporter, with two distinct and mutually exclusive binding sites for Na+ and H+. The rapid stimulation of the Na+/H+ antiporter in G0/G1-arrested CC139 cells upon addition of growth factors, together with the fact that intracellular H+ concentration is, under physiological conditions, around the apparent K0.5 of the system, strongly suggests a key role of this antiport in pHi regulation and mitogen action.     

Isolation and properties of fibroblast mutants overexpressing an altered Na+/H+ antiporter

A new method based on the toxicity of low intracellular pH (pHi) was developed to isolate fibroblast variants overexpressing Na+/H+ antiport activity. Chinese hamster lung fibroblasts (CCL39) were incubated for 60 min in medium containing 50 mM NH4Cl. Removal of external NH+4 induced a rapid and lethal intracellular acidification when the Na+/H+ antiporter was inhibited during the 60 min of the pHi recovery phase. The inhibition was provoked either by adding 5-(N-methyl,N-propyl)amiloride (MPA, LD50 = 0.3 microM) or by reducing external [Na+] (LD50 = 25 mM). Progressively increasing the MPA concentration during the acid-load selection led to the isolation of two stable variants: AR40 and AR300, resistant, respectively, to 40 and 300 microM MPA. In response to an acid-load, these variants display a much higher rate of pHi recovery due to an overexpression of Na+/H+ antiport activity. In addition, AR40 and AR300 have an altered Na+/H+ antiporter: in AR300 cells K0.5 of MPA for inhibiting Na+/H+ exchange is shifted from 5 X 10(-8) to 1.5 X 10(-6) M, Km (Na+) is decreased 2-fold, and Vmax is increased 4.5-fold. Alternatively reducing Na+ concentration of the pHi recovery saline medium in a stepwise manner led to the selection of another class of variants (DD8 and DD12) also characterized by an altered Na+/H+ antiporter and an increased expression level. The 10-fold increased rate of amiloride-sensitive Na+ influx of DD12 is accounted for by a 4-fold increase in Vmax and a 2.5-fold increase in affinity for Na+ or Li+ at the external site. Interestingly, the affinity for the amiloride analog MPA and for external H+ is unchanged in DD12. In conclusion, the genetic approach presented here: provides a general and specific method for selecting variants of the Na+/H+ antiporter with increased expression levels and/or with structural alterations and demonstrates that the external Na+- and amiloride-binding sites are not identical, since they can be genetically altered independently of each other.     

Mechanism of proline transport in Escherichia coli K12. III. Inhibition of membrane potential-driven proline transport by syn-coupled ions and evidence for symmetrical transition states of the 2H+/proline symport carrier

Specific inhibition of 2H+/proline symport by syn-coupled ions (Na+, Li+, and H+) was investigated using cytoplasmic membrane vesicles prepared from the proline carrier-overproducing strain MinS/ pLC4 -45 of Escherichia coli K12. The 2H+/proline symport driven by the membrane potential generated via respiration with 20 mM ascorbate/Tris, 0.1 mM phenazine methosulfate was specifically inhibited by Na+. The inhibition by Na+ was described by a fully noncompetitive mechanism, and the apparent Ki for Na+ was 15 mM. A linear correlation between the apparent Vmax and the apparent Kd was observed. Li+ stimulated the transport activity 2-fold at 10 mM and inhibited it at concentrations above 50 mM. H+ caused fully noncompetitive inhibition of 2H+/proline symport, and its apparent Ki was 0.6 microM. These results indicate that the concentrations of Na+ and H+ strictly and independently regulate the amount of the active C state carrier responsible for 2H+/proline symport driven by the membrane potential by inhibiting the transition from the C* state carrier which exhibits Na+- and H+-dependent binding of proline and is predominant in nonenergized conditions.     

Inhibition of Na+-dependent Ca2+ efflux from heart mitochondria by amiloride analogues

The Na+-induced release of accumulated Ca2+ from heart mitochondria is inhibited by amiloride, benzamil and several other amiloride analogues. These drugs do not affect uptake or release of Ca2+ mediated by the ruthenium red-sensitive uniporter and their effects, like those of diltiazem and other Ca2+-antagonists, appear to be localized principally at the Na+/Ca2+ antiporter of the mitochondrion. Benzamil inhibits Na+/Ca2+ antiport non-competitively with respect to [Na+] with a Ki of 167 microM. In the presence of 1.5 mM Pi the Ki for benzamil inhibition of this reaction is decreased to 87 microM.     

Two uncompetitive, activated, and transport sites of the Na+/H+ exchanger for pH regulation in perfused rat kidney

The purpose of this study is to assess the effect of an apparent alteration in intracellular pH and the effect of amiloride on the activity of the Na+/H+ antiporter in perfused rat kidney. Rat kidney-Na+ retention was determined using tracer 22Na in perfusate composed of HCl-glycine buffer (pH 3.80 to pH 5.92) or NH4OH-glycine buffer (pH 6.22-7.95) containing Na+ to match physiologic concentrations. Plotting renal Na+ retention for 10 min versus pH in absence of amiloride showed two classical uncompetitive activator curves for H+, one curve from pH 4.19 to 5.10 and another from pH 6.22 to 7.95. H+ acts as an uncompetitive reversible binding substrate with the receptor triggering activation of the exchanger already sequestered with Na+, thus yielding two Ka values for the exchanger suggesting non-first order kinetics. Using an equation derived for uncompetitive-activation binding of Nao+ and Hi+, plotting [mM Na+ mg protein-1 10 min-1]-1 versus [H+], two linear plots are observed on Cartesian coordinates with abscissa intersecting at 47 +/- 1 microM, pKa = 4.32 +/- 0.02 (pH 4.19-5.10) and 4.21 +/- 0.02 microM, pKa = 5.38 +/- 0.01 (pH 6.22-7.95), respectively. Perfusing buffer containing 2 mM amiloride, completely inactivated the antiporter showing stronger inhibition between pH 3.80 and 5.92. Results suggest the presence of two uncompetitive binding sites for H+ with the Na+/H+ exchanger. One is a high affinity binding site at physiological intracellular apparent pH, and another is a low affinity binding site at ischaemic apparent pH, implying the existence of two titration sites for intracellular pH regulation.     

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.     

Na+ for H+ exchange in rabbit erythrocytes

The effect of a transmembrane pH gradient on the ouabain, bumetanide, and phloretin resistant H+ efflux was studied in rabbit erythrocytes. Proton equilibration was reduced by the use of DIDS (125 microM) and acetazolamide (1 mM). H+ efflux from acid loaded erythrocytes (pHi = 6.1) was measured in a K+ (145 mM) medium, pH0 = 8.0, in the presence and absence of 60 microM 5,N,N-dimethyl-amiloride (DMA). The H+ efflux rate in a K+-containing medium was 116.38 +/- 4.5 mmol/l cell X hr. Substitution of Nao+ for Ko+ strongly stimulated H+ efflux to 177.89 +/- 7.9 mmol/l cell X hr. The transtimulation of H+ efflux by Nao+ was completely abolished by DMA falling to values not different from controls with an ID50 of about 8.6 X 10(-7) M. The sequence of substrate selectivities for the external transport site were Na greater than greater than greater than Li greater than choline, Cs, K, and Glucamine. The transport system has no specific anion requirement, but is inhibited by NO3-. The DMA sensitive H+ efflux was a saturable function of [Na+]o, with an apparent Km and Vmax of about 14.75 +/- 1.99 mM and 85.37 +/- 7.68 mmol/l cell X hr, respectively. However, the Nao+-dependent and DMA-sensitive H+ efflux was sigmoidally activated by [H+]i, suggesting that Hi+ interacts at both transport and modifier sites. An outwardly directed H+ gradient (pHi 6.1, pH = 8.0) also promoted DMA sensitive Na+ entry (61.2 +/- 3.0 mmol/l cell X hr) which was abolished when pHo was reduced to 6.0. The data is therefore consistent with the presence of a Na+/H+ exchange system in rabbit erythrocytes.     

Intracellular acidification-induced alkali metal cation/H+ exchange in human neutrophils

Pretreatment of isolated human neutrophils (resting pHi congruent to 7.25 at pHo 7.40) with 30 mM NH4Cl for 30 min leads to an intracellular acidification (pHi congruen to 6.60) when the NH4Cl prepulse is removed. Thereafter, in 140 mM Na+ medium, pHi recovers exponentially with time (initial rate, approximately 0.12 pH/min) to reach the normal resting pHi by approximately 20 min, a process that is accomplished mainly, if not exclusively, though an exchange of internal H+ for external Na+. This Na+/H+ countertransport is stimulated by external Na+ (Km congruent to 21 mM) and by external Li+ (Km congruent to 14 mM), though the maximal transport rate for Na+ is about twice that for Li+. Both Na+ and Li+ compete as substrates for the same translocation sites on the exchange carrier. Other alkali metal cations, such as K+, Rb+, or Cs+, do not promote pHi recovery, owing to an apparent lack of affinity for the carrier. The exchange system is unaffected by ouabain or furosemide, but can be competitively inhibited by the diuretic amiloride (Ki congruent to 8 microM). The influx of Na+ or Li+ is accompanied by an equivalent counter-reflux of H+, indicating a 1:1 stoichiometry for the exchange reaction, a finding consistent with the lack of voltage sensitivity (i.e., electroneutrality) of pHi recovery. These studies indicate that the predominant mechanism in human neutrophils for pHi regulation after intracellular acidification is an amiloride-sensitive alkali metal cation/H+ exchange that shares a number of important features with similar recovery processes in a variety of other mammalian cell types.     

Cytoplasmic pH regulation in thymic lymphocytes by an amiloride- sensitive Na+/H+ antiport

The mechanisms underlying cytoplasmic pH (pHi) regulation in rat thymic lymphocytes were studied using trapped fluorescein derivatives as pHi indicators. Cells that were acid-loaded with nigericin in choline+ media recovered normal pHi upon addition of extracellular Na+ (Nao+). The cytoplasmic alkalinization was accompanied by medium acidification and an increase in cellular Na+ content and was probably mediated by a Nao+/Hi+ antiport. At normal [Na+]i, Nao+/Hi+ exchange was undetectable at pHi greater than or equal to 6.9 but was markedly stimulated by internal acidification. Absolute rates of H+ efflux could be calculated from the Nao+-induced delta pHi using a buffering capacity of 25 mmol X liter-1 X pH-1, measured by titration of intact cells with NH4+. At pHi = 6.3, pHo = 7.2, and [Na+]o = 140 mM, H+ extrusion reached 10 mmol X liter-1 X min-1. Nao+/Hi+ exchange was stimulated by internal Na+ depletion and inhibited by lowering pHo and by addition of amiloride (apparent Ki = 2.5 microM). Inhibition by amiloride was competitive with respect to Nao+. Hi+ could also exchange for Lio+, but not for K+, Rb+, Cs+, or choline+. Nao+/Hi+ countertransport has an apparent 1:1 stoichiometry and is electrically silent. However, a small secondary hyperpolarization follows recovery from acid-loading in Na+ media. This hyperpolarization is amiloride- and ouabain-sensitive and probably reflects activation of the electrogenic Na+-K+ pump. At normal Nai+ values, the Nao+/Hi+ antiport of thymocytes is ideally suited for the regulation of pHi. The system can also restore [Na+]i in Na+-depleted cells. In this instance the exchanger, in combination with the considerable cytoplasmic buffering power, will operate as a [Na+]i- regulatory mechanism.     

Ion specificity of cardiac sarcolemmal Na+/H+ antiporter

In bovine cardiac sarcolemmal vesicles, an outward H+ gradient stimulated the initial rate of amiloride-sensitive uptake of 22Na+, 42K+, or 86Rb+. Release of H+ from the vesicles was stimulated by extravesicular Na+, K+, Rb+, or Li+ but not by choline or N-methylglucamine. Uptakes of Na+ and Rb+ were half-saturated at 3 mM Na+ and 3 mM Rb+, but the maximal velocity of Na+ uptake was 1.5 times that of Rb+ uptake. Na+ uptake was inhibited by extravesicular K+, Rb+, or Li+, and Rb+ uptake was inhibited by extravesicular Na+ or Li+. Amiloride-sensitive uptake of Na+ or Rb+ increased with increase in extravesicular pH and decrease in intravesicular pH. In the absence of pH gradient, there were stimulations of Na+ uptake by intravesicular Na+ and K+ and of Rb+ uptake by intravesicular Rb+ and Na+. Similarly, there were trans stimulations of Na+ and Rb+ efflux by extravesicular alkali cations. The data suggest the existence of a nonselective antiporter catalyzing either alkali cation/H+ exchange or alkali cation/alkali cation exchange. Since increasing Na+ caused complete inhibition of Rb+/H+ exchange, but saturating K+ caused partial inhibitions of Na+/H+ exchange and Na+/Na+ exchange, the presence of a Na(+)-selective antiporter is also indicated. Although both antiporters may be involved in pH homeostasis, a role of the nonselective antiporter may be in the control of Na+/K+ exchange across the cardiac sarcolemma.     

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.     

Voltage-dependent modulation of ion binding and translocation in the cardiac Na(+)-Ca2+ exchange system

The transport of Na+ and Ca2+ ions in the cardiac Na(+)-Ca2+ exchanger can be described as separate events (Khananshvili, D. (1990) Biochemistry 29, 2437-2442). Thus, the Na(+)-Na+ and Ca(2+)-Ca2+ exchange reactions reflect reversible partial reactions of the transport cycle. The effect of diffusion potentials (K(+)-valinomycin) on different modes of the Na(+)-Ca2+ exchanger (Na(+)-Ca2+, Ca(2+)-Ca2+, and Na(+)-Na+ exchanges) were tested in reconstituted proteoliposomes, obtained from the Triton X-100 extracts of the cardiac sarcolemmal membranes. The initial rates of the Nai-dependent 45Ca-uptake (t = 1 s) were measured in EGTA-entrapped proteoliposomes at different voltages. At the fixed values of voltage [45 Ca]o was varied from 4 to 122 microM, and [Na]i was saturating (150 mM). Upon varying delta psi from -94 to +91 mV, the Vmax values were increased from 9.5 +/- 0.5 to 26.5 +/- 1.5 nmol.mg-1.s-1 and the Km from 17.8 +/- 2.5 to 39.1 +/- 5.2 microM, while the Vmax/Km values ranged from only 0.53 +/- 0.08 to 0.73 +/- 0.17 nmol.mg-1.s-1.microM-1. The equilibrium Ca(2+)-Ca2+ exchange was voltage sensitive at very low [Ca]o = [Ca]i = 2 microM, while at saturating [Ca]o = [Ca]i = 200 microM the Ca(2+)-Ca2+ exchange became voltage-insensitive. The rates of the equilibrium Na(+)-Na+ exchange appears to be voltage insensitive at saturating [Na]o = [Na]i = 160 mM. Under the saturating ionic conditions, the rates of the Na(+)-Na+ exchange were at least 2-3-fold slower than the Ca(2+)-Ca2+ exchange. The following conclusions can be drawn. (a) The near constancy of the Vmax/Km for Na(+)-Ca2+ exchange at different voltages is compatible with the ping-pong model proposed previously. (b) The effects of voltage on Vmax of Na(+)-Ca2+ exchange are consistent with the existence of a single charge carrying transport step. (c) It is not yet possible to clearly assign this step to the Na+ or Ca2+ transport half of the cycle although it is more likely that 3Na(+)-transport is a charge carrying step. Thus, the unloaded ion-binding domain contains either -2 or -3 charges (presumably carboxyl groups). (d) The binding of Na+ and Ca2+ appears to be weakly voltage-sensitive. The Ca(2+)-binding site may form a small ion-well (less than 2-3 A).     

Chemotactic factor-induced activation of Na+/H+ exchange in human neutrophils. I. Sodium fluxes

The nature of Na+ fluxes in resting and in chemotactic factor-activated human neutrophils was investigated. In resting cells, ouabain-insensitive unidirectional 22Na+ in- and effluxes represented passive electrodiffusional fluxes through ion channels: they were nonsaturable and voltage-dependent (PNa = 4.3 X 10(-9) cm/s). Amiloride (1 mM) had little effect on resting 22Na+ influx (approximately 0.8 meq/liter X min), thereby suggesting a minor contribution of Na+/H+ exchange and a lack of amiloride-sensitive Na+ channels. When neutrophils were exposed to the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP, 0.1 microM), 22Na+ influx was stimulated approximately 30-fold (initial rate approximately 22 meq/liter X min). The FMLP-induced 22Na+ influx was saturable with respect to external Na+ (Km 26-35 mM, Vmax approximately 28 meq/liter X min), was electroneutral, and could be competitively inhibited by amiloride (Ki 10.6 microM). From a resting value of approximately 30 meq/liter of cell water, internal Na+ in FMLP-stimulated cells rose exponentially to reach a concentration of approximately 60 meq/liter by 10-15 min. This uptake was blocked by amiloride. FMLP also stimulated the efflux of 22Na+ which followed a single exponential time course (rate coefficient approximately 0.16 min-1). The FMLP-induced 22Na+ fluxes were similar to those observed with 10 microM monensin, a known Na+/H+ exchanging ionophore. The data indicate that FMLP activates an otherwise quiescent, amiloride-sensitive Na+/H+ exchange. Furthermore, all of the FMLP-induced 22Na+ fluxes can be satisfactorily accounted for by transport through the exchanger, leaving little room for an appreciable increase in Na+ conductance.     

Regulation of intracellular pH in cultured hippocampal neurons by an amiloride-insensitive Na+/H+ exchanger.

Regulation of intracellular pH (pHi) in single cultured rat hippocampal neurons was investigated using the fluorescent pHi indicator dye bis-carboxyethylcarboxyfluorescein. Resting pHi was dependent on the presence of bicarbonate and external Na+ but was not altered significantly by removal of Cl- or treatment with the anion exchange inhibitor diisothiocyanatostilbene-2,2'-disulfonate. Recovery of pHi from acute acid loading was due, in large part, to a pharmacologically distinct variant of the Na+/H+ antiporter. In nominally HCO3(-)-free solutions, this recovery exhibited a saturable dose dependence on extracellular Na+ (Km = 23-26 mM) or Li+. The antiporter was activated by decreasing pHi and was unaffected by collapse of the membrane potential with valinomycin. Like the Na+/H+ antiporter described in other cell systems, the hippocampal activity was inhibited by harmaline, but in sharp contrast, neither amiloride nor its more potent 5-amino-substituted analogues were able to prevent the recovery from an acid load. These data indicate that Na(+)-dependent mechanisms dominate pHi regulation in hippocampal neurons and suggest a role for a novel variant of the Na+/H+ antiporter.     

Chemotactic factor-induced activation of Na+/H+ exchange in human neutrophils. II. Intracellular pH changes

The intracellular pH (pHi) changes resulting from chemotactic factor-induced activation of Na+/H+ exchange in isolated human neutrophils were characterized. Intracellular pH was measured from the equilibrium distribution of [14C]-5,5-dimethyloxazolidine-2,4-dione and from the fluorescence of 6-carboxyfluorescein. Exposure of cells to 0.1 microM N-formyl-methionyl-leucyl-phenylalanine (FMLP) in 140 mM Na+ medium at extracellular pH (pHo) 7.40 led to a rise in pHi along an exponential time course (rate coefficient approximately 0.55 min-1). By 10 min, a new steady-state pHi was reached (7.75-7.80) that was 0.55-0.60 units higher than the resting pHi of control cells (7.20-7.25). The initial rate of H+ efflux from the cells (approximately 15 meq/liter X min), calculated from the intrinsic intracellular buffering power of approximately 50 mM/pH, was comparable to the rate of net Na+ influx (approximately 17 meq/liter X min), an observation consistent with a 1:1 stoichiometry for Na+/H+ exchange. This counter-transport could be inhibited by amiloride (apparent Ki approximately 75 microM). When either the external ([Na+]o) or internal Na ([Na+]i) concentrations, pHo, or pHi were varied independently, the new steady-state [Na+]i and pHi values in FMLP-stimulated cells were those corresponding to a chemical equilibrium distribution of Na+ and H+ across the cell membrane. By analogy to other activated cells, these results indicate that an alkalinization of pHi in human neutrophils is mediated by a chemotactic factor-induced exchange of internal H+ for external Na+.