Unidirectional Na+, Ca2+, and K+ fluxes through the bovine rod outer segment Na-Ca-K exchanger

The properties of the Na-Ca exchanger in the plasma membrane of rod outer segments isolated from bovine retinas (ROS) were studied. Unidirectional Ca2+, Na+, and K+ fluxes were measured with radioisotopes and atomic absorption spectroscopy. We measured K+ fluxes associated with the Ca-Ca self-exchange mode of the Na-Ca exchanger to corroborate our previous conclusion that the ROS Na-Ca exchanger differs from Na-Ca exchangers in other tissues by its ability to transport K+ (Schnetkamp, P. P. M., Basu, D. K. & Szerencsei, R. T. (1989) Am. J. Physiol. 257, C153-C157). The Na-Ca-K exchanger was the only functional cation transporter in the plasma membrane of bovine ROS with an upper limit of a flux of 10(5) cations/ROS/s or a current of 0.01 pA contributed by other cation channels, pumps, or carriers; cation fluxes via the Na-Ca-K exchanger amounted to 5 x 10(6) cations/ROS/s or a current of 1 pA. Ca2+ efflux via the forward mode of the Na-Ca-K exchanger did not operate with a fixed single stoichiometry. 1) The Na/Ca coupling ratio was increased from three to four when ionophores were added that could provide electrical compensation for the inward Na-Ca exchange current. 2) The K/Ca coupling ratio could vary by at least 2-fold as a function of the external Na+ and K+ concentration. The results are interpreted in terms of a model that can account for the variable Ca/K coupling ratio: we conclude that the Ca2+ site of the exchanger can translocate independent of translocation of the K+ site, whereas translocation of the K+ site requires occupation of the Ca2+ site, but not its translocation. The results are discussed with respect to the physiological role of Na-Ca-K exchange in rod photoreceptors.     

Sodium ions selectively eliminate the fast component of guanosine cyclic 3',5'-phosphate induced Ca2+ release from bovine rod outer segment disks

Guanosine cyclic 3',5'-phosphate (cGMP) induced Ca2+ release from bovine rod outer segment (ROS) disks showed two kinetic components that could be distinguished in three ways: (1) The slow component (half-rise time of about 30 s) was blocked by 1-cis diltiazem [cf. Koch, K. W., & Kaupp, U. B. (1985) J. Biol. Chem. 260, 6788-6800], whereas the fast component (half-rise time of less than 1 s) was not affected by 1-cis diltiazem. (2) The slow component required the presence of alkali cations, whereas the fast component did not. (3) Preincubation with Na+ (50 mM) selectively eliminated the fast component, whereas the slow component was not affected. The action of Na+ appeared to be caused by Na-Ca exchange removing Ca2+ from a pool that can also be accessed by cGMP. The slow component of cGMP-induced Ca2+ release was not affected by Na+ and, hence, appears to reside in disks that do not contain a functional Na-Ca exchanger. The local anesthetic tetracaine blocked both the slow and fast component of cGMP-induced Ca2+ release from bovine ROS disks.     

Cation selectivity of and cation binding to the cGMP-dependent channel in bovine rod outer segment membranes

The properties of the cGMP-dependent channel present in membrane vesicles prepared from intact isolated bovine rod outer segments (ROS) were investigated with the optical probe neutral red. The binding of neutral red is sensitive to transport of cations across vesicular membranes by the effect of the translocated cations on the surface potential at the intravesicular membrane/water interface (Schnetkamp, P. P. M. J. Membr. Biol. 88: 249-262). Only 20-25% of ROS membrane vesicles exhibited cGMP-dependent cation fluxes. The cGMP-dependent channel in bovine ROS carried currents of alkali and earth alkali cations, but not of organic cations such as choline and tetramethylammonium; little discrimination among alkali cations (K greater than Na = Li greater than Cs) or among earth alkali cations (Ca greater than Mn greater than Sr greater than Ba = Mg) was observed. The cation dependence of cGMP-induced cation fluxes could be reasonably well described by a Michaelis-Menten equation with a dissociation constant for alkali cations of about 100 mM, and a dissociation constant for Ca2+ of 2 mM. cGMP-induced Na+ fluxes were blocked by Mg2+, but not by Ca2+, when the cations were applied to the cytoplasmic side of the channel. cGMP-dependent cation fluxes showed a sigmoidal dependence on the cGMP concentration with a Hill coefficient of 2.1 and a dissociation constant for cGMP of 92 microM. cGMP-induced cation fluxes showed two pharmacologically distinct components; one component was blocked by both tetracaine and L-cis diltiazem, whereas the other component was only blocked by tetracaine.     

Purification of the bovine rod outer segment Na+/Ca2+ exchanger

Optimal conditions for solubilization and stabilization of the Na+/Ca2+ exchanger from rod outer segments were examined. The exchanger was found to be most stable at low detergent concentrations (7.5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate), greater than or equal to 100 mM NaCl, pH 7.0-7.5, and with 0.1% added soybean asolectin. The sulfhydryl-modifying reagent, dithiothreitol, caused a loss of exchanger activity and was omitted throughout the purification procedure. These conditions were used to purify the Na+/Ca2+ exchanger from rod outer segments by a combination of selective solubilization, ion exchange, and wheat germ agglutinin chromatography. The procedure achieves a 336-fold increase in exchanger specific activity. The presence of exchanger activity most closely correlates with a polypeptide of molecular mass 215-kDa. Exchanger activity in both the crude rod outer segments and the purified exchanger is specifically dependent upon the presence of K+ in the assay medium; neither choline nor Li+ can substitute for K+.     

Effect of Monovalent Cations on Na+/Ca2+ Exchange and ATP-Dependent Ca2+ Transport in Synaptic Plasma Membranes

Two Ca2+ transport systems were investigated in plasma membrane vesicles isolated from sheep brain cortex synaptosomes by hypotonic lysis and partial purification. Synaptic plasma membrane vesicles loaded with Na+ (Na+i) accumulate Ca2+ in exchange for Na+, provided that a Na+ gradient (in leads to out) is present. Agents that dissipate the Na+ gradient (monensin) prevent the Na+/Ca2+ exchange completely. Ca2+ accumulated by Na+/Ca2+ exchange can be released by A 23187, indicating that Ca2+ is accumulated intravesicularly. In the absence of any Na+ gradient (K+i-loaded vesicles), the membrane vesicles also accumulate Ca2+ owing to ATP hydrolysis. Monovalent cations stimulate Na+/Ca2+ exchange as well as the ATP-dependent Ca2+ uptake activity. Taking the value for Na+/Ca2+ exchange in the presence of choline chloride (external cation) as reference, other monovalent cations in the external media have the following effects: K+ or NH4+ stimulates Na+/Ca2+ exchange; Li+ or Cs+ inhibits Na+/Ca2+ exchange. The ATP-dependent Ca2+ transport system is stimulated by increasing K+ concentrations in the external medium (Km for K+ is 15 mM). Replacing K+ by Na+ in the external medium inhibits the ATP-dependent Ca2+ uptake, and this effect is due more to the reduction of K+ than to the elevation of Na+. The results suggest that synaptic membrane vesicles isolated from sheep brain cortex synaptosomes possess mechanisms for Na+/Ca2+ exchange and ATP-dependent Ca2+ uptake, whose activity may be regulated by monovalent cations, specifically K+, at physiological concentrations.     

A role for Na+/Ca2+ exchange in the generation of superoxide radicals by human neutrophils

A Na+/Ca2+ exchange mechanism has been recently described in human neutrophils that constitutes the principal pathway for Ca2+ influx into resting cells. The potential role of this system in regulating the respiratory burst in response to activation by the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine was explored. In the presence of 1 mM Ca2+, a variety of di- and trivalent cations suppressed the generation of O(-2) radicals in a series of decreasing efficacy: La3+ approximately Zn2+ much greater than Sr2+ approximately Cd2+ greater than Ba2+ greater than Co2+ greater than Ni2+ approximately Mg2+. This sequence is similar to their rank order of activity in inhibiting 45Ca2+ influx via Na+/Ca2+ counter-transport. Benzamil, phenamil, and 2',4'-dichlorobenzamil, analogues of amiloride which selectively block Na+/Ca2+ exchange in neutrophils, likewise suppressed the release of O(-2) with apparent Ki values of approximately 30 microM. The effect of the cations was competitive with Ca2+, while the interaction between the benzamil derivatives and Ca2+ appeared to be noncompetitive in nature. Both the divalent cations and benzamil also inhibited the rise in cytoplasmic Ca2+ as monitored by fura-2 fluorescence: these agents reduced peak cytosolic Ca2+ levels after N-formyl-methionyl-leucyl-phenylalanine stimulation to values seen in the absence of extracellular Ca2+. These results are compatible with the hypothesis that the influx of Ca2+ via Na+/Ca2+ exchange contributes to the transient elevation in intracellular free Ca2+. The polyvalent cations block the entry of critical Ca2+ ions by competing with Ca2+ for binding to the translocation site on the exchange carrier, while benzamil acts by lowering the maximal transport rate. These studies emphasize that Na+/Ca2+ exchange through its effects on cytoplasmic Ca2+ plays a major regulatory role in activation of the respiratory burst in chemotactic factor-stimulated neutrophils.     

Effect of potassium ions and membrane potential on the Na-Ca-K exchanger in isolated intact bovine rod outer segments

Two recent studies reported that Na-Ca exchange in the outer segments of tiger salamander rod photoreceptors (Cervetto, L., Lagnado, L., Perry, R. J., Robinson, D. W., and McNaughton, P. A. (1989) Nature 337, 740-743) and of bovine rod photoreceptors (Schnetkamp, P. P. M., Basu, D. K., and Szerencsei, R. T. (1989) Am. J. Physiol. 257, C153-157) requires and transports K+ in a 4Na/(1Ca+1K) stoichiometry. In this study, we have examined the effects of K+ ions and membrane potential on the kinetics of Na-Ca and Ca-Ca exchange in rod outer segments isolated from bovine retinas. The objective was to establish the ion selectivity and voltage dependence of the different cation binding sites on the Na-Ca-K exchange protein. Potassium ions activated Na-Ca exchange when present on the Ca2+ side, although the extent of activation decreased with decreasing Na+ concentration. Potassium ions inhibited Na-Ca exchange when present on the Na+ side; inhibition arose from competition between Na+ and K+ for a common single cation-binding site. Activation of Na-Ca exchange by K+ displayed a different ion selectivity than that observed for inhibition of Na-Ca exchange by K+. The results are interpreted in terms of a three-site model for the rod Na-Ca-K exchanger. The rate of forward Na-Ca exchange decreased by 1.75-fold for a 60 mV depolarization of the plasma membrane but only at lower Na+ concentrations. The rate of Ca-Ca exchange was not affected by changes in membrane potential.     

Calcium-hydrogen exchange in isolated bovine rod outer segments

We have measured Ca-H exchange in rod photoreceptors with different preparations of rod outer segments isolated from bovine retinas (ROS). One preparation contained ROS with an intact plasma membrane (intact ROS), and in the other preparation, the plasma membrane was leaky to small solutes (leaky ROS) and the cytoplasmic space was freely accessible to externally applied solutes. Addition of Ca2+ to Ca2+-depleted ROS (both intact and leaky) resulted in uptake of Ca2+ that was accompanied by the release of protons when catalytic amounts of the ionophore A23187 were present. This ionophore mediates Ca-H exchange transport across ROS membranes and serves to gain access to the intracellular compartment where Ca-H exchange appears to take place. Two protons were ejected for each calcium ion taken up. Conversely, when protons were added to Ca2+-enriched ROS, Ca2+ was released in the presence of A23187. The majority of this Ca-H exchange was observed only when A23187 was present in both intact and leaky ROS. We conclude that Ca-H exchange occurs predominantly in the intradiskal space and at the surface of the disk membrane rather than across the disk membrane. These exchange binding sites can accommodate 10 mol of Ca2+/mol of rhodopsin at physiological pH. We were unable to detect any Ca2+ release when a proton gradient was rapidly established across the disk membrane in the absence of A23187. These results are discussed in relation to the hypothesis that protons produced by the light-induced hydrolysis of cGMP cause the release of Ca2+ into the cytoplasm of rod photoreceptor cells.     

Transport of K+ by Na(+)-Ca2+, K+ exchanger in isolated rods of lizard retina.

Transport of K+ by the photoreceptor Na(+)-Ca2+, K+ exchanger was investigated in isolated rod outer segments (OS) by recording membrane current under whole-cell voltage-clamp conditions. Known amounts of K+ were imported in the OS through the Ca(2+)-activated K+ channels while perfusing with high extracellular concentration of K+, [K+]o. These channels were detected in the recordings from the OS, which probably retained a small portion of the rest of the cell. The activation of forward exchange (Na+ imported per Ca2+ and K+ extruded) by intracellular K+, Ki+, was described by first-order kinetics with a Michaelis constant, Kapp(Ki+), of about 2 mM and a maximal current, Imax, of about -60 pA. [Na+]i larger than 100 mM had little effect on Kapp(Ki+) and Imax, indicating that Nai+ did not compete with Ki+ for exchange sites under physiological conditions, and that Na+ release at the exchanger intracellular side was not a rate-limiting step for the exchange process. Exchanger stoichiometry resulted in one K+ ion extruded per one positive charge imported. Exchange current was detected only if Ca2+ and K+ were present on the same membrane side, and Na+ was simultaneously present on the opposite side. Nonelectrogenic modes of ion exchange were tested taking advantage of the hindered diffusion found for Cai2+ and Ki+. Experiments were carried out so that the occurrence of a putative nonelectrogenic ion exchange, supposedly induced by the preapplication of certain extracellular ion(s), would have resulted in the transient presence of both Cai2+ and Ki+. The lack of electrogenic forward exchange in a subsequent switch to high Nao+, excluded the presence of previous nonelectrogenic transport.     

Optical measurements of Na-Ca-K exchange currents in intact outer segments isolated from bovine retinal rods

The properties of Na-Ca-K exchange current through the plasma membrane of intact rod outer segments (ROS) isolated from bovine retinas were studied with the optical probe neutral red. Small cellular organelles such as bovine ROS do not offer an adequate collecting area to measure Na-Ca-K exchange currents with electrophysiological techniques. This study demonstrates that Na-Ca-K exchange current in bovine ROS can be measured with the dye neutral red and dual-wavelength spectrophotometry. The binding of neutral red is sensitive to transport of cations across the plasma membrane of ROS by the effect of the translocated cations on the surface potential of the intracellular disk membranes (1985. J. Membr. Biol. 88: 249-262). Electrogenic Na+ fluxes through the ROS plasma membrane were measured with a resolution of 10(5) Na+ ions/ROS per s, equivalent to a current of approximately 0.01 pA; maximal electrogenic Na-Ca-K exchange flux in bovine ROS was equivalent to a maximal exchange current of 1-2 pA. Electrogenic Na+ fluxes were identified as Na-Ca-K exchange current based on a comparison between electrogenic Na+ flux and Na(+)-stimulated Ca2+ release with respect to flux rate, Na+ dependence, and ion selectivity. Neutral red monitored the net entry of a single positive charge carried by Na+ for each Ca2+ ion released (i.e., monitored the Na-Ca-K exchange current). Na-Ca-K exchange in the plasma membrane of bovine ROS had the following properties: (a) Inward Na-Ca-K exchange current required internal Ca2+ (half-maximal stimulation at a free Ca2+ concentration of 0.9 microM), whereas outward Na-Ca-K exchange current required both external Ca2+ (half-maximal stimulation at a free Ca2+ concentration of 1.1 microM) and external K+. (b) Inward Na-Ca-K exchange current depended in a sigmoidal manner on the external Na+ concentration, identical to Na(+)-stimulated Ca2+ release measured with Ca(2+)-indicating dyes. (c) The neutral red method was modified to measure Ca(2+)-activated K+ fluxes (half-maximal stimulation at 2.7 microM free Ca2+) via the Na-Ca-K exchanger in support of the notion that the rod Na-Ca exchanger is in effect a Na-Ca-K exchanger. (d) Competitive interactions between Ca2+ and Na+ ions on the exchanger protein are described.     

The effects of manganese and changes in internal calcium on Na-Ca exchange fluxes in the intact squid giant axon.

The effects of manganese chloride were studied on Na-Ca exchange fluxes from intact squid axons. Ca uptakes and Cao-dependent sodium efflux were inhibited half-maximally by 3-7 mM MnCl2. Mn inhibition appears less during Nao-Cai exchange (half-maximal inhibition; 30 mM) than that during Cao-Nai exchange, even when both fluxes were activated with 100 mM Na. The effects of changes in [Ca2+i], effected by Ca-EGTA injection or inhibition of mitochondrial Ca uptake by ruthenium red, were examined on the reverse (Cao-Nai) exchange mode. Ca-EGTA mixtures, designed to raise [Ca2+i] above 2 microM, inhibited Cao-Nai exchange fluxes. Ruthenium red inhibited mitochondrial Ca buffering to effect increases in Cai in the absence of Ca chelators; it activated Nao-Cai exchange fluxes but had little effect on Cao-Nai exchange despite similar reported Km for Cai. The results reflect the difficulty in demonstrating the stimulatory effect of [Ca2+i] on Cao-Nai exchange fluxes in intact axons.     

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.     

The effect of Li+ on Na-Ca exchange in cardiac sarcolemmal vesicles

The effects of Li+ on Na-Ca exchange in bovine cardiac sarcolemmal vesicles were examined. The initial rate of Na(+)-dependent Ca2+ uptake and efflux was inhibited by Li+ in a dose dependent manner. The initial rate of Na(+)-dependent Ca2+ uptake was inhibited 49.8 +/- 2.9% (S.E.) (n = 6) in the presence of Li+ compared to activity in external K+ or choline+. Kinetic analysis indicated that Li+ increased the Km for Ca2+ (96.3 microM) compared to K+ and choline+ (25.5 and 22.9 microM respectively) while Vmax (1.4, 1.2 and 1.1 nmol Ca2+/mg protein/sec respectively) remained unchanged. Li+ did not alter the experimentally derived stoichiometry of the exchange reaction of 3 Na+ for 1 Ca2+.     

Transmembrane Na+ and Ca2+ electrochemical gradients in cardiac muscle and their relationship to force development

Na+- and CA2+-sensitive microelectrodes were used to measure intracellular Na+ and Ca2+ activities (alpha iCa) of sheep ventricular muscle and Purkinje strands to study the interrelationship between Na+ and Ca2+ electrochemical gradients (delta muNa and delta muCa) under various conditions. In ventricular muscle, alpha iNa was 6.4 +/- 1.2 mM and alpha iCa was 87 +/- 20 nM ([Ca/+] = 272 nM). A graded decrease of external Na+ activity (alpha oNa) resulted in decrease of alpha iNa, and increase of alpha iCa. There was increase of twitch tension in low- alpha oNa solutions, and occasional increase of resting tension in 40% alpha oNa. Increase of external Ca2+ (alpha oCa) resulted in increase of alpha iCa and decrease of alpha iNa. Decrease of alpha oCa resulted in decrease of alpha iCa and increase of alpha iNa. The apparent resting Na-Ca energy ratio (delta muCa/delta muNa) was between 2.43 and 2.63. When the membrane potential (Vm) was depolarized by 50 mM K+ in ventricular muscle, Vm depolarized by 50 mV, alpha iNa decreased, and alpha iCa increased, with the development of a contracture. The apparent energy coupling ratio did not change with depolarization. 5 x 10(-6) M ouabain induced a large increase in alpha iNa ad alpha iCa, accompanied by an increase in twitch and resting tension. Under the conditions we have studied, delta muNa and delta muCa appeared to be coupled and n was nearly constant at 2.5, as would be expected if the Na-Ca exchange system was able to set the steady level of alpha iCa. Tension threshold was about 230 nM alpha iCa. The magnitude of twitch tension was directly related to alpha iCa.     

Na+/Ca2+ exchange in plasma membrane vesicles from a glucose-responsive insulinoma.

Plasma membrane vesicles from a glucose-responsive insulinoma exhibited properties consistent with the presence of a membrane Na+/Ca2+ exchange. The exchange was rapid, reversible, and was dependent on the external Ca2+ concentration (Km = 4.1 +/- 1.1 microM). External Na+ inhibited the uptake in a dose-dependent manner (IC50 = 15 mM). Dissipation of the Na+ gradient by 10 microM monensin decreased Na+/Ca2+ exchange from 0.74 +/- 0.17 nmoles/mg protein/s to 0.11 +/- 0.05 nmoles/mg protein/s. Exchange was not influenced by veratridine, tetrodotoxin and ouabain, or by modifiers of cAMP. No effect was seen using the calcium channel blockers, nitrendipine or nifedipine. Glucose had no direct effect on Na+/Ca2+ exchange, while glyceraldehyde, glyceraldehyde-3-phosphate and dihydroxyacetone inhibited the exchange. Na+ induced efflux of calcium was seen in Ca2+ loaded vesicles and was half maximal at [Na+] of 11.1 +/- 0.75 mM. Ca2+ efflux was dependent on [Na+], with a Hill coefficient of 2.7 +/- 0.07 indicating that activation of Ca2+ release involves a minimum of three sites. The electrogenicity of this exchange was demonstrated using the lipophilic cation tetraphenylphosphonium [( 3H]-TPP), a membrane potential sensitive probe. [3H]-TPP uptake increased transiently during Na+/Ca2+ exchange indicating that the exchange generated a membrane potential. These results show that Na+/Ca2+ exchange operates in the beta cell and may be an important regulator of intracellular free Ca2+ concentrations.     

Frog rod outer segments with attached inner segment ellipsoids as an in vitro model for photoreceptors on the retina

Purified suspensions of frog rod outer segments still attached to the mitochondria-rich inner segment portion of the receptor cell (OS-IS) can be obtained in quantities (0.1 mg/retina) sufficient for chemical analysis. In oxygenated glucose-bicarbonate Ringer's medium with added Percoll, they display normal dark currents, light sensitivity, and photocurrent kinetics for several hours. Two millimolar cytoplasmic levels of ATP and GTP are maintained, fivefold higher than in isolated OS. The levels are not altered by abolition of the dark current with ouabain. Nucleoside triphosphates are more effectively buffered than in isolated OS, and their levels remain constant during changes in external calcium levels. 32Pi is incorporated into endogenous ATP and GTP pools twice as efficiently as in isolated OS, and is used in the phosphorylation of rhodopsin. OS-IS take up and release 45Ca++ by Na+-, Ca++-, and IBMX-sensitive mechanisms. Illumination causes release of 45Ca++, which confirms retinal studies by other groups using Ca++-sensitive electrodes. Thus, OS-IS suspensions model the behavior of photoreceptors still attached to the living retina. Their availability permits the simultaneous assay and correlation of electrophysiological and chemical changes occurring during excitation and adaptation.     

Na+-H+ exchange in isolated renal brush-border membrane vesicles in response to metabolic acidosis. Kinetic effects

Chronic metabolic acidosis increased the Na+-H+ exchange activity in isolated renal brush-border membrane vesicles. Treatment altered the initial rate of Na+ uptake by increasing Vm (acidotic, 15.3 +/- 0.7 nmol of Na+ X mg-1 X 2 s-1; normal, 11.3 +/- 0.9 nmol of Na+ X mg-1 X 2 s-1), and not the apparent affinity KNa+ (acidotic, 10.2 +/- 0.5 mM; normal 10.2 +/- 0.6 mM). Metabolic acidosis resulted in the proportional increase in 1 mM Na+ uptake at every intravesicular pH measured. A positive cooperative effect on Na+ uptake was found with increased intravesicular acidity in vesicles from both normal and acidotic rats. When the data were analyzed by the Hill equation, it was found that metabolic acidosis did not change the n (acidotic, 1.33 +/- 0.13; normal, 1.43 +/- 0.07) or the K'H+ (acidotic, 0.27 +/- 0.05 microM; normal, 0.28 +/- 0.06 microM), but increased the apparent Vm (acidotic, 1.10 +/- 0.08 nmol of Na+ X mg-1 X 2 s-1; normal, 0.81 +/- 0.07 nmol of Na+ X mg-1 X 2 s-1). The uptake of Na+ in exchange for H+ in membrane vesicles from normal and acidotic animals was not influenced by membrane potential. We conclude that metabolic acidosis leads to either an increase in the number of functioning exchangers or an increase in the turnover rate of the limiting step in the exchange.     

Effects of internal and external cations and of ATP on sodium-calcium and calcium-calcium exchange in squid axons.

Calcium-45 efflux was measured in squid axons whose internal solute concentration was controlled by internal dialysis. Most of the Ca efflux requires either external Na (Na-Ca exchange) or external Ca plus in alkali metal ion (Ca-Ca exchange; cf. Blaustein & Russell, 1975). Both Na-Ca and Ca-Ca exchange are apparently mediated by a single mechanism because both are inhibited by Sr and Mn, and because addition of Na to an external medium optimal for Ca-Ca exchange inhibits Ca efflux. The transport involves simultaneous (as opposed to sequential) ion counterflow because the fractional saturation by internal Ca (Cai) does not affect the external Na (Nao) activation kinetics; also, Nao promotes Ca efflux whether or not an alkali metal ion is present inside, whereas Ca-Ca exchange requires alkali metal ions both internally and externally (i.e., internal and external sites must be appropriately loaded simultaneously). ATP increases the affinity of the transport mechanism for both Cai and Nao, but it does not affect the maximal transport rate at saturating [Ca2+]i and [Na+]o; this suggest that ATP may be acting as a catalyst of modulator, and not as an energy source. Hill plots of the Nao activation data yield slopes congruent to 3 for both ATP-depleted and ATP-fueled axons, compatible with a 3 Na+-for-1 Ca2+ exchange. With this stoichiometry, the Na electrochemical gradient alone could provide sufficient energy to maintain ionized [Ca2+]i in the physiological range (about 10(-7) M).     

Intravesicular pH changes in submitochondrial particles induced by monovalent cations: relationship to the Na+/H+ and K+/H+ antiporters

The fluorescence of internalized fluorescein isothiocyanate dextran has been used to monitor the intravesicular pH of submitochondrial particles (SMP). Respiring SMP maintain a steady-state delta pH (interior acid) that results from the inwardly directed H+ flux of respiration and an opposing passive H+ leak. Addition of K+, Na+, or Li+ to SMP results in a shift to a more alkaline interior pH (pHi) in both respiring and nonrespiring SMP. The K+-dependent change in pHi, like the K+/H+ antiport in intact mitochondria, is inhibited by quinine and by dicyclohexylcarbodiimide. The Na+-dependent reaction is only partially inhibited by these reagents. Both the Na+- and the K+-dependent pH changes are sensitive to amiloride derivatives. The Km for both Na+ and K+ is near 20 mM whereas that for Li+ is closer to 10 mM. The K+/H+ exchange reaction is only slightly inhibited by added Mg2+, but abolished when A23187 is added with Mg2+. The passive exchange is optimal at pHi 6.5 with either Na+ or K+, and cannot be detected above pHi of 7.2. Both the Na+/H+ and the K+/H+ exchange reactions are optimal at an external pH of 7.8 in respiring SMP (pHi 7.1). Valinomycin stimulates the K+-dependent pH change in nonrespiring SMP, as does nigericin. It is concluded that SMP show K+/H+ antiport activity with properties distinct from those of Na+/H+ antiport. However, the properties of the K+/H+ exchange do not correspond in all respects to those of the antiport in intact mitochondria. Donnan equilibria and parallel uniport pathways for H+ and cations appear to contribute to cation-dependent pH changes in SMP.     

Sodium-calcium exchange in membrane vesicles from Artemia

Membrane vesicles were prepared from Artemia nauplii (San Francisco Bay variety) 45 h after hydration of the dry cysts. Na+-loaded vesicles accumulated up to 10 nmol Ca2+/mg protein when diluted 50-fold into 160 mM KCl containing 15 microM CaCl2. Practically no accumulation of Ca2+ was observed if the vesicles were diluted into 160 mM NaCl instead of KCl, or if they were treated with monensin, a Na+ ionophore, for 30 s prior to addition of CaCl2 to the KCl medium. These observations indicate that the Artemia vesicles exhibit Na-Ca exchange activity. The velocity of Ca2+ accumulation by the vesicles in KCl was stimulated 2.6-fold by the K+ ionophore valinomycin, suggesting that the exchange system is electrogenic, with a stoichiometry greater than 2Na+ per Ca2+. Km,Ca and Vmax values were 15 microM and 7.5 nmol/mg protein.s, respectively. Exchange activity in the Artemia vesicles was inhibited by benzamil (IC50 approximately equal to 100 microM) and by quinacrine (IC50 approximately equal to 250 microM), agents that also inhibit exchange activity in cardiac sarcolemmal vesicles. Unlike cardiac vesicles, however, exchange activity in Artemia was not stimulated by limited proteolysis, redox reagents, or intravesicular Ca2+. This indicates that the two exchange systems are regulated by different mechanisms. Vesicles were prepared from Artemia at various times after hydration of the dry cysts and examined for exchange activity. Activity was first observed at approximately 10 h after hydration and increased to a maximal value by 30-40 h; hatching of the free swimming nauplii occurred at 18-24 h. The results suggest that hatching Artemia nauplii might be a particularly rich source of mRNA coding for the Na+-Ca2+ exchange carrier.