Abnormal rapid Ca2+ release from sarcoplasmic reticulum of malignant hyperthermia susceptible pigs.

Using the rapid filtration technique to investigate Ca2+ movements across the sarcoplasmic reticulum (SR) membrane, we compare the initial phases of Ca2+ release and Ca2+ uptake in malignant hyperthermia susceptible (MHS) and normal (N) pig SR vesicles. Ca2+ release is measured from passively loaded SR vesicles. MHS SR vesicles present a 2-fold increase in the initial rate of calcium release induced by 0.3 microM Ca2+ (20.1 +/- 2.1 vs. 6.3 +/- 2.6 nmol mg-1 s-1). Maximal Ca2+ release is obtained with 3 microM Ca2+. At this optimal concentration, rate of Ca2+ efflux in absence of ATP is 55 and 25 nmol mg-1 s-1 for MHS and N SR, respectively. Ca(2+)-induced Ca2+ release is inhibited by Mg2+ in a dose-dependent manner for both MHS and N pig SR vesicles (K1/2 = 0.2 mM). Caffeine (5 mM) and halothane (0.01% v/v) increase the Ca2+ sensitivity of Ca(2+)-induced Ca2+ release. ATP (5 mM) strongly enhances the rate of Ca2+ efflux (to about 20-40-fold in both MHS and N pig SR vesicles). Furthermore, both types of vesicles do not differ in their high-affinity site for ryanodine (Kd = 12 nM and Bmax = 6 pmol/mg), lipid content, ATPase activity and initial rate of Ca2+ uptake (0.948 +/- 0.034 vs. 0.835 +/- 0.130 mumol mg-1 min-1 for MHS and N SR, respectively). Our results show that MH syndrome is associated to a higher rate of Ca2+ release in the earliest phase of the calcium efflux.     

Distinction between the two basic mechanisms of cation transport in the cardiac Na(+)-Ca2+ exchange system

In order to distinguish between the Ping-Pong and sequential mechanisms of cation transport in the cardiac Na(+)-Ca2+ exchange system, the initial rates of the Nai-dependent 45Ca uptake (t = 1 s) were measured in reconstituted proteoliposomes, loaded with a Ca chelator. Under "zero-trans" conditions ([Na]o = [Ca]i = 0) at a fixed [Na]i = 10-160 mM with varying [45Ca]o = 2.5-122 microM for each [Na]i, the Km and Vmax values increased from 7.7 to 33.5 microM and from 2.3 to 9.0 nmol.mg-1.s-1, respectively. The Vmax/Km values show a +/- 2-10% deviation from the average value of 0.274 nmol.mg-1.s-1.microM-1 over the whole range of [Na]i. These deviations are within the standard error of Vmax (+/- 3-7%), Km (+/- 11-17%), and Vmax/Km (+/- 11-19%). This suggests that, under conditions in which Vmax and Km are [Na]i dependent and vary 4-5-fold, the Vmax/Km values are constant within the experimental error. In the presence of K(+)-valinomycin the Vmax/Km values are 0.85 +/- 0.17 and 1.08 +/- 0.18 nmol.mg-1.s-1.microM-1 at [Na]i = 20 and 160 mM, respectively, suggesting that under conditions of "short circuit" of the membrane potential the Vmax/Km values still exhibit the [Na]i independence. At a very low fixed [45Ca]o = 1.1 microM with varying [Na]i = 10-160 mM, the initial rates were found to be [Na]i independent. At a high fixed [45Ca]o = 92 microM the initial rates show a sigmoidal dependence on the [Na]i with Vmax = 13.8 nmol.mg-1.s-1, KmNa = 21 mM, and Hill coefficient nH = 1.5. The presented data support a Ping-Pong (consecutive) mechanism of cation transport in the Na(+)-Ca2+ exchanger.     

Na+-H+ exchange in cardiac sarcolemmal vesicles

The transport of Na+ by a purified sarcolemmal vesicular preparation from canine ventricular tissue was studied as a function of both internal and external pH. The uptake of Na+ into sarcolemmal vesicles increased upon raising the extravesicular pH of the reaction medium. Half-maximal uptake of Na+ was observed at a pHo of about 8.1 and maximal uptake occurred at pH 8.6. The uptake of Na+ by sarcolemma was also dependent upon the intravesicular pH. Na+ uptake into sarcolemmal vesicles was greatly attenuated in the absence of a H+ gradient across the membrane. Transport of Na+ was potently inhibited by amiloride, a known blocker of Na+-H+ exchange. LiCl was also an effective inhibitor of Na+ transport. In the presence of optimal H+ gradients, Na+ uptake was linear for the first 5 seconds of the reaction and exhibited a Vmax of 290 nmol Na+/mg per min and a KNa of 3.5 mM. These experiments strongly indicate the presence of a Na+-H+ exchange system in cardiac sarcolemma. This activity appeared to be relatively specific for this membrane fraction. The identification of Na+-H+ exchange activity in a sarcolemmal vesicular fraction from the heart will permit extensive characterization of the regulation and kinetics of this antiporter in future investigations.     

Demonstration of a Na+/H+ exchange activity in purified canine cardiac sarcolemmal vesicles

Purified canine cardiac sarcolemmal membrane vesicles exhibit a sodium ion for proton exchange activity (Na+/H+ exchange). Na+/H+ exchange was demonstrated both by measuring rapid 22Na uptake into sarcolemmal vesicles in response to a transmembrane H+ gradient and by following H+ transport in response to a transmembrane Na+ gradient with use of the probe acridine orange. Maximal 22Na uptake into the sarcolemmal vesicles (with starting intravesicular pH = 6 and extravesicular pH = 8) was approximately 20 nmol/mg protein. The extravesicular Km of the Na+/H+ exchange activity for Na+ was determined to be between 2 and 4 mM (intravesicular pH = 5.9, extravesicular pH = 7.9), as assessed by measuring the concentration dependence of the 22Na uptake rate and the ability of extravesicular Na+ to collapse an imposed H+ gradient. All results suggested that Na+/H+ exchange was reversible and tightly coupled. The Na+/H+ exchange activity was assayed in membrane subfractions and found most concentrated in highly purified cardiac sarcolemmal vesicles and was absent from free and junctional sarcoplasmic reticulum vesicles. 22Na uptake into sarcolemmal vesicles mediated by Na+/H+ exchange was dependent on extravesicular pH, having an optimum around pH 9 (initial internal pH = 6). Although the Na+/H+ exchange activity was not inhibited by tetrodotoxin or digitoxin, it was inhibited by quinidine, quinacrine, amiloride, and several amiloride derivatives. The relative potencies of the various inhibitors tested were found to be: quinacrine greater than quinidine = ethylisopropylamiloride greater than methylisopropylamiloride greater than dimethylamiloride greater than amiloride. The Na+/H+ exchange activity identified in purified cardiac sarcolemmal vesicles appears to be qualitatively similar to Na+/H+ exchange activities recently described in intact cell systems. Isolated cardiac sarcolemmal vesicles should prove a useful model system for the study of Na+/H+ exchange regulation in myocardial tissue.     

Ca2+ transport through the brush border membrane of human placenta syncytiotrophoblasts.

The calcium (Ca2+) uptake by brush border membrane vesicles isolated from fresh human placentas has been characterized. This process was saturable and time- and concentration-dependent. It exhibited a double Michaelis-Menten kinetics, with apparent Km values of 0.17 +/- 0.03 and 2.98 +/- 0.17 mM Ca2+, and Vmax values of 0.9 +/- 0.13 and 2.51 +/- 0.45 pmol.micrograms-1.5 s-1. It was not influenced by the presence of Na+ or Mg2+ in the incubation medium. It was not increased by K+ or anion diffusion potentials, inside negative. At a steady state of 1 mM Ca2+ uptake, a large proportion (approximately 94%) of the Ca2+ was bound to the internal surface of the membranes. Preincubation of these membrane vesicles with voltage-dependent Ca2+ channel blockers (nifedipine and verapamil) had no influence on Ca2+ uptake. However, this uptake was very sensitive to pH. In the absence of a pH gradient, the Ca2+ uptake increased with alkalinity. When the intravesicular pH was kept constant while the pH of the incubation medium was increased, Ca2+ uptake was also stimulated by alkaline pH. In contrast, when the pH of the incubation medium was kept constant and the intravesicular pH was progressively increased, Ca2+ uptake was diminished with alkaline pH. Therefore, H+ gradient (H+ in trans-position greater than H+ in cis-position) favored Ca2+ transport, suggesting a H+/Ca2+ exchange mechanism. Finally, in contrast to the basal plasma membrane, the brush border membrane did not show any ATP-dependent Ca2+ transport activity.     

Na+-Ca2+ exchange in squid optic nerve membrane vesicles is activated by internal calcium

The role of intracellular Ca2+ as essential activator of the Na+-Ca2+ exchange carrier was explored in membrane vesicles containing 67% right-side-out and 10% inside-out vesicles, isolated from squid optic nerves. Vesicles containing 100 microM free calcium exhibited a 2-fold increase in the initial rate of Na+i-dependent Ca2+ uptake as compared with vesicles where intravesicular calcium was chelated by 2 mM EGTA or 10 mM HEDTA. The activatory effect exerted by intravesicular Ca2+ on the reverse mode of Na+-Ca2+ exchange (i.e. Na+i-Ca2+o exchange) is saturated at about 100 microM Ca2+i and displays an apparent K 1/2 of 12 microM. Intravesicular Ca2+ produced activation of Na+i-Ca2+i exchange activity rather than an increase in Ca2+ uptake due to Ca2+-Ca2+ exchange. The presence of Ca2+i was essential for the Na+i-dependent Na+ influx, a partial reaction of the Na+-Ca2+ exchanger. In fact, the Na+ influx levels in vesicles loaded with 2 mM EGTA were close to those expected from diffusional leak while in vesicles containing Ca2+i an additional Na+-Na+ exchange was measured. The results suggest that in nerve membrane vesicles Ca2+ at the inner aspect of the membrane acts as an activator of the Na+-Ca2+ exchange system.     

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.     

H+ transport by reconstituted gastric (H+ + K+)-ATPase

Gastric (H+ + K+)-ATPase was reconstituted into artificial phosphatidylcholine/cholesterol liposomes by means of a freeze-thaw-sonication technique. Upon addition of MgATP, active H+ transport was observed, with a maximal rate of 2.1 mumol X mg-1 X min-1, requiring the presence of 100 mM K+ at the intravesicular site. However, in the absence of ATP an H+-K+ exchange with a maximal rate of 0.12 mumol X mg-1 X min-1 was measured, which could be inhibited by the well-known ATPase inhibitors vanadate and omeprazole, giving the first evidence of a passive K+-H+ exchange function of gastric (H+ + K+)-ATPase. An Na+-H+ exchange activity was also measured, which was fully inhibited by 1 mM amiloride. Simultaneous reconstitution of Na+/H+ antiport and (H+ + K+)-ATPase could explain why reconstituted ATPase appeared less cation-specific than the native enzyme (Rabon, E.C., Gunther, R.B., Soumarmon, A., Bassilian, B., Lewin, M.J.M. and Sachs, G. (1985) J. Biol. Chem. 260, 10200-10212).     

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.     

Liver plasma membrane calcium transport. Evidence for a Na+-dependent Ca2+ flux

Plasma membrane vesicles isolated from rat liver exhibited an azide-insensitive Mg2+-ATP-dependent Ca2+ pump which accumulated Ca2+ at a rate of 5.1 +/- 0.5 nmol of calcium/mg of protein/min and reached a total accumulation of 33.2 +/- 2.6 nmol of calcium/mg of protein in 20 microM Ca2+ at 37 degrees C. Equiosmotic addition of 50 mM Na+ resulted in a loss of accumulated calcium. Measurement of Mg2+-ATP-dependent Ca2+ uptake in the presence of 50 mM Na+ revealed no effect of Na+ on the initial rate of Ca2+ uptake, but a decrease in the total accumulation. The half-maximal effect of Na+ on Ca2+ accumulation was achieved at 14 mM. The Ca2+ efflux rate constant in the absence of Na+ was 0.16 +/- 0.01 min-1, whereas the efflux rate constant in the presence of 50 mM Na+ was 0.25 +/- 0.02 min-1. Liver homogenate sedimentation fractions from 1,500 to 105,000 X g were assayed for azide-insensitive Mg2+-ATP-dependent Ca2+ accumulation. Na+-sensitive Ca2+ uptake activity was found to specifically co-sediment with the plasma membrane-associated enzymes, 5'-nucleotidase and Na+/K+-ATPase, whereas Na+-insensitive Ca2+ uptake was found to co-sediment with the endoplasmic reticulum-associated enzyme, glucose-6-phosphatase. The plasma membrane Ca2+ pump was also distinguished from the endoplasmic reticulum Ca2+ pump by its sensitivity to inhibition by vanadate. Half-maximal inhibition of plasma membrane Ca2+ uptake occurred at 0.8 microM VO4(3-), whereas half-maximal inhibition of microsomal Ca2+ uptake occurred at 40 microM.     

Mechanism of cis- and trans-substrate interactions at the tetraethylammonium/H+ exchanger of rabbit renal brush-border membrane vesicles

The kinetic basis for trans-effects of intravesicular substrates on the uptake of the organic cation, tetraethylammonium (TEA), into rabbit renal brush-border membrane vesicles (BBMV) was studied. Preloading BBMV with 1, 2, or 4 mM TEA stimulated the initial rate of uptake and the total net accumulation of 0.1 mM [3H]TEA. The stimulatory effect of intravesicular TEA on the initial rate of uptake was a saturable function of the trans-TEA concentration, with a half-maximal effect noted at an intravesicular concentration of 0.28 mM. A 1 mM trans-concentration of TEA increased the Jmax of [3H]TEA uptake (from 4.3 to 6.8 nmol.mg-1.min-1) without affecting the apparent Kt. An outwardly directed H+ gradient also increased Jmax (to 10.7 nmol.mg-1.min-1), although the addition of an outwardly directed TEA gradient did not produce further increases in the rate of TEA uptake. External H+ acted as a competitive inhibitor of TEA uptake, and an increase in external [H+] (from 32 nM to 100 nM) produced an increase in the apparent Kt for TEA transport (from 0.12 to 0.26 mM) without affecting the Jmax. The results suggested that TEA and H+ compete for a common site or set of mutually exclusive sites on the cytoplasmic and luminal aspects of TEA/H+ exchanger in the renal brush border, and that these sites have a similar affinity for TEA.     

Vasopressin, insulin and peroxide(s) of vanadate (pervanadate) influence Na+ transport mediated by (Na+, K+)ATPase or Na+/H+ exchanger of rat liver plasma membrane vesicles

Uptake of 22Na+ by liver plasma membrane vesicles, reflecting Na+ transport by (Na+, K+)ATPase or Na+/H+ exchange was studied. Membrane vesicles were isolated from rat liver homogenates or from freshly prepared rat hepatocytes incubated in the presence of [Arg8]vasopressin or pervanadate and insulin. The ATP dependence of (Na+, K+)ATPase-mediated transport was determined from initial velocities of vanadate-sensitive uptake of 22Na+, the Na(+)-dependence of Na+/H+ exchange from initial velocities of amiloride-sensitive uptake. By studying vanadate-sensitive Na+ transport, high-affinity binding sites for ATP with an apparent Km(ATP) of 15 +/- 1 microM were observed at low concentrations of Na+ (1 mM) and K+ (1mM). At 90 mM Na+ and 60 mM K+ the apparent Km(ATP) was 103 +/- 25 microM. Vesiculation of membranes and loading of the vesicles prepared from liver homogenates in the presence of vasopressin increased the maximal velocities of vanadate-sensitive transport by 3.8-fold and 1.9-fold in the presence of low and high concentrations of Na+ and K+, respectively. The apparent Km(ATP) was shifted to 62 +/- 7 microM and 76 +/- 10 microM by vasopressin at low and high ion concentrations, respectively, indicating that the hormone reduced the influence of Na+ and K+ on ATP binding. In vesicles isolated from hepatocytes preincubated with 10 nM vasopression the hormone effect was conserved. Initial velocities of Na+ uptake (at high ion concentrations and 1 mM ATP) were increased 1.6-1.7-fold above control, after incubation of the cells with vasopressin or by affinity labelling of the cells with a photoreactive analogue of the hormone. The velocity of amiloride-sensitive Na+ transport was enhanced by incubating hepatocytes in the presence of 10 nM insulin (1.6-fold) or 0.3 mM pervanadate generated by mixing vanadate plus H2O2 (13-fold). The apparent Km(Na+) of Na+/H+ exchange was increased by pervanadate from 5.9 mM to 17.2 mM. Vesiculation and incubation of isolated membranes in the presence of pervanadate had no effect on the velocity of amiloride-sensitive Na+ transport. The results show that hormone receptor-mediated effects on (Na+, K+)ATPase and Na+/H+ exchange are conserved during the isolation of liver plasma membrane vesicles. Stable modifications of the transport systems or their membrane environment rather than ionic or metabolic responses requiring cell integrity appear to be involved in this regulation.     

Mixed type inhibition of the renal Na+/H+ antiporter by Li+ and amiloride. Evidence for a modifier site

We studied the interactions of Na+, Li+, and amiloride on the Na+/H+ antiporter in brush-border membrane vesicles from rabbit renal cortex. Cation-mediated collapse of an outwardly directed proton gradient (pHin = 6.0; pHout = 7.5) was monitored with the fluorescent amine, acridine orange. Proton efflux resulting from external addition of Na+ or Li+ exhibited simple saturation kinetics with Hill coefficients of 1.0. However, kinetic parameters for Na+ and Li+ differed (Km for Li+ = 1.2 +/- 0.1 mM; Km for Na+ = 14.3 +/- 0.8 mM; Vmax for Li+ = 2.40 +/- 0.07 fluorescence units/s/mg of protein; Vmax for Na+ = 7.10 +/- 0.24 fluorescence units/s/mg of protein). Inhibition of Na+/H+ exchange by Li+ and amiloride was also studied. Li+ inhibited the Na+/H+ antiporter by two mechanisms. Na+ and Li+ competed with each other at the cation transport site. However, when [Na+] was markedly higher than [Li+], [( Na+] = 90 mM; [Li+] less than 1 mM), we observed noncompetitive inhibition (Vmax for Na+/H+ exchange reduced by 25%). The apparent Ki for this noncompetitive inhibition was congruent to 50 microM. In addition, 2-30 mM intravesicular Li+, but not Na+, resulted in trans inhibition of Na+/H+ exchange. Amiloride was a mixed inhibitor of Na+/H+ exchange (Ki = 30 microM, Ki' = 90 microM) but was only a simple competitive inhibitor of Li+/H+ exchange (Ki = 10 microM). At [Li] = 1 mM and [amiloride] less than 100 microM, inhibition of Na+/H+ exchange by a combination of the two inhibitors was always less than additive. These results suggest the presence of a cation-binding site (separate from the cation-transport site) which could be a modifier site of the Na+/H+ antiporter.     

Na+ translocation by complex I (NADH:quinone oxidoreductase) of Escherichia coli

Following on from our previous discovery of Na+ pumping by the NADH:ubiquinone oxidoreductase (complex I) of Klebsiella pneumoniae, we show here that complex I from Escherichia coli is a Na+ pump as well. Our study object was the Escherichia coli mutant EP432, which lacks the Na+/H+ antiporter genes nhaA and nhaB and is therefore unable to grow on LB medium at elevated Na+ concentrations. During growth on mineral medium, the Na+ tolerance of E. coli EP432 was influenced by the organic substrate. NaCl up to 450 mM did not affect growth on glycerol and fumarate, but growth on glucose was inhibited. Correlated to the Na+ tolerance was an increased synthesis of complex I in the glycerol/fumarate medium. Inverted membrane vesicles catalysed respiratory Na+ uptake with NADH as electron donor. The sodium ion transport activity of vesicles from glycerol/fumarate-grown cells was 40 nmol mg-1 min-1 and was resistant to the uncoupler carbonyl-cyanide m-chlorophenylhydrazone (CCCP), but was inhibited by the complex I-specific inhibitor rotenone. With an E. coli mutant deficient in complex I, the Na+ transport activity was low (1-3 nmol mg-1 min-1), and rotenone was without effect.     

Pantothenate-sodium cotransport in renal brush-border membranes

The mechanism of pantothenate transport into rabbit renal brush-border membrane vesicles was studied. Under voltage-clamped conditions, an inward NaCl gradient induced the transient accumulation of pantothenate against its concentration gradient, indicating Na+/pantothenate cotransport. K+, Rb+, Li+, NH4+, and choline+ were ineffective in replacing Na+. Pantothenate analogs, D-glucose, and various carboxylic acids did not inhibit Na+-dependent pantothenate transport, suggesting that this system is specific for pantothenate. Kinetic analysis of the Na+-dependent pantothenate uptake revealed a single transport system which obeyed Michaelis-Menten kinetics (Km = 16 microM and Vmax = 6.7 pmol X mg-1 X 10 s-1). Imposition of an inside-negative membrane potential caused net uphill pantothenate accumulation in the presence of Na+ but absence of a Na+ gradient, indicating that Na+/pantothenate cotransport is electrogenic. The relationship between extravesicular Na+ concentration and pantothenate transport measured under voltage-clamped conditions was sigmoidal: a Hill coefficient (napp) of 2 and a [Na+]0.5 of 55 mM were calculated. It is suggested that an anionic pantothenate1- molecule is cotransported with two Na+ to give a net charge of +1. The coupling of pantothenate transport to the Na+ electrochemical gradient may provide an efficient mechanism for reabsorption of pantothenate in the kidney.     

The mechanism of decreased Na+-dependent D-glucose transport in brush-border membrane vesicles from rabbit kidneys with experimental Fanconi syndrome

In our previous paper (Yanase, M. et al. (1983) Biochim. Biophys. Acta 733, 95-101) we reported that the Na+-dependent D-glucose uptake into brush-border membrane vesicles is decreased in rabbits with experimental Fanconi syndrome (induced by anhydro-4-epitetracycline). In the present paper we investigate the mechanism underlying this decrease. D-Glucose is taken up into the osmotically active space in anhydro-4-epitetracycline-treated brush-border membrane vesicles and exhibits the same distribution volume and the same degree of nonspecific binding and trapping as in control brush-border membrane vesicles. The passive permeability properties of control and anhydro-4-epitetracycline-treated brush-border membrane vesicles are shown to be the same as measured by the time-dependence of L-glucose efflux from brush-border membrane vesicles. D-Glucose flux was measured by the equilibrium exchange procedure at constant external and internal Na+ concentrations and zero potential. Kinetic analyses of Na+-dependent D-glucose flux indicate that Vmax in anhydro-4-epitetracycline-treated brush-border membrane vesicles (79.3 +/- 7.6 nmol/min per mg protein) is significantly smaller than in control brush-border membrane vesicles (141.3 +/- 9.9 nmol/min per mg protein), while the Km values in the two cases are not different from each other (22.3 +/- 0.9 and 27.4 +/- 1.8 mM, respectively). These results suggest that Na+-dependent D-glucose carriers per se are affected by anhydro-4-epitetracycline, and that this disorder is an important underlying mechanism in the decreased Na+-dependent D-glucose uptake into anhydro-4-epitetracycline-treated brush-border membrane vesicles.     

Site density of the sodium-calcium exchange carrier in reconstituted vesicles from bovine cardiac sarcolemma

The site density of the Na2+-Ca2+ exchanger in bovine cardiac sarcolemma was estimated from measurements of the fraction of reconstituted proteoliposomes exhibiting exchange activity. Sarcolemmal vesicles were solubilized with 1% Triton X-100 in the presence of either 100 mM NaCl or 100 mM KCl; after a 20-40-min incubation period on ice, sufficient KCl, NaCl, CaCl2, and soybean phospholipids were added to each extract to give final concentrations of 40 mM NaCl, 120 mM KCl, 0.1 mM CaCl2, and 10 mg/ml phospholipid. These mixtures were then reconstituted into proteoliposomes, and the rate of 45Ca2+ isotopic exchange was measured under equilibrium conditions. Control studies showed that Na+-Ca2+ exchange activity was completely lost if Na+ was not present during solubilization. The difference in 45Ca2+ uptake between vesicles initially solubilized in the presence or absence of NaCl therefore reflected exchange activity and corresponded to 3.1 +/- 0.3% of the total 45Ca2+ uptake by the entire population of vesicles, as measured in the presence of the Ca2+ ionophore A23187. Assuming that each vesicle with exchange activity contained 1 molecule of the Na+-Ca2+ exchange carrier, a site density of 10-20 pmol/mg of protein for the exchanger was calculated. The Vmax for Na+-Ca2+ exchange activity in the proteoliposomes was approximately 20 nmol/mg of protein.s which indicates that the turnover number of the exchange carrier is 1000 s-1 or more. Thus, the Na+-Ca2+ exchanger is a low density, high turnover transport system.     

Na+-H+ exchange in luminal-membrane vesicles from rabbit proximal convoluted and straight tubules in response to metabolic acidosis.

Na+-H+-exchanger activity of pars convoluta and pars recta luminal-membrane vesicles prepared from the proximal tubule of acidotic and control rabbits were assayed by a rapid-filtration technique and an Acridine Orange method. Both experimental approaches revealed the existence of an antiporter, sensitive to metabolic acidosis, in pars convoluta membrane vesicles. Kinetic data, obtained with the pH-sensitive dye, showed that the Km for Na+ transport was unchanged by acidosis, whereas Vmax. for exchanger activity was increased, on an average, by 44%. The fluorescence method, in contrast with the rapid-filtration technique, was able to detect exchanger activity in pars recta membrane vesicles. The Km value for the antiporter located in pars recta is comparable with that calculated for pars convoluta membrane vesicles. By contrast, the Vmax. of this exchanger is only about 25% of that found for pars convoluta. Furthermore, metabolic acidosis apparently does not increase Na+-H+-exchanger activity of pars recta luminal-membrane vesicles.     

Negative regulation of the platelet Na+/H+ exchanger by trimeric G-proteins.

Human platelets contain a Na+/H+ exchanger (NHE) that regulates the cytosolic pH. The role of trimeric G-proteins in NHE control was investigated in plasma membrane vesicles by measuring exchange of intravesicular protons for extravesicular Na+. Exchange was saturable, independent of membrane potential and inhibited by ethylisopropyl amiloride (Ki 0.05 micromol.L-1), demonstrating the involvement of NHE-1. The G-protein activators AlF4- and GMP-P(NH)P reduced exchange by increasing the Km for Na+ from 11.3 +/- 2.1 mM to 21.6 +/- 1.4 mM (AlF4-) and 19.8 +/- 1.1 mM (GMP-P(NH)P), leaving Vmax and the Hill coefficient unchanged. This effect was abolished by inhibitors of Gi-proteins (N-ethylmaleimide, holoenzyme- and A-protomer of pertussis toxin) and by an anti-Galpha Ig and GDP(beta)S. Activation of Gi-proteins by mastoparan and its synthetic analogue Mas7 also strongly reduced NHE activity. These data show that in platelets NHE-1 is under negative control of the Gi-family of trimeric G-proteins.     

The role of chloride in taurine transport across the human placental brush-border membrane.

Taurine, a sulfated beta-amino acid, is conditionally essential during development. A maternal supply of taurine is necessary for normal fetal growth and neurologic development, suggesting the importance of efficient placental transfer. Uptake by the brush-border membrane (BBM) in several other tissues has been shown to be via a selective Na(+)-dependent carrier mechanism which also has a specific anion requirement. Using BBM vesicles purified from the human placenta, we have confirmed the presence of Na(+)-dependent, carrier-mediated taurine transport with an apparent Km of 4.00 +/- 0.22 microM and a Vmax of 11.72-0.36 pmol mg-1 protein 20 s-1. Anion dependence was examined under voltage-clamped conditions, in order to minimize the contribution of membrane potential to transport. Uptake was significantly reduced when anions such as thiocyanate, gluconate, or nitrate were substituted for Cl-. In addition, a Cl(-)-gradient alone (under Na(+)-equilibrated conditions) could energize uphill transport as evidenced by accelerated uptake (3.13 +/- 0.8 pmol mg-1 protein 20 s-1) and an overshoot compared to Na+, Cl- equilibrated conditions (0.60 +/- 0.06 pmol mg-1 protein 20 s-1). A Cl(-)-gradient (Na(+)-equilibrated) also stimulated uptake of [3H]taurine against its concentration gradient. Analysis of uptake in the presence of varying concentrations of external Cl- suggested that 1 Cl- ion is involved in Na+/taurine cotransport. We conclude that Na(+)-dependent taurine uptake in the placental BBM has a selective anion requirement for optimum transport. This process is electrogenic and involves a stoichiometry of 2:1:1 for Na+/Cl-/taurine symport.