Role of sodium ions in p-aminohippurate transport by newt kidney.

1. The effect of external Na+ concentration on p-aminohippurate uptake by isolated kidneys of newt (Triturus pyrrhogaster) was studied kinetically and electrophysiologically. 2. p-Aminohippurate uptake conformed to Michaelis-Menten type kinetics in regard to both p-aminohippurate and Na+ concentrations in the incubation medium. Kinetic studies revealed that reduction of Na+ concentration increased the values of Kt without altering the maximal rate (V) of p-aminohippurate uptake. The values of Kt were a linear function of the reciprocal of Na+ concentration. These results suggest the presence of interaction between p-aminohippurate and Na+ at the carrier level, i.e. Na+-coupled cotransport. 3. p-Aminohippurate had no effect on the electrical potential difference across the peritubular membrane in both 10 and 100 mM Na+ solutions, suggesting that p-aminohippurate is transported across the peritubular membrane in a form of electrically neutral carrier complex. This is consistent with the results of the kinetic studies. 4. p-Aminohippurate uptake was proportional to the electrochemical potential gradient of Na+ (delta mu Na) across the peritubular membrane. This result indicates that the maintenance of sufficient delta mu Na appears to be necessary for the accumulation of p-aminohippurate against its electrochemical potential gradient, supporting Na+ gradient hypothesis.     

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.     

Coupling of Li+ distribution to the plasma membrane potential of rat cortical synaptosomes

The effect of the plasma membrane potential delta psi p on the transport rate and steady state distribution of Li+ was assessed in rat cortical synaptosomes. Up to 15 mM Li+ failed to saturate Li+ influx into polarized synaptosomes in a Na+-based medium with 3 mM external K+. Veratridine increased and tetrodotoxin, ouabain, or high external K+ decreased the rate of Li+ influx. At steady state, Li+ was concentrated about 3-fold in resting synaptosomes at 0.3 to 1 mM Li+ externally. Subsequent depolarization of the plasma membrane by veratridine or high external K+ induced an immediate release of Li+. When graded depolarizations were imposed onto the plasma membrane by varying concentrations of ouabain, veratridine, or external K+, steady state distribution of Li+ was linearly related with K+ distribution or electrochemical activity coefficients. It was concluded that uptake rate and steady state distribution of Li+ depend significantly on delta psi p. However, Li+ gradients were lower than predicted from delta psi p, suggesting that (secondary) active transport systems counteracted passive equilibration by uphill extrusion of Li+. The electrochemical potential difference delta mu Li+ maintained at a delta psi p of -72 mV was calculated to 4.2 kJ/mol of Li+. At physiological external K+, Li+ was not actively transported by the sodium pump. The ouabain sensitivity resulted from the coupling of Li+ uptake to the pump-dependent K+ diffusion potential. In low K+ and K+-free media, however, active transport of Li+ by the sodium pump contributed to total uptake. In the absence of K+, Li+ substituted for K+ in generating a delta psi p of -64 mV maximally, as calculated from TPMP+ distribution at 40 mM external Li+. Since Li+ gradients were far too low to account for a diffusion potential, it was assumed that Li+ gave rise to an electrogenic pump potential.     

Roles of sodium and potassium ions on p-aminohippurate transport in rabbit kidney slices.

This investigation was principally undertaken to test the ionic gradient hypothesis as applied to active p-aminohippurate uptake in the rabbit kidney cortical slice preparation. Efflux of p-aminohippurate from the slice was shown to be independent of external Na+ concentration. Transferring slices from a low sodium preincubation to a high sodium incubation medium containing p-aminohippurate increased intracellular concentrations of both Na+ and K+, and p-aminohippurate accumulation occurred. Transferring slices from a low sodium preincubation to a high sodium incubation medium containing ouabain and p-aminohippurate resulted in a net increase in intracellular Na+ concentration but no p-aminohippurate accumulation occurred. Different combinations of preincubation and incubation media gave a high to low array of intracellular Na+ concentrations and these directly reflected their respective p-aminohippurate uptake. These results suggest that the Na+ gradient hypothesis does not adequately explain the transport of organic acids in rabbit kidney. These results also suggest that Na+ possibly has an intracellular role through its stimulation of (Na+ + K+)-ATPase channeled to energizing the p-aminohippurate accumulative mechanism.     

Studies on the transport of carnitine in the brain using synaptosomes isolated from guinea-pig cerebral cortex

Synaptosomes isolated from guinea pig cerebral cortex accumulate L-carnitine from the medium in an active process, dependent on the sodium gradient across the plasma membrane and on (Na+ + K+)-ATPase activity. L-Carnitine uptake is inhibited by oxidative phosphorylation uncouplers and by ouabain, a known inhibitor of (Na+ + K+)-ATPase. In addition, the omission of Na+ or its replacement by Li+ inhibited the transport, which was also competitively inhibited by gamma-aminobutyrate. The kinetics of carnitine uptake show that the overall process would consist of two components: a passive diffusion and a carrier-mediated transport which is saturated at 1-2 mM carnitine concentration.     

Active amino acid transport in plasma membrane vesicles from Simian virus 40-transformed mouse fibroblasts. Characteristics of electrochemical Na+ gradient-stimulated uptake.

Selectively permeable membrane vesicles isolated from Simian virus 40-transformed mouse fibroblasts catalyzed Na+ gradient-coupled active transport of several neutral amino acids dissociated from intracellular metabolism. Na+-stimulated alanine transport activity accompanied plasma membrane material during centrifugation in discontinuous dextran 110 gradients. Carrier-mediated transport into the vesicle was demonstrated. When Na+ was equilibrated across the membrane, countertransport stimulation of L-[3H]alanine uptake occurred in the presence of accumulated unlabeled L-alanine, 2-aminoisobutyric acid, or L-methionine. Competitive interactions among neutral amino acids, pH profiles, and apparent Km values for Na+ gradient-stimulated transport into vesicles were similar to those previously described for amino acid uptake in Ehrlich ascites cells, which suggests that the transport activity assayed in vesicles is a component of the corresponding cellular uptake process. Both the initial rate and quasi-steady state of uptake were stimulated as a function of a Na+ gradient (external Na+ greater than internal Na+) applied artificially across the membrane and were independent of endogenous (Na+ + K+)-ATPase activity. Stimulation by Na+ was decreased when the Na+ gradient was dissipated by monensin, gramicidin D or Na+ preincubation. Na+ decreased the apparent Km for alanine, 2-aminoisobutyric acid, and glutamine transport. Na+ gradient-stimulated amino acid transport was electrogenic, stimulated by conditions expected to generate an interior-negative membrane potential, such as the presence of the permeant anions NO3- and SCN-. Na+-stimulated L-alanine transport was also stimulated by an electrogenic potassium diffusion potential (K+ internal greater than K+ external) catalyzed by valinomycin; this stimulation was blocked by nigericin. These observations provide support for a mechanism of active neutral amino acid transport via the "A system" of the plasma membrane in which both a Na+ gradient and membrane potential contribute to the total driving force.     

Phenylalanine uptake in isolated renal brush border vesicles.

The uptake of L-phenylalanine into brush border microvilli vesicles and basolateral plasma membrane vesicles isolated from rat kidney cortex by differential centrifugation and free flow electrophoresis was investigated using filtration techniques. Brush border microvilli but not basolateral plasma membrane vesicles take up L-phenylalanine by an Na+-dependent, saturable transport system. The apparent affinity of the transport system for L-phenylalanine is 6.1 mM at 100 mM Na+ and for Na+ 13mM at 1 mM L-phenylalanine. Reduction of the Na+ concentration reduces the apparent affinity of the transport system for L-phenylalanine but does not alter the maximum velocity. In the presence of an electrochemical potential difference of Na+ across the membrane (etaNao greater than etaNai) the brush border microvilli accumulate transiently L-phenylalanine over the concentration in the incubation medium (overshoot pheomenon). This overshoot and the initial rate of uptake are markedly increased when the intravesicular space is rendered electrically more negative by membrane diffusion potentials induced by the use of highly permeant anions, of valinomycin in the presence of an outwardly directed K+ gradient and of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence of an outward-directed proton gradient. These results indicate that the entry of L-phenylalanine across the brush border membrane into the proximal tubular epithelial cells involves cotransport with Na+ and is dependent on the concentration difference of the amino acid, on the concentration difference of Na+ and on the electrical potential difference. The exit of L-phenylalanine across the basolateral plasma membranes is Na+-independent and probably involves facilitated diffusion.     

Effect of the electrochemical sodium gradient on alanine transport in 3T6 and CHO cells

The dependence of L-alanine uptake by 3T6 and CHO-K1 cells on Na+ electrochemical gradient has been studied. The Na+ chemical gradient was changed by a short-term (partial or complete) replacement of Na+ for choline. The membrane potential change was achieved by addition of potassium ionophore--valinomycin (10 microM) into the medium. It is determined that the value of Km for alanine uptake by 3T6 cells increases from 2 mM, with 140 mM Na+ in the medium, up to 30 mM, if the replacement of Na+ for choline is complete. Similar results are obtained for CHO cells. The membrane potential increase under the influence of valinomycin leads to the increase in the value of Vmax of the uptake. The data obtained are interpreted on the basis of the well known scheme of Na+ alanine complex transfer, where Na+ increases the affinity of the carrier to the amino acid, and the membrane potential increases the carrier mobility.     

Active transport of alanine by the neutral amino-acid exchanger ASCT1

ASCT1 protein is a member of the glutamate transporter superfamily, which shows system ASC selectivity and properties and has been characterized as a Na+-dependent neutral amino-acid exchanger. Here, by using ASCT1-expressing oocytes, the uptake of alanine and glutamate was measured to investigate ASCT1's ability to mediate a concentrative transport of alanine, ASCT1's sodium dependence, and the influence of pH on the mutual inhibition between alanine and glutamate. Alanine uptake was measured after 30 min incubation. Kinetic analysis of the Na+ dependence of alanine uptake showed an apparent K0.5 (affinity constant) value for Na+ of 23.1 +/- 4.3 mM (mean +/- SE). Concentration dependence of alanine uptake was tested at 100 and 1 mM Na+, with apparent K0.5 values of 0.16 +/- 0.04 and 1.8 +/- 0.4 mM, respectively, at pH 7.5, and 0.21 +/- 0.06 and 1.9 +/- 0.3 mM at pH 6. Vmax was not modified between 100 and 1 mM Na+ at either pH. ASCT1 actively transports alanine and accumulates it in the cytosol even when the Na+ concentration in the medium was as low as 1-3 mM. 22Na uptake studies revealed that Na+ transport was stimulated by the presence of alanine in the medium. Our results demonstrate that ASCT1 is able to mediate a concentrative transport of alanine, which is Na+-dependent but not coupled to the Na+ gradient.     

Kidney epithelial cells of monkey origin (BSC-1) express a sodium bicarbonate cotransport. Characterization by 22Na+ flux measurements

Na movement across the plasma membranes of confluent monolayers of monkey kidney epithelial cells (BSC-1) was studied using 22Na+ uptake and efflux techniques in the presence of 10(-4) M ouabain. In the presence of 28 mM bicarbonate, uptake was inhibited by both 10(-3) M amiloride and 10(-3) M 4,4'diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). In DIDS-pretreated cells, 10(-3) M amiloride led to a further reduction of 22Na+ uptake, while 10(-5) furosemide was ineffective. DIDS also inhibited sodium efflux, indicating that the DIDS-sensitive pathway mediates both influx and efflux of 22Na+. DIDS-sensitive 22Na+ uptake, as studied in the presence of both 10(-4) M ouabain and 10(-3) M amiloride, was abolished by the absence of bicarbonate, which could not be substituted by other plasma membrane-permeable buffers. In 28 mM HCO3-, DIDS-sensitive uptake of 28 mM Na+ was cis-inhibited by 124 mM Na+, but no significant inhibition by K+ or Li+ was found. DIDS-sensitive 22Na+ uptake was a saturable function of both Na+ concentration (apparent Km between 20 and 40 mM at 28 mM HCO3-) and HCO3- concentration (apparent Km between 7 and 14 mM at 151 mM Na+). Intracellular microelectrode measurements showed that net Na+ transport in the presence of HCO3- is electrogenic, i.e. that there is anion cotransport with Na+. This effect is abolished by 1 mM DIDS. It is concluded that monkey kidney epithelial cells possess a stilbene-sensitive, electrogenic sodium bicarbonate symport, which may play an important role in bicarbonate reabsorption in the mammalian kidney.     

Characterization of the myo-inositol transport system in Trypanosoma cruzi.

myo-inositol is a growth factor for mammalian cells as well as for the pathogenic protozoa Trypanosoma cruzi. Most of the cell surface molecules in this organism rely on myo-inositol as the biosynthetic precursor for phosphoinositides and glycosylated phosphatidylinositols. The aim of this work was to investigate the process of myo-inositol translocation across the parasite cell membrane. myo-Inositol uptake was concentration-dependent in the concentration range 0.1-10 microM with maximal transport obtained at 8 microM. Using sodium-free buffers, where Na+ was replaced by choline or K+, myo-inositol uptake was inhibited by 50%. Furosemide, an inhibitor of the ouabain-insensitive Na+-ATPase, inhibited the Na+-dependent and Na+-independent myo-inositol uptake by 68 and 33%, respectively. In contrast, ouabain, an (Na++/K+) ATPase inhibitor, did not affect transport. Part of the myo-inositol uptake is mediated by active transport as it was inhibited when energy metabolism inhibitors such as carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (34%), 2,4-dinitrophenol (50%), KCN (71%) and NaN3 (69%) were added to the medium, or the temperature of the medium was lowered to 4 degrees C. The addition of glucose (5-50 mM) or mannose (10 mM) did not change the myo-inositol uptake, whereas the addition of 10 mM nonlabeled myo-inositol totally inhibited this transport, indicating that the transporter is specific for myo-inositol. Phloretin (0.3 mM) and phoridzin (5 mM), but not cytochalasin B, were efficient inhibitors of myo-inositol uptake. A portion of the accumulated myo-inositol is converted to inositol phosphates and phosphoinositides. These data show that myo-inositol transport in T. cruzi epimastigotes is mediated by at least two specific transporters - one Na+-dependent and the other Na+-independent.     

The influence of cellular amino acids and the Na+ : K+ pump on the membrane potential of the Ehrlich ascites tumor cell.

The membrane potential of the Ehrlich ascites tumor cell was shown to be influenced by its amino acid content and the activity of the Na+ :K+ pump. The membrane potential (monitored by the fluorescent dye, 3,3'-dipropylthiodicarbocyanine iodide) varied with the size of the endogenous amino acid pool and with the concentration of accumulated 2-aminoisobutyrate. When cellular amino acid content was high, the cells were hyperpolarized; as the pool declined in size, the cells were depolarized. The hyperpolarization seen with cellular amino acid required cellular Na+ but not cellular ATP. Na+ efflux was more rapid from cells containing 2-aminoisobutyrate than from cells low in internal amino acids. These observations indicate that the hyperpolarization recorded in cells with high cellular amino acid content resulted from the electrogenic co-efflux of Na+ and amino acids. Cellular ATP levels were found to decline rapidly in the presence of the dye and hence the influence of the pump was seen only if glucose was added to the cells. When the cells contained normal Na+ (approx. 30mM), the Na+ :K+ pump was shown to have little effect on the membrane potential (the addition of ouabain had little effect on the potential). When cellular Na+ was raised to 60mM, the activity of the pump changed the membrane potential from the range -25 to -30 mV to -44 to -63 mV. This hyperpolarization required external K+ and was inhibited by ouabain.     

Localization of the Na+/K+-ATPase and of an amiloride sensitive Na+ uptake on thyroid epithelial cells

The Na+/K+-ATPase was localized using purified specific antibodies, on the basolateral membranes of rat thyroid epithelial cells and of cultured porcine thyroid cells, by immunofluorescence and immunoelectron microscopy. No staining was observed on the apical membranes. When cultured cells formed monolayers, with their apical pole in contact with the culture medium, 22Na+ uptake was inhibited by amiloride. Inhibition was dependent upon extracellular Na+ concentration, half maximal inhibition was obtained with 0.7 microM amiloride in the presence of 5 mM Na+. Ouabain was ineffective on Na+ uptake into intact monolayers. A brief treatment of the monolayers with ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) opened the tight junctions and allowed the access of ouabain to the basal pole of the cells. In this condition ouabain increased Na+ uptake. When cells were reorganized into follicle-like structures, with their basal pole in contact with the culture medium, Na+ uptake was not modified by amiloride but was increased by ouabain. We conclude that in thyroid cells, the Na+/K+-ATPase is present on the basolateral domain of the plasma membrane whereas an amiloride sensitive sodium uptake occurs at the apical surface.     

The recovery from the relaxant effect by ouabain and the metabolic dependency of K+ contractions in guinea-pig taenia coli.

1. Ouabain (2.5 x 10(-5) M) inhibited preferentially the tonic response to 40 mM K+ medium (containing enough Na+) without affecting the phasic in taenia coli. When 11 mM lactate was added to the medium (pH 6.5) in the presence of ouabain, the tonic phase to 40 mM K+ recovered markedly. 2. Ouabain (2.5 x 10(-5) M) did not affect the tonic tension in 152 mM K+ medium (Na+ 0 mM). However, ouabain inhibited the recovered tension by the addition of 50 mM Na+ in the 152 mM K+ medium. But ouabain failed to inhibit the marked recovered tension by the addition of 11 mM lactate which utilized, even in the absence of external Na+, in 152 mM K+ medium. 3. Ouabain partly inhibited the shortening to 40 mM K+ (containing enough Na+) at light load; however, it inhibited markedly the shortening at heavy load. 4. There is a possibility that ouabain inhibits active transport of glucose depending on external Na+ in taenia coli of smooth muscle. Ouabain could not inhibit the tension by lactate which utilized under conditions of independent on Na+. Furthermore, it is suggested that ouabain inhibits the contraction which depends on aerobic metabolism; however, it has only a slight effect on contraction which depends on aerobic metabolism; however, it has only a slight effect on contraction which was not so dependent on aerobic metabolism.     

Correlation of the effect of Ca+2 on Na+ and K+ permeability and membrane potential of Ehrlich ascites tumor cells

The effect of Ca+2 on the transport and intracellular distribution of Na+ and K+ in Ehrlich ascites tumor cells was investigated in an effort to establish the mechanism of Ca+2-induced hyperpolarization of the cell membrane. Inclusion of Ca+2 (2 mM) in the incubation medium leads to reduced cytoplasmic concentrations of Na+, K+ and Cl- in steady cells. In cells inhibited by ouabain, Ca+2 causes a 41% decrease in the rate of net K+ loss, but is without effect on the rate of net Na+ accumulation. Net K+ flux is reduced by 50%, while net Na+ flux is unchanged in the transport-inhibited cells. The membrane potential of cells in Ca+2-free medium (-13.9 +/- 0.8 mV) is unaffected by the addition of ouabain. However, the potential of cells in Ca+2-containing medium (-23.3 +/- 1.2 mV) declines in one hour after the addition of ouabain to values comparable to those of control cells (-15.2 +/- 0.7 mV). The results of these experiments are consistent with the postulation that Ca+2 exerts two effects on Na+ and K+ transport. First, Ca+2 reduces the membrane permeability to K+ by 25%. Second, Ca+2 alters the coupling of the Na/K active transport mechanism leading to an electrogenic hyperpolarization of the membrane.     

Leucine transport in relation to the activities of Na+-H+ antiporter and Na+/K+ pump stimulated by serum and a tumor promoter

Fetal calf serum and 12-O-tetradecanoylphorbol 13-acetate (TPA) increased the rate of leucine uptake by Chang liver cells in Na+-containing medium. Addition of monensin to the incubation medium also increased the leucine uptake. All these agents were capable of raising the cytoplasmic pH, which was blocked by a prior addition of amiloride or removing Na+ from assay medium, suggesting activation of Na+-H+ exchange across the cell membrane by fetal calf serum and TPA. The stimulation of leucine uptake by monensin and fetal calf serum was blocked completely or incompletely by addition of ouabain or amiloride. The basal and fetal-calf-serum- or TPA-stimulated leucine uptake was extensively depressed by the presence of an excess of 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid in the incubation medium. Based on these results it is proposed that the transport of leucine by the system L is stimulated by fetal calf serum and TPA with a high concentration of Na+ outside the cells as a result of alkalinization of the cytoplasm and coordinated activation of (Na+ + K+)-ATPase by these stimulators to maintain the transmembrane Na+ gradient and also hyperpolarize the cell membrane.     

The Gibbs-Donnan near-equilibrium system of heart

The gradients of the major inorganic ions across the plasma membrane of heart were examined to determine the factors controlling the extent and direction of the changes induced during injury, certain diseases, and electrolyte disturbances. The ionic environment was altered by changing only the concentration of inorganic phosphate, [sigma Pi]o, from 0 to 1.2 to 5 mM in the Krebs-Henseleit buffer perfusing working rat hearts. Raising [sigma Pi]o from 1.2 to 5 mM resulted in a decrease in total Mg2+ content and calculated free cytosolic [Mg2+] from 0.44 to 0.04 mM, conversion of 4 mmol of MgATP2- to ATP4- and a decrease in measured intracellular [Cl-]i from 41 to 16 mM. At all levels of [sigma Pi]o, both the [Na+]i and [K+]i were invariant at about 3 mM and 130 mM, respectively, as was the energy of hydrolysis of the terminal phosphate bond of sigma ATP, delta GATP Hydr, of -13.2 kcal/mol. The relationship maintained between the ions on both sides of the plasma membrane by the 3Na+/2K(+)transporting ATPase (EC 3.6.1.37) and an open K+ channel was: (formula; see text) The energy of the gradients of the other inorganic ions across the plasma membrane, delta G[ion]o/i, exhibited three distinct quanta of energy derived from the prime quantum of delta GATP Hydr of -13.2 kcal/mol. The second quantum was about one-third of delta GATP Hydr or +/- 4.4 kcal/mol and comprised the delta G[Na+]o/i, delta G[Mg2+]o/i, and delta G[HPO42-]o/i. These results indicated near-equilibrium was achieved by the reactants of the 3Na+/2K(+)-ATPase, the K+ channel, the Na(+)-Pi co-transporter, and a postulated net Mg2+/H2PO4- exchanger. The third quantum was one-third of delta G[Na+]o/i or about +/- 1.5 kcal/mol and comprised delta G[H+]o/i, delta G[HCO3-]o/i, and delta G[Cl-]o/i. The delta G[K+]o/i was 0, indicating near-equilibrium between the chemical energy of [K+]o/i and the E across the plasma membrane of -83 mV. It is concluded that the gradients of the major inorganic ions across the plasma membrane and the potential across that membrane constitute a Gibbs-Donnan equilibrium system catalyzed by transport enzymes sharing common substrates. The chemical and electrical energies of those gradients are equal in magnitude and opposite in sign to the chemical energy of ATP hydrolysis.     

Characterization of glutamate transport system in hydrophobic protein (H protein) of Bacillus subtilis.

Hydrophobic protein (H protein) was isolated from membrane fractions of Bacillus subtilis and constituted into artificial membrane vesicles with lipid of B. substilis. Glutamate was accumulated into the vesicle when a Na+ gradient across the membrane was imposed. The maximum effect of Na+ on the transport was achieved at a concentration of about 40 mM, while the apparent Km for Na+ was approximately 8 mM. On the other hand, Km for glutamate in the presence of 50 mM Na+ was about 8 micro M. Increasing the concentration of Na+ resulted in a decrease in Km for glutamate, maximum velocity was not affected. The transport was sensitive to monensin (Na+ ionophore). Glutamate was also accumulated when pH gradient (interior alkaline) across the membrane was imposed or a membrane potential was induced with K+-diffusion potential. The pH gradient-driven glutamate transport was sensitive to carbonylcyanide m-chlorophenylhydrazone and the apparent Km for glutamate was approximately 25 microM. These results indicate that two kinds of glutamate transport system were present in H protein: one is Na+ dependent and the other is H+ dependent.     

Regulation of pantothenic acid transport in the heart. Involvement of a Na+-cotransport system

Pantothenic acid transport was studied in the isolated perfused rat heart and isolated sheep cardiac sarcolemmal vesicles. In the perfused heart, pantothenic acid transport was significantly greater if hearts were perfused as working hearts rather than Langendorff hearts, but was unaffected by the perfusion substrates used (11 mM glucose or 1.2 mM palmitate). Uptake rates of pantothenic acid in working hearts are dependent on perfusate concentrations of pantothenic acid (a Vmax of 418 nmol/g dry weight/30 min and a Km for pantothenic acid of 10.7 mircoM were obtained). Reduction in perfusate Na+ concentration from 145 to 105 mM (the Na+ was replaced with 40 mM choline) resulted in a small but significant decrease in pantothenic acid uptake. At 145 mM Na+, addition of a mixture of amino acids, whose uptake is Na+-dependent, resulted in a significant decrease in pantothenic acid uptake by the heart (173 +/- 5 to 132 +/- 12 nmol/g dry weight). If an inward Na+ gradient in isolated, purified sarcolemmal vesicles, was imposed, a rapid uptake of pantothenic acid was observed. Uptake rates are markedly reduced if Na+ was replaced by equimolar concentrations of K+ or if external Na+ was reduced below 40 mM. In the presence of Na+, increasing pantothenic acid concentrations resulted in an increase in pantothenic acid uptake by the vesicles. Combined, these data demonstrate that pantothenic acid is transported across the myocardial sarcolemmal membrane by a Na+-dependent mechanism, which may be common to a number of small molecules.     

Studies of insulin action on the amphibian oocyte plasma membrane using NMR, electrophysiological and ion flux techniques

Insulin (0.1-10 microM) reinitiates the meiotic divisions in Rana oocytes and produces a 14-20 mV negative-going hyperpolarization of the plasma membrane as well as a 0.25 unit increase in intracellular pH during the first 90 min. During hyperpolarization, the Na+ conductance of the membrane decreases by 40-50% with a concomitant increase in 22Na+ uptake from the medium. The increased uptake of Na+ during a period of decreasing Na+ conductance is apparently due to an increase in fluid phase turnover associated with insulin-mediated endocytosis. Both membrane hyperpolarization and increase in pHi are Na+-dependent and are blocked by the serine proteinase inhibitor, phenylmethylsulfonyl fluoride. The membrane potential of the prophase oocyte has a significant electrogenic component with potential but not conductance sensitive to glycosides and substitution of Li+ for Na+. Insulin hyperpolarizes Li+ or glycoside-treated oocytes whereas glycosides do not affect insulin-hyperpolarized oocytes. [3H]Ouabain binding by the plasma membrane of the untreated oocyte shows at least two K+-sensitive components (Kd = 42 and 2000 nM) linked to inhibition of the Na+ pump. Insulin-treated oocytes show a single class of intermediate-affinity ouabain sites (Kd = 490 nM) which appear to result from insulin-induced internalization of membrane-bound ouabain. [125I]Insulin binding to the plasma membrane shows a class of high-affinity sites (Kd = 87 nM) with 40-50 pump sites per insulin-binding site. Our results suggest that insulin-induced mediator peptides stimulate Na+-H+ exchange resulting in an increase in intracellular pH and Na+ uptake concomitant with an increase in receptor-mediated endocytosis and a decrease in Na+ conductance and associated membrane hyperpolarization. The net result appears to be a down-regulation of the Na+ pump which together with a decrease in Na+ conductance may divert high-energy phosphate compounds from cation regulation to anabolic processes of meiosis.