Kinetic Analysis Of Na Research Articles

Ionic liquid-induced low temperature graphitization of cellulose-derived biochar for high performance sodium storage

Abstract The graphitization of biochar by high temperature carbonization above 2000 °C or metal-based catalytic approaches posed specific hindrances to the industrial production of high-quality graphitic carbon from biomass. The use of imidazolium-based ionic liquids (ILs) to induce fast graphitization of biochar at a low temperature range has not yet been reported. In this work, the carbonization of microcrystalline cellulose and 1-butyl-3-methylimidazolium acetate (BMIMAcO) at the temperature range of 750 °C–1400 °C led to enhanced graphitization of the biochar in comparison with the carbonization of microcrystalline cellulose alone. The incorporation of intact imidazolium rings into carbon skeleton played a critical role in the formation of graphitic structure with high nitrogen content. The IL-induced cellulose carbon obtained at 1000 °C (ICC-1000) with 5.67 at.% of nitrogen-doping presented interconnected graphitic nanosheets with 0.488 nm interlayer spacing and abundant mesopores and macropores on the surface. When used as anodes of sodium-ion batteries (SIBs), the ICC-1000 exhibited stable reversible capacity around 391 mAh g−1 at 100 mA g−1 for 100 cycles and 136 mAh g−1 at 500 mA g−1 for 1000 cycles, showing satisfactory sodium storage performance. Kinetic analysis of Na+ storage revealed that the ICC-1000 showed obvious capacitive characteristics and improved electric conductivity. DFT calculations suggested an interlayer spacing of 4.9 A for optimal Na+ intercalation in multilayer graphene, and the capacity of pristine multilayer graphene was greatly improved from 69.8 to 527.3 mAh g−1 after 4.8 at.% of N-doping.

Constructing N-Doped porous carbon confined FeSb alloy nanocomposite with Fe-N-C coordination as a universal anode for advanced Na/K-ion batteries

Sb-based anodes have shown great potential in sodium-ion batteries (SIBs) and potassium-ion batteries (PIBs) owing to their high theoretical capacities and applicable voltage platforms, but they usually suffer from severe volume change and sluggish ion diffusion kinetic. Herein, a novel N-doped three-dimension porous carbon network confined nanosized FeSb alloy composite (3D FeSb@NC) with a strong Fe-N-C bond is elaborately designed and fabricated, this unique structure sufficiently relieves the volume change of Sb during cycling, reduces the diffusion length for both ion/electrons, enhances the electronic conductivity, and provides abundant active sites for Na+/K+ storage. Moreover, the strong Fe-N-C bond can strengthen the interface adhesion between carbon matrix and alloy particle for sustaining structure integrity. The 3D FeSb@NC anodes delivers outstanding electrochemical performances in both SIBs and PIBs, in terms of ultralong cycling life (capacity retention of 85% after 750 cycles for SIBs and 80% after 1000 cycles for PIBs) and excellent rate capability (231 mAh g−1 at 5 A g−1 for SIBs and 119.7 mAh g−1 at 2 A g−1 for PIBs). The kinetic analysis of Na+/K+ storage reveal that the extrinsic pseudo-capacitive contribution accounts for the high rate performance. This work provides an effective strategy to develop nanocomposite anode with superior structural stability and durability for SIBs/PIBs.

Estragole blocks neuronal excitability by direct inhibition of Na+ channels

Estragole is a volatile terpenoid, which occurs naturally as a constituent of the essential oils of many plants. It has several pharmacological and biological activities. The objective of the present study was to investigate the mechanism of action of estragole on neuronal excitability. Intact and dissociated dorsal root ganglion neurons of rats were used to record action potential and Na+ currents with intracellular and patch-clamp techniques, respectively. Estragole blocked the generation of action potentials in cells with or without inflexions on their descendant (repolarization) phase (Ninf and N0 neurons, respectively) in a concentration-dependent manner. The resting potentials and input resistances of Ninf and N0 cells were not altered by estragole (2, 4, and 6 mM). Estragole also inhibited total Na+ current and tetrodotoxin-resistant Na+ current in a concentration-dependent manner (IC50 of 3.2 and 3.6 mM, respectively). Kinetic analysis of Na+ current in the presence of 4 mM estragole showed a statistically significant reduction of fast and slow inactivation time constants, indicating an acceleration of the inactivation process. These data demonstrate that estragole blocks neuronal excitability by direct inhibition of Na+ channel conductance activation. This action of estragole is likely to be relevant to the understanding of the mechanisms of several pharmacological effects of this substance.

Designing high-capacity cathode materials for sodium-ion batteries

Na-rich layered oxides as cathode materials for sodium-ion batteries were designed using an electrochemical method based on Li-rich layered oxides. The materials show high specific capacity that can reach 234mAh/g at a current of 5mA/g. The energy density of this material (644Wh/kg) is even higher than those of commercial cathodes for lithium-ion batteries, such as LiFePO4 and LiMn2O4. Kinetic analysis of Na+ insertion/extraction into/from the Na-rich layered oxide reveals that the Na+ diffusion coefficient is about 10−14 cm2/s.

Kinetic evidence for the existence and mechanism of formation of a barrel stave structure from pore-forming dendrimers.

A dendritic approach to the construction of a homologous series of pore-forming amphiphiles has been developed, based on the use of spermidine, spermine, lysine, and cholic acid. A kinetic analysis of Na+ transport across bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine by three dendritic amphiphiles has provided the strongest evidence to date for a barrel stave structure.

In Vivo and In Vitro Effects of Acrylamide on Synaptosomal Neurotransmitter Uptake and Release

Evidence suggests acrylamide (ACR) neurotoxicity is mediated by impaired presynaptic transmission. To assess the effects of ACR on nerve terminal function, [ 3H ]glutamate release and uptake were determined in brain synaptosomes isolated from intoxicated rats (50 mg/kg per day×8 days, i.p. or 21 mg/kg per day×21 days, p.o.). Regardless of ACR dose-rate, a significant reduction in synaptosomal K +-stimulated, Ca 2+-dependent release was detected, whereas kinetic analysis of Na +-dependent uptake did not reveal consistent changes. Immunoblot analysis showed normal protein levels (e.g. SNAP-25) in dysfunctional synaptosomes isolated from ACR-intoxicated rats. This suggests that defective release does not involve changes in protein synthesis and/or anterograde delivery of presynaptic constituents. To identify potential targets, synaptosomes were exposed in vitro to [ 14 C]-ACR and radiolabeled proteins were separated by gel electrophoresis and detected by autoradiography. [ 14 C]-ACR labeling of distinct synaptosomal protein bands (10.5–154,000 kDa) was blocked by the sulfhydryl alkylating agent, N-ethylmaleimide (NEM; 4 mM) but not by the non-neurotoxic structural analog propionamide (10 mM). In vitro characterization of synaptosomal [ 3H ]glutamate uptake and release showed that ACR, NEM and iodoacetic acid (IAA) produced concentration-dependent decreases in each parameter that were highly correlated to reductions in free sulfhydryl content. All three chemicals were equiefficacious with respect to reducing sulfhydryl content and neurotransmitter uptake/release, although the relative potencies differed; NEM> IAA> ACR. Kinetic analysis of uptake showed that in vitro exposure to ACR, IAA or NEM at their respective IC 50’s caused similar reductions in V max. These data suggest that ACR-induced synaptic dysfunction involves adduction of presynaptic protein thiol groups and subsequent reduction in neurotransmitter release.

The characterization of ion regulation in Amazonian mosquito larvae: evidence of phenotypic plasticity, population-based disparity, and novel mechanisms of ion uptake.

This study is the first step in characterizing ion uptake mechanisms of mosquito larvae from the Amazon region of Brazil. Hemolymph NaCl levels and rates of unidirectional Na(+) and Cl(-) uptake were measured in larvae of Aedes aegypti and Culex quinquefasciatus in a series of environmental manipulations that are known to challenge ion regulation in other aquatic animals. Despite being reared for numerous generations in dilute media (20 micromol L(-1) NaCl), both species were able to maintain high hemolymph NaCl concentrations, a departure from previous studies. Exposure to distilled water or high-NaCl media did not affect hemolymph ion levels, but pH 3 caused significant decreases in hemolymph Na(+) and Cl(-) levels in both species. Exposure to water from Rio Negro (pH 5.5), an organically rich but ion-poor body of water, did not disturb hemolymph Na(+) and Cl(-) levels or the uptake of these ions. Acute exposure to control media or Rio Negro water titrated to pH 3.5 caused inhibition of Na(+) uptake and stimulation of Cl(-) uptake in C. quinquefasciatus, but A. aegypti larvae experienced only a significant reduction of Na(+) uptake in Rio Negro/pH 3.5 treatment. The stimulation of Cl(-) uptake at low pH has been documented only in aquatic insects and differs from all other invertebrate and vertebrate species. A similar pattern of Na(+) uptake inhibition and Cl(-) uptake stimulation was observed in A. aegypti larvae exposed to bafilomycin A(1), a blocker of V-type H(+) ATPase. Culex quinquefasciatus larvae were unaffected by this drug. Both Na(+) and Cl(-) uptake were reduced when C. quinquefasciatus larvae were exposed to acetazolamide, indicating that H(+) and HCO(3)(-), derived from hydration of CO(2), are involved with Na(+) and Cl(-) uptake. Kinetic analysis of Na(+) and Cl(-) uptake in C. quinquefasciatus, A. aegypti, and Anopheles nuneztovari larvae indicate that these Amazonian species share similar high-capacity and high-affinity mechanisms. Comparison of the Amazonian C. quinquefasciatus with a Californian population provided evidence of both phenotypic plasticity and population disparity in Na(+) and Cl(-) uptake, respectively. When the California population of C. quinquefasciatus was reared in a medium similar to that of the Amazonian group (60 micromol L(-1) NaCl) instead of 4,000 micromol L(-1) NaCl, larvae increased both Na(+) uptake capacity (J(max)) and affinity (i.e., reduced K(m)), yet Cl(-) uptake did not change from its nonsaturating, low-capacity pattern. In the reverse experiment, Amazonian C. quinquefasciatus demonstrated plasticity in both Na(+) and Cl(-) uptake by significantly reducing rates when held in 4,000 micromol L(-1) NaCl for 3 d.

Ionoregulatory specializations for exceptional tolerance of ion-poor, acidic waters in the neon tetra (Paracheirodon innesi).

To better understand how fish are able to inhabit dilute waters of low pH, we examined ionoregulation in exceptionally acid-tolerant neon tetras (Paracheirodon innesi), which are native to the ion-poor, acidic Rio Negro, Amazon. Overall ion balance was unaffected by 2-wk exposure to pH 4.0 and 3.5. Measurements of unidirectional Na+ fluxes during exposure to pH 3.5 showed that tetras experienced only a mild, ionic disturbance of short duration (</=24 h) as a result of a stimulation of Na+ efflux. At pH 3.25, Na+ efflux was almost fourfold greater (all fish died within 6-8 h). At both pHs, active Na+ uptake was not inhibited, and in fact, at pH 3.5, uptake was stimulated. Kinetic analysis of Na+ uptake at pH 6.5 and 3.5 produced virtually identical low Km values and high maximum-transport values. These results confirmed the pH insensitivity of the uptake mechanism and revealed a mechanism well designed to operate in the dilute, acidic waters of the Rio Negro. Na+ influx was only mildly sensitive to amiloride (a Na+ channel blocker), which, along with the pH insensitivity, suggests that Na+ uptake may occur by a novel mechanism. Na+ efflux was reduced by addition of Ca2+ to the test water at pH 6.5, but the effect disappeared at pH 3.5. Exposure to LaCl3 (a strong Ca2+ displacer) also stimulated Na+ efflux. These results suggest that Ca2+ plays a role in determining branchial ion permeability at high pH but that, at low pH, where Na+ efflux is stimulated, alternate, Ca2+-independent mechanisms are employed to control Na+ efflux.

Ion regulation in ion-poor acidic water by the blackskirt tetra (Gymnocorymbus ternetzi), a fish native to the Amazon River.

We examined the ionoregulatory capabilities of the blackskirt tetra (Gymnocorymbus ternetzi), which is native to ion-poor acidic waters of the Amazon River. Examination of Na+ uptake, which was only slightly sensitive to the uptake blocker amiloride, revealed several specializations for uptake in these waters. Kinetic analysis of Na+ uptake (at pH 6.5) revealed a high maximum rate of uptake and a low Michaelis-Menten constant, which allows the tetras to take up Na+ at high rates even at very low water levels. At pH 4.5, a pH where they experience sizable ion disturbances, they displayed several mechanisms to restore balance. Kinetic analysis at pH 4.5 revealed that the maximum uptake rate rose 67% while the Michaelis-Menten constant remained unchanged. Further tests showed that the upregulation of Na+ uptake occurred within 12 h in response to a doubling of Na+ efflux. Despite these specializations of the Na+ uptake mechanism, blackskirt tetras were not especially tolerant of low pH. Upon exposure to pH 4.0, they experienced a massive loss of Na+ due to a fourfold increase of Na+ efflux (relative to pH 5.0) and an 80% inhibition of uptake. Measurement of Na+ efflux in waters with different Ca2+ levels and in the presence of LaCl, a strong Ca2+ competitor, correlated the stimulation of Na+ efflux at low pH with a low branchial affinity for Ca2+. These tests indicate that blackskirt tetras possess abilities to resist the disruptive effects of moderately low pH but cannot survive in waters with a pH of 4.0 or less because of leaching of Ca2+ from branchial tight junctions, which stimulates ion losses.

Aspartate 55 in the Na+/proline permease of Escherichia coli is essential for Na+-coupled proline uptake.

Four acidic residues in the N-terminal domain of Na+/proline permease of Escherichia coli (Asp33, Asp34, and Asp55 in putative loop 2, Glu75 in putative transmembrane domain II) were individually replaced with neutral or charged amino acid residues. Replacement of Glu75, the only residue in the permease presumed to be in the middle of a transmembrane domain, Asp33, or Asp34 had little or no influence on the kinetics of Na+-coupled proline transport. In contrast, removal of the carboxylate at position 55 (Asp55 --> Asn or Asp55 --> Cys permease) impaired proline uptake completely while lengthening of the side chain at this position by one methylene group (Asp55 --> Glu permease) allowed transport at a reduced initial rate. Importantly, all permease molecules were present in the membrane at concentrations comparable to the wild-type protein. Kinetic analysis of Na+-coupled proline transport catalyzed by Asp55 --> Glu permease revealed a 5-fold increase of the K(m) for proline and a 30-fold decrease of the V(max) compared to wild-type. Remarkably, replacement of Asp55 by Glu led to a 50-fold decrease of the apparent affinity of the permease for Na+. Furthermore, replacement of Asp55 with Cys or Asn blocked proline-induced Na+ uptake whereas significant Na+ transport was observed with Asp55 --> Glu permease. In addition, transport of proline down its concentration gradient was not detectable with deenergized cells containing Asp55 --> Glu permease at low Na+ concentrations. However, downhill transport activity was observed in the presence of high Na+ concentrations. Replacement of Asp55 with Asn or Cys impaired downhill transport under all conditions tested. The observations demonstrate that a carboxylate at position 55 of proline permease is essential for Na+-coupled proline transport. It is suggested that Asp55 may be involved in binding of the coupling ion.

Na+/H+ antiport activity and cell growth in cultured skin fibroblasts of IDDM patients with nephropathy.

IDDM patients with incipient and overt nephropathy have been found to exhibit an overactivity of RBC sodium-lithium countertransport. To explore the physiological relevance of this finding, we measured the activity of Na+/H+ antiport in serially passaged cultured skin fibroblasts from IDDM patients with and without nephropathy and from normal, nondiabetic control subjects. Na+/H+ antiport activity (measured as the rate of amiloride-sensitive Na+ influx at pHi = 6.4, extracellular pH = 8.0, and [Na+] = 1 mM) was elevated significantly in IDDM patients with nephropathy compared with IDDM patients without nephropathy and nondiabetic control subjects (13.35 +/- 3.8 vs. 8.54 +/- 2.0 vs. 7.33 +/- 2.3 nmol Na+.mg protein-1.min-1; P less than 0.006 and P less than 0.001, respectively). A kinetic analysis of Na+/H+ antiport activity showed that the raised activity in IDDM patients with nephropathy was caused by an increased Vmax for extracellular Na+. Km values were similar in the three groups. pH-stimulated amiloride-sensitive Na+ influx also was higher under baseline conditions and after serum stimulation in cells from IDDM patients with nephropathy. pHi values were significantly higher, both during active proliferation and after 10-min exposure to serum, in cells from IDDM patients with nephropathy, compared with IDDM patients without nephropathy and nondiabetic control subjects. Serum-stimulated incorporation of [3H]thymidine into DNA was greater in IDDM patients with nephropathy than in the other two groups (35.7 +/- 18.9- vs. 17.4 +/- 7.5- vs. 11.9 +/- 8.7-fold stimulation above baseline; P less than 0.01 for both.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of in vitro metabolic acidosis on luminal Na+/H+ exchange and basolateral Na+:HCO3- cotransport in rabbit kidney proximal tubules.

The aim of this study was to evaluate the role of the kidney in mediating the signals involved in adaptive changes in luminal Na+/H+ exchange and basolateral Na+:HCO3- cotransport systems in metabolic acidosis. Proximal tubular suspensions were prepared from rabbit kidney cortex and incubated in acidic (A) or control (C) media (pH 6.9 vs 7.4, 5% CO2) for 2 h. Brush border membrane (BBM) and basolateral membrane (BLM) vesicles were isolated from the tubular suspensions and studied for the activity of Na+/H+ exchange and Na+:HCO3- cotransport. Influx of 1 mM 22Na at 10 s (pH6 7.5, pH(i) 6.0) into BBM vesicles was 68% higher in group A compared to group C. The increment in Na+ influx in the group A was amiloride sensitive, suggesting that Na+/H+ exchange was responsible for the observed differences. Kinetic analysis of Na+ influx showed a Km of 8.1 mM in C vs 9.2 in A and Vmax of 31 nmol/mg protein per min in group C vs 57 in A. Influx of 1 mM 22Na at 10 s (pH0 7.5, pH(i) 6.0, 20% CO2, 80% N2) into BLM vesicles was 83% higher in the group A compared to C. The HCO3-dependent increment in 22Na uptake in group A was 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid sensitive, suggesting that Na+:HCO3- cotransport accounted for the observed differences. Kinetic analysis of Na+ influx showed a Km of 11.4 mM in C vs 13.6 in A and Vmax of 35 nmol/mg protein per min in C vs 64 in A. The presence of cyclohexamide during incubation in A medium had no effect on the increments in 22Na uptake in group A. We conclude that the adaptive increase in luminal Na+/H+ exchange and basolateral Na+:HCO3- cotransport systems in metabolic acidosis is acute and mediated via direct signal(s) at the level of renal tubule.

Potassium depletion increases luminal Na+/H+ exchange and basolateral Na+:CO3=:HCO3- cotransport in rat renal cortex.

Most HCO3- reabsorption in proximal tubules occurs via electroneutral Na+/H+ exchange in brush border membranes (BBMS) and electrogenic Na+:CO3=:HCO3- cotransport in basolateral membranes (BLMS). Since potassium depletion (KD) increases HCO3- reabsorption in proximal tubules, we evaluated these transport systems using BBM and BLM vesicles, respectively, from control (C) and KD rats. Feeding rats a potassium deficient diet for 3-4 wk resulted in lower plasma [K+] (2.94 mEq/liter, KD vs. 4.47 C), and higher arterial pH (7.51 KD vs. 7.39 C). KD rats gained less weight than C but had higher renal cortical weight. Influx of 1 mM 22Na+ at 5 s (pHo 7.5, pHi 6.0, 10% CO2, 90% N2) into BLM vesicles was 44% higher in the KD group compared to C with no difference in equilibrium uptake. The increment in Na+ influx in the KD group was DIDS sensitive, suggesting that Na+:CO3=:HCO3- cotransport accounted for the observed differences. Kinetic analysis of Na+ influx showed a Km of 8.2 mM in KD vs. 7.6 mM in C and Vmax of 278 nmol/min/mg protein in KD vs. 177 nmol/min/mg protein in C. Influx of 1 mM 22Na+ at 5 s (pHo 7.5, pHi 6.0) into BBM vesicles was 34% higher in the KD group compared to C with no difference in equilibrium uptake. The increment in Na+ influx in the KD group was amiloride sensitive, suggesting that Na+/H+ exchange was responsible for the observed differences. Kinetic analysis of Na+ influx showed a Km of 6.2 mM in KD vs. 7.1 mM in C and Vmax of 209 nmol/min/mg protein in KD vs. 144 nmol/min/mg protein in C. Uptakes of Na(+)-dependent [3H]glucose into BBM and [14C]succinate into BLM vesicles were not different in KD and C groups, suggesting that the Na+/H+ exchanger and Na+:CO3=:HCO3- cotransporter activities were specifically altered in KD. We conclude that adaptive increases in basolateral Na+:CO3=:HCO3- cotransport and luminal Na+H+ exchange are likely responsible for increased HCO3- reabsorption in proximal tubules of KD animals.

Taurocholate transport and Na+-K+-ATPase activity in fetal and neonatal rat liver plasma membrane vesicles.

The ontogenesis of Na+-K+-ATPase activity and Na+-taurocholate cotransport was studied in basolateral plasma membrane vesicles from fetal and neonatal rat liver. Membrane vesicles from each age group were 30-fold enriched in the basolateral marker enzyme Na+-K+-ATPase, 4- to 7-fold enriched in the bile canalicular membrane marker enzymes alkaline phosphatase and Mg2+ ATPase, and not significantly enriched in activities of marker enzymes for intracellular organelles. Na+-K+-ATPase activity was significantly lower in basolateral membranes from late fetal (day 21-22) and neonatal (day 1) rat liver. Kinetic analysis of Na+-K+-ATPase activity at various concentrations of ATP revealed that the maximum velocity of enzyme reaction (Vmax) for Na+-K+-ATPase was 70 and 90% of adult activity in the fetus and the neonate, respectively. The ATP Km was significantly lower in the neonate than the adult, suggesting a higher affinity of the neonatal enzyme for ATP. In contrast to the early maturation of Na+-K+-ATPase, transport of taurocholate was markedly lower in both fetal and neonatal vesicles compared with the adult. Taurocholate uptake on day 19 of gestation did not differ in the presence of a Na+ or K+ gradient, and uphill transport, as indicated by an overshoot, did not occur. On day 20 taurocholate uptake was stimulated by a Na+ compared with a K+ gradient, and accumulation of isotope above equilibrium was demonstrated. Na+-dependent transport of taurocholate by late fetal (day 22) and neonatal vesicles was saturable but the Vmax at each age was significantly lower and the apparent Km higher in developing compared with adult membrane vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of Na +/phosphate cotransport in LLC-PK 1 cells by 12- O-tetradecanoylphorbol 13-acetate (TPA)

We have tested for the effect of the phorbol ester 12- O-tetradecanoylphorbol 13-acetate (TPA) on Na +/phosphate cotransport in an established epithelial cell line of renal origine (LLC-PK 1). Incubation of LLC-PK 1 cells with TPA produced an increase in Na +/phosphate (P i) cotransport. The maximal response was reached at a TPA concentration of 10 ng/ml. Other phorbol esters which have no potency or a smaller one to activate protein kinase C had no effect on Na +/P i cotransport. Incubation of LLC-PK 1 cells with 10 ng/ml TPA for 8 h led to a 300% increase in Na +/P i cotransport; in the presence of cycloheximide the increase amounted only to a 100% and was reached within 2 h. Kinetic analysis of Na +/P i contransport indicated an increase in the apparent V max without an effect on the apparent K m. The increased P i transport was retained in isolated apical vesicles. Na +-dependent alanine transport into LLC-PK 1 monolayers was affected by TPA administration in a similar manner. TPA had under the chosen experimental conditions no effect on [ 3H]thymidine incorporation into DNA excluding a general proliferative effect. We conclude that TPA via activation of protein kinase C regulates the number of operating transport systems. As also other Na +-coupled transport systems are influenced, the TPA effect appears to be related to the expression of a general ‘adaptive’ alteration of membrane transport in LLC-PK 1 cells.

Mechanisms of Ca2+ transport in plasma membrane vesicles prepared from cultured pituitary cells. I. Characterization of Na+/Ca2+ exchange activity.

GH3 rat anterior pituitary cells possess a Na+/Ca2+ exchange transport mechanism which is present in purified plasma membrane vesicles prepared from these cells. Imposition of an outwardly directed Na+ gradient in vesicles results in a marked concentrative uptake of Ca2+ which is abolished by the Ca2+ ionophore A23187. Transport activity depends on a sustained Na+ gradient. Dissipation of the driving force by treatment with Na+ ionophores or by passive gradient collapse abolished transport activity. The exchange reaction is completely reversible since addition of extravesicular Na+ enhances Ca2+ efflux from Ca2+ loaded vesicles. A kinetic analysis of Na+/Ca2+ exchange indicates saturation kinetics for both substrates with apparent values of Km for Na+ and Ca2+ of 17 mM and 5 microM, respectively, and a Vmax of about 8 nmol/min/mg of protein for Ca2+ uptake at 25 degrees C. In addition to Na+/Ca2+ exchange, the transporter functions in a Ca2+/Ca2+ exchange mode with an apparent Km of 20 microM and Vmax of 16 nmol/min/mg of protein for Ca2+ influx. Na+/Ca2+ exchange is not inhibited by protonophores indicating that Ca2+ flux does not occur via coupled Na+/H+, Ca2+/H+ exchange. Transport is inhibited by derivatives of the pyrazine diuretic amiloride. The pH dependency of Ca2+ uptake displays a sigmoidal relationship with stimulation of activity at alkaline pH and inhibition at acid pH. Furthermore, the reaction is electrogenic (i.e. more than 2 Na+ transported per Ca2+) as demonstrated by stimulated uptake of lipophilic cations during exchange and by effects of artificially imposed membrane potentials on the rate of Ca2+ transport. Plasma membrane vesicles prepared from bovine anterior pituitary glands also display Na+/Ca2+ exchange with many of the same characteristics. These results support the notion that Na+/Ca2+ exchange functions in Ca2+ homeostasis in pituitary cells.

Amino acid transport and rubidium-ion uptake in monolayer cultures of hepatocytes from neonatal rats.

Amino acid and K(+) transport during development has been investigated in hepatocyte monolayer cultures with either alpha-amino[1-(14)C]isobutyrate or (86)Rb(+) used as a tracer for K(+). Parenchymal cells from neo- and post-natal rat livers have been isolated by an improved non-perfusion technique [Bellemann, Gebhardt & Mecke (1977)Anal.Biochem.81, 408-415], and the resulting hepatocyte suspensions purified from non-hepatocytes before inoculation. In the presence of Na(+) (Na(+)-dependent component), the rates of amino acid uptake in neonatal hepatocytes were markedly enhanced compared with cells from 30-day-old rats. When Na(+) was replaced by choline (Na(+)-independent component) the accumulation of alpha-aminoisobutyrate was decreased and it was not affected by the age of the animals. Kinetic analysis of Na(+)-dependent alpha-aminoisobutyrate transport revealed the existence of a high-affinity low-K(m) component (K(m)0.91mm) with a V(max.) of 2.44nmol/mg of protein per 4min, which later declined gradually with progressive development. Rates of Rb(+) transport were concomitantly enhanced in neonatal hepatocytes and thereafter declined with postnatal age. The increased Rb(+) influx was effectively inhibited by ouabain and reflected elevated activity of the electrogenic Na(+)/K(+)-pump during early stages of development. Kinetic evaluation of the enhanced rates of Rb(+) uptake indicates multiple and co-operative binding sites of the enzyme involved in the Rb(+) uptake, and the transport system is positively co-operative (the Hill coefficient h is >1.0). In short, amino acid transport in neonatal rat hepatocytes is increased as a result of an existing low-K(m) component for the Na(+)-dependent alpha-aminoisobutyrate uptake, which endows the hepatocytes with a high capability for concentrating amino acids at low ambient values. The concomitant enhancement of K(+) transport reflects changes in the electrochemical gradient for Na(+) across the hepatocellular membrane and, along with this, presumably alterations in the membrane potential; the latter might be the driving force for the enhanced alpha-aminoisobutyrate transport in the alanine-preferring system during postnatal age.