Monovalent cation and ouabain effects on PAH uptake by rabbit kidney slices.

The effects of K+, Na+, and ouabain on para-aminohippurate (PAH) accumulation by rabbit kidney slices have been further examined. The present studies show that for maximum uptake of PAH to occur in Na+,K+-depleted slices extra-cellular Na+ and K+ ions are required together. Kinetic studies revealed that increasing the external K+ from 0 to 5 mM caused a decrease in the apparent Km of transport. The Vmax values were not changed significantly. In the presence of 5 mM K+, increasing Na+ concentration in the bathing medium from 0 to 145 mM produced an increase in the V max while the apparent Km remained constant. In the presence of 5 mM external K+, ouabain inhibition of PAH uptake was progressively and entirely antagonized by decreasing external Na+. Studies on kidney slices treated with ouabain in a Na+,K+-free medium in order to inhibit active electrolyte transport showed that PAH uptake under anaerobic conditions was specifically enhanced by Na+. This stimulation occurred when cell [Na+] was less than medium [Na+]. This Na+-dependent and energy-independent accumulation of PAH was inhibited by hippuric acid but not by N-methylnicotinamide.     

Catecholamine release from bovine chromaffin cells: the role of sodium-calcium exchange in ouabain-evoked release

Spontaneous catecholamine (CA) release from bovine chromaffin cells maintained in primary tissue culture has been measured after pre-loading the cells with [3H]noradrenaline. Ouabain inhibited 86Rb+ uptake and increased 3H release in a concentration-dependent manner during a 60 min incubation period. Low external Na+ (5 mM: Li+ substitution) also increased 3H release. Whereas the 3H-releasing action of ouabain was maintained, the Li(+)-evoked release decreased with time. The effects of both ouabain and low Na+ solution on 3H release were completely inhibited by removal of Ca2+ from the external medium even though in Ca2(+)-free solution ouabain further inhibited 86Rb+ uptake into the cells. Readmission of Ca2+ to Na(+)-loaded cells (10-4 M-ouabain in Ca2(+)-free-1 mM-EGTA solution for 60 min) markedly increased the release of 3H. In the additional presence of diphenylhydantoin (DPH, 10-4 M) 3H release was significantly less on Ca2+ readmission. The 3H release from Na(+)-loaded cells was proportional to the concentration of Ca2+ readmitted. The 3H release was further increased from Na(+)-loaded cells in response to Ca2+ readmission when [Na+]o was lowered from 149 to 5 mM (Li+, choline+, Tris+ or sucrose substitution) though Li+ was less effective than the other Na+ substitutes. Potassium removal from the external medium significantly inhibited the 3H release evoked by Ca2+ readmission to Na(+)-loaded cells, even when [Ca2+]o was greater than normal (7.5 mM) or if Ca2+ was readmitted in low [Na+]o solution. Rb+, Cs+ or Li+ could substitute for K+ with the order of potency: Rb+ greater than or equal to K+ greater than Cs+ greater than Li+. A slight increase of external K+ (10.8 mM) potentiated the 3H release from Na(+)-loaded cells on Ca2+ readmission, but a higher concentration of K+ (149.4 mM) had the opposite action. The data is consistent with the hypothesis that ouabain-evoked CA release from bovine chromaffin cells is, in part, a consequence of an internal Na(+)-dependent Ca2+ influx. The evidence also suggests that there is Na(+)-Ca2+ competition at the external arm of the exchanger together with a monovalent cation activation site.     

Studies on the lithium transport across the red cell membrane

1. Extracellular potassium bicarbonate, ouabain, dipyridamole and the Na+ distribution between red cells and plasma influence Li+ transport across the human red cell membrane. The significance of these parameters for the intraindividual variability of the steady-state ratio of external to internal Li+ was studied in vitro. 2. Elevation of external K+ in the physiological concentration range increases the steady-state distribution ratio Lie+/Lii+ indirectly by increasing the ratio Nae+/Nai+ through activation of the Na+-K+ pump, and directly by inhibiting ouabain-sensitive Li+ uptake. 3. A rise in bicarbonate concentration decreases the Li+ ratio directly by accelerating Li+ uptake through a leak, and indirectly by increasing the Na+ leak, thus reducing the Na+ ratio. 4. Dipyridamole blocks both bicarbonate effects. 5. Ouabain decreases the Na+ ratio and inhibits Li+ uptake by the Na+-K+ pump, thereby exerting two opposite effects on the Li+ distribution ratio. 6. The results confirm the previous observation that the steady-state Li+ distribution depends strongly on the Na+ distribution ratio, i-e., the driving force for Na+-dependent Li+ uphill countertransport. It is concluded that the Na+ distribution between red cells and plasma and the concentrations of K+ and bicarbonate in plasma need to be considered as factors influencing the in vivo Li+ distribution. However, the considerable interindividual differences of Li+ distribution cannot be ascribed to variations in these parameters.     

Furosemide-sensitive thallium fluxes in smooth muscle of rabbit uterus

The furosemide-sensitive uptake of thallium represents approximately equal to 50% of the total uptake of thallium by rabbit uterus and requires Cl- and Na+. The furosemide-sensitive uptake of thallium is stimulated by other ions at low concentrations with the rank order Li+ greater than Tl+ greater than K+ = Rb+ greater than Cs+ and is inhibited by these ions at high concentrations with the rank order Tl+ greater than K+ = Rb+ greater than Cs+ greater than Li+, suggesting multiple cation binding sites on the carrier. Uptake of 36Cl- is inhibited by furosemide in the presence of ouabain. Thallium efflux and 36Cl efflux in the presence of ouabain is inhibited by furosemide. The chloride concentration regulates the proportion of thallium uptake that is ouabain sensitive and furosemide sensitive without altering the total uptake. It is suggested that the furosemide-sensitive uptake of thallium reflects a Na+-Cl- -K+ exchange system that could be classified as a cotransport or countertransport of any two of these ions and also could be the smooth muscle chloride pump.     

Interrelation between membrane transport and the contents of alkali metal cations in HeLa cells

The relation of membrane transport of alkali cations to their external concentrations or to their cellular contents was studied in HeLa cells. Chilling the cells at 0 degrees C reversed cell Na+ and K+ to a mirror image of the normal pattern. Upon rewarming to 37 degrees C the ouabain-sensitive Rb+ uptake became 2-fold faster than the control. A kinetic analysis revealed that the stimulation was due to an increase in the maximal rate of Rb+ uptake, Jmax. The increase in apparent Km was relatively small. The analysis also showed that the ouabain-sensitive cation transport system seemed to have two binding sites for Rb+. The stimulation of Rb+ uptake was related to an increase in cell Na+, and an addition of ouabain abolished such a relation. Net Na+ flux which was in the direction from inside the cells to the medium at hypernormal cell Na+ was iiincreased when cell Na+ ncreased. In contrast, net Na+ flux which was in the opposite direction in the presence of ovabain was reduced and became almost 0 at cell Na+ of 900 nmol/mg of protein. The Na+/Rb+ coupling ratio in the ouabain-sensitive cation transport was apparently less than 1 at nearly physiological cell Na+, but it approached 1.5 when cell Na+ was sufficiently high. The sum of cell K+ plus Rb+ varied inversely with cell Na+, and this relation was unaffected upon treatment with ouabain. When Rb+ uptake declined below 80% of the control, cell K+ plus Rb+ was reduced, however, 40% of the sum of cell cations was still preserved even after complete inhibition of the cation pumps by ouabain treatment of 2 hr. Interrelations of these results are discussed.     

K(+)-induced reversal of astrocyte glutamate uptake is limited by compensatory changes in intracellular Na+.

Glutamate uptake is coupled to counter-transport of K+, and high external K+ concentrations can induce reversal of glutamate uptake in whole-cell patch-clamp and isolated membrane preparations. However, high external K+ causes little or no reversal of glutamate uptake in intact astrocytes, suggesting a regulatory mechanism not evident in membrane preparations. One mechanism by which intact cells could limit the effects of altered extracellular ion concentrations on glutamate transport is by compensatory changes in intracellular Na+ concentrations. This possibility was examined using astrocyte cultures treated in two ways to reduce the driving force for glutamate uptake: incubation in high K+ (with reciprocal reduction in Na+), and incubation with metabolic inhibitors to induce ATP depletion. ATP depletion produced a rise in intracellular Na+, a collapse of the membrane sodium gradient and a massive reversal of glutamate uptake. By contrast, incubation in high K+/low Na+ medium did not significantly alter the sodium gradient and did not induce glutamate uptake reversal. The sodium gradient was shown to be maintained under these conditions by compensatory reductions in intracellular Na+ that approximately matched the reductions in extracellular Na+. These findings suggest a mechanism by which astrocytes may limit reversal of glutamate uptake under high K+/low Na+ conditions, and further suggest a general mechanism by which Na(+)-dependent transport processes could be shielded from fluctuating extracellular ion concentrations.     

A Na+-dependent D-mannose transporter in the apical membrane of chicken small intestine epithelial cells

The presence of a Na+/D-mannose cotransporter in brush-border membrane vesicles (BBMV) isolated from chicken small intestine was examined. In the presence of an electrochemical gradient for Na+, but not in its absence, D-mannose was accumulated transiently by the BBMV. D-Mannose uptake into the BBMV was energized by both the membrane potential and the chemical gradient for Na+. The relationship between D-mannose transport and external D-mannose concentration was described by an equation that represented the superposition of a saturable component (Michaelis-Menten constant Km 12.5 microM) and another component unsaturatable up to 80 microM D-mannose. D-Mannose uptake was inhibited by various substances in the following order of potency: D-mannose>D-glucose>phlorizin>phloretin>D-fructose. For the uptake of alpha-methyl-glucopyranoside the order was D-glucose=phlorizin>phloretin=D-fructose=D-mannose. The initial rate of D-mannose uptake increased as the extravesicular [Na+] increased, with a Hill coefficient of 1, suggesting that the Na+:D-mannose cotransport stoichiometry is 1:1. It is concluded that the intestinal apical membrane has a saturable, electrogenic and concentration- and Na+-dependent mannose transport mechanism that differs from the sodium-dependent glucose transporter SGLT1.     

The coupling of downhill ion movements associated with reversal of the sodium pump in human red cells

1. Previous work on the incorporation of inorganic phosphate (P(i)) into ATP has suggested reversal of the chemical reactions of the Na pump in human red cells. A study has now been made of the associated movements of Na and K.2. The efflux of K, and the influx and efflux of Na were measured. When the Ringer was without K, the loss of cell K was inhibited by ouabain, and the ouabain-sensitive component of K efflux (0.36 mu-equiv.ml.(-1).hr(-1)) required external Na.3. The exchange of Na was also inhibited by ouabain. When influx and efflux of Na were measured simultaneously in K-free Ringer there was an excess of ouabain-sensitive influx over efflux of about 0.36 mu-equiv.ml.(-1).hr(-1). This difference balanced the ouabain-sensitive K efflux, and was not found with 10 mM-external K. The Na and K movements appear to be coupled and to be mediated by reversal of the Na pump.4. The net uptake of Na sensitive to ouabain was 0.38 mu-equiv.ml.(-1).hr(-1) for red cells incubated in K-free Ringer, and the net loss of K under the same conditions was 0.58 mu-equiv.ml.(-1).hr(-1) in rough keeping with the unidirectional flux values.5. Oligomycin decreased Na influx and efflux to the same extent as ouabain.6. There appears to be a coupled downhill movement of Na and K that is abolished both by inhibitors of the Na pump and by external K which promotes normal transport of Na outwards and K inwards.     

Cation effects on acid secretion in rabbit gastric glands

The ability of isolated gastric glands to produce acid as a function of intra- and extracellular concentrations of K+ and Na+ was investigated. Ouabain inhibited acid formation as measured by the aminopyrine (AP) accumulation technique in a dose-dependent manner with an ED50 of 2 X 10(-6) M at 5.4 mM extracellular K+. This inhibition was counteracted by increasing extracellular K+ or decreasing extracellular Na+. In K+-free solutions with regular Na+, the AP accumulation was almost totally abolished; the readdition of K+ rapidly restored the response with a K0.5 of 1 mM extracellular K+. In the absence of Na+, with or without ouabain, there was always a significant residual AP accumulation, but the K0.5 of extracellular K+ required for acid formation increased to about 20 mM. Despite repeated washings in Na+-K+-free solutions, the intracellular K+ content could not be decreased below 24 mM above the apparent K0.5 for K+. It was found that the intracellular K+ could be depleted without disturbance of the acid secretory function if the glands were treated with the neutral ionophore amphotericin B and ouabain in K+-Na+-free solutions. Complete dose-response curves of H+ secretion as a function of K+ concentration could now be obtained. Thus the K0.5 for K+ activation of AP accumulation was 16.5 +/- 0.9 mM; upon histamine stimulation of the glands, the K0.5 decreased to 10.4 +/- 0.7 mM. Intracellular K+ concentrations above approximately 60 mM inhibited AP accumulation. Na+ dose dependently inhibited the K+-induced response. At low extracellular K+ concentrations, a ouabain-insensitive K+ accumulation mechanism was unmasked. This K+ uptake was partly inhibited y p-chloromercuribenzene sulfonic acid and by inhibitors of the gastric H+-K+-ATPase and amplified by inhibitors of K+ conductance channels such as Ba2+. The K+ content of gastric glands is mainly regulated by the Na+-K+-ATPase, but additional K+-uptake pathways are present. The affinity for K+ is increased in histamine-stimulated glands, which might indicate one mechanism of stimulating the secretory response. Na+ inhibits secretion possibly by binding to the cytosolic cation site of the gastric ATPase. The cation relationship to acid secretion in the rabbit parietal cell is similar to that described for vesicles isolated from hog gastric mucosa.     

Lithium activates mammalian Na+/H+ exchangers: isoform specificity and inhibition by genistein

Replacement of external NaCl with LiCl induced cytoplasmic alkalinization in CCL-39 cells and rat L6 myoblasts expressing the endogenous Na+/H+ exchanger isoform NHE1. This Li+-induced alkalinization is due to activation of the Na+/H+ exchanger because it was completely inhibited by 100 microM ethylisopropylamiloride (apparent Kd=1 microM) and because it did not occur in exchanger-deficient PS120 cells. The effect of Li+ was not mimicked by Na+, K+, Cs+ and choline+. Li+ caused cytoplasmic alkalinization in PS120 cells expressing NHE1 or NHE2, but not NHE3, when Li+ was added to cells at a concentration high enough to saturate their external transport sites as predicted from Li+ affinities. Li+ did not induce phosphatidylinositol (PI) turnover or intracellular Ca2+ mobilization. Li+-induced alkalinization was not affected by protein kinase C down-regulation, loss of glycogen synthase kinase 3beta caused by antisense oligonucleotide treatment, or pretreatment with calphostin C, pertussis toxin, MEK inhibitor PD98059 and PI3-kinase inhibitor LY294002. However, it was markedly suppressed by the tyrosine kinase inhibitor genistein (10 microM). Thus, externally added Li+ activates NHE1 and NHE2 via a mechanism possibly involving a tyrosine kinase, causing an increase in cytoplasmic pH that could potentially affect various cell functions.     

Sodium-bicarbonate cotransport current in identified leech glial cells.

1. The membrane current associated with the cotransport of Na+ and HCO3- was investigated in neuropil glial cells in isolated ganglia of the leech Hirudo medicinalis L. using the two-electrode voltage-clamp technique. 2. The addition of 5% CO2-24 mM HCO3- evoked an outward current, which slowly decayed, and which was dependent upon the presence of external Na+. Removal of CO2-HCO3- elicited a transient inward current. Re-addition of Na+ to Na(+)-free saline in the presence of CO2-HCO3- also produced an outward current. Under these conditions an intracellular alkalinization and a rise in intracellular [Na+] were recorded using triple-barrelled, ion-sensitive microelectrodes. Addition or removal of HCO3-, in the absence of external Na+, caused little or no change in membrane voltage, membrane current and intracellular pH, indicating that the glial membrane has a very low HCO3- conductance. 3. Voltage steps revealed nearly linear current-voltage relationships both in the absence and presence of CO2-HCO3-, with an intersection at the assumed reversal potential of the HCO(3-)-dependent current. These results suggest a cotransport stoichiometry of 2HCO3-: 1 Na+. The HCO(3-)-dependent current could be inhibited by diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). 4. Simultaneous recording of current and intracellular pH showed a correlation of the maximal acid-base flux with the transient HCO(3-)-dependent current during voltage steps in the presence of CO2-HCO3-. The maximum rate of acid-base flux and the HCO(3-)-dependent peak current showed a similar dependence on membrane voltage. Lowering the external pH from 7.4 to 7.0 produced an inward current, which increased twofold in the presence of CO2-HCO3-. This current was largely inhibited by DIDS, indicating outward-going electrogenic Na(+)-HCO3- cotransport during external acidification. 5. When external Na+ was replaced by Li+, a similar outward current and intracellular alkalinization were observed in the presence of CO2-HCO3-. The Li(+)-induced intracellular alkalinization was not inhibited by amiloride, a blocker of Na+(Li+)-H+ exchange, but was sensitive to DIDS. These results suggest that Li+ could, at least partly, substitute for Na+ at the cotransporter site. 6. Our results indicate that the Na(+)-HCO3- cotransport produces a current across the glial cell membrane in both directions with a reversal potential near the membrane resting potential, rendering pHi a function of the glial membrane potential.     

Alterations of 86Rb+ fluxes in poliovirus-infected HeLa cells and their dependence on virus replication

Components of the 86Rb+ influx were investigated subsequent to poliovirus infection in the presence and absence of guanidine-HCl, both under normal steady-state conditions and after Na+ preloading of the cells. Measurements of the ouabain-sensitive 86Rb+ uptake indicated a biphasic change in the activity of the Na+, K+ pump in the course of virus infection: a transient increase in the second hour postinfection, that was detectable only after Na+ preloading and inhibition after 3 hr. The enhanced activity of the Na+, K+ pump was not affected, while the decrease later was fully prevented by the antiviral agent guanidine-HCl. The piretanide-sensitive 86Rb+ uptake due to the Na+, K+, 2 Cl- cotransport system also became strongly inhibited beginning in the second hour postinfection. The inhibition of this transport system was partially antagonized by guanidine-HCl. The remaining 86Rb+ influx in the presence of ouabain and piretanide increased in the third hour postinfection. The latter change in 86Rb+ influx, indicating an increased permeability to monovalent cations was completely abolished by guanidine-HCl.     

Rubidium transport in human erythrocyte suspensions monitored by 87Rb NMR with aqueous chemical shift reagents

Dysprosium(III) triethylenetraamine-N,N,N',N",N"',N"'-hexaacetic acid (DyTTHA3-) was used as an aqueous chemical shift reagent in conjunction with high-resolution nuclear magnetic resonance (NMR) spectroscopy to monitor 87-rubidium (87Rb+) transport in human erythrocyte suspensions. NMR spectra demonstrated two resonances which were assigned to the intra- and extracellular compartments of the erythrocyte suspension. Uptake of 87Rb+ was shown to proceed via the [Na,K]-ATPase dependent pump as evidenced by the inhibition of uptake in the presence of ouabain. The steady state intra- to extracellular concentration ratio of 87Rb was 3.00 and 1.13 in the absence and presence of ouabain, respectively. The rate of uptake of 87Rb+ in the absence and presence of ouabain was found to be 1.3 and 0.5 mmol Rb+/L erythrocytes/h at 18 mM Rb+, respectively. Data are also presented which indicate that the intracellular component of 87Rb is less than 100% NMR visible.     

Glutamate release from microglia via glutamate transporter is enhanced by amyloid-beta peptide.

In the present study, we found that amyloid-beta peptide enhanced glutamate release from primary cultured rat microglia via the Na+-dependent glutamate transporter, which was activated by extracellular K+. Glutamate transport current was measured by a conventional whole-cell patch recording mode under voltage-clamp conditions. With the pipette solution containing 10 mM glutamate and 100 mM Na+, an increase of the external K+ concentration from 0 to 10 mM evoked an outward current, resulting from co-extrusion of glutamate and Na+. The inward current, reflecting forward glutamate transport, was also activated by external glutamate. Both these reverse and forward glutamate transport currents were three-fold greater in microglia incubated with a relatively low concentration of amyloid-beta peptide (25-35) (5 microM) for four days. The glutamate-activated inward current was blocked by D,L-threo-beta-hydroxyaspartate in a dose-dependent manner (ranging from 0.001 to 1 mM), but not by a high concentration of kainate (1 mM). The glutamate concentration released from microglia upon high-K+ stimulation was also significantly increased (up to 170 microM) after treatment with amyloid-beta peptide (25-35). These results suggest that, at the pathological sites where extracellular K+ concentration may increase, the activation of microglia by amyloid-beta peptide causes an increase in extracellular glutamate concentration via reverse glutamate transporter, and therefore this mechanism may contribute to the pathogenesis of neuronal dysfunction and death in Alzheimer's disease.     

Myocardial interstitial choline and glutamate levels during acute myocardial ischaemia and local ouabain administration

AIM: Noradrenaline (NA) uptake transporters are known to reverse their action during acute myocardial ischaemia and to contribute to ischaemia-induced myocardial interstitial NA release. By contrast, functional roles of choline and glutamate transporters during acute myocardial ischaemia remain to be investigated. Because both transporters are driven by the normal Na+ gradient across the plasma membrane in a similar manner to NA transporters, the loss of Na+ gradient would affect the transporter function, which would in turn alter myocardial interstitial choline and glutamate levels. The aim of the present study was to examine the effects of acute myocardial ischaemia and the inhibition of Na+,K+-ATPase on myocardial interstitial glutamate and choline levels. METHODS: In anaesthetized cats, we measured myocardial interstitial glutamate and choline levels while inducing acute myocardial ischaemia or inhibiting Na+,K+-ATPase by local administration of ouabain. RESULTS: The choline level was not changed significantly by ischaemia (from 0.93 +/- 0.06 to 0.82 +/- 0.13 microm, mean +/- SE, n = 6) and was decreased slightly by ouabain (from 1.30 +/- 0.06 to 1.05 +/- 0.07 microm, P < 0.05, n = 6). The glutamate level was significantly increased from 9.5 +/- 1.9 to 34.7 +/- 6.1 microm by ischaemia (P < 0.01, n = 6) and from 8.9 +/- 1.0 to 15.9 +/- 2.3 microm by ouabain (P < 0.05, n = 6). Inhibition of glutamate transport by trans-L-pyrrolidine-2,4-dicarboxylate (t-PDC) suppressed ischaemia- and ouabain-induced glutamate release. CONCLUSION: Myocardial interstitial choline level was not increased by acute myocardial ischaemia or by Na+,K+-ATPase inhibition. By contrast, myocardial interstitial glutamate level was increased by both interventions. The glutamate transporter contributed to glutamate release via retrograde transport.     

Recovery of intracellular pH in cortical brain slices following anoxia studied by nuclear magnetic resonance spectroscopy: role of lactate removal, extracellular sodium and sodium/hydrogen exchange.

[31P]- and [1H]nuclear magnetic resonances recorded in an interleaved fashion were used in order to quantify high-energy phosphates, intracellular pH and lactate in cortical brain slices of the guinea-pig superfused in a CO2/HCO3(-)-buffered medium during and after anoxic insults. The volume-averaged intracellular pH and energy status of the preparation following anoxia were determined. In the presence of external Na+, intracellular pH normalized in 3 min and was significantly more alkaline from 10 to 12 min of recovery, but lactate remained elevated for 12 min of reoxygenation following anoxia. The amount of lactate removed was only 40% of the quantity of acid extruded showing operation of H+ neutralizing transmembrane mechanisms other than transport of lactic acid. Amiloride (1 or 2 mM) did not prevent the recovery of intracellular pH, but it blocked the "overshoot" of the alkalinization at 10-12 min of recovery. In a medium containing 70 mM K+, 60 mM Na+ and 0.1 mM Ca2+, the recovery of pH, but not lactate washout, was significantly delayed. Removal of external Na+ caused severe energetic failure, decreases both in oxygen uptake and in N-acetyl aspartate concentration, indicating loss of viable tissue. In Na(+)-free superfusion, lactic acidosis caused a more severe drop in intracellular pH than in the presence of Na+. Complexing of extracellular Ca2+ in the Na(+)-free medium inhibited the acidification by 0.38 pH units during anoxia which is as much as the acidification caused by lactate accumulation in the absence of Na+. In Na(+)-free medium intracellular pH recovered, however, from an anoxic level to a normoxic value in 6 min. Metabolic damage of the slice preparation induced by anoxia in the absence of Na+ was as profound in the presence as in the absence of Ca2+ showing that accumulation of Ca2+ is not the only reason for the damage. It is concluded that recovery of intracellular pH from lactic-acidosis can occur independently of energetic recovery and involves acid extrusion mechanism(s) that is(are) dependent on external Na+ and sensitive to high K+.     

Na-dependent Li-transport in primary nerve cell cultures

Primary cultures from chick embryonic brain were used to study the steady state distribution of lithium. The intra/extracellular Li+ ratio decreased by enhancing the external Na+ concentration. Ouabain did not influence this unequality. A phloretin-sensitive component was revealed in the Li uptake at low Na+ concentration. The findings might suggest the existence of a Na+-dependent Li+ countertransport system in these brain cell cultures.     

Axonal transport of Na+,K+-ATPase identified as a ouabain binding site in rat sciatic nerve

Na+,K+-ATPase levels were measured in different segments of rat sciatic nerves by in vitro binding of [3H]ouabain. Binding sites were found to accumulate on both sides of a ligature tied on the sciatic nerve, indicating an anterograde and retrograde axoplasmic transport of Na+,K+-ATPase. Accumulation of Na+,K+-ATPase at the ligature was time dependent and appeared to occur through fast axoplasmic transport mechanisms. This accumulation on both sides of the ligature was also visualized by autoradiographic studies in longitudinal section of sciatic nerves using [3H]ouabain.     

Ion movements in human red cells independent of the sodium pump

1. A study was made of the dependence on external Na of the movements of Na and K in human red cells. Special attention was given to ouabain-insensitive movements. The effect of internal Na on Na influx, and the influence of some sulphydryl inhibitors on ion movements and metabolism was also investigated.2. External Na stimulated ouabain-insensitive Na efflux and K influx. There was also a ouabain-insensitive component of Na influx that was raised on increasing the internal Na concentration. Exchange diffusion of Na appears to occur in the presence of ouabain and external K.3. Net transport of Na and K in the presence of ouabain was independent of external Na, as was also lactate production.4. Ethacrynic acid partially inhibited the Na pump; the Na-dependent components of Na efflux and K influx in the presence of ouabain were completely inhibited by ethacrynic acid. Both ouabain-sensitive and ouabain-insensitive adenosinetriphosphatase activities were inhibited by ethacrynic acid indicating a non-specific effect of this compound. Iodoacetamide decreased only the ouabain-insensitive ATPase activity.5. The results suggest that when the Na pump is blocked by ouabain, part of the residual ion movements can be attributed to exchange diffusion.     

Calcium-independent GABA release from striatal slices: the role of calcium channels

We have investigated the role of Ca2+ and Ca2+ channels in the modulation of GABA release. Brain slices prepared from rat striatum were preincubated with [3H]GABA, superfused with Krebs bicarbonate buffer, and exposed to electrical field stimulation (2 Hz for 3 min). Tritium efflux was measured as an index of GABA release. Both resting and evoked efflux were greatly accelerated by deleting Ca2+ from the medium and adding EGTA (1 mM). However, when the concentration of Mg2+ in the buffer was elevated to 10 mM, no effect of the Ca2(+)-deficiency was observed on resting release and its impact on evoked overflow was diminished. Moreover, addition of verapamil (10 microM), a Ca2+ channel blocking agent, reduced evoked overflow even in the absence of external Ca2+, while 4-aminopyridine (10 microM), a K+ channel inhibitor, enhanced GABA efflux in normal buffer but had no effect in the absence of Ca2+. Finally, we have shown previously that nipecotic acid, an inhibitor of high affinity GABA transport, increases GABA overflow in normal buffer, but blocks it in Ca2(+)-free buffer. Collectively, these results suggest that Ca2+ channels may play two roles in the regulation of depolarization-induced GABA release. Firstly, these channels permit a depolarization-induced influx of Ca2+ which then promotes GABA release. In addition, these channels influence GABA release through a mechanism that does not involve external Ca2+. Although the precise nature of this latter involvement is unclear, we propose that the Ca2+ channels serve to permit an influx of Na+, which in turn promotes Ca2(+)-independent release through an influence on the high affinity GABA transport system.