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⁺ 13 mM 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 for Na⁺ across the membrane (η_Na0 >η_Na1) the brush border microvilli accumulate transiently l-phenylalanine over the concentration in the incubation medium (overshoot phenomenon). 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.     

Calcium ions in the presence of exogenous phosphatidylserine interfere with GABA diffusion through the Deiters' neuron membrane

Diffusion of GABA through the plasma membrane of GABA-acceptive neurons might be a mechanism of importance for the termination of its synaptic action. In the present investigation we studied the effects of phosphatidylserine (PS) (10^–4–10^–3 M), Ca²⁺ 2 mM and PS+2 mM Ca²⁺ on such a process. The method involved the use of single microdissected Deiters' membranes which were put between two small microchambers in order to study the passage of GABA across the membrane. The results show that whereas PS and Ca²⁺ by themselves have no effect on such a process, PS+2 mM CaCl₂ give a significant, although slight, inhibition. The hypothesis that Calcium ion + PS effect is due to a disturbance of the interaction between GABA and endogenous PS molecules of the membrane is discussed.     

Resistance of the intact and reconstituted adipocyte hexose transport system to irreversible inhibition by sulfhydryl and amino reagents

Sensitivity of the adipocyte D-glucose transport system in intact plasma membranes or following solubilization and reconstitution into phospholipid vesicles to several protein-modifying reagents was investigated. When intact plasma membranes were incubated with N-ethylmaleimide (20 mM) or fluorodinitrobenzene (4 mM), D-glucose transport activity was virtually abolished. However, washing the membranes free of unreacted reagents restored transport activity, indicating that covalent interaction with the membranes did not mediate the transport inhibition. Reaction of [³H] N-ethylmaleimide with plasma membranes under similar conditions resulted in extensive labeling of all protein fractions resolved on dodecyl sulfate gels. Similarly, addition of N-ethyl-maleimide to cholate-solubilized membrane protein had no effect on transport activity in artifical phospholipid vesicles reconstituted under conditions where the membrane protein was free of unreacted N-ethylmaleimide. Transport activity in plasma membranes was also inhibited by both reduced and oxidized dithiothreitol or glutathione (15 mM) in a readily reversible manner, consistent with a noncovalent mode of inhibition. Thus, the insulin-responsive adipocyte D-glucose transport system differs from the red cell hexose transport system in its remarkable insensitivity to modulation by covalent blockade of sulfhydryal or amino groups by the reagents studied.     

Mode of transport and possible mechanism of action of l-phenylalanine benzyl ester as an anti-sickling agent

l-Phenylalanine benzyl ester (Phe-Bz) and a number of ester analogues prevent sickling of erythrocytes from sickle cell disease patients. The compounds tested exhibit anti-sickling activity in the concentration range 0.5–3.0 mM. A general feature of these compounds is the presence of two aromatic rings in their molecular structure. The anti-sickling agents rapidly enter the erythrocyte and are hydrolysed to their component molecules. Incubation of human erythrocytes with 3.0 mM l-phenylalanine for 30 min at 37°C results in accumulation of 2.0 mmol l-phenyalanine/l cells, while incubation of erythrocytes with 3.0 mM Phe-Bz under similar conditions results in the production of 4.0 mmol l-phenylalanine/l cells and an equivalent amount of benzyl alcohol. Both l-phenylalanine and benzyl alcohol are inhibitors of the gelation of deoxyhaemoglobin S (deoxy-HbS) in vitro. Moreover, Phe-Bz and related anti-sickling agents fluidize the lipid bilayer of the erythrocyte membrane, inhibiting several transport systems, including those for l-phenylalanine, uridine and sulphate ions, as well as the Na⁺ pump and the Na⁺/K⁺ cotransporter, but increasing the passive influx and efflux of both cations and anions. The accumulation of Phe-Bz hydrolysis products within the erythrocyte together with the effects of Phe-Bz on cation permeability result in the influx of water causing the cell to swell. Thus, treatment of erythrocytes with 3.0 mM Phe-Bz at 37°C for 30 min causes an increase in mean cell volume of 14.8%, decreasing the mean intracellular haemoglobin concentration from 34 to 29.6 g%. The increase in cell volume caused by Phe-Bz and its analogues together with the direct effects of their hydrolysis products on HbS probably act in concert to bring about the anti-sickling effect.     

Effect of furosemide on GABA<Subscript>A</Subscript>-induced <Superscript>36</Superscript>Cl transport and Cl--ATPase activity in synaptic membranes of carp brain (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.)

We studied the effect of furosemide on GABA_A-induced ³⁶Cl transport and GABA_A-induced Cl^--ATPase activity in synaptic membranes of fish brain. At physiological pH 7.4, GABA (0.1–100 µM) stimulated ³⁶Cl influx in synaptoneurosomes and Cl^--ATPase activity in synaptic membranes. Furosemide (0.1–0.5 mM) removed the activating effect of the mediator on chloride transport and enzyme activity (I₅₀ equaled 0.16 and 0.12 mM, respectively). In the absence of the mediator, picrotoxin (50 µM) activated the basal ³⁶Cl influx in synaptoneurosomes and the basal Mg²⁺-ATPase activity of synaptic membranes. Furosemide (1 mM) removed the activating effect of picrotoxin on both biochemical processes. The obtained data demonstrated similar sensitivities of GABA_A-induced transport of ³⁶Cl in synaptoneurosomes and of GABA_A-induced Cl^--ATPase activity in the synaptic membranes to furosemide and indicated the involvement of the ATPase in GABA_A-induced processes. The soluble ATPase, recovered by sodium deoxycholate solubilization of the membranes, remained sensitive to GABA_A-ergic ligands, which suggested proximity of their binding sites with ATP hydrolysis sites in the protein molecule and their structural coupling.Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 1, 2005, pp. 18–22.Original Russian Text Copyright © 2005 by Menzikov, Menzikova.     

ATP-dependent Cl- uptake by plasma membrane vesicles from the rat brain

Uptake of Cl- by plasma membrane vesicles from the rat brain was stimulated by ATP at 37 degrees C, but not by beta, gamma-methylene ATP or at 0 degrees C. The addition of Triton X-100 or sucrose to the incubation medium diminished the ATP-stimulated Cl- uptake, suggesting that Cl- was transported across the membranes into the intravesicular space. This ATP-stimulated Cl- uptake was not affected by 1 mM ouabain. 1 microM oligomycin, 0.1 mM gamma-aminobutyric acid or 0.1 mM picrotoxin. Thus, non-mitochondrial ATP-driven Cl- transport through a system other than Na, K-ATPase or Cl- channels occurs in neuronal plasma membrane vesicles.     

Renal palmitate transport: possible sites for interaction with a plasma membrane fatty acid-binding protein

Summary In previous studies from this laboratory [14], a mediated transport system for long chain fatty acids was observed in rat renal basolateral membrane vesicles. Transport was measured in the absence of albumin and indicated the presence of a Na⁺ independent anion exchange mechanism. The present experiments were done to characterize renal transport of fatty acids derived from fatty acid-albumin complexes. ³H-palmitate uptake by brush border (BBMV) and basolateral membrane vesicles (BLMV) isolated from rat renal cortex was determined using a rapid filtration technique. All incubation media contained 100 µM ³H-palmitate complexed to 100 µM bovine serum albumin. Up to 65% of initially bound fatty acid-albumin complexes were displaceable by washing with solution containing 0.1% albumin. Total palmitate uptake was measured as the remaining non-displaceable radioactivity. In BBMV in low ionic strength (300 mM mannitol) or ionic buffers (100 mM mannitol + 100 mM NaCl or KCl), total palmitate uptake at 15 sec did not differ from equilibrium (60 min) values of 10–11 nmoles/mg protein. Uptake was primarily due to binding. A similar pattern was seen with BLMV in 300 mM mannitol buffer: In BLMV in 100 mM NaCl or KCl buffers, equilibrium uptake was 10-fold lower than at 15 sec. This suggests binding followed by cation-dependent translocation. If a putative FABP_PM is involved in transport only, its presence should be confined to BLMV.     

Kinetics of 3-O-methyl-D-glucose transport in isolated rat hepatocytes.

3-O-Methyl-D-glucose transport across the plasma membrane of isolated rat hepatocytes was followed for net entry of the sugar into sugar-free cells (zero trans entry), net exit of sugar into sugar-free medium (zero trans exit) and for unidirectional entry and exit fluxes when cells had been equilibrated with sugar in the extracellular medium (equilibrium exchange entry and exit). These measurements were performed at 20 degrees C and pH 7.4 by the use of simple manual methods. Initial rates of transport showed a Michaelis--Menten dependency on the sugar concentration at the cis side of the membrane over the range of concentrations tested (100 microM to 100 mM). Transport was found to be symmetrical with no evidence of substrate stimulation of transport from the trans side of the membrane. Parameters (mean values +/- S.E.M.) of transport were estimated as Vmax. 86.2 +/- 9.7 mmol/litre of cell water per min and Km 18.1 +/- 5.9 mM for exchange entry, Vmax. 78.8 +/- 5.3 mmol/litre of cell water per min and Km 17.6 +/- 3.5 mM for exchange exit, Vmax. 84.1 +/- 8.4 mmol/litre of cell water per min and Km 16.8 +/- 4.6 mM for zero trans exit.     

Differential accumulation of Glut1 in the non-DRM domain of the plasma membrane in response to the inhibition of oxidative phosphorylation

Approximately 50% of Glut1 in the plasma membrane of Clone 9 cells is localized to the detergent-resistant membrane (DRM) fraction. Acute exposure (90 min) to 5mM azide stimulated glucose transport by approximately 4.7-fold and increased the abundance of Glut1 in the non-DRM fraction of the plasma membrane by approximately 2.9-fold while the abundance of Glut1 in the DRMs was not changed. In parallel experiments, approximately 17 h exposure to azide further increased the rate of glucose transport over that observed at 90 min by approximately 33% and increased plasma membrane Glut1 content by approximately 3.5-fold over control. The increase in total plasma membrane Glut1 reflected a approximately 4.7-fold increase of Glut1 content in the non-DRM fraction and a approximately 2.6-fold increase in the DRMs. We conclude that acute exposure to azide increases Glut1 content in the non-DRM fractions, while prolonged exposure to azide increases the Glut1 content in both non-DRM and DRM fractions. These changes may play an important role in the stimulation of glucose transport in response to the inhibition of oxidative phosphorylation.     

Differences in L-alanine uptake by livers of Wistar and lean Zucker rats

The L-alanine uptake by livers of Wistar and lean Zucker rats has been studied. The hepatic uptake and fractional extraction rates of alanine were estimated in 50–55 day old rats. No significant differences in amino acid concentrations and blood flows in afferent and efferent liver vessels were seen in lean Zucker rats when compared with Wistar rats. However, the hepatic uptake (1.6±0.1 and 0.7±0.1 mol/min/100 g bw, p<0.01) and the fractional extraction (26.8±2.1 and 15.2±3.1%, p<0.05) were much lower in Zucker than in Wistar rats. The hepatic active transport of L-alanine was determinedin vitro using isolated plasma membrane vesicles. Vesicles isolated from livers of lean Zucker rats showed similar values of Km (2.5±0.7 vs 2.0±0.5 mM for Wistar and Zucker respectively, N.S.), but lower values of Vmax when compared with Wistar rats (1.1±0.1 vs 0.6±0.005 nmol/mg prot 5 s, p<0.01, for Wistar and lean Zucker rats respectively). These results indicate that, the liver of lean Zucker rats concentrates alanine less efficiently than the liver of Wistar rats. This fact correlates well with a lower capacity of the Na⁺-dependent L-alanine trasport in liver plasma membrane vesicles from lean Zucker rats.     

Mechanism of mitochondrial transport of thallous ions

Rat liver mitochondria were found to swell under nonenergized conditions when suspended in media containing 30–40 mM TINO₃. Respiration on succinate caused a rapid contraction of mitochondria swollen under nonenergized conditions. In the presence of thallous acetate, there was a rapid initial swelling under nonenergized conditions until a plateau was reached; respiration on succinate then caused a further swelling. Trace amounts of²⁰⁴Tl (less than 100 µM) equilibrated fairly rapidly across the mitochondrial membrane. The influx of Tl⁺ was able to promote the decay not only of a valinomycin-induced K⁺-diffusion potential but also of respiration-generated fields in the inner membrane in accordance with the electrophoretic nature of Tl⁺ movement. Efflux of Tl⁺ showed a half-time of about 10 sec at 20°C and was not affected appreciably by the energy state. Efflux was retarded by Mg²⁺ and by lowering the temperature. The data indicate that Tl⁺ when present at high concentrations, 30 mM or more, distributes across the mitochondrial inner membrane both in response to electrical fields and to pH. In energized mitochondria the uptake of Tl⁺ would occur electrophoretically, while Tl⁺/H⁺ exchange would constitute a leak. In the presence of NO ₃ ^– , the movements of Tl⁺ are determined by that of NO ₃ ^– , indicating short-range coupling of electrical forces. At low concentrations of Tl⁺, 5 mM or less, there was no indication of a Tl⁺/H⁺ exchange, which appears to be induced by high concentrations of Tl⁺.     

Effects of semen preservation on boar spermatozoa head membranes

Head plasma membranes were isolated from the sperm-rich fraction of boar semen and from sperm-rich semen that had been subjected to three commercial preservation processes: Ex tended for fresh insemination (extended), prepared for freezing but not frozen (cooled), and stored frozen for 3-5 weeks (frozen-thawed). Fluorescence polarization was used to determine fluidity of the membranes of all samples for 160 min at 25°C and also for membranes from the sperm-rich and extended semen during cooling and reheating (25 to 5 to 40°C, 0.4°C/min). Head plasma membranes from extended semen were initially more fluid than from other sources (P < 0.05). Fluidity of head membranes from all sources decreased at 25°C, but the rate of decrease was significantly lower for membranes from cooled and lower again for membranes from frozen-thawed semen. Cooling to 5°C reduced the rate of fluidity change for plasma membranes from the spernvrich fraction, while heating over 30°C caused a signifi cantly greater decrease. The presence of Ca⁺⁺ (10 mM) lowered the fluidity of the head plasma membranes from sperm-rich and extended semen over time at 25°C but did not affect the membranes from the cooled or frozen-thawed semen. The change in head plasma membrane fluidity at 25°C may reflect the dynamic nature of spermatozoa membranes prior to fertilization. Extenders, preservation processes and temperature changes have a strong influence on head plasma membrane fluidity and therefore the molecular organization of this membrane.     

Mode of transport and possible mechanism of action of l-phenylalanine benzyl ester as an anti-sickling agent

l-Phenylalanine benzyl ester (Phe-Bz) and a number of ester analogues prevent sickling of erythrocytes from sickle cell disease patients. The compounds tested exhibit anti-sickling activity in the concentration range 0.5–3.0 mM. A general feature of these compounds is the presence of two aromatic rings in their molecular structure. The anti-sickling agents rapidly enter the erythrocyte and are hydrolysed to their component molecules. Incubation of human erythrocytes with 3.0 mM l-phenylalanine for 30 min at 37°C results in accumulation of 2.0 mmol l-phenyalanine/l cells, while incubation of erythrocytes with 3.0 mM Phe-Bz under similar conditions results in the production of 4.0 mmol l-phenylalanine/l cells and an equivalent amount of benzyl alcohol. Both l-phenylalanine and benzyl alcohol are inhibitors of the gelation of deoxyhaemoglobin S (deoxy-HbS) in vitro. Moreover, Phe-Bz and related anti-sickling agents fluidize the lipid bilayer of the erythrocyte membrane, inhibiting several transport systems, including those for l-phenylalanine, uridine and sulphate ions, as well as the Na⁺ pump and the Na⁺/K⁺ cotransporter, but increasing the passive influx and efflux of both cations and anions. The accumulation of Phe-Bz hydrolysis products within the erythrocyte together with the effects of Phe-Bz on cation permeability result in the influx of water causing the cell to swell. Thus, treatment of erythrocytes with 3.0 mM Phe-Bz at 37°C for 30 min causes an increase in mean cell volume of 14.8%, decreasing the mean intracellular haemoglobin concentration from 34 to 29.6 g%. The increase in cell volume caused by Phe-Bz and its analogues together with the direct effects of their hydrolysis products on HbS probably act in concert to bring about the anti-sickling effect.     

Isolation of a cDNA from Saccharomyces cerevisiae that encodes a high affinity sulphate transporter at the plasma membrane

Resistance to selenate and chromate, toxic analogues of sulphate, was used to isolate a mutant of Saccharomyces cerevisiae deficient in the capacity to transport sulphate into the cells. A clone which complements this mutation was isolated from a cDNA library prepared from S. cerevisiae poly(A)⁺ RNA. This clone contains an insert which is 2775 by in length and has a single open reading frame that encodes a 859 amino acid polypeptide with a molecular mass of 96 kDa. Sequence motifs within the deduced amino acid sequence of this cDNA (SUL1) show homology with conserved areas of sulphate transport proteins from other organisms. Sequence analysis predicts the position of 12 putative membrane spanning domains in SUL1. When the cDNA for SUL1 was expressed in S. cerevisiae, a high affinity sulphate uptake activity (K_m = 7.5 ± 0.6 M for SO ₄ ^2– ) was observed. A genomic mutant of S. cerevisiae in which 1096 by were deleted from the SUL1 coding region was constructed. This mutant was unable to grow on media containing less than 5 mM sulphate unless complemented with a plasmid containing the SUL1 cDNA. We conclude that the SUL1 cDNA encodes a S. cerevisiae high affinity sulphate transporter that is responsible for the transfer of sulphate across the plasma membrane from the external medium.     

Altered coupling states between calcium transport and (Ca2+, Mg2+)-ATPase in the AS-30D Ascites hepatocarcinoma plasma membrane

Plasma membrane fractions from normal, regenerating liver and the AS-30D ascites hepatocarcinoma exhibited a high degree of enrichment when a set of plasma membrane enzyme markers were studied in comparison to the ones associated to the mitochondrial and cytosolic compartments. While the (Ca²⁺, Mg²⁺)-ATPase observed for the plasma membrane fraction isolated from normal liver showed an activity of 1.2 µmoles/mg/min, the regenerating liver and the AS-30D plasma membrane fractions presented a much lower ATPase activity (0.3 and 0.22 µmoles/mg/min respectively). Despite the differences in ATPase activity observed between models, the plasma membrane fraction from the AS-30D hepatocarcinoma presented a calcium transport activity similar to the value observed for the normal system (5.9 and 5.5 nmoles Ca²⁺/mg/10min, respectively). Interestingly, the ATP Pi exchange experiments carried out with the different plasma membrane fractions revealed that the (Ca²⁺, Mg²⁺)-ATPase contained in the plasma membrane from the AS-30D cells shows an exchange activity of 26 nmoles ATP Pi/mg/min, similar to the one observed for the enzyme from normal liver (30 nmoles ATP Pi/mg/min). Our results suggest that the plasma membrane from the transformed model presents a more efficient mechanism to regulate the movement of calcium through the calcium pump, with an optimum expenditure of energy.Dedicated to the memory of Catalina Mas Oliva and Valentín Mas Morera.     

Inhibition of Mrp2- and Ycf1p-mediated transport by reducing agents: evidence for GSH transport on rat Mrp2

Mammalian Mrp2 and its yeast orthologue, Ycf1p, mediate the ATP-dependent cellular export of a variety of organic anions. Ycf1p also appears to transport the endogenous tripeptide glutathione (GSH), whereas no ATP-dependent GSH transport has been detected in Mrp2-containing mammalian plasma membrane vesicles. Because GSH uptake measurements in isolated membrane vesicles are normally carried out in the presence of 5-10 mM dithiothreitol (DTT) to maintain the tripeptide in the reduced form, the present study examined the effects of DTT and other sulfhydryl-reducing agents on Ycf1p- and Mrp2-mediated transport activity. Uptake of S-dinitrophenyl glutathione (DNP-SG), a prototypic substrate of both proteins, was measured in Ycf1p-containing Saccharomyces cerevisiae vacuolar membrane vesicles and in Mrp2-containing rat liver canalicular plasma membrane vesicles. Uptake was inhibited in both vesicle systems in a concentration-dependent manner by DTT, dithioerythritol, and β-mercaptoethanol, with concentrations of 10 mM inhibiting by ∼40%. DTT’s inhibition of DNP-SG transport was noncompetitive. In contrast, ATP-dependent transport of [³H]taurocholate, a substrate for yeast Bat1p and mammalian Bsep bile acid transporters, was not significantly affected by DTT. DTT also inhibited the ATP-dependent uptake of GSH by Ycf1p. As the DTT concentration in incubation solutions containing rat liver canalicular plasma membrane vesicles was gradually decreased, ATP-dependent GSH transport was now detected. These results demonstrate that Ycf1p and Mrp2 are inhibited by concentrations of reducing agents that are normally employed in studies of GSH transport. When this inhibition was partially relieved, ATP-dependent GSH transport was detected in rat liver canalicular plasma membranes, indicating that both Mrp2 and Ycf1p are able to transport GSH by an ATP-dependent mechanism.     

Endothelial Cell Swelling by Aldosterone

There is accumulating evidence that mineralocorticoids not only act on kidney but also on the cardiovascular system. We investigated the response of human umbilical venous endothelial cells (HUVECs) to aldosterone at a time scale of 20 minutes in absence and presence of the aldosterone antagonist spironolactone or other transport inhibitors. We applied atomic force microscopy (AFM), which measures cell volume and volume shifts between cytosol and cell nucleus. We observed an immediate cell volume increase (about 10%) approximately 1 min after addition of aldosterone (0.1 µmol/l), approaching a maximum (about 18%) 10 min after aldosterone treatment. Cell volume returned to normal 20 min after hormone exposure. Spironolactone (1 µmol/l) or amiloride (1 µmol/l) prevented the late aldosterone-induced volume changes but not the immediate change observed 1 min after hormone exposure. AFM revealed nuclear swelling 5 min after aldosterone addition, followed by nuclear shrinkage 15 min later. The Na⁺/H⁺ exchange blocker cariporide (10 µmol/l) was ineffective. We conclude: (i) Aldosterone induces immediate (1 min) swelling independently of plasma membrane Na⁺ channels and intracellular mineralocorticoid receptors followed by late mineralocorticoid receptor- and Na⁺-channel-dependent swelling. (ii) Intracellular macromolecule shifts cause the changes in cell volume. (iii) Both amiloride and spironolactone may be useful for medical applications to prevent aldosterone-induced vasculopathies.     

Effects of membrane potential on calcium fluxes of Pelvetia eggs

⁴⁵Ca²⁺ fluxes across the plasma membrane of zygotes of the fucoid alga, Pelvetia fastagiata (J. Ag.) De Toni, were studied in artificial sea waters of various potassium concentrations. Except for two cases, hyperpolarization of the cell membrane (with low [K⁺]) increases, and depolarization (with high [K⁺]) decreases the influx of Ca²⁺ over the range of [K⁺] studied (1–100 mM). The fractional increases of influx during hyperpolarization are close to the fractional increases in membrane potential but the decreases during depolarization are much smaller than those in membrane potential. In two anomalous cases, the influxes of ⁴⁵Ca²⁺ at a potassium concentration of 30 mM were about 20% higher than the control value instead of being 10% lower.The effluxes of ⁴⁵Ca²⁺ are increased by both hyperpolarization and by depolarization. On balance (and excepting the two anomalous cases) the net result of hyperpolarization should be to increase and that of depolarization to decrease intracellular [Ca²⁺].     

Na-dependent transport of tricarboxylic acid cycle intermediates by renal brush border membranes. Effects on fluorescence of a potential-sensitive cyanine dye

The effect of the transport of tricarboxylic acid cycle intermediates on the membrane potential of renal brush border vesicles was studied using fluorescence of the cyanine dye, 3,3′-dipropylthiadicarbocyanine iodide. The behavior of the dye in the preparation was established with valinomycin-induced K⁺-diffusion potentials; increases in fluorescence were associated with depolarizing conditions. Addition of 1 mM succinate or citrate to membrane/dye suspensions produced transient increases in fluorescence, indicative of a depolarizing event(s) associated with the transport of these substrates. The transient response in fluorescence was Na⁺ dependent, of greater magnitude under Na⁺-gradient as compared to Na⁺-equilibrium conditions, and was a saturable function of substrate concentration. The specificity of the fluorescence response was identical to that obtained from studies of the competitive inhibition of succinate transport by tricarboxylic acid cycle intermediates and analogs. We conclude that the major tricarboxylic acid cycle intermediates are transported via a common Na⁺-dependent transport system in renal brush border membranes.     

“Intracellular” GABA affects the equilibrium distribution of Cl− across the plasma membrane of a GABA acceptive neuron

The permeability of Cl^– ions through single microdissected plasma membranes from Deiters' neurons was studied by a microtechnique. In particular, the time course of the passage of³⁶Cl^– ions from a microchamber, M₁, to another one, M₂, across the membrane was followed. This study was performed with or without -amino-butyric acid (GABA) in the two microchambers. The results suggest that in basal conditions the high intracellular concentration normally present in these neurons, 3.3 mM (1), causes a higher permeability of Cl^– in the direction inside outside in the respect of the plasma membrane. Extracellular GABA, 0.1 mM, is able to abolish this imbalance in Cl^– permeability in the two opposite directions. This event appears to be the basis for GABA induced hyperpolarization of these neurons.