Transport of l-leucine hydroxy analogue and l-lactate in rabbit small-intestinal brush-border membrane vesicles

Substitution of the -amino group of amino acids by hydroxyl groups yields hydroxy analogues (HA), which have been ascribed beneficial effects in nitrogensparing diets for uremic patients. In this study, intestinal uptake of l-leucine HA (l-LeuHA) and l-lactate into rabbit jejunal brush-border membrane vesicles was investigated. An inward-directed H⁺ or Na⁺ gradient stimulated uptake of both labelled substrates in a voltageclamped assay. The H⁺ gradient was the major driving force of uptake as compared with the Na⁺ gradient, and it led to a transient accumulation of both l-LeuHA and l-lactate. The proton ionophore carbonylcyanide p trifluoromethoxyphenylhydrazone (FCCP) reduced the initial H⁺-gradient-driven uptake rates of both substrates, but was without effect on Na⁺-gradient-driven uptakes. The H⁺-gradient-driven l-LeuHA uptake was saturable (apparent K_t = 15.4 mM). -HA of l-leucine, l-isoleucine, l-valine, d-leucine, d-valine or l-lactate inhibited the H⁺-gradient-driven l-LeuHA or l-lactate uptakes whereas free branched-chain amino acids had no effect. Preloading the vesicles with one of the l-or d-HA of branched-chain amino acids or with l-lactate stimulated tracer l-LeuHA and also tracer l-lactate uptakes in the presence of a H⁺ gradient. It is concluded that H⁺-gradient-driven transport of l- and d-stereoisomeric HA of branched-chain amino acids as well as of l-lactate across rabbit intestinal brush-border membranes is mediated by the same carrier. Furthermore, there exists a Na⁺gradient-driven l-lactate transport system in the rabbit intestinal brush-border membrane.     

Uphill transport of pyrimidine nucleosides in renal brush border vesicles

The uptake of cytidine, of thymidine and of uridine in brush border vesicles prepared from the cortex of rat kidney has been studied by the technique of rapid filtration. The nucleosides were not metabolized in the vesicles. The time-courses of uptake in the presence of inwardly directed gradients of Na⁺ and of K⁺ showed an overshoot, indicating uphill transport. The overshoot was much more pronounced with Na⁺ than with K⁺; it was not observed when Na⁺ was at equilibrium across the membrane. The uptake of the nucleosides was stimulated by an inside negative potential in the presence of Na⁺. These results provide evidence for a cotransport of pyrimidine nucleosides with Na⁺. The apparentK _m's for the uptake of cytidine, of thymidine and of uridine were 3.76 mol · l^–1, 4.18 mol · l^–1 and 7.21 mol · l^–1 respectively. The uptake of the pyrimidine nucleosides was insensitive to 6-nitrobenzylthioinosine. This insensitivity as well as the high affinity for the nucleosides and the capacity for uphill transport indicate that the nucleoside carrier(s) is renal brush border is (are) different from the carriers found in most other cell types.Abbreviations HEPES N-2-hydroxymethylpiperazine-N-2-ethanesulfonic acid - MES 2-(N-morpholino)ethanesulfonic acid - NBMPR 6-nitrobenzylthioinosine - Tris tris(hydroxymethyl)-aminomethane     

Increased sodium-dependent D-glucose transport in the jejunal brush-border membrane of spontaneously hypertensive rat

The current studies explore the effect of hypertension on D-glucose transport into jejunal brush-border membrane vesicles (BBMV). Spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats, as a control group, were used. The purity of the BBMV from both groups of animals was validated by the finding that the specific activity of brush-border enzyme marker, sucrase, was severalfold greater in membrane vesicles compared with corresponding values in mucosal homogenate. D-glucose uptake was Na⁺ dependent in both groups of animals, with a transient increase in the intravesicular concentration of D-glucose. However, the initial rate and the magnitude of the accumulation of Na⁺-dependent D-glucose was significantly higher in SHR compared with WKY rats. In order to investigate the mechanism(s) for the increase in Na⁺-dependent D-glucose transport in SHR, several experiments were performed: (1) an experiment that indicated ²²Na uptake, as an indicator for Na⁺ permeability, was similar between SHR and WKY rats, (2) kinetic studies that indicated that V _max values of SHR were significantly greater that those of WKY rats. In contrast, similar K _m values for glucose were found between SHR and WKY rats, (3) Na⁺-dependent phlorizin binding measurements that were not altered by hypertension and (4) a study of the brush-border membrane lipid composition that showed a significant increase in the free cholesterol/phospholipid ratio in SHR. We conclude that altered membrane cholesterol content and consequently altered lipid fluidity could be, at least in part, responsible for the observed increase in Na⁺-dependent D-glucose transport in SHR. Received: 27 October 1995/Received after revision and accepted: 23 January 1996     

Asymmetry in the transport of lactate by basolateral and brush border membranes of rat kidney cortex

The uptake ofl(+)lactate into rat renal cortical brush border (BBV) and basolateral (BLV) membrane vesicles, isolated through differential centrifugation and free flow electrophoresis, were studied using a rapid filtration technique. In contrast to the lactate transport into the BBV, that into the BLV: 1) was found to proceed only towards equilibrium, 2) showed Na⁺-independent coupling of the influx ofl(+)lactate and the efflux ofl(+) but not to the efflux ofd(–)lactate, 3) was not inhibited byd(–)lactate, 2-thiolactate or 3-phenyl-lactate, but 4) was inhibited by 3-thiolactate and -hydroxybutyrate and 5) was accelerated by changes in inwardly directed ionic gradients or by increases in cation conductance both of which led to increased intravesicular positivity. The latter changes had the opposite effect on the uptake ofl(+)lactate by BBV. Thus, while thel(+)lactate transport system present in BBV showed the characteristics of Na-dependent electrogenic cotransport system, that in the BLV was consistent with a carrier mediated Na-independent, facilitated diffusion system.     

Na+ and pH dependence of proline and beta-alanine absorption in rat small intestine

Aims: Early characterization of intestinal absorption of imino acids in mammals has demonstrated the existence of a Na⁺‐dependent, Cl^?‐independent transport system in rat small intestine, which is the only carrier for β‐alanine. Based on the substrate selectivity, it was proposed that the Proton Amino Acid Transporter 1 (PAT1) could be the same as this imino acid carrier. The present study characterizes the pH and Na⁺ dependence of proline and β‐alanine uptake in rat small intestine. Methods: Intestinal uptake of radiolabelled l ‐proline or β‐alanine was measured in brush border membrane vesicles and everted intestinal rings, in the presence and absence of Na⁺ and at different pH values. Results: The existence of an inwardly directed H⁺ gradient in the absence of Na⁺ enhanced the initial entry of proline and β‐alanine in brush border membrane vesicles, that reached a transient overshoot with maximal value around 30 s. In the absence of pH gradient, no overshoot was shown. In entire tissue, there was an increase of proline and β‐alanine uptake at acidic pH that was higher in the presence of Na⁺ than in its absence. This ion dependence and pH effect of the amino acids uptake also increased with the incubation period. Substrate inhibition studies confirmed that intestinal proline absorption in rat occurs mainly by system B and PAT1‐like transporter. Conclusions: There is a Na⁺‐independent, H⁺‐dependent transporter of amino acids at the apical membrane of the rat enterocytes.     

Intravesicular NAD has no effect on sodium-dependent phosphate transport in isolated renal brush border membrane vesicles

The effects of intravesicular NAD on Na⁺-dependent³²P_i uptake were investigated in isolated rat kidney brush border membrane vesicles (BBMV). NAD was introduced into the vesicles by osmotic shock, and extravesicular NAD was removed by passing the vesicles through a anion exchange column. The effectiveness of the osmotic shock procedure and the hydrolysis of extra- and intravesicular NAD were controlled by enzymatic analysis and thin layer chromatography. ADP-ribosylation of the membrane proteins was analyzed in vesicles osmotically shocked in the presence of either [adenylate-³²P]-NAD or [adenine-2,8-³H]-NAD by SDS-polyacrylamide gel electrophoresis.It was found that the Na⁺-dependent P_i uptake was inhibited when the BBMV were incubated with NAD at alkaline pH, which resulted in rapid NAD hydrolysis. When NAD was present in the intravesicular space only, the Na⁺-dependent P_i uptake was not inhibited.³²P from NAD was rapidly incorporated into a number of brush border membrane proteins, but no incorporation of³H-adenine could be detected.The results provide evidence that NAD does not inhibit P_i transport by a direct interaction with the cytoplasmic side of the brush border membrane. No evidence of ADP-ribosylation of the brush border membrane protein(s) was found.     

The stimulus-secretion coupling of amino acid-induced insulin release

l-Glutamine enhances insulin release evoked byl-leucine in isolated rat pancreatic islets. The enhancing action ofl-glutamine, which is a rapid but steadily increasing and not rapidly reversible phenomenon, is not attributable to any major change in either K⁺ or Ca²⁺ outflow from the islet cells. It coincides with an apparent increase in Ca²⁺ inflow rate and, hence, with Ca accumulation in the islets. The initial ionic response tol-leucine is not qualitatively altered by the presence ofl-glutamine. In their combined capacity to stimulate⁴⁵Ca net uptake in the islets,l-glutamine can be replaced byl-asparagine but not byl-glutamate, whereasl-leucine can be replaced byl-norvaline orl-isoleucine, but not byl-valine, glycine orl-lysine. Such a specificity is identical to that characterizing the effect of these various amino acids upon insulin release. It is postulated that the release of insulin evoked by the combination ofl-leucine andl-glutamine involves essentially the same remodelling of ionic fluxes as that evoked by other nutrient secretagogues with, however, an unusual time course for the functional response tol-glutamine.     

Effects of semistarvation on transintestinald-glucose transport andd-glucose uptake in brush border and basolateral membranes of rat enterocytes

The present work shows that semistarvation (8–10 g of food for 10 days) increases netd-glucose, Na⁺ and water transport in the everted and perfused rat jejunum. A lincar and positive correlation between cell sugar concentration and transport was found in control and semistarved rats, but the phenomenon was more relevant only in semistarved animals. Membrane vesicle experiments showed that semistarvation increases sugar overshoot only in brush border membrane vesicles, while this situation does not occur in basolateral membrane vesicles. The effect of partial food deprivation seems to enhance net sugar transport by mereasing sugar entry across the apical membrane of enterocytes.     

Polar distribution of sodium-dependent and sodium-independent transport system forl-lactate in the plasma membrane of rat enterocytes

The uptake ofl-lactate by rat small intestinal brush-border and basal-lateral plasma membrane vesicles has been studied.l-Lactate uptake by the isolated membrane vesicles is osmotically sensitive and represents predominantly transport into an intravesicular space and not binding to the membranes.The transport ofl-lactate across the brush-border membrane is stimulated by sodium, whereas the transport across the basal-lateral plasma membrane is sodium-independent. In both types of membrane vesiclesl-lactate is transported faster thand-lactate andl-lactate transport is inhibited by -cyano-cinnamic acid.l-Lactate transport across basal-lateral membranes is inhibited byd-lactate and pyruvate and transstimulated byl-lactate and pyruvate.The polar distribution of transport system forl-lactate in the plasma membrane of rat enterocytes—a Na⁺/l-lactate cotransport system in the brush-border membrane and a facilitated diffusion system in the basal-lateral membrane — can explain the fact that in the intact epitheliuml-lactate produced by cell metabolism is preferentially released on the serosal side and could enable the cell to perform vectorial, secondary active transport ofl-lactate from the intestinal lumen to the serosal compartment.     

Defective jejunal and colonic salt absorption and alteredNa+/H+ exchanger 3 (NHE3) activity in NHE regulatory factor 1 (NHERF1) adaptor protein-deficient mice

We investigated the role of the Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) on intestinal salt and water absorption, brush border membrane (BBM) morphology, and on the NHE3 mRNA expression, protein abundance, and transport activity in the murine intestine. NHERF1-deficient mice displayed reduced jejunal fluid absorption in vivo, as well as an attenuated in vitro Na⁺ absorption in isolated jejunal and colonic, but not of ileal, mucosa. However, cAMP-mediated inhibition of both parameters remained intact. Acid-activated NHE3 transport rate was reduced in surface colonocytes, while its inhibition by cAMP and cGMP was normal. Immunodetection of NHE3 revealed normal NHE3 localization in the BBM of NHERF1 null mice, but NHE3 abundance, as measured by Western blot, was significantly reduced in isolated BBM from the small and large intestines. Furthermore, the microvilli in the proximal colon, but not in the small intestine, were significantly shorter in NHERF1 null mice. Additional knockout of PDZK1 (NHERF3), another member of the NHERF family of adaptor proteins, which binds to both NHE3 and NHERF1, further reduced basal NHE3 activity and caused complete loss of cAMP-mediated NHE3 inhibition. An activator of the exchange protein activated by cAMP (EPAC) had no effect on jejunal fluid absorption in vivo, but slightly inhibited NHE3 activity in surface colonocytes in vitro. In conclusion, NHERF1 has segment-specific effects on intestinal salt absorption, NHE3 transport rates, and NHE3 membrane abundance without affecting mRNA levels. However, unlike PDZK1, NHERF1 is not required for NHE3 regulation by cyclic nucleotides. N. Broere and M. Chen share first authorship and are listed in alphabetical order.     

The Na+-dependent binding of [3H]l-aspartate in thaw-mounted sections of rat forebrain

Binding of [³H]l-aspartate to thaw-mounted coronal sections of frozen rat forebrain was strong in grey regions of telencephalon (neocortex, hippocampus and neostriatum), but it was weaker and unevenly distributed in diencephalon. At low nanomolar concentrations of ligand used in the present studies, [³H]l-aspartate binding was strongly inhibited by l-threo-3-hydroxyaspartate and l-trans-pyrrolidine-2,4-dicarboxylate, compounds known to be substrate/inhibitors of the high affinity uptake of l-glutamate and l-aspartate. None of the typical ligands for the glutamate and aspartate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), N-methyl-d-aspartate and kainate, produced a strong enough inhibition (only CNQX at 100 M weakly inhibited) of the Na⁺-dependent [³H]l-aspartate binding to suggest that [³H]l-aspartate was bound to the receptor binding sites. Furthermore, the binding was absolutely dependent on the presence of Na⁺ in the incubation medium. It is concluded that [³H]l-aspartate is a ligand suitable for autoradiographic studies of the distribution of Na⁺-dependent, high affinity uptake of acidic amino acids in the central nervous system (CNS). However, feasibility of using [³H]l-aspartate as a specific marker of glutamatergic and/or aspartergic synapses in the CNS requires further investigation.     

Phosphate transport in brush border membrane vesicles isolated from renal cortex of young growing and adult rats

Recent clearance studies have demonstrated that the maximal tubular reabsorption of inorganic phosphate (Pi) per ml of glomerular filtrate (max. TRPi/ml GF) of the whole kidney is markedly lower in adult than in young growing rats fed either normal (0.8 g%) or low (0.2 g%) phosphorus diet. In addition, in adult rats clearance studies indicate that enhancement of max. TRPi/ml GF is observed 21 days but not 8 days after starting the low (0.2%) phosphorus diet. In the present work we have studied in the same experimental condition the Na⁺-dependent Pi uptake in brush border membrane vesicles (BBMV) isolated from renal cortex of either young growing or adult rats. The results of this study indicate that under the low (0.2%) but not under the normal (0.8%) phosphorus diet the Na⁺-dependent Pi uptake by BBMV was significantly depressed in adult as compared to young growing rats. In adult rats the Pi transport response to Pi restriction monitored at the brush border membrane level was different from that observed by clearance studies in the whole kidney. Indeed, the Pi uptake by BBMV was already enhanced after 8 days of Pi restriction and it did not increase further when studied 21 days after starting the low (0.2%) phosphorus diet. These results suggest that the regulation of the overall transfer of Pi across the renal epithelium may involve other additional modulating factors than the Na⁺-dependent Pi transport system present in the luminal membrane of the proximal tubule.     

Effect of starvation on neutral amino acid transport in isolated small-intestinal cells from guinea pigs

The effects of starvation on neutral amino acid transport were examined in isolated enterocytes. Starvation stimulated L-alanine transport by the Na⁺-dependent system A and the Na⁺-independent system L without producing any changes in either the Na⁺-dependent systems ASC or the passive non-mediated uptake. Starvation produces a twofold increase in V _max of system A without any change in K _t. Starvation produces an increase in V _max of system L of 1.7 times without any change in K _t. Activation of systems A and L by starvation was reversible with subsequent refeeding. The effects of a series of amino acids on systems A and L were evaluated. A different inhibition pattern was found in starved animals as compared to controls. Starvation increases Na⁺-dependent L-alanine uptake and Na⁺-independent cycloleucine uptake by small-intestinal brushborder membrane vesicles. These results suggest that starvation stimulates amino acid transport across the apical plasma membrane of the enterocytes by inducing specific carrier units.     

Phosphate uptake by superficial and deep nephron brush border membranes

Dietary phosphate restriction and acute parathyroidectomy in rat are known to be associated with a selective increase in phosphate uptake by renal cortical brush border membranes (BBM). Conversely, phosphate loading and parathyroid hormone (PTH) administration result in a decrease of this uptake.In the present study, we investigated whether the response of the membrane to these various stimuli implies similar or different modifications of the kinetic properties of this membrane, whether these modifications affect one or both of the two systems of phosphate transport previously described, whether both superficial and deep nephron populations are involved, and whether the two stimuli: dietary phosphate, and parathyroid activity, are additive or not.Kinetic studies of phosphate (PO₄) uptake by BBM vesicles were performed in seven groups of rats: control (N), acutely thyroparathyroidectomized (TPTX), PTH loaded (PTH), phosphate loaded (P⁺), phosphate depleted (P^–) phosphate depleted with acute thyroparathyroidectomy (P^–TPTX), and phosphate depleted-PTH loaded (P^–PTH). In each of these experimental conditions, superficial and deep nephrons were investigated.Results indicate that 1. BBM from deep nephrons present a greater capacity for PO₄ transport than those from superficial nephrons; 2. Whereas a dual system of PO₄ uptake is observed in superficial BBM, deep BBM present only one single system; 3. Phosphate in the diet influences PO₄ uptake by BBM to a greater extent in the deep than in the superficial nephrons; 4. PTH status on the contrary, equally influences both populations; 5. TPTX does not significantly enhance PO₄ uptake in phosphate depleted rats; however, PTH loading curtails this uptake; 6. In the deep BBM neither the PTH status nor the phosphate content of the diet modify the apparentK _m. In the superficial BBM, the apparentK _m of the high affinity system (low substrate concentrations) varies with the PTH activity.     

Transport ofl-lysine by rat renal brush border membrane vesicles

l-³H-lysine uptake into brush border membrane vesicles was measured by a rapid filtration technique. A significant binding ofl-lysine at the vesicle interior was observed. Extrapolating initial linear uptake to zero incubation time did not indicate binding of the amino acid to the external membrane surface.Sodium stimulated thel-lysine uptake specifically. Experiments in the presence of potassium/valinomycin induced diffusion potentials, and experiments with a potential sensitive fluorescent dye documented an electrogenic uptake mechanism forl-lysine only in the presence of sodium. Sodium independent uptake proceeds via an electroneutral pathway. Transstimulation experiments show carrier mediated uptake in the presence and absence of sodium. An outwardly directed proton-gradient stimulatedl-lysine uptake in the presence and absence of sodium.Saturation ofl-lysine uptake was observed in the presence and absence of sodium. In the absence of sodium,l-lysine uptake was inhibited byl-arginine,l-cystine,l-phenylalanine andl-methionine. The sodium dependent uptake was inhibited byl-arginine andl-cysteine; small inhibition byl-phenylalanine was observed. In the presence or absence of sodium,l-lysine uptake was inhibited neither byd-lysine nor byl-glutamic acid.These results document carrier mediated transport ofl-lysine via (a) transport mechanism(s) not obligatory requiring sodium.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - Tris Tris(hydroxymethy)aminomethane - EGTA ethyleneglycol-bis-(-aminoethyl-ether)-N,N-tetraacetic acid - diamide azodicarboxylic acid[bisdimethylamide] - FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - MES 2-(N-morpholino) ethanesulfonic acid     

Membrane currents and cytoplasmic sodium transients generated by glutamate transport in Bergmann glial cells

Effects of glutamate and kainate (KA) on Bergmann glial cells were investigated in mouse cerebellar slices using the whole-cell configuration of the patch-clamp technique combined with SBFI-based Na⁺ microfluorimetry. l-Glutamate (1 mM) and KA (100 μM) induced inward currents in Bergmann glial cells voltage-clamped at −70 mV. These currents were accompanied by an increase in intracellular Na⁺ concentration ([Na⁺]_i) from the average resting level of 5.2 ± 0.5 mM to 26 ± 5 mM and 33 ± 7 mM, respectively. KA-evoked signals (1) were completely blocked in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 μM), an antagonist of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/KA ionotropic glutamate receptors; (2) reversed at 0 mV, and (3) disappeared in Na⁺-free, N-methyl-D-glucamine (NMDG⁺)-containing solution, but remained almost unchanged in Na⁺-free, Li⁺-containing solution. Conversely, l-glutamate-induced signals (1) were marginally CNQX sensitive (∼10% inhibition), (2) did not reverse at a holding potential of +20 mV, (3) were markedly suppressed by Na⁺ substitution with both NMDG⁺ and Li⁺, and (4) were inhibited by d,l-threo-β-benzyloxyaspartate. Further, d-glutamate, l-, and d-aspartate were also able to induce Na⁺-dependent inward current. Stimulation of parallel fibres triggered inward currents and [Na⁺]_i transients that were insensitive to CNQX and MK-801; hence, we suggested that synaptically released glutamate activates glutamate/Na⁺ transporter in Bergmann glial cells, which produces a substantial increase in intracellular Na+ concentration.     

Transport ofl-lysine by rat intestinal brush border membrane vesicles

Brush border membranes were isolated from rat jejunum by a divalent cation precipitation method.³H-l-Lysine uptake was measured by a rapid filtration technique. Uptake after prolongued incubation periods was osmotically insensitive and represented almost exclusively binding to the vesicles. Extrapolating initial linear uptake to a zero incubation time indicated no binding of the amino acid to the external membrane surface.Sodium did not significantly alter the initial uptake rate.l-Lysine transport respresents a carrier mediated uptake in the presence and absence of sodium as indicated by the transstimulation experiments. The transport mechanism operates stereospecifically and is inhibited by other basic amino acids andl-leucine andl-phenylalanine. Saturation experiments result in aK _m of 0.26 mmoles/l and aV _max of 272 pmoles/mg protein/10 s.Inside negative anion diffusion potentials and inside negative potassium diffusion potentials (valinomycin) were unable to increase the transport rate. Transmembrane pH-gradients were also unable to alter transport.Abbreviations HEPES N-2-hydroxethylpiperazine-N-ethane sulfonic acid - Tris tris(hydroxymethyl)aminomethane - EGTA ethyleneglycolbis-(-aminoethyl-ether)-N,N tetraacetic acid - MES 2-(N-morpholino)-ethanesulfonic acid     

45Ca2+ uptake by dispersed pancreatic islet cells: Effects ofd-glucose and the calcium probe,chlorotetracycline

Uptake of⁴⁵Ca²⁺ was studied in dispersed pancreatic islet cells from non-inbredob/ob-mice. Like whole islets the dispersed cells responded to 20 mMd-glucose with a markedly increased⁴⁵Ca²⁺-labeling of both the lanthanum-nondisplaceable and the lanthanum-displaceable calcium pools. The pronounced effect ofd-glucose could not be reproduced with 3-O-methyl-d-glucose,l-glucose,d-mannose,l-leucine, ord-leucine; however,⁴⁵Ca²⁺ uptake was greater in the presence ofl-leucine as compared withd-leucine.⁴⁵Ca²⁺ uptake by dispersed cells or whole islets was stimulated severalfold by 100 M or more chlorotetracycline. At the concentration of only 10 M, chlorotetracycline had no effect on whole islets and partially inhibited⁴⁵Ca²⁺ uptake by the dispersed cells. The ability ofd-glucose to stimulate⁴⁵Ca²⁺ uptake by islets or dispersed cells remained in the presence of 10 M chlorotetracyline. Islet cell suspensions apparently represent a valid model for studying how Ca²⁺ interacts with the cells. However, when using chlorotetracycline as fluorescent Ca²⁺ probe, attention must be paid to its potential ionophoric activity. At only 10 M, the drug seems to monitor a peripheral pool of Ca²⁺, some of which may reside in normal transport channels.     

Sodium gradient-energized concentrative transport of adenosine in renal brush border vesicles

The uptake of adenosine in brush border vesicles of the proximal tubule of the rat kidney has been studied with a filtration technique. The initial rate of uptake was almost 6 times greater in the presence of NaCl than in the presence of KCl. The stimulatory effect of Na⁺ was strictly dependent on a gradient of Na⁺ (out>in). The time course of uptake showed an overshoot with a maximum at 20 s with a gradient of NaCl, but not with KCl. Inosine and 5-AMP were produced from adenosine within the vesicles. In the presence of an inhibitor or adenosine deaminase adenosine was not significantly metabolized during the first 20 s of uptake. Thus, kinetic parameters of transport could be studied in the absence of interferences with metabolism. AK _m of 1.1 M and aV _max of 232 pmol · min^–1 · mg protein^–1 were calculated for the Na⁺ gradient-dependent transport. The dependency on a Na⁺ gradient, the capacity for uphill transport and the high affinity for adenosine situate this transport system apart from the mechanisms of transport of nucleosides described so far. It may be relevant in regard to the role of adenosine in the regulation of glomerular filtration.Abbreviations used EHNA erythro-9-(2-hydroxy-3-nonyl)adenine - FCCP carbonylcyanide p-trifluoromethoxy-phenylhydrazone - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Tris tris (hydroxymethyl)-aminomethane     

Effect of rabbit duodenal mRNA on phosphate transport in Xenopus laevis oocytes: dependence on 1,25-dihydroxy-vitamin-D3

Expression of Na⁺-dependent transport of phosphate (P_i) was analysed in Xenopus laevis oocytes after injection of poly(A)-rich RNA isolated from the duodenal mucosa of rabbits with increased levels of 1,25-(OH)₂-VitD₃ (injection of vitamin D₃ or low-P_i diet) or from control animals. In parallel, the effect of elevated levels of vitamin D₃ was studied in isolated duodenal brush-border membrane vesicles. In brush-border membrane vesicles, the rate of Na⁺-P_i cotransport was found to be doubled after 1,25-(OH)₂-VitD₃ injections while Na⁺ — d-glucose cotransport (measured as a control) was not altered. In X. laevis oocytes, Na⁺-dependent P_i uptake was increased after injection of poly(A)-rich RNA isolated from duodenal mucosa of animals with increased levels of 1,25-(OH)₂-VitD₃ but not after injection of poly(A)-rich RNA isolated from control animals; between the two groups of mRNA no difference in the expression of the Na⁺ — d-glucose transport system was observed. Sucrose density gradient fractionation suggests that mRNA species related to the increased Na⁺-dependent P_i uptake are of average chain lengths between 2×10³ and 3×10³ bases (2–3 kb). It is concluded that in duodenal enterocytes 1,25-(OH)₂-VitD₃ increases the content of mRNA species of 2–3 kb that might be involved either directly in Na-P_i cotransport or at least in controlling its activity.