G. Stange

Transport of sulphate in rat jejunal and rat proximal tubular basolateral membrane vesicles

Basolateral membrane vesicles were isolated by a Percoll density gradient centrifugation method from small intestinal and renal proximal tubular epithelial cells. Transport of sulphate across the basolateral membrane was analyzed by measuring the uptake of tracer sulphate.In both membrane preparations, preloading the vesicles with sulphate-or hydroxyl-anions stimulated tracer sulphate uptake (trans-stimulation); an inwardly directed sodium gradient did not stimulate sulphate influx whether in the absence or in the presence of sulphate- or hydroxyl-iontrans-stimulation. Under sulphate trans-stimulation conditions, DIDS (10−4 mol/l) inhibited sulphate influx.In intestinal membranes, trans-stimulation of sulphate influx was obtained by preloading the vesicles with chloride, in renal membranes by preloading with bicarbonate. Under sulphate trans-stimulation conditions, in intestinal membranes, sulphate influx was strongly inhibited by chloride, in renal membranes, chloride inhibition was absent. Under bicarbonate trans-stimulation conditions, in renal membranes, sulphate transport was inhibited by lactate.It is concluded that small intestinal and renal proximal tubular basolateral membrane vesicles contain a transport mechanism for sulphate that cannot be energized by a sodium gradient. The transport system in small intestinal basolateral membranes seems to be different from that in renal membranes. It is suggested that the observed interaction between inorganic and organic anion transport in renal basolateral membranes is indirect.

Identification of a cDNA/protein leading to an increased P-i-uptake in Xenopus laevis oocytes

Abstract. In a previous report we documented an increased Na+-dependent transport of inorganic phosphate (Pi) in Xenopus laevis oocytes injected with mRNA isolated from rabbit duodenum (Yagci et al., Pfluegers Arch.422:211–216, 1992; ref 24). In the present study we have used expression cloning in oocytes to search for the cDNA/mRNA involved in this effect. The identified cDNA (provisionally named PiUS; for Pi-uptake stimulator) lead to a 3-4-fold stimulation of Na+-dependent Pi-uptake (10ng cRNA injected, 3–5 days of expression). Na+-independent uptake of Pi was also affected but transport of sulphate and l-arginine (in the presence or absence of sodium) remained unchanged. The apparent Km-values for the induced Na+-dependent uptake were 0.26 ± 0.04 mm for Pi and 14.8 ± 3.0 mm for Na+. The 1796 bp cDNA codes for a protein of 425 amino acids. Hydropathy analysis suggests a lack of transmembrane segments. In vitro translation resulted in a protein of 60 kDa and provided no evidence of glycosylation. In Northern blots a mRNA of ∼2 kb was recognized in various tissues including different intestinal segments, kidney cortex, kidney medulla, liver and heart. Homology searches showed no similarity to proteins involved in membrane transport and its control. In conclusion, we have cloned from a rabbit small intestinal cDNA library a novel cDNA encoding a protein stimulating Pi-uptake into Xenopus laevis oocytes, but which is not a Pi-transporter itself.

Transport ofl-lysine by rat renal brush border membrane vesicles

Abstractl-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.

Transport ofl-cystine by rat renal brush border membrane vesicles

Brush border membranes were isolated from rat renal cortex by a divalent cation precipitation method.l-35S-cystine uptake into the vesicles was measured by a rapid filtration method. Covalent incorporation of tracer into membrane proteins was observed after prolonged incubations. At short incubation periods (1 min) binding was small and allowed an analysis of transmembrane transport. To guarantee transport ofl-cystine, the experiments were performed in the presence of the oxidant diamide.Sodium stimulatedl-cystine uptake specifically. A potassium/valinomycin induced inside negative diffusion potential stimulated sodium dependentl-cystine transport. Thus, transport is potential sensitive in the presence of sodium. At low substrate and inhibitor concentrations,l-cystine transport was inhibited byl-lysine,l-ornithine andl-arginine but not byd-lysine in the presence and absence of sodium. At higher inhibitor concentration, the neutral amino acidsl-phenylalanine andl-leucine also inhibitedl-cystine uptake, but only the sodium dependent uptake. These inhibition experiments suggest thatl-cystine is transported by the brush border membrane by a transport system for basic amino acids not necessarily requiring sodium. In addition, transport ofl-cystine can also procee via sodium dependent transport pathways for neutral amino acids.In the concentration range tested (up to 0.225 mmoles/l), no saturation ofl-cystine transport was observed in the presence and absence of sodium.

Transport ofl-cysteine by rat renal brush border membrane vesicles

SummaryBrush border membranes were isolated from rat renal cortex by a divalent cation precipitation method.l-35S-cysteine uptake into the vesicles was measured by a rapid filtration method. Only minimal binding of the amino acid to the vesicles was observed. Sodium stimulatesl-cysteine uptake specifically. Anion replacement experiments, experiments in the presence of potassium/valinomycin-induced diffusion potential as well as experiments with a potential-sensitive fluorescent dye document an electrogenic sodium-dependent uptake mechanism forl-cysteine. Tracer replacement experiments as well as the fluorescence experiments indicate a preferential transport ofl-cysteine. Transport ofl-cysteine is inhibited byl-alanine andl-phenylalanine but not byl-glutamic acid and thel-basic amino acids. Initial, linear influx kinetics provide evidence for the existence of two transport sites. The results suggest (a) sodium-dependent mechanism(s) forl-cysteine shared by other neutral amino acids.

Calcium uptake into rat small intestinal brush border membrane vesicles: Characterization of transmembrane calcium transport at short initial incubation times

Calcium transport into brush border vesicles from rat small intestine was investigated by determining uptake rates at very short incubation periods. At incubation times up to 1 second a linear relationship between calcium uptake and time was observed at free calcium concentrations ranging from 1 microM to 5 mM. At time points above 1 second calcium uptake deviates progressively from linearity. Several lines of evidences (EGTA-wash, dependency on membrane potential, temperature sensitivity and effect of the calcium ionophore A23187) suggest transmembrane transport rather than extravesicular binding of calcium as being responsible for calcium uptake. Saturation experiments performed under initial linear and curvilinear uptake conditions show a saturable transport component in the mu molar and only a tendency to saturate in the molar concentration range. It is concluded that uptake values far from equilibrium are characteristic for transmembrane flux of calcium. Transmembrane flux of calcium is mediated by multiple and potential-sensitive mechanisms.

Modulation of renal type IIa Na^+/Pi cotransporter kinetics by the arginine modifier phenylglyoxal

The effects of the arginine-modifying reagent phenylglyoxal on the kinetics of the type IIa Na + /Pi cotransporter expressed in Xenopus, oocytes were studied by means of 32Pi uptake and electrophysiology. Phenylglyoxal incubation induced up to 60% loss of cotransport function but only marginally altered the Na+-leak. Substrate activation and pH dependency remained essentially unaltered, whereas the voltage dependency of Pi-induced change in electrogenic response was significantly reduced. Presteady-state charge movements were suppressed and the equilibrium charge distribution was shifted slightly towards hyperpolarizing potentials. Charge movements in the absence of external Na+ were also suppressed, which indicated that the empty-carrier kinetics were modified. These effects were incorporated into an ordered alternating access model for NaPi-IIa, whereby the arginine modification by phenylglyoxal was modeled as altered apparent electrical distances moved by mobile charges, together with a slower rate of translocation of the electroneutral, fully loaded carrier.

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

The effects of intravesicular NAD on Na+-dependent32Pi 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 Pi 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 Pi 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 Pi 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.