Metabolism of NAD by isolated rat renal brush border membranes

NAD and NADH are metabolized to adenosine, ribosyl nicotinamide and P_i by isolated rat renal brush border membranes (BBM). The similar distribution like the BBM marker enzyme alkaline phosphatase indicates that nucleotide pyrophosphatase and 5-nucleotidase are BBM enzymes.     

Phosphate transport by isolated renal brush border vesicles

Summary A sodium dependent specific transport system for phosphate is present in the brush border microvilli but absent from the basal-lateral plasma membranes. The apparent affinity of this transport system for phosphate is 0.08 mM at 100 mM sodium and pH 7.4. It is inhibited competitively by arsenate with an apparent inhibitor constant of 1.1 mM (100 mM sodium, pH 7.4). Sodium dependent phosphate uptake is two times higher at pH 8 compared to the uptake observed at pH 6. The apparent affinity of the transport system for sodium is also pH-dependent, half-maximal stimulation of uptake is found at pH 6 with 129 mM sodium, at pH 7.4 with 60 mM sodium and at pH 8 with 50 mM sodium. Under all conditions a nonhyperbolic dependence of phosphate uptake on the sodium concentration is observed. The uptake of phosphate by brush border microvilli vesicles shows a typical overshoot phenomenon in the presence of sodium gradient across the membrane {\text{ }}C_{Na_i } )$$ " align="middle" border="0"> . The amount of phosphate taken up after 2 min is about twice the equilibrium value reached after 2 h of incubation. At pH 7.4 the initial rate of uptake is increased only slightly (12%) by inside negative membrane diffusion potentials and inhibited to the same extent by inside positive membrane diffusion potentials.These results indicate that the entry of phosphate across the brush border membrane into the epithelial cell of the proximal tubule is coupled to the entry of sodium. The transfer of phosphate is dependent on its concentration gradient and on the concentration difference of sodium. The data are best explained by the following hypothesis: Both the primary phosphate as well as the secondary phosphate are transported in cotransport with sodium. The divalent form however seems to be transported preferentially. Its transport occurs electroneutral with 2 sodium ions; the monovalent phosphate also enters the cell together with 2 sodium ions but as a positively charged complex.The exit of phosphate across the contraluminal cell border is sodium independent and is favoured by the high intracellular phosphate concentration and the inside negative membrane potential.Part of these data have been presented at the Spring Meeting of the German Physiological Society at Bochum 1975 (Pflügers Arch.355, R 98, 1975).     

Phosphate transport by rat renal brush border membrane vesicles: Influence of dietary phosphate,thyroparathyroidectomy, and 1,25-dihydroxyvitamin D3

In the present work we have investigated whether the changes in the renal handling of inorganic phosphate (Pi) induced by 1) dietary Pi, 2) removal of parathyroid glands and 3) 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], are associated with alterations in the Na-dependent Pi uptake by brush border membrane vesicles (BBMV) isolated from renal cortex. Shamoperated (SHAM) or thyroparathyroidectomized (TPTX) rats treated or not with 26 pmol/day of 1,25 (OH)₂D₃i.p. were fed low (0.2%) or high (1.2%)P diet for 7 days. The results showed that in SHAM, TPTX and TPTX+1,25 (OH)₂D₃ the Pi uptake by BBMV was greater after low than high Pi diet. It was greater in TPTX than in SHAM counterparts fed either diets. In TPTX fed low or high Pi diet 1,25 (OH)₂D₃ decreased the Pi uptake to the level observed in SHAM. A striking parallelism was found between variations in Pi uptake by BBMV and in the tubular Pi reabsorption of the whole kidney. The Na-dependent glucose, the mannitol uptake by BBMV, and the alkaline phosphatase activity in cortical homogenates and BBMV were not affected by the various treatments. Thus, dietary Pi, chronic TPTX and 1,25 (OH)₂D₃ appear to specifically affect the Na-dependent Pi transport system bound to the brush border membranes of renal cortical tubules. The alterations observed at this membrane level could account, at least in part, for the changes induced by these three factors on the overall tubular reabsorption of Pi.     

Demonstration of sodium-dependent,electrogenic substrate transport in rat small intestinal brush border membrane vesicles by a cyanine dye

The cyanine dye DiS-C₂(5) was tested as an indicator for changes in membrane potential of subfractionated rat jejunal brush border membrane vesicles. The fluorescence of this dye increased with inside positive and decreased with inside negative potentials. The sensitivity to inside negative potentials was greater than to inside positive potentials. The addition ofl-alanine,l-phenylalanine,l-methionine,d-galactose andd-glucose in the presence of sodium provoked a transient fluorescence increase indicating an inside positive membrane potential due to electrogenic, sodium-coupled transport. Besides the sodium-dependence, the dye reflected stereo-specificity and saturability ofd-glucose transport. Whend-glucose loaded vesicles were incubated ind-glucose-free medium, a decrease in fluorescence was observed indicating thatd-glucose efflux is also electrogenic.Abbreviations Dis-C₂(5) 3,3-diethylthiadicarbocyanine iodide - DiS-C₃(5) 3,3-dipropylthiadicarbocyanine iodide - HEPES N-2-hydroxyethylpiperazine-N-ethane sulfonic acid - Tris tris(hydroxymethyl)aminomethane - EGTA ethyleneglycol-bis-(-aminoethyl-ether)-N,N-tetraacetic acid     

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     

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     

Sodium-dependent adenosine transport is diminished in brush border membrane vesicles from hypothyroid rat kidney

 Studies of the uptake of [3H]adenosine ([³H]ADO) were performed using brush border membrane vesicles (BBMV) from normal (N) and hypothyroid (Tx) rat kidneys, to test if decreased Na⁺ reabsorption in hypothyroidism might be associated with abnormalities in ADO transport. [³H]ADO uptake (1–10 μmol) for both conditions was measured in the presence of Na⁺ (10–150 mmol/l); the effects of dipyridamole (10 μmol/l) and 1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX, 10 μmol/l) were also studied. Na⁺-stimulated ADO uptake was decreased in Tx BBMV. Michaelis–Menten constants showed a decreased ADO carrier affinity (K _m 2.46 ± 0.14 in N, vs K _m 4.46 ± 0.88 μmol/l in Tx, P<0.05), with no change in the number of carriers (V _max 295 ± 25 in N, vs 229.2 ± 56 pmol·min^–1·mg protein in Tx). Na⁺ affinity remained unchanged (K _Na+ 11.5 ± 3.5 in N, vs K _Na+ 12.72 ± 0.7 mmol/l in Tx). Inhibition of Na⁺-dependent ADO transport was 50% in N as opposed to 58% in Tx with dipyridamole, and 72% in N versus 47% in Tx with PACPX. These results suggest that decreased Na⁺-dependent ADO cotransport contributes to the diminished tubular reabsorption that occurs in hypothyroidism. Received: 17 June 1996 / Received after revision: 9 September 1996 / Accepted: 16 September 1996     

On the correlation between alkaline phosphatase and phosphate transport in rat renal brush border membrane vesicles

Alkaline phosphatase activity in renal cortex homogenates and in isolated brush border membrane vesicles was compared with the rates of sodium-dependent transport of inorganic phosphate (P_i) by isolated brush border membranes. Brush border membrane vesicles were isolated from renal cortical homogenates of rats adapted during a period of 5–7 weeks to diets with different dietary contents of P_i (low P_i diet=0.15 g%, high P_i diet=2.0 g%).Alkaline phosphatase activity was not increased in the low P_i diet group as compared to the standard P_i diet group but was reduced in the high P_i diet group. Sodium-dependent transport of P_i was increased 2–3-fold in the low P_i diet group as compared to the standard P_i diet group, whereas transport activity was only unsignificantly decreased in the high P_i diet group.Studying kinetik parameters in the two extreme dietary groups it has been found that these differences are due to alteredV _max of the transport activity as well as of alkaline phosphatase activity. TheK _m for both activities remained unaltered.Alkaline phosphatase activity and transport of P_i in brush border membrane vesicles were also compared in the presence of EDTA or Zn²⁺ at concentrations which inhibit alkaline phosphatase activity. Transport of P_i was not affected by the inhibitors even when alkaline phosphatase was inhibited by more than 70% (0.5 mmol/l Zn²⁺) or completely (0.5 mmol/l EDTA).The experiments suggest that no correlation between alkaline phosphatase activity and transport of P_i exists in isolated brush border membrane vesicles.     

Mechanism of rapid phosphate (Pi) transport adaptation to a single low Pi meal in rat renal brush border membrane

Previous studies have shown that the adaptive response of tubular inorganic phosphate (Pi) transport to Pi deprivation is detectable in the whole kidney 24 h after switching rats from a high (HPD) to a low (LPD) Pi diet. In the present work we report on a more rapid adaptive response of the sodium (Na)-dependent Pi transport system located in the luminal membrane of the proximal tubule and its relation with changes in phosphatemia an parathyroid hormone status. Rats were fed HPD and trained to eat their daily ration within 1 h. After two weeks of equilibration half of the animals received a single LPD ration. 1, 2 and 4 h after the end of food consumption the animals were either sacrificed for renal cortical brush border membrane vesicle (BBMV) isolation or used for determining plasma Pi concentration, urinary excretion of Pi and cAMP. The results indicate that 2 and 4 h after the end of feeding, the Na-dependent Pi transport in BBMV was stimulated by 70 and 140% respectively in intact rats exposed for the first time to LPD. This response was preceded by a significant fall in plasma Pi concentration (HPD: 2.46±0.03, LPD: 2.04±0.05 mM), in the urinary excretion of Pi (HPD: 899.0±68.1; LPD: 6.5±3.3 mol/ml GFR) and cAMP (HPD: 76.9±7.4, LPD: 48.2±1.4 pmol/ml GF). This last result suggested a rapid inhibition of PTH after one single LPD feeding. In thyroparathyroidectomized (TPTX) rats the Na-dependent Pi transport system was also stimulated 4 h after LPD, but to a slightly less extent than in intact rats.In conclusion, the Na-dependent Pi transport located in the luminal membrane of the proximal tubule reacts within hours to dietary Pi restriction. This specific tubular response coupled with the hypophosphatemia should account for the rapid decrease in urinary Pi excretion. Although the adaptive response at the BBMV level is also expressed in TPTX rats, inhibition of the PTH-cAMP system could contribute to the rapid adaptation observed in intact animals.     

Adaptation of phosphate transport to low phosphate diet in renal and intestinal brush border membrane vesicles: influence of sodium and pH

The possible role of changes in the sodium (Na) affinity of the carrier for inorganic phosphate (P_i) in the adaptation of P_i transport to low P_i diet was examined in both renal and intestinal brush border membranes vesicles (BBMV) obtained from the same animal. This role was assessed by measuring the Na concentration resulting in half maximal activation of P_i transport (K _0.5Na) in renal and intestinal BBMV prepared from animals adapted to either low (LPD) or high (HPD) phosphorus diet for 7 days. TheK _0.5Na was not modified by dietary P_i, in both renal and intestinal BBMV. LPD increased maximal P_i transport from 1794.8±198.0 to 296.4±362.0 in renal and from 28.2±3.4 to 80.5±7.2 pmol/mg 10 s in intestinal BBMV. For both LPD and HPD lowering pH from 7.4 to 6 dramatically increasedK _{0.5 Na} in renal and intestinal BBMV. As compared to pH 7.4, it was enhanced by approximately 200% in both renal and intestinal membranes. This change of Na affinity with acidic pH prevented the expression of P_i transport adaptation at 100 mM Na concentration. However, at saturating Na concentrations (500 mM for renal, 300 mM for intestinal membranes), P_i transport adaptation was equally expressed at pH 6 and 7.4 in both types of membranes. Hill coefficient analysis indicates a 2:1 stoichiometry of Na to P_i in renal and intestinal membranes isolated from high or low P_i diet animals. This ratio was not modified by changes of the medium pH.     

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     

Glutathione-dependent inactivation of sodium-dependent phosphate transport across rat renal brush-border membrane

Thiol/disulfide is fundamental in protein function; we previously observed an inhibitory effect of thiol oxidants on the Na-dependent phosphate (Pi) uptake into renal brush border membrane vesicles (BBMV). We examined whether oxidation of glutathione (GSH) is involved in the mechanism. Vesicular thiols were measured by liquid chromatography. BBMV were incubated with reagents before an influx of Pi. Diamide (5 mM) reduced the capacity of the Pi uptake. Subsequent treatment with dithiothreitol (5 mM) blocked the inhibitory effect of diamide. Vesicular GSH was not modified only by the incubation, whereas it was oxidized by the treatment with diamide, and reduced by dithiothreitol. Furthermore, in vivo treatment with cAMP provided GSH-depleted BBMV without any influence on Pi uptake. Diamide did not inhibit the transport of Pi into GSH-depleted vesicles, but it did inhibit the uptake when GSH was introduced into the vesicles. In conclusion, a GSH-dependent mechanism is involved in the inhibitory effect of diamide on sodium-dependent Pi transport across the renal brush-border membrane.     

Effect of feeding a high protein diet on amino acid uptake into rat intestinal brush border membrane vesicles

Uptake of the neutral amino acidl-leucine into isolated rat intestinal brush border membrane (=BBM) vesicles and into a jejunal mucosa preparation as affected by the protein content of the diet was investigated. Adult rats fed either a high carbohydrate (HC) diet (11% protein) or a high protein (HP) diet (77% protein) for several weeks were used for the experiments.The time course ofl-leucine uptake into BBM vesicles prepared from the small intestine of HC-or HP-rats was studied under conditions of an inwardly directed Na⁺-gradient and under Na⁺-equilibrium conditions. Furthermore, in one series of experiments the Na⁺-equilibrium was replaced by a K⁺-equilibrium. l-leucine uptake under Na⁺-gradient conditions displayed the overshoot phenomenon typically associated with Na⁺-gradient-dependent active transport processes in BBM vesicles and the overshoot in group HP exceeded that in group HC significantly.Under both Na⁺-and K⁺-equilibrium conditionsl-leucine uptake into the BBM-vesicles also was faster in group HP.Finallyl-leucine uptake into jejunal mucosa in group HP exceeded that in group HC, too.The results therefore indicate that Na⁺-dependent and Na⁺-independent transport of neutral amino acids across the intestinal brush border membrane adapts to the dietary protein level.Some of the results were reported in a preliminary form [16]     

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.     

Zinc inhibition of glucose uptake in brush border membrane vesicles from pig small intestine

The effect of zinc on sodium coupled glucose uptake was studied in pig intestinal brush border membrane vesicles. In this system zinc inhibited glucose uptake and appeared to have a K _i of 0.25 mM. When tested by spectrophotometry, electron microscopy and protein determination following centrifugation, no evidence of significant vesicle aggregation was found with 0.5 mM zinc treatment. Zinc inhibition of glucose uptake persisted when the vesicle membrane potential was clamped with identical KCl concentrations inside and outside the vesicles in the presence of valinomycin. Variation of the glucose and sodium concentrations gave results indicating that zinc reduces glucose affinity for the carrier but not sodium binding to the transporter. The glucose inhibitory effect was not due to a rapid dissipation of the sodium gradient as zinc failed to affect sodium uptake in the absence of glucose. Zinc also failed to inhibit glucose efflux from vesicles under isotopic exchange conditions, when glucose and sodium concentrations were identical inside and outside vesicles. The t1/2 of glucose inhibition by zinc was relatively long, i.e. 6 min. We conclude that zinc acts as an inhibitor of glucose transport by interacting with the sodium-glucose co-transporter. The long zinc incubation time required to achieve maximal inhibition of glucose transport suggests that this interaction takes place within vesicles.     

The influence of pH on phosphate transport into rat renal brush border membrane vesicles

Isometric contracture responses of normal and denervated inferior oblique muscles (IO) of the rabbit have been investigated in vitro at 35°C. The threshold concentration for eliciting potassium contractures was about 20 mM K⁺. In normal IO low potassium concentrations up to about 50 mM K⁺ evoked only sustained contractures, higher concentrations were responded by contractures with an initial transient component. The transient tension development was maximal at about 100 mM K⁺ the sustained component at 80 mM K⁺. After denervation the characteristic time course of the contractures was not changed, but the tension output of the preparations was diminished and long-term denervated IO have a somewhat lowered threshold. In normal IO acetylcholine (ACh), succinylcholine (SCh) and choline (Ch) caused also sustained contractures, the threshold doses were about 5 M for ACh and SCh and 500 M for Ch. The ACh sensitivity of the preparations was increased by physostigmine and decreased or abolished byd-tubocurarine. Denervation increased the drug sensitivity but the shape of the contractures was hardly influenced. The properties of slow tonic muscle fibres in mammalian extraocular muscles (EOM) probably responsible for sustained contractures and their changes after denervation are discussed.     

Transport of organic cations in brush border membrane vesicles from rabbit kidney cortex

The transport of three organic cations, tetraethylammonium (TEA), morphine and N¹-methylnicotinamide (NMN) was studied in brush border membrane vesicles from rabbit kidney cortex under voltage clamp conditions. A proton gradient (pH_i=6.0, pH_o=7.4) produced a large stimulation of TEA and morphine uptake, yielding a transient overshoot of 190 and 220% respectively, as compared to equilibrium uptake values. No overshoot was observed under pH equilibrium conditions (pH_i=pH_o=7.4, control). These data suggest the presence of a proton-organic cation exchange mechanism in the rabbit renal cortical brush border membrane. Identical experimental conditions (proton gradient) failed however to stimulate significantly NMN transport above control values measured under pH equilibrium conditions. Proton gradient driven TEA transport showed an inhibition of 21% in the presence of NMN (1 mM) and of 63% in the presence of TEA (1 mM), and TEA transport was stimulated by preloading the vesicles with 1 mM TEA (305%) but not with 1 mM NMN (128%). NMN transport showed an inhibition of 39% in the presence of 1 mM TEA and of 27% in the presence of 1 mM NMN and its transport was stimulated by preloading the vesicles with 1 mM TEA (228%) and 1 mM NMN (178%). Our data suggest that TEA, NMN and morphine are transported by a common transport mechanism for which NMN has only a low affinity.     

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     

Ca2+-dependent protein phosphorylation in brush border membranes of rat kidney proximal tubules

Ca²⁺-dependent protein phosphorylation was studied in isolated brush border membrane vesicles from rat kidney cortex. Phosphorylation of about 20 target proteins with -³²P-ATP was observed after opening the vesicles transiently by detergent treatment or by hypotonic shock indicating that phosphorylation takes place from the cytoplasmic side. Five of these polypeptides (M_r 12, 19, 29, 97 and 138 kD) showed increased phosphorylation in the presence of micromolar calcium.Addition of exogenous calmodulin did not enhance the calcium induced phosphorylation significantly, nor did trifluoperazine depress it, suggesting that Ca²⁺-calmodulin-dependent protein kinase is not involved in the studied Ca²⁺-induced phosphorylation.12-O-tetradecanoylphorbol-13-acetate (TPA) minimized the Ca²⁺ dependency for the 12 and 97 kD polypeptides. Cytotoxin I inhibited the incorporation of phosphate into the 12 and 97 kD polypeptides in a dose-dependent manner. Excess phosphatidylserine could reverse this inhibition but stimulated also the phosphorylation of the 19 kD polypeptide. These findings suggest that at least the 12 and the 97 kD polypeptides are substrates for an endogenous protein kinase C.When studied under conditions where Ca²⁺ increases membrane phosphorylation, no effects could be observed on the kinetic parameters (K _m andV _max) of the sodium-dependent phosphate uptake.Abbreviations BBMV Brush border membrane vesicle - EDTA ethylenediamine-tetraacetic acid - EGTA ethyleneglycol (-aminoethylether)-N,Ntetra-acetic acid - HEDTA N-hydroxyethylene-diamine triacetic acid - HEPES N-2-hxdroxyethylpiperazine-N-2-ethanesulfonic acid - P_i inorganic phosphate - PTH parathyroid hormone - PS phosphatidylserine - TFP trifluoperazine - Tris Tris-(hydroxymethyl)aminomethane - TPA 12-O-tetradecanoylphorbol-13-acetate     

Unidirectional influx of phosphate across the mucosal membrane of rabbit small intestine

The influx of phosphate across the mucosal border of different regions of rabbit small intestine was investigated using the technique of Schultz et al., J. Gen. Physiol.50, 1241–1260 (1967). In the duodenum, the phosphate influx consisted of two components: 1. a saturable part, inhibited competitively by the presence of arsenate in the mucosal solution and strongly dependent on the mucosal Na concentration, and 2. a Na-independent part, linearly related to the mucosal phosphate concentration. In the jejunum and the ileum, the phosphate influx was a linear function of the mucosal phosphate concentration. In these regions arsenate had no effect on the influx, supporting the idea of a diffusional transport. HgCl₂ (0.5 mM) reduced the phosphate influx in the duodenum, at 140 mM Na, to the levels under Na-free conditions. The Na-independent influx was only slightly decreased by HgCl₂, suggesting that this agent affects mainly the Na-dependent phosphate influx. In the ileum HgCl₂ decreased the influx by about the same amount as under Na-free conditions in the duodenum. Thus, in the rabbit, the duodenum only appears to have a Na-dependent, carrier mediated phosphate transport mechanism at the mucosal membrane. In the jejunum and the ileum the phosphate uptake seems to be by simple diffusion.Presented in part at the Second Meeting of the European Intestinal Transport Group, Pont-à-Mousson, France, September 6–9, 1978. Abstract published in Gastroenterol. Clin. Biol.3, 173 (1979)