Experssion of chronic thyroparathyroidectomy on phosphate transport in whole kidney and proximal luminal membranes during phosphate deprivation

Both parathyroid hormone (PTH) and the dietary supply of inorganic phosphate (Pi) influence the tubular Pi reabsorptive capacity. In this study we have assessed how removal of parathyroid gland affects the response of Pi transport to Pi restriction in the whole kidney and in brush border membrane vesicles (BBMV) isolated from the renal cortex. Intact and thyroparathyroidectomized (TPTX) rats were switched from normal to low Pi diet. The maximal tubular Pi reabsorption per volume of glomerular filtrate (max. TRPi/ml GF) and the initial (3, 6 and 9 s) Na⁺-dependent Pi uptake by BBMV were determined before and after 1, 2, 3 and 8 days of Pi restriction. The results indicate that before Pi restriction max. TRPi/ml GF was 1.94±0.04 and 4.03±0.14 mol/ml GF in intact and TPTX rats respectively. The corresponding Na⁺-dependent Pi uptake (3 s value) was 137±23 in sham and 272±41 pmol/mg protein in TPTX rats. During Pi restriction the initial difference in max. TRPi/ml GF remained constant throughout the time course study. At 8 days max. TRPi/ml GF was in intact 3.08±0.09 and in TPTX 5.04±0.18 mol/ml GF. In sharp contrast, in BBMV the initial difference between intact and TPTX became completely abolished 8 days after starting the Pi restriction (SHAM=425±69; TPTX=434±pmol/mg protein). In conclusion, the overall tubular Pi transport capacity and the Na⁺-dependent Pi transport system located in the luminal membrane of the proximal tubule appear to be quite differently affected by changes in the PTH status during Pi restriction.     

Effect of diphosphonate treatment on phosphate transport by renal brush border vesicles

Treatment with disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) given in doses of 10 mg P/kg s.c. for 7 days inhibits bone mineral retention and decreases the tubular capacity to reabsorb inorganic phosphate (Pi) in thyroparathyroidectomized (TPTX) rats. In the present work we show that pretreatment with EHDP depressed Na+-dependent Pi uptake by brush border membrane vesicles (BBMV) isolated from renal cortex of TPTX rats. The effect of EHDP was observed after feeding both high (1.2%) and low (0.2%) phosphorus diets. The EHDP-induced changes observed in vitro at the brush border level parallel the variations in the overall tubular Pi transport capacity as assessed by clearance techniques in conscious rats. Na+-dependent glucose uptake by BBMV, as well as alkaline phosphatase activity in cortical homogenates and in the BBMV were not affected by EHDP treatment. Accordingly, EHDP, given in doses that block bone mineral retention, appears to specifically affect the Na+-dependent transport of Pi across the luminal membrane of proximal tubules of renal cortex.     

Tubular adaptation to Pi restriction in hypophysectomized rats

The renal tubule adapts its tubular transport capacity for inorganic phosphate (Pi) in response to a reduction in the Pi supply. In order to assess whether growth hormone plays a critical role in this adaptive response we have studied the change in the tubular handling of Pi which follows Pi restriction in hypophysectomized (HPX) rats and compared it to that occurring in intact counterparts. HPX and intact rats were maintained either on a 1.2 g/100 g phosphorus diet or fed a 0.2 g/100 g phosphorus diet for 3, 6 or 12 days. HPX rats received ACTH and thyroxine in doses which normalize their low glomerular filtration rate (GFR). Then the maximal net Pi reabsorption per volume of glomerular filtrate (max. TRPi/ml GFR) were determined during acute Pi infusion by clearance technique. The results indicate that HPX rats responded to Pi restriction by raising their tubular capacity to reabsorb Pi. However, the rapidity and the magnitude of the adaptive response was significantly less in HPX than in intact rats. The adaptation to Pi restriction was also observed in HPX rats after thyroparathyroidectomy. It is concluded that growth hormone and other pituitary hormones do not play a key role in the adaptive response to Pi restriction. The reduced adaptive response observed in HPX rats with intact thyroparathyroid glands could be due to the decreased Pi demand consecutive to impaired growth.     

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.     

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.     

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.     

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]     

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     

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     

Secreted frizzled-related protein-4 reduces sodium–phosphate co-transporter abundance and activity in proximal tubule cells

The phosphatonin, secreted frizzled-related protein-4 (sFRP-4), induces phosphaturia and inhibits 25-hydroxyvitamin D 1-hydroxylase activity normally induced in response to hypophosphatemia. To determine the mechanism by which sFRP-4 alters renal phosphate (P_i) transport, we examined the effect of sFRP-4 on renal brush border membrane (BBMV) Na⁺-dependent P_i uptake, and the abundance and localization of the major Na⁺–P_i-IIa co-transporter in proximal tubules and opossum kidney (OK) cells. Infusion of sFRP-4 increased renal fractional excretion of P_i and decreased renal -catenin concentrations. The increase in renal P_i excretion with sFRP-4 infusion was associated with a 21.9±3.4% decrease in BBMV Na⁺-dependent P_i uptake (P<0.001) compared with a 39.5±2.1% inhibition of Na⁺-dependent P_i transport in renal BBMV induced by PTH (P<0.001). sFRP-4 infusion was associated with a 30.7±4.8% decrease in Na⁺–P_i-IIa co-transporter protein abundance (P<0.01) assessed by immunoblotting methods compared to a 45.4±8.8% decrease induced by PTH (P<0.001). In OK cells, sFRP-4 reduced surface expression of a heterologous Na⁺–P_i-IIa co-transporter. We conclude that sFRP-4 increases renal P_i excretion by reducing Na⁺–P_i-IIa transporter abundance in the brush border of the proximal tubule through enhanced internalization of the protein.     

Sodium-taurine cotransport in reptilian renal brush-border membrane vesicles

The coupled transport of Na⁺ with taurine into snake renal brush-border membrane vesicles (BBMV) was studied using 5-s uptake conditions. Taurine transport into snake renal BBMV involved two parallel processes, one saturable (Na⁺-dependent) and one (Na⁺-independent) that behaved like passive diffusion. Below 1 mM taurine concentration, the Na⁺-dependent system accounted for 60% of total taurine uptake. Over both low (0.001–0.80 mM) and high (0.8–5.0 mM) taurine concentration ranges, the Na⁺-dependent taurine uptake within each range showed Michaelis-Menten kinetics, suggesting the presence of two independent saturable Na⁺-dependent transport systems for taurine. The high-affinity, low-capacity system saturated above 100 M with a K _m of 71.4±45.7 M and a maximum velocity (V _max) of 21.9±3.77 pmol (mg protein)^–1 (5 s)^–1. The low-affinity, high-capacity system saturated above 1 mM, with a K _m of 1.11±0.63 mM and a V _max of 252±47 pmol (mg protein)^–1 (5 s)^–1. The stoichiometric relationship between external Na⁺ concentration and taurine uptake (at 10 M) by the high-affinity BBMV transport system was examined by the activation method under short-circuited conditions. The 5-s rate of taurine transport was a sigmoid function of increasing extravesicular Na⁺ concentration. Kinetic analysis of the interaction of Na⁺ with the high-affinity taurine transport system suggested that 3 Na⁺ ions (3.2±0.7) may be involved with 1 taurine molecule in the transport event. The data suggest the presence of a highly efficient and high-affinity reabsorptive taurine transport system on the luminal membrane of the kidney of the garter snake, a species that can secrete taurine in vivo.     

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.     

Tetraethylammonium transport by snake renal brush-border membrane vesicles

Transport of tetraethylammonium (TEA) by snake (Thamnophis spp.) renal brush-border membrane vesicles (BBMV) was studied. An outwardly directed proton gradient (pH 6.0 in, pH 7.5 out) stimulated uptake of TEA into BBMV and supported concentrative accumulation. Uptake of radioactively labeled TEA was also stimulated by outwardly directed gradients of unlabeled TEA and choline. The initial rate of TEA uptake was a saturable process that was adequately described by Michaelis-Menten kinetics. TEA uptake was not influenced by changes in the electrical potential difference across the membranes. Although uptake of TEA was stimulated by an outwardly directed Na⁺ gradient and inhibited by an inwardly directed Na⁺ gradient, these effects were probably secondary to the generation of proton gradients via a Na⁺/H⁺ exchanger demonstrated in these same BBMV. In agreement with previous studies with intact snake renal tubules, the present results indicate that TEA transport across the brush-border membrane involves electroneutral countertransport for protons or organic cations.     

Relation between intestinal alkaline phosphatase activity and brush border membrane transport of inorganic phosphate,D-glucose,andD-glucose-6-phosphate

In order to evaluate the role of the alkaline phosphatase in intestinal transport processes, we studied the influence of known modulators of the alkaline phosphatase (polyclonal anti-calf AP antibodies, theophylline and zinc ions) on the absorption rate of glucose, inorganic phosphate and glucose liberated from glucose-6-phosphate into calf duodenal brush border membrane vesicles. Our results allow the following conclusions: First a direct involvement of the AP in the Na⁺-dependent glucose absorption is unlikely. Indeed, theophylline inhibits strongly the AP activity but rather stimulates the glucose uptake; second the AP is indirectly involved in glucose absorption from glucose-6-phosphate, if its enzymatic hydrolysis is the only source of glucose. In that case the Na⁺-dependent uptake of glucose was completely suppressed either by phosphatase specific antibodies or by theophylline; third the positive correlation found with calf intestinal BBMV between the inhibition of AP by AP antibodies or by theophylline and the decrease of rate of Na⁺-dependent P_i uptake rate suggests that the enzyme plays some role in the P_i absorption.It appears from the present study that the AP is probably not a carrier protein itself, but its hydrolytic activity might nevertheless be important for intestinal absorption. After hydrolysis of phosphoric esters the alcohol residues and P_i can be supplied to their specific carriers. Furthermore, the high P_i affinity of the enzyme at physiological pH values, could even favour a transient sequestration of phosphate, which then could be transferred to the P_i carrier.Abbreviations used AP alkaline phosphatase - pNPP paranitrophenylphosphate - Gle-6-ph glucose-6-phosphate - P phosphate ion - IgG protein A purified immunoglobulins G - TCA trichloracetic acid - DEA diethanolamine - MgAc₂ magnesium acetate - ZnAc₂ zinc acetate - BBMV brush border membrane vesicles - 1,25(OH)₂D₃ 1,25-dihydroxycholecalciferol - Mannitol-buffer buffer-containing per liter 300 mmol mannitol and 10 mmol HEPES/Tris, pH 7.4     

Pi transport, phosphorylation, and dephosphorylation in renal membranes from HYP/Y mice

To ascertain whether cAMP-dependent phosphorylation could be demonstrated in brush border membrane vesicles (BBMV) isolated from kidneys of mice with X-linked hypophosphatemic rickets (HYP/Y) and normal littermates (+/Y) and, if so, to determine whether the absence of dephosphorylation might underlie differences in Na+-dependent 32Pi transport in BBMV, we measured 1) 32Pi transport, 2) cAMP-dependent phosphorylation, and 3) dephosphorylation in BBMV from +/Y and HYP/Y mice. Na+ gradient-dependent 32Pi transport was decreased in BBMV from HYP/Y mice as reflected in a decreased apparent Vmax. cAMP-dependent phosphorylation of a 62,000 Mr protein was demonstrated in sodium dodecyl sulfate polyacrylamide gels of BBMV from +/Y and HYP/Y mice and was associated with decreased Na+-dependent 32Pi transport. Dephosphorylation of the 62,000 Mr band was demonstrable in both types of membranes. Thus, both cAMP-dependent protein kinase and phosphoprotein phosphatase activities were demonstrable in BBMV isolated from +/Y and HYP/Y mice. These results are consistent with the renal tubular defect in the HYP/Y mouse reflecting an intrinsic abnormality of Pi transport in the brush border membrane independent from mediation of the phosphaturic effect of parathyroid hormone.     

Stimulation of enterocyte enzymatic activities,MHC class II expression and other immunological factors after oral treatment with Nocardia delipidated cell mitogen in germ-free rats

Nocardia delipidated cell mitogen (NDCM) was given intragastrically (200 μg/animal) to 2-month-old germ-free (GF) and conventionally (CV) reared AVN rats. On day 4, enzymatic activities of enterocyte brush border membrane vesicles (BBMV) isolated from jejunal scraping were measured. The results indicated that activities of sucrase, lactase and glucoamylase in BBMV were stimulated following NDCM treatment. The most pronounced increase of enzymatic activities was found in GF rats. In contrast to CV rats, GF rats do not express MHC class II on epithelial cells during their life. NDCM treatment induced MHC class II expression in enterocytes from GF rats. The levels of IgA and IgG in sera from NDCM-treated CV rats did not change significantly. The level of IgG in sera of GF rats was found to be enhanced after NDCM treatment. Markedly increased incorporation of ³H-thymidine by spleen lymphocytes stimulated with Con A in vitro was observed only in NDCM-treated GF rats. ³H-uridine incorporation into Con A-stimulated lympocytes of GF rats was decreased after NDCM treatment.     

Mechanism of Cl− transport in eel intestinal brush-border membrane vesicles

Cl^– transport was studied in a preparation of brush-border membrane vesicles (BBMV) from seawater eel intestine. ³⁶Cl^– uptake appeared to be stimulated by a positive inside membrane diffusion potential generated (a) by a concentration gradient of salts, the cations of which are more permeable than the anions, (b) by a K⁺ diffusion potential obtained by imposing a K⁺ concentration gradient (C _out>C_in) in the presence of valinomycin, (c) an inwardly directed H⁺ ion concentration gradient. The membrane-potential-driven Cl^– transport was inhibited by 1 mM 5-nitro-2-(4-phenylpropylamino)-benzoic acid. Arachidonic acid also inhibited Cl^– uptake in eel intestinal BBMV, but the effect appeared to be unspecific, as the unsaturated fatty acid also affected the Na⁺ dependent D-glucose uptake. The effect of arachidonic acid was reversed in the presence of bovine serum albumin. Cl^– influx was the same in the presence of inwardly directed gradients of Li⁺, Na⁺ or K⁺, arguing against the presence of Na⁺-Cl^–, as well as K⁺-Cl^– cotransport. The absence of a significant contribution of the Na⁺-K⁺-2Cl^– cotransport mechanism to the Cl^– uptake in seawater eel intestinal BBMV was indicated from the observations that Cl^– uptake was not stimulated by the simultaneous presence of inwardly directed Na⁺ and K⁺ gradients, and that it was nearly insensitive to 1 mM bumetanide in the presence of extravesicular Na⁺ and K⁺. Furthermore, no evidence for the dependence of Cl^– uptake on the Na⁺ gradient was obtained under a short-circuited membrane diffusion potential, i.e. in the presence of equilibrated K⁺ and valinomycin. The finding that the Cl^– uptake in the presence of a H⁺ gradient was not inhibited by 1 mM SITS and was significantly reduced in the presence of [K⁺]_in=[K⁺]_out and valinomycin, suggests that no anion exchanger is present in our experimental system. We conclude that Cl^– uptake in eel intestinal BBMV does not occur via an electroneutral Na⁺-dependent Cl^– transport mechanism (either cotransport or double exchange) and is realized by a Cl^– conductance.     

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.     

Compensatory regulation of the sodium/phosphate cotransporters NaPi-IIc (SCL34A3) and Pit-2 (SLC20A2) during Pi deprivation and acidosis

The role of four Pi transporters in the renal handling of Pi was analyzed using functional and molecular methods. The abundance of NaPi-IIa, NaPi-IIc, and Pit-2 was increased by 100% in kidney from rats on a 0.1% Pi diet, compared to a 0.6% Pi diet. Pit-1 was not modified. Type II-mediated Pi uptake in Xenopus oocytes increased as the pH of the uptake medium increased, and the opposite occurred with Pit-1 and Pit-2. At pH 6.0, Pi uptake mediated through type II was ≈10% of the uptake at pH 7.5, but the uptake through Pit-2 was 250% of the activity at pH 7.5. Real brush-border membrane vesicles (BBMV) responded to pH changes following the same pattern as type II transporters. Adaptation to a 0.1% Pi diet was accompanied by a 65% increase in the V _max of BBMV Pi transport at pH 7.5, compared to a 0.6% Pi diet. The increase was only 11% at pH 6.0. Metabolic acidosis increased the expression of NaPi-IIc and Pit-2 in animals adapted to a low Pi diet, and phosphaturia was only observed in control diet animals. The combination of the pH effect, Pi adaptation, and metabolic acidosis suggests very modest involvement of Pit-2 in renal Pi handling. Real-time PCR and mathematical analyses of transport findings suggest that NaPi-IIa RNA accounts for 95% of all Pi transporters and that type II handles 97% of Pi transport at pH 7.5 and 60% of Pi transport at pH 6.0, depending on the pH and the physiological conditions.     

Expression of rat ileal Na+-sulphate cotransport in Xenopus laevis oocytes: functional characterization

Small-intestinal sulphate absorption is a Na⁺-dependent process having its highest rate in the ileum; it involves brush-border membrane Na⁺-sulphate cotransport. Injection of rat ileal mRNA into Xenopus laevis oocytes induced Na⁺-dependent sulphate uptake in a dose-dependent manner, with no apparent effect on Na⁺-independent sulphate uptake. For mRNA-induced transport, the apparent K _m value for sulphate interaction was 0.6±0.2 mM and that for sodium interaction was 25±2 mM (Hill coefficient: 2.3±0.3). mRNA-induced transport, was inhibited by thiosulphate, but not by phosphate or 4,4,′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS). Using a rat renal Na⁺-sulphate cotransporter cDNA as a probe [NaSi-1; Markovich et al. (1993) Proc Natl Acad Sci USA 90:8073–8077], the highest hybridization signals (2.3 kb and 2.9 kb) were obtained in size fractions showing the highest expression of Na⁺-dependent sulphate transport in oocytes. Hybrid depletion experiments using antisense oligonucleotides (from the NaSi-1 cDNA sequence), provided further evidence that rat small-intestinal (ileal) Na⁺-sulphate cotransport is closely related to rat proximal-tubular brushborder membrane Na⁺-sulphate cotransport.