Biotin uptake mechanisms in brush-border and basolateral membrane vesicles isolated from rabbit kidney cortex

Biotin transport was studied using brush-border and basolateral membrane vesicles isolated from rabbit kidney cortex. An inwardly directed Na+ gradient stimulated biotin uptake into brush-border membrane vesicles and a transient accumulation of the anion against its concentration gradient was observed. In contrast, uptake of biotin by basolateral membrane vesicles was found to be Na+-gradient insensitive. Generation of a negative intravesicular potential by valinomycin-induced K+ diffusion potentials or by the presence of Na+ salts of anions of different permeabilities enhanced biotin uptake by brush-border membrane vesicles, suggesting an electrogenic mechanism. The Na+ gradient-dependent uptake of biotin into brush-border membrane vesicles was saturable with an apparent Km of 28 microM. The Na+-dependent uptake of tracer biotin was significantly inhibited by 50 microM biotin, and thioctic acid but not by 50 microM L-lactate, D-glucose, or succinate. Finally, the existence in both types of membrane vesicles of a H+/biotin- cotransport system could not be demonstrated. These results are consistent with a model for biotin reabsorption in which the Na+/biotin- cotransporter in luminal membranes provides the driving force for uphill transport of this vitamin.     

Bile-salt inhibition of sodium ion-coupled D-glucose and L-alanine accumulation by brush-border-membrane vesicles from hamster jejunum.

The effects of bile salts on Na+-coupled accumulation of D-glucose and L-alanine by brush-border-membrane vesicles isolated from hamster jejunum were investigated. The approximate percentage inhibition of Na+-coupled D-glucose accumulation produced by various bile salts at a concentration of 1 mM were: deoxycholate and chenodeoxycholate, 60%; glycine and taurine conjugates of deoxycholate and chenodeoxycholate, 40--50%; lithocholate, 45%; cholate and its glycine and taurine conjugates, less than 10%. Inhibition of Na+-coupled accumulation of D-glucose was rapid, reversible and not due to dissolution of the vesicles. Na+-coupled accumulation of L-alanine was also inhibited by deoxycholate. Deoxycholate but not cholate enhanced (1) the rate of Na+ influx, (2) the rate of influx of D-glucose and L-alanine in the absence of a Na+ gradient and (3) the rate of efflux of D-glucose and L-alanine from vesicles preloaded with this sugar or amino acid. Deoxycholate-stimulated efflux of D-glucose was not blocked by phlorizin, which completely prevented efflux in the absence of this bile salt. These results suggest that selected bile salts inhibit Na+-coupled accumulation of D-glucose and L-alanine by enhancing the rate of dissipation of the Na+ gradient required for substrate accumulation. In addition, bile salts may also decrease D-glucose and L-alanine accumulation by increasing the rate of efflux of these substrates across the brush-border plasma membrane.     

Amino acid transport in brush-border-membrane vesicles isolated from human small intestine.

Uptake of L-alanine and L-phenylalanine by purified bursh-border-membrane vesicles isolated from human small intestine was investigated by using a rapid-filtration technique. L-Alanine entered the same osmotically reactive space as D-glucose, indicating that transport into the vesicle rather than binding to the membranes was being observed. The uptake rate for L-alanine was higher in the presence of a Na+ gradient than in the presence of a K+ gradient. In the presence of a Na+ gradient, the lipophilic anion SCN- caused an increase in L-alanine transport, whereas the nearly impermeant SO42- anion decreased the uptake of L-alanine compared with its uptake in the presence of Cl-. The uptake of L-phenylalanine into the brush-border-membrane vesicle was also stimulated by Na+. The results indicate co-transport of Na+ and neutral amino acids inthe human intestinal brush-border membrane.     

Effect of parathyrin on the transport properties of isolated renal brush-border vesicles.

The transport properties of brush-border membrane vesicles isolated by a calcium-precipitation method from the renal cortex of normal and parathyrin (parathyroid hormone)-treated rats were studied by a rapid-filtration technique. Parathyrin elicited a dose-dependent decrease in the Na+-dependent phosphate uptake by the brush-border membrane vesicles, but the uptake of D-glucose, Na+ and mannitol was not affected. A maximum inhibition of 30% was observed after the application of 30 U.S.P. units intramuscularly 1 h before the animals were killed. Intravenous infusion of dibutyryl cyclic AMP (0.5-1.5 MG) also decreased the phosphate uptake by the brush-border vesicles. Both dibutyryl cyclic AMP and parathyrin were ineffective when added in vitro to brush-border membrane vesicles isolated from normal rats. These data suggest that parathyrin exerts its action on the phosphate reabsorption in the renal proximal tubule by affecting the Na+/phosphate co-transport system in the brush-border membrane. The effects of parathyrin on Na+ and glucose transport, however, seem to be due to alterations to the driving forces for transport and not to the brush-border transport systems.     

Enzymatic removal of alkaline phosphatase from renal brush-border membranes. Effect on phosphate transport and on phosphate binding

Brush-border membrane vesicles prepared from rabbit kidney cortex were incubated at 37 degrees C for 30 min with phosphatidylinositol-specific phospholipase C. This maneuver resulted in a release of approx. 85% of the brush-border membrane-linked enzyme alkaline phosphatase as determined by its enzymatic activity. Transport of inorganic [32P]phosphate (100 microM) by the PI-specific phospholipase C-treated brush-border membrane vesicles was measured at 20-22 degrees C in the presence of an inwardly directed 100 mM Na+ gradient. Neither initial uptake rates, as estimated from 10-s uptake values (103.5 +/- 6.8%, n = 7 experiments), nor equilibrium uptake values, measured after 2 h (102 +/- 3.4%) were different from controls (100%). Control and PI-specific phospholipase C-treated brush-border membrane vesicles were extracted with chloroform/methanol to obtain a proteolipid fraction which has been shown to bind Pi with high affinity and specificity (Kessler, R.J., Vaughn, D.A. and Fanestil, D.D. (1982) J. Biol. Chem. 257, 14311-14317). Phosphate binding (at 10 microM Pi) by the extracted proteolipid was measured. No significant difference in binding was observed between the two types of preparations: 31.0 +/- 9.37 in controls and 29.8 +/- 8.3 nmol/mg protein in the proteolipid extracted from PI-specific phospholipase C-treated brush-border membrane vesicles. It appears therefore that alkaline phosphatase activity is essential neither for Pi transport by brush-border membrane vesicles nor for Pi binding by proteolipid extracted from brush-border membrane. These results dissociate alkaline phosphatase activity, but not brush-border membrane vesicle transport of phosphate, from phosphate binding by proteolipid.     

D-glucose uptake in intestinal brush-border membrane vesicles of rachitic rats

We have previously reported the metabolic consequences of feeding rats Steenbock and Black's rickets-inducing diet, deficient in vitamin D and with an altered Ca/P ratio. Using isolated brush-border membrane vesicles prepared from the jejunum, ileum and duodenum of control and rachitic rats, we have demonstrated a marked decrease of Na+-dependent D-glucose uptake at jejunum-ileum level of rachitic rats. At duodenum level Na+-dependent D-glucose transport was not influenced by rickets. A lack of any significant difference between the two animal groups was observed studying the facilitated transport of D-glucose, the diffusion of L-glucose and the Na+-dependent uptake of phenylalanine and aspartate.     

Sugar transport by renal plasma membrane vesicles. Characterization of the systems in the brush-border microvilli and basal-lateral plasma membranes.

Uptake studies of D- and L-glucose were performed on vesicles derived from brush-border and basal-lateral membranes. The uptake of the sugars into the vesicles was osmotically sensitive and independent of glucose metabolism. In brush-border vesicles D-glucose but not L-glucose transport was Na+ -dependent, was inhibited by phlorizin, and showed a transitory vesicle/medium ratio greater than 1, in the presence of an initial Na+ gradient. Basal-lateral membranes take up D-glucose faster than L-glucose, but the D-glucose uptake is significantly less sensitive to sodium removal and only moderately inhibited by phlorizin as compared to the brush-border fraction.     

Effect of 25-hydroxyvitamin D3 on Na+-dependent D-glucose transport in rachitic rats

We have previously reported that feeding rats on Steenbock and Black's rickets-inducing diet, deficient in vitamin D and with an altered Ca/P ratio, leads to metabolic consequences and a marked decrease of Na+-dependent D-glucose uptake at the jejunum-ileum level. To clarify the relationship between experimental rickets and D-glucose uptake, 25-hydroxyvitamin D3 (25-OH-D3) was given to rats fed on the rickets inducing diet. In the jejunum-ileum of these animals Na+-dependent D-glucose uptake returned to the values of the controls while the decrease in D-glucose uptake in the brush-border membrane vesicles prepared from kidney cortex of rachitic animals was not corrected by the administration of 25-OH-D3.     

The mechanism of decreased Na+-dependent D-glucose transport in brush-border membrane vesicles from rabbit kidneys with experimental Fanconi syndrome

In our previous paper (Yanase, M. et al. (1983) Biochim. Biophys. Acta 733, 95-101) we reported that the Na+-dependent D-glucose uptake into brush-border membrane vesicles is decreased in rabbits with experimental Fanconi syndrome (induced by anhydro-4-epitetracycline). In the present paper we investigate the mechanism underlying this decrease. D-Glucose is taken up into the osmotically active space in anhydro-4-epitetracycline-treated brush-border membrane vesicles and exhibits the same distribution volume and the same degree of nonspecific binding and trapping as in control brush-border membrane vesicles. The passive permeability properties of control and anhydro-4-epitetracycline-treated brush-border membrane vesicles are shown to be the same as measured by the time-dependence of L-glucose efflux from brush-border membrane vesicles. D-Glucose flux was measured by the equilibrium exchange procedure at constant external and internal Na+ concentrations and zero potential. Kinetic analyses of Na+-dependent D-glucose flux indicate that Vmax in anhydro-4-epitetracycline-treated brush-border membrane vesicles (79.3 +/- 7.6 nmol/min per mg protein) is significantly smaller than in control brush-border membrane vesicles (141.3 +/- 9.9 nmol/min per mg protein), while the Km values in the two cases are not different from each other (22.3 +/- 0.9 and 27.4 +/- 1.8 mM, respectively). These results suggest that Na+-dependent D-glucose carriers per se are affected by anhydro-4-epitetracycline, and that this disorder is an important underlying mechanism in the decreased Na+-dependent D-glucose uptake into anhydro-4-epitetracycline-treated brush-border membrane vesicles.     

Hydrogen ion-coupled transport of D-glucose by phlorizin-sensitive sugar carrier in intestinal brush-border membranes

In rabbit intestinal brush-border membrane vesicles, Na+-independent D-glucose uptake in the presence of an inside-negative transmembrane potential was found to be stimulated by an imposed pH gradient. Na+-independent, pH-dependent and phlorizin-sensitive D-glucose-evoked potentials could be recorded from isolated toad intestine. The obtained data suggest that phlorizin-sensitive D-glucose carriers of intestinal brush-border membrane can interact with H+ when Na+ is absent.     

Towards the mechanism of altered nutrients uptake in renal brush border membrane vesicles from pyelonephritic rats

Affinity constant (Km) of D-glucose, L-alanine, L-aspartate, L-lysine, L-proline and nutrients coupled Na+ were determined in renal brush border membrane vesicles prepared from control and pyelonephritic rats. The Km of D-glucose, amino acids and nutrients coupled Na+ was noted to be significantly increased (p less than 0.001) in experimental animals. The Vmax of D-glucose and amino acids was determined at different concentrations of nutrients keeping extravesicular Na+ constant or at different concentrations of extravesicular Na+ keeping nutrient concentration constant. In the experimental rats the Vmax decreased significantly (p less than 0.01) when compared to control. The increased Km and decreased Vmax may be one of the underlying mechanism leading to decrease in the uptake of D-glucose and amino acids.     

Hydrolysis and transport of proline-containing peptides in renal brush-border membrane vesicles from dipeptidyl peptidase IV-positive and dipeptidyl peptidase IV-negative rat strains

In this investigation, we have demonstrated that the renal brush-border membrane of Fischer 344 rats from the Japanese Charles River Inc. specifically lacks dipeptidyl peptidase IV (DPP IV) activity, whereas the renal brush-border membrane of Fischer 344 rats from three different sources within the United States possesses normal levels of DPP IV activity. Comparison of the brush-border proteins between Charles River (U.S.A.) Fischer 344 rats (DPP IV positive) and Japanese Charles River Fischer 344 rats (DPP IV negative) revealed that a protein band (Mr = 100,000), apparently identical with DPP IV, was absent in the membranes from Japanese Charles River Fischer 344 rats. We examined the handling of radiolabeled beta-casomorphin fragment 1-5 (Tyr-Pro-[3H]Phe-Pro-Gly), a specific substrate for DPP IV, in renal brush-border membrane vesicles isolated from DPP IV-positive and DPP IV-negative rats. Although the membrane vesicles from DPP IV-positive rats were able to hydrolyze the pentapeptide to di- and tripeptides with the subsequent active transport of these products via the H+ gradient-dependent peptide transport system, the membrane vesicles from DPP IV-negative rats failed to hydrolyze the pentapeptide and hence lacked the ability to transport the radiolabel actively from the parent peptide. The H+ gradient-dependent glycyl-sarcosine uptake and the Na+ gradient-dependent proline uptake, however, were normal in DPP IV-negative rats. Urine analysis revealed that the DPP IV-negative rats excreted proline- and hydroxyproline-containing peptides in significantly increased amounts in their urine compared with control rats. Furthermore, following intravenous administration of Tyr-Pro-Phe-Pro-NH2, a peptide that is exclusively hydrolyzed by DPP IV, urinary excretion of the peptide in the intact form was many-fold greater in DPP IV-negative rats than in control rats. These data provide conclusive evidence for the obligatory role of DPP IV in the renal handling of proline (and hydroxyproline)-containing peptides.     

Increased Na(+)-dependent D-glucose transport and altered lipid composition in renal cortical brush-border membrane vesicles from bile duct-ligated rats.

Erythrocyte membranes of patients with liver disease are characteristically enriched in cholesterol, a change known to impair several carrier-mediated membrane transport functions. In the present study we have assessed whether experimental liver disease can affect the membrane lipid composition and transport function of kidney epithelial cells. Small (about 5%) but significant (P less than 0.01) increases were found in the cholesterol-to-phospholipid molar ratio (C/PL) of rat renal cortical brush-border membrane (BBM) vesicles 3, 8, and 15 days after bile duct ligation which correlated closely with increased fluorescence polarization, i.e., decreased membrane fluidity (r = 0.75, P less than 0.001; n = 27). A lipoprotein-mediated pathogenesis was suggested by the close relationship between BBM C/PL and plasma C/PL (r = 0.69, P less than 0.001). The mean high-affinity Na(+)-coupled D-glucose uptake by BBM vesicles was higher 1, 3, 8, and 15 days after ligation than in non-operated rats, significantly so at 3 and 8 days (611 +/- 37 and 593 +/- 22 vs. 507 +/- 21 pmol/mg protein per 4 sec; P less than 0.05), and was positively correlated with BBM C/PL (r = 0.58, P less than 0.01) and fluorescence polarization (r = 0.41, P less than 0.05). Brief incubation of BBM vesicles from normal rats with cholesterol-rich phospholipid liposomes simultaneously increased BBM C/PL and Na(+)-dependent D-glucose uptake. Stimulation of BBM Na(+)-glucose cotransport in ligated rats was not due to delayed dissipation of the Na+ gradient or to a more rapid development of membrane potential. High-affinity Na(+)-dependent D-glucose uptake kinetics in 3-day bile duct-ligated rats showed a lower Kt, without an alteration in maximum velocity, Vmax, compared to sham-operated animals (0.298 +/- 0.015 vs. 0.382 +/- 0.029 mM; P less than 0.05), whilst the binding dissociation constant, Kd of high-affinity phlorizin binding sites was reduced by ligation (0.453 +/- 0.013 vs. 0.560 +/- 0.015 microM; P less than 0.001). We conclude that an early effect of bile duct ligation is to enrich renal cortical brush-border membranes in cholesterol, thereby decreasing membrane fluidity and stimulating Na(+)-dependent D-glucose uptake by increasing the affinity of the carrier.     

Regulation of amino acid and glucose transport activity expressed in isolated membranes from untransformed and SV 40-transformed mouse fibroblasts.

Membrane vesicles isolated from untransformed Balb/c and Swiss mouse fibroblasts and their SV 40-transformed derivatives were shown to catalyze carrier-mediated, intravesicular uptake of alpha-aminoisobutyric acid and D-glucose. Concentrative uptake of alpha-aminoisobutyric acid required the presence of a Na+-gradient (external greater than internal) and could occur independently of endogenous (Na+ + K+)ATPase activity. A K+ diffusion gradient (internal greater than external) in the presence of valinomycin, or the addition of the Na+ salt of a highly permeant anion, conditions expected to create an interior-negative membrane potential stimulated Na+-gradient-dependent uptake, suggesting this process is electrogenic. D-Glucose uptake was nonconcentrative and did not require ion gradients or metabolic conversion. Na+ gradient-dependent transport of alpha-aminoisobutyric acid was reduced both in initial rate and extent of uptake in vesicles from confluent untransformed cells and increased in those from SV 40-transformed cells, compared with activities observed in vesicles from proliferating untransformed cells. No changes in D-glucose carrier activity were observed when assayed at low glucose concentrations.     

1-O-n-octyl-beta-D-glucopyranoside as a competitive inhibitor of Na+-dependent D-glucose cotransporter in the small intestine brush-border membrane

1-O-n-Octyl-beta-D-glucopyranoside is a competitive inhibitor of the Na+-dependent D-glucose uptake into rabbit, rat and human intestinal brush-border membrane vesicles. The lack of effect on the equilibrium uptake demonstrates that the detergent does not act by rupturing the vesicles; no membrane leakiness was apparent at the concentrations of octylglucopyranoside used, since D-glucose uptake is not inhibited even in the absence of the Na+ gradient (in K+ solution). There is a competitive interaction between octylglucopyranoside and D-glucose, as shown by Dixon and by Hunter and Down plots. The selectivity of the detergent effect is confirmed by its modest influence on amino acid uptake.     

1,25-Dihydroxycholecalciferol-related Na+/D-glucose transport in brush-border membrane vesicles from embryonic chick jejunum. Modulation by triiodothyronine

1,25-Dihydroxycholecalciferol, when present at and above 10 nM in an organ-culture system of embryonic chick jejunum, approximately doubled the rate of Na(+)-gradient-driven D-glucose uptake by brush-border membrane vesicles, but had no effect on Na(+)-independent D-glucose transfer. The sterol also had no effect on Na+ influx along an outside/inside Na+ gradient ([Na+]o = 100 mM; [Na+]i = 0 mM). This renders it unlikely that in embryonic intestine, calcitriol raises Na(+)-dependent D-glucose transport through changes in the electrochemical Na+ gradient. D-[U-14C]Glucose tracer exchange, measured under voltage-clamp condition at Na+/D-glucose equilibrium, revealed that addition of calcitriol to the culture medium approximately doubled the activity of the Na+/D-glucose transporter in the brush-border membrane. This was also reflected by an corresponding increase in the maximal velocity of the transfer process. Increased [3H]phlorizin binding after calcitriol treatment suggests that the steroid hormone activates Na+/D-glucose transport through increasing the number of carrier molecules in the brush-border membrane. 10 nM triiodothyronine, which by itself has no effect on Na(+)-dependent D-glucose transport, potentiated the effect of 1,25-dihydroxycholecalciferol such that in the presence of both hormones, Na+/D-glucose-carrier activity was increased fourfold above control levels.     

Influence of amino acid side-chain modification on the uptake system for beta-lactam antibiotics and dipeptides from rabbit small intestine

The influence of chemical modification of functional amino acid side-chains in proteins on the H(+)-dependent uptake system for orally active alpha-amino-beta-lactam antibiotics and small peptides was investigated in brush-border membrane vesicles from rabbit small intestine. Neither a modification of cysteine residues by HgCl2, NEM, DTNB or PHMB and of vicinal thiol groups by PAO nor a modification of disulfide bonds by DTT showed any inhibition on the uptake of cephalexin, a substrate of the intestinal peptide transporter. In contrast, the Na(+)-dependent uptake systems for D-glucose and L-alanine were greatly inhibited by the thiol-modifying agents. With reagents for hydroxyl groups, carboxyl groups or arginine the transport activity for beta-lactam antibiotics also remained unchanged, whereas the uptake of D-glucose and L-alanine was inhibited by the carboxyl specific reagent DCCD. A modification of tyrosine residues with N-acetylimidazole inhibited the peptide transport system and did not affect the uptake systems for D-glucose and L-alanine. The involvement of histidine residues in the transport of orally active alpha-amino-beta-lactam antibiotics and small peptides (Kramer, W. et al. (1988) Biochim. Biophys. Acta 943, 288-296) was further substantiated by photoaffinity labeling studies using a new photoreactive derivative of the orally active cephalosporin cephalexin, 3-[phenyl-4-3H]azidocephalexin, which still carries the alpha-amino group being essential for oral activity. 3-Azidocephalexin competitively inhibited the uptake of cephalexin into brush-border membrane vesicles. The photoaffinity labeling of the 127 kDa binding protein for beta-lactam antibiotics with this photoprobe was decreased by the presence of cephalexin, benzylpenicillin or dipeptides. A modification of histidine residues in brush-border membrane vesicles with DEP led to a decreased labeling of the putative peptide transporter of Mr 127,000 compared to controls. This indicates a decrease in the affinity of the peptide transporter for alpha-amino-beta-lactam antibiotics by modification of histidine residues. The data presented demonstrate an involvement of tyrosine and histidine residues in the transport of orally active alpha-amino-beta-lactam antibiotics across the enterocyte brush-border membrane.     

Factors affecting the transport of beta-amino acids in rat renal brush-border membrane vesicles. The role of external chloride

The effect of a variety of ions and other solutes on the accumulation of the beta-amino acid, taurine, was examined in rat renal brush-border membrane vesicles. Initial taurine uptake (15 and 30 s) is sodium-dependent with a typical overshoot. This Na+ effect was confirmed by exchange diffusion and gramicidin inhibition of taurine uptake. External K+ or Li+ do not increase taurine accumulation more than Na+-free mannitol, except that the combination of external K+ and Na+ in the presence of nigericin enhances uptake. Of all anions tested, including more permeant (SCN- and NO3-) or less permeant (SO4(2-)), chloride supported taurine accumulation to a significantly greater degree. Preloading vesicles with choline chloride reduced taurine uptake, suggesting that external Cl- stimulates uptake. Since this choline effect could be related to volume change, due to the slow diffusion of choline into vesicles, brush-border membrane vesicles were pre-incubated with LiCl, LiNO3 and LiSO4. Internal LiCl, regardless of the final Na+ anion mixture, reduced initial rate (15 and 60 s) and peak (360 s) taurine uptake. Internal LiNO3 or LiSO4 with external NaCl resulted in similar or higher values of uptake at 15, 60 and 360 s, indicating a role for external Cl- in taurine uptake in addition to Na+ effect. Although uptake by vesicles is greatest at pH 8.0 and inhibited at acidic pH values (pH less than 7.0), an externally directed H+ gradient does not influence uptake. Similarly, amiloride, an inhibitor of the Na+/H+ antiporter, had no influence on taurine accumulation over a wide variety of concentrations or at low Na+ concentrations. Taurine uptake is blocked only by other beta-amino acids and in a competitive fashion. D-Glucose and p-aminohippurate at high concentrations (greater than 10(-3) M) reduce taurine uptake, possibly by competing for sodium ions, although gramicidin added in the presence of D-glucose inhibits taurine uptake even further. These studies more clearly define the nature of the renal beta-amino acid transport system in brush-border vesicles and indicate a role for external Cl- in this uptake system.     

Insulin regulates Na+/glucose cotransporter activity in rat small intestine

In order to examine the involvement of insulin in the activity of Na+/glucose cotransporter in rat small intestine, we compared Na(+)-dependent uptake of D-glucose by brush-border membrane vesicles prepared from control, streptozotocin-induced diabetic, insulin-treated diabetic and starved diabetic rats. In four groups, the uptake of D-glucose showed a transient overshoot in the presence of Na+ gradient between medium and vesicles (medium greater than vesicles). The overshoot magnitude was increased (1.8-fold of controls) in diabetic brush border membrane vesicles and recovered to the control level by the treatment of diabetic rats with insulin. In contrast, increased uptake of D-glucose in diabetic rats was not recovered by the starvation of diabetic rats although the blood glucose level was the same as that of controls. Furthermore, we attempted to examine phlorizin binding activities among four groups. Scatchard analysis indicated that phlorizin binding to diabetic brush border membrane vesicles was increased (1.6-fold of controls) without a change of the affinity for phlorizin as compared with controls. Increased binding of phlorizin to diabetic brush border membrane vesicles was also recovered to the control level by the treatment of diabetic rats with insulin, but not by starvation. These results suggested that the increased activity of Na+/glucose cotransporter in diabetic rats was due to the increase of the number of cotransporter and that intestinal cotransporter was physiologically controlled by insulin, but not by blood glucose levels.     

Cyclic AMP-dependent protein phosphorylation in canine renal brush-border membrane vesicles is associated with decreased phosphate transport

It is known that the administration of parathyroid hormone to dogs results in phosphaturia and decreased phosphate transport in brush-border vesicles isolated from the kidneys of those dogs. Parathyroid hormone has been shown to activate adenylate cyclase at the basal-lateral membrane of the renal proximal tubular cell. It has been postulated that parathyroid hormone-induced phosphaturia is effected through phosphorylation of brush-border protein by membrane-bound cAMP-dependent protein kinase. An experimental system was designed such that phosphorylation of brush-border vesicles and Na+-stimulated solute transport could be studied in the same preparations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane vesicles revealed cAMP-dependent phosphorylation of 2 protein bands (Mr = 96,000 and 62,000), which was enhanced by exposure of the inside of the membrane vesicles to ATP and cAMP. Cyclic AMP-dependent phosphorylation of brush-border vesicles was accompanied by inhibition of Na+-stimulated Pi but not D-glucose transport or 22Na+ uptake. When renal brush-border vesicles from parathyroidectomized and normal dogs were phosphorylated in vitro in the presence and absence of cAMP, both the cAMP-dependent phosphorylation and inhibition of Na+-stimulated Pi transport were greater in vesicles isolated from kidneys of parathyroidectomized dogs relative to control animals. We conclude that the cAMP-dependent phosphorylation of brush-border membrane-vesicle proteins is associated with specific inhibition of Na+-stimulated Pi transport. The phosphaturic action of parathyroid hormone (PTH) could be mediated through the cAMP-dependent phosphorylation of specific brush-border membrane proteins.