Kidney Brush-border Membrane Vesicles Research Articles

Phosphate transport in kidneys: effect of transmembrane electrical potential

The effect of a transmembrane electrical potential on phosphate transport by kidney brush-border membrane vesicles was studied. The initial rate of Na(+)-dependent phosphate influx was twice as high as that of efflux. Generation of a negative transmembrane potential had a stimulatory effect on the rate of influx but had no effect on efflux. The Na+ saturation curve for phosphate influx was sigmoidal, and the Hill coefficients were similar, in the presence and absence of a transmembrane potential. The membrane potential increased both the affinity for phosphate and the maximal velocity (Vmax) of the transporter. In the absence of a Na+ gradient, the stimulation by the potential was 1.78-fold. When a proton gradient (in greater than out) was the driving force, the electrical potential stimulated phosphate transport 1.71-fold. Internal Na+ (trans) inhibited phosphate influx whether a potential was present or not. Internal phosphate (trans) stimulated phosphate influx in the absence of a potential but not in its presence. These results indicate that the electrical potential is an important driving force for the Na(+)-phosphate carrier and that the translocation of the carrier is a potential-dependent step.

Analysis of the pH Dependence of Folate Binding and Transport by Rat Kidney Brush Border Membrane Vesicles

Folate reabsorption by the mammalian kidney occurs following a tight binding reaction with the renal brush border membrane. Previous studies have shown that transport of folic acid (PteGlu) by rat kidney brush border membrane vesicles occurs maximally at pH 5.6 via a saturable system that is associated with a binding component. The present studies have shown that the pH dependency of transport was due to the development of the transmembrane pH gradient (7.3 in/5.6 out), not to the acidic pH per se. The pH gradient-mediated transport was stimulated by an inwardly directed ionic gradient, either of NaCl or choline chloride. These gradients also stimulated the membrane binding of PteGlu suggesting that NaCl and choline chloride may have increased PteGlu transport by altering binding to the brush border membrane. Renal brush border membrane vesicular transport of PteGlu was not affected by induction of a relatively positive intravesicular space. Transport was inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, an anion exchange inhibitor. The results suggest that rat kidney brush border membrane transport of PteGlu is initiated by association with a specific membrane protein, followed by transfer of folate across the membrane. The overall activity is influenced by a transmembrane pH gradient.

Comparison of GABA uptake by brain and kidney preparations

The uptake ofγ-aminobutyric acid (GABA) was measured in rat cerebral cortical synaptosomes and rat kidney brush-border membrane vesicles (BBMV). Three GABA uptake systems (Km = 1.3, 50 and 3246µM respectively) were present in synaptosomes, but only two uptake systems (Km = 11 and 1203µM respectively) were detectable in BBMV. The uptake systems in the two types of tissue preparations were similar in that every system was inhibited byp-hydroxymercuribenzoate and by the action of neuraminidase, thereby indicating that, irrespective of the tissue, both sulfhydryl and sialyl groups were necessary components of all the GABA uptake systems. In contrast, differences were observed between synaptosomal and BBMV uptake systems with respect to their sensitivity to inhibition by GABA structural analogs. While the synaptosomal GABA uptake systems were strongly inhibited by nipecotic acid, diaminobutyric acid,β-alanine and 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO), none of these compounds significantly inhibited the BBMV uptake systems. It is therefore concluded that the GABA uptake systems in brain and kidney tissues are quite different entities. While the role of GABA transport in the inactivation of the neurotransmitter function of GABA is well documented, the role of the kidney GABA uptake system is unclear, and the possibility exists that the latter systems are present in kidney tissues primarily to transport compounds other than GABA. The present study does however highlight a difference in drug susceptibility of the brain and kidney uptake systems, a phenomenon which may have some therapeutic relevance with respect to the use of GABA analogs as anticonvulsant agents.

Vitamin B6 Uptake by Rat Kidney Cells and Brush‐Border Membrane Vesicles

Annals of the New York Academy of SciencesVolume 585, Issue 1 p. 106-109 Vitamin B6 Uptake by Rat Kidney Cells and Brush-Border Membrane Vesicles BARBARA B. BOWMAN, BARBARA B. BOWMAN Department of Biochemistry Emory University School of Medicine Atlanta, Georgia 30322Search for more papers by this authorDONALD B. McCORMICK, DONALD B. McCORMICK Department of Biochemistry Emory University School of Medicine Atlanta, Georgia 30322Search for more papers by this authorELIZABETH R. SMITH, ELIZABETH R. SMITH Department of Biochemistry Emory University School of Medicine Atlanta, Georgia 30322Search for more papers by this author BARBARA B. BOWMAN, BARBARA B. BOWMAN Department of Biochemistry Emory University School of Medicine Atlanta, Georgia 30322Search for more papers by this authorDONALD B. McCORMICK, DONALD B. McCORMICK Department of Biochemistry Emory University School of Medicine Atlanta, Georgia 30322Search for more papers by this authorELIZABETH R. SMITH, ELIZABETH R. SMITH Department of Biochemistry Emory University School of Medicine Atlanta, Georgia 30322Search for more papers by this author First published: May 1990 https://doi.org/10.1111/j.1749-6632.1990.tb28046.xCitations: 7AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume585, Issue1Vitamin B6May 1990Pages 106-109 RelatedInformation

Sulfated bile acids inhibit Na(+)-H+ antiport in human kidney brush-border membrane vesicles

Patients with obstructive jaundice are at a high risk for acute renal failure after surgery. Direct toxic membrane effects of bile acids or bilirubin have been discussed as possible causes. Therefore, we investigated the influence of bile acids and conjugated bilirubin on Na(+)-H+ antiport and ion permeabilities in brush-border membrane vesicles isolated from the human kidney. Brush-border membrane vesicles were prepared by Mg2+ precipitation. These were highly purified as estimated from the 14-fold enrichment in the specific activity of alanine aminopeptidase. The pH-sensitive dye acridine orange was used to study the properties of proton uptake under different conditions. The brush-border membrane vesicles from human kidney cortex exhibited Na+ and K+ conductances, which were small compared with H+ conductance. Furthermore, these membranes possess an Na(+)-H+ antiporter that is sensitive to amiloride. Various bile acids (30 microM) had no significant effect on Na(+)-H+ antiport. However, the addition of sulfated bile acids resulted in a significant inhibition (greater than 50%) of the Na(+)-H+ antiporter. A nonspecific effect of sulfated bile acids on the vesicles was excluded by the use of ionophores to determine vesicle integrity and to estimate the various ion conductances. Therefore specific inhibition of the human renal Na(+)-H+ antiporter by sulfated bile acids occurs. This could result in an impaired cellular pH regulation and might play a role in postoperative acute renal failure in patients with obstructive jaundice.

Stimulation of Canine Kidney BBMV ATPase Activity by Acidic pH in the Presence of Zn2+: An ATPase Activity Distinct from Transport ATPases and Alkaline Phosphatase That May Be an Ecto-ATPase

Renal brush border membrane vesicles (BBMV) of the dog possess at least two ATPase activities. In the present study, we have examined the effect of pH, ions, and inhibitors on the activity of ATPase in BBMV. Two different sets of conditions were identified that produced stimulation of ATPase activity. A unique stimulation of BBMV ATPase activity occurred at acidic pH in the presence of 1 mM ZnCl2. In the absence of Zn2+, a second ATPase activity was stimulated by alkaline pH values with peak stimulation occurring between pH 8.5 and 9.0. The results suggest that the alkaline pH-stimulated hydrolysis of ATP probably represents the activity of BBMV alkaline phosphatase. The unique acidic pH + Zn2(+)-stimulated ATPase activity must represent the activity of a second protein other than the alkaline phosphatase, since purified alkaline phosphatase did not show this activity. The biochemical identity and physiological function of this renal BBMV ATPase activity remain to be determined, but it may be an ecto-ATPase.

Thiobarbituric acid-reactive substance formation of rat kidney brush border membrane vesicles induced by ferric nitrilotriacetate

An iron chelate, ferric nitrilotriacetate (Fe 3+-NTA), is nephrotoxic and also carcinogenic to the kidney in experimental animals. Iron-promoted lipid peroxidation in the proximal tubules is thought to be responsible for the pathologic process. In the present study, iron-promoted lipid peroxidation, with thiobarbituric acid (TBA) formation as an indication, in the tubular surface was simulated in vitro using rat kidney brush border membrane vesicles and the results were compared with those using linoleate micelles and rat liver microsomal lipid liposomes. Addition of ascorbate, cysteine, or dithiothreitol to the Fe 3+-NTA solution resulted in consumption of dissolved oxygen and promoted the lipid peroxidation in the micelles and in the liposomes. In contrast, addition of glutathione to the Fe 3+-NTA solution caused only sluggish oxygen consumption and far less peroxidation in these lipid systems. When the brush border membrane vesicles were used for the peroxidation substrate, Fe 3+-NTA and glutathione could promote TBA formation at a rate comparable to that elicited by Fe 3+-NTA with cysteine or dithiothreitol. Acivicin, a γ-glutamyl transpeptidase inhibitor, suppressed the peroxidation of the brush border membrane vesicles promoted by Fe 3+-NTA and glutathione. These results suggest the following mechanism of proximal tubular cell lipid peroxidation promoted by Fe-NTA: Fe 3+-NTA filtered through glomeruli is rapidly reduced by cysteine and Fe 2+-NTA starts lipid peroxidation at the site, leading to proximal tubular necrosis. Cysteine is amply supplied by the decomposition of glutathione within the lumen by the action of γ-glutamyl transpeptidase and dipeptidase situated at the proximal tubular brush border membrane.

Brush-border membrane cation conducting channels from rat kidney proximal tubules

This is a description and kinetic characterization of cation channels from rat kidney brush-border membrane vesicles and from apical membranes of proximal tubule cells in culture. Channel activity was demonstrated and characterized in both artificial phospholipid bilayers and in tissue culture. Intermediate conductance, approximately 50 pS, cation-selective channels were observed by both methods. Channels were characterized by a Na permeability (PNa)/K permeability (PK) of 1-5:1. Open-channel current-voltage curves were linear in symmetric 300 mM NaCl. In tissue culture the gating kinetics are described by two open-time constants and two closed-time constants. Channel activity was neither voltage nor Ca2+ dependent and the probability of being in the open state ranged from 0.6 to 0.95. In tissue culture experiments the channel demonstrated nonstationary gating activity. A second, 15-pS cation channel, seen in planar bilayers, demonstrated a higher selectivity for Na+ with a (PNa/PK ratio of greater than 10).

Gamma-Aminobutyric acid uptake by rat kidney brush-border membrane vesicles.

Brush-border membrane vesicles (BBMV) from rat kidney cortex possessed two uptake systems for gamma-aminobutyric acid (GABA), a high affinity system (Km = 10.9 microM) and a low affinity system (Km = 1203 microM). Both uptake systems were inhibited by p-hydroxymercuribenzoic acid and ouabain, and by the action of neuraminidase, whereas the GABA analogs nipecotic acid, beta-alanine, 2,4-diaminobutyric acid and 4,5,6,7-tetrahydroisoxazolo-[4,5 c]-pyridin-3-ol had no effect on the GABA uptake activity. The BBMV uptake systems were clearly different from the GABA transport systems present in brain tissue.

Mechanism of acridine orange interaction with phospholipids and proteins in renal microvillus vesicles.

The mechanism of interaction of acridine orange (AO), a fluorescent, weak base, with rabbit kidney brush border membrane vesicles (BBMV) has been studied by absorption, and steady-state and time-resolved fluorescence spectroscopy. Equilibrium binding experiments indicate that AO binds to an apparent single class of sites on BBMV with a dissociation constant of 90 microM and site stoichiometry of 810 nmol/mg protein. The absorption spectra AO indicate that BBMV induces aggregation of AO; experiments with lipid vesicles show that the aggregation requires BBMV membrane proteins. Fluorescence stopped-flow experiments in which 0.15 mg/ml BBMV is mixed with increasing concentrations of AO result in a time course of fluorescence enhancement for [AO] less than 1.5 microM, and of fluorescence quenching for [AO] greater than 1.5 microM. Similar stopped-flow experiments with phosphatidylcholine lipid vesicles result only in a fluorescence enhancement time course. These results indicate the presence of two parallel pathways for AO binding to BBMV: one for AO binding to BBMV lipid, the other for AO binding to BBMV protein. Nanosecond lifetime measurements and fluorescence titration experiments confirm the presence of two environments for AO in BBMV. Fluorescence stopped-flow experiments indicate that AO responds to the imposition of an outwardly directed proton gradient by a rapid (less than 0.5 s) decrease in fluorescence, corresponding to re-equilibration of AO into the acidic intravesicular compartment, followed by an increase in fluorescence, corresponding to proton flux across the membrane. These findings have been incorporated into a stepwise mechanism for AO interaction with BBMV which have direct implications for the use of AO as a pH indicator in biological systems.

Determination of solute reflection coefficients in kidney brush-border membrane vesicles by light scattering: influence of the refractive index

Solute reflection coefficients σ of cell membrane vesicles or liposomes are commonly determined by comparison of the water flow induced by a gradient of the studied solute and that of a reference molecule using light scattering techniques. However, variations in scattered light which are mainly related to change in vesicle volume are also influenced by the refractive index of the surrounding medium. Therefore comparing kinetics of vesicle shrinkage induced by hyperosmotic solutions which have different refractive indexes might lead to an under or over estimation of σ. We determined σ NaCl in rat kidney brush-border membrane vesicles by two different approaches using mannitol, a poorly permeant molecule, as reference. (1) The refractive index of the hyperosmotic NaCl solution was adjusted to that of mannitol by addition of polyvinyl pyrrolidone ( M r 40 000), without a significant increase in osmolality. Thereby the change in scattered light intensity induced by osmotic vesicle shrinkage due to gradients of NaCl and mannitol were comparable and led to a σ NaCl value close to one instead of the previously published value of 0.53. (2) The reflection coefficient was calculated from the lifetime of vesicle shrinkage which is not refractive index-dependent. Again σ NaCl was not different from one. These results suggest that the water proteic pathways found in the luminal membrane of proximal tubules are not shared by salts.

Inhibition of Na +-dependent phosphate transport by group-specific covalent reagents in rat kidney brush border membrane vesicles : Evidence for the involvement of tyrosine and sulfhydryl groups on the interior of the membrane

The effects of tyrosine- and sulfhydryl-specific reagents on the Na +-dependent transport of phosphate in brush border membrane vesicles prepared from rat renal cortex were investigated. This study is the first to show that the tyrosine-specific reagents 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and tetranitromethane inactivate the transporter in a concentration- and time-dependent fashion while the membrane impermeant tyrosine reagent, N-acetylimidazole, has no effect on phosphate uptake. The membrane permeant sulfhydryl reagent N-ethylmaleimide also caused a time and concentration-dependent inactivation of this transport process but the membrane impermeant reagents 7-chloro-4-sulfobenzo-2-oxa-1,3-diazole and eosin-5-malemide had little effect on phosphate uptake. The inhibitory effects of both tyrosine- and sulfhydryl-specific reagents were additive, but no protection from inactivation by tyrosine-specific reagents could be achieved by preincubation of the vesicles with the substrates of the transporter or with competitive inhibitors of the transport process. These results suggest that the amino acids modified by these agents are located either within the membrane or on the cytosolic surface of the transporter. These residues may not participate in substrate binding, but may be important for the conformational change of the transporter necessary for the translocation of phosphate across these membranes. This study also shows that Na +-dependent phosphate transport can be inactivated by other reagents which covalently modify histidine, carboxyl, and amino groups on proteins.

Folate binding and transport by rat kidney brush-border membrane vesicles

[ 3H]Pteroylglutamic acid (PteGlu) uptake was studied using brush-border membrane vesicles isolated from rat kidney. Results on the uptake of [ 3H]PteGlu by brush-border membrane vesicles incubated in media of increasing osmolarities demonstrated that uptake was contributed by two components, intravesicular transport and membrane binding. Both the components of the uptake exhibited similar pH dependence, with maxima at pH 5.6, and were found to be saturable mechanisms with K m values of 6.7 · 10 −7 and 11.2 · 10 −7 M, respectively. These studies show that PteGlu is transported by isolated rat kidney brush-border membrane vesicles in a manner consistent with a saturable system and that a binding component may be functionally associated with this.

Diethylpyrocarbonate inhibition of sodium-glucose cotransport in kidney brush-border membrane vesicles

Exposure of kidney brush-border membrane vesicles to the acylating reagent diethylpyrocarbonate resulted in inactivation of the glucose transporter, as demonstrated by inhibition of sodium-coupled d-glucose transport and phlorizin binding. The transport site(s) was protected against inactivation by the simultaneous presence of sodium ions and d-glucose, and were partially protected by phlorizin. Transport activity was not restored by hydroxylamine; this rules out the possibility of diethylpyrocarbonate interaction with histidine, serine or tyrosine transporter residues. Dithiothreitol, a thiol protector, slightly prevented diethylpyrocarbonate inactivation. It is therefore suggested that (an) amino group(s) in the translocation complex is involved, at the level of the sugar transport site and the preferential protection of d-glucose against diethylpyrocarbonate inactivation related to a conformation change caused by the simultaneous binding of sodium and d-glucose to the cotransporter.

Separation and reconstitution of sodium-dependent glucose transport activity from renal brush-border membranes using gel-filtration chromatography

Pig kidney brush-border membrane vesicles were solubilized using a final concentration of 1% Triton X-100, found optimal for quantitative reconstitution of d-glucose transport into liposomes. Using reconstituted proteoliposomes, selective permeability towards d-glucose compared to other sugars tested was shown as well as the main features of d-glucose transport in native membranes, namely sodium dependence and phlorizin inhibition of d-glucose accumulation. After removal of Triton X-100 from the detergent extract, some membrane proteins (about 40%), which are insoluble in the absence of detergent, were isolated. Among these proteins resolubilized by 1% Triton X-100, the component catalyzing the d-glucose transport was located by gel-filtration chromatography separation, using reconstitution of transport as the assay. The active fraction displayed a molecular size of 50 Å; when analyzed on SDS polyacrylamide gel electrophoresis, it contained one major protein subunit with an apparent molecular weight close to 65 000. We conclude that this protein fraction is involved in d-glucose transport by renal brush borders.

Gentamicin inhibits Na +-dependent d-glucose transport in rabbit kidney brush-border membrane vesicles

We studied the effect of gentamicin on Na +-dependent d-glucose transport into brush-border membrane vesicles isolated from rabbit kidney outer cortex (early proximal tubule) and outer medulla (late proximal tubule) in vitro. We found the same osmotically active space and nonspecific binding between control and gentamicin-treated brush-border membrane vesicles. There was no difference in the passive permeability properties between control and gentamicin-treated brush-border membrane vesicles. Kinetic analyses of d-glucose transport into 1 mM gentamicin-treated brush-border membrane vesicles demonstrated that gentamicin decreased V max in the outer cortical preparation, while it did not affect V max in the outer medullary preparation. With regard to K m, there was no effect of gentamicin in any vesicle preparation. When brush-border membrane vesicles were incubated with higher concentrations of gentamicin, Na +-dependent d-glucose transport was inhibited dose-dependently in both outer cortical and outer medullary preparations. Dixon plots yield inhibition constant K i = 4 mM in the outer cortical preparation and K i = 7 mM in the outer medullary preparation. These results indicate that the Na +-dependent d-glucose transport system in early proximal tubule is more vulnerable to gentamicin toxicity than that in late proximal tubule.

Phosphonocarboxylic acids as specific inhibitors of Na+-dependent transport of phosphate across renal brush border membrane.

We investigated interactions of phosphonoformic acid (PFA), phosphonoacetic acid (PAA), and other phosphonyl derivatives with the Na+ gradient [Na+ extravesicular greater than Na+ intravesicular; Nao+ greater than Na+i]-dependent transport system for phosphate (Pi) in renal cortical brush border membrane vesicles (BBMV). PFA and PAA inhibited in a dose-dependent manner the Na+ gradient [Na+o greater than Na+i]-dependent uptake of Pi by rat kidney BBMV. PFA was a more potent inhibitor than PAA while phosphonopropionic acid, hydroxymethylphosphonic acid, and phenylphosphonic acid had no effect on Pi transport. The inhibitory effect of PFA was competitive (Ki approximately equal to 4.6 X 10(-4) M) and reversible upon dilution. The uptake of Pi by BBMV in the absence of Na+ gradient [Nao+ = Na+i] was also inhibited by PFA. The PFA had no effect on uptake of L-[3H]proline, D-[3H]glucose, or 22Na+ by BBMV nor did it alter intravesicular volume of BBMV. The relative (%) extent of inhibition by PFA was not altered by changes in the extravesicular pH or changes in the steepness of the Na+ gradient [Nao+ greater than Na+i]. The inhibition of PFA was analogous in renal BBMV from rats, mice, rabbits, or dogs. Unlike other known inhibitors of brush border membrane (BBM) transport of Pi, e.g. arsenate, NAD, and ethane-1-hydroxy-1,1-diphosphonate, PFA and PAA had no inhibitory effect on BBM-bound or solubilized alkaline phosphatase. Also, PFA did not interfere with the activity of renal cortical (Na-K)ATPase. Administration of PFA (0.5 g/kg/day, intraperitoneally) to thyroparathyroidectomized rats fed a low Pi diet elicited an increase in urinary excretion of Pi, but did not change the excretion of Na+, K, and Ca2+. The results show that the PFA, and to a lesser degree PAA, are specific competitive inhibitors of the Na+-Pi cotransport in renal cortical BBM and are suitable probes for studies of this transport system.

Osmotic water permeability and solute reflection coefficients of rat kidney brush-border membrane vesicles

Solute reflection coefficients, σ i , of rat kidney brush-border membrane vesicles were determined by the comparison of water flows induced by equiosmolal gradients of sucrose and NaCl, KCl or mannitol. The values of 0.53 for σ NaCl and 0.56 for σ KCl when compared with 0.92 for σ mannitol suggested some interactions between salt and water pathways. Altering the membrane proteins with 0.4 mM HgCl 2 decreased the osmotic water permeability of the vesicles by 70 to 80% and brought σ NaCl and σ KCl to a value not different from 1. This argued in favor of water protein pathways in the luminal membrane of kidney proximal cells which are partly accessible to NaCl and KCl.

Divalent metal is required for both phosphate transport and phosphate binding to phosphorin, a proteolipid isolated from brush-border membrane vesicles.

The Na+-dependent phosphate transport system in the brush border of rabbit kidney exhibits a positive requirement for a divalent metal ion. Treatment of the brush-border membrane vesicles (BBMV) with a divalent metal chelator in combination with the divalent metal ionophore A23187 dramatically and selectively decreased the Na+-dependent uptake of phosphate; Na+-independent uptake of phosphate was not affected. The combination of chelator plus A23187 also inhibited uptake of phosphate in the presence of Na+ but in the absence of a gradient for sodium across the BBMV. This indicates that the inhibitor is not a result of an alteration in the Na+ gradient by chelator plus ionophore. The inhibited Na+ gradient-dependent transport of phosphate was restored by removing the chelator and adding Mn2+ to the BBMV. The phosphate-binding proteolipid (phosphorin) isolated from rabbit kidney BBMV binds inorganic phosphate with high affinity and specificity. Binding of phosphate to phosphorin is also inhibited by divalent metal chelators and can be restored by addition of a divalent metal. We conclude that a divalent metal ion is required both for the Na+-dependent phosphate transport in BBMV and for the binding of phosphate to the proteolipid phosphorin. These findings are consistent with our suggestion that phosphorin is a component of the Na+-dependent phosphate transport system in renal brush-border membranes.

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

The effects of intravesicular NAD on Na+-dependent 32Pi 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 of 3H-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.