Preparation Of Brush Border Membrane Vesicles Research Articles

G-protein stimulation inhibits amiloride-sensitive Na/H exchange independently of cyclic AMP

G-proteins are heterotrimeric proteins involved in many transmembrane signaling events. Both the renal basolateral membrane and the renal brush border membrane contain large quantities of these proteins. G-proteins appear related to hormonal signaling in the basolateral membrane and presumably affect ion gating in the brush border. We investigated the influence of G-proteins on the amiloride-sensitive Na/H exchanger, the activity of which is regulated at least in part by cAMP-dependent protein kinase, by measuring the amiloride-sensitive component of [22Na+] uptake in rat renal brush border membrane vesicles (BBMV) in the presence of a pH gradient. Incubation of vesicles with AlF4- (10 microM Al3+, 10 mM F-) resulted in significant inhibition of amiloride-sensitive [22Na+] uptake at both 20 seconds and 5 minutes of incubation. Incorporation of GTP gamma S into BBMV by transient hypotonic lysis also resulted in significantly reduced amiloride-sensitive [22Na+] uptake compared to controls at both time points. This inhibition could be reversed by GDP beta S. Similar lysis in the presence of 10 microM GDP beta S alone had no significant effect. When Na(+)-dependent [14C]-D-glucose uptake into BBMV was studied no significant effect of these G-protein modulating agents was observed. Adenylate cyclase activity could not be stimulated in these BBMV preparations using standard techniques. Furthermore, cAMP-dependent protein kinase activity, strongly stimulated in these BBMV by exogenously added cAMP, was not stimulated by 10 microM GTP gamma S alone. These findings suggest that the amiloride-sensitive Na/H exchanger can be regulated by G-proteins independently of adenylate cyclase and cAMP-dependent protein kinase.

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 (Cout greater than Cin) 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-phenylpropyl-amino)-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.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of bursh border membrane vesicles from rat renal cortex by size-exclusion chromatography

Size-exclusion chromatography with controlled pore glass (CPG) was used in the further purification of renal brush border membrane vesicles (BBMV) isolated by the Ca precipitation method. The BBMV obtained had an almost spherical shape and their average diameter was about 95 nm in isotonic solution. The specific activities of alkaline phosphate and leucine aminopeptidase in the BBMV preparation were increased 18- and 17-fold, respectively, over those in the crude homogenate. The uptake of d-glucose by the purified BBMV in the presence of a sodium gradient reached 8.53 nmol/mg protein at 20 s. These results indicate that CPG chromatography is a suitable procedure by which to obtain purified renal BBMV of homogenous size and with high specific marker enzyme activity for use in the study of membrane transport.

Incorporation into a planar lipid bilayer of K channels from the luminal membrane of rabbit proximal tubule

The presence of ionic channels at the apical membrane of rabbit proximal tubule cells was investigated by fusion of brush-border membrane vesicles (BBMV) with a planar lipid bilayer (PE/PC, 1:1). The BBMV obtained from native membranes showed poor fusogenic properties. The probability of vesicles fusion with a planar bilayer was, however, enhanced by preincubating the BBMV with liposomes made of azolectin. We report here the presence in BBMV preparations of two K +-selective channels of 65 pS and 40 pS, respectively, in asymmetrical 200/ /50 mM KCl solutions. The channel of 65 pS appeared highly selective to K + over Na + and Cl − ions, while the 40 pS channel discriminated poorly between K + and Na + with a permeability ratio P K/ P Na = 4. The open probability P 0 of both channels was found to be voltage-independent with the potential range −60 mV to +60 mV. These K + channels may be related to channels identified using other methods.

Characterization of the Early Ultrastructural and Biochemical Events Occurring in Dichloromethane Diphosphonate Nephrotoxicity

A chelator, dichloromethane diphosphonate (Cl2MDP), used to treat for malignancy-induced hypercalcemia, has nephrotoxic potential. An acute animal model developed to examine the mechanism was used to further characterize the renal effects. NAG enzymuria appears to be an early premonitor of injury. Ultrastructurally, an increase in size and number of protein-containing phagolysosomal reabsorption droplets in proximal convoluted tubules associated with proteinuria precedes advent of tubular cell necrosis indicating these organelles to be a potential target site for Cl2MDP in the kidney. In vitro studies using rabbit cortical tubules and rat brush border membrane vesicle preparations suggest that the renal toxicity is not due to perturbation of phosphate transport or oxidative metabolism. An operational hypothesis emerges indicating that Cl2MDP may be protein bound affecting carrier protein charge facilitating glomerular leakage with tubular accumulation via protein transport. Cl2MDP may induce critical cation perturbation at the subcellular level as the mechanism of cell death.

Dopamine inhibits Na(+)-H+ exchanger activity in renal BBMV by stimulation of adenylate cyclase

To determine a renal tubular mechanism for the natriuretic effect of dopamine (DA) and DA-1 agonists, we measured Na(+)-H+ exchange activity (amiloride sensitive) in rat renal cortical brush-border membrane vesicles (BBMV). Renal cortical tissues were preincubated with ligands before BBMV preparation to study Na(+)-H+ exchange activity in the absence of the added ligands that may compete for ion binding sites of the exchanger. DA and DA-1 agonist-inhibited Na(+)-H+ exchange activity was concentration and time dependent. The inhibitory effect was not due to increased permeability, collapse of the proton gradient, or change in vesicle size and did not extend to Na(+)-glucose symport. DA-2 agonists had no effect, whereas alpha-adrenergic agonists increased Na(+)-H+ exchange activity. Kinetic analysis revealed that the DA-1 agonist inhibited Na(+)-H+ exchange activity by a noncompetitive process. 2',5'-Dideoxyadenosine inhibited adenylate cyclase activity and reversed the inhibitory effect of DA-1 agonist on the exchanger. H4, an isoquinoline sulfonamide, which inhibits protein kinase A, also reversed the inhibitory effect of DA-1 agonist on the exchanger. Thus the DA-1 agonist-mediated inhibition of Na(+)-H+ exchange activity in BBMV is a receptor-mediated adenylate cyclase-linked process.

The Signal Transducer for the Dopamine-1 Regulated Sodium Transport in Renal Cortical Brush Border Membrane Vesicles

We have reported the presence of dopamine-1 (DA-1) and dopamine-2 (DA-2) receptors in renal brush border and basolateral membranes. DA-1 agonists stimulate adenylate cyclase (AC) and phospholipase C (PLC) activity in both membranes. Moreover, the ability of a DA-1 agonist (fenoldopam) to stimulate PLC activity is independent of AC activity. A DA-2 agonist (LY171555) by itself was without effect and did not enhance the ability of the DA-1 agonist to stimulate PLC activity. The DA-1 but not DA-2 agonists inhibit Na+/H+ exchange activity in brush border membrane vesicles (BBMV) and Na+/K(+)-ATPase activity in basolateral membranes. However, cAMP inhibits, while protein kinase C (presumably via PLC activity) stimulates, Na+/H+ exchange activity. We therefore determined the effect of DA-1 agonists on Na+/H+ exchange activity when PLC or AC activity was blocked using neomycin or dideoxyadenosine, respectively. The drugs were incubated with minced renal cortex prior to preparation of BBMV by differential centrifugation and MnCl2 precipitation. Enrichment of BBMV was not affected by drug treatment. The Na+/H+ exchange activity was assessed by measuring amiloride (1 mmol/L) sensitive 22Na+ uptake in BBMV (pHi = 5.5, pHo = 7.5, Nai+ = O, Nao+ = 1 mmol/L). Neomycin inhibited DA and DA-1-stimulated PLC activity in BBMV in a concentration dependent manner (10(-6) to 10(-4) mol/L). Neomycin (10(-4) mol/L) completely blocked the ability of DA and DA-1 agonist to stimulate PLC activity but had no consistent effect on DA-1 inhibited Na+/H+ exchange activity. Dideoxyadenosine inhibited DA and DA-1 simulated AC activity without affecting DA-1 stimulated PLC activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparation and properties of brush-border membrane vesicles from human small intestine

This study describes a simple and rapid method for the preparation of brush-border membrane vesicles from intestinal biopsies. The specific activities of sucrase, amino peptidase N, and alkaline phosphatase in these vesicles were the same as those in vesicles prepared from intestinal segments. The vesicles from all the regions of the small intestine can transport d-glucose in an Na+-dependent manner. The rates of transport of d-glucose presented here are far higher than previously reported. The method should have a wide applicability to studies of transport mechanisms and the distribution of transport processes within the intestine.

Brush border membrane non-esterified fatty acids. Physiological levels and significance for mucosal iron uptake in mouse proximal intestine.

Brush border membrane vesicles prepared using divalent cation precipitation methods can contain unphysiological levels of non-esterified fatty acids. Fatty acid production from endogenous lipid during brush border membrane vesicle preparation is effectively prevented by the lipase inhibitor diethyl 4-nitrophenylphosphate plus cooling. Vesicles prepared using this procedure have variable levels of non-esterified fatty acids (range 22-193 nmol mg-1 protein). Changes in non-esterified fatty acid levels in brush border membrane vesicles parallel Fe uptake by vesicles from Fe/ascorbate solutions. Brush border membrane vesicle fatty acids appear to be derived from the diet but hypoxic mice are able to maintain high brush border membrane non-esterified fatty acid levels despite reduced dietary intake. Non-esterified fatty acids in brush border membrane may thus provide a physiological mechanism of mucosal Fe uptake.

Carrier-mediated transport of pyroglutamyl-histidine in renal brush border membrane vesicles.

These studies were performed to determine if a transmembrane carrier for pyroglutamyl-histidine (pGlu-His) is present in the luminal membrane of renal proximal tubular cells. Previous studies have suggested the intact transepithelial transport of pGlu-His, a dipeptide formed by the hydrolysis of luteinizing hormone-releasing hormone by enzymes associated with the brush border in the proximal nephron. With the use of a renal brush border membrane vesicle preparation, pGlu-His showed H+-stimulated, Na-independent, saturable transport into an osmotically active space. High-pressure liquid chromatographic analysis of both the intravesicular and extravesicular fluids indicated intact uptake of the dipeptide. The transport constant (Kt) and Vmax for pGlu-His transport were 9.3 X 10(-8) M and 6.1 X 10(-12) mol.mg-1.min-1, respectively. Transport of pGlu-His was not inhibited by the dipeptides glycyl-proline, glycyl-sarcosine, and N-beta-alanyl-L-histidine, which have been previously shown to be transported into renal brush border vesicles via a single, low-affinity, high-capacity, Na-independent, and H+-stimulated peptide carrier. In addition, the gamma-glutamyl-containing peptides gamma-glutamyl-histidine and N(N-L-gamma-glutamyl-L-cysteinyl)glycine and the tripeptide pyroglutamyl-histidyl-prolinamide were without an inhibitory effect. In contrast, transport of pGlu-His was inhibited by the dipeptide pyroglutamyl-alanine. This study demonstrates the existence of a high-affinity, low-capacity H+ cotransport system for pGlu-His in the proximal tubular luminal plasmalemma, which appears to be specific for pyroglutamyl-containing dipeptides. The data indicate that multiple dipeptide carriers are present in the proximal nephron.

Effect of age on renal conservation of phosphate in the rat.

Renal handling of phosphate (Pi) was examined in male Wistar-derived rats, 2-3, 6, 12, 18, and 24 mo of age. We observed a significant age-related phosphaturia [i.e., elevated urinary excretion (UPi V) and fractional excretion (FEPi)] in rats fed a normal phosphorus diet (NPD; 0.5% Pi). Concomitantly, plasma Pi decreased significantly and progressively with age. The mechanism of this age-related decrement in Pi conservation was examined by determining the initial (5 s) rate of Na+ gradient-dependent uptake of Pi in renal brush-border membrane vesicles (BBMV). Pi uptake significantly declined with increasing age. No consistent age-related decrease was seen in the Na+ gradient-dependent uptakes of glucose and proline by the same BBMV preparations, demonstrating the specificity of the Pi transport decrement. Pi transport kinetics revealed a significant age-related decrease in Vmax. No difference in Km of Pi was seen between age groups. These kinetic findings suggest either a decreased number of Pi carriers or decreased turnover of Pi carriers. Elevated parathyroid hormone did not explain the alteration in Pi conservation since urinary cAMP was not elevated in the intact senescent rat, and Pi uptake was not normalized in 24-mo-old rats 3 days after parathyroidectomy. The senescent 24-mo-old rat as well as the young adult 6-mo-old animal adapted to a low-phosphorus diet (LPD; 0.1% Pi) with a striking (greater than 100%) increase in Pi uptake by BBMV compared with NPD. thus the senescent kidney retained the capacity to respond appropriately to a LPD.(ABSTRACT TRUNCATED AT 250 WORDS)

Vitamin D status and brush border membrane vesicles: 1,25-dihydroxyvitamin D3 induced destabilization.

Purified chick duodenal brush border membrane vesicles (BBMV) were used to assess the effect of vitamin D on intestinal Ca2+ transport and membrane stability. BBMV preparations are right-side-out as judged by a nine-fold increase in accessibility of lactoperoxidase to core material actin in the presence of Triton X-100. Freshly prepared BBMV from vitamin D-deficient chicks support both sodium-dependent glucose transport and Ca2+ uptake. In vivo treatment with 1,25(OH)2D3 results in an 85% increase in the Vmax of Ca2+-uptake from 2.2 to 3.9 nmol/min/mg protein. The Km of Ca2+-uptake (0.9 mM) is independent of the vitamin D status of the chick. The majority of BBMV derived from vitamin D-replete chicks were destabilized and rendered incapable of supporting either sodium-dependent glucose uptake or Ca2+ uptake if they were held at 0-4 degrees C for 2 to 24 h. In 40 separate experiments, 80% of membranes derived from vitamin D-replete chicks showed characteristics of destabilization, whereas only 24% of all control membranes exhibited a lack of viability.

Effect of Lactose on Phosphate Transport into Rat Intestinal Brush Border Membrane Vesicles

Brush border membrane vesicles (BBMV) were used to evaluate the influence of lactose on phosphate (Pi) transport in the intestine of the rat. BBMV were prepared from duodenum, jejunum and ileum of 3-, 4- and 7-wk-old rats. Na+-dependent active transport of Pi was investigated in the presence of an Na+ gradient; Na+-independent, diffusional transport was investigated by substitution of choline chloride for NaCl. In 3-wk-old rats active Pi transport dominated, whereas in 4-wk-old rats a decrease in active transport and an increase of the diffusional component of total Pi transport were observed. Furthermore, in 7-wk-old rats, Pi was transported by simple diffusion alone. Addition of lactose to the preincubation medium had no effect on Pi transport in duodenal and jejunal BBMV isolated from 3-wk-old rats. In the remaining BBMV preparations, lactose caused a twofold enhancement of diffusional Pi uptake. Glucose and galactose had no effect on diffusional Pi uptake into BBMV. These results indicate that lactose may directly interact with luminal membranes, thus leading to increased diffusional transport. The membranes with a decreased Na+-dependent Pi transport activity are most susceptible to the influence of lactose.

Sulfate transport by mouse renal cortical slices does not represent uptake by brush-border membrane

We measured uptake of isotopically 35S-labelled sulfate anion by slices and by brush-border membrane vesicles prepared from mouse renal cortex to identify: (i) whether metabolic incorporation of anion influences net transport; (ii) which membrane is primarily exposed in the renal cortex slice. Slices accumulated sulfate without significant incorporatoin into metabolic pools. Net uptake of sulfate at 0.1 mM by the slice occurred against an electrochemical gradient as determined by mesurement of free intracellular sulfate concentration, the isotopic distribution ratio at steady-state, and the distribution of lipophilic ions (TPP + and SCN −). Carrier mediation of sulfate transport in the slice was confirmed by observing concentration-dependent saturation of net uptake and counter-transport stimulation of efflux. Anion uptake was Na +-independent, K +- and H +-stimulated, and inhibited by disulfonated stilbenes. Brush-border membrane vesicles accumulated sulfate by a saturable mechanism dependent on a Na + gradient (outside > inside); others have shown that uptake of sulfate by brush-border membrane vesicles is insensitive to inhibition by disulfonated stilbenes. These findings indicate that different mechanisms serve sulfate transport in renal cortex slice and brush-border membrane vesicle preparations. They also imply that the slice exposes an epithelial surface different from the brush-border, presumably the basolateral membrane, or its equivalent, since sulfate transport by slices resembles that obserbed with isolated basolateral membrane vesicles.

Na/H- and Cl/OH-exchange in rat jejunal and rat proximal tubular brush border membrane vesicles. Studies with acridine orange.

The quenching of the acridine orange fluorescence was used to monitor the formation and/or dissipation of a delta pH in brush border vesicles isolated from rat kidney cortex or rat jejunum. Similar findings were obtained with both brush border membrane vesicle preparations. Acridine orange fluorescence was quenched by a preset delta pH (intravesicular acid) or by the ionophore (valinomycin/CCCP) dependent development of a delta pH (intravesicular acid) under conditions of potassium efflux. Under sodium efflux conditions, an acidification of the intravesicular space occurred: a) due to indirect (electrical) coupling of sodium and proton fluxes; b) due to directly coupled sodium/proton exchange. The initial rate of the dissipation of a preset delta pH was accelerated by pulse injections of sodium in a saturable manner; lithium partially replaced sodium. The sodium dependent acceleration in the rate of dissipation of a preset delta pH was not altered by replacing gluconate with chloride. Amiloride was an inhibitor of directly coupled sodium/proton exchange. An inwardly directed chloride gradient did not induce intravesicular acidification. The initial rate of the dissipative proton fluxes (preset delta pH) was slightly accelerated by an outwardly directed chloride gradient. Sodium/proton exchange dependent acidification of the intravesicular space was not altered by replacing gluconate with chloride. These results clearly document the existence of sodium/proton exchange in both renal and intestinal brush border membrane vesicles. In contrast, Cl/OH exchange--under our experimental conditions--must have a much smaller rate than Na/H exchange.

The static head method for determining the charge stoichiometry of coupled transport systems Applications to the sodium-coupled d-glucose transporters of the renal proximal tubule

The static head method for determining the charge stoichiometry (the number of moles of charge translocated per mole of substrate) of a coupled transport system is presented. The method involves establishing experimental conditions under which a membrane potential exactly balances the thermodynamic driving force of a known substrate gradient. The charge stoichiometry can then be calculated from thermodynamic principles. In contrast to the usual steady-state method for determining charge stoichiometry in cell suspensions and vesicle preparations, the static head method is applicable to systems which are not capable of maintaining a constant membrane potential over time. The charge stoichiometries of two renal sodium coupled d-glucose transporters previously identified in brush-border membrane vesicle preparations from the outer cortex (early proximal tubule) and outer medulla (late proximal tubule) are determined. The charge stoichiometries of these transporters are in good agreement with their sodium/glucose coupling ratios arguing against the possibility that glucose transport is coupled to ions other than sodium in these membranes.

Transport of inorganic and organic substances in the renal proximal tubule

The transport through the epithelial cell layer of the renal proximal tubule proceeds in principle by passive paracellular and active transcellular transport. The active transcellular transport is mostly secondary active. This means it proceeds coupled with the flux of Na+ ions, whereby the transcellular gradient of sodium, created by the (Na+ + K+)-ATPase, located at the contraluminal cell side, provides the main driving force. Once in the cell the substances leave the other cell side by a Na+ -independent, but carrier-mediated transport system. Using microperfusion and electrophysiological techniques as well as brush border membrane vesicle preparation the Na+ -H+ countertransport and the Na+-cotransport of amino acids, phosphate, sulfate, thiosulfate, bile acids, aliphatic-aromatic monocarboxylic acids (lactate) and dicarboxylic acids was studied. Special emphasis will be given to the bidirectional transport of thiosulfate as well as to the specificity of the monocarboxylic acid and dicarboxylic acid transport system.

Pathways of cycloleucine transport in killifish small intestine.

By using blocking concentrations of competitors, i.e., concentrations of other neutral amino acids that cause maximal inhibition, cycloleucine transport into slices or everted sacs of killifish (fundulus heteroclitus) small intestine could be partitioned into three pathways. One is apparently not mediated, a second is inhibited by all neutral amino acids tested (component 1), and a third, is inhibited by alpha-aminocarboxylic acids (component 2), but not by beta-alanine or taurine. Both mediated pathways were Na dependent, and each yielded a linear double reciprocal plot of initial slice uptake vs. cycloleucine concentration. Apparent Kt and Vmax values for component 1 were 0.03 mM and 33 pmol/mg tissue per 3 min, respectively; corresponding values for component 2 were 0.12 mM and 28 pmol/mg tissue per 3 min. Additional experiments with an intestinal brush border membrane vesicle preparation indicate that these mediated components reflect true differences in carrier specificity rather than the differential effects of inhibitors on metabolism or on the Na gradient that drives cycloleucine transport.

Virus isolation with glass of controlled pore size: MS2 bacteriophage and Kilham virus

Size-exclusion chromatography with controlled pore glass (CPG) was used in the further purification of renal brush border membrane vesicles (BBMV) isolated by the Ca precipitation method. The BBMV obtained had an almost spherical shape and their average diameter was about 95 nm in isotonic solution. The specific activities of alkaline phosphate and leucine aminopeptidase in the BBMV preparation were increased 18- and 17-fold, respectively, over those in the crude homogenate. The uptake of d-glucose by the purified BBMV in the presence of a sodium gradient reached 8.53 nmol/mg protein at 20 s. These results indicate that CPG chromatography is a suitable procedure by which to obtain purified renal BBMV of homogenous size and with high specific marker enzyme activity for use in the study of membrane transport.