Rat Renal Brush-border Membrane Vesicles Research Articles

In-vitro potencies of histamine H2-receptor antagonists on tetraethylammonium uptake in rat renal brush-border membrane vesicles.

The histamine H2 antagonists cimetidine, ranitidine and famotidine are organic bases that are cleared from the body by active renal tubular secretion involving the organic cation transporter in the proximal tubule. To determine the potential for competition for the transporter between these drugs and other drugs, their inhibitory potencies were assessed in-vitro, using rat renal brush-border membrane vesicles and tetraethylammonium as the substrate. The concentration-dependent effect of cimetidine, ranitidine and famotidine on the 15-s proton-stimulated uptake of tetraethylammonium into the membrane vesicles was studied using five different rat kidneys. The order of inhibition potencies was: cimetidine (mean IC50 = 1.07 microM) > famotidine (2.43 microM) < ranitidine (55.4 microM). The results indicate the potential for drug interactions in the kidney, especially for cimetidine and famotidine.

The mechanism of excretion of trientine from the rat kidney: trientine is not recognized by the H+/organic cation transporter.

Trientine dihydrochloride is used to treat Wilson's disease by chelating copper and increasing its urinary excretion. The mechanism of renal excretion of trientine has been investigated in-vivo and in-vitro. Trientine clearance in the rat-was significantly faster than creatinine clearance. When trientine and the same number of moles of copper ions were administered simultaneously to the rat, however, trientine clearance decreased to almost the same level as the creatinine clearance. To clarify this active excretion system for trientine, the uptake of trientine and a physiological polyamine compound, spermine, was investigated using rat renal brush-border membrane vesicles. Although, because trientine and spermine are organic cations, the H+/organic cation transporter is expected to recognize these compounds, neither an outwardly directed H+ gradient nor an inward Na+ gradient stimulated trientine uptake. [14C]Spermine uptake was, nevertheless, trans-stimulated by both unlabelled spermine and trientine and the trans-stimulating effect of spermine on trientine uptake was, furthermore, completely abolished by addition of copper ions to the incubation medium. These results suggest that there is a specific transport system for spermine and trientine on the renal brushborder membrane. This transport system contributes to the secretion of trientine in the kidney proximal tubule but does not recognize the trientine-copper complex.

Effect of lamivudine on uptake of organic cations by rat renal brush-border and basolateral membrane vesicles.

The effect of lamivudine on uptake of a representative organic cation, tetraethylammonium (TEA), by rat renal brush-border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) has been investigated. The pH-driven uptake of TEA by BBMV (pHin = 6.0, pHout = 7.5) was inhibited by lamivudine. The IC50 value (concentration resulting in 50% inhibition) for the concentration-dependent effect of lamivudine on TEA uptake by BBMV after 30 s was 2668 microM whereas IC50 values for cimetidine and trimethoprim were < 2.5 microM and < 25 microM, respectively. The early uptake of TEA by BLMV was also reduced significantly by lamivudine. The IC50 value for the concentration-dependent effect of lamivudine on uptake of TEA by BLMV at 30 s was > 25 mM, whereas the IC50 values for cimetidine and trimethoprim were 2116 microM and 445 microM, respectively. These findings suggest that compared with other cationic drugs, such as trimethoprim and cimetidine, lamivudine is a weak inhibitor of organic cation transport into the tubules by the brush-border and basolateral membranes of renal epithelial cells. It is unlikely lamivudine will have any significant effect on the excretion of co-administered cationic drugs by the renal tubules.

The presence of an Na+/spermine antiporter in the rat renal brush-border membrane.

This study was aimed at determining the driving force for spermine transport in rat renal proximal tubular brush-border membrane. The uptake of spermine and trientine, a spermine-like drug used for treating Wilson's disease, into rat renal brush-border membrane vesicles was significantly stimulated by an outwardly directed Na+ gradient. The Na+-dependent uptake was temperature dependent and saturable. A kinetic analysis of the initial uptake of spermine with an Na+ gradient gave a Km value of 1.44 microM and a Vmax value of 6.31 pmol (mg protein)(-1)/30s. The Na+ dependent uptake of [3H]spermine was inhibited by spermine, trientine and tetraethylene-pentamine. Substrates of the H+/organic cation transporter (cimetidine and tetraethyl-ammonium), physiological polyamines (putrescine and spermidine) with 2 or 3 amino groups and aminoglycosides (amikacin and tobramicin) with 4 or 5 cationic amines did not affect the uptake of spermine in the presence of an outwardly directed Na+ gradient. These results suggest that the renal tubular secretion of spermine is mediated by an Na+/spermine antiport system which is specific for a straight-chain polyamine compound with more than 4 amino groups.

Identification of Essential Histidine and Cysteine Residues of the H+/Organic Cation Antiporter Multidrug and Toxin Extrusion (MATE)

Multidrug and toxin extrusion 1 (MATE1) has been isolated as an H(+)/organic cation antiporter located at the renal brush-border membranes. Previous studies using rat renal brush-border membrane vesicles indicated that cysteine and histidine residues played critical roles in H(+)/organic cation antiport activity. In the present study, essential histidine and cysteine residues of MATE1 family were elucidated. When 7 histidine and 12 cysteine residues of rat (r)MATE1 conserved among species were mutated, substitution of His-385, Cys-62, and Cys-126 led to a significant loss of tetraethylammonium (TEA) transport activity. Cell surface biotinylation and immunofluorescence analyses with confocal microscopy indicated that rMATE1 mutant proteins were localized at plasma membranes. Mutation of the corresponding residues in human (h)MATE1 and hMATE2-K also diminished the transport activity. The transport of TEA via rMATE1 was inhibited by the sulfhydryl reagent p-chloromercuribenzenesulfonate (PCMBS) and the histidine residue modifier diethyl pyrocarbonate (DEPC) in a concentration-dependent manner. The PCMBS-caused inhibition of the transport via rMATE1 was protected by an excess of various organic cations such as TEA, suggesting that cysteine residues act as substrate-binding sites. In the case of DEPC, no such protective effects were observed. These results suggest that histidine and cysteine residues are required for MATE1 to function and that cysteine residues may serve as substrate-recognition sites.

Morin (3,5,7,2′,4′-Pentahydroxyflavone) Exhibits Potent Inhibitory Actions on Urate Transport by the Human Urate Anion Transporter (hURAT1) Expressed in Human Embryonic Kidney Cells

In allopurinol-allergic patients, uricosuric agents are often used in the treatment of hyperuricemia. The existing uricosuric agents are not without problems and the availability of better and safer alternatives is highly desirable. Our previous study (J Pharmacol Exp Ther (2006) 316:169-175) has demonstrated that morin (3,5,7,2',4'-pentahydroxyflavone), which occurs in the twigs of Morus alba L. documented in traditional Chinese medicinal literature for treatment of conditions akin to gout, is a potent inhibitor of urate uptake in rat renal brush-border membrane vesicles. It is also effective in lowering uric acid level in a hyperuricemic rat model in vivo. Whether morin is an equally effective uricosuric agent in human requires verification. The human urate anion transporter (hURAT1) has recently been cloned and identified to be the organic anion transporter that mediates renal urate reabsorption in the human kidney. In the present investigation, human embryonic kidney cells were transfected with hURAT1 and the expression was validated by reverse transcription-polymerase chain reaction and subcellular distribution of the exogenously introduced transporter by confocal microscopy. The inhibitory actions of morin on human renal urate reabsorption were demonstrated using this system. The IC50 value of the inhibition by morin was determined to be 2.0 microM, compared with 50 microM for probenecid, 100 microM for sulfinpyrazone, and 0.3 microM for benzbromarone. Kinetic analysis of the uptake inhibition by morin indicates that this compound is a competitive inhibitor of urate uptake on the human urate transporter with a K(i) value of 5.74 microM.

Oppositely directed H+ gradient functions as a driving force of rat H+/organic cation antiporter MATE1

Recently, we have isolated the rat (r) H(+)/organic cation antiporter multidrug and toxin extrusion 1 (MATE1) and reported its tissue distribution and transport characteristics. Functional characterization suggested that an oppositely directed H(+) gradient serves as a driving force for the transport of a prototypical organic cation, tetraethylammonium, by MATE1, but there is no direct evidence to prove this. In the present study, therefore, we elucidated the driving force of tetraethylammonium transport via rMATE1 using plasma membrane vesicles isolated from HEK293 cells stably expressing rMATE1 (HEK-rMATE1 cells). A 70-kDa rMATE1 protein was confirmed to exist in HEK-rMATE1 cells, and the transport of various organic cations including [(14)C]tetraethylammonium was stimulated in intracellular acidified HEK-rMATE1 cells but not mock cells. The transport of [(14)C]tetraethylammonium in membrane vesicles from HEK-rMATE1 cells exhibited the overshoot phenomenon only when there was an outwardly directed H(+) gradient, as observed in rat renal brush-border membrane vesicles. The overshoot phenomenon was not observed in the vesicles from mock cells. The stimulated [(14)C]tetraethylammonium uptake by an H(+) gradient [intravesicular H(+) concentration ([H(+)](in)) > extravesicular H(+) concentration ([H(+)](out))] was significantly reduced in the presence of a protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). [(14)C]tetraethylammonium uptake was not changed in the presence of valinomycin-induced membrane potential. These findings definitively indicate that an oppositely directed H(+) gradient serves as a driving force of tetraethylammonium transport via rMATE1, and this is the first demonstration to identify the driving force of the MATE family. The present experimental strategy is very useful in identifying the driving force of cloned transporters whose driving force has not been evaluated.

Characterization of Prostaglandin E1 Transport in Rat Renal Brush-border Membrane

Transport of prostaglandin E(1) (PGE(1)) was investigated in rat renal brush-border membrane vesicles. The uptake of [(3)H]PGE(1) was sensitive to osmosis and temperature. This uptake was saturable and mediated by high-affinity (K(m)=2.1 microM)/low-capacity (V(max)=17.4 pmol/mg protein/30 sec) and low-affinity (K(m)=526.5 microM)/high-capacity (V(max)=1,032.5 pmol/mg protein/30 sec) transport systems. [(3)H]PGE(1) uptake was Na(+)-independent and inhibited by various eicosanoids including PGE(2) and PGF(2alpha). Bromcresol green and sulfobromophthalein, potent inhibitors of prostaglandin transporter (PGT), significantly decreased [(3)H]PGE(1) uptake. Uptake was also inhibited by indomethacin and probenecid, which reportedly have little effect on PGT. Benzylpenicillin and taurocholate decreased the uptake of [(3)H]PGE(1). Like p-[(14)C]aminohippurate (PAH) uptake by vesicles, the uptake of [(3)H]PGE(1) was stimulated by an inside-positive membrane potential, created by applying an inward K(+) gradient and valinomycin. However, the uptake of [(3)H]PGE(1) was not inhibited by PAH, suggesting that PAH and PGE(1) are transported by separate transport systems. [(3)H]PGE(1) uptake was not stimulated by outwardly directed gradients of Cl(-) nor unlabeled PGE(1), indicating that an anion exchanger may not be involved in PGE(1) transport. These findings suggest that the transport of PGE(1) in rat renal brush-border membrane is mediated by specific transport system(s), at least in part, by a potential-sensitive transport system.

The Dual Actions of Morin (3,5,7,2′,4′-Pentahydroxyflavone) as a Hypouricemic Agent: Uricosuric Effect and Xanthine Oxidase Inhibitory Activity

Hyperuricemia is associated with a number of pathological conditions such as gout. Lowering of elevated uric acid level in the blood could be achieved by xanthine oxidase inhibitors and inhibitors of renal urate reabsorption. Some natural compounds isolated from herbs used in traditional Chinese medicine have been previously demonstrated to possess xanthine oxidase inhibitory activities. In the present investigation, morin (3,5,7,2',4'-pentahydroxyflavone), which occurs in the twigs of Morus alba L. documented in traditional Chinese medicinal literature to treat conditions akin to gout, was demonstrated to exert potent inhibitory action on urate uptake in rat renal brush-border membrane vesicles, indicating that this compound acts on the kidney to inhibit urate reabsorption. Lineweaver-Burk transformation of the inhibition kinetics data demonstrated that the inhibition of urate uptake was of a competitive type, with a K(i) value of 17.4 microM. In addition, morin was also demonstrated to be an inhibitor of xanthine oxidase. Lineweaver-Burk analysis of the enzyme kinetics indicated that the mode of inhibition was of a mixed type, with K(i) and K(ies) values being 7.9 and 35.1 microM, respectively. Using an oxonate-induced hyperuricemic rat model, morin was indeed shown to exhibit an in vivo uricosuric action, which could explain, in part at least, the observed hypouricemic effect of morin in these rats. The potential application of this compound in the treatment of conditions associated with hyperuricemia was discussed.

Phenolsulfonphthalein transport by potential-sensitive urate transport system

The purpose of this study was to elucidate the transporter-mediated secretion systems for phenolsulfonphthalein in brush-border membranes. In human and rat renal brush-border membranes, a potential-sensitive transport system has been shown to be involved in the efflux of organic anions. The uptake of phenolsulfonphthalein into rat renal brush-border membrane vesicles was stimulated by an inside-positive membrane potential. This potential-sensitive uptake of phenolsulfonphthalein was inhibited by probenecid, pyrazinoate and urate. p-Aminohippurate had no effect on the potential-sensitive uptake of phenolsulfonphthalein. Moreover, urate competitively inhibited the uptake of phenolsulfonphthalein. On the other hand, the uptake of phenolsulfonphthalein was slightly increased in the presence of an outward Cl- gradient. These results suggest that phenolsulfonphthalein has high affinity for the potential-sensitive urate transport system but has low affinity for an anion exchanger.

Na +-dependent fructose transport via rNaGLT1 in rat kidney

We found a system of Na +-dependent uptake of fructose by rat renal brush-border membrane vesicles. It consisted of two saturable components, and was thought to involve at least two transporters. rNaGLT1, a novel glucose transporter in rat kidney, showed fructose uptake as well as α-methyl- D-glucopyranoside uptake by transfected HEK293 cells. The features of the lower affinity type of fructose transporter in the brush-border membranes, such as affinity and substrate recognition, were very comparable with those of rNaGLT1-transfected HEK293 cells. These results indicated that rNaGLT1 is a primary fructose transporter in rat renal brush-border membranes.

Distinct transport activity of tetraethylammonium from l-carnitine in rat renal brush-border membranes

We investigated the contribution of the Na+/l-carnitine cotransporter in the transport of tetraethylammonium (TEA) by rat renal brush-border membrane vesicles. The transient uphill transport of l-carnitine was observed in the presence of a Na+ gradient. The uptake of l-carnitine was of high affinity (Km=21 μM) and pH dependent. Various compounds such as TEA, cephaloridine, and p-chloromercuribenzene sulfonate (PCMBS) had potent inhibitory effects for l-carnitine uptake. Therefore, we confirmed the Na+/l-carnitine cotransport activity in rat renal brush-border membranes. Levofloxacin and PCMBS showed different inhibitory effects for TEA and l-carnitine uptake. The presence of an outward H+ gradient induced a marked stimulation of TEA uptake, whereas it induced no stimulation of l-carnitine uptake. Furthermore, unlabeled TEA preloaded in the vesicles markedly enhanced [14C]TEA uptake, but unlabeled l-carnitine did not stimulate [14C]TEA uptake. These results suggest that transport of TEA across brush-border membranes is independent of the Na+/l-carnitine cotransport activity, and organic cation secretion across brush-border membranes is predominantly mediated by the H+/organic cation antiporter.

Development and characterization of an animal model of carnitine deficiency

Mammals cover their carnitine needs by diet and biosynthesis. The last step of carnitine biosynthesis is the conversion of butyrobetaine to carnitine by butyrobetaine hydroxylase. We investigated the effect of N-trimethyl-hydrazine-3-propionate (THP), a butyrobetaine analogue, on butyrobetaine hydroxylase kinetics, and carnitine biosynthesis and body homeostasis in rats fed a casein-based or a vegetarian diet. The K(m )of butyrobetaine hydroxylase purified from rat liver was 41 +/- 9 micromol x L(-1) for butyrobetaine and 37 +/- 5 micromol x L(-1) for THP, and THP was a competitive inhibitor of butyrobetaine hydroxylase (K(i) 16 +/- 2 micromol x L(-1)). In rats fed a vegetarian diet, renal excretion of total carnitine was increased by THP (20 mg.100 g(-1) x day(-1) for three weeks), averaging 96 +/- 36 and 5.3 +/- 1.2 micromol x day(-1) in THP-treated and control rats, respectively. After three weeks of treatment, the total carnitine plasma concentration (8.8 +/- 2.1 versus 52.8 +/- 11.4 micromol x L(-1)) and tissue levels were decreased in THP-treated rats (liver 0.19 +/- 0.03 versus 0.59 +/- 0.08 and muscle 0.24 +/- 0.04 versus 1.07 +/- 0.13 micromol x g(-1)). Carnitine biosynthesis was blocked in THP-treated rats (-0.22 +/- 0.13 versus 0.57 +/- 0.21 micromol x 100 g(-1) x day(-1)). Similar results were obtained in rats treated with the casein-based diet. THP inhibited carnitine transport by rat renal brush-border membrane vesicles competitively (K(i) 41 +/- 3 micromol x L(-1)). Palmitate metabolism in vivo was impaired in THP-treated rats and the livers showed mixed steatosis. Steady-state mRNA levels of the carnitine transporter rat OCTN2 were increased in THP-treated rats in skeletal muscle and small intestine. In conclusion, THP inhibits butyrobetaine hydroxylase competitively, blocks carnitine biosynthesis in vivo and interacts competitively with renal carnitine reabsorption. THP-treated rats develop systemic carnitine deficiency over three weeks and can therefore serve as an animal model for human carnitine deficiency.

Regulation of NHE3 activity by G protein subunits in renal brush-border membranes.

NHE3 activity is regulated by phosphorylation/dephosphorylation processes and membrane recycling in intact cells. However, the Na(+)/H(+) exchanger (NHE) can also be regulated by G proteins independent of cytoplasmic second messengers, but the G protein subunits involved in this regulation are not known. Therefore, we studied G protein subunit regulation of NHE3 activity in renal brush-border membrane vesicles (BBMV) in a system devoid of cytoplasmic components and second messengers. Basal NHE3 activity was not regulated by G(s)alpha or G(i)alpha, because antibodies to these G proteins by themselves were without effect. The inhibitory effect of D(1)-like agonists on NHE3 activity was mediated, in part, by G(s)alpha, because it was partially reversed by anti-G(s)alpha antibodies. Moreover, the amount of G(s)alpha that coimmunoprecipitated with NHE3 was increased by fenoldopam in both brush-border membranes and renal proximal tubule cells. Furthermore, guanosine 5'-O-(3-thiotriphosphate) but not guanosine 5'-O-(2-thiodiphosphate), the inactive analog of GDP, increased the amount of G(s)alpha that coimmunoprecipitated with NHE3. The alpha(2)-adrenergic agonist, UK-14304 or pertussis toxin (PTX) alone had no effect on NHE3 activity, but UK-14304 and PTX treatment attenuated the D(1)-like receptor-mediated NHE3 inhibition. The ability of UK-14304 to attenuate the D(1)-like agonist effect was not due to G(i)alpha, because the attenuation was not blocked by anti-G(i)alpha antibodies or by PTX. Anti-Gbeta(common) antibodies, by themselves, slightly inhibited NHE3 activity but had little effect on D(1)-like receptor-mediated NHE3 inhibition. However, anti-Gbeta(common) antibodies reversed the effects of UK-14304 and PTX on D(1)-like agonist-mediated NHE3 inhibition. These studies provide concrete evidence of a direct regulatory role for G(s)alpha, independent of second messengers, in the D(1)-like-mediated inhibition of NHE3 activity in rat renal BBMV. In addition, beta/gamma dimers of heterotrimeric G proteins appear to have a stimulatory effect on NHE3 activity in BBMV.

Cadmium Binding and Sodium-Dependent Solute Transport in Renal Brush-Border Membrane Vesicles

Exposure to cadmium (Cd) impairs renal transport systems for glucose, amino acids, phosphate, and dicarboxylates. To investigate if these changes are directly related to a Cd binding to the renal brush-border membrane, Cd binding and the Na+-dependent uptakes ofd-glucose,l-alanine, phosphate, and succinate were determined in rat renal brush-border membrane vesicles (BBMV) exposed to CdCl2. Cd uptake by BBMV showed time and concentration dependence. Changes in medium osmolality had no effect on Cd uptake, indicating that the process primarily involves binding of Cd to the membrane. Scatchard analysis indicated the presence of two types of Cd binding sites, differing in affinity and number. Increasing the medium Cd concentration from 50 to 200 μM resulted in a progressive increase in Cd binding to the membrane and decrease in Na+-dependent transport ofd-glucose,l-alanine, inorganic phosphate, and succinate. In all cases, the inhibition of transport was directly proportional to the total amount of Cd binding to the membrane. These results suggest that, during chronic exposure to Cd, free Cd ions liberated in renal tubular cells may directly interact with brush-border membranes and impair Na+-dependent solute transports.

Effects of interactions between drugs on the renal excretion of trientine in rats--acetazolamide and furosemide increase trientine excretion.

To elucidate the effects of drug interactions on the urinary excretion of trientine in rats. Trientine and various other drugs were intravenously administered to rats and the urinary excretion of trientine was investigated. To clarify the mechanisms of drug-drug interactions, we also investigated the effects of various drugs on spermine uptake by rat renal brushborder membrane vesicles. Cimetidine, a substrate of the H+/organic cation antiporter, and aminoglycoside antibiotics did not affect trientine excretion, while acetazolamide and furosemide, which increase the concentration of sodium ions in renal proximal tubules, increased the excretion of trientine. However, trichlormethiazide, which acts in renal distal tubules, did not affect trientine excretion. Acetazolamide and furosemide did not directly affect the Na+/spermine transporter because these diuretics had no effect on the uptake of spermine into the rat renal brush-border membrane vesicles. There is no interaction between trientine and the substrate of the H+/organic cation antiporter or aminoglycoside antibiotics. However, drugs that change the concentration of sodium ions in renal proximal tubules, such as diuretics, can increase the trientine excretion since the increase in the luminal concentration of sodium ion accelerates the Na+/spermine antiporter.

Absence of vacuolar H+-ATPases from rat small intestine brush-border membranes.

Rat small intestinal brush-border membrane (BBM) vesicles were solubilized to test for the presence of an intravesicular H+-ATPase. Several detergents that in rat renal BBM vesicles revealed a bafilomycin-sensitive H+-ATPase failed to expose a similar ATPase in small intestinal vesicles. Antibodies against the 31-kDa and 70-kDa H+-ATPase subunits labelled respective antigens in the BBM of renal proximal tubules, but not in the BBM of enterocytes. The results demonstrate that a bafilomycin-sensitive, vacuolar-type H+-ATPase is absent from the BBM of enterocytes and, hence, cannot contribute to H+ secretion.

Sodium-dependent high-affinity binding of carnitine to human placental brush border membranes

The interaction of carnitine with human placental brush-border membrane vesicles was investigated. Carnitine was found to associate with the membrane vesicles in a Na +-dependent manner. The time course of this association did not exhibit an overshoot, which is typical of a Na + gradient-driven transport process. The absolute requirement for Na + was noticeable whether the association of carnitine with the vesicles was measured with a short time incubation or under equilibrium conditions, indicating Na +-dependent binding of carnitine to the human placental brush-border membranes. The binding was saturable and was of a high-affinity type with a dissociation constant of 1.37 ± 0.03 μM. Anions had little or no influence on the binding process. The binding process was specific for carnitine and its acyl derivatives. Betaine also competed for the binding process, but other structurally related compounds did not. Kinetic analyses revealed that Na + increased the affinity of the binding process for carnitine and the Na +/carnitine coupling ration for the binding process was 1. The dissociation constant for the interaction of Na + with the binding of carnitine was 24 ± 4 mM. This constitutes the first report on the identification of Na +-dependent high-affinity carnitine binding in the plasma membrane of a mammalian cell. Studies with purified rat renal brush-border membrane vesicles demonstrated the presence of Na + gradient-driven carnitine transport but no Na +-dependent carnitine binding in these membrane vesicles. In contrast, purified intestinal brush-border membrane vesicles possess neither Na + gradient-driven carnitine transport nor Na +-dependent carnitine binding.

An Effect of Ca 2+ on the Intrinsic Cl − -conductance of Rat Kidney Cortex Brush Border Membrane Vesicles

Brush-border membrane vesicles (BBMV) were prepared from superficial rat renal cortex by a divalent(2+)-precipitation technique using either CaCl2 or MgCl2. The dependence of the initial [14C]-D-glucose (or [3H]-L-proline) uptake rate and the extent of the overshoot of D-glucose or L-proline uphill accumulation from solutions containing 100 mM Na+ salt, was found to be dependent upon the precipitating divalent cation. With Mg2+ precipitation the initial uptake and overshoot accumulation of either D-glucose or L-proline were enhanced compared to BBMV prepared by Ca2+ precipitation. When the anion composition of the media was varied (uptake in Cl- media in comparison to gluconate(-)-containing media) it was found that the Cl(-)-dependent component of the initial uptake was markedly depressed with Ca(2+)-prepared BBMV (104.99 +/- 33.31 vs. 13.83 +/- 1.44 pmoles/sec/mg protein for Mg2+ and Ca2+ prepared vesicles respectively). When Ca2+ was loaded into Mg2+ prepared BBMV using a freeze-thaw technique, it was found that the magnitude and Cl- enhancement of D-glucose transport was reduced in a dose-dependent manner. Neomycin, an inhibitor of phospholipase C, had no effect on the reduction of D-glucose uptake by Ca2+ in Mg2+ prepared vesicles. In contrast, phosphatase inhibitors such as vanadate and fluoride were able to partially reverse the Ca2+ inhibition of D-glucose uptake and restore the enhancement due to Cl- media. In addition, inhibitors of protein phosphatase 2B, deltamethrin (50 nM) and trifluoperazine (10 microM), caused partial reversal of Ca2(+)-dependent inhibition of D-glucose uptake. Direct measurement of changes in the bi-ionic (Cl-vs. gluconate-) transmembrane electrical potential differences using the cyanine dye, 3,3'-dipropylthiodicarbocyanine iodide DiSC3-(5) confirmed that Cl- conductance was reduced in Ca(2+)-prepared vesicles. We conclude that a Cl- conductance coexists with Na+ cotransport in rat renal BBMV and this may be subject to negative regulation by Ca2+ via stimulation of protein phosphatase (PP2B).

Micromolar Concentrations of Bromosulfophthalein Affect Transport of &lt;i&gt;D&lt;/i&gt;-Glucose, &lt;i&gt;L&lt;/i&gt;-Alanine and &lt;i&gt;p&lt;/i&gt;-Aminohippurate in Rat Renal Brush-Border Membranes

The effect of bromosulfophthalein (BSP) on potential-stimulated p-aminohippurate (PAH) uptake as well as on Na+-D-glucose and Na+-L-alanine cotransport was investigated in rat renal brush-border membrane vesicles. All three transport mechanisms were inhibited by BSP in the following order: PAH > D-glucose > L-alanine. Half-maximal inhibition of initial uptakes was observed at approximately 10,75 and 250 µM BSP, respectively. The inhibition of initial Na+-D-glucose uptake could not be attributed to increased cation permeability, since equilibrium exchange was equally affected by BSP. The nonspecific short-term inhibition of unrelated transport systems by BSP suggests a more general effect, such as a change in membrane fluidity. In addition, long-term incubation with 0.5 mM BSP resulted in a loss of vesicle integrity.