Rat Kidney Brush-border Membrane Research Articles

Novel polymyxin derivatives are effective in treating experimental Escherichia coli peritoneal infection in mice

Novel synthetic polymyxin derivatives including NAB737 and NAB739 are as effective as polymyxin B in vitro against the common opportunistic pathogen Escherichia coli. Another derivative, NAB7061, lacks direct antibacterial action but sensitizes E. coli to several other antibacterial agents including macrolides. The renal handling of NAB739 and NAB7061 in rats differs from that of polymyxin B. Furthermore, the affinities of NAB739 and NAB7061 for isolated rat kidney brush border membrane are significantly lower than that of polymyxin B. Here we investigate the in vivo antibacterial effect of these compounds. The polymyxin derivatives were evaluated in an experimental murine peritonitis model. Immunocompetent mice were infected intraperitoneally with E. coli IH3080 and were subcutaneously treated with NAB737, NAB739 or NAB7061. A >4.0 log(10) reduction in bacterial load compared with saline control was achieved 6 h after initiation of treatment with 1 mg/kg of NAB739 twice or 4 mg/kg of NAB737 twice. Combination therapy with NAB7061 (5 mg/kg) twice and erythromycin (10 mg/kg) resulted during the same time course in a >2.0 log(10) reduction in bacterial load compared with saline control. Neither NAB7061 nor erythromycin was effective as monotherapy. Together with the ability to reduce bacterial load, the NAB compounds also improved the clinical status of the mice. We found that the three novel synthetic polymyxin B derivatives had a potent in vivo bactericidal effect against E. coli.

Novel Polymyxin Derivatives Carrying Only Three Positive Charges Are Effective Antibacterial Agents

The lack of novel antibiotics against gram-negative bacteria has reinstated polymyxins as the drugs of last resort to treat serious infections caused by extremely multiresistant gram-negative organisms. However, polymyxins are nephrotoxic, and this feature may complicate therapy or even require its discontinuation. Like that of aminoglycosides, the nephrotoxicity of polymyxins might be related to the highly cationic nature of the molecule. Colistin and polymyxin B carry five positive charges. Here we show that novel polymyxin derivatives carrying only three positive charges are effective antibacterial agents. NAB739 has a cyclic peptide portion identical to that of polymyxin B, but in the linear portion of the peptide, it carries the threonyl-D-serinyl residue (no cationic charges) instead of the diaminobutyryl-threonyl-diaminobutyryl residue (two cationic charges). The MICs of NAB739 for 17 strains of Escherichia coli were identical, or very close, to those of polymyxin B. Furthermore, NAB739 was effective against other polymyxin-susceptible strains of Enterobacteriaceae and against Acinetobacter baumannii. At subinhibitory concentrations, it dramatically sensitized A. baumannii to low concentrations of antibiotics such as rifampin, clarithromycin, vancomycin, fusidic acid, and meropenem. NAB739 methanesulfonate was a prodrug analogous to colistin methanesulfonate. NAB740 was the most active derivative against Pseudomonas aeruginosa. NAB7061 (linear portion of the peptide, threonyl-aminobutyryl) lacked direct antibacterial activity but sensitized the targets to hydrophobic antibiotics by factors up to 2,000. The affinities of the NAB compounds for isolated rat kidney brush border membrane were significantly lower than that of polymyxin B.

Nephrotoxicity of uranyl acetate: effect on rat kidney brush border membrane vesicles

Since the Gulf war exposure to depleted uranium, a known nephrotoxic agent, there is a renewed interest in the toxic effects of uranium in general and its mechanism of nephrotoxicity which is still largely unknown in particular. In order to investigate the mechanism responsible for uranium nephrotoxicity and the therapeutic effect of urine alkalization, we utilized rat renal brush border membrane vesicles (BBMV). Uranyl acetate (UA) caused a decrease in glucose transport in BBMV. The apparent K (i) of uranyl was 139+/-30 microg uranyl/mg protein of BBMV. Uranyl at 140 microg/mg protein of BBMV reduced the maximal capacity of the system to transport glucose [V (max) 2.2+/-0.2 and 0.96+/-0.16 nmol/mg protein for control and uranyl treated BBMV (P<0.001), respectively] with no effect on the apparent K (m) (1.54+/-0.33 and 1.54+/-0.51 mM for control, and uranyl treated BBMV, respectively). This reduction in V(max) is at least partially due to a decrease in the number of sodium-coupled glucose transporters as apparent from the reduction in phlorizin binding to the uranyl treated membranes, V (max) was reduced from 247+/-13 pmol/mg protein in control BBMV to 119+/-3 pmol/mg protein in treated vesicles (P<0.001). The pH of the medium has a profound effect on the toxicity of UA on sodium-coupled glucose transport in BBMV: higher toxicity at neutral pH (around pH 7.0), and practically no toxicity at alkaline pH (7.6). This is the first report showing a direct inhibitory dose and pH dependent effect of uranyl on the glucose transport system in isolated apical membrane from kidney cortex.

Study of bradykinin metabolism by rat lung tissue membranes and rat kidney brush border membranes by HPLC with inductively coupled plasma—mass spectrometry and orthogonal acceleration time-of-flight mass spectrometry

The coupling of the techniques, high-performance liquid chromatography (HPLC), orthogonal acceleration time-of-flight mass spectrometry (OATOF-MS) and inductively coupled plasma mass spectrometry (ICP-MS) provides a very powerful method for identifying and quantifying the products of bradykinin metabolism. In this study, we were able to identify the major metabolites of bradykinin degradation reported in the literature. In addition, a new bradykinin metabolite corresponding to bradykinin 5,9 fragment (BK-(5,9)-fragment) was identified as a product of neutral endopeptidase (NEP) activity. This finding establishes that NEP cleaves bradykinin simultaneously at the positions 4-5 and 7-8. We also demonstrate the equivalent participation of NEP and angiotensin-converting enzyme (ACE) within the rat lung tissue membranes (RLTM) in bradykinin degradation, suggesting its suitability as a model for the assay of dual ACE/NEP inhibitors. On the contrary, in rat kidney brush border membranes (KBBM), ACE is not significantly involved in bradykinin metabolism, with NEP being the major enzyme.

Nateglinide uptake by a ceftibuten transporter in the rat kidney brush-border membrane

Nateglinide, a novel oral hypoglycemic agent, possesses a carbonyl group and a peptide-type bond in its structure. We previously reported that nateglinide transport occurs via a single system that may be identical to the ceftibuten/H + cotransport system by the rat small intestine. We speculated that the absorption system present on the intestinal epithelium may be similar to that found on the renal tubular epithelium. The aim of this study was to characterize the transporters on the apical side of the kidney that may contribute to the reabsorption of ceftibuten and nateglinide. The uptake of nateglinide by rat renal brush-border membranes is associated with an H +-coupled transport system. Ceftibuten competitively inhibited H +-dependent nateglinide uptake. In contrast, Gly-Sar, cephradine and cephalexin had no effect on nateglinide uptake. Nateglinide competitively inhibited H +-driven transporter-mediated ceftibuten uptake. We conclude that nateglinide transport occurs via a single system that is H +-dependent and may be identical to the ceftibuten/H + cotransport system.

EP24.15 interacts with the angiotensin II type I receptor and bradykinin B2 receptor.

The carboxyl-terminal cytoplasmic domain of the angiotensin II type 1 receptor (AT1) is known to interact with several classes of intracellular proteins that may modulate receptor function. Employing yeast two-hybrid screening of a human embryonic kidney cDNA library with the carboxyl-terminal cytoplasmic domain of the AT1 receptor as a bait, we have isolated EP24.15 (EC 3.4.24.15, thimet oligopeptidase) as a potentially interacting protein. EP24.15 is widely distributed and is known to degrade bioactive peptides such as angiotensin I and II and bradykinin. In addition, EP24.15 was previously identified as a putative soluble angiotensin II binding protein. Two-hybrid screening also determined that EP24.15 can interact with the B2 bradykinin receptor. Transient expression of EP24.15 in a porcine kidney epithelial cell line stably expressing full length AT1 and full length B2 followed by affinity chromatography and co-immunoprecipitation confirmed EP24.15 association with both AT1 and B2 receptors. EP24.15 was also co-immunoprecipitated with AT1 and B2 in rat kidney brush border membranes (BBM) and basolateral membranes (BLM). Both AT1 and B2 undergo ligand-induced endocytosis. Analysis of endosomal fractions following immunoprecipitation with AT1 or B2 antibodies detected strong association of EP24.15 with the receptors in both light and heavy endosomal populations. Therefore, the present study indicates that EP24.15 associates with AT1 and B2 receptors both at the plasma membrane and after receptor internalization and suggests a possible mechanism for endosomal disposition of ligand that may facilitate receptor recycling.

Spatial-Temporal Studies of Membrane Dynamics: Scanning Fluorescence Correlation Spectroscopy (SFCS)

Giant unilamellar vesicles (GUVs) have been widely used as a model membrane system to study membrane organization, dynamics, and protein-membrane interactions. Most recent studies have relied on imaging methods, which require good contrast for image resolution. Multiple sequential image processing only detects slow components of membrane dynamics. We have developed a new fluorescence correlation spectroscopy (FCS) technique, termed scanning FCS (i.e., SFCS), which performs multiple FCS measurements simultaneously by rapidly directing the excitation laser beam in a uniform (circular) scan across the bilayer of the GUVs in a repetitive fashion. The scan rate is fast compared to the diffusion of the membrane proteins and even small molecules in the GUVs. Scanning FCS outputs a “carpet” of timed fluorescence intensity fluctuations at specific points along the scan. In this study, GUVs were assembled from rat kidney brush border membranes, which included the integral membrane proteins. Scanning FCS measurements on GUVs allowed for a straightforward detection of spatial-temporal interactions between the protein and the membrane based on the diffusion rate of the protein. To test for protein incorporation into the bilayers of the GUVs, antibodies against one specific membrane protein (NaPi II cotransporter) were labeled with ALEXA-488. Fluorescence images of the GUVs in the presence of the labeled antibody showed marginal fluorescence enhancement on the GUV membrane bilayers (poor image contrast and resolution). With the application of scanning FCS, the binding of the antibody to the GUVs was detected directly from the analysis of diffusion rates of the fluorescent antibody. The diffusion coefficient of the antibody bound to NaPi II in the GUVs was ∼200-fold smaller than that in solution. Scanning FCS provided a simple, quantitative, yet highly sensitive method to study protein-membrane interactions.

Characterization of D-fructose transport by rat kidney brush-border membrane vesicles: changes in hypertensive rats.

D-fructose transport was characterized in renal brush-border membrane vesicles (BBMVs) from both spontaneously hypertensive rats (SHR) and normotensive genetic control Wistar-Kyoto (WKY) rats. Kinetic studies indicated that the maximal rate (Vmax) of D-fructose transport was significantly lower in SHR compared with WKY rats. No differences were observed in the Michaelis constant (Km) or the diffusion constant (Kd) between the two groups of animals. D-fructose inhibited its own transport, whereas the presence of D-glucose, D-galactose, phlorizin, and cytochalasin B did not inhibit the transport of D-fructose in either animal group. To explain the reduction in D-fructose transport in SHR, the density of the D-fructose transporter, GLUT5, was analyzed by Western blot. GLUT5 levels were lower in SHR, a reduction similar to that of the Vmax. Thus, there appears to be a high-affinity, low-capacity, GLUT5-type fructose carrier in the apical membranes of rat kidney cortex, and the decrease in the Vmax of D-fructose transport in renal BBMVs from hypertensive rats correlates well with a reduction in the expression of GLUT5 protein.

Characterization of L-carnitine transport into rat skeletal muscle plasma membrane vesicles.

Transport of L-carnitine into skeletal muscle was investigated using rat sarcolemmal membrane vesicles. In the presence of an inwardly directed sodium chloride gradient, L-carnitine transport showed a clear overshoot. The uptake of L-carnitine was increased, when vesicles were preloaded with potassium. When sodium was replaced by lithium or cesium, and chloride by nitrate or thiocyanate, transport activities were not different from in the presence of sodium chloride. However, L-carnitine transport was clearly lower in the presence of sulfate or gluconate, suggesting potential-dependent transport. An osmolarity plot revealed a positive slope and a significant intercept, indicating transport of L-carnitine into the vesicle lumen and binding to the vesicle membrane. Displacement experiments revealed that approximately 30% of the L-carnitine associated with the vesicles was bound to the outer and 30% to the inner surface of the vesicle membrane, whereas 40% was unbound inside the vesicle. Saturable transport could be described by Michaelis-Menten kinetics with an apparent Km of 13.1 microM and a Vmax of 2.1 pmol.(mg protein-1).s-1. L-Carnitine transport could be trans-stimulated by preloading the vesicles with L-carnitine but not with the carnitine precursor butyrobetaine, and was cis-inhibited by L-palmitoylcarnitine, L-isovalerylcarnitine, and glycinebetaine. On comparing carnitine transport into rat kidney brush-border membrane vesicles and OCTN2, a sodium-dependent high-affinity human carnitine transporter, cloned recently from human kidney also expressed in muscle, the Km values are similar but driving forces, pattern of inhibition and stereospecificity are different. This suggests the existence of more than one carnitine carrier in skeletal muscle.

Carnitine transport and its inhibition by sulfonylureas in human kidney proximal tubular epithelial cells

The kidney plays an important role in the homeostasis of carnitine by its ability to reabsorb carnitine almost completely from the glomerular filtrate. The transport process responsible for this reabsorption has been investigated thus far only in laboratory animals. Here we report on the characteristics of carnitine uptake in a proximal tubular epithelial cell line derived from human kidney. The uptake process was found to be obligatorily dependent on Na + with no involvement of anions. The process was saturable, with a Michaelis–Menten constant of 14 ± 1 μM. The Na +:carnitine stoichiometry was 1:1. The same process also was found to be responsible for the uptake of acetylcarnitine and propionylcarnitine, two acyl esters of carnitine with potential for therapeutic use in humans. The uptake process was specific for carnitine and its acyl esters. Betaine, a structural analog of carnitine, interacted with the uptake process to a significant extent. The present studies also showed that sulfonylureas, oral hypoglycemic agents currently used in the management of type 2 diabetes, inhibited the carnitine uptake system. Among the sulfonylureas tested, glibenclamide was the most potent inhibitor. The inhibition was competitive. Glibenclamide inhibited the uptake not only of carnitine but also of acetylcarnitine and propionylcarnitine. The inhibition most likely was the result of direct interaction of the compound with the carnitine transporter because the inhibition could be demonstrated in purified rat kidney brush border membrane vesicles.

Inhibition of the hydrolytic and transpeptidase activities of rat kidney γ‐glutamyl transpeptidase by specific monoclonal antibodies

Monoclonal antibodies (mAb) against the native form of rat kidney gamma-glutamyl transpeptidase (GGT) were isolated by screening hybridomas with rat kidney brush-border membrane vesicles. They were directed against protein rather than sugar epitopes in that each recognized all GGT isoforms. All of them inhibited partially the enzyme activity of GGT. They were specific in that they inhibited the rat enzyme, but not the mouse or human enzyme. Kinetic analyses were carried out with free GGT and GGT-mAb complexes with d-gamma-glutamyl-p-nitroanilide in the presence or absence of maleate, or in the presence or absence of alanine, cysteine, cystine or glycylglycine as gamma-glutamyl acceptors. mAbs 2A10 and 2E9 inhibited the hydrolytic and glutaminase activities of GGT and had little effect on the transpeptidation activity of the enzyme, whereas mAbs 4D7 and 5F10 inhibited transpeptidation, but not hydrolytic or glutaminase activities. mAb 5F10 mimicked the effect of maleate on GGT, in that it inhibited transpeptidation, enhanced the glutaminase activity and increased the affinity of the donor site of GGT for acivicin. Such mAbs may be useful for long-term studies in tissue cultures and in vivo, and for the identification of GGT epitopes that are important for the hydrolytic and transpeptidase activities.

Taurocholate induces preferential release of phosphatidylcholine from rat liver canalicular vesicles.

Biliary phospholipid secretion involves predominant segregation of canalicular phosphatidylcholine into bile. We tested the hypothesis that micellar concentrations of the major physiologic bile salt taurocholate can preferentially solubilize phosphatidylcholine from the canalicular rat liver plasma membrane. Subcellular fractions from rat liver and kidney were isolated with standardized procedures, incubated in vitro with taurocholate or 3-[(3-cholamidopropyl)dimethylammonio]-propane-1-sulphonate (CHAPS) and released phospholipids determined after centrifugation. After incubation of canalicular (cLPM) and basolateral (blLPM) rat liver plasma membrane vesicles with 6 and 8 mM taurocholate, the proportion of phosphatidylcholine released was about two-fold higher as compared with its relative contribution to the overall lipid composition of the membranes. Quantitatively, this taurocholate-induced preferential phosphatidylcholine release was about four-fold higher in cLPM (117 nmol) as compared with blLPM (28 nmol). Comparison of membranes from different organs showed that increased sphingomyelin content reduced taurocholate-induced phosphatidylcholine release. Furthermore, phosphatidylcholine release from cLPM did not fit an inverse exponential relationship between membrane sphingomyelin content and phosphatidylcholine release from different starting material, indicating that cLPM is especially prone to taurocholate-induced phosphatidylcholine release. In contrast, in rat liver microsomes and kidney brush border membranes, taurocholate released phospholipids in proportion of their membrane contents, indicating an unspecific membrane solubilizing effect only. Similarly, CHAPS had an unselective lipid solubilizing effects in cLPM and blLPM. These results support the concept that the very last step of canalicular phospholipid secretion is mediated in vivo by bile salt-induced vesiculation of phosphatidylcholine-enriched microdomains from the outer leaflet of cLPM.

Solubilization and reconstitution characteristics of the carrier protein(s) responsible for the transport of ceftibuten, a substrate for the oligopeptide transporters, in rat renal brush-border membrane

Optimal procedures for the reconstitution of the transport activity of ceftibuten, a dianionic ß-lactam antibiotic, from rat kidney brush-border membrane were developed. The uptake activity into reconstituted proteoliposomes appeared to be particularly sensitive to the extraction conditions, and to the lipid composition used for reconstitution. Changes in the concentration of octyl glucoside significantly affected the extraction of ceftibuten transport activity, and optimal extraction was achieved at a concentration of 60 mM. Optimal reconstitution was achieved using a lipid composition of asolectin, cholesterol and phosphatidylserine in a w/w percent ratio of 60:30:10, respectively, and with a lipid-to-protein ratio of 10. The uptake of ceftibuten into the resulting proteoliposomes showed temperature and pH dependency, was inhibitable by a range of cephem antibiotics, oligopeptides and the organic anion PAH, and was trans-stimulated by cephalexin and dipeptides. This reconstitution system will likely prove useful in future studies on the functional analysis of the peptide transport system in a purified form.

Proton/solute cotransport in rat kidney brush-border membrane vesicles: relative importance to both d-glucose and peptide transport

We have determined the relative importance of the transmembrane proton electrochemical gradient to the transport of d-[ 14C]glucose and [ 14C]glycylsarcosine (gly-sar) in rat kidney brush-border membrane vesicles (BBMV) from superficial renal cortex. Electrogenic [ 14C]gly-sar transport was first optimised by imposing a pH gradient (pH o = 5.7, pH i = 8.4) and an interior negative p.d. (using outwardly directed K + gradient plus valinomycin). Under identical conditions (pH o = 5.7, pH i = 8.4), an acceleration f of initial d-[ 14C]glucose (at 100 μM) transport by 2.0 ± 0.7-fold was observed compared to no proton gradient (pH o = 8.4, pH i = 8.4). This increase was due primarily to an effect of external protons, since acidic conditions (pH o = pH i = 5.7) also resulted in ann acceleration of d-glucose influx (2-fold). The increase in d-glucose transport in the presence of external acidity was reduced by the uncoupler FCCP, even in the absence of a proton gradient. Furthermore, the increased d-glucose transport with external acidity in the presence of a proton gradient was insensitive to a K + gradient-driven diffusion potential in the presence of valinomycin. In no instance was an overshoot accumulation of d-[ 14C]glucose observed in H + gradient conditions. H +-stimulated d-[ 14C]glucose transport showed a linear dependence on d-glucose concentration up to 20 mM d-glucose, unlike electrogenic Na +-dependent d-glucose transport, whose K m was 1.77 ± 0.35 mM. In contrast, the initial rate of [ 14C]gly-sar (100 μM) transport by the renal H +/di-tripeptide transporter was accelerated 15.7 ± 3.3-fold and stimulated a marked overshoot of 5.1 ± 0.4-fold over equilibrium values. Conversely, the electrogenic, Na +/glucose transporter could be readily demonstrated, whilst [ 14C]gly-sar transport could not be energised by an inward Na + gradient. The absence of electrogenic d-glucose transport in H + gradient conditions is clear evidence against H +/glucose cotransport in Na +-free conditions mediated by SGLT2 (sodium-glucose transporter, renal cortex). Furthermore, since a proton gradient does not increase brush-border membrane d-glucose uptake in Na +-rich media, it is unlikely that in vivo renal d-glucose transport mediated via SGLT2 may be energised by the transmembrane proton gradient.

Membrane association and oligomeric organization of the alpha and beta subunits of mouse meprin A.

Meprins are oligomeric cell surface metalloproteinases of the "astacin family." They consist of two types of subunits (alpha and beta), which are evolutionarily related and whose cDNA sequences predict a similar domain structure. The present work shows that reducing agents solubilized meprin alpha subunits (approximately 90%), but not beta subunits, from mouse kidney brush border membranes. In addition, immunoblotting of membranes or purified meprins with an antibody raised to the alpha subunit epidermal growth factor-like domain, predicted to be near the COOH terminus from the cDNA-deduced amino acid sequence, indicated that this domain is not present in the mature alpha subunit. By contrast, an epitope predicted to be near the COOH terminus of the beta subunit was present in the mature form of beta. When meprins were solubilized from brush border membranes by papain, the size of the alpha subunit (approximately 90 kDa) did not change, while the beta subunit decreased from 110 to 90 kDa with concomitant loss of the COOH-terminal epitope. These data indicate that beta is a type I transmembrane protein, while alpha does not transverse the membrane and its association is dependent on disulfide bonds. The oligomeric organization of purified meprin A (EC 3.4.24.18), examined by sedimentation equilibrium analysis and native gradient gel electrophoresis, is that of disulfide-bridged dimers which aggregate noncovalently to form higher molecular weight complexes, predominantly tetramers. Western blotting of ICR kidney brush border membrane proteins identified alpha 2 homodimers and alpha beta heterodimers. Treatment of mouse or rat kidney brush border membranes with 7 M urea solubilized alpha 2, but not alpha beta dimers. Thus, the mature alpha subunit exists in alpha 2 and alpha beta disulfide-linked dimers which form tetramers, and the alpha 2 homodimers associate with the membrane through noncovalent interactions with alpha beta.

Interaction of rat kidney P-glycoprotein with a urinary component and various drugs including cyclosporin A

The interaction of rat renal P-glycoprotein with various drugs and a hydrophobic component found in rat urine was studied to gain an understanding of both its transport function in kidney and its potential role in drug secretion and drug-induced nephrotoxicity. Rat kidney brush-border membranes (BBM) were photolabeled with [3H]azidopine, a calcium-channel blocker that covalently labeles P-glycoprotein. P-glycoprotein was immunoprecipitated with a rabbit polyclonal antibody against the human MDR1 protein (multidrug resistance gene class 1). The amount of [3H]azidopine incorporated into P-glycoprotein was quantitated following gel electrophoresis and fluorography. Photolabeling inhibition assays were conducted with a panel of drugs known to interact with P-glycoprotein in multidrug-resistant cells. Verapamil or quinidine [half-maximal inhibition constant (K0.5) = 1 microM], vinblastine (K0.5 = 3 microM), and doxorubicin or daunomycin (K0.5 = 10 microM) all blocked [3H]azidopine photolabeling of renal P-glycoprotein. Of the drugs tested, the immunosuppressant drug, cyclosporin A, interacted with kidney P-glycoprotein with the highest affinity (K0.5 = 50 nM). However, the cardiac glycoside, digoxin, failed to inhibit P-glycoprotein photolabeling. A hydrophobic rat urine extract prepared by reverse-phase chromatography also blocked photolabeling of renal P-glycoprotein. Our current hypothesis is that various drugs may inhibit urinary excretion of an endogenous substrate by virtue of their ability to bind with high affinity to P-glycoprotein. A hypothesis of drug-induced nephrotoxicity based on the interaction of various compounds like cyclosporin A with P-glycoprotein is presented.

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.

Vascular A10 cell membranes contain an endothelin metabolizing neutral endopeptidase

We have investigated the possible presence of endothelin-metabolizing neutral endopeptidase (NEP, EC 3.4.24.11) on A10 cell membranes using [ 125I]-ET-1 binding and direct measurements of NEP. NEP activity of A10 cell membranes has been compared to that of solubilized rat kidney brush border membranes (KNEP). Specific [ 125I]-ET-1 (50 pM) binding (defined with 100 nM ET-1) to A10 cell membranes was increased in a concentration dependent manner by the selective NEP inhibitors thiorphan, phosphoramidon, and SQ 28,603 {(±)-N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]-β-alanine} with EC 50 values of 9.4, 28.4, and 5.7 nM respectively. At equilibrium (150 min), 70% more specific binding was apparent in the presence of these inhibitors. Phosphoramidon (2 μM) did not alter B max values, but it decreased the apparent K D for [ 125I] ET-1 from 63 (±3) to 27 (±2) pM. Thiorphan, phosphoramidon, and SQ 28,603 inhibited A10 cell NEP activity with IC 50 values of 5.3, 36.5, and 6.0 nM respectively, which was similar to values obtained with KNEP (3.6, 22.6, and 3.5 nM). ET-1 inhibited A10 cell NEP, and KNEP with IC 50 values of 30 and 21.3 μM respectively. The order of inhibitory potencies: ET-3 > ET-1 = ET-2 ≥ sarafotoxin-6b was similar for both systems. These data suggest A10 cell membranes contain a NEP which has similar characteristics to NEP 24.11, and which actively metabolizes [ 125I]-ET-1.

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.

Effects of dimethylsulfoxide and mercurial sulfhydryl reagents on water and solute permeability of rat kidney brush border membranes

The effects of dimethylsulfoxide, DMSO, and mercurial sulfhydryl reagents have been studied on water and small solute permeability of rat renal brush border membrane vesicles. Water and solute permeability was measured by mixing membrane vesicles with hypertonic solutions in a stopped-flow apparatus and following osmotically-induced changes in vesicular volume via changes in scattered light intensity. The rate constant of the fast osmotic shrinkage is proportional to the osmotic water permeability, while the rate constant of the slow reswelling phase is proportional to the solute permeability. Using mannitol as the osmotic agent, the osmotic shrinkage of rat renal brush border membrane vesicles followed a biphasic time course. 80% of the vesicles shrunk with a rate constant of approx. 50 s −1 and 20% with a rate constant of approx. 2 s −1, DMSO decreased dose-dependently the amplitude of the fast osmotic shrinkage, without affecting its rate constant. In contrast to DMSO, HgCl 2 decreased the rate constant but not the amplitude of the fast osmotic shrinkage of renal brush border vesicles. Between 40–50 μM HgCl 2, the inhibition of the fast osmotic shrinkage was completed. DMSO and HgCl 2 increase the activation energy of water permeation in renal membranes from 3 to 12–15 kcal/mol. DMSO and HgCl 2 did not affect the rate constant of the slow osmotic shrinkage of renal membrane vesicles and were also without effect on osmotic shrinkage of small intestinal brush border and pure phospholipid vesicles. In renal brush border membranes, HgCl 2 at low concentrations (<10 μM) increased by 15-fold the permeability to NaCl and urea but not to mannitol, an effect which precedes the inhibition of water permeability at higher HgCl 2 concentrations. The increase in small solute permeability was irreversible while the inhibition of water permeability could be reversed with cysteine and dithiothreitol. We conclude that water and small solute pathways in rat renal brush border membranes are completely separate entities, which are effected differently by DMSO and HgCl 2. These pathways for water and solutes must be membrane proteins since neither DMSO nor HgCl 2 affect the permeability properties of pure phospholipid vesicles.