Dicarboxylate transport in renal basolateral and brush-border membrane vesicles.

Characteristics of succinate transport were determined in basolateral and brush-border membrane vesicles (BLMV and BBMV, respectively) isolated in parallel from rabbit renal cortex. The uptake of succinate was markedly stimulated by the imposition of an inwardly directed Na+ gradient, showing an "overshoot" phenomenon in both membrane preparations. The stimulation of succinate uptake by an inwardly directed Na+ gradient was not significantly affected by pH clamp or inhibition of Na(+)-H+ exchange. The Na(+)-dependent and -independent succinate uptakes were not stimulated by an outwardly directed pH gradient. The Na dependence of succinate uptake exhibited sigmoidal kinetics, with Hill coefficients of 2.17 and 2.38 in BLMV and BBMV, respectively. The Na(+)-dependent succinate uptake by BLMV and BBMV was stimulated by a valinomycin-induced inside-negative potential. The Na(+)-dependent succinate uptake by BLMV and BBMV followed a simple Michaelis-Menten kinetics, with an apparent Km of 22.20 +/- 4.08 and 71.52 +/- 0.14 microM and a Vmax of 39.0 +/- 3.72 and 70.20 +/- 0.96 nmol/(mg.min), respectively. The substrate specificity and the inhibitor sensitivity of the succinate transport system appeared to be very similar in both membranes. These results indicate that both the renal brush-border and basolateral membranes possess the Na(+)-dependent dicarboxylate transport system with very similar properties but with different substrate affinity and transport capacity.     

Ontogeny of rabbit proximal tubule urea permeability

Urea transport in the proximal tubule is passive and is dependent on the epithelial permeability. The present study examined the maturation of urea permeability (P(urea)) in in vitro perfused proximal convoluted tubules (PCT) and basolateral membrane vesicles (BLMV) from rabbit renal cortex. Urea transport was lower in neonatal than adult PCT at both 37 and 25 degrees C. The PCT P(urea) was also lower in the neonates than the adults (37 degrees C: 45.4 +/- 10.8 vs. 88.5 +/- 15.2 x 10(-6) cm/s, P < 0.05; 25 degrees C: 28.5 +/- 6.9 vs. 55.3 +/- 10.4 x 10(-6) cm/s; P < 0.05). The activation energy for PCT P(urea) was not different between the neonatal and adult groups. BLMV P(urea) was determined by measuring vesicle shrinkage, due to efflux of urea, using a stop-flow instrument. Neonatal BLMV P(urea) was not different from adult BLMV P(urea) at 37 degrees C [1.14 +/- 0.05 x 10(-6) vs. 1.25 +/- 0.05 x 10(-6) cm/s; P = not significant (NS)] or 25 degrees C (0.94 +/- 0.06 vs. 1.05 +/- 0.10 x 10(-6) cm/s; P = NS). There was no effect of 250 microM phloretin, an inhibitor of the urea transporter, on P(urea) in either adult or neonatal BLMV. The activation energy for urea diffusion was also identical in the neonatal and adult BLMV. These findings in the BLMV are in contrast to the brush-border membrane vesicles (BBMV) where we have previously demonstrated that urea transport is lower in the neonate than the adult. Urea transport is lower in the neonatal proximal tubule than the adult. This is due to a lower rate of apical membrane urea transport, whereas basolateral urea transport is the same in neonates and adults. The lower P(urea) in neonatal proximal tubules may play a role in overall urea excretion and in developing and maintaining a high medullary urea concentration and thus in the ability to concentrate the urine during renal maturation.     

Water transporting properties of hepatocyte basolateral and canalicular plasma membrane domains

Previous work from our laboratory supports an important role for aquaporins (AQPs), a family of water channel proteins, in bile secretion by hepatocytes. To further define the pathways and molecular mechanisms for water movement across hepatocytes, we directly assessed osmotic water permeability (Pf) and activation energy (Ea) in highly purified, rat hepatocytes basolateral membrane vesicles (BLMV) and canalicular membrane (CMV) vesicles by measuring scattered light intensity using stopped-flow spectrophotometry. The time course of scattered light for BLMV and CMV fit well to a single-exponential function. In BLMV, Pf was 108 +/- 4 mum.s-1 (25 degrees C) with an Ea of 7.7 kcal/mol; in CMV, Pf was 86 +/- 5 mum.s-1 (25 degrees C) with an Ea of 8.0 kcal/mol. The AQP blocker, dimethyl sulfoxide, significantly inhibited the Pf of both basolateral (81 +/- 4 mum.s-1; -25%) and canalicular (59 +/- 4 mum.s-1; -30%) membrane vesicles. When CMV were isolated from hepatocytes treated with dibutyryl cAMP, a double-exponential fit was needed, implying two functionally different vesicle populations; one population had Pf and Ea values similar to those of CMV from untreated hepatocytes, but the other population had a very high Pf (655 +/- 135 mum.s-1, 25 degrees C) and very low Ea (2.8 kcal/mol). Dimethyl sulfoxide completely inhibited the high Pf value in this second vesicle population. In contrast, Pf and Ea of BLMV were unaltered by cAMP treatment of hepatocytes. Our results are consistent with the presence of both lipid- and AQP-mediated pathways for basolateral and canalicular water movement across the hepatocyte plasma membrane barrier. Our data also suggest that the hepatocyte canalicular membrane domain is rate-limiting for transcellular water transport and that this domain becomes more permeable to water when hepatocytes are exposed to a choleretic agonist, presumably by insertion of AQP molecules. These data suggest a molecular mechanism for the efficient coupling of osmotically active solutes and water transport during canalicular bile formation.     

Characteristics of tetraethylammonium transport in rabbit renal plasma-membrane vesicles.

Transport of [14C]tetraethylammonium (TEA), an organic cation, was studied in brush-border (BBMV) and basolateral (BLMV) membrane vesicles isolated from rabbit kidney cortex. In BBMV, the presence of an outwardly directed H+ gradient induced a marked stimulation of TEA uptake against its concentration gradient (overshoot phenomenon), whereas a valinomycin-induced inside-negative potential had no effect on TEA uptake. In BLMV, TEA uptake was significantly stimulated by the presence of an outwardly directed H+ gradient and by an inside-negative potential, but the effect of H+ gradient was absent when the vesicles were chemically 'voltage clamped'. In BBMV, internal H+ stimulated TEA uptake in a non-competitive manner by binding at a site with apparent pKa of 6.87. External H+ inhibited TEA uptake through a direct interaction with the putative H+/organic-cation exchanger at a site with apparent pKa of 6.78. Changing external pH while maintaining the pH gradient constant produced a result similar to that obtained by changing external pH alone. Increasing external H+ showed a mixed-type inhibition of TEA uptake. These results suggest that in the rabbit TEA transport across the basolateral membranes is driven by an inside-negative potential and that transport across the brush-border membrane is driven by a H+ gradient via an electroneutral H+/TEA antiport system.     

Lipid-phase structure in epithelial cell membranes: comparison of renal brush border and basolateral membranes

The lipid-phase structures of brush border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) isolated from rabbit renal cortex were compared by steady-state and phase-modulation measurements of diphenylhexatriene (DPH) and trans- and cis-parinaric acid (tPnA and cPnA) fluorescence. A temperature-scanning system was used which gave reproducible temperature profiles of steady-state and dynamic fluorescence parameters with a resolution of 0.1 degrees C. Steady-state anisotropy of DPH showed a triphasic dependence on temperature with slope discontinuities at 22 +/- 4 and 47 +/- 3 degrees C (BBMV) and at 23 +/- 2 and 48 +/- 1 degrees C (BLMV). At all temperatures, DPH anisotropy in BBMV was greater than that in BLMV. Ground-state heterogeneity analysis of tPnA and cPnA fluorescence lifetime data demonstrated the presence of long (greater than 12 ns) and short (less than 5 ns) lifetime components, interpreted in terms of solid-phase and fluid-phase lipid domains. The fraction of solid-phase phospholipid decreased from 0.9 to 0.1 for BBMV and from 0.7 to 0.3 in BLMV with increasing temperature (10-50 degrees C). In both membranes, tryptophan-PnA fluorescence energy-transfer measurements showed that membrane proteins were surrounded by a fluidlike phospholipid phase. These results demonstrate the inadequacy of steady-state DPH anisotropy data in defining the structural characteristics of complex biological membranes. Results obtained with the phase-sensitive parinaric acid probes demonstrate major differences in the phase structure of the two opposing cell membranes in both the bulk lipid and the lipid microenvironment around membrane proteins.     

Proton nuclear magnetic resonance measurement of diffusional water permeability in suspended renal proximal tubules.

Diffusional water permeability was measured in renal proximal tubule cell membranes by pulsed nuclear magnetic resonance using proton spin-lattice relaxation times (T1). A suspension of viable proximal tubules was prepared from rabbit renal cortex by Dounce homogenization and differential sieving. T1 measured in a tubule suspension (22% of exchangeable water in the intracellular compartment) containing 20 mM extracellular MnCl2 was biexponential with time constants 1.8 +/- 0.1 ms and 8.3 +/- 0.2 ms (mean +/- SD, n = 8, 37 degrees C, 10 MHz). The slower time constant, representing diffusional exchange of water between intracellular and extracellular compartments, increased to 11.6 +/- 0.6 ms (n = 6) after incubation of tubules with 5 mM parachloromercuribenzene sulfonate (pCMBS) for 60 min at 4 degrees C and was temperature dependent with activation energy Ea = 2.9 +/- 0.4 kcal/mol. To relate T1 data to cell membrane diffusional water permeabilities (Pd), a three-compartment exchange model was developed that included intrinsic decay of proton magnetization in each compartment and apical and basolateral membrane water transport. The model predicted that the slow T1 was relatively insensitive to apical membrane Pd because of low luminal/cell volume ratio. Based on this analysis, basolateral Pd (corrected for basolateral membrane surface convolutions) is 2.0 X 10(-3) cm/s, much lower than corresponding values for basolateral Pf (10-30 X 10(-3) cm/s) measured in the intact tubule and in isolated basolateral membrane vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Maturational Changes in Rabbit Renal Basolateral Membrane Vesicle Osmotic Water Permeability

We have recently demonstrated that while the osmotic water permeability (P _f ) of neonatal proximal tubules is higher than that of adult tubules, the P _f of brush-border membrane vesicles from neonatal rabbits is lower than that of adults. The present study examined developmental changes in the water transport characteristics of proximal tubule basolateral membranes by determining aquaporin 1 (AQP1) protein abundance and the P _f in neonatal (10–14 days old) and adult rabbit renal basolateral membrane vesicles (BLMV). At 25°C the P _f of neonatal BLMV was significantly lower than the adult BLMV at osmotic gradients ranging from 40 to 160 mOsm/kg water. The activation energies for osmotic water movement were identical in the neonatal and adult BLMV (8.65 ± 0.47 vs. 8.86 ± 1.35 kcal · deg⁻¹· mol⁻¹). Reflection coefficients for sodium chloride and sodium bicarbonate were identical in both the neonatal and adult BLMV and were not different from one. Mercury chloride (0.5 mm) reduced osmotic water movement by 31.3 ± 5.5% in the adult BLMV, but by only 4.0 ± 4.0% in neonatal vesicles (P < 0.01). Adult BLMV AQP1 abundance was higher than that in the neonate. These data demonstrate that neonatal BLMV have a lower P _f and AQP1 protein abundance than adults and that a significantly greater fraction of water traverses the basolateral membrane lipid bilayer and not water channels in neonates compared to adults. The lower P _f of the neonatal BLMV indicates that the basolateral membrane is not responsible for the higher transepithelial P _f in the neonatal proximal tubule. Received: 8 July 1999/Revised: 9 November 1999     

Inhibition of intestinal glucose uptake by aporphines and secoaporphines

The effects of aporphines and secoaporphines on glucose uptake by isolated intestinal brush-border membrane vesicles (BBMV) or basolateral membrane vesicles (BLMV) and glucose absorption during in situ intestinal perfusion were studied. Of the tested compounds, N-allylsecoboldine was the most potent glucose uptake inhibitor, with IC50 values of 159 microM and 121 microM, respectively, for uptake by BBMV and BLMV. While thaliporphine competitively inhibited glucose uptake by both membrane preparations, inhibition by N-allylsecoboldine was competitive using BBMV and noncompetitive using BLMV. In addition, N-allylsecoboldine significantly reduced both glucose absorption during in situ intestinal perfusion and blood glucose levels in the oral glucose tolerance test. The results demonstrate that levels of both aporphines and secoaporphines achievable by oral administration have an inhibitory effect on intestinal glucose uptake and suggest that the hypoglycemic effects of these compounds merit attention.     

Proteinases inhibit H(+)-ATPase and Na+/H+ exchange but not water transport in apical and endosomal membranes from rat proximal tubule.

A marked increase in water permeability can be induced in Xenopus oocytes by injection of mRNA from tissues that express water channels, suggesting that the water channel is a protein. In view of this and previous reports which showed that proteinases may interfere with mercurial inhibition of water transport in red blood cells (RBC), we examined the influence of trypsin, chymotrypsin, papain, pronase, subtilisin and thermolysin on water permeability as well as on ATPase activity, H(+)-pump, passive H+ conductance, and Na+/H+ exchange in apical brush-border vesicles (BBMV) and endosomal (EV) vesicles from rat renal cortex. H+ transport was measured by Acridine orange fluorescence quenching and water transport by stopped-flow light scattering. As measured by potential-driven H+ accumulation in BBMV and EV, proteinase treatment had little effect on vesicle integrity. In BBMV, ecto-ATPase activity was inhibited by 15-30%, Na+/H+ exchange by 20-55%, and H+ conductance was unchanged. Osmotic water permeability (Pf) was 570 microns/s and was inhibited 85-90% by 0.6 mM HgCl2; proteinase treatment did not affect Pf or the HgCl2 inhibition. In EV, NEM-sensitive H+ accumulation and ATPase activity were inhibited by greater than 95%. Pf (140 microns/s) and HgCl2 inhibition (75-85%) were not influenced by proteinase treatment. SDS-PAGE showed selective digestion of multiple polypeptides by proteinases. These results confirm the presence of water channels in BBMV and EV and demonstrate selective inhibition of ATPase function and Na+/H+ exchange by proteinase digestion. The lack of effect of proteinases on water transport by mercurials. We conclude that the water channel may be a small integral membrane protein which, unlike the H(+)-ATPase and Na+/H+ exchanger, has no functionally important membrane domains that are sensitive to proteolysis.     

Functional colocalization of water channels and proton pumps in endosomes from kidney proximal tubule

The apical membrane of mammalian proximal tubule undergoes rapid membrane cycling by exocytosis and endocytosis. Osmotic water and ATP- driven proton transport were measured in endocytic vesicles from rabbit and rat proximal tubule apical membrane labeled in vivo with the fluid phase marker fluorescein-dextran. Osmotic water permeability (Pf) was determined from the time course of fluorescein-dextran fluorescence after exposure of endosomes to an inward osmotic gradient in a stopped- flow apparatus. Pf was 0.009 (rabbit) and 0.029 cm/s (rat) (23 degrees C) and independent of osmotic gradient size. Pf in rabbit endosomes was inhibited reversibly by HgCl2 (KI = 0.2 mM) and had an activation energy of 6.4 +/- 0.5 kcal/mol (15-35 degrees C). Endosomal proton ATPase activity was measured from the time course of internal pH, measured by fluorescein-dextran fluorescence, after the addition of external ATP. Endosomes contained an ATP-driven proton pump that was sensitive to N-ethylmaleimide and insensitive to vanadate and oligomycin. In response to saturating [ATP] the pump acidified the endosomal compartment at a rate of 0.17 (rat) and 0.029 pH unit/s (rabbit); at an external pH of 7.4, the steady-state pH was 6.4 (rat) and 6.5 (rabbit). To examine whether water channels and the proton ATPase were present in the same endosome, the time course of fluorescein-dextran fluorescence was measured in response to an osmotic gradient in the presence and absence of ATP. ATP did not alter endosome Pf, but decreased the amplitude of the fluorescence signal by 43 +/- 3% (rabbit) and 47 +/- 4% (rat).(ABSTRACT TRUNCATED AT 250 WORDS)     

Transport of divalent transition-metal ions is lost in small-intestinal tissue of b/b Belgrade rats

Belgrade rats exhibit microcytic, hypochromic anemia and systemic iron deficiency due to a glycine-to-arginine mutation at residue 185 in a metal ion transporter of a divalent metal transporter/divalent cation transporter/solute carrier 11 group A member 2 or 3 (DMT1/DCT1/SLC11A2), a member of the natural-resistance-associated macrophage protein (Nramp) family. By use of rabbit duodenal tissue, a calcein fluorescence assay has previously been developed to assess transport of divalent metal ions across the small-intestinal brush border membrane (BBM). The assay was readily applied here to rat BBM to learn if it detects DMT1 activity. The results demonstrate protein-mediated transport across the BBM of all tested ions: Mn(2+), Fe(2+), and Ni(2+). Transport into BBM vesicles (BBMV) from (b/b) Belgrade rats was below the detection limit. BBMV of +/b origin had substantial activity. The kinetic rate constant for Ni(2+) membrane transport for +/b BBMV was within the range for normal rabbit tissue. Vesicles from +/b basolateral membranes (BLM) showed similar activity to BBMV while b/b BLM vesicles (BLMV) lacked transport activity. Immunoblots using isoform-specific antibodies demonstrated that intestinal levels of b/b DMT1 were increased compared to +/b DMT1, reflecting iron deficiency. Immunoblots on BBMV indicated that lack of activity in b/b vesicles was not due to a failure of DMT1 to localize to the BBMV; an excess of specific isoforms was present compared to +/b BBMV or duodenal extracts. Immunoblots from BLMV also exhibited enrichment in DMT1 isoforms, despite their distinct origin. Immunofluorescent staining of thin sections of b/b and +/b proximal intestines confirmed that DMT1 localized similarly in mutant and control enterocytes and showed that DMT1 isoforms have distinct distributions within intestinal tissue.     

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

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

Signals that regulate GLUT4 translocation

We have shown that there is a maturational increase in osmotic water permeability (Pf) of rabbit renal brush border membrane vesicles (BBMV). The purpose of the present study was to further investigate the changes in proximal tubule water transport that occur during postnatal development. Diffusional water permeability (PDW) has not been measured directly in adult or neonatal BBMV. We validated the method described by Ye and Verkman (Simultaneous optical measurement of osmotic and diffusional water permeability in cells and liposomes. Biochemistry 28:824-829, 1989) to measure PDW in red cell ghosts and liposomes, to examine the maturational changes in PDW in BBMV. This method utilizes the sensitivity of 8-aminonaphtalene-1,3,6-trisulfonic acid (ANTS) fluorescence to the D2O-H2O content of the solvent. ANTS-loaded neonatal (11 days old) and adult BBMV were rapidly mixed with two volumes of isoosmotic D2O solution using a stopped-flow apparatus at 5 degrees -37 degrees C. PDW was lower in neonatal than adult BBMV at 5 degrees (3.77 +/- 0.34 vs. 5.35 +/- 0.43 mm/sec, respectively, p<0.05) and 20 degrees C (7.03 +/- 0.40 vs. 9.04 +/- 0.25 mm/sec, respectively, p<0.001), but was not different at 30 degrees and 37 degrees C. The activation energy (Ea) was higher in neonatal than in adult BBMV (9.29 +/- 0.56 kcal/mol vs. 6.46 +/- 0.56 kcal/mol, p<0.001). In adult BBMV, PDW was inhibited by 0.5 mM HgCl2 by 46.6 +/- 3.6%, while it was not affected in neonatal BBMV (p<0.001). The results indicate that PDW can be measured in rabbit renal BBMV. There are significant changes in water transport across the apical membrane during postnatal development, consistent with a maturational increase in channel-mediated water transport.     

Urate/alpha-ketoglutarate exchange in avian basolateral membrane vesicles

Membrane transport pathways for transcellular secretion of urate across the proximal tubule were investigated in avian kidney. The presence of coupled urate/alpha-ketoglutarate exchange was investigated in basolateral membrane vesicles (BLMV) by [(14)C]urate and [alpha-(3)H]ketoglutarate flux measurements. An inward Na gradient induced accumulation of alpha-ketoglutarate of sufficient magnitude to suggest a Na-dicarboxylate cotransporter. An inward Na gradient also induced concentrative accumulation of urate in the presence of alpha-ketoglutarate but not in its absence, suggesting urate/alpha-ketoglutarate exchange. alpha-Ketoglutarate-dependent stimulation of urate uptake was not observed in brush-border membrane vesicles. An outward urate gradient induced concentrative accumulation of alpha-ketoglutarate. alpha-Ketoglutarate-coupled urate uptake was specific for alpha-ketoglutarate, Cl dependent, and insensitive to membrane potential. alpha-Ketoglutarate-coupled urate uptake was inhibited by increasing p-aminohippurate (PAH) concentrations, and alpha-ketoglutarate-coupled PAH uptake was observed. alpha-Ketoglutarate-coupled PAH uptake was inhibited by increasing urate concentrations, and an outward urate gradient induced concentrative accumulation of PAH. These results suggest a Cl-dependent, alpha-ketoglutarate-coupled anion exchange mechanism as a pathway for active urate uptake across the basolateral membrane of urate-secreting proximal tubule cells.     

Characterization of sodium-dependent and sodium-independent nucleoside transport systems in rabbit brush-border and basolateral plasma-membrane vesicles from the renal outer cortex.

The transport of uridine into rabbit renal outer-cortical brush-border and basolateral membrane vesicles was compared at 22 degrees C. Uridine was taken up into an osmotically active space in the absence of metabolism for both types of membrane vesicles. Uridine influx by brush-border membrane vesicles was stimulated by Na+, and in the presence of inwardly directed gradients of Na+ a transient overshoot phenomenon was observed, indicating active transport. Kinetic analysis of the saturable Na+-dependent component of uridine flux indicated that it was consistent with Michaelis-Menten kinetics (Km 12 +/- 3 microM, Vmax. 3.9 +/- 0.9 pmol/s per mg of protein). The sodium:uridine coupling stoichiometry was found to be consistent with 1:1 and involved the net transfer of positive charge. In contrast, uridine influx by basolateral membrane vesicles was not dependent on the cation present and was inhibited by nitrobenzylthioinosine (NBMPR). NBMPR-sensitive uridine transport was saturable (Km 137 +/- 20 microM, Vmax. 5.2 +/- 0.6 pmol/s per mg of protein). Inhibition of uridine flux by NBMPR was associated with high-affinity binding of NBMPR to the basolateral membrane (Kd 0.74 +/- 0.46 nM). Binding of NBMPR to these sites was competitively blocked by adenosine and uridine. These results indicate that uridine crosses the brush-border surface of rabbit proximal renal tubule cells by Na+-dependent pathways, but permeates the basolateral surface by NBMPR-sensitive facilitated-diffusion carriers.     

Solvent drag measurement of transcellular and basolateral membrane NaCl reflection coefficient in kidney proximal tubule

The NaCl reflection coefficient in proximal tubule has important implications for the mechanisms of near isosmotic volume reabsorption. A new fluorescence method was developed and applied to measure the transepithelial (sigma NaClTE) and basolateral membrane (sigma NaClcl) NaCl reflection coefficients in the isolated proximal straight tubule from rabbit kidney. For sigma NaClTE measurement, tubules were perfused with buffers containing 0 Cl, the Cl-sensitive fluorescent indicator 6-methoxy-N-[3-sulfopropyl] quinolinium and a Cl-insensitive indicator fluorescein sulfonate, and bathed in buffers of differing cryoscopic osmolalities containing NaCl. The transepithelial Cl gradient along the length of the tubule was measured in the steady state by a quantitative ratio imaging technique. A mathematical model based on the Kedem-Katchalsky equations was developed to calculate the axial profile of [Cl] from tubule geometry, lumen flow, water (Pf) and NaCl (PNaCl) permeabilities, and sigma NaClTE. A fit of experimental results to the model gave PNaCl = (2.25 +/- 0.2) x 10(-5) cm/s and sigma NaClTE = 0.98 +/- 0.03 at 23 degrees C. For measurement of sigma NaClbl, tubule cells were loaded with SPQ in the absence of Cl. NaCl solvent drag was measured from the time course of NaCl influx in response to rapid (less than 1 s) Cl addition to the bath solution. With bath-to-cell cryoscopic osmotic gradients of 0, -60, and +30 mosmol, initial Cl influx was 1.23, 1.10, and 1.25 mM/s; a fit to a mathematical model gave sigma NaClbl = 0.97 +/- 0.04. These results indicate absence of NaCl solvent drag in rabbit proximal tubule. The implications of these findings for water and NaCl movement in proximal tubule are evaluated.     

NaCl reflection coefficients in proximal tubule apical and basolateral membrane vesicles. Measurement by induced osmosis and solvent drag.

Two independent methods, induced osmosis and solvent drag, were used to determine the reflection coefficients for NaCl (sigma NaCl) in brush border and basolateral membrane vesicles isolated from rabbit proximal tubule. In the induced osmosis method, vesicles loaded with sucrose were subjected to varying inward NaCl gradients in a stopped-flow apparatus. sigma NaCl was determined from the osmolality of the NaCl solution required to cause no initial osmotic water flux as measured by light scattering (null point). By this method sigma NaCl was greater than 0.92 for both apical and basolateral membranes with best estimates of 1.0. sigma NaCl was determined by the solvent drag method using the Cl-sensitive fluorescent indicator, 6-methoxy-N-[3-sulfopropyl]quinolinium (SPQ), to detect the drag of Cl into vesicles by inward osmotic water movement caused by an outward osmotic gradient. sigma NaCl was determined by comparing experimental data with theoretical curves generated using the coupled flux equations of Kedem and Katchalsky. By this method we found that sigma NaCl was greater than 0.96 for apical and greater than 0.98 for basolateral membrane vesicles, with best estimates of 1.0 for both membranes. These results demonstrate that sigma NaCl for proximal tubule apical and basolateral membranes are near unity. Taken together with previous results, these data suggest that proximal tubule water channels are long narrow pores that exclude NaCl.     

Glucose and fructose uptake by <Emphasis Type="Italic">Limulus polyphemus</Emphasis> hepatopancreatic brush border and basolateral membrane vesicles: evidence for Na<Superscript>+</Superscript>-dependent sugar transport activity

[³H]-fructose and [³H]-glucose transport activities were determined in brush border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) from Limulus polyphemus (horseshoe crab) hepatopancreas. Glucose transport was equilibrative in the absence of sodium and sodium dependent in the presence of sodium in BBMV, suggesting GLUT-like and SGLT-like transport activity. Glucose transport by BLMV was equilibrative and sodium independent. Fructose uptake by BBMV and BLMV was equilibrative in the absence of sodium and sodium dependent in the presence of sodium. Western blot analysis using a rabbit anti-mouse SGLT-1 polyclonal antibody indicated the presence of a cross-reacting horseshoe crab BBMV protein of similar molecular weight to the mammalian SGLT1. Sequence alignment of the mouse SGLT-4 and SGLT1 with a translated, horseshoe crab-expressed sequence tag also indicated significant identity between species. Fructose and glucose uptake in the absence and presence of sodium by hepatopancreas BBMV and BLMV indicated the presence of sodium-dependent transport activity for each sugar that may result from the presence of transporters similar to those described for other species.     

Glucocorticoids increase osmotic water permeability (Pf) of neonatal rabbit renal brush border membrane vesicles

During postnatal maturation, there is an increase in renal brush border membrane vesicle (BBMV) osmotic water permeability and a parallel increase in aquaporin-1 (AQP1) protein abundance. The mechanisms responsible for these changes remain unknown. Because serum glucocorticoid levels rise postnatally and have previously been linked to other maturational changes in renal function, we examined the effects of glucocorticoids on osmotic (Pf) and diffusional (P(DW)) water permeability and AQP1 protein abundance of renal BBMV. Neonatal rabbits were treated with dexamethasone (10 microg/100 g) for three days and compared with control neonates and adults. Pf and P(DW) were measured at 20 degrees C with a stopped-flow apparatus using light-scattering and aminonaphthalene trisulfonic acid (ANTS) fluorescence, respectively. Pf was significantly higher in BBMV from dexamethasone-treated neonates compared with vehicle-treated neonates, but remained lower than in BBMV from adults (P<0.05). P(DW) in dexamethasone and vehicle-treated neonatal BBMV was lower than in adult BBMV. Pf/P(DW) ratio increased from neonate (5.1+/-0.3) to dexamethasone (7.0+/-0.1) and adult BBMV (6.3+/-0.1). AQP1 expression was increased by dexamethasone treatment to adult levels. Membrane fluidity, which is inversely related to generalized polarization (GP) of steady-state laurdan fluorescence, was significantly higher in neonatal BBMV than both dexamethasone and adult BBMV (GP: neonate 0.285+/-0.002, dexamethasone treatment 0.302+/-0.006, and adult 0.300+/-0.005; P<0.05). These combined results show that dexamethasone-treatment during days 4-7 of life increases BBMV water permeability despite a decrease in membrane fluidity. This occurs by increasing channel-mediated water transport, as reflected in an increase in AQP1 protein abundance and a higher Pf/P(DW) ratio. This mimics the maturational changes and suggests a physiological role for glucocorticoids in maturation of proximal tubule water transport.     

Expression of mRNA coding for kidney and red cell water channels in Xenopus oocytes

The existence and identity of protein water transporters in biological membranes has been uncertain. Osmotic water permeability (Pf) was measured in defolliculated Xenopus oocytes microinjected with water or mRNA from kidney cortex, kidney papilla, reticulocyte, brain, and muscle. Pf was measured by quantitative image analysis from the time course of oocyte swelling in response to an osmotic gradient. When assayed at 10 degrees C, Pf in water-injected oocytes increased from (3.6 +/- 0.9) x 10(-4) cm/s (S.D., n = 16) to 74 x 10(-4) cm/s with addition of amphotericin B, showing absence of unstirred layers. At 48-72 h after injection of 50 ng of unfractionated mRNA, Pf (in cm/s x 10(-4] was: 4.0 +/- 1.5 (rabbit brain, n = 15), 4.2 +/- 1.8 (rabbit muscle, n = 10), 18.4 +/- 6.3 (rabbit reticulocyte, n = 20), 16.1 +/- 5.6 (rat renal papilla, n = 24), 12.9 +/- 6.3 (rat renal cortex, n = 20), 14.4 +/- 6.1 (rabbit renal papilla, n = 15), and 11.8 +/- 3.4 (rabbit renal cortex, n = 8). In oocytes injected with mRNA from rat renal papilla, Pf was inhibited reversibly by 0.3 mM HgCl2 (4.1 +/- 1.6, n = 10); expressed water channels from kidney and red cell had activation energies of less than 4 kcal/mol. These results show functional oocyte expression of water channels from red cell, kidney proximal tubule (cortex), and the vasopressin-sensitive kidney collecting tubule (papilla), indicating that water channels are proteins, and providing an approach for the expression cloning of water channels.