Evidence for electroneutral chloride transport in rabbit renal cortical brush border membrane vesicles

1984 ◽  
Vol 247 (5) ◽  
pp. F837-F847 ◽  
Author(s):  
D. Shiuan ◽  
S. W. Weinstein
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Chloride Transport ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Ph Gradient ◽  
Diffusion Potential ◽  
Proton Diffusion ◽  
Proximal Tubular ◽  
Sodium Glucose Cotransport

Controversial evidence exists as to whether electroneutral Cl-OH exchange occurs across the proximal tubular brush border membrane of the mammalian kidney. To help resolve this controversy, we carried out a series of experiments using rabbit renal cortical brush border membrane vesicles to determine the effect of changes in pH on 36Cl uptake across this membrane. Reducing pH equally inside and outside of the vesicle stimulated Cl-Cl exchange as well as chloride uptake. A proton gradient (out greater than in) stimulated and caused an overshoot in unidirectional Cl uptake. All three of these processes were inhibited by 4-acetoamido-4-isothiocyano-2,2-disulfonic stilbene. Valinomycin with K+ out-in added to shunt any proton diffusion potential, minimally inhibited pH gradient-dependent Cl uptake, and carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP), added to increase the proton diffusion potential, minimally stimulated pH gradient-dependent Cl uptake. Sodium-glucose cotransport (an electrogenic system) was used to assess the efficacy of these ionophores. FCCP markedly enhanced and valinomycin markedly inhibited pH gradient stimulated sodium-glucose cotransport. Valinomycin added to vesicles with a 180/0 meq K+ out greater than in gradient stimulated less Cl uptake than a 6.0/7.5 pH out greater than in gradient. We conclude that these results provide strong evidence for the existence of an electroneutral Cl-OH exchanger or Cl-H symporter in this membrane.

Na+ and H+ transport in human jejunal brush-border membrane vesicles

1988 ◽  
Vol 255 (2) ◽  
pp. G206-G211
Author(s):  
J. G. Kleinman ◽  
J. M. Harig ◽  
J. A. Barry ◽  
K. Ramaswamy
Keyword(s):  
Fluorescence Quenching ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Ph Gradient ◽  
Diffusion Potential ◽  
Voltage Clamping ◽  
22Na Uptake ◽  
Na Uptake

We have examined pH gradient-driven Na+ uptake and Na+-driven H+ transport in brush-border membrane vesicles prepared from jejunal tissue obtained from organ donors by measuring the influx of 22Na and the fluorescence quenching of acridine orange (AO). Vesicle preparation by either Ca2+ or Mg2+ precipitation showed no difference in 22Na uptake or AO fluorescence quenching and dissipation. An outward H+ gradient [intravesicular pH (pHi) 5.5; extravesicular pH (pHo) 7.5] induced a Na+ uptake “overshoot” of threefold over equilibrium, whereas the absence of an H+ gradient (at either pH 5.5 or 7.5) did not produce an overshoot. Voltage clamping by Ki+ = Ko+ plus valinomycin reduced the overshoot by 50%. The initial rate of pH-driven Na+ uptake in voltage-clamped vesicles was related to [Nao+] (Km = 29 mM and Vmax = 9.5 nmol.mg protein-1.3 s-1). Amiloride inhibited this uptake in voltage-clamped vesicles (Ki = 99 microM). Dissipation of AO fluorescence quench in vesicles with a preformed internal acid gradient was hastened by Nao+ as well as voltage clamping in the absence of Na+. In vesicles without a pH gradient, internal Na+, as well as a diffusion potential (Ki+ 100; Ko+ 0 plus valinomycin) in the absence of Na+, induced AO quenching. External Na+ and Li+, but not choline, acted to dissipate AO quenching induced by a diffusion potential, and the rate of dissipation was unaffected by the presence of Cl-.Li+ and NH4+, but not Cs+, K+, Rb+, or choline+, inhibited pH gradient-driven 22Na uptake. We conclude that human jejunal brush-border membrane vesicles contain conductive pathways for both Na+ and H+ and an Na+-H+ exchanger.

Low-affinity transport of pyroglutamyl-histidine in renal brush-border membrane vesicles

1989 ◽  
Vol 257 (5) ◽  
pp. C971-C975 ◽  
Author(s):  
H. A. Skopicki ◽  
K. Fisher ◽  
D. Zikos ◽  
G. Flouret ◽  
D. R. Peterson
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Luminal Membrane ◽  
Renal Brush Border Membrane ◽  
Proximal Tubular ◽  
Renal Brush Border

These studies were performed to determine if a low-affinity carrier is present in the luminal membrane of proximal tubular cells for the transport of the dipeptide, pyroglutamyl-histidine (pGlu-His). We have previously described the existence of a specific, high-affinity, low-capacity [transport constant (Kt) = 9.3 X 10(-8) M, Vmax = 6.1 X 10(-12) mol.mg-1.min-1] carrier for pGlu-His in renal brush-border membrane vesicles. In the present study, we sought to demonstrate that multiple carriers exist for the transport of a single dipeptide by determining whether a low-affinity carrier also exists for the uptake of pGlu-His. Transport of pGlu-His into brush-border membrane vesicles was saturable over the concentration range of 10(-5)-10(-3) M, yielding a Kt of 6.3 X 10(-5) M and a Vmax of 2.2 X 10(-10) mol.mg-1.min-1. Uptake was inhibited by the dipeptides glycyl-proline, glycyl-sarcosine, and carnosine but not by the tripeptide pyroglutamyl-histidyl-prolinamide. We conclude that 1) pGlu-His is transported across the luminal membrane of the proximal tubule by multiple carriers and 2) the lower affinity carrier, unlike the higher affinity carrier, is nonspecific with respect to other dipeptides.

pH gradient-stimulated phosphate transport in outer medullary brush-border membranes

1989 ◽  
Vol 257 (4) ◽  
pp. F639-F648
Author(s):  
G. A. Quamme ◽  
J. J. Walker ◽  
T. S. Yan
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Phosphate Uptake ◽  
Membrane Vesicles ◽  
Phosphate Transport ◽  
Brush Border Membrane Vesicles ◽  
Ph Gradient ◽  
Disulfonic Acid ◽  
Maximal Velocity ◽  
Medullary Tissue

Phosphate transport was studied in brush-border membrane vesicles prepared from outer medullary tissue of the porcine kidney. Phosphate uptake studies were performed in the absence of sodium at 21 degrees C. A 1.2- to 12-fold overshoot, above equilibrium values, was present with intracellular pH (pHin) equal to 8.0 and extracellular pH (pHout) equal to 6.5, which was not evident at pHin = pHout. Concentration-dependence of the pH-stimulate uptake was determined by the difference of uptake in the absence of a pH gradient (pHin = pHout) from that in the presence of a pH gradient over a large range of phosphate concentrations. The uptake was consistent with a single facilitative system characterized by apparent kinetic parameters; with Michaelis constant 149 +/- 11 microM and maximal velocity 4.9 +/- 0.4 nmol.mg protein-1.min-1, n = 3. Phosphate uptake was inhibited by the stilbene derivative 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid with a mean inhibition constant (Ki) value of 0.15 mM (n = 2). In addition, pH gradient-stimulated phosphate uptake was sensitive to furosemide and bumetanide; Ki values of 0.50 +/- 0.05 and 0.11 +/- 0.04 mM, respectively. Arsenate (1 mM) and phosphonoformate (1 mM) inhibited pH-dependent phosphate uptake, whereas sulfate (5 mM), bicarbonate (25 mM), and chloride (100 mM) were without effect, indicating that the transport system is relatively specific to phosphate and its close analogues. pH gradient-stimulated phosphate uptake was not influenced by potassium-diffusional gradients. The data provide evidence for a facilitative process in brush-border membrane vesicles isolated from outer medullary tissue of the pig kidney that is capable of transporting phosphate in the absence of sodium.

Effects of cations on pH gradient-stimulated sulfate transport in rabbit ileal brush-border membrane vesicles

1985 ◽  
Vol 249 (5) ◽  
pp. G614-G621 ◽  
Author(s):  
C. M. Schron ◽  
R. G. Knickelbein ◽  
P. S. Aronson ◽  
J. Della Puca ◽  
J. W. Dobbins
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Disodium Salt ◽  
Brush Border Membrane Vesicles ◽  
Ph Gradient ◽  
Disulfonic Acid ◽  
Rabbit Ileum ◽  
External K

In brush-border membrane vesicles from rabbit ileum, we previously reported pH gradient-stimulated SO4 uptake and presented evidence that this represents carrier-mediated SO4-OH exchange. In the present study inhibitors of SO4-OH exchange (H-SO4 cotransport) were shown not to inhibit Na-SO4 cotransport, suggesting that these are two separate carrier-mediated transport mechanisms. While pH gradient-stimulated SO4 uptake was inhibited 87% by 0.1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, disodium salt (DIDS) and 79% by 1.0 mM furosemide, Na+-stimulated SO4 uptake was only inhibited 11 and 0%, respectively. K+ (20 mM), Cl (5 mM), and oxalate (0.25 mM) inhibited pH gradient-stimulated SO4 uptake (38-65%) but had no effect on Na+-stimulated SO4 uptake. Finally, at Na+ concentrations (10 mM) significantly less than that required for Na+-stimulated SO4 uptake (60-100 mM), external Na+ inhibited pH gradient-stimulated SO4 uptake, suggesting two independent effects of this cation. SO4 uptake was also inhibited by external K+ both in the presence and absence of a pH gradient. A Dixon plot of the DIDS-sensitive SO4 uptake under pH gradient conditions yielded a straight line, indicating a single site of interaction between external K+ and the SO4-OH carrier (apparent Ki = 7.2 mM). In contrast to the inhibition by external K+, internal K+ stimulated SO4 uptake. This effect was DIDS sensitive and not enhanced by valinomycin, suggesting an interaction of internal K+ with the SO4-OH exchanger independent of a K+-induced electrical potential. SO4 uptake and the effects of K+ were pH modulated with less SO4 uptake and less K+ effect at higher pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple transport systems for organic cations in renal brush-border membrane vesicles

1989 ◽  
Vol 256 (4) ◽  
pp. F540-F548 ◽  
Author(s):  
Y. Miyamoto ◽  
C. Tiruppathi ◽  
V. Ganapathy ◽  
F. H. Leibach
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Diffusion Potential ◽  
Transport Systems ◽  
Organic Cations ◽  
Uptake Kinetic ◽  
Carrier Systems ◽  
Renal Brush Border

The characteristics of guanidine uptake in brush-border membrane vesicles isolated from rabbit renal cortex were investigated. Guanidine uptake was markedly stimulated by an outwardly directed H+ gradient, resulting in a transient uphill transport. This stimulation was not due to an inside-negative, H+-diffusion potential because an ionophore-induced H+-diffusion potential and a K+-diffusion potential (both inside-negative) failed to enhance guanidine uptake. The H+ gradient itself appeared to be the driving force for the uptake. These data suggest that guanidine-H+ antiport (or guanidine-OH- symport) is the mechanism of guanidine uptake in these membrane vesicles. Guanidine uptake was only minimally inhibited by organic cations such as tetraethylammonium, N1-methylnicotinamide, and choline, but many other organic cations such as amiloride, clonidine, imipramine, and harmaline caused considerable inhibition. Uptake of radiolabeled guanidine was inhibited more effectively by guanidine than by tetraethylammonium, whereas uptake of radiolabeled tetraethylammonium was inhibited more effectively by tetraethylammonium than by guanidine. beta-Lactam antibiotics did not inhibit guanidine uptake but did inhibit tetraethylammonium uptake. Kinetic analysis showed that there were at least two kinetically distinct carrier systems for guanidine uptake, whereas tetraethylammonium uptake occurred via a single carrier system. These data provide evidence that renal brush-border membranes possess multiple carrier systems for organic cations.

scholarly journals Electroneutral, HCO3--independent, pH gradient-dependent uphill transport of Cl- by ileal brush-border membrane vesicles. Possible role in the pathogenesis of chloridorrhea

1989 ◽  
Vol 263 (3) ◽  
pp. 775-784 ◽  
Author(s):  
M Vasseur ◽  
M Caüzac ◽  
F Alvarado
Keyword(s):  
Membrane Potential ◽  
Brush Border ◽  
Metal Ion ◽  
Brush Border Membrane ◽  
Competitive Inhibition ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Ph Gradient ◽  
Influx Rate ◽  
Dependent Inhibition

By applying a rapid filtration technique to isolated brush border membrane vesicles from guinea pig ileum, 36Cl uptake was quantified in the presence and absence of electrical, pH and alkali-metal ion gradients. A mixture of 20 mM-Hepes and 40 mM-citric acid, adjusted to the desired pH with Tris base, was found to be the most suitable buffer. Malate and Mes could be used to replace the citrate, but succinate, acetate and maleate proved to be unsuitable. In the absence of a pH gradient (pHout:pHin = 7.5:7.5), Cl- uptake increased slightly when an inside-positive membrane potential was applied, but uphill transport was never observed. A pH gradient (pHout:pHin = 5.0:7.5) induced both a 400% increase in the initial Cl- influx rate and a long-lasting (20 to 300 s) overshoot, indicating that a proton gradient can furnish the driving force for uphill Cl- transport. Under pH gradient conditions, initial Cl- entry rates had the following characteristics. (1) They were unaffected by cis-Na+ and/or -K+, indicating the absence of Cl-/K+, Cl-/Na+ or Cl-/K+/Na+ symport activity. (2) Inhibition by 20-100 mM-trans-Na+ and/or -K+ occurred, independent of the existence of an ion gradient. (3) Cl- entry was practically unaffected by short-circuiting the membrane potential with equilibrated potassium and valinomycin. (4) Carbonyl cyanide m-chlorophenylhydrazone was strongly inhibitory and so, to a lesser extent, was 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonic acid [(SITS)], independent of the sign and size of the membrane potential. (5) Cl- entry was negligibly increased (less than 30%) by either trans-Cl- or -HCO3-, indicating the absence of an obligatory Cl-/anion antiport activity. In contrast, the height of the overshoot at 60 s was increased by trans-Cl-, indicating time-dependent inhibition of 36Cl efflux. That competitive inhibition of 36Cl fluxes by anions is involved here is supported by initial influx rate experiments demonstrating: (1) the saturability of Cl- influx, which was found to exhibit Michaelis-Menten kinetics; and (2) competitive inhibition of influx by cis-Cl- and -Br-. Quantitatively, the conclusion is warranted that over 85% of the total initial Cl- uptake energized by a pH gradient involves an electroneutral Cl-/H+ symporter or its physicochemical equivalent, a Cl-/OH- antiporter, exhibiting little Cl- uniport and either Cl-/Cl- or Cl-/HCO3- antiport activities.(ABSTRACT TRUNCATED AT 400 WORDS)

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