Effects of membrane fluidizing agents on renal brush border proton permeability

1985 ◽  
Vol 249 (6) ◽  
pp. F933-F940 ◽  
Author(s):  
H. E. Ives ◽  
A. S. Verkman
Keyword(s):  
Water Transport ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Fold Increase ◽  
Dependent Manner ◽  
Transport Pathway ◽  
Ph Gradients ◽  
Orange Fluorescence ◽  
Dose Dependent

H+ permeability (PH) of brush border membrane vesicles isolated from rabbit renal cortex was measured from the rate of collapse of preformed pH gradients using acridine orange fluorescence quenching. n-Alkanols increased PH from 0.005 to 0.1 cm/s in a dose-dependent manner. At 25 degrees C, PH increased to 0.01 cm/s at [n-alkanol] = 90 mM (butanol), 30 mM (pentanol), 7 mM (hexanol), and 1.8 mM (heptanol). Activation energy (Ea) of PH was 21.6 kcal/mol (5-50 degrees C), which decreased to 18.5 kcal/mol in the presence of either 200 mM butanol or 12 mM hexanol. Membrane fluidity was estimated from diphenylhexatriene anisotropy (r). n-Alkanols decreased r from 0.25 to 0.18 in a dose-dependent manner. At 25 degrees C, r = 0.22 at [n-alkanol] = 200 mM (butanol), 27 mM (pentanol), 9.5 mM (hexanol), and 2 mM (heptanol). The effects of n-alkanols on PH and r correlated well with known n-alkanol lipid-water partition coefficients. Similar increases in PH and decreases in r were observed for nonalkanol lipid anesthetics. The effects of n-alkanols on the Na+-H+ antiporter and on osmotically driven water transport were also studied. At concentrations of n-alkanol that resulted in a 10-fold increase in PH, there was no significant effect on either Na+-H+ exchange or water transport. These results suggest a lipid pathway for brush border H+ diffusion that is distinct from both the Na+-H+ antiporter and the water transport pathway.

High-affinity paroxetine binding to the human placental serotonin transporter

1990 ◽  
Vol 259 (2) ◽  
pp. C196-C204 ◽  
Author(s):  
D. R. Cool ◽  
F. H. Leibach ◽  
V. Ganapathy
Keyword(s):  
Serotonin Transporter ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Binding Capacity ◽  
Membrane Vesicles ◽  
Dependent Manner ◽  
High Affinity ◽  
Paroxetine Binding ◽  
Dose Dependent ◽  
Dissociation Constant Kd

We investigated the interaction of paroxetine, a nontricyclic antidepressant, with the serotonin transporter of the human placental brush-border membrane. Paroxetine bound to the purified placental brush-border membranes with a high affinity [dissociation constant (Kd) = 72 pM]. The maximal binding capacity (Bmax) was 3.9 pmol/mg protein. Imipramine, desipramine, and serotonin inhibited the binding in a dose-dependent manner with inhibition constant (Ki) values of 4.4 nM, 48.7 nM, and 1.77 microM, respectively, whereas reserpine, ketanserin, and 5-hydroxytryptophan did not have any effect. Imipramine and serotonin inhibited paroxetine binding by increasing the Kd with essentially no effect on Bmax. Binding of paroxetine to the membranes increased hyperbolically with increasing concentrations of Na+ in the assay medium. Cl- had little effect on the binding. The effect of Na+ was primarily to increase the affinity of the transporter for paroxetine with no effect on Bmax. The association constant (Ka) increased hyperbolically as the concentration of Na+ increased, indicating a 1Na+:1paroxetine stoichiometry. The maximal value for Ka was 12.1 +/- 2.5 x 10(12) M-1, and Kd for Na+ was 10.0 +/- 3.5 mM. Treatment of the membranes with tyrosyl group-specific reagents reduced the Na(+)-dependent binding, suggesting the involvement of tyrosyl residues in the binding process. This inhibition was, however, significantly reduced when treatment with the reagent was performed in the presence of Na+, suggesting that the reactive tyrosyl residues were located at or near the Na(+)-binding site. Paroxetine inhibited NaCl gradient-dependent serotonin uptake in placental brush-border membrane vesicles both at pH 6.5 and 7.5.(ABSTRACT TRUNCATED AT 250 WORDS)

Na+/H+ antiporter of brush border vesicles: studies with acridine orange uptake

1982 ◽  
Vol 242 (6) ◽  
pp. F733-F739 ◽  
Author(s):  
D. G. Warnock ◽  
W. W. Reenstra ◽  
V. J. Yee
Keyword(s):  
Fluorescence Quenching ◽  
Acridine Orange ◽  
Brush Border ◽  
Membrane Vesicles ◽  
Sodium Concentration ◽  
Sodium Influx ◽  
Ph Gradients ◽  
Orange Fluorescence ◽  
Acridine Orange Fluorescence ◽  
Rate Limiting

Fluorescence quenching of acridine orange was used to characterize the generation and collapse of pH gradients by the Na+/H+ antiporter of brush border membrane vesicles prepared from rabbit renal cortex. Quenching was observed when acridine orange, a weak base, was taken up to an acidic intravesicular space. Na+/H+ exchange was examined with both Na+ uptake and efflux studies. Acridine orange fluorescence quenching demonstrated the cation specificity of the Na+/H+ antiporter (i.e., sodium and lithium) and was inhibited by amiloride. Parallel studies with nigericin, a K+/H+ antiporter, demonstrated that acridine orange responded very rapidly to pH gradients. Therefore, acridine orange equilibration was not rate limiting in our studies of the Na+/H+ antiporter. Initial rate measurements were made to obtain kinetic parameters for the Na+/H+ antiporter. In sodium influx studies, the half-maximal rate of acridine orange fluorescence change was obtained with an external sodium concentration of 13.3 +/- 0.5 mM.

A sodium-hydrogen exchange system in isolated apical membrane from LLC-PK1 epithelia

1986 ◽  
Vol 251 (6) ◽  
pp. F1003-F1008
Author(s):  
A. Moran ◽  
J. Biber ◽  
H. Murer
Keyword(s):  
Hydrogen Exchange ◽  
Brush Border Membrane ◽  
Cultured Cells ◽  
Apical Membrane ◽  
Short Circuit ◽  
Membrane Vesicles ◽  
Ph Gradients ◽  
Noncompetitive Inhibition ◽  
Carbonyl Cyanide ◽  
Orange Fluorescence

We have monitored transmembrane pH gradients using acridine orange fluorescence quenching and traced Na+ flux to study the properties of Na+-H+ exchange in apical membrane vesicles isolated from LLC-PK1 epithelia. The membranes have low conductance for Na+, H+, and K+ ions. An outwardly directed K+ gradient in the presence of valinomycin and carbonyl cyanide p-trifluoromethoxyphenyl hydrazone produced intravesicular acidification. This pH gradient was collapsed by addition of extravesicular Na+ or Li+ ions but not by tetramethylammonium. Amiloride (10(-4) M) inhibited the effect of both Na+ and Li+ . An outwardly directed Na+ gradient stimulated H+ influx, which was also inhibited by 10(-4) M amiloride. Membrane short-circuit conditions affected neither Na+ nor H+ flux, consistent with transport mediated by an electroneutral process. The interaction of amiloride and sodium is consistent with noncompetitive inhibition with Ki = 100+/- 10 microM for amiloride and an apparent Km for Na+ of approximately 20 mM. This finding is in agreement with previous studies of intact LLC-PK1 epithelia but differs from observations in brush-border membrane vesicles isolated from kidney proximal tubule in which competitive and mixed inhibition have been reported. These observed differences can be reconciled if two types of Na+-H+ exchange systems exist along the nephron, one with competitive and the other with noncompetitive inhibition, and if only the latter is expressed in the homogeneous cultured cells.

scholarly journals Selenium (selenate) transport by human placental brush border membrane vesicles

1988 ◽  
Vol 59 (1) ◽  
pp. 13-19 ◽  
Author(s):  
D. B. Shennan
Keyword(s):  
Brush Border ◽  
Incubation Medium ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Competitive Inhibitor ◽  
Brush Border Membrane Vesicles ◽  
Exchange Inhibitor ◽  
Dose Dependent Fashion ◽  
Microvillus Membrane ◽  
Dose Dependent

1. Selenate uptake by human placental brush-border-membrane vesicles was studied in order to establish whether this anion shares a pathway with sulphate.2. Selenate uptake was found to be saturable with respect to medium selenate and was inhibited by the anion exchange inhibitor 4,4′-diisothiocyano-stilbene-2,2′-disulphonate (DIDS).3. Anions which have a similar tetrahedral shape to selenate, e.g. chromate, molybdate, tungstate and sulphate, were effective inhibitors of selenate uptake when added to the incubation medium.4. Sulphate inhibited selenate influx in a dose-dependent fashion; moreover sulphate was found to be a competitive inhibitor of selenate uptake.5. It is concluded that selenate and sulphate share a pathway for transport in the human placental microvillus membrane.

Effect of ethanol on disaccharidases of hamster jejunal brush border membrane.

1979 ◽  
Vol 237 (1) ◽  
pp. E68
Author(s):  
P K Dinda ◽  
R O Hurst ◽  
I T Beck
Keyword(s):  
Alkaline Phosphatase ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Kinetic Studies ◽  
Time Dependent ◽  
Acute Exposure ◽  
Dependent Manner ◽  
Brush Border Enzyme ◽  
Dose Dependent

This study was undertaken to investigate the effect of alcohol on the activity of jejunal disaccharidases (DS). The activity of DS in a preparation of purified brush border membrane of hamster jejunum was measured in the absence and in the presence (0.8 to 6.4% wt/vol) of ethanol. To compare the effect of alcohol on DS with its action on a brush border enzyme of a different group, we also measured the activity of alkaline phosphatase (AP) under similar conditions. Ethanol depressed the activity of sucrase, maltase, and lactase in a dose-dependent and time-dependent manner, but it stimulated the activity of AP. The ethanol-induced inhibition of DS was completely reversible. Kinetic studies indicate that ethanol depressed the Vmax and increased the Km of sucrase and lactase. The Vmax of maltase also decreased, but the Km of this hydrolase was not affected by ethanol. From the results of this study it would appear that acute exposure of the jejunal brush border to ethanol depresses the DS activity of the membrane and that (because the AP was not depressed) the ethanol-induced inhibition of DS is not the result of a general inhibition of all enzymes of the brush border.

Na+-H+ exchange in rat colonic brush-border membrane vesicles

1986 ◽  
Vol 250 (6) ◽  
pp. G781-G787
Author(s):  
E. S. Foster ◽  
P. K. Dudeja ◽  
T. A. Brasitus
Keyword(s):  
Membrane Potential ◽  
Acridine Orange ◽  
Large Intestine ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Exchange Process ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Proton Efflux ◽  
Ph Gradients

To demonstrate the presence of a Na+-H+ exchange process in brush-border membrane vesicles from rat colonocytes, the fluorescence response of acridine orange was used to monitor the formation and dissipation of pH gradients. An inwardly directed Na+ gradient stimulated the outward flux of H+, whereas proton influx was stimulated by an outwardly directed Na+ gradient. Since the ionophore valinomycin in the presence of a K+ gradient did not alter Na+-stimulated proton efflux, the interrelationship of Na+ and H+ movement could not be explained solely on the basis of a membrane potential. Na+-stimulated proton efflux was saturable with a Km for Na+ of 20.1 +/- 1.6 mM. Inwardly directed Li+ gradients also stimulated proton efflux, and the Km for Li+ was 30.2 +/- 1.7 mM. In contrast, impermeant cations failed to stimulate the outward flux of H+. Amiloride (1 mM) inhibited both Na+-stimulated proton efflux and influx. Therefore, Na+-H+ exchange is present in rat colonic brush-border membranes and has characteristics similar to other Na+-H+ antiporters. This exchange process may be an important mechanism for Na+ absorption in the large intestine.

Sodium-phosphate transporter adaptation to dietary phosphate deprivation in normal and hypophosphatemic mice

1995 ◽  
Vol 268 (6) ◽  
pp. G917-G924 ◽  
Author(s):  
J. F. Collins ◽  
N. Bulus ◽  
F. K. Ghishan
Keyword(s):  
Brush Border ◽  
Molecular Mechanisms ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Fold Increase ◽  
Xenopus Laevis Oocytes ◽  
Protein Levels ◽  
Renal Brush Border Membrane ◽  
Renal Tubular Reabsorption ◽  
Renal Tubular

The X-linked hypophosphatemic (Hyp) mouse is a model for hypophosphatemic vitamin D-resistant rickets and is a homologue of human X-linked hypophosphatemia. The defect in the Hyp mouse appears to be related to decreased renal tubular reabsorption of P(i) via the renal brush-border membrane (Na(+)-P(i)) transporter. Dietary P(i) deprivation upregulates Na(+)-P(i) transport activity in brush-border membrane vesicles (BBMV) isolated from both normal and Hyp mice; however, the molecular mechanisms underlying this phenomenon are not known. The current studies were designed to investigate the effect of P(i) deprivation on the renal Na(+)-P(i) transporter. Low P(i) diet upregulated Na(+)-P(i) transporter activity in isolated BBMV by 2.1-fold in normal and Hyp mice (n = 3, P = 0.01). Low P(i) diet also induced a 1.9 +/- 0.3-fold increase in normal mice and 2.9 +/- 0.4-fold increase in Hyp mice in Na(+)-P(i) transporter message levels (n = 3, P = 0.028). The increase in message level encoding the Na(+)-P(i) transporter stimulated increased Na(+)-dependent P(i) uptake by Xenopus laevis oocytes when poly(A)+ RNA was injected into them from mice on low P(i) diet (approximately 1.67-fold in normal mice and 1.33-fold in Hyp mice). Immunoreactive protein levels increased 2.3 +/- 0.4-fold in normal mice and 8.2 +/- 0.5 in the Hyp mouse kidney cortexes (n = 3, P = 0.0001) in response to dietary P(i) deprivation.(ABSTRACT TRUNCATED AT 250 WORDS)

MPP+ is transported by the TEA(+)-H+ exchanger of renal brush-border membrane vesicles

1990 ◽  
Vol 258 (3) ◽  
pp. F597-F605 ◽  
Author(s):  
K. D. Lazaruk ◽  
S. H. Wright
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Organic Cations ◽  
Michaelis Constant ◽  
Transport Pathway ◽  
Renal Brush Border Membrane ◽  
Competitive Inhibitors ◽  
Renal Brush Border

Rabbit renal brush-border membrane vesicles (BBMV) were used to study the transport of the cationic neurotoxin, 1-methyl-4-phenylpyridinium (MPP+). An outwardly directed H(+)-gradient stimulated MPP+ uptake and led to the development of an active accumulation of MPP+ within the vesicles. H(+)-gradient driven MPP+ transport was saturable, with a maximal transport rate of 3 nmol.mg-1.min-1 and an apparent Michaelis constant (Kt) of 8 microM. MPP+ and tetraethylammonium (TEA) behaved as competitive inhibitors of one another's transport in renal BBMV, suggesting the presence of a common transport pathway for these organic cations. At an ambient pH of 7.5, preloading BBMV with MPP+ failed to stimulate TEA uptake, although trans TEA did stimulate MPP+ uptake. Increasing ambient pH to 8.5 (i.e., reducing competition between H+ and these organic cations for a common transport pathway) led to a clear reciprocal trans stimulation of TEA and MPP+ fluxes. With an equilibrium-shift protocol, a trans concentration of MPP+ energized uphill transport of TEA. We conclude that MPP+ and TEA share a common organic cation-H+ exchange pathway in the renal brush border, although turnover of an MPP(+)-loaded exchanger is slow compared with that for a TEA or H(+)-loaded exchanger.

Mechanism of urate and p-aminohippurate transport in rat renal microvillus membrane vesicles

1983 ◽  
Vol 245 (2) ◽  
pp. F151-F158 ◽  
Author(s):  
A. M. Kahn ◽  
S. Branham ◽  
E. J. Weinman
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Initial Rate ◽  
Organic Anion ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Dependent Manner ◽  
Total Uptake ◽  
Hydroxyl Ion ◽  
Ion Gradient

The transport of urate and p-aminohippurate (PAH) was evaluated in brush border membrane vesicles from the rat renal cortex. The binding of urate to the membranes was 6% of total uptake and no conversion of urate to allantoin was detected. The binding of PAH to the membranes was 24% of total uptake. In the presence of an outwardly directed hydroxyl ion gradient (pHi = 7.5, pHo = 6.0), the uptake of urate and PAH was stimulated relative to the absence of a hydroxyl ion gradient (pHi = pHo = 7.5) and the influx of urate resulted in a transient overshoot of the equilibrium value. The hydroxyl ion gradient-stimulated uptake of urate and PAH was not solely due to a change in membrane potential. Probenecid, DIDS, furosemide, and pyrazinoate inhibited the hydroxyl ion gradient-stimulated uptake of urate and PAH in a dose-dependent manner. The uptake of [14C]urate and [3H]PAH could be cis-inhibited and trans-stimulated by either unlabeled urate or PAH. In the presence of an outwardly directed bicarbonate gradient and 10% CO2 (outside HCO-3 = 5.4 mM, inside HCO-3 = 54 mM, pHo = 6.5, pHi = 7.5), the initial rate of urate uptake was faster and the initial rate of urate efflux was slower compared with vesicles that had the same pH gradient without bicarbonate or CO2. The effects of bicarbonate gradients on organic anion transport were not dependent on diffusion potentials. Finally, 100 mM extravesicular Na+, K+, Li+, or Cs+ did not affect urate or PAH uptake. These results indicate that brush border membrane vesicles from the rat kidney contain an anion exchange transport system with affinity for urate, PAH, hydroxyl ions, and bicarbonate. In addition there is no evidence for a sodium-urate or sodium-PAH cotransport mechanism in these membranes.

Effect of ethanol on peptidases of hamster jejunal brush-border membrane

1982 ◽  
Vol 242 (5) ◽  
pp. G442-G447
Author(s):  
P. K. Dinda ◽  
I. T. Beck
Keyword(s):  
Conformational Changes ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Kinetic Studies ◽  
Inhibitory Effect ◽  
Time Dependent ◽  
Peptidase Activity ◽  
Dependent Manner ◽  
Dose Dependent ◽  
Hydrolysis Of

This study was undertaken to investigate the effect of ethanol on the brush-border activity of the small intestine. Brush-border membrane isolated from hamster jejunum was incubated with L-phenylalanylglycine (Phe-Gly), L-leucylglycine (Leu-Gly), or glycyl-L-tyrosine (Gly-Tyr) in the absence and presence of 1-5% (wt/vol) ethanol, and the L-amino acids liberated were determined. Ethanol was found to depress the hydrolysis of all peptides in a dose-dependent manner. The inhibitory effect of ethanol on the peptidases does not appear to be time dependent. The ethanol-induced inhibition of peptidase activity is completely reversible. Kinetic studies indicate that ethanol caused a decrease in the Vmax of the enzymes responsible for the hydrolysis of the Phe-Gly and Gly-Tyr but did not have any effect on their Km. In the hydrolysis of Leu-Gly, two enzymes were involved, and ethanol depressed the Vmax of both without affecting the Km of either. These findings suggest that ethanol produces conformational changes of the peptidases involved in the hydrolysis of these three dipeptides.

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