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R Laprade

Publications and source records attributed to R Laprade.

At least 73 records · Page 4Linked to original sources
PubMed1986

Characterization of the apical membrane ionic permeability of the rabbit proximal convoluted tubule.

Basolateral membrane potential (psi BL), transepithelial potential (psi T), and the ratio of apical to basolateral membrane resistance (RA/RBL) were measured in rabbit proximal convoluted tubules (PCT) perfused in vitro. Analysis of RA/RBL changes using several luminal perfusates indicates that the cotransport of Na with glucose and alanine would represent 19% of the apical conductance in normal conditions; the cotransport of Na with acetate, citrate, sulfate, and phosphate would represent 7%, whereas Na, K, and Cl diffusion would represent 10, 4, and 0% of this apical conductance, respectively. On the other hand, psi BL values can also be analyzed using the equivalent circuit of the epithelium to obtain the apical membrane equivalent electromotive force (EA) in the presence of each perfusate. These values, as well as the preceding values obtained from RA/RBL measurements, indicate that in the absence of cotransported solutes the transference number for Na diffusion is several times larger than for K diffusion. Among the conductance pathways studied, the transference number sequence would be as follows: Na cotransport with alanine and glucose greater than Na cotransport with anions greater than Na diffusion greater than K diffusion greater than Cl diffusion. This study also suggests the presence of another important but unidentified apical ionic permeation pathway, since the total of the transference numbers obtained from RA/RBL analysis represents only 40% of the total apical membrane conductance and the absolute values of EA are difficult to account for using only the tested apical membrane permeation pathways.

Acetates↗
PubMed1984

Carrier-mediated ion transport in lipid bilayer membranes.

The electrical properties predicted by a widely accepted model for carrier-mediated ion transport in lipid bilayers are described. The different steps leading to ion transport and their associated rate constants are reaction at the interface between an ion in the aqueous phase and a carrier in the membrane (kRi), followed by translocation of the ion-carrier complex across the membrane interior (kis) and its dissociation at the other interface (kDi) after which the free carrier crosses back the membrane interior (ks). Results on glyceryl monooleate (GMO) membranes for a family of homologue carriers, the macrotetralide actin antibiotics (nonactin, monactin, dinactin, trinactin, and tetranactin) and a variety of ions (Na+, Cs+, Rb+, K+, NH4+, and Tl+) are presented. Internally consistent data obtained from steady-state electrical measurements (zero-current potential and conductance, current-voltage relationship) allow us to obtain the equilibrium permeability ratios for the different ions and show that for a given carrier kRi is relatively invariant from one ion to the other, except for Tl+ (larger), which implies that the ionic selectivity is controlled by the dissociation of the complex. The values of the individual rate constants obtained from current relaxation experiments are also presented and confirm the findings from steady-state measurements, as well as the isostericity concept for complexes of different ions with the same carrier (kis invariant). These also allow us to determine the aqueous phase membrane and torus membrane partition coefficients. Finally, the observed increase in kis from nonactin to tetranactin and, for all homologues, from GMO-decane to solvent-free GMO membranes, together with the concomitant decrease in kDi, can be explained in terms of modifications of electrostatic energy profiles induced by variations in carrier size and membrane thickness.

Anti-Bacterial Agents↗
PubMed1984

Luminal and peritubular ionic substitutions and intracellular potential of the rabbit proximal convoluted tubule.

Transepithelial (psi T) and basolateral (psi BL) potential difference was measured in rabbit proximal convoluted tubules perfused in vitro. In control solution without protein, the mean psi BL was -54 +/- 2.2 mV (n = 57). Luminal substitution of K by Na had no effect. Complete luminal substitution of glucose and alanine, 110 mM substitution of Na or NaCl produced transient hyperpolarizations of psi BL of 14, 10, and 13 mV, respectively, with a return close to the control value within 4-8 min in all cases. Returning to control solution produced similar time-course transient depolarizations of psi BL of 17, 11, and 16 mV, respectively, again with a return to the control value in 4-10 min. Omission of glucose and alanine in the perfusate produced a decrease in cell volume of 14% that was maximal in 4 min with a complete recovery in the post-control period. A 110 mM luminal or peritubular substitution of Cl by cyclamate produced no significant effect on psi BL after taking into account the large psi T generated by the diffusion of Cl across the paracellular pathway. On the other hand, complete peritubular substitution of K by Na and 110 mM substitution of Na or NaCl produced sustained but reversible depolarizations of psi BL of 37.5, 10.2, and 20.4 mV, respectively. The transient nature of the hyperpolarization following luminal substitution of glucose, alanine, or Na can be interpreted in terms of changes in the intracellular sodium activity that would affect the Na-K-ATPase pump. Similarly, the sustained depolarization seen after a peritubular substitution of K and Na would also be compatible with a decrease in the basolateral ionic pump activity.

Alanine↗
PubMed1984

Transepithelial and cell membrane electrical resistances of the rabbit proximal convoluted tubule.

A technique using double-barreled perfusion pipettes and intracellular microelectrodes was developed to measure transepithelial, apical, and basolateral membrane electrical resistances in isolated rabbit proximal convoluted tubules (PCT). This technique has been tested successfully with respect to cable analysis: the transepithelial resistance (RT) did not change with tubule length and the measured core resistance of the lumen (RC) varied according to prediction with lumen diameter and perfusate resistivity. In control solutions, a linear I-V relationship was observed at the entry of the tubule for current varying from -300 to +300 nA. The mean RT was 1,050 +/- 70 omega X cm (n = 33) (a specific resistance of 8.2 omega X cm2). Bath proteins and large variations in transtubular hydrostatic pressure had no significant effect on RT, whereas RT was not systematically related to transepithelial PD or to the sodium-to-chloride permeability ratio (n = 22). Perfusate substitution of 50 mM NaCl by mannitol increased RT by 21% (n = 7) but the same maneuver in the peritubular solution had no significant effect after a 5-min equilibration period. The ratio of apical to basolateral cell membrane resistance (RA/RBL) determined with intracellular microelectrodes was 3.1 +/- 0.3 (n = 27) in control solutions and increased within 1 min by 36% (n = 8) when glucose and alanine were replaced by mannitol in the perfusate solution. Using simultaneous initial changes in transepithelial and basolateral potential differences when glucose and alanine were removed, the individual values of RA and RBL were determined. Mean RBL was 4,900 +/- 990 omega X cm (39 +/- 1.3 omega X cm2) and mean RA was 15,000 +/- 4,300 omega X cm (118 +/- 33 omega X cm2).

Amino Acids↗
PubMed1983

Liquid junctions and isolated proximal tubule transepithelial potentials.

Accurate evaluation of the unavoidable liquid junction potentials (LJP) involved whenever bath (B) and perfusate (P) solutions differ in ionic composition is of fundamental importance in the determination of proximal convoluted tubule (PCT) transepithelial potential, psi T, and its active and diffusion components, psi A and psi D. Therefore, a precise method of measuring these LJP, using Ag/AgCl electrodes, has been developed and successfully tested. The measured LJP, psi B-P, between artificial bath serum containing 6 g/100 ml albumin and ultrafiltrate-like perfusate is +2.6 mV. The average measured potential difference (PD) in rabbit PCT with these solutions was -3.0 mV, psi T thus being only -0.4 mV. Three maneuvers known to abolish psi A (hypothermia, 10(-4) ouabain, or K-free bath) brought the PD close to the reference potential, indicating that psi A congruent to PD and that psi D congruent to psi B-P. From dilution potentials, after correction for LJP (4.3 in the absence and 6.2 mV in the presence of proteins), PNa/PCl was calculated to be 0.9 for midcortical and 1.1 for juxtamedullary PCT in the absence of proteins and slightly lower in their presence. These values of LJP obtained with Ag/AgCl electrodes are between 30 and 100% larger than the ones measured using a saturated KCl bridge or the ones in previous studies, indicating that in these studies psi T as well as PNa/PCl has been overestimated.

Animals↗
PubMed1982

Mechanism of neomycin stimulation of D-glucose uptake in rabbit intestinal brush border membrane.

In order to study the effect of the antibiotic neomycin on the intestinal epithelium, D-glucose was used as a probe molecule and its transport into rabbit brush border membrane vesicles was measured by a rapid filtration method. Treatment of the epithelium with neomycin sulfate prior to the preparation of the brush border membrane enhanced the D-glucose uptake, whereas neutral N-acetylated neomycin did not. This action of neomycin was related to its polycationic character and not to its bactericidal action. No significant difference could be demonstrated between the protein content or disaccharidase-specific activities of the brush border fractions from treated or non-treated intestines. Electrophoretic protein patterns of SDS-solubilized membrane were not significantly different after neomycin treatment. To gain more information on the mechanism involved in the stimulation of D-glucose transport, experiments were conducted on phosphatidyl glycerol artificial membranes and the results compared with those obtained with brush border membrane. At a concentration of 10(-7) M, neomycin decreased the nonactin-induced K+ conductance by a factor of approx. 100. The membrane conductance was linearly dependent on the neomycin concentration and the conductance in 10(-2) M KCl was 10 times that in 10(-3) M KCl. The valence of neomycin was estimated, from the slope of these curves, to be between 6 and 4. In contrast, acetylated neomycin had no effect on the nonactin-induced K+ membrane conductance. Therefore, the effect of neomycin on artificial membrane is related to its 4 to 6 positive charges. It is proposed that the stimulation of sugar transport in brush border membrane is related to screening of the membrane negative charges by the positively-charged neomycin. Accumulation of anions at the membrane surface then occurs and their diffusion into the intravesicular space would increase the transmembrane potential which, in turn, stimulates the entry of D-glucose.

Animals↗
PubMed1982

Effects of variation of ion and methylation of carrier on the rate constants of macrotetralide-mediated ion transport in lipid bilayers.

The effects of methylation on the rate constants of carrier-mediated ion transport have been studied on monooleindecane bilayers with K+, Rb+, NH4+, and Tl+ ions, using the series of homologue carriers, nonactin, monactin, dinactin, trinactin, and tetranactin, each member of the series differing from the previous one by only one methyl group. Measurements of the amplitude and time constant of the current relaxation after a voltage jump over a large domain of voltage and permeant ion concentration, together with a computer curve-fitting procedure, have allowed us, without the help of steady-state current-voltage data, to deduce and compare the values of the various rate constants for ion transport: formation (kRi) and dissociation (kDi) of the ion-carrier complex at the interface, translocation across the membrane interior of the carrier (ks) and the complex (kis). With the additional information from steady-state low-voltage conductance measurements, we have obtained the value of the aqueous phase-membrane and torus-membrane partition coefficient of the carrier (gammas and gammas). From nonactin to tetranactin with the NH4+ ion, kis, and gammas are found to increase by factors of 5 and 3, respectively, kDi and gammas to decrease respectively by factors 8 and 2, while kRi and ks are practically invariant. Nearly identical results are found for K+, Rb+, and Tl+ ions. kRi, ks and kis are quite invariant from one ion to the other except for Tl+ were kRi is about five times larger. On the other hand, kDi depends strongly on the ion, indicating that dissociation is the determining step of the ionic selectivity of a given carrier. The systematic variations in the values of the rate constants with increasing methylation are interpreted in terms of modification of energy barriers induced by the carrier increasing size. Within this framework, we have been able to establish and verify a fundamental relationship between the variations of kis and kDi with methylation.

Anti-Bacterial Agents↗
PubMed1982

Kinetics of carrier-mediated ion transport in two new types of solvent-free lipid bilayers.

In contrast with the usual glyceryl-monooleate/decane (GMO-D) bilayer lipid membranes, new membranes, formed from a mixture of GMO in squalene (GMO-S) or from a mixture of GMO in triolein (GMO-T), seem to be almost solvent free. Our results from voltage-jump relaxation studies, using these "solvent-free" membranes with the homologue carriers, nonactin, monactin, dinactin, trinactin, and tetranactin, are compared with the corresponding ones for GMO-D membranes. With all homologues, solvent-free membranes show an increase of the free carrier translocation rate, ks, by a factor of 2.5, a decrease in the dissociation rate constant of the complex, kDi, by a factor of 1.5 and no significant change in its formation rate constant, kRi. However, the principal effect of the absence of solvent in these membranes is an increase by a factor of approximately 10 of the translocation rate constant for moving the complex across the membrane, kis. This increase varies regularly from a factor of 7-15 with decreasing carrier size, and is always larger for GMO-T than for GMO-S membranes. These solvent-free effects are interpreted in terms of modifications of electrostatic and hydrophobic energy profiles in the membrane.

Ammonia↗
PubMed1982

Surface potential and conductance induced in lipid bilayers by the negatively charged ionophore Br-X537A.

The adsorption of Br-X537Z and its effect on the surface potential of monoolein lipid bilayers was measured using the nonactin conductances as a probe to determine the surface charge density. Because of the pH dependence of this adsorption, it was concluded that not only the negatively charged molecules X- could induce a surface charge but also a dimer HX2- made from X- and the neutral molecule HX. Also an important bilayer conductance was induced by Br-X537A. From the BrX537A concentration dependence of this conductance, the effect of pH, and the induced surface potential, it was found that two charged complexes are transported across the bilayer depending on pH. At pH greater than or equal to 7 the conducting molecule is X- and at pH less than or equal to 5 the complex is H2X3-. A quantitative model is obtained to calculate both the induced surface potential and the conductance.

Adsorption↗
PubMed1982

Cation and local anesthetic conductance induced by the negatively charged ionophore Br-X537A in lipid bilayers.

The cation conductance induced by Br-X537A was measured for K+ and Ca2+ at different pH and different Br-X537A concentrations. The K+-concentration dependence was different depending on the pH of the solution. At pH 4.3, the conductance depended linearly on [K+] while at pH 7 it depended on [K+]2. About the same dependence on [Br-X537A] was found at these pH values. From these results it was concluded that two complexes were transporting K+; at pH greater than or equal to 7 the complex is mostly K2X3- and at pH less than or equal to 5 the complex is mostly KHX3-. From the conductance dependence on [Ca2+], pH, and [Br-X537A] it was found that the only conducting complex is CaX3-. It was also observed that Br-X537A could transport tetracaine. Taking into account the surface charge induced by tetracaine, the conductance depended linearly on the tetracaine concentration. Correcting for the surface charged induced by Br-X537A, the tetracaine conductance was found to depend on [Br-X537A]2. Consequently, the conducting complex is TX2- at pH greater than or equal to 7. No other conducting complexes are formed at the pH is decreased and at pH 4 tetracaine is not conducted. It was found that procaine was not conducted between pH 8 and 4.

Anesthetics, Local↗
PubMed1982

Cell volume regulation in the proximal convoluted tubule.

To evaluate the effect of hyper- and hypotonicity on proximal convoluted tubule (PCT) cell volume, nonperfused PCT were studied in vitro with hypertonic solutions containing sodium chloride, urea, or mannitol (450 mosmol/kg H2O) and with hypotonic low sodium chloride solutions (160 mosmol/kg H2O). When the tubules were subjected to hypertonic peritubular solutions containing NaCl, cell volume immediately decreased by 15.5% and remained constant throughout the experimental period (60 min). With mannitol, the initial decrease was identical to that with NaCl (17.7%), but the PCT volume increased slightly during the experimental period. With urea, the decrease in cell volume was smaller (7%) and transient. In hypotonicity, the PCT swelled rapidly, but this swelling was followed by a rapid regulatory phase in which PCT volume nearly returned to control values after less than 10 min. With a potassium-free peritubular medium or 10(-3) M ouabain, the regulatory phase of hypotonicity completely disappeared, whereas the cells did not maintain their reduced volume in NaCl-induced hypertonicity. These results suggest that Na-K-ATPase plays an important role in the maintenance of a reduced cellular volume in hypertonicity and in the regulatory phase of hypotonicity, probably by an active extrusion of sodium and water from the cell.

Animals↗
PubMed1982

[Comparison of the kinetics of ionophore-induced ion transport in conventional and solventless membranes].

In contrast to conventional membranes prepared from a mixture of glyceryl-monooleate (GMO) and decane (GMO-D) which still contain decane in the final state, the membranes formed from a mixture of GMO and squalene (GMO-S) or triolein (GMO-T) contain finally only GMO and are said to be "solventless". The use of the electric current relaxation technique following a voltage jump has allowed us to characterize the effect of the absence of solvent on the various steps of ion transport induced by nonactin. We have measured the rates of formation (kri) of dissociation (kDi) and of translocation (kis) of the ion-ionophore complex as well as the rate of translocation of the ionophore (ks). With the GMO-S and GMO-T membranes, kis is about ten times higher, ks, twice as high, and kDi twice as small than with the GMO-D membranes. The observed effects are always more prominent with the GMO-T membranes. These results can be interpreted in terms of modifications of the energy barriers in the membrane due to the absence of solvent.

Glycerides↗
PubMed1976

Spin labels as probes for tetraphenylboron ion interaction with liposomes.

The effects of tetraphenylboron (TFB) on the molecular organization of lipids within phosphatidylcholine (PC) liposomes were investigated using the spin-labeled method. Perturbations at the surface of the lipid were probed using stearamide and cholestane spin labels; perturbations in the hydrophobic-portion were probed with spin-labeled amphiphilic fatty esters.

Binding Sites↗
PubMedDate not supplied

Fractionation of renal brush border membrane proteins with Triton X-114 phase partitioning.

Analysis of brush border membrane proteins by gel electrophoresis has revealed a complex polypeptide composition. We have investigated the use of Triton X-114 phase partitioning to fractionate such proteins on the basis of their degree of hydrophobicity. Each of the fractions was composed of a complex but distinct set of proteins. Most proteins were solubilized by Triton X-114 and partitioned into the detergent-poor fraction. Trehalase, gamma-glutamyl transpeptidase, and leucine aminopeptidase were well solubilized (greater than 80%) and enriched 5.1-, 3.9-, and 2.5-fold in the detergent-rich fraction. In contrast, alkaline phosphatase and 5'-nucleotidase were poorly solubilized. The specific activities of these enzymes were increased 2.7- and 2.3-fold in the insoluble protein fraction. Maltase was almost completely solubilized and partitioned into the detergent-poor fraction with a small enrichment factor (1.3). These results suggest that Triton X-114 phase partitioning could be useful as a first step in the purification of many brush border membrane proteins.

5'-Nucleotidase↗
PubMedDate not supplied

Effect of cholate on H(+)-ATPase and other proteins of dog renal brush-border membrane.

A short treatment of dog renal brush-border membrane vesicles (BBMV) with sodium cholate, followed by dialysis of the detergent, reorients the polarity of H(+)-ATPase in the membrane and exposes its ATP binding sites to the extravesicular space, as previously shown with pig BBMV. In cholate-pretreated vesicles, the H(+)-ATPase remains fully active, but is inserted under the reversed polarity in sealed vesicles. A large spontaneous N-ethylmaleimide-sensitive ATPase activity is thus observed, as well as a steep intravesicular acidification upon external ATP addition, two findings absent in native vesicles. The ability of nitrate plus ATP to dissociate the hydrolytic subunits ot the proton pump in cholate-pretreated vesicles, but not in native vesicles, demonstrates that most of the ATP binding subunits are accessible to ATP following cholate treatment. The sensitivity of the cytoplasmic domain of the H(+)-ATP activity to trypsin also confirms the reorientation of the enzyme in cholate-pretreated vesicles. The H(+)-ATPase and alkaline phosphatase remain largely associated with the membranes after the treatment with cholate, but gamma-glutamyltranspeptidase, aminopeptidase N, and neutral endopeptidase are largely solubilized. Upon dialysis of cholate, all these enzymes are in part reinserted in the membrane according to their original polarity. The reorientation process is however specific for the H(+)-ATPase. Cholate treatment does not increase the formation of inside-out vesicles. Thus the treatment with cholate really reorients the polarity of the H(+)-ATPase in vesicles and allows for study of the proton pumping capacity of vacuolar H(+)-ATPase of proximal tubules.

Adenosine Triphosphate↗