Mikihisa Takano

Carrier-mediated transport systems of tetraethylammonium in rat renal brush-border and basolateral membrane vesicles.

Transport of [3H]tetraethylammonium, an organic cation, has been studied in brush-border and basolateral membrane vesicles isolated from rat kidney cortex. Some characteristics of carrier-mediated transport for tetraethylammonium were demonstrated in brush-border and basolateral membrane vesicles; the uptake was saturable, was stimulated by the countertransport effect, and showed discontinuity in an Arrhenius plot. In brush-border membrane vesicles, the presence of an H+ gradient ( [H+]i greater than [H+]o) induced a marked stimulation of tetraethylammonium uptake against its concentration gradient (overshoot phenomenon), and this concentrative uptake was completely inhibited by HgCl2. In contrast, the uptake of tetraethylammonium by basolateral membrane vesicles was unaffected by an H+ gradient. Tetraethylammonium uptake by basolateral membrane vesicles was significantly stimulated by a valinomycin-induced inside-negative membrane potential, while no effect of membrane potential was observed in brush-border membrane vesicles. These results suggest that tetraethylammonium transport across brush-border membranes is driven by an H+ gradient via an electroneutral H+-tetraethylammonium antiport system, and that tetraethylammonium is transported across basolateral membranes via a carrier-mediated system and this process is stimulated by an inside-negative membrane potential.

A simple method for the isolation of basolateral plasma membrane vesicles from rat kidney cortex Enzyme activities and some properties of glucose transport

A procedure for preparing basolateral membrane vesicles from rat renal cortex was developed by differential centrifugation and Percoll density gradient centrifugation, and the uptake of D-[3H] glucose into these vesicles was studied by a rapid filtration technique. (Na+ + K+)-ATPase, the marker enzyme for basolateral membranes, was enriched 22-fold compared with that found in the homogenate. The rate of D-glucose uptake was almost unaffected by Na+ gradient (no overshoot).

Carrier-mediated transport of cephalexin via the dipeptide transport system in rat renal brush-border membrane vesicles

Carrier-mediated transport of aminocephalosporin antibiotics by renal brush-border membrane vesicles has been studied in relation to the transport systems for dipeptides and amino acids. Dipeptides such as L-carnosine (beta-alanyl-L-histidine) and L-phenylalanylglycine competitively inhibited the uptake of cephalexin, but amino acids did not. Cephalexin uptake was stimulated by the countertransport effect of L-carnosine in the normal and papain-treated vesicles, and by the effect of L-phenylalanylglycine only in the papain-treated vesicles. In the papain-treated vesicles, the hydrolysis of dipeptides was markedly decreased, and the specific activity for cephalexin transport was increased approx. 2-fold because of the partial removal of membrane proteins. These results suggest that carrier-mediated transport of cephalexin can be transported by the system for dipeptides in renal brush-border membranes.

Cimetidine transport in rat renal brush border and basolateral membrane vesicles

The transport of cimetidine by rat renal brush border and basolateral membrane vesicles has been studied in relation to the transport system of organic cation. Cimetidine inhibited [3H]tetraethylammonium uptake by basolateral membrane vesicles in a dose dependent manner, and the degree of the inhibition was almost the same as that by unlabeled tetraethylammonium. In contrast, cimetidine inhibited the active transport of [3H]tetraethylammonium by brush border membrane vesicles more strongly than unlabeled tetraethylammonium did. In agreement with the transport mechanism of tetraethylammonium in brush border membranes, the presence of an H+ gradient ([H+]i greater than [H+]o) induced a marked stimulation of cimetidine uptake against its concentration gradient (overshoot phenomenon), and this concentrative uptake was inhibited by unlabeled tetraethylammonium. These results suggest that cimetidine can share common carrier transport systems with tetraethylammonium in renal brush border and basolateral membranes, and that cimetidine transport across brush border membranes is driven by an H+ gradient via an H+-organic cation antiport system.

Carrier-mediated transport of amino-cephalosporins by brush border membrane vesicles isolated from rat kidney cortex.

The uptake of cephalosporin antibiotics by brush border membrane vesicles isolated from rat renal cortex has been studied by a rapid filtration technique, demonstrating a carrier-mediated transport system for amino-cephalosporins such as cephalexin and cephradine. The antibiotics were taken up into an osmotically reactive intravesicular space. The uptake of cephalexin was saturable (apparent Km2.2 mM), was inhibited by structural analogues and sulfhydryl reagents, and was stimulated by the countertransport effect, although the Na+ gradient did not affect the uptake. This transport system was essentially different from the transport system for p-aminohippurate in brush border membranes. The uptake properties for cephradine in brush border membrane vesicles appeared to be similar to those for cephalexin. The present results suggest the existence of a carrier-mediated transport system for amino-cephalosporins in brush border membranes. This system may be a part of the mechanism of tubular reabsorption of these antibiotics.

In-vivo clearance study of vancomycin in rats

The renal handling of vancomycin in rats and the effects of various drugs (probenecid, cimetidine and quinidine) on urinary excretion of the antibiotic were investigated by in‐vivo clearance.

Relationship between expression level of P-glycoprotein and daunorubicin transport in LLC-PK1 cells transfected with human MDR1 gene

P-Glycoprotein-mediated transcellular transport and intracellular accumulation of [3H]daunorubicin were examined in cell monolayers with different levels of P-glycoprotein. The porcine kidney epithelial cell line LLC-PK1 was transfected with MDR1 cDNA, and four sublines, LLC-GA5, LLC-GA5-VLB4, LLC-GA5-COL10, and LLC-GA5-COL150, were obtained by culturing the cells in the absence or in the presence of 4 ng/mL vinblastine, 10 ng/mL colchicine, and 150 ng/mL colchicine, respectively. Western blot analysis showed a large difference in P-glycoprotein expression within these sublines. The degree of drug resistance was dependent on the expression level of P-glycoprotein. The amount of the unidirectional transport of [3H]daunorubicin by P-glycoprotein corresponded to the expression level of P-glycoprotein, which was followed by the decrease in intracellular accumulation of the agent. The concentration of cyclosporin A required for the inhibition of P-glycoprotein-mediated transport of [3H]daunorubicin was higher in cells with a high expression of P-glycoprotein. These findings suggest that the transport of daunorubicin by P-glycoprotein and its inhibition by cyclosporin A correspond to the expression level of P-glycoprotein.

Transport of procainamide in a kidney epithelial cell line LLC-PK1

Transport of procainamide, an anti-arrhythmic drug, was investigated in LLC-PK1 kidney epithelial cell line. The uptake of procainamide by LLC-PK1 monolayers cultured in plastic dishes was temperature-dependent, saturable and inhibited by organic cations such as cimetidine and N-acetylprocainamide. An aminocephalosporin antibiotic, cephalexin, also inhibited procainamide uptake, but an organic anion, p-aminohippurate, did not. The uptake of procainamide was greater at an alkaline external pH than at an acidic pH. In addition, procainamide uptake increased when intracellular pH was decreased and the uptake decreased when the intracellular pH was increased by ammonium chloride treatment, indicating the involvement of an H+/procainamide antiport system in apical membrane. The basolateral to apical flux of procainamide across LLC-PK1 monolayers cultured on permeable supports was 2.5-times larger than the apical to basolateral flux, and only the former process was inhibited by other organic cations. These findings suggest that LLC-PK1 cells can transport procainamide by the organic cation transport system and that procainamide is transported unidirectionally from basolateral to apical side across the cell monolayers.

Role of chloride on carrier-mediated transport of p-aminohippurate in rat renal basolateral membrane vesicles

Effect of inorganic anions on p-amino[3H]hippurate transport in renal basolateral membranes has been studied using the vesicles preloaded with unlabeled p-aminohippurate (countertransport condition). The uptake of p-amino[3H]hippurate was stimulated by the outward gradient of unlabeled p-aminohippurate and the labeled substrate was accumulated into the vesicles against its concentration gradient in the presence of Cl-. The substitution of SCN- and SO4(2-) for Cl- in both sides of the vesicles depressed the initial rate and the overshoot magnitude of p-amino[3H]hippurate uptake. These results suggest that Cl- may play an important role for the carrier-mediated transport system of organic anion in renal basolateral membranes.

Enzyme activities and sodium-dependent active d-glucose transport in apical membrane vesicles isolated from kidney epithelial cell line (LLC-PK1)

Apical membrane vesicles were isolated from the confluent LLC-PK1 cells by nitrogen cavitation and Mg/EGTA precipitation methods. The specific activities of marker enzymes for apical membranes were enriched 8- to 18-fold relative to those in the homogenate. D-[3H]Glucose uptake into the vesicles was stimulated in the presence of Na+ gradient (overshoot phenomenon), and the values of apparent Km and Vmax for Na+-dependent component of D-glucose uptake were 0.3 mM and 5.8 nmol/mg protein per min, respectively.