Trans-stimulation effect on tetraethylammonium (an organic cation) transport was examined in rat renal brush-border membrane vesicles. The uptake of [14C]tetraethylammonium at pH 6.0-8.5 was stimulated by preloading the membrane vesicles with unlabeled tetraethylammonium. When the uptake was measured in preloaded membrane vesicles in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, stimulation was observed at high pH but not at low pH. These results suggest that the mechanisms of the trans-stimulation effect on organic cation transport are different depending on the pH. When pH is low, the trans-stimulation is due to generation of an outward H+ gradient, which in turn stimulates [14C]tetraethylammonium uptake by H(+)-[14C]tetraethylammonium exchange. In contrast, when pH is high, the stimulation is due to direct exchange of tetraethylammonium for [14C]tetraethylammonium.
Effect of sulfhydryl reagents on the transport of tetraethylammonium, an organic cation, has been studied in brush border membrane vesicles isolated from rat renal cortex. H+ gradient-dependent uptake of tetraethylammonium by the vesicles was inhibited by various sulfhydryl reagents in a dose-dependent manner, and the potency of the reagents was followed in the order of HgCl2 greater than p-chloromercuribenzoate, p-chloromercuribenzene sulfonate (PCMBS) greater than N-ethylmaleimide. In the absence of H+ gradient, tetraethylammonium uptake and efflux also were inhibited by p-chloromercuribenzoate and PCMBS. The sulfhydryl reagents did not affect the dissipation rate of H+ gradient across the membranes. Pretreatment of brush border membranes with PCMBS resulted in an inhibition of tetraethylammonium uptake in the presence and absence of H+ gradient, and this inhibition was reversed by subsequent treatment of the vesicles with thiols such as dithiothreitol, glutathione and cysteine. The inhibitory effect by PCMBS pretreatment was protected in the preincubation with unlabeled tetraethylammonium. These results suggest that sulfhydryl reagents inhibit the transport of tetraethylammonium by their specific interaction with the active sites of the carrier, and that sulfhydryl groups are essential for organic cation transport system in renal brush border membranes.
p-Aminohippurate (PAH) transport in apical and basolateral membranes of OK cells was studied to characterize each membrane process and to determine which process is important for the vectorial transport of PAH across the OK cell monolayers. PAH uptake from the basal side into OK cells was much higher than from the apical side, and was inhibited almost completely by anion transport inhibitors (probenecid and furosemide) and low temperature. On the other hand, PAH efflux from OK cells to the apical side was higher than to the basal side. The efflux across the apical membrane also was inhibited by the transport inhibitors and low temperature. The uptake of PAH across the basolateral membrane was inhibited by dicarboxylates such as alpha-ketoglutarate, and kinetic analysis showed that the inhibition was a mixed-type. These findings suggest that PAH transport in apical and basolateral membranes of OK cells is a specifically mediated process, and both processes contribute to the vectorial transport of PAH across the cell monolayers. PAH/dicarboxylate exchange may be involved in PAH transport in the basolateral membrane of OK cells.
P-glycoprotein modulators are respected to be multidrug resistance reversing agents in cancer chemotherapy. Some calcium channel blockers, calmodulin inhibitors or immunosuppressive agents have been used in clinical studies, although the dose of these drugs required to test in vitro experimental data might cause potent pharmacological effects which are not desirable in patients. By using LLC-GA5-COL150 cells that express P-glycoprotein specifically on the apical membranes, we examined the transport of anticancer drugs mediated by P-glycoprotein. Cepharanthin, a biscoclaurine alkaloid, potently inhibits the transport of vinblastine and daunorubicin, both commonly used anticancer agents. The 50% inhibitory concentration of cepharanthin on daunorubicin transport was 2.06 microM. Combined inhibitory effects on daunorubicin transport were observed when cepharanthin was used together with cyclosporin A, a potent immunosuppressive agent and P-glycoprotein modulator. Cepharanthin itself was transported by P-glycoprotein. Transcellular transport of cepharanthin across LLC-GA5-COL150 cell monolayers was saturable when its concentration was under 5 microM, and the transport was inhibited by P-glycoprotein modulators. These results indicate that cepharanthin can reverse multidrug resistance, and proper combination with other P-glycoprotein modulators could potentiate its inhibitory effect on expelling the anticancer drugs out of the cell via P-glycoprotein.
Bestatin [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-leucine], a potent inhibitor of aminopeptidase B and leucine aminopeptidase, enhances the immune response to activate the defense mechanism of the living organism and suppresses the growth and metastasis of cancer. Bestatin has been effectively used by p.o. administration, but the mechanisms of intestinal absorption remain to be solved. The present study was undertaken to examine whether bestatin, a dipeptide containing an unusual amino acid, is transported via dipeptide carriers in intestinal brush-border membranes, by using cephradine as a probe for the H+/dipeptide cotransport system. The initial uptake of cephradine in the presence or absence of an inward H+ gradient, driving force, was inhibited by bestatin and this inhibition occurred in a competitive manner (Ki = 0.47 mM). The uptake of cephradine was stimulated by the countertransport effect of bestatin, the definitive criterion for ascertaining a common transport system. These findings indicate that bestatin, as well as cephradine and other p.o. cephalosporins, can be transported via dipeptide carriers in intestinal brush-border membranes.
The characteristics of gentamicin transport have been studied by using cultured kidney epithelial cell line LLC-PK1. The uptake of gentamicin by the LLC-PK1 cells appeared to be linear for 30 min and reached the equilibrium at day 1. Marked stimulation of gentamicin uptake was observed on the development of a confluent cell density, accompanied by the increases of marker enzyme activities and Na+-dependent D-glucose transport in the apical membranes. Gentamicin uptake was inhibited by metabolic inhibitors such as rotenone and 2,4-dinitrophenol, and was inhibited competitively in the presence of other aminoglycosides. Depending on the external calcium concentration, calcium ionophore A23187 stimulated gentamicin uptake, whereas ethylene glycol bis(beta-aminoethyl ether)N,N1-tetraacetic acid, a calcium chelator, inhibited gentamicin uptake. These results suggest that gentamicin uptake by the LLC-PK1 cells may be mediated via specialized transport system, and calcium ion movement may play an important role as a regulatory factor for this transport system.
The pharmacokinetics of zonisamide was studied using routine therapeutic drug monitoring data from 68 epileptic patients. The 266 serum concentration data at steady-state after repetitive oral administration were analyzed using the nonlinear mixed effects model (NONMEM) program designed for estimation of population pharmacokinetic parameters. A one-compartment model with dose-dependent clearance was used for the pharmacokinetic analysis of zonisamide. The volume of distribution (V) was estimated to be 1.27 l/kg in a typical 33-kg patient, assuming that the bioavailability of orally administered zonisamide is 100%. The maximal daily dose to be cleared (Vmax) and the concentration giving half maximal clearance (a Michaelis-Menten constant) was 27.6 mg/d/kg and 45.9 μg/ml, respectively. The parameter of a power function of weight to adjust V and Vmax was estimated to be 0.741. In addition, Vmax for zonisamide appears to be 13% increased in patients receiving carbamazepine concurrently. The population pharmacokinetic parameters of zonisamide will be useful for designing dosage regimens in epileptic patients.
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