Transport of an organic anion, p-aminohippurate (PAH), was investigated in the OK kidney epithelial cell line using the cells grown on permeable supports. Specifically mediated transcellular transport of PAH across OK cell monolayers occurred only from basal side to apical side, and the accumulation of PAH in the cells was much larger from basal side than from apical side. Both the basal-to-apical transport and the accumulation from basal side were temperature dependent, saturable, and inhibited by anion transport inhibitors such as probenecid. In contrast, such a specific transport of PAH was not observed in LLC-PK1 cells. To further characterize the transcellular transport of PAH across OK cell monolayers, PAH transport in apical and basolateral membranes of the cells was studied. Initial uptake of PAH from basal side was much higher than from apical side. In contrast, PAH efflux from the cells to apical side was higher than to basal side. Both uptake from basal side and efflux to apical side were temperature dependent and inhibited by anion transport inhibitors. In addition, basolateral uptake of PAH was inhibited by dicarboxylates. These results suggest that both basolateral and apical transports of PAH are vectorial processes, which results in vectorial, basal-to-apical transport of PAH across OK cell monolayers. PAH/dicarboxylate exchange is probably involved in PAH transport across basolateral membrane in OK cells. Thus, OK cells should provide a potent in vitro model system for the study of active secretion of organic anion in the kidney.
The mechanisms by which decanoic acid enhances the permeation of fluorescein isothiocyanate dextran 4400 (FD-4) through Caco-2 cell monolayers were studied.
In the presence of glucose, the enhancing effect of decanoic acid was inhibited by N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide hydrochloride (W7, a calmodulin antagonist) by about 50%. In contrast, the permeation-enhancing effect of decanoic acid was smaller and was not affected by W7 in the presence of 2-deoxyglucose, although it was almost completely inhibited by 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride (H7, a protein kinase C inhibitor). A linear relationship between the permeation rate of FD-4 and reciprocal of the transepithelial electrical resistance (TEER) indicated that decanoic acid facilitated the permeation of FD-4 via a paracellular route under all experimental conditions examined.
Thus, two different mechanisms (calmodulin-dependent or protein kinase C-dependent) appear to be involved in the permeation-enhancing effect of decanoic acid across Caco-2 cell monolayers via paracellular routes.
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.
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. The vancomycin-to-inulin excretion ratio (ER) was greater than unity at various infusion rates of vancomycin. Quinidine, co-administered with vancomycin, significantly decreased the total, renal and net secretory clearance of the antibiotic. Cimetidine also decreased, though not significantly, the secretory clearance of vancomycin by about 20%, but probenecid did not. These results suggested that vancomycin is secreted in renal tubules in rats, and that quinidine decreases the total clearance of vancomycin partly by inhibiting its tubular secretion in the kidney.
Nuclear factor-κB (NF-κB) is a key regulator of cancer progression and the inflammatory effects of disease. To identify inhibitors of DNA binding to NF-κB, we developed a new homogeneous method for detection of sequence-specific DNA-binding proteins. This method, which we refer to as DSE-FRET, is based on two phenomena: protein-dependent blocking of spontaneous DNA strand exchange (DSE) between partially double-stranded DNA probes, and fluorescence resonance energy transfer (FRET). If a probe labeled with a fluorophore and quencher is mixed with a non-labeled probe in the absence of a target protein, strand exchange occurs between the probes and results in fluorescence elevation. In contrast, blocking of strand exchange by a target protein results in lower fluorescence intensity. Recombinant human NF-κB (p50) suppressed the fluorescence elevation of a specific probe in a concentration-dependent manner, but had no effect on a non-specific probe. Competitors bearing a NF-κB binding site restored fluorescence, and the degree of restoration was inversely correlated with the number of nucleotide substitutions within the NF-κB binding site of the competitor. Evaluation of two NF-κB inhibitors, Evans Blue and dehydroxymethylepoxyquinomicin ([-]-DHMEQ), was carried out using p50 and p52 (another form of NF-κB), and IC50 values were obtained. The DSE-FRET technique also detected the differential effect of (-)-DHMEQ on p50 and p52 inhibition. These data indicate that DSE-FRET can be used for high throughput screening of anticancer drugs targeted to DNA-binding proteins.
Keloids and hypertrophic scars, especially keloids, are clinically intractable scars caused by an abnormal proliferation of fibroblasts and excessive production of collagen. The present clinical therapy for such scars is described briefly, and the feasibility of iontophoretic therapy with tranilast was examined in hairless rats and patients with scars. A drug electrode containing 12 mg tranilast, which was dissolved in 1.5 ml of ethanol/water (8 : 2 v/v) mixture, was placed on the dorsal skin surface of anesthetized rats or the affected parts of patients, and connected to the negative pole. An electric current was pulsed through at one min intervals. The in vivo current density was almost comparable between intact skin surfaces of healthy volunteers and keloids/hypertrophic scars of patients. Tranilast given iontophoretically (2 mA) a period of 30 min a week reduced the patients' complaints of pain and itching after only one or two treatments. Thus, the transdermal iontophoretic delivery of tranilast may be a useful treatment for keloid and hypertrophic scars, particularly for relieving pain and itching, and is more beneficial than tranilast given orally. Some discussions were also made in the present report.
ABSTRACT The lack of novel antibiotics against gram-negative bacteria has reinstated polymyxins as the drugs of last resort to treat serious infections caused by extremely multiresistant gram-negative organisms. However, polymyxins are nephrotoxic, and this feature may complicate therapy or even require its discontinuation. Like that of aminoglycosides, the nephrotoxicity of polymyxins might be related to the highly cationic nature of the molecule. Colistin and polymyxin B carry five positive charges. Here we show that novel polymyxin derivatives carrying only three positive charges are effective antibacterial agents. NAB739 has a cyclic peptide portion identical to that of polymyxin B, but in the linear portion of the peptide, it carries the threonyl- d -serinyl residue (no cationic charges) instead of the diaminobutyryl-threonyl-diaminobutyryl residue (two cationic charges). The MICs of NAB739 for 17 strains of Escherichia coli were identical, or very close, to those of polymyxin B. Furthermore, NAB739 was effective against other polymyxin-susceptible strains of Enterobacteriaceae and against Acinetobacter baumannii . At subinhibitory concentrations, it dramatically sensitized A. baumannii to low concentrations of antibiotics such as rifampin, clarithromycin, vancomycin, fusidic acid, and meropenem. NAB739 methanesulfonate was a prodrug analogous to colistin methanesulfonate. NAB740 was the most active derivative against Pseudomonas aeruginosa . NAB7061 (linear portion of the peptide, threonyl-aminobutyryl) lacked direct antibacterial activity but sensitized the targets to hydrophobic antibiotics by factors up to 2,000. The affinities of the NAB compounds for isolated rat kidney brush border membrane were significantly lower than that of polymyxin B.
