A sensitive enzyme-linked immunosorbent assay (ELISA) for rat interleukin 8/cytokine-induced neutrophil chemoattractant (CINC) has been established by using biotin-conjugated anti-CINC rabbit IgG. The biotin-streptavidin sandwich ELISA detected CINC at concentrations from 3 pg/ml up to 30 ng/ml. The concentration of CINC in the pouch fluid (exudate) of rat carrageenin-induced inflammation was measured by the ELISA. After a time lag of about 2 h, neutrophils steadily accumulated in the carrageenin/air-pouch until 8 h. Similarly, the CINC level of exudate increased after about a 2-h lag, and reached a maximum (134 ng/ml) at 8 h, and thereafter decreased to a negligible concentration at 24 h after carrageenin injection. In association with the rise in CINC level, the concentration of exudate 96-kDa gelatinase corresponding to neutrophil gelatinase/type IV collagenase increased with time. The results suggest that CINC contributes, at least in part, to the neutrophil migration into the inflammatory lesion of the carrageenin-induced inflammation in rats

View full abstract

Effect of repetitive administration of phenytoin (PHT) on the single-dose pharmacokinetics of primidone (PRM) was investigated in 3 healthy male subjects. The peak concentration of unchanged PRM was achieved at 12 and 8 h after the administration of PRM in the absence and the presence of PHT, respectively. The elimination half-life of PRM was decreased from 19.4±2.2 (mean±S. E.) to 10.2±5.1 h (p<0.05) and the total body clearance was increased from 24.6±3.1 to 45.1±5.1 ml/h/kg (p<0.01) in the presence of PHT. No significant change was observed for the apparent volume of distribution between the two treatments. In the absence of PHT, the measurable amount (≤0.1 μmol/l) of phenobarbital (PB) and phenylethylmalonamide (PEMA) did not appear in the serum until 5.3 and 1.3 h after the PRM administration, and the peak concentrations of PB and PEMA were achieved at 52 and 36 h, but the concentrations of both metabolites were very low (PB 1.3 μmol/l ; PEMA 1.7 μmol/l). In the presence of PHT, within 0.8 and 0.5 h after the administration of PRM, the derived PB and PEMA appeared in the serum. About a 6-fold increase in the peak concentrations of both the metabolites were observed (PB 8.2 μmol/l ; PEMA 11.0 μmol/l). No significant changes were observed for the elimination half-lives of both PB and PEMA in the absence and presence of PHT. These results indicate that the induction of the enzyme system responsible for the oxidation of PRM to PB and PEMA mainly contribute to the alteration in the PRM disposition by PHT in the single-dose condition.

View full abstract

The effect of glutathione depletor diethylmaleate on rat hepatic glutathione S-transferase and glutathione peroxidase was studied in vivo and in vitro. When diethylmaleate (600 mg/kg) was given i. p. to rats, liver glutathione was depleted within 2 h and recovered to the control level 5 h after diethylmaleate treatment. Both glutathione S-transferase and peroxidase activities in microsomes, not in cytosol, were markedly increased during glutathione depletion and only glutathione S-transferase activity remained at high levels after recovery of the glutathione content. The increase in microsomal glutathione S-transferase and peroxidase activities with concomitant exhaustion of glutathione was also observed by perfusion of the isolated liver with diethylmaleate (10 mM). When liver microsomes were incubated with diethylmaleate in vitro at 37°C, glutathione S-transferase, but not peroxidase, activity was increased ; the increase was not reversed by dithiothreitol. These results indicate that diethylmaleate activates microsomal glutathione S-transferase by direct reaction to the enzyme during glutathione depletion and suggest that glutathione S-transferase activity and glutathione peroxidase activity in the microsomal enzyme may be differently regulated.

View full abstract

In order to clarify the mechanism of intestinal absorption of an antibiotic, fosfomycin (FOM), the uptakes of FOM by rabbit and human small intestinal brush-border membrane vesicles (BBMV) were studied. The initial uptake of FOM by BBMV at 15 s was saturable at a higher concentration of FOM. The kinetic parameters at 37°C of the saturable uptake expressed by the Michaelis-Menten equation were K₁=5.17 mM and J_max=3.88 nmol/15 s/mg protein for rabbits, and K₁=4.03 mM and J_max=1.90 nmol/15 s/mg protein for humans. The most efficient uptake was observed in the presence of both inward-directed Na⁺-and H⁺-gradients in both mammals. The uptake of FOM was inhibited by inorganic phosphate, FOM glycol which is a degradation product of FOM in the gastric juice and specific inhibitors of phosphate transport such as arsenate and phosphonoacetic acid. These findings confirmed that FOM absorption from rabbit and human small intestines is associated with the phosphate transport system. These transport phenomena of FOM are in close agreement with those obtained previously in rat BBMV studies. Judging from the results obtained for three mammalian species, rat, rabbit and human, it was concluded that carrier-mediated transport via the phosphate transport system is a very important pathway of intestinal absorption of FOM.

View full abstract

We have shown previously that recombinant human interleukin 1 (IL-1) and interleukin 6 (IL-6) inhibited the proliferation of a mouse myeloid leukemic cell line (M1), and that IL-6 induced differentiation of the cells into macrophage-like cells and that IL-1 augmented this differentiation. Using this model we investigated the action mechanisms of IL-1 and IL-6, IL-6, but not IL-1, stimulated prostaglandin E2 (PGE2) production. The differentiative effect of IL-6 however, was not suppressed by indomethacin, although PGE2 induction by IL-6 was completely inhibited. Exogenously added PGE2 neither augmented the differentiative effect of IL-6 nor induced differentiation in combination with IL-1. Therefore, stimulation of PGE2 production did not appear to be essential for differentiative effects of these cytokines. Dibutyryl cAMP, 8-Br-cAMP and two adenylate cyclase-activating reagents, cholera toxin (CT) and forskolin (FK), all exhibited the similar augmenting effects as IL-1. These reagents augmented M1 cell differentiation by IL-6, and they did not induce differentiation in combination with IL-1. cAMP derivatives, CT, FK, IL-1 and IL-6 all inhibited the proliferation of M1 cells. CT and FK increased the intracellular cAMP levels. However, neither IL-1 nor IL-6 increased the cAMP levels. In contrast to the cAMP derivatives and reagents that activate adenylate cyclase activity, phorbol 12-myristate 13-acetate (PMA) and calcium ionophore neither induced nor augmented the differentiation in combination with either IL-1 or IL-6. Intracellular Ca²⁺ concentration was not altered by IL-1 or IL-6 suggesting that Ca²⁺/Calmodulin kinase and protein kinase C activation are not involved in this signal transduction pathway. Therefore, the present study suggests that IL-1 exhibits an effect similar to that of cAMP without affecting intracellular cAMP level.

View full abstract

The chromosomal aberration test using a Chinese hamster lung cell line (CHL) was carried out on 19 aporphine alkaloids including apomorphine with and without rat liver homogenates (S9) mix. Einghteen of 19 alkaloids tested induced chromosomal aberration in the presence of absence of S9 mix. Among the alkaloids tested, anolobine, liriodenine and 4, 5-dioxodehydrocrebanine induced chromosomal aberrations at relatively low concentrations and as low as 2.5, 5 and 3.13 μg/ml, respectively. Dicentrine, anolobine and 4, 5-dioxodehydrocrebanine induced chromosomal aberrations with high frequencies, Apomorphine induced 10% aberrant cells at 12.5 μg/ml in the direct method and 12% at 100 μg/ml with S9 mix in the S9 method. Liriodenine which was the most potent mutagen for TA100 with S9 mix and roemerine which was the most potent for TA98 with S9 mix in Ames test were also clastogenic with and without S9 mix.

View full abstract

Urinary excretion of enzymes by rats was assessed after glutathione (GSH) was depleted by treatment with a mixture of the GSH depletors D, L-buthionine-S, R-sulfoximine (BSO) and diethylmaleate (DEM). Renal GSH was low 2 h after treatment and later returned to the control level. The urinary excretion of γ-glutamyltranspeptidase (γ-GTP) and N-acetyl-β-D-glucosaminidase (NAG) remained high for at least 3 d after the injection of BSO (100 mg/kg) and DEM (0.5 ml/kg), with no effect on the blood urea nitrogen level. N, N'-Dimethylthiourea (DMTU), a scavenger of oxygen free radicals, inhibited this increase in the urinary excretion of γ-GTP. DMTU also inhibited the increase in cisplatin-induced NAG excretion caused by the GSH depletors. These results suggested that the urinary excretion of these enzymes is an index of renal tubular injury caused by shortterm depletion of renal GSH, and that the generation of free radicals may be involved in renal tubular injury during GSH depletion or caused by cisplatin together with GSH depletors.

View full abstract

Release of endogenous taurine by electrical stimulation of slices of the hippocampus, cerebral cortex, cerebellum and medulla oblongata of the rat was studied and compared with that of alanine and/or γ-aminobutyric acid (GABA). Electrical stimulation caused a calcium-dependent release of taurine from slices of the hippocampus, cerebral cortex and cerebellum but not from slices of the medulla oblongata. The stimulus-evoked release of taurine in the hippocampus was rapid in onset and declined to baseline fast, which was essentially similar to the time course pattern of the stimulusevoked release of GABA. In addition, there were distinct regional differences in the relative amounts of taurine released. Electrical stimulation did not release alanine from any regions examined. These results support the hypothesis that taurine plays a neurotransmitter role in the hippocampus, cerebral cortex and cerebellum of the rat.

View full abstract

A new compound, 1-[2-(decylthio) ethyl] azacyclopentan-2-one (HPE-101) was synthesized, and its skin penetration enhancing activity was examined by using ¹⁴C-indomethacin as a penetrant. A solution of HPE-101 and indomethacin was applied to a cloth pad affixed onto an adhesive tape to give a HPE-101 patch, and the patch was applied to hairless mouse skin. The amount of percutaneously absorbed indomethacin was determined by measuring the radioactivity excreted in urine for 24 h after application. 1) Azone and decylmethyl sulfoxide, enhanced markedly the percutaneous absorption of indomethacin when the propylene glycol-ethanol (9 : 1 v/v) mixture was used as the solvent. 2) Among various penetration enhancers dissolved in the indomethacin solutions and applied to screen for penetration enhancing activity, HPE-101 was found to be the most prominent. 3) Solvents containing more than 3% (w/w) of HPE-101 produced a plateau level of the penetration enhancing activity. 4) Daily application of 1% (w/w) solutions of HPE-101 or Azone increased the daily excretion of indomethacin significantly above the level excreted on the previous day. However, repeated daily application beyond 3 d gave a steady state excretion of indomethacin. 5) The mouse skin was pretreated with 3% (w/w) solutions of HPE-101 or Azone for 24 h on the 1st day, and the indomethacin solution was applied for 24 h on the 3rd day and 7th day to examine the recovery of skin barrier function. Enhanced excretion of indomethacin was still noted on the 3rd day, but enhancement was not observed on the 7th day.

View full abstract

The aim of the study was to compare two methods classically used in rats to determine the fractional catabolic rate (FCR) of labeled high or low density lipoproteins : constant infusion and single pulse. The FCR of [¹⁴C]-sucrose HDL (High density lipoprotein) was studied. For the short term experiment (8 hours), both methods gave similar FCR determined 8 hours after HDL constant infusion (9.4%. h^-1±0.6) or single pulse (8.5%. h^-1±0.4), values significantly higher than those measured 24 hr after the single pulse (6.2%. h^-1±0.3). The identification and simulation of the model representing HDL movements between an intravascular and extravascular pool, after single pulse and constant infusion methods, demonstrated that FCR of lipoproteins cannot be precisely measured with techniques involving excessively short observation periods.

View full abstract