Potential role for S100A4 in the disruption of the blood-brain barrier in collagen-induced arthritic mice, an animal model of rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease associated with chronic inflammation of the joints. RA has been shown to increase the morbidity of and mortality due to cardiovascular and cerebrovascular diseases. We recently reported that cerebrovascular permeability was increased in mice with collagen-induced arthritis (CIA), an animal model of RA. S100A4, a member of the S100 family, is up-regulated in synovial fluid and plasma from RA patients. This study was aimed at evaluating a role of S100A4 in the mediation of blood-brain barrier (BBB) dysfunction in CIA mice. CIA was induced by immunization with type II collagen in mice. Cerebrovascular permeability was assessed by measurement of sodium fluorescein (Na-F) levels in the brains of control and CIA mice. Serum S100A4 concentrations in control and CIA mice were measured by enzyme-linked immunosorbent assays (ELISA). Accumulation of Na-F in the brain and serum levels of S100A4 were increased in CIA mice. Increased S100A4 levels in the serum are closely correlated with hyperpermeability of the cerebrovascular endothelium to Na-F. We investigated whether S100A4 induces BBB dysfunction using mouse brain capillary endothelial cells (MBECs). S100A4 decreased the transendothelial electrical resistance and increased Na-F permeability in the MBECs. S100A4 reduced the expression of occludin, a tight junction protein, and stimulated p53 expression in MBECs. These findings suggest that S100A4 increases paracellular permeability of MBECs by decreasing expression levels of occludin, at least in part, via p53. The present study highlights a potential role for S100A4 in BBB dysfunction underlying cerebrovascular diseases in patients with RA.

Ovariectomy aggravates convulsions and hippocampal gamma-aminobutyric acid inhibition induced by cyclosporin A in rats.

The possible cyclosporin A application for rheumatoid arthritis that develops preferentially in middle-aged women raises concerns about adverse effects of cyclosporin A, including neurotoxicity in patients with climacterium. The present study was aimed at elucidating the effect of cyclosporin A on the convulsive activity and gamma-aminobutyric acid (GABA) neural activity of the hippocampus in ovariectomized rats, as a menopause/climacterium model. Ovariectomy markedly aggravated the effect of repeated administration of cyclosporin A (40 mg/kg, once a day for 5 or 6 days), convulsions and reduction of the basal GABA levels and aminooxyacetic acid-evoked GABA accumulation. These aggravations were blocked by estradiol replacement. The present findings demonstrated that ovariectomy increased the susceptibility to cyclosporin A-induced convulsions by accelerating an inhibitory action of cyclosporin A on GABA neural activity in the hippocampus, this being blocked by estrogen replacement. Menopause/climacterium is, therefore, included in the risk factors for cyclosporin A-induced neurotoxicity and this risk is lowered by estrogen replacement therapy.

Pharmacological characterization of cyclosporine A-induced kaolin intake in rats.

Kaolin intake behavior of rats is known to be one of the useful animal models to evaluate the emetic and antiemetic actions of drugs. The present study was aimed at elucidating the pharmacological characterization of cyclosporine A (CsA)-induced kaolin intake in rats. Subchronic treatment (once a day for 3 days) with CsA produced a dose- and time-dependent increase in kaolin intake. Scopolamine (muscarinic antagonist), mepyramine (selective histamine H(1) antagonist) and diphenhydramine (H(1) and muscarinic antagonist) but neither domperidone (dopamine D(2) antagonist) nor ondansetron (serotonin 5-HT(3) antagonist) significantly inhibited CsA-induced kaolin intake. These findings suggest that an activation of central muscarinic and H(1) receptor is closely associated with CsA-induced kaolin intake in rats. Use of scopolamine and/or diphenhydramine may be possible regimens to alleviate and avoid nausea and vomiting in patients with CsA therapy.

Cyclosporine enhances alpha1-adrenoceptor-mediated nitric oxide production in C6 glioma cells.

The present study was aimed at elucidating the effect of cyclosporine on phenylephrine-evoked nitric oxide (NO) production in C6 glioma cells using direct electrochemical NO monitoring. Phenylephrine (0.1-10 microM) dose-dependently stimulated NO production (0.8-12.9 microM) and this was blocked by NO synthase inhibitor, prazosin, Ca2+-depletion and Xestospongin C (a blocker of the inositol 1,4,5-trisphosphate (IP3) receptor), suggesting that the alpha1-adrenoceptor signaling pathway mediates NO production in C6 cells. Cyclosporine (approximately 10 microM) failed to evoke NO production but increased phenylephrine-evoked NO production by 20-120% of phenylephrine alone in a dose-dependent manner (1-5 microM). Xestospongin C, at a concentration which showed no effect on phenylephrine-induced NO production, significantly inhibited the cyclosporine-enhanced phenylephrine response. This finding suggests that cyclosporine may increase phenylephrine-induced NO production by accelerating IP3 receptor function in the alpha1-adrenoceptor signaling pathway in C6 cells. This enhanced NO production in glial cells may be operative for the occurrence of cyclosporine neurotoxicity including convulsions and encephalopathy.

Protein binding and the metabolism of thiamylal enantiomers in vitro.

UNLABELLED: Thiamylal, a chiral thiobarbiturate, is marketed as a racemic product. We studied the serum protein binding and microsomal metabolism of thiamylal enantiomers in vitro. The unbound fraction of R(+)-thiamylal was greater than that of S(-)-thiamylal. The analysis of binding data revealed that both enantiomers bound to human serum albumin through only one site. In displacement studies with site-specific probes, dansylsarcosine, but not warfarin, significantly decreased the binding of both enantiomers. The bindings of enantiomers were also decreased by octanoate and a large concentration of oleate. These findings suggest that both enantiomers bind to Site II of albumin with higher affinity for S(-)-enantiomer. R(+)-thiamylal was metabolized more rapidly than S(-)-enantiomer by human liver microsomes. An experiment with isoform-selective inhibitors and cytochrome P-450 (CYP) isoforms showed that CYP2C9 had the highest activity for the metabolism of both enantiomers, the activity being 7 to 10 times that of CYP2E1 and CYP3A4. CYP2C9 showed a significantly rapid metabolism of R(+)-enantiomer, suggesting that CYP2C9 is mainly involved in the enantioselective metabolism of thiamylal. IMPLICATIONS: Because clinically marketed thiamylal is a racemic compound, a pharmacokinetic study of each enantiomer may be beneficial. We found that the enantioselectivity of thiamylal existed in protein binding and metabolism. This may be caused by the differences in the affinities of enantiomers for albumin and cytochrome P-450 isoform.

Potentiation of vancomycin-induced histamine release by muscle relaxants and morphine in rats.

The intravenous injection of vancomycin sometimes causes anaphylactoid reactions, in which histamine release may play a major role. These reactions are more frequently manifested when vancomycin is injected into anesthetized patients. We examined the vancomycin-induced histamine release and the interaction of vancomycin with muscle relaxants or opioid in rats. In an in vitro study with rat peritoneal mast cells, treatment with vancomycin at concentrations of greater than 1.25 mM produced significant histamine release. Tubocurarine, vecuronium, pancuronium, succinylcholine, and morphine up to concentrations of 0.25, 1, 5, 30, and 5 mM, respectively, produced no significant histamine release. However, the nonsignificant histamine release induced by 0.5 mM vancomycin was clearly enhanced by combining vancomycin with any of these agents. In the in vivo study, the intravenous injection of vancomycin significantly increased the plasma histamine levels in rats when vancomycin was injected at 200 mg/kg of body weight (63.2 +/- 34.0 ng/ml [mean +/- standard deviation]) but not when it was injected at 100 mg/kg (30.8 +/- 20.2 ng/ml) compared with that in the saline-treated rats (22.5 +/- 11.4 ng/ml). Although the subcutaneous administration of morphine (10 mg/kg) never increased the plasma histamine levels, the intravenous injection of vancomycin (100 mg/kg) 30 min after this morphine treatment markedly increased the plasma histamine levels (56.0 +/- 26.9 ng/ml). These findings provide experimental evidence that the combination of muscle relaxants or an opioid with vancomycin may increase the risk of anaphylactoid reactions by enhancing the release of histamine.

Inhibition of GABA system involved in cyclosporine-induced convulsions.

In this study, we attempted to clarify the mechanisms mediating cyclosporine-evoked convulsions. Cyclosporine (50 mg/kg, i.p.) significantly enhanced the intensity of convulsions induced by bicuculline (GABA receptor antagonist), but not those induced by strychnine (glycine receptor antagonist), N-methyl-D-aspartic acid, quisqualic acid or kainic acid (glutamate receptor agonists). Bicuculline plus cyclosporine-induced convulsions were significantly suppressed by an activation of GABAergic transmission with diazepam, phenobarbital and valproate. The GABA turnover estimated by measuring aminooxyacetic acid-induced GABA accumulation in the mouse brain was significantly inhibited by cyclosporine (50 mg/kg, i.p.). When cultured rat cerebellar granule cells were exposed to 1 microM cyclosporine for 24 hr, the specific [3H]muscimol (10 nM) binding to intact granule cells decreased to 53% of vehicle controls. The present study provides the first evidence suggesting that cyclosporine inhibits GABAergic neural activity and binding properties of the GABAA receptor. These events are closely related to the occurrence of adverse central effects including tremors, convulsions, coma and encephalopathy under cyclosporine therapy.

Improved method of determining thiamylal enantiomers in human serum by high-performance liquid chromatography.

Thiamylal, a widely used anesthetic drug, has two enantiomers. We developed a simple and rapid method for measuring the thiamylal enantiomers in human serum. The method involves a liquid-liquid extraction procedure followed by chiral resolution using a 5 microm silica-bonded alpha1-acid glycoprotein column (Chiral-AGP). The thiamylal enantiomers and internal standard were eluted within 15 min and were well-resolved. At concentrations of 1, 5 and 20 microg ml(-1), the relative standard deviations of R(+)- and S(-)-thiamylal were 1.35-2.88% and 1.37-3.01%, respectively, for the intra-day assay, and 2.93-4.46% and 2.46-4.84%, respectively, for the inter-day assay. This method facilitates the routine monitoring and pharmacokinetic studies of thiamylal enantiomers.

Inhibition of neuronal dopamine uptake by some antiallergic drugs.

The effects of 10 antiallergic drugs (astemizole, azelastine, ebastine, emedastine, epinastine, ketotifen, oxatomide, terfenadine, pemirolast and tranilast) on neuronal dopamine uptake were examined. Some drugs examined showed a concentration-dependent inhibition of [3H]dopamine uptake into synaptosomal preparations of the rat striatum. The inhibition constant (Ki) values were 231-876 nM for ebastine, terfenadine, oxatomide and astemizole. The specific binding of [3H] (1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine) (GBR12935) to the rat striatal membranes was also inhibited by these antiallergic drugs. There was a good correlation between the degrees of inhibition of [3H]dopamine uptake and [3H]GBR12935 binding. Then, the behavioral excitement induced by L-DOPA (100 mg/kg, s.c.) plus pargyline hydrochloride (80 mg/kg, i.p.) in mice was significantly enhanced by i.p. treatment with ebastine (10 mg/kg) and astemizole (5 mg/kg). These results suggest that the neuronal dopamine uptake is inhibited by some antiallergic drugs, especially ebastine.

Direct and continuous electrochemical measurement of noradrenaline-induced nitric oxide production in C6 glioma cells.

1. Nitric oxide (NO) production in C6 glioma cells was directly monitored in real time by electrochemical detection with a NO-specific biosensor. 2. We present here the first direct evidence that noradrenaline elicits long-lasting NO production in C6 cells pretreated with lipopolysaccharide and interferon-gamma, an effect blocked by NG-monomethyl-L-arginine, a NO synthase inhibitor. 3. This direct electrochemical measurement of glia-derived NO should facilitate our understanding of the kinetics of glial signaling in glia-glia and glia-neuron networks in the brain.

Determination of salicylic acid in human serum with capillary zone electrophoresis.

The determination of salicylic acid (SA), a metabolite of aspirin, in human serum was developed using capillary zone electrophoresis (CZE) with diode array detection. The reproducibility of separation and quantification with CZE analysis of the extract of SA from human serum was appropriate for the intra- and inter-day assay coefficients. A high correlation was revealed between the serum SA levels in volunteers determined by CZE and those determined by a fluorescence polarization immunoassay (r=0.973, n = 12), although the former values were slightly higher than the latter. There were no peaks interfering with the assay of SA by internal standard method. This CZE method could provide a simple and efficient method for monitoring SA in patients.

Enhancement of serotonergic neural activity contributes to cyclosporine-induced tremors in mice.

A single cyclosporine injection (50 mg/kg, i.p.) significantly enhanced harmine- but not oxotremorine-induced tremors in mice. This potentiation became more apparent when cyclosporine (50 mg/kg, i.p.) was administered once a day for seven days. These findings suggest an involvement of monoaminergic mechanisms in cyclosporine-induced tremors. The effects of cyclosporine were examined on the dynamics of noradrenaline, dopamine and serotonin in the mouse brain. Both single and repeated treatment with cyclosporine significantly facilitated the serotonin turnover as estimated from the probenecid-induced accumulation of 5-hydroxyindoleacetic acid, but either mode of treatment failed to change the contents of monoamines and their metabolites or the turnover of noradrenaline and dopamine. Therefore, the cyclosporine-enhanced activity of serotonin neurons may be interpreted as producing adverse central effects, including tremors.

Repeated interferon-alpha administration inhibits dopaminergic neural activity in the mouse brain.

In vivo effects of single and repeated interferon-alpha administrations on the dynamics of noradrenaline, dopamine and 5-hydroxytryptamine were investigated in the mouse brain. Single interferon-alpha administration (15, 30 and 60 X 10(6) U/kg i.p.) had no significant effect on the levels of monoamines and their metabolites or monoamine turnover. When interferon-alpha (15 X 10(6) U/kg i.p.) was administered once a day for 5 days, however, both dopamine and 3,4-dihydroxyphenylacetic acid levels were significantly decreased and alpha-methyl-p-tyrosine-induced dopamine depletion was significantly suppressed. These results suggest that repeated interferon-alpha administration inhibits dopaminergic neural activity. This inhibitory action of interferon-alpha in dopamine neurons may be involved in adverse central effects, such as parkinsonism and depression with suicidal potential.

Application of high-performance thin-layer chromatography for the detection of organophosphorus insecticides in human serum after acute poisoning.

We developed a rapid and simple method for identifying 25 commonly used organophosphorus insecticides in human serum using high-performance thin-layer chromatography (HPTLC). These organophosphates were separated on plates with three different developing systems within 6-18 min and detected by means of ultraviolet radiation and coloring reactions with 4-(4-nitrobenzyl)pyridine-tetraethylenepentamine reagent (NT reagent) or palladium chloride reagent (PdCl2 reagent). Each organophosphate was accurately identified by means of the R(F) x 100 value and the spot color in three systems. The detection limits of dichlorvos, fenitrothion, malathion, methidathion, parathion and trichlorfon in serum by the liquid-liquid extraction method were 1.1, 0.12, 0.12, 0.05, 0.6 and 0.1 microg/ml, respectively. These sensitivities may be sufficient to detect those organophosphates in patient serum after acute poisoning.

Instability of standard calibrators may be involved in overestimating vancomycin concentrations determined by fluorescence polarization immunoassay.

Fluorescence polarization immunoassay (FPIA) is widely used to determine serum vancomycin concentrations, and it has been shown to over-estimate vancomycin concentrations in sera from renally impaired patients. This phenomenon has generally been thought to result from interference by vancomycin crystalline degradation products (CDP-1). In this study, we confirmed that serum vancomycin concentrations in various patients determined by FPIA were higher than those determined by high-performance liquid chromatography (HPLC) or enzyme multiplied immunoassay (EMIT). However, the quantitative differences in the serum vancomycin concentrations determined by FPIA versus HPLC were higher than the CDP-1 concentrations, even when the cross-reactivity of FPIA to CDP-1 is assumed to be 100%. When the vancomycin calibrators for FPIA were stored at 4 degrees C for 30 days, their concentrations determined by FPIA and HPLC decreased by 14 and 20%, respectively, and CDP-1 corresponding to 20% of primary vancomycin was formed. When stored at 25 degrees C, the degradation of vancomycin was more marked. We concluded that not only the cross-reactivity of FPIA to CDP-1 but also the instability of calibrators may cause the overestimation of serum vancomycin concentrations determined by FPIA.

Cerebral blood flow velocity and failure of autoregulation in neonates: their relation to outcome of birth asphyxia.

Using the continuous wave Doppler technique, we examined the pulsatility index (PI) of the anterior cerebral artery in 14 asphyxiated infants. We also measured the blood pressure (BP) and fontanel pressure (FP) and calculated the cerebral perfusion pressure (CPP). According to the neurological prognosis, we divided the 14 infants into two groups and studied correlation of each factor. In the good-prognosis group (n = 11), PIs are within normal limits. There is a negative correlation between BP, CPP and PI, suggesting that the autoregulation of cerebral blood flow has been lost. On the other hand, there is no significant correlation between BP, CPP and PI in the poor-prognosis group (n = 3). These infants are thought to have lost the autoregulation, but their cerebral blood flow is not pressure-passive. Not only BP but also brain edema, vasodilation, and possibly other factors may contribute to determine the cerebral blood flow. Concerning FP, no remarkable correlations are found between two groups. It is therefore very important to monitor the PI, BP, FP in asphyxiated infants even if the degree of asphyxia is mild.

Ipsilateral and contralateral recordings of auditory brainstem responses to monaural stimulation.

The developmental changes of the ipsilateral and contralateral auditory brainstem responses (ABRs) were studied in 105 normal infants and children. In both ipsilateral and contralateral recordings, the peak and interpeak latencies shortened with increasing age, while the amplitudes of wave V had a tendency to become higher. Contralateral ABR amplitudes were always smaller than those of ipsilateral ABRs. In the contralateral recording, wave I was absent and the contralateral wave II and wave III complex began to separate after birth (25%); separation percentage reached 80-100% at 7 months of age. Our results suggest that the contralateral recording of ABRs is a useful measure of developmental changes in infant auditory pathways.