Takeshi Shibuya

Simultaneous determination of α-tocopherol and α-tocopherolquinone by high-performance liquid chromatography and coulometric detection in the redox mode

Abstract A simple, selective and highly sensitive assay method for the simultaneous determination of α-tocopherol and α-tocopherolquinone in plasma or erythrocyte membrane by high-performance liquid chromatography (HPLC) with a series of multiple coulometric working electrodes (CWE) was investigated. For good separation of α-tocopherol and α-tocopherolquinone, an MC MEDICAL C18 reversed-phase column and a mobile phase consisting of 96% methanol [methanol-HPLC-grade distilled water (96:4, v/v)] with 40 mM sodium perchlorate were used. Also, selective, highly sensitive and simultaneous detection of these substances was performed in redox mode using a series of four CWE. In this detection mode, the first, second and third CWE were set at −0.45 V for pre-reaction and to prevent interference, the fourth CWE was used as an electrode for actual measurement with its potential set at +0.40 V against a palladium reference electrode. The detection limits were 50–100 pg. Excellent chromatograms of α-tocopherol and α-tocopherolquinone were obtained within 8 min. The usefulness of reversed-phase HPLC with the redox detection mode was confirmed by application to the determination of the concentrations of α-tocopherol and α-tocopherolquinone in a crude ethanol-hexane extract of rat plasma or erythrocyte membrane. These findings suggest that reversed-phase HPLC with the redox detection mode using a series of four CWE is applicable to study the preventive effect of α-tocopherol on lipid peroxidation.

Involvement of nitric oxide in intracerebroventricular β-endorphin-induced neuronal release of methionine-enkephalin

Previous work has suggested that the antinociceptive effect of nitrous oxide (N2O) in rats is mediated, at least in part, by beta-endorphin (beta-EP) and that centrally administered beta-EP stimulates release of methionine-enkephalin (ME) in the rat spinal cord. Since inhibition of central nitric oxide (NO) production has been found to suppress N2O antinociception, we examined the possible involvement of NO in the release of spinal cord ME by i.c.v. beta-EP. Urethane-anesthetized, male Sprague-Dawley rats were intrathecally (i.t.) perfused with artificial cerebrospinal fluid (aCSF) and fractions of perfusate were assayed for immunoreactive (i.r.) ME. The beta-EP-induced increase in ME concentration in the i.t. perfusate was significantly suppressed by perfusing the animal with aCSF containing 100 microM L-NG-nitro arginine (L-NOARG), an inhibitor of NO synthase (NOS). The further addition of 50 microM L-arginine (L-ARG), but not D-arginine (D-ARG), to the aCSF reversed the suppression of the ME change by L-NOARG. However, the potency of L-ARG decreased with increasing concentrations of L-ARG. On the other hand, increasing the concentration of L-NOARG in the aCSF to 250 microM failed to produce a greater suppression of the beta-EP-induced increase in ME. These findings suggest that NO may mediate the beta-EP-induced release of ME in the spinal cord and that interference with this mechanism might be an explanation for the antagonism of N2O antinociception in rats by NOS inhibitors.

Prenatal and postnatal exposure to diazepam: effects on opioid receptor binding in rat brain cortex.

The effects of chronic perinatal or postnatal administrations of diazepam on opiate receptor development in cerebral cortex and striatum were studied at two different post-treatment ages, using tritiated ethylketocyclazocine, the prototypic kappa-opiate, for measurements of opiate binding site ontogeny. Prenatal plus postnatal exposure to diazepam markedly decreased the total number of binding sites for [3H]ethylketocyclazocine in the rat cerebral cortex and striatum at 14 days of age. Postnatal diazepam treatment alone did not alter [3H]ethylketocyclazocine binding site development in cortex or striatum. These results suggest that development of opiate receptors in rat brain can be altered by prenatal exposure to diazepam.

Time-course alterations of monoamine levels and cerebral blood flow in brain regions after subarachnoid hemorrhage in rats.

To investigate the possible correlation between changes in monoaminergic neuronal activity and cerebral blood flow (CBF) in the same brain region after subarachnoid hemorrhage (SAH), monoamine levels were analyzed by both HPLC-ECD and fluorohistochemistry techniques, and CBF was measured by using colored microspheres. At the second day of SAH, significant and nonsignificant reductions in blood flow were seen in the examined brain regions with a marked increase in CBF appearing in the telencephalon and hypothalamus on the third day. Significant reductions of monoamine levels in most brain regions were also observed on the second day after SAH, whereas norepinephrine (NE) levels in midbrain increased to 1.5 times compared to the normal level. These reductions were sustained until the fourth day of SAH, although at the third day, serotonin (5-HT) and dopamine levels in the hippocampus and 5-HT levels in the cerebelium were significantly elevated. In fluorohistochemical studies, the fluoro-intensities of monoamines, particularly catecholamines, in the midbrain dorsal NE bundle were enhanced at the second day after SAH. These NE neurons originated from the A5 cell group close to the area where homologous blood was applied through the cisterna magna. The results obtained after SAH show an apparent correlation between changes in monoamine levels and CBF in norepinephrine (NE)-rich areas. These results suggest that SAH-induced neuronal dysfunctions, particularly with NE neurons, are caused not only by reductions of blood flow but also by hemorrhage.

Schistosoma mansoni: the role of calcium in the stimulation of cercarial proteinase release.

We investigated the role of calcium mobilization in the induction of proteinase release from cercarial preacetabular glands. Proteinase release was measured by the ability of cercariae to break down a 3H-labeled proline extracellular fibroblast matrix and calcium influx was measured using 45Ca2+. The role of calcium in the activation of cercarial proteinase was examined by investigating the effects of calcium addition and removal on linoleate-induced matrix degradation, the ability of various calcium modulators (Verapamil, fendiline, nifedipine, SK-525A, BAY K-8644, Ryanodine, and SK-7171A) to stimulate or inhibit linoleate-induced proteinase release, the ability of calcium modulators directly to induce cercarial proteinase release, and the ability of various stimulants of proteinase release to induce calcium influx or efflux from cercariae. The results of these studies indicate that proteinase release is dependent on external calcium concentration, voltage-operated channels are either nonexistent in cercariae or have a minimal role in overall calcium influx, and that activation of Ca2+ influx can be caused by both free fatty acids and calcium modulators by a hypothesized receptor-operated channel. Although calcium uptake is important in cercarial proteinase release, it is not the only factor involved. Calcium uptake alone does not guarantee that proteinase will be secreted. On the other hand, if Ca2+ influx does not occur, proteinase will not be secreted.

Effect of opioid peptide antisera on nitrous oxide antinociception in rats

This study was performed to examine the effects of ICV injection of antiserum against beta-endorphin (beta-EP) or methionine-enkephalin (ME) on nitrous oxide-induced antinociception in rats using the hot plate test. The injection of beta-EP antiserum reversed the antinociceptive effect of nitrous oxide in a dose-related manner up to 200 micrograms/rat. However, antagonism of nitrous oxide by 400 micrograms beta-EP antiserum was comparable to that produced by 200 micrograms. On the other hand, similar amounts of ME antiserum had little effect against nitrous oxide antinociception. These findings suggest that beta-EP may play an important role in the antinociceptive effect of nitrous oxide.

Neuropsychopharmacological studies of a Ca2+ channel blocker on the modulation of brain Ca2+ mobilization of spontaneously hypertensive rats under mild stress.

The psychotropic effects of a calcium channel blocker (Ca antagonist) were examined in behavioral studies following changes in 45Ca2+ influx in synaptosomal fractions of brain tissues using spontaneously hypertensive rats (SHR). Under a novel circumstance utilizing 85-dB noise, SHR demonstrated hyperactivity and a significant increase in 45Ca2+ uptake into synaptosomal fractions of frontal cortex (FC) and hippocampus. Such hyperactivity may be caused not only be seeking behavior but also by stress-induced anxiety. Such hyperactivity was significantly blocked after 10 days of repeated administration of diazepam (DZP), tandospirone (SM-3997; SM), a 5-HT1A anxiolytic, and nitrendipene (Nit), a Ca antagonist. Moreover, repeated administration of DZP, SM and Nit reduced the maximum binding density of 3H-PN200-110 and reduced the 45Ca2+ uptake in FC of SHR. In hippocampus, midbrain, hypothalamus and striatum, the increased ratio of 45Ca2+ uptake was reduced after repeated administration of Nit or SM. These results suggest that the hyperactivity induced by this novel circumstances was reduced by DZP, SM and Nit and may be attributed to inhibition of voltage-dependent Ca channel activities in FC. In addition, Nit may induce anti-anxiety through the modulation of Ca2+ mobilization in the central nervous system.

Different effects of methylxanthines on central serotonergic postsynaptic neurons in a mouse behavioral model.

Effects of the four methylxanthines (100 mg/kg, IP)--caffeine, theophylline, theobromine, and pentoxifylline--on the central serotonergic neuron were studied in mice using a behavioral model, the head-twitch response. The four methylxanthines potentiated the head twitches induced by 5-hydroxytryptophan (5-HTP) in pargyline-pretreated mice; pentoxifylline was the most potent. The potentiating effect of pentoxifylline was increased by paroxetine, the selective inhibitor of uptake of 5-hydroxytryptamine (5-HT), but those of the other drugs were not. In nontreated animals, caffeine directly induced head-twitch responses, which were not affected by pargyline pretreatment but were increased by prior treatment with 5,7-dihydroxytryptamine (5,7-DHT). The number of head twitches produced by caffeine in 5,7-DHT-treated mice was increased twofold by p-chlorophenylalanine (p-CPA), the tryptophan hydroxylase inhibitor. In mice treated with both 5,7-DHT and p-CPA, theophylline induced the responses, although much less potently than caffeine. Theobromine and pentoxifylline produced even fewer responses. From the results of the present study, it may be concluded that the methylxanthines possess qualitatively different actions on the central serotonergic neuron; caffeine and theophylline appear to have direct effects on the postsynaptic neuron, but theobromine and pentoxifylline do not.

Penetration of Schistosoma mansoni cercariae into a living skin equivalent.

We evaluated the use of a living skin equivalent (LSE) as a suitable membrane for Schistosoma mansoni cercarial penetration. LSE is a living artificial skin composed of a dermal layer containing human dermal fibroblasts embedded in a collagen lattice and an epidermal layer consisting of differentiated human keratinocytes. The keratinocytes differentiate into a stratum corneumlike layer, whereas the dermal-epidermal junction forms a layer similar, but not identical to, the basement membrane. We exposed LSE to 50 cercariae for 0, 3, 6, 20, and 30 hr at 37 C, and the percentage of penetration was evaluated by counting cercariae remaining on the LSE surface. No cercarial penetration was observed in the first 15 min of exposure; however, penetration was detected at all other times. Maximum penetration rates were observed at 20 hr (80%). In other experiments LSE was pretreated topically with 0 or 4 micrograms/cm2 linoleic acid, then exposed to between 800 and 1,000 cercariae for 18-20 hr at 37 C. LSE pretreated with linoleate had significantly higher penetration rates than untreated membranes (81% +/- 2.51% vs. 65.9% +/- 6.97%, P = 0.03). Increasing linoleate concentrations from 10 to 40 micrograms/cm2 gradually decreased the ability of cercariae to penetrate the membrane. Some LSE membranes also were processed for light microscopy, and we present photomicrographs showing schistosomulae within the epidermal and dermal layers of the LSE. We conclude that despite the time it takes for cercariae to penetrate LSE, these membranes may allow investigators to examine, in vitro, host-parasite interactions at the level of the skin.

Relation between long-lasting amounts of excitatory amino acid and its neuronal uptake system in cultured cerebellar granule cells under hypoglycemia

1. Basal release of amino acids (Glu, Gln, Gly and Tau) in cultures of rat cerebellar granule cells was detected at 3 days in vitro (DIV). The amounts of Gln and Gly released increased according to days of culture. Moreover, the amounts of Glu in a glia poor culture of granule cells tended to be higher than those in glia rich cultured cells, while Gln, Gly and Tau concentrations were lower in glia poor cells than in glia rich cells. 2. After depolarization induced by high KCl, amounts of all measured amino acids significantly rose to more than 1.5 times their basal values. The increased values obtained in a glia rich culture of granule cells were higher than those in a glia poor culture of cells throughout all cultured days. 3. Under deprivation of glucose, most concentrations of amino acids in the medium, especially Gln concentration, increased by 50 mM KCl were lower than those seen under normal conditions. Such lesser efflux examined under hypoglycemia was much more clearly recognized in glia rich cultures than in glia poor cultures. However, the amounts of Glu at 10 and 14 DIV, and also Gly and Tau amounts at 10 DIV were significantly higher than those seen in the cultures of glia poor cells under normal conditions. 4. The dosage of 10 microM Glu-induced [Ca2+]i accumulation was inhibited by several different types of Ca antagonists and scopolamine. Meanwhile, the dosages of the tested drugs, except for scopolamine, required for blocking Glu-induced [Ca2+]i accumulation under hypoglycemia were less than those required under normal conditions. These results suggest that neuronal death induced hypoglycemia might be caused by the dysfunction of the neuronal, but not glial, Glu uptake system, and that some Ca antagonists might be useful in preventing the neuronal death caused by hypoglycemia.