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Anesthetic treatment has been associated with widespread apoptotic neurodegeneration in the neonatal rodent brain. It has recently been suggested that propofol, a short-acting intravenous anesthetic agent, may have a potential as a neuroprotective agent. An apoptotic pathway mediated through endoplasmic reticulum (ER) stress has been attracting attention. ER stress is associated with accumulation of unfolded or misfolded proteins in ER, and ER stress-induced apoptosis is implicated in a wide range of diseases, including ischemia/reperfusion injury, neurodegeneration, and diabetes. We investigated whether thapsigargin-induced ER stress is prevented by propofol in human neuroblastoma SH-SY5Y cells. SH-SY5Y cells were pretreated with various concentrations of propofol (1-10 μM) for 3h before co-treatment with 0.5 μM thapsigargin and propofol for 20 h. Levels of ssDNA, specific evidence of apoptosis, and biomarkers of ER stress (mRNA expression of Chop and sXbp-1) were determined. We also assayed calpain and caspase-4 activities and intracellular Ca(2+) ([Ca(2+)]i) levels. Thapsigargin-induced increases in ssDNA levels, expressions of ER stress biomarkers, activities of caspase-4 and calpain, and level of [Ca(2+)]i were suppressed by co-incubation with propofol. Our data indicate the possibility that propofol inhibits the Ca(2+) release from ER at clinically employed dose levels. These results demonstrate that propofol suppresses the ER stress-induced apoptosis in this cell system, and may have the neuroprotective potency. It may also be a promising agent for preventing damage from cerebral ischemia or edema.

Aims In recent years, there has been an increase in patients with arteriosclerosis and the risk of lifestyle-related diseases. However, the pathogenesis and medication of atherosclerosis have not been elucidated. We developed a rat model of lifestyle-related diseases by feeding a high-fat diet and 30% sucrose solution (HFDS) to spontaneously hypertensive hyperlipidemic rats (SHHR) and reported that this model is a useful model of early atherosclerosis. In order to elucidate the pathogenesis of early atherosclerosis, we searched for atherosclerosis-related genes by microarray analysis using the aortic arch rat model of lifestyle-related diseases. Main methods Four-month-old male Sprague–Dawley rats and SHHR were each divided into two normal diet (ND) groups and two HFDS groups. After a four-month treatment, the expression of mRNA in the aortic arch was detected using the oligo DNA microarray one-color method and quantified using real-time PCR. Key findings In this study, we detected 39 genes in microarray analysis. Esm1, Retnlb Mkks, and Grem2 showed particularly marked changes in gene expression in the SHHR-HFDS group. Compared with the SD-ND group, the SHHR-HFDS group had an increase in Mkks gene expression of about 26-fold and an approximately 22-fold increase in the expression of Grem2. Similarly, the expression of Esm1 increased by about 12-fold and that of Retnlg by about 10-fold as shown by quantitative real-time PCR. Significance This study suggested that these four genes might be important in early atherosclerosis development.

The presence of hypertension and hyperlipidemia accelerates atherosclerosis and increases the risk of ocular disease. Since there were few rat models for atherosclerosis, spontaneously hypertensive rats (SHRs) and spontaneously hyperlipidemic rats (HLRs) were crossbred to obtain a new model: the spontaneously hypertensive hyperlipidemic rat (SHHR). Matrix metalloproteinases (MMPs) play an important role in ocular degeneration. The purpose of this study is to investigate changes in the MMP activities in vitreous and plasma as well as MMP expression in the retinas of SHHRs, which served as a model of vascular degeneration. We used 8-month-old Wistar–Kyoto (WKY) rats and Sprague–Dawley (SD) rats, SHRs, HLRs, and SHHRs. The MMP-2 and MMP-9 activities in plasma and vitreous were examined by zymography. The mRNA expression of MMP-2, MMP-9, and tissue inhibitor of metalloproteinases-3 (TIMP-3) in retina was examined by quantitative PCR. The localized expression of MMP-9 in the retinas was examined by immunostaining. The MMP-9 activity increased significantly in SHHRs compared with all other rats. MMP-9 was observed mainly at the superficial layer of the retina on immunostaining. The MMP-2, MMP-9, and TIMP-3 mRNA in retina was not significantly different in SHHRs as compared with all other rats. Increased MMP-9 activity in vitreous was influenced more intensely from plasma than retina because there was no change in MMP-9 expression in retina, and MMP-9 immunostaining was observed mainly at the surface of the retina, where blood vessels are present. In this study, the complications of hypertension and hyperlipidemia induced increased MMP-9 activity in vitreous and plasma. It is therefore suggested that MMP-9 may be involved in causing this result and in the development of retinal disease.

The accumulation of hydrophobic bile acid, such as glycochenodeoxycholic acid (GCDCA), in the liver has been thought to induce hepatocellular damage in human chronic cholestatic liver diseases. We previously reported that GCDCA-induced apoptosis was promoted by both mitochondria-mediated and endoplasmic reticulum (ER) stress-associated pathways in rat hepatocytes. In this study, we elucidated the relationship between these pathways in GCDCA-induced apoptotic HepG2 cells. HepG2 cells were treated with GCDCA (100-500microM) with or without a caspase-8 inhibitor, Z-IETD-fluoromethyl ketone (Z-IETD-FMK) (30microM) for 3-24h. We demonstrated the presence of both apoptotic pathways in these cells; that is, we showed increases in cleaved caspase-3 proteins, the release of cytochrome c from mitochondria, and the expression of ER resident molecular chaperone Bip mRNA and ER stress response-associated transcription factor Chop mRNA. On the other hand, pretreatment with Z-IETD-FMK significantly reduced the increases, compared with treatment with GCDCA alone. Immunofluorescence microscopic analysis showed that treatment with GCDCA increased the cleavage of BAP31, an integral membrane protein of ER, and pretreatment with Z-IETD-FMK suppressed the increase of caspase-8 and BAP31 cleavage. In conclusion, these results suggest that intact activated caspase-8 may promote and amplify the ER stress response by cleaving BAP31 in GCDCA-induced apoptotic cells.

In chronic cholestatic liver diseases, accumulation of hydrophobic bile acids is thought to damage hepatocytes. The mechanism of how cells die has been an open debate, but apoptotic pathways are known to involve activation of death receptors and mitochondrial dysfunction. Recently apoptosis via an endoplasmic reticulum (ER) stress-mediated pathway was also found. In this study, we examined whether ER stress is induced in rat hepatocytes by treatment with glycochenodeoxycholic acid (GCDCA, 50–300 μM for 1–24 h), and if so, whether ER stress-mediated apoptosis occurs in this system. We determined mobility of intracellular calcium ion, activities of calpain and caspase-12, specific to ER stress-mediated apoptosis, and Bip and Chop mRNA expressions, biomarkers of ER stress. We found that GCDCA induces ER-related calcium release within about ten seconds. Significant increases in activities of calpain and caspase-12 were observed after 15 h of GCDCA treatment. Bip and Chop mRNA expressions were increased with the treated GCDCA dose and incubation time. Cytochrome c release from mitochondria peaked in about 2 h of incubation. These results suggest that ER stress is actually induced by GCDCA, though its role in hepatocellular apoptosis may be smaller than mitochondria-mediated pathway. The presence of ER stress might be important in pathogenesis of cholestatic liver diseases.

Ursodeoxycholic acid (UDCA) has been widely used for treating cholestatic liver diseases. However, in a recent review of clinical trial articles, its therapeutic benefits were not proven. Therefore, we investigated whether UDCA prevents or potentiates glycochenodeoxycholic acid (GCDCA)-induced apoptosis in isolated rat hepatocytes. Hepatocellular cytotoxicity was assessed by lactate dehydrogenase (LDH) release, and apoptosis evaluated by DNA fragmentation, caspase activities, release of cytochrome C from mitochondria, and mitochondrial membrane potential change (Deltapsi). When hepatocytes were co-incubated with GCDCA and UDCA for a short time (2-6 h), GCDCA-induced LDH release was significantly reduced, while prolonged co-incubation (12-20 h) increased it. Similarly, the same co-incubation for a short time resulted in the inhibition of caspase activities and cytochrome C release, while prolonged incubation enhanced them compared with the incubation with GCDCA alone. Furthermore, UDCA significantly promoted the GCDCA-induced Deltapsi decline after 4h of incubation. These results demonstrated that UDCA reduced GCDCA-induced apoptosis in short incubation, but potentiated it in prolonged incubation. Based on these, we propose a hypothesis that induction of Deltapsi decrease from earlier stage of incubation may be responsible for the aggravation of GCDCA-induced apoptosis in long-term exposure, and would like to raise caution about clinical long-term use of UDCA.

A new series of diphenylalkyl piperazine derivatives with high affinities for the dopamine transporter (DAT), which were modified at both the diphenylalkyl moiety and the phenyl ring in the phenylamino moiety of 1-[4,4-bis(4-fluorophenyl)butyl]-4-[2-hydroxy-3-(phenylamino)propyl]piperazine 1 , was evaluated for their inhibitory activities against auto-oxidative lipid peroxidation in canine brain homogenates. Some of these were approximately equivalent in activity to α-tocopherol as a potent antioxidant with IC 50 values of low micromolar order, and the 4-hydroxyphenyl derivative 11 showed the most potent antioxidative activity with an IC 50 value of 0.32 μM, exhibiting approximately 5-fold more potent activity than α-tocopherol. The structure–activity relationship (SAR) studies of the antioxidative activity of these derivatives are presented.

A new series of diphenyl piperazine derivatives containing the phenyl substituted aminopropanol moiety, which were modified at sites between the diphenyl and piperazine moieties, was prepared and evaluated for dopamine transporter binding affinity with [(3)H]GBR12935 in rat striatal membranes. These synthesized compounds showed apparent dopamine transporter binding affinities (IC(50)

Aim: The purpose of the study is to investigate whether hypercoagulation in diabetes is observed not only in increased plasma levels of the coagulative factors but also in increased hepatic mRNA levels. Materials and methods: The age-related changes of coagulation factors were determined using KK and KK-Ay mice as a model for human type 2 diabetes mellitus. Expression of the α-, β- and γ-chains of fibrinogen-mRNA and prothrombin-mRNA from the mouse liver was examined by reverse transcription–polymerase chain reaction (RT-PCR). Results: Hemoglobin A1c (HbA1C), plasma fibrinogen, triglyceride and insulin levels increased apparently, especially in KK-Ay mouse at 4 months old. The mRNA levels of γ-chain of fibrinogen significantly increased at 3 and 4 months of age in both mice. The mRNA levels of α- and β-chains of fibrinogen and prothrombin were significantly increased in 4-month-old KK-Ay mouse. Conclusions: These results suggest that the increase in hepatic mRNA expression of coagulant factors contributes to the hypercoagulable state in type 2 diabetic mice.

We examined the properties of voltage-dependent Ca(2+) channels (VDCCs) mediating 1-methyl-4-phenylpyridinium (MPP(+))-evoked [3H]DA release from rat striatal slices. In some cases, the Ca(2+)-independent efflux of neurotransmitters is mediated by the high-affinity neurotransmitter-uptake systems. To determine whether such a mechanism might be involved in MPP(+)-evoked [3H]DA release. MPP(+) (1,10 and 100 microM) evoked the release of [3H]DA from rat striatal slices in a concentration-dependent manner. In the absence of Ca(2+), MPP(+) (10 and 100 microM)-evoked [3H]DA release was significantly decreased to approximately 50% of control (a physiological concentration of Ca(2+)). In the presence of Ca(2+), nomifensine (0.1,1 and 10 microM) dose-dependently and significantly inhibited the MPP(+)-evoked release of [3H]DA. Nomifensine (1 and 10 microM) also dose-dependently and significantly inhibited the MPP(+)-evoked release of [3H]DA under Ca(2+)-free conditions. MPP(+)-evoked [3H]DA release was partly inhibited by nicardipine (1 and 10 microM), an L-type Ca(2+) channel blocker. On the other hand, the N-type Ca(2+) channel blocker omega-conotoxin-GVIA (omega-CTx-GVIA) (1 and 3 microM) did not affect this release. omega-agatoxin-IVA (omega-Aga-IVA) at low concentrations (0.1 microM), which are sufficient to block P-type Ca(2+) channels alone, also had no effect. On the other hand, MPP(+)-evoked [3H]DA release was significantly decreased by high concentrations of omega-Aga-IVA (0.3 microM) that would inhibit Q-type Ca(2+) channels. In addition, application of the Q-type Ca(2+) channel blocker omega-conotoxin-MVIIC (omega-CTx-MVIIC) (0.3 and 1 microM) also significantly inhibited MPP(+)-evoked [3H]DA release. These results suggest that MPP(+)-evoked [3H]DA release from rat striatal slices is largely mediated by Q-type Ca(2+) channels, and the Ca(2+)-independent component is mediated by reversal of the DA transport system.

We have previously identified 204 partial cDNA fragments (ADRG1–204) as antidepressant related genes/expressed sequence tags. Then, we developed our original cDNA microarrays, on which the 194 clones out of ADRG1–204 were spotted. With this ADRG microarray, we found that the expression of a spot, ADRG55, which representing cysteine string protein (CSP), was significantly increased in rat brain after chronic treatment with a selective serotonin reuptake inhibitor, sertraline. In the present study, reverse transcription-polymerase chain reaction analysis confirmed the induction of CSP at mRNA levels in rat frontal cortex after chronic treatment with two different classes of antidepressants, imipramine or sertraline. Western blot analysis also revealed that CSP-immunoreactivity was increased after antidepressant treatment. In conclusion, our data suggest that CSP is one of the common functional molecules induced after chronic antidepressant treatment.

We examined the characteristics of dopamine (DA) uptake and its regulation by neurotrophic factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) in cultured rat astrocytes. In the presence of inhibitors of monoamine oxidase (MAO) and catechol-O-methyl-transferase (COMT), astrocytes took up DA by Na + -dependent and Na + -independent mechanisms that were sensitive to a reduction in temperature. The Na + -dependent and Na + -independent components increased linearly with increasing [ 3 H]DA concentration (1–1000 μM), and showed no saturation. Na + -dependent DA uptake was significantly inhibited by ouabain, a Na + -K + ATPase inhibitor. In bFGF-treated astrocytes, [ 3 H]DA uptake increased in a time-dependent manner until 48 h, and declined after 72 h in both the presence and absence of Na + . In EGF-treated astrocytes, [ 3 H]DA uptake increased in a time-dependent manner until 72 h in both the presence and absence of Na + . This enhancement of DA uptake induced by EGF or bFGF was significantly inhibited when the cells were cultured with actinomycin D, cycloheximide, or brefeldin A. Actinomycin D and brefeldin A also significantly inhibited the basal uptake of [ 3 H]DA into astrocytes. These results suggest the existence of Na + -dependent and Na + -independent DA uptake in cultured rat astrocytes, and that EGF or bFGF might stimulate the expression and translocation of the extraneuronal DA transporter.

The effects of Sasa senanensis Rehder extract (SE) by alkaline hydrolysis on histamine release from rat peritoneal exudate cells (PECs) were examined. Preincubation with SE for 5 min suppressed calcium-ionophore A23187-induced histamine release in a concentration-dependent manner. A23187 evoked a quick increase in cytoplasmic free calcium ([Ca2+]i) levels in the presence of extracellular calcium. Preincubation with SE also had an inhibitory effect on calcium influx increases induced by A23187. These results indicate that SE prevents degranulation from rat PECs by inhibiting [Ca2+]i level elevation.

We investigated the effects of glutamate on cell proliferation and the expression of basic fibroblast growth factor (bFGF) and its receptor (FGF-R1) mRNA in cultured rat retinal pigment epithelial (RPE) cells. The number of primary RPE cells was significantly higher after treatment with 0.2 to 1.0 mM glutamate (maximum at 1.0 mM) for 7 days than in controls. Glutamate-stimulated cell proliferation was abolished by (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), but not by 6,7-dinitroquinoxaline-2,3-dione or l (+)-2-amino-3-phosphonopropionic acid. Proliferation was increased to a similar extent by N-methyl- d -aspartate (NMDA), but not by kainate, α-amino-3-hydroxy-3-methyl-4-isoxazolepropionic acid or trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid. NMDA-receptor-like immunoreactivity was detected in most cells cultured. Treatment of cells with glutamate increased the level of bFGF mRNA and, to a lesser extent, that of FGF-R1 mRNA, which peaked 2 and 4 days, respectively, after glutamate was added. The increase in bFGF mRNA induced by glutamate was inhibited by MK-801. These findings suggest that glutamate might stimulate proliferation of RPE cells through activation of NMDA receptors and expression of bFGF and further suggest that glutamate may be involved in the proliferative changes of RPE cells in retinal wound healing.

We investigated the behavioral changes induced by mild stress in animals that may be relatively susceptible to a depressive-like state, the Fischer 344 rat strain. The mild stress of repeated handling and intraperitoneal (IP) injections with saline (2 ml/kg, twice a day for 14 days) elicited a moderate suppression of body weight gain, a decrease in open field activity, and a prolonged immobility during the tail suspension test in Fischer 344 rats compared with Sprague–Dawley rats. Chronic treatment of Fischer 344 rats with imipramine (10 mg/kg IP, twice a day for 14 days) effectively suppressed open field activity and prolonged immobility. These results suggest that repeated saline injections may be a mild stressor in these rats. In the Fischer 344 strain, which may be vulnerable to the effects of mild stressors, repeated saline injections might induce a depressive-like state and could presumably represent an experimental model for depression.

We investigated the effects of citalopram, a selective serotonin reuptake inhibitor (SSRI), using an animal model for a depressive state. In Fischer 344 rats, known as emotional animals, repeated stress by twice-daily intraperitoneal (i.p.) saline injections for 14 days elicited a depressive state characterized by a decreased open-field activity and a prolonged immobility during the tail-suspension test. Concomitantly, suppression of norepinephrine (NE)-induced cAMP formation was found in the cerebral cortical slices of the stress-exposed rats without changes in adrenergic alpha 1- or beta-receptors. The difference in cAMP formation between the intact and the stress groups was totally abolished under the blockade of the alpha 1-receptor system or by the stimulation with isoproterenol or forskolin, whereas the suppressed response in the stress group was also observed in combination with isoproterenol and phenylephrine. From these results, we confirmed that the potentiation of the beta-receptor-stimulated cAMP formation by the alpha 1-receptor is attenuated following repeated stress. Chronic i.p. administration of citalopram dissolved in saline improved both the suppressed open-field activity and the prolonged immobility in the tail-suspension test. The animals treated with citalopram exhibited a comparable alpha 1-potentiation effect as observed in the intact rats. However, another SSRI, paroxetine, was less effective on the attenuation of the alpha 1-potentiation in spite of its behavioral improvement in the depressive state. These findings suggest that citalopram has a protective effect against the repeated stress-induced depressive state by mechanisms besides the serotonin reuptake inhibition.

Effects of Ca2+ on [3H]5-hydroxytryptamine (5-HT) uptake into rat cortical synaptosomes were studied. The uptake was enhanced in the presence of Ca2+ in Krebs-Ringer medium and the uptake at 0.3-5 mM Ca2+ was 2.4-2.7 times greater than that observed in the absence of Ca2+. The maximal increase at the concentration of 1 mM Ca2+ was achieved after 2 min preincubation. Ca(2+)-dependent enhancement of the [3H]5-HT uptake reflected an increase in Vmax of the uptake process. However, Kd and Bmax values for [3H]paroxetine were not significantly changed in the presence of 1 mM Ca2+ compared with Ca(2+)-free condition. On the other hand, uptake was still enhanced after synaptosomes were washed with Ca(2+)-free after preincubation with 1 mM Ca2+. Staurosporine (a protein kinase C inhibitor) and wortmannin (a myosin light chain kinase inhibitor) did not affect Ca(2+)-dependent enhancement of the uptake, whereas 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazin e (KN-62, an inhibitor of Ca2+ /calmodulin-dependent kinase II) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7, a calmodulin antagonist) significantly reduced it. Moreover, L-type, but not P- or N-type, voltage-dependent Ca(2+)-channel blockers suppressed enhancement of the uptake. These results indicate that Ca(2+)-dependent enhancement of [3H]5-HT uptake is mediated by activation of calmodulin-dependent protein kinases, suggesting a possibility of calmodulin-dependent regulation of in vivo 5-HT uptake.

We investigated the behavioral and neurochemical features of Fischer 344 strain rats in which a depressive state was induced by repeated handling and saline injections as a mild Stressor. The repeated intraperitoneal injections of saline (2 ml/kg, twice a day for 14 days) elicited a moderate suppression of body weight gain, a decrease in their open field activity and a prolonged immobility in the tail suspension test. In the stress-exposed rats, the tissue content of norepinephrine (NE) was increased in the cerebral cortex and hypothalamus, whereas that of dopamine or serotonin was not affected. Although the stress exposure did not affect the binding properties of either the alpha-1 or beta adrenoceptors, it suppressed cAMP formation stimulated by NE, but not by isoproterenol or forskolin, in the cerebral cortical slices. In the presence of prazosin or phorbol ester, the difference in NE-stimulated cAMP formation between the control and the stress groups was totally abolished. Phenylephrine enhanced isoproterenol-stimulated cAMP formation in the control but not in the stress group. From these results, it is postulated that the alpha-1 potentiation of beta adrenoceptor-stimulated cAMP formation was attenuated in the stress group. These findings suggest that the manipulation of mild Stressor with repeated handling and saline injections to Fischer 344 rats elicits a depressive state characterized by the behavioral changes and the attenuated alpha-1 potentiation in the cerebral cortex, and that this manipulation might be available for the study of the stress-induced depressive state as a generally acceptable mild stress model.

In vivo central effects of some dopamine uptake inhibitors were evaluated in both brain microdialysis and behavioural studies in rats, and compared with their in vitro affinities to dopamine uptake sites. IC50 values of GBR12909 (1-[2- bis(4-fluorophenyl)methoxy]ethyl]-4-(3- phenylpropyl)piperazine), diclofensine, mazindol, amfonelic acid and nomifensine for inhibiting 1 nM [3H]GBR12935 (1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine) binding to rat striatal membrane were 7.0, 36, 81, 187 and 290 nM, respectively. In the brain microdialysis study, dopamine levels in the striatal dialysates were increased to 16.3- (GBR12909), 14.1- (nomifensine), 4.8- (diclofensine) and 1.9-fold (amfonelic acid) the respective basal levels 40-60 min after i.p. administration (0.1 mmol/kg) and thereafter decreased slowly but remained at the elevated levels for a further 3 h, while mazindol gradually increased dopamine levels though less pronouncedly than others (1.7-fold 200 min after administration). Remarkable and comparable stereotyped behaviours (licking and forepaw treading) were continuously observed at least for 3 h after administration of GBR12909, nomifensine and amfonelic acid, while stereotypies induced by diclofensine and mazindol were moderate and marginal, respectively. In vivo potencies of dopamine uptake inhibitors to increase the extracellular dopamine levels in the striatum tended to correlate with their in vitro affinities to dopamine uptake sites except in the case of nomifensine, and correlated significantly with their potencies to induce stereotyped behaviours except in the case of amfonelic acid. Based on these findings, pharmacological characteristics of these dopamine uptake inhibitors are discussed.

1. Enzyme properties of monoamine oxidase (MAO) in the frontal cortex and liver of the gerbil were investigated using 5-hydroxytryptamine (5-HT), benzylamine (Bz) and tyramine (Tyr) as substrates. 2. The Km values of MAO towards the three substrates were almost similar to the values in other species. The Vmax value of MAO towards Bz was much lower than the value towards 5-HT. 3. In the inhibition studies with selective MAO-A and MAO-B inhibitors, clorgyline and deprenyl, deamination of 5-HT, Bz and Tyr in both tissues was induced by MAO-A alone, MAO-B alone and both forms of the enzyme, respectively, indicating the same substrate specificity as that in rats. 4. The apparent proportion of MAO-A to MAO-B activities in the gerbil liver was approximately 6:4, whereas MAO-A in the frontal cortex of the gerbil was exclusively predominant, consistent with the previous data in the golden hamster which belongs to the same family as the gerbil.

The effect of a single treatment of electroacupuncture (Acu) at early or late stages of the efferent phase on 2, 4, 6-trinitrochlorobenzene (TNCB)-induced delayed type hypersensitivity (DTH) was studied in intact and hypophysectomized (HPX) mice. Acu (2.5 Hz, 15 min) applied to the acu-point equivalent to GV4 at 0, 3, 18 or 21 h after TNCB challenge induced significant suppression (45–73%) of the maximal extent of ear swelling at 24 h after TNCB challenge. An immunosuppressive and antiinflammatory drug, prednisolone 10 mg/kg i.p., also suppressed the DTH to the same extent. Pretreatment with intracisternal injection of naloxone hydrochloride (2 μg) significantly blocked the Acu-evoked DTH suppression when Acu treatment was done at 0 or 3 h. On the contrary, naloxone did not block the effect of Acu treatment given at 21 h. In order to examine the potential involvement of the pituitary in the suppression of DTH by Acu, the DTH reaction was examined in HPX mice. Acu failed to produce suppressive response in the HPX mice unless given at 0 h. These findings indicate that Acu treatment at acu-point GV4 during the efferent phase of induced DTH can suppress the DTH through central opioidergic or nonopioidergic systems. The pituitary is apparently pivotal in this immunosuppression and it is suggested that the DTH suppression by Acu may be mediated via activation of the neuroendocrine system.

We studied the mechanisms of hypolipidemic effects of NIP-200 (3,5-dimethyl-4,6-diphenyltetrahydro-2H-1,3,5-thiadiazine- 2-thione), a potent hypolipidemic compound, in cholesterol biosynthesis in the liver, cholesterol absorption in small intestine, and cholesterol catabolism and excretion in rats. NIP-200 did not reduce cholesterol biosynthesis and had no effects on cholesterol absorption in the small intestine. In the cholesterol catabolism and excretion, NIP-200 induced increases in cholesterol and bile acids levels in the bile and acidic steroids in the feces, and it enhanced cholesterol 7 alpha-hydroxylase activity in the liver. These results suggest that NIP-200 increases the synthesis of bile acids as a result of the activation of cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme in the conversion of cholesterol to bile acids. Therefore, it is considered that one of the probable mechanisms of the serum total cholesterol lowering action of NIP-200 involves the enhancement of catabolism and excretion of cholesterol in the liver.

We investigated the changes in striatal monoaminergic functions, focusing on the release and metabolism, in a cerebral ischemic model induced by a 5-min bilateral occlusion of the carotid arteries (BOCA) and reperfusion in anesthetized gerbils. In the microdialysis study, the striatal extracellular level of dopamine (DA) markedly increased (144-fold) immediately after BOCA. Although norepinephrine (NE) and 5-hydroxytryptamine (5-HT) could not be detected in the dialysates throughout the baseline period, they increased to detectable levels after BOCA. On the contrary, the tissue contents of NE and 5-HT decreased or tended to decrease up to 4 hr following reperfusion. Striatal DA contents did not show any changes in the early period after ischemia-reperfusion and slightly increased at 4 hr or later. Tissue contents of 3-methoxytyramine (3-MT), a metabolite of DA by catechol-O-methyltransferase (COMT), increased 0 and 5 min after reperfusion. Normethanephrine (NMN), which is a metabolite of NE by COMT, also increased not only 5 min after but also up to 4 hr after ischemia-reperfusion, indicating a sustained increase in NE release. These results suggested that the neuronal activity of NE, which is supposed to exert a protective effect on ischemic damage, was enhanced for a longer period than that of DA after transient ischemia.

To investigate in vivo effects of energy failure on functions of dopaminergic neurons, we administered 0 (control), 0.2, 1 or 2 mM sodium cyanide (NaCN) dissolved in Ringer's solution for 60 min into the rat striatum through a brain microdialysis membrane. During NaCN perfusion, a transient and concentration-dependent increase in dopamine (DA) levels in the dialysate was observed. The maximum DA level during 2 mM NaCN perfusion was found to be 63-fold higher than the control levels. Leves of 3,4-dihydroxyphenylacetic acid and homovanillic acid were continuously lowered during and after NaCN perfusion. These data suggest that suppression of ATP production by NaCN induces an abrupt and remarkable increase in dopamine release from the nerve terminal in the striatum.

We reported previously that electroacupuncture (Acu) at an acu-point equivalent to GV-4 in mice either enhanced or suppressed the delayed type hypersensitivity (DTH) to 2,4,6-trinitrochlorobenzene (TNCB, picryl chloride) depending on the time of treatment. We report here the suppression of the efferent phase of DTH to TNCB by Acu in mice. In 7- to 9-week-old male BALB/c, C57BL/6 and ddY mice, significant suppression of the DTH was observed when Acu had been applied once per day for three consecutive days before TNCB challenge. When Acu had been applied a single, significant suppression also occurred. Application to another point (at a middle area of the femoral muscle) failed to suppress the DTH to TNCB. This Acu-evoked DTH suppression was blocked dose-relatedly by pretreatment of systemic naloxone hydrochloride, indicating that opioid receptor-mediated mechanisms are involved in this immune response.

Brain lipid composition was studied in thiamine deficient rats treated with thiamine antimetabolites (oxythiamine: OT, and pyrithiamine: PT) and thiamine deficient diet (TDD). After intraperitoneal injection of OT (40 mg/kg/day) or TDD feeding for 6 days, body weight gain decreased. However, the PT (500 micrograms/kg/day) treated rats or the pair fed control (PFC: TDD + thiamine of 5 mg/kg, i.p.) showed no decrease in body weight gain compared with the regular diet control (C). Brain lipid levels (total lipid, total cholesterol, triglyceride, phospholipid, sphingomyelin and cerebroside) were examined in four brain regions (cerebral cortex, subcortical structure, brain stem and cerebellum). Total lipid level increased in four regions in OT or TDD treated rats, but total lipid level in the cerebellum in PT treated rats decreased. Total cholesterol level increased in all treated rats, while the triglyceride level in the brain stem decreased dramatically in OT or TDD treated rats. Cerebroside levels of four regions in the PT, OT or TDD group remarkably decreased, and PFC rats showed a significant improvement of the decrease in cerebroside level. It is conceivable that these changes in brain lipid composition provided some clues for the histological and morphological changes of the brain as manifested by the myelin degradation in acute thiamine deficiency.

Type 1 ryanodine receptor (RyR1) is a Ca2+ release channel in the sarcoplasmic reticulum and plays a pivotal role in excitation-contraction coupling in skeletal muscle. RyR1 is the major target for muscle diseases, e.g., malignant hyperthermia (MH) and central core disease (CCD). To date, over 200 mutations have been identified in the RyR1 gene of MH and CCD patients. It is widely believed that MH and CCD mutations cause hyperactivation of the Ca2+-induced Ca2+ release (CICR), resulting in abnormal Ca2+ homeostasis in skeletal muscle. CICR shows biphasic Ca2+ dependence, thus the activity can be determined by three parameters: sensitivity to activating Ca2+, sensitivity to inactivating Ca2+, and the gain (i.e., peak activity). However, it remains unclear how the disease-associated mutations affects these parameters. In this study, we expressed several RyR1 channels carrying different MH/CCD mutations at amino-terminal region in HEK cells and tested their CICR by live-cell Ca2+ imaging and [3H]ryanodine binding. Our results suggest that the amino-terminal disease-associated mutations divergently affects the parameters of CICR depending on the sites for mutation. The underlying molecular mechanism will be discussed.

The type 1 ryanodine receptor (RyR1) is a Ca2+ release channel found in the sarcoplasmic reticulum of skeletal muscle and plays a pivotal role in excitation-contraction coupling. The RyR1 channel is activated by a conformational change of the dihydropyridine receptor upon depolarization of the transverse tubule, or by Ca2+ itself, i.e., Ca2+-induced Ca2+ release (CICR). The molecular events transmitting such signals to the ion gate of the channel are unknown. The S4-S5 linker, a cytosolic loop connecting the S4 and S5 transmembrane segments in six-transmembrane type channels, forms α-helical structure and mediates signal transmission in a wide variety of channels. To address the role of S4-S5 linker in the RyR1 channel gating, we performed alanine substitution scan of amino (N)-terminal half of the putative S4-S5 linker (Thr4825-Ser4829) that exhibits high helix probability. The mutant RyR1 was expressed in HEK cells and the CICR activity was investigated by caffeine-induced Ca2+ release, single-channel current recordings, and [3H]ryanodine binding. Four mutants (T4825A, I4826A, S4828A and S4829A) had reduced CICR activity without changing the Ca2+ sensitivity, whereas the L4827A mutant formed a constitutive active channel. T4825I, a disease-associated mutation for malignant hyperthermia, exhibited enhanced CICR activity. An α-helical wheel representation of the N-terminal S4-S5 linker provides a rational explanation to the observed activities of the mutants. These results suggest that N-terminal half of the S4-S5 linker may form α-helical structure and play an important role in the RyR1 channel gating.