Methamphetamine (MA) addicts have been reported to reveal a deficit in cognitive performance, which is related to the function of the prefrontal cortex. Although it is well known that the medial prefrontal cortex (mPFC) in rats, which is crucial for cognitive function, shows long-term potentiation (LTP) in the projections from the hippocampus, there is no study on the influence of MA on synaptic plasticity of the hippocampal-mPFC pathway. In the present experiments, using sodium pentobarbitone-anesthetized rats, we investigated the effect of repeated MA treatment (4 mg/kg, 4 days, i. p.) on hippocampus-induced LTP in the mPFC. In MA-treated rats, LTP in the hippocampal-mPFC pathway was occluded compared with saline-treated rats. To further investigate this MA-induced reduction of LTP in the hippocampal-mPFC pathway, we administered D1 and D2 antagonists prior to MA injections. The MA-induced occlusion of hippocampal-mPFC LTP was prevented by D1 but not D2 antagonists. These findings suggest that D1 but not D2 receptors have a significant role in the MA-induced deterioration of synaptic plasticity in the hippocampal-mPFC circuits. (This work was supported by a grant from the Japan Health Sciences Foundation.) [Jpn J Physiol 54 Suppl:S248 (2004)]

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Serum albumin can be classified as either reduced albumin (Alb-SH) or oxidized albumin (Alb-SO). Alb-SH decreases with aging and in renal disease. In hemodialysis patients, Alb-SO is increased before dialysis, but during hemodialysis, Alb-SO decreases as it is converted to Alb-SH. At present, the only available assay for Alb-SH has been HPLC, thus limiting its use in medical technology. Therefore, serum total SH (T-SH) was measured by a DTNB assay instead of Alb-SH.In this study, we measured T-SH in hemodialysis patients before and after dialysis. Total protein, albumin, BUN, and creatinine were also measured and compared with T-SH. With hemodialysis, T-SH increased, while BUN and creatinine decreased. There were significant inverse correlations between changes in T-SH and BUN, and between changes in T-SH and creatinine. Our findings suggest that measurement of T-SH may be a useful parameter in patients undergoing hemodialysis. [Jpn J Physiol 54 Suppl:S249 (2004)]

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Various anti-allergic drugs are used for an allergic disease for the improvement and the reduction of allergic symptoms in clinical. To evaluate the effects of azelastine hydrochloride on the IgE-producing hybridoma cells, we asked whether or not azelastine hydrochloride decreases Cε mRNA level and the level of IgE FE-3 secreted in the cultured medium of IgE-producing hybridoma cells, FE-3. FE-3 cells were treated with various concentrations of azelastine hydrochloride for 24 h, and then re-cultured for 0.5 to 3 h in the growth medium after the cell number was adjusted to be equal among samples. Cε mRNA levels of FE-3 cells were determined using Northern blot analysis and IgE FE-3 levels in cultured medium were determined using IgE-capture ELISA. It was observed that the expression levels of Cε mRNA were significantly lower at 1 h after re-start of culture in the 10⁻⁵ M of azelastine hydrochloride-exposed group than in the control group. IgE FE-3 levels in cultured medium were significantly lower at 2 and 3 h after re-start of culture in the 10⁻⁵ M of azelastine hydrochloride-exposed group than in the control group. The electrophoretic mobility shift assay presented the possibility that the decrease of Cε mRNA level as shown here may be, at least in part, through the reduction of NF-κB DNA binding activity on the treatment with azelastine hydrochloride. These findings suggest that azelastine hydrochloride might exert its anti-allergic effect through the direct inhibition of IgE mRNA expression. [Jpn J Physiol 54 Suppl:S249 (2004)]

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We previously observed, under a video-enhanced contrast-differential interference contrast (VEC-DIC) microscope, that glutamate induces mitochondrial dysfunction and nuclear granulation within 20 min in neurons. The granulation corresponds to DNA fragmentation following the increase in nucleoplasmic Ca²⁺ concentration ([Ca²⁺]_n). In the present study, to determine the role of mitochondria in developing nuclear granulation, we examined, in cultured hippocampal neurons, the morphological changes and the increase in [Ca²⁺]_n associated with nuclear changes following an application of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP). The morphological changes were observed with a VEC-DIC microscope, and [Ca²⁺]_n assessed with fluo-3 under a confocal laser microscope. We observed that: 1) FCCP (10 μM) induced the nuclear changes identical to those induced by glutamate; 2) FCCP persistently increased [Ca²⁺]_n before inducing the nuclear changes; 3) the removal of extracellular Ca²⁺ did not affect these processes; and 4) following FCCP exposure inositol trisphosphate (IP₃) concentration in the neuron increased, which was detected with green fluorescent protein that translocates from the plasma membrane to the cytoplasm when IP₃ increases. We conclude that mitochondrial dysfunction stimulates the IP₃ pathway, increased [Ca²⁺]_n, and induced rapid DNA fragmentation, which aggravates the process of glutamate excitotoxicity. [Jpn J Physiol 54 Suppl:S249 (2004)]

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Excitotoxic neuronal death induced by glutamate is generally divided into two types; one is acute and the other is delayed. The mechanisms underlying the delayed neuronal death involve apoptotic process. Since caspase-3 is a central downstream effector of caspase cascade executing the apoptosis program, glutamate may activate caspase-3. However, the time-course of activation remains unclear. In the present study, we examined the effects of brief (20 min) and continuous exposure to glutamate on the activation of caspase-3 in cultured rat hippocampal neurons. Neurons were prepared from 1-day-old rats. Necrotic and apoptotic cells were detected with Annexin-V-FITC and propidium iodide, respectively. Morphological changes in the neurons were observed with a video-enhanced contrast-differential interference contrast microscope. The activations of caspase-3 were analyzed by fluorescence imaging with a confocal laser microscope using a cell-permeable caspase-3 substrate FAM-DEVD-FMK. Our results showed that 20-min-exposure to glutamate induced delayed neuronal death and activated caspase-3 significantly in 6 h in a dose-dependent manner. Interestingly, the continuous exposure for 1 h, which induced necrosis, also significantly activated caspase-3. Our data indicated that caspase-3 pathway is activated in the process of acute as well as delayed neuronal death induced by glutamate. Even in the process of necrosis, apoptotic signal is already activated but covered with the rapid cell death. [Jpn J Physiol 54 Suppl:S249 (2004)]

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We have been studying the mechanisms underlying motor hyperactivity caused by the deficit in dopaminergic neurons. In the present study, we examined the effects of endocrine disruptors (EDs) on the central nervous system. EDs are mainly derived from industrial products and are detected in the human umbilical cord and cord serum. Male Wistar rats received intracisternal injection of EDs at 5 days of age. Some phenols and phthalates caused a significant increase in spontaneous motor activity in both the dark and light phases at 4 weeks of age, while 6-hydroxydopamine (6-OHDA) induced hyperactivity in the dark phase. Bisphenol A, octylphenol and p-nonylphenol diminished tyrosine hydroxylase (TH) immunoreactivity in the midbrain. Gene expression profiles determined by DNA array in the midbrain and striatum varied with EDs and differed from that of 6-OHDA. Neonatal treatment with EDs may affect several factors including dopaminergic neurons. We further examined the effects of EDs injected into the unilateral substantia nigra in 9-week-old rats. Circling behavior was found towards the side ipsilateral to ED injection after methamphetamine treatment, and TH immunoreactivity was attenuated on the injection side. These results suggest that neonatal exposure to EDs may provide an animal model of developmental disorders, such as attention-deficit hyperactivity disorder that show clinical variation. Treatment with EDs in adulthood may also generate an animal model of neurodegenerative disorders, such as Parkinson's disease. [Jpn J Physiol 54 Suppl:S250 (2004)]

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Statins, HMG CoA reductase inhibitors, have pleiotropic effects on vascular diseases. We therefore investigated whether simvastatin ameliorates the development of pulmonary hypertension(PH) and vascular changes in the monocrotaline(MCT)-treated rats. Methods: Rats treated with a single subcutaneous injection of MCT(60mg/kg) or saline, were injected intraperitoneally with simvastatin(10mg/kg) or saline once daily from day –3 to day 16. At the end of experiment, awake mean pulmonary arterial pressure was determined through pulmonary artery catheter. The heart was dissected to weigh the right ventricle, and the lungs were obtained for vascular mophometric analysis. To correlate VCAM1 expression to the vasculoprotective effects of simvastatin, immunohistochemical study was performed. Results: MCT rats developed PH, right ventricular hypertrophy(RVH) and pulmonary vascular disease, which was accompanied by upregulation of VCAM1 expression. Simvastatin decreased PH , RVH, pulmonary arterial wall thickness and the proportion of muscular arteries in the peripheral arteries, which was associated with suppression of VCAM1 expression. Conclusion: Simvatatin ameliorated the vascular changes associated with PH in rats. These findings suggest that simvastatin could be a new therapeutic agent against PH at least in part by modifying oxidant-related signaling pathways. [Jpn J Physiol 54 Suppl:S250 (2004)]

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Protein transduction therapy using poly-arginine peptide can deliver biologically active proteins, peptides and compounds. We showed that 11 poly-arginine fused p53 protein (11R-p53) effectively penetrates across the plasma membrane and inhibits the proliferation of cancer cells. However, the intracellular half-life of the delivered protein is less than 24 h. Therefore, development of a protein therapy to transduce stable protein or a method to stabilize the delivered protein is important to consider clinical trial of protein therapy. Here, we generated two kinds of p53 mutants,which were resistant to Mdm2-mediated ubiquitination and the expressions were more stable than wild-type 11R-p53. Moreover, the mutant 11R-p53s displayed a higher transcriptional activity and a more powerful inhibition of the proliferation of glioma cells compared with wild-type 11R-p53. These results suggest that long-expressing p53 mutant protein therapy may overcome the disadvantage of 11R-p53 and become a new effective cancer therapy. [Jpn J Physiol 54 Suppl:S250 (2004)]

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We reported that a novel protein, mast cell surface antigen-1 (MASA-1), was expressed specifically on human mast cells in the lung lesional tissues of chronic inflammatory diseases such as bronchiolitis obliterans organizing pneumonia (BOOP) and sarcoidosis. In this study, we investigated the expression of MASA-1 on mast cells by immunofluorescence method in bronchoalveolar lavage fluid (BALF) obtained from patients with respiratory diseases (n=72). Levels of SCF and TGF-β1 were measured by ELISA. MASA-1 was found to be expressed in mast cells in patients with BOOP, interstitial pneumonia (IP) and sarcoidosis, though it was not expressed in mast cells in BALF obtained from the lung without lesion in patients with lung cancer. The number of MASA-1 positive cells was significantly increased in patients with chronic inflammatory diseases such as BOOP and IP compared to patients with acute pneumonia and bronchiolitis. Moreover, levels of TGF-β1 and SCF in BOOP and IP were higher than in acute pneumonia and bronchiolitis. There were good correlations between the number of MASA-1 positive cells and levels of TGF-β1 and SCF (r=0.82, r=0.83). These results suggest that human MASA-1 is specifically expressed in mast cells in BALF of chronic inflammatory diseases and that its expression is induced by inflammatory cytokines. MASA-1 should be a marker for activated mast cells in chronic respiratory diseases. [Jpn J Physiol 54 Suppl:S250 (2004)]

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Although mast cells are pivotal effector cells in allergic inflammatory reactions, their role of mast cells in the onset and progression of atopic dermatitis is not fully understood. We have recently cloned a novel protein, mast cell surface antigen-1 (MASA-1), and reported that the MASA-1 expression is specifically induced by inflammatory cytokines such as TGF-β1 in chronic inflammatory lung diseases. In this study, the kinetics of mast cell increase in relation to MASA-1 expression was investigated in an animal model of atopic dermatitis. NC/Nga mice were repeatedly treated with 0.1% picryl chloride (PiCl) once a week according to the method reported by Matsuda et al. The scratching behavior was more frequently observed in PiCl-treated mice compared to non-treated mice (control), and the thickness of PiCl-treated ear gradually increased up to 2 months. The symptoms of dermatitis such as dry skin and flare in PiCl-treated mice were also increasingly developed. Histologically, infiltration of mononuclear cells was evident in the lesional tissues, and the number of mast cells was gradually increased. MASA-1 was specifically expressed on mast cells in the skin lesions of atopic dermatitis in PiCl-treated mice, while in the control mice few mast cells showed its expression. These results suggest that MASA-1 may be involved in the pathogenesis of atopic dermatitis. [Jpn J Physiol 54 Suppl:S251 (2004)]

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Argyrophilic dark neurons (DNs) reflect an early histopathological state of neuronal damage in various brain insults. In the present study, we examined the fate of DNs following two kinds of brain insults: an injection of ibotenic acid (IA) into the hippocampus and a stressful swimming exercise (SWIM). Wistar rats were unilaterally injected IA into the hippocampus CA1 or were made to swim in a pool for 3 h. Argyrophil III (DNs)-, activated caspase-3 immuno-, TUNEL- and H-E-, stainings and ultrastractural examinations were done after these insults. IA injection; typical DNs were densely detected around the injection site at 1-3 h after injection, and argyrophilic only in soma with pycnotic nucleus at 12-24 h. Caspase-3 activation was detected 3 h to 3 d, TUNEL positive cells were detected at 3-7 d after, and the decrease in cell number was detected. SWIM; no typical DNs, but "brown" neurons, were detected in the hippocampus, striatum, amygdala, cortex etc (most frequently in CA1 pyramidal cell layer) up to 3 days. A light activation of caspase-3 was detected but no TUNEL positive cells. EM study revealed that, after IA injection, silver particles aggregated diffusely in dendrites and cytoplasm. After SWIM, silver particles distributed mainly on mitochondria. These data suggest that DNs reflect a various degree of neuronal damages. [Jpn J Physiol 54 Suppl:S251 (2004)]

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In the dental or periodontal treatment, laser is commonly used to relieve pain. We have reported the direct effects of the Nd:YAG laser sweeping irradiation on nerve discharges of Xenopus tactile nerve fibers. Nerve discharges were reversibly suppressed with the sweeping irradiation. But the effect of sweeping irradiation is not clear. In this study, we tried to clarify the effect of sweeping irradiation by comparison with non-sweeping irradiation. Dorsal surface skin of Xenopus was removed together with fine tactile nerve bundles. Chinese ink was painted on a dissected fine nerve bundle. From this bundle, nerve discharges with tactile stimuli on the skin surface were recorded by a silver electrode, and counted by the spike counter. After non contact Nd:YAG laser sweeping irradiation or after non-sweeping irradiation, tactile responses were periodically recorded with paying enough attention to stimulate the same surface area under the dissection microscope. Results were followings. 1) With sweeping irradiation, nerve discharges were reversibly suppressed. 2) With non-sweeping irradiation, nerve discharges were suppressed within a few seconds. And it is very difficult to recover from the suppression. In most cases, the suppression was irreversible. These results show the efficacy of the sweeping irradiation. [Jpn J Physiol 54 Suppl:S251 (2004)]

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We have developed a new type of olfactory stimulator triggered by air pressure for use in recording of olfactory evoked potential (OEP). This olfactory stimulator is equipped with air regulators, tenperature controls, flowmeters, a digital pressure sensor, a moisture regulator, solenoid valves, one touch joints and Teflon tubing. Except the moisture regulator, all of the parts are contained within a steel box. The operative switches for olfactory stimulus (OS), air stimulus and air wash are set in the top panel, which includes a temperature display and power switch. The olfactory switch feeds clean air mixed with an odorant to the nose of the subject for OS, and the air switch feeds clean air only for control stimulus. A program was written and installed in a controller to operate the device. The airflow is set at the level of 7.0L/min and the air pressure is set at 0.065 Mpa at a room temperature of 24.5°C. In order to test the usefulness of this olfactory stimulator, we recorded OEP at Fz on the scalps of healthy volunteers who were administered amyl acetate odorant with the device. Impedances were kept below 2 Kω. Singals were collected with a band-pass of 0.05-30 Hz and digitized at a rate of 1024 Hz. Their OEP components contained a large negative deflection peak with a latency of approximately 600 msec. In conclusion, this olfactory stimulator device is effective for use in OEP recording. [Jpn J Physiol 54 Suppl:S252 (2004)]

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The present investigation attempted to clarify whether intensity of exercise influences functional sympatholysis during mild rhythmic handgrip exercise (RHG). In eleven subjects, we measured muscle oxygenation both exercising and non-exercising muscle in the same arm by near infrared spectroscopy (NIRS), heart rate and blood pressure. We obtained the total labile signal to assess a relative evaluation for muscle oxygenation by occlusion for 6 minutes at rest. Subjects performed RHG (20 times / minute) for 6 minutes at 10%, 20%, and 30% of maximal voluntary contraction (MVC) at random. We used nonhypotensive LBNP of -20 mmHg for 2 minutes to elicit muscle sympathetic nerve activity at rest and during RHG. LBNP caused a decrease of 16.4% (pre), 18.6% (post), respectively, in oxygenation at rest. The attenuation of decrease in oxygenation in non-exercising muscle to LBNP during RHG did not elicit. On the other hand, the decrease in oxygenation in exercising muscle attenuated progressively as exercise intensity increased, when LBNP was applied during exercise. The attenuation of decrease in oxygenation to LBNP during RHG at 20% and 30% exhibited differences from the rest (p<0.05). These findings indicate that an increase in the intensity of exercise cause functional sympatholysis during mild RHG. [Jpn J Physiol 54 Suppl:S252 (2004)]

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Leptin is known to regulate appetite and energy expenditure, Which may have a powerful influence on bone. There is some conflicting evidence that leptin may decrease bone formation via a central nervous system and may stimulate both bone formation and bone resorption via direct peripheral effect. We studied the relationship of effect of leptin and other factors, serum estrogen and treadmill running, on bone. Leptin receptor deficient Zucker (fa/fa) rats and lean controls were used.Twenty eight female Zucker (fa/fa) rats and twenty eight lean controls were assigned to four groups respectively: sham operated group, sham operated and exercise group. Ovariectomized group, ovariectomized and exercise group. Exercise groups were assigned to treadmill running (20 m/min), 30 min/day, 5 days/week, for 8 weeks. Right femur was used to measure bone strength determined by the three-point bending test. Bone mineral density of left femur was measured by dual energy X-ray system and that of tibia was measured by peripheral quantitative computed tomography (pQCT). In lean controls, especially, ovariectomized group, exercise increase bone strength. However, in Zucker (fa/fa) rat, bone strength was decreased in exercize group. Differences were not observed among the groups in BMD. We observed positive effect of exercise on bone in existence leptin signal, however, in Zucker (fa/fa) rats, exercise did not influence on bone. These result suggested that leptin could play important part in bone metabolism in rats. [Jpn J Physiol 54 Suppl:S252 (2004)]

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Hepatic stellate cells (HSCs) constitute liver sinusoids and have contractility, thereby regulating portal blood flow. Although it has been hypothesized that NO production by HSCs may also contribute to the regulation of portal circulation, there is no direct evidence for this novel function of HSCs, mainly because possible contamination of sinusoidal endothelial cells could not be avoided completely. Here we obtain the direct evidence of NO production in HSCs, using single-cell system andα-SMA as a marker of HSCs. Confocal images of immunostaining showed thatα-SMA-positive rat HSCs in primary culture expressed eNOS and caveolin-1, both of which were colocalized in the cell membrane at rest. In contrast, ATP and sphingomyelinase (SMase), which produces a lipid messenger, ceramide, induced the translocation of eNOS from caveolin-localizing cell membrane to the cytosol, as being compatible with the notion that eNOS activation requires its dissociation from caveolin, which binds and inhibits eNOS in the cell membrane at rest. Simultaneous fluorometric measurements of cytosolic Ca2+ concentration ([Ca2+]i) and NO production in single HSCs revealed that SMase induced NO production without [Ca2+]i elevation, whereas ATP elevated the both parameters. Western blotting showed the presence of eNOS, but not iNOS. Our results also indicate that the activation of eNOS in HSCs is mediated by not only a well-known Ca2dependent mechanism but also by a novel Ca2independent, probably ceramide-mediated, one. [Jpn J Physiol 54 Suppl:S252 (2004)]

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Acquired long QT syndrome is most often caused by block of cardiac HERG K⁺ channels by commonly used medications. We determined the structural basis for high-affinity block of HERG channels by class III antiarrhythmic agents, including dofetilide, E-4031, MS-551, amiodarone, dronedarone and bepridil. We mutated to alanine individual residues of the S6 transmembrane domain (L646-Y667) and the few residues of the pore helix (L622-V625) predicted to line the inner channel cavity based on homology with the solved crystal structure of the KcsA channel. Block of each mutant channel expressed in Xenopus oocytes was determined using a concentration of drug equal to 10-times the IC₅₀ value (2.2 μM dofetilide, 5.7 μM E-4031, 58 μM MS-551, 18 μM amiodarone, 28 μM drondedarone and 30 μM bepridil). Currents were measured during repetitive 5-s depolarizing steps to 0 mV, applied at 0.166 Hz. At these concentrations, each drug caused ~ 85% inhibition of wild-type HERG current and most mutant channels. Block of HERG current by dofetilide, E-4031 and MS-551 was less for 3 pore helix mutants (T623A, S624A and V625A) and 3 S6 domain mutants (G648A, F656A and V659A). Y652A and F656A mutation in the S6 domain and the mutations in the pore helix reduced block by most drugs. These results confirm that residues in the S6 domain and base of the pore helix form the drug binding site on the HERG channel, and the central importance of F656 and Y652. [Jpn J Physiol 54 Suppl:S253 (2004)]

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