Biomedical subjects
C Kubo
Publications and source records attributed to C Kubo.
Presynaptic 5-HT3 receptor-mediated modulation of synaptic GABA release in the mechanically dissociated rat amygdala neurons.
Nystatin-perforated patch recordings were made from mechanically dissociated basolateral amygdala neurons with preserved intact native presynaptic nerve terminals to study the mechanism of 5-HT3 receptor-mediated serotonergic modulation of GABAergic inhibition. The specific 5-HT3 agonist mCPBG (1 microM) rapidly facilitated the frequency of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) and this facilitation desensitized within 1 min. Tropisetron (30 nM), a specific 5-HT3 antagonist, blocked the mCPBG effect. mCPBG augmented mIPSC amplitude. However, no direct postsynaptic serotonergic currents were evoked by mCPBG. Neither GABA-evoked current amplitude nor the kinetics of individual GABAergic mIPSCs were affected by mCPBG. Therefore, the augmentation is unlikely to be due to postsynaptic effects evoked by mCPBG. At higher concentrations mCPBG produced shorter-duration facilitation of miniature events. While mCPBG increased the mIPSC frequency in calcium-containing solution with Cd2+, this increase was absent in Ca2+-free external solution. It appears that the Ca2+ influx through voltage-dependent calcium channels was not as crucial as that through 5-HT3 receptors for synaptic GABA release. When two pulses of mCPBG (each 1 microM, 1 min) were given, the response to the second pulse elicited full recovery when the interval between pulses was at least 9 min. Protein kinase A (PKA) activation by 8-Br-cAMP (300 microM) shortened and PKA inhibition by Rp-cAMP (100 microM) prolonged the recovery time. PKA activity did not affect the time course of fast desensitization. Our results suggest that a 5-HT3-specific agonist acts on presynaptic nerve terminals facilitating synaptic GABA release without postsynaptic effects. The facilitation requires calcium influx through presynaptic 5-HT3 receptors. PKA modulates the recovery process from desensitization of presynaptic 5-HT3 receptor-mediated regulation of synaptic GABA release.
Influence of short-term repeated fasting on the longevity of female (NZB x NZW)F1 mice.
Caloric restriction in rodents is well known to retard the rate of aging, increase mean and maximum life-spans, and inhibit the occurrence of many age-associated diseases. However, little is known about the influence of short-term repeated fasting on longevity. In this study, female (NZB x NZW)F1 mice were used to test the physiological effect of short-term repeated fasting (4 consecutive days, every 2 weeks). The results showed that fasting mice survived significantly longer than the full-fed mice, in spite of the fasting group having a heavier body weight than the control group. Mean survival times for fasting and control mice were 64.0+/-15.3 and 47.9+/-9.4 weeks, respectively. Short-term repeated fasting manipulation was also effective on the prolongation of life-span in autoimmune-prone mice.
Proteolysis and phosphorylation-mediated regulation of thrombin receptor activity in in situ endothelial cells.
The regulatory mechanism of thrombin receptor responsiveness in in situ endothelial cells was investigated by evaluating elevations of cytosolic Ca(2+) concentration ([Ca(2+)](i)) in fura-2-loaded porcine aortic valvular strips. Once stimulated with thrombin, endothelial cells did not respond to the second thrombin stimulation within 90 min. However, applying thrombin receptor activating peptide (TRAP7) at 15 min after the thrombin stimulation caused [Ca(2+)](i) elevation, which was smaller than that seen without preceding stimulation. After 90 min, response to TRAP7 recovered to the control level. When stimulated with TRAP7, the subsequent responses to thrombin and TRAP7 were attenuated at 15 min, and fully recovered after 90 min. Staurosporine partially prevented the TRAP7-induced desensitization. The recovery of responsiveness was inhibited completely by calyculin-A and partially by okadaic acid. Proteolysis and phosphorylation thus play an important role in thrombin receptor desensitization in in situ endothelial cells. Both cleaved and uncleaved receptors were desensitized through phosphorylation in part by staurosporine-sensitive kinase, and restored the responsiveness through dephosphorylation by type 1 phosphatase. The mechanism of regulation of thrombin receptor activity in in situ endothelial cells differed from those reported in cultured endothelial cells. We suggest that the cell-specific regulatory mechanism may be altered by culture conditions.
Stimulus-specific alteration of the relationship between cytosolic Ca(2+) transients and nitric oxide production in endothelial cells ex vivo.
1. To investigate the quantitative relationship between elevation in the intracellular Ca(2+) concentration ([Ca(2+)](i)) and nitric oxide (NO) production, the changes in [Ca(2+)](i) and NO production were determined in parallel, using fluorimetry of fura-2 and 2, 3-diaminonaphthalene, respectively, in endothelial cells ex vivo of pig aortic valves. 2. The extent of [Ca(2+)](i) elevation was quantitatively assessed by two parameters: the level of peak [Ca(2+)](i) elevation and the area under the [Ca(2+)](i) curve during treatment (the integrated [Ca(2+)](i) elevation). The amount of NO production was expressed as a percentage of that obtained with 10 microM ATP for 3 min. 3. ATP, bradykinin, thrombin, and ionomycin were used as stimulation to induce NO production, and all these caused [Ca(2+)](i) increases and NO production in a concentration-dependent manner. 4. The relationships between the peak [Ca(2+)](i) and NO production or between the integrated [Ca(2+)](i) elevation and NO production were well described by a straight line. However, the slope value of the linear relationship in both cases varied with the type of stimulation, with thrombin giving the greatest value, followed by ATP, bradykinin and ionomycin. 5. These data suggest that in endothelial cells ex vivo: (1) [Ca(2+)](i) elevation regulates NO production, but (2) the peak [Ca(2+)](i) elevation- or the integrated [Ca(2+)](i) elevation-NO production relationships varies depending on the type of agonists. Our results thus demonstrate the presence of the agonists-dependent modulation of the relationship between [Ca(2+)](i) elevation and NO production in endothelial cells ex vivo.
Dietary nucleic acids promote a shift in Th1/Th2 balance toward Th1-dominant immunity.
BACKGROUND: Dietary sources of nucleic acids and their relative components are known to affect host immune function; however, it has not yet been clarified whether such dietary nucleic acids influence the pathogenesis of allergic reaction. OBJECTIVE: The purpose of this study is to elucidate the effect of dietary nucleic acids on Th1/Th2 balance. METHODS: Both human flora-associated and specific pathogen-free BALB/c mice were maintained on either nucleic acid-free, or -supplemented diets. The effects of nucleic acids on both in vivo antibody levels and in vitro splenocyte cytokine production were compared using these mice. RESULTS: Supplementation of nucleic acids caused a reduction in the serum antibody levels of total IgM, IgG, IgG1, and IgE in the human flora-associated mice without affecting the composition of intestinal flora. In contrast, there was no significant difference of the serum IgG2a levels between nucleic acid-free and -supplemented mice. Such a phenomenon as that, the supplementation of dietary nucleic acids reduces the serum IgE or IgG1 levels, but not the IgG2a level, was also seen in the specific pathogen free mice. Moreover, when the mice were systematically challenged with ovalbumin, the supplementation of nucleic acids also suppressed the serum ovalbumin-specific IgE and IgG1 antibody levels as well as in vitro IL-4 and IL-10 secretion, while enhancing both the serum ovalbumin-specific IgG2a antibody levels and in vitro IFN gamma secretion. CONCLUSION: These results suggested that dietary nucleic acids may play an important role in promoting a shift in Th1/Th2 balance toward Th1-dominant immunity.
Induction of mammalian cell transformation and genotoxicity by 2-methoxyestradiol, an endogenous metabolite of estrogen.
2-methoxyestradiol (2-MeOE(2)) is an endogenous metabolite of 17beta-estradiol and a proposed inhibitor of tumor growth and angiogenesis. However, 2-MeOE(2) is also an inhibitor of microtubule assembly and other microtubule inhibitors, e.g. colcemid and diethylstilbestrol, induce aneuploidy and cell transformation in cultured mammalian cells. To assess the in vitro carcinogenicity and related activity of 2-MeOE(2), the abilities of this metabolite to induce cell transformation and genetic effects were studied simultaneously using Syrian hamster embryo (SHE) fibroblasts. Growth of these cells was reduced by treatment with 2-MeOE(2) at 0.1-1.0 microg/ml in a concentration-dependent manner. Treatment of SHE cells with 2-MeOE(2) at 0.3 or 1.0 microg/ml for 2-48 h also resulted in a concentration- and treatment time-related increase in the mitotic index and the percentage of multinucleated cells. Treatment with 2-MeOE(2) at 0.1-1.0 microg/ml for 48 h induced a statistically significant increase in the frequencies of morphological transformation of SHE cells in a concentration-dependent manner. A statistically significant increase in the frequencies of somatic mutations at the Na(+)/K(+) ATPase or hprt locus was also observed in cells treated with 2-MeOE(2) for 48 h at 0.1 or 0.3 microg/ml, respectively. Treatment of SHE cells with 2-MeOE(2) at 0.3 or 1.0 microg/ml for 24 h induced chromosome aberrations, mainly breaks, exchanges and chromosome pulverization. The incidence of chromosome aberrations was not affected by co-treatment with alpha-naphthoflavone, an inhibitor of 2-hydroxylase that inhibits oxidative conversion of 2-MeOE(2) to 2-hydroxyestradiol, but the incidence was slightly increased by co-treatment with L-ascorbic acid. Numerical chromosomal changes in the near diploid range and in the tetraploid and near tetraploid ranges were also detected in 2-MeOE(2)-treated cells. These findings indicate that 2-MeOE(2) has cell transforming and genotoxic activities in cultured mammalian cells and potential carcinogenic activity.
Changes in the leukocyte distribution and surface expression of adhesion molecules accompanied with hypothalamically induced restlessness in the cat.
One type of emotional behavior called restlessness occurs when the anteromedial hypothalamus is stimulated in cats. We examined the changes in the distribution and surface expression of adhesion molecules in leukocytes accompanied with restlessness. Mature female cats were used for this study. The cats were stimulated with 60 Hz sine wave train pulses (20-90 microA, 10 s in duration, at 5-min intervals) for 60 min. Samples of blood were collected from 30 min before stimulation up to several hours after the final stimulation. The number of granulocytes increased just after stimulation, while at the same time the expression of L-selectin decreased. On the other hand, the number of CD4+ and CD8+ T lymphocytes decreased at 1-2 h after the end of the stimulation, while the expression of L-selectin increased. In addition, the expression of LFA-1 and VLA-4 did not change. These data suggest that hypothalamically elicited restlessness is thus accompanied by a leukocyte distribution change, which might be mediated by changes in the expression of L-selectin on leukocytes. Plasma cortisol increased during stimulation in restlessness. However, during in vitro culture experiments, cortisol did not alter the expression of leukocyte L-selectin which thus indicated that cortisol does not directly affect the surface expression of L-selectin. These results thus suggest that hypothalamically induced restlessness is a useful stress model for psychoneuroimmunological studies.
Presynaptic serotonergic inhibition of GABAergic synaptic transmission in mechanically dissociated rat basolateral amygdala neurons.
1. The basolateral amygdala (ABL) nuclei contribute to the process of anxiety. GABAergic transmission is critical in these nuclei and serotonergic inputs from dorsal raphe nuclei also significantly regulate GABA release. In mechanically dissociated rat ABL neurons, spontaneous miniature inhibitory postsynaptic currents (mIPSCs) arising from attached GABAergic presynaptic nerve terminals were recorded with the nystatin-perforated patch method and pharmacological isolation. 2. 5-HT reversibly reduced the GABAergic mIPSC frequency without affecting the mean amplitude. The serotonergic effect was mimicked by the 5-HT1A specific agonist 8-OH DPAT (8-hydroxy-2-(di-n-propylamino)tetralin) and blocked by the 5-HT1A antagonist spiperone. 3. The GTP-binding protein inhibitor N-ethylmaleimide removed the serotonergic inhibition of mIPSC frequency. In either K+-free or Ca2+-free external solution, 5-HT could inhibit mIPSC frequency. 4. High K+ stimulation increased mIPSC frequency and 8-OH DPAT inhibited this increase even in the presence of Cd2+. 5. Forskolin, an activator of adenylyl cyclase (AC), significantly increased synaptic GABA release frequency. Pretreatment with forskolin prevented the serotonergic inhibition of mIPSC frequency in both the standard and high K+ external solution. 6. Ruthenium Red (RR), an agent facilitating the secretory process in a Ca2+-independent manner, increased synaptic GABA release. 5-HT also suppressed RR-facilitated mIPSC frequency. 7. We conclude that 5-HT inhibits GABAergic mIPSCs by inactivating the AC-cAMP signal transduction pathway via a G-protein-coupled 5-HT1A receptor and this intracellular pathway directly acts on the GABA-releasing process independent of K+ and Ca2+ channels in the presynaptic nerve terminals.
Differences between bulimia nervosa and binge-eating disorder in females with type 1 diabetes: the important role of insulin omission.
This study explored the differences between bulimia nervosa ("BN," n=22) and binge-eating disorder ("BED," n=11) in type 1 diabetic females and the factors most predictive of poor glycemic control in patients suffering from these disorders. These two groups and a control group without eating disorders (n=32) were compared across a number of demographic, psychological, and medical variables. BN manifested significantly more severe disturbances related to eating disorders, depression, anxiety, a higher rate of co-occurring mental disorders, and poorer psychosocial functioning compared with BED. BN also showed poorer glycemic control. Multivariate analysis indicated that higher serum glycosylated hemoglobin (HbA1c) levels were most associated with the presence of severe insulin omission in type 1 diabetic females with binge eating. Clinicians may be able to determine the psychological/medical severity of illness in these patients by identifying the presence of compensatory behaviors to prevent weight gain such as severe insulin omission, as described in the DSM-IV.
The restoration of the antitumor T cell response from stress-induced suppression using a traditional Chinese herbal medicine Hochu-ekki-to (TJ-41:Bu-Zhong-Yi-Qi-Tang).
We previously reported that restraint stress impairs the antitumor immune responses through its suppressive effect on the Th1-type cytokine production from CD4+ T cells. In this study, we investigated a potential of Hochu-ekki-to (TJ-41:Bu-Zhong-Yi-Qi-Tang) to restore stress-induced immunosuppression. The oral administration of TJ-41 was able to improve a decreased cellularity in the lymph node and spleen and to improve an inhibition of tumor-specific Th1-type cytokine production, both of which were induced by repeated restraint stress in tumor-bearing mice. The oral administration of TJ-41 also induced a partial recovery of the antitumor cytolytic activity in the stress-burdened tumor-bearing mice. More importantly, the growth of tumors in stress-burdened preimmunized mice was obviously inhibited by TJ-41, and resulted in tumor-free state in 75% of the mice. Regarding the mechanisms by which TJ-41 restored the antitumor responses in stress-burdened mice, we found that the serum levels of corticosterone and interleukin-12 were normalized by TJ-41. In addition, the expression of CD80 and CD86, which both decreased in the stress-burdened mice, was restored to the normal level by TJ-41. Taken together, our results indicate that the oral administration of TJ-41 is able to restore the antitumor T cell responses in stress-burdened tumor-bearing mice by normalizing the serum corticosterone, interleukin-12 and the expression of costimulatory molecules.
Prostaglandin E(2) has antinociceptive effect through EP(1) receptor in the ventromedial hypothalamus in rats.
The effects of microinjection of prostaglandin E(2) (PGE(2)) (50 fg-50 ng/0.2 microl) into the ventromedial hypothalamus (VMH) on nociception were studied using a hot-plate test in rats. Microinjection of PGE(2) (5-500 pg and 50 ng/0.2 microl) into the VMH significantly prolonged the paw-withdrawal latency on a hot plate 5 and 10 min after injection, respectively. Maximal prolongation was obtained 5 min after the injection of PGE(2) at 5 pg. Subsequently, to determine whether the PGE(2) receptor subtype EP(1) is involved in the PGE(2)-induced antinociceptive effect in the VMH, we observed the changes in nociception after intraVMH microinjection of SC19220, an EP(1) receptor antagonist, and 17-phenyl-omega-trinor PGE(2), an EP(1) receptor agonist. Simultaneous injection of SC19220 (150 ng) with PGE(2) (500 pg) into the VMH blocked the PGE(2)-induced prolongation of the paw-withdrawal latency. Moreover, an intraVMH microinjection of 17-phenyl-omega-trinor PGE(2) (500 pg) prolonged it. These results indicate that PGE(2) in the VMH has antinociceptive effect through its actions on EP(1) receptors in rats.
Esophageal motility and psychiatric factors in functional dyspepsia patients with or without pain.
Subtypes of functional dyspepsia (FD), including refluxlike dyspepsia, ulcerlike dyspepsia, dysmotility-like dyspepsia, and nonspecific dyspepsia, have been described and are widely used clinically. However, these symptom patterns often overlap, and the terms are insufficient for indicating all FD symptoms. In this study, we divided 71 FD patients into two groups: patients with or without pain. Group I, the pain dyspepsia group, included patients in whom the main symptoms were epigastralgia and/or chest pain. Group II, the painless dyspepsia group, included patients without pain, in whom the symptoms were nausea, vomiting, and heartburn. We examined the relationship between esophageal function and psychiatric factors in the test groups and compared them with a control group. Of the FD patients, 19.7% [8 (25%) of 32 group I patients, 6 (15.4%) of 39 group II patients] had esophageal motility disorders, such as nutcracker esophagus and diffuse esophageal spasm. The LES pressure of group I was higher than that of group II by esophageal manometry (P < 0.05). In 17 (53.1%) of 32 group I patients and 31 (79.5%) of 39 group II patients, psychiatric disorders (38.0% had depressive disorder and 21.1% had an anxiety disorder) were diagnosed following DSM III-R criteria. Group II tended to be more depressive than group I (P = 0.0508). Psychological assessment scores, STAI-I and STAI-II, were higher in groups I and II than in the control group (P < 0.001). Long-term distress, anxiety, and depression seem to influence the symptoms of FD patients. Esophageal dysmotility may be an important functional abnormality of FD.
Antidepressant treatment of patients with diffuse esophageal spasm: a psychosomatic approach.
The cause of diffuse esophageal spasm (DES) has not been clearly established, and effective treatment is lacking. To determine whether a psychosomatic approach can be effective in treating DES patients, nine patients and 26 healthy volunteers were studied. Esophageal manometry and psychological testing were performed in both groups. The psychological background of the DES patients was investigated. Psychiatric diagnoses were made according to the criteria outlined in the Diagnostic and Statistical Manual of Mental Disorders, 3rd edition, revised. The authors started psychosomatic treatment with isosorbide dinitrate for 1 month, then prescribed serotonin reuptake inhibitor antidepressants for an additional month. Anxiety and depression scores were substantially higher for the DES group than for the control group. Five of the nine DES patients (56%) were diagnosed as having major psychiatric disorders. Only one patient showed improvement with isosorbide dinitrate, and eight patients improved with antidepressants. These initial observations suggest that psychosomatic treatment with antidepressants may be effective in the treatment of DES.
Restraint stress-induced immunosuppression by inhibiting leukocyte migration and Th1 cytokine expression during the intraperitoneal infection of Listeria monocytogenes.
In this study, a murine model of Listeria monocytogenes infection was used to investigate effects of restraint stress (RST) on host defense. We observed that the L. monocytogenes infection as well as RST induced an elevation of endogenous corticosterone (CORT) levels and RST synergistically enhanced endogenous CORT levels during the listerial infection. RST suppressed the migration of leukocytes including macrophages, neutrophils, NK cells and lymphocytes into the peritoneal cavities after the intraperitoneal inoculation of L. monocytogenes. RST also suppressed the increase of the surface MHC class II antigen expression in both peritoneal macrophages and B cells during the listerial infection. Interestingly, gene expression of iNOS, MCP-1 (JE) and Th1-type cytokines including IFN-gamma and IL-12 was down-regulated but Th2-type cytokine (IL-4 and IL-6) gene expression in the PEC was rather up-regulated on day 7 after infection, indicating that Th2-type immune response is more resistant to the elevated endogenous CORT levels than Th1-type response. Treatment of mice with RU486, a glucocorticoid receptor antagonist, restored the immune responses suppressed by RST to their normal levels in the infected mice, suggesting that the RST-induced elevation of endogenous corticosterone levels is mainly responsible for the induction of the immunosuppressive events during L. monocytogenes infection.
Mechanism of endothelium-dependent relaxation induced by thrombin in the pig coronary artery.
The mechanism of thrombin-induced endothelium-dependent relaxation was investigated using fura-2 front-surface fluorometry. Thrombin induced an endothelium-dependent relaxation during U46619-induced contractions in pig coronary arterial strips. The relaxation consisted of two components: the early phasic component with a transient decrease in [Ca2+]i of smooth muscle and the subsequent sustained tonic component without [Ca2+]i decrease. The phasic relaxation was inhibited by a combination of N(omega)-nitro-L-arginine and K+-depolarization, while the tonic component was inhibited by either indomethacin or K+-depolarization. Thrombin induced a transient [Ca2+]i increase and nitric oxide (NO) production in pig aortic valvular endothelial cells, which expressed NO synthase as determined by reverse transcription and polymerase chain reaction. Thus, it was concluded that NO and hyperpolarizing factor were involved in the phasic component of thrombin-induced relaxation and that hyperpolarizing factor and prostacyclin were involved in the tonic component.