Takeshi Kawahara

Involvement of γ-aminobutyric acid neurotransmission in phencyclidine-induced dopamine release in the medial prefrontal cortex

The present study was designed to examine the possible involvement of γ-aminobutyric acid (GABA) neurotransmission in the mechanism of phencyclidine (1-(1-phenylcyclohexyl)piperidine; PCP)-induced dopamine release in the medial prefrontal cortex, using in vivo microdialysis in awake, freely moving rats. Local perfusion via the dialysis probe into the medial prefrontal cortex with PCP (100 and 500 μM) and dizocilpine ((+)-5-methyl-10,11-dihydroxy-5-H-dibenzo(a,d)cyclo-heptan-5,10-imine; MK-801, 10 and 50 μM), a selective non-competitive NMDA receptor antagonist, was found to increase extracellular dopamine levels. Co-perfusion with NMDA (1 mM) or the GABAA receptor agonist muscimol (50 μM) attenuated the effects of PCP (500 μM) and MK-801 (50 μM) on extracellular dopamine levels. The dopamine reuptake inhibitor nomifensine (50 μM) also produced an increase in extracellular dopamine levels in the medial prefrontal cortex, but this effect was not affected by co-perfusion with muscimol (50 μM). On the other hand, local perfusion with PCP (100 and 500 μM) and MK-801 (10 and 50 μM), but not nomifensine (50 μM), reduced extracellular GABA levels in the medial prefrontal cortex. Co-perfusion with NMDA (1 mM) reduced the effects of PCP (500 μM) and MK-801 (50 μM) on extracellular GABA levels. These results suggest that PCP may facilitate dopamine release in the medial prefrontal cortex, at least in part, by the inhibition of GABA release via the antagonism of NMDA receptors.

Protective mechanism of reduced water against alloxan-induced pancreatic β-cell damage: Scavenging effect against reactive oxygen species

Reactive oxygen species (ROS) cause irreversible damage to biological macromolecules, resulting in many diseases. Reduced water (RW) such as hydrogen-rich electrolyzed reduced water and natural reduced waters like Hita Tenryosui water in Japan and Nordenau water in Germany that are known to improve various diseases, could protect a hamster pancreatic β cell line, HIT-T15 from alloxan-induced cell damage. Alloxan, a diabetogenic compound, is used to induce type 1 diabetes mellitus in animals. Its diabetogenic effect is exerted via the production of ROS. Alloxan-treated HIT-T15 cells exhibited lowered viability, increased intracellular ROS levels, elevated cytosolic free Ca2+ concentration, DNA fragmentation, decreased intracellular ATP levels and lowering of glucose-stimulated release of insulin. RW completely prevented the generation of alloxan-induced ROS, increase of cytosolic Ca2+ concentration, decrease of intracellular ATP level, and lowering of glucose-stimulated insulin release, and strongly blocked DNA fragmentation, partially suppressing the lowering of viability of alloxan-treated cells. Intracellular ATP levels and glucose-stimulated insulin secretion were increased by RW to 2–3.5 times and 2–4 times, respectively, suggesting that RW enhances the glucose-sensitivity and glucose response of β-cells. The protective activity of RW was stable at 4 °C for over a month, but was lost by autoclaving. These results suggest that RW protects pancreatic β-cells from alloxan-induced cell damage by preventing alloxan-derived ROS generation. RW may be useful in preventing alloxan-induced type 1-diabetes mellitus.

Stimulatory Effect of a Dietary Casein Phosphopeptide Preparation on the Mucosal IgA Response of Mice to Orally Ingested Lipopolysaccharide from Salmonella typhimurium

The effect on immunoglobulin production of a commercially available casein phosphopeptide preparation (CPP-III) consisting mainly of bovine αs2-casein (1-32) and β-casein (1-28) in mice that had orally ingested lipopolysaccharide (LPS) from Salmonella typhimurium was investigated. No significant difference in body weight gain was observed between the mice fed on the CPP-III-added diet and those fed on the control diet. The mice fed on the CPP-III-added diet exhibited similar serum and intestinal IgG, IgM, and IgE responses towards LPS to those fed on the control diet. In contrast, fecal and intestinal anti-LPS IgA and total IgA in mice fed on the CPP-III-added diet were significantly higher than in those fed on the control diet. Spleen cells from mice fed on the CPP-III-added diet produced larger amounts of IgA, IL-5, and IL-6 than cells from mice fed on the control diet. These results suggest that dietary casein phosphopeptide may protect a host from invasion of the intestinal mucosa by food-born pathogenic microorganisms.

Anti-Allergic Effect of a Hot-Water Extract of Quince (Cydonia oblonga)

We examined the effect of a crude hot-water extract (HW) of quince (Cydonia oblonga Miller) fruit on type I allergy in vivo and in vitro. The oral administration of the quince HW-added diet to NC/Nga mice for 63 d showed a significant decrease in the development of atopic dermatitis-like skin lesions under conventional conditions. The concentration of IgE in the serum collected from mice fed with quince HW was also lowered in a dose-dependent manner. Moreover, we found that quince HW inhibited the release of β-hexosaminidase from rat basophilic leukemia cell line RBL-2H3 after a 24-h treatment. The quince HW fraction of less than 3 kDa reduced the mRNA expression of the high-affinity IgE receptor (FcεRI) γ subunit. These results suggest that quince HW had an inhibitory effect on type I allergy by suppressing IgE production and IgE-mediated degranulation.

Stimulatory effect of lactic acid bacteria from commercially available nozawana-zuke pickle on cytokine expression by mouse spleen cells

We investigated the effect of lactic acid bacteria (LAB) isolated from eight samples of commercially available Nozawana-zuke, a traditional Japanese pickle, on cytokine expression by mouse spleen cell cultures. The 12 isolated strains of LAB (Nz1–Nz12), which were identified as genus Lactobacillus or Leuconostoc by the API50CHL test, enhanced the expression of interferon (IFN)-γ with 6 h of culture. Ten of these 12 LAB, particularly Nz8, enhanced interleukin (IL)-12 p40 expression. The actinase E- and Benzonase-treated or untreated cell wall fraction of Nz8 enhanced both IFN-γ and IL-12 p40 expression, while the cell plasma fraction had little effect. In the presence of anti-toll like receptor 4 antibody, the effect of the cell wall fraction of Nz8 was significantly abrogated. These results suggest that some LAB from Nozawana-zuke have a T helper 1-type immunoenhancing effect.

Effect of β-Casein (1-28) on Proliferative Responses and Secretory Functions of Human Immunocompetent Cell Lines

The effect of bovine β-casein (1-28) purified from commercial casein phosphopeptide preparations on human T, B, and monocyte cell lines was evaluated. Beta-casein (1-28) enhanced the proliferation of the following: T cell lines HUT-78, Jurkat Clone E6-1, and MOLT-4; B cell lines BALL, KHM-1B, and U266B1; and monocyte cell lines U937 and HL-60. Moreover, β-casein (1-28) stimulated IgA production by KHM-1B over 96 h of culture. Semiquantitative reverse transcriptional-polymerase chain reaction analysis indicated that β-casein (1-28) enhanced mRNA expression of interleukin (IL)-6 in U266B1 and KHM-1B. These results suggest that β-casein (1-28) exerts a mitogenic effect on human T, B, and monocyte cells, and an IgA-enhancing effect on B cells.

Stimulatory Effects of Casein Phosphopeptide (CPP-III) on mRNA Expression of Cytokines in Caco-2 Cells

The effect of CPP-III, a commercially available casein phosphopeptide, on the mRNA expression of cytokines in Caco-2 cells was investigated. CPP-III enhanced the mRNAs of interleukin (IL)-6 and tumor necrosis factor-α while IL-1β was not affected. The mRNA expression of IL-6 was stronger in the presence of both CPP-III and bacterial components such as peptidoglycan from Lactobacillus acidophilus and lipopolysaccharide from Salmonella typhimurium. These results suggest that CPP-III influences the expression of cytokines in intestinal epithelial cells.

Effects of the serotonin2A/2C receptor agonist and antagonist on phencyclidine-induced dopamine release in rat medial prefrontal cortex.

1. Systemic administration of PCP (7.5 mg/kg, i.p.) produced a greater increase in extracellular DA levels in the mPFC than in the STR and NAC, as determined by in vivo microdialysis of awake, freely moving rats. Preferential activation by PCP of prefrontal DA neurons may be, at least in part, the basis for the pathophysiology of PCP-induced psychosis as well as schizophrenia. 2. Recent studies suggest a possible involvement of 5-HT2A receptors in the pathophysiology and treatment of schizophrenia. This study was designed to examine whether and how 5-HT2A receptors modulate PCP-induced DA release in the mPFC. 3. The 5-HT2A/2C receptor agonist (+/-)-DOI (2.5 mg/kg, but not 0.75 mg/kg, i.p.), administered 60 min prior to PCP, significantly attenuated the PCP-induced increase in extracellular DA levels. Pretreatment of the 5-HT2A/2C receptor antagonist ritanserin (1.0 and 5.0 mg/kg, i.p.), administered 60 min prior to PCP, did not influence the PCP-induced increase. When administered alone, neither DOI (2.5 mg/kg) nor ritanserin (1.0 mg/kg) affected basal extracellular DA levels in the mPFC. 4. The NMDA receptor antagonist MK-801 (1.0 mg/kg, i.p.) also increased extracellular DA levels in the mPFC, but this effect was unaffected by pretreatment with DOI (2.5 mg/kg). 5. These results suggest that the stimulation of 5-HT2A/2C receptors may inhibit DA release in the mPFC when it is facilitated by PCP. Other than the NMDA receptor-mediated mechanism may also be involved in the neurochemical interaction between 5-HT2A receptors and PCP in the mPFC.

Ceruletide inhibits phencyclidine-induced dopamine and serotonin release in rat prefrontal cortex.

Phencyclidine (PCP; 5.0 mg/kg, i.p.) produced a greater increase in extracellular dopamine (DA) levels in the prefrontal cortex than in the striatum, while PCP increased the extracellular 5-hydroxytryptamine (serotonin; 5-HT) levels in the prefrontal cortex but not the striatum, as determined by in vivo microdialysis in awake, freely moving rats. The cholecystokinin (CCK)-related decapeptide ceruletide (120 and 400 microg/kg, i.p.), administered 60 min prior to PCP, significantly attenuated the PCP-induced increase in the extracellular levels of DA and 5-HT in the prefrontal cortex, but not in the striatum. These effects were reversed by PD 135,158, a selective CCK-B receptor antagonist (0.1 mg/kg, s.c.), administered 5 min prior to ceruletide. When administered alone, ceruletide (400 microg/kg, i.p.) significantly increased basal extracellular DA levels only in the prefrontal cortex. The selective N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine (0.5 mg/kg, i.p.) also increased extracellular DA levels in the prefrontal cortex, but this effect was unaffected by ceruletide pretreatment. These results suggest that ceruletide may differentially modulate basal and PCP-induced release of DA and 5-HT in the prefrontal cortex.

Inhibitory effect of heat-killed Lactobacillus strain on immunoglobulin E-mediated degranulation and late-phase immune reactions of mouse bone marrow-derived mast cells.

This study investigated the in vitro effect of Lactobacillus strains, a major group of probiotic lactic acid bacteria, on immunoglobulin E (IgE)- and antigen-induced mast cell degranulation and subsequent gene expression. Bone marrow-derived mast cells (BMMCs) from DBA/2 mice were cultured with heat-killed Lactobacillus strains for 24 h. Some strains significantly inhibited IgE- and antigen-induced β-hexosaminidase release from BMMCs. Furthermore, Lactobacillus reuteri NBRC 15892, which exhibited the strongest inhibitory activity, significantly reduced the elevated interleukin (IL)-4, IL-13, tumor necrosis factor-α, and cyclooxygenase-2 expression levels that was induced by 1-2 h of stimulation with IgE and antigens. The suppressive effect of NBRC 15892 strain on BMMC degranulation was significantly reduced in the presence of a toll-like receptor (TLR)2-neutralizing antibody. In addition, downregulation of cell surface FcεRIα expression was observed after 6 h of NBRC 15892 treatment. These results suggest that some Lactobacillus strains inhibited IgE-mediated mast cell degranulation and subsequent late-phase reactions involving mast cells via a TLR2-dependent mechanism with FcεRIα downregulation.