Recent evidence for rapid synthesis and action of oestrogens during auditory processing in a songbird.

It is now clear that oestrogens are not only circulating reproductive hormones, but that they also have neurotransmitter-like properties in a wide range of brain circuits. The view of oestrogens as intrinsic neuromodulators that shape behaviour has been bolstered by a series of recent developments from multiple vertebrate model systems. Here, we review several recent findings from studies of songbirds showing how the identified neural circuits that govern auditory processing and sensorimotor integration are modulated by the local and acute production of oestrogens. First, studies using in vivo microdialysis demonstrate that oestrogens fluctuate in the auditory cortex (30-min time bin resolution) when songbirds are hearing song and interacting with conspecifics. Second, oestrogens rapidly boost the auditory-evoked activity of neurones in the same auditory cortical region, enhancing auditory processing. Third, local pharmacological blockade of oestrogen signalling in this region impairs auditory neuronal responsiveness, as well as behavioural song preferences. Fourth, the rapid actions of oestrogens that occur within the auditory cortex can propagate downstream (trans-synaptically) to sensorimotor circuits to enhance the neural representation of song. Lastly, we present new evidence showing that the receptor for the rapid actions of oestradiol is likely in neuronal membranes, and that traditional nuclear oestrogen receptor agonists do not mimic these rapid actions. Broadly speaking, many of these findings are observed in both males and females, emphasising the fundamental importance of oestrogens in neural circuit function. Together, these and other emergent studies provide support for rapid, brain-derived oestrogen signalling in regulating sensorimotor integration, learning and perception.

NQ-Y15 inhibits the calcium mobilization by elevation of cyclic AMP in rat platelets.

2-1(4-Cyanophenyl)aminol-3-chloro-1,4-naphthalenedione (NQ-Y15) is a dual action drug which acts as a thromboxane A2 (TXA2) synthase inhibitor and TXA2/PGH2 receptor antagonist. In the present study, we examined the effects of NQ-Y15 on Ca2+ mobilization, which is the common event in various types of platelet activation, in arachidonic acid (AA)-stimulated rat platelets. The elevation of cytosolic Ca2+ concentration ([Ca2+]i) induced by AA was inhibited by NQ-Y15 in a concentration-dependent manner. This inhibition-effect of NQ-Y15 was found to be based on the suppression of the rise in [Ca2+]i by the inhibition of both Ca2+ release from internal stores and Ca2+ influx from the extracellular space. Our successive trial was focused on the role of cyclic AMP (cAMP) in the action of NQ-Y15, because cAMP was reported to be increased by dual action drugs such as picotamide and to inhibit the increase in [Ca2+]i. NQ-Y15 was confirmed to increase cAMP in AA-stimulated rat platelets. These results suggested that NQ-Y15 might inhibit the rise in [Ca2+]i in AA-treated rat platelets by increasing cAMP, which is involved in the inhibition of platelet activation.

Carbofuran suppresses T-cell-mediated immune responses by the suppression of T-cell responsiveness, the differential inhibition of cytokine production, and NO production in macrophages.

The effects of carbofuran (2,3-dihydro-2,2-dimethyl-7-benzo-furanol N-methylcarbamate) on the functions of T cells in splenocytes and peritoneal macrophages were examined in view of T-cell-mediated immune response (CMIR) in male C57BL/6 mice. Intraperitoneal administration of carbofuran (0.075, 0.15 and 0.3 mg/kg body weight) resulted in significant suppression of delayed type hypersensitivity (DTH), indicating that it caused the suppression of CMIR. Carbofuran decreased Concanavalin A (Con A)- and alloantigen-induced proliferation, and interleukin (IL)-2 production of splenocytes. In vitro addition of rIL-2 could not completely restore the suppressed T-cell proliferation, and IL-2-induced proliferation of Con A-activated splenocytes was also suppressed, which implied that carbofuran caused defects in IL-2 production and responsiveness of splenocytes to IL-2, leading to the suppression of T-cell proliferation. Con A-induced production of interferon-gamma (IFN-gamma) was significantly suppressed by carbofuran, while that of IL-4 was not affected. The production of transforming growth factor-beta from splenocytes was also significantly inhibited by carbofuran. Judging from these results, carbofuran might directly suppress the cytokine production in T helper 1 (Th1) cells. In addition, IFN-gamma-induced production of nitric oxide (NO) in macrophages was also inhibited by carbofuran, which might be one of the important mechanisms of carbofuran-induced CMIR suppression in mice. Collectively, the present study suggests that carbofuran might suppress CMIR through the suppression of T-cell responsiveness, IFN-gamma production in Th1 cells, and NO generation in macrophages.

Brazilin augments cellular immunity in multiple low dose streptozotocin (MLD-STZ) induced type I diabetic mice.

Brazilin, an active principle of Caesalprenia sappan, was examined for its immunopotentiating effects in multiple low dose streptozotocin (MLD-STZ) induced type diabetic mice. Brazilin was intraperitoneally administered for 5 consecutive days to MLD-STZ induced type I diabetic mice. Delayed type hypersensitivity, Con A-induced proliferation of splenocytes and mixed lymphocyte reaction, which had been decreased in diabetic mice, were significantly recovered by the administration of brazilin. Brazilin increased IL-2 production without affecting suppressor cell activity. Con A-induced and IL-2-induced expression of high affinity IL-2 receptors were also enhanced by brazilin. These results indicate that brazilin augments cellular immune responses, which are suppressed in the MLD-STZ induced type I diabetic mice, by increasing IL-2 production and responsiveness of immune cells to IL-2.

Effects of brazilin on GLUT4 recruitment in isolated rat epididymal adipocytes.

The effects of brazilin on glucose transport into isolated rat epididymal adipocytes were investigated. Brazilin increased [3H]2-deoxy-D-glucose uptake, which was characterized by an increase in Vmax with no effect on the Km value. Phenylarsine oxide, which inhibits the translocation of glucose transporters, decreased brazilin-stimulated glucose transport to the basal level. The inhibition of phosphatidylinositol 3-kinase (PI3-kinase) with wortmannin also blocked brazilin-stimulated glucose transport. Western blot analysis with an anti-GLUT4 antibody revealed that brazilin increased the translocation of GLUT4 from intracellular pools to the plasma membrane. Brazilin, in combination with phorbol ester, showed an additive effect on glucose transport. The stimulating effect of phorbol ester on glucose transport was inhibited by staurosporine, but the effect of brazilin remained unchanged. Protein kinase C activity was not influenced by brazilin treatment. The inhibition of protein synthesis showed no effect on brazilin-stimulated glucose transport, and GLUT4 content in the total membrane fraction was not altered as a result of treatment with brazilin for 4 hr. Metabolic labeling of GLUT4 with [35S]methionine showed that de novo synthesis of GLUT4 was not induced by brazilin. These data suggest that brazilin may increase glucose transport by recruitment of GLUT4 from intracellular pools to the plasma membrane of adipocytes via the activation of PI3-kinase. However, the effect of brazilin may not be mediated by GLUT4 synthesis and protein kinase C activation.

Effects of Brazilin on induction of immunological tolerance by sheep red blood cells in C57BL/6 female mice.

Brazilin was examined for its effects on the induction of immunological tolerance. Brazilin was administered to C57BL/6 female mice for 2 consecutive days before the immunization with high dose SRBC (10(9) cells) which can produce immunological tolerance. Delayed type hypersensitivity, IgM plaque forming cells, ConA induced IL-2 production and mitogen- or antigen-induced proliferation of lymphocytes were measured as evaluation parameters. Administration of brazilin prior to immunization could keep the DTH and IL-2 production almost optimally immunized levels. Brazilin also inhibited the elevation of non-specific suppressor cell activity. ConA induced proliferation of splenocytes in high dose SRBC immunized mice was significantly decreased by pretreatment of brazilin. And this might be one of the reason for augmentation of DTH by brazilin. However, IgM plaque forming cells were not affected by the treatment of brazilin. These results indicate that brazilin prevents the induction of immunological tolerance caused by high dose SRBC by suppressing the elevation of suppressor cell activity and by inhibiting the decrease in IL-2 production in C57BL/6 female mice.

Effects of Brazilin on the phospholipase A2 activity and changes of intracellular free calcium concentration in rat platelets.

Brazilin (7,11b-dihydrobenz[b]indeno[1,2-d]pyran-3,6a,9,10 (6 H)-tetrol) inhibited thrombin-,collagen- and ADP-induced aggregation of washed rat platelets. Thrombin- and collagen-induced ATP release were also inhibited by brazilin in a concentration-dependent manner. Brazilin inhibited the formation of platelet thromboxane A2 caused by thrombin, whereas it had no effect on the prostaglandin D2 formation. Brazilin inhibited [3H]-arachidonic acid liberation from membrane phospholipids of thrombin-stimulated platelets. Brazilin inhibited the rise of intracellular free calcium caused by thrombin. These results indicate that the inhibition of phospholipase (PLA2) activity and [Ca2+]i elevation might be at least a part of antiplatelet mechanism of brazilin.

Brazilin inhibits activities of protein kinase C and insulin receptor serine kinase in rat liver.

Hypoglycemic action of brazilin was found to be based on the improvement of peripheral glucose utility, and this action might be correlated with the insulin action pathway. In the present study we investigated the effect of brazilin on the insulin receptor autophosphorylation, protein kinase C (PKC), protein phosphatase and insulin receptor serine kinase in order to confirm whether the hypoglycemic mechanism is concerned with insulin action pathway. Brazilin was found to inhibit PKC and insulin receptor serine kinase, which are involved in the regulation of insulin signal pathway. But any significant effect was not shown on insulin receptor tyrosine kinase activity, autophosphorylation and phosphatase activity. These findings suggest that brazilin might enhance insulin receptor function by decreasing serine phosphorylation, which might mediate hypoglycemic effect of brazilin.

Brazilin modulates immune function mainly by augmenting T cell activity in halothane administered mice.

Previously we reported that brazilin, the main principle of Caesalpinia sappan, was able to improve the altered immune functions caused by halothane administration in mice. To elucidate the mechanisms of its immunomodulating activities, the effects of brazilin on the functions of T cells and splenic cellularity were investigated. Brazilin decreased splenic cellularity and IL-2 production which had been augmented in mice treated with halothane (21.5% in olive oil, 10 mmol/kg) for 4 consecutive days whereas the reduced expression of IL-2 receptors by ConA or standard IL-2 was increased by brazilin treatment. These data indicate that halothane induced a dysfunction of T cells resulting in abnormal immune responses and these altered immune functions might be improved mainly by affecting the function of T cells.