Relationship between LTP and the nuclear protein induced by calcineurin

Results demonstrate that calcineurin plays a role in long term potentiatibn (LTP) in the hippocampus. A 45 ku enzyme was administered, which exhibits Ca²⁺ /calmodulin independent activity to rats, and the protein components of nuclear extracts from the cell-free system of rat hippocampus were tested. It is found that the level of a component significantly increased in nuclear extracts following the activation of calcineurin. The concentration of the component in the nucleus is also markedly increased following hippocampal LTP elicited by ginsenosides. These results suggest that the activation of calcineurin induces a preexisting protein component to translocate to the nucleus in the hippocampus. The nuclear translocation of the component may be required for LTP in the hippocampus.     

Inositol-1,4,5-trisphosphate receptor regulates hepatic gluconeogenesis in fasting and diabetes

In the fasted state, increases in circulating glucagon promote hepatic glucose production through induction of the gluconeogenic program. Triggering of the cyclic AMP pathway increases gluconeogenic gene expression via the de-phosphorylation of the CREB co-activator CRTC2 (ref. 1). Glucagon promotes CRTC2 dephosphorylation in part through the protein kinase A (PKA)-mediated inhibition of the CRTC2 kinase SIK2. A number of Ser/Thr phosphatases seem to be capable of dephosphorylating CRTC2 (refs 2, 3), but the mechanisms by which hormonal cues regulate these enzymes remain unclear. Here we show in mice that glucagon stimulates CRTC2 dephosphorylation in hepatocytes by mobilizing intracellular calcium stores and activating the calcium/calmodulin-dependent Ser/Thr-phosphatase calcineurin (also known as PP3CA). Glucagon increased cytosolic calcium concentration through the PKA-mediated phosphorylation of inositol-1,4,5-trisphosphate receptors (InsP(3)Rs), which associate with CRTC2. After their activation, InsP(3)Rs enhanced gluconeogenic gene expression by promoting the calcineurin-mediated dephosphorylation of CRTC2. During feeding, increases in insulin signalling reduced CRTC2 activity via the AKT-mediated inactivation of InsP(3)Rs. InsP(3)R activity was increased in diabetes, leading to upregulation of the gluconeogenic program. As hepatic downregulation of InsP(3)Rs and calcineurin improved circulating glucose levels in insulin resistance, these results demonstrate how interactions between cAMP and calcium pathways at the level of the InsP(3)R modulate hepatic glucose production under fasting conditions and in diabetes.     

Feedback inhibition of calcineurin and Ras by a dual inhibitory protein Carabin

Feedback regulation of adaptive immunity is a fundamental mechanism for controlling the overall output of different signal transduction pathways, including that mediated by the T-cell antigen receptor (TCR). Calcineurin and Ras are known to have essential functions during T-cell activation. However, how the calcineurin signalling pathway is terminated in the process is still largely unknown. Although several endogenous inhibitors of calcineurin have been reported, none fulfils the criteria of a feedback inhibitor, as their expression is not responsive to TCR signalling. Here we identify an endogenous inhibitor of calcineurin, named Carabin, which also inhibits the Ras signalling pathway through its intrinsic Ras GTPase-activating protein (GAP) activity. Expression of Carabin is upregulated on TCR signalling in a manner that is sensitive to inhibitors of calcineurin, indicating that Carabin constitutes part of a negative regulatory loop for the intracellular TCR signalling pathway. Knockdown of Carabin by short interfering RNA led to a significant enhancement of interleukin-2 production by antigen-specific T cells in vitro and in vivo. Thus, Carabin is a negative feedback inhibitor of the calcineurin signalling pathway that also mediates crosstalk between calcineurin and Ras.     

Calcineurin/NFAT signalling regulates pancreatic beta-cell growth and function

The growth and function of organs such as pancreatic islets adapt to meet physiological challenges and maintain metabolic balance, but the mechanisms controlling these facultative responses are unclear. Diabetes in patients treated with calcineurin inhibitors such as cyclosporin A indicates that calcineurin/nuclear factor of activated T-cells (NFAT) signalling might control adaptive islet responses, but the roles of this pathway in beta-cells in vivo are not understood. Here we show that mice with a beta-cell-specific deletion of the calcineurin phosphatase regulatory subunit, calcineurin b1 (Cnb1), develop age-dependent diabetes characterized by decreased beta-cell proliferation and mass, reduced pancreatic insulin content and hypoinsulinaemia. Moreover, beta-cells lacking Cnb1 have a reduced expression of established regulators of beta-cell proliferation. Conditional expression of active NFATc1 in Cnb1-deficient beta-cells rescues these defects and prevents diabetes. In normal adult beta-cells, conditional NFAT activation promotes the expression of cell-cycle regulators and increases beta-cell proliferation and mass, resulting in hyperinsulinaemia. Conditional NFAT activation also induces the expression of genes critical for beta-cell endocrine function, including all six genes mutated in hereditary forms of monogenic type 2 diabetes. Thus, calcineurin/NFAT signalling regulates multiple factors that control growth and hallmark beta-cell functions, revealing unique models for the pathogenesis and therapy of diabetes.     

Noradrenaline and beta-adrenoceptor agonists increase activity of voltage-dependent calcium channels in hippocampal neurons

The predominance of unconventional transmitter release sites at noradrenaline-containing synapses and the diffuse projections of noradrenaline-containing fibres originating in locus coeruleus have led to speculation that noradrenaline may act as a neuromodulator in the central nervous system. Evidence suggests that it has a modulatory function in the plasticity of the developing nervous system, in controlling behavioural states of an organism, and in learning and memory. Recently, Hopkins and Johnston demonstrated that noradrenaline enhances the magnitude, duration and probability of induction of long-term potentiation (LTP) at mossy fibre synapses in the hippocampal formation, and LTP is widely believed to be a cellular substrate for aspects of memory. To investigate the membrane effects of noradrenaline on central neurons, we used a newly developed preparation in which patch-clamp techniques can be applied to exposed adult cortical neurons. We report here that noradrenaline produces an enhancement in the activity of voltage-dependent calcium channels in granule cells of the hippocampal dentate gyrus. This action appears to be mediated by beta-adrenoceptors and can be mimicked by cyclic AMP.     

大鼠在空间辨别性学习记忆时海马CA1,CA3区和DG的ACh能纤维和星形胶质细胞的变化

目的通过建立大鼠空间辨别性学习记忆的动物模型,观察大鼠海马CA1,CA3区和齿状回(DG)的乙酰胆碱(ACh)能纤维密度和含量的变化以及星形胶质细胞的数量及其胶质原纤维酸性蛋白(GFAP)表达的变化,从形态学上探讨中枢ACh和星形胶质细胞与空间辨别性学习记忆的关系.方法建立空间辨别性学习记忆的动物模型,采用乙酰胆碱酯酶(AChE)的组织化学染色及GFAP标记星形胶质细胞.并用显微镜方格目测系统和图像分析仪对大鼠海马CA1,CA3区及DG的GFAP免疫阳性星形胶质细胞的数量及其GFAP表达进行检测.结果模型组与对照组相比大鼠各观察部位的AChE阳性纤维的密度和含量均增高,有显著性差异(P<0.05).而空白组和游水组相比大鼠各观察部位的AChE阳性纤维的密度和含量均无显著性差异(P>0.05),模型组内各组间两两比较大鼠各观察部位的AChE阳性纤维的密度和含量均无显著性差异(P>0.05);模型组与对照组相比大鼠各观察部位的星形胶质细胞的数量及其GFAP的表达均增加,有显著性差异(P<0.05).而空白组和游水组相比大鼠各观察部位的星形胶质细胞的数量及其GFAP的表达均无显著性差异(P>0.05),模型组内各组间两两比较大鼠各观察部位的星形胶质细胞的数量及其GFAP的表达均无显著性差异(P>0.05).结论在空间辨别性学习记忆过程中,大鼠各观察部位的AChE阳性纤维的密度和含量增高,说明中枢ACh参与空间辨别性学习记忆过程;在空间辨别性学习记忆过程中,大鼠各观察部位的星形胶质细胞的数量及其GFAP表达的增加,说明星形胶质细胞参与空间辨别性学习记忆过程.     

Regulation of calcium signalling in T lymphocytes by the second messenger cyclic ADP-ribose

Cyclic ADP-ribose (cADPR) is a natural compound that mobilizes calcium ions in several eukaryotic cells. Although it can lead to the release of calcium ions in T lymphocytes, it has not been firmly established as a second messenger in these cells. Here, using high-performance liquid chromatography analysis, we show that stimulation of the T-cell receptor/CD3 (TCR/CD3) complex results in activation of a soluble ADP-ribosyl cyclase and a sustained increase in intracellular levels of cADPR. There is a causal relation between increased cADPR concentrations, sustained calcium signalling and activation of T cells, as shown by inhibition of TCR/CD3-stimulated calcium signalling, cell proliferation and expression of the early- and late-activation markers CD25 and HLA-DR by using cADPR antagonists. The molecular target for cADPR, the type-3 ryanodine receptor/calcium channel, is expressed in T cells. Increased cADPR significantly and specifically stimulates the apparent association of [3H]ryanodine with the type-3 ryanodine receptor, indicating a direct modulatory effect of cADPR on channel opening. Thus we show the presence, causal relation and biological significance of the major constituents of the cADPR/calcium-signalling pathway in human T cells.     

Dendritic spines as individual neuronal compartments for synaptic Ca2+ responses.

The possibility that postsynaptic spines on neuronal dendrites are discrete biochemical compartments for Ca(2+)-activated processes involved in synaptic plasticity is a widely proposed concept that has eluded experimental demonstration. Using microfluorometry on CA3 neurons in hippocampal slices, we show here that with weak presynaptic stimulation of associative/commissural fibres, Ca2+ accumulates in single postsynaptic spines but not in the parent dendrite. Stronger stimulation also promotes changes in dendrites. The NMDA-receptor antagonist AP-5 blocks changes in Ca2+ in spines. Sustained steep Ca2+ gradients between single spines and the parent dendrite, often lasting several minutes, develop with repeated stimulation. The observed compartmentalization allows for the specificity, cooperativity and associativity displayed by memory models such as long-term potentiation.