Glucose represses connexin36 in insulin-secreting cells.

The gap-junction protein connexin36 (Cx36) contributes to control the functions of insulin-producing cells. In this study, we investigated whether the expression of Cx36 is regulated by glucose in insulin-producing cells. Glucose caused a significant reduction of Cx36 in insulin-secreting cell lines and freshly isolated pancreatic rat islets. This decrease appeared at the mRNA and the protein levels in a dose- and time-dependent manner. 2-Deoxyglucose partially reproduced the effect of glucose, whereas glucosamine, 3-O-methyl-D-glucose and leucine were ineffective. Moreover, KCl-induced depolarization of beta-cells had no effect on Cx36 expression, indicating that glucose metabolism and ATP production are not mandatory for glucose-induced Cx36 downregulation. Forskolin mimicked the repression of Cx36 by glucose. Glucose or forskolin effects on Cx36 expression were not suppressed by the L-type Ca(2+)-channel blocker nifedipine but were fully blunted by the cAMP-dependent protein kinase (PKA) inhibitor H89. A 4 kb fragment of the human Cx36 promoter was identified and sequenced. Reporter-gene activity driven by various Cx36 promoter fragments indicated that Cx36 repression requires the presence of a highly conserved cAMP responsive element (CRE). Electrophoretic-mobility-shift assays revealed that, in the presence of a high glucose concentration, the binding activity of the repressor CRE-modulator 1 (CREM-1) is enhanced. Taken together, these data provide evidence that glucose represses the expression of Cx36 through the cAMP-PKA pathway, which activates a member of the CRE binding protein family

Critical role of the transcriptional repressor neuron-restrictive silencer factor in the specific control of connexin36 in insulin-producing cell lines.

Connexin36 (Cx36) is specifically expressed in neurons and in pancreatic beta-cells. Cx36 functions as a critical regulator of insulin secretion and content in beta-cells. In order to identify the molecular mechanisms that control the beta-cell expression of Cx36, we initiated the characterization of the human 5' regulatory region of the CX36 gene. A 2043-bp fragment of the human CX36 promoter was identified from a human BAC library and fused to a luciferase reporter gene. This promoter region was sufficient to confer specific expression to the reporter gene in insulin-secreting cell lines. Within this 5' regulatory region, a putative neuron-restrictive silencer element conserved between rodent and human species was recognized and binds the neuron-restrictive silencing factor (NRSF/REST). This factor is not expressed in insulin-secreting cells and neurons; it functions as a potent repressor through the recruitment of histone deacetylase to the promoter of neuronal genes. The NRSF-mediated repression of Cx36 in HeLa cells was abolished by trichostatin A, confirming the functional importance of histone deacetylase activity. Ectopic expression, by viral gene transfer, of NRSF/REST in different insulin-secreting beta-cell lines induced a marked reduction in Cx36 mRNA and protein content. Moreover, mutations in the Cx36 neuron-restrictive silencer element relieved the low transcriptional activity of the human CX36 promoter observed in HeLa cells and in INS-1 cells expressing NRSF/REST. The data showed that cx36 gene expression in insulin-producing beta-cell lines is strictly controlled by the transcriptional repressor NRSF/REST indicating that Cx36 participates to the neuronal phenotype of the pancreatic beta-cells

Comparative effects of estrogens and prolactin on nigral and striatal GAD activity

The comparative effects of a 10 day estrogen treatment and estrogen independent hyperprolactinemia on nigral and striatal glutamic acid decarboxylase (GAD, EC 4.1.1.15) activity were investigated in male rats. Data obtained show that estrogen treatment decreases GAD activity in substantia nigra, while an increase was observed in conditions of hyperprolactinemia induced by modulation of strio-nigral GABAergic system by estrogens and prolactin is suggested. © 1982

Expression and functional analysis of glutamate receptors in glial cells

The brain consists of a complex network in which neurones and glial cells are structurally and functionally interwoven. Astrocytes, the most numerous member of the glial family, were originally considered, along with the whole glial population, to be only of structural importance (Virchow, 1846). For example, during development the radial glia, the precursors of astrocytes, serve as a scaffold at which neurones migrate to form the layered structure of different brain regions such as the cortex, the hippocampus or the cerebellum. During the last two decades, considerable knowledge about astrocytes has accumulated regarding their physiological function. One exciting function is their contribution to the regulation of the extracellular space and, thereby, also of brain excitability (Walz, 1989). Qualities such as their capacity for uptake and metabolism of transmitters, buffering capacity of ions and ability to convey external signals via surface receptors to biological responses within the cells indicate an intimate crosstalk between glial cells and neurones. The other major glial population in the brain are the oligodendrocytes. As small cells with few processes they form the myelin sheath, a highly lipid enriched stack of cell membranes enwrapping 50 to 300αm long axonal segments to enhance the conduction of electrical signals and to inhibit electrical crosstalk between individual axons. Oligodendrocytes are capable of myelinating up to 50 axonal segments simultaneously. Mature oligodendrocytes develop from progenitors originating from the subventricular zone as the germinative layer (Miller, 1996). In vertebrates, progenitors start to migrate to their final destination regions, the presumptive white matter, during the first postnatal week

Expression of connexin 43 in the human epileptic and drug-resistant cerebral cortex.

Gap junctions are specialized channels composed of several connexins, membrane proteins that mediate electrical and metabolic coupling between cells. Previous data have suggested that changes in the expression of Cx43, the main astrocytic Cx isoform, may be involved in seizure activity in human epileptic tissue. However, Cx43 has never been examined in focal cortical dysplasia (FCD) and in other human refractory epilepsies.We analyzed Cx43 protein localization and Cx43 mRNA levels in surgical specimens of cortex from a cohort of patients with intractable epilepsy vs control nonepileptic tissue. Samples had neuropathologically defined diagnosis of cryptogenic epilepsy or epilepsy secondary to FCD.Cx43 immunoreactivity, which labeled punctate elements, did not reveal distinctive features in cryptogenic epilepsy and FCD type IA and IIA. A peculiar pattern of immunolabeling was instead observed in FCD type IIB, in which large aggregates of Cx43-immunopositive puncta were clustered around subsets of balloon cells and astrocytes. Further characterization revealed that these balloon cells do not express markers of precursor cells, such as CD34. Quantitative real-time reverse transcriptase PCR showed elevated levels of Cx43 transcript in a subgroup (25\%) of cryptogenic epilepsy specimens compared to control and FCD ones.Our study points out that a rearrangement of Cx43-positive elements is part of abnormal tissue organization in FCD type IIB, and that cryptogenic epilepsies include forms with increased Cx43 mRNA expression. The data implicate functional consequences of altered Cx43 expression, and therefore of altered gap junctional coupling, in abnormal network properties of subtypes of human refractory epilepsies

Expression of metabotropic glutamate receptors in rat and human testis

12noreservedThe G protein-coupled receptor kinase type 4 mediates the homologous desensitisation of type-1 metabotropic glutamate (mGlu1) receptors and is predominantly expressed in the testis. Hence, we searched for the expression of mGlu1 or other mGlu receptor subtypes in rat and human testes. RT-PCR analysis showed the presence of mGlu1, -4 and -5 (but not -2 or -3) receptor mRNA in the rat testis. The presence of mGlu1 and -5 (but not mGlu2/3) receptor proteins was also demonstrated by Western blot analysis. In the rat testis, both mGlu1a and -5 receptors were highly expressed in cells of the germinal line. It is likely that these receptors are functional, because the agonist, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid, was able to stimulate inositol phospholipid hydrolysis in slices prepared from rat testes. Immunocytochemical analysis of bioptic samples from human testes showed a high expression of mGlu5 receptors inside the seminiferous tubuli, whereas mGlu1a immunoreactivity was restricted to intertubular spaces. mGlu5 receptors were also present in mature spermatozoa, where they were localised in the mid-piece and tail. This localisation coincided with that of beta -arrestin, a protein that is critically involved in the homologous desensitisation and internalisation of G protein-coupled receptors. Taken collectively, these results offer the first evidence for the expression of any glutamate receptor in testes, and suggest that at least mGlu5 receptors are present and functionally active in mature human sperm.mixedSTORTO, M.; SALLESE, M.; SALVATORE, L.; POULET, R.; CONDORELLI, L.; DELL'ALBANI, P.; MARCELLO, M.; ROMEO, R.; PIOMBONI, P.; BARONE, N.; NICOLETTI, F.; DE BLASI, A.Storto, M.; Sallese, M.; Salvatore, L.; Poulet, R.; Condorelli, L.; Dell'Albani, P.; Marcello, M.; Romeo, R.; Piomboni, P.; Barone, N.; Nicoletti, F.; DE BLASI, A

A neural-specific hypomethylated domain in the 5' flanking region of the glial fibrillary acidic protein gene

In the present study we examined the methylation status of the dial fibrillary acidic protein (GFAP) gene promoter, analyzing various CG sites in both the human and rat gene in GFAP-expressing and nonexpressing tissues, Moreover, we studied the methylation of specific CG sites in different rat brain areas during postnatal development. in cell cultures highly enriched in specific neural-or non-neural-cell types (fibroblasts), and in human gliomas. The obtained results do not support a simple correlation between demethylation and expression of the GFAP gene but help to identify a cluster of CG sites in the 5' flanking region (from -1176 to -1471 in the rat) that are hypomethylated in neural cell types and localized in a region highly conserved between rat, mouse and human GFAP promoters, Neural-specific hypomethylation of this conserved zone can be observed also in the human GFAP gene both in normal brain tissue and neoplastic dial cells, A higher demethylation of the -1176 site at early stage of postnatal life was observed in specific rat brain areas, such as hippocampus and cerebellum. The most dramatic differences were observed in the cerebellum where a peak of demethylation of the -1176 site was detected at 15 days of postnatal life, followed by an intense remethylation of this site, Results of experiments in the CG4 dial progenitor cell line showed that demethylation of the -1176 site is already established before transcriptional activation of the GFAP gene, Moreover, results of experiments in primary cell cultures show that in neuronal cell types, such as cerebellar granule cells and embryonic cerebral hemisphere neurons, the level of demethylation of the -1176 site is comparable to that observed in cultured astrocytes, In contrast a high level of methylation can be observed in cultured non-neural cell types (fibroblasts), Such neural-specific hypomethylation could be established in a very early stage in the progression along the neural cell lineage and could play a role in maintaining a local open chromatin conformation which is then necessary to allow the interaction with specific regulatory factors present in astroglial cells