The reactive nitrogen species, nitric oxide (NO), plays an important role in the pathogenesis of neurodegenerative diseases. The suppression of NO production may be fundamental for survival of neurons. Here, we report that pretreatment of human ramified microglial cells with nearly physiological levels of exogenous NO prevents lipopolysaccharide (LPS)/tumor necrosis factor ␣ (TNF␣)-inducible NO synthesis, because by affecting NF-B activation it inhibits inducible Ca 2؉ -independent NO synthase isoform (iNOS) mRNA expression. Using reverse transcriptase polymerase chain reaction, we have found that both NO donor sodium nitroprusside (SNP) and authentic NO solution are able to inhibit LPS/ TNF␣-inducible iNOS gene expression; this effect was reversed by reduced hemoglobin, a trapping agent for NO. The early presence of SNP during LPS/TNF␣ induction is essential for inhibition of iNOS mRNA expression. Furthermore, SNP is capable of inhibiting LPS/ TNF␣-inducible nitrite release, as determined by Griess reaction. Finally, using electrophoretic mobility shift assay, we have shown that SNP inhibits LPS/TNF␣-elicited NF-B activation. This suggests that inhibition of iNOS gene expression by exogenous NO may be ascribed to a decreased NF-B availability. Nitric oxide (NO)1 is a major messenger molecule playing key roles in many physiological and pathological processes (1). NO production is catalyzed by at least two major forms of the NO synthase (NOS) enzyme: a constitutive Ca 2ϩ -dependent NOS isoform (cNOS) and an inducible Ca 2ϩ -independent NOS isoform (iNOS), which is expressed after stimulation with Escherichia coli lipopolysaccharide (LPS) and cytokines. Recently, we have demonstrated that LPS and/or TNF␣ are able to induce iNOS in human ramified microglia leading to a high NO output (2). On the other hand, NO release from mouse microglia is thought to play an important role in neuronal cell death (3-5). In a recent work, Meda et al. (6) suggested a possible involvement of NO produced by rat microglia after activation with ␤-amyloid protein and IFN-␥ in the pathogenesis of neuronal degradation occurring with age and in Alzheimer's disease.Preserving iNOS gene from its undesirable induction may be important for neuronal survival. Down-regulation of iNOS expression was reported to be achieved by some factors such as dexamethasone, interleukin-4, transforming growth factor-␤, and basic fibroblast growth factor (7-9). Recently, Griscavage et al. However, little is known about the regulatory effects on the mechanism by the variable low concentrations of the available NO before iNOS induction. Recently, we have observed that sodium nitroprusside (SNP), a well known NO donor, elicited inhibition of LPS-induced iNOS expression in rat neutrophils, suggesting a possible suppressive effect on iNOS gene expression by exogenous NO (12). The promoter region of human iNOS gene in vascular smooth muscle cells was shown to contain the consensus sequences for the binding of NFB, a nuclear transcriptional factor (13), and iNOS transc...

To determine whether insulin secretion is affected by a blockage of gap junctions between B cells, we have studied the secretion of rat pancreatic islets of Langerhans, primary dispersed islet cells, and cells of the RINm5F line, during shortterm exposure to heptanol. Within minutes, this alkanol blocked gap junctions between the B cells of intact islets and abolished their normal secretory response to glucose. These two changes were rapidly and fully reversible after return of the islets to control medium. We further found that heptanol had no significant effect on the glucose-stimulated secretion of single B cells but inhibited that of B cell pairs. In the clone of RINm5F cells, whose junctional coupling and D-glyceraldehyde-induced stimulation of insulin release by aggregated cells were also inhibited by heptanol, this alkanol did not perturb intracellular pH and Ca2+ and the most distal steps of the secretion pathway. In summary, a gap junction blocker affected the secretion of insulin-producing cells by a mechanism which is dependent on cell contact and is not associated with detectable pleiotropic perturbations of the cell secretory machinery. The data provide evidence for the involvement of junctional coupling in the control of insulin secretion. (J. Clin.

The regulation of neurotrophin (NT) secretion is critical for many aspects of NT-mediated neuronal plasticity. Neurons release NTs by activity-regulated secretion pathways, initiated either by neurotransmitters and͞or by existing NTs by a positive-feedback mechanism. This process depends on calcium release from intracellular stores. Little is known, however, about potential pathways that down-regulate NT secretion. Here we demonstrate that nitric oxide (NO) induces a rapid down-regulation of brain-derived neurotrophic factor (BDNF) secretion in cultured hippocampal neurons. Similar effects occur by activating a downstream target of intracellular NO, the soluble guanylyl cyclase, or by increasing the levels of its product, cGMP. Furthermore, down-regulation of BDNF secretion is mediated by cGMP-activated protein kinase G, which prevents calcium release from inositol 1,4,5-trisphosphate-sensitive stores. Our data indicate that the NO͞cGMP͞protein kinase G pathway represents a signaling mechanism by which neurons can rapidly down-regulate BDNF secretion and suggest that, in hippocampal neurons, NT secretion is finely tuned by both stimulatory and inhibitory signals.N eurotrophins (NTs), such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-4͞5 (NT-4͞5), and neurotrophin-3 (NT-3), regulate neuronal survival and differentiation during embryonic development (1, 2). In addition to their trophic role, NTs are thought to participate in certain brain functions such as modulation of synaptic transmission and memory formation (3-6). NTs have been shown to modulate synaptic transmission across a broad temporal spectrum ranging from short-term modulation, which occurs in the order of seconds to minutes (7-17), to a prolonged effect that persists for many hours, such as the long-term potentiation (LTP) (18-23) or long-term depression (24-27) response. In fact, NTs are required for the maintenance of LTP in hippocampal slices, because inhibition of BDNF signaling by using receptor bodies applied early after LTP induction restored potentiated synaptic transmission to baseline levels (22). In addition, pretreatment of hippocampal neuron slices with anti-NT receptor antiserum prevented the late phase of the LTP (22). It has been suggested that BDNF concentrations in CA3͞ CA1 hippocampal slices must reach a critical threshold level to initiate and maintain the LTP response (18). This phenomenon has been demonstrated in heterozygous BDNF-defective mice (18,20) that, having impaired endogenous NT production, require either the exogenous administration (20) or local reexpression (19) of BDNF to initiate the LTP response. These observations emphasize the important role played by NTs in modulating synaptic activity and the need to understand better the mechanisms that regulate NT secretion.Recent studies have investigated how neuronal activity can modulate NT secretion. NGF and BDNF secretion is induced in hippocampal slices and cultured hippocampal neurons in response to excitatory neurotransmitters ...

Cardiac myocytes undergo apoptosis under condition of ischemia. Little is known, however, about the molecular pathways that mediate this response. We show that serum deprivation and hypoxia, components of ischemia in vivo, resulted in apoptosis of rat ventricular myoblast cells H9c2. Hypoxia alone did not induce signi¢cant apoptosis for at least 48 h, but largely increased the proapoptotic action of serum deprivation. H9c2 cells apoptosis is evidenced by an increase in terminal (TdT)-mediated dUTP nick end-labeling-positive nuclei and by activation of caspases 3, 6, 7 and 9, and loss of mitochondrial functions. In this model of simulated ischemia, represented by serum deprivation plus hypoxia, cardiomyoblasts apoptosis was associated with a p53-independent Bax accumulation and with a down-regulation of Bcl-xL, whereas the levels of cIAP-1, cIAP-2 and X-IAP proteins did not change. Phorbol-12-myristate-13-acetate signi¢cantly reduced the induction of apoptosis, inhibiting caspase 3 cleavage, Bax accumulation, Bcl-xL downregulation as well as restoring cell viability. ß

Graphene-based materials are the focus of intense research efforts to devise novel theranostic strategies for targeting the central nervous system. In this work, we have investigated the consequences of long-term exposure of primary rat astrocytes to pristine graphene (GR) and graphene oxide (GO) flakes. We demonstrate that GR/GO interfere with a variety of intracellular processes as a result of their internalization through the endolysosomal pathway. Graphene-exposed astrocytes acquire a more differentiated morphological phenotype associated with extensive cytoskeletal rearrangements. Profound functional alterations are induced by GO internalization, including the upregulation of inward-rectifying K channels and of Na-dependent glutamate uptake, which are linked to the astrocyte capacity to control the extracellular homeostasis. Interestingly, GO-pretreated astrocytes promote the functional maturation of cocultured primary neurons by inducing an increase in intrinsic excitability and in the density of GABAergic synapses. The results indicate that graphene nanomaterials profoundly affect astrocyte physiology in vitro with consequences for neuronal network activity. This work supports the view that GO-based materials could be of great interest to address pathologies of the central nervous system associated with astrocyte dysfunctions.