Abstract: NO-sensitive guanylyl cyclase (GC(NO)), the major NO target, is involved in important regulatory functions in the cardiovascular, gastrointestinal and central nervous systems. GC(NO) exists as heterodimers of alpha(1/2) and beta1 subunits. Deletion of the obligate beta1 dimerizing partner abrogates NO/cGMP signaling and shortens the life span of KO mice. Localization studies in the CNS have shown that beta1 is more widespread than alpha subunits and in some areas is the only GC(NO) subunit expressed, suggesting that beta1 may have GC(NO)-independent functions. GC(NO) is predominantly cytosolic, but association to membranes and other intracellular structures has been described. Here, we show localization of beta1 in cytoplasm and nucleus of cells expressing alpha subunits and GC(NO) activity (astrocytes, C6 cells), as well as in cells devoid of alpha subunits and GC(NO) activity (microglia). In both cell types beta1 associates peripherally to chromosomes in all phases of mitosis. Immunodepletion of beta1 in C6 cells enhances chromatin condensation in an in vitro assay. Moreover, silencing beta1 by siRNA induces cell cycle re-entry as determined by flow cytometry, and increases proliferation rate in a MTT-assay, whereas infection with beta1-containing adenovirus has the opposite effect. These actions are independent of cGMP formation. We postulate that beta1 is a multifunctional protein that regulates chromatin condensation and cell cycle progression, in addition to being an obligate monomer in functional GC(NO) heterodimers.
Abstract: We previously showed that treatment with bacterial lipopolysaccharide (LPS) or pro-inflammatory cytokines decreases NO-sensitive guanylyl cyclase (GC(NO)) activity in astrocytes by decreasing the half-life of the obligate GC(NO) beta1 subunit in a NO-independent but transcription- and translation-dependent process. Here we show that LPS-induced beta1 degradation requires proteasome activity and is independent of NFkappaB activation or beta1 interaction with HSP90. Immunocytochemistry and confocal microscopy analysis revealed that LPS promotes colocalization of the predominantly soluble beta1 protein with ubiquitin and the 20S proteasome in nuclear aggregates that present characteristics of clastosomes, nuclear bodies involved in proteolysis via the ubiquitin-proteasome system. Proteasome and protein synthesis inhibitors prevented LPS-induced clastosome assembly and nuclear colocalization of beta1 with ubiquitin and 20S proteasome, strongly supporting a role for these transient nuclear structures in GC(NO) down-regulation during neuroinflammation.
Abstract: Reactive gliosis is a prominent feature of CNS injury that involves dramatic changes in glial cell morphology together with increased motility, phagocytic activity, and release of inflammatory mediators. We have recently demonstrated that stimulation of the cGMP-protein kinase G (PKG) pathway by NO or atrial natriuretic peptide (ANP) regulates cytoskeleton dynamics and motility in rat astrocytes in culture. In this work, we show that the cGMP-PKG pathway stimulated by ANP, but not by NO, regulates microglial cell morphology by inducing a dramatic reorganization in the actin cytoskeleton. Both ANP (0.01-1.0 microM) and the permeable cGMP analog, dibutyryl-cGMP (1-100 microM), promote a rapid (maximal at 30 min) and concentration-dependent increase in size, rounding, and lamellipodia and filopodia formation in rat brain cultured microglia. These morphological changes involve an augment and redistribution of F-actin and result in increased phagocytic activity. ANP-induced rearrangements in actin cytoskeleton and inert particle phagocytosis are prevented by the PKG inhibitor, Rp-8-Br-PET-cGMPS (0.5 microM), and involve inhibition of RhoA GTPase and activation of Rac1 and Cdc42. However, ANP does not induce NO synthase Type 2 (NOS-2) or tumor necrosis factor-alpha expression and is able to decrease lipopolysaccharide (LPS)-elicited induction of these inflammatory genes. The morphological changes and the decrease of LPS-induced NOS-2 expression produced by ANP in cultured microglia are also observed by immunostaining in organotypic cultures from rat hippocampus. These results suggest that stimulation of the ANP-cGMP-PKG pathway in microglia could play a beneficial role in the resolution of neuroinflammation by removing dead cells and decreasing levels of proinflammatory mediators.
Abstract: A large body of evidence supports a role for the NO-cGMP-protein kinase G pathway in the regulation of synaptic transmission and plasticity, brain development and neuroprotection. Circumstantial evidence implicates natriuretic peptide-stimulated cGMP formation in the same CNS functions. In addition to neurons, both cGMP-mediated pathways are functional in glial cells and an increasing number of reports indicate that they may control important aspects of glial cell physiology relevant to neuronal function. In this article we briefly review the regulation of cGMP formation in glial cells and summarize recent evidence indicating that cGMP-mediated pathways can play important roles in astroglial and microglial function in normal and diseased brain.
Abstract: NO-sensitive guanylyl cyclase or soluble guanylyl cyclase (sGC) is the major target for NO and cyclic GMP the mediator of its vasodilating and neuromodulatory actions. Studies on the mechanism of nitrovasodilator-induced tolerance have shown that in smooth muscle cells sGC is down-regulated by prolonged exposure to exogenous or endogenous NO. Increased expression of NO synthase (NOS) in CNS glial cells is a landmark of acute and chronic neuroinflammation. Our studies in cultured astroglial cells demonstrate that exposure to neuroinflammatory agents leads to a long-lasting down-regulation of sGC that occurs by NO-dependent and independent mechanisms. Decreased expression of the enzyme at the protein and mRNA level is evident in the brain of adult rats after intracerebral injection of inflammatory compounds. A decreased cGMP synthesizing capacity may contribute to the neurodegenerative process associated to neuroinflammation.
Abstract: In Alzheimer's disease (AD) brains increased NO synthase (NOS) expression is found in reactive astrocytes surrounding amyloid plaques. We have recently shown that treatment with beta-amyloid peptides or IL-1beta down-regulates NO-sensitive soluble guanylyl cyclase (sGC) in cultured astrocytes and in adult rat brain. In this work, we have examined sGC activity and expression in postmortem brain tissue of AD patients and matched controls. No significant alteration was observed in basal or NO-stimulated sGC activity, nor in sGC beta1 and alpha1 subunit levels in cortical extracts of AD brains. Immunohistochemistry showed intense and widespread labeling of sGC beta1 in cortical and hippocampal neurons and white matter fibrillar astrocytes, while grey matter astrocytes were faintly stained. In AD, expression of sGC in neurons and fibrillar astrocytes is not altered but is markedly reduced in reactive astrocytes surrounding amyloid plaques. Immunostaining for sGC beta1 was also lacking in reactive astrocytes in cortex and subcortical white matter in Creutzfeldt-Jakob disease brains and in subacute and chronic plaques in multiple sclerosis (MS) brains. Thus, induction of astrocyte reactivity is associated with decreased capacity to generate cGMP in response to NO both in vitro and in vivo. This effect may be related to the development of the astroglial inflammatory response.
Abstract: The human immunodeficiency virus type-1 (HIV-1) coat glycoprotein gp120 has been proposed as a likely etiologic agent of HIV-associated dementia (HAD). The pathogenic mechanisms underlying HAD have not yet been fully elucidated, but different evidences indicate that glial cells play an essential role in the development and amplification of the disease. The NO/cyclic GMP (cGMP) system is a widespread signal transduction pathway in the CNS involved in numerous physiological and pathological functions. Increased expression of NO synthase has been reported in the brain of AIDS patients and in cultured rodent glial cells exposed to gp120. The aim of this study was to investigate if gp120 could cause alterations in the metabolism of the NO physiological messenger cGMP that could contribute to the pathogenesis of HAD. Here, we show that long-term treatment (more than 24 h) of rat cerebellar astrocyte-enriched cultures with gp120 (10 nM) induces changes in the cultured cells--astrocyte stellation and proliferation of ameboid microglia--compatible with the acquisition of a reactive phenotype and reduces the capacity of the astrocytes to accumulate cGMP in response to NO in a time-dependent manner (maximal after 72 h). Measurements in cell extracts show that gp120 enhances Ca2+-independent cGMP phosphodiesterase activity by 80-100% without significantly affecting soluble guanylyl cyclase (sGC). Experiments in whole cells using specific phosphodiesterase inhibitors indicate that the viral protein increases the activity of cGMP specific phosphodiesterase 5.
Abstract: In the CNS, NO is an important physiological messenger involved in the modulation of brain development, synaptic plasticity, neuroendocrine secretion, sensory processing, and cerebral blood flow [Annu. Rev. Physiol. 57 (1995) 683]. These NO actions are largely mediated by cyclic GMP (cGMP) formed by stimulation of soluble guanylyl cyclase (sGC). NO has also been recognized as a neuropathological agent in conditions such as epilepsy, stroke and neurodegenerative disorders. In these conditions, NO may contribute to excitotoxic cell death and neuroinflammatory cell damage [Brain Res. Bull. 41 (1996) 131; Glia 29 (2000) 1]. NO can be formed in every type of CNS parenchymal cell, however, cGMP appears to be formed mainly in neurons and astroglia [Annu. Rev. Physiol. 57 (1995) 683]. There is a large body of information about the regulation of NO formation in brain cells under both normal and pathological conditions but much less is known about the control of cGMP generation, in particular during neuroinflammation when there is a high NO output. Here we briefly review our present knowledge on the regulation of NO-dependent cGMP formation in brain cells under inflammatory conditions.
Abstract: We previously showed that soluble guanylyl cyclase (sGC) is down-regulated in astroglial cells after exposure to LPS. Here, we show that this effect is not mediated by released IL-1beta but that this cytokine is also able to decrease NO-dependent cGMP accumulation in a time- and concentration-dependent manner. The effect of IL-1beta is receptor-mediated, mimicked by tumor necrosis factor-alpha and involves a decrease in sGC activity and protein. IL-1beta and LPS decrease the half-life of the sGC beta1 subunit by a NO-independent but transcription- and translation-dependent mechanism. Additionally, both agents induce a NO-dependent decrease of sGC subunit mRNA. Decreased sGC subunit protein and mRNA levels are also observed in adult rat brain after focal injection of IL-1beta or LPS.
Abstract: In astroglial cells beta-amyloid peptides (betaA) induce a reactive phenotype and increase expression of NO synthase. Here we show that treatment of rat brain astrocytes with betaA decreases their capacity to accumulate cyclic GMP (cGMP) in response to NO as a result of a decreased expression of soluble guanylyl cyclase (sGC) at the protein and mRNA levels. Potentiation of betaA-induced NO formation by interferon-gamma did not result in a larger decrease in cGMP formation and inhibition of NO synthase failed to reverse down-regulation of sGC, indicating that NO is not involved. The betaA effect was prevented by the protein synthesis inhibitor cycloheximide. Intracerebral betaA injection also decreased sGC beta1 subunit mRNA levels in adult rat hippocampus and cerebellum. A loss of sGC in reactive astrocytes surrounding beta-amyloid plaques could be a mechanism to prevent excess signalling via cGMP at sites of high NO production.
Abstract: In rat brain astroglia-enriched cultures long-term treatment with interleukin-1beta induces NO release and stimulation of soluble guanylyl cyclase. The cGMP formed is recovered in the extracellular medium but not in the cell monolayer. The interleukin-1beta effect is mediated by type I receptor and potentiated by interferon-gamma. In cells treated with bacterial endotoxin a larger NO-dependent cGMP accumulation occurs only intracellularly, however a significant cGMP egression is observed when cells are co-treated with interleukin-1beta. The non-selective anion transport inhibitors probenecid and verapamil block cGMP efflux, indicating that interleukin-1beta stimulates a cGMP transporter.
Abstract: Induction of nitric oxide (NO) synthase (NOS) type 2 (NOS-2) in glial cells after exposure to bacterial endotoxin [lipopolysaccharide (LPS)] or inflammatory cytokines has been repeatedly demonstrated both in vitro and in vivo. However, little is known about effects of these agents on NO-dependent cyclic GMP (cGMP) formation. In this work, we show that treatment of rat cerebellar astrocyte-enriched primary cultures with LPS decreases NO donor-stimulated cGMP formation with a similar initial time course (up to 9-12 h) and concentration dependency (0.1-1 ng/ml) as for induction of NOS-2. This effect appears to be due to a down-regulation of soluble guanylyl cyclase (sGC) because LPS treatment decreases sGC activity and sGC beta1 subunit levels. In contrast, cGMP phosphodiesterase activity and stimulation of the particulate guanylyl cyclase by atrial natriuretic peptide are not affected. Incubation of astroglial cultures with a transcription inhibitor (actinomycin D) or a protein synthesis inhibitor (cycloheximide) for 18-20 h does not decrease sGC activity but totally prevents LPS-induced desensitization of sGC. Inhibition of NOS-2 activity with N(G)-monomethyl-L-arginine or inhibition of NOS-2 induction with the synthetic glucocorticoid dexamethasone failed to prevent the inhibitory effect of LPS on sGC, indicating that NO production is not involved. Moreover, after removal of LPS the time for recovery of sGC responsiveness is much longer than that for NOS-2 return to basal levels. LPS impairment of cGMP formation also occurs in cortical astrocytes but not in cerebellar granule neurons. The decreased responsiveness of sGC to NO stimulation following LPS challenge may prevent inappropriate astroglial cGMP signaling caused by excess production of NO by adjacent activated glial cells. Key Words: Astroglia-Neurons-Nitric oxide-Soluble guanylyl cyclase-Lipopolysaccharide.
Abstract: In cultured rat cerebellar astroglia kainate induces cGMP formation with low potency (EC50 310 microM). In the presence of cyclothiazide, a blocker of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor desensitization, the effect of kainate was potentiated and glutamate and AMPA elicited large increases (> 100-fold) in cGMP levels. The response to all three agonists was abolished by the nitric oxide synthase inhibitor N(G)-nitro-L-arginine and required extracellular calcium. Uptake of Co2+ was induced by AMPA in a limited population of astroglial cells and this effect was potentiated by cyclothiazide. These results indicate that calcium-permeable AMPA receptors mediate stimulation of nitric oxide formation in cerebellar astroglia. This effect may be relevant for glutamate-dependent synaptic plasticity processes in the cerebellum.
Abstract: Ca2+ entry induced by N-methyl-D-aspartate (NMDA) in neurons and by noradrenaline (NA) in astrocytes is known to increase intracellular cyclic GMP (cGMP) levels through stimulation of the Ca2+-dependent nitric oxide synthase type I (NOS-I). The possibility that Ca2+ entry could also down-regulate intracellular cGMP by activating a Ca2+/calmodulin-dependent phosphodiesterase (CaM-PDE) has been investigated here in primary cultures enriched in granule neurons or in astroglia from rat cerebellum. We show that the same agonists that stimulate nitric oxide (NO) formation (NMDA and NA at 100 microM) and the Ca2+ ionophore A23187 (10 microM) decrease cGMP generated in response to direct stimulation of soluble guanylyl cyclase (sGC) by NO donors in both cell types. This effect requires extracellular Ca2+ and is prevented by the calmodulin inhibitor W7 (100 microM). Membrane depolarization, manipulations of the Na+ gradient, and intracellular Ca2+ mobilization also decrease NO donor-induced cGMP formation in granule cells. In astroglia Ca2+ entry additionally down-regulates cGMP generated by stimulation of the particulate GC by atrial natriuretic peptide (ANF). Decreases in cGMP produced by A23187 were more pronounced in the absence than in the presence of the PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX; 1 mM), indicating that a CaM-PDE was involved. We also show that astroglial cells can accumulate similar amounts of cGMP than neurons in response to NO donors when IBMX is present but much lower levels in its absence. This may result from a lower ratio of sGC to PDE activities in astroglia.
Abstract: We have previously reported that stimulation of astrocyte cultures by particular agonists and calcium ionophores induces cyclic GMP formation through activation of a constitutive nitric oxide synthase (NOS) and that astrocytes from cerebellum show the largest response. In the present work we have used rat cerebellar astrocyteenriched primary cultures to identify and characterise the isoform of NOS expressed in these cells. The specific NOS activity in astrocyte homogenates, determined by conversion of [3H]arginine to [3H]citrulline, was ten times lower than in homogenates from cerebellar granule neurons. Upon centrifugation at 100,000 g, the astroglial activity was recovered in the supernatant, whereas in neurons around 30% of the activity remained particulate. The cytosolic NOS activities of both astrocytes and granule neurons displayed the same Km for L-arginine, dependency of calcium, and sensitivity to NOS inhibitors. Expression of NOS-I in astrocyte cytosolic fractions was revealed by Western blot with a specific polyclonal antiserum against recombinant NOS-I. Double immunofluorescence labelling using anti-glial fibrillary acidic protein (GFAP) and anti-NOS-I antibodies revealed that a minor population of the GFAP-positive cells, usually in clusters, presented a strong NOS-I immunostaining that was predominantly located around the nuclei and had a granular appearance, indicating association with the endoplasmic reticulum-Golgi system. Astrocytes of stellate morphology also showed immunoreactivity in the processes. Similar staining was observed with the avidin-biotin-peroxidase complex using different anti-NOS-I antisera. With this method the majority of cells showed a weak NOS-I immunoreactivity around the nuclei and cytosol. A similar pattern was observed with the NADPH-diaphorase reaction. These results demonstrate that the NOS-I expressed in astrocytes presents the same biochemical characteristics as the predominant neuronal isoform but may differ in intracellular location.
Abstract: Treatment of rat cerebellar astrocyte-enriched primary cultures with dexamethasone enhances the nitric oxide-dependent cyclic GMP formation induced by noradrenaline in a time-(> 6 h) and concentration-dependent manner (half-maximal effect at 1 nM). Stimulation of cyclic GMP formation by the calcium ionophore A23187 is similarly enhanced. In contrast, cyclic GMP accumulation in cells treated with lipopolysaccharide is inhibited by dexamethasone. The potentiating effect of dexamethasone is prevented by the protein synthesis inhibitor cycloheximide and is not due to increased soluble guanylate cyclase activity. Agonist stimulation of [3H]arginine to [3H]citrulline conversion is enhanced by dexamethasone in astrocytes but not in cerebellar granule cells. These results indicate that glucocorticoids may up-regulate astroglial calcium-dependent nitric oxide synthase while preventing expression of inducible nitric oxide synthase and are the first report of a differential long-term regulation of the expression of neuronal and astroglial constitutive nitric oxide synthase activities.
Abstract: We have previously demonstrated nitric oxide (NO)-dependent cyclic GMP (cGMP) formation in response to noradrenaline (NA) and glutamate (GLU) in astrocyte-enriched cultures from rat cerebrum. In the present work we show heterogeneity in agonist responses in astrocyte cultures from cerebellum, hippocampus and cortex. The response to NA was higher in cells from cerebellum, intermediate in cultures from hippocampus and low in cortical astrocytes. GLU had no significant effect in cortical and cerebellar cultures and presented lower effects than NA in cells from hippocampus. The NO donor sodium nitroprusside (SNP) produced much higher cGMP levels than agonists and the order of efficacies was cerebellum > cortex > hippocampus. Responses to NA and SNP in cerebellar astrocytes were sensitive to culture conditions decreasing when cells were seeded at low density or subcultured. Microglial cells were the main contaminants of the cerebellar astrocyte cultures but did not contribute to the NA or the SNP responses. No soluble guanylyl cyclase or calcium-dependent NO synthase (cNOS) activities were detected in microglial cultures. The effect of NA in cerebellar astrocytes was blocked by L-arginine analogues and by the alpha 1-adrenoceptor antagonist prazosin. The calcium ionophore A23187 mimicked the effect of NA and omission of calcium from the medium prevented both responses. NA did not elicit cGMP formation in granule cell cultures. These results support an astroglial location of the alpha 1-adrenoceptors and the cNOS that mediate NA stimulation of cGMP formation in cerebellum.
