Agustina García

Different receptors mediate stimulation of nitric oxide-dependent cyclic GMP formation in neurons and astrocytes in culture

The ability of various compounds to stimulate cyclic GMP accumulation was studied in neuronal and astrocyte-enriched primary cultures from rat cerebrum. Glutamate was the only agonist eliciting a response in neurons whereas several agonists had an effect in astrocytes but only those due to norepinephrine and glutamate were of considerable magnitude. The responses were markedly inhibited by the nitric oxide synthase inhibitor NG-monomethyl-L-arginine. The effect of glutamate appears to be mediated predominantly by NMDA receptors in neurons and by quisqualate AMPA-insensitive receptors in astrocytes.

Effect of zinc, copper and glucocorticoids on metallothionein levels of cultured neurons and astrocytes from rat brain

The knowledge of brain metallothionein (MT) regulation and especially of MT presence in specific cell types is scarce. Therefore, the effect of several well-known MT inducers, measured by radioimmunoassays using antibodies that cross-react with MT-I and MT-II or specific for MT-I and which do not cross-react with human growth inhibitory factor (GIF or MT-III), has been studied in primary cultures of neurons or astrocytes obtained from rat cerebrum. MT-I levels in glial cells were about ten times higher than those in neuronal cells (538 +/- 194 vs. 49 +/- 16 pg MT-I/micrograms protein, mean +/- S.D. from three separate cell preparations). Increasing the concentration of Zn in the bovine serum albumin (BSA)-containing culture medium up to 50 microM significantly increased MT-I levels by up to 3.5-fold in neurons and 2.5-fold in astrocytes. In contrast, Cu up to 50 microM increased MT-I levels in a saturable manner in both neurons (up to 5-fold) and astrocytes (up to 1.5-fold), the maximum effect occurring at 5 microM Cu. In general, the combination of Zn and Cu further increased MT-I levels. The effect of the metals on MT-I appeared to reflect metal uptake, since MT-I induction was less marked when the BSA concentration in the medium was increased from 2 to 10 mg/ml. Dexamethasone increased MT-I levels in both neurons and astrocytes in vitro in a concentration-dependent manner. Endotoxin, IL-1 and IL-6 did not have a significant effect on glial MT levels at the concentrations studied. The administration of dexamethasone to rats increased MT-I levels in non-frontal cortex, cerebellum, pons+medulla, midbrain and hippocampus, but not in hypothalamus, frontal cortex and striatum. Endotoxin increased liver but not brain MT-I levels. Immunocytochemical studies in adult rat brain preparations with a polyclonal antibody that cross-reacts with MT-I and MT-II indicated that immunostaining was always nuclear in glial cells, whereas in neurons it was nuclear in the cerebral cortex, hippocampus and the granular layer of the cerebellum, and nuclear plus cytoplasmic in Purkinje cells in the cerebellum, hypothalamic nuclei and gigantocellular reticular nucleus in the brain stem. Meninges, choroidal plexus, ependymal and endothelial cells were also MT-immunoreactive.

The cyclic GMP-protein kinase G pathway regulates cytoskeleton dynamics and motility in astrocytes

We have previously demonstrated that inflammatory compounds that increase nitric oxide (NO) synthase expression have a biphasic effect on the level of the NO messenger cGMP in astrocytes. In this work, we demonstrate that NO‐dependent cGMP formation is involved in the morphological change induced by lipopolysaccharide (LPS) in cultured rat cerebellar astroglia. In agreement with this, dibutyryl‐cGMP, a permeable cGMP analogue, and atrial natriuretic peptide, a ligand for particulate guanylyl cyclase, are both able to induce process elongation and branching in astrocytes resulting from a rapid, reversible and concentration‐dependent redistribution of glial fibrillary acidic protein (GFAP) and actin filaments without significant change in protein levels. These effects are also observed in astrocytes co‐cultured with neurons. The cytoskeleton rearrangement induced by cGMP is prevented by the specific protein kinase G inhibitor Rp‐8Br‐PET‐cGMPS and involves downstream inhibition of RhoA GTPase since is not observed in cells transfected with constitutively active RhoA. Furthermore, dibutyryl‐cGMP prevents RhoA‐membrane association, a step necessary for its interaction with effectors. Stimulation of the cGMP‐protein kinase G pathway also leads to increased astrocyte migration in an in vitro scratch‐wound assay resulting in accelerated wound closure, as seen in reactive gliosis following brain injury. These results indicate that cGMP‐mediated pathways may regulate physio‐pathologically relevant responses in astroglial cells.

Cyclic GMP inhibition of stimulated phosphoinositide hydrolysis in neuronal cultures

We used a radioreceptor binding assay to analyse the effect of dibutyryl cyclic GMP (dbcGMP) and the nitric oxide (NO) donor sodium nitroprusside (SNP) on inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) mass accumulation in response to the muscarinic receptor agonist carbachol and K(+)-induced depolarization in neuronal primary cultures. In forebrain neurones carbachol induced a transient rise in Ins(1,4,5)P3 with a maximal increase of about 2.5-fold at 5 s. At this time 30 mM K+ induced a somewhat lower stimulation. Both carbachol and high K+ stimulations were significantly inhibited by dbcGMP and by SNP. The effect of SNP was blocked by haemoglobin. dbcGMP inhibition of the carbachol effect was also observed in cerebellar granule cell cultures. These results show for the first time that the NO/cGMP generating system can modulate phosphoinositide turnover in CNS neurones in a manner similar to that previously reported to occur in peripheral neurones and non-neuronal tissues.

Calcium-dependent nitric oxide formation in glial cells

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.

Differential expression of metallothioneins in the CNS of mice with experimental autoimmune encephalomyelitis

Multiple sclerosis is an inflammatory, demyelinating disease of the CNS. Metallothioneins-I+II are antioxidant proteins induced in the CNS by immobilisation stress, trauma or degenerative diseases which have been postulated to play a neuroprotective role, while the CNS isoform metallothionein-III has been related to Alzheimers disease. We have analysed metallothioneins-I-III expression in the CNS of mice with experimental autoimmune encephalomyelitis. Moreover, we have examined the putative role of interferon-gamma, a pro-inflammatory cytokine, in the control of metallothioneins expression during experimental autoimmune encephalomyelitis in interferon-gamma receptor knockout mice with two different genetic backgrounds: 129/Sv and C57BL/6x129/Sv. Mice with experimental autoimmune encephalomyelitis showed a significant induction of metallothioneins-I+II in the spinal cord white matter, and to a lower extent in the brain. Interferon-gamma receptor knockout mice suffered from a more severe experimental autoimmune encephalomyelitis, and interestingly showed a higher metallothioneins-I+II induction in both white and grey matter of the spinal cord and in the brain. In contrast to the metallothioneins-I+II isoforms, metallothionein-III expression remained essentially unaltered during experimental autoimmune encephalomyelitis; interferon-gamma receptor knockout mice showed an altered metallothionein-III expression (a slight increase in the spinal cord white matter) only in the C57BL/6x129/Sv background. Metallothioneins-I+II proteins were prominent in areas of induced cellular infiltrates. Reactive astrocytes and activated monocytes/macrophages were the sources of metallothioneins-I+II proteins. From these results we suggest that metallothioneins-I+II but not metallothionein-III may play an important role during experimental autoimmune encephalomyelitis, and indicate that the pro-inflammatory cytokine interferon-gamma is unlikely an important factor in this response.

Norepinephrine increases cyclic GMP in astrocytes by a mechanism dependent on nitric oxide synthesis

Norepinephrine induces a rapid and concentration-dependent rise (EC50 = 1.21 +/- 0.33 microM) in cyclic GMP levels in astrocyte-enriched cultures from rat brain. The response is partially mediated by alpha 1-adrenoceptors since a marked inhibition is observed in the presence of prazosin while the beta-antagonist propranolol shows a smaller effect and the alpha 2-antagonist yohimbine is ineffective. L-NG-methylarginine, an inhibitor of nitric oxide synthesis from L-arginine, blocks the norepinephrine-induced cyclic GMP accumulation. This effect is reversed by L-arginine.

Characteristics of nitric oxide synthase type I of rat cerebellar astrocytes.

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 astrocyte‐enriched 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 100,000g, 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.

Sildenafil (Viagra) ameliorates clinical symptoms and neuropathology in a mouse model of multiple sclerosis

Cyclic GMP (cGMP)-mediated pathways regulate inflammatory responses in immune and CNS cells. Recently, cGMP phosphodiesterase inhibitors such as sildenafil, commonly used to treat sexual dysfunction in humans including multiple sclerosis (MS) patients, have been reported to be neuroprotective in animal models of stroke, Alzheimer’s disease, and focal brain lesion. In this work, we have examined if sildenafil ameliorates myelin oligodendrocyte glycoprotein peptide (MOG35–55)-induced experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. We show for the first time that treatment with sildenafil after disease onset markedly reduces the clinical signs of EAE by preventing axonal loss and promoting remyelination. Furthermore, sildenafil decreases CD3+ leukocyte infiltration and microglial/macrophage activation in the spinal cord, while increasing forkhead box transcription factor 3-expressing T regulatory cells (Foxp3 Tregs). However, sildenafil treatment did not significantly affect MOG35–55-stimulated proliferation or release of Th1/Th2 cytokines in splenocytes but decreased ICAM-1 in spinal cord infiltrated cells. The presence of reactive astrocytes forming scar-like structures around infiltrates was enhanced by sildenafil suggesting a possible mechanism for restriction of leukocyte spread into healthy parenchyma. These results highlight novel actions of sildenafil that may contribute to its beneficial effects in EAE and suggest that treatment with this widely used and well-tolerated drug may be a useful therapeutic intervention to ameliorate MS neuropathology.

Regulation by calcium of the nitric oxide/cyclic GMP system in cerebellar granule cells and astroglia in culture.

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 μM) and the Ca2+ ionophore A23187 (10 μM) 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 μM). 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. J. Neurosci. Res. 49:333–341, 1997.