Chronic hyperammonemia impairs the glutamate–nitric oxide–cyclic GMP pathway in cerebellar neurons in culture and in the rat in vivo

The aim of this work was to assess whether ammonia concentrations similar to the increase found in the brain of hyperammonemic rats (100 μm ), impair N-methyl-d -aspartate (NMDA) receptor-mediated signal transduction. We first measured glutamate neurotoxicity, which in these neurons is mediated by activation of NMDA receptors, as an initial parameter reflecting activation of NMDA receptor-mediated pathways. Long-term treatment of cultured neurons with ammonia prevents glutamate-induced neuronal death. The EC₅₀ was 20 μm , and at 100 μm the protection was complete. The induction of the protective effect was not immediate, but took several hours. Treatment with 100 μm ammonia did not prevent a glutamate- or NMDA-induced rise of intracellular calcium. Ammonia impaired the glutamate–nitric oxide–cGMP (3′,5′-cyclic guanosine monophosphate) pathway in a dose- and time-dependent manner. Glutamate-induced formation of cGMP was reduced by 42%, while activation of nitric oxide synthase was not affected. Ammonia reduced by 31% cGMP formation induced by S-nitroso-N-acetyl-penicillamine (SNAP), a NO-generating agent, confirming that the interference occurs at the level of guanylate cyclase activation by nitric oxide. To assess whether chronic moderate hyperammonemia in vivo also impairs the glutamate–nitric oxide–cGMP pathway, we determined by in vivo brain microdialysis in freely moving rats the formation of cGMP induced by NMDA. In hyperammonemic rats, the formation of cGMP induced by NMDA and SNAP was reduced by ca. 60 and 41%, respectively, indicating that chronic hyperammonemia in the animal in vivo also impairs the glutamate–nitric oxide–cGMP pathway. Impairment of this pathway can contribute to the neurological alterations found in hyperammonemia and hepatic encephalopathy.     

Prenatal exposure to polybrominated diphenylether 99 enhances the function of the glutamate-nitric oxide-cGMP pathway in brain in vivo and in cultured neurons

Polybrominated diphenylethers (PBDEs) are widely used as flame retardants. Significant amounts of PBDEs are present in the milk of lactating women. The possible neurotoxic effects of PBDEs are not well known. Perinatal exposure to PBDEs affects both motor and cognitive functions by mechanisms that remain unclear. Some types of learning depend on N-methyl-D-aspartate receptor activation, which increases intracellular calcium that binds to calmodulin and activates nitric oxide synthase, increasing nitric oxide formation that activates guanylate cyclase, increasing cGMP formation. Part of this cGMP is released to the extracellular fluid. We studied whether prenatal exposure of rats to PBDE99 alters the function of this glutamate-nitric oxide-cGMP pathway in rat brain in vivo. At 10 weeks of age, rats treated with PBDE99 showed increased function of the glutamate-nitric oxide-cGMP pathway in brain in vivo, as assessed by microdialysis in freely moving rats. The increased function of the pathway was reproduced in primary cultures of cerebellar neurons prepared from rats prenatally exposed to PBDE99 as well as in neurons cultured from normal rats and treated in vitro with PBDE99. Increased calmodulin content and activation of soluble guanylate cyclase by nitric oxide contributed to the increased function of the pathway.     

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 persent 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 α₁-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 α₁-adrenoceptors and the cNOS that mediate NA stimulation of cGMP formation in cerebellum.     

Nitric oxide promotes survival of cerebellar granule neurons cultured in vitro through the Akt pathway

In this study,cerebellar granule neurons were used to examine the role of nitric oxide on cell survival.The N-methyl-D-aspartic acid receptor antagonist,MK-801,and the soluble guanylate cyclase antagonist,1H-[1,2,4]oxadiazolo-[4,3-a] quinoxalin-1-one,decreased cell viability,induced caspase-3,and decreased phosphorylated-Akt levels,suggesting that blockade of nitric oxide production promotes apoptosis of differentiating cerebellar granule neurons.After administration of sodium nitroprusside,an endogenous nitric oxide donor,cell viability recovered,caspase-3 expression was decreased,and phosphorylated-Akt levels increased.This study provides direct evidence that nitric oxide can sustain the survival of developing cerebellar granule neurons in vitro through the nitric oxide-Akt pathway.Moreover,endogenous nitric oxide exerts these effects in a cyclic guanosine monophosphate-dependent manner while exogenous nitric oxide does so in a cyclic guanosine monophosphate-independent manner.     

Polychlorinated Biphenyls PCB 153 and PCB 126 Impair the Glutamate–Nitric Oxide–cGMP Pathway in Cerebellar Neurons in Culture by Different Mechanisms

Polychlorinated biphenyls (PCBs) are persistent organic pollutants present in human blood and milk. Exposure to PCBs during pregnancy and lactation leads to cognitive impairment in children. Perinatal exposure to PCB 153 or PCB 126 impairs the glutamate–nitric oxide–cGMP pathway in cerebellum in vivo and learning ability in adult rats. The aims of this work were: (1) to assess whether long-term exposure of primary cultures of cerebellar neurons to PCB 153 or PCB 126 reproduces the impairment in the function of the glutamate–nitric oxide–cGMP pathway found in rat cerebellum in vivo; (2) to provide some insight on the steps of the pathway affected by these PCBs; (3) to assess whether the mechanisms of interference of the pathway are different for PCB 126 and PCB 153. Both PCB 153 and PCB 126 increase basal levels of cGMP by different mechanisms. PCB 126 increases the amount of soluble guanylate cyclase while PCB 153 does not. PCB 153 reduces the amount of calmodulin while PCB 126 does not. Also both PCBs impair the function of the glutamate–nitric oxide–cGMP pathway by different mechanisms, PCB 153 impairs nitric oxide-induced activation of soluble guanylate cyclase and increase in cGMP while PCB 126 does not. PCB 126 reduces NMDA-induced increase in calcium while PCB 153 does not. When PCB 153 and PCB 126 exhibit the same effect, PCB 126 was more potent than PCB 153, as occurs in vivo.     

Carnitine prevents NMDA receptor-mediated activation of MAP-kinase and phosphorylation of microtubule-associated protein 2 in cerebellar neurons in culture

Activation of N-methyl-D-aspartate (NMDA) receptors leads to increased phosphorylation of the microtubule-associated protein MAP-2 by a mechanism that involves activation of nitric oxide synthase and nitric oxide-induced activation of mitogen-activated protein kinase (MAP-kinase). We have assessed the effects of carnitine on this signal transduction pathway in primary cultures of rat cerebellar neurons. We show that carnitine inhibits NMDA-induced phosphorylation of MAP-2 and that this is due to decreased activation of MAP-kinase. This effect is not due to inhibition by carnitine of NMDA-induced activation of nitric oxide synthase or to quenching of the nitric oxide formed, which are not affected by carnitine. Carnitine also inhibits the increase in phosphorylation of MAP-2 induced by the nitric oxide-generating agent S-nitroso-N-acetylpenicillamine, but not nitric oxide-induced activation of soluble guanylate cyclase. These results indicate that carnitine interferes with NMDA-induced, nitric oxide mediated activation of MAP-kinase at a step subsequent to nitric oxide formation.     

Predominant localization in glial cells of freel-arginine. Immunocytochemical evidence

Nitric oxide has been recently identified as an endogenous activator of the soluble guanylate cyclase in the brain as well as in vascular endothelial cells and macrophages. In the present study, we determined the localization of free arginine in the brain because nitric oxide was formed from the terminal guanido group ofl-arginine. Anti-arginine antiserum was raised in guinea pigs by repeated injection ofl-arginine covalently conjugated to guinea pig serum albumin via glutaraldehyde. Specicic anti-arginine antibody was purified from the antiserum by using an affinity gel coupled withl-arginine. Arginine-like immunoreactivity in the rat brain and spinal cord was found concentrated mainly in astrocytes including Bergmann glial cells in the cerebellum and processes of astrocytes around blood vessels. The present results suggest that glial cells, particularly astrocytes, are the main locus ofl-arginine, a nitric oxide precursor, in the brain.     

Predominant localization in glial cells of free L-arginine. Immunocytochemical evidence

Nitric oxide has been recently identified as an endogenous activator of the soluble guanylate cyclase in the brain as well as in vascular endothelial cells and macrophages. In the present study, we determined the localization of free arginine in the brain because nitric oxide was formed from the terminal guanido group of L-arginine. Anti-arginine antiserum was raised in guinea pigs by repeated injection of L-arginine covalently conjugated to guinea pig serum albumin via glutaraldehyde. Specific anti-arginine antibody was purified from the antiserum by using an affinity gel coupled with L-arginine. Arginine-like immunoreactivity in the rat brain and spinal cord was found concentrated mainly in astrocytes including Bergmann glial cells in the cerebellum and processes of astrocytes around blood vessels. The present results suggest that glial cells, particularly astrocytes, are the main locus of L-arginine, a nitric oxide precursor, in the brain.     

The effect of acute and repeated hyperammonemia on γ-glutamyl transpedtidase in homogenates and capillaries of various rat brain regions

The effect of hyperammonemia of varying degree and duration on the γ-glutamyl-transpeptidase (GGT) activity was studied in the homogenates and capillaries of different brain regions of the rat. “Acute” hyperammonemia (750 and 600 mg of ammonium acetate per kg b. w. were injected i. p. at 30 min interval, and the animals were decapitated immediately), in which blood ammonia was increased 14-fold, and brain ammonia six-fold above the control level, produced a 20% increase of the enzyme activity in cerebellum, and a 17% decrease in gyrus dentatus, but had no effect in the frontal cortex and the CA1 and CA3 regions of hippocampus. “Subchronic” hyperammonemia (two injectons of 600 mg ammonium acetate/kg were given at 24 h intervals, and tissue samples were removed 24 h later), that was accompanied by only a 60% increase of blood or brain ammonia, increased the activity in cerebellum to 38% above control, but produced no effect in the other brain regions. “Chronic” hyperammonemia (three injections of 600 mg ammonium acetate/kg at 24 h intervals and excision of tissue samples 30 min after the last injection), in which blood and brain ammonia were, respectively, 60 and 100% higher than in control animals, elevated the GGT, activity in the cerebellum by 57%, in CA1 by 15%, and in CA3 by 21%, but produced no effect in the frontal cortex or gyrus dentatus. By contrast, “chronic” hyperammonemia produced a 30% increase of GGT activity in cerebral cortical capillaries, but only a 10% increase in hippocampal capillaries, and no change in cerebellar capillaries. The results suggest that, hyperammonemia of relatively long duration may contribute to the enhancement of brain GGT activity observed in chronic forms of hepatic encephalopathy. However, ammonia does not appear to activate the enzyme directly.     

Protein kinases, nitric oxide and long-term depression of synapses in the cerebellum

We have analysed the effects of polymyxin B, a potent inhibitor of calcium-dependent protein kinases, of L-N-monomethylarginine, an inhibitor of nitric oxide synthesis, and of methylene blue which prevents activation of soluble guanylate cyclase, on long-term depression of parallel fibre-mediated EPSPs of rat cerebellar Purkinje cells in slices maintained in-vitro. In control conditions, a long-term depression of parallel fibre-mediated EPSPs was consistently induced by their pairing with calcium spikes directly elicited in the postsynaptic cells. This long-term change in synaptic strength was not observed in the presence of polymyxin B, of L-N-monomethylarginine, or of methylene blue, suggesting that calcium-dependent protein kinases and nitric oxide are both involved.     

Whole brain spheroid cultures as a model to study the development of nitric oxide synthase-guanylate cyclase signal transduction

Whole brain spheroids provide a suitable model to study neurodevelopment. In the literature a role for the nitric oxide (NO)-cyclic guanosine 3',5'-monophosphate (cGMP) signalling pathway during development has frequently been suggested. In this study we investigated whether functional cGMP pathways were present in differentiated spheroids. In 3-week-old spheroids soluble guanylate cyclase was stimulated with N-methyl D-aspartic acid or sodium nitroprusside (NO donor). The results showed that the NO synthase-cGMP pathway is present in the culture system. Soluble guanylate cyclase-dependent cGMP formation was found in NO synthase containing neurons, in neurons of the GABAergic, glutamatergic and cholinergic system, and in astroglia and oligodendroglia. Activation of particulate guanylate cyclase by atrial natriuretic peptide also triggered an increase in cGMP production. Particulate guanylate cyclase was found in astroglia and in microglia as well as in glutamic acid decarboxylase and calbindin containing structures and neuronal NO synthase containing neurons. Chronic inhibition of NO synthase during culture development had no effect on soluble or particulate guanylate cyclase functioning. Similarly, inhibition of soluble guanylate cyclase during culture development did not have any effect on NO synthase and particulate guanylate cyclase functioning. It is concluded that NO synthase and both soluble and particulate guanylate cyclase are present in whole brain spheroid cultures and that their activity can be influenced by several stimuli. The spheroid culture system constitutes a suitable model to study the NO-cGMP pathway during brain development in mammals.     

Hyperammonemia impairs long-term potentiation in hippocampus by altering the modulation of cGMP-degrading phosphodiesterase by protein kinase G

Hyperammonemia impairs long-term potentiation (LTP) in hippocampus, by an unknown mechanism. LTP in hippocampal slices requires activation of the soluble guanylate cyclase (sGC)-protein kinase G (PKG)-cGMP-degrading phosphodiesterase pathway. The aim of this work was to assess whether hyperammonemia impairs LTP by impairing the tetanus-induced activation of this pathway. The tetanus induced a rapid cGMP rise, reaching a maximum at 10 s, both in the absence or presence of ammonia. The increase in cGMP is followed in control slices by a sustained decrease in cGMP due to PKG-mediated activation of cGMP-degrading phosphodiesterase, which is required for maintenance of LTP. Hyperammonemia prevents completely tetanus-induced cGMP decrease by impairing PKG-mediated activation of cGMP-degrading phosphodiesterase. Addition of 8Br-cGMP to slices treated with ammonia restores both phosphodiesterase activation and maintenance of LTP. Impairment of LTP in hyperammonemia may be involved in the impairment of the cognitive function in patients with hepatic encephalopathy.     

CNTF protects oligodendrocytes from ammonia toxicity: intracellular signaling pathways involved

In pediatric patients, hyperammonemia can provoke irreversible damages to developing CNS like cortical atrophy, ventricular enlargement, demyelination or gray and white matter hypodensities which are concordant with alterations of neurons and oligodendrocytes. Cerebral injury triggers endogenous protective mechanisms that can prevent or limit brain damage. Understanding these mechanisms may lead to new therapeutic strategies. We investigated whether ciliary neurotrophic factor (CNTF), a cytokine-like protein expressed by astrocytes and described as an injury-associated survival factor, was up-regulated by ammonia in developing reaggregated 3D brain cell cultures. We showed that CNTF is up-regulated by ammonia exposure, through mediation of p38 MAPK activation in astrocytes. We also observed that SAPK/JNK and Erk1/2 activations in oligodendrocytes and neurons, respectively, also play indirect roles in CNTF synthesis by astrocytes. Co-treatment with exogenous CNTF demonstrated strong protective effects on oligodendrocytes, but not on neurons, against ammonia toxicity. These protective effects involved JAK/STAT, SAPK/JNK and c-jun proteins.     

Intercellular action of nitric oxide increases cGMP in cerebellar Purkinje cells

cGMP is thought to play a role in cerebellar signalling yet its production within Purkinje cells has never been detected. In the present study, the hydrolysis of a fluorescent substrate analogue, 2'-O-anthranyloyl cyclic GMP, by type 5 phosphodiesterase was monitored within Purkinje cells in slices and in culture. Nitric oxide, either endogenously released from adjacent neurons or pharmacologically applied, accelerated the rate of hydrolysis in a manner that was dependent on soluble guanylyl cyclase, demonstrating that nitric oxide triggers cyclic GMP production in Purkinje cells, which in turn activates type 5 phosphodiesterase. We conclude that NO acts as an intercellular messenger in the cerebellar cortex and that parallel fibre terminals are a probable source of nitric oxide.     

Role of nitric oxide and cyclic GMP in glutamate-induced neuronal death

Glutamate is the main excitatory neurotransmitter in mammals. However, excessive activation of glutamate receptors is neurotoxic, leading to neuronal degeneration and death. In many systems, including primary cultures of cerebellar neurons, glutamate neurotoxicity is mainly mediated by excessive activation of NMDA receptors, leading to increased intracellular calcium which binds to calmodulin and activates neuronal nitric oxide synthase (NOS), increasing nitric oxide (NO) which in turn activates guanylate cyclase and increases cGMP. Inhibition of NOS prevents glutamate neurotoxicity, indicating that NO mediates glutamate-induced neuronal death in this system. NO generating agents such as SNAP also induce neuronal death. Compounds that can act as “scavengers” of NO such as Croman 6 (CR-6) prevent glutamate neurotoxicity. The role of cGMP in the mediation of glutamate neurotoxicity remain controversial. Some reports indicate that cGMP mediates glutamate neurotoxicity while others indicate that cGMP is neuroprotective. We have studied the role of cGMP in the mediation of glutamate and NO neurotoxicity in cerebellar neurons. Inhibition of soluble guanylate cyclase prevents glutamate and NO neurotoxicity. There is a good correlation between inhibition of cGMP formation and neuroprotection. Moreover 8-Br-cGMP, a cell permeable analog of cGMP, induced neuronal death. These results indicate that increased intracellular cGMP is involved in the mechanism of neurotoxicity. Inhibitors of phosphodiesterase increased extracellular but not intracellular cGMP and prevented glutamate neurotoxicity. Addition of cGMP to the medium also prevented glutamate neurotoxicity. These results are compatible with a neurotoxic effect of increased intracellular cGMP and a neuroprotective effect of increased extracellular cGMP.     

Distribution and different activation of adenylate cyclase by naf and of guanylate cyclase by NaN3 in neuronal and glial cells separated from rat cerebral cortex

Distribution of adenylate cyclase and guanylate cyclase activities in neuronal perikarya and glial cells separated from rat brain, and cellular differences in activation between of adenylate cyclase by NaF and of guanylate cyclase by NaN3 have been studied. Adenylate cyclase activity was higher in the glial cells than in the neuronal fraction, while guanylate cyclase activity was equally detected in both cell fractions. Adenylate cyclase was mainly derived from the particulate fraction of both brain cell homogenates, whereas the major portion of guanylate cyclase activity was found in their soluble rather than in the particulate fractions. Although bulk-separated neurons and glial cells almost failed to change intracellular cyclic nucleotide levels in response to some putative neurotransmitters, activation of adenylate cyclase by NaF was found to be greater in neuronal than in glial cell fractions, and was observed more clearly in the soluble than in the particulate fractions. Sodium azide greatly increased guanylate cyclase in the particulate fraction, but did not affect it considerably in the soluble one. Addition of catalase to the reaction mixture together with NaN3 further stimulated guanylate cyclase both int he soluble and the particulate fractions. These results suggest that adenylate cyclase and guanylate cyclase without intimate coupling to the transmitter-receptor system, but with activation by NaF or NaN3, may be distributed ubiquitously in the cells separated from rat cerebral cortex.     

Age-related Changes in the NMDA Receptor/Nitric Oxide/cGMP Pathway in the Hippocampus and Cerebellum of Freely Moving Rats Subjected to Transcerebral Microdialysis

The N-methyl-d -aspartate (NMDA) receptor/nitric oxide synthase/guanylate cyclase pathway was studied during aging by monitoring extracellular cGMP in the rat hippocampus and cerebellum during in vivo microdialysis. In the hippocampus the basal cGMP efflux decreased by 50% from 3 to 12 months of age, whereas it remained constant with age in the cerebellum. Locally perfused NMDA (1 mM) evoked remarkable cGMP responses in 3-month-old rats; in the hippocampus the cGMP production was already dramatically reduced at 12 months, whereas in the cerebellum a similar impairment occurred much later (24 months). The nitric oxide donor S-nitroso-N-penicillamine (1 mM) elicited cGMP responses which slightly decreased from 3 to 12–24 months in the hippocampus, while no significant decrement with age could be seen in the cerebellum. Local perfusion of the phosphodiesterase inhibitor 3-isobutyl-1 methylxanthine (IBMX, 1 mM) produced large increases in hippocampal cGMP levels. The response decreased at 12 and 24 months, apparently in parallel with the fall in the basal level of cGMP. No significant differences across ages were observed following IBMX infusion in the cerebellum. The decreases in basal outflow and in the NMDA-evoked cGMP response seen in the aged hippocampus were not compensated for by supplying l -arginine. Infusion of d -serine (1 mM) enhanced (150–200%) extracellular cGMP in the cerebellum with no age-related differences. The activity in vitro, of hippocampal nitric oxide synthase at 24 months was 33% lower than at 3 months, whereas the cerebellar enzyme did not show any age-related decay. Aging seems therefore to affect differentially the NMDA receptor/nitric oxide synthase/cGMP pathway in the rat hippocampus versus the cerebellum. In the hippocampus the early fall in the NMDA-evoked cGMP response seems to originate from deficits in NMDA receptor function and nitric oxide synthase and guanylate cyclase activities; in the cerebellum, the decreased response to NMDA in the old animals seems essentially to be due to impairment of NMDA receptor function.