cyclic-gmp and Hepatic-Encephalopathy

Hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome that results from liver failure and is characterized by a wide range of symptoms such as alteration in the sleep-waking cycle, neuromuscular coordination, mood, and cognition. The deregulation of nitric oxide (NO)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) signaling pathway is thought to play an important role in the etiology and progression of neurodegenerative diseases, and several studies pointed that the cGMP signaling is impaired in patients with HE and experimental models of chronic hyperammonemia. This review aimed to briefly present the current knowledge of the cGMP signaling pathways in neuroinflammation, neurogenesis, and memory in hepatic encephalopathy and its potential therapeutic role.

Topics: Animals; Cognition; Cyclic GMP; Disease Models, Animal; Hepatic Encephalopathy; Humans; Memory; Mice; Neurogenic Inflammation; Signal Transduction

Effects of ammonia on astrocytes play a major role in hepatic encephalopathy, acute liver failure and other diseases caused by increased arterial ammonia concentrations (e.g., inborn errors of metabolism, drug or mushroom poisoning). There is a direct correlation between arterial ammonia concentration, brain ammonia level and disease severity. However, the pathophysiology of hyperammonemic diseases is disputed. One long recognized factor is that increased brain ammonia triggers its own detoxification by glutamine formation from glutamate. This is an astrocytic process due to the selective expression of the glutamine synthetase in astrocytes. A possible deleterious effect of the resulting increase in glutamine concentration has repeatedly been discussed and is supported by improvement of some pathologic effects by GS inhibition. However, this procedure also inhibits a large part of astrocytic energy metabolism and may prevent astrocytes from responding to pathogenic factors. A decrease of the already low glutamate concentration in astrocytes due to increased synthesis of glutamine inhibits the malate-aspartate shuttle and energy metabolism. A more recently described pathogenic factor is the resemblance between NH

Topics: Ammonia; Animals; Astrocytes; Brain; Cyclic GMP; Energy Metabolism; Glutamine; Hepatic Encephalopathy; Humans; Hyperammonemia; Lactic Acid; Liver Failure, Acute; Potassium; Pyruvic Acid; Sodium-Potassium-Exchanging ATPase; Solute Carrier Family 12, Member 2

Cyclic GMP (cGMP) modulates important cerebral processes including some forms of learning and memory. cGMP pathways are strongly altered in hyperammonemia and hepatic encephalopathy (HE). Patients with liver cirrhosis show reduced intracellular cGMP in lymphocytes, increased cGMP in plasma and increased activation of soluble guanylate cyclase by nitric oxide (NO) in lymphocytes, which correlates with minimal HE assessed by psychometric tests. Activation of soluble guanylate cyclase by NO is also increased in cerebral cortex, but reduced in cerebellum, from patients who died with HE. This opposite alteration is reproduced in vivo in rats with chronic hyperammonemia or HE. A main pathway modulating cGMP levels in brain is the glutamate-NO-cGMP pathway. The function of this pathway is impaired both in cerebellum and cortex of rats with hyperammonemia or HE. Impairment of this pathway is responsible for reduced ability to learn some types of tasks. Restoring the pathway and cGMP levels in brain restores learning ability. This may be achieved by administering phosphodiesterase inhibitors (zaprinast, sildenafil), cGMP, anti-inflammatories (ibuprofen) or antagonists of GABAA receptors (bicuculline). These data support that increasing cGMP by safe pharmacological means may be a new therapeutic approach to improve cognitive function in patients with minimal or clinical HE.

Topics: Animals; Brain; Cyclic GMP; Glutamic Acid; Hepatic Encephalopathy; Humans; Hyperammonemia; Learning Disabilities; Nitric Oxide; Phosphodiesterase Inhibitors; Rats; Signal Transduction

Ammonia is a major neurotoxin implicated in hepatic encephalopathy (HE). Here we discuss evidence that many aspects of ammonia toxicity in HE-affected brain are mediated by glutamine (Gln), synthesized in excess from ammonia and glutamate by glutamine synthetase (GS), an astrocytic enzyme. The degree to which Gln is increased in brains of patients with HE was found to positively correlate with the grade of HE. In animals with HE, a GS inhibitor, methionine sulfoximine (MSO), reversed a spectrum of manifestations of ammonia toxicity, including brain edema and increased intracranial pressure, even though MSO itself increased brain ammonia levels. MSO inhibited, while incubation with Gln reproduced the oxidative stress and cell swelling observed in ammonia-exposed cultured astrocytes. Recent studies have shown that astrocytes swell subsequent to Gln transport into mitochondria and its degradation back to ammonia, which then generates reactive oxygen species and the mitochondrial permeability transition. This sequence of events led to the formulation of the "Trojan Horse" hypothesis. Further verification of the role of Gln in the pathogenesis of HE will have to account for: (1) modification of the effects of Gln by interaction of astrocytes with other CNS cells; and (2) direct effects of Gln on these cells. Recent studies have demonstrated a "Trojan Horse"-like effect of Gln in microglia, as well as an interference by Gln with the activation of the NMDA/NO/cGMP pathway by ammonia as measured in whole brain, a process that likely also involves neurons.

Topics: Ammonia; Animals; Astrocytes; Brain; Cyclic GMP; Glutamate-Ammonia Ligase; Glutaminase; Glutamine; Hepatic Encephalopathy; Humans; Methionine Sulfoximine; Mitochondria; Nitric Oxide

The NMDA type of glutamate receptors modulates learning and memory. Excessive activation of NMDA receptors leads to neuronal degeneration and death. Hyperammonemia and liver failure alter the function of NMDA receptors and of some associated signal transduction pathways. The alterations are different in acute and chronic hyperammonemia and liver failure. Acute intoxication with large doses of ammonia (and probably acute liver failure) leads to excessive NMDA receptors activation, which is responsible for ammonia-induced death. In contrast, chronic hyperammonemia induces adaptive responses resulting in impairment of signal transduction associated to NMDA receptors. The function of the glutamate-nitric oxide-cGMP pathway is impaired in brain in vivo in animal models of chronic liver failure or hyperammonemia and in homogenates from brains of patients died in hepatic encephalopathy. The impairment of this pathway leads to reduced cGMP and contributes to impaired cognitive function in hepatic encephalopathy. Learning ability is reduced in animal models of chronic liver failure and hyperammonemia and is restored by pharmacological manipulation of brain cGMP by administering phosphodiesterase inhibitors (zaprinast or sildenafil) or cGMP itself. NMDA receptors are therefore involved both in death induced by acute ammonia toxicity (and likely by acute liver failure) and in cognitive impairment in hepatic encephalopathy.

Topics: Adenosine Triphosphate; Animals; Brain; Cyclic GMP; Free Radicals; Hepatic Encephalopathy; Humans; Hyperammonemia; Liver Failure; Nitric Oxide; Receptors, N-Methyl-D-Aspartate; Sodium-Potassium-Exchanging ATPase

Patients with liver disease may present hepatic encephalopathy (HE), a complex neuropsychiatric syndrome covering a wide range of neurological alterations, including cognitive and motor disturbances. HE reduces the quality of life of the patients and is associated with poor prognosis. In the worse cases HE may lead to coma or death. The mechanisms leading to HE which are not well known are being studied using animal models. The neurological alterations in HE are a consequence of impaired cerebral function mainly due to alterations in neurotransmission. We review here some studies indicating that alterations in neurotransmission associated to different types of glutamate receptors are responsible for some of the cognitive and motor alterations present in HE. These studies show that the function of the signal transduction pathway glutamate-nitric oxide-cGMP associated to the NMDA type of glutamate receptors is impaired in brain in vivo in HE animal models as well as in brain of patients died of HE. Activation of NMDA receptors in brain activates this pathway and increases cGMP. In animal models of HE this increase in cGMP induced by activation of NMDA receptors is reduced, which is responsible for the impairment in learning ability in these animal models. Increasing cGMP by pharmacological means restores learning ability in rats with HE and may be a new therapeutic approach to improve cognitive function in patients with HE. However, it is necessary to previously assess the possible secondary effects. Patients with HE may present psychomotor slowing, hypokinesia and bradykinesia. Animal models of HE also show hypolocomotion. It has been shown in rats with HE that hypolocomotion is due to excessive activation of metabotropic glutamate receptors (mGluRs) in substantia nigra pars reticulata. Blocking mGluR1 in this brain area normalizes motor activity in the rats, suggesting that a similar treatment for patients with HE could be useful to treat psychomotor slowing and hypokinesia. However, the possible secondary effects of mGluR1 antagonists should be previously evaluated. These studies are setting the basis for designing therapeutic procedures to specifically treat the individual neurological alterations in patients with HE.

Topics: Animals; Brain; Cognition; Cyclic GMP; Glutamic Acid; Hepatic Encephalopathy; Humans; Motor Activity; Nitric Oxide; Receptors, Glutamate; Receptors, Metabotropic Glutamate; Receptors, N-Methyl-D-Aspartate; Signal Transduction

Rats with chronic hyperammonemia reproduce the cognitive and motor impairment present in patients with hepatic encephalopathy. It has been proposed that enhanced GABAergic neurotransmission in hippocampus may contribute to impaired learning and memory in hyperammonemic rats. However, there are no direct evidences of the effects of hyperammonemia on GABAergic neurotransmission in hippocampus or on the underlying mechanisms. The aims of this work were to assess if chronic hyperammonemia enhances the function of GABA

Topics: Animals; Cyclic GMP; GABA Plasma Membrane Transport Proteins; gamma-Aminobutyric Acid; Hepatic Encephalopathy; Hippocampus; Humans; Hyperammonemia; Rats; Rats, Wistar; Receptors, GABA-A; Synaptic Transmission

Alterations in brain nitric oxide (NO)/cGMP synthesis contribute to the pathogenesis of hepatic encephalopathy (HE). An increased asymmetrically dimethylated derivative of L-arginine (ADMA), an endogenous inhibitor of NO synthases, was observed in plasma of HE patients and animal models. It is not clear whether changes in brain ADMA reflect its increased local synthesis therefore affecting NO/cGMP pathway, or are a consequence of its increased peripheral blood content. We measured extracellular concentration of ADMA and symmetrically dimethylated isoform (SDMA) in the prefrontal cortex of control and thioacetamide (TAA)-induced HE rats. A contribution of locally synthesized dimethylarginines (DMAs) in their extracellular level in the brain was studied after direct infusion of the inhibitor of DMAs synthesizing enzymes (PRMTs), S-adenosylhomocysteine (AdoHcy, 2 mM), or the methyl donor, S-adenosylmethionine (AdoMet, 2 mM), via a microdialysis probe. Next, we analyzed whether locally synthesized ADMA attains physiological significance by determination of extracellular cGMP. The expression of PRMT-1 was also examined. Concentration of ADMA and SDMA, detected by positive mode electrospray LC-DMS-MS/MS, was greatly enhanced in TAA rats and was decreased (by 30 %) after AdoHcy and AdoMet infusion. TAA-induced increase (by 40 %) in cGMP was unaffected after AdoHcy administration. The expression of PRMT-1 in TAA rat brain was unaltered. The results suggest that (i) the TAA-induced increase in extracellular DMAs may result from their effective synthesis in the brain, and (ii) the excess of extracellular ADMA does not translate into changes in the extracellular cGMP concentration and implicate a minor role in brain NO/cGMP pathway control.

Topics: Animals; Arginine; Cyclic GMP; Disease Models, Animal; Extracellular Space; Hepatic Encephalopathy; Liver Failure, Acute; Male; Prefrontal Cortex; Protein-Arginine N-Methyltransferases; Rats, Sprague-Dawley; RNA, Messenger; S-Adenosylhomocysteine; S-Adenosylmethionine; Signal Transduction

Peripheral inflammation contributes to the neurological alterations in hepatic encephalopathy (HE). Neuroinflammation and altered GABAergic neurotransmission mediate cognitive and motor alterations in rats with HE. It remains unclear (a) if neuroinflammation and neurological impairment in HE are a consequence of peripheral inflammation and (b) how neuroinflammation impairs GABAergic neurotransmission. The aims were to assess in rats with HE whether reducing peripheral inflammation with anti-TNF-α (1) prevents cognitive impairment and motor in-coordination, (2) normalizes neuroinflammation and extracellular GABA in the cerebellum and also (3) advances the understanding of mechanisms linking neuroinflammation and increased extracellular GABA.. Rats with HE due to portacaval shunt (PCS) were treated with infliximab. Astrocytes and microglia activation and TNF-α and IL-1β were analyzed by immunohistochemistry. Membrane expression of the GABA transporters GAT-3 and GAT-1 was analyzed by cross-linking with BS3. Extracellular GABA was analyzed by microdialysis. Motor coordination was tested using the beam walking and learning ability using the Y maze task.. PCS rats show peripheral inflammation, activated astrocytes, and microglia and increased levels of TNF-α and IL-1β. Membrane expression of GAT-3 and extracellular GABA are increased, leading to impaired motor coordination and learning ability. Infliximab reduces peripheral inflammation, microglia, and astrocyte activation and neuroinflammation and normalizes GABAergic neurotransmission, motor coordination, and learning ability.. Neuroinflammation is associated with altered GABAergic neurotransmission and increased GAT-3 membrane expression and extracellular GABA (a); peripheral inflammation is a main contributor to the impairment of motor coordination and of the ability to learn the Y maze task in PCS rats (b); and reducing peripheral inflammation using safe procedures could be a new therapeutic approach to improve cognitive and motor function in patients with HE

Topics: Animals; Anti-Inflammatory Agents; Cerebellum; Cyclic GMP; Cytokines; Dinoprostone; Disease Models, Animal; Extracellular Fluid; GABA Plasma Membrane Transport Proteins; gamma-Aminobutyric Acid; Glial Fibrillary Acidic Protein; Hepatic Encephalopathy; Inflammation; Infliximab; Learning Disabilities; Male; Maze Learning; Psychomotor Disorders; Rats; Rats, Wistar

Patients with liver cirrhosis show disturbances in sleep and in its circadian rhythms which are an early sign of minimal hepatic encephalopathy (MHE). The mechanisms of these disturbances are poorly understood. Rats with porta-caval shunt (PCS), a model of MHE, show sleep disturbances reproducing those of cirrhotic patients. The aims of this work were to characterize the alterations in circadian rhythms in PCS rats and analyze the underlying mechanisms. To reach these aims, we analyzed in control and PCS rats: (a) daily rhythms of spontaneous and rewarding activity and of temperature, (b) timing of the onset of activity following turning-off the light, (c) synchronization to light after a phase advance and (d) the molecular mechanisms contributing to these alterations in circadian rhythms. PCS rats show altered circadian rhythms of spontaneous and rewarding activities (wheel running). PCS rats show more rest bouts during the active phase, more errors in the onset of motor activity and need less time to re-synchronize after a phase advance than control rats. Circadian rhythm of body temperature is also slightly altered in PCS rats. The internal period length (tau) of circadian rhythm of motor activity is longer in PCS rats. We analyzed some mechanisms by which hypothalamus modulate circadian rhythms. PCS rats show increased content of cGMP in hypothalamus while the activity of cGMP-dependent protein kinase was reduced by 41% compared to control rats. Altered cGMP-PKG pathway in hypothalamus would contribute to altered circadian rhythms and synchronization to light.

Topics: Activity Cycles; Animals; Behavior, Animal; Body Temperature Regulation; Chronobiology Disorders; Circadian Rhythm; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Disease Models, Animal; Down-Regulation; Hepatic Encephalopathy; Hypothalamus; Male; Motor Activity; Photoperiod; Portacaval Shunt, Surgical; Rats, Wistar; Running; Sleep; Time Factors

Liver failure in experimental animals or in human cirrhosis elicits neuroinflammation. Prolyl oligopeptidase (PREP) has been implicated in neuroinflammatory events in neurodegenerative diseases: PREP protein levels are increased in brain glial cells upon neuroinflammatory insults, but the circulating PREP activity levels are decreased in multiple sclerosis patients in a process probably mediated by bioactive peptides. In this work, we studied the variation of PREP levels upon liver failure and correlated it with several inflammatory markers to conclude on the relation of PREP with systemic and/or neuroinflammation.. PREP enzymatic activity and protein levels measured with immunological techniques were determined in the brain and plasma of rats with portacaval shunt (PCS) and after treatment with ibuprofen. Those results were compared with the levels of PREP measured in plasma from cirrhotic patients with or without minimal hepatic encephalopathy (MHE). Levels of several pro-inflammatory cytokines and those of NO/cGMP homeostasis metabolites were measured in PCS rats and cirrhotic patients to conclude on the role of PREP in inflammation.. In PCA rats, we found that PREP levels are significantly increased in the hippocampus, striatum and cerebellum, that in the cerebellum the PREP increase was significantly found in the extracellular space and that the levels were restored to those measured in control rats after administration of an anti-inflammatory agent, ibuprofen. In cirrhotic patients, circulatory PREP activity was found to correlate to systemic and neuroinflammatory markers and had a negative correlation with the severity of the disease, although no clear relation to MHE.. These results support the idea that PREP levels could be used as indicators of cirrhosis severity in humans, and using other markers, it might contribute to assessing the level of neuroinflammation in those patients. This work reports, for the first time, that PREP is secreted to the extracellular space in the cerebellum most probably due to glial activation and supports the role of the peptidase in the inflammatory response.

Topics: Adult; Aged; Animals; Anti-Inflammatory Agents, Non-Steroidal; Brain; Cyclic GMP; Cytokines; Disease Models, Animal; Fibrosis; Hepatic Encephalopathy; Humans; Ibuprofen; Liver Failure; Lymphocytes; Male; Middle Aged; Nitric Oxide; Portacaval Shunt, Surgical; Prolyl Oligopeptidases; Rats; Rats, Wistar; Serine Endopeptidases; Systemic Inflammatory Response Syndrome

In a previous study by our group memory impairment in rats with minimal hepatic encephalopathy (MHE) was associated with the inhibition of the glutamate‑nitric oxide‑cyclic guanosine monophosphate (Glu‑NO‑cGMP) pathway due to elevated dopamine (DA). However, the effects of DA on the Glu‑NO‑cGMP pathway localized in primary cortical astrocytes (PCAs) had not been elucidated in rats with MHE. In the present study, it was identified that when the levels of DA in the cerebral cortex of rats with MHE and high‑dose DA (3 mg/kg)‑treated rats were increased, the co‑localization of N‑methyl‑d‑aspartate receptors subunit 1 (NMDAR1), calmodulin (CaM), nitric oxide synthase (nNOS), soluble guanylyl cyclase (sGC) and cyclic guanine monophosphate (cGMP) with the glial fibrillary acidic protein (GFAP), a marker protein of astrocytes, all significantly decreased, in both the MHE and high‑dose DA‑treated rats (P<0.01). Furthermore, NMDA‑induced augmentation of the expression of NMDAR1, CaM, nNOS, sGC and cGMP localized in PCAs was decreased in MHE and DA‑treated rats, as compared with the controls. Chronic exposure of cultured cerebral cortex PCAs to DA treatment induced a dose‑dependent decrease in the concentration of intracellular calcium, nitrites and nitrates, the formation of cGMP and the expression of NMDAR1, CaM, nNOS and sGC/cGMP. High doses of DA (50 µM) significantly reduced NMDA‑induced augmentation of the formation of cGMP and the contents of NMDAR1, CaM, nNOS, sGC and cGMP (P<0.01). These results suggest that the suppression of DA on the Glu‑NO‑cGMP pathway localized in PCAs contributes to memory impairment in rats with MHE.

Topics: Animals; Astrocytes; Behavior, Animal; Calmodulin; Cerebral Cortex; Cyclic GMP; Disease Models, Animal; Dopamine; Dose-Response Relationship, Drug; Glial Fibrillary Acidic Protein; Glutamates; Guanylate Cyclase; Hepatic Encephalopathy; Memory; N-Methylaspartate; Nitric Oxide; Nitric Oxide Synthase; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Receptors, N-Methyl-D-Aspartate; Signal Transduction; Soluble Guanylyl Cyclase

To assess whether non invasive blood flow measurement by arterial spin labeling in several brain regions detects minimal hepatic encephalopathy.. Blood flow (BF) was analyzed by arterial spin labeling (ASL) in different brain areas of 14 controls, 24 cirrhotic patients without and 16 cirrhotic patients with minimal hepatic encephalopathy (MHE). Images were collected using a 3 Tesla MR scanner (Achieva 3T-TX, Philips, Netherlands). Pulsed ASL was performed. Patients showing MHE were detected using the battery Psychometric Hepatic Encephalopathy Score (PHES) consisting of five tests. Different cognitive and motor functions were also assessed: alterations in selective attention were evaluated using the Stroop test. Patients and controls also performed visuo-motor and bimanual coordination tests. Several biochemical parameters were measured: serum pro-inflammatory interleukins (IL-6 and IL-18), 3-nitrotyrosine, cGMP and nitrates+nitrites in plasma, and blood ammonia. Bivariate correlations were evaluated.. In patients with MHE, BF was increased in cerebellar hemisphere (P = 0.03) and vermis (P = 0.012) and reduced in occipital lobe (P = 0.017). BF in cerebellar hemisphere was also increased in patients without MHE (P = 0.02). Bimanual coordination was impaired in patients without MHE (P = 0.05) and much more in patients with MHE (P < 0.0001). Visuo-motor coordination was impaired only in patients with MHE (P < 0.0001). Attention was slightly affected in patients without MHE and more strongly in patients with MHE (P < 0.0001). BF in cerebellar hemisphere and vermis correlated with performance in most tests of PHES [(number connection tests A (NCT-A), B (NCT-B)and line tracing test] and in the congruent task of Stroop test. BF in frontal lobe correlated with NCT-A. Performance in bimanual and visuomotor coordination tests correlated only with BF in cerebellar hemisphere. BF in occipital lobe correlates with performance in the PHES battery and with CFF. BF in cerebellar hemisphere correlates with plasma cGMP and nitric oxide (NO) metabolites. BF in vermis cerebellar also correlates with NO metabolites and with 3-nitrotyrosine. IL-18 in plasma correlates with BF in thalamus and occipital lobe.. Non invasive BF determination in cerebellum using ASL may detect MHE earlier than the PHES. Altered NO-cGMP pathway seems to be associated to altered BF in cerebellum.

Topics: Aged; Ammonia; Attention; Biomarkers; Blood Flow Velocity; Cerebellum; Cerebrovascular Circulation; Cognition; Cyclic GMP; Early Diagnosis; Female; Hepatic Encephalopathy; Humans; Inflammation Mediators; Liver Cirrhosis; Magnetic Resonance Imaging; Male; Middle Aged; Motor Activity; Nitric Oxide; Perfusion Imaging; Predictive Value of Tests; Psychometrics; Regional Blood Flow; Retrospective Studies; Stroop Test

Cirrhotic patients with minimal hepatic encephalopathy (MHE) show impaired driving ability and increased vehicle accidents. The neurological deficits contributing to impair driving and the underlying mechanisms are poorly understood. Early detection of driving impairment would help to reduce traffic accidents in MHE patients. It would be therefore useful to have psychometric or biochemical parameters reflecting driving impairment. The aims of this work were as follows: (i) to shed light on the neurological deficits contributing to impair driving; (ii) to assess whether some psychometric test or biochemical parameter is a good indicator of driving impairment.. We assessed in 22 controls, 36 cirrhotic patients without and 15 with MHE, driving performance using a driving simulator (SIMUVEG) and Driver Test. MHE was diagnosed using the psychometric hepatic encephalopathy score (PHES). Psychometric tests assessing different neurological functions (mental processing speed, attention, visuo-spatial and bimanual coordination) were performed. Blood ammonia and parameters related with nitric oxide-cGMP metabolism, IL-6, IL-18 and 3-nitrotyrosine were measured.. Patients with MHE showed impaired driving ability correlating with MHE grade, with impaired vehicle lateral control in spite of reduced driving speed. Patients with MHE show psychomotor slowing, longer reaction times, impaired bimanual and visuo-spatial coordination and concentrated attention and slowed speed of anticipation and increased blood ammonia, cGMP, IL-6, IL-18 and 3-nitrotyrosine.. Impaired mental processing speed, attention and alterations in visuo-spatial and motor coordination seem main contributors to impaired driving ability in patients with MHE. Increased serum 3-nitrotyrosine is associated with impaired driving ability.

Topics: Adult; Aged; Analysis of Variance; Automobile Driving; Biomarkers; Chemokines; Cyclic GMP; Flicker Fusion; Hepatic Encephalopathy; Humans; Liver Cirrhosis; Middle Aged; Nitric Oxide; Psychometrics; Tyrosine

The pathogenesis of hepatic encephalopathy (HE) is associated with hyperammonemia (HA) and subsequent exposure of the brain to excess of ammonia. Alterations of the NO/cGMP pathway and increased glutamine (Gln) content are collectively responsible for many HE symptoms, but how the two events influence each other is not clear. Previously we had shown that Gln administered intracerebrally inhibited the NO/cGMP pathway in control rats and even more so in rats with HA, and we speculated that this effect is due to inhibition by Gln of arginine (Arg) transport (Hilgier et al., 2009). In this study we demonstrate that a 3-day HA in the ammonium acetate model increases the expression in the brain of y(+)LAT2, the heteromeric transporter which preferentially stimulates Arg efflux from the cells in exchange for Gln. The expression of the basic amino acid transporter CAT1, transporting Arg but not Gln remained unaffected by HA. Multiple parameters of Arg or Gln uptake and/or efflux and their mutual dependence were altered in the cerebral cortical slices obtained from HA rats, in a manner indicating enhanced y(+)LAT2-mediated transport. HA elevated Gln content and decreased cGMP content as measured both in the cerebral cortical tissue and microdialysates. Intracortical administration of 6-diazo-5-oxo-L-norleucine (DON), which inhibits Gln fluxes between different cells of the CNS, attenuated the HA-induced decrease of cGMP in the microdialysates of HA rats, but not of control rats. The results suggest that, reduced delivery of Arg due to enhanced y(+)LAT2-mediated exchange of extracellular Gln for intracellular Arg may contribute to the decrease of NO/cGMP pathway activity evoked in the brain by HA.

Topics: Amino Acid Transport System y+; Animals; Cerebral Cortex; Cyclic GMP; Fusion Regulatory Protein 1, Light Chains; Hepatic Encephalopathy; Hyperammonemia; Male; Nitric Oxide; Organ Culture Techniques; Rats; Rats, Sprague-Dawley; Rats, Wistar; Signal Transduction

Recently we reported a decrease of C-type natriuretic peptide (CNP)-dependent, natriuretic peptide receptor 2 (NPR2)-mediated cyclic GMP (cGMP) synthesis in a non-neuronal compartment of cerebral cortical slices of hyperammonemic rats [Zielińska, M., Fresko, I., Konopacka, A., Felipo, V., Albrecht, J., 2007. Hyperammonemia inhibits the natriuretic peptide receptor 2 (NPR2)-mediated cyclic GMP synthesis in the astrocytic compartment of rat cerebral cortex slices. Neurotoxicology 28, 1260-1263]. Here we accounted for the possible involvement of cerebral capillary endothelial cells in this response by measuring the effect of ammonia on the CNP-mediated cGMP formation and intracellular calcium ([Ca2+]i) accumulation in a rat cerebral endothelial cell line (RBE-4). We first established that stimulation of cGMP synthesis in RBE-4 cells was coupled to protein kinase G (PKG)-mediated Ca2+ influx from the medium which was inhibited by an L-type channel blocker nimodipine. Ammonia treatment (1h, 5mM NH4Cl) evoked a substantial decrease of CNP-stimulated cGMP synthesis which was related to a decreased binding of CNP to NPR2 receptors, and depressed the CNP-dependent [Ca2+]i accumulation in these cells. Ammonia also abolished the CNP-dependent Ca2+ accumulation in the absence of Na+. In cells incubated with ammonia in the absence of Ca2+ a slight CNP-dependent increase of [Ca2+]i was observed, most likely representing Ca2+ release from intracellular stores. Depression of CNP-dependent cGMP-mediated [Ca2+]i accumulation may contribute to cerebral vascular endothelial dysfunction associated with hyperammonemia or hepatic encephalopathy.

Topics: Ammonia; Aniline Compounds; Animals; Binding Sites; Binding, Competitive; Calcium; Calcium Channel Blockers; Calcium Signaling; Cell Line; Cell Membrane; Cerebral Arteries; Cerebrovascular Circulation; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Endothelial Cells; Hepatic Encephalopathy; Hyperammonemia; Natriuretic Peptide, C-Type; Protein Binding; Rats; Receptors, Atrial Natriuretic Factor; Xanthenes

Patients with liver cirrhosis with normal neurological and mental status examination may present minimal forms of hepatic encephalopathy, showing intellectual function impairment that cannot be detected through general clinical examination but can be unveiled using specific neuropsychological or neurophysiological examination. Evaluation of minimal hepatic encephalopathy (MHE) in cirrhotic patients would have prognostic value. The psychometric hepatic encephalopathy score (PHES) has been recommended as the "gold standard" in the diagnosis of MHE. Altered modulation of cyclic GMP (cGMP) levels in the brain seems to be responsible for the impairment of some types of cognitive function in liver disease. In animal models of liver disease, some of the alterations in modulation of cGMP levels in the brain are reproduced in lymphocytes. The aim of the present work was to assess whether there is a correlation between the alterations in different parameters involved in modulation of cGMP levels and the presence of MHE in patients with liver disease. We studied in 46 patients with liver cirrhosis and 26 controls the performance in the PHES battery of psychometric tests and the critical flicker frequency (CFF), the concentration of cGMP in plasma and lymphocytes, activation of guanylate cyclase by nitric oxide (NO) in lymphocytes, and several parameters likely involved in altered cGMP homeostasis in liver disease such as ammonia, NO metabolites, and atrial natriuretic peptide (ANP). Activation of guanylate cyclase by NO in lymphocytes and cGMP in plasma were higher and CFF lower in patients with MHE than in patients without MHE. Ammonia, ANP, and metabolites of NO were higher in patients than in controls but were no different in patients with or without MHE. Alteration in activation of guanylate cyclase by NO in lymphocytes correlates with PHES performance, CFF, and ammonia levels. This suggests that altered modulation of guanylate cyclase by NO in lymphocytes would reflect a parallel alteration in the brain occurring in patients with MHE that would be involved in their cognitive impairment.

Topics: Adult; Aged; Case-Control Studies; Cyclic GMP; Enzyme Activation; Female; Guanylate Cyclase; Hepatic Encephalopathy; Humans; Liver Cirrhosis; Liver Failure; Lymphocytes; Male; Middle Aged; Nitric Oxide; Nitric Oxide Donors; Penicillamine; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase

Topics: Biomarkers; Central Nervous System; Cyclic GMP; Hepatic Encephalopathy; Humans; Magnetic Resonance Imaging; Nitric Oxide; Protons

One of the neurological alterations in patients with minimal or overt hepatic encephalopathy is cognitive impairment. This impairment is reproduced in rats with chronic liver failure due to portacaval shunt (PCS). These rats show decreased ability to learn a conditional discrimination task in a Y-maze, likely due to reduced function of the glutamate-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway in brain. It has been proposed that inflammation exacerbates the neuropsychological alterations induced by hyperammonemia, suggesting that inflammation-associated alterations may contribute to cognitive impairment in hepatic encephalopathy. This study assessed whether treatment with an anti-inflammatory drug, ibuprofen, is able to restore the function of the glutamate-NO-cGMP pathway in cerebral cortex in brain in vivo and/or learning ability in PCS rats. We show that PCS rats have increased levels of interleukin-6 and increased activities of cyclooxygenase and of inducible NO synthase in cerebral cortex, indicating the presence of inflammation. Chronic treatment with ibuprofen normalizes cyclooxygenase and inducible NO synthase activities but not interleukin-6 levels. Moreover, ibuprofen normalizes the function of the glutamate-NO-cGMP pathway in cerebral cortex in vivo and completely restores the ability of rats with chronic liver failure to learn the Y-maze task. This supports that inflammation contributes to the cognitive impairment in hepatic encephalopathy.. the results reported point to the possible therapeutic utility of decreasing inflammation in the treatment of the cognitive deficits in patients with minimal or overt hepatic encephalopathy.

Topics: Ammonia; Animals; Anti-Inflammatory Agents, Non-Steroidal; Cyclic GMP; Hepatic Encephalopathy; Ibuprofen; Inflammation; Male; Maze Learning; Nitric Oxide; Nitric Oxide Synthase Type II; Portacaval Shunt, Surgical; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Wistar

Topics: Ammonia; Animals; Anti-Inflammatory Agents, Non-Steroidal; Cyclic GMP; Cytokines; Hepatic Encephalopathy; Inflammation; Nitric Oxide; Nitric Oxide Synthase; Prostaglandin-Endoperoxide Synthases; Rats

Intellectual function is impaired in patients with hyperammonemia and hepatic encephalopathy. Chronic hyperammonemia with or without liver failure impairs the glutamate-nitric oxide-cGMP pathway function in brain in vivo and reduces extracellular cGMP in brain as well as the ability of rats to learn a Y maze conditional discrimination task. We hypothesized that the decrease in extracellular cGMP may be responsible for the impairment in learning ability and intellectual function and that pharmacological modulation of the levels of cGMP may restore learning ability. The aim of this work was to try to reverse the impairment in learning ability of hyperammonemic rats by pharmacologically increasing extracellular cGMP in brain. We assessed whether learning ability may be restored by increasing extracellular cGMP in brain by continuous intracerebral administration of: (1) zaprinast, an inhibitor of the phosphodiesterase that degrades cGMP or (2) cGMP. We carried out tests of conditional discrimination learning in a Y maze with control and hyperammonemic rats treated or not with zaprinast or cGMP. Learning ability was reduced in hyperammonemic rats, which needed more trials than control rats to learn the task. Continuous intracerebral administration of zaprinast or cGMP restored the ability of hyperammonemic rats to learn this task. Pharmacological modulation of extracellular cGMP levels in brain may be a useful therapeutic approach to improve learning and memory performance in individuals in whom cognitive abilities are impaired by different reasons, for example in patients with liver disease who present hyperammonemia and decreased intellectual function.

Topics: Animals; Brain; Cyclic GMP; Disease Models, Animal; Extracellular Fluid; Hepatic Encephalopathy; Hyperammonemia; Learning Disabilities; Male; Maze Learning; Memory Disorders; Phosphodiesterase Inhibitors; Purinones; Rats; Rats, Wistar; Recovery of Function; Treatment Outcome; Up-Regulation

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.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Ammonia; Animals; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 5; Down-Regulation; Electric Stimulation; Guanylate Cyclase; Hepatic Encephalopathy; Hippocampus; Hyperammonemia; In Vitro Techniques; Long-Term Potentiation; Male; Rats; Rats, Wistar; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Soluble Guanylyl Cyclase; Synaptic Transmission

It is shown that the glutamate-NO-cGMP pathway is impaired in cerebellum of rats with portacaval anastomosis in vivo as assessed by in vivo brain microdialysis in freely moving rats. NMDA-induced increase in extracellular cGMP in the cerebellum was significantly reduced (by 27%) in rats with portacaval anastomosis. Activation of soluble guanylate cyclase by the NO-generating agent S-nitroso-N-acetyl-penicillamine and by the NO-independent activator YC-1 was also significantly reduced (by 35-40%), indicating that portacaval anastomosis leads to remarkable alterations in the modulation of guanylate cyclase in cerebellum. Moreover, the content of soluble guanylate cyclase was increased ca. two-fold in the cerebellum of rats with portacaval anastomosis. Activation of soluble guanylate cyclase by NO was higher in lymphocytes isolated from rats with portacaval anastomosis (3.3-fold) than in lymphocytes from control rats (2.1-fold). The results reported show that the content and modulation of soluble guanylate cyclase are altered in brain of rats with hepatic failure, resulting in altered function of the glutamate-NO-cGMP pathway in the rat in vivo. This may lead to alterations in cerebral processes such as intercellular communication, circadian rhythms, including the sleep-waking cycle, long-term potentiation, and some forms of learning and memory.

Topics: Animals; Cerebellum; Cyclic GMP; Disease Models, Animal; Enzyme Activators; Excitatory Amino Acid Agonists; Glutamic Acid; Guanylate Cyclase; Hepatic Encephalopathy; Hyperammonemia; Immunoblotting; Indazoles; Lymphocytes; Male; Microdialysis; N-Methylaspartate; Nitric Oxide; Nitric Oxide Donors; Penicillamine; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; S-Nitroso-N-Acetylpenicillamine; Synaptic Transmission

Chronic hyperammonemia impairs the glutamate-nitric oxide-cGMP pathway in rat brain in vivo. The aims of this work were to assess whether hyperammonemia impairs modulation of soluble guanylate cyclase, and to look for a peripheral marker for impairment of this pathway in brain. We activated the pathway at different steps using glutamate, SNAP, or YC-1. In control neurons these compounds increased cGMP by 7.4-, 9.7- and 7.2-fold, respectively. In ammonia-treated neurons formation of cGMP induced by glutamate, SNAP, and YC-1 was reduced by 50%, 56%, and 52%, respectively, indicating that hyperammonemia impairs activation of guanylate cyclase. This enzyme is also present in lymphocytes. Activation of guanylate cyclase by SNAP or YC-1 was impaired in lymphocytes from hyperammonemic rats. These results suggest that determination of the activation of soluble guanylate cyclase in lymphocytes could serve as a peripheral marker for impairment of the neuronal glutamate-nitric oxide-cGMP pathway in brain.

Topics: Ammonia; Animals; Biomarkers; Cells, Cultured; Chronic Disease; Cyclic GMP; Enzyme Activation; Excitatory Amino Acid Antagonists; Glutamic Acid; Guanylate Cyclase; Hepatic Encephalopathy; Indazoles; Lymphocytes; Neurons; Penicillamine; Rats; Rats, Wistar; S-Nitroso-N-Acetylpenicillamine; Time Factors

Patients with fulminant hepatic failure (FHF) have a severe microcirculatory disturbance causing tissue hypoxia. Infusion of acetylcysteine improves survival and reduces the incidence of multiorgan failure by enhancing tissue oxygenation. Because the observed circulatory effects of acetylcysteine in FHF are similar to and synergistic with those produced by the microcirculatory vasodilator prostacyclin, we postulated that acetylcysteine might potentiate an endogenous vasodilator. Nitric oxide, a vasodilator that activates soluble guanylate cyclase, is a possible candidate as plasma cyclic 3',5'-guanosine monophosphate (cGMP) is raised in FHF, and in vitro acetylcysteine has been found to enhance soluble guanylate cyclase activity. To investigate this possible mechanism further, plasma cGMP was measured before and after acetylcysteine infusion in 24 patients with FHF and again in 6 patients after recovery from acute illness. cGMP levels were high in FHF during acute illness (median, 7.0 nmol/L [interquartile range, 2.6-10.0]) in comparison with levels taken after recovery (1.5 nmol/L [1.0-1.9]; P < .05). Levels rose further after acetylcysteine infusion in the FHF cases (mean increase, 204% [95% CI; 49 to +360]; P < .01) but not in the cases after recovery (38% [-7 to +84]). There were no significant changes in levels of plasma atrial natriuretic peptide (ANP) or cyclic adenosine monophosphate (cAMP) (mean increases, 8% [-6 to +22] and 17% [-9 to +43], respectively). The findings further support the hypothesis that the beneficial hemodynamic effects of acetylcysteine in FHF are mediated by enhancing the activity of the nitric oxide/soluble guanylate cyclase enzyme system.

Topics: Acetylcysteine; Adolescent; Adult; Atrial Natriuretic Factor; Cyclic AMP; Cyclic GMP; Enzyme Activation; Female; Guanylate Cyclase; Hemodynamics; Hepatic Encephalopathy; Humans; Male; Middle Aged; Nitric Oxide; Oxygen; Oxygen Consumption

Nitric oxide and atrial natriuretic peptides are the main activators of guanylyl cyclases, which transform GTP into cyclic GMP and thereby contribute to the decrease of vascular tone. To investigate the increase, if any, of plasma cyclic GMP concentrations in human patients with hyperdynamic circulation resulting from acute liver failure and to ascertain whether guanylyl cyclase activation is involved in the decline of systemic vascular resistance that occurs in this pathophysiological condition, we simultaneously recorded hemodynamic data and cyclic GMP levels in patients with fulminant liver failure before and after liver transplantation and in normokinetic patients undergoing abdominal nonseptic surgery. We also compared these data with those recorded in patients with hyperkinetic shock resulting from gram-negative sepsis or nitric oxide-independent vasomotor agent (carbamate) over-dose. In all these patients we simultaneously studied kidney function, platelet counts and atrial natriuretic peptides. Patients with fulminant liver failure had higher cyclic GMP concentrations than did control patients undergoing abdominal surgery (11.02 +/- 1.55 pmol.ml-1 vs. 1.77 +/- 0.18 pmol.ml-1, p < 0.001). At similar heart-loading conditions these concentrations were lower than those in gram-negative septic shock (18.2 +/- 1.35 pmol.ml-1, p < 0.05) but higher than those in carbamate-induced shock (3.6 +/- 0.7 pmol.ml-1, p < 0.01). In addition, cyclic GMP concentrations significantly decreased from the fulminant liver failure period to the posttransplantation period, although atrial natriuretic peptide levels did not change significantly and kidney function worsened.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adolescent; Adult; Atrial Natriuretic Factor; Blood Circulation; Carbamates; Cyclic GMP; Female; Guanylate Cyclase; Hemodynamics; Hepatic Encephalopathy; Humans; Kidney; Liver Failure, Acute; Liver Transplantation; Male; Middle Aged; Platelet Count; Prospective Studies; Shock; Shock, Septic; Vascular Resistance