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Keywords:

  • cognitive function;
  • cGMP;
  • hepatic encephalopathy;
  • neurological alterations;
  • nitric oxide

Abstract

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

It has been proposed that impairment of the glutamate-nitric oxide-cyclic guanosine monophosphate (cGMP) pathway in brain contributes to cognitive impairment in hepatic encephalopathy. The aims of this work were to assess whether the function of this pathway and of nitric oxide synthase (NOS) are altered in cerebral cortex in vivo in rats with chronic liver failure due to portacaval shunt (PCS) and whether these alterations are due to hyperammonemia. The glutamate-nitric oxide-cGMP pathway function and NOS activation by NMDA was analysed by in vivo microdialysis in cerebral cortex of PCS and control rats and in rats with hyperammonemia without liver failure. Similar studies were done in cortical slices from these rats and in cultured cortical neurons exposed to ammonia. Basal NOS activity, nitrites and cGMP are increased in cortex of rats with hyperammonemia or liver failure. These increases seem due to increased inducible nitric oxide synthase expression. NOS activation by NMDA is impaired in cerebral cortex in both animal models and in neurons exposed to ammonia. Chronic liver failure increases basal NOS activity, nitric oxide and cGMP but reduces activation of NOS induced by NMDA receptors activation. Hyperammonemia is responsible for both effects which will lead, independently, to alterations contributing to neurological alterations in hepatic encephalopathy.

Abbreviations used
cGMP

cyclic guanosine monophosphate

eNOS

endothelial nitric oxide synthase

iNOS

inducible nitric oxide synthase

nNOS

neuronal nitric oxide synthase

NO

nitric oxide

PCS

porto-caval shunt

SNAP

S-nitroso-N-acetylpenicillamine

The molecular bases for the neurological alterations in patients with hepatic encephalopathy remain unclear. Hyperammonemia is considered a main factor in some neurological alterations. Hepatic encephalopathy may be a consequence of altered glutamatergic neurotransmission (Butterworth 1992; Felipo and Butterworth 2002). Both hyperammonemia and liver failure alter glutamatergic neurotransmission at different steps (reviews; Michalak et al. 1997; Felipo and Butterworth 2002; Monfort et al. 2002).

Activation of NMDA receptors increases intracellular calcium which binds to calmodulin and activates nitric oxide synthase (NOS), increasing production of nitric oxide (NO), which activates soluble guanylate cyclase (sGC), increasing cyclic guanosine monophosphate (cGMP) formation, part of which is released to the extracellular fluid. This glutamate-NO-cGMP pathway modulates cerebral processes such as the sleep-waking cycle, long-term potentiation (Boulton et al. 1995; Hawkins 1996), and some forms of learning and memory. Some of these processes are altered in patients with hepatic encephalopathy. (O’Carroll et al. 1991; Garfinkel and Zisapel 1996; Cordoba et al. 1998; Pantiga et al. 2003; Weissenborn et al. 2003).

The function of the glutamate-NO-cGMP pathway is impaired in cerebellum of rats with chronic liver failure due to portacaval anastomosis or with chronic hyperammonemia (Hermenegildo et al. 1998; Monfort et al. 2001). Activation of guanylate cyclase by NO is impaired in cerebellum in vivo (Hermenegildo et al. 1998). Modulation of guanylate cyclase by NO is also altered in brain from patients died due to liver cirrhosis (Corbalán et al. 2002). Activation of guanylate cyclase by NO is decreased in cerebellum homogenates but increased in cerebral cortex homogenates compared with controls (Corbalán et al. 2002).

These results support that liver failure impairs the glutamate-NO-cGMP pathway in cerebellum. However, it remains unclear whether the function of the whole pathway is altered in cerebral cortex. This matter may have implications in the development and understanding of new therapeutic treatments for the neurological alterations in hepatic encephalopathy. The impairment of the glutamate-NO-cGMP pathway may be responsible for some cognitive alterations in patients with hepatic encephalopathy (Erceg et al. 2005a,b). Increasing extracellular cGMP in brain restores learning ability in rats with portacaval anastomosis or with hyperammonemia (Erceg et al. 2005a,b). This supports that decreased cGMP formation in response to NMDA receptors activation would be involved in the impairment of cognitive function in hepatic encephalopathy and that increasing cGMP would restore some types of cognitive function.

However, if the function of the glutamate-NO-cGMP pathway were increased in brain areas where activation of guanylate cyclase by NO is increased, such as cerebral cortex, increasing cGMP would be beneficial for functions modulated by the pathway in cerebellum but detrimental for those modulated in cerebral cortex.

The aim of this work was to assess whether the function of the whole glutamate-NO-cGMP pathway is altered in cerebral cortex in vivo in rats with chronic liver failure due to portacaval anastomosis. To assess the contribution of hyperammonemia to the alterations, we also analysed the function of the pathway in hyperammonemic rats without liver failure. The function of the pathway is impaired in cerebral cortex in vivo in both models. We also analysed the mechanisms involved in this impairment.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Portacaval anastomosis

It was constructed according to Lee and Fisher (1961) using male Wistar rats (220–240 g). Control rats were sham operated. Experiments were performed 4 weeks after surgery.

Hyperammonemic rats without liver failure

Male Wistar rats (120–140 g) were made hyperammonemic by feeding them an ammonium-containing diet for 4  weeks as previously described (Azorin et al. 1989).

Analysis of the function of the glutamate-nitric oxide-cGMP pathway in rat cortex by in vivo microdialysis

Rats were anesthetized using halotane and a microdialysis guide was implanted in the cerebral cortex 3 mm above the final coordinates: AP +3.7 mm, ML −0.8 mm and DV −1.5 mm, according to (Paxinos and Watson 1996). After 48 h a microdialysis probe was implanted. Probes were perfused (3 μL/min) with artificial cerebrospinal fluid (aCSF): (in mmol/L): NaCl, 145; KCl, 3.0; CaCl2, 2.26; buffered at pH 7.4 with 2 mmol/L phosphate buffer. After a 2–3 h stabilization period, 30 min samples were collected. NMDA or the nitric oxide-generating agent S-nitroso-N-acetylpenicillamine (SNAP) were perfused through the microdialysis probe as indicated in Figures. Samples were made 4 mmol/L in EDTA and stored at −80°C until analysis of cGMP or nitrites content.

Determination of cGMP

cGMP was measured by using the BIOTRAK cGMP enzyme immunoassay kit from Amersham (Amersham Pharmacia Biotech, Buckinghamshire, UK).

Treatment of cortical neurons in culture with ammonia

Primary cultures of neurons from cerebral cortex were prepared as previously described (Rodrigo et al. 2005a). As much as 100 µmol/L NH4Cl was added to the culture medium 24 h after seeding.

Changes in intracellular calcium in cultured neurons

Changes in intracellular Ca2+ were monitored in single neurons by confocal microscopy using Fluo-3/AM as previously described (Marcaida et al. 1995).

Activation of the glutamate-NO-cGMP pathway in cortical neurons

Ten days after seeding neurons were washed with Locke’s solution without magnesium (see above). Treatment with 0.3 mmol/L NMDA was for 5 min at 37°C. Then neurons were resuspended in acetate buffer containing 4 mmol/L EDTA and disrupted by sonication. Samples were centrifuged (14 000 g, 5 min) and cGMP or nitrites were measured in the supernatant. Pellets were resuspended in 250 μL 0.25 mol/L NaOH and protein was measured by the bichinconic acid method. cGMP was determined using the BIOTRAK cGMP enzymeimmunoassay kit from Amersham.

Determination of nitrites and nitrates

Nitrites and nitrates were measured in cortical neurons, in microdialysis samples and in cortical slices by the Griess method (Verdon et al. 1995) using nitrate reductase. As much as 100 µL of culture supernatant, 50 μL of slice supernatant or 90 µL of microdialysis samples were mixed with equal volumes of Griess reagent. After 10 min at 20–25°C, absorbance was measured at 540 nm.

Determination of nitric oxide synthase activity in cortical neurons

The conversion of [14C]arginine to [14C]citrulline was determined as described by Kiedrowski et al. (1992). Ten days after seeding neurons were washed twice with Locke’s solution without magnesium and [14C]arginine (1.7 µmol/L, 0.25 µCi) was added. After 5 min, 0.3 mmol/L NMDA was added and the incubation continued for 5 min. The medium was removed and the neurons washed three times with 2 mL cold Locke’s solution and resuspended in 1 mL 0.3 mol/L H3ClO4. After centrifugation [14C]citruline was determined in the supernatant and protein in the pellet. [14C]Citruline was separated from [14C]arginine by chromatography through a Dowex AG50WX-8 (Na+ form) column (Kiedrowski et al. 1992). For each sample, a blank treated with 100 µmol/L nitroarginine to inhibit NOS was carried out. NOS activity is expressed as the difference between [14C]citruline formed in absence and presence of nitroarginine.

Activation of nitric oxide synthase in cortical slices

Rats were decapitated and their brains were transferred into ice-cold Krebs buffer (in mmol/L): NaCl 119, KCl 2.5, KH2PO4 1, NaHCO3 26.2, CaCl2 2.5 and glucose 11, aerated with 95% O2 and 5% CO2 at pH 7.4. Cortex were dissected and transversal slices (400 μm) were obtained using a manual chopper, transferred to incubation wells and incubated for 30 min at 35.5°C in Krebs buffer. Activation of NOS was determined by two procedures:

  • (i)
    Determination of nitrites formation: the slices were incubated for 10 min in the presence or the absence of 0.3 mmol/L NMDA, collected and homogenized in 300 μL of acetate buffer. Samples were centrifuged (14 000 g, 5 min) and nitrites + nitrates were measured in the supernatant as above. Pellets were resuspended in 300 μL of 0.25 mol/L NaOH and protein was measured by the bichinconic acid method.
  • (ii)
    Conversion of [14C]arginine to [14C]citrulline (Kiedrowski et al. 1992). Cortical slices (400 μm) were incubated for 30 min at 35.5°C in Krebs buffer and [14C]arginine (1.7 µmol/L, 0.25 µCi) was added. After 5 min, 0.3 mmol/L NMDA was added and the incubation continued for 5 min. The buffer was removed and slices washed three times with 2 mL cold Krebs buffer and homogenized in 1 mL 0.3 mol/L H3ClO4. After centrifugation (14 000 g, 5 min) [14C]citruline was determined as described for neurons.

Determination of inducible nitric oxide synthase activity

Inducible nitric oxide synthase (iNOS) activity was determined as the activity of NOS that is calcium-independent. Calcium-independent NOS activity was measured by the conversion of [14C]arginine to [14C]citrulline as described by Kiedrowski et al. (1992). Rats were killed by decapitation and cerebral cortex was homogenized in five volumes of ice-cold buffer (A) (20 mmol/L HEPES, 0.32 mol/L sucrose, 1 mmol/L dithiotreitol, 10 mg/L leupeptin, 10 mg/L pepstatin A and 1 mmol/L EGTA to chelate endogenous calcium; pH 7.4). Homogenates (25 μL) were incubated for 60 min at 37°C with 100 μL of reaction mixture containing 200 µmol/L NADPH, 100 µmol/L tetrahidro-biopterin, 2 mmol/L EDTA, 1.2 mmol/L EGTA and 1 μCi/mL L- [14C]arginine monohydrochloride in buffer A and 75 μL of distilled water. The reaction was stopped by adding 1 mL of ice-cold buffer containing 30 mmol/L HEPES and 3 mmol/L EDTA (pH 5.5). [14C]Citruline was separated from [14C]arginine by chromatography through a Dowex AG50WX-8 (Na+ form) column. Protein concentration was quantified by bichinconic acid method.

Immunohistochemistry

Rats were anesthetized with ether and perfused transcardally with 2.5% paraformaldehyde in phosphate buffer (pH 7.2). Brain was removed and post-fixed in the same fixative for 4 h at 4°C. Tissue blocks were dehydrated and embedded in paraffin. Contiguous 8 μm sections were obtained and processed for the immunodetection of iNOS or GFAP. The same number of sections from each group was included in every immunostaining batch to avoid variability in staining.

Sections were deparaffinated and treated for 30 min with non-immune serum diluted 1 : 30 in Tris buffer (pH 7.6) containing 0.2% Triton X-100 and then separately with polyclonal iNOS sera (Chemicon, Temecula, CA, USA) diluted 1 : 5000 in Tris buffer-Triton X-100 for 3 days at 4°C. Following three 5 min washes in Tris buffer, the sections were incubated with biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA, USA) following the manufacturer’s instructions. The peroxidase activity was demostrated using 0.03% DAB (Sigma-Aldrich, Tres Cantos, Madrid, Spain) in 0.05 mol/L Tris buffer with 0.005% H2O2. Samples were washed twice in graded ethanol series, and mounted in DePeX. Some sections were incubated with preimmune serum at 1 : 30 dilution as the primary antiserum; these control sections showed no immunoreactive product.

Double immunofluorescence

For simultaneous detection of iNOS and GFAP, double immunofluorescence was performed on the sections to visualize cellular co-localization. The sections were incubated 3 days at 4°C with the first primary antibody (polyclonal iNOS, Chemicon, at 1 : 5000 dilution), washed several times and incubated with the specific secondary antibody FITC-conjugated goat anti-rabbit IgG (Chemicon, at 1 : 100 dilution). After washing sections were incubated 24 h at 4°C with the second primary antibody (monoclonal GFAP, Sigma-Aldrich, at 1 : 500), followed by incubation with the secondary antibody TRICT-conjugated goat anti-mouse IgG (Chemicon, at 1 : 100 dilution). After being washed, the labeled sections were coverslipped with the Vectashield mounting medium (Vector Laboratory). As control for our double-labeling experiments, the first primary antibodies were omitted or replaced with normal serum and no double-labeling could be seen. Sections were examined by the use of an fluorescence microscope (Carl Zeiss Micro Imaging S. L, Barcelona, Spain).

Immunoblotting

Cortex was homogenized in 66 mmol/L Tris-HCl (pH 7.4), 1% SDS, 1 mmol/L EGTA, 10% glycerol, 1 mmol/L sodium orthovanadate and 1 mmol/L sodium fluoride. Samples were subjected to electrophoresis and immunoblotting as previously described (Felipo et al. 1988) using an antibody against neuronal nitric oxide synthase (nNOS) (1 : 1000) from BD Transduction Laboratories (Erembodegem, Belgium). The images were captured using the gelprinter plus System, TDI (Madrid, Spain) and band intensities quantified using the Alpha Imager 2200, version 3.1.2 (AlphaInnotech Corporation, San Francisco, CA, USA).

RNA extraction and quantification of inducible nitric oxide synthase and neuronal nitric oxide synthase mRNA by real-time PCR

Cortex was homogenized in quiazol reagent (Quiagen, Valencia, CA, USA) and RNA was extracted using the RNeasy lipid tissue Minikit (Quiagen). RNA concentration was determined using Quant-iT Ribogreen RNA kit (Molecular Probes, Invitrogen). Quantitative RT-PCR was developed in two steps. The RT reaction was performed in 100 μL. Two μg of RNA were reverse transcribed using random hexanucleotides with the TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed in triplicate in 25 μL reactions containing Taqman Universal PCR Master Mix (Applied Biosystems) and 1 μL of cDNA on a GeneAmp® 5700 Sequence Detection System (Applied Biosystems). The following primer/probe sets for rat from Applied Biosystems were used, 00583793_m1 for nNOS and 00561646_m1 for iNOS.

After cDNA amplification Ct values for cDNA samples were obtained. β-actin was used as control gene to calculate ΔCt. The relative amount of xNOS/β-actin transcripts was calculated using the 2(−ΔΔCt) method as described previously (Livak and Schmittgen 2001).

All animal procedures were approved by the Institute and met the guidelines of the European Union for care and management of experimental animals.

Statistical analysis

Data were analysed by analysis of variance (anova) followed when appropriate by Newman–Keuls’s post hoc test. When only two values were compared the Student t-test was used. Significance levels were set at α = 0.05.

Results

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

To study the contribution of hyperammonemia to the alterations induced by liver failure we carried out similar studies in rats with chronic liver failure due to portacaval anastomosis (PCS rats) and in rats with hyperammonemia without liver failure (hyperammonemic rats). For clarity we present the results obtained in both groups of rats in parallel. The control rats for the PCS rats were sham operated while the control rats for hyperammonemic rats were non-operated rats.

Basal extracellular cGMP was higher in PCS rats (129 ± 8 pmol/L) than in sham-operated controls (89 ± 5 pmol/L) (Fig. 1a). It was also higher in hyperammonemic rats (144 ± 16 pmol/L) than in control rats (83 ± 4 pmol/L) (Fig. 2a).

image

Figure 1.  Chronic liver failure increases the basal concentration of extracellular cGMP and impairs the function of the glutamate-nitric oxide-cGMP pathway in cerebral cortex in vivo. The function of the whole pathway was analysed by in vivo microdialysis in freely moving rats. Microdialysis probes were inserted in the cerebral cortex of control rats (SHAM) or of rats with chronic liver failure (PCS), perfused at 3 μL/min and samples were taken every 30 min. (a) Basal concentration of cGMP was determined in fractions 1–5. (b) NMDA (0.3 mmol/L) was administered in the perfusion stream for 30 min at the time indicated by the horizontal bar. Data are presented as percentage of basal values. Values are the mean SEM from 10 rats per group. Values significantly different (p < 0.05) from basal cGMP before administration of NMDA are indicated by (i) for control and by (ii) for rats with chronic liver failure. Values that are significantly different from control rats are indicated by asterisks, *p < 0.05.

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image

Figure 2.  Chronic hyperammonemia increases basal concentration of extracellular cGMP and impairs the function of the glutamate-nitric oxide-cGMP pathway in cerebral cortex in vivo. The function of the whole pathway was analysed by in vivo microdialysis in freely moving rats. Microdialysis probes were inserted in the cerebral cortex of control or hyperammonemic rats, perfused at 3 μL/min and samples were taken every 30 min. (a) Basal concentration of cGMP was determined in fractions 1–5. (b) NMDA (0.3 mmol/L) was administered in the perfusion stream for 30 min at the time indicated by the horizontal bar. Data are presented as percentage of basal values. Values are the mean SEM from 10 different rats per group. Values significantly different (p < 0.05) from basal cGMP before perfusion of NMDA are indicated by (i) for control rats and by (ii) for hyperammonemic rats. Values that are significantly different from control rats are indicated by asterisks, *p < 0.05.

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To analyze the function of the glutamate-NO-cGMP pathway, we determined NMDA-induced increase in extracellular cGMP. In sham controls NMDA increased cGMP significantly to 281 ± 18% of basal. In PCS rats cGMP also increased to 171 ± 6% of basal (Fig. 1b). The NMDA-induced increase in extracellular cGMP was significantly lower in PCS rats than in sham controls, indicating a significant 62% reduction in the function of the whole glutamate-NO-cGMP pathway.

The function of the whole pathway was also impaired in hyperammonemic rats. NMDA increased extracellular cGMP in control rats to 294 ± 54% of basal and in hyperammonemic rats to 177 ± 5% of basal (Fig. 2b), indicating a significant 61% reduction in the function of the whole glutamate-NO-cGMP pathway.

We then assessed activation of guanylate cyclase by NO in vivo. In control rats SNAP increased extracellular cGMP to 261 ± 40% of basal. The increase was significantly higher in PCS rats, reaching 459 ± 74% of basal (Fig. 3a). A similar effect was observed in hyperammonemic rats. In control rats SNAP increased extracellular cGMP to 185 ± 12% of basal. The increase was significantly higher in hyperammonemic rats, reaching 327 ± 28% of basal (Fig. 3b).

image

Figure 3.  Chronic liver failure or chronic hyperammonemia increase activation of soluble guanylate cyclase by nitric oxide in cerebral cortex in vivo. Activation of soluble guanylate cyclase by NO was analysed by in vivo microdialysis in freely moving rats. Microdialysis probes were inserted in the cerebral cortex of control rats (SHAM) and rats with chronic liver failure (PCS) (a) or in the cerebral cortex of control rats or hyperammonemic rats (b) perfused at 3 μL/min and samples were taken every 30 min. SNAP (0.5 mmol/L) was administered in the perfusion stream for 30 min at the time indicated by the horizontal bar. Data are presented as percentage of basal values. Values are the mean SEM from 10 different rats per group. Values significantly different (p < 0.05) from basal cGMP before perfusion of SNAP are indicated by (i) for control and by (ii) for PCS or hyperammonemic rats. Values that are significantly different from controls are indicated by asterisks, *p < 0.05.

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The above results show that both in PCS and in hyperammonemic rats the function of the whole glutamate-NO-cGMP pathway is reduced while activation of guanylate cyclase by NO is increased. This suggests that a previous step should be inhibited. We analyzed activation of NOS in vivo following NMDA administration by measuring nitrites and nitrates in extracellular fluid.

Basal concentration of nitrites in the extracellular fluid was higher in PCS rats (7.3 ± 1.5 µmol/L) than in sham controls (4.8 ± 0.2 µmol/L) (Fig. 4a). Nitrites were also higher in hyperammonemic rats (7.2 ± 0.8 µmol/L) than in control rats (2.2 ± 0.4 µmol/L) (Fig. 5a).

image

Figure 4.  Chronic liver failure increases basal concentration of extracellular nitrites and impairs NMDA-induced formation of nitric oxide in cerebral cortex in vivo. Activation of nitric oxide synthase by NMDA was analysed by in vivo microdialysis in freely moving rats. Microdialysis probes were inserted in the cerebral cortex of control rats (SHAM) or of rats with chronic liver failure (PCS), perfused at 3 μL/min and samples were taken every 30 min. (a) Basal concentration of nitrites was determined in fractions 1–5. (b) NMDA (0.3 mmol/L) was administered in the perfusion stream for 30 min at the time indicated by the horizontal bar. Data are presented as percentage of basal values. Values are the mean SEM from 10 different rats per group. Values significantly different (p < 0.05) from basal nitrites before perfusion of NMDA are indicated by (i) for control rats. Values that are significantly different from controls are indicated by asterisks, *p < 0.05.

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image

Figure 5.  Chronic hyperammonemia increases basal concentration of extracellular nitrites and impairs NMDA-induced formation of nitric oxide in cerebral cortex in vivo. Activation of nitric oxide synthase by NMDA was analysed by in vivo microdialysis in freely moving rats. Microdialysis probes were inserted in the cerebral cortex of control or hyperammonemic rats, perfused at 3 μL/min and samples were taken every 30 min. (a) Basal concentration of nitrites was determined in fractions 1–5. (b) NMDA (0.3 mmol/L) was administered in the perfusion stream for 30 min at the time indicated by the horizontal bar. Data are presented as percentage of basal values. Values are the mean SEM from 10 different rats per group. Values significantly different (p < 0.05) from basal nitrites before perfusion of NMDA are indicated by (i) for control rats. Values that are significantly different from control rats are indicated by asterisks, *p < 0.05.

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We then analyzed the activation of NOS in vivo following NMDA administration. NMDA activated NOS and increased extracellular nitrites in sham controls to 159 ± 18% of basal. However, in PCS rats NMDA did not increase extracellular nitrites, which remained at 103 ± 6% of basal (Fig. 4b). A similar impairment in activation of NOS was found in hyperammonemic rats. NMDA activated NOS and increased extracellular nitrites in control rats to 272 ± 68% of basal. However, in hyperammonemic rats NMDA did not increase significantly extracellular nitrites, which remained at 137 ± 33% of basal (Fig. 5b).

To further confirm that activation of NOS following NMDA administration is impaired in cerebral cortex of rats with PCS or with hyperammonemia we measured NOS activity in cortical slices using two approaches:

  • (i)
    Determination of nitrites formation as above.
  • (ii)
    Determination of NOS activity by measuring the formation of radioactive citrulline from radioactive arginine.

As occurs for extracellular nitrites in vivo (Figs 4 and 5), basal levels of nitrites were higher in slices from PCS rats than from sham-operated controls (Fig. 6a) and in slices from hyperammonemic rats than from controls (Fig. 7a). This suggests that both in PCS and in hyperammonemic rats basal NOS activity is higher that in control rats.

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Figure 6.  Chronic liver failure increases basal levels of nitrites and impairs NMDA-induced activation of nitric oxide synthase in slices from cerebral cortex. Activation of nitric oxide synthase (NOS) by NMDA was analysed by measuring formation of nitrites (a) or the formation of [14C]-citrulline from [14C]-arginine (b). Cortical slices were freshly prepared from control rats (SHAM) or from rats with chronic liver failure (PCS) and incubated in the absence or the presence of NMDA as described in the methods section. (a) The levels of nitrites under basal conditions and 10 min after addition of NMDA. (b) The activity of NOS under basal conditions and 5 min after addition of NMDA. Values are the mean SEM from 10 different rats per group. Values significantly different (p < 0.05) from basal nitrites or basal activity of NOS before addition of NMDA are indicated by (i) for control rats. Values that are significantly different from control rats are indicated by asterisks, *p < 0.05; **p < 0.01.

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image

Figure 7.  Chronic hyperammonemia increases basal levels of nitrites and impairs NMDA-induced activation of nitric oxide synthase in slices from cerebral cortex. Activation of nitric oxide synthase (NOS) by NMDA was analysed by measuring formation of nitrites (a) or the formation of [14C]-citrulline from [14C]-arginine (b). Cortical slices were freshly prepared from control or hyperammonemic rats and incubated in the absence or the presence of NMDA as described in the methods section. (a) The levels of nitrites under basal conditions and 10 min after addition of NMDA. (b) The activity of NOS under basal conditions and 5 min after addition of NMDA. Values are the mean SEM from 10 different rats per group. Values significantly different (p < 0.05) from basal nitrites or basal activity of NOS before addition of NMDA are indicated by (i) for control rats. Values that are significantly different from control rats are indicated by asterisks, *p < 0.05; **p < 0.01.

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We then determined activation of NOS following activation of NMDA receptors. NMDA increased nitrites in control rats. However, as occurs in vivo, in slices from PCS rats NMDA did not increase nitrites (Fig. 6a). NMDA-induced activation of NOS was also impaired in hyperammonemic rats. NMDA increased nitrites in control rats but not in hyperammonemic rats (Fig. 7a).

We then measured NOS activity by determining the conversion of [14C]-arginine to [14C]-citrulline. Basal NOS activity was significantly higher in slices from PCS rats than from sham controls (Fig. 6b). It was also higher in slices from hyperammonemic rats than from control rats (Fig. 7b).

NMDA increased NOS activity in slices from sham controls threefold and in slices from non-operated controls 2.6-fold. However, NMDA did not increase NOS activity in slices from PCS rats or from hyperammonemic rats (Figs 6b and 7b).

The above results indicate that in cerebral cortex of both PCS and hyperammonemic rats the glutamate-NO-cGMP pathway is impaired at the level of NOS activation.

Both NMDA receptors and nNOS involved in this pathway are located in neurons. To confirm that hyperammonemia impairs NMDA-induced activation of NOS in neurons we used cultures of cortical neurons and assessed whether chronic exposure to ammonia reproduces the effects found in cortex of PCS and hyperammonemic rats.

We analyzed the effects on the whole pathway by adding NMDA and measuring cGMP. As shown in Fig. 8, chronic exposure to ammonia does not affect basal cGMP concentration which was 4.4 ± 0.3 pmol/mg protein in control neurons and 3.8 ± 0.5 pmol/mg protein in neurons exposed to ammonia.

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Figure 8.  The function of the glutamate-nitric oxide-cGMP pathway is impaired in cortical neurons chronically exposed to ammonia. Primary cultures of cortical neurons were prepared as indicated in methods; 24 h after seeding, 100 µmol/L ammonium chloride was added to the culture medium of one half of the plates (Ammonia); the other half were the controls (Control). Cultures were used 10 days after seeding. Neurons were treated with NMDA (0.3 mmol/L) for 5 min. The content of cGMP in neurons treated or not (basal) with NMDA was measured. Values are the mean ± SEM of triplicate samples from seven different cultures. Values significantly different (p < 0.05) from basal cGMP before addition of NMDA are indicated by (i) for control neurons and by (ii) for neurons exposed to ammonia. Values that are significantly different in neurons exposed to ammonia from control neurons are indicated by asterisks, *p < 0.05.

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Addition of NMDA increased cGMP to 14 ± 1.1 pmol/mg protein in control neurons and the increase was significantly lower in neurons exposed to ammonia, reaching only 9.6 ± 1.2 pmol/mg protein (Fig. 8).

To assess whether activation of NOS following NMDA administration is impaired in cortical neurons we measured NOS activity using two approaches:

  • (i)
    Determination of nitrites formation.
  • (ii)
    Determination of NOS activity by measuring the formation of radioactive citrulline from radioactive arginine.

The basal content of nitrites was similar in control neurons (1.7 ± 0.2 nmol/mg protein) and in neurons exposed to ammonia (1.9 ± 0.2 nmol/mg protein). Addition of 0.3 mmol/L NMDA increased nitrites to 3.4 ± 0.2 nmol/mg protein in control neurons. However, in neurons exposed to ammonia NMDA did not increase nitrites, which remained at 2.0 ± 0.2 nmol/mg protein (Fig. 9a).

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Figure 9.  NMDA-induced activation of nitric oxide synthase is impaired in cortical neurons chronically exposed to ammonia. Activation by a Ca2+ ionophore is also impaired. Primary cultures of cortical neurons were prepared and treated with ammonia as in Fig. 10. Cultures were used 10 days after seeding. (a) The levels of nitrites under basal conditions, 5 min after addition of 300 µmol/L NMDA and 15 min after addition of 10 µmol/L ionomycin, a Ca2+ ionophore. (b) The activity of nitric oxide synthase (NOS) under basal conditions and 5 min after addition of NMDA. Values are the mean ± SEM of triplicate samples from seven different cultures. Values significantly different (p < 0.05) from basal nitrites or basal activity of NOS before addition of NMDA are indicated by (i) for control neurons and by (ii) for ammonia-treated neurons. Values that are significantly different in neurons exposed to ammonia from control neurons are indicated by asterisks, *p < 0.05.

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We measured NOS activity by determining the conversion of [14C]-arginine to [14C]-citrulline. Basal NOS activity was similar in control neurons and in neurons exposed to ammonia. Addition of NMDA increased NOS activity in control neurons 3.5-fold. However, in neurons exposed to ammonia NMDA increased NOS activity only twofold (Fig. 9b).

The impairment of NMDA-induced activation of NOS may be due to reduced increase in intracellular calcium or to impairment of NOS activation per se. To discern between these possibilities we measured NMDA-induced increase in calcium in cortical neurons. As shown in Fig. 10, chronic exposure to ammonia did not affect NMDA-induced increase in calcium.

image

Figure 10.  Chronic exposure of cortical neurons to ammonia does not affect NMDA-induced increase of intracellular calcium. Primary cultures of cortical neurons were prepared and treated with ammonia as in Fig. 9. Cultures were used 10 days after seeding. Free intracellular calcium content was followed with fluo-3/AM using a confocal microscope. The basal calcium level was recorded for 200 s, then 0.3 mmol/L NMDA was added and the fluorescence was recorded for 1600 s. Typical traces are shown in (a) and (b). The increase in calcium at 1600 s compared with 200 s was similar in control neurons (a) or ammonia-treated neurons (b). The values (mean ± SEM) of quadruplicate samples from six different cultures are shown in (c).

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This indicates that the impairment of the pathway occurs at the level of NOS activation. To further confirm this possibility we tested whether the activation of NOS in response to a Ca2+ ionophore is also impaired in neurons exposed to ammonia. As shown in Fig. 9a addition of ionomycin increased NO formation by 323 ± 45% in control neurons and the increase was significantly lower (92 ± 40%) in neurons exposed to ammonia. This confirms that exposure to ammonia leads to impaired activation of NOS by calcium-calmodulin.

To assess the possible contribution of increased nNOS or iNOS expression to the increase in basal NOS activity and in nitrites concentration we analysed the content of nNOS and iNOS mRNA in cerebral cortex of hyperammonemic and PCS rats. The amount of nNOS mRNA was not affected in any of the groups. The contents of nNOS mRNA for each group, expressed as percentage of control non-operated rats were 109 ± 10%, 97 ± 7% and 107 ± 11% for hyperammonemic, sham-operated and PCS rats, respectively (Fig. 11a).

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Figure 11.  Content of neuronal nitric oxide synthase (nNOS) in cerebral cortex from rats with chronic liver failure or hyperammonemia without liver failure and in neurons chronically exposed to ammonia. Cerebral cortex from control rats (C), hyperammonemic rats (H), rats with chronic liver failure (PCS, P) and sham-operated controls (SHAM, S) were homogenized as described in methods for mRNA isolation (a) and immunoblotting (b and c). Cortical neurons in culture exposed (a) or not (c) to ammonia were also homogenized for immunoblotting. Quantitative real-time RT-PCR was carried out as described in the methods section using beta-actin mRNA as an internal reference. The amount of nNOS mRNA in each sample was referred to beta-actin mRNA in the same sample. Expression of of nNOS mRNA (a) is given as percentage of the value for control non-operated rats. The homogenates (50 μg of protein for cerebral cortex and for cultured neurons) were subjected to electrophoresis and the content of nNOS was analyzed by immunoblotting. Some representative immunoblottings are shown in (b). The different numbers indicate different animals or cells cultures from the same group. (c) The intensities of the bands were quantified and expressed as percentage of controls. Values are the mean ± SEM of at least seven experiments. Values that are significantly different from controls are indicated by asterisks *p < 0.05.

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We also analysed the nNOS protein amount in cerebral cortex of PCS rats, hyperammonemic rats and neuronal cultures. We found a very slight increase in nNOS in cortex of PCS rats (16%) and in cortical neurons exposed to ammonia (27%). However, nNOS was reduced (28%) in cerebral cortex of hyperammonemic rats without liver failure (Fig. 11b,c).

Control, non-operated rats showed very low but measurable levels of iNOS mRNA. The expression of iNOS was significantly increased to 140 ± 13% in hyperammonemic rats. In sham operated rats the expression of iNOS was significantly higher (150 ± 9%) than in control non-operated rats. In PCS rats iNOS expression was significantly higher than in sham operated controls, reaching 213 ± 22% of controls (Fig. 12a,b).

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Figure 12.  Chronic hyperammonemia or liver failure increase the expression and activity of inducible nitric oxide synthase (iNOS) in cerebral cortex. Cerebral cortices from control rats (C), rats with chronic hyperammonemia (H), rats with chronic liver failure (PCS, P) and sham-operated controls (SHAM, S) were homogenized in quiazol reagent and the RNA was extracted. Quantitative real-time RT-PCR was carried out as described in the methods section using beta-actin mRNA as an internal reference. The amount of iNOS mRNA in each sample was referred to beta-actin mRNA in the same sample. The reaction products after quantitative PCR for iNOS were loaded in 2% agarose gels, stained with ethidium bromide and are shown in (a). The different numbers indicate different animals from the same group. Expression of iNOS mRNA (b) is given as percentage of the value for control non-operated rats. The activity of iNOS (c) was determined as calcium-independent NOS activity and is given as percentage of conversion of arginine to citrulline per min per mg protein. Values are the mean ± SEM of triplicate samples from 10 rats per group in (b) and from six rats per group in (c). Values that are significantly different from control rats are indicated by asterisks, *p < 0.05; **p < 0.01.

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These data indicate that iNOS expression is induced in cortex of PCS rats. To assess in which type of cells is iNOS induced we analyzed by immunohistochemistry its location. As shown in Fig. 13a, iNOS immunoreactivity was found in the cytoplasm of pyramidal neurons and in the main dendrites in the cortex of PCS rats but was completely absent in control rats. To assess whether iNOS is also induced in astrocytes, we used double labelling immunofluorescence of GFAP and iNOS. As shown in Fig. 13b, GFAP and iNOS colocalize in some astrocytes. These data confirm that iNOS is induced in cortex of PCS rats and show that it is induced both in some neurons and in some astrocytes.

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Figure 13.  Chronic liver failure increase the content of inducible nitric oxide synthase (iNOS) in astrocytes and neurons in cerebral cortex. iNOS content was analyzed by immunohistochemistry (a and b) and immunoblotting in cerebral cortex from rats with chronic liver failure. Cerebral cortex from sham-operated control rats (SHAM, S) and rats with chronic liver failure (PCS, P) were immunostained for iNOS or GFAP as described in methods. (a) iNOS immunoreactive granular structures in the cytoplasm of pyramidal-like neuorns and the main dendrites in cortex from rats with chronic liver failure. (b) A double immunofluorescence of GFAP and iNOS. GFAP is shown in red fluorescence (rhodamine), and iNOS is represented by green fluorescence (fluorescein isothiocyanate). Co-localization appears as follow in astrocytes ([RIGHTWARDS ARROW]), indicating that iNOS is also strongly induced in astrocytes from rats with chronic liver failure. Cerebral cortex from control rats (C), hyperammonemic rats (H), rats with chronic liver failure (PCS, P) and sham-operated controls (SHAM, S) were homogenized as described in methods. As much as 100 μg of protein was subjected to electrophoresis and the content of iNOS was analyzed by immunoblotting. A representative immunoblotting is shown in (c). The different numbers indicate different animals from the same group. Protein content is significantly higher in cortex of rats with chronic liver failure.

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The iNOS protein amount was analyzed in cerebral cortex of PCS and of hyperammonemic rats. It was increased by ca. 20-fold in PCS rats and by ca. fourfold in hyperammonemic rats as analyzed by immunoblotting (Fig. 13c).

We also determined the activity of iNOS (calcium-independent NOS activity) in cerebral cortex from hyperammonemic and PCS rats. As shown in Fig. 12c, iNOS activity was significantly increased in hyperammonemic (131 ± 19%) and PCS (148 ± 40%) rats. This suggests that most of the increase in basal NOS activity in hyperammonemic and PCS rats would be due to the increase in iNOS activity.

Discussion

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Activation of the glutamate-NO-cGMP pathway is altered in brain in vivo animal models of hyperammonemia and liver failure (Hermenegildo et al. 1998; Monfort et al. 2001). To design proper treatments to normalize the function of the pathway it is important to know which steps are altered and should be the target of these treatments. We have previously shown that activation of soluble guanylate cyclase by NO is altered in vivo in the above animal models and in vitro in brain homogenates from patients died in hepatic encephalopathy (Corbalán et al. 2002). We show now that activation of guanylate cyclase by NO is increased in cerebral cortex in vivo in hyperammonemic as well as PCS rats. The mechanisms underlying this enhanced activation are not clear by now. The mechanisms that regulate the extent of activation of sGC by NO remain unclear. Endogenous allosteric modulators of sGC with the ability to alter its sensitivity to NO have been proposed (Koesling 1998), one of which has been partially purified (Kim and Burstyn 1994). It is not known whether hyperammonemia or liver failure could alter the amount of these modulators. In addition, sGC activity is regulated by phosphorylation by either protein kinase C (Zwiller et al. 1985) or cAMP-dependent protein kinase (Zwiller et al. 1981) both of which result in modifications of enzyme activity (Louis et al. 1993). It is not known whether sGC phosphorylation is modified in brain in chronic liver failure.

Another important step of the glutamate-NO-cGMP pathway which may modulate its function is activation of NOS by calcium-calmodulin. However, the effects of chronic hyperammonemia and liver failure on the modulation of NOS activity in brain in vivo have not been previously studied.

The results reported here show that in cerebral cortex in vivo in rats with chronic liver failure due to portacaval anastomosis there are two different alterations in the modulation of NOS activity:

  • (i)
    activity of NOS and formation of NO and cGMP under basal conditions are increased, and
  • (ii)
    activation of NOS induced by activation of NMDA receptors is strongly reduced.

Both effects will lead, independently, to different alterations that will have important consequences on cerebral function and contribute to the neurological alterations of hepatic encephalopathy, as discussed below.

The effects are the same in rats with chronic liver failure or with hyperammonemia without liver failure, indicating that hyperammonemia is responsible both for increased basal NOS activity and for impaired activation of NOS by NMDA receptors.

The increase in basal cGMP would be a consequence of both increased basal NO formation and increased activation of guanylate cyclase by NO.

The increase in NO formation under basal conditions would be due to increased basal activity of some of the NOS. An increase in NOS activity in vitro in homogenates from cerebral cortex and cerebellum of PCS rats has been reported (Rao et al. 1995, 1997a). We show here that NOS activity is also increased in vivo in cerebral cortex of rats with liver failure.

There are three types of NOS: neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). We show here that iNOS expression is increased in cerebral cortex of both hyperammonemic and PCS rats. This agrees with the report of Schliess et al. (2002) showing that high ammonia levels induce iNOS expression in cultured astrocytes. iNOS expression is also induced in glial cells in cerebellum of PCS rats (Suarez et al. 2005). We also show by immunohistochemistry that iNOS is present at considerable levels both in neurons and in astrocytes in cerebral cortex of in rats with chronic liver failure but not in control rats. Moreover, the activity of iNOS is also negligible in control rats but is significantly increased in cortex of hyperammonemic rats. All these data suggest that the increase in basal NOS activity in rats with liver failure or hyperammonemia must be due to the increase in iNOS content and activity. To assess whether increased iNOS would explain the increase in basal NOS activity and formation of nitrites in PCS and hyperammonemic rats, we analysed whether there is a correlation between the increase in iNOS mRNA and the increase in basal nitrites. There is an excellent correlation (r = 0.996, p = 0.002) between both parameters, further supporting that increased iNOS would be responsible for the increase in basal activity of NOS and in basal nitrites.

Rao et al. (1997b) reported a twofold increase in nNOS in PCS rats. We have not found any change in nNOS mRNA expression (Fig. 12c). Moreover, the changes in nNOS protein content in cerebral cortex of PCS rats, hyperammonemic rats and neuronal cultures are very small and do not correlate with changes in basal activity of NOS, suggesting that changes in nNOS content would not be responsible for increased basal NOS activity.

Increased basal NOS activity and NO production can led to nitrosylation and altered function of important proteins and result in altered cerebral function that may contribute to the neurological alterations in hepatic encephalopathy. This can contribute to the increased cerebral blood flow observed both in rats (Gjedde et al. 1978) and human patients (Bianchi Porro et al. 1969) with portocaval anastomosis. Increased NO production could also be responsible for the nocturnal worsening of hemodynamic parameters in patients with liver cirrhosis (Genesca et al. 2000).

The second important alteration reported here is that activation of NOS induced by activation of NMDA receptors is strongly reduced in cerebral cortex in vivo in rats with chronic liver failure or hyperammonemia. This effect is reproduced in cortical neurons chronically exposed to ammonia, indicating that the impairment of NMDA-induced activation of NOS would occur mainly in neurons and affect essentially nNOS. This is not surprising as NMDA receptors are expressed essentially in neurons.

As a consequence of impaired NOS activation the function of the whole glutamate-NO-cGMP pathway is impaired. These results clearly show that the different steps of the pathway are modulated in different ways. In cerebral cortex of hyperammonemic or PCS rats activation of guanylate cyclase by NO is higher than in control rats. However, the function of the whole pathway is reduced because activation of NOS is impaired.

We have previously shown that activation of guanylate cyclase by NO is increased in cerebral cortex but reduced in cerebellum from PCS rats and from patients died in hepatic encephalopathy (Monfort et al. 2001; Corbalán et al. 2002). The present results show that, in spite of this regional difference in modulation of guanylate cyclase, the function of the whole glutamate-NO-cGMP pathway is impaired both in cerebellum (Hermenegildo et al. 1998; Monfort et al. 2001) and in cerebral cortex, as shown in the present work.

The impairment in the function of the pathway in cerebral cortex is a consequence of the impairment in activation of nNOS by calcium following activation of NMDA receptors. The mechanisms by which hyperammonemia and liver failure impair the activation of nNOS by calcium are not clear for the moment. Two main mechanisms have been described that modulate activation of nNOS by calcium: regulation of spatial localization and phosphorylation of nNOS. There are several proteins bearing PDZ domains that directly associate with nNOS and regulate its spatial localization. These proteins include PSD 95 (Ziff 1997) and CAPON (Jaffrey et al. 1998, 2002). PSD-95 binds to both the NR2 subunit of NMDA receptor and to nNOS at excitatory synapses. This close association of NMDA receptors and nNOS allows a more efficient activation of nNOS by Ca2+ entering through the NMDA receptors (Sattler et al. 1999). Some members of the dystrophin family of proteins, PIN, and caveolin-3 also interact with and modulate nNOS.

Activity of nNOS is also modulated by phosphorylation. Several protein kinases (e.g. protein kinase C, cAMP-dependent protein kinase and CaM kinases) phosphorylate nNOS at different sites affecting its catalytic activity. The protein kinases CaMKI and CaMKII phosphorylate nNOS at Ser 741 and Ser 847, respectively, reducing nNOS activity (Komeima et al. 2000). nNOS is also phosphorylated at Thr 1296 (Song et al. 2005) attenuating its activity.

The mechanisms by which chronic hyperammonemia or liver failure affect modulation of nNOS activity by calcium may include: alterations in phosphorylation or in the content or function of the proteins that modulate nNOS localization. Further studies are required to clarify this point.

The impairment of the function of the glutamate-NO-cGMP pathway will led to alterations in cerebral processes modulated by this pathway, including cognitive function (Danysz et al. 1995; Chen et al. 1997; Prickaerts et al. 1997; Ingram et al. 1998a,b; Meyer et al. 1998; Zou et al. 1998). Impairment of this pathway may be responsible for some cognitive alterations in patients with hepatic encephalopathy (Erceg et al. 2005a,b). Pharmacological treatments that normalize the function of the glutamate-NO-cGMP pathway and extracellular cGMP in brain restore learning ability in rats with chronic liver failure or hyperammonemia (Erceg et al. 2005a,b). This clearly supports that impaired NOS activation following activation of NMDA receptors and the associated alteration in the glutamate-NO-cGMP pathway contributes to some of the cognitive alterations in hepatic encephalopathy.

Understanding the mechanisms by which hyperammonemia and liver failure increase basal NOS activity and impair NMDA receptor-induced activation of NOS could help to design more specific treatments to normalize its function which, in turn, may serve to treat some of the neurological alterations in patients with hepatic encephalopathy.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by grants from the Ministerio de Ciencia y Tecnología (SAF2002-00851 and SAF2005-06089) and from Ministerio de Sanidad (Red G03-155 and FIS PI050253) of Spain and by grants from Consellería de Empresa, Universidad y Ciencia, Generalitat Valenciana (Grupos03/001, GV04B-055, GV04B-012 and GVS05/082).

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  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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