SEARCH

SEARCH BY CITATION

Keywords:

  • Nitric oxide synthase;
  • CNS;
  • Cerebral ischemia

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. NOS activity
  5. mRNA analysis
  6. Characterization of NOS isoforms by western blot
  7. LDH assay
  8. Synthesis of ONOO-
  9. RESULTS
  10. NOS activity in rat forebrain slices exposed to OGD
  11. Effects of dexamethasone, cycloheximide, and HbO2 on NOS activity and protein in rat forebrain slices exposed to OGD
  12. Effect of NO donors and ONOO- on calcium-dependent NOS activity from rat forebrain slices
  13. Quantification of nNOS and iNOS protein in rat forebrain slices exposed to OGD or an NO donor
  14. Levels of nNOS mRNA in rat forebrain slices exposed to OGD and an NO donor
  15. LDH efflux
  16. DISCUSSION
  17. Acknowledgements

Abstract : The precise role that nitric oxide (NO) plays in the mechanisms of ischemic brain damage remains to be established. The expression of the inducible isoform (iNOS) of NO synthase (NOS) has been demonstrated not only in blood and glial cells using in vivo models of brain ischemia-reperfusion but also in neurons in rat forebrain slices exposed to oxygen-glucose deprivation (OGD). We have used this experimental model to study the effect of OGD on the neuronal isoform of NOS (nNOS) and iNOS. In OGD-exposed rat forebrain slices, a decrease in the calcium-dependent NOS activity was found 180 min after the OGD period, which was parallel to the increase during this period in calcium-independent NOS activity. Both dexamethasone and cycloheximide, which completely inhibited the induction of the calcium-independent NOS activity, caused a 40-70% recovery in calcium-dependent NOS activity when compared with slices collected immediately after OGD. The NO scavenger oxyhemoglobin produced complete recovery of calcium-dependent NOS activity, suggesting that NO formed after OGD is responsible for this down-regulation. Consistently, exposure to the NO donor (Z)-1-[(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NONOate) for 180 min caused a decrease in the calcium-dependent NOS activity present in control rat forebrain slices. Furthermore, OGD and DETA-NONOate caused a decrease in level of both nNOS mRNA and protein. In summary, our results indicate that iNOS expression down-regulates nNOS activity in rat brain slices exposed to OGD. These studies suggest important and complex interactions between NOS isoforms, the elucidation of which may provide further insights into the physiological and pathophysiological events that occur during and after cerebral ischemia.

There is increasing evidence that nitric oxide (NO) may play complex roles in the pathophysiology of cerebral ischemia. After the initial observation that NMDA receptor activation generates NO (Garthwaite et al., 1988), it has been postulated that an overproduction of this molecule derived from the excessive stimulation of the neuronal isoform (nNOS) of NO synthase (NOS) is the link between the actions of excitatory amino acids and the subsequent cell damage (Dawson et al., 1991 ; Nowicki et al., 1991). However, the precise role that NO plays in the mechanisms of ischemic brain damage has been a source of controversy, as this agent might be either beneficial or detrimental to the ischemic brain (for review, see Iadecola, 1997). NO production is catalyzed by at least two major isoforms of the NOS enzyme : nNOS and endothelial NOS, which are constitutively expressed, require calcium and calmodulin for activation, and produce NO for short intervals, and a high-output inducible isoform of NOS (iNOS), which is independent of calcium and calmodulin and is expressed after exposure to cytokines and/or lipopolysaccharide (for review, see Knowles and Moncada, 1994). It has therefore been reported to mediate cytotoxicity in many cell systems (Moncada et al., 1991 ; Gross and Wolin, 1995).

In this context, we have recently shown that iNOS is expressed in neurons and other CNS cell types after oxygen-glucose deprivation (OGD) in rat forebrain slices and that this expression occurs in short intervals, suggesting that NO can play an important pathogenic role in the tissue damage that occurs after cerebral ischaemia (Moro et al., 1998). We have now used this experimental model to study the effect of OGD on nNOS and iNOS isoforms. Our results show an important decrease in nNOS content under these conditions that might be due to an overall NO synthesis dependent on the expression of iNOS.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. NOS activity
  5. mRNA analysis
  6. Characterization of NOS isoforms by western blot
  7. LDH assay
  8. Synthesis of ONOO-
  9. RESULTS
  10. NOS activity in rat forebrain slices exposed to OGD
  11. Effects of dexamethasone, cycloheximide, and HbO2 on NOS activity and protein in rat forebrain slices exposed to OGD
  12. Effect of NO donors and ONOO- on calcium-dependent NOS activity from rat forebrain slices
  13. Quantification of nNOS and iNOS protein in rat forebrain slices exposed to OGD or an NO donor
  14. Levels of nNOS mRNA in rat forebrain slices exposed to OGD and an NO donor
  15. LDH efflux
  16. DISCUSSION
  17. Acknowledgements

Preparation and incubation of slices

Male Sprague-Dawley rats (weighing 200-250 g) were killed by decapitation (according to procedures approved by the Committee of Animal Care at the Universidad Complutense of Madrid), and forebrain slices were prepared as described (Moro et al., 1998). In brief, slices were preincubated in sucrose-free preincubation solution equilibrated with 95% O2/5% CO2, in a shaking water bath at 37°C for 45 min. After the preincubation period, slices were incubated in a modified Krebs-Henseleit solution (incubation solution) containing 120 mM NaCl, 2 mM KCl, 2 mM CaCl2, 26 mM NaHCO3, 1.19 mM MgSO4, 1.18 mM KH2PO4, 11 mM glucose, and 10 μM 5,6,7,8-tetrahydrobiopterin bubbled with 95% O2/5% CO2. The slices corresponding to the control group were then incubated an additional 20 min in the same conditions. Slices corresponding to the “ischemic” group were incubated 20 min in incubation solution without glucose and equilibrated with 95% N2/5% CO2 to mimic an ischemic condition (OGD). After these periods of 20 min, the medium was replaced with fresh incubation solution equilibrated with 95% O2/5% CO2 to simulate a reperfusion period. In some experiments, dexamethasone (1 μM) and cycloheximide (10 μM) were included in the incubation solution during both OGD and reperfusion periods. In another set of experiments, the NO donors (Z)-1-[(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NONOate ; 1 mM) or 3-(4-morpholinyl)sydnonimine (SIN-1 ; 1 mM), a bolus addition of peroxynitrite (ONOO- ; 300 μM), or the NO scavenger oxyhemoglobin (HbO2 ; 10 μM) was added to some slices in normal incubation solution during the 180 min after the OGD period. Slices were taken out 180 min after the OGD or control period, except for the time course experiments, in which slices were taken out at 60, 120, and 180 min after the 20-min OGD period. All these tissues were frozen immediately with liquid nitrogen. Incubation solution was collected for lactate dehydrogenase (LDH) assay. To study the direct effect of NO donors on NOS enzymatic activity, some slices were frozen immediately after cutting.

NOS activity

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. NOS activity
  5. mRNA analysis
  6. Characterization of NOS isoforms by western blot
  7. LDH assay
  8. Synthesis of ONOO-
  9. RESULTS
  10. NOS activity in rat forebrain slices exposed to OGD
  11. Effects of dexamethasone, cycloheximide, and HbO2 on NOS activity and protein in rat forebrain slices exposed to OGD
  12. Effect of NO donors and ONOO- on calcium-dependent NOS activity from rat forebrain slices
  13. Quantification of nNOS and iNOS protein in rat forebrain slices exposed to OGD or an NO donor
  14. Levels of nNOS mRNA in rat forebrain slices exposed to OGD and an NO donor
  15. LDH efflux
  16. DISCUSSION
  17. Acknowledgements

NOS activity was determined after sonication of the forebrain slice (Labsonic) at 4°C in 5 volumes of buffer containing 320 mM sucrose, 1 mM EDTA, 1 mMdl-dithiothreitol, 10 μg/ml leupeptin, 100 μg/ml phenylmethylsulfonyl fluoride, 10 μg/ml soybean trypsin inhibitor, 2 μg/ml aprotinin, and 50 mM Tris, brought to pH 7.0 at 20°C with HCl. The homogenate was centrifuged at 5°C at 12,000 g for 20 min, and the pellet was discarded. NOS activity was then determined in cell extracts under conditions (substrate and calcium concentration) of maximal activity, to assess indirectly the amount of enzyme present by monitoring the conversion of l-[U-14C]arginine into [U-14C]citrulline in the postmitochondrial supernatant. The activity of the calcium-dependent NOS was calculated from the difference between the [14C]citrulline produced from control samples and samples containing 1 mM EGTA ; the calcium-independent activity was determined from the difference between samples containing 1 mM EGTA and samples containing 1 mM EGTA and 1 mMNG-monomethyl-l-arginine (Salter et al., 1991).

The protein content of the homogenate from each slice was determined using bicinchoninic acid (Hill and Straka, 1988).

mRNA analysis

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. NOS activity
  5. mRNA analysis
  6. Characterization of NOS isoforms by western blot
  7. LDH assay
  8. Synthesis of ONOO-
  9. RESULTS
  10. NOS activity in rat forebrain slices exposed to OGD
  11. Effects of dexamethasone, cycloheximide, and HbO2 on NOS activity and protein in rat forebrain slices exposed to OGD
  12. Effect of NO donors and ONOO- on calcium-dependent NOS activity from rat forebrain slices
  13. Quantification of nNOS and iNOS protein in rat forebrain slices exposed to OGD or an NO donor
  14. Levels of nNOS mRNA in rat forebrain slices exposed to OGD and an NO donor
  15. LDH efflux
  16. DISCUSSION
  17. Acknowledgements

Total RNA was extracted from forebrain slices by the guanidinium isothiocyanate method (Chirgwin et al., 1979). Aliquots of RNA (10 μg) were size-fractionated by electrophoresis (20 mA for 15 h) in a 0.9% agarose gel containing 2% formaldehyde and the 3-(N-morpholino)propanesulfonic acid buffering system (Chomczynski and Sacchi, 1987). After transference of the RNA to Nytran membranes (NY 13-N ; Schleicher and Schüell, Dassel, Germany), the level of iNOS and nNOS mRNA was determined by hybridization using as the probe, respectively, an EcoRI-HindII fragment from the murine iNOS cDNA (kindly donated by Dr. Q.-W. Xie and Dr. C. Nathan, Cornell University) labeled with [α-32P]dCTP (Random Primed labeling kit ; Amersham, Buckinghamshire, U.K.) and an EcoRI fragment from the rat brain nNOS cDNA (kindly donated by Dr. I. G. Charles and Dr. S. Moncada). On hybridization the membranes were exposed to an x-ray film (Kodak-X-OMAT), and the bands were quantitated by laser densitometry (Molecular Dynamics, Sunnyvale, CA, U.S.A.). Hybridization with a probe specific for ribosomal 18S was used to normalize the RNA lane charge of the blot.

Characterization of NOS isoforms by western blot

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. NOS activity
  5. mRNA analysis
  6. Characterization of NOS isoforms by western blot
  7. LDH assay
  8. Synthesis of ONOO-
  9. RESULTS
  10. NOS activity in rat forebrain slices exposed to OGD
  11. Effects of dexamethasone, cycloheximide, and HbO2 on NOS activity and protein in rat forebrain slices exposed to OGD
  12. Effect of NO donors and ONOO- on calcium-dependent NOS activity from rat forebrain slices
  13. Quantification of nNOS and iNOS protein in rat forebrain slices exposed to OGD or an NO donor
  14. Levels of nNOS mRNA in rat forebrain slices exposed to OGD and an NO donor
  15. LDH efflux
  16. DISCUSSION
  17. Acknowledgements

Slices were homogenized in lysis buffer [10 mM Tris (pH 8.0), 0.2% Nonidet P-40, and 1 mM dithioerythritol], and after centrifugation in a microcentrifuge for 15 min, the proteins present in the supernatant were loaded (10 μg) and sizeseparated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (50 mA). The gels were blotted onto a PVDF membrane (Millipore, Barcelona, Spain) and incubated, respectively, with a specific polyclonal anti-iNOS antibody [1 : 1,000 dilution ; Transduction Laboratories, Lexington, KY, U.S.A. (Lowenstein et al., 1992)] or a specific polyclonal anti-nNOS antibody [kindly donated by Prof. J. Rodrigo (Rodrigo et al., 1994)]. NOS isoforms were revealed by the ECL kit following the manufacturer's instructions (Amersham).

Synthesis of ONOO-

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. NOS activity
  5. mRNA analysis
  6. Characterization of NOS isoforms by western blot
  7. LDH assay
  8. Synthesis of ONOO-
  9. RESULTS
  10. NOS activity in rat forebrain slices exposed to OGD
  11. Effects of dexamethasone, cycloheximide, and HbO2 on NOS activity and protein in rat forebrain slices exposed to OGD
  12. Effect of NO donors and ONOO- on calcium-dependent NOS activity from rat forebrain slices
  13. Quantification of nNOS and iNOS protein in rat forebrain slices exposed to OGD or an NO donor
  14. Levels of nNOS mRNA in rat forebrain slices exposed to OGD and an NO donor
  15. LDH efflux
  16. DISCUSSION
  17. Acknowledgements

ONOO- was synthesized by the reaction of acidified NaNO2 (2.1 M) with H2O2 (2.1 M) in a quenched flow reactor (Blough and Zafiriou, 1985). The reaction was quenched with 4.2 M NaOH, and any unreacted H2O2 was removed with solid MnO2. ONOO- was stored at -20°C before use. The concentration was determined before each experiment by measuring the absorbance at 302 nm [ε302 nm = 1,670 M-1 cm-1 (Hughes and Nicklin, 1968)]. Typical concentrations of ONOO- were 220-350 mM.

Chemicals and statistical analyses

l-[U-14C]Arginine was obtained from Amersham, 5,6,7,8-tetrahydro-l-biopterin was obtained from Research Biochemicals International (Natick, MA, U.S.A.), and other chemicals were from Sigma (Spain) or as indicated in the previous sections. HbO2 was prepared according to the procedure of Paterson et al. (1976). Results are expressed as mean ± SEM values of the indicated number of experiments. Newman-Keuls test was used to determine the significance of differences between means, and p < 0.05 was considered as statistically significant.

NOS activity in rat forebrain slices exposed to OGD

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. NOS activity
  5. mRNA analysis
  6. Characterization of NOS isoforms by western blot
  7. LDH assay
  8. Synthesis of ONOO-
  9. RESULTS
  10. NOS activity in rat forebrain slices exposed to OGD
  11. Effects of dexamethasone, cycloheximide, and HbO2 on NOS activity and protein in rat forebrain slices exposed to OGD
  12. Effect of NO donors and ONOO- on calcium-dependent NOS activity from rat forebrain slices
  13. Quantification of nNOS and iNOS protein in rat forebrain slices exposed to OGD or an NO donor
  14. Levels of nNOS mRNA in rat forebrain slices exposed to OGD and an NO donor
  15. LDH efflux
  16. DISCUSSION
  17. Acknowledgements

OGD caused a time-dependent decrease in calcium-dependent NOS activity (120 and 180 vs. 0 min, p < 0.05, n = 16 ; Fig. 1). The time course of this decrease was parallel to the increase of the calcium-independent NOS activity in these tissues (60, 120, and 180 vs. 0 min, p < 0.05, n = 16 ; Fig. 1), which appeared ~60 min after the OGD period and reached maximal activity 180 min after the onset of the “reperfusion” period. During the same interval calcium-independent and -dependent NOS activities in control forebrain slices remained unchanged (p > 0.05, n = 16 ; Fig. 1).

image

Figure 1. Time course of calcium-dependent and -independent NOS activity in control and oxygen-glucose-deprived rat forebrain slices. NOS activity was measured by monitoring the conversion of l-[U-14C] arginine into l-[U-14C] citrulline (see materials and Methods). Data are mean ± SEM (bars) values (n = 16). *p < 0.05 versus 0 min.

Download figure to PowerPoint

Effects of dexamethasone, cycloheximide, and HbO2 on NOS activity and protein in rat forebrain slices exposed to OGD

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. NOS activity
  5. mRNA analysis
  6. Characterization of NOS isoforms by western blot
  7. LDH assay
  8. Synthesis of ONOO-
  9. RESULTS
  10. NOS activity in rat forebrain slices exposed to OGD
  11. Effects of dexamethasone, cycloheximide, and HbO2 on NOS activity and protein in rat forebrain slices exposed to OGD
  12. Effect of NO donors and ONOO- on calcium-dependent NOS activity from rat forebrain slices
  13. Quantification of nNOS and iNOS protein in rat forebrain slices exposed to OGD or an NO donor
  14. Levels of nNOS mRNA in rat forebrain slices exposed to OGD and an NO donor
  15. LDH efflux
  16. DISCUSSION
  17. Acknowledgements

Addition of either a transcriptional inhibitor of iNOS expression, dexamethasone (1 = μM), or a protein synthesis inhibitor, cycloheximide (10 μM), blocked the appearance of calcium-independent NOS activity measured in rat forebrain slices exposed to OGD (Fig. 2). Concomitantly, these compounds produced a significant recovery in the calcium-dependent NOS activity (p < 0.05, n = 16 ; Fig. 2). When OGD-exposed slices were incubated with the NO scavenger HbO2 (10 μM), there was also a recovery of the calcium-dependent NOS activity present in slices exposed to OGD (OGD vs. OGD + HbO2 slices, respectively, n = 6-16, p < 0.05 ; Fig. 2). In this case, calcium-independent NOS activity was not significantly affected (OGD vs. OGD + HbO2, n = 6-16, p > 0.05).

image

Figure 2. Effect of cycloheximide, dexamethasone, and HbO2 on calcium-dependent and -independent NOS activities in rat forebrain slices exposed to OGD. Slices were collected at time 0 (OGD0) or 180 min (OGD180) after the end of the OGD period. Enzymatic activity was determined as described in the legend to Fig. 1. Data are mean ± SEM (bars) values (n = 6-16). *p <0.05 versus OGD180 (open columns), #p < 0.05 versus OGD180 (gray columns).

Download figure to PowerPoint

Furthermore, addition of dexamethasone (1 μM) produced a decrease in the levels of iNOS protein and an increase in the levels of nNOS protein in rat forebrain slices exposed to OGD (Fig. 3).

image

Figure 3. Left panel : Western blot analysis of nNOS and iNOS in soluble extracts from control and OGD- and OGD plus dexamethasone (DEX ; 1 μM)-treated rat forebrain slices. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the levels of nNOS and iNOS were measured by western blotting. Right panel : Laser densitometric analysis of nNOS and iNOS expression. Data are mean ± SEM (bars) values of the band intensities (n = 3).

Download figure to PowerPoint

Effect of NO donors and ONOO- on calcium-dependent NOS activity from rat forebrain slices

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. NOS activity
  5. mRNA analysis
  6. Characterization of NOS isoforms by western blot
  7. LDH assay
  8. Synthesis of ONOO-
  9. RESULTS
  10. NOS activity in rat forebrain slices exposed to OGD
  11. Effects of dexamethasone, cycloheximide, and HbO2 on NOS activity and protein in rat forebrain slices exposed to OGD
  12. Effect of NO donors and ONOO- on calcium-dependent NOS activity from rat forebrain slices
  13. Quantification of nNOS and iNOS protein in rat forebrain slices exposed to OGD or an NO donor
  14. Levels of nNOS mRNA in rat forebrain slices exposed to OGD and an NO donor
  15. LDH efflux
  16. DISCUSSION
  17. Acknowledgements

Exposure to the NO donor DETA-NONOate (1 mM) but not to SIN-1 (1 mM) or ONOO- (300 μM) caused a decrease in calcium-dependent NOS activity present in control rat forebrain slices (Fig. 4A). On the other hand, calcium-dependent NOS activity from brain homogenates assayed in the presence of 1 mM DETA-NONOate was partially inhibited as compared with the activity found in the absence of this compound (Fig. 4B).

image

Figure 4. Effect of NO donors or ONOO- on calcium-dependent NOS activity in rat forebrain slices. A : Effect of exposure to DETA-NONOate (1 mM), SIN-1 (1 mM), or ONOO- (300 μM) on calcium-dependent NOS activity in rat forebrain slices collected 180 min after the end of the exposure period. B : Calcium-dependent NOS activity in rat forebrain slices, determined in the absence and presence of DETA-NONOate (1 mM). NOS activity was determined as described in the legend to Fig. 1. Data are mean ± SEM (bars) values (n = 4). *p < 0.05 versus control.

Download figure to PowerPoint

Quantification of nNOS and iNOS protein in rat forebrain slices exposed to OGD or an NO donor

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. NOS activity
  5. mRNA analysis
  6. Characterization of NOS isoforms by western blot
  7. LDH assay
  8. Synthesis of ONOO-
  9. RESULTS
  10. NOS activity in rat forebrain slices exposed to OGD
  11. Effects of dexamethasone, cycloheximide, and HbO2 on NOS activity and protein in rat forebrain slices exposed to OGD
  12. Effect of NO donors and ONOO- on calcium-dependent NOS activity from rat forebrain slices
  13. Quantification of nNOS and iNOS protein in rat forebrain slices exposed to OGD or an NO donor
  14. Levels of nNOS mRNA in rat forebrain slices exposed to OGD and an NO donor
  15. LDH efflux
  16. DISCUSSION
  17. Acknowledgements

nNOS protein was strongly detected in extracts obtained from control slices as detected by western blot analysis using specific anti-nNOS antibody. OGD caused a significant decrease in the amount of nNOS protein (Fig. 5). Moreover, treatment with DETA-NONOate (1 mM) decreased the amount of nNOS protein present in control slices (Fig. 5). It is interesting that OGD caused the expression of iNOS protein in rat forebrain slices (Fig. 5), whereas DETA-NONOate barely increased iNOS levels in these preparations (Fig. 5).

image

Figure 5. Left panel : Western blot analysis of nNOS and iNOS in soluble extracts from control and OGD- or DETA-NONOate (1 mM)-treated rat forebrain slices. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the levels of nNOS and iNOS were measured by western blotting. Right panel : Laser densitometric analysis of nNOS and iNOS expression. Data are mean ± SEM (bars) values of the band intensities (n = 3).

Download figure to PowerPoint

Levels of nNOS mRNA in rat forebrain slices exposed to OGD and an NO donor

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. NOS activity
  5. mRNA analysis
  6. Characterization of NOS isoforms by western blot
  7. LDH assay
  8. Synthesis of ONOO-
  9. RESULTS
  10. NOS activity in rat forebrain slices exposed to OGD
  11. Effects of dexamethasone, cycloheximide, and HbO2 on NOS activity and protein in rat forebrain slices exposed to OGD
  12. Effect of NO donors and ONOO- on calcium-dependent NOS activity from rat forebrain slices
  13. Quantification of nNOS and iNOS protein in rat forebrain slices exposed to OGD or an NO donor
  14. Levels of nNOS mRNA in rat forebrain slices exposed to OGD and an NO donor
  15. LDH efflux
  16. DISCUSSION
  17. Acknowledgements

OGD caused a significant decrease in nNOS mRNA levels present in rat forebrain slices (Fig. 6A). In these conditions, there was an expression of the gene encoding iNOS in rat forebrain slices, as assessed by the detection of iNOS message (Fig. 6A). It is interesting that when control slices were exposed to 1 mM DETA-NONOate, a decrease in nNOS mRNA levels (39% with respect to control) was found (Fig. 6A).

image

Figure 6a. Northern blot analysis of nNOS and iNOS mRNA in control and OGD- or DETA-NONOate (1 mM)-treated rat forebrain slices, collected 180 min after the end of the OGD period. B : RNA pattern after size-separation in an agarose gel. C : Laser densitometric analysis of nNOS and iNOS bands after normalization with the corresponding amount of 18S ribosomal RNA. Data are mean ± SEM (bars) values (n = 3).

Download figure to PowerPoint

Also, the integrity of the RNA was well preserved during the time of incubation as deduced by the RNA pattern after size-separation in an agarose gel (Fig. 6B). The densitometric analysis dt the nNOS and iNOS bands after normalization with the corresponding amount of 18S ribosomal RNA is shown in Fig. 6C.

LDH efflux

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. NOS activity
  5. mRNA analysis
  6. Characterization of NOS isoforms by western blot
  7. LDH assay
  8. Synthesis of ONOO-
  9. RESULTS
  10. NOS activity in rat forebrain slices exposed to OGD
  11. Effects of dexamethasone, cycloheximide, and HbO2 on NOS activity and protein in rat forebrain slices exposed to OGD
  12. Effect of NO donors and ONOO- on calcium-dependent NOS activity from rat forebrain slices
  13. Quantification of nNOS and iNOS protein in rat forebrain slices exposed to OGD or an NO donor
  14. Levels of nNOS mRNA in rat forebrain slices exposed to OGD and an NO donor
  15. LDH efflux
  16. DISCUSSION
  17. Acknowledgements

LDH levels in the incubation solution of oxygen-glucose-deprived slices were significantly higher than those found in control slices during the whole period of reperfusion (714 ± 100 vs. 193 ± 17 mOD/min for OGD and control slices, respectively, p < 0.05, n = 8). However, when control slices were exposed to 1 mMDETA-NONOate, LDH release to the medium was not significantly different from that in control slices in the absence of this compound (217 ± 19 vs. 193 ± 17mOD/min for DETA-NONOate and control slices, respectively, p > 0.05, n = 8).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. NOS activity
  5. mRNA analysis
  6. Characterization of NOS isoforms by western blot
  7. LDH assay
  8. Synthesis of ONOO-
  9. RESULTS
  10. NOS activity in rat forebrain slices exposed to OGD
  11. Effects of dexamethasone, cycloheximide, and HbO2 on NOS activity and protein in rat forebrain slices exposed to OGD
  12. Effect of NO donors and ONOO- on calcium-dependent NOS activity from rat forebrain slices
  13. Quantification of nNOS and iNOS protein in rat forebrain slices exposed to OGD or an NO donor
  14. Levels of nNOS mRNA in rat forebrain slices exposed to OGD and an NO donor
  15. LDH efflux
  16. DISCUSSION
  17. Acknowledgements

We have previously shown that neurons and glial cells express iNOS after OGD in rat forebrain slices, as early as 120 min after the hypoxic insult (Moro et al., 1998). We have now found that, in these conditions, a decrease in content of nNOS occurs in parallel to iNOS expression. Our results showing direct measurements of enzyme activity indicate the presence of a calcium-independent NOS activity in rat forebrain slices after OGD. We have previously shown that this enzymatic activity is due to the expression of the inducible isoform, as demonstrated by the immunodetection by histochemistry as well as by the quantification of both iNOS message and protein by northern and western blot analysis, respectively (Moro et al., 1998). Different authors have found iNOS expression in in vivo models of ischemia-reperfusion, but this expression took place not before 1-3 days (Endoh et al., 1994 ; Iadecola et al., 1995 ; Yoshida et al., 1995). Proinflammatory cytokines are known to cause the induction of iNOS in several cell systems. It is interesting that interleukin-1 β, tumor necrosis factor-α, and interferon-γ are rapidly (within a few hours) induced in the brain following ischemia (Minami et al., 1992 ; Liu et al., 1993, 1994). These cytokines have been involved in the expression of iNOS in several cell types, including neurons and glial cells (Galea et al., 1992 ; Simmons and Murphy, 1992 ; Minc-Golomb et al., 1994). In our model, it is likely that these cytokines induce iNOS expression, thus mimicking the in vivo situation, although other mechanisms can be also implicated.

We have now found that the calcium-dependent NOS activity is down-regulated in rat forebrain slices exposed to OGD. This activity mainly arises from the nNOS isoform, because endothelial NOS protein was scarcely detectable in our samples (data not shown). Two mechanisms might account for this nNOS down-regulation : It might be a direct consequence of the ischemic damage, by which cytosolic proteins are lost owing to disruption of the plasma membrane ; alternatively, it might derive from an autoinhibition caused by an excessive formation of NO resulting from iNOS expression. The first possibility is unlikely owing to several reasons : (a) Calcium-dependent NOS activity is maintained in tissues exposed to OGD but in which calcium-independent NOS expression had been blocked with cycloheximide or with dexamethasone, suggesting that the former is somehow down-regulated by the latter. (b) Calcium-dependent NOS activity is significantly recovered in OGD-exposed slices in which NO had been scavenged with HbO2, supporting further the hypothesis implicating NO formation as responsible for the decrease in calcium-dependent NOS activity. (c) LDH efflux is increased after OGD but not after exposure to the NO donor DETA-NONOate, consistent with the idea that down-regulation of calcium-dependent NOS activity is due to the exposure to NO and not to necrotic damage ; moreover, the integrity of the RNA after OGD is maintained. It is interesting that the exposure of control slices to the NO donor SIN-1, which decomposes to form both NO and O2- in simple salt solutions, thus generating the powerful oxidant ONOO- (Feelisch et al., 1989 ; Beckman et al., 1990), or directly to ONOO-, did not affect calcium-dependent NOS activity in these preparations. The key factor for comparing the effect of short-lived species as ONOO- is exposure measured in terms of concentration multiplied by time. Because it has been shown that 1 mM SIN-1 provides a steady-state concentration of ONOO- of 0.26 μM at 37°C (Brunelli et al., 1995), we have used a bolus addition of 300 μM ONOO- to ensure that enough ONOO- was used. Our results may suggest that the down-regulation of nNOS reported in this article is not due to ONOO-, although further studies should be performed to clarify this point.

Our results showing inhibition of constitutive NOS when calcium-independent NOS activity is expressed are consistent with previous findings in human megakaryoblastic cells (Lelchuk et al., 1992), human vascular smooth muscle cells and endothelial cells (MacNaul and Hutchinson, 1993), and rat glomeruli (Schwartz et al., 1997), although the mechanism by which NO itself is responsible for the suppression of nNOS is not clear. It has been reported that NO is able to decrease NOS content at two different levels : First, it has been reported that NO production from both constitutive NOS and iNOS may be regulated by a direct effect of NO on the activity of NOS (Rogers and Ignarro, 1992 ; Buga et al., 1993 ; Griscavage et al., 1993 ; Rengasamy and Johns, 1993) by forming a ferrous-nitrosyl complex (Abu-Soud et al., 1995). Second, it has been shown that NO inhibits the gene expression of NOS isoforms such as iNOS (Colasanti et al., 1995). Our results show that addition of an NO donor to the enzymatic assay performed on a fresh slice homogenate causes an attenuation of calcium-dependent NOS activity as a result of a direct action of NO on NOS catalytic activity ; however, this direct inhibition has been reported to be reversible (Rogers and Ignarro, 1992), and therefore it would not be expected to contribute when the enzymatic assay is performed on slices in which iNOS is inhibited in the presence of EGTA. Moreover, the direct inhibition caused by DETA-NONOate present in the enzymatic assay was ~35%, whereas the decrease of calcium-dependent NOS activity in slices exposed to OGD after 180 min of “reperfusion” was ~70% of the activity present immediately after the OGD period, indicating that the direct action of NO on catalytic NOS activity does not account for the decrease reported in this article.

The decrease in calcium-dependent NOS activity caused by OGD and its recovery induced by those pharmacological tools that inhibit iNOS expression, the quantification of nNOS by western blotting showing a decrease in the amount of nNOS protein and its recovery by dexamethasone, and the decrease in nNOS message in rat forebrain slices exposed to OGD or to the NO donor DETA-NONOate suggest that this inhibition mainly occurs at a transcriptional or a posttranscriptional level, either by inhibition of nNOS gene expression in these tissues or by an increase in nNOS mRNA degradation, which would eventually lead to a decrease in a basal nNOS de novo synthesis in these tissues during the experiment. The fact that cycloheximide only partially preserved nNOS activity when compared with control tissues might be due to a balance between inhibition of the nNOS synthesis and degradation (Casado et al., 1997).

In summary, our results indicate that iNOS expression down-regulates nNOS activity in rat brain slices exposed to OGD. These studies suggest important and complex interactions between NOS isoforms, the elucidation of which may provide further insights into the physiological and pathophysiological events that occur during and after cerebral ischemia.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. NOS activity
  5. mRNA analysis
  6. Characterization of NOS isoforms by western blot
  7. LDH assay
  8. Synthesis of ONOO-
  9. RESULTS
  10. NOS activity in rat forebrain slices exposed to OGD
  11. Effects of dexamethasone, cycloheximide, and HbO2 on NOS activity and protein in rat forebrain slices exposed to OGD
  12. Effect of NO donors and ONOO- on calcium-dependent NOS activity from rat forebrain slices
  13. Quantification of nNOS and iNOS protein in rat forebrain slices exposed to OGD or an NO donor
  14. Levels of nNOS mRNA in rat forebrain slices exposed to OGD and an NO donor
  15. LDH efflux
  16. DISCUSSION
  17. Acknowledgements

We thank Mr. Oscar G. Bodelón for skillful technical assistance. This work was supported by grants PM95-0070 and PM95-0007 from the DGICYT. J.D.A. and A.C. are recipients of fellowships from the Spanish Ministry of Education and Culture and the Universidad Complutense, respectively.

  • 1
    Abu-Soud H.M., Wang J., Rousseau D.L., Fukuto J.M., Ignarro L.J., Stuehr D.J. (1995) Neuronal nitric oxide synthase self-inactivates by forming a ferrous-nitrosyl complex during aerobic catalysis.J. Biol. Chem. 270,2299723006.
  • 2
    Beckman J.S., Beckman T.W., Chen J., Marshall P.A., Freeman B.A. (1990) Apparent hydroxyl radical production by peroxynitrite : implications for endothelial injury from nitric oxide and superoxide.Proc. Natl. Acad. Sci. USA 87,16201624.
  • 3
    Blough N.V. & Zafiriou O.C. (1985) Reaction of superoxide with nitric oxide to form peroxynitrite in alkaline aqueous solution.Inorg. Chem. 24,35023504.
  • 4
    Brunelli L., Crow J.P., Beckman J.S. (1995) The comparative toxicity of nitric oxide and peroxynitrite to Escherichia coli. Arch. Biochem. Biophys. 316,327334.
  • 5
    Buga G.M., Griscavage J.M., Rogers N.E., Ignarro L.J. (1993) Negative feedback regulation of endothelial cell function by nitric oxide.Circ. Res. 73,808812.
  • 6
    Casado M., Diaz-Guerra M.J.M., Boscá L., Martin-Sanz P. (1997) Differential regulation of nitric oxide synthase mRNA expression by lipopolysaccharide and pro-inflammatory cytokine in fetal hepatocytes treated with cycloheximide.Biochem. J. 327,819823.
  • 7
    Chirgwin J.M., Przybyla A.E., MacDonald R.J., Rutter W.J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.Biochemistry 18,52945299.
  • 8
    Chomczynski P. & Sacchi N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal. Biochem. 162,156159.DOI: 10.1006/abio.1987.9999
  • 9
    Colasanti M., Persichini T., Menegazzi M., Mariotto S., Giordano E., Caldarera C.M., Sogos V., Lauro G.M., Suzuki H. (1995) Induction of nitric oxide synthase mRNA expression. Suppression by exogenous nitric oxide.J. Biol. Chem. 270,2673126733.
  • 10
    Dawson V.L., Dawson T.M., London E.D., Bredt D.S., Snyder S.H. (1991) Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures.Proc. Natl. Acad. Sci. USA 88,63686371.
  • 11
    Endoh M., Maiese K., Wagner J. (1994) Expression of the inducible form of nitric oxide synthase by reactive astrocytes after transient global oxygen and glucose deprivation.Brain Res. 651,92100.
  • 12
    Feelisch M., Ostrowski J., Noack E. (1989) On the mechanism of NO release from sydnonimines.J. Cardiovasc. Pharmacol. 14 (Suppl. 11) , S13S22.
  • 13
    Galea E., Feinstein D.L., Reis D.J. (1992) Induction of calcium-independent nitric oxide synthase activity in primary rat glial cultures.Proc. Natl. Acad. Sci. USA 89,1094510949.
  • 14
    Garthwaite J., Charles S.L., Chess-Williams R. (1988) Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain.Nature 336,385388.
  • 15
    Griscavage J.M., Rogers N.E., Sherman M.P., Ignarro L.J. (1993) Inducible nitric oxide synthase from a rat alveolar macrophage cell line is inhibited by nitric oxide.J. Immunol. 151,63296337.
  • 16
    Gross S.S. & Wolin M.S. (1995) Nitric oxide : pathophysiological mechanisms.Annu. Rev. Physiol. 57,737769.
  • 17
    Hill H.D. & Straka J.G. (1988) Protein determination using bicinchoninic acid in the presence of sulfhydryl reagents.Anal. Biochem. 170,203208.
  • 18
    Hughes M.N. & Nicklin H.G. (1968) The chemistry of pernitrites. Part I. Kinetics of decomposition of pernitrous acid.J. Chem. Soc. A,450452.
  • 19
    Iadecola C. (1997) Bright and dark sides of nitric oxide in ischemic brain injury.Trends Neurosci. 20,132139.
  • 20
    Iadecola C., Zhang F., Xu S., Casey R., Ross M.E. (1995) Inducible nitric oxide synthase gene expression in brain following cerebral ischemia.J. Cereb. Blood Flow Metab. 15,378384.
  • 21
    Knowles R.G. & Moncada S. (1994) Nitric oxide synthases in mammals.Biochem. J. 298,249258.
  • 22
    Koh J.Y. & Choi D.W. (1987) Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay.J. Neurosci. Methods 20,8390.
  • 23
    Lelchuk R., Radomski M.W., Martin J.F., Moncada S. (1992) Constitutive and inducible nitric oxide synthases in human megakaryoblastic cells.J. Pharmacol. Exp. Ther. 262,12201224.
  • 24
    Liu T., McDonnell P.C., Young P.R., White R.F., Siren A.L., Lallenbeck J.M., Barone F.C., Feuerstein G.Z. (1993) Interleukin-1 beta mRNA expression in ischemic rat cortex.Stroke 24,17461750.
  • 25
    Liu T., Clark R.K., McDonnell P.C., Young P.R., White R.F., Barone F.C., Feuerstein G.Z. (1994) Tumor necrosis factor-alpha expression in ischemic neurons.Stroke 25,14811488.
  • 26
    Lowenstein C.J., Glatt C.S., Bredt D.S., Snyder S.H. (1992) Cloned and expressed macrophage nitric oxide synthase contrasts with the brain enzyme.Proc. Natl. Acad. Sci. USA 89,67116715.
  • 27
    MacNaul K.L. & Hutchinson N.I. (1993) Differential expression of iNOS and cNOS mRNA in human vascular smooth muscle cells and endothelial cells under normal and inflammatory conditions.Biochem. Biophys. Res. Commun. 196,13301334.DOI: 10.1006/bbrc.1993.2398
  • 28
    Minami M., Kuraishi Y., Yabuuchi K., Yamazaki A., Satoh M. (1992) Induction of interleukin-1β mRNA in rat brain after transient forebrain ischemia.J. Neurochem. 58,390392.
  • 29
    Minc-Golomb D., Tsarfaty I., Schwartz J.P. (1994) Expression of inducible nitric oxide synthase by neurons following exposure to endotoxin and cytokine.Br. J. Pharmacol. 112,720722.
  • 30
    Moncada S., Palmer R.M.J., Higgs E.A. (1991) Nitric oxide : physiology, pathophysiology, and pharmacology.Pharmacol. Rev. 43,109142.
  • 31
    Moro M.A., De Alba J., Leza J.C., Lorenzo P., Fernández A.P., Bentura M.L., Boscá L., Rodrigo J., Lizasoain I. (1998) Neuronal expression of inducible nitric oxide synthase after oxygen-glucose deprivation in rat forebrain slices.Eur. J. Neurosci. 10,445456.
  • 32
    Nowicki J.P., Duval D., Poignet H., Scatton B. (1991) Nitric oxide mediates neuronal death after focal cerebral ischaemia in the mouse.Eur. J. Pharmacol. 204,339340.
  • 33
    Paterson R.A., Eagles P.A.M., Young D.A.B., Beddell C.R. (1976) Rapid preparation of large quantities of human haemoglobin with low phosphate content by counter-flow dialysis.Int. J. Biochem. 7,117118.
  • 34
    Rengasamy A. & Johns R.A. (1993) Regulation of nitric oxide synthase by nitric oxide.Mol. Pharmacol. 44,124128.
  • 35
    Rodrigo J., Springall D.R., Uttenthal O., Bentura M.L., AbadiaMolina F., Riveros-Moreno V., Martínez-Murillo R., Polak J.M., Moncada S. (1994) Localization of nitric oxide synthase in the adult rat brain.Philos. Trans. R. Soc. [Biol.] 345,175221.
  • 36
    Rogers N.E. & Ignarro L.J. (1992) Constitutive nitric oxide synthase from cerebellum is reversibly inhibited by nitric oxide formed from l-arginine. Biochem. Biophys. Res. Commun. 189,242249.
  • 37
    Salter M., Knowles R.G., Moncada S. (1991) Widespread tissue distribution, species distribution and changes in activity of Ca2+-dependent and Ca2+-independent nitric oxide synthases. FEBS Lett. 291,145149.
  • 38
    Schwartz D., Mendonca M., Schwartz I., Xia Y., Satriano J., Wilson C.B., Blantz R.C. (1997) Inhibition of constitutive nitric oxide synthase (NOS) by nitric oxide generated by inducible NOS after lipopolysaccharide administration provokes renal dysfunction in rats.J. Clin. Invest. 100,439448.
  • 39
    Simmons M.L. & Murphy S. (1992) Induction of nitric oxide synthase in glial cells.J. Neurochem. 59,897905.
  • 40
    Yoshida T., Waeber C., Huang Z., Moskowitz M.A. (1995) Induction of nitric oxide synthase activity in rodent brain following middle cerebral artery occlusion.Neurosci. Lett. 194,214218.