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Keywords:

  • calmodulin;
  • cerebral ischemia;
  • nitric oxide synthase;
  • nitrotyrosine;
  • peroxynitrite;
  • tamoxifen

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Tamoxifen (TAM), a widely used non-steroidal anti-estrogen, has recently been shown to be neuroprotective in a rat model of reversible middle cerebral artery occlusion (rMCAo). Tamoxifen has several potential mechanisms of action including inhibition of the release of excitatory amino acids (EAA) and nitric oxide synthase (NOS) activity. The question addressed in this study was whether TAM reduces ischemia-induced production of nitrotyrosine, considered as a footprint of the product of nitric oxide and superoxide, peroxynitrite. In rat brain, 2 h rMCAo produced a time-dependent increase in nitrotyrosine content in the cerebral cortex, as measured by Western blot analysis. Compared with vehicle, TAM significantly reduced nitrotyrosine levels in the ischemic cortex at 24 h. The neuronal (n)NOS inhibitor, 7-nitroindazole also tended to reduce nitrotyrosine, but this reduction was not statistically significant. Immunostaining for nitrotyrosine was seen in cortical neurons in the MCA territory and this immunostaining was reduced by TAM. In vitro, TAM and the calmodulin inhibitor trifluoperazine inhibited, with similar EC50 values, the activity of recombinant nNOS as well as NOS activity in brain homogenates, measured by conversion of [3H]arginine to [3H]citrulline. There was marginal inhibition of recombinant inducible (i)NOS activity up to 100 µm TAM. These data suggest that TAM is an effective inhibitor of Ca2+/calmodulin-dependent NOS and the derived peroxynitrite production in transient focal cerebral ischemia and this may be one mechanism for its neuroprotective effect following rMCAo.

Abbreviations used
ALS

amyotrophic lateral sclerosis

EAA

excitatory amino acids

eNOS

endothelial nitric oxide synthase

iNOS

inducible nitric oxide synthase

nNOS

neuronal nitric oxide synthase

rMCAo

reversible middle cerebral artery occlusion

TAM

tamoxifen

TTC

triphenyltetrazolium chloride

Cerebral ischemia leads to a massive release of excitatory amino acids (EAAs) into the extracellular space which triggers neuronal and tissue damage (Benveniste et al. 1984; Hagberg et al. 1985). Neurotoxicity may arise from an increase in calcium ion (Ca2+) influx that occurs by several mechanisms, including activation of NMDA and AMPA receptors (Choi 1988, 1995), and failure of the Na+/Ca2+ exchanger due to intracellular Na+ overload (Siesjo and Bengtsson 1989). Pathological elevation in [Ca2+]i leads to Ca2+/calmodulin-dependent activation of many enzymes, including nitric oxide synthase (NOS), calcineurin, phospholipases and proteases, which lead to damage to proteins, nucleic acids, lipids, failure of cellular metabolism and death of the cell (Siesjo and Bengtsson 1989; Choi 1995; Tymianski and Tator 1996; Bolanos and Almeida 1999).

Tamoxifen (TAM) is widely used for the treatment of breast cancer as a non-steroidal anti-estrogen (Butta et al. 1992; Jordan 1993; MacGregor and Jordan 1998). It also inhibits swelling-activated anion release (Kirk and Kirk 1994), and we have previously found that blockers of swelling-activated anion channels suppress EAA release during ischemia (Seki et al. 1999). Phillis et al. (1998) showed that TAM inhibited ischemia-induced EAA release in a cortical superfusion model. In our laboratory, TAM was found to be neuroprotective in rat reversible middle cerebral artery occlusion (rMCAo) (Kimelberg et al. 2000). However, the mechanism of its action did not seem likely to be due only to inhibition of EAA release during ischemia. Tamoxifen protected even if given after ischemia (Kimelberg et al. 2000) when extracellular EAA levels have returned toward normal (Seki et al. 1999). Also, TAM-induced neuroprotection was large relative to the incomplete inhibition of ischemia-induced EAA release seen with TAM (Phillis et al. 1998), or with other anion channel blockers (Seki et al. 1999). Therefore, we looked for alternative mechanisms ‘downstream’ of release of EAAs. Because TAM has been reported to be a potent inhibitor of calmodulin (Lam 1984; Lopes et al. 1990) and neuronal (n)NOS (Renodon et al. 1997), we explored whether TAM inhibits NOS activity in vivo by measuring its effects on the production of nitrotyrosine, considered to be a footprint of peroxynitrite (ONOO) (Coeroli et al. 1998; Eliasson et al. 1999) in rMCAo.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Materials

l-[2,3-3H]Arginine (1 mCi/mL) was purchased from Du Pont-NEN Research Products (Boston, MA, USA). Recombinant rat constitutive nNOS and recombinant mouse inducible (i)NOS were from Calbiochem (San Diego, CA, USA). 7-Nitroindazole, trifluoperazine and other chemicals, unless specified otherwise, were purchased from Sigma Chemicals (St Louis, MO, USA).

Focal ischemia model

All animal procedures were in accordance with guidelines for the care and use of laboratory animals and were approved by the institutional animal care and use committee. Anesthesia was induced in male Sprague−Dawley rats (Taconic, body weight 300–350 g) using methohexital sodium (50 mg/kg, i.p.) after atropine sulfate (50 mg/kg, i.m.). Animals were then intubated and ventilated with a gas mixture of 1.0% halothane in 30% O2/balance N2. Temperature was monitored with a rectal probe and a probe inserted under the temporalis muscle and maintained between 37.0 and 37.5°C with a heating pad and lamp. The left femoral artery was exposed and catheterized with polyethylene tubing (PE-50) to allow blood sampling and the monitoring of arterial blood pressure during ischemia and initial reperfusion. Arterial blood gases were examined before injection of drug, just before occlusion of the middle cerebral artery and 30 min after the start of reperfusion.

Tamoxifen (5 mg/kg) and 7-nitroindazole (50 mg/kg) were given i.p. in 300 µL of peanut oil and sonicated before injection. Drug or vehicle administration was always carried out 30 min prior to MCAo.

Reversible middle cerebral artery occlusion was performed as described by Longa et al. (1989). Briefly, a 4-0 nylon intraluminal suture coated with a mixture of silicone resin (Xantopren; Heraeus Dental, Osaka, Japan) and a hardener (Activator NF; Heraeus Dental) was introduced into the internal carotid artery and advanced until it blocked blood flow into the middle cerebral artery. The suture was left in place for 2 h and then withdrawn from the external carotid artery. After 30 min of reperfusion the femoral catheter was removed and the surgical wounds were closed. Anesthesia was then turned off and the animal kept on a heating blanket with the temperature monitored and maintained until the animal was fully recovered from anesthesia at which time it was returned to the cage.

Rats were killed at 12 or 24 h after ischemia with an overdose of methohexital sodium. To measure nitrotyrosine by Western blot analysis or immunohistochemistry, brains were removed and sectioned at 2-mm thickness from 3 mm posterior of the anterior tip of the frontal lobe. Four cross-sections (from 3- to 11-mm anterior coordinate) were selected and the regions corresponding to the penumbra, core-of-infarct and contralateral regions collected. The penumbra, core-of-infarct and contra-lateral regions were based on anatomical rather than pathophysiological criteria, with the core defined as the lateral cortex and the penumbra as the more medial cortex as described by Fukuyama et al. (1998). The region selected as core was the anatomical region that was consistently the most damaged cortex as assessed by 2,3,5-triphenyltetrazolium chloride (TTC) staining and histological criteria in our previous studies (Kimelberg et al. 2000). Although this region appeared paler than the surrounding tissue no pathophysiologically quantitative assessment aside from nitrotyrosine measurement was carried out.

Experimental design

To determine the time course of nitrotyrosine accumulation, animals were killed 12 h (vehicle treated, 2 h rMCAo, n = 5) or 24 h after rMCAo (vehicle treated, 2 h rMCAo, n = 5). These were compared with control animals (n = 3) with no treatment or surgical manipulation. For comparison of drug treatments, animals were treated with TAM (n = 8) or 7-nitroindazole (n = 7) and compared with vehicle-treated animals (n = 5) and vehicle-treated sham-operated animals (n = 3, surgical manipulations performed but the suture was not advanced), all killed at 24 h after ischemia.

Western blotting

For immunoblot analysis of proteins, samples from regions corresponding to penumbra and core-of-infarct in ipsilateral and contralateral areas were freshly excised. They were then homogenized at 4°C in 50 mm Tris−HCl (pH 7.4), 1 mm EDTA and 1 mm phenylmethylsulfonyl fluoride and centrifuged at 15 000 g for 15 min to remove any insoluble material. The supernatant was retained. The protein concentration of the supernatant was determined using Protein Assay ESL reagent (Boehringer Mannheim) with bovine serum albumin as the standard. Solubilized proteins (10 µg/lane) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS−PAGE; 12% acrylamide, 1.5-mm-thick slab gels) and the proteins were then transferred to a poly(vinylidene difluoride) membrane. The membrane was blocked with 5% non-fat skim milk overnight at 4°C and incubated with mouse monoclonal nitrotyrosine antibody (1 : 1000 dilution, Upstate Biotechnology, Lake Placid, NY, USA) for 2 h. After washing, the membrane was incubated with goat antimouse IgG conjugated to horseradish peroxidase (1 : 4000) for 2 h. The horseradish peroxidase product was visualized using an enhanced chemiluminescence western blotting detection system (Amersham). The band intensities were quantitated by densitometric scanning using the NIH image program.

Immunohistochemistry

Rats were killed 24 h after recirculation (n = 3 in each group: sham rMCAo, TAM-treated rMCAo and vehicle-treated rMCAo) by perfusing via the ascending aorta under deep anesthesia with cold 0.1 m phosphate-buffered saline (pH 7.4) followed by phosphate-buffered saline containing 4% paraformaldehyde. Brains were removed, post-fixed for 1.5 h in the same fixative, and placed in 0.1 m phosphate-buffered saline containing 15% sucrose. Serial coronal cryostat sections (20 µm) were collected on silanated slides. Non-specific endogenous peroxidase activity was prevented by incubating the sections in 0.3% hydrogen peroxide in 10% methanol. Sections were blocked with normal goat serum containing 2% bovine serum albumin for 1 h. They were incubated overnight at 4°C in a humidified chamber with affinity-purified rabbit polyclonal antinitrotyrosine antibody (Upstate Biotechnology) at a concentration of 30 µg/mL in a Tris-buffered saline containing 150 mm NaCl, 0.05% Tween-20, 20 mm Tris (pH 7.4) and additionally 1% bovine serum albumin. The secondary antirabbit IgG biotinylated antibodies (1 : 500 dilution) were visualized with avidin−biotin peroxidase (Vectastain Elite ABC kit, Vector Labs, Burlingame, CA, USA). The sections were then incubated with fresh diaminobenzidine tetrahydrochloride solution at room temperature. Negative controls were carried out by pre-incubating the primary antibody with 10 mm nitrotyrosine in phosphate-buffered saline (1 h, 20°C). Sections were counterstained with hematoxylin.

Nitric oxide synthase activity assays

Recombinant nNOS activity was measured by the conversion of l-[3H]arginine to l-[3H]citrulline as described by Bredt and Snyder (1989). Enzymatic reactions were conducted at 37°C for 15 min. Recombinant rat nNOS enzyme was dissolved in 50 mm HEPES plus 1 mm dithiothreitol. The incubation mixture contained 125 µL of 50 mm HEPES (pH 7.4), 1 mm dithiothreitol, 1 mmβ-nicotinamide adenine dinucleotide phosphate reduced form (NADPH), 1 mm CaCl2, 17 µg/mL calmodulin, 20 µm tetrahydrobiopterin, 1 µm FAD, 1 µm FMN and 0.6 µL of l-[3H]arginine (sp. activity 1 mCi/mL). The reaction was started by adding nNOS solution (25 µL; 0.2 Units) and was stopped by addition of a stop solution containing 20 mm HEPES, 2 mm EDTA and 0.2 mm EGTA (pH 5.5). The mixture was then applied to anion-exchange columns containing Dowex AG50WX-8 (Na+ form; Bio-Rad, Richmond, VA, USA) and eluted with 2 mL of distilled water. [3H]Citrulline in eluates was measured by scintillation spectrometry (Packard Tri-Carb 1900TR; Packard Instrument Co., Meriden, CT, USA). Control samples without NOS were included for background determinations. Recombinant iNOS activity assays were performed identically except CaCl2 and calmodulin were omitted in an assay buffer.

To determine Ca2+-dependent NOS activity in brain samples, rat brains were homogenized in 4 vol. buffer containing 50 mm HEPES, 1 mm dithiothreitol and 0.5 mm EDTA (pH 7.4). Homogenates were centrifuged for 15 min at 18 000 g (4°C). Supernatants were collected, diluted an additional five times with the same buffer, and frozen at −80°C. Twenty-five-microliter supernatant aliquots were used in assays of brain Ca2+-dependent NOS activity. The assays were performed as described above for recombinant nNOS. Final tissue dilution factor was 1 : 120 to 1 : 125 and final protein content was 100–150 µg/assay.

Statistical analysis

Data are expressed as mean ± SEM. Statistical analysis was performed by one-way anova followed by the Bonferroni-Dunn method for multiple comparisons. Statistical significance was accepted at the p < 0.05 level. Determinations of IC50 values were performed using sigmoidal fitting in origin 6.0 (Microcal, Northampton, MA, USA).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Physiological parameters

Physiologic parameters of animals were unaltered by either 7-nitroindazole or TAM (Table 1). There were no significant differences among any of the experimental groups.

Table 1.  Physiologic variables (mean ± SEM) measured prior to drug administration (pre-drug), after drug administration prior to rMCAo (pre-rMCAo) and during reperfusion prior to recovery from anesthesia (post-rMCAo)
TreatmentVehicleVehicleVehicleTamoxifen7-Nitroindazole
IschemiarMCAorMCAoshamrMCAorMCAo
Time of sacrifice12 h24 h24 h24 h24 h
Number55387
Mean arterial blood pressure (mmHg)
 Pre-drug115 ± 7106 ± 7105 ± 6106 ± 5107 ± 5
 Pre-rMCAo111 ± 7103 ± 6108 ± 7104 ± 7108 ± 6
 Post-rMCAo119 ± 8114 ± 6117 ± 7106 ± 5115 ± 6
pH
 Pre-drug7.46 ± 0.027.44 ± 0.037.47 ± 0.017.44 ± 0.017.45 ± 0.01
 Pre-rMCAo7.45 ± 0.027.42 ± 0.037.45 ± 0.017.43 ± 0.017.44 ± 0.02
 Post-rMCAo7.37 ± 0.027.39 ± 0.027.42 ± 0.037.41 ± 0.017.39 ± 0.02
PaCO2 (mmHg)
 Pre-drug35.6 ± 1.436.6 ± 3.231.3 ± 2.036.6 ± 0.733.3 ± 1.6
 Pre-rMCAo36.2 ± 2.138.6 ± 2.333.0 ± 1.037.2 ± 0.934.1 ± 1.6
 Post-rMCAo41.4 ± 1.942.0 ± 2.235.3 ± 4.936.6 ± 1.839.9 ± 1.9
PaO2 (mmHg)
 Pre-drug127.2 ± 7.3120.6 ± 9.5132.3 ± 7.9110.7 ± 2.7118.7 ± 5.3
 Pre-rMCAo111.0 ± 3.8115.0 ± 6.0123.3 ± 1.8113.5 ± 1.9120.3 ± 3.0
 Post-rMCAo122.0 ± 6.5115.0 ± 4.2127.7 ± 15.3114.1 ± 4.5111.3 ± 2.3

Detection of nitrotyrosine in brain samples by Western blot analysis

Blotting with antinitrotyrosine monoclonal antibody detected two major protein bands, corresponding to ≈ 68 and 50 kDa. The 68-kDa band was stained more intensely than the 50-kDa band. Densitometric quantification of the 68-kDa nitrotyrosine band demonstrated an increase in signal from 12 to 24 h after reperfusion in both the penumbra and core area (Fig. 1). In the control samples or samples from the contralateral side nitrotyrosine staining was marginal.

image

Figure 1. Nitrotyrosine production in the ischemic rat brain. (Upper) Representative western blots of cerebral cortex extracts with a monoclonal antibody against nitrotyrosine. Lanes 1, 2 and 3 represent 10 µg/lane protein samples from control sham-operated animals from brain regions corresponding to penumbra, core and contralateral cortex, respectively. Lanes 4, 5 and 6 are penumbra, core and contralateral cortex, respectively, 12 h after rMCAo. Lanes 7, 8 and 9 are penumbra, core and contralateral cortex, respectively, 24 h after rMCAo. (Lower) Quantification of nitrotyrosine staining in the postischemic rat brain. Bar graphs represent means ± SEM of densitometric measurements (optical density, O.D.) of nitrotyrosine staining of the ≈ 68 kDa protein. Number of animals is given in parentheses.

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The density of nitrotyrosine in penumbra and infarct core was compared between animals pretreated with vehicle, TAM or 7-nitroindazole. Tamoxifen significantly reduced the nitrotyrosine signal in the penumbra and core area compared with the vehicle-treated group (79 and 70% reduction, respectively, p < 0.05, Fig. 2). 7-Nitroindazole also reduced the signal, but its effect was not statistically significantly different from the vehicle treated group (28% reduction in both penumbra and core). No significant nitrotyrosine signal was detected at 24 h in the cortex of sham-operated animals.

image

Figure 2. Efects of TAM and 7-nitroindazole on nitrotyrosine production after MCA occlusion. (Upper) Representative western blots of cerebral cortex extracts using a monoclonal antibody against nitrotyrosine. Lanes 1 and 2 are samples (10 µg protein/lane) from brain regions corresponding to penumbra and core, respectively, in a sham-operated animal. Lanes 3 and 4 are penumbra and core, respectively, 24 h after rMCAo treated with vehicle. Lanes 5 and 6 are penumbra and core, respectively, 24 h after rMCAo treated with TAM. Lanes 7 and 8 are penumbra and core, respectively, 24 h after rMCAo treated with 7-nitroindazole. (Lower) Effects of TAM and 7-nitroindazole on nitrotyrosine staining of the ≈ 68 kDa protein 24 h after rMCAo. Results are mean ± SEM of densitometric measurements (optical density, O.D.). Number of animals is given in parentheses. *Significantly different from vehicle-treated group, p < 0.05.

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Anti-nitrotyrosine immunohistochemistry in ischemic brain

As illustrated in Fig. 3(a), the cytoplasm of pyramidal cells in the cerebral cortex on the ischemic side showed strong immunoreactivity with the antinitrotyrosine antibody in all vehicle-treated animals 24 h after rMCAo. Tamoxifen reduced antinitrotyrosine staining in the cells of the ischemic cortex (Fig. 3b). Little or no immunostaining was detected in the ipsilateral cortex of the sham-operated animals (Fig. 3c) or in the cerebral cortex contralateral to the occlusion (data not shown). As a negative control, antibody preabsorbed with nitrotyrosine showed no staining in sections from animals 24 h after rMCAo (data not shown).

image

Figure 3. Representative immunohistochemical stainings of nitrotyrosine in cerebral cortex 24 h after rMCAo. Cell nuclei were counterstained with hematoxylin. (a) Cortical penumbra in vehicle treated animal; (b) cortical penumbra in a TAM-treated animal; (c) region corresponding to penumbra after sham operation. Arrows point to antinitrotyrosine staining of pyramidal neurons, the reduced nitrotyrosine staining of neuronal bodies in penumbra of TAM-treated animals is marked by asterisks. Bar in (a) corresponds to 100 µm.

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Effect of TAM on NOS activity in vitro

To our knowledge, only one previous study has directly shown inhibition of recombinant nNOS by TAM (Renodon et al. 1997). To compare the dose–response curve for nNOS inhibition by TAM with other inhibitors, we measured recombinant nNOS activity in vitro. Recombinant nNOS activity was suppressed completely in the presence of 10 µmNG-nitro-l-arginine (data not shown) and 10 µm 7-nitroindazole (Fig. 4a), both well-known NOS inhibitors (Furfine et al. 1993; Mesenge et al. 1998). Tamoxifen and the calmodulin inhibitor trifluoperazine showed a dose-dependent inhibition of nNOS with identical slopes and similar apparent IC50 values of 6.8 and 7.6 µm for TAM and trifluoperazine, respectively (Fig. 4a). In contrast to recombinant nNOS, recombinant iNOS enzyme was essentially insensitive to TAM up to 100 µm(Fig. 4a, open symbols). We found no inhibition with 32 µm TAM and marginal 14% inhibition with 100 µm TAM which was not statistically significant (n = 4, p = 0.06; Fig. 4a).

image

Figure 4. Dose–response curves for the effects of TAM (●), 7-nitroindazole (▪) and trifluoperazine (▴) on the recombinant nNOS activity (a). Effects of TAM on recombinant iNOS activity are shown by (○). (b) Endogenous NOS activity in brain homogenates measured as conversion of l-[3H]arginine to l-[3H]citrulline. Data are means ± SEM of between two and three experiments and at least four independent determinations (see text for details).

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Calcium-dependent NOS activity in brain homogenates was also efficiently inhibited by TAM and trifluoperazine with estimated IC50 values of 25 and 10.6 µm, respectively (Fig. 4b). A shift in TAM sensitivity, compared with recombinant nNOS, may be partially explained by absorption of the hydrophobic TAM molecule on proteins and lipids in brain homogenate, so that actual concentrations are less than those added.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Nitric oxide synthase inhibition and TAM neuroprotection

The novel finding in this study is that TAM is a potent inhibitor of NOS activity, as measured by inhibition of nitrotyrosine production, in rMCAo in the rat, an ischemic model in which we have recently shown that TAM confers marked neuroprotection (Kimelberg et al. 2000). TAM also inhibited recombinant nNOS and Ca2+-dependent NOS activity in brain homogenates at concentrations that are likely to be achieved in the brain after injection, assuming total body equilibration. This effect of tamoxifen is likely due to inhibition of calmodulin (Lam 1984; Lopes et al. 1990), and our data show that inhibition of recombinant nNOS catalytic activity by TAM was practically identical to the effect of trifluoperazine, a well-known calmodulin inhibitor (Massom et al. 1990). In brain homogenates NOS sensitivity to TAM was somewhat reduced compared with recombinant enzyme, which may be due to changes in assay conditions (increased calmodulin and total protein concentration plus lipid in homogenates). Calmodulin antagonists have been found to be protective against cardiac ischemia (Otani et al. 1989; Sargent et al. 1992), and against transient cerebral ischemia when given 5 min after induction of 2 h of rMCAo (Kuroda et al. 1997), or 1 h after induction of 1 h rMCAo (Sato et al. 1999).

Calmodulin-dependent neuronal NOS is upregulated as early as 15 min and peaks ≈ 1 h after initiation of permanent focal cerebral ischemia (Zhang et al. 1994). Increased NOS activity is associated with peroxynitrite formation when superoxide radical is present, as is likely in reperfusion after cerebral ischemia (Beckman and Koppenol 1996; Coeroli et al. 1998; Mesenge et al. 1998; Eliasson et al. 1999). Thus, protection by inhibition of neuronal NOS activity through the Ca2+-calmodulin system would be expected within a few hours of the onset of cerebral ischemia, consistent with our finding that TAM provided effective neuroprotection when infusion was begun at up to 3 h but not at 4 h after onset of 2 h of reversible focal ischemia (Kimelberg et al. 2000). We measured the reduction by TAM of nitrotyrosine levels 12 and 24 h after reperfusion to achieve sufficient levels of nitrotyrosine for accurate quantitation on western blots. As shown in Fig. 1., levels of nitrotyrosine increase with time after rMCAo. Nitrotyrosine is considered a long-lived footprint of NO and ONOO (Smith et al. 1997; Coeroli et al. 1998; Eliasson et al. 1999; Xu et al. 2000). A considerable portion of this nitrotyrosine might be expected to be due to iNOS activation (Hirabayashi et al. 2000), but we found (Fig. 4) that recombinant iNOS was insensitive to TAM up to a concentration ≈ 10-fold greater than the concentration likely to be present in the CNS at the doses used.

Taken together, these findings suggest that one neuroprotective mechanism for TAM may be through reduction of nNOS activity and inhibition of peroxynitrite production. This is consistent with recent evidence that inhibitors of nNOS and nNOS gene knockouts are protective against cerebral ischemia (Huang et al. 1994; Yoshida et al. 1994; Hara et al. 1996). In our experiments, intraperitoneal injection of the nNOS inhibitor 7-nitroindazole, despite its high potency in vitro, inhibited nitrotyrosine levels less than TAM. This may be explained by more efficient crossing of the blood−brain barrier by TAM (Biegon et al. 1996).

In contrast to nNOS, nitric oxide production by endothelial NOS (eNOS) is considered to be largely neuroprotective (Lo et al. 1996). The beneficial role for eNOS in cerebral ischemia is attributed to the vascular effects of NO, including vasodilation and reduced neutrophil accumulation (Lo et al. 1996; Batteur-Parmentier et al. 2000). This enzyme may also be inhibited by TAM, because eNOS is also calmodulin dependent. However, eNOS is only thought to be responsible for a minor part of the nitrotyrosine formation in ischemia (Hirabayashi et al. 2000) and its effects may be localized to the vasculature. Also, estrogens and the TAM analog raloxifene have been reported to activate eNOS in endothelial cells indirectly via a phosphoinositol-3 kinase pathway (Haynes et al. 2000; Simoncini and Genazzani 2000). It is not known whether TAM increases eNOS activity like its analog raloxifene, but such an action would be a further mechanism for neuroprotection, However, in our previous work we found that TAM did not significantly alter cerebral blood flow during ischemia (Kimelberg et al. 2000).

Although the third form of NOS, inducible NOS (iNOS) is also thought to participate in delayed brain damage in ischemia (Iadecola et al. 1996, 1997), this enzyme is Ca2+-insensitive, and we found it to be essentially insensitive to inhibition by up to 100 µm TAM (Fig. 4), confirming the results of Renodon et al. (1997). Therefore, inhibition of iNOS appears unlikely to contribute to the reduction of nitrotyrosine levels observed after rMCAo in this study. Also, iNOS is induced 12–24 h after reversible MCAo (Iadecola et al. 1996; Hirabayashi et al. 2000), beyond the therapeutic window of TAM and therefore is also unlikely to be responsible for any of the previously described TAM neuroprotection (Kimelberg et al. 2000).

Other possible mechanisms for neuroprotection by TAM

The degree of neuroprotection by TAM in rMCAo (Kimelberg et al. 2000) appears to exceed the protective effects of NOS inhibitors and nNOS gene deletion in the same model of cerebral ischemia (Huang et al. 1994; Yoshida et al. 1994; Hara et al. 1996). This suggests other neuroprotective effects of TAM in addition to nNOS inhibition. Although TAM is best known for its use in the treatment of breast cancer where it works via its antagonism of estrogen receptors (Butta et al. 1992; Jordan 1993; MacGregor and Jordan 1998), TAM also shows estrogen-agonist effects depending on tissue, dose and duration of dose (Love et al. 1991; Jordan 1993; MacGregor and Jordan 1998). The neuroprotective effects of estrogens after cerebral ischemia have been widely demonstrated (Simpkins et al. 1997; Toung et al. 1998; Wang et al. 1999; Sawada et al. 2000). However, in most cases neuroprotection was demonstrated after chronic treatment with estrogens. Recent studies have reported that estrogens are effective when given acutely after ischemia, for example up to 3 h after induction of permanent MCAo in the ovariectomized rat (Yang et al. 2000). In this study when 17β-estradiol (E2) was injected intravenously, an estrogen silicone elastomer pellet was also implanted to maintain plasma levels of E2. E2 and other estrogens have a multitude of effects and their neuroprotective ability does not seem to be only, or perhaps at all, related to effects at estrogen receptors (Green and Simpkins 2000). Estradiol, at concentrations up to 10−5 m, does not inhibit nNOS (Hayashi et al. 1994) so the protective effects of estrogen seem unlikely to resemble TAM in inhibiting NOS. However, some estrogen effects at supraphysiological concentrations, such as antioxidant activity, may be shared by TAM. It may well be that estrogen and tamoxifen actions at estrogen receptors are unrelated to their respective neuroprotective effects. The neuroprotective effects of both estrogens (Toung et al. 1998; Wang et al. 1999) and tamoxifen (Kimelberg et al. 2000) appear unrelated to increases in blood flow. Further work needs to be done to determine to what extent estrogens and tamoxifen share common mechanisms of action and whether any of the neuroprotective effects of TAM include actions at the estrogen receptors.

Tamoxifen is also a highly effective inhibitor of swelling activated amino acid release (Kirk and Kirk 1994), and reduces EAA release from the ischemic cortex by ≈ 40% (Phillis et al. 1998). This effect is likely attributable to calmodulin inhibition (Kirk and Kirk 1994). In a recent study (Mongin et al. 1999), we found that the calmodulin inhibitor trifluoperazine potently blocked amino acid release in primary astrocyte cultures with an IC50 of ≈ 10 µm, a concentration very close to the effective concentrations of TAM and trifluoperazine for nNOS inhibition seen in this study. We had previously reported inhibition of d-[3H]aspartate release by 10 µm TAM in primary astrocyte cultures (Rutledge et al. 1998).

A further possible mode for tamoxifen action is its antioxidant activity (Wiseman et al. 1993). The antioxidant action of TAM has been suggested as the main reason for its cardioprotective effects (Love et al. 1991; Wiseman et al. 1993) and TAM may well attenuate oxidative damage in the CNS. This potential mechanism of TAM neuroprotection needs further evaluation.

Possible sites for tyrosine nitration

Peroxynitrite produced in the reaction of nitric oxide and superoxide anion non-specifically nitrates protein tyrosine residues (Beckman and Koppenol 1996; Smith et al. 1997; Eliasson et al. 1999). Our western blot analysis detected nitrated proteins primarily at 68 and 50 kDa after cerebral ischemia. Although we did not identify the protein targets of tyrosine nitration in ischemic brain, relatively selective staining in western blots and intense staining of neuronal bodies in the penumbra suggests limited sites of protein nitration. Recent studies have shown that the low molecular weight neurofilament proteins are susceptible to peroxynitrite-mediated nitration in sporadic amyotrophic lateral sclerosis (ALS) (Abe et al. 1995; Strong et al. 1998). The axons of pyramidal cells of the cerebral cortex contain a 68-kDa neurofilament protein (Shaw et al. 1981) which we speculate may account wholly or in part for the intense staining of neuronal bodies and processes in pyramidal cells observed in our study.

In conclusion, this report demonstrates that TAM inhibits the production of nitrotyrosine in vivo after reversible focal cerebral ischemia. The effect is likely due to inhibition of calmodulin-dependent nNOS production and may be one mechanism of TAM neuroprotection in reversible focal ischemia (Kimelberg et al. 2000).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank Yiqiang Jin and Carol Charniga for expert technical assistance. This work was supported by NIH grant NS35205 to HKK and a grant from Mizuho America Inc to KO and BIT.

References

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  6. Acknowledgements
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