- Top of page
- Materials and methods
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.
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.