Tamoxifen inhibits nitrotyrosine formation after reversible middle cerebral artery occlusion in the rat

Tamoxifen (TAM), a widely used non-steroidal anti-estrogen, has recently be en shown to be neuroprotective in a rat model of reversible middle cerebral artery occlusion (rMCAo). Tamoxifen has several potential mechanisms of ac tion including inhibition of the release of excitatory amino acids (EAA) an d nitric oxide synthase (NOS) activity. The question addressed in this stud y was whether TAM reduces ischemia-induced production of nitrotyrosine, con sidered as a footprint of the product of nitric oxide and superoxide, perox ynitrite. In rat brain, 2 h rMCAo produced a time-dependent increase in nit rotyrosine content in the cerebral cortex, as measured by Western blot anal ysis. Compared with vehicle, TAM significantly reduced nitrotyrosine levels in the ischemic cortex at 24 h. The neuronal (n)NOS inhibitor, 7-nitroinda zole also tended to reduce nitrotyrosine, but this reduction was not statis tically 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 si milar EC50 values, the activity of recombinant nNOS as well as NOS activity in brain homogenates, measured by conversion of [H-3]arginine to [H-3]citr ulline. There was marginal inhibition of recombinant inducible (i)NOS activ ity up to 100 muM TAM. These data suggest that TAM is an effective inhibito r 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.