ABSTRACT
The excessive activation of the N-methyl-D-aspartate receptor
(NMDAR)/nitric oxide (NO) pathway has been proposed to be
involved in the neuropathology of various neurodegenerative
disorders. In this study, NO was found to mediate glutamateinduced
excitotoxicity in primary cultured neurons. Compared
with the NO synthase (NOS) inhibitor, NG-monomethyl-L-arginine
(L-NMMA), and the NMDAR antagonist memantine, bis(7)-
tacrine was found to be more potent in reducing NO-mediated
excitotoxicity and the release of NO caused by glutamate.
Moreover, like L-NMMA but not like 5H-dibenzo[a,d]cyclohepten-
5,10-imine (MK-801) and memantine, bis(7)-tacrine showed
greater neuroprotection and inhibition on NO release when
neurons were pretreated for a prolonged time between 0 and
24 h and remained quite potent even when neurons were
post-treated 1 h after the glutamate challenge. Bis(7)-tacrine
was additionally found to be as moderately potent as memantine
in competing with [3H]MK-801, inhibiting NMDA-evoked
currents and reducing glutamate-triggered calcium influx,
which eventually reduced neuronal NOS activity. More importantly,
at neuroprotective concentrations, bis(7)-tacrine substantially
reversed the overactivation of neuronal NOS caused
by glutamate without interfering with the basal activity of NOS.
Furthermore, in vitro pattern analysis demonstrated that bis(7)-
tacrine competitively inhibited both purified neuronal and inducible
NOS with IC50 values at 2.9 and 9.3 M but not endothelial
NOS. This result was further supported by molecular
docking simulations that showed hydrophobic interactions between
bis(7)-tacrine and three NOS isozymes. Taken together,
these results strongly suggest that the substantial neuroprotection
against glutamate by bis(7)-tacrine might be mediated
synergistically through the moderate blockade of NMDAR and
selective inhibition of neuronal NOS.