Abstract
Microspectrofluorimetric measurements of excitatory amino acid-evoked rises in intracellular free calcium concentration ([Ca2+]i), electrophysiological measurements of currents through single NMDA receptor-operated ion channels and estimates of cellular viability following NMDA challenge were employed to examine the interactions of (-)- and (+)-beta-cyclazocine with the NMDA receptor-channel complex in cultured rat hippocampal neurons. Rises in [Ca2+]i evoked by NMDA, but not those evoked by kainate, AMPA or 50 mM K+, were reduced by (-)-beta-cyclazocine in a concentration- and use-dependent manner with an estimated IC50 value of 272 nM. In outside-out patches, (-)-beta-cyclazocine did not change the magnitudes of unitary NMDA-evoked currents but diminished both the frequency of channel openings and their mean open time. The IC50 for (-)-beta-cyclazocine against NMDA channel open state probability was estimated at 84 nM. The actions of (-)-beta-cyclazocine were consistent with a voltage-dependent open channel block of the NMDA channel with a blocking rate constant of 7.03.10(7) M-1.s-1 at -40 mV. Neurons exposed to a high concentration of NMDA in vitro were protected from death by 1 and 10 microM (-)-beta-cyclazocine. In all of the above assays, (+)-beta-cyclazocine was considerably less potent an NMDA antagonist and neuroprotective agent than (-)-beta-cyclazocine; the IC50 for (+)-beta-cyclazocine against channel open state probability was estimated at 14 microM. The results demonstrate that (-)-beta-cyclazocine is a potent and selective inhibitor of NMDA-evoked responses in cultured rat hippocampal neurons and an effective neuroprotective agent in vitro.
Published Version
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