Blockade by sigma site ligands of N‐methyl‐D‐aspartate‐evoked responses in rat and mouse cultured hippocampal pyramidal neurones

Abstract

1 The effects of a range of structurally-dissimilar compounds which possess affinity for ai binding sites were examined on the responses of cultured hippocampal pyramidal neurones to the excitatory amino acid analogues N-methyl-D-aspartate (NMDA), kainate and (RS)-a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). 2 In mouse hippocampal neurones under whole-cell voltage-clamp, the compounds tested reversibly attenuated NMDA-, but not kainate-or AMPA-, evoked currents with a rank order potency (ICm3 The attenuation of NMDA-evoked responses was not mediated through interactions with the agonist, glycine (except haloperidol) or polyamine (except ifenprodil) binding sites on the NMDA receptor-channel complex but, in the light of the voltage-and, in some cases, use-dependent nature of their antagonism, an interaction with the ion channel appears to be a likely mechanism of action for many of the compounds. 4 Micromolar concentrations of selected ai site ligands also reduced NMDA-evoked rises in intracellular free calcium concentration in Fura-2-loaded cultured hippocampal neurones of the rat with the same rank order potency as observed in the electrophysiological studies. 5The data indicate that, at micromolar concentrations, the ai site ligands tested act as NMDA receptor antagonists, an action which does not appear to be mediated by high-affinity binding site(s). The functional effects of micromolar concentrations of ai site ligands cannot, therefore, be attributed exclusively to interactions with high-affinity ai binding sites.

Blockade by sigma site ligands of high voltage‐activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones

“…The NMDA antagonist activities of the same range of compounds as those tested in the present study, with the exception of L 687,384 (examined by McLarnon et al, 1994), were described in the preceding paper (Fletcher et al, 1995). There is little apparent similarity between either the absolute or the rank order NMDA antagonist and Ca2`channel blocking potencies (as assessed under whole-cell voltage-clamp conditions) of the compounds tested, indicating that the antagonist binding sites at HVA Ca2" channels and at the NMDA receptor-channel complex have different pharmacological specificities.…”

“…The micromolar concentrations of the compounds required to block HVA Ca2" channels (Table 1) are substantially higher than those nanomolar concentrations associated with competition for high-affinity a binding sites labelled with tritiated a ligands or non-opioid antitussives (see Fletcher et al, 1995). Furthermore, the rank order potency of the compounds tested as antagonists of HVA Ca2" channels (Table 1) does not parallel that of their affinities for high-affinity a binding sites.…”

“…After loading with Fura-2, coverslips were placed in a chamber at 20-240C and continuously superfused at 1.5 ml min' with a solution containing (mm): NaCl 136.5, KCl 3, NaH2PO4 1.5, MgSO4 1.5, D-glucose 10, /Ba (nA) a sIte Ugands block Ca2* channels use of the same logistic equation as in the electrophysiological studies (see above). Other experimental details are provided in the preceding paper (Fletcher et al, 1995).…”

“…In the preceding paper (Fletcher et al, 1995), the N-methyl-Daspartate (NMDA) antagonist actions of a series of a site ligands were described. We suggested that the appreciable NMDA antagonist activity of many of the compounds tested might underlie, at least in part, their antiepileptiform and neuroprotective effects which, in vitro, are observed at micromolar concentrations.…”

“…Final IC50 values (the concentration of test compound resulting in a 50% inhibition of the control response) and percentage reductions of control responses are expressed as mean + s.e.mean, with n the number of neurones tested. To derive IC50 values, data points were fitted to the logistic equation described in the companion paper (Fletcher et al, 1995).…”