Study of potency, kinetics of block and toxicity of NMDA receptor antagonists using fura-2
Introduction
High-affinity uncompetitive NMDA receptor antagonists like MK-801 bind within the activated NMDA-gated ion channel and impede the flow of Ca2+ into neurons. Significant neurobehavioral side effects limit the therapeutic value of these high-affinity, uncompetitive antagonists (Kemp et al., 1987). In contrast, low-affinity, uncompetitive NMDA receptor antagonists, like memantine, have reduced toxicities, possibly due to faster rates of unblocking and equilibrium block (Rogawski, 1993). Likewise, the low-affinity uncompetitive NMDA receptor antagonists, ARL 15896AR (formerly FPL 15896AR; (S)-α-phenyl-2-pyridine-ethanamine dihydrochloride) and the desglycinyl metabolite (ARL 12495AA; 1,2-diphenyl-2-propylamine monohydrochloride; DGR) of remacemide HCl (ARL 12924AA; 2-amino-N-(1-methyl-1,2-diphenylethyl)acetamide hydrochloride), block NMDA-induced toxicity in cortical cultures, and rapidly reduce NMDA-induced increases in [Ca2+]i (Black et al., 1995, Black et al., 1996). This rapid block of the [Ca2+]i response differs from the high-affinity antagonists which exhibit a slower block of the NMDA-induced rise in [Ca2+]i (Black et al., 1996). The slow blocking rate of MK-801 allows significant Ca2+ influx such that very high concentrations of MK-801 are required to completely block NMDA-triggered [Ca2+]i responses, producing long-lasting, maximal blockade of the channel (Kemp et al., 1987).
In this report we describe a simple system to determine the kinetics of block of uncompetitive NMDA receptor antagonists to facilitate screening novel compounds. We show that there is an inverse relationship between the high- and low-affinity uncompetitive NMDA receptor antagonists and their rates of block of NMDA-induced [Ca2+]i responses.
Section snippets
Reagents
We purchased MK-801, ketamine, dextrorphan, dextromethorphan and NMDA from Research Biochemicals International (Natick, MA, USA), memantine from Tocris Cookson (St. Louis, MO, USA) and obtained ADCI ((±)-5-aminocarbonyl-10,11-dihydro-5H-dibenzo[a,d]cyclo-hepten-5,10-imine) and PCA (1-phenylcyclohexylamine) from Dr. A. Thurkauf (Neurogen, Branford, CT, USA) and desglycinyl remacemide, ARL 15896AR and ARL 16247AA (the same chemical structure as CNS-1102; Cerestat; N-(1-napthyl)-N′-(3-ethylphenyl)-
Block of NMDA-triggered [Ca2+]i responses
Memantine, a low-affinity antagonist, reduced the peak NMDA-induced [Ca2+]i to a suprabasal plateau level for as long as NMDA and the antagonist were perfused (Fig. 1B) as did the low-affinity antagonists ARL 15896AR (Black et al., 1995) and desglycinyl remacemide (Black et al., 1996). A second pattern of blockade was observed with the high-affinity antagonists, MK-801 (0.1 μM; Fig. 1C), PCP (1–10 μM; data not shown) and ARL 16247AA (1 and 5 μM; data not shown). All allowed an initial [Ca2+]i
Discussion
In this report, we compare the kinetics of block and potency of uncompetitive NMDA antagonists in blocking NMDA-triggered increaes in [Ca2+]i in rat cortical neurons. Our data suggest that the effects of antagonists at the NMDA receptor ion channel can be evaluated qualitatively and quantitatively by this method. The IC50 values (plateau) for the block of NMDA-induced [Ca2+]i responses by the NMDA receptor antagonists compare favourably to values reported previously (Rogawski, 1993).
Acknowledgements
We thank Drs. Eric Harris and Dorothea Sanchez for helpful discussions. M.B. was supported by Astra Arcus USA. We acknowledge support from the Canada/Astra Fight Stroke Program funded by the National Research Council of Canada and Astra Arcus USA (formerly Fisons Pharmaceuticals).
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2009, NeuropharmacologyCitation Excerpt :Below, we summarize how we believe certain biophysical properties of memantine, all of which were confirmed in the present studies on human NMDA receptors, are important for its mechanism of action in the treatment of Alzheimer's disease. Memantine and other well tolerated open-channel blockers show much faster open-channel blocking/unblocking kinetics than compounds burdened with negative psychotropic effects such as (+)MK-801 or phencyclidine (Rogawski et al., 1991; Chen et al., 1992; Rogawski, 1993; Parsons et al., 1993, 1999; Black et al., 1996). The kinetics of (+)MK-801 and phencyclidine are too slow to allow them to leave the channel upon depolarization, which is often reflected in apparently weaker functional voltage-dependency.
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2007, NeuropharmacologyCitation Excerpt :These theories are discussed below and presented in a graphic form in several figures. Most hypotheses are based on the widely documented and accepted fact that memantine and other well-tolerated open channel blockers show much faster open channel blocking/unblocking kinetics than compounds burdened with psychotropic side-effects such as (+)MK-801 or phencyclidine (Rogawski et al., 1991; Chen et al., 1992; Rogawski, 1993; Parsons et al., 1993; Black et al., 1996; Parsons et al., 1999b) (see Fig. 5). Unblocking kinetics are directly related to affinity, i.e. less potent compounds have faster unblocking kinetics whereas the on-rate of blockade does not seem to depend on affinity, at least when expressed as Kon (Parsons et al., 1995).