Inhibition Of N-methyl-D-aspartate Receptors By Bis(7)-tacrine And Underlying Mechanisms

Part 1 Comparison of the inhibition by tacrine, memantine and bis(7)-tacrine of N-methyl-D-aspartate-activated currents in cultured rat hippocampal neuronsThe cellular mechanism of Alzheimer's disease (AD) is closely related to cholinergic disfunction and neuronal apoptosis. In treating AD, especially in preventing AD and other neurodegenerative diseases, acetylcholinesterase inhibitors, such as bis(7)-tacrine, which possess anti-apoptosis functions should be more effective than compounds that only have pure acetylcholinesterase inhibitory properties. The present study was carried out to determine the inhibitory mechanisms of bis(7)-tacrine by comparing it with tacrine and memantine in primary cultured rat hippocampal neurons using whole-cell patch-clamp techniques. The results indicate that, all three drugs selectively produced a concentration-dependent inhibition of NMDA-activated current (IC50 values of 3.61±0.78μM, 112.33±19.83μM, 8.82±1.03μM for bis(7)-tacrine, tacrine and memantine, respectively). Both tacrine and memantine were'open channel blockers', acted at the inner of NMDA receptor channels, showing'agonist-dependency'and'voltage-dependency'. The former had quicker association and disassociation rate with NMDA receptor than the latter. Tacrine at a concentration above 25μM inhibited NMDA-evoked current by enhancing receptor desensitization, followed by a delayed current peak just after cessation of drug application. However, the inhibition of NMDA-activated currents by bis(7)-tacrine was different from that of tacrine and memantine: inhibition was enhanced largely by 1 s preapplication of bis(7)-tacrine, but longer preapplication (2-90 s) had no more inhibitory effect. The percentage inhibition of NMDA-activated current by bis(7)-tacrine was not significantly different at the holding potentials ranging from -50 to +50 mV, without changing the reversal potential too. Bis(7)-tacrine did not significantly change the EC₅₀ value of NMDA-activated current [49.48±2.93μM in the absence vs 57.32±8.43μM in the presence of bis(7)-tacrine; ANOVA, Pï¹¥0.05;n = 7-8], but decreased the E_max of NMDA current by 40% (ANOVA, P﹤0.05; n = 7-8), showing that the inhibition of NMDA-induced currnet by bis(7)-tacrine was non-competitive. These results also suggest that bis(7)-tacrine, a novel dimeric acetylcholinesterase inhibitor, more potently inhibits NMDA receptor function than tacrine and memantine by a slow onset, non-competitive mechanism, which may have better clinical efficiency and safety properties.Part 2 Investigation of N-methyl-D-aspartate receptor modulatory sites possibly involved in the bis(7)-tacrine inhibition in cultured rat hippocampal neuronsBis(7)-tacrine, a novel dimeric acetylcholinesterase (AChE) inhibitor, has been proposed as one of the most promising agents to treat Alzheimer's disease. Recently, the agent was found to prevent glutamate-induced neuronal apoptosis by inhibiting N-methyl-D-aspartate (NMDA) receptors in cultured rat cerebellar granule neurons in addition to causing an inhibitory effect on acetylcholinesterase. In the present study, the possible modulatory site of bis(7)-tacrine on NMDA receptors was investigated using whole-cell patch-clamp recording in cultured rat hippocampal neurons. The inhibitory rates of bis(7)-tacrine were neither altered by changing the concentrations of glycine (0.1-10μM) or proton (pH 8.1-6.7) in the external solution, nor by adding ditiothreitol (2 mM), spermine (10μM), Mg²⁺ (10-100μM), or Zn²⁺ (5-20μM) to the external solution. 25μM bis(7)-tacrine in the recording pipette solution did not alter the inhibition rate of 30μM NMDA-activated current by 2.5μM bis(7)-tacrine applied externally (37±3% vs coltrol of 36±4%, Pï¹¥0.05; n = 4). However, 2.5μM bis(7)-tacrine and 5μM dizocilpine (MK-801) inhibited NMDA-activated currents by 36% and 22%, respectively; co-application of these two drugs only inhibited NMDA-activated currents by 37%. The results suggest that, although bis(7)-tacrine is very unlikely acting at the MK-801 site, MK-801 could negatively modulate the inhibition of NMDA receptor function by bis(7)-tacrine.Part 3 Inhibition of N-methyl-D-aspartate-activated current by bis(7)-tacrine in HEK-293 cells expressing NR1/NR2A or NR1/NR2B receptorsIn normal rat forebrain the NR1/NR2A and NR1/NR2B dimmers, but not the NR1/NR2A/NR2B trimer, are the main constitutional forms of NMDA receptors. The present study was carried out to determine the functional properties of heteromeric NMDA receptor subunits composed by NR1/NR2A or NR1/NR2B expressed in HEK- 293 cells and their inhibition by bis(7)-tacrine using whole-cell patch-clamp techniques. The results demonstrate that, when co-applied to HEK-293 cells expressing NR1/NR2A receptors, 1μM bis(7)-tacrine inhibited 30μM NMDA- and 1000μM NMDA-activated steady-state current by 46% and 40%, respectively (ANOVA, Pï¹¥0.05; n = 5), suggesting that the inhibition of bis(7)-tacrine doesn't depend on NMDA concentration which is consitant with a non-competitive mechanism of inhibition. But for the NR1/NR2B receptor, 1μM bis(7)-tacrine inhibited 30μM NMDA- and 1000μM NMDA-activated steady-state current by 61% and 13%, respectively (ANOVA, P﹤0.05; n = 6), showing that it appears to be competitive with NMDA. In addition, simultaneous application of 1μM bis(7)-tacrine and 1000μM NMDA produced a moderate inhibition of peak NMDA-activated current, followed a gradual decline of the current to a steady-state. However, when 1μM bis(7)-tacrine was applied for 5 s before NMDA, the peak current was almost completely inhibited. These results show that bis(7)-tacrine inhibition of NMDA current on NR1/NR2B was slow onset, and it did not depend on the existing of agonist. With holding potential ranging from -50 to +50 mV, the bis(7)-tacrine inhibition rate of NMDA current was the same, and the reversal potential did not change too. The results are consitant with what we have observed in cultured hippocampal neurons, showing that bis(7)-tacrine inhibits NR1/NR2B receptors in a non-competitive, agonist-independent and voltage-independent manner. These results also indicate that the NR1/NR2A and NR1/NR2B receptors could be used to study the molecular mechanism of bis(7)-tacrine inhibition.