Rammes G, Swandulla D, Spielmanns P, Parsons C G
Department of Molecular Pharmacology, Institute for Experimental and Clinical Pharmacology and Toxicology, University of Erlangen, Germany.
Neuropharmacology. 1998 Oct-Nov;37(10-11):1299-320. doi: 10.1016/s0028-3908(98)00111-7.
In outside-out patches from cultured hippocampal neurones, glutamate (1 mM) applied for 1 ms evoked currents which rose rapidly (tau(on) 451 +/- 31 micros) to a peak and then deactivated with slower kinetics (1.95 +/- 0.13 ms). Offset time constants were significantly slower with longer application durations (tau(off) 3.10 +/- 0.19, 3.82 +/- 0.25, 4.80 +/- 0.65 and 7.56 +/- 0.65 ms with 10, 20, 100 and 500 ms applications respectively). Desensitization was complete within 100 ms with a similar rate for all application durations (4.74 +/- 0.34 ms with 100 ms applications). GYKI 52466 reduced inward peak currents with an IC50 of 11.7 +/- 0.6 microM and had similar potency on steady-state currents to longer glutamate applications. GYKI 52466 had no significant effect on desensitization or deactivation time constants but caused a modest and significant prolongation of onset kinetics at higher concentrations. Cyclothiazide (100 microM) potentiated steady-state currents 25-fold at 100 ms and caused a modest but significant slowing in onset kinetics (601 +/- 49 micros with 1 ms applications) but a more pronounced prolongation of deactivation time constants (5.55 +/- 0.66 ms with 1 ms applications). In 50% of neuronal patches cyclothiazide completely eliminated desensitization. In those patches with residual desensitization, the rate was not significantly different to control (5.36 +/- 0.43 ms with 100 ms applications). Following 100 ms applications of glutamate, GYKI 52466 had IC50s of 11.7 +/- 1.1 microM and 75.1 +/- 7.0 microM in the absence and presence of cyclothiazide (100 microM) respectively. Onset kinetics were slowed from 400 +/- 20 micros to 490 +/- 30 micros by cyclothiazide (100 microM) and then further prolonged by GYKI 52466 (100 microM) to a double exponential function (tau(on1) 1.12 +/- 0.13 ms and tau(on2) 171.5 +/- 36.5 ms). GYKI 52466 did not re-introduce desensitization but concentration-dependently weakened cyclothiazide's prolongation of deactivation time constants (1 ms applications: 5.01 +/- 0.71, 4.47 +/- 0.80 and 2.28 +/- 0.64 ms with GYKI 52466 30, 100 and 300 microM respectively). NBQX reduced peak current responses with an IC50 of 28.2 +/- 1.3 nM. Paradoxically, steady-state currents with 500 ms applications of glutamate were potentiated from 3.3 +/- 1.2 pA to 29.4 +/- 6.4 pA by NBQX (1 nM). Higher concentrations of NBQX then antagonized this potentiated response. The potency of NBQX in antagonizing steady-state currents to 500 ms applications of glutamate (IC50 120.9 +/- 30.2 nM) was 2-fold less than following 100 ms applications (IC50 67.7 +/- 2.6 nM). NBQX had no effect on rapid onset, desensitization or deactivation time constants. However, a slow relaxation of inhibition was seen with longer applications. NBQX was 2-5-fold less potent against inward currents in the presence of cyclothiazide (100 microM) depending on the application duration but had no effect on the rapid onset, desensitization or deactivation time constants. The same relaxation of inhibition was seen as with NBQX alone. NBQX (1 microM) reduced AMPA receptor-mediated EPSC amplitude to 7 +/- 1% of control with no effect on kinetics. Cyclothiazide (330 microM) caused a 2.8-fold prolongation of the decay time constant (control 26.6 +/- 2.2 ms, cyclothiazide 74.2 +/- 7.6 ms, n = 9). Additional application of NBQX (1 microM) partly reversed this prolongation to 1.9 fold (47.7 +/- 2.5 ms, n = 5). These results support previous findings that cyclothiazide also allosterically influences AMPA receptor agonist/antagonist recognition sites. There were no interactions between NBQX and cyclothiazide on desensitization or deactivation time constants of glutamate-induced currents but clear interactions on EPSC deactivation kinetics. This raises the possibility that the interactions of NBQX, GYKI 52466 and cyclothiazide on AMPA-receptor-mediated EPSC kinetics observed are due to modulation of glutamate-release at presynaptic AMPA receptors.
在培养的海马神经元的外向膜片上，施加1 mM谷氨酸1 ms可诱发电流，该电流迅速上升（开启时间常数τ(on)为451±31微秒）至峰值，然后以较慢的动力学过程失活（1.95±0.13 ms）。随着施加持续时间延长，关闭时间常数显著减慢（分别施加10、20、100和500 ms时，τ(off)为3.10±0.19、3.82±0.25、4.80±0.65和7.56±0.65 ms）。脱敏在100 ms内完成，且所有施加持续时间的速率相似（施加100 ms时为4.74±0.34 ms）。GYKI 52466使内向峰值电流降低，IC50为11.7±0.6微摩尔，对稳态电流的效力与较长时间的谷氨酸施加相似。GYKI 52466对脱敏或失活时间常数无显著影响，但在较高浓度下使起始动力学适度且显著延长。环噻嗪（100微摩尔）在100 ms时使稳态电流增强25倍，并使起始动力学适度但显著减慢（施加1 ms时为601±49微秒），但使失活时间常数更显著延长（施加1 ms时为5.55±0.66 ms）。在50%的神经元膜片中，环噻嗪完全消除了脱敏。在那些有残余脱敏的膜片中，速率与对照无显著差异（施加100 ms时为5.36±0.43 ms）。在施加100 ms谷氨酸后，在不存在和存在环噻嗪（100微摩尔）时，GYKI 52466的IC50分别为11.7±1.1微摩尔和75.1±7.0微摩尔。环噻嗪（100微摩尔）使起始动力学从400±20微秒减慢至490±30微秒，然后GYKI 52466（100微摩尔）进一步将其延长为双指数函数（τ(on1)为1.12±0.13 ms，τ(on2)为171.5±36.5 ms）。GYKI 52466未重新引入脱敏，但浓度依赖性地减弱了环噻嗪对失活时间常数的延长（施加1 ms时：分别用30、100和300微摩尔的GYKI 52466时，失活时间常数为5.01±0.71、4.47±0.80和2.28±0.64 ms）。NBQX使峰值电流反应降低，IC50为28.2±1.3 nM。矛盾的是，在施加500 ms谷氨酸时，NBQX（1 nM）使稳态电流从3.3±1.2 pA增强至29.4±6.4 pA。更高浓度的NBQX随后拮抗这种增强的反应。NBQX拮抗500 ms谷氨酸施加的稳态电流的效力（IC50为120.9±30.2 nM）比施加100 ms时低2倍（IC50为67.7±2.6 nM）。NBQX对快速起始、脱敏或失活时间常数无影响。然而，随着施加时间延长，可观察到抑制的缓慢解除。在存在环噻嗪（100微摩尔）时，NBQX对内向电流的效力根据施加持续时间降低2 - 5倍，但对快速起始、脱敏或失活时间常数无影响。与单独使用NBQX时一样，可观察到抑制的相同解除。NBQX（1微摩尔）使AMPA受体介导的兴奋性突触后电流（EPSC）幅度降低至对照的7±1%，对动力学无影响。环噻嗪（330微摩尔）使衰减时间常数延长2.8倍（对照为26.6±2.2 ms，环噻嗪为74.2±7.6 ms，n = 9）。额外施加NBQX（1微摩尔）部分逆转了这种延长，至1.9倍（47.7±2.5 ms，n = 5）。这些结果支持先前的发现，即环噻嗪也通过变构作用影响AMPA受体激动剂/拮抗剂识别位点。NBQX和环噻嗪在谷氨酸诱导电流的脱敏或失活时间常数上没有相互作用，但在EPSC失活动力学上有明显相互作用。这增加了一种可能性，即观察到的NBQX、GYKI 52466和环噻嗪对AMPA受体介导的EPSC动力学的相互作用是由于对突触前AMPA受体处谷氨酸释放的调节。
