Characterisation of N-methyl-D-aspartate receptor-specific[3H]ifenprodil binding to recombinant human NR1a/NR2B receptors compared with native receptors in rodent brain membranes
Characterisation of N-methyl-D-aspartate receptor-specific[3H]ifenprodil binding to recombinant human NR1a/NR2B receptors compared with native receptors in rodent brain membranes
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DOI：
10.1046/j.1471-4159.2000.0752455.x
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发表时间：
2000-12-01
影响因子：
4.7
通讯作者：
Hutson, PH
中科院分区：
文献类型：
--
作者：
Grimwood, S;Richards, P;Hutson, PH
我们利用在L(tk-)细胞中稳定表达的重组人NR1a/NR2B受体，在特定的测定条件下进行了[H-3]异丙苯地尔结合实验，首次详细描述了NMDANR1/NR2B受体上的异丙苯地尔位点的药理作用。与大鼠皮层/海马膜相比，[H-3]ifenprodil与人重组NR1a/NR2B受体和天然大鼠受体上的单个可饱和位点相结合，其B-max分别为1.83和2.45pmol/mg蛋白质，K-D值分别为33.5和24.8 nM。伊芬地尔中心的各种配体--伊利普地尔、(R*，R*)-4-羟基-α-(4-羟基苯基)-β-甲基-4-苯基-1-哌啶乙醇[(+/-)-CP-101,606]、顺-3-[4-(4-氟苯基)-4-羟基-1-piperidinyl]-3，4-dihydro-2H-1-benzopyran-4，7-diol[(+/-)-CP-283,097]和(R*，(S*)-α-(4-羟基苯基)-β-甲基-4-(苯甲基)-1-哌啶丙醇[(+/-)-RO25-6981]对[H-3]ifenprodil与重组人NR1a/NR2B和天然大鼠受体结合的抑制作用非常相似，而多胺配体(精胺、精胺和Arcaine)对[H-3]ifenprodil结合的变构抑制作用比天然受体低约两倍。与天然大鼠受体相比，谷氨酸位点配体对[H-3]ifenprodil与重组NR1a/NR2B受体结合的调节作用较弱，本文还将所描述的NMDA受体特异性结合条件应用于体外实验，以确定系统给予的ifenprodil位点配体[ifenprodil，(+/-)-CP-101,606，(+/-)-CP-283,097，和(+/-)RO25-6981]的受体占有率。
We have performed [H-3]ifenprodil binding experiments under NMDA receptor-specific assay conditions to provide the first detailed characterisation of the pharmacology of the ifenprodil site on NMDA NR1/NR2B receptors, using recombinant human NR1a/NR2B receptors stably expressed in L(tk-) cells, in comparison with rat cortex/hippocampus membranes, [H-3]lfenprodil bound to a single, saturable site on both human recombinant NR1a/NR2B receptors and native rat receptors with B-max values of 1.83 and 2.45 pmol/mg of protein, respectively, and K-D values of 33.5 and 24.8 nM, respectively. The affinity of various ifenprodil site ligands-eliprodil, (R*,R*)-4-hydroxy-alpha-(4-hydroxyphenyl)-beta -methyl-4-phenyl-1-piperidineethanol [(+/-)-CP-101,606], cis-3-[4-(4-fluorophenyl)-4-hydroxy-1- piperidinyl]-3,4-dihydro-2H-1-benzopyran-4,7-diol [(+/-)-CP-283,097], and (R*,S*)-alpha-(4-hydroxyphenyl)-beta -methyl-4-(phenylmethyl)-1-piperidinepropanol [(+/-)-Ro 25-6981] was very similar for inhibition of [H-3]ifenprodil binding to recombinant human NR1a/NR2B and native rat receptors, whereas allosteric inhibition of [H-3]ifenprodil binding by polyamine site ligands (spermine, spermidine, and arcaine) showed approximately twofold lower affinity for recombinant receptors compared with native receptors. Glutamate site ligands were less effective at modulating [H-3]ifenprodil binding to recombinant NR1a/NR2B receptors compared with native rat receptors, The NMDA receptor-specific [H-3]ifenprodil binding conditions described were also applied to ex vivo experiments to determine the receptor occupancy of ifenprodil site ligands [ifenprodil, (+/-)-CP-101,606, (+/-)-CP-283,097, and (+/-)Ro 25-6981] given systemically.