RAT-LIVER AND KIDNEY CONTAIN HIGH-DENSITIES OF SIGMA(1) AND SIGMA(2) RECEPTORS - CHARACTERIZATION BY LIGAND-BINDING AND PHOTOAFFINITY-LABELING
RAT-LIVER AND KIDNEY CONTAIN HIGH-DENSITIES OF SIGMA(1) AND SIGMA(2) RECEPTORS - CHARACTERIZATION BY LIGAND-BINDING AND PHOTOAFFINITY-LABELING
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DOI：
10.1016/0922-4106(94)90115-5
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发表时间：
1994-06-15
期刊：
影响因子：
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通讯作者：
BOWEN, WD
中科院分区：
文献类型：
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作者：
HELLEWELL, SB;BRUCE, A;BOWEN, WD
通过放射性配体与三种高选择性 σ 探针结合以及使用 [H-3] 叠氮基-二邻甲苯基胍 ([H-3] 叠氮基-DTG) 进行光亲和标记，研究了大鼠肝脏和肾脏是否存在 σ 受体亚型。 [H-3](+)-喷他佐辛是一种高选择性 sigma(1) 探针，与肝膜位点结合，K-d = 7.5 nM，B-max = 2929 fmol/mg 蛋白质。肾脏中的 [H-3](+)-喷他佐辛结合位点的 K-d = 23.3 nM，B-max = 229 fmol/mg 蛋白质。 [H-3]1,3-二邻甲苯基胍 ([H-3]DTG) 和 [H-3](+)-3-(3-羟基苯基)-N-(1-丙基)哌啶 ([H-3](+)-3-PPP) 标记 sigma(1) 和 sigma(2) 受体。肝脏中 [H-3]DTG 的参数为 K-d = 17.9 nM 和 B-max = 11895 fmol/mg 蛋白质。 [H-3](+)-3-PPP 观察到类似的参数，K-d = 51.9 nM 和 B-max = 11070 fmol/mg 蛋白质。 [H-3]DTG 与大鼠肾脏结合，K-d = 45.8 nM，B-max = 1190 fmol/mg 蛋白质。 [H-3]DTG 或 [H-3](+)-3-PPP 和 [H-3](+)-3-PPP 标记的位点数量相对于 [H-3](+)-喷他佐辛的观察结果表明肝脏和肾脏含有 σ 受体的两种亚型。这通过与 [H-3](+)-喷他佐辛和 [H-3]DTG 的竞争研究得到证实（存在右旋洛芬以掩盖 sigma(1) 位点）。在这两种组织中，[H-3](+)-喷他佐辛标记的位点对氟哌啶醇具有高亲和力，并且对 (+)-苯并吗啡烷具有高于 (-)-苯并吗啡烷的对映选择性。两种组织中的 [H-3]DTG + 右旋洛啡烷标记位点对氟哌啶醇也具有高亲和力，但具有对 (+)-苯并吗啡烷具有低亲和力的特征 sigma(2)，以及对 (-)-苯并吗啡烷相对于相应 (+)-异构体的对映选择性。使用[H-3](+)-3-PPP + 右洛啡烷获得了类似的结果。几种新型芳基二胺，例如 1S,2R-cis-N-[2-(3,4-二氯苯基乙基]-N-甲基-2-(1-吡咯烷基)环己胺 (BD737) 和 N-[2-(3,4-二氯苯基)乙基]-N-甲基-2-(1-吡咯烷基)乙胺 (BD1008)，以高亲和力与这两个位点结合。用 10 nM [H-3]azido-DTG 标记可特异性标记 25 kDa 和 21.5 kDa 的多肽（100 nM 或 500 nM），完全阻断 25 kDa 多肽的标记，但对较低分子量蛋白质的标记没有影响。 (+)-10,11-二氢-5-甲基-5H-二苯并[a,d]环庚烯-5,10-亚胺((+)-MK-801) 对任一多肽的标记均无影响。这些数据与 25 kDa 和 21.5 kDa 蛋白质分别代表 sigma(1) 和 sigma(2) 受体的观点一致。 sigma(1) 和 sigma(2) 受体，其中 sigma(2) 位点占总 sigma 群体的 75-80% sigma 受体亚型的高密度可能表明肝和肾组织中的重要功能。
Rat liver and kidney were investigated for the presence of sigma (sigma) receptor subtypes by radioligand binding with three highly selective sigma probes and by photoaffinity labeling using [H-3]azido-di-o-tolylguanidine ([H-3]azido-DTG). [H-3](+)-Pentazocine, a highly selective sigma(1) probe, bound to sites in liver membranes with K-d = 7.5 nM and B-max = 2929 fmol/mg protein. [H-3](+)-Pentazocine binding sites in kidney had K-d = 23.3 nM and B-max = 229 fmol/mg protein. [H-3]1,3-Di-o-tolylguanidine ([H-3]DTG) and [H-3](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ([H-3](+)-3-PPP) label both sigma(1) and sigma(2) receptors. Parameters for [H-3]DTG in the liver were K-d = 17.9 nM and B-max = 11895 fmol/mg protein. Similar parameters were observed for [H-3](+)-3-PPP, K-d = 51.9 nM and B-max = 11070 fmol/mg protein. [H-3]DTG bound to rat kidney with K-d = 45.8 nM and B-max = 1190 fmol/mg protein. The observation that either [H-3]DTG or [H-3](+)-3-PPP and [H-3](+)-3-PPP labeled a higher number of sites relative to [H-3](+)-pentazocine suggested that liver and kidney contain both subtypes of sigma receptor. This was confirmed by competition studies vs. [H-3](+)-pentazocine and [H-3]DTG (in the presence of dextrallorphan to mask sigma(1) sites). In both tissues, [H-3](+)-pentazocine labeled sites with high affinity for haloperidol and enantioselectivity for (+)-benzomorphans over (-)-benzomorphans. [H-3]DTG + dextrallorphan labeled sites in both tissues which also had high affinity for haloperidol, but which had the characteristic sigma(2) properly of low affinity for (+)-benzomorphans and enantioselectivity for (-)-benzomorphans over the corresponding(+)-isomer. Similar results were obtained with [H-3](+)-3-PPP + dextrallorphan. Several novel aryl diamines, such as 1S,2R-cis-N-[2-(3,4-dichlorophenylethyl]-N-methyl-2-(1-pyrrolidinyl)cyclohexylamine (BD737) and N-[2-(3,4-dichlbrophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine (BD1008), bound to both sites with high affinity. Photoaffinity labeling with 10 nM [H-3]azido-DTG resulted in specific labeling of polypeptides of 25 kDa and 21.5 kDa. Dextrallorphan (100 nM or 500 nM) completely blocked labeling of the 25 kDa polypeptide, but had no effect on labeling of the lower molecular weight protein. (+)-10,11-Dihydro-5-methyl-5H-dibenzo[a,d]cyclohepten-5,10-imine((+)-MK-801) had no effect on labeling of either polypeptide. These data are consistent with the notion that the 25 kDa and 21.5 kDa proteins represent sigma(1) and sigma(2) receptors, respectively. Thus, rat liver and kidney contain high densities of sigma(1) and sigma(2) receptors, with sigma(2) sites comprising 75-80% of the total sigma population. The high density of sigma receptor subtypes may indicate important functions in hepatic and renal tissues.