Hellewell S B, Bruce A, Feinstein G, Orringer J, Williams W, Bowen W D
Unit on Receptor Biochemistry and Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892.
Eur J Pharmacol. 1994 Jun 15;268(1):9-18. doi: 10.1016/0922-4106(94)90115-5.
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 [3H]azido-di-o-tolylguanidine ([3H]azido-DTG). 3H-Pentazocine, a highly selective sigma 1 probe, bound to sites in liver membranes with Kd = 7.5 nM and Bmax3 = 2929 fmol/mg protein. 3H-Pentazocine binding sites in kidney had Kd = 23.3 nM and Bmax = 229 fmol/mg protein. [3H]1,3-Di-o-tolylguanidine ([3H]DTG) and 3H-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine (3H-3-PPP) label both sigma 1 and sigma 2 receptors. Parameters for [3H]DTG in the liver were Kd = 17.9 nM and Bmax = 11,895 fmol/mg protein. Similar parameters were observed for 3H-3-PPP, Kd = 51.9 nM and Bmax = 11,070 fmol/mg protein. [3H]DTG bound to rat kidney with Kd = 45.8 nM and Bmax = 1190 fmol/mg protein. The observation that either [3H]DTG or 3H-3-PPP and 3H-3-PPP labeled a higher number of sites relative to 3H-pentazocine suggested that liver and kidney contain both subtypes of sigma receptor. This was confirmed by competition studies vs. 3H-pentazocine and [3H]DTG (in the presence of dextrallorphan to mask sigma 1 sites). In both tissues, 3H-pentazocine labeled sites with high affinity for haloperidol and enantioselectivity for (+)-benzomorphans over (-)-benzomorphans. [3H]DTG + dextrallorphan labeled sites in both tissues which also had high affinity for haloperidol, but which had the characteristic sigma 2 property of low affinity for (+)-benzomorphans and enantioselectivity for (-)-benzomorphans over the corresponding (+)-isomer. Similar results were obtained with 3H-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-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine (BD1008), bound to both sites with high affinity. Photoaffinity labeling with 10 nM [3H]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.(ABSTRACT TRUNCATED AT 400 WORDS)
通过使用三种高选择性σ探针进行放射性配体结合以及使用[³H]叠氮二邻甲苯基胍（[³H]叠氮-DTG）进行光亲和标记，对大鼠肝脏和肾脏中的σ受体亚型进行了研究。³H-喷他佐辛是一种高选择性σ1探针，与肝细胞膜上的位点结合，Kd = 7.5 nM，Bmax3 = 2929 fmol/mg蛋白质。肾脏中³H-喷他佐辛结合位点的Kd = 23.3 nM，Bmax = 229 fmol/mg蛋白质。[³H]1,3-二邻甲苯基胍（[³H]DTG）和³H-3-(3-羟基苯基)-N-(1-丙基)哌啶（³H-3-PPP）可同时标记σ1和σ2受体。肝脏中[³H]DTG的参数为Kd = 17.9 nM，Bmax = 11,895 fmol/mg蛋白质。³H-3-PPP也观察到类似参数，Kd = 51.9 nM，Bmax = 11,070 fmol/mg蛋白质。[³H]DTG与大鼠肾脏结合，Kd = 45.8 nM，Bmax = 1190 fmol/mg蛋白质。相对于³H-喷他佐辛，[³H]DTG或³H-3-PPP和³H-3-PPP标记的位点数量更多，这一观察结果表明肝脏和肾脏中含有两种σ受体亚型。与³H-喷他佐辛和[³H]DTG的竞争研究（在右美沙芬存在以掩盖σ1位点的情况下）证实了这一点。在这两种组织中，³H-喷他佐辛标记的位点对氟哌啶醇具有高亲和力，对(+)-苯并吗啡烷的对映选择性高于(-)-苯并吗啡烷。[³H]DTG + 右美沙芬标记的两种组织中的位点对氟哌啶醇也具有高亲和力，但具有σ2的特征性质，即对(+)-苯并吗啡烷亲和力低，对(-)-苯并吗啡烷的对映选择性高于相应的(+)-异构体。³H-3-PPP + 右美沙芬也得到了类似结果。几种新型芳基二胺，如1S,2R-顺式-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），与这两个位点都具有高亲和力。用10 nM [³H]叠氮-DTG进行光亲和标记导致25 kDa和21.5 kDa多肽的特异性标记。右美沙芬（100 nM或500 nM）完全阻断了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）对这两种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σ1和σ2受体的观点一致。（摘要截断于400字）
