Rothman R B, Bykov V, de Costa B R, Jacobson A E, Rice K C, Brady L S
Unit on Receptor Studies, NIMH, Bethesda, MD 20892.
Peptides. 1990 Mar-Apr;11(2):311-31. doi: 10.1016/0196-9781(90)90088-m.
Guinea pig brain membranes depleted of mu and delta receptors by pretreatment with the site-directed acylating agents, 2-(4-ethoxybenzyl)-1- diethylaminoethyl-5-isothiocyanatobenzimidazole.HCl (BIT) and N-phenyl-N-[1-(2-(4-isothiocyanato)phenethyl)-4- piperidinyl]-propanamide.HCl (FIT), were used in this study to test the hypothesis that guinea pig brain possesses subtypes of kappa receptors. Pretreatment of membranes with either (-)-(1S,2S)-U50,488 or the kappa selective acylating agent, (1S,2S)-trans-2-isothiocyanato-N-methyl-N-[2-(1- pyrrolidinyl)cyclohexyl]benzeneacetamide, caused a wash-resistant inhibition of kappa 1 binding sites labeled by [3H]U69,593 binding, but not kappa 2 binding sites labeled by [3H]bremazocine. Binding surface analysis of [3H]bremazocine binding resolved two binding sites, termed kappa 2 and kappa 2b, present at densities of 212 and 225 fmol/mg protein, which had low affinity for (-)-(1S,2S)-U50,488 and U69,593. The kappa 2b site had high affinity for beta-endorphin(1-31) (Kd = 5.5 nM) and [D-Ala2,D-Leu5]enkephalin (Kd = 14 nM), and lower affinity for [D-Ala2-MePhe4,Gly-ol5]enkephalin (Kd = 147 nM) and [Leu5]enkephalin (Kd = 46.0 nM). Binding surface analysis of [3H]U69,593 binding also resolved two binding sites, termed kappa 1a and kappa 1b, present at densities of 6.0 and 40.0 fmol/mg protein. The kappa 1a binding site was characterized by very high affinity for alpha-neoendorphin. Quantitative autoradiographic studies demonstrated that kappa 2a and kappa 2b binding sites are heterogeneously distributed in guinea pig brain, and that the anatomical distribution of kappa 1 binding sites reported in the literature is different from that observed in this study for the kappa 2 binding sites. Viewed collectively, these data provide evidence for four kappa receptor subtypes in guinea pig brain.
通过用位点导向的酰化剂2-(4-乙氧基苄基)-1-二乙氨基乙基-5-异硫氰酸苯并咪唑盐酸盐（BIT）和N-苯基-N-[1-(2-(4-异硫氰酸基)苯乙基)-4-哌啶基]-丙酰胺盐酸盐（FIT）进行预处理，去除了豚鼠脑膜中的μ和δ受体，本研究使用这些脑膜来检验豚鼠脑具有κ受体亚型的假设。用(-)-(1S,2S)-U50,488或κ选择性酰化剂(1S,2S)-反式-2-异硫氰酸基-N-甲基-N-[2-(1-吡咯烷基)环己基]苯乙酰胺对脑膜进行预处理，导致对[3H]U69,593标记的κ1结合位点产生耐洗脱抑制，但对[3H]布马佐辛标记的κ2结合位点无抑制作用。[3H]布马佐辛结合的结合表面分析解析出两个结合位点，称为κ2和κ2b，其密度分别为212和225 fmol/mg蛋白质，它们对(-)-(1S,2S)-U50,488和U69,593具有低亲和力。κ2b位点对β-内啡肽(1-31)（Kd = 5.5 nM）和[D-Ala²,D-Leu⁵]脑啡肽（Kd = 14 nM）具有高亲和力，对[D-Ala²-MePhe⁴,Gly-ol⁵]脑啡肽（Kd = 147 nM）和[Leu⁵]脑啡肽（Kd = 46.0 nM）具有较低亲和力。[3H]U69,593结合的结合表面分析也解析出两个结合位点，称为κ1a和κ1b，其密度分别为6.0和40.0 fmol/mg蛋白质。κ1a结合位点对α-新内啡肽具有非常高的亲和力。定量放射自显影研究表明，κ2a和κ2b结合位点在豚鼠脑中呈异质性分布，并且文献中报道的κ1结合位点的解剖分布与本研究中观察到的κ2结合位点的分布不同。综合来看，这些数据为豚鼠脑中四种κ受体亚型提供了证据。
