Tanaka Takashi, Zhang Li, Suzuki Fumiko, Muramatsu Ikunobu
Department of Pharmacology, School of Medicine, Fukui Medical University, Matsuoka, Fukui 910-1193, Japan.
Br J Pharmacol. 2004 Feb;141(3):468-76. doi: 10.1038/sj.bjp.0705627. Epub 2004 Jan 12.
The binding kinetics of [3H]-prazosin were measured using intact segments of rat tail artery (RTA) and thoracic aorta (RAO), and the data were compared with those obtained using a conventional membrane ligand-binding method. In intact RTA and RAO segments, [3H]-prazosin bound reversibly in a time-dependent and receptor-specific manner at 4 degrees C to alpha-1 adrenoceptors (ARs) of the plasma membrane, with affinities (pKD): 9.5 in RTA; 9.9 in RAO) that were in agreement with values estimated by a conventional membrane ligand-binding method. However, nonspecific binding was considerably higher in RAO than RTA, failing to detect clearly the specific binding at high concentrations (>300 pm) of [3H]-prazosin in binding experiments with RAO segments and membranes. The abundance of receptor in the RTA and RAO (Bmax mg-1) of total tissue protein), estimated using the tissue segment-binding approach (527+/-14 fmol mg-1 for RTA; 138+/-4 fmol mg-1 for RAO), was about 25-fold higher than values estimated using a conventional membrane-binding method (22+/-5 fmol mg-1) for RTA; 5+/-1 fmol mg-1 for RAO). Binding competition experiments using intact tissue segments or membranes derived from RTA tissue yielded comparable data, indicating a coexistence of alpha-1A AR (high affinity for prazosin, KMD-3213 and WB4101 and low affinity for BMY 7378) and alpha-1B AR (high affinity for prazosin but low affinity for KMD-3213, WB4101 and BMY 7378). In RAO tissue, careful evaluation of the tissue segment-binding assay revealed the coexpression of alpha-1B AR (high affinity for prazosin, but low affinity for KMD-3213 and BMY 7378) and alpha-1D AR (high affinity for prazosin and BMY 7378, but low affinity for KMD-3213), whereas the membrane-binding approach failed to detect these receptor subtypes with certainty. The present study indicates that previous estimates of alpha-1 AR density and alpha-1 AR subtypes obtained by a conventional membrane-binding approach, as opposed to our improved tissue segment-binding assay, may have substantially underestimated the abundance of receptors present in arterial tissues, and may have failed to identify accurately the presence of receptor subtypes. Advantages and disadvantages of the tissue segment-binding approach are discussed.British Journal of Pharmacology (2004) 141, 468-476. doi:10.1038/sj.bjp.0705627
使用大鼠尾动脉（RTA）和胸主动脉（RAO）的完整节段测量了[3H]-哌唑嗪的结合动力学，并将数据与使用传统膜配体结合方法获得的数据进行了比较。在完整的RTA和RAO节段中，[3H]-哌唑嗪在4℃下以时间依赖性和受体特异性方式可逆地结合到质膜的α-1肾上腺素能受体（ARs）上，其亲和力（pKD）：RTA中为9.5；RAO中为9.9），这与通过传统膜配体结合方法估计的值一致。然而，RAO中的非特异性结合明显高于RTA，在使用RAO节段和膜进行的结合实验中，当[3H]-哌唑嗪浓度较高（>300 pm）时，无法清楚地检测到特异性结合。使用组织节段结合方法估计的RTA和RAO中总组织蛋白的受体丰度（Bmax mg-1）（RTA为527±14 fmol mg-1；RAO为138±4 fmol mg-1）比使用传统膜结合方法估计的值（RTA为22±5 fmol mg-1；RAO为5±1 fmol mg-1）高约25倍。使用来自RTA组织的完整组织节段或膜进行的结合竞争实验产生了可比的数据，表明α-1A AR（对哌唑嗪、KMD-3213和WB4101具有高亲和力，对BMY 7378具有低亲和力）和α-1B AR（对哌唑嗪具有高亲和力，但对KMD-3213、WB4101和BMY 7378具有低亲和力）共存。在RAO组织中，对组织节段结合试验的仔细评估揭示了α-1B AR（对哌唑嗪具有高亲和力，但对KMD-3213和BMY 7378具有低亲和力）和α-1D AR（对哌唑嗪和BMY 7378具有高亲和力，但对KMD-3213具有低亲和力）的共表达，而膜结合方法无法确定地检测到这些受体亚型。本研究表明，与我们改进的组织节段结合试验相反，通过传统膜结合方法获得的先前对α-1 AR密度和α-1 AR亚型的估计可能大大低估了动脉组织中存在的受体丰度，并且可能未能准确识别受体亚型的存在。讨论了组织节段结合方法的优缺点。《英国药理学杂志》（2004年）141卷，468 - 476页。doi:10.1038/sj.bjp.0705627
