Edwards S J, Rattigan S, Colquhoun E Q, Woodcock E A, Clark M G
Department of Biochemistry, University of Tasmania, Hobart, Australia.
J Mol Cell Cardiol. 1988 Nov;20(11):1025-34. doi: 10.1016/0022-2828(88)90579-2.
The characteristics of alpha 1-adrenergic receptors were investigated in perfused rat hearts at 37 degrees C. [3H]Prazosin was bound in a time-dependent manner and reached equilibrium at 15 min. Scatchard analysis of the specific binding isotherm for [3H]prazosin indicated a population of high affinity sites (Kd = 0.41 nM, Bmax = 13.2 pmol/g wet wt). Prazosin binding was displaced by epinephrine as well as by the adrenergic antagonists prazosin greater than phentolamine greater than yohimbine greater than propranolol. Specific prazosin binding was defined as that portion of the binding inhibited by 10 microM phentolamine; phentolamine and epinephrine displaced 3H-prazosin to the same level. [3H]Prazosin was not metabolized by the heart. When pre-labelled hearts were perfused at 37 degrees C with prazosin-free medium non-specific binding of [3H]prazosin decreased more rapidly (t0.5 = 4 min) than specific binding (t0.5 = 38 min). Perfusion of the heart at lower temperatures (less than 10 degrees C) decreased the rate of loss of nonspecific binding and prevented the loss of specific binding. Fractionation of [3H]prazosin perfused hearts at 0 degrees C, when dissociation was minimal, led to a loss of binding so that sarcolemma-enriched fractions contained approximately 2% of the binding sites present in the perfused heart. The binding characteristics of sarcolemma-enriched fractions (Kd 0.10 nM, Bmax 300 fmol/mg protein) differed significantly from those of the perfused heart. Exposure of the heart to 10 min of ischaemia prior to binding studies did not alter the characteristics of the [3H]prazosin binding sites. It is concluded that the perfused rat heart contains a population of alpha 1-adrenoceptors which differ from those of isolated sarcolemma preparations perhaps because of alterations that occur during sarcolemma isolation. The perfused heart should be an appropriate model system in which to study the relationship between receptor occupancy and biological response as well as the direct effects of perturbations such as ischaemia.
在37摄氏度的灌注大鼠心脏中研究了α1 - 肾上腺素能受体的特性。[3H]哌唑嗪以时间依赖的方式结合，并在15分钟时达到平衡。对[3H]哌唑嗪特异性结合等温线的Scatchard分析表明存在一群高亲和力位点（Kd = 0.41 nM，Bmax = 13.2 pmol/g湿重）。肾上腺素以及肾上腺素能拮抗剂哌唑嗪＞酚妥拉明＞育亨宾＞普萘洛尔均可取代哌唑嗪的结合。特异性哌唑嗪结合定义为被10μM酚妥拉明抑制的结合部分；酚妥拉明和肾上腺素将3H - 哌唑嗪取代至相同水平。[3H]哌唑嗪未被心脏代谢。当用无哌唑嗪的培养基在37摄氏度灌注预先标记的心脏时，[3H]哌唑嗪的非特异性结合下降速度（t0.5 = 4分钟）比特异性结合（t0.5 = 38分钟）更快。在较低温度（低于10摄氏度）下灌注心脏可降低非特异性结合的丧失速率，并防止特异性结合的丧失。在0摄氏度对[3H]哌唑嗪灌注的心脏进行分级分离，此时解离最小，导致结合丧失，因此富含肌膜的级分含有灌注心脏中存在的结合位点的约2％。富含肌膜级分的结合特性（Kd 0.10 nM，Bmax 300 fmol/mg蛋白质）与灌注心脏的结合特性有显著差异。在结合研究之前使心脏缺血10分钟并未改变[3H]哌唑嗪结合位点的特性。结论是，灌注的大鼠心脏含有一群α1 - 肾上腺素能受体，它们与分离的肌膜制剂中的受体不同，这可能是由于肌膜分离过程中发生的改变。灌注心脏应该是一个合适的模型系统，用于研究受体占据与生物学反应之间的关系以及诸如缺血等干扰的直接影响。
