Dillon J S, Nayler W G
Br J Pharmacol. 1987 Jan;90(1):99-109. doi: 10.1111/j.1476-5381.1987.tb16829.x.
The [3H]-verapamil binding activity of rat cardiac sarcolemmal fragments was studied, using membranes harvested from non-perfused, aerobically-perfused and ischaemic hearts. Glass-fibre filters were found to contain specific, high affinity--(KD 38 +/- 3.1 nM) [3H]-verapamil binding sites--making them unsuitable for use in [3H]-verapamil binding studies. Incubation of membranes from non-perfused hearts in a medium containing 150 mM NaCl, 1 mM CaCl2 and 50 mM Tris revealed two populations of [3H]-verapamil binding sites. When centrifugation instead of filtration was used to separate bound and free [3H]-verapamil, high affinity sites with a KD of 0.57 +/- 0.19 microM and a Bmax of 38 +/- 5.2 pmol mg-1 protein, and low affinity sites with a KD of 78 +/- 27.5 microM and a Bmax of 2.9 +/- 1.3 nmol mg-1 protein were detected. However, only low affinity binding sites could be detected in membranes which had been incubated in a cation-free medium containing 50 mM Tris. [3H]-verapamil binding to the low and high affinity sites was saturable, reversible, stereospecific and displaceable by D600 greater than diltiazem greater than Ca2+ but not by nifedipine, nitrendipine, nisoldipine or prazosin. The two populations of binding sites survived aerobic perfusion and 60 min ischaemia at 37 degrees C. Ischaemia reduced the Bmax and KD but selectivity was maintained.
利用从非灌注、有氧灌注和缺血心脏采集的膜片，研究了大鼠心肌肌膜片段的[3H] - 维拉帕米结合活性。发现玻璃纤维滤器含有特异性、高亲和力（KD为38±3.1 nM）的[3H] - 维拉帕米结合位点，这使得它们不适用于[3H] - 维拉帕米结合研究。将非灌注心脏的膜片在含有150 mM氯化钠、1 mM氯化钙和50 mM Tris的培养基中孵育，发现有两类[3H] - 维拉帕米结合位点。当使用离心而非过滤来分离结合型和游离型[3H] - 维拉帕米时，检测到KD为0.57±0.19 microM、Bmax为38±5.2 pmol mg-1蛋白的高亲和力位点，以及KD为78±27.5 microM、Bmax为2.9±1.3 nmol mg-1蛋白的低亲和力位点。然而，在含有50 mM Tris的无阳离子培养基中孵育的膜片中，仅能检测到低亲和力结合位点。[3H] - 维拉帕米与低亲和力和高亲和力位点的结合具有饱和性、可逆性、立体特异性，且可被D600取代，取代能力为D600＞地尔硫䓬＞Ca2+，但不能被硝苯地平、尼群地平、尼索地平或哌唑嗪取代。这两类结合位点在37℃的有氧灌注和60分钟缺血条件下依然存在。缺血降低了Bmax和KD，但选择性得以维持。
