利用SR 33557鉴定大鼠脑中一个新的钙拮抗剂结合位点。

Identification of a novel calcium antagonist binding site in rat brain by SR 33557.

作者信息

Kenny B A, Fraser S, Spedding M

机构信息

Department of Pharmacology, Syntex Research Centre, Riccarton, Edinburgh.

出版信息

Br J Pharmacol. 1993 Jan;108(1):93-9. doi: 10.1111/j.1476-5381.1993.tb13445.x.

DOI:10.1111/j.1476-5381.1993.tb13445.x

PMID:7679034

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1907724/

Abstract

In K(+)-depolarized taenia preparations from guinea-pig caecum SR 33557 was a potent antagonist of Ca(2+)-induced contractions and antagonized the effect of the calcium channel activator Bay K 8644. 2. SR 33557 displayed high affinity (pKi 9.54 +/- 0.04, nH 1.01) for the [3H]-(+/-)-PN 200-110 binding site in rat cerebral cortex membranes. In the presence of 5 mM Ca2+ this affinity was reduced (pKi 8.82 +/- 0.01, nH 1.05) whilst the affinity of nitrendipine was unaffected by this concentration of Ca2+. 3. Saturation binding experiments in rat cerebral cortex carried out in the absence and presence of SR 33557 (0.1-1.0 nM) indicated an apparently competitive interaction at the dihydropyridine site, in that SR 33557 reduced the KD of [3H]-(+/-)-PN 200-110 binding without any effect on Bmax. In kinetic experiments, the rate of dissociation of [3H]-(+/-)-PN 200-110 from rat cerebral cortex was unchanged in the presence of SR 33557 (5 nM). 4. D-cis-diltiazem fully reversed the inhibition [3H]-nitrendipine binding to rat cerebral cortex produced by SR 33557 indicating the site of action of SR 33557 to be distinct from the dihydropyridine (DHP) binding site. 5. Saturation analysis indicated that [3H]-SR 33557 (0.01-0.8 nM) labelled a single class of binding sites in rat cerebral cortex membranes with high affinity (KD 0.12 +/- 0.01, Bmax 222 +/- 20 fmol mg-1 protein), although kinetic data indicated the existence of negative cooperativity between the binding sites. 6.In competition studies, a variety of different calcium antagonists displayed similar affinity for [3H]-SR 33557 and [3H]-(+/-)-PN 200-110 sites. The [3H]-SR 33557 site was sensitive to the inhibitory effect of divalent cations. The affinity of Cd2+ was 0.026 +/- 0.015 mM and the rank order of affinity was Cd2+ >Ca2+ >Mn2+ >Mg2+ >Na+.7. We propose that SR 33557 labels a distinct site in rat cerebral cortex. The coupling between the SR 33557 and DHP site appears to be very close, resulting in apparently competitive interactions in some experimental protocols but can be revealed as negatively allosteric in other circumstances.

摘要

在豚鼠盲肠K⁺去极化的绦虫制剂中，SR 33557是Ca²⁺诱导收缩的强效拮抗剂，并拮抗钙通道激活剂Bay K 8644的作用。2. SR 33557对大鼠大脑皮层膜中[³H]-(±)-PN 200 - 110结合位点显示出高亲和力（pKi 9.54 ± 0.04，nH 1.01）。在5 mM Ca²⁺存在下，这种亲和力降低（pKi 8.82 ± 0.01，nH 1.05），而尼群地平的亲和力不受该Ca²⁺浓度的影响。3. 在不存在和存在SR 33557（0.1 - 1.0 nM）的情况下对大鼠大脑皮层进行的饱和结合实验表明，在二氢吡啶位点存在明显的竞争性相互作用，即SR 33557降低了[³H]-(±)-PN 200 - 110结合的KD，而对Bmax没有影响。在动力学实验中，在SR 33557（5 nM）存在下，[³H]-(±)-PN 200 - 110从大鼠大脑皮层的解离速率没有变化。4. D-顺式地尔硫卓完全逆转了SR 33557对大鼠大脑皮层[³H]-尼群地平结合的抑制作用，表明SR 33557的作用位点与二氢吡啶（DHP）结合位点不同。5. 饱和分析表明，[³H]-SR 33557（0.01 - 0.8 nM）在大鼠大脑皮层膜中标记了一类具有高亲和力的单一结合位点（KD 0.12 ± 0.01，Bmax 222 ± 20 fmol mg⁻¹蛋白质），尽管动力学数据表明结合位点之间存在负协同性。6. 在竞争研究中，多种不同的钙拮抗剂对[³H]-SR 33557和[³H]-(±)-PN 200 - 110位点显示出相似的亲和力。[³H]-SR 33557位点对二价阳离子的抑制作用敏感。Cd²⁺的亲和力为0.026 ± 0.015 mM，亲和力的顺序为Cd²⁺＞Ca²⁺＞Mn²⁺＞Mg²⁺＞Na⁺。7. 我们提出SR 33557在大鼠大脑皮层中标记了一个独特的位点。SR 33557和DHP位点之间的偶联似乎非常紧密，在某些实验方案中导致明显的竞争性相互作用，但在其他情况下可显示为负变构作用。