Hamon M, Berthaut J, Wisner A, Dray F, Descomps B, Dao T H
Unite 58 INSERM, Montpellier, France.
Prostaglandins. 1993 Sep;46(3):251-68. doi: 10.1016/0090-6980(93)90008-u.
We studied PGE2 specific binding sites in human myometrial microsomes prepared from uterine specimens obtained by hysterectomy (women between 38 and 55 years of age). Competition experiments showed that the potency order for various prostaglandins (PGs) was: PGE2 > or = PGE1 >> PGF2 alpha > Iloprost > or = Carbacyclin >> ZK 110841 (PDG2 analogue). These relative affinities indicated that the receptor was of the EP type. In kinetic experiments GTP, GppNHp and GTP gamma S increased the rate of PGE2 binding (steady state was reached more rapidly in the presence of nucleotides) but maximal specific binding was not significantly different. Complete dissociation could not be obtained, even in the presence of GTP. Only 50% of maximal binding was readily dissociable. The dissociation rate was 4.56.10(-4) sec-1 (half time of about 660 sec) and in the presence of GTP analogues it was slightly increased (k-1 = 7.16 10(-4) sec-1, half time 420 sec.). Scatchard analysis of saturation curves showed an increase in ligand receptor affinity in the presence of GTP or nucleotide analogues: the Kd shifted from 9.66 +/- 2.8.10(-9) M to 4.96 +/- 1.25.10(-9) M, but the number of binding sites did not change significantly (310 +/- 37 to 350 +/- 17 fmol/mgP). The effect of GTP was observed at a concentration of 5.10(-4)M. GppNHp and GTP gamma S were effective at 1.10(-5) M. Pretreatment of myometrial membranes with pertussis or cholera toxins had no effect on PGE2 binding to membrane sites. Our conclusion is that GTP induced conversion of a population of low affinity sites into a population of higher affinity sites. This effect of guanine nucleotides was described in adipocytes and kidney medulla. Competition studies with PGE2 analogues (sulprostone, 17-phenyl-omega-trinor PGE2, M&B 28,767, misoprostol, butaprost) showed that this receptor mediates a contractile response and is probably an EP3 subtype.
我们研究了从子宫切除标本（38至55岁女性）制备的人子宫肌层微粒体中的前列腺素E2（PGE2）特异性结合位点。竞争实验表明，各种前列腺素（PGs）的效力顺序为：PGE2≥PGE1>>PGF2α>伊洛前列素≥卡前列环素>>ZK 110841（PDG2类似物）。这些相对亲和力表明该受体属于EP类型。在动力学实验中，鸟苷三磷酸（GTP）、鸟苷5'-三磷酸（GppNHp）和鸟苷5'-O-（3-硫代三磷酸）（GTPγS）增加了PGE2的结合速率（在核苷酸存在下更快达到稳态），但最大特异性结合没有显著差异。即使在GTP存在下也无法实现完全解离。只有50%的最大结合是易于解离的。解离速率为4.56×10⁻⁴秒⁻¹（半衰期约为660秒），在GTP类似物存在下略有增加（k⁻¹ = 7.16×10⁻⁴秒⁻¹，半衰期420秒）。饱和曲线的Scatchard分析表明，在GTP或核苷酸类似物存在下，配体受体亲和力增加：解离常数（Kd）从9.66±2.8×10⁻⁹M变为4.96±1.25×10⁻⁹M，但结合位点数量没有显著变化（310±37至350±17飞摩尔/毫克蛋白）。在5×10⁻⁴M的浓度下观察到GTP的作用。GppNHp和GTPγS在1×10⁻⁵M时有效。用百日咳毒素或霍乱毒素预处理子宫肌层膜对PGE2与膜位点的结合没有影响。我们的结论是，GTP诱导了一群低亲和力位点转化为一群高亲和力位点。鸟嘌呤核苷酸的这种作用在脂肪细胞和肾髓质中已有描述。用PGE2类似物（舒前列素、17-苯基-ω-三降PGE2、M&B 28767、米索前列醇、布他前列素）进行的竞争研究表明，该受体介导收缩反应，可能是EP3亚型。
