Gopalakrishnan V, McNeill J R, Sulakhe P V, Triggle C R
Department of Pharmacology, University of Saskatchewan, Saskatoon, Canada.
Endocrinology. 1988 Aug;123(2):922-31. doi: 10.1210/endo-123-2-922.
Previously, we reported that magnesium (Mg2+) enhanced the binding affinity of arginine vasopressin [( 3H]AVP) to a single class of sites in rat liver microsomes. In the present study we have examined the effects of divalent cations and guanine nucleotides on the binding characteristics of both the nonselective agonist and the V1 receptor-selective antagonist, d(CH2)5Tyr(Me)-[3H]AVP, to microsomal and plasma membrane fractions of rat liver. At a subsaturating concentration (100 pM) of [3H]AVP, divalent cations increased specific binding in a concentration-dependent manner with the following rank order of potency: Co2+ greater than Mn2+ greater than Ni2+ greater than Mg2+ greater than Ca2+ = control. The maximal effect for Mg2+ was evident at 1 mM, a physiologically relevant concentration. In contrast, binding of the V1 receptor antagonist (at a subsaturating concentration of 10 pM) was inhibited by divalent cations, the rank order of potency being Mn2+ greater than Co2+ greater than Ca2+ greater than Mg2+ greater than Ni2+. The inhibitory effects of divalent cations were of lesser magnitude (up to 60%) compared to the stimulation of agonist binding (up to 700%). Mg2+ enhanced the affinity of [3H]AVP (Kd was decreased from approximately 2 nM to 133 pM), while the affinity of the [3H]V1 antagonist was decreased (Kd was increased from 10 to 95 pM). Scatchard analysis of saturation data (Mg2+ present) revealed similar maximum binding values for the binding of radiolabeled agonist and antagonist, indicating that AVP receptors in rat liver are mostly of the V1 subtype. Competition experiments between V1/V2-specific AVP analogs with either the radiolabeled agonist or antagonist also indicated the presence of predominantly V1 receptor sites in rat liver microsomes. The properties of plasma membrane receptor sites were similar to those of the microsomal sites, except that the density of receptors was higher in the former. In both equilibrium and competitive inhibition experiments GTPase-resistant analogs of guanine nucleotides, GTP gamma S and GDP beta S, decreased the affinity of the agonist for the receptor, but not that of the antagonist. Treatment of membranes with 0.2 mM N-ethylmaleimide (NEM) reduced the maximum binding of [3H]AVP and abolished the GTP gamma S-evoked decrease in agonist-binding affinity. In contrast, antagonist binding was unaffected by NEM. NEM pretreatment failed to influence the divalent cation-dependent increase in agonist-binding affinity. The results provide direct evidence for the existence of a high and a low affinity state of the hepatic V1 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)
此前，我们报道镁离子（Mg2+）增强了精氨酸加压素（[3H]AVP）与大鼠肝微粒体中单一类型位点的结合亲和力。在本研究中，我们检测了二价阳离子和鸟嘌呤核苷酸对非选择性激动剂和V1受体选择性拮抗剂d(CH2)5Tyr(Me)-[3H]AVP与大鼠肝微粒体和质膜部分结合特性的影响。在[3H]AVP的亚饱和浓度（100 pM）下，二价阳离子以浓度依赖的方式增加特异性结合，其效力顺序如下：Co2+＞Mn2+＞Ni2+＞Mg2+＞Ca2+ = 对照。Mg2+在1 mM（生理相关浓度）时达到最大效应。相比之下，二价阳离子抑制V1受体拮抗剂（在10 pM的亚饱和浓度下）的结合，效力顺序为Mn2+＞Co2+＞Ca2+＞Mg2+＞Ni2+。与激动剂结合的刺激作用（高达700%）相比，二价阳离子的抑制作用较小（高达60%）。Mg2+增强了[3H]AVP的亲和力（Kd从约2 nM降至133 pM），而[3H]V1拮抗剂的亲和力降低（Kd从10增至95 pM）。对饱和数据（存在Mg2+）的Scatchard分析显示，放射性标记激动剂和拮抗剂结合的最大结合值相似，表明大鼠肝中的AVP受体大多为V1亚型。V1/V2特异性AVP类似物与放射性标记激动剂或拮抗剂之间的竞争实验也表明大鼠肝微粒体中主要存在V1受体位点。质膜受体位点的特性与微粒体位点相似，只是前者的受体密度更高。在平衡和竞争性抑制实验中，鸟嘌呤核苷酸的GTP酶抗性类似物GTPγS和GDPβS降低了激动剂对受体的亲和力，但未降低拮抗剂的亲和力。用0.2 mM N-乙基马来酰亚胺（NEM）处理膜降低了[3H]AVP的最大结合，并消除了GTPγS引起的激动剂结合亲和力降低。相比之下，拮抗剂结合不受NEM影响。NEM预处理未能影响二价阳离子依赖性的激动剂结合亲和力增加。这些结果为肝V1受体存在高亲和力和低亲和力状态提供了直接证据。（摘要截断于400字）
