Schwartzentruber R S, Omeljaniuk R J
Department of Biology, Lakehead University, Thunder Bay, Ontario, Canada.
Gen Comp Endocrinol. 1995 Feb;97(2):209-19. doi: 10.1006/gcen.1995.1020.
Binding parameters of radiolabeled pGlu-3-Me-His2-Pro-NH2 ([3H]MeTRH) to pituitary TRH receptors of rainbow trout (Oncorhynchus mykiss) and goldfish (Carassius auratus) were investigated. Washed pituitary membranes were incubated with [3H]MeTRH in the absence (B0) or presence of TRH or TRH analogs under various paradigms. Specific binding (Bsp) was thermolabile and tissue dependent. Association of Bsp was slow (k + 1 = 1.43 x 10(7) M-1 min-1) achieving equilibrium binding after 60 min, and remaining stable for at least 60 min. Thereafter, equilibrium-bound [3H]MeTRH was dissociated by addition of excess MeTRH; estimated dissociation rate constant (k - 1) and half-life (t1/2) were 6.74 x 10(-2) min-1 and 10.2 min, respectively, with a kinetically derived dissociation constant (k - 1/k + 1) of 4.71 x 10(-9) M. [3H]MeTRH binding was differentially displaced by TRH and TRH analogs. LIGAND analysis of multiple homologous (MeTRH) and heterologous (TRH) displacement experiments indicates the presence of a single class of binding sites with nanomolar affinity. Data pooled from multiple heterologous (TRH) displacement experiments suggested the presence of an additional class of higher-affinity (picomolar) binding sites. Additionally, examination of separate preparations of neurointermediate lobes and pars distalis from trout and goldfish suggested that these two classes of binding sites were differentially located between the two lobes. Specificity analysis of the trout pituitary TRH receptor indicates that the rank order of potency for [3H]MeTRH displacement was MeTRH > TRH > pGlu-Phe-Pro-NH2. Analogs with N- or C-terminal substitutions had little competitive potential. This study indicates the presence and binding characteristics of specific TRH receptors in the salmonid and cyprinid pituitary gland.
研究了放射性标记的焦谷氨酸-3-甲基组氨酸2-脯氨酸-氨基（[3H]甲基促甲状腺激素释放激素）与虹鳟（Oncorhynchus mykiss）和金鱼（Carassius auratus）垂体促甲状腺激素释放激素受体的结合参数。在不同模式下，将洗涤后的垂体膜与[3H]甲基促甲状腺激素释放激素在不存在（B0）或存在促甲状腺激素释放激素或促甲状腺激素释放激素类似物的情况下孵育。特异性结合（Bsp）对热不稳定且依赖于组织。Bsp的结合缓慢（k + 1 = 1.43×10(7) M-1 min-1），60分钟后达到平衡结合，并至少稳定60分钟。此后，通过加入过量的甲基促甲状腺激素释放激素使平衡结合的[3H]甲基促甲状腺激素释放激素解离；估计的解离速率常数（k - 1）和半衰期（t1/2）分别为6.74×10(-2) min-（1）和10.2分钟，动力学推导的解离常数（k - 1/k + 1）为4.71×10(-9) M。[3H]甲基促甲状腺激素释放激素的结合被促甲状腺激素释放激素和促甲状腺激素释放激素类似物以不同方式取代。对多个同源（甲基促甲状腺激素释放激素）和异源（促甲状腺激素释放激素）取代实验的LIGAND分析表明存在一类具有纳摩尔亲和力的单一结合位点。从多个异源（促甲状腺激素释放激素）取代实验汇总的数据表明存在另一类高亲和力（皮摩尔）结合位点。此外，对来自虹鳟和金鱼的神经中间叶和远侧部的单独制剂的检查表明，这两类结合位点在两个叶之间的定位不同。虹鳟垂体促甲状腺激素释放激素受体特异性分析表明，[3H]甲基促甲状腺激素释放激素取代的效力顺序为甲基促甲状腺激素释放激素>促甲状腺激素释放激素>焦谷氨酸-苯丙氨酸-脯氨酸-氨基。具有N-或C-末端取代的类似物几乎没有竞争潜力。本研究表明鲑科和鲤科垂体中存在特异性促甲状腺激素释放激素受体及其结合特性。
