被DuP753识别的血管紧张素II受体调节两条不同的鸟嘌呤核苷酸结合蛋白信号通路。

Angiotensin II receptor recognized by DuP753 regulates two distinct guanine nucleotide-binding protein signaling pathways.

作者信息

Crawford K W, Frey E A, Cote T E

机构信息

Uniformed Services University of the Health Sciences, Department of Pharmacology, Bethesda, Maryland 20889-4799.

出版信息

Mol Pharmacol. 1992 Jan;41(1):154-62.

PMID:1310139

Abstract

The 7315c cell, derived from a rat anterior pituitary tumor, expresses an angiotensin II (AII) receptor. [3H]AII binds to 7315c membranes specifically and saturably (Kd = 2.1 +/- 0.6 x 10(-6) M, Bmax = 282 +/- 33 fmol/mg of protein). GTP diminished the affinity of the membranes for [3H]AII (Kd = 4.1 +/- 0.4 x 10(-9) M, Bmax = 210 +/- 26 fmol/mg of protein). [3H]AII binding was displaced by AII (Ki = 1.3 +/- 0.6 x 10(-9) M), angiotensin III (AIII) (Ki = 0.9 +/- 0.4 x 10(-9) M), and the nonpeptide AII antagonist DuP753 (Ki = 1.4 +/- 0.6 x 10(-8) M). In contrast, a second nonpeptide AII ligand, PD123177, did not compete for [3H]AII binding sites. In intact cells, AII and AIII stimulated inositol trisphosphate (IP3) production (EC50 = 1.1 +/- 0.6 x 10(-8) M and 1.1 +/- 0.5 x 10(-8) M, respectively); this response to AII was antagonized by DuP753 (Ki = 1.7 +/- 0.3 x 10(-7) M). Pertussis toxin treatment failed to affect the ability of AII to stimulate IP3 production. In a crude membrane preparation, GTP was required for maximal AII-induced IP3 stimulation; guanosine thio-diphosphate abolished the agonist-GTP stimulation of IP3 production, in a concentration-dependent fashion. AII and AIII also inhibited adenylyl cyclase (EC50 = 2.9 +/- 1.1 x 10(-8) M and 6.0 +/- 1.0 x 10(-8) M, respectively). DuP753 antagonized the inhibition by AII of adenylyl cyclase (Ki = 2.8 +/- 0.4 x 10(-8) M). PD123177 failed to antagonize AII-induced cyclase inhibition. Pertussis toxin treatment abolished the AII and AIII inhibition of adenylyl cyclase. GTP was required for AII-induced inhibition of adenylyl cyclase. These data suggest that, in 7315c cells, a single subtype of AII receptor, identified by DuP753, is capable of regulating two different guanine nucleotide-binding protein (G protein) signalling pathways; one G protein, which is insensitive to pertussis toxin, stimulates IP3 production and the other G protein, which is sensitive to pertussis toxin, inhibits adenylyl cyclase.

摘要

源自大鼠垂体前叶肿瘤的7315c细胞表达血管紧张素II（AII）受体。[3H]AII特异性且饱和性地结合到7315c细胞膜上（解离常数Kd = 2.1±0.6×10⁻⁶ M，最大结合容量Bmax = 282±33 fmol/mg蛋白质）。鸟苷三磷酸（GTP）降低了细胞膜对[3H]AII的亲和力（Kd = 4.1±0.4×10⁻⁹ M，Bmax = 210±26 fmol/mg蛋白质）。[3H]AII的结合被AII（抑制常数Ki = 1.3±0.6×10⁻⁹ M）、血管紧张素III（AIII）（Ki = 0.9±0.4×10⁻⁹ M）和非肽类AII拮抗剂DuP753（Ki = 1.4±0.6×10⁻⁸ M）所取代。相反，另一种非肽类AII配体PD123177不竞争[3H]AII结合位点。在完整细胞中，AII和AIII刺激肌醇三磷酸（IP3）的产生（半数有效浓度EC50分别为1.1±0.6×10⁻⁸ M和1.1±0.5×10⁻⁸ M）；DuP753拮抗AII引起的这种反应（Ki = 1.7±0.3×10⁻⁷ M）。百日咳毒素处理未能影响AII刺激IP3产生的能力。在粗制细胞膜制剂中，最大程度的AII诱导的IP3刺激需要GTP；鸟苷硫代二磷酸以浓度依赖的方式消除了激动剂 - GTP对IP3产生的刺激。AII和AIII也抑制腺苷酸环化酶（EC50分别为2.9±1.1×10⁻⁸ M和6.0±1.0×10⁻⁸ M）。DuP753拮抗AII对腺苷酸环化酶的抑制作用（Ki = 2.8±0.4×10⁻⁸ M）。PD123177未能拮抗AII诱导的环化酶抑制作用。百日咳毒素处理消除了AII和AIII对腺苷酸环化酶的抑制作用。AII诱导的腺苷酸环化酶抑制作用需要GTP。这些数据表明，在7315c细胞中，由DuP753鉴定的单一亚型的AII受体能够调节两种不同的鸟嘌呤核苷酸结合蛋白（G蛋白）信号通路；一种对百日咳毒素不敏感的G蛋白刺激IP3产生，另一种对百日咳毒素敏感的G蛋白抑制腺苷酸环化酶。