Laitinen J T, Jokinen M
Department of Physiology, University of Kuopio, Finland.
J Neurochem. 1998 Aug;71(2):808-16. doi: 10.1046/j.1471-4159.1998.71020808.x.
Histamine elicits its biological effects via three distinct G protein-coupled receptors, termed H1, H2, and H3. We have used guanosine 5'-(gamma-[35S]thio)triphosphate (GTPgamma[35S]) autoradiography to localize histamine receptor-dependent G protein activation in rat brain tissue sections. Initial studies revealed that in basal conditions, adenosine was present in tissue sections in sufficient concentrations to generate an adenosine A1 receptor-dependent GTPgamma[35S] signal in several brain regions. All further incubations therefore contained 8-cyclopentyl-1,3-dipropylxanthine (10 microM), a selective A1 receptor antagonist. Histamine elicited dose-dependent increments in GTPgamma[35S] binding to discrete anatomical structures, most notably the caudate putamen, cerebral cortex, and substantia nigra. The overall anatomical pattern of the histamine-evoked binding response closely reflects the known distribution of H3 binding sites and was faithfully mimicked by N(alpha)-methylhistamine, (R)-alpha-methylhistamine, and immepip, three H3-selective agonists. In all regions examined, the GTPgamma[35S] signal was reversed with thioperamide and clobenpropit, two potent H3-selective antagonists, whereas mepyramine, a specific H1 antagonist, and cimetidine, a prototypic H2 antagonist, proved ineffective. These data indicate that in rat brain tissue sections, GTPgamma[35S] autoradiography selectively detects H3 receptor-dependent signaling in response to histamine stimulation. As the existing evidence suggests that GTPgamma[35S] autoradiography preferentially reveals responses to G(i/o)-coupled receptors, our data indicate that most, if not all, central H3 binding sites represent functional receptors coupling to G(i/o), the inhibitory class of G proteins. Besides allowing more detailed studies on H3 receptor signaling within anatomically restricted regions of the CNS, GTPgamma[35S] autoradiography offers a novel approach for functional in vitro screening of H3 ligands.
组胺通过三种不同的G蛋白偶联受体发挥其生物学效应，分别称为H1、H2和H3。我们使用鸟苷5'-（γ-[35S]硫代）三磷酸（GTPγ[35S]）放射自显影技术，在大鼠脑组织切片中定位组胺受体依赖性G蛋白激活。初步研究表明，在基础条件下，腺苷在组织切片中的浓度足以在几个脑区产生腺苷A1受体依赖性GTPγ[35S]信号。因此，所有后续孵育均含有8-环戊基-1,3-二丙基黄嘌呤（10 microM），一种选择性A1受体拮抗剂。组胺引起GTPγ[35S]与离散解剖结构的结合呈剂量依赖性增加，最显著的是尾状壳核、大脑皮层和黑质。组胺诱发的结合反应的整体解剖模式密切反映了已知的H3结合位点分布，并被三种H3选择性激动剂N（α）-甲基组胺、（R）-α-甲基组胺和依美哌啶如实地模拟。在所有检查的区域中，GTPγ[35S]信号被两种强效H3选择性拮抗剂硫丙酰胺和氯苯丙哌嗪逆转，而特异性H1拮抗剂美吡拉敏和原型H2拮抗剂西咪替丁则无效。这些数据表明，在大鼠脑组织切片中，GTPγ[35S]放射自显影技术可选择性检测组胺刺激后H3受体依赖性信号传导。由于现有证据表明GTPγ[35S]放射自显影技术优先揭示对G（i/o）偶联受体的反应，我们的数据表明，大多数（如果不是全部）中枢H3结合位点代表与G（i/o）偶联的功能性受体，即G蛋白的抑制类。除了允许对中枢神经系统解剖学受限区域内的H3受体信号传导进行更详细的研究外，GTPγ[35S]放射自显影技术还为H3配体的功能性体外筛选提供了一种新方法。
