Zgombick J M, Branchek T A
Synaptic Pharmaceutical Corporation, Paramus, NJ 07652, USA.
Naunyn Schmiedebergs Arch Pharmacol. 1998 Nov;358(5):503-8. doi: 10.1007/pl00005285.
The opossum kidney (OK) cell line has been shown previously to express endogenous 5-HT1B receptors which negatively couple to adenylate cyclase. Since other Gi-linked receptors have been shown to inhibit adenylate cyclase and to elevate intracellular calcium concentrations ([Ca2+]i), studies were initiated to determine whether native opossum 5-HT1B receptors could also display dual coupling to these signal transduction mechanisms. Saturation studies using 125I-iodocyanopindolol ([125I]CYP) to radiolabel the 5-HT1B receptor in OK cell membranes (in the presence of 3 microM (-)-isoproterenol to mask beta-adrenergic receptors) yielded an equilibrium dissociation constant (pKd) of 10.04 and binding site density (Bmax) of 55 fmol/mg protein. Exposure of intact OK cells to 5-HT, CP 93,129, a selective rodent 5-HT1B receptor agonist, and (+/-)-cyanopindolol, a mixed 5-HT1A/1B receptor agonist/antagonist, produced concentration-dependent inhibitions of forskolin (3 MM)-stimulated cAMP accumulation (FSCA; Emax=90-95%) and elevations of [Ca2+]i (Emax approximately 200 nM increase above basal levels). Agonist potencies (pEC50) ranged from 9.7 to 8.1 and were comparable between the two second messenger assays, although slightly higher agonist potencies (approximately three-fold) were observed in the cAMP assay. GR 127,935, a selective 5-HT1B/1D receptor antagonist, behaved as a strong partial agonist in both the cAMP and calcium assays, with an intrinsic activity of 0.7 relative to 5-HT. Methiothepin, a nonselective 5-HT receptor antagonist, competitively antagonized the inhibitory cAMP response elicited by CP 93,129, yielding an apparent pKb value of 7.3. Methiothepin (10 microM) completely antagonized the stimulatory calcium response evoked by a saturating concentration of CP 93,129 (100 nM). Pertussis toxin pretreatment blocked the CP 93,129-induced inhibition of FSCA and elevation of [Ca2+]i in OK cells, indicating the involvement of Gi/o proteins in transducing these second messenger responses. The agonist properties of (+/-)-cyanopindolol and GR 127,935 observed in both second messenger assays suggests that a large degree of receptor reserve may be present, even though 5-HT1B receptor expression is low in OK cells. The OK cell line continues to serve as a model system to investigate 5-HT1B receptor-mediated signaling events.
先前已表明负鼠肾（OK）细胞系表达内源性5-HT1B受体，该受体与腺苷酸环化酶负偶联。由于已证明其他与Gi偶联的受体可抑制腺苷酸环化酶并提高细胞内钙浓度（[Ca2+]i），因此开展了研究以确定天然负鼠5-HT1B受体是否也能与这些信号转导机制进行双重偶联。使用[125I]（-）-碘氰吲哚洛尔（[125I]CYP）对OK细胞膜中的5-HT1B受体进行放射性标记的饱和研究（在存在3 microM（-）-异丙肾上腺素以掩盖β-肾上腺素能受体的情况下）得出平衡解离常数（pKd）为10.04，结合位点密度（Bmax）为55 fmol/mg蛋白质。将完整的OK细胞暴露于5-羟色胺、CP 93,129（一种选择性啮齿动物5-HT1B受体激动剂）和（+/-）-氰吲哚洛尔（一种5-HT1A/1B受体激动剂/拮抗剂），会产生浓度依赖性抑制福斯高林（3 mM）刺激的cAMP积累（FSCA；Emax = 90 - 95%）以及[Ca2+]i升高（Emax比基础水平增加约200 nM）。激动剂效力（pEC50）范围为9.7至8.1，在两种第二信使测定中相当，尽管在cAMP测定中观察到的激动剂效力略高（约三倍）。GR 127,935（一种选择性5-HT1B/1D受体拮抗剂）在cAMP和钙测定中均表现为强效部分激动剂，相对于5-羟色胺的内在活性为0.7。美替拉嗪（一种非选择性5-HT受体拮抗剂）竞争性拮抗CP 93,129引起的抑制性cAMP反应，表观pKb值为7.3。美替拉嗪（10 microM）完全拮抗饱和浓度的CP 93,129（100 nM）引起的刺激性钙反应。百日咳毒素预处理可阻断CP 93,129诱导的OK细胞中FSCA的抑制和[Ca2+]i的升高，表明Gi/o蛋白参与转导这些第二信使反应。在两种第二信使测定中观察到的（+/-）-氰吲哚洛尔和GR 127,935的激动剂特性表明，即使OK细胞中5-HT1B受体表达较低，可能仍存在大量受体储备。OK细胞系继续作为研究5-HT1B受体介导的信号转导事件的模型系统。
