Zgombick J M, Borden L A, Cochran T L, Kucharewicz S A, Weinshank R L, Branchek T A
Synaptic Pharmaceutical Corporation, Paramus, New Jersey 07652.
Mol Pharmacol. 1993 Sep;44(3):575-82.
The second messenger coupling of cloned human 5-hydroxytryptamine (5-HT)1D alpha and 5-HT1D beta receptors stably expressed in murine fibroblasts (LM (tk-)) was investigated. Clonal cell lines expressing similar receptor densities (Bmax = 750-950 fmol/mg) were used in this study. 5-HT (EC50 = 1.5-2.0 nM) and sumatriptan (EC50 = 6-14 nM), a selective 5-HT1D agonist, produced dose-dependent inhibition of forskolin-stimulated cAMP accumulation in intact cells transfected with the 5-HT1D alpha or 5-HT1D beta receptor gene. The maximal inhibitory responses elicited by these agonists were slightly greater with the 5-HT1D alpha receptor (approximately 90%) than the 5-HT1D beta receptor (approximately 80%). 5-HT (EC50 = 1.7-2.4 nM) and sumatriptan (EC50 = 8-18 nM) also evoked dose-dependent elevations in intracellular calcium concentrations ([Ca2+]i), with EC50 values that were indistinguishable from those for inhibition of forskolin-stimulated cAMP accumulation. Cells expressing 5-HT1D beta receptors displayed significantly larger 5-HT-induced increases in [Ca2+]i than did cells expressing 5-HT1D alpha receptors (206 nM versus 114 nm increase; p < 0.01). Dose-dependent elevations in inositol phosphates (IP) were also observed after application of 5-HT (EC50 = 29-54 nM) or sumatriptan (EC50 = 73-481 nM); the maximal increases in IP accumulation were modest (51-69%) for both 5-HT1D subtypes. In contrast to the cAMP and calcium responses, the concentration-response curves for IP accumulation were shifted to the right at least 10-fold. Methiothepin, a nonselective 5-HT1 antagonist, competitively antagonized the cAMP response, yielding an apparent dissociation constant (Kb) of 3-4 nM for the 5-HT1D receptors. Methiothepin (10 microM) significantly reduced the elevations in [Ca2+]i (> 90%) and IP (> 75%) evoked by saturating concentrations (1 microM) of agonists. All three functional responses were significantly attenuated (> 90%) by pretreatment with 100 ng/ml pertussis toxin. The sumatriptan-induced elevation of [Ca2+]i via activation of the 5-HT1D subtypes may provide a molecular mechanism of action by which sumatriptan could directly constrict cerebral blood vessels and alleviate migraine symptoms.
研究了稳定表达于鼠成纤维细胞（LM(tk-)）中的克隆人5-羟色胺（5-HT）1Dα和5-HT1Dβ受体的第二信使偶联。本研究使用了表达相似受体密度（Bmax = 750 - 950 fmol/mg）的克隆细胞系。5-HT（EC50 = 1.5 - 2.0 nM）和舒马曲坦（EC50 = 6 - 14 nM，一种选择性5-HT1D激动剂）对用5-HT1Dα或5-HT1Dβ受体基因转染的完整细胞中福司柯林刺激的cAMP积累产生剂量依赖性抑制。这些激动剂引起的最大抑制反应在5-HT1Dα受体（约90%）比5-HT1Dβ受体（约80%）时略大。5-HT（EC50 = 1.7 - 2.4 nM）和舒马曲坦（EC50 = 8 - 18 nM）也引起细胞内钙浓度（[Ca2+]i）的剂量依赖性升高，其EC50值与抑制福司柯林刺激的cAMP积累的EC50值无差异。表达5-HT1Dβ受体的细胞比表达5-HT1Dα受体的细胞显示出5-HT诱导的[Ca2+]i增加显著更大（增加206 nM对114 nM；p < 0.01）。应用5-HT（EC50 = 29 - 54 nM）或舒马曲坦（EC50 = 73 - 481 nM）后也观察到肌醇磷酸（IP）的剂量依赖性升高；两种5-HT1D亚型的IP积累最大增加幅度适中（51 - 69%）。与cAMP和钙反应相反，IP积累的浓度-反应曲线至少向右移动了10倍。美噻吨，一种非选择性5-HT1拮抗剂，竞争性拮抗cAMP反应，对5-HT1D受体产生的表观解离常数（Kb）为3 - 4 nM。美噻吨（10 μM）显著降低激动剂饱和浓度（1 μM）引起的[Ca2+]i升高（> 90%）和IP升高（> 75%）。所有三种功能反应在用100 ng/ml百日咳毒素预处理后均显著减弱（> 90%）。舒马曲坦通过激活5-HT1D亚型诱导的[Ca2+]i升高可能提供了一种作用分子机制，通过该机制舒马曲坦可直接收缩脑血管并缓解偏头痛症状。
