Center for Addiction Research, University of Texas Medical Branch, Galveston, TX 77555, USA.
BMC Neurosci. 2012 Mar 7;13:25. doi: 10.1186/1471-2202-13-25.
The serotonin (5-HT) 2A and 2C receptors (5-HT2AR and 5-HT2CR) are involved in a wide range of physiological and behavioral processes in the mammalian central and peripheral nervous systems. These receptors share a high degree of homology, have overlapping pharmacological profiles, and utilize many of the same and richly diverse second messenger signaling systems. We have developed quantitative assays for cells stably expressing these two receptors involving minimal cell sample manipulations that dramatically improve parallel assessments of two signaling responses: intracellular calcium (Cai++) changes and activation (phosphorylation) of downstream kinases. Such profiles are needed to begin to understand the simultaneous contributions from the multiplicity of signaling cascades likely to be initiated by serotonergic ligands.
We optimized the Cai++ assay for stable cell lines expressing either 5-HT2AR or 5-HT2CR (including dye use and measurement parameters; cell density and serum requirements). We adapted a quantitative 96-well plate immunoassay for pERK in the same cell lines. Similar cell density optima and time courses were observed for 5-HT2AR- and 5-HT2CR-expressing cells in generating both types of signaling. Both cell lines also require serum-free preincubation for maximal agonist responses in the pERK assay. However, 5-HT2AR-expressing cells showed significant release of Cai++ in response to 5-HT stimulation even when preincubated in serum-replete medium, while the response was completely eliminated by serum in 5-HT2CR-expressing cells. Response to another serotonergic ligand (DOI) was eliminated by serum-replete preincubation in both cells lines.
These data expand our knowledge of differences in ligand-stimulated signaling cascades between 5-HT2AR and 5-HT2CR. Our parallel assays can be applied to other cell and receptor systems for monitoring and dissecting concurrent signaling responses.
血清素（5-HT）2A 和 2C 受体（5-HT2AR 和 5-HT2CR）参与哺乳动物中枢和外周神经系统的广泛生理和行为过程。这些受体具有高度同源性，具有重叠的药理学特征，并利用许多相同的和丰富多样的第二信使信号转导系统。我们已经开发了用于稳定表达这些两种受体的细胞的定量测定法，涉及最小的细胞样本操作，这些操作极大地改善了两种信号反应的平行评估：细胞内钙（Cai++）变化和下游激酶的激活（磷酸化）。需要这种谱来开始理解可能由 5-羟色胺能配体引发的多重信号级联的同时贡献。
我们优化了用于稳定表达 5-HT2AR 或 5-HT2CR 的细胞系的 Cai++测定法（包括染料使用和测量参数；细胞密度和血清要求）。我们在相同的细胞系中适应了用于 pERK 的定量 96 孔板免疫测定法。在产生两种类型的信号时，观察到 5-HT2AR 和 5-HT2CR 表达细胞的相似细胞密度最佳值和时间过程。这两种细胞系在 pERK 测定中也需要无血清预孵育以获得最大激动剂反应。然而，即使在富含血清的培养基中预孵育，5-HT2AR 表达细胞也会对 5-HT 刺激表现出显着的 Cai++释放，而在 5-HT2CR 表达细胞中，该反应则完全被血清消除。另一种 5-羟色胺能配体（DOI）的反应也被两种细胞系的富含血清预孵育消除。
这些数据扩展了我们对 5-HT2AR 和 5-HT2CR 之间配体刺激信号级联差异的认识。我们的平行测定法可应用于其他细胞和受体系统，用于监测和剖析并发信号反应。
