DataSheet1_Fingerprinting heterocellular β-adrenoceptor functional expression in the brain using agonist activity profiles.PDF

Noradrenergic projections from the brainstem locus coeruleus drive arousal, attentiveness, mood, and memory, but specific adrenoceptor (AR) function across the varied brain cell types has not been extensively characterized, especially with agonists. This study reports a pharmacological analysis of brain AR function, offering insights for innovative therapeutic interventions that might serve to compensate for locus coeruleus decline, known to develop in the earliest phases of neurodegenerative diseases. First, β-AR agonist activities were measured in recombinant cell systems and compared with those of isoprenaline to generate Δlog(Emax/EC50) values, system-independent metrics of agonist activity, that, in turn, provide receptor subtype fingerprints. These fingerprints were then used to assess receptor subtype expression across human brain cell systems and compared with Δlog(Emax/EC50) values arising from β-arrestin activation or measurements of cAMP response desensitization to assess the possibility of ligand bias among β-AR agonists. Agonist activity profiles were confirmed to be system-independent and, in particular, revealed β2-AR functional expression across several human brain cell types. Broad β2-AR function observed is consistent with noradrenergic tone arising from the locus coeruleus exerting heterocellular neuroexcitatory and homeostatic influence. Notably, Δlog(Emax/EC50) measurements suggest that tested β-AR agonists do not show ligand bias as it pertains to homologous receptor desensitization in the system examined. Δlog(Emax/EC50) agonist fingerprinting is a powerful means of assessing receptor subtype expression regardless of receptor expression levels or assay readout, and the method may be applicable to future use for novel ligands and tissues expressing any receptor with available reference agonists.

脑干蓝斑核（locus coeruleus）发出的去甲肾上腺素能投射可调控觉醒、注意力、情绪与记忆，但目前针对不同脑细胞类型中肾上腺素能受体（adrenoceptor, AR）的具体功能，尤其是激动剂介导的功能，尚未得到充分解析。本研究对脑部肾上腺素能受体功能开展了药理学分析，为创新性治疗干预提供了理论依据——这些干预手段或可弥补神经退行性疾病早期阶段即可出现的蓝斑核功能减退。首先，本研究在重组细胞体系中检测了β肾上腺素能受体（β-AR）激动剂的活性，并与异丙肾上腺素（isoprenaline）的活性进行比对，以此计算Δlog(Emax/EC50)值——该指标为不依赖实验体系的激动剂活性量化参数，可用于构建受体亚型的特征指纹图谱。随后，利用这些特征指纹图谱评估人类脑细胞体系中各受体亚型的表达情况，并将结果与由β抑制蛋白（β-arrestin）激活或环磷酸腺苷（cAMP）反应脱敏实验得到的Δlog(Emax/EC50)值进行比对，以此研判β-AR激动剂是否存在配体偏倚现象。实验证实，激动剂活性谱具有系统无关性，尤其明确了β2肾上腺素能受体（β2-AR）在多种人类脑细胞类型中存在功能性表达。本研究观测到的广泛β2-AR功能活性，与蓝斑核介导的去甲肾上腺素能张力所发挥的跨细胞神经兴奋及稳态调控作用相一致。值得注意的是，Δlog(Emax/EC50)检测结果显示，在所研究的实验体系中，受试β-AR激动剂并未呈现出与同源受体脱敏相关的配体偏倚。基于Δlog(Emax/EC50)的激动剂指纹图谱分析法，是一种无需考虑受体表达水平或实验读值即可精准评估受体亚型表达的高效手段，该方法未来可推广应用于带有可用参考激动剂的新型配体及表达任意受体的组织样本研究。