Kukkonen J P, Näsman J, Ojala P, Oker-Blom C, Akerman K E
Department of Biochemistry and Pharmacy, Abo Akademi University, BioCity, Turku, Finland.
J Pharmacol Exp Ther. 1996 Nov;279(2):593-601.
The human muscarinic ACh receptor subtypes m1, m3 and m5 have been expressed in Sf9 cells using the baculovirus expression system. Stimulation of all three subtypes with CCh caused an increase in inositol-1,4,5-trisphosphate and intracellular Ca++. The increase in cytosolic free Ca++ was to a large extent due to influx. The levels of receptors (< 0.1-1 pmol/mg protein) increased with infection time in a narrow time span (24-36 h). The changes in the receptor densities did not significantly affect the EC50 values of CCh-mediated Ca++ mobilization with the m3 or the m5 subtype. The EC50 value was higher with the m1 receptor at low expression levels (approximately 100 fmol/mg protein), and it decreased with an increase in receptor density. The receptor subtypes displayed no gross differences in their response to oxotremorine-M, which behaved as a full agonist, or to oxotremorine and pilocarpine, which were less active. With the m3 subtype, there was an increase in the maximal response to oxotremorine with longer infection times. The results demonstrate that the recombinant muscarinic receptors, expressed in Sf9 cells, show many of the characteristics of endogenously expressed receptors when studied at low expression levels and that the receptor density may significantly affect the receptor pharmacology.
利用杆状病毒表达系统，已在Sf9细胞中表达了人毒蕈碱型乙酰胆碱受体亚型m1、m3和m5。用氯化乙酰胆碱刺激所有这三种亚型会导致1,4,5-三磷酸肌醇和细胞内钙离子增加。胞质游离钙离子的增加在很大程度上是由于钙离子内流。受体水平（<0.1 - 1 pmol/mg蛋白质）在狭窄的时间跨度（24 - 36小时）内随感染时间增加。受体密度的变化对m3或m5亚型的氯化乙酰胆碱介导的钙离子动员的EC50值没有显著影响。在低表达水平（约100 fmol/mg蛋白质）时，m1受体的EC50值较高，并且随着受体密度的增加而降低。这些受体亚型对作为完全激动剂的氧化震颤素-M，或对活性较低的氧化震颤素和毛果芸香碱的反应没有明显差异。对于m3亚型，随着感染时间延长，对氧化震颤素的最大反应增加。结果表明，在Sf9细胞中表达的重组毒蕈碱受体在低表达水平研究时表现出许多内源性表达受体的特征，并且受体密度可能会显著影响受体药理学。
