A Stable Cell Line Co-expressing hTRPV1 and GCaMP6s: A Novel Cell-based Assay For High-throughput Screening of hTRPV1 Agonists
- Authors: Shi J.1, Chen X.1, Zhang Y.1, Shi T.1, Zhang R.1, Zhu S.1, Zong X.1, Wang C.1, Li L.1
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Affiliations:
- Department of Laboratory of Pharmacology, State Key Laboratory of NBC Protection for Civilians
- Issue: Vol 27, No 2 (2024)
- Pages: 298-306
- Section: Chemistry
- URL: https://rjpbr.com/1386-2073/article/view/644626
- DOI: https://doi.org/10.2174/1386207326666230511143259
- ID: 644626
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Abstract
Background:Transient receptor potential vanilloid-1 (TRPV1) is a non-selective cation channel capable of integrating various noxious chemical and physical stimuli. Recently, human TRPV1 (hTRPV1) has attracted wide attention from researchers because it is closely related to pain, inflammation, temperature perception, and tumors. Our study was aimed at generating a stable cell line co-expressing hTRPV1 receptor and GCaMP6s calcium indicator protein and, based on this, developing high-throughput screening methods for targeting hTRPV1 agonists.
Methods:The CHO-hTRPV1-GCaMP6s cell line stably expressing hTRPV1 and GCaMP6s was generated by co-transfection of hTRPV1 and GCaMP6s into Chinese hamster ovary (CHO) cells. The high-throughput screening methods were developed based on detecting the concentration of intracellular calcium ions ([Ca2+]i) by using chemically synthesized dyes and genetically encoded calcium indicator (GECI). Meanwhile, the sensitivity and adaptability of these methods in the evaluation of capsaicinoids were also compared.
Results:A stable cell line co-expressing hTRPV1 and GCaMP6s was generated and used to establish a functional high-throughput screening assay based on the measurement of [Ca2+]i by fluorometric imaging plate reader (FLIPR). The GECI exhibited a higher sensitivity and applicability than that of chemically synthesized dyes in detecting the changes in [Ca2+]i induced by capsaicin. The CHO-hTRPV1-GCaMP6s cell line was further used to detect the dose-dependent relationships of various hTRPV1 agonists (comparison of EC50 values: capsaicin (39 ± 1.67 nM) & nonivamide (67 ± 3.05 nM) $#60; piperine (9222 ± 1851 nM)), and this order is consistent with the pharmacological properties of hTRPV1 activation by these agonists.
Conclusion:The successful establishment of the CHO-hTRPV1-GCaMP6s cell lines and their application in high-throughput screening of hTRPV1 agonists.
About the authors
Jingjing Shi
Department of Laboratory of Pharmacology, State Key Laboratory of NBC Protection for Civilians
Email: info@benthamscience.net
Xuejun Chen
Department of Laboratory of Pharmacology, State Key Laboratory of NBC Protection for Civilians
Email: info@benthamscience.net
Yi Zhang
Department of Laboratory of Pharmacology, State Key Laboratory of NBC Protection for Civilians
Email: info@benthamscience.net
Tong Shi
Department of Laboratory of Pharmacology, State Key Laboratory of NBC Protection for Civilians
Email: info@benthamscience.net
Ruihua Zhang
Department of Laboratory of Pharmacology, State Key Laboratory of NBC Protection for Civilians
Email: info@benthamscience.net
Siqing Zhu
Department of Laboratory of Pharmacology, State Key Laboratory of NBC Protection for Civilians
Email: info@benthamscience.net
Xingxing Zong
Department of Laboratory of Pharmacology, State Key Laboratory of NBC Protection for Civilians
Email: info@benthamscience.net
Chen Wang
Department of Laboratory of Pharmacology, State Key Laboratory of NBC Protection for Civilians
Author for correspondence.
Email: info@benthamscience.net
Liqin Li
Department of Laboratory of Pharmacology, State Key Laboratory of NBC Protection for Civilians
Author for correspondence.
Email: info@benthamscience.net
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