Establishment of a Cell Model for Dynamic Monitoring of Intracellular Calcium Concentration and High-Throughput Screening of P2Y2 Regulators

P2Y receptors are G-protein-coupled receptors (GPCRs) for extracellular nucleotides. The P2Y2 receptor subtype is expressed in a variety of cell types and plays an important role in physiological and pathophysiological processes such as inflammatory responses and neuropathic pain. Based on this, the P2Y2 has been identified as an important drug target. The specificity of current P2Y2 receptor modulators is relatively poor, and currently, specific and efficient P2Y2 receptor modulators and efficient screening strategies are lacking. In this study, a cell model based on calcium-activated chloride channels (CaCCs) was established that can detect changes in intracellular calcium concentrations and can be used to high-throughput screen for P2Y2 receptor-specific regulators. This screening strategy is suitable for screening of most G-protein-coupled receptor regulators that mediate increases in intracellular calcium signals. The cell model consists of three components that include the endogenously expressed P2Y2 receptor protein, the exogenously expressed calcium-activated chloride channel Anoctamin-1 (Ano1), and a yellow fluorescent protein mutant expressed within the cell that is highly sensitive to iodine ions. This model will allow for high-throughput screening of GPCR regulators that mediate increased intracellular calcium signaling using the calcium-activated transport of iodide ions by Ano1. We verified the ability of the model to detect intracellular calcium ion concentration using fluorescence quenching kinetic experiments by applying existing P2Y2 agonists and inhibitors to validate the screening function of the model, and we also evaluated the performance of the model in the context of high-throughput screening studies. The experimental results revealed that the model could sensitively detect intracellular calcium ion concentration changes and that the model was accurate in regard to detecting P2Y2 modulators. The resultant value of the Z-factor was 0.69, thus indicating that the model possesses good sensitivity and specificity.

[1]  S. Yamada,et al.  Effects of saw palmetto extract on the vanilloid receptor TRPV1 , 2021, Lower urinary tract symptoms.

[2]  C. Coddou,et al.  Purinergic P2Y2 and P2X4 Receptors Are Involved in the Epithelial-Mesenchymal Transition and Metastatic Potential of Gastric Cancer Derived Cell Lines , 2021, Pharmaceutics.

[3]  M. Sanderson-Smith,et al.  Autocrine regulation of wound healing by ATP release and P2Y2 receptor activation. , 2021, Life Science.

[4]  R. Boucher,et al.  Purinergic receptors in airway hydration. , 2020, Biochemical pharmacology.

[5]  L. Ooi,et al.  P2Y2 and P2X4 Receptors Mediate Ca2+ Mobilization in DH82 Canine Macrophage Cells , 2020, International journal of molecular sciences.

[6]  A. Alhowail,et al.  Role of the purinergic P2Y2 receptor in hippocampal function in mice. , 2020, European review for medical and pharmacological sciences.

[7]  Ying-zheng Zhao,et al.  Bioactive Factors-imprinted Scaffold Vehicles for Promoting Bone Healing: The Potential Strategies and the Confronted Challenges for Clinical Production , 2020, BIO Integration.

[8]  S. Bai,et al.  CUDC‐907, a novel dual PI3K and HDAC inhibitor, in prostate cancer: Antitumour activity and molecular mechanism of action , 2020, Journal of cellular and molecular medicine.

[9]  Zhiyu Li,et al.  Current knowledge on the nucleotide agonists for the P2Y2 receptor. , 2018, Bioorganic & medicinal chemistry.

[10]  H. Donahue,et al.  The Roles of P2Y2 Purinergic Receptors in Osteoblasts and Mechanotransduction , 2014, PloS one.

[11]  Kyle A. Brown,et al.  Synthesis of Extended Uridine Phosphonates Derived from an Allosteric P2Y2 Receptor Ligand , 2014, Molecules.

[12]  S. Skalicky,et al.  P2Y2 receptor agonists for the treatment of dry eye disease: a review , 2014, Clinical ophthalmology.

[13]  S. Offermanns,et al.  Platelet-derived nucleotides promote tumor-cell transendothelial migration and metastasis via P2Y2 receptor. , 2013, Cancer cell.

[14]  R. Faria,et al.  Action of Natural Products on P2 Receptors: A Reinvented Era for Drug Discovery , 2012, Molecules.

[15]  P. Kongsuphol,et al.  Expression and function of epithelial anoctamins , 2012, Experimental physiology.

[16]  S. Cianchetta,et al.  Cell-based imaging of sodium iodide symporter activity with the yellow fluorescent protein variant YFP-H148Q/I152L. , 2007, American journal of physiology. Cell physiology.

[17]  D. Kellerman,et al.  Inhaled P2Y2 receptor agonists as a treatment for patients with Cystic Fibrosis lung disease. , 2002, Advanced drug delivery reviews.

[18]  A. Hopkins,et al.  The druggable genome , 2002, Nature Reviews Drug Discovery.

[19]  A. S. Kritchenkov,et al.  Enhancing the in vivo stability of polycation gene carriers by using PEGylated hyaluronic acid as a shielding system , 2022, BIO Integration.

[20]  C. Müller,et al.  P 2 Y 2 and G q / G 11 control blood pressure by mediating endothelial mechanotransduction , 2019 .