Development of a Real-time cAMP Assay to Study TRP Channel Modulation of cAMP Signalling

Abstract

Background and Purpose Class A GPCR activity is regulated by the negative allosteric action of extracellular sodium ions that bind to a conserved sodium pocket within the receptor. When GPCR is activated, the pocket collapses leading to sodium ions exit from the pocket into the cytoplasm. It is unknown if changes in the intracellular sodium concentration can alter sodium occupancy in the pocket to modify GPCR activity. TRP channels are an important sources of sodium entry into cells and their activity increases intracellular sodium concentration. The longer term aim of this study is to determine the influence of TRP activity and related changes in intracellular sodium on GPCR activity. This study describes the first steps in this project, to develop a real-time cAMP assay in MDCK II cells that endogenously express various GPCRs, such as vasopressin receptors, and TRPA1 channels.

Experimental Approach TRPA1 expression in MDCK cells was determined pharmacologically by examining the effects of TRPA1 agonists and a TRPA1 antagonist (A967079) on intracellular calcium concentrations using a Fura-2 based assay. Transfection of MDCK cells with a plasmid encoding the luminescent cAMP sensor, GloSensor, was used to provide a real-time readout of changes in cAMP levels in response to the adenylyl cyclase activator forskolin. Various combinations (ratios) of GloSensor plasmid and Lipofectamine 2000 or Lipofectamine 3000 transfection reagents were used to identify conditions for optimal transfection efficiency. In parallel, attempts were made to develop a stable monoclonal cell line expressing GloSensor. The optimal concentration of the antibiotic, hygromycin B, to select for transfected cells was determined and single cell cloning was initiated.

Key Results Challenge of MDCK II cells with AITC, carvacrol and menthol evoked increases in intracellular calcium concentrations with mean EC50 values of 33±0.3 µM, 154.3±1.2 µM and 2.35±0.24 mM, respectively. The responses to all 3 compounds were inhibited by A967079 with a similar IC50 values (~0.3 µM). Gq- coupled V1 expression was tested functionally using vasopressin which showed no effect on intracellular calcium levels. Changing the ratios of DNA:Lipofectame or amount of DNA had little to no effect on transient transfection efficiency and resulted in weak forskolin-mediated Glosensor luminescent responses. Cell viability assays showed that the optimal selection concentration of hygromycin B to select for growth of transfected cells is 300 µg/mL. After hygromycin B selection, polyclonal MDCK II cells expressing GloSensor produced larger concentration-dependent responses to forskolin, and measurable responses to vasopressin indicating Gs-coupled V2 expression. Single cell colonies were grown but had to be discarded after fungal infection.

Conclusion MDCK II cells endogenously express TRPA1 channels and vasopressin V2 receptors suitable for transfection with the cAMP sensor, GloSensor. Transient transfection of MDCK II cells with GloSensor does not offer a suitable tool to measure changes in cAMP levels. In contrast, antibiotic selection of Glosensor transfected MDCK II cells followed by single cell cloning to establish monoclonal cell lines provides a promising approach for sensitive real-time cAMP assays.