Assay of the enzymatic activity of Rh-PDE in mammalian cells (GloSensor assay)

HEK293 cells were purchased from JCRB Cell Bank and cultured in E-MEM media with l-Glutamine and Phenol Red (Wako), containing 10% (vol/vol) fetal bovine serum (FBS) and penicillin–streptomycin. The cells were co-transfected with the plasmid carrying the Rh-PDE variant and the pGloSensor-22F cAMP vector (Promega), by using Lipofectamine 2000 (Invitrogen). Changes in the intracellular cAMP concentration of the HEK293 cells were measured by the GloSensor assay (Promega). The transfected cells were incubated with or without 0.5 μM all-trans-retinal (Toronto Research Chemicals). Before measurements, the culture medium was replaced with a CO₂-independent medium, containing 10% (vol/vol) FBS and 2% (vol/vol) GloSensor cAMP stock solution (Promega). The cells were then incubated for 2 h at room temperature in the dark. The intracellular cAMP level was observed by monitoring the luminescence, using a microplate reader (Corona Electric) at 27 °C. The cells were treated with 3.5 μM forskolin (Wako), a direct activator of adenylyl cyclase, to elevate the intracellular cAMP level. The cells were illuminated with a xenon lamp (LAX-103, Asahi Spectra Co., Ltd., Japan) through an interference filter (510 nm). The light intensity was adjusted to 2.28 µW/mm², and measured by an LP1 power meter (Sanwa Electric Instruments Co., Ltd., Japan).