Phosphodiesterases (PDEs) regulate the intracellular levels of cAMP and cGMP by hydrolyzing cAMP and cGMP to their inactive 5 monophosphates. These cyclic nucleotides play an important role as second messengers in diverse physiological functions. PDE1B plays a role in signal transduction by regulating the intracellular concentration of cyclic nucleotides. PDE1B hydrolyzes both cAMP and cGMP to nucleoside 5-prime monophosphate. Although it prefers cGMP at low substrate levels, it has a Vmax that is approximately equal for both cGMP and cAMP (Polli, J.W. and Koncaid, R.L., 1994; Yu, J. et al., 1997).
The PDE1B Cell-Based Reporter Assay Kit is designed for identification of PDE1B inhibitors in cultured cells. In the PDE1B reporter assay, PDE1B-HEK293 cells are transiently transfected with expression vectors for atrial natriuretic peptide (ANP) receptor A (ANPRA, a guanylate cyclase), cyclic nucleotide-gated cation channel CNGA2, and the photoprotein aequorin. When cells are activated with ANP, ANP induces the production of cGMP through ANPRA. cGMP can activate the CNGA2 channel and induces Ca2+ influx, resulting in aequorin luminescence. Therefore, the intracellular cGMP level is monitored via aequorin luminescence induced by Ca2+ influx through CNGA2, acting as the intracellular cGMP sensor. PDE1B can reduce the ANP-induced cGMP level in the cells, causing lowered aequorin luminescence. When PDE1B activity is inhibited (for example, by the PDE1 inhibitor BAY607550), then the ANP-induced cGMP level increases, resulting in calcium influx that stimulates luminescence signals.
The PDE1B Cell-Based Reporter Assay Kit is designed for screening inhibitors of PDE1B and for monitoring PDE1B/ cGMP/ Ca2+ channel signaling pathway activity in vivo.
Store cell line in liquid nitrogen immediately upon receipt. Store other components as recommended. Components are stable at least 12 months from date of receipt, when stored as directed.
PDE1B-HEK293 cell line: 1 vial; liquid nitrogen ANPRA/aequorin Expression vector*: 20 µg (80 ng DNA/ µl); -20°C CNGA2 Expression vector*: 20 µg (80 ng DNA/ µl); -20°C Atrial natriuretic peptide (ANP), 10 µM: 50 µl; -20°C Coelenterazine, 5 mM: 50 µl; -20°C Ca2+ free assay buffer: 100 mL; +4°C BSA (100X): 500 µl; -20°C CaCl2, 1M: 200 µl; +4°C *These vectors are suitable for transient transfection. They are NOT suitable for transformation and amplification in bacteria.