Inhibition of PDE1 Results in Death of Y79–Rb Cells in vitro

Abstract

Abstract: : Purpose: To characterize phoshodiesterases (PDEs) in cultured retinoblastoma cells (Y79–Rb) cultured with dibutyryl cyclic AMP (dbcAMP), an agent that causes differentiation and cell death, and to study the physiological effects of PDE inhibition. Methods:Y79–Rb cells were incubated in the presence or absence of dbcAMP for 6 days. Proteins in cell homogenates were separated using FPLC anion exchange chromatography. PDE activity was assayed using ³H– cGMP or cAMP as substrates in the presence or absence of Ca²⁺ and calmodulin (CaM), PDE inhibitors, or anti–CaM (ACC). Peak fractions of PDE activity were analyzed by western blot with isozyme–specific antibodies. RT–PCR was performed using RNA isolated from treated and untreated Y79–Rb cells and PDE isozyme–specific primers. Results: Treatment of Y79–Rb cells with dbcAMP increased enzymatic activity of two distinct PDE gene families (PDE1 and PDE9). PDE1 hydrolyses both cAMP and cGMP, can be activated by Ca²⁺/CaM, is inhibited specifically by vinpocetine and Ks–505a, and can be immunoabsorbed by ACC. The Y79–Rb PDE1 is recognized by anti–PDE1C. The Y79–Rb PDE9 hydrolyzes only cGMP, is Ca²⁺/CaM independent, is not inhibited by vinpocetine or E–4021, and is recognized by anti–PDE9. Quantitative RT–PCR reveals PDE1C and PDE9 transcripts, however the expression is independent of dbcAMP. Treatment of Y79 cells with pharmacological concentrations of vinpocetine resulted in a rapid decrease in cell metabolism (monitored by Alamar blue) and cell death (monitored by trypan blue exclusion). Conclusions: dbcAMP increases soluble PDE1 and PDE9 enzymatic activities in Y79–Rb cells. Inhibition of PDE1C results in Y79–Rb cell death.

Keywords: enzymes/enzyme inhibitors • protein purification and characterization • retinoblastoma