Functional identification of phosphodiesterase activity in human trabecular meshwork cells

The phosphodiesterases (PDE) activity in human trabecular meshwork cells (H TM-3) was investigated in this study in order to better understand the sign al transduction pathways in the conventional outflow tract of the eye. Agon ists (isoproterenol or nitroprusside) were used to stimulate adenylyl cycla se and guanylyl cyclase, respectively, in the absence and presence of nonse lective IBMX or PDES specific inhibitors E4021 (1). The subcellular distrib ution of cAMP and cGMP PDEs was determined directly by PDE enzyme assays us ing HTM-3 cells. Levels of cyclic nucleotides were measured in the same cel ls by radioimmunoassay (RIA). Isoproterenol alone elevated cAMP levels, and this response was enhanced by IBMX. Nitroprusside alone caused no increase in basal cGMP levels but, in the presence of E4021, nitroprusside produced significant, dose-related elevation of cGMP levels. Subcellular distributi on experiments indicated that the greatest activity for PDEs resided in the supernatant fraction. In conclusion, HTM-3 cells contain PDEs that degrade both cyclic nucleotides. The PDE activities reside predominantly in the su pernatant, but the PDE activity for degrading cGMP is more pronounced. More over, results with E4021 suggest that PDES activity could play a critical r ole in modulating cGMP-related activity in the trabecular meshwork.