Drugs Targeting Specific Phosphodiesterase Families: Selectivity Toward Photoreceptor PDE6 and Effects on Intact Photoreceptors

Abstract

Abstract: : Purpose:PDE6 is the central enzyme in visual transduction. Disruptions of cGMP metabolism in photoreceptors can cause visual defects, including blindness. Inhibitors specific to members of the phosphodiesterase (PDE) superfamily (e.g., sildenafil) are important as therapeutic agents, but the effects of PDE inhibitors on the photoreceptor PDE (PDE6) and on photoreceptor cells are poorly understood. To remedy this, we studied the potency and selectivity of PDE inhibitors on rod and cone PDE6 and on cGMP metabolism in isolated rod photoreceptors. Methods:Bovine rod and cone PDE6 were purified from frozen bovine retinas as described [Pentia et al., Meth. Mol. Biol., in press (2005)]. PDE6 activity in the presence of PDE inhibitors was assayed with a radiotracer assay. Intact frog rod outer segment (ROS) were purified on a discontinuous Percoll density gradient [Cote, Meth. Enzymol. 315, 646 (2000)]. Frog rod PDE6 was studied in its membrane–associated state. Results:A set of inhibitors specific for PDE1–5 were tested with bovine rod and cone PDE6. Whereas tadalafil (K_I = 2100 nM) is >200–fold selective for PDE5 over PDE6, sildenafil (K_I = 11 nM) and vardenafil (K_I = 0.7 nM) show similar potency for PDE5 and rod PDE6. Zaprinast’s 10–fold selectivity of PDE6 over PDE5 qualifies it as a PDE6–selective inhibitor. PDE1 inhibitors were equipotent in blocking catalysis of PDE1 and PDE6. No inhibitors discriminated rod and cone PDE6. IBMX, sildenafil and vardenafil all elevate cGMP levels in intact dark–adapted ROS, but none were able to inhibit all PDE6 activity. The potency of PDE inhibitors to elevate cGMP levels in dark–adapted ROS was much lower than the inhibitory potency toward the purified enzyme, particularly for PDE5/6–selective inhibitors. Part of this difference represents competition between the drug and the PDE6 inhibitory γ subunit for the active site of the holoenzyme. Under certain conditions, low concentrations of PDE5/6–selective inhibitors (but not IBMX) can stimulate PDE6 hydrolysis. Conclusions:The drug–binding site in the rod and cone PDE6 catalytic pocket shares similarities with the corresponding region of both PDE1 and PDE5. Multiple factors in addition to intrinsic drug binding affinity (including restricted diffusion across the plasma membrane, the high concentration of PDE6 in ROS, and competition with Pγ) determine the effectiveness of PDE inhibitors to disrupt cGMP metabolism in photoreceptors.

Keywords: photoreceptors • pharmacology • enzymes/enzyme inhibitors