Efficacy and selectivity of phosphodiesterase-targeted drugs in inhibiting photoreceptor phosphodiesterase (PDE6) in retinal photoreceptors

Abstract

Purpose: Phosphodiesterase (PDE) inhibitors are important therapeutic agents, but their effects on photoreceptor PDE (PDE6) and photoreceptor cells are poorly understood. The potency and selectivity of various classes of PDE inhibitors on purified rod and cone PDE6 and on intact rod outer segments (ROS) were characterized.

Methods: The inhibition constant (K(i)) of isozyme-selective PDE inhibitors was determined for purified rod and cone PDE6. Perturbations of cGMP levels in isolated ROS suspensions by PDE inhibitors were quantitated by a cGMP enzyme-linked immunoassay.

Results: Most PDE5-selective inhibitors were excellent PDE6 inhibitors. Vardenafil, a potent PDE5 inhibitor (K(i) = 0.2 nM), was the most potent PDE6 inhibitor tested (K(i) = 0.7 nM). Zaprinast was the only drug that inhibited PDE6 more potently than did PDE5. PDE1-selective inhibitors were equally effective in inhibiting PDE6. In intact ROS, PDE inhibitors elevated cGMP levels, but none fully inhibited PDE6. Their potency for elevating cGMP levels in ROS was much lower than their ability to inhibit the purified enzyme. Competition between PDE5/6-selective drugs and the inhibitory gamma-subunit for the active site of PDE6 is proposed to reduce the effectiveness of drugs at the enzyme-active site.

Conclusions: Several classes of PDE inhibitors inhibit PDE6 equally as well as the PDE family to which they are targeted. In intact ROS, high PDE6 concentrations, binding of the gamma-subunit to the active site, and calcium feedback mechanisms attenuate the effectiveness of PDE inhibitors to inhibit PDE6 and disrupt the cGMP signaling pathway during visual transduction.

Fig. 1. Synergistic effect of inhibiting PDE6 and buffering intracellular Ca2+ on cGMP levels in intact ROS.

Purified ROS suspensions ([rhodopsin] = 4.8 μM) were prepared in complete darkness as described in Methods. Two portions were incubated with either 1.09 mM EGTA ([Ca²⁺]_free = 500 nM; squares) or 400 μM IBMX (triangles). A third sample was first pre-incubated for 10 min with EGTA followed by IBMX addition (circles). Portions were quenched at the indicated times with 50% HCl/ethanol. The cGMP concentration was determined by an enzyme-linked immunoadsorbant assay, and reported relative to the rhodopsin content of the sample. Data points at the mean ± S.D. of three experiments.

Fig. 2. Time course of drug-induced increases in cGMP concentration in intact ROS.

Purified ROS (0.008 mol cGMP/mol rhodopsin) were pre-incubated with 1.09 mM EGTA for 10 min to buffer the free calcium concentration. At time zero, 400 μM IBMX (circles), 50 μM sildenafil (triangles) or 100 μM vardenafil (squares) were added, and samples quenched in 50% HCl/ethanol for cGMP determinations. The results were normalized to the average maximum stimulation for each drug (units: mol cGMP per mol rhodopsin): IBMX, 0.040; vardenafil, 0.035, and; sildenafil, 0.014. Data points are the mean of three experiments in which the standard deviation was ≤ 13% of the mean value.

Fig 3. PDE inhibitors induce dose-dependent elevations of ROS cGMP levels that do not saturate.

Ca²⁺-bufferedROS suspensions (10 min pre-incubation with 1.09 mM EGTA) were treated with the indicated concentration of IBMX (circles), sildenafil (triangles) or vardenafil (squares) for 10 min, and then acid-quenched for cGMP extraction and quantitation. The data points are the mean ± S.D. of three experiments.

Fig 4. PDE6 holoenzyme is less susceptible to inhibition by PDE inhibitors.

ROS suspensions were homogenized, and soluble proteins and metabolites were separated from PDE6-containing ROS membranes by centrifugation (see Methods). The ROS membranes were further depleted of endogenous nucleoside triphosphates by incubation at 22ºC for 30 min. Nonactivated PDE6 holoenzyme (4 nM; open symbols) was incubated with vardenafil (panel A) or IBMX (panel B) for 15 min before addition of 10 μM [³H]cGMP to assay catalytic activity. Activated PDE6 (20 pM; filled symbols) was incubated with vardenafil (panel A) or IBMX (panel B) for 15 min before adding 1 μM [³H]cGMP. The results shown here are typical of at least three other experiments.

Fig 5. PDE5/6-selective inhibitors—but not IBMX—can stimulate catalysis at high cGMP concentrations.

ROS membranes (2.0 nM PDE6 concentration) depleted of soluble proteins and nucleotides were incubated with IBMX (circles), sildenafil (triangles) or vardenafil (squares) for 15 min. Catalytic activity was determined by a colorimetric assay with 2 mM cGMP. The data was normalized to the basal PDE6 activity for plotting. The data represent the mean ± S.D. (n = 3). * Statistically significant, p<0.05.