Purchase this article with an account.
Teemu Turunen, Ari O Koskelainen; Electrophysiological determination of inhibition constants for phosphodiesterase (PDE6) inhibitors.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5591.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
PDE inhibitors are used to treat a variety of diseases but the lack of inhibitor specificity among different PDE families can inflict adverse side effects. The inhibition efficiencies of various PDE inhibitors towards photoreceptor PDE (PDE6) are mostly determined in vitro on enzymatically (trypsin) activated PDE6. In addition, the potencies of inhibitors towards trypsin-activated and thermally activated PDE6 have been shown to differ. However, methods to quantitatively study the inhibition potency of drugs towards light-activated PDE6 in natural surroundings are not available. This study proposes an ex vivo approach for quantitative determination of the inhibition constant towards light-activated PDE6 and thermally activated PDE6.
Transretinal electroretinography (ERG) and local ERG across the rod outer segments were used to record photoresponses from WT and GCAPs-/- dark-adapted mouse retinas at 37°C. Isolated retinas were superfused with solutions containing 2mM aspartate and 50µM BaCl2 to separate the rod photoresponses. The inhibition constants for 3-isobutyl-1-methylxanthine (IBMX), sildenafil, and zaprinast towards light-activated PDE6 (Ki,light) were determined based on the decrease of phototransduction gain. In addition, a novel experimental paradigm was developed for electrophysiological determination of the inhibition constant of PDE inhibitors towards thermally activated PDE (Ki,thermal).
The inhibition constants towards light-activated PDE6 (Ki,light) were 13.4 ± 0.7 µM for IBMX (n = 16 retinas, Fig. 1), 0.56 ± 0.09 µM for sildenafil (n = 4 retinas), and 0.97 ± 0.07 µM for zaprinast (n = 4 retinas). The Ki,thermal of IBMX was 15.2 ± 0.2 µM (n=6 retinas, mean ± SEM).
This novel ex vivo approach allows a precise testing of the inhibition potency of drugs towards PDE6. Our results suggest that the Ki of IBMX has the same value for both the light-activated and the thermally activated forms of PDE6.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
Determination of inhibition constant of IBMX towards light-activated PDE6.
This PDF is available to Subscribers Only