Purchase this article with an account.
Teemu Tapio Turunen, Anni Aleksandra Villentytär Antikainen, Ari O Koskelainen; Determination of basal PDE activity in WT, GCAPs-/- and GCAPs-/- rec-/- mice rods. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):992.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Absorption of a photon activates rhodopsin (R*) which starts a G-protein signaling cascade leading to an activation of phosphodiesterase (PDE*) hydrolyzing cGMP. Due to thermal energy, there is always some PDE activity present even in darkness. The amount of PDE* in darkness determines the rate of spontaneous cGMP hydrolysis, i.e. the basal PDE activity (βdark), which is one of the main factors setting the kinetics of photoresponse recovery. Here we introduce and test a novel approach to determine βdark.
PDE inhibitors decrease the catalytic activity of PDE. A decrease in the basal PDE activity can be compensated by increasing [PDE*] with light. We assume that the amount of light-induced PDE activity needed to keep the [cGMP]in, and thus the photoreceptor signal, constant after introducing PDE inhibitor to the retina matches with the decrease in βdark. This was accomplished with a PID controlled background light feedback. βdark was calculated based on the activation factor of the cascade (determined), the lifetimes of R* (from literature) and PDE* (determined), the inhibition constant of the inhibitor (determined) and the amount of light needed for the compensation. Local ERG was used to determine βdark from isolated WT, GCAPs-/- and GCAPs-/- rec-/- mouse retinas in HEPES buffered solution. The PDE inhibitor IBMX was used at concentrations of 10, 20 and 40 µM.
The βdark values obtained from a linear fit to data (value ± SER) were 1.7 ± 0.01 s-1 for WT, 2.2 ± 0.2 s-1 for GCAPs-/- and 1.8 ± 0.1 s-1 for GCAPs-/- rec-/- mice. The time constant of an exponential fit to the late recovery phase of dim flash responses of GCAPs-/- believed to be set mainly by basal PDE activity was 440 ms, corresponding to βdark of 2.3 s-1.
βdark was determined for the first time for WT mice and the βdark values were comparable between the mouse strains used. βdark obtained from light compensation (≈ 2 s-1) and from dim flash recovery (2.3 s-1) are somewhat smaller than previously obtained for GCAPs-/- mice (4.1 s-1, Gross et al., Biophys. J., vol. 102, 2012).
This PDF is available to Subscribers Only