Phosphodiesterase-6 (PDE6) is an enzyme catalyzing the hydrolysis of cyclic guanosine monophosphate (cGMP), a second messenger molecule participating in a vast number of biological processes. Vertebrate photoreceptor cells react to light through a phototransduction cascade, where the activation of PDE6 leads to a decrease in the intracellular cGMP concentration and closure of cation channels in the photoreceptor outer segment plasma membrane. The channel closure causes a decrease in the intracellular calcium concentration, providing photoreceptor cells with negative feedback for light-induced sensitivity regulation, i.e., light adaptation, via calcium sensor proteins. In rod photoreceptor cells, the calcium sensors guanylate cyclase-activating proteins (GCAPs) and recoverin are the dominant mediators of light adaptation. In publication I of this thesis, a novel source of calcium-mediated light adaptation was discovered in rods by recording transretinal ex vivo electroretinography (TERG) across dark-adapted isolated mouse retinas. The focus of the rest of the thesis work was to characterize this novel calcium-dependent sensitivity regulation mechanism with the leading hypothesis that the regulation might originate from the calcium-mediated control of the basal PDE6 activity (βdark). To test the hypothesis, novel paradigms and methods were developed for the determination of βdark. In publication II, a device for the simultaneous recording of TERG and local ERG across the photoreceptor outer segment layer (LERG-OS) was developed to obtain quantitative information on rod phototransduction. The work demonstrated that the TERG recordings, although affected by signal components from photoreceptor inner segments, correspond well with those registered by LERG-OS. These ex vivo ERG methods, TERG and LERG-OS, enabled the quantitative investigation of PDE6 and its inhibition in the living retina. Traditionally, PDE6 inhibitors have been examined using biochemically activated purified PDE6 molecules. In publication III of this thesis, a new method was developed to quantify the inhibition constants for PDE6 inhibitors acting on the naturally occurring light-activated and spontaneously activated forms of PDE6. According to the results, the inhibition constants of different PDE inhibitors can vary substantially against these two PDE6 forms. In publication IV, the inhibition constants were utilized for cGMP clamp experiments, a novel paradigm which allowed the determination of βdark in wild type mouse rods for the first time. In publication V, the methods, together with mathematical modeling of photoresponses, were used to investigate the calcium-mediated light adaptation mechanisms in knockout mice lacking the dominant GCAPs- and recoverin-mediated adaptation pathways. It was found that βdark increases by ~ 20 – 30% when rod extracellular calcium concentration is reduced below its normal physiological range. This finding introduces a novel mechanism contributing to rod light adaptation and to the functional regulation of PDE enzymes.
|Translated title of the contribution||Fosfodiesteraasin tausta-aktiivisuuden vaikutus hiiren sauvasolujen valovasteiden säädössä|
|Publication status||Published - 2020|
|MoE publication type||G5 Doctoral dissertation (article)|
- light adaptation