Comparative Effects of Phosphodiesterase Inhibitors on Detached Rod Outer Segment Function
In vertebrate retinal rods light reduces a standing inward current that flows into the outer segment in darkness. Current is carried primarily by sodium ( 70%) and calcium ( 15%) ions which pass through channels in the outer segment surface membrane that are opened by cGMP (Yau and Baylor, 1989). A photoproduct (Rh *) formed by isomerization of rhodopsin catalyzes GTP-GDP exchange to activate a G protein (transducin, T) which stimulates cGMP-specific phosphodiesterase (PDE) causing cGMP hydrolysis, channel closure and a decrease in dark current. The recovery of current following light exposure involves inactivation of the light stimulated PDE and an increase in cGMP synthesis by activation of guanylate cyclase (Hodgkin and Nunn, 1988; Detwiler et al., 1989). The sequential activation and inactivation of PDE in the onset and recovery of rod light responses has been probed using IBMX, a membrane permeant phosphodiesterase inhibitor (Beavo et al., 1970; Capovilla et al., 1983; Hodgkin and Nunn, 1988). While such studies have provided useful information about the orchestration of transduction IBMX is not an ideal inhibitor. It is not very potent (K i = 1 × 10−5M) which makes it practically impossible to use to fully inhibit light-stimulated PDE. It is also non-specific in that it inhibits many kinds of cyclic nucleotide phosphodiesterases which could potentially complicate the interpretation of results. Assays on isolated enzymes have shown two compounds (dipyridamole and M&B:22,948) selectively inhibit cGMP phosphodiesterases (Beavo, 1988; Gillespie and Beavo, 1989) and are 20 to 100 times more potent than IBMX (K t = 3.8 × 10−7M and 1 × 10−7M respectively). This short paper compares the effects of IBMX, dipyridamole (FitzGerald, 1987) and M&B:22,948 (Broughton et al., 1974) on dark current and light responses recorded from detached rod outer segments during whole-cell voltage clamp. Our results show that there are significant differences in the action of the three inhibitors, which suggests that they may affect more than PDE. In the course of these experiments we also discovered that in nucleoside triphosphate depleted outer segments steps of bright light cause an increase rather than a decrease in dark current. Since these cells contain little or no GTP and dark current is maintained by an exogenous source of cGMP, the production of inward or inverted light responses suggests that steps of strong light can inhibit PDE possibly through a G protein independent pathway.
KeywordsHydrolysis DMSO Adenosine Retina Trypsin
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