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Patricia Veiga-Crespo, Hodan Abdshill, Birgitta Sandström, Volkhard Kaever, Valeria Marigo, Francois Paquet-Durand, Frank Schwede, Andreas Rentsch, Per A R Ekström; Degenerating photoreceptors in Retinitis pigmentosa models release cGMP. A way of self protection?. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2731.
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© 2017 Association for Research in Vision and Ophthalmology.
The inherited retinal degeneration Retinitis pigmentosa (RP) has unknown mechanisms. Many RP models show increased levels of photoreceptor cGMP, suggesting high cGMP as a degeneration factor. We speculated that RP photoreceptors may try to extrude excess cGMP as a means of self protection, and hence here analysed the presence of cGMP in medium conditioned by degenerating RP retinas.
Relevantly aged mouse RP model or healthy retinas were cultured as organotypic explants for various timepoints, with medium changed every second day. Spent medium was used for cGMP measurements via ELISA based kits. cGMP in the medium was confirmed by HPLC and mass spectrometry. Compounds with effects on tentative cGMP transporters, or on cGMP-dependent protein kinase (PKG) or cyclic nucleotide gated channels (CNGs), were tried on the cultures. Conditioned medium from cultures of rod-like cells from retinal stem cells was also tested
Medium conditioned by retinas from rd1, rd10 and rd2 mice, all known to accumulate photoreceptor cGMP, had elevated levels of cGMP, with different release profiles. The cGMP into medium/retina was for rd1 measured to 277±3 pmoles (mean±SEM, n=4) between 4 and 6 days in vitro (DIV) from postnatal day 5, and for healthy retina to 57±5 (n=9, p<0.001). Treatment between 2 and 6 DIV with probenecid, a blocker of some transporter proteins, reduced rd1 medium cGMP to about 22 % of untreated at 6 DIV. Furthermore, cGMP analogues with effects on CNGs and/or PKG also altered the cGMP level in the medium. Highest values for rd2 (PN5+9DIV, n=4) and rd10 (PN7+10DIV, n=4) were estimated to 200±12 and 244±5 pmoles/retina, respectively. Conditioned medium from rod-like cells likewise contained cGMP.
Compared to healthy retinas, explants from three RP models all increased the levels of cGMP in their conditioned medium, suggesting that cGMP finds its way out of the diseased photoreceptor cells. This could occur via some transporter mechanism, since probenecid reduced the extracellular cGMP levels, and may thus represent a controlled release rather than a passive leakage. The release also has a relation to cGMP interactions, since cGMP analogues were able to modulate the extracellular cGMP levels. cGMP release could be a component in the degeneration machinery, with possible relevance for the outcome of the disease.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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