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(Investigative Ophthalmology and Visual Science. 2007;48:978-984.)
© 2007 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.06-0543
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Localization of the Cyclic ADP-Ribose-Dependent Calcium Signaling Pathway in Bovine Rod Outer Segments

Isabella Panfoli,1 Silvia Ravera,1 Andrea Fabiano,1 Raffaella Magrassi,2,3 Alberto Diaspro,2,3 Alessandro Morelli,1 and Isidoro M. Pepe1

1From the Department of Biology, 2LAMBS (MicroScoBiO Research Center and Department of Physics), University of Genoa, Genova, Italy; and the 3INFM-CNR (Istituto Nazionale per la Fisica della Materia and Centro Nazionale delle Ricerche), Genova, Italy.

PURPOSE. Calcium ions play a pivotal role in phototransduction. In this study, the presence and functional role of the adenosine diphosphoribosyl (ADPR)-cyclase-cyclic ADP-ribose (cADPR) system in bovine retinal rod outer segments (ROS) was investigated.

METHODS. A Ca2+ release from osmotically intact ROS discs elicited by cADPR was studied in the presence of the Ca2+ tracer fluo-3. Endogenous cyclic guanosine diphosphate ribose (cGDPR) formation in discs was investigated by spectrophotometric detection of its synthesis from nicotinamide guanine dinucleotide (NGD+). ADPR-cyclase was also investigated at a structural level on mildly denaturing SDS-PAGE by production of cyclic inosine diphosphate ribose from nicotinamide hypoxantine dinucleotide (NHD+). Western immunoblot analysis with a specific antibody was conducted to verify the presence of ryanodine-sensitive Ca2+ channels (RyRs) in ROS discs.

RESULTS. cADPR-dependent Ca2+ release was a linear function of extravesicular free Ca2+ concentration, between 200 and 900 nM Ca2+. When free Ca2+ was 203 ± 10 nM the mean Ca2+ release was 23 ± 3 pmol/mL per milligram protein. The average rate of cGDPR production was 13 ± 2 nmol cGDPR/min per milligram protein, by a putative enzyme with an apparent molecular mass of 53 ± 1 kDa. ROS ADPR-cyclase was localized in the membranous fraction. No nicotinamide adenine dinucleotide glycohydrolase (NADase) activity was detected. The presence of RyR channels in pure disc preparations was confirmed by confocal laser scanning microscopy.

CONCLUSIONS. A cADPR metabolism may be present in retinal ROS discs, which may be Ca2+ stores operated by cADPR. A model is proposed for the physiological role of cADPR-mediated Ca2+ release in bovine ROS.






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