Localization of the cyclic ADP-ribose-dependent calcium signaling pathway in bovine rod outer segments.
OBJECTIVE
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 Ca(2+) release from osmotically intact ROS discs elicited by cADPR was studied in the presence of the Ca(2+) 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 Ca(2+) channels (RyRs) in ROS discs.


RESULTS
cADPR-dependent Ca(2+) release was a linear function of extravesicular free Ca(2+) concentration, between 200 and 900 nM Ca(2+). When free Ca(2+) was 203 +/- 10 nM the mean Ca(2+) 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 Ca(2+) stores operated by cADPR. A model is proposed for the physiological role of cADPR-mediated Ca(2+) release in bovine ROS.
Panfoli
Isabella
I
Department of Biology, University of Genoa, Genova, Italy. isabella.panfoli@unige.it
Ravera
Silvia
S
Fabiano
Andrea
A
Magrassi
Raffaella
R
Diaspro
Alberto
A
Morelli
Alessandro
A
Pepe
Isidoro M
IM
D036541:ADP-ribosyl Cyclase / Q000502:physiology*; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D002417:Cattle; D036563:Cyclic ADP-Ribose / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D006156:Guanosine Diphosphate Sugars / Q000378:metabolism; D007292:Inosine Nucleotides / Q000378:metabolism; D018613:Microscopy, Confocal; D009244:NAD+ Nucleosidase / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000201:enzymology* / Q000648:ultrastructure; D013054:Spectrophotometry, Atomic; D014785:Vision, Ocular
D015220:Calcium Channels; D006156:Guanosine Diphosphate Sugars; D007292:Inosine Nucleotides; C107267:cyclic IDP-ribose; C090657:cyclic guanosine diphosphate-ribose; D036563:Cyclic ADP-Ribose; D012243:Rhodopsin; D036541:ADP-ribosyl Cyclase; D009244:NAD+ Nucleosidase; D002118:Calcium