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.

Localization of the cyclic ADP-Ribose-dependent calcium signaling pathway in bovine rod outer segments.

PANFOLI, ISABELLA;RAVERA, SILVIA;DIASPRO, ALBERTO GIOVANNI;MORELLI, ALESSANDRO;I. M. Pepe
2007-01-01

Abstract

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.
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11567/392341
Citazioni
  • ???jsp.display-item.citation.pmc??? 2
  • Scopus 9
  • ???jsp.display-item.citation.isi??? 9
social impact