March 2007
Volume 48, Issue 3
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Biochemistry and Molecular Biology  |   March 2007
Localization of the Cyclic ADP-Ribose-Dependent Calcium Signaling Pathway in Bovine Rod Outer Segments
Author Affiliations
  • Isabella Panfoli
    From the Department of Biology,
  • Silvia Ravera
    From the Department of Biology,
  • Andrea Fabiano
    From the Department of Biology,
  • Raffaella Magrassi
    LAMBS (MicroScoBiO Research Center and Department of Physics), University of Genoa, Genova, Italy; and the
    INFM-CNR (Istituto Nazionale per la Fisica della Materia and Centro Nazionale delle Ricerche), Genova, Italy.
  • Alberto Diaspro
    LAMBS (MicroScoBiO Research Center and Department of Physics), University of Genoa, Genova, Italy; and the
    INFM-CNR (Istituto Nazionale per la Fisica della Materia and Centro Nazionale delle Ricerche), Genova, Italy.
  • Alessandro Morelli
    From the Department of Biology,
  • Isidoro M. Pepe
    From the Department of Biology,
Investigative Ophthalmology & Visual Science March 2007, Vol.48, 978-984. doi:10.1167/iovs.06-0543
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      Isabella Panfoli, Silvia Ravera, Andrea Fabiano, Raffaella Magrassi, Alberto Diaspro, Alessandro Morelli, Isidoro M. Pepe; Localization of the Cyclic ADP-Ribose-Dependent Calcium Signaling Pathway in Bovine Rod Outer Segments. Invest. Ophthalmol. Vis. Sci. 2007;48(3):978-984. doi: 10.1167/iovs.06-0543.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

Ca2+ signaling is the most ubiquitous signal transduction mechanism, mediating the intracellular effects of extracellular signals into a diverse array of specific cellular responses. 1 Several metabolites of nicotinamide adenine dinucleotide (NAD+) are second messengers in cell calcium signaling. Cyclic adenosine diphosphoribose (cyclic ADP-ribose, cADPR) modulates calcium movements from ryanodine-sensitive endoplasmic reticulum stores. 2 A novel Ca2+-release mechanism activated by nicotinic acid adenine dinucleotide phosphate (NAADP+) has been shown to act on different stores of calcium (for review, see Ref. 3 ). ADPR has been shown to modulate the activity of a member of the transient receptor potential channels family (TRPM2). 4 cADPR, abundant in the brain, 5 has been shown to modulate calcium movements from ryanodine-sensitive stores, in both neurons 6 7 and astrocytes. 8  
Ryanodine-sensitive Ca2+ receptors (RyR) are homotetrameric peptides 9 modulating calcium release from intracellular stores. Together with inositol 1,4,5-trisphosphate (IP3) receptors, 10 they belong to a family displaying calcium-induced calcium release (CICR), 11 the process accounting for cytosolic free Ca2+ spatiotemporal dynamics. Three RyR isoforms are known, 10 11 12 all found in the nervous system. 13 cADPR is involved in calcium-dependent neural processes, including synaptic transmission, plasticity, and neuronal excitability. 6 8 14 15 16 cADPR-dependent signaling has been identified within the nucleus in several cell types. 17 The cADPR-synthesizing enzymes known to date form a growing family of conserved proteins. 18 The first identified enzyme of this family was isolated from the synaptic terminal cytosol of the mollusc Aplysia californica. 19 In mammalian systems, the ectoenzymes CD38 and CD157 (or BST-1), membrane-associated 20 and -anchored, 21 proteins, respectively appear to be the major enzymes with ADP-ribosyl cyclase (ADPR-cyclase) activity. 18 The two proteins share approximately 30% sequence identity with the Aplysia cyclase. CD38 is expressed in the nervous system, 20 principally in the soma of neural cells, and so it is unlikely to play a significant role in relation to ryanodine-sensitive internal stores. Recently, the existence of a novel endocellular mammalian ADPR-cyclase, involved in brain development and functioning, 22 was demonstrated in neural cells from Cd38 −/− mice. 22 23 Basile et al. 24 demonstrated that, in addition to producing cADPR, ADPR, and NAADP, ADPR-cyclases can generate other adenine dinucleotides that are known to exist naturally and to have biological activity. Therefore, there is incipient evidence of the existence of other types of ADPR-cyclases in mammalian cells. 
Vertebrate retina, with its laminar composition, has been considered a model of the central nervous system, from which it derives. In vertebrate visual phototransduction, free Ca2+ concentration has long been known to play a pivotal role, mediating guanosine 3′,5′-cyclic monophosphate (cGMP) metabolism, 25 26 and light adaptation in rods and cones. 27 We have characterized an active (45Ca2+) uptake in ROS discs, a sarcoendoplasmic reticulum type of Ca2+-ATPase (SERCA) in bovine ROS discs 28 29 and a Ca2+ion-sensitive adenylate kinase activity associated with disc membranes. 30 The cited results are consistent with the notion of discs as dynamic organelles able to store and release calcium ions, consistent with the idea that the nearly all calcium measured in the ROS is sequestered within the discs. 28 31  
In the present study, we investigated the presence and functional role of the ADPR-cyclase-cADPR system in ROS. Toward this end, subfractions of ROS, i.e., purified discs, crude discs, and cytosolic fractions were screened. Results suggest that discs (1) possess a consistent ADPR-cyclase activity; (2) are able to release Ca2+ in response to cADPR; and, (3) contain RyR Ca2+ channels. Based on these data, a role for cADPR-mediated Ca2+ signaling in mammalian phototransduction is proposed. 
Methods
Chemicals
cADPR, nicotinamide guanine dinucleotide (NGD+), nicotinamide hypoxantine dinucleotide (NHD+), calmodulin (CaM), and A23187 were purchased from Sigma-Aldrich (St. Louis, MO); fluo-3 from Molecular Probes (Eugene, OR); goat polyclonal antibody IgG, (200 μg/mL) against ryanodine-sensitive Ca2+ receptors from Santa Cruz Biotechnology (sc-8169; Santa Cruz, CA); and mouse monoclonal antibody raised against rabbit Na/K ATPase α1 (C464.6) from Santa Cruz Biotechnology (sc-21712). All other reagents were of analytical grade. 
ROS and Disc Preparations
ROS were isolated from 20 bovine retinas (from a local abattoir), by sucrose gradient centrifugation 32 in the presence of protease inhibitor cocktail (Sigma-Aldrich). Osmotically intact discs were obtained, after bursting ROS for 3 hours in a 5% single-density gradient (Ficoll; Sigma-Aldrich) solution in distilled water with 5 mM dithiothreitol (DTT) and 70 μg/mL leupeptin, by collecting them at the solution surface after centrifuging for 2 hours at 25,000 rpm in a rotor (FW-27; Beckman, Fullerton, CA). 33 To obtain crude disc membranes and cytosolic fractions, ROS were homogenized in a glass homogenizer (1 mL) and centrifuged for 4 minutes at 14,500 rpm in a centrifuge (Eppendorf, Fremont, CA), and the supernatant (cytosol) and membranes were separated. 
Rhodopsin Determination
Rhodopsin concentration was determined spectrophotometrically (molar extinction coefficient of 41,000 M−1 · cm−1) 34 by measuring the difference in 500 nm absorption between spectra recorded before and after exhaustive bleaching (green light) for 5 minutes. The A280/A500 ratio was 2.9 ± 0.6 for ROS and 1.8 ± 0.2 (average ± SD) for discs, respectively. Absorbance spectra were recorded with a dual-beam spectrometer (UV2; Unicam, Cambridge, UK). 
Calcium Measurements by Flame Atomic Absorption Spectrometry
Total calcium content of osmotically intact discs was assayed by flame atomic absorption spectrometry (AAS; Perkin Elmer, Wellesley, MA) on 0.3 mL of packed discs (0.8 mM rhodopsin) with a hollow cathode lamp for Ca (422.7 nm), as previously described. 35  
Transmission Electron Microscopy of Osmotically Intact Disc Preparations
For ultrastructural analysis, osmotically intact discs (1.2 mg total protein) were centrifuged at 6000 rpm for 2 minutes in the centrifuge (Eppendorf). The pellet was fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 10 minutes. Then the sample was centrifuged and washed with 0.1 M cacodylate buffer (pH 7.4) and 2% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.4) was added, for 10 minutes. After the osmium solution was discarded, the sample was treated with a 1% aqueous solution of uranyl acetate, dehydrated through a graded ethanol series, then embedded in Epon resin and polymerized at 42°C, overnight followed by 48 hours at 70°C. Ultrathin sections were obtained by using a microtome (Ultracut E; Leica, Heidelberg, Germany) and analyzed with an electron microscope 36 (FEI CM10 or Tecnai 12 G2 (FEI, Eindhoven, the Netherlands). 
Sample Preparation and Ca2+ Background Tuning for Fluorometric Free Ca2+ Measurements
Freshly prepared disc suspensions (11 mg protein, 0.8 mM rhodopsin concentration) were left overnight at 4°C (freezing caused disc damage) in 2 mM CaCl2 (200 μL discs plus 4 μL of 0.1 M CaCl2), as discs tend to loose intravesicular Ca2+ in the hours after their isolation. Preloading was not necessary when disc suspensions were used immediately after preparation. It was impossible to load disc vesicles during the experiment, as the Ca2+-ATPase of ROS is inactive at nanomolar calcium concentrations (K 0.5 for Ca2+ = 10 μM). 28 29 35 Before experiments, 0.2 mL discs were rapidly centrifuged, the supernatant discarded, and the remnant (∼40 μL) washed in resuspended in double-distilled water (10 times the volume). The procedure was repeated twice (605-fold dilution), and the discs were resuspended in 0.4 mM rhodopsin at a 6 mg/mL protein concentration. 
Fluorometric Free Ca2+ Measurements
Protein disc suspension (0.15 mL of 6 mg/mL) was added to the incubation medium (0.45 mg/mL final). The corresponding dilution of initial Ca2+ in the suspension was 8067-fold, and so in the experiment, free Ca2+ was ∼0.2 μM, which is the background level over which it is possible to observe cADPR-evoked Ca2+ release. 37 Incubation was conducted at room temperature in the presence of 30 mM HEPES (pH 7.1), 120 mM KCl, 10 μg/mL CaM, and 2.5 μM fluo-3. Recordings (excitation, 505 nm; emission, 534 nm) were performed with a spectrophotometer (LS 50B; Perkin Elmer) with continuous stirring. Seeking to study CICR, we determined the dependence of the entity of cADPR-evoked Ca2+ release from discs on the extravesicular free Ca2+ concentration ([Ca2+]) in the experimental mixture, producing an elevation of background [Ca2+] in the medium by addition of a few picomoles of CaCl2. These additions randomly modified [Ca2+] (ranging from ∼200–900 nM). [Ca2+] quantitation was achieved by probe calibration at the end of each experiment. 37 Water, first deionized by passage through two consecutive ion-exchange columns and second passed through a water purification system (Milli-Q Ultrapure Water Purification System; Millipore Corp., Billerica, MA), was used throughout. 
Western Blot Analysis
ROS homogenates were subjected to SDS-PAGE. 38 Proteins were transferred onto nitrocellulose membranes (Hybond ECL; GE Healthcare, Uppsala, Sweden) in a mini transblot device (Bio-Rad Laboratories, Hercules, CA). Sheets were blocked for 1 hour at room temperature in TBS-BSA 1% (25 mM Tris-HCl [pH 7.4], 150 mM NaCl plus 1% BSA). 39  
Overlay Assays
Samples on nitrocellulose were probed with specific antibody diluted 1:1000 in TBS-BSA 3.5%. After extensive washing, the binding of the antibody was revealed by an enhanced chemiluminescence (ECL) detection system (Roche Diagnostics, Mannheim, Germany) using HRP-conjugated monoclonal secondary anti-goat antibody (GE Healthcare) diluted 1:1000 in TBS-BSA 1%. Blots were then autoradiographed (Hyperfilm ECL; GE Healthcare) and images acquired. 
Assays for ADPR-Cyclase Activity
Putative ADPR-cyclase was assayed by using NGD+, a β-NAD+ analogue, as a substrate, according to Graeff et al. 40 Aliquots of bovine ROS homogenates, or purified disc suspensions (300 μg protein) were incubated with the same reaction mixture as was used for Ca2+ measurements (pH 7.2). The reaction was started by addition of 1 mM NGD+, conducted at 37°C, and stopped by addition of 50 μL of 25% perchloric acid (PCA). Absorbance of neutralized (50 μL 2 M K2CO3) and clarified supernatants was read at 300 nm and optical densities converted to rates of cGDPR production (molar extinction coefficient of 3758 M−1 · cm−1). 
Detection of ADPR-Cyclase Activity by SDS-PAGE
Samples were incubated at 25°C for 5 minutes, with a modified Laemmli sample buffer containing 1.5% SDS (instead of the usual 8%) and no β-mercaptoethanol, and run on SDS-PAGE (12% polyacrylamide). 41 To remove excess SDS, gels were washed in 50 mM Tris-HCl (pH 7.0), with repeated buffer changes. NHD+ (0.2 mM) in 50 mM Tris-HCl (pH 7.0) was then added, and after the gels were incubated for 15 minutes in the dark at 20°C, the fluorescent bands, corresponding to locally produced cyclic inosine diphosphate ribose, were visualized under UV light and images acquired (Electrophoresis Documentation and Analysis System EDAS 290; Eastman Kodak, Rochester, NY). 
Assay for NAD+ Glycohydrolase Activity
NADase activity was assayed by spectrophotometric measures of decrement of β-NAD+, according to Wu et al., 42 with minor modifications. Briefly, the reaction mixture contained 0.05 M potassium phosphate buffer (pH 7.3), 5 mM β-NAD, 1 mM MgCl2 and 200 μg of either crude or purified disc preparations in a total volume of 1 mL. The reaction was terminated by addition of 0.2 mL 25% PCA. Clarified and neutralized supernatants were added of 0.25 M Tris-HCl, 35 mM semicarbazide, 1% (vol/vol) ethanol. Initial absorbance at 340 nm was recorded, and then 60 μg alcohol dehydrogenase (ADH, EC 1.1.1.1; Roche) were added and the increase in A340 monitored. 
Skeletal Muscle Preparations
Three grams of mouse muscle were homogenized in 10 mL of 0.25 M sucrose, 5 mM HEPES buffer, 1 mM EDTA (pH 7.2), and protease inhibitor cocktail (Sigma-Aldrich). Homogenate was centrifuged for 10 minutes at 500g, the precipitate discarded, and the supernatant centrifuged for 20 minutes at 20,000g (Heraeus centrifuge, Hanau, Germany). A microsomal pellet was collected after the 20,000g supernatant was centrifuged for 60 minutes at 100,000g (SW40 Rotor; Beckman). 
Confocal Laser Scanning Microscopy
Image acquisition was performed with a confocal microscope (SP2 AOBS TCS; Leica) equipped with a 405-nm 10-mW laser diode. Images were collected with a 100× oil immersion numeric aperture 1/4 1.4 objective lens (HCX PL APO; Leica Microsystems SpA, Milan, Italy). Images of the sample were obtained with the 514-nm line of a 20-mW argon ion laser. To detect the presence or absence of the red dye (CY3), we analyzed the images to determine the related mean intensity values. A spectral evaluation was made possible by the spectral characteristics of the microscope scanning head (SP2 AOBS; Leica). The spectral window used for collecting fluorescence was 500 to 700 nm, according to reported emission spectra. 43 The resultant images were acquired, stored, elaborated, and visualized with the confocal software (Leica). 
Protein Assay
Protein content was determined by bicinchoninic acid (BCA) protein assay kit from Pierce Biotechnology, Inc. (Rockford, IL), with BSA as the standard. 
Results
Osmotically intact disc suspensions purified from bovine ROS by single-density gradient flotation (Ficoll; Sigma-Aldrich), were characterized for purity and rhodopsin content. For osmotically intact discs, the A280/A500 ratio was found to be 1.8 ± 0.1 (average ± SD). Rhodopsin concentration in the original preparation of the preloaded discs was 0.8 mM, whereas it was 0.4 mM after dilution. Mitochondrial contamination of the purified disc fractions was negligible, as shown by transmission electron microscopy (TEM) imaging (Fig. 1) . An immunoblot analysis of ROS and discs with a mouse monoclonal antibody raised against rabbit sodium/potassium ATPase α1, a ubiquitous protein of the inner segment, was conducted. Figure 2shows that, with respect to the muscle homogenates used as the positive control, cross-reactivity with Na/K ATPase antibody was present in the ROS but not in the purified discs. 
Disc suspensions (0.45 mg/mL) displayed a Ca2+-releasing activity of cADPR (10 μM final concentration), monitored in the presence of the calcium-reporting dye fluo-3 (Fig. 3) . Initial free Ca2+ concentrations in the incubation medium were ∼200 nM. To vary the Ca2+ background levels, we added a few picomoles of CaCl2 (see the Methods section). In the case of Figure 3A , 0.1 nmol CaCl2 was added. Then at the 247-nM free Ca+2 level, the first cADPR addition was performed and elicited a release of 46 pmol/mL per milligram protein. When free [Ca2+] was 203 ± 10 (mean ± SD) nM, Ca2+ release was 23.0 ± 3.0 pmol/mL per milligram protein (mean ± SD, n = 5). Addition of the calcium ionophore A23187 at the end of the experiment for calibration (not shown, for clarity) showed that the Ca2+ plateau was far from probe saturation. The stored Ca2+ released by A23187 was 75.00 nM (i.e., 150 nanomoles). Total Ca2+ stored in the 0.150 mL discs in the experiment was 160 nanomoles, being total Ca2+ concentration in the original preloaded preparation 2 mM, as measured by flame atomic absorption spectrometry. Therefore, the fraction of the stored Ca2+ released by the A23187 was >90%. The ratio of Ca2+ to rhodopsin was 2.4. 
cADPR-evoked Ca2+ release was strengthened by increasing extravesicular free [Ca2+]. Experiments from the same preparation as shown in Figure 3Ashowed that Ca2+ release induced by cADPR was a linear function of extravesicular free [Ca2+] (Fig. 3B)in approximately the physiological range. A comparison of Figure 3Awith 3B shows that, at 247 nM, Ca released was 84 nM, and at 336 nM was 70 nM, whereas it would be expected to be 250 nM at 336 nM. So, desensitization to sequential additions of cADPR was little. 
To investigate endogenous cADPR formation, a spectrophotometric assay of GDP-ribosyl cyclase (GCPR-cyclase) activity in ROS discs was conducted (Fig. 4) . This method is based on NGD+ conversion in abundance by most known ADPR cyclases into a cyclic derivative, cGDPR, resistant to hydrolysis. 40 41 Both crude and purified ROS discs contained readily detected ADPR-cyclase activity, with an average initial rate of 13 ± 2 nanomoles cGDPR/min per milligram protein (mean ± SD, n = 7). In samples in which proteins were denatured by adding 25% PCA just before addition of NGD+, there was no cGDPR production. 
A putative NAD+ glycohydrolase activity, spectrophotometrically assayed in both crude and purified ROS discs measuring the decrement of β-NAD+, was not found (0.0 nmol β-NAD consumed/min per milligrams). The enzyme was tested with the purpose of better understanding whether the putative ROS enzyme was similar to the novel enzyme from brain which, in contrast with CD38, does not display NADase activity. 22 23  
To obtain conclusive evidence of the presence of ADPR-cyclase in bovine discs, we investigated the enzyme at a structural level. For this purpose, ADPR-cyclase of discs was detected on mildly denaturing SDS-PAGE by way of its production of fluorescent cyclic inosine diphosphate ribose from NHD+. 41 One gel representative of at least 10 separated experiments is shown in Figure 5 , where black and white are inverted for the sake of clarity. The resultant apparent molecular mass (MW) of the enzyme, seen as a fluorescent band in the gel was 53 ± 1 (mean ± SD). To seek the subcellular localization of the ADPR-cyclase, we tested three fractions (Fig. 5) : osmotically intact discs (lane 3), crude discs (lane 2), and cytosolic ROS fractions (lane 1). ADPR-cyclase activity appeared to be located only within membranes, being detectable in both crude and purified discs and not detectable in the soluble ROS fraction. 
The dependence of cADPR-elicited Ca2+ release on free extradiscal [Ca2+] (Fig. 3B)prompted us to investigate the presence in the discs of the RyR channels to which cADPR is known to bind. Samples were screened with an antibody (Ab) against an epitope mapping at the C-terminal region of human RyR, where the calcium release channel activity resides. Pictures obtained in confocal microscopy (Fig. 6A)show the presence in purified discs of RyR channels, stained by indirect immunocytochemistry (in red, a secondary Ab coupled to Cy3). The spectrum of Cy3-labeled secondary Ab is shown in Figure 6B . Incubation of discs with secondary Ab yielded only negligible immunoreactivity, confirming the specificity of disc surface interaction of the Ab with the RyRs (data not shown). In Figure 6C , Western blot analysis with the same Ab shows that a protein with the apparent mass of 520.0 ± 60 kDa (mean ± SD) was present in osmotically intact discs (lane 1) but not in ROS cytosol. Mouse skeletal muscle microsomes, used as a positive control (lane 1) showed a band of 560 ± 75 kDa (mean ± SD). 
Discussion
The results presented in this work point to the existence of a cADPR metabolism in retinal ROS. cADPR elicited a Ca2+ release from osmotically intact discs that was modulated by extravesicular [Ca2+] (Fig. 3) . Osmotically intact bovine ROS disc preparations were shown to be devoid of mitochondria and endoplasmic reticulum (Fig. 1)and negligibly contaminated by inner segments, as suggested by the lack of cross-reactivity with a mouse monoclonal antibody raised against rabbit Na/K ATPase in purified discs (Fig. 2) . The trend of the dependence was linear (Fig. 3B)around the physiological levels in dark-adapted ROS. 44 45 Saturation is likely reached for higher Ca2+ concentrations that cannot be explored by means of fluo-3. In our experimental conditions, Ca2+ reuptake into discs was not observed (Fig. 3) , because the ATP-driven Ca2+ pump of ROS discs does not operate at nanomolar Ca2+ concentrations. 28 29 Data indicate that in the discs, self-inactivation due to consecutive additions of cADPR was minimal (Fig. 3)
CaM is one of the accessory proteins that modulating calcium release from RyR channels along with cADPR, calcium and magnesium ions. 12 CaM and CaM-like proteins were found in ROS. 25  
A GDPR-cyclase activity is present in both crude and purified ROS discs, with initial rate of ∼13 ± 1 namomoles cGDPR formed/min per milligram of protein (Fig. 4) . Such a result is comparable to the activity found in pancreatic homogenate (35.5 ± 4 nanomoles cGDPR formed/min per milligram of protein) 46 and in mouse brain synaptosomes, where the initial reaction rate was 25 nanomoles cADPR formed/min per milligram of protein. 22 In isolated cardiac myocytes 47 and scallop muscle supernatants, 37 activity was lower (∼2.5 nanomoles/mg per minute). The mouse brain enzyme 22 23 was unable to catalyze the cyclization reaction with NGD+, similar to an ADPR-cyclase identified in T-cell lymphocytes. 48 Conversely, the ROS putative ADPR-cyclase can use the NAD-analogue NGD+ as a substrate as can most ADPR-cyclases (CD38, CD157, and the Aplysia cyclase 39 40 49 ). When the ADPR-cyclase activity of discs was investigated on mildly denaturing SDS-PAGE with NHD+, a fluorescent band was observed on gels at the apparent molecular mass of 53 ± 1 kDa (Fig. 4) . Mr of both CD38 and BST1, as observed in Western Blot analyses, is 45 kDa, whereas Mr of Aplysia ovotestis cyclase is approximately 35 kDa. 19  
Confocal laser imaging of intact disc preparations showed that RyR channels are expressed in purified discs of the bovine retina (Fig. 6A) . To explore the molecular mass of RyRs we performed Western immunoblot analysis by means of the same Ab on both intact and crude discs. Results (Fig. 6B)suggest that discs possess RyRs with a molecular mass similar to that of most RyRs. 
ADPR-cyclase is a ubiquitous enzyme. 49 In lower metazoan tissues, such as Aplysia ovotestis, ADPR-cyclase activity is typically cytosolic 2 19 50 whereas, in sea urchin eggs, it is localized partially in the cytosol and partially in the membranes. 51 In vertebrate cells and tissues, the ADPR-cyclase activity was found to be associated with plasma membranes. 2 This association also occurs for both the ectocellular multifunctional CD38-ADPR-cyclase glycoprotein 2 14 15 and the BST-1/BF-3 antigen. 8  
Recently, scientists have sought to identify ADPR-cyclase activities located in intracellular membranous structures, not in contact with the vertebrate cell surface. Ceni et al. 22 23 have described the cited novel endocellular ADPR-cyclase inside synaptosomes from Cd38−/− mouse brain. Purified ROS discs are exquisitely intracellular organelles, whose protein composition has been shown to differ from that of the plasma membrane. 52 The ROS disc ADPR-cyclase activity was detectable only in crude or purified discs but not in the cytosolic ROS fraction, and so the enzyme appears to be intracellular, but not cytosolic (Fig. 5) . It has been shown that the Cd38 −/− mouse ADPR-cyclase displays high intracellular ADPR-cyclase activity and a very low NADase activity in vitro. 22 Of note, the ROS enzyme does not display NAD-glycohydrolase activity, unlike CD38 and CD157. More experiments that go beyond the scope of the present work are needed to compare the biochemical characteristics of the enzyme found in the ROS with the other ADPR cyclases described up to now. 
We propose that a novel mammalian ROS ADPR-cyclase regulates the production of cADPR and therefore calcium levels within ROS. In the dark, the cGMP-gated channel mediates an inflow of Ca2+, keeping ROS cytosolic [Ca2+] relatively high. 44 During the excitation phase of the photoresponse, light stimulates an enzymatic cascade culminating in cGMP hydrolysis leading to the closure of light-sensitive plasma membrane channels, generating the nervous signal. 25 26 In resting bovine ROS cADPR would elicit a [Ca2+]-dependent Ca2+ release from discs, thanks to the relatively high (∼500 nM) 44 cytosolic dark-adapted ROS free Ca2+ concentration, which would keep RyRs in an activatable state. This hypothesis is in accordance with our results showing that the disc RyR channels are in an activated state over which desensitization to cADPR is partially ineffective (Fig. 3A) . The physiological role of the cADPR pool generated within the ROS in the dark would therefore be to implement Ca2+ release from discs in the dark, contributing to the maintenance of elevated free [Ca2+] in the dark. 
After light absorption, the decrease in internal [Ca2+] plays a key role in the recovery of photoreceptors through the regulation of guanylyl cyclases (GC1 and -2) catalyzing the conversion of GTP to cGMP. 25 26 [Ca2+] inside the photoreceptor was calculated to drop to the theoretical level of 10−10 M 53 ; however, experiments have shown that, after illumination, Ca2+ drops to approximately 140 nM. 45 Light was also thought to increase [Ca2+] initially. 54 Moreover, Schnetkamp 55 reported that the internal Ca2+ concentration is maintained at relatively high levels (83 nM) in isolated bovine ROS kept in Ca2+-free medium, by a combined inactivation of the Na+/Ca2+-K+ exchanger coupled with Ca2+release from discs. 55 It is tempting to presume that the cADPR-dependent calcium extrusion keeps the extradiscal Ca2+ decline less steep immediately after illumination, considering that, at 200 nM, RyR channels are still sensitive to cADPR (Fig. 3) . Ultimately, somewhere below this value it is reasonable to think that the fall in cytosolic ROS free [Ca2+] drives the RyRs to a nonresponsive state; otherwise Ca2+ would never reach the measured level of 83 to 140 nM. 
cGMP was found to stimulate cADPR synthesis by activating its synthetic enzyme through the cGMP-dependent protein kinase type I (cGKI). 56 Mammals have two cGKs: cGKI, expressed in some brain regions and in retina, 57 58 and cGKII, widely distributed in the mammalian brain. Fluctuations in cGMP concentration, higher in ROS than in any other vertebrate cell, 25 may further control ADPR-cyclase activity. cADPR has been reported to operate either as a second messenger in Ca2+ signaling through the activation of its synthetic enzyme, or as a RyR channel modulator, as in muscle cells. 2 In fact, in skeletal muscle sarcoplasmic reticulum cADPR is bound to RyR1, because basal ADPR-cyclase activity and Ca2+ ion dynamics cause the rapid switching on and off of the channels. Instead, according to our model, cADPR acts in ROS as a messenger. In conclusion, ROS discs would be Ca2+ stores operated by cADPR. 
 
Figure 1.
 
Morphologic analysis of osmotically intact ROS disc membranes. Osmotically intact disc suspensions were observed by TEM. The fraction mainly contained closed membrane vesicles, in which mitochondria, endoplasmic reticulum, melanosomes, and other cellular subfractions are absent. Because of elevated protein concentration, some membranes did not appear as vesicles but as more or less parallel membrane sheets. The length of the membrane profiles was ∼1 μm.
Figure 1.
 
Morphologic analysis of osmotically intact ROS disc membranes. Osmotically intact disc suspensions were observed by TEM. The fraction mainly contained closed membrane vesicles, in which mitochondria, endoplasmic reticulum, melanosomes, and other cellular subfractions are absent. Because of elevated protein concentration, some membranes did not appear as vesicles but as more or less parallel membrane sheets. The length of the membrane profiles was ∼1 μm.
Figure 2.
 
Western blot with anti Na/K-ATPase. The presence of Na/K-ATPase was determined by Western blot analysis with a mouse monoclonal antibody raised against rabbit Na/K-ATPase-α1. (B). Thirty micrograms protein was loaded in each lane for SDS-PAGE. (A) Ponceau-S staining of proteins after blot and dots of secondary antibody made for and overlay.
Figure 2.
 
Western blot with anti Na/K-ATPase. The presence of Na/K-ATPase was determined by Western blot analysis with a mouse monoclonal antibody raised against rabbit Na/K-ATPase-α1. (B). Thirty micrograms protein was loaded in each lane for SDS-PAGE. (A) Ponceau-S staining of proteins after blot and dots of secondary antibody made for and overlay.
Figure 3.
 
(A) cADPR evoked Ca2+ release from retinal rod disc vesicles. cADPR-induced Ca2+ release was performed on a suspension (2 mL) of osmotically intact bovine ROS discs (0.45 mg/mL). Ca2+ release was conducted at pH 7.2 in the presence of the Ca2+ tracer Fluo-3. The fluorometric trace from a typical experiment depicts an increase in free Ca2+ after addition of 0.2 picomoles CaCl2 and after two consecutive additions of cADPR (20 nmol each) to the disc suspension (arrows). (B) [Ca2+]-dependent Ca2+ release from retinal rod disc vesicles. A plot of cADPR-dependent peak Ca2+ release as a function of free extravesicular Ca2+ levels. Osmotically intact disc vesicles were incubated in the presence of the Ca2+ tracer fluo-3 in experiments similar to those in (A). Peak Ca2+ release data show a linear trend (R 2 = 0.85).
Figure 3.
 
(A) cADPR evoked Ca2+ release from retinal rod disc vesicles. cADPR-induced Ca2+ release was performed on a suspension (2 mL) of osmotically intact bovine ROS discs (0.45 mg/mL). Ca2+ release was conducted at pH 7.2 in the presence of the Ca2+ tracer Fluo-3. The fluorometric trace from a typical experiment depicts an increase in free Ca2+ after addition of 0.2 picomoles CaCl2 and after two consecutive additions of cADPR (20 nmol each) to the disc suspension (arrows). (B) [Ca2+]-dependent Ca2+ release from retinal rod disc vesicles. A plot of cADPR-dependent peak Ca2+ release as a function of free extravesicular Ca2+ levels. Osmotically intact disc vesicles were incubated in the presence of the Ca2+ tracer fluo-3 in experiments similar to those in (A). Peak Ca2+ release data show a linear trend (R 2 = 0.85).
Figure 4.
 
Assay of ADPR-cyclase activity in osmotically intact bovine ROS discs. The time course of generation of cGDPR by crude discs suspended with 1 mM NGD+ in the same reaction mixture as for the Ca2+ measurements (•). cGDPR production was assayed by spectrophotometric analysis of absorbance of neutralized supernatants at 300 nm wavelength. Optical densities were converted to rates of cGDPR production (ε = 3758 M−1· cm−1). A deproteinized sample showed no variation after NGD+ addition (▪). Each point represents the mean ± SD of results in five different experiments.
Figure 4.
 
Assay of ADPR-cyclase activity in osmotically intact bovine ROS discs. The time course of generation of cGDPR by crude discs suspended with 1 mM NGD+ in the same reaction mixture as for the Ca2+ measurements (•). cGDPR production was assayed by spectrophotometric analysis of absorbance of neutralized supernatants at 300 nm wavelength. Optical densities were converted to rates of cGDPR production (ε = 3758 M−1· cm−1). A deproteinized sample showed no variation after NGD+ addition (▪). Each point represents the mean ± SD of results in five different experiments.
Figure 5.
 
Detection of ADPR-cyclase activity on mildly denaturing SDS-PAGE. Shown are the fluorescent bands (inverted for clarity) corresponding to cyclic inosine diphosphate ribose locally produced by ADPR-cyclase activity, as visualized under UV light. Samples (60 μg/lane) were loaded onto a 12% polyacrylamide gel and run on a modified Laemmli SDS-PAGE. The gel was then incubated in the dark for 15 minutes with 0.2 mM NHD+. Lane 1: cytosolic ROS fraction; lane 2: crude discs; lane 3: osmotically intact discs. Apparent molecular mass of the enzyme was 53 ± 1 (average ± SD, n = 10).
Figure 5.
 
Detection of ADPR-cyclase activity on mildly denaturing SDS-PAGE. Shown are the fluorescent bands (inverted for clarity) corresponding to cyclic inosine diphosphate ribose locally produced by ADPR-cyclase activity, as visualized under UV light. Samples (60 μg/lane) were loaded onto a 12% polyacrylamide gel and run on a modified Laemmli SDS-PAGE. The gel was then incubated in the dark for 15 minutes with 0.2 mM NHD+. Lane 1: cytosolic ROS fraction; lane 2: crude discs; lane 3: osmotically intact discs. Apparent molecular mass of the enzyme was 53 ± 1 (average ± SD, n = 10).
Figure 6.
 
Localization of RyR channels with isoform-specific antibody on ROS discs. (A) Confocal fluorescence imaging of intact discs stained with a secondary anti-mouse antibody CY3 bound to a goat polyclonal antibody against RyR Ca2+ receptors. The resultant image was taken in the 512 × 512 format for a scanned area of 150 μm2. White box: a magnified view of a single disc. As shown by the spectral analysis (inset), elaborated from several spectral acquisitions within the 560- to 660-nm range with a wavelength resolution of 5 nm, the fluorescence intensity of the discs is due only to the red CY3 (plotted in B). (C, D) Western blot analysis. Samples (80 μg/lane) were separated by 7% SDS-PAGE, transferred to nitrocellulose membranes, and then probed with the same antibody as used in (A). Lane 1: mouse skeletal microsomes, used as positive control; lane 2: crude discs; lane 3: osmotically intact discs; lane 4: cytosolic ROS fraction. Antibody recognized a protein with the apparent mass of 500 ± 70 kDa (average ± SD; n = 3). (D) Colloidal Coomassie blue staining of proteins after the run in (C).
Figure 6.
 
Localization of RyR channels with isoform-specific antibody on ROS discs. (A) Confocal fluorescence imaging of intact discs stained with a secondary anti-mouse antibody CY3 bound to a goat polyclonal antibody against RyR Ca2+ receptors. The resultant image was taken in the 512 × 512 format for a scanned area of 150 μm2. White box: a magnified view of a single disc. As shown by the spectral analysis (inset), elaborated from several spectral acquisitions within the 560- to 660-nm range with a wavelength resolution of 5 nm, the fluorescence intensity of the discs is due only to the red CY3 (plotted in B). (C, D) Western blot analysis. Samples (80 μg/lane) were separated by 7% SDS-PAGE, transferred to nitrocellulose membranes, and then probed with the same antibody as used in (A). Lane 1: mouse skeletal microsomes, used as positive control; lane 2: crude discs; lane 3: osmotically intact discs; lane 4: cytosolic ROS fraction. Antibody recognized a protein with the apparent mass of 500 ± 70 kDa (average ± SD; n = 3). (D) Colloidal Coomassie blue staining of proteins after the run in (C).
The authors thank Carlo Tacchetti and Consuelo Venturi (University of Genova, Italy) for performing the TEM imaging. 
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Figure 1.
 
Morphologic analysis of osmotically intact ROS disc membranes. Osmotically intact disc suspensions were observed by TEM. The fraction mainly contained closed membrane vesicles, in which mitochondria, endoplasmic reticulum, melanosomes, and other cellular subfractions are absent. Because of elevated protein concentration, some membranes did not appear as vesicles but as more or less parallel membrane sheets. The length of the membrane profiles was ∼1 μm.
Figure 1.
 
Morphologic analysis of osmotically intact ROS disc membranes. Osmotically intact disc suspensions were observed by TEM. The fraction mainly contained closed membrane vesicles, in which mitochondria, endoplasmic reticulum, melanosomes, and other cellular subfractions are absent. Because of elevated protein concentration, some membranes did not appear as vesicles but as more or less parallel membrane sheets. The length of the membrane profiles was ∼1 μm.
Figure 2.
 
Western blot with anti Na/K-ATPase. The presence of Na/K-ATPase was determined by Western blot analysis with a mouse monoclonal antibody raised against rabbit Na/K-ATPase-α1. (B). Thirty micrograms protein was loaded in each lane for SDS-PAGE. (A) Ponceau-S staining of proteins after blot and dots of secondary antibody made for and overlay.
Figure 2.
 
Western blot with anti Na/K-ATPase. The presence of Na/K-ATPase was determined by Western blot analysis with a mouse monoclonal antibody raised against rabbit Na/K-ATPase-α1. (B). Thirty micrograms protein was loaded in each lane for SDS-PAGE. (A) Ponceau-S staining of proteins after blot and dots of secondary antibody made for and overlay.
Figure 3.
 
(A) cADPR evoked Ca2+ release from retinal rod disc vesicles. cADPR-induced Ca2+ release was performed on a suspension (2 mL) of osmotically intact bovine ROS discs (0.45 mg/mL). Ca2+ release was conducted at pH 7.2 in the presence of the Ca2+ tracer Fluo-3. The fluorometric trace from a typical experiment depicts an increase in free Ca2+ after addition of 0.2 picomoles CaCl2 and after two consecutive additions of cADPR (20 nmol each) to the disc suspension (arrows). (B) [Ca2+]-dependent Ca2+ release from retinal rod disc vesicles. A plot of cADPR-dependent peak Ca2+ release as a function of free extravesicular Ca2+ levels. Osmotically intact disc vesicles were incubated in the presence of the Ca2+ tracer fluo-3 in experiments similar to those in (A). Peak Ca2+ release data show a linear trend (R 2 = 0.85).
Figure 3.
 
(A) cADPR evoked Ca2+ release from retinal rod disc vesicles. cADPR-induced Ca2+ release was performed on a suspension (2 mL) of osmotically intact bovine ROS discs (0.45 mg/mL). Ca2+ release was conducted at pH 7.2 in the presence of the Ca2+ tracer Fluo-3. The fluorometric trace from a typical experiment depicts an increase in free Ca2+ after addition of 0.2 picomoles CaCl2 and after two consecutive additions of cADPR (20 nmol each) to the disc suspension (arrows). (B) [Ca2+]-dependent Ca2+ release from retinal rod disc vesicles. A plot of cADPR-dependent peak Ca2+ release as a function of free extravesicular Ca2+ levels. Osmotically intact disc vesicles were incubated in the presence of the Ca2+ tracer fluo-3 in experiments similar to those in (A). Peak Ca2+ release data show a linear trend (R 2 = 0.85).
Figure 4.
 
Assay of ADPR-cyclase activity in osmotically intact bovine ROS discs. The time course of generation of cGDPR by crude discs suspended with 1 mM NGD+ in the same reaction mixture as for the Ca2+ measurements (•). cGDPR production was assayed by spectrophotometric analysis of absorbance of neutralized supernatants at 300 nm wavelength. Optical densities were converted to rates of cGDPR production (ε = 3758 M−1· cm−1). A deproteinized sample showed no variation after NGD+ addition (▪). Each point represents the mean ± SD of results in five different experiments.
Figure 4.
 
Assay of ADPR-cyclase activity in osmotically intact bovine ROS discs. The time course of generation of cGDPR by crude discs suspended with 1 mM NGD+ in the same reaction mixture as for the Ca2+ measurements (•). cGDPR production was assayed by spectrophotometric analysis of absorbance of neutralized supernatants at 300 nm wavelength. Optical densities were converted to rates of cGDPR production (ε = 3758 M−1· cm−1). A deproteinized sample showed no variation after NGD+ addition (▪). Each point represents the mean ± SD of results in five different experiments.
Figure 5.
 
Detection of ADPR-cyclase activity on mildly denaturing SDS-PAGE. Shown are the fluorescent bands (inverted for clarity) corresponding to cyclic inosine diphosphate ribose locally produced by ADPR-cyclase activity, as visualized under UV light. Samples (60 μg/lane) were loaded onto a 12% polyacrylamide gel and run on a modified Laemmli SDS-PAGE. The gel was then incubated in the dark for 15 minutes with 0.2 mM NHD+. Lane 1: cytosolic ROS fraction; lane 2: crude discs; lane 3: osmotically intact discs. Apparent molecular mass of the enzyme was 53 ± 1 (average ± SD, n = 10).
Figure 5.
 
Detection of ADPR-cyclase activity on mildly denaturing SDS-PAGE. Shown are the fluorescent bands (inverted for clarity) corresponding to cyclic inosine diphosphate ribose locally produced by ADPR-cyclase activity, as visualized under UV light. Samples (60 μg/lane) were loaded onto a 12% polyacrylamide gel and run on a modified Laemmli SDS-PAGE. The gel was then incubated in the dark for 15 minutes with 0.2 mM NHD+. Lane 1: cytosolic ROS fraction; lane 2: crude discs; lane 3: osmotically intact discs. Apparent molecular mass of the enzyme was 53 ± 1 (average ± SD, n = 10).
Figure 6.
 
Localization of RyR channels with isoform-specific antibody on ROS discs. (A) Confocal fluorescence imaging of intact discs stained with a secondary anti-mouse antibody CY3 bound to a goat polyclonal antibody against RyR Ca2+ receptors. The resultant image was taken in the 512 × 512 format for a scanned area of 150 μm2. White box: a magnified view of a single disc. As shown by the spectral analysis (inset), elaborated from several spectral acquisitions within the 560- to 660-nm range with a wavelength resolution of 5 nm, the fluorescence intensity of the discs is due only to the red CY3 (plotted in B). (C, D) Western blot analysis. Samples (80 μg/lane) were separated by 7% SDS-PAGE, transferred to nitrocellulose membranes, and then probed with the same antibody as used in (A). Lane 1: mouse skeletal microsomes, used as positive control; lane 2: crude discs; lane 3: osmotically intact discs; lane 4: cytosolic ROS fraction. Antibody recognized a protein with the apparent mass of 500 ± 70 kDa (average ± SD; n = 3). (D) Colloidal Coomassie blue staining of proteins after the run in (C).
Figure 6.
 
Localization of RyR channels with isoform-specific antibody on ROS discs. (A) Confocal fluorescence imaging of intact discs stained with a secondary anti-mouse antibody CY3 bound to a goat polyclonal antibody against RyR Ca2+ receptors. The resultant image was taken in the 512 × 512 format for a scanned area of 150 μm2. White box: a magnified view of a single disc. As shown by the spectral analysis (inset), elaborated from several spectral acquisitions within the 560- to 660-nm range with a wavelength resolution of 5 nm, the fluorescence intensity of the discs is due only to the red CY3 (plotted in B). (C, D) Western blot analysis. Samples (80 μg/lane) were separated by 7% SDS-PAGE, transferred to nitrocellulose membranes, and then probed with the same antibody as used in (A). Lane 1: mouse skeletal microsomes, used as positive control; lane 2: crude discs; lane 3: osmotically intact discs; lane 4: cytosolic ROS fraction. Antibody recognized a protein with the apparent mass of 500 ± 70 kDa (average ± SD; n = 3). (D) Colloidal Coomassie blue staining of proteins after the run in (C).
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