Investigative Ophthalmology & Visual Science Cover Image for Volume 42, Issue 3
March 2001
Volume 42, Issue 3
Free
Immunology and Microbiology  |   March 2001
Mechanisms of Photoreceptor Cell Death in Cancer-Associated Retinopathy
Author Affiliations
  • Tadao Maeda
    From the Departments of Ophthalmology,
  • Akiko Maeda
    From the Departments of Ophthalmology,
  • Ikuyo Maruyama
    Department of Ophthalmology, Hirosaki University School of Medicine, Japan.
  • Kei-ichi Ogawa
    From the Departments of Ophthalmology,
  • Yoshio Kuroki
    Biochemistry (Section 1), and
  • Hiroeki Sahara
    Pathology (Section 1), Sapporo Medical University School of Medicine, Japan; and
  • Noriyuki Sato
    Pathology (Section 1), Sapporo Medical University School of Medicine, Japan; and
  • Hiroshi Ohguro
    Department of Ophthalmology, Hirosaki University School of Medicine, Japan.
Investigative Ophthalmology & Visual Science March 2001, Vol.42, 705-712. doi:
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Tadao Maeda, Akiko Maeda, Ikuyo Maruyama, Kei-ichi Ogawa, Yoshio Kuroki, Hiroeki Sahara, Noriyuki Sato, Hiroshi Ohguro; Mechanisms of Photoreceptor Cell Death in Cancer-Associated Retinopathy. Invest. Ophthalmol. Vis. Sci. 2001;42(3):705-712.

      Download citation file:


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

      ×
  • Supplements
Abstract

purpose. In a previous study, both recoverin and heat shock cognate protein (hsc) 70 were recognized as autoantigens by sera from patients with cancer-associated retinopathy (CAR), and retinal dysfunction similar to CAR was inducible by intravitreous injection of anti-recoverin and anti-hsc 70 antibodies to Lewis rat. The purpose of the present study was to elucidate the effects of these antibodies on retinal photoreceptor cell functions, the contribution of caspase during the photoreceptor degeneration, and the roles of aberrant expression of recoverin in tumor cells.

methods. As photoreceptor functions, rhodopsin phosphorylation using freshly prepared rod outer segments (ROS) and electroretinogram (ERG) were studied. Expression of recoverin in several kinds of tumors was examined by reverse transcription–polymerase chain reaction and Western blot analysis. The effects of recoverin on calcium-dependent protein phosphorylation were studied using the A549 lung adenocarcinoma cell line, which does not express recoverin.

results. Rhodopsin phosphorylation in bovine ROS was significantly promoted by the addition of anti-recoverin antibody. Similar effects on rhodopsin phosphorylation and ERG impairment were observed in rat eyes treated with anti-recoverin antibody. Co-injection of caspase inhibitors with anti-recoverin antibody inhibited ERG impairment and significantly suppressed the antibody-induced enhancement of rhodopsin phosphorylation. Aberrant expression of recoverin was found in 15 of 30 tumor tissues from patients with cancer without CAR. Profiles of calcium-dependent protein phosphorylation of cell lysate from A549 cells were modulated by the presence of purified recoverin.

conclusions. These observations suggest that anti-recoverin antibody is incorporated into rod photoreceptor cells and modulates rhodopsin phosphorylation, which in turn produces activation of caspase-dependent apoptotic pathways. Regarding antibody generation in CAR, a high incidence of aberrant expression of recoverin in cancer tissues is important, as suggested previously.

Cancer-associated retinopathy (CAR) is a paraneoplastic retinal photoreceptor degeneration found in patients with small-cell carcinoma of the lung and other malignant tumors. 1 2 3 4 5 6 7 Clinically, CAR is characterized by photopsia, progressive visual loss with ring scotoma, attenuated retinal arterioles, and abnormalities of the a- and b-waves of the ERG. 8 It has been suggested that CAR may be caused by autoimmune reactions to retinal antigens, including recoverin, 9 10 65-kDa heat shock cognate (hsc) protein 70, 6 11 12 48-kDa protein, 8 enolase, 13 and neurofilaments. 7 Among these antigens, recoverin has been most extensively studied for its pathologic association with CAR, and it has been shown that direct intravitreal administration of anti-recoverin antibody causes a decrease in electroretinogram (ERG) responses and apoptotic cell death within the retinal outer nuclear layers (ONLs). 14 In addition, our group revealed that this recoverin-induced retinal dysfunction is greatly enhanced by co-injection with anti-hsc 70 antibody. 14 Therefore, these observations suggest that anti-recoverin and anti-hsc 70 antibodies are involved together in the pathogenesis of CAR. Regarding anti-recoverin antibody generation, it has been suggested that the aberrant expression of recoverin in cancer cells is a critical step for triggering an autoimmune reaction that causes retinal degeneration. However, within the molecular pathology of CAR, we still do not know how these antibodies cause retinal photoreceptor cell death and what mechanism is involved in the antibody’s generation in patients with malignant tumors. 
In the present study, to further investigate the pathologic effects of anti-recoverin and anti-hsc70 antibodies in CAR, the function of recoverin was estimated by measuring levels of rhodopsin phosphorylation after preincubation of rod outer segment (ROS) homogenate with these antibodies, and by injecting these antibodies into the vitreous cavity of Lewis rats. Whether apoptotic cell death caused by these antibodies is caspase dependent was also examined using caspase inhibitors. In addition, to elucidate the effects of recoverin aberrantly expressed in cancer cells and to determine the percentage of aberrant expression of recoverin in malignant tumors, profiles were evaluated of calcium-dependent protein phosphorylation of cell lysate from the A549 lung adenocarcinoma cell line, which does not express recoverin, 15 and reverse transcription–polymerase chain reaction (RT-PCR) of recoverin was performed using several cancerous tissues. 
Materials and Methods
All experimental procedures were designed to conform to the ARVO Statement for Use of Animals in Ophthalmic and Vision Research and the tenets of the Declaration of Helsinki, as well as our own institution’s guidelines. Unless otherwise stated, all procedures were performed at 4°C or on ice using ice-cold solutions. Recoverin was purified from frozen bovine retinas, as described by Polans et al. 16 The A549 cell line, established from human lung adenocarcinoma, was purchased from the American Type Culture Collection (ATCC; Rockville, MD). This cell line was maintained in RPMI 1640 containing 10% fetal bovine serum and antibiotics. Tumor tissues from various organs (30 samples) were biopsy specimens and surgical materials obtained mainly at the Sapporo Medical University Hospital. The donors of these tumors had no history of visual symptoms corresponding with those of CAR, and none of them had serum antibody against recoverin. As a normal tissue control, lymphocytes from four healthy volunteers were used. 
Antibodies
Preparation of anti-recoverin serum and affinity purification of anti-recoverin IgG using protein G Sepharose column chromatography were performed as described previously. 14 As a control, preimmune rabbit serum was also subjected to the IgG purification. Anti-hsc 70 serum was purchased from StressGen (Sidney, British Columbia, Canada). The specificity and titers examined by Western blot analysis using a bovine retina soluble fraction were demonstrated in our recent study. 15 All antibodies were diluted with phosphate-buffered saline (PBS) to adjust the IgG concentration to 1 mg/ml. 
Vitreous Injection of Antibodies and Caspase Inhibitors to Lewis Rats
Six-week-old Lewis rats (approximately 180 g) reared in cyclic light conditions (12 hours on; 12 hours off) were used. Rats were anesthetized by intramuscular injection of a mixture of ketamine (80–125 mg/kg) and xylazine (9–12 mg/kg), as described in our previous study. 14 In rats under anesthesia, a 5- to 10-μl PBS solution containing preimmune IgG, anti-recoverin IgG, and/or anti-hsc 70 serum was injected into the vitreous cavity of the rat eye, as described previously. 14 The caspase inhibitors, Z-Val-Ala-Asp (OMe)-fluoromethylketone (Z-VAD-FMK) and Z-Asp (OMe)-Glu (OMe)-Val-Asp (OMe)-fluoromethyl ketone (Z-DEVD-FMK), were purchased from Enzyme Systems Products (Livermore, CA). A 20-mM solution of these inhibitors was prepared in dimethyl sulfoxide, and 1μ l of the solution was co-injected with antibodies. Animals showing apparent traumatic changes after vitreous injection, such as cataract, vitreous hemorrhage, and retinal detachment were excluded from the present study. After the surgery, a drop of 0.05% ofloxacin was administered to avoid infection. 
Electroretinography
ERG measurements were performed in rats, as described previously. 14 Briefly, the anesthetized animals were kept in dark adaptation for at least 1 hour in an electrically shielded room. The pupils were dilated with drops of 0.5% tropicamide. The scotopic ERG response was recorded with a contact electrode equipped with a suction apparatus to fit on the cornea (Kyoto Contact Lens, Kyoto, Japan). A grounding electrode was placed on the ear. Responses evoked by white flashes (3.5 × 102 lux, 200-msec duration) were recorded by an evoked potential measuring system (Neuropack MES-3102; Nihon Kohden, Tokyo, Japan). 
Rhodopsin Phosphorylation
Rhodopsin phosphorylation was studied as a retinal photoreceptor function, by using freshly prepared bovine ROS membranes, as described previously. 17 Briefly, ROS homogenate (containing rhodopsin at a final concentration of 2 mg/ml) was preincubated in 300μ l of 100 mM Na-phosphate buffer (pH 7.2) containing 5 mM MgCl2 in the presence of either 0.1 mM CaCl2 or 1 mM EGTA and antibody (anti-recoverin IgG or rabbit serum IgG, 50 μg per rat) on ice for 1 hour in the dark. After preincubation, 0.5 mM [γ-32P] adenosine triphosphate (ATP; 300 counts per minute per nanomole[ cpm/nanomole]) was added to the mixture, and rhodopsin phosphorylation was performed at 30°C in the dark for 10 minutes after illumination by a 150-W lamp for 1 second from a distance of 20 cm. After incubation, the reaction was terminated by the addition of 10% trichloroacetic acid (TCA), after which the mixture was washed with fresh 10% TCA three times, and radioactivity was counted using a scintillation cocktail. 
Rhodopsin phosphorylation in rat eyes was also studied using freshly isolated ROS of rat retinas, as described previously, 17 with some modifications. Briefly, after dark adaptation of enucleated eyeballs (four to six eyes for each condition) for 1 hour on ice, retinas were dissected and homogenized in 1 ml 45% sucrose in buffer A (100 mM NaPi buffer, [pH 7.2] containing 5 mM MgCl2 and 100 mM potassium fluoride[ KF]). After centrifugation at 13,000 rpm for 5 minutes, the supernatant was diluted twice with buffer A and centrifuged again at 13,000 rpm for 5 minutes. The pellet was dissolved in 200 μl of buffer A containing 0.5 mM [γ-32P] ATP (300 cpm/nanomole) and incubated at 30°C for 5 minutes under a 150-W lamp from a distance of 20 cm in the presence of either 0.1 mM CaCl2 or 1 mM EGTA. The reaction was terminated by addition of 200 mM NaPi buffer B (pH 7.2), containing 5 mM adenosine, 100 mM KF, 200 mM KCl, and 200 mM EDTA, and centrifuged at 13,000 rpm for 5 minutes. The pellet was dissolved in 50 μl of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and analyzed by SDS-PAGE using 12.5% gel. The gels were stained with Coomassie blue and dried, followed by performance of an autoradiogram. The band corresponding with rhodopsin was cut out and dissolved with 0.5 ml of H2O2, and radioactivities were counted in scintillation cocktail. 
In Vitro Protein Phosphorylation of A549 Cell Lysate
A549 cells (1 × 106) derived from lung adenocarcinoma were suspended in 1 ml of 10% sucrose containing 1 mM benzamidine and passed through a 250-μl microsyringe (Hamilton, Reno, NV) 10 times on ice. The suspension was centrifuged at 700g to remove the nucleus, and the supernatant was collected for phosphorylation experiments. The reaction mixture (400μ l) was composed of buffer A, [γ-32P] ATP (0.5 mM, 300 cpm/nanomole), purified recoverin (0 or 50 μg/ml), and either CaCl2 (0 or 0.1 mM) or EGTA (0 or 1 mM). The phosphorylation reaction was performed on ice for 20 minutes and was quenched by addition of buffer B. Phosphorylated and nonphosphorylated proteins were precipitated by the chloroform-methanol method 18 and subjected to SDS-PAGE followed by autoradiography. 
RT-PCR Analysis
Total RNA from tumor tissues was isolated using a reagent (Isogen; Nippon Gene, Tokyo, Japan), according to the procedure described by the manufacturer, and was reverse-transcribed, by using reverse transcriptase with oligo (dT) primer (Superscript II; Gibco–Life Technologies, Rockville, MD). The incubation was performed at 42°C for 50 minutes and at 70°C for 15 minutes. The PCR amplifications were performed using 4.4 μl for recoverin or 2.2μ l for β-actin from the RT reaction mixture in 50 μl of PCR mixture containing 50 picomoles of sense and antisense primers. After the initial incubation at 94°C for 4 minutes, 30 cycles of amplification were conducted with denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute, and extension at 72°C for 2 minutes. The following primer pairs were used for RT-PCR analysis: 5′-TGTGTTCCGCAGCTTCGATT-3′ as the sense primer and 5′-TGAGGCTCAACTAACTGGATCAG-3′ as the antisense primer for recoverin, with an expected PCR product of 369 bp; and 5′-CTGTCTGGCGGCACCACCAT-3′ as the sense primer and 5′-GCAACTAAGTCATAGTCCGC-3′ as the antisense primer for β-actin, with an expected PCR product of 254 bp. The amplified PCR products were electrophoresed on a 1.5% agarose gel containing ethidium bromide, and densitometric analysis of the bands was performed (Epi-Light UVF500 densitometer; Aisin Cosmos R&D, Tokyo, Japan). 
To confirm the identity of the bands, the PCR product for recoverin was cloned into a vector with a TA cloning kit (pCRII; Invitrogen, Carlsbad, CA). The nucleotide sequences of the clones were determined by using a kit (ABI Genetically analyzed PRIM 310; AmpliCycle sequencing kit; Perkin Elmer–Applied Biosystems, Foster City, CA). 
Aberrant expression of recoverin in several cancerous tissues was also confirmed by Western blot analysis. Briefly, approximately 0.1 g of tumor was dissolved in 1 ml of 100 mM Tris-HCl buffer (pH 8.0), containing 0.1% SDS and 5 mM 2-mercaptoethanol, and the mixture was sonicated. After centrifugation at 13,000 rpm for 5 minutes, the supernatant was subjected to Western blot analysis, by using affinity purified anti-recoverin antibody (1:2000 dilutions), as described previously. 12  
Western Blot Analysis
Western blot analysis was performed as described previously. 15 Briefly, the protein fraction isolated by the reagent (Isogen; Nippon Gene) according to the manufacturer’s procedure was analyzed by SDS-PAGE), using a 12.5% polyacrylamide gel. Separated proteins in a gel were electrotransferred to polyvinylidene (PVDF) membranes in 10 mM bis-tris phosphate buffer (pH 8.4), containing 10% methanol. After blocking with 5% skim milk in PBS, the membrane was probed successively with the anti-peptide antibody and horseradish peroxidase (HRP)–labeled anti-rabbit IgG (Funakoshi, Tokyo, Japan). Immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Pharmacia, Buckinghamshire, UK) according to the method described by the manufacturer. 
Statistical Analysis
The data are shown as mean ± SD. P < 0.05 was considered significantly different, as assessed by Student’s t-test. 
Results
To elucidate the molecular mechanisms causing apoptosis of retinal photoreceptors by anti-recoverin antibody, the effects were examined of the anti-recoverin antibody on light-dependent rhodopsin phosphorylation in anti-recoverin antibody–treated rat eyes. As a control, freshly isolated bovine ROS homogenates were preincubated with anti-recoverin antibody for 1 hour in the dark on ice in the presence or absence of Ca2+. Thereafter, 0.5 mM[γ -32P] ATP was added to the mixture and incubated at 30°C for 10 minutes in the dark after exposure to a flash. After incubation, the phosphorylation reaction was terminated by addition of 10% TCA, and the mixture washed with fresh 10% TCA followed by scintillation counting. As shown in Figure 1 , rhodopsin phosphorylation levels were suppressed by the addition of Ca2+ in the presence of preimmune rabbit IgG (condition A). Anti-hsc 70 antibody had no effects on rhodopsin phosphorylation (condition B). In contrast, such Ca2+-dependent suppression of rhodopsin phosphorylation was abolished and the level of rhodopsin phosphorylation was significantly enhanced by the presence of anti-recoverin IgG (condition C). These effects of anti-recoverin IgG were considered likely to have resulted from a substantial inhibition of the function of recoverin. 
Next, ROS phosphorylation of anti-recoverin antibody–treated rat eyes was examined. In our previous study, we found that anti-recoverin antibody was incorporated within the inner parts of the retina at 3 hours, and then antibody localization shifted toward the outer parts of the retina at 12 hours after administration. Between 12 and 24 hours, the antibody accumulated within both the ONL and photoreceptor layer, and thereafter the antibody disappeared slowly from the retina within 6 days. 16 Therefore, eyes were enucleated 36 hours after administration of either preimmune rabbit IgG or anti-recoverin antibody and incubated on ice for 1 hour in the dark followed by separation of ROS by the sucrose gradient centrifugation method. ROS was then homogenized and incubated with 0.5 mM[γ -32P] ATP in 100 mM Na-phosphate buffer (pH 7.2) containing 5 mM MgCl2 in the presence or absence of Ca2+ for 10 minutes at 30°C under 150-W lamp illumination. Phosphorylated ROS was analyzed by SDS-PAGE in which the radioactivity of the corresponding rhodopsin band was counted (Fig. 2A ). Intravitreal treatment with anti-recoverin antibody did not affect the protein concentrations of rhodopsin, rhodopsin kinase, and other proteins (Fig. 2A) . However, rhodopsin phosphorylation levels of anti-recoverin IgG–treated retinas were significantly higher than those of control in the presence of Ca2+, in both the 36-hour (Fig 2B left, open columns) and 3-week preparations (Fig. 2B left, shaded columns; conditions A and D). This enhancement of rhodopsin phosphorylation by anti-recoverin antibody was not significantly changed by the co-injection of anti-hsc 70 serum (Fig. 2B , left panel, condition E). Intravitreous injection of anti-hsc 70 serum alone had no effect on rhodopsin phosphorylation (Fig. 2B , left panel, condition C). To exclude the possibility that nonincorporated anti-recoverin antibody outside the cell affected rhodopsin phosphorylation, retinas dissected from eyes intravitreously injected with PBS were mixed with anti-recoverin antibody in the ROS preparation and subjected to rhodopsin phosphorylation analysis (Fig. 2B , condition B). No effects of the presence of anti-recoverin IgG on the levels of rhodopsin phosphorylation were found in the ROS preparation. 
It has been suggested that an apoptotic cell death process is involved in the anti-recoverin antibody–induced retinal dysfunction. To clarify whether this process is caspase dependent, caspase inhibitors were co-injected with anti-recoverin antibody, and retinal functions were evaluated by ERG and rhodopsin phosphorylation. Enhancement of rhodopsin phosphorylation by anti-recoverin antibody was markedly suppressed by the co-injection of caspase inhibitors, Z-DEVD-FMK and Z-VAD-FMK (Fig. 2B , conditions F and G) in both the 36-hour and 3-week preparations. In experiments using bovine ROS, these caspase inhibitors did not affect the levels of rhodopsin phosphorylation (Fig. 1 , conditions D and E). In ERG measurements, the caspase inhibitors also suppressed anti-recoverin antibody–induced reduction (Fig. 2B , right). 
To understand the molecular pathology of the onset of CAR, we studied the mechanisms of the aberrant expression of recoverin in various cancer tissues. mRNA expression of recoverin was examined by RT-PCR in carcinoma tissues from various organs, including lung small-cell carcinoma, lung adenocarcinoma, gastric cancer, breast cancer, colon cancer, ovarian cancer, uterine cancer, embryonic cancer, malignant melanoma, or leukemia. As shown in Figures 3 and 4 , mRNA expression and immunoreactivity of recoverin were recognized in 15 of 30 tumor tissues, and sequences of the PCR products corresponded to those from human recoverin (data not shown). 
We next examined whether recoverin might have some particular physiological role in the cancer cells beside that of an immunogenic autoantigen. Recoverin has been found to play an important role in the regulation of light-dependent rhodopsin phosphorylation within the photoreceptor cells. 19 20 In view of these observations, we speculated that recoverin might be involved in the regulation of protein phosphorylation in cancer cells. To test our hypothesis, we studied protein phosphorylation, by using A549 cells derived from lung adenocarcinoma, which do not express recoverin. 15 We then incubated postnuclear supernatant with 1 mM [γ-32P] ATP in the presence or absence of purified recoverin. In the absence of recoverin, protein phosphorylation was significantly enhanced in a calcium-dependent manner (Fig. 5) . However, profiles of protein phosphorylation determined by autoradiograms in the presence or absence of calcium were markedly altered by adding the purified recoverin. These observations suggest that recoverin may modulate some pathways in calcium signaling of cancer cells. 
Discussion
Many investigators agree that anti-recoverin antibody plays a major role in the molecular origin of CAR, causing retinal photoreceptor degeneration. Adamus et al. 21 reported that anti-recoverin antibody induced apoptotic cell death in a retinal cell culture system and that intravitreous administration of a monoclonal antibody against recoverin induced apoptosis of ONL detected by TdT-dUTP terminal nick-end labeling (TUNEL) assay, DNA fragmentation, and electron microscopic features in Lewis rat eyes in vivo. 22 In addition, our group independently reported that intravitreous administration of anti-recoverin antibody induced a decrease in ERG amplitudes and apoptotic cell death in the ONL in Lewis rat eyes and that these effects were significantly promoted by the presence of anti-hsc 70 antibody. 14 However, we still do not know how anti-recoverin antibody binds with the target molecules and causes cell death by apoptotic processes, because recoverin is known to be present within the cytosol. With regard to antibody internalization, much experimental evidence has been reported in other paraneoplastic disorders 23 and autoimmune diseases. 24 25 26 Furthermore, Adamus et al. 22 identified internalization of anti-recoverin antibody into retinal cells in vitro resulting in apoptotic cell death. If this is possible, anti-recoverin antibody must block the function of recoverin, which regulates rhodopsin phosphorylation in a Ca2+-dependent manner. 19 20 In the present study, we found that levels of rhodopsin phosphorylation were significantly enhanced by the intravitreous administration of the anti-recoverin antibody. This result corresponded with that obtained by the experiment using freshly isolated bovine ROS homogenate. It remains to be clarified whether these changes in rhodopsin phosphorylation after the internalization of the anti-recoverin antibody into photoreceptor cells is related to an apoptotic process effected through the caspase-dependent pathway. 
In the present study, we found that enhancement of rhodopsin phosphorylation in rat eyes treated by anti-recoverin antibody was greatly reduced by the presence of Z-DEVD-FMK and Z-VAD-FMK, which are used as specific inhibitors for caspases 1, 3, and 4 and caspases 3, 6, 7, 8,and 10, respectively. However, these peptide inhibitors did not in themselves have any effect on rhodopsin phosphorylation in bovine ROS homogenate. Therefore, these observations suggest that these caspase inhibitors may have a secondary effect on rhodopsin phosphorylation. Although the exact mechanisms of the inhibitors’ effect on rhodopsin phosphorylation are unclear, we speculate that a caspase-dependent apoptotic process may facilitate the antibody’s internalization in photoreceptor cells. As another possibility, we also speculate that caspase-dependent apoptosis may facilitate destruction of specific proteins including recoverin. In fact, we observed significantly high levels of rhodopsin phosphorylation 3 weeks after administration of the antibody, although it was thought that the antibody probably would have been abolished by then. In addition, these inhibitors effectively improved ERG responses induced by intravitreous administration of anti-recoverin antibody. So far, there are at least 14 known caspases, and most of them are involved in the process of apoptosis. 27 Recently, Katai et al. 28 reported that caspases 1 and 2 and caspases 1 and 3 were central in the apoptotic cell death process of retinal cells found in Royal College of Surgeons (RCS) rats 28 and ischemia–reperfusion models, 29 respectively. It was also suggested that similar caspase-dependent roles were involved in the apoptosis of rat retina induced by intravitreous injection of N-methyl-d-aspartate (NMDA) 30 or anti-heat shock protein (hsp) 27 antibody as a glaucoma model. 31 Therefore, our present data strongly suggest that a similar caspase pathway, as mentioned earlier, was involved in our rat model of CAR, although we do not know which caspase proteases are involved after the internalization of anti-recoverin antibody in the photoreceptors. 
Regarding the pathologic effects of anti-hsc 70 antibody, we suggest that it internalizes into photoreceptor cells and blocks biologic protection by chaperon functions of hsc 70 to suppress protein aggregation, denaturation, and misfolding under several stress conditions, and this mechanism may promote anti-recoverin antibody–mediated retinal degeneration. 12 This speculation was supported by a previous finding showing that vitreous injection of anti-hsc 70 antibody does not induce the ERG responses but significantly enhances the changes in the responses that are induced by the anti-recoverin antibody, 14 and by present data revealing that intravitreal administration of anti-hsc 70 antibody did not effect rhodopsin phosphorylation. 
Other important questions are what mechanisms are involved in the antibody generation in CAR, and what are the physiological roles of recoverin in cancer cells? Regarding the generation of autoantibody toward recoverin, it was identified that recoverin is aberrantly expressed in the cancer cells or their cell lines obtained from patients with CAR, and this may trigger the autoimmune reaction. 32 33 34 Preliminary studies have revealed that such aberrant expression of retina-specific recoverin is not identified in cancer cells without retinopathy. These observations suggest that aberrant expression of recoverin in cancer cells is an initial and critical step in the cause of retinopathy. However, in contrast, we found aberrant expression of recoverin in approximately 50% of cancer tissues from patients who had cancer without CAR. This rate of aberrant expression of recoverin in cancer tissues is almost identical with that reported recently using several types of cancer cell lines. 15 Therefore, unknown mechanisms additional to the aberrant expression of recoverin must be involved in the antibody generation in CAR. 
The reactions caused by aberrantly expressed recoverin in cancer cells are presently unknown. However, the functional role of recoverin (regulation of rhodopsin phosphorylation in a calcium-dependent manner in photoreceptor cells) allowed us to speculate that recoverin may effect calcium-dependent protein phosphorylation in cancer cells. In the present study, protein phosphorylation patterns were modulated by the presence of recoverin in a calcium-dependent manner. We do not know the mechanisms causing the changes in protein phosphorylation by recoverin in cancer cells. However, we think that recoverin may regulate some G-protein–coupled receptor kinases in a calcium-dependent manner. In fact, it has been found that calcium-binding proteins belong to the neuronal calcium sensor (NCS) gene family, including such genes as S-modulin, neurocalcin hippocalcin frequenin, vilip1, vilip2, vilip3, visinin, HLP2, and NCS-1, which share functional and structural homologies with recoverin and are widely distributed within the nervous system. 35 These members in the family were identified to regulate rhodopsin phosphorylation in a calcium-dependent manner, suggesting that they may function to regulate the phosphorylation of G-protein–coupled receptors. 35 In addition, we also found a significant reduction in cell proliferation of A549 cells after transfection of human recoverin cDNA. 15 Therefore, all evidence taken together, we speculate that aberrantly expressed recoverin may play a role in a calcium-signaling pathway in cancer cells. 
In conclusion, based on the current observations, we propose the molecular pathologic mechanisms of retinal photoreceptor degeneration in CAR shown in Figure 6 . First, recoverin aberrantly expressed in cancerous tissues is recognized by immunocytes by some unknown mechanism, and specific antibody toward recoverin is produced. Second, the anti-recoverin antibody reaches the retina through the peripheral circulation and is taken up into photoreceptor cells. Last, the antibody blocks recoverin function, and enhancement of rhodopsin phosphorylation induces retinal apoptosis. 
 
Figure 1.
 
Effects of anti-recoverin and anti-hsc 70 antibodies or caspase inhibitors on rhodopsin phosphorylation in bovine ROS homogenate. Freshly isolated bovine ROS homogenate was preincubated with antibody, 5 μg preimmune rabbit IgG (A), anti-hsc 70 serum (containing 5 μg IgG) (B), 5 μg anti-recoverin IgG (C), and 1 mM caspase inhibitors, Z-VAD-FMK (D) or Z-DEVD-FMK (E). Rhodopsin phosphorylation was then performed by addition of 0.5 mM [γ-32P] ATP in the presence of 0.1 mM CaCl2 (open bars) or 1 mM EGTA (shaded bars) under flash illumination. The reaction was terminated by addition of 10% TCA and the radioactivity was counted in scintillation cocktail, and the radioactivities were plotted. Experiments were performed in triplicate.
Figure 1.
 
Effects of anti-recoverin and anti-hsc 70 antibodies or caspase inhibitors on rhodopsin phosphorylation in bovine ROS homogenate. Freshly isolated bovine ROS homogenate was preincubated with antibody, 5 μg preimmune rabbit IgG (A), anti-hsc 70 serum (containing 5 μg IgG) (B), 5 μg anti-recoverin IgG (C), and 1 mM caspase inhibitors, Z-VAD-FMK (D) or Z-DEVD-FMK (E). Rhodopsin phosphorylation was then performed by addition of 0.5 mM [γ-32P] ATP in the presence of 0.1 mM CaCl2 (open bars) or 1 mM EGTA (shaded bars) under flash illumination. The reaction was terminated by addition of 10% TCA and the radioactivity was counted in scintillation cocktail, and the radioactivities were plotted. Experiments were performed in triplicate.
Figure 2.
 
Effects of antibodies and caspase inhibitors on rhodopsin phosphorylation and ERG in rat eyes. (A) Preimmune IgG (5μ g; lane A), PBS (lane B), anti-hsc 70 serum (5μ g IgG; lane C), 5 μg anti-recoverin IgG (lane D), or anti-recoverin IgG and anti-hsc 70 serum (5 μg IgG each) without (lane E) or with (lanes F, G) caspase inhibitors was injected intravitreously into the eyes of Lewis rats. (B) Thirty-six hours (open bars) or 3 weeks (shaded bars) after administration, ROS was prepared, and light dependent phosphorylation by [γ-32P] ATP (left) and ERG measurement (right) were examined. In condition B, 5 μg anti-recoverin IgG was mixed with fresh dissected retinas followed by the ROS preparation. After the reaction, samples were analyzed by SDS-PAGE (A, top) followed by autoradiogram (A, middle) or Western blot analysis using anti-rhodopsin kinase antibody 36 (1:3000 dilution; A, bottom). The radioactivity of rhodopsin bands in SDS-PAGE was counted in a scintillation cocktail, and the radioactivities were plotted (B, left). Experiments were performed in triplicate. ERG measurements were performed in 10 eyes in each condition, and the amplitudes of b-wave were plotted (B, right).
Figure 2.
 
Effects of antibodies and caspase inhibitors on rhodopsin phosphorylation and ERG in rat eyes. (A) Preimmune IgG (5μ g; lane A), PBS (lane B), anti-hsc 70 serum (5μ g IgG; lane C), 5 μg anti-recoverin IgG (lane D), or anti-recoverin IgG and anti-hsc 70 serum (5 μg IgG each) without (lane E) or with (lanes F, G) caspase inhibitors was injected intravitreously into the eyes of Lewis rats. (B) Thirty-six hours (open bars) or 3 weeks (shaded bars) after administration, ROS was prepared, and light dependent phosphorylation by [γ-32P] ATP (left) and ERG measurement (right) were examined. In condition B, 5 μg anti-recoverin IgG was mixed with fresh dissected retinas followed by the ROS preparation. After the reaction, samples were analyzed by SDS-PAGE (A, top) followed by autoradiogram (A, middle) or Western blot analysis using anti-rhodopsin kinase antibody 36 (1:3000 dilution; A, bottom). The radioactivity of rhodopsin bands in SDS-PAGE was counted in a scintillation cocktail, and the radioactivities were plotted (B, left). Experiments were performed in triplicate. ERG measurements were performed in 10 eyes in each condition, and the amplitudes of b-wave were plotted (B, right).
Figure 3.
 
Expression of mRNA for recoverin in various tumor tissues. RNA (2 μg) from 30 tumor tissues from various organs and 4 normal lymphocyte samples from healthy volunteers were reverse-transcribed to generate cDNA pools, and 4.5 μl from a 22-μl cDNA pool was used for PCR using specific primers. PCR products were evaluated by agarose gel electrophoresis and ethidium bromide staining.
Figure 3.
 
Expression of mRNA for recoverin in various tumor tissues. RNA (2 μg) from 30 tumor tissues from various organs and 4 normal lymphocyte samples from healthy volunteers were reverse-transcribed to generate cDNA pools, and 4.5 μl from a 22-μl cDNA pool was used for PCR using specific primers. PCR products were evaluated by agarose gel electrophoresis and ethidium bromide staining.
Figure 4.
 
Aberrant expression of recoverin in various tumor tissues by Western blot analysis. Tumor tissues with or without mRNA expression of recoverin were confirmed by Western blots, by using a specific antibody toward recoverin. The numbered lanes correspond with diseases and lymphocytes as shown in Figure 3 . rec, purified bovine recoverin.
Figure 4.
 
Aberrant expression of recoverin in various tumor tissues by Western blot analysis. Tumor tissues with or without mRNA expression of recoverin were confirmed by Western blots, by using a specific antibody toward recoverin. The numbered lanes correspond with diseases and lymphocytes as shown in Figure 3 . rec, purified bovine recoverin.
Figure 5.
 
In vitro protein phosphorylation of A549 cells in the presence or absence of recoverin. A549 cell lysate was composed of buffer A, containing [γ-32P] ATP, purified recoverin (0 or 50μ g/ml), CaCl2 (0 or 0.1 mM), and EGTA (0 or 1 mM). The phosphorylation reaction was performed on ice for 20 minutes and thereafter was quenched by addition of 200 mM NaPi buffer (pH 7.2) containing 5 mM adenosine, 100 mM KF, 200 mM KCl, and 200 mM EDTA. Proteins were precipitated by the chloroform-methanol method and subjected to SDS-PAGE followed by autoradiography.
Figure 5.
 
In vitro protein phosphorylation of A549 cells in the presence or absence of recoverin. A549 cell lysate was composed of buffer A, containing [γ-32P] ATP, purified recoverin (0 or 50μ g/ml), CaCl2 (0 or 0.1 mM), and EGTA (0 or 1 mM). The phosphorylation reaction was performed on ice for 20 minutes and thereafter was quenched by addition of 200 mM NaPi buffer (pH 7.2) containing 5 mM adenosine, 100 mM KF, 200 mM KCl, and 200 mM EDTA. Proteins were precipitated by the chloroform-methanol method and subjected to SDS-PAGE followed by autoradiography.
Figure 6.
 
Presumable molecular mechanisms of retinal photoreceptor degeneration in CAR. R, rhodopsin; rec, recoverin; RK, rhodopsin kinase; p, phosphoric acid; ATP, adenosine 5′-triphosphate; ADP, adenosine 5′-diphosphate; APC, antigen-presenting cell.
Figure 6.
 
Presumable molecular mechanisms of retinal photoreceptor degeneration in CAR. R, rhodopsin; rec, recoverin; RK, rhodopsin kinase; p, phosphoric acid; ATP, adenosine 5′-triphosphate; ADP, adenosine 5′-diphosphate; APC, antigen-presenting cell.
Sawyer RA, Selhorse JB, Zimmerman LE. Blindness caused by photoreceptor degeneration as a remote effect of cancer. Am J Ophthalmol. 1976;90:606–613.
Kornguth SE, Klein R, Appen R, Choate J. Occurrence of anti-retinal ganglion cell antibodies in patients with small cell cancer of the lung. Cancer. 1982;50:1289–1293. [CrossRef] [PubMed]
Keltner JL, Roth AM, Chang RS. Photoreceptor degeneration: possible autoimmune disorder. Arch Ophthalmol. 1983;101:564–569. [CrossRef] [PubMed]
Buchanan TAS, Gardiner TA, Archer DB. An ultrastructural study of retinal photoreceptor degeneration associated with bronchial cancer. Am J Ophthalmol. 1984;97:277–287. [CrossRef] [PubMed]
Klingele TG, Burde RM, Rappazzo JA, Isserman MJ, Burgess D, Kantor O. Paraneoplastic retinopathy. J Clin Neurol Ophthalmol. 1984;4:239–245.
Grunwald GB, Klein R, Simmonds MA, Kornguth SE. Autoimmune basis for visual paraneoplastic syndrome in patients with small-cell cancer. Lancet. 1985;1(8430)658–661. [PubMed]
Korngruth SE, Kalinke T, Grunwald GB, Schutta H, Dahl D. Anti-neurofilament antibodies in the sera of patients with small cell carcinoma of the lung and with visual paraneoplastic syndrome. Cancer Res. 1986;46:2588–2595. [PubMed]
Jacobson DM, Thirkill CE, Tipping SJ. A clinical triad to diagnose paraneoplastic retinopathy. Ann Neurol. 1990;28:162–167. [CrossRef] [PubMed]
Thirkill CE, Roth AM, Keltner JL. Cancer-associated retinopathy. Arch Ophthalmol. 1987;105:372–375. [CrossRef] [PubMed]
Crofts JW, Bachynski BN, Odel JG. Visual paraneoplastic syndrome associated with undifferentiated endometrial carcinoma. Can J Ophthalmol. 1988;23:128–132. [PubMed]
Thirkill CE, FitzGerald P, Sergott RC, Roth AM, Tyler NK, Keltner JL. Cancer-associated retinopathy (CAR) with autoantibodies reacting with retinal optic nerve, cancer cells. N Engl J Med. 1989;321:1589–1594. [CrossRef] [PubMed]
Ohguro H, Ogawa K, Nakagawa T. Recoverin and hsc 70 are found as autoantigens in patients with cancer-associated retinopathy. Invest Ophthalmol Vis Sci. 1999;40:82–89. [PubMed]
Adamus G, Aptsiauri N, Guy J, Heckenlively J, Flannery J, Hargrave PA. The occurrence of serum autoantibodies against enolase in cancer-associated retinopathy. Clin Immunol Immunopathol. 1996;78:120–129. [CrossRef] [PubMed]
Ohguro H, Ogawa K, Maeda T, Maeda A, Maruyama I. Cancer-associated retinopathy induced by both anti-recoverin and anti-hsc 70 antibodies in vivo. Invest Ophthalmol Vis Sci. 1999;40:3160–3167. [PubMed]
Maeda A, Ohguro H, Maeda T, et al. Aberrant expression of photoreceptor-specific calcium-binding protein (recoverin) in cancer cell lines. Cancer Res. 2000;60:1914–1920. [PubMed]
Polans AS, Crabb J, Palczewski K. Calcium-binding proteins in the retina. Methods Neurosci. 1993;15:248–260.
Ohguro H, Johnson RS, Ericsson LH, Walsh KA, Palczewski K. Control of rhodopsin multiple phosphorylation. Biochemistry. 1994;33:1023–1028. [CrossRef] [PubMed]
Wessel D, Flugge UI. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem. 1984;138:141–143. [CrossRef] [PubMed]
Kawamura S. Rhodopsin phosphorylation as a mechanism of cyclic GMP phosphodiesterase regulation by S-modulin. Nature. 1991;62:855–857.
Ohguro H, Rudnicka–Nawrot M, Buczylko J, et al. Structural and enzymatic aspects of rhodopsin phosphorylation. J Biol Chem. 1996;271:5215–5224. [CrossRef] [PubMed]
Adamus G, Machnicki M, Seigel GM. Apoptotic retinal cell death induced by antirecoverin autoantibodies of cancer-associated retinopathy. Invest Ophthalmol Vis Sci. 1997;38:283–291. [PubMed]
Adamus G, Machnicki M, Elerding H, Sugden B, Blocker YS, Fox DA. Antibodies to recoverin induce apoptosis of photoreceptor and bipolar cells in vivo. J Autoimmun. 1998;11:523–533. [CrossRef] [PubMed]
Furneaux HM, Rosenblum MK, Dalmau J, et al. Selective expression of Purkinje-cell antigens in tumor tissue from patients with paraneoplastic cerebellar degeneration. N Engl J Med. 1990;322:1844–1851. [CrossRef] [PubMed]
Minota S, Koyasu S, Yahara I, Winfield J. Autoantibodies to the heat-shock protein hsp90 in systemic lupus erythematosus. J Clin Invest. 1988;81:106–109. [CrossRef] [PubMed]
Jarjour W, Jeffries B, Davis J, Welch W, Miura T, Winfield JB. Autoantibodies to human stress proteins. Arthritis Rheum. 1991;34:1133–1138. [CrossRef] [PubMed]
Mairesse N, Kahn MF, Appelboom T. Antibodies to the constitutive 73 kDa heat shock protein: a new marker of mixed connective tissue disease. Am J Med. 1993;6:595–600.
Wolf BB, Green DR. Suicidal tendencies: apoptotic cell death by caspase family proteinases. J Biol Chem. 1999;274:20049–20052. [CrossRef] [PubMed]
Katai N, Kikuchi T, Shibuki H, et al. Caspaselike proteases activated in apoptotic photoreceptors of Royal College of Surgeons rats. Invest Ophthalmol Vis Sci. 1999;40:1802–1807. [PubMed]
Katai N, Yoshimura N. Apoptotic retinal neuronal death by ischemia-reperfusion is executed by two distinct caspase family proteases. Invest Ophthalmol Vis Sci. 1999;40:283–291.
Lam TT, Abler AS, Kwong JM, Tso MO. N-methyl-d-aspartate (NMDA)–induced apoptosis in rat retina. Invest Ophthalmol Vis Sci. 1999;40:2391–2397. [PubMed]
Tezel G, Wax MB. Inhibition of caspase activity in retinal cell apoptosis induced by various stimuli in vitro. Invest Ophthalmol Vis Sci. 1999;40:2660–2667. [PubMed]
Polans AS, Witkowska D, Haley TL, Amundson D, Baizer L, Adamus G. Recoverin, a photoreceptor-specific calcium-binding protein, is expressed by the tumor of a patient with cancer-associated retinopathy. Proc Natl Acad Sci USA. 1995;92:9176–9180. [CrossRef] [PubMed]
Yamaji Y, Matsubara S, Yamadori I, et al. Characterization of a small-cell-lung-carcinoma cell line from a patient with cancer-associated retinopathy. Int J Cancer. 1996;65:671–676. [CrossRef] [PubMed]
Matsubara S, Yamaji Y, Sato M, Fujita J, Takahara J. Expression of a photoreceptor protein, recoverin, as a cancer-associated retinopathy autoantigen in human lung cancer cell lines. Br J Cancer. 1996;74:1419–1422. [CrossRef] [PubMed]
De Castro E, Nef S, Fiumelli H, Lenz SE, Kawamura S, Nef P. Regulation of rhodopsin phosphorylation by a family of neuronal calcium sensors. Biochem Biophys Re Commun. 1995;216:133–140. [CrossRef]
Zhao X, Huang J, Khuani SC, Palczewski SK/FNM>. Molecular forms of human rhodopsin kinase (GRK1). J Biol Chem. 1998;273:5124–5131. [CrossRef] [PubMed]
Figure 1.
 
Effects of anti-recoverin and anti-hsc 70 antibodies or caspase inhibitors on rhodopsin phosphorylation in bovine ROS homogenate. Freshly isolated bovine ROS homogenate was preincubated with antibody, 5 μg preimmune rabbit IgG (A), anti-hsc 70 serum (containing 5 μg IgG) (B), 5 μg anti-recoverin IgG (C), and 1 mM caspase inhibitors, Z-VAD-FMK (D) or Z-DEVD-FMK (E). Rhodopsin phosphorylation was then performed by addition of 0.5 mM [γ-32P] ATP in the presence of 0.1 mM CaCl2 (open bars) or 1 mM EGTA (shaded bars) under flash illumination. The reaction was terminated by addition of 10% TCA and the radioactivity was counted in scintillation cocktail, and the radioactivities were plotted. Experiments were performed in triplicate.
Figure 1.
 
Effects of anti-recoverin and anti-hsc 70 antibodies or caspase inhibitors on rhodopsin phosphorylation in bovine ROS homogenate. Freshly isolated bovine ROS homogenate was preincubated with antibody, 5 μg preimmune rabbit IgG (A), anti-hsc 70 serum (containing 5 μg IgG) (B), 5 μg anti-recoverin IgG (C), and 1 mM caspase inhibitors, Z-VAD-FMK (D) or Z-DEVD-FMK (E). Rhodopsin phosphorylation was then performed by addition of 0.5 mM [γ-32P] ATP in the presence of 0.1 mM CaCl2 (open bars) or 1 mM EGTA (shaded bars) under flash illumination. The reaction was terminated by addition of 10% TCA and the radioactivity was counted in scintillation cocktail, and the radioactivities were plotted. Experiments were performed in triplicate.
Figure 2.
 
Effects of antibodies and caspase inhibitors on rhodopsin phosphorylation and ERG in rat eyes. (A) Preimmune IgG (5μ g; lane A), PBS (lane B), anti-hsc 70 serum (5μ g IgG; lane C), 5 μg anti-recoverin IgG (lane D), or anti-recoverin IgG and anti-hsc 70 serum (5 μg IgG each) without (lane E) or with (lanes F, G) caspase inhibitors was injected intravitreously into the eyes of Lewis rats. (B) Thirty-six hours (open bars) or 3 weeks (shaded bars) after administration, ROS was prepared, and light dependent phosphorylation by [γ-32P] ATP (left) and ERG measurement (right) were examined. In condition B, 5 μg anti-recoverin IgG was mixed with fresh dissected retinas followed by the ROS preparation. After the reaction, samples were analyzed by SDS-PAGE (A, top) followed by autoradiogram (A, middle) or Western blot analysis using anti-rhodopsin kinase antibody 36 (1:3000 dilution; A, bottom). The radioactivity of rhodopsin bands in SDS-PAGE was counted in a scintillation cocktail, and the radioactivities were plotted (B, left). Experiments were performed in triplicate. ERG measurements were performed in 10 eyes in each condition, and the amplitudes of b-wave were plotted (B, right).
Figure 2.
 
Effects of antibodies and caspase inhibitors on rhodopsin phosphorylation and ERG in rat eyes. (A) Preimmune IgG (5μ g; lane A), PBS (lane B), anti-hsc 70 serum (5μ g IgG; lane C), 5 μg anti-recoverin IgG (lane D), or anti-recoverin IgG and anti-hsc 70 serum (5 μg IgG each) without (lane E) or with (lanes F, G) caspase inhibitors was injected intravitreously into the eyes of Lewis rats. (B) Thirty-six hours (open bars) or 3 weeks (shaded bars) after administration, ROS was prepared, and light dependent phosphorylation by [γ-32P] ATP (left) and ERG measurement (right) were examined. In condition B, 5 μg anti-recoverin IgG was mixed with fresh dissected retinas followed by the ROS preparation. After the reaction, samples were analyzed by SDS-PAGE (A, top) followed by autoradiogram (A, middle) or Western blot analysis using anti-rhodopsin kinase antibody 36 (1:3000 dilution; A, bottom). The radioactivity of rhodopsin bands in SDS-PAGE was counted in a scintillation cocktail, and the radioactivities were plotted (B, left). Experiments were performed in triplicate. ERG measurements were performed in 10 eyes in each condition, and the amplitudes of b-wave were plotted (B, right).
Figure 3.
 
Expression of mRNA for recoverin in various tumor tissues. RNA (2 μg) from 30 tumor tissues from various organs and 4 normal lymphocyte samples from healthy volunteers were reverse-transcribed to generate cDNA pools, and 4.5 μl from a 22-μl cDNA pool was used for PCR using specific primers. PCR products were evaluated by agarose gel electrophoresis and ethidium bromide staining.
Figure 3.
 
Expression of mRNA for recoverin in various tumor tissues. RNA (2 μg) from 30 tumor tissues from various organs and 4 normal lymphocyte samples from healthy volunteers were reverse-transcribed to generate cDNA pools, and 4.5 μl from a 22-μl cDNA pool was used for PCR using specific primers. PCR products were evaluated by agarose gel electrophoresis and ethidium bromide staining.
Figure 4.
 
Aberrant expression of recoverin in various tumor tissues by Western blot analysis. Tumor tissues with or without mRNA expression of recoverin were confirmed by Western blots, by using a specific antibody toward recoverin. The numbered lanes correspond with diseases and lymphocytes as shown in Figure 3 . rec, purified bovine recoverin.
Figure 4.
 
Aberrant expression of recoverin in various tumor tissues by Western blot analysis. Tumor tissues with or without mRNA expression of recoverin were confirmed by Western blots, by using a specific antibody toward recoverin. The numbered lanes correspond with diseases and lymphocytes as shown in Figure 3 . rec, purified bovine recoverin.
Figure 5.
 
In vitro protein phosphorylation of A549 cells in the presence or absence of recoverin. A549 cell lysate was composed of buffer A, containing [γ-32P] ATP, purified recoverin (0 or 50μ g/ml), CaCl2 (0 or 0.1 mM), and EGTA (0 or 1 mM). The phosphorylation reaction was performed on ice for 20 minutes and thereafter was quenched by addition of 200 mM NaPi buffer (pH 7.2) containing 5 mM adenosine, 100 mM KF, 200 mM KCl, and 200 mM EDTA. Proteins were precipitated by the chloroform-methanol method and subjected to SDS-PAGE followed by autoradiography.
Figure 5.
 
In vitro protein phosphorylation of A549 cells in the presence or absence of recoverin. A549 cell lysate was composed of buffer A, containing [γ-32P] ATP, purified recoverin (0 or 50μ g/ml), CaCl2 (0 or 0.1 mM), and EGTA (0 or 1 mM). The phosphorylation reaction was performed on ice for 20 minutes and thereafter was quenched by addition of 200 mM NaPi buffer (pH 7.2) containing 5 mM adenosine, 100 mM KF, 200 mM KCl, and 200 mM EDTA. Proteins were precipitated by the chloroform-methanol method and subjected to SDS-PAGE followed by autoradiography.
Figure 6.
 
Presumable molecular mechanisms of retinal photoreceptor degeneration in CAR. R, rhodopsin; rec, recoverin; RK, rhodopsin kinase; p, phosphoric acid; ATP, adenosine 5′-triphosphate; ADP, adenosine 5′-diphosphate; APC, antigen-presenting cell.
Figure 6.
 
Presumable molecular mechanisms of retinal photoreceptor degeneration in CAR. R, rhodopsin; rec, recoverin; RK, rhodopsin kinase; p, phosphoric acid; ATP, adenosine 5′-triphosphate; ADP, adenosine 5′-diphosphate; APC, antigen-presenting cell.
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×