Preservation of Retinal Morphology and Functions in Royal College Surgeons Rat by Nilvadipine, a Ca²⁺ Antagonist

Abstract

purpose. The Royal College of Surgeons (RCS) rat is the most extensively studied animal model for understanding the molecular pathology in inherited retinal degeneration, such as retinitis pigmentosa (RP). The purpose of the present study was to evaluate the pharmacologic effects of several Ca²⁺ antagonists on the retinal degeneration of RCS rats.

methods. Several Ca²⁺ antagonists, diltiazem, nicardipine, nilvadipine, and nifedipine, were intraperitoneally administered and retinal morphology and functions analyzed.

results. Among the Ca²⁺ antagonists, only intraperitoneally administered nilvadipine preserved retinal morphology and electroretinogram responses in RCS rats during the initial stage of retinal degeneration. Studies using immunohistochemistry, RT-PCR, and Western blot analysis revealed significant enhancement of rhodopsin kinase and αA-crystallin expression and suppression of caspase 1 and 2 expression in the retina of nilvadipine-treated rats.

conclusions. These data suggest that nilvadipine is beneficial for the preservation of photoreceptor cells in RCS rats and can be used to treat some patients with RP.

Retinitis pigmentosa (RP) is a disease of inherited retinal degeneration characterized by nyctalopia, ring scotoma, and bone-spicule pigmentation of the retina. So far, no effective therapy has been available for RP. Several animal models with inherited retinal degeneration have been studied to elucidate the pathologic molecular characteristics of RP and to design an effective therapy for it. The Royal College of Surgeons (RCS) rat, in which the retinal pigment epithelial (RPE) cell is affected by the retinal dystrophy (rdy) mutation and continuously expresses the rdy phenotype, ¹ has been the most widely used animal model for the study of RP. In terms of the molecular characteristics of retinal degeneration, it has been suggested that failure of the RPE to phagocytose the shed tips of rod outer segments (ROS) debris in the RCS rat is primarily involved. ^{2 3 4}  

Recently, D’Cruz et al. ⁵ used a positional cloning approach to study the rdy locus of the RCS rat, and they discovered a small deletion of RCS rat DNA that disrupts the gene encoding the receptor tyrosine kinase Mertk, which may be a molecular target for ingestion of outer segments by RPE cells. It has been shown that mutations in Mertk cause human RP. ⁶ In contrast, RCS rat photoreceptor cells are considered to be normal in their structure and functions, because they can survive after retinal RPE transplantation. ^{7 8} However, it has been found that several changes occur in RCS rat ROS, including protein phosphorylation levels of opsin, ⁹ arrestin, ^{10 11} and ROS, ¹² which may affect quenching of the phototransduction pathway in RCS rats. In fact, we recently found significantly lower levels of mRNA expression of αA-crystallin and rhodopsin kinase (RK), which are thought to be involved in post-Golgi processing of opsin and rhodopsin phosphorylation, respectively, in RCS rats than those in the control rats at the age of 3 to 4 weeks. In contrast, expression of other photoreceptor cell-specific proteins including rhodopsin, transducin, arrestin, and recoverin were almost comparable between RCS and control rats at the age of 3 to 4 weeks. ¹³  

Therefore, based on these observations, we suggested that low levels of rhodopsin phosphorylation might cause misregulation of phototransduction pathways in rod photoreceptor cells, resulting in their degeneration. This idea is supported by experimental evidence that absence of rhodopsin phosphorylation in transgenic mice carrying rhodopsin mutations causes retinal degeneration. ^{14 15} Because recoverin, a retina-specific Ca²⁺-binding protein, negatively regulates rhodopsin phosphorylation by rhodopsin kinase in a Ca²⁺-dependent manner, ¹⁶ it is plausible that suppression of recoverin-dependent inhibition of rhodopsin kinase by the lowering of intracellular Ca²⁺ levels by some drugs may be effective in the preservation of photoreceptor cells in RCS rats. Frasson et al. ¹⁷ recently reported the interesting finding of rod photoreceptor rescue by d- cis-diltiazem, a Ca²⁺ channel blocker in a different animal model of RP, the rd mouse, in which the gene encoding cGMP phosphodiesterase is affected. In consideration of all these data, we assume that the regulation of intracellular Ca²⁺ levels may have potential as a therapy to prevent progressive retinal degeneration in RP and in animal models. 

To test our hypothesis, several kinds of Ca²⁺ antagonists used in clinical practice ^{18 19 20} were administered to RCS rats at 3 weeks after birth—the time when degenerative changes in photoreceptor cells are known to begin. Then, the retinal function was evaluated by electroretinography (ERG), and histologic studies including light microscopy, immunohistochemistry, and electron microscopy were performed. 

Materials and Methods

All experimental procedures were designed to conform to both the ARVO Statement for Use of Animals in Ophthalmic and Vision Research and our own institution’s guidelines. Unless otherwise stated, all procedures were performed at 4°C or on ice, using ice-cold solutions. Nilvadipine, diltiazem, nicardipine, and nifedipine were generous gifts from Fujisawa Pharmaceutical Co., Tokyo, Japan; Tanabe Pharmaceutical Co., Osaka Japan; Yamanouchi Pharmaceutical Co., Tokyo, Japan; and Bayer Pharmaceutical Co., Osaka, Japan, respectively. Anti-human RK monoclonal antibody (G8), ²¹ in which immunoreactivities to rat rhodopsin kinase were confirmed in our previous article, ¹³ was generously provided by Krzysztof Palczewski (Department of Ophthalmology, University of Washington, Seattle, Washington). Anti-αA-crystallin antibody, and anti-caspase 1 and -caspase 2 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The specificity and titers of all antibodies were examined by Western blot analysis and ELISA, using rat retina soluble fractions. 

Anesthesia

In the present study, 3- to 5-week-old inbred RCS (rdy ^−/−) rats (Crea, Tokyo, Japan) reared in cyclic light conditions (12 hours on-12 hours off) were used. For anesthesia induction, rats inhaled diethyl ether. Once unconscious, the animals were injected intramuscularly with a mixture of ketamine (80–125 mg/kg) and xylazine (9–12 mg/kg). The adequacy of the anesthesia was tested by tail clamping, and supplemental doses of the mixture were administered intramuscularly, if needed. 

Drug Administration

Nilvadipine and nifedipine were dissolved in a mixture of ethanol, polyethylene glycol 400, and distilled water (2:1:7) at a concentration of 0.1 mg/mL, diluted twice with physiological saline before use, and injected intraperitoneally (1.0 mL/kg) into anesthetized rats every day early in the morning for 2 weeks. In control rats, the same solution without nilvadipine or nifedipine (vehicle solution) was administered similarly. Nicardipine and diltiazem were dissolved in PBS at 0.25 mg/mL and 1 mg/mL, respectively, and injected intraperitoneally (1.0 mL/kg), similarly to the other agonists. As a control, the same volume of a mixture of ethanol, polyethylene glycol 400, and distilled water (2:1:7) or PBS was administered. Before administration, the pH of all drug solutions was adjusted to approximately 7.4. The concentrations of these drugs administered to RCS rats were determined by their concentrations in oral administration to human patients with hypertension for 1 day in our clinical practice (nilvadipine, 0.05–0.3 mg/kg; nifedipine, 0.1–0.5 mg/kg; nicardipine, 0.2–1.0 mg/kg; and diltiazem, 0.3–3 mg/kg). ²²  

Light Microscopy

Five RCS rats each (age, 3–5 weeks; weight, 150–200 g) were studied for the control and four different Ca²⁺ antagonist administration conditions. Under deep anesthesia, animals were perfused through the initial portion of the aorta with 300 mL 4% paraformaldehyde in 0.1 M PBS (pH 7.4), and retinas were dissected and embedded in paraffin. Posterior segments (5 × 5 mm² containing the optic disc at center) cut from the enucleated eyes were embedded in paraffin. Retinal sections were cut vertically through the optic disc at 4 μm thickness, nasally to temporally, mounted on subbed slides, and dried. The sections were processed with hematoxylin-eosin staining after deparaffinization with graded ethanol and xylene solutions. Retinal sections were photographed, and the thickness of each retinal layer was measured at temporal and nasal points 1 mm away from the optic disc (two points per section) in five different sections from five rats in each condition. Thickness of retinal layers is shown as mean ± SD. Significant differences between groups were found using the Mann-Whitney test with a significance level of P < 0.05. 

Electron Microscopy

Nilvadipine-treated and untreated RCS rats (three animals for each condition) were used. Under deep anesthesia, each animal was perfused through the initial portion of the aorta with 300 mL 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Tissues were dissected, postfixed in phosphate-buffered 1% osmium tetroxide (pH 7.4), dehydrated in an ascending series of ethanol solutions, and passed through propylene oxide. The blocks were embedded in Epon 812. Thin sections were stained in uranyl and lead salt solutions. 

Electroretinography

Details of preparation, recording technique, and measurements of ERG responses have been described elsewhere. ²³ Five RCS rats were studied (age, 3–5 weeks; weight, 150–200 g) each for control and four Ca²⁺ antagonists. Under anesthesia, each rat was laid on its side on a heating pad (at 37°C), with its head fixed in place with surgical tape in an electrically shielded room, and dark-adapted for at least 2 hours. The pupils were dilated with drops of 0.5% tropicamide. ERGs were recorded with a contact electrode equipped with a suction apparatus to fit on the cornea (Kyoto Contact Lens Co., Kyoto, Japan). A grounding electrode was placed on the ear. Response evoked by a white flash (3.5 × 10² lux, 200-ms duration) were recorded (Neuropack, model MES-3102; Nihon Kohden, Tokyo, Japan). The a-wave amplitude was determined from the baseline to the bottom of the a-wave. The b-wave amplitude was determined from the bottom of the a-wave to the top of the b-wave. ERG amplitudes are shown as mean ± SD. Significant differences between groups were found using the Mann-Whitney test with a significance level of P < 0.05. 

Immunofluorescence Microscopy

Normal retinas of Sprague-Dawley rats (SD; 5 weeks old, three rats), nilvadipine-treated and untreated RCS rats (4 and 5 weeks old; three animals in each group) were analyzed. Animals under deep anesthesia were perfused through the initial portion of the aorta with 300 mL 4% paraformaldehyde in 0.1 PBS (pH 7.4), and retinas were dissected, embedded in optimal cutting temperature (OCT) compound (Tissue-Tek, Tokyo, Japan), and sectioned vertically through the optic disc into 14-μm-thick sections with a cryostat. Before application of the primary antibodies, sections were blocked with PBS containing 5% goat serum and 3% bovine serum albumin for 1 hour and then incubated overnight with anti-rhodopsin kinase (1:1000), anti-αA crystallin (1:500), anti-caspase 1 (1:500), or anti-caspase 2 (1:500) antibodies at 4°C. Sections were washed and incubated with fluorescein-isothiocyanate (FITC)-conjugated antibodies to mouse, goat, or rabbit IgG (Cappel, Durham, NC) for 1 hour at room temperature. Specificity control experiments were performed by omitting the primary antibodies. Sections were counterstained with iodide (TOTO-3; Molecular Probes, Eugene, OR) and observed with a confocal laser microscope (Radians 2000; Bio-Rad Laboratories, Hertfordshire, UK). Photographs were taken 1 to 2 mm from the disc. 

RT-PCR Analysis and Relative Amount of α-Crystallins and RK

Total RNA from retinas was isolated using an extraction reagent (Isogen; Nippon Gene, Tokyo, Japan), according to the procedure recommended by the manufacturer. The cDNAs were generated from 2 μg retinal RNA in a 12-μL reaction, using 1 μL oligo(dT) primer (0.5 mg/mL; Gibco-BRL, Life Technologies, Inc., Rockville, MD). The reaction mixture was denatured at 70°C for 10 minutes. Four microliters first-strand buffer (250 mM Tris-HCl, 375 mM KCl, 15 mM MgCl₂; Superscript; Gibco), 2 μL dithiothreitol (DTT, 0.1 M; Gibco), 1 μL deoxyribonucleoside triphosphate (dNTP, 10 mM; Gibco), 1 μL RNase inhibitor (40 U/μL; Gibco), and 1 μL reverse transcriptase (200 U/μL; Superscript II; Gibco) were added to the mix. The incubation was performed at 42°C for 50 minutes and at 70°C for 15 minutes. The PCR amplifications were performed with 4 μL of the RT reaction, 5 μL 10× PCR buffer (200 mM Tris-HCl, 500 mM KCl), 2 μL MgCl₂ (50 mM), 1 μL dNTP, 5 μL sense and antisense primers (10 pM/μL), and 0.5 μL Taq polymerase (5 U/μL; Gibco). The PCR mix was denatured at 94°C for 4 minutes and then run for 28 cycles of 94°C for 1 minute, 55°C for 1 minute, and 72°C for 2 minutes. 

The primers used for RT-PCR were as follows: 5′-ATGGACGTCACCATCCAGCA-3′, corresponding to bases 158 to 178 of the cDNA sequence and 5′-AGCTGGGCTTCTCCTCCCGT-3′ corresponding to bases 713 to 732 of the cDNA sequence for αA-crystallin, ²⁴ with expected PCR products of 485 bp; 5′-ATGGACATAGCCATCCACCACCCCTGGAT-3′ corresponding to bases 21 to 49 of the cDNA sequence and 5′-AATCTACTTCTTAGGGGCTGCAGTGACAGC-3′ corresponding to bases 522 to 551 of the cDNA sequence for αB-crystallin, ²⁵ with an expected PCR product of 531 bp; 5′-AAGACCAAGGGCTATGCAGGGA-3′ corresponding to bases 1226 to 1247 of the cDNA sequence and 5′-CTAGGAGATGAGACACATCCCTGA-3′ corresponding to bases 1856 to 1879 of the cDNA sequence for rhodopsin kinase, ²⁶ with an expected PCR product of 654 bp; 5′-GTATGGAATCCTGTGGCATCC-3′ corresponding to bases 2683 to 2703 of the genomic DNA sequence and 5′-TACGCAGCTCAGTAACAGTCC-3′ corresponding to bases 3135 to 3155 of the genomic DNA sequence for β-actin, ²⁷ with an expected PCR product of 349 bp. The amplified PCR fragments were electrophoresed on a 1.5% agarose gel containing ethidium bromide. 

SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis

SDS-PAGE was performed by the method of Laemmli ²⁸ using a 12.5% SDS-PAGE slab gel and a minigel apparatus (Hoeffer, San Francisco, CA). Western blot analysis was performed as described previously. ²⁹ Briefly, after SDS-PAGE of the ROS soluble protein sample, separated proteins in the gel were electrotransferred to polyvinylidene fluoride (PVDF) membranes in 10 mM bis-tris-propane buffer (pH 8.4) containing 10% (wt/vol) methanol solution. After nonspecific binding was blocked by 5% (wt/vol) skim milk in PBS, the membrane was probed successively with antibodies and horseradish peroxidase (HRP)-labeled secondary antibodies (Funakoshi Co., Tokyo, Japan). Specific antigen-antibody binding was visualized with an enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech, Amersham, UK), according to the method of the manufacturer. 

Results

To study the effects of Ca²⁺ antagonists on retinal morphology in the RCS rat, four Ca²⁺ antagonists—diltiazem, nifedipine, nicardipine, and nilvadipine—all of which are used in clinical practice, and their vehicle solutions were systemically administered to 3-week-old RCS rats every day for 2 weeks (n = 5 rats, 10 eyes in each condition), and then the thickness of each retinal layer was compared among the groups. As shown in Figure 1 , no significant differences were observed in thickness of retinal layers in rats treated with diltiazem, nifedipine, or nicardipine and the control rats during the 2 weeks. However, in contrast, the retinal the outer nuclear layer (ONL) and the outer segment (OS) were significantly thicker in 4- and 5-week-old RCS rats given nilvadipine than those in control rats and rats in other drug groups. This suggests that nilvadipine affords significant protection against thinning of retinal layers in the RCS rat during retinal degeneration. Electron microscopy showed that marked irregularity in the photoreceptor OS in the untreated retina (Fig. 2a) . On the contrary, the structure was more preserved in nilvadipine-treated retinas (Fig. 2b) . The outer plexiform layer (OPL) of untreated retinas was thinner than in the nilvadipine-treated retinas, showing the progress of the structural destruction (Figs. 3a 3b) . Although the degeneration was still observed in the OPL of nilvadipine-treated retinas, preservation of the synapse together with synaptic ribbons was noted (Fig. 3b) . 

These morphologic differences between the nilvadipine-treated and untreated RCS rats were correlated with differences in retinal function by ERGs(Fig.4) . Nilvadipine-treated retina showed a significant preservation of a- and b-wave amplitudes compared with the control at 4 weeks (P < 0.001). The preservation effect on ERG response by nilvadipine was less, but still significant (P < 0.01) at 5 weeks. However, other Ca²⁺ antagonists had no effect on the ERG responses. Therefore, taken together, these data strongly suggest that administration of nilvadipine produces significant preservation of retinal morphology and function. 

To further understand nilvadipine neuroprotection, RK, αA-crystallin, and caspase immunolabeling and mRNA expression were studied. In immunofluorescence microscopy, nonspecific immunoreactivity was not detected in the specificity control retinas. Although histologic disorder was observed, nilvadipine-treated retinas showed better preservation of intensities of RK and αA-crystallin expression, which has been shown to be specifically decreased in RCS rat, than untreated RCS retinas in the OS of the photoreceptor layer, both at 4 and 5 weeks(Fig. 5) . These data were confirmed by RT-PCR and/or Western blot analysis. That is, as shown in Figures 6 and 7 , expression of RK and αA-crystallin in retinas of 4- and 5-week-old RCS rats was enhanced by the administration of nilvadipine compared with expression in control. In contrast, expression of αB-crystallin and β-actin were almost comparable between nilvadipine-treated and untreated RCS rats (Fig. 6) . Immunoreactivities to caspase 1 and 2 were detected in the cytoplasm of the ONL in the untreated RCS retina (4 weeks old), whereas the nilvadipine-treated retina showed smaller number of immunopositive cells in the ONL (Fig. 5) . In 5-week-old rats, untreated RCS retina showed weaker immunopositivity than that at 4 weeks, and few positive cells were detected in nilvadipine-treated retinas. Caspase 1 and 2 immunoreactivities were not observed in the intact retina (Fig. 5) . These changes between nilvadipine-treated and untreated RCS retinas were also noted in Western blot analysis (Fig. 7) . 

Discussion

In the present study, we found that intraperitoneal administration of nilvadipine, a Ca²⁺ antagonist, had a protective effect on photoreceptor cells during retinal degeneration of RCS rat as follows: (1) In the morphology, significant preservation of the thickness of retinal outer layers was observed in light microscopic examination of photoreceptor OS structures and synapse-synaptic ribbon formation within the ONL was observed in electron microscopic examination. (2) Functionally, amplitudes of a- and b-waves of ERGs were preserved. (3) Preservation of expressions of αA-crystallin and RK were noted in immunohistochemistry, RT-PCR, and Western blot analysis. (4) Expression of caspases 1 and 2 was suppressed. However in contrast, such neuroprotective effects were not observed with other Ca²⁺ antagonists, including diltiazem, nifedipine, and nicardipine. These observations suggest that nilvadipine protects the structure and functions of photoreceptor cells by inhibition of the apoptotic process in RCS rat retinas. 

Ca²⁺ antagonists, which have been widely used as treatments for systemic hypertension, inhibit the entry of calcium ion intracellularly, relax vascular smooth muscle cells, and increase regional blood flow in several organs. ^{18 19 20} The dihydropyridine (DHP) derivatives nifedipine, nicardipine, and nilvadipine; the benzothiazepine derivative diltiazem; and the phenylalkylamine derivative verapamil are the major Ca²⁺ antagonists used in clinical practice. ³⁰ It has been shown that these drugs have different properties in the specificity and kinetics of blocking Ca²⁺ channels. On the basis of kinetics and voltage-dependent properties, the Ca²⁺ channels have been classified into two groups: One is activated by small depolarizations and is then subsequently inactivated and is called a low-voltage-activated (LVA) Ca²⁺ channel, whereas the other is activated by larger depolarizations, shows little inactivation, and is called a high-voltage-activated (HVA) Ca²⁺ channel. Based on pharmacologic properties, HVA Ca²⁺ channels can be separated further into four types (L, N, P/Q, and R). ^{31 32} Regarding retinal Ca²⁺ channels, it has been revealed that L-type HVA Ca²⁺ channel currents have been identified in photoreceptors of amphibians and fish and are sensitive to DHPs. ^{33 34 35} Similarly, L-type currents have been found in mammalian cone photoreceptors. ^{36 37} Recently, a novel Ca²⁺ channel gene, CACN1F, encoding α_1F, a retina-specific α₁ subunit of L-type HVA Ca²⁺ channels was identified, ^{38 39} and its immunolocalization was observed within the ONL and OPL in rat retina. ⁴⁰ It has been reported that mutations in CACN1F cause incomplete X-linked congenital stationary night blindness (CSNB2), ^{38 38} in which neurotransmission between the photoreceptors and retinal bipolar cells is impaired. ⁴¹ Taken together with the fact that most DHPs and diltiazem are L-type HVA Ca²⁺ channel blockers, ³⁰ we can reasonably speculate that these drugs react with retinal L-type HVA Ca²⁺ channels and presumably prevent photoreceptor cell death. In fact, it has recently been reported that rod photoreceptors of rd mice were rescued by d- cis-diltiazem. ¹⁷  

Among the several Ca²⁺ channel blockers, three DHPs (nilvadipine, nicardipine, and nifedipine) and diltiazem, it is not known why only nilvadipine was effective in the current study in the preservation of RCS photoreceptors, but there are several possible explanations to consider. 

Preferable Transmission of Nilvadipine to the Central Nervous System, Including Retina

It has been shown that nilvadipine is a much higher hydrophobic chemical than nifedipine and nicardipine. ^{42 43} A pharmacokinetic study showed that [¹⁴C]-nilvadipine is well distributed in various types of tissue, including brain, after intraperitoneal administration. ⁴⁴ Functionally, nilvadipine increased vertebrate blood flow more effectively than nifedipine or nicardipine in dogs ⁴⁵ and increased blood velocity and blood flow in the optic nerve head as well as in the choroid and retina in rabbits. ⁴⁶ In the present study, we injected nilvadipine intraperitoneally at a concentration of 0.1 mg/kg. This concentration of nilvadipine is almost comparable to the concentrations (0.05–0.3 mg/kg) in oral administration to human patients with hypertension for 1 day. ²² These observations suggest that systemic administration of nilvadipine, by being able to cross the blood-brain barrier, should be able to reach cytoprotective levels within the central nervous system (CNS), including the retina. Because of this preferable transmission to the CNS, nilvadipine has in fact been used clinically in Japan for protection against neuronal cell death after the onset of CNS diseases, such as cerebral infarction. ⁴⁷  

LVA Ca²⁺ Channel Blocking by Nilvadipine

It has been shown that nilvadipine is an LVA and L-type HVA Ca²⁺ channel blocker. ⁴⁸ However, in contrast, nifedipine, nicardipine, and diltiazem have much less effect on LVA Ca²⁺ channels. ⁴⁹ This is another possible reason for nilvadipine’s effect, because the presence of LVA Ca²⁺ channels has been reported in the retina. ⁵⁰ In terms of the blocking of other types of HVA Ca²⁺ channels by these antagonists, Diochot et al. reported that the DHPs verapamil and diltiazem similarly block N-, P/Q- and R-type calcium currents, using sensory and motor neurons. ⁵¹ Therefore, the difference in ability to block N-, P/Q- and R-type calcium channels among these Ca²⁺ antagonists may not be involved. As another possible mechanism, it has been speculated that these Ca²⁺ antagonists may affect channels other than Ca²⁺ channels, such as Na⁺ channels, differently. In fact, it has been found that diltiazem is an Na⁺ channel blocker in ventricular myocytes. ⁵² However, no study has been available so far to determine the effect of nilvadipine on Na⁺ channels. Such a study is needed in the near future. 

An apoptotic mechanism of retinal ganglion cell death has been mainly involved in the pathologic molecular characteristics of glaucoma. ⁵³ It has also been suggested that some of the calcium antagonists effectively retard the progression of visual field defects in some patients with glaucoma, ^{54 55 56 57} and especially in normal-tension glaucoma, owing to its vasodilating effects on intraocular blood flow. ^{54 57} In terms of the preservation effects of nilvadipine on retinal degeneration in RCS rats, we do not presently know which mechanism of action is more important, its vasodilating effect or the lowering of intracellular Ca²⁺ levels. However, the recent observation of rod photoreceptor rescue of the rd mouse by d- cis-diltiazem, another Ca²⁺ antagonist with less vasodilating action in retinal blood vessels, ¹⁷ led us to speculate that the lowering of intracellular Ca²⁺ levels by nilvadipine may be more important. If so, we can reasonably hypothesize that misregulation of Ca²⁺-binding proteins induced by an influx of Ca²⁺ into the photoreceptor cells may be normalized by administration of a Ca²⁺ antagonist, because it is known that Ca²⁺ regulation by Ca²⁺-binding proteins, including recoverin, guanylate cyclase-activating protein, and calmodulin, is pivotal in signal transduction mechanisms in photoreceptors. ⁵⁸ We speculate that nilvadipine may also affect Ca²⁺-dependent regulation in RPE cells in RCS rats, because signal transduction pathways regulated by tyrosine kinases, including Mertk, are known to be regulated by Ca²⁺. ⁵⁹  

As another possibility, the suppression of the Ca²⁺-dependent apoptotic process by the Ca²⁺ antagonist was considered. In fact, it has been reported recently that caspase-dependent apoptotic pathways (caspase 1 and 2) are activated during retinal degeneration in RCS rats. ⁶⁰ Similarly, a caspase 3-dependent mechanism has been shown to be involved in photoreceptor cell death in transgenic rats with the rhodopsin Ser334ter mutation. ⁶¹ This rhodopsin mutant is not phosphorylated by RK because of elimination of all the possible phosphorylation sites. ⁶² Therefore, it is thought that this mutant is functionally similar to RCS rats in which low levels of mRNA expression of RK has been shown. ¹³ However, we observed no immunoreactivities toward caspase 3 in nilvadipine-treated and untreated retinas in RCS rats (data not shown). 

Recently, Bush et al. ⁶³ reported that the Ca²⁺ antagonist d- cis-diltiazem had no effects on photoreceptor degeneration in the rhodopsin P23H rat. Patients with RP and mouse models of the P23H rhodopsin mutation are known to show delayed photoresponse recovery, suggesting that the quenching and adaptation processes of rhodopsin phosphorylation and its related reactions may be impaired. ⁶⁴ Because these reactions are Ca²⁺-dependent, it has been thought that Ca²⁺ antagonists may also have a protective effect on retinal degeneration in the rhodopsin P23H rat. However, protective effects by d- cis-diltiazem were not so significant in the rhodopsin P23H mutant rat. In our present study, nilvadipine preserved photoreceptor cell function and structure in the RCS rat, but retinal degeneration still progressed. Therefore, it seems likely that Ca²⁺ channel blockers have protective effects against retinal degeneration in some disease models, but these effects may be variable among different models, species, diseases’ stages, and Ca²⁺ antagonists. 

In conclusion, our findings suggest that nilvadipine may be effective in the preservation of photoreceptor cells during retinal degeneration in RCS rats. Taken together with the presence of mutations in Mertk in patients with RP, identical with those found in RCS rats, ⁶ our present observations strongly suggest that the clinical application of nilvadipine has potential benefit in the treatment of RP. 

 Supported by Grants-in-Aid for Scientific Research B-11470361, B-12557145, and C-13671846 from the Japanese Ministry of Health and Welfare; by the Japanese Ministry of Education, Sports and Culture; Naito Memorial Foundation; the Ciba-Geigy Foundation for the Promotion of Science; the Mochida Memorial Foundation for Medical and Pharmaceutical Research; Uehara Memorial Foundation; Karouji Memorial Foundation for Medical Research, and the JRPS Research Foundation.

 Submitted for publication September 10, 2001; revised December 3, 2001; accepted December 12, 2001.

 Commercial relationships policy: N.

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Effects of several Ca²⁺ antagonists on thickness of retinal layers of RCS rat. Hematoxylin-eosin staining of retinal sections at 1 mm from optic disc in 4- and 5-week old RCS rat eyes treated with Ca²⁺ antagonists, diltiazem, nifedipine, nicardipine, nilvadipine, or vehicle solution. Photographs of the sections were taken (a), and each of the retinal layers were measured at temporal and nasal points 1 mm from the optic disc in five retinas (two points per eye, total of 10 points) and the results plotted (b). Lanes 1–5: 4 weeks; lanes 6–10: 5 weeks; lanes 1 and 6: vehicle solution; lanes 2 and 7: diltiazem; lanes 3 and 8: nifedipine; lanes 4 and 9: nicardipine; lanes 5 and 10: nilvadipine. *P < 0.01 (Mann-Whitney test). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; OS, outer segment.

Figure 2.

 

Electron micrographs of photoreceptor outer segment of untreated (a) and nilvadipine-treated (b) retina (5 weeks old). The disc arrangement is more irregular in untreated (a) than nilvadipine-treated retina (b). Scale bars, 1 μm.

Figure 3.

 

Electron micrographs of the ONL, OPL, and INL of untreated (a) and nilvadipine-treated (b) retina (4 weeks old). The INL is situated at the top and the ONL at the bottom in each micrograph. (a) In untreated retina, the decrease in thickness and the structural destruction were visible in the OPL. Apoptotic nuclei (N) are seen in the ONL. (b) The thickness of the OPL was more preserved in nilvadipine-treated retina, and synapse ribbons were visible in some photoreceptor terminals (arrows). INL, inner nuclear layer. Scale bars, 1 μm.

Figure 4.

 

Effects of Ca²⁺ antagonists on scotopic ERG in RCS rats. RCS rats treated with diltiazem (⋄), nifedipine (▵), nicardipine (▿), nilvadipine (○), or their vehicle solutions (□), respectively, every day after 3 weeks of age. During the 2 weeks after the injection, scotopic ERG was recorded once a week. (a) Typical ERG responses of RCS rat eyes at 3 weeks of age (before operation) and at 4 and 5 weeks of age after administration of nilvadipine or vehicle solution. (b) ERG measurements were performed in 10 eyes (5 rats) in each condition, and the amplitudes of a-and b-waves were plotted. *P < 0.01, **P < 0.001 (Mann-Whitney test).

Figure 5.

 

Immunohistochemical localization of RK, αA-crystallin (Crys), caspase 1 (Casp 1), and caspase 2 (Casp 2) in untreated (RCS) and nilvadipine-treated (nilv) rats 4 and 5 weeks of age. Immunohistochemical analysis of RK and αA-crystallin was also performed in the normal intact retina (intact). Scale bars, 20 μm.

Figure 6.

 

Expressions of mRNA for RK, αA-crystallin, αB-crystallin, and β-actin in retina and RPE from RCS rats administered nilvadipine (+) or vehicle solution (−). RNA (2 μg) from retinas of 3- to 5-week old RCS rats administered nilvadipine or vehicle solution was reverse-transcribed to generate a cDNA pool, and 2.2 μL from 22 μL of the cDNA pool was used for PCR, using specific primers. PCR products were evaluated by agarose gel electrophoresis and ethidium bromide staining.

Figure 7.

 

Analysis of ROS proteins in RCS and control rats by Western blot analysis using antibody against RK, αA-crystallin, caspase 1 and 2. Two retinas of 3- to 5-week-old RCS rats administered nilvadipine (+) or its vehicle solution (−) were homogenized in 100 μL of 10 mM HEPES buffer (pH 7.5) containing 2% Tween-20. An aliquot (10 μL) was mixed with the sample buffer (10 μL) and loaded on an SDS-PAGE gel (a) and then electrotransferred to PVDF membrane. (b) Western blot analysis was performed using anti-RK mAb (1:3000) or anti-α-A-crystallin (1:2000), anti-caspase I (1:2000), or anti-caspase 2 (1:2000) polyclonal antibodies.