Retinoid cycles in the cone-dominated chicken retina

In past decades, the role of retinoids in support of rod photopigment regeneration has been extensively characterized. In the rhodopsin cycle,retinal chromophore from bleached rod pigments is reduced to retinol and transferred to the retinal pigment epithelium (RPE) to store as all-trans retinyl ester. This ester pool is subsequently utilized for visual pigment regeneration. However, there is a lack of information on the putative cone visual cycle. In the present study, we provide experimental evidence in support of a novel retinoid cycle for cone photopigment regeneration. In the cone-rich chicken, light exposure resulted in the accumulation of 11-cis retinyl esters to the retina and all-trans retinyl esters to the RPE. Both the rate of increase and the amount of 11-cis retinyl esters in the retina far exceeded those of the all-trans retinyl esters in the RPE. In response to dark adaptation, this 11-cis retinyl ester pool in the retina depletes at a rate several times faster than the all-trans retinyl ester pool in the RPE. In vitro, isolated, dark-adapted retinas devoid of RPE show both an accumulation of 11-cis retinyl ester and a concomitant reduction of 11-cis retinal chromophore in response to light exposure. Finally, we provide experimental results to elucidate a cone visual cycle in chicken by relating the change in retinoids (retinal and retinyl ester) with time during light and dark adaptation. Our results support a new paradigm for cone photopigment regeneration in which the 11-cisretinyl ester pool in the retina serves as the primary source of visual chromophore for cone pigment regeneration.

During dim light conditions, vertebrates depend upon rod photoreceptors for vision. In the rod visual cycle, the kinetics and metabolism involved in light and dark adaptation have been established in past decades (for a review, see Lamb and Pugh, 2004). In the rod outer segment (ROS), 11-cis retinal is the chromophore of rhodopsin. Upon illumination, 11-cis retinal undergoes a conformational shift to yield free all-trans retinal. To regenerate rhodopsin, rods are dependent on the retinal pigment epithelium (RPE) for a supply of 11-cis retinal. 11-cis retinal is generated from all-trans retinol derived from bleached chromophore in a series of reactions termed the visual cycle (for a review, see McBee et al., 2001). All-trans retinal released from rhodopsin is reduced in the ROS and travels to the RPE, where it is immediately esterified by lecithin retinol:acyltransferase (LRAT) to yield all-trans retinyl ester(Saari and Bredberg, 1993). All-trans retinyl esters are isomerized to 11-cis retinol by isomerohydrolase (Moiseyev et al.,2003; Gollapalli and Rando,2003) by a reaction that is highly dependent on cellular retinal binding protein (CRALBP; Winston and Rando, 1998; Stecher et al.,1999) and RPE65 (Redmond et al., 1998). 11-cis retinol is then oxidized by retinol dehydrogenase to 11-cis retinal before returning to the ROS. Dowling(1960) first defined the dynamics of this cycle during light and dark adaptation in the rod-dominated albino rat. During light conditions, retinal in the retina (as chromophore)diminishes while retinol (as retinyl ester) increases in the RPE. During dark conditions, the retinyl ester pool in the RPE depletes and retinal is regenerated in the retina. Therefore, rods are reliant on the all-trans retinyl ester pool in the RPE for sustained chromophore regeneration.

In humans, daylight vision is primarily mediated by cone photoreceptors. Cone chromophore regeneration is several fold faster than rod chromophore regeneration in light conditions (Perry and McNaughton, 1991). Cone, but not rod, visual pigment from isolated frog retina can spontaneously regenerate(Goldstein, 1967; Hood and Hock, 1973). Mata et al. (2002) have shown that membrane fractions prepared from cone-dominated retina contain three key visual cycle enzymatic activities, including those of retinol isomerase,11-cis retinyl ester synthase and 11-cis retinol dehydrogenase. Data from our laboratory and others have shown that the retinae of cone-dominated species such as chicken and ground squirrel store a larger amount of retinyl ester than those in the RPE, of which the 11-cisisomer is more abundant (Rodriguez and Tsin, 1989; Das et al.,1992; Mata et al.,2002). These experiments suggest that at least some vitamin A chromophore for cone pigment (iodopsin) may come from a location within the retina and may follow a separate visual cycle pathway(Jin et al., 1994; Bustamante et al., 1995).

In the present study, to further the understanding of this proposed cone visual cycle, we quantitatively characterize the change in retinoids in chicken retina and RPE during light and dark adaptation. We show that light exposure increases 11-cis retinyl esters in the chicken retina and all-trans retinyl esters in the RPE. Upon dark adaptation, the 11-cis retinyl ester pool in the retina declines at a rate about nine times faster than that of the all-trans retinyl pool in the RPE. Furthermore, isolated retinae devoid of RPE in vitro show similar light-initiated accumulation of 11-cis retinyl esters in concert with a depletion of 11-cis retinal, confirming the RPE independence of the accumulation of this 11-cis retinyl ester pool. Finally, to begin the characterization of the kinetics of such a cone visual cycle, we have collected experimental evidence to show a light-dependent accumulation/depletion cycle of 11-cis retinyl esters in the chicken retina during visual pigment bleaching and regeneration. Based on the amount of 11-cis retinyl esters and its fast depletion rate, we conclude that this 11-cis retinyl ester pool is the primary source of the visual chromophore for cone pigment regeneration.

Heads from freshly decapitated adult chicken (Gallus domesticusL.; ∼1 kg; from Tyson Foods, Inc., Seguin, TX, USA) were immediately placed on ice and transported in the dark (2 h) to the research laboratory. For light adaptation, eyelids were removed, and heads (on ice) were exposed to bright illumination (about 2000 lux from two 90 W halogen bulbs at 35 cm from sample). After light or dark adaptation, retina was dissected free of the RPE(i.e. retinae containing visible RPE contamination were discarded) under light or dim red light, respectively. Ocular tissues were homogenized in Tyrode's avian buffer (containing, in g l^-1, NaCl 8.0; KCl 0.2:MgCl₂ 0.05; CaCl₂ 0.2; NaH₂CO₄0.33; NaHCO₃ 1.0; glucose 1.0; Wilson, 1979) under dim red light immediately after dissection. Adult chicken retinae contained 3.10±0.11 mg (N=40) of total protein and, for RPE, 0.9±0.14 mg(N=32).

Retinae were dissected (under dim red light) from dark-adapted (4 h) adult chicken eyes (from Tyson Foods, Inc.). Tissue was placed in Tyrode's avian buffer and exposed to different durations of light exposure (bleached 80%visual pigments; ∼2500 lux from two 90 W halogen bulbs at 30 cm from sample) in vitro. Tissues were homogenized in Tyrode's avian buffer under dim red light.

Young chickens (∼60 g) were purchased from Producer's Coop (Seguin,Texas, USA) and dark adapted overnight before being exposed to two 90 W halogen bulbs (bleached 70% visual pigments; about 2000 lux) for the indicated durations. Chicken were anesthetized by CO₂, decapitated, and,after eye removal, retina was dissected free of RPE. Tissues were homogenized in Tyrode's avian buffer under dim red light.

Ocular tissues were homogenized in 10 mmol l^-1 Tris-HCl (pH 7.5), and retinoids were extracted by the addition of ethanol and hexane. Retinyl esters were analyzed by gradient HPLC (0.2 to 10% dioxane in hexane, 2 ml min^-1; 4.6×250 cm, 5 μm silica column) according to the method described by Mata et al.(2002; see retinyl esters in Fig. 1). Gradient HPLC results indicated no detectable amounts of retinol in light- or in dark-adapted chicken retina or RPE. Therefore, hexane extracts prepared from retina and RPE samples in subsequent experiments were first saponified in strong base (0.33 mol l^-1 KOH in ethanol), and the retinol products (derived from retinyl ester) were analyzed by isocratic HPLC (717plus Waters autosampler,Waters 515 pump with a photodiode array detector; Milford, MA, USA) with 10%dioxane/hexane eluting through a 4.6×250 cm, 5 μm silica column at 2 ml min^-1 (i.e. retinyl esters in Figs 2, 3). To measure 11-cisand all-trans retinals, retinoids were extracted by the formaldehyde method (Suzuki et al., 1988; Okajima and Pepperberg, 1997)as well as by the hydroxylamine method(Groenendijk et al., 1980)before being analyzed on isocratic HPLC (same HPLC system except eluting at 1%dioxane/hexane at 2 ml min^-1). Both methods yielded similar results. 11-cis retinyl esters and retinols were monitored at 318 nm,and all-trans retinyl esters and retinols were monitored at 325 nm. Retinals were monitored at 365 nm. Retinoids were identified by comparison of their retention times to authentic standards, and their online photodiode array UV spectra. Quantification was performed by comparing peak areas of retinoids with those from calibration curves obtained from authentic standards(Waters Millennium Software).

Fig. 1A shows HPLC tracing of total retinyl esters extracted from the chicken retina after 2 h of dark adaptation (top) and 2 h of light adaptation (bottom). Only 11-cisretinyl ester (peak I with a retention time of 5.8 min; Fig. 1A) was eluted from gradient HPLC when the absorbance (in optical density units) was monitored at 318 nm. Peak I coeluted with authentic 11-cis retinyl palmitate standard and also exhibited its UV spectrum (see inset of Fig. 1A). Light adaptation resulted in an approximately 6-fold increase in the height of peak I,suggesting that light induces a significant increase in 11-cisretinyl ester in the retina (see also Fig. 1C).

By contrast, Fig. 1B shows that the primary isomer in the light- and dark-adapted RPE is all-trans retinyl ester (peak II with a retention time of 6.5 min,monitored at 325 nm; Fig. 2B). A photodiode array spectrum shows that peak II has an absorption maximum of 325 nm, with an inflection at 323 nm, which is consistent with the spectrum of pure all-trans retinyl ester. Peak II also co-eluted with authentic all-trans retinyl palmitate, further confirming the identity of this HPLC component. Light exposure resulted in a 3-fold increase in the height of peak II, suggesting that light significantly increased the quantity of this all-trans retinyl ester in the chicken RPE.

When the same experiment was repeated by sampling at multiple time points under light and dark conditions (see Fig. 1C,D), the 11-cis retinyl ester pool in the retina increased nearly 6-fold in 2 h (open circles, Fig. 1C), while subsequent dark adaptation induced a rapid depletion of this pool to its original level within 1 h. Meanwhile, the all-trans retinyl ester pool in the retina remained relatively low and unchanged (filled circles, Fig. 1C). In distinctive contrast, upon illumination, the all-trans retinyl ester pool in the RPE increased about 3-fold (filled circles, Fig. 1D) while the 11-cis retinyl ester pool remained low (open circles, Fig. 1D). During the dark phase of these experiments, the rate of depletion of all-trans retinyl esters in the RPE was significantly lower than the depletion rate of the 11-cis retinyl esters in the retina (compare Fig. 1C,D).

A summary of measured rates of accumulation and depletion of the retinyl ester pools in the retina and RPE is shown in Table 1. During light and dark adaptation, the 11-cis retinyl ester pool in the retina accumulated(1.17 nmol h^-1) and was depleted (-2.45 nmol h^-1) more rapidly than the all-trans retinyl ester pool in the RPE, which increased during light adaptation at 0.32 nmol h^-1 and decreased during dark adaptation at -0.27 nmol h^-1. This suggests that the 11-cis retinyl ester pool was preferentially utilized for chromophore regeneration in dark conditions.

When monitored at wavelengths of 325 nm and 318 nm, small amounts of additional 11-cis and all-trans retinyl esters (i.e. other than 11-cis and all-trans retinyl palmitates) were noted. Although they constituted a small portion of the total retinyl esters (i.e. less than 10% total), it would be appropriate to include them in our subsequent experiments to study the change in retinyl esters with time. Therefore, retinoid extracts were first saponified and the released retinols were measured isocratically by HPLC (as explained in the Materials and methods). Results from gradient HPLC in previous experiments did not reveal any significant level of free retinols in the hexane extract prepared from light- or dark-adapted retina and RPE.

To demonstrate that the 11-cis retinyl ester pool in the retina was not derived from retinoids in the RPE, we first dissected dark-adapted retina free of RPE. Retinae suspended in avian buffer were then exposed to strong bleaching conditions (2500 lux from two 90 W halogen bulbs) for 0.5 and 1.0 h. Within 30 min of bleaching, the 11-cis retinyl ester pool increased ∼3-fold from ∼0.07 to 0.26 nmol mg^-1 while the 11-cis retinal pool decreased from ∼0.18 to 0.04 nmol mg^-1, indicating that ∼80% of all visual pigments were bleached in the isolated retinae in vitro(Fig. 2). This provides strong evidence to suggest that bleaching of visual pigment (i.e. decrease in 11-cis retinal) resulted in an increase in the accumulation of 11-cis retinyl esters in the retina. The accumulation of 11-cis retinyl ester upon bleaching, rather than all-transretinyl ester, also suggests strongly that an isomerase activity is present in the isolated retina. All-trans retinol was stable during dark and light conditions at ∼0.07 nmol mg^-1 while all-transretinyl ester increased from ∼0.01 to 0.1 nmol mg^-1 after 1 h(Fig. 2). The accumulation of these all-trans retinoids may be the result of an impaired retinol dehydrogenase (to convert all-trans retinal to all-transretinol) in the isolated retinae and the lack of RPE to which all-trans retinol can be transferred and esterified. Nevertheless,data from these experiments on isolated retinae clearly show that 11-cis retinyl ester was generated from within the retina. However,the type of retinal cells where 11-cis retinyl ester is synthesized or stored remains to be determined.

To define the visual cycle in the cone-dominated chicken, we quantitatively measured changes in the level of the 11-cis, all-transretinyl esters and retinals in the chicken retina and RPE at different durations of light and dark adaptation(Fig. 3). Retina was dissected free of RPE after light and dark treatments (see Materials and methods). In the fully dark-adapted state, we found an average of ∼0.56 nmol of 11-cis retinal in the retina. Because 11-cis retinal exists in a 1:1 stoichiometric ratio with cone opsin, our data are consistent with previous reports that the chicken retina contains ∼0.4-0.5 nmol of photopigments (rhodopsin and iodopsin) per eye(Wald et al., 1955; Bridges et al., 1987). Within 5 min of light exposure, the amount of 11-cis retinal dropped from∼0.5 nmol eye^-1 to 0.23 nmol eye^-1. After an additional 15 min of bleaching, only 0.15 nmol of 11-cis retinal per eye was recovered (i.e. ∼30% of the dark-adapted value). After 5 min of dark adaptation, the original level of chromophore was restored (∼0.5 nmol eye^-1). This regeneration is consistent with the kinetics of bleached cone pigment in the human eye fovea, which regenerate 100% of cone pigment within 3 min of dark adaptation after strong bleaching conditions(fig. 5 in Mahroo and Lamb,2004). During light conditions, all-trans retinal increased to ∼0.15 nmol eye^-1 from dark adapted levels of∼0.04 nmol eye^-1 (Fig. 3B). This reflects a continuing active processing of bleached chromophore to other retinoids throughout light conditions.

After 5 min of strong light conditions, the amount of 11-cisretinyl ester in the retina increased from ∼0.67 to ∼1.5 nmol eye^-1. This pool steadily increased to ∼2.1 nmol eye^-1 after 20 min of light conditions. The chicken eye, therefore,produced 11-cis retinyl esters in the retina at about 4 molar equivalent to the amount of photopigment in the eye within this short time period (see Discussion on explanation of molar excess). This pool declined rapidly within the first 5 min of dark adaptation from ∼2.1 to ∼1.0 nmol eye^-1. This depletion readily accounts for the net amount of regenerated pigment in the same time window (∼0.4 nmol 11-cisretinal per eye).

A steady increase of the all-trans retinyl ester pool in the RPE was also seen after 20 min of light adaptation (∼0.17 to ∼0.52 nmol eye^-1) (Fig. 3B). Interestingly, the net depletion of this pool within the first 5 min of dark adaptation (∼0.27 nmol eye^-1) cannot account for the increase of 11-cis retinal (∼0.4 nmol eye^-1) during the same time period, suggesting that photopigment regeneration must be dependent on a retinoid supply from the 11-cis retinyl ester pool. It should also be noted that a significant part of this all-trans ester pool may be contributing to the rod visual cycle in chicken much to the same extent that it contributes to the rod visual cycle in rat(Dowling, 1960).

Our data provide evidence that a novel light-driven cone visual cycle exists in the cone-rich chicken retina(Bustamante et al., 1995; Mata et al., 2002). Our work suggests that this cone visual cycle in the chicken retina possesses significantly different features from those in the classical rhodopsin cycle. Similar to the rhodopsin cycle, light exposure in the cone visual cycle bleaches visual pigments and significantly increases retinyl esters, while dark adaptation (subsequent to light exposure) leads to pigment regeneration and significantly reduces retinyl esters. By contrast, light exposure induces a significant accumulation of 11-cis retinyl esters in the retina,rather than all-trans retinyl esters in the RPE. Furthermore, dark adaptation (subsequent to light exposure) leads to a rapid accumulation of 11-cis retinal (5 min in chicken vs 50 min in rat),consistent with the kinetics of cone (not rod) pigment regeneration(Dowling, 1960). Results from isolated retina experiments show that the 11-cis retinyl ester pool originates within the retina. Therefore, cone chromophore regeneration is dependent on the 11-cis retinyl ester pool in the retina and is independent of the all-trans retinyl ester pool in the RPE. A schematic of retinoid partitioning in the cone retinoid cycle is provided in Fig. 4. Bleached chromophore is the main precursor for 11-cis retinyl ester, while 11-cisretinyl ester is the main precursor for regenerated cone chromophore. The exact cellular mechanism of this partitioning remains to be investigated.

Chicken possess a 4 molar equivalent of 11-cis retinyl esters to photopigment during light conditions (Fig. 3A), in stark contrast to the 1:1 molar equivalent of retinyl ester in the RPE and chromophore in the albino rat(Dowling, 1960). Further studies should elucidate the location(s) of the 11-cis retinyl ester pool(s). Interestingly, we have also observed that there is a limited capacity of this 11-cis retinyl ester pool in the retina (A. T. C. Tsin, R. T. Villazana-Espinoza and A. Hatch, unpublished results).

An estimated bleaching rate of 0.1 nmol min^-1 can be derived from data obtained during the first 5 min of bleaching in our experiments with light- and dark-adapted chicken (Fig. 3A). Assuming that this rate continues throughout light exposure,the amount of released chromophore is ∼2.0 nmol in 20 min. The 11-cis and all-trans retinyl esters in the retina increase a net 2.0 nmol during this same time period. This suggests that the 11-cis retinal released from pigment bleaching can account for the accumulation of retinyl esters.

Data from our work provides an estimated

(i.e. 50% of the total time to reach reaction maximum) of 2.5 min for 11-cisretinal regeneration in the chicken retina(Fig. 3A). A

\(t_{\frac{1}{2}}\)

of ∼1 min for the regeneration of human fovea photopigment (after a strong bleach) can be deduced from the work of Mahroo and Lamb(2004). Given that the

\(t_{\frac{1}{2}}\)

is ∼6 min for rod pigment regeneration (fig. 9 in Lamb and Pugh, 2004), our data are consistent with the kinetics of cone regeneration. It is probable that our estimated

\(t_{\frac{1}{2}}\)

of 2.5 min for chicken retinal chromophore regeneration deviates from ∼1 min(for human cone pigment regeneration) because our data are derived from a bleach of the mixed chicken photoreceptor population (i.e. 30-40% rod and 60-70% cone; see Meyer and May,1973), as opposed to the 100% fovea cone photoreceptor population used in Mahroo and Lamb's studies (Mahroo and Lamb, 2004).

The newly identified visual cycle enzymatic activities (retinol isomerase,11-cis retinyl ester synthase and 11-cis retinol dehydrogenase) in the cone-dominated retina(Mata et al., 2002), their specific locale(s) and their relationship to the kinetics described here should be the subject of future investigations. Das and colleagues were the first to suggest that Müller glia may contribute substantially to chromophore regeneration by demonstrating the isomerization of retinol by chicken Müller cells in culture (1992). Cellular retinol binding protein,cellular retinal binding protein(Bunt-Milam and Saari, 1983; Eisenfeld et al., 1985) and retinal G-protein coupled-receptor (Pandey et al., 1994) are involved in vitamin A metabolism and have been shown to be expressed in Müller cells. Müller apical processes associate with the cell bodies of photoreceptors (for a review, see Newman and Reichenbach, 1996). Interestingly, cones, but not rods, can transport 11-cis retinal from the cell body to the outer segment (Jin et al., 1994). Is it possible that Müller glia isomerize retinol and provide an 11-cis retinoid to the cone inner segment for transport to the cone outer segment (COS)? If this is the case, Müller glia may acquire additional chromophore precursor from vascular tissue through end feet processes. This mechanism would be somewhat analogous to the RPE obtaining vitamin A from the choroidal capillaries and could possibly account for the excess retinoids in the retina (4 molar equivalent of retinyl esters to photopigment) observed during light adaptation of the chicken retina.

Recently, the isomerization of all-trans retinyl ester in the RPE has been implicated to be tightly controlled by RPE65, a protein essential to this process (Redmond et al.,1998). Soluble (sRPE65) and palmitoylated (mRPE65) isoforms of RPE65 have been identified in the RPE (Ma et al., 2001). RPE65 has been shown to bind both all-trans retinyl esters (Ma et al., 2001) and all-trans retinol(Xue et al., 2004). Hypothetically, Xue et al.(2004) have suggested that, in light conditions, the influx of all-trans retinol in the RPE is shuttled to LRAT by sRPE65, where it is immediately esterified to produce all-trans retinyl palmitate. All-trans retinyl palmitate may donate acyl groups via LRAT to sRPE65, thereby generating mRPE65,which can facilitate the isomerization reaction and generate 11-cisretinol. In the retina, RPE65 protein is expressed in the mammalian and amphibian COS but is absent in rods (Ma et al., 1998; Znoiko and Crouch,2002). This suggests that RPE65 may be serving one of its proposed functions in the COS. The LRAT transcript and protein are absent from chicken retina (Mata et al., 2005). Since mRPE65 contains a higher propensity to donate acyl moieties for 11-cis retinol over all-trans retinol via LRAT(Xue et al., 2004), it may also provide this function in cone photoreceptors via 11-cisester synthase. Hypothetically, 11-cis retinol supplied by Müller glia could be stored as 11-cis retinyl ester in the COS during constant light conditions. Conversely, if sRPE65 is in the COS, it may be shuttling retinol to another location for isomerization. Further studies are needed to address these questions.

We thank Dr Don Allen, Dr Nathan Mata, Dr Jian-xing Ma, Dr Vijay Sarthy and Dr John E. Dowling for critical review of this manuscript. This study was supported by NIH grant GM 08194 and grant GM 07717.