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From the Departments of 1 Ophthalmology, 5 Cell Biology, and 6 Biochemistry and Molecular Biology, and 2 Oklahoma Center for Neuroscience, University of Oklahoma Health Sciences Center, Oklahoma City; and 3 Dean A. McGee Eye Institute, Oklahoma City, Oklahoma; and 4 James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York.
Abstract
PURPOSE. Dogs affected with progressive rod-cone degeneration (prcd) have reduced levels of docosahexaenoic acid (DHA, 22:6n-3) in their plasma and rod photoreceptor outer segments (ROS). Dietary supplementation of DHA has failed to increase the ROS DHA levels to that of unaffected control dogs. The present study was undertaken to test the hypothesis that prcd-affected dogs have a reduced capacity for the synthesis and/or release of DHA in retinal pigment epithelial (RPE) cells.
METHODS. RPE cells (first passage cultures) from prcd-affected and normal dogs were incubated with [3H]eicosapentaenoic acid (EPA, 20:5n-3) for 24 and 72 hours. After incubation, the radiolabeled fatty acids in the cells and media were analyzed.
RESULTS. DHA and all its metabolic intermediates were detected in RPE cells from prcd-affected and normal dogs. No significant difference was found in the amount of products (including DHA) synthesized between normal and affected RPE cells at either time point. In the culture media, RPE cells from prcd-affected dogs released significantly more DHA than cells from normal dogs after 72-hour incubation, but not after 24-hour incubation.
CONCLUSIONS. RPE cells from prcd-affected dogs can synthesize and release DHA at least as efficiently as cells from normal dogs. Therefore, synthesis of DHA from its precursor and its release from RPE cells does not appear to contribute to the reduction in ROS DHA levels found in prcd-affected animals.
Although gene mutations for photoreceptor-specific proteins are generally considered the primary causative factor for retinitis pigmentosa, abnormalities in systemic levels of essential long-chain polyunsaturated fatty acids, particularly docosahexaenoic acid (DHA, 22:6n-3), frequently have been reported to be associated with various forms of retinitis pigmentosa in humans. Dogs with progressive rod-cone degeneration (prcd) have reduced DHA levels in the plasma,1 similar to the findings in humans with retinitis pigmentosa, and also have lower DHA levels (approximately 20%) in their rod photoreceptor outer segments (ROS).2 Although ROS DHA levels in humans with retinitis pigmentosa have not yet been examined, pronounced reduction in DHA levels has been observed in the sperm (the only other body fluid or tissue in the body, besides retina and brain, that is enriched in DHA) of male patients affected with retinitis pigmentosa.3 Thus, the prcd-affected dogs are an excellent animal model for the human disease to study the mechanisms underlying the lower DHA phenotype and its cause-and-effect relationship with the disease process.
In an effort to correct or slow down the progressive loss of photoreceptor cells, we previously gave daily DHA supplements to prcd-affected dogs for 5 months and assessed subsequently the prcd disease phenotype.2 We found that, although DHA supplementation produced a sustained elevation in plasma and liver DHA levels, the ROS DHA levels in the affected animals remained lower than those in control dogs, and the prcd disease phenotype was not changed by this dietary manipulation. These results suggest that the synthesis of DHA-containing phospholipid molecular species in the retinas or the delivery of DHA from circulation to the retina could be downregulated in the affected animals.
In the present study, we examined the possible contribution by retinal pigment epithelial (RPE) cells to the lower ROS DHA phenotype. Our study indicates that RPE cells from affected animals have a similar capability for synthesis of DHA from eicosapentaenoic acid (20:5n-3) and for release of DHA to the surrounding media, compared to those cells from normal animals.
Materials and Methods
Animals
The dogs used in this study were derived from miniature poodles
and were homozygous normal or affected with the prcd
mutation; the dogs were bred and raised as part of a colony supported
by an NEI/NIH grant (EY06855, "Models of Hereditary Retinal
Degeneration"). All dogs were maintained in the same animal care
facility (Retinal Disease Studies Facility, Kennett Square, PA) with
controlled cyclic illumination (12-hour light–dark). Four
prcd-affected animals (2–3 months old) and three
age-matched normal dogs were used for the studies. The
prcd-affected animals at this age show no morphologic
evidence of retinal disease. All studies described were performed in
compliance with ARVO Resolution on the Use of Animals in Ophthalmic and
Vision Research and followed protocols approved by the animal protocol
review committees of Cornell University and University of Oklahoma
Health Sciences Center. The dogs were euthanatized with a barbiturate
overdose, and the eyes were enucleated and transported to the
laboratory in a chilled Puck F saline with calcium and antibiotics.
RPE Cultures
RPE cells from normal and prcd-affected animals were
isolated as previously described.4
Briefly, after lens,
vitreous, and retinas were removed aseptically, the RPE cells were
released by repeated trypsinization. Dissociated cells were placed in
DMEM medium containing 15% fetal bovine serum (FBS) and plated in
35-mm dishes at a density of 2 x 105
cells/dish. The cells were allowed to attach to the plate and grow. At
confluence, RPE cells were subcultured (1:3 ratio) by trypsinization.
The confluent first passage (P1) cultures from both normal and affected
RPE cells were used for the following isotope incubations.
Incubation Conditions
Elongation and desaturation of [3H]20:5n-3
(American Radiolabeled Chemicals, St. Louis, MO) were studied in
confluent P1 RPE cultures grown in HL-1 medium (Hycor Biomedical,
Irvine, CA) containing 10% FBS on 35-mm dishes. Incubations were
carried out for 24 and 72 hours in duplicate with 2 ml culture medium
containing 15 µCi (0.75 nmol) of [3H]20:5n-3
(conjugated with delipidated bovine serum albumin [BSA] at a molar
ratio of 2:1). The cells were maintained at 37°C in an incubator
containing 5% CO2. At the end of the incubation
period, the media were removed from the cells and centrifuged to remove
cellular debris; the cells were rinsed twice with ice-cold HL-1 medium
containing 10 µM fatty acid–free BSA and harvested by
trypsinization. Both cells and clarified media were stored at –80°C
until analysis.
Lipid Extraction and Saponification
Before lipid extraction, the culture media were centrifuged at
100,000g for 1 hour to remove subcellular membrane
contaminants. Total lipids from the cells and media were extracted
twice with chloroform/methanol. Lipid extracts were dried under
nitrogen and suspended in a known volume of ethanol. Aliquots were
taken for total radioactivity determination; the remaining lipid
extracts were suspended in 1 ml of 2% KOH (wt/vol) in ethanol and
saponified at 100°C for 30 minutes. After cooling to room
temperature, 1 ml water and 75 µl concentrated HCl were added, and
the released free fatty acids were extracted three times with 2 ml
hexane.
HPLC Analysis of Fatty Acids
The extracted free fatty acids were converted to phenacyl esters
and separated by high-performance liquid chromatography (HPLC) on a
Sulpeco LC-18 column (25 cm x 4.6 mm).5
The fatty
acid phenacyl esters were eluted at a flow rate of 2 ml/min with a
linear gradient of acetonitrile/water from 80/20 (vol/vol) to 92:8
(vol/vol) for 45 minutes, followed by holding at 92:8 (vol/vol) for 10
minutes and returning to 80:20 (vol/vol) for 5 minutes. The
radioactivity profile was monitored by online scintillation counting
(Flo-one A250; Radiomatic, Tampa, FL) using Ultima-Flo M (Packard
Instrument, Downers Grove, IL) at 2.5:1 (vol/vol) ratio of cocktail to
mobile phase. The phenacyl esters were monitored at an absorbance of
242 nm. Identities of individual fatty acids have been previously
determined5
and further confirmed by catalytic
hydrogenation and HPLC analysis of hydrogenated products.
Results
RPE cells from normal and prcd-affected dogs grew well in culture and maintained a nice, distinguishable epithelial cell morphology. The affected cells appeared the same as normal cells, except that they grew and reached confluency slightly faster than normal cells.
Labeling in the Cells
The labeling profiles from both normal and affected RPE cells
after 24- and 72-hour incubation with
[3H]20:5n-3 are shown in Figure 1 . Elongation and desaturation products of 20:5n-3, namely 22:5n-3,
24:5n-3, 24:6n-3, and 22:6n-3, were all detected. The identities of
these labeled compounds were confirmed by HPLC analysis of their
respective hydrogenation products (data not shown).
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Discussion
Dogs with prcd have abnormalities in long-chain polyunsaturated fatty acid metabolism, with reduced DHA levels (approximately 20%–25%) in plasma and ROS and slightly elevated levels in the liver, compared to unaffected dogs.1 2 The mechanism for the reduction in ROS DHA levels in prcd-affected animals is not clear. It was initially thought that synthesis of DHA in the liver and/or transport to the target tissues could be affected. We tested this hypothesis previously and tried to bypass the possible defect in DHA production in the liver by providing preformed DHA to affected and normal dogs for up to 5 months.2 We found that supplementation caused a rapid and sustained elevation in plasma DHA levels in both groups, but had no effect on the progression of the disease in prcd-affected animals. The prcd-affected dogs fed a DHA-enriched diet still had significantly lower DHA levels in ROS compared to control dogs.2 In dogs supplemented with DHA for up to 24 days, levels of DHA were lower in ROS and higher in the liver from prcd-affected dogs. These studies indicate that the lower content of ROS DHA in prcd-affected dogs is not the result of a dietary deficiency of DHA and its precursor fatty acids. Besides a possible problem in packaging DHA into lipoproteins in the liver,6 these results suggest the possibility that the problem in prcd-affected animals could be in DHA trafficking between the blood and the retina, possibly involving passage through RPE cells.
In this study, we investigated a possible defect in RPE of
prcd-affected dogs in the synthesis and release of DHA to
the extracellular space, by comparing these two activities in the RPE
of prcd-affected and normal animals. This is important
because RPE cells could use 22:5n-3, which is slightly elevated in the
plasma of affected animals,1
to synthesize DHA to
compensate for the reduced DHA levels in ROS of
prcd-affected animals. RPE cells of frogs have been shown to
readily synthesize DHA from its precursors.7
Also, because
RPE cells are the major nutrient supplier for the photoreceptors and
play central roles in recycling of DHA from daily shed ROS tips back to
photoreceptors,8
9
it is conceivable that alterations in
DHA metabolism in the RPE could affect photoreceptor DHA content. We
found that RPE cells from both prcd-affected and normal dogs
can elongate 20:5n-3 to 22:5n-3 and further convert 22:5n-3 to 22:6n-3
through Sprecher’s 
4-desaturase independent pathway10
(22:5n-3 
24:5n-3 24:6n-3 22:6n-3). Rates for DHA synthesis
appeared only slightly higher for affected RPE compared to normal
cells. Thus, in agreement with our previous in vivo
studies,11
there is no defect in DHA synthesis in the RPE
of prcd-affected dogs. Furthermore, under our experimental
conditions, RPE cells from prcd-affected animals showed no
apparent defect in releasing DHA, inasmuch as the level of DHA in the
culture media from affected cells was clearly not lower than that from
normal cells.
It is noteworthy that the labeling pattern in the culture media did not simply reflect that of the cells. When the distribution of radioactivity was compared, clearly more DHA and less 22:5n-3 was found in the media than in the cells. Thus, dog RPE cells appeared to favor the release of DHA over 22:5n-3, although both are similar in structure, with 22:5n-3 having one less cis double bond. Evidently the types of fatty acids in the media are not the result of equilibration, but rather of cellular regulation, which also has been demonstrated in retinal and cerebral microvascular endothelial cells.12 13
In light of our earlier2 11 14 and present studies on prcd-affected dogs, it is possible that lower ROS DHA levels could be due to cellular upregulation of DHA catabolism and/or downregulation of DHA incorporation into membranes to reduce disease-originated metabolic (perhaps oxidative) stress. DHA provides optimal lipid environment for rhodopsin and probably other membrane proteins in photoreceptors and thus is essential for optimal photoreceptor function. On the other hand, DHA is highly unsaturated (six double bonds) and easily peroxidized, especially in the photoreceptor cells, where high levels of oxygen and unsaturated fatty acid content and light exposure provide the ideal environment for lipid peroxidation. Toxic factors from lipid hydroperoxides derived from DHA could affect photoreceptor enzyme activities and damage photoreceptor membranes, as has been reported for light damage in albino rats.15 16 17 Biological adaptation, such as shortened ROS length, reduced levels of rhodopsin and DHA-containing glycerolipids, and increased antioxidants vitamin E and vitamin C and glutathione-dependent enzyme activities, as well as neurotrophic factors, in response to light stress has been shown in the retinas of rats raised in bright cyclic light compared to controls raised in dim cyclic light.16 17 18 19 Similarly, oxidative stress achieved through the stimulation of endogenous oxidant generation in human spermatozoa can cause DNA fragmentation and loss in their capacities for movement and oocyte fusion.20 Therefore, it is possible that reduction in DHA levels in retinas and plasma could be part of a biological adaptation to metabolic stress, possibly oxidative, caused by different mutations in retinitis pigmentosa. We currently are testing the hypothesis that mutations in a number of genes encoding retina-specific proteins can cause lower DHA phenotypes.
Footnotes
Supported by the National Institutes of Health (Grants EY06855, EY00871, and EY04149); Research to Prevent Blindness, Inc.; The Foundation Fighting Blindness; Samuel Roberts Noble Foundation; and Presbyterian Health Foundation. REA is a Senior Scientific Investigator for Research to Prevent Blindness, Inc.
Submitted for publication December 10, 1998; revised April 7, 1999; accepted May 5, 1999.
Proprietary interest category: N.
Corresponding author: Robert E. Anderson, Dean A. McGee Eye Institute, 608 Stanton L. Young Boulevard, Room 409, Oklahoma City, OK 73104. E-mail: robert-anderson@ouhsc.edu
References
This article has been cited by other articles:
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  N. G. Bazan Synaptic lipid signaling: significance of polyunsaturated fatty acids and platelet-activating factor J. Lipid Res., December 1, 2003; 44(12): 2221 - 2233. [Abstract] [Full Text] [PDF]
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 B. Mirnikjoo, S. E. Brown, H. F. S. Kim, L. B. Marangell, J. D. Sweatt, and E. J. Weeber Protein Kinase Inhibition by omega -3 Fatty Acids J. Biol. Chem., March 30, 2001; 276(14): 10888 - 10896. [Abstract] [Full Text] [PDF]
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