The photoreceptor cells and RPE cells are intermittently exposed to potentially adverse conditions such as light, high oxygen consumption, active fluxes of polyunsaturated fatty acids, and the overall active metabolism of these cells. These conditions trigger reactive oxygen species formation in abnormal quantities, as well as lipid peroxidation. It is remarkable that cellular integrity can be maintained in photoreceptor and RPE cells for several decades, so long as homeostasis is not broken (38
). Among the factors sustaining homeostasis are antioxidants, including the carotenoids zeaxanthin and lutein, which accumulate in the macula. Similar risks of cellular damage occur in the brain, where the relationship of neurons to astrocytes is extensive. This is particularly evident in the synapses, which are literally “wrapped up” by astrocytes. In addition, astrocytes are part of the neurovascular unit and participate in the retrieval of DHA from the bloodstream.
We designed experiments to test the potential significance of NPD1 in the photoreceptor/RPE cell relationship. Using an in vitro cellular model, we mimicked what may occur in the eye when homeostasis is challenged by exposure to oxidative stress.
RPE cells fed with bovine outer segments become more resistant to oxidative stress than cells that do not phagocytize rod outer segments (). Neither outer segments nor microspheres alone trigger Hoechst positive cells; outer segments combined with oxidative stress markedly decrease oxidative-stress-induced apoptosis, unlike microspheres.
Figure 3 Photoreceptor outer segment phagocytosis attenuates oxidative stress-induced apoptosis with concomitant synthesis of NPD1. ARPE-19 cells were plated and maintained in culture for 72 h followed by 8 h of serum starvation before the addition of photoreceptor (more ...)
The RPE cell recycles DHA from phagocytized disc membranes back to the inner segment of the photoreceptor cell through the interphotoreceptor matrix (31
). Thus, the bulk of DHA in RPE cells is a component of photoreceptor disc membrane phospholipids that, after phagocytosis, is recycled as a part of outer segment renewal. The RPE cell contributes to enrich photoreceptor cells in DHA by taking up this fatty acid from the bloodstream through the choriocapillaris (40
). Since DHA is the initial precursor of NPD1 synthesis, we explored the hypothesis that part of the DHA arriving during the photoreceptor renewal may be used for NPD1 synthesis. Free DHA accumulates in RPE cells and media, 6 h after the onset of phagocytosis, and oxidative stress results in further increases of free DHA (35
). This approach has identified a remarkable phagocytosis-dependent NPD1 synthesis in the presence of oxidative stress. Rod outer segment tips are the biologically relevant ligand for the retinal pigment epithelium. When RPE cells undergoing photoreceptor phagocytosis were subjected to oxidative stress, accumulation of NPD1 was observed (no accumulation was observed). This NPD1 increase was several times higher than that observed in RPE cells that phagocytized microspheres, amounting to a 36.6-fold NPD1 increase for the outer segment-treated cells after oxidative stress. Control cells showed only a 15-fold increase after oxidative stress (). This enhanced synthesis of NPD1 after outer segment phagocytosis is concomitant with outer segment-induced attenuation of oxidative stress-mediated apoptosis (). Although ARPE-19 cells also phagocytized, the biologically inert polystyrene microspheres during these studies, NPD1 content was not affected in RPE cells or in the incubation media (). Moreover, although oxidative stress did stimulate NPD1 accumulation, this was also not affected by microsphere phagocytosis. These results correlate with the observed lack of cytoprotection offered by microsphere phagocytosis (). In addition, outer segment-mediated retinal pigment epithelium protection against oxidative stress, with concurrent NPD1 synthesis, takes place in low passage primary human RPE cells, prepared from human eyes supplied by the National Development and Research Institutes, Inc. (Bazan NG, et al., unpublished studies).
Unlike non-specific, non-biological ligand microsphere phagocytosis, outer segments also have been reported to trigger early-response gene induction in the retinal pigment epithelium (43
), including COX-2 (44
) and peroxisome proliferator-activated receptor gamma (PPARγ) expression (45
). Whether any of these events are related to the NPD1 survival signaling described here remains to be ascertained.
We simultaneously measured free DHA pool size by LC-MS/MS, and found that it increases as a function of time of exposure to oxidative stress in ARPE-19 cells (35
). Free DHA in cells showed a moderate increase after 6 h when cells were subjected only to outer segment phagocytosis (10.5-fold increase). Oxidative stress, however, strongly enhanced free DHA accumulation in a time-dependent fashion, peaking at 16 h. Interestingly, although there was an overall 10-fold increase, outer segment phagocytosis kept the DHA pool size at a constant 2.4-fold increased level. This implies that NPD1 synthesis reflects an event other than enhanced, overall availability of free DHA upon phagocytosis. There is a correlation between increases in free DHA pool size and increases in NPD1 synthesis. Outer segment phagocytosis stimulates NPD1 synthesis at 3–6 h in cells and accumulation in media after 16 h (), while free DHA increases earlier and keeps accumulating up to 16 h. These enhancements in DHA and NPD1 pool size are much larger when outer segment phagocytosis takes place on RPE cells exposed to oxidative stress. Interestingly, microsphere phagocytosis does not cause enhanced changes in DHA (35
) and NPD1 (). Thus, a very specific DHA pool may be the precursor for NPD1. The studies illustrated in demonstrate a correlation between enhanced free DHA and increased NPD1 content in ARPE-19 cells undergoing oxidative stress. ARPE-19 cells incubated with 2
-DHA show that 2
-NPD1 is formed. This approach allows us to follow DHA conversion specifically because the deuterium is on the methylene carbons 21 and 22, which are not metabolically altered. Also, the products are heavier than the same non-deuterated molecule (by a mass unit of 1) and can be detected by tandem mass spectrometry (35
). illustrates the characterization of 2
-NPD1 (negative molecular ion m/z 364.2), as well as endogenous non-deuterated NPD1 (negative molecular ion m/z 359.2). These observations support the notion that as free DHA accumulates in the ARPE-19 cells during photoreceptor phagocytosis, it is a substrate for NPD1 synthesis.
Figure 4 Deuterated DHA, added to the ARPE-19 cell medium, is incorporated and converted to deuterated NPD1. (A) Time course of synthesis of d5-NPD1 in ARPE-19 cells and medium, after 100nM [2H5] DHA was added to cells at the onset of oxidative stress. Results (more ...)
AA is also a precursor of bioactive lipids, including prostaglandins and lipoxygenase products, which have been correlated with photoreceptor phagocytosis (46
). That AA is released under the present experimental conditions (data not shown), led us to explore some of the AA cascade members. We found that lipoxin A4
) HETE, and 15(S
) HETE were unchanged during outer segment phagocytosis (35
), thereby suggesting that NPD1 is selectively synthesized during this fundamental event that underlies photoreceptor cell renewal essential to sustaining its integrity.