Light Exposue Activates Retina Ganglion Cell Lysophosphatidic Acid Acyl Transferase and Phosphatidic Acid Phosphatase by a c-fos-Dependent Mechanism

Authors

  • G. A. de Arriba Zerpa,

    1. CIQUIBIC (CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba * INIBIBB (CONICET), Universidad Nacional del Sur, Bahia Blanca, Argentina
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  • M. E. Guido,

    1. CIQUIBIC (CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba * INIBIBB (CONICET), Universidad Nacional del Sur, Bahia Blanca, Argentina
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  • D. F. Bussolino,

    1. CIQUIBIC (CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba * INIBIBB (CONICET), Universidad Nacional del Sur, Bahia Blanca, Argentina
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  • S. J. Pasquare,

    1. CIQUIBIC (CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba * INIBIBB (CONICET), Universidad Nacional del Sur, Bahia Blanca, Argentina
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  • P. I. Castagnet,

    1. CIQUIBIC (CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba * INIBIBB (CONICET), Universidad Nacional del Sur, Bahia Blanca, Argentina
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  • N. M. Giusto,

    1. CIQUIBIC (CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba * INIBIBB (CONICET), Universidad Nacional del Sur, Bahia Blanca, Argentina
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  • B. L. Caputto

    1. CIQUIBIC (CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba * INIBIBB (CONICET), Universidad Nacional del Sur, Bahia Blanca, Argentina
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  • Abbreviations used : AT II, lysophosphatidate acyl transerase ; LPA, lysophosphatidic acid ; NEM, N-ethylmaleimide ; PA, phosphatidic acid ; PAPase, phosphatidate phosphohydrolase ; PBS, sodium phosphate buffer ; PSS, phosphatidylserine synthase ; PTA, phosphotungstic acid ; PtdCho, phosphatidylcholine ; PtdEtn, phosphatidylethanolamine ; PtdIns, phosphatidylinositol ; PtdSer, phosphatidylserine ; TCA, trichloroacetic acid.

Address correspondence and reprint requests to Dr. B. L. Caputto at Departamento de Química Biológica, Facultad de Ciencias Químicas, Pabellón Argentina, Ciudad Universitaria, 5000 Córdoba, Argentina.

Abstract

Abstract : We previously reported that the biosynthesis of phospholipids in the avian retina is altered by light stimulation, increasing significantly in anglion cells in light and in photoreceptor cells in dark. In the present work, we have determined that light significantly increases the incorporation of [3H]glycerol into retina ganglion cell glycerophospholipids in vivo by a Fos-dependent mechanism because an oligonucleotide antisense to c-fos mRNA substantially blocked the light-dark differences. We also studied in viro the enzyme activities of phosphatidate phosphohydrolase (PAPase), lysophosphatidate acyl transferase (AT II), and phosphatidylserine synthase from retinas of chickens exposed to light or dark. Higher PAPase I and AT II activities were found in incubations of retinal ganglion cells from animals exposed to light ; no increase was observed in preparations obtained from light-exposed animals reated with the c-fos antisense oligonucleotide. No light-dark differences were found in phosphatidylserine synthase activity. These findings support the idea that a coordinated photic regulation of PAPase I and AT II is taking place in retina ganglion cells. This constitutes a reasonable mechanism to obtain an overall increased synthesis of glycerophospholipids in stimulated cells that is mediated by the expression of Fos-like proteins.

Phospholipids exert functions at both the intracellular and extracellular surface of cell membranes, acting as reservoirs for the precursors of first and second messengers such as acetylcholine, eicosanoids, diacylglycerol, and inositol 1, 4, 5-trisphosphate. The pioneering work conducted by E. P. Kennedy (see review by Kennedy, 1961) identified the biosynthetic pathway for the major phospholipids, but the complex molecular and cellular processes underlying the regulation of phospholipid biosynthesis are still being elucidated in the nervous system.

Phospholipid labeling is increased by light stimulation in calf (Urban et al., 1973), Bufo arenarum (Bazan and Bazan, 1976), and rat (Anderson and Hollyfield, 1981 ; Anderson et al., 1983, 1985 ; Schmidt, 1983abc ; Birkle and Bazan, 1989) retinas in vitro. The newly synthesized membranes are disributed in the fast wave of axonal transport to the rest of the cell (Toews et al., 1979 ; Grafstein and Forman, 1980 ; Toews and Morell, 1981 ; Ledeen, 1985).

We previously proposed that phospholipid metabolism is regulated by the action of proteins from the Fos-related family of immediate early genes (Guido et al., 1996 ; Bussolino et al., 1998). This hypothesis results from observations obtained using both in vivo (Guido and Caputto, 1990) and in vitro experimental designs in which light stimulation increases 32P labeling of phospholipids in chick retina ganglion cells (Guido et al., 1996) and decreases it in photoreceptor cells (Bussolino et al., 1998) in a Fos-dependent manner. Transcription and transduction of Fos protein participate in the regulation of the rate of synthesis of phospholipids (Guido et al., 1996 ; Bussolino et al., 1998) as no light-promoted differences in phospholipid labeling can be observed either in photoreceptor cells or in ganglion cells when the expression of Fos-like proteins is specifically inhibited by means of an oligonucleotide antisense to c-fos mRNA.

For our understanding of the molecular mechanisms that underlie the regulation of phospholipid biosynthesis, it is essential to examine the activation status of phospholipid-synthesizing enzymes under conditions of light stimulation. In the present report we have determined the activity of several enzymes of the pathway of phospholipid biosynthesis in isolated retina ganglion cells obtained from stimulated and nonstimulated animals. We found that two key enzymes of this pathway—the acyl transferase that converts lysophosphatidic acid (LPA) into phosphatidic acid (PA) and the phosphatidate phosphatase that converts PA into diacylglycerol—are activated by light stimulation. Furthermore, the activation of both enzymes is dependent on the expression of c-fos as no light-dark differences are observed in the ganglion cells in the presence of an oligonucleotide antisense to c-fos mRNA. In contrast, phosphatidylserine (PtdSer) synthase (PSS) is not regulated in these cells by light stimulation.

MATERIALS AND METHODS

Animal handling and in vivo glycerol labeling

Cobb Hardig chicks were reared after hatching until 8 days of age on a 12-h light-12-h dark cycle with water and food adlibitum. The animals were then subjected to a 48-h dark adaptation period as described by Caputto et al. (1982). One hour before being separated into the light and dark groups, each animal received a 10-μl intraocular injection containing one of the following solutions : saline for the conrols ; 50 μg of an oligonucleotide antisense to c-fos (5′-TGC-GTT-GAA-GCC-CGA-GAA-3′) ; or 50 μg of the corresponding oligonucleotide sense to c-fos. When the in vivo incorporation from [3H]glycerol was to be determined, before separating the animals into the light and dark groups, a second 10-μl injection containing 8.5 μCi of [3H]glycerol was administered. Then the animals, singly caged, were exposed to 1,000 lux of light stimulation or remained in the dark for 1 h. The animals were killed, the eyes were excised, and the eye cups were immediately frozen in liquid N2. Manipulations of the animals were done under dim red light up to the step of freezing the eye cups for the dark group of animals or up to starting the light exposure for the light-stimulated group of chicks. All animal handling was done in agreement with the standards stated in the Guide to the Care and Use of Experimental Animals published by the Canadian Council on Animal Care and were approved by the local animal care committee (Exp. 15-99-39796).

Preparation of retina ganglion cells and inner nuclear layer cells

The retinal ganglion cells and inner nuclear cells were isolated from the rest of the retina as described previously (Caputto et al., 1982 ; Guido et al., 1996). In brief, the frozen eye cups were lyophilized, and the retinas were glued by their ganglion cell layer to a cellophane tape. The different cell types were separated by covering the first tape with another piece of tape, pressing, and separating both tapes. After repeating this procedure six to eight times, a cell preparation highly enriched in ganglion cells remains glued to the first tape, whereas the inner nuclear layer is obtained attached to the fourth tape.

Ganglion cells or cells from the inner nuclear layer were resuspended from the corresponding tape with 40 μl of water per retina cell layer. Homogenates were prepared by applying a 20-s sonication in a Branson Sonifier, and protein content was determined according to the method of Read and Northcote (1981).

Immunocytochemical procedures

The eyes excised from chicks from both illumination conditions were fixed for 2 h in 4% paraformaldehyde in sodium phosphate buffer (PBS ; pH 7.4) at 4°C and immersed in 30% sucrose in PBS at 4°C for 2-3 days until they sank. Manipulation of the eyes from animals in the dark was performed under a dim red light until the eyes sank. The retinas were embedded in frozen embedding compound (Tissue-Tek), and a series of 8-μm-thick transversal sections cut on a cryostat were mounted onto polylysine-coated slides. The sections were pretreated with 5% albumin in PBS and incubated for 2 h at room temperature with a polyclonal antibody against c-Fos (Bussolino et al., 1998) diluted 1 : 100. The slices were then incubated for 2 h at room temperature with crystalline tetramethylrhodamine isothiocyanate-conjugated anti-rabbit IgG. After washing, coverslips were mounted in Fluorsave Reagent (Calbiochem) and observed for epifluorescence on a Zeiss Axioplan microscope with the filter for the rhodamine fluorochrome.

In vitro incorporation of 32P

In vitro labeling of phospholipids was determined as described previously (Guido et al., 1996). In brief, reaction mixtures contained, in a final volume of 1 ml, 140 mM NaCl, 4.5 mM KCl, 0.5 mM MgCl2, 5.6 mM glucose, 0.2 mM K2HPO4, 75 mM Tris-HCl, and 50 μCi of [32P]orthophosphate. Reactions were started by addition of 100-250 μg of protein from ganglion cell homogenates and were incubated for 1 h at 37°C. Reactions were stopped by addition of 1 ml of 10% trichloroacetic acid (TCA) conaining 1% phosphotungstic acid (PTA). Phospholipid 32P labeling was determined using the TCA-PTA method as described by Guido and Caputto (1990). In brief, TCA and PTA were added to the reactions to a final concentration of 5 and 0.5%, respectively. Suspensions were centrifuged for 15 min at 1,000 g, the supernatants were discarded, and the pellets were resuspended in 5% TCA-0.5% PTA. This procedure was repeated five times, the pellets were washed once with water, and lipids were extracted from the pellet with chloroform/methanol (2 : 1, vol/vol). Radioactivity was determined in the dried extracts by liquid scintillation.

In vitro determination of acyl-CoA acyltransferase activity

The activity of ganglion cell acyl-CoA acyltransferase was determined by measuring the incorporation of [14]oleate from [14C]oleoyl-CoA into exogenous LPA acceptors as described by Castagnet and Giusto (1997). In brief, the standard incubation medium contained 1 mM MgCl2, 10 mM ATP, 60 mM Tris-HCl (pH 7.8), [14C]oleoyl-CoA (0.1 μCi per assay) diluted to 20 μM with unlabeled oleoyl-CoA, 150 μM LPA, and 200 μg of ganglion cell protein in a final volume of 250 μl. The reaction was started by addition of the radioactive substrate or of the ganglion cell preparation. The assay mixture was sonicated for 30 s and then incubated for 5 min in a shaking bath at 37°C. The reaction was stopped by addition of 5 ml of chloroform/methanol (2 : 1, vol/vol). Blanks prepared by using membranes boiled for 5 min and incubated as described above yielded an activity <0.5% of the activity observed in the experimental samples with addition of exogenous acceptors. Lipids were extracted by the method of Folch et al. (1957), and the lipid extract was dried under N2, resuspended in chloroform/methanol (2 : 1 vol/vol), and spotted on silica gel H plates. The chromatograms were developed in a solvent system containing chloroform/methanol/acetic acid/water (50/37.5/3.5/2, by volume) and visualized by exposing the plates to iodine vapors. The spots corresponding to phosphatidylethanolamine (PtdEtn), PtdSer, and phosphatidylinositol (PtdIns) were scraped off, and radioactivity was determined by liquid scintillation.

Determination of phosphatidate phosphohydrolase (PAPase) I and II activities in isolated ganglion cell preparations

The PAPase I and II activities present in the ganglion cell preparations obtained from chicks exposed to light or maintained in the dark were determined by measuring the rate of release of 1,2-diacyl-[2-3H]glycerol from [3H]PA as described by Pasquaré and Giusto (1993). PAPase activities were differentiated on the basis of Mg2+ dependency and N-ethylmaleimide (NEM) sensitivity (Jamal et al., 1991). In brief, PAPase Mg2+ -dependent activity was determined in a medium containing, in a final volume of 0.5 ml, 50 mM Tris-maleate buffer (pH 6.4), 1 mM dithiothreitol, 1 mM Mg2+, and 200 μg of ganglion cell preparation protein. The reaction was started by adding 0.6 mM [3H]PA (0.1-0.2 μCi/μmol) plus 0.4 mM dipalmitoyl PtdCho prepared as a sonicated dispersion. Reactions were incubated for 30 min at 37°C. The assay for Mg2+ -independent forms of PAPase was carried out in the presence of 1 mM EDTA plus 1 mM EGTA. The difference between PAPase activity in the presence of Mg2+ and the PAPase activity in the presence of 1 mM EDTA plus 1 mM EGTA was taken as Mg2+ -dependent activity.

For the determination of NEM-insensitive PAPase activity, each assay contained 50 mM Tris-maleate buffer (pH 6.5), 1 mM dithiothreitol, 1 mM EDTA, 1 mM EGTA, 4.2 mM NEM, and 200 μg of ganglion cell preparation protein. The reaction was started by adding 0.6 mM [3H]PA (0.1-0.2 μCi/μmol). NEM-sensitive activity was determined in the same medium containing 1 mM Mg2+ but in which NEM was omitted. Parallel incubations were performed after preincubating the enzyme preparations for 10 min with 4.2 mM NEM. The reaction was started by adding 0.6 mM [3H]PA plus 0.4 mM PtdCho. The difference between the two activities was taken as NEM-sensitive PAPase. Assays were carried out at 37°C for 30 min and stopped by addition of chloroform/methanol (2 : 1, by volume). Blanks were prepared identically except that the membranes were boiled for 5 min before use.

For PAPase activity product determination, neutral lipids were separated by gradient-thickness TLC on silica gel G (Giusto and Bazan, 1979) and developed with hexane/diethyl ether/acetic acid (36 : 65 : 1.1, by volume) as the first solvent system and hexane/diethyl ether/acetic acid (20 : 80 : 2, by volume) as the second solvent system. PAPase activity was expressed as the sum of labeled monoacylglycerol plus diacylglycerol per hour per milligram of protein. PAPase I activity was very similar irrespective of the method used for its determination. Consequently, the mean of activity was calculated from the pooled data from both methods.

Determination of PSS I and II activities in ganglion cell preparations

The PSS I and II activities present in ganglion cell preparations obtained from chicks exposed to light or maintained in the dark was determined by measuring the rate of incorporation of [3H]serine into the endogenous PtdCho and PtdEtn phosphatidyl donors in vitro as described by Vance and Vance (1988). In brief, the incubation system contained, in a final volume of 0.5 ml, 10 mM CaCl2, 4 mM hydroxylamine, 25 mM HEPES (pH 7.4), 25 mM 0.01% Triton X-100, 0.4 mM [3H]serine (25 μCi/μmol), and 100 μg of ganglion cell protein. Incubations were carried out for 30 min at 37°C, and the reaction was stopped by addition of 0.5 ml of TCA-PTA (10% and 1%, respectively). 3H-Phospholipid labeling was determined by the TCA-PTA method (Guido and Caputto, 1990).

Statistical analysis

Experimental data were analyzed throughout using the Student’s two-tailed t test.

RESULTS

fos Expression and 32P labeling of phospholipids in vitro

The in vitro labeling of phospholipids with 32P was determined in ganglion cells isolated from retinas obtained from dark-adapted chicks that received an intraocular injection containing 50 μg of a c-fos mRNA antisense oligonucleotide into one eye (antisense) and 50 μg of the corresponding sense oligonucleotide into the other eye (sense). One hour after oligonucleotide administration, the animals were exposed to a 1-h period of a 1,000-lux light stimulation (light) or remained for the same interval in the dark (dark). In accordance with previous results, fos expression (Bussolino et al., 1998) and phospholipid labeling (Guido et al., 1996) increased in light with respect to dark (p < 0.001 for phospholipid labeling ; Fig. 1, right panel). Activation of phospholipid labeling is dependent on c-fos expression as no light-dark differences were observed in ganglion cells either in Fos protein (Fig. 1, left panel) or in phospholipid labeling (Fig. 1, right panel) in the antisense eyes, indicating that the lack of differences between antisense light and dark were due to the specific inhibition of c-fos expression.

Figure 1.

Phospholipid labeling and c-fos expression in retina ganglion cells. Left panel : Immunofluorescent expression of c-Fos protein in light and dark retinas isolated from chicks intraocularly injected with 50 μg of an oligonucleotide antisense to c-fos (5′-TGC-GTT-GAA-GCC-CGA-GAA-3′ ; ANTISENSE) or 50 μg of the corresponding sense oligonucleotide (SENSE). Right panel : The in vitro 32P labeling of phospholipids in ganglion cells obtained from the same experimental condition as described in the left panel. Data are mean ± SD values of 10 independent determinations. *p < 0.001 as determined by Student’s two-tailed t test. The arrow points to the ganglion cell layer of the retina preparations.

FIG. 1.

fos expression and [3H]glycerol labeling of phospholipids in vivo

To determine if the increased labeling of phospholipids is due to an increase in the de novo synthesis of these lipids, the in vivo labeling of phospholipids with intraocularly administered [3H]glycerol was determined in light and dark retina ganglion cells. Table 1 shows that phospholipid labeling from [3H]glycerol in vivo was significantly higher (p < 0.005) in light than in dark. However, if 1 h before being separated into the light and dark groups, the chicks received the antisense oligonucleotide into one eye and the sense oligonucleotide into the other, no differences were found between light and dark in the labeling of phospholipids in the eye that received the antisense oligonucleotide. The same light-dark difference as in control animals that received saline was observed in the eyes that received the sense oligonucleotide. These results indicate the participation of fos in the establishment of the light-dark differences in the de novo synthesis of glycerophospholipids (Table 1).

Table 1. Effect of an intraocular injection containing a c-fos antisense or sense oligonucleotide on [3H]glycerol labeling of light and dark retina cell layers in vivoChicks received an intraocular injection containing one of the following : saline ; 50 μg of oligonucleotide. One hour later, the animals received [3H]glycerol inoculated intraocularly and were exposed to light or remained in the dark. The in vivo labeling of phospholipids was determined in ganglion cells and in inner nuclear layer cells. The same animal was used for the sense and the antisense experimental conditions, with one eye for each condition. Data are means ± SD of 22-24 individual animals processed independently. Statistical analysis was performed by Student’s two-tailed t test.
  Animal condition
Intraocular injectionCell layerDarkLight
   (cpm/μg of protein)
  1. ap < 0.005.

SalineGanglion7.01 ± 0.348.31 ± 0.35a
fos antisense oligonucleotideGanglion7.44 ± 0.357.43 ± 0.19
fos sense oligonucleotideGanglion6.91 ± 0.048.43 ± 0.64a
SalineInner nuclear7.25 ± 0.547.30 ± 0.34

TABLE 1.

To examine the specificity of the light effect with regard to other retinal cell types, glycerol labeling of phospholipids was determined in the inner nuclear cell layer in the animals injected intraocularly with [3H]glycerol. In contrast to the findings in the ganglion cell preparations, no differences were found in the inner nuclear cell layer between the lights and dark conditions (Table 1).

Activities of different phospholipid-synthesizing enzymes in dark and light

To elucidate possible steps of the pathway of synthesis of phospholipids that are regulated by light-induced fos expression, the following individual reactions of the pathway (as schematized in Fig. 2) were determined.

Figure 2.

Pathways of biosynthesis of phospholipids. Shown are the pathways of biosynthesis of the major phospholipids. Pathways not considered in the present study (via CDP-diacylglycerol) have been omitted. Bold arrows in b and c denote the light-activated steps that are dependent on Fos expression.

FIG. 2.

Acyl-CoA acyltransferase.

Acyl-CoA acyltransferase I catalyzes the first committed step in glycerophospholipid synthesis, that is, the esterification of glycerol-3-phosphate at the sn-1 position with a fatty acid to form 1-acyl-sn-glycerol-3-phosphate (LPA). LPA is further esterified at sn-2 position by 1-acyl-sn-glycerol-3-phosphate acyltransferase [LPA acyl transferase (AT II)] to form 1,2-diacylglycerol-3-phosphate (PA) (Fig. 2).

The activity of ganglion cell AT II was determined by measuring the in vitro incorporation of [14C]oleate from [14C]oleoyl-CoA into an exogenous LPA acceptor. Linear conditions with respect to time of incubation and protein concentration were previously determined using a mixture of membranes obtained from animals in light and dark conditions (data not shown).

It was found that the AT II activity in ganglion cells was significantly higher (p < 0.005) in the retina ganglion cells from the light condition compared with those from the dark condition (Fig. 3). However, if these determinations were performed in ganglion cells from antisense and sense conditions, the light-dark differences observed in the AT II activity disappeared in the antisense group (Fig. 3, antisense). In all cases, [14C]PA accounted for >90% of the reaction product as determined by TLC.

Figure 3.

Oleoyl-CoA acyltransferase (AT II) activity in retina ganglion cells. AT II activity was determined as indicated in Materials and Methods in retina ganglion cells isolated from chicks in light (open columns) and dark (solid columns) conditions. Saline, antisense, and sense were as in Table 1. Data are mean ± SD (bars) values of three independent experiments with 10 chicks in each experimental condition per experiment. *p < 0.005 as determined by Student’s two-tailed t test.

FIG. 3.

PAPase.

The next step in the pathway of conversion of PA into more complex phospholipids such as PtdCho, PtdEtn, and PtdSer is its dephosphorylation. This reaction is catalyzed by PAPases and yields diacylglycerol (arrow c of Fig. 2). Two PAPase isozymes have been described. PAPase I activity is considered to be primarily involved in lipid synthesis in the endoplasmic reticulum (Aridor-Piterman et al., 1992 ; Gomez and Muñoz et al., 1992a, b), whereas the PAPase II isozyme seems to be involved in plasma membrane phospholipid remodeling associated with signal transduction events and to cell growth and differentiation (Kai et al., 1997). PAPase I activity was detected in ganglion cells isolated both from chicks subjected to the light conditions and those from the dark condition. The activity of light was significantly higher (p < 0.005) than that of dark (Fig. 4). However, when the animals were subjected to the antisense and to the sense conditions, no light-dark differences were observed in the PAPase I in the antisense eyes (Fig. 4). In contrast, the same light-dark differences as in the control animals were observed in PAPase I activity of the sense eyes (Fig. 4).

Figure 4.

PAPase I activity in isolated retina ganglion cells. PAPase I activity was determined as indicated in Materials and Methods in retina ganglion cells isolated from dark (filled columns) and light (open columns) conditions. Saline, antisense, and sense were as in Fig. 3. Data are mean ± SD (bars) values of three independent experiments with 10 chicks in each experimental condition per experiment. *p < 0.005 with respect to dark, saline as determined by Student’s two-tailed t test.

FIG. 4.

The ganglion cell preparations used to assay PAPase I activity were also assayed for PAPase II activity, as described in MATERIALS AND METHODS, but no detectable activity was found either in the light or in the dark group.

PSS I and II.

PtdSer synthesis is believed to occur through a Ca2+-stimulated, energy-independent, base-exchange reaction (Borkenhagen et al., 1961) (Fig. 2). PSS I catalyzes the serine and choline base exchange, and PSS II catalyzes that of serine and ethanolamine. No differences were found in the in vitro rate of [3H]serine exchange with endogenous PtdCho and PtdEtn phosphatidyl donors between light and dark ganglion cell preparations (Fig. 5).

Figure 5.

PSSI I and II activities, determined in retina ganglion cells isolated from light and dark conditions as described in MATERIALS AND METHODS. Data are mena ± SD (bars) values of three independent experiments with 10 chicks in each experimental condition per experiment. No light-dark differences were observed between the two groups of animals, as determined by Student’s two-tailed t test.

FIG. 5.

DISCUSSION

Although an increasing body of evidence suggests that in growing axons, a significant fraction of the phospholipids supplied to the membrane may be synthesized locally within the axon (Vance et al., 1991, 1994), it is generally accepted that in mature neurons, phospholipids are mainly provided by the cell body, where they are synthesized and distributed to the rest of the cell by anterograde axonal transport (Ledeen, 1985 ; Marcheselli and Giusto, 1986 ; Guido and Caputto, 1990). In retina ganglion cells, the amount of lipids transported to the optic tectum is proportional to the amount synthesized in the cell soma in the retina (Guido and Caputto, 1990 ; Caputto, 1991). To approach the elucidation of how the rate of synthesis of phospholipids is regulated, we studied if light stimulation applied under physiological conditions (in vivo) is capable of modifying enzymatic activities in individual retina cell types. The activities were measured under controlled experimental conditions (in vitro) in which membrane permeability, systemic effectors, etc., are not influencing the activity measurements. Using this in vivo-in vitro approach, we previously demonstrated that the in vitro 32P-phospholipid labeling is regulated by light both in retina ganglion cells and in photoreceptor cells (Guido et al., 1996 ; Bussolino et al., 1998). This regulation is in an opposite direction for each cell type : Light stimulation increases phospholipid labeling in the ganglion cells and decreases it in the photoreceptor cells, in parallel with the electric activity of each neuronal cell type. We further demonstrated that this light-promoted regulation is dependent on the expression of c-fos or Fos-related proteins (Guido et al., 1996 ; Bussolino et al., 1998).

We have now determined the possible light-promoted regulation of various activities of the pathway of phospholipid biosynthesis considered critical for the establishment and maintenance of the molecular lipid species. We also determined the influence that the expression of Fos-related proteins has on these activities. The first reaction of the pathway measured was the acylation of LPA to form PA, which is the precursor to triacylglycerol and more complex phospholipids such as PtdCho, PtdEtn, and PtdSer. The AT II activity was significantly higher in ganglion cells from light-exposed animals compared with the dark-maintained controls. This light-promoted activation is dependent on the expression of Fos-related proteins because no light-dark differences are observed if an oligonucleotide antisense to c-fos mRNA is inoculated intraocularly previous to separating the animals into the light and dark groups.

PA is in a branching point in the pathway of synthesis as it can be dephosphorylated by the PAPase to form diacylglycerol or, alternatively, in the presence of CTP, can form CDP-diacylglycerol, the precursor for phosphatidylglycerol and phosphatidylinositol. Jamal et al. (1991) found that at least two forms of PAPases exist in rat liver homogenates. The PAPase I enzyme present in the cytosol and endoplasmic reticulum is dependent on Mg2+ and inhibited by SH-reactive reagents like NEM. The PAPase II enzyme is tightly bound to plasma membrane, is independent of Mg2+, and is insensitive to NEM. Under the experimental conditions used, only PAPase I activity was detectable in ganglion cells from both light-exposed and dark-maintained chicks. This lack of activity may indicate either that these cells do not contain PAPase II activity or, taking into consideration the experimental procedure used to isolate the ganglion cells (lyophilization, separation, and resuspension of the cells), that this activity was lost during the procedure. As occurred with AT II, this light-promoted activation of PAPase I is dependent on the expression of Fos-related proteins as no light-dark differences are observed if an oligonucleotide antisense to c-fos mRNA is present in the cell when these enzyme activities are to be stimulated.

The levels of the major phospholipids of a given cell are maintained in such a way that the ratio of PtdCho/PtdEtn/PtdSer is kept more or less constant (for review, see Araki and Wurtman, 1998). In ganglion cells, the pattern of labeling of phospholipids shows no marked difference between light and dark (Guido et al., 1996). The coordinated regulation by c-fos or by Fos-related proteins of AT II and PAPase I observed herein seems a resonable mechanism to obtain an overall increased synthesis of phospholipids.

Although c-fos expression has been shown to be a mediator of delayed light-induced apoptosis of photoreceptor cells (Hafezi et al., 1997, 1998), its exact role in retinal physiology needs to be clarified. The mechanism by which the protein product of c-fos is regulating the phospholipid metabolism in cells under physiological stimulation is also unknown. The previous observation that after 15 min of light stimulation, phospholipid labeling is activated by a Fos-dependent mechanism points to a new activity of c-fos as the timing of these activation events is not compatible with a transcription factor activity of c-fos or Fos-related proteins. Further studies will be required to establish if this gene has a nuclear function in the activation of lipid metabolism in stimulated cells or if its action is exerted in its way to the nucleus concomitantly or immediately after its translation. Even though a body of information is starting to explain the process of regulation of lipid content and composition, undoubtedly more studies are required to describe fully this complex phenomenon. This includes examination of whether other proteins of the immediate early genes have the capability to modulate enzyme activities of the pathway of biosynthesis of phospholipids.

Acknowledgements

This work has been supported by FONCyT (Agencia Nacional de Promoción Científica y Tecnológica), CONICET (Consejo Nacional de Investigaciones Científicas y Tecnológicas de la República Argentina), Fundación Antorchas, SeCyT-UNC (Secretarín de Ciencia y Técnica de la Universidad Nacional de Córdoba), and CONICOR (Consejo de Investigaciones Científicas de la Provincia de Córdoba). The authors thank H. J. F. Maccioni for discussion of the manuscript.

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