Neuroprotectin D1 is synthesized in the cone photoreceptor cell line 661W and elicits protection against light-induced stress.

Docosahexaenoic acid (DHA), an omega-3 fatty acid family member, is obtained by diet or synthesized from dietary essential omega-3 linolenic acid and delivered systemically to the choriocapillaris, from where it is taken up by the retinal pigment epithelium (RPE). DHA is then transported to the inner segments of photoreceptors, where it is incorporated in phospholipids during the biogenesis of outer segment disk and plasma membranes. As apical photoreceptor disks are gradually shed and phagocytized by the RPE, DHA is retrieved and recycled back to photoreceptor inner segments for reassembly into new disks. Under uncompensated oxidative stress, the docosanoid neuroprotectin D1 (NPD1), a potent mediator derived from DHA, is formed by the RPE and displays its bioactivity in an autocrine and paracrine fashion. The purpose of this study was to determine whether photoreceptors have the ability to synthesize NPD1, and whether or not this lipid mediator exerts bioactivity on these cells. For this purpose, 661W cells (mouse-derived photoreceptor cells) were used. First we asked whether these cells have the ability to form NPD1 by incubating cells with deuterium (d4)-labeled DHA exposed to dark and bright light treatments, followed by LC-MS/MS-based lipidomic analysis to identify and quantify d4-NPD1. The second question pertains to the potential bioactivity of these lipids. Therefore, cells were incubated with 9-cis-retinal in the presence of bright light that triggers cell damage and death. Following 9-cis-retinal loading, DHA, NPD1, or vehicle were added to the media and the 661W cells maintained either in darkness or under bright light. DHA and NPD1 were then quantified in cells and media. Regardless of lighting conditions, 661W cells acquired DHA from the media and synthesized 4-9 times as much d4-NPD1 under bright light treatment in the absence and presence of 9-cis-retinal compared to cells in darkness. Viability assays of 9-cis-retinal-treated cells demonstrated that 34 % of the cells survived without DHA or NPD1. However, after bright light exposure, DHA protected 23 % above control levels and NPD1 increased protection by 32 %. In conclusion, the photoreceptor cell line 661W has the capability to synthesize NPD1 from DHA when under stress, and, in turn, can be protected from stress-induced apoptosis by DHA or NPD1, indicating that photoreceptors effectively contribute to endogenous protective signaling mediated by NPD1 under stressful conditions.

Calcium-independent phospholipase A2 regulates retinal pigment epithelium proliferation and may be important in the pathogenesis of retinal diseases.

Calcium-independent phospholipase A2, group VIA (iPLA2-VIA) is involved in cell proliferation. This study aimed to evaluate the role of iPLA2-VIA in retinal pigment epithelium (RPE) cell proliferation and in retinal diseases involving RPE proliferation. A human RPE cell line (ARPE-19) was used to explore this role in vitro. Proliferating ARPE-19 cells had increased expression and activity of iPLA2-VIA. iPLA2-VIA was found in the nuclei of proliferating ARPE-19 cells, whereas in confluent ARPE-19 cells, with limited proliferation, iPLA2-VIA was primarily found in the cytosol. Inhibition of iPLA2-VIA decreased the rate of proliferation, whereas over expression of iPLA2-VIA increased the rate of proliferation. Using an experimental porcine model of RPE proliferation we demonstrated significant nuclear upregulation of iPLA2-VIA in proliferating RPE cells in vivo. We furthermore evaluated the expression of iPLA2-VIA in proliferative vitreoretinopathy (PVR). PVR membranes revealed nuclear expression of iPLA2-VIA in the RPE cells which had migrated and participated in the formation of the membranes. Overall, the present results point to an important role of iPLA2-VIA in the regulation of RPE proliferation suggesting that iPLA2-VIA may be considered as a possible pharmaceutical target in retinal diseases involving RPE proliferation and migration.

Enhancement of hippocampal excitatory synaptic transmission by platelet-activating factor.

The biologically active lipid platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine; PAF) is a mediator of inflammatory and immune responses, and it accumulates in the brain during convulsions or ischemia. We have examined whether PAF may play a second messenger role in the central nervous system by studying effects on synaptic transmission in cultured hippocampal neurons. Carbamyl-PAF, a nonhydrolyzable PAF analog with a similar pharmacologic profile, augmented glutamate-mediated, evoked excitatory synaptic transmission and increased the frequency of spontaneous miniature excitatory synaptic events without increasing their amplitude or altering their time course. This compound had no significant effect on gamma-aminobutyric acid-mediated inhibitory synaptic responses. Lyso-PAF, the biologically inactive metabolic intermediate, had no effect on synaptic transmission. Moreover, the enhancement of excitatory synaptic transmission by carbamyl-PAF was blocked by a PAF receptor antagonist. These results indicate a specific presynaptic effect of PAF in enhancing excitatory synaptic transmission in cultured rat hippocampal neurons.

Docosanoids are multifunctional regulators of neural cell integrity and fate: significance in aging and disease.

The identification of neuroprotectin D1 (NPD1), a biosynthetic product of docosahexaenoic acid (DHA), in brain and retina as well as the characterization of its bioactivity, is generating a renewed interest in the functional role and pathophysiological significance of omega-3 fatty acids in the central nervous system. Neurotrophins, particularly pigment epithelium-derived factor (PEDF), induce NPD1 synthesis and its polarized apical secretion, implying paracrine and autocrine bioactivity of this lipid mediator. Also, DHA and PEDF synergistically activate NPD1 synthesis and antiapoptotic protein expression and decreased proapoptotic Bcl-2 protein expression and caspase 3 activation during oxidative stress. In experimental stroke, endogenous NPD1 synthesis was found to be upregulated, and the infusion of the lipid mediator into the brain under these conditions revealed neuroprotective bioactivity of NPD1. The hippocampal CA1 region from Alzheimer's disease (AD) patients (rapidly sampled) shows a major reduction in NPD1. The interplay of DHA-derived neuroprotective signaling aims to counteract proinflammatory, cell-damaging events triggered by multiple, converging cytokine and amyloid peptide factors, as in the case of AD. Generation of NPD1 from DHA thereby appears to redirect cellular fate toward successful preservation of retinal pigment epithelial (RPE)-photoreceptor cell integrity and brain cell aging. The Bcl-2 pro- and antiapoptotic proteins, neurotrophins, and NPD1, lie along a cell fate-regulatory pathway whose component members are highly interactive, and have potential to function cooperatively in cell survival. Agents that stimulate NPD1 biosynthesis, NPD1 analogs, or dietary regimens may be useful as new preventive/therapeutic strategies for neurodegenerative diseases.

Effect of K+ depolarization on the synthesis of prostaglandins and hydroxyeicosatetra(5,8,11,14)enoic acids (HETE) in the rat retina. Evidence for esterification of 12-HETE in lipids.

[14C]Arachidonic acid is metabolized to prostaglandins and hydroxyeicosatetraenoic acids in the rat retina. After intravitreal injection of [14C]arachidonic acid, 25% of the injected radiolabel was recovered in the retinal lipids. Phosphatidylcholine and phosphatidylinositol were most actively labeled; however, all glycerolipids incorporated arachidonic acid. The synthesis of prostaglandins E2, F2 alpha, D2, 6-keto-F1 alpha, thromboxane B2 and hydroxyeicosatetraenoic acids was measured by high-performance liquid chromatography. The identity of 12-HETE was confirmed by gas chromatography-mass spectrometry. Incubation of prelabeled retinas in vitro promoted the release of [14C]arachidonic acid from glycerolipids. A 12-fold increase in the synthesis of hydroxyeicosatetraenoic acids occurred with no change in the synthesis of prostaglandins. Incubation in a depolarizing medium (45 mM K+) resulted in a selective increase in hydroxyeicosatetraenoic acids, an effect that was blocked by nordihydroguaiaretic acid (1 microM) and eicosatetraynoic acid (10 microM). 12-[3H8]Hydroxyeicosatetraenoic acid, intravitreally injected, was incorporated into retinal lipids with a distribution similar to arachidonic acid. When retinas labeled with 12-[3H8]hydroxyeicosatetraenoic acid were incubated, there was a large release of the incorporated radioactivity, and metabolism to other products with the chromatographic properties of dihydroxyeicosatetraenoic acids. The release of 12-hydroxyeicosatetraenoic acid was not affected by depolarizing conditions (45 mM K+); however, the conversion of 12-hydroxyeicosatetraenoic acid to dihydroxy isomers was stimulated by K+. These experiments demonstrate active pathways for the generation of eicosanoids in the rat retina that are sensitive to membrane depolarization and lipoxygenase inhibitors.

De novo biosynthesis of docosahexaenoyl-phosphatidic acid in bovine retinal microsomes.

Lysophosphatidic acid stimulated several-fold the formation of docosahexaenoyl-phosphatidic acid from 14C-labeled docosahexaenoic acid (22:6 (n-3] in the bovine retina. 1-Palmitoyl- and 1-oleoyl-sn-glycerol 3-phosphate were the preferred acceptors. Most of the activity was localized in the 105 000 X g microsomal fraction. Despite the very high content of 22:6 in the phospholipids of photoreceptor membranes, only about 1% of the microsomal activity was found in discs isolated from rod outer segments. The newly synthesized docosahexaenoyl-phosphatidic acid was further metabolized to diacylglycerols, triacylglycerols, phosphatidylcholine and phosphatidylserine. The de novo synthesis of docosahexaenoyl-phosphatidylcholine was stimulated by 1 mM CDPcholine. Lysophosphatidic acid and lysophosphatidylcholine up to 50 microM do not compete with each other for 22:6 in the formation of their respective diacylated lipids. This suggests that this fatty acid is introduced into phosphatidic acid and phosphatidylcholine via different acylation systems. We conclude that, in addition to the deacylation-acylation cycle, there is also an active pathway for the acylation of 22:6 into glycerolipids during the de novo biosynthesis of phosphatidic acid.

Changes in triacylglycerol, diacylglycerol and free fatty acids after fertilization in developing toad embryos.

The content and fatty-acid composition of triacylglycerols, diacylglycerols and free fatty acids were analyzed from the unfertilized oocyte stage to the gastrula stage in the toad Bufo arenarum Hensel. Fertilization triggered a 30% and a 40% decrease in triacylglycerol and diacylglycerol, respectively. In contrast, free fatty acid increased continuously from oocyte to gastrula stage with an accumulation of palmitate predominating. However, the observed increase in free fatty acid was too small to account for the decreases in both neutral glycerides. The decrease in triacylglycerol might be a reflection of the activation of lipolytic enzymes and the subsequent oxidation of fatty acids to meet the increased metabolic energy requirements brought on by fertilization. The diminished diacylglycerol content due to fertilization may be accounted for, at least in part, by the utilization of diacylglycerol in the synthesis of membrane phospholipids, inasmuch as their decrease occurred simultaneously with an increase in phosphatidic acid. When cell-free homogenates taken from the three stages of development (unfertilized, fertilized and gastrula) were incubated in Tris-Ringer buffer for 90 min, free fatty acid accumulated. Triacylglycerol and diacylglycerol did not change substantially during this incubation period. This fact indicates that the free fatty acid released during incubation was not derived from neutral glycerides, but probably from membrane phospholipids. The release of free fatty acid was significantly greater in samples from the fertilized oocyte stage. The results described in this paper suggest that the synthesis of membrane phospholipids, including an enhanced turnover of the acyl moiety, plays a significant role in the metabolic events activated by fertilization.

Membrane lipids composition and metabolism during early embryonic development. Phospholipid subcellular distribution and 32P labeling.

Phospholipid composition and 32P metabolism were studied in oocytes and early developing embryos of the toad, Bufo arenarum, Hensel. The content and distribution of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, sphingomyelin, phosphatidylserine, and diphosphatidylglycerol in embryos, whole oocytes, and the subcellular fractions of both were determined. Phosphatidylcholine and phosphatidylethanolamine were the major constituents of yolk platelet. Diphosphatidylglycerol was confined to the mitochondrial fraction, where it represented about 7% of the total phosphoacylglycerols. Relatively large amounts of sphingomyelin were found in microsomal and postmicrosomal supernatants. After in vivo labeling with 32P, the early development of individual phospholipids in subcellular fractions and in whole eggs was followed. The greatest uptake was found in mitochondrial and yolk platelet fractions. A steady increase in the amount of 32P present in phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol was seen in the whole embryo from oocyte to late gastrula stage and in all subcellular fractions. Phosphatidic acid exhibited a slight decrease in specific activity, except in the yolk platelet fraction. This high 32P incorporation would indicate a rapid and uneven polar headgroup turnover determined by phospholipid class and subcellular fraction. At the same time, the phospholipid content of the subcellular fractions studied remained unchanged during early embryogenesis. Moreover, 32P was actively incorporated into the individual phospholipids in the absence of measurable net synthesis.

Chain elongation and desaturation of eicosapentaenoate to docosahexaenoate and phospholipid labeling in the rat retina in vivo.

The metabolism of [1-14C]eicosapentaenoic acid in the retina after intravitreal injections in the adult rat eye was studied. The acylation of eicosapentaenoic acid and the appearance of labeled docosapentaenoate and docosahexaenoate in individual phospholipids was observed at 3, 5 and 30 min after injection. The elongation and desaturation products represented about 8 and 4%, respectively, of the total radioactivity of phospholipids 3 min after injection. The highest labeling was found in phosphatidylcholine, phosphatidylinositol and phosphatidic acid. The uneven labeling profiles and the specific activities in individual phospholipids suggested that, in addition to the deacylation-acylation route for the introduction of polyenoic acyl groups into phospholipids, acylation may also take place during the synthesis of phosphatidic acid, followed by channeling to phospholipids.

Propranolol increases the biosynthesis of phosphatidic acid, phosphatidylinositol and phosphatidylserine in the toad retina. Studies in the entire and subcellular fractions.

1. Intact toad retinas incubated for short periods of time with [2-3H]glycerol were subject to subcellular fractionation. 2. The composition and labeling of glycerolipids were studied in the following subcellular fractions: rod outer segments (ROS), nuclear-photoreceptor inner segment synaptic body (P1), synaptosomal-mitochondrial (P2), microsomal and cytosolic. 3. It was concluded that the biosynthetic de novo route was followed by [2-3H]glycerol in the toad retina since radioactive was located solely in the glycerol backbone of lipids and phosphatidic acid specific activity was the highest. 4. Propranolol produces an increase in the biosynthesis of acidic phospholipids and inhibition in the biosynthesis of zwitterionic lipids in the entire toad retina. The effect was mainly located in microsomes and in the soluble fraction during the first minutes of incubation, being spread afterwards to other fractions. 5. These data are consistent with the view that enzymes of the biosynthesis of glycerolipids are modified in the retinal endoplasmic reticulum by propranolol, providing a useful tool to evaluate the regulation of the pathway.

Phosphatidic acid, phosphatidylinositol, phosphatidylserine and cardiolipin in the course of early embryonic development. Fatty acid composition and content in whole toad embryos and in mitochondrial fractions.

The fatty acid composition and content of phosphatidylinositol, phosphatidylserine and phosphatidic acid have been studied during the early development of toad embryos. Acidic phospholipids have been analyzed in whole oocytes and embryos and in the following subcellular fractions: yolk platelets, mitochondria and microsomes. Also cardiolipin, a mitochondrial phospholipid, has been analyzed. Gastrula stage embryos have shown, mainly in the mitochondrial fraction, an increase in the content of phosphatidic acid, phosphatidylserine and phosphatidylinositol with respect to unfertilized oocytes. Changes in the distribution of acyl groups of phosphatidic acid have been detected when different subcellular fractions are compared. On the other hand, the phosphatidylserine composition remains unmodified. Arachidonate and stearate are the principal components of phosphatidylinositol. Cardiolipin shows the same composition up to gastrulation and linoleate comprises about 50% of the total acyl groups.

High content of 22:6 (docosahexaenoate) and active [2-3H]glycerol metabolism of phosphatidic acid from photoreceptor membranes.

This study describes the content, fatty acid composition and [2-3H]glycerol metabolism of phosphatidic acid of rod outer segment membranes from vertebrate retinas. A relatively high content of phosphatidic acid was observed in rod outer segment membranes isolated from rat, toad and bovine retinas. In bovine retinas, about 65% of the acyl groups of phosphatidic acid were composed of docosahexaenoate. Arachidonate and docosapentaenoate represented about 4 and 5%, respectively, of the total, whereas stearate was the most common saturated acyl chain. An active [2-3H]glycerol metabolism in the phosphatidic acid of these membranes was found when whole retinas were incubated with the precursor for short periods prior to subcellular fractionation. Our results suggested that the pool of phosphatidic acid enriched in docosahexaenoate may arise from de novo biosynthesis or from phospholipid degradation by a phospholipase D enzyme, and that it is not metabolically related, in any major fashion, to the diacylglycerols of rod outer segment membranes.

NPD1-mediated stereoselective regulation of BIRC3 expression through cREL is decisive for neural cell survival.

Neuroprotectin D1 (NPD1), a docosahexaenoic acid (DHA)-derived mediator, induces cell survival in uncompensated oxidative stress (OS), neurodegenerations or ischemic stroke. The molecular principles underlying this protection remain unresolved. We report here that, in retinal pigment epithelial cells, NPD1 induces nuclear translocation and cREL synthesis that, in turn, mediates BIRC3 transcription. NPD1 activates NF-κB by an alternate route to canonical signaling, so the opposing effects of TNFR1 and NPD1 on BIRC3 expression are not due to interaction/s between NF-κB pathways. RelB expression follows a similar pattern as BIRC3, indicating that NPD1 also is required to activate cREL-mediated RelB expression. These results suggest that cREL, which follows a periodic pattern augmented by the lipid mediator, regulates a cluster of NPD1-dependent genes after cREL nuclear translocation. BIRC3 silencing prevents NPD1 induction of survival against OS. Moreover, brain NPD1 biosynthesis and selective neuronal BIRC3 abundance are increased by DHA after experimental ischemic stroke followed by remarkable neurological recovery. Thus, NPD1 bioactivity governs key counter-regulatory gene transcription decisive for retinal and brain neural cell integrity when confronted with potential disruptions of homeostasis.

Essential versus accessory aspects of cell death: recommendations of the NCCD 2015.

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as 'accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. 'Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death.

Excitable membranes, lipid messengers, and immediate-early genes. Alteration of signal transduction in neuromodulation and neurotrauma.

The physical nature of neuronal cells, particularly in the functional and morphological segregation of synapse, soma, and dendrites, imparts special importance on the integrity of their cell membranes for the localization of function, generation of intrinsic second messengers, and plasticity required for adaptation and repair. The component phospholipids of neural membranes are important sources of bioactive mediators that participate in such diverse phenomena as memory formation and cellular damage following trauma. A common role for PAF in these processes is established through the suppressive effects of its antagonists. Furthermore, being both an extracellular and intracellular agonist of phospholipase activation, in addition to being a product of phospholipase activity, PAF assumes a centralized role in the cellular metabolism following neural stimulation. The linkage of PAF to neural immediate-early gene expression, both in vitro and in vivo, suggests that its effects are initiating to long-term formative and reparative processes. Such a common link between destructive and plastic responses provides an important view of cellular and tissue maintenance in the nervous system.

Cationic amphiphilic drugs inhibit the synthesis of long-chain fatty acyl coenzyme A in rat brain microsomes.

The effect of cationic amphiphilic drugs (CAD) on the synthesis of thiol esters of coenzyme A with long-chain fatty acids was studied in microsomes of rat brain in vitro. The results indicate that propranolol, tetracaine and to a lesser extent, chloroquine, inhibit enzyme activity. Procaine and lidocaine did not inhibit enzyme activity in concentrations up to 0.8 mM. This inhibition seems to be directed primarily to the synthesis of polyunsaturated fatty acyl coenzyme A. The results also suggest that this inhibition may be due to the action of CAD on the microsomal membrane and not to an interaction of these drugs with the fatty acid substrates.

Visualization of [3H]docosahexaenoic acid trafficking through photoreceptors and retinal pigment epithelium by electron microscopic autoradiography.

PURPOSE: [3H]docosahexaenoic (DHA) acid was followed through the retinal pigment epithelial cells and photoreceptors for up to 5 days after injection to specifically determine which membrane systems of the retinal pigment epithelial cells are used in the handling of [3H]DHA after shedding and phagocytosis of rod tips. METHODS: Frogs (Rana pipiens) were injected with [3H]DHA in the dorsal lymph sacs, and maintained for up to 5 days. Retinas were processed for electron microscopic autoradiography, stored for various periods of time, and then analyzed by transmission electron microscopy. RESULTS: After 1 day, [3H]DHA had accumulated within photoreceptor ellipsoids, and had begun to appear as dense label in newly formed discs. By day 5, the basal region of dense label had expanded apically. Newly shed rod outer segment tips were diffusely labeled; but occasionally after several hours, they acquired additional label as they moved near Bruch's membrane. Retinal pigment epithelial cytoplasm maintained a constant level of label, with myeloid bodies sometimes slightly labeled. Oil droplets of the retinal pigment epithelium accumulated dense label throughout this study. CONCLUSIONS: When [3H]DHA enters the retinal pigment epithelium, some is retained within oil droplets, whereas the rest is passed on to the photoreceptors. [3H]DHA is initially taken up by inner segments and then dispersed to photoreceptor synaptic terminals as well as to ellipsoids where discs are assembled. Phagosomal labeling exactly matches rod outer segment tips, but occasionally increases as degradation occurs near Bruch's membrane. Normally, density of label remains constant throughout the degradation process.

Platelet-activating factor induces the expression of metalloproteinases-1 and -9, but not -2 or -3, in the corneal epithelium.

PURPOSE: The inflammatory mediator platelet-activating factor (PAF) induces the expression of interstitial collagenase (metalloproteinase-1) messenger RNA in rabbit corneal epithelium. In this study, the authors investigated the effect of PAF on gene expression and protein activity of other matrix metalloproteinases (MMPs) in the cornea. METHODS: Rabbit corneas were incubated in an organ culture with 100 nM of cPAF (a nonhydrolyzable PAF analog), PAF, or lyso-PAF, an inactive metabolite of PAF. In some experiments, the corneas were preincubated for 1 hour with 10 microM BN50730, a PAF antagonist, before cPAF was added to the medium. Corneal epithelial cells and/or conditioned medium were collected at different times for analysis. Also, in vivo experiments were done by injecting 2 micrograms of cPAF intrastromally into rabbit eyes and collecting the epithelium 8 hours later for study. Northern blot analysis and zymography were performed to determine the mRNA abundance and/or enzyme activity of 92 kd gelatinase (MMP-9), 72 kd gelatinase (MMP-2), and stromelysin (MMP-3). The activity of MMP-1 was tested by collagenase assays. RESULTS: cPAF induced the expression of MMP-9 mRNA, but not MMP-3 mRNA. The message was induced at 4 hours and remained elevated at 48 hours, with a peak at 36 hours. In corneas preincubated with BN50730, MMP-9 mRNA activation by cPAF was inhibited. In vivo injection of cPAF also induced the expression of MMP-9. Furthermore, cPAF increased MMP-9 activity in the epithelial cells and in the conditioned media. The effect was blocked by BM50730. cPAF did not affect MMP-2 activity. Finally, cPAF also increased MMP-1 collagenolytic activity of the corneal epithelium, which was blocked by the PAF antagonist. CONCLUSION: These results suggest a novel mechanism by which PAF activates MMPs. The lipid mediator selectively enhances the expression of MMP-1 and MMP-9 in rabbit corneal epithelium. This activation by PAF may be involved in the remodeling mechanisms of the cornea after injury and, when overexpressed, may lead to the formation of corneal ulcers. Specific PAF antagonists could therapeutically deter corneal ulcer formation and facilitate corneal wound healing.

Platelet-activating factor enhances urokinase-type plasminogen activator gene expression in corneal epithelium.

PURPOSE: To determine whether platelet-activating factor (PAF), a lipid mediator that is accumulated in the cornea after alkali burn, induces the gene expression of urokinase-type plasminogen activator (uPA) in the corneal epithelium. Possible signaling mechanisms of uPA gene induction by PAF also were examined. METHODS: Rabbit corneas were cultured with or without PAF. One hour before stimulation, PAF antagonists or other modulators were added to PAF. In some experiments, the corneas were permeabilized to introduce guanosine triphosphate analogs into the corneal epithelial cells. Corneal epithelia were then harvested for Northern blot analysis, nuclear runoff transcription assay, and zymography. RESULTS: Platelet-activating factor induced uPA mRNA expression in the corneal epithelium. New protein synthesis was not required for the induction of uPA mRNA. The induction was at the level of transcription as shown by nuclear runoff assays. Additionally, both actinomycin D and alpha-amanitin inhibited the increase in uPA mRNA by PAF. The message was translated into protein, which was secreted into the conditioned medium. An antagonist with high affinity for intracellular PAF binding sites (BN 50730) inhibited uPA gene expression and cellular secretion of the protein. The effect of PAF was not mediated by G proteins and was independent of protein kinase C- and cyclic adenosine monophosphate-dependent signal transduction pathways. Okadaic acid increased the expression of uPA and, at longer times, augmented the effect of PAF, suggesting that a signaling pathway that requires phosphorylation is involved in activated uPA mRNA synthesis. CONCLUSIONS: After corneal injury and inflammation, PAF may be an important initiator of the proteolytic cascade, leading to epithelial defects and corneal ulceration. Antagonists of PAF could be useful in the prevention of these diseases.