Localization of lipocalin-type prostaglandin D synthase (beta-trace) in iris, ciliary body, and eye fluids.

PURPOSE: Prostaglandin (PG) D synthase is present in neural tissues and cerebrospinal fluid (beta-trace). This enzyme belongs to the lipocalin family which consists of transporter proteins for lipophilic substances in the extracellular space. PGD synthase is found in retinal pigment epithelium, from where it is secreted into the interphotoreceptor matrix. The authors have undertaken the localization of this unique enzyme within the tissues and spaces of the anterior segment of the eye. METHODS: Iris, ciliary body, lens, and aqueous and vitreous humors were collected from adult rats and mice. PGD synthase activity was determined, and the protein was quantified by Western blot analysis and localized immunohistochemically. Finally, in situ hybridization was performed to localize PGD synthase mRNA. RESULTS: PGD synthase was most abundant in the aqueous and vitreous humors. It was less abundant in tissue cytosolic fractions; these fractions had almost 10-fold as much as their corresponding membrane-bound fractions. Lens tissue had the lowest amount observed. PGD synthase was localized to the epithelial cells of the iris and the ciliary body and to the adjacent extracellular chambers, but PGD synthase mRNA was found only within the epithelial cells. Several glycosylated forms of PGD synthase were also detected. CONCLUSIONS: PGD synthase was synthesized within the epithelial cells of the iris and the ciliary body and was then secreted into the aqueous and vitreous humors, where it accumulated as an active enzyme.

Arachidonic acid metabolism and cerebral blood flow in the normal, ischemic, and reperfused gerbil brain. Inhibition of ischemia-reperfusion-induced cerebral injury by a platelet-activating factor antagonist (BN 52021).

Cerebral ischemia and ischemia-reperfusion induced cerebral injury results in the accumulation of free fatty acids and diacylglycerols as a result of increased activity of phospholipases A and C. We have evaluated the incorporation of 14C arachidonic acid into the whole brain and synaptoneurosomes, the effect of cerebral ischemia on 14C incorporation, and the effect of a PAF antagonist (BN 52021) on cerebral blood flow, free fatty acids, diacylglycerols, and polyphosphoinositides. Peak incorporation of 14C arachidonic acid into the whole brain and synaptoneurosomal fractions occurred 30 minutes following intraventricular injection. Peak incorporation into cerebellar synaptoneurosomal fractions was at 60 minutes following intraventricular injection. Turnover in phospholipid pools was similar in the whole brain and synaptoneurosomes (PI greater than PC greater than PE). Considering phosphatidylinositol content in the gerbil brain, the specific activity of 14C arachidonic acid was 22 times greater in PI than PC. Five minutes of bilateral carotid artery ligation resulted in decreased phosphatidylinositol and polyphosphoinositols. Bilateral carotid artery ligation resulted in systemic arterial hypertension, complete forebrain ischemia (CBF less than 7 ml/100 gm/min) and a 20% to 50% reduction in midbrain CBF. Reperfusion resulted in cerebral reactive hyperemia and systemic hypotension. BN 52021 inhibited the maturation of ischemia-reperfusion induced cerebral injury. Cerebral blood flow was improved. Free fatty acids were decreased, suggesting inhibition of phospholipase A activity. Decreased DAG pools with increased PIP2 pools suggest a possible coinhibition of phospholipase C.

Platelet-activating factor and polyunsaturated fatty acids in cerebral ischemia or convulsions: intracellular PAF-binding sites and activation of a fos/jun/AP-1 transcriptional signaling system.

Platelet-activating factor (PAF) is a lipid mediator formed in the early response of the central nervous system to ischemia or convulsions. Free polyunsaturated fatty acids and arachidonic and docosahexaenoic acids are accumulated along with PAF. Antagonists of PAF have been found to improve cerebral blood flow and partially block the rise in free fatty acids, an effect that may arise by way of inhibition of PAF receptors or stimulation of the reacylation of free fatty acids released upon insult. Three intracellular PAF-binding sites have been identified in rat cerebral cortex. These very high-affinity binding sites are inhibited by PAF antagonists, with certain antagonists exhibiting specificity for a particular binding site. This specificity indicates heterogeneity in these binding sites. Ischemia or stimulation also leads to protooncogene transcriptional activation. Here, we discuss studies with cells in culture showing that PAF promotes transcriptional activation of immediate-early genes. PAF activates the transcription of the immediate-early genes fos and jun, whose gene products are regulators of the transcription of other genes. Transcription of fos is also activated by convulsion or ischemia in the central nervous system. The activation of these genes by PAF can be inhibited by PAF antagonists, and is apparently accomplished by way of an AP-1 transcription regulatory sequence in the promoter region of the target genes. Studies with deletion mutants show that PAF can also exert its activating properties by way of cyclic adenosine-3',5'-monophosphate-(cAMP) and Ca(2+)-responsive elements, and suggest that PAF is involved in an interconnected network of cell signaling that may coordinate short-term and long-term responses of cells to stimulus and injury.

Experimental models and their use in studies of diabetic retinal microangiopathy.

Diabetes produces dramatic changes in retinal microvasculature, triggering endothelial cell proliferation and microaneurysms. Capillaries become weakened, releasing blood into vitreal and retinal spaces. Photoreceptors become occluded and separated from the choriocapillaris, resulting in visual acuity decline, detachment and cell death. Several models have been developed that have proved useful for the study of this disease, resulting in a better understanding of the processes involved. Streptozotocin treatment affects the pancreatic beta cells, rapidly reducing them until insulin is no longer synthesized in sufficient amounts. The galactosemic model shifts metabolism away from glucose, increasing aldose reductase and retinal polyol metabolism. Finally, two weeks of cycled oxygen from high to low tension every 24 hours, followed by return to room air, triggers microangiogenesis in developing retinas. Use of these models, separately or in combination, as well as electroretinographic analysis, has begun to reveal the events taking place as diabetic retinopathy progresses. Endothelial cells become separated from pericytes as basement membranes thicken, and vascular endothelial growth factor increases, triggering their proliferation. Finally, early changes occurring within photoreceptors can now be studied.

Platelet-activating factor is a messenger in the electroconvulsive shock-induced transcriptional activation of c-fos and zif-268 in hippocampus.

Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), undetectable in resting neural tissue, accumulates in brain during seizures. A hetrazepine, BN-50730, is shown here to displace [3H]PAF-specific binding from microsomal, but not from synaptosomal membranes, indicating selectivity for a high affinity intracellular binding site. Rats pretreated with BN-50730 by intraperitoneal or intracerebroventricular injection exhibited an inhibition of the electroconvulsive shock (ECS)-induced expression of c-fos and zif-268 in hippocampus. A much more pronounced, dose-dependent inhibition of ECS-induced zif-268 mRNA in hippocampus by intracerebroventricular injection of BN-50730 was observed. It is concluded that, in the hippocampus, PAF is a mediator of the expression of zif-268 and, to a lesser extent, c-fos through an intracellular specific binding site. Thus, PAF may be a messenger in signal regulated zinc-finger transcription factors, and in other immediate-early genes involved in long-term synaptic plasticity changes.

Composition and labeling by [3H]glycerol and [3H]serine of phospholipids of the chicken retina and optic tectum.

The content and fatty acid composition of phospholipids and the in vivo labeling of lipids by [3H]glycerol and [3H]serine was studied in the retina and the optic tectum of young chickens. The tectum had a higher content of phospholipids and a significantly lower ratio of choline (CGP) to ethanolamine (EGP) glycerophospholipids than the retina. Lipids of the chicken optic system were characterized by a high proportion of polyenoic fatty acids of the n-6 series compared to other species. Intravitreally injected [3H]glycerol was incorporated into all glycerol-containing lipids of the retina, especially in CGP and EGP. Most of the label from [3H]serine was found in serine glycerophospholipids (SGP). The time-dependent distribution of both precursors among retinal lipids was consistent with de novo synthesis as well as metabolic interconversions of lipids. Thus, [3H] from serine also appeared in EGP and CGP, indicating the presence and activity of SGP decarboxylase and EGP-n-methyl transferase. Lipids labeled with both precursors in retina were subsequently found in the tectum, via axoplasmic transport. Even though different lipid classes were labelled by each precursor the proportion of lipids transported to the tectum was similar in both cases (about 1% of the label present in retina).

Sustained induction of prostaglandin endoperoxide synthase-2 by seizures in hippocampus. Inhibition by a platelet-activating factor antagonist.

Prostaglandin G/H synthase-2 and zif-268 mRNA expression is transiently induced in rat brain by kainic acid (KA)-induced seizures and by a single electroconvulsive shock. Induction of both genes by KA shows neuroanatomical specificity in the order hippocampus > cerebral cortex > striatum > brain stem > cerebellum. Nuclear run-on and Western blotting shows that both genes are transcriptionally activated, and that kainic acid up-regulation of prostaglandin G/H synthase-2 mRNA expression in hippocampus matches increased protein levels. Whereas the magnitude of hippocampal zif-268 mRNA induction is similar in both seizure models, peak induction of prostaglandin G/H synthase-2 mRNA is 7-fold greater in the kainic acid model than in the electroconvulsive shock model and is much more prolonged. Pretreatment of animals by intracerebroventricular injection with the intracellular platelet-activating factor receptor antagonist BN 50730 strongly attenuates kainic acid and electroconvulsive shock induction of prostaglandin G/H synthase-2 expression. The drug partially inhibits electroconvulsive shock induction of zif-268, but is relatively ineffective against kainic acid-induced zif-268 expression. Seizure-induced expression of both genes involves platelet-activating factor, but the mechanisms of induction must be otherwise distinct. The selectively elevated induction of hippocampal prostaglandin G/H synthase-2 by kainic acid correlates with a neuroanatomical region in which the agonist induces neuronal damage.

Effects of a platelet activating factor antagonist (BN 52021) on free fatty acids, diacylglycerols, polyphosphoinositides and blood flow in the gerbil brain: inhibition of ischemia-reperfusion induced cerebral injury.

The effects of a platelet activating factor antagonist (BN 52021) on cerebral ischemia-reperfusion was studied in the gerbil. Following ten minutes of bilateral carotid artery ligation, gerbils were reperfused and injected intraperitoneally with either BN 52021 or vehicle-dimethylsulfoxide. Cerebral blood flow and systemic arterial pressure were monitored until 90 minutes of reperfusion. Free fatty acids, diacylglycerols and polyphosphoinositides were then analyzed in forebrains and midbrains. BN 52021 inhibited the maturation of ischemic injury. Cerebral blood flow increased following 60 to 90 minutes of reperfusion. Free fatty acid levels were reduced likely by inhibition of phospholipase A. Phospholipase activity may likely be decreased since there was a tendency to increase phosphatidylinositol-4',5'-bisphosphate and diacylglycerols in BN 52021-treated animals.

Distinct platelet-activating factor binding sites in synaptic endings and in intracellular membranes of rat cerebral cortex.

The binding of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC or PAF, platelet-activating factor) to synaptic plasma membranes, microsomal membranes, and other rat cerebral cortex subcellular fractions was studied. Using several PAF-binding antagonists, three distinct sites were identified. Two of them were in intracellular membranes (microsomes) and one in synaptic plasma membranes. Microsomal membranes were prepared after obtaining a 43,500 x g pellet from the postmitochondrial supernatant and subsequent centrifugation at 105,000 x g of the resulting supernatant. Most plasma membrane markers were retained in the 43,500 x g pellet (Sun, G.Y., Huang, H.-M., Kelleher, J.A., Stubbs, E.B., Sun, A. Y. (1988) Neurochem. Int. 12, 69-77). Microsomes were purified by density-gradient centrifugation and marker enzymes showed relatively very low contamination by plasma membrane markers. Myelin and mitochondria were devoid of specific PAF binding. A site displaying the highest PAF-binding affinity reported to date in all cells and membranes (KD = 22.5 +/- 1.7 pM and Bmax 8.75 = fmol/mg protein), was found in the microsomal fraction. There was a second binding site in microsomal fractions (KD = 25.0 +/- 0.8 nM and Bmax = 0.96 pmol/mg protein. Ca2+ decreases PAF affinity for the microsomal binding sites. The third binding site displays relatively low specific PAF binding and is present in synaptosomal plasma membranes. Moreover, displacement curves by a wide variety of PAF antagonists indicated different affinities for each of the binding sites described here. These results indicate that PAF-binding sites are heterogeneous in rat cerebral cortex, and they imply that the microsomal membrane sites may be involved, at least in part, in intracellular events such as gene expression.

Lipocalin-type prostaglandin D synthase (beta-trace) is located in pigment epithelial cells of rat retina and accumulates within interphotoreceptor matrix.

Glutathione-Independent prostaglandin D synthase, identical to beta-trace, (a major CSF protein), is localized in the CNS. This enzyme, lipocalin-type prostaglandin D synthase, is a member of the lipocalin family of secretory proteins that transport small lipophilic substances. This enzyme's activity in adult rat retina was enriched sixfold in retinal pigment epithelium (RPE) and even more in interphotoreceptor matrix (IPM), all higher than brain. Western blots with anti-lipocalin-type prostaglandin D synthase showed three distinct immunoreactive bands. In the retinal cytosolic fraction, only one band was observed (M(r) 25,000); in IPM, the larger component occurred (M(r), 26,000). The RPE membrane-bound fraction showed two bands (M(r) 20,000 and 23,000), indicating synthesis, and the cytosolic fraction contained two bands (M(r) 23,000 and 26,000), indicating modification for release into IPM. At least two glycosylation sites occurred on the prostaglandin D synthase moiety, explaining the three immunoreactive bands in Western blots. Immunohistochemistry with polyclonal antibodies against this lipocalin-type enzyme showed intense localization in RPE, but less in photoreceptor outer and inner segments. In situ hybridization showed mRNA specifically expressed in RPE. Thus, lipocalin-type prostaglandin D synthase is predominantly expressed in RPE and actively accumulated in IPM. This may demonstrate gene sharing because, while catalyzing prostaglandin D2 synthesis, it may perform an additional, unrelated role in IPM. This enzyme is secreted from the RPE into IPM from which it is then taken up by photoreceptors. However, the nature of its ligand(s) is not known; they may be retinoids and/or docosahexanoic acid.

Diacylglycerol kinase epsilon regulates seizure susceptibility and long-term potentiation through arachidonoyl- inositol lipid signaling.

Arachidonoyldiacylglycerol (20:4-DAG) is a second messenger derived from phosphatidylinositol 4,5-bisphosphate and generated by stimulation of glutamate metabotropic receptors linked to G proteins and activation of phospholipase C. 20:4-DAG signaling is terminated by its phosphorylation to phosphatidic acid, catalyzed by diacylglycerol kinase (DGK). We have cloned the murine DGKepsilon gene that showed, when expressed in COS-7 cells, selectivity for 20:4-DAG. The significance of DGKepsilon in synaptic function was investigated in mice with targeted disruption of the DGKepsilon. DGKepsilon(-/-) mice showed a higher resistance to electroconvulsive shock with shorter tonic seizures and faster recovery than DGKepsilon(+/+) mice. The phosphatidylinositol 4,5-bisphosphate-signaling pathway in cerebral cortex was greatly affected, leading to lower accumulation of 20:4-DAG and free 20:4. Also, long-term potentiation was attenuated in perforant path-dentate granular cell synapses. We propose that DGKepsilon contributes to modulate neuronal signaling pathways linked to synaptic activity, neuronal plasticity, and epileptogenesis.

KID-1, a protein kinase induced by depolarization in brain.

Membrane depolarization leads to changes in gene expression that modulate neuronal plasticity. Using representational difference analysis, we have identified a previously undiscovered cDNA, KID-1 (kinase induced by depolarization), that is induced by membrane depolarization or forskolin, but not by neurotrophins or growth factors, in PC12 pheochromocytoma cells. KID-1 is an immediate early gene that shares a high degree of sequence similarity with the family of PIM-1 serine/threonine protein kinases. Recombinant KID-1 fusion protein is able to catalyze both histone phosphorylation and autophosphorylation. KID-1 mRNA is present in a number of unstimulated tissues, including brain. In response to kainic acid and electroconvulsive shock-induced seizures, KID-1 is induced in specific regions of the hippocampus and cortex.