Canonical phospholipase D isoforms in visual function and ocular response to stress.

Classical phospholipase D (PLD) isoforms, PLD1 and PLD2, catalyze the hydrolysis of phosphatidylcholine (PC) to generate phosphatidic acid (PA) which can be further dephosphorylated to diacylglycerol (DAG). Through the generation of these lipid messengers, the PLD pathway can modulate several cellular events, such as proliferation, membrane trafficking, autophagy and the inflammatory response, among many others. This review summarizes the participation of canonical PLD isoforms in physiological and pathological responses in the eye. Although the role of the PLD pathway in ocular and retinal response to stress has not been fully elucidated, pharmacological inhibition of these signaling enzymes seems to be a promising therapeutic tool to avoid inflammatory processes in the retina, abnormal cellular proliferation on the ocular surface and pathological neovascularization. On the contrary, the modulation of classical PLDs may potentiate corneal healing. In summary, the knowledge of the role of PLD1 and PLD2 in the molecular basis of ocular inflammatory and degenerative diseases opens new avenues for potential therapeutic exploration.

Lipid Signaling in Retinal Pigment Epithelium Cells Exposed to Inflammatory and Oxidative Stress Conditions. Molecular Mechanisms Underlying Degenerative Retinal Diseases.

The retinal pigment epithelium (RPE) is a monolayer of pigmented cells whose function is essential for the integrity of the retina and for visual function. Retinal diseases that eventually end in vision loss and blindness involve inflammation, oxidative stress (OS), and alterations in the RPE-photoreceptor cellular partnership. This chapter summarizes the role of lipid signaling pathways and lipidic molecules in RPE cells exposed to inflammatory and OS conditions. The modulation of these pathways in the RPE, through either enzyme inhibitors or receptor stimulation or blockage, could open new therapeutic strategies for retinal degenerative diseases.

High glucose-induced phospholipase D activity in retinal pigment epithelium cells: New insights into the molecular mechanisms of diabetic retinopathy.

Chronic hyperglycemia, oxidative stress and inflammation are key players in the pathogenesis of diabetic retinopathy (DR). In this work we study the role of phospholipase D (PLD) pathway in an in vitro model of high glucose (HG)-induced damage. To this end, we exposed human retinal pigment epithelium (RPE) cell lines (ARPE-19 and D407) to HG concentrations (16.5 or 33 mM) or to normal glucose concentration (NG, 5.5 mM) for 4, 24 or 72 h. Exposure to HG increased reactive oxygen species levels and caspase-3 cleavage and reduced cell viability after 72 h of incubation. In addition, short term HG exposure (4 h) induced the activation of early events, that involve PLD and ERK1/2 signaling, nuclear factor kappa B (NFκB) nuclear translocation and IκB phosphorylation. The increment in pro-inflammatory interleukins (IL-6 and IL-8) and cyclooxygenase-2 (COX-2) mRNA levels was observed after 24 h of HG exposure. The effect of selective pharmacological PLD1 (VU0359595) and PLD2 (VU0285655-1) inhibitors demonstrated that ERK1/2 and NFκB activation were downstream events of both PLD isoforms. The increment in IL-6 and COX-2 mRNA levels induced by HG was reduced to control levels in cells pre-incubated with both PLD inhibitors. Furthermore, the inhibition of PLD1, PLD2 and MEK/ERK pathway prevented the loss of cell viability and the activation of caspase-3 induced by HG. In conclusion, our findings demonstrate that PLD1 and PLD2 mediate the inflammatory response triggered by HG in RPE cells, pointing to their potential use as a therapeutic target for DR treatment.

2-Arachidonoylglycerol metabolism is differently modulated by oligomeric and fibrillar conformations of amyloid beta in synaptic terminals.

Alzheimer's disease (AD) is the most prevalent disorder of senile dementia mainly characterized by amyloid-beta peptide (Aß) deposits in the brain. Cannabinoids are relevant to AD as they exert several beneficial effects in many models of this disease. Still, whether the endocannabinoid system is either up- or down-regulated in AD has not yet been fully elucidated. Thus, the aim of the present paper was to analyze endocannabinoid 2-arachidonoylglycerol (2-AG) metabolism in cerebral cortex synaptosomes incubated with Aß oligomers or fibrils. These Aß conformations were obtained by "aging" the 1-40 fragment of the peptide under different agitation and time conditions. A diminished availability of 2-AG resulting from a significant decrease in diacylglycerol lipase (DAGL) activity was observed in the presence of large Aß1-40 oligomers along with synaptosomal membrane damage, as judged by transmission electron microscopy and LDH release. Conversely, a high availability of 2-AG resulting from an increase in DAGL and lysophosphatidic acid phosphohydrolase activities occurred in the presence of Aß1-40 fibrils although synaptosomal membrane disruption was also observed. Interestingly, neither synaptosomal mitochondrial viability assayed by MTT reduction nor membrane lipid peroxidation assayed by TBARS formation measurements were altered by Aß1-40 oligomers or fibrils. These results show a differential effect of Aß1-40 peptide on 2-AG metabolism depending on its conformation.

Phospholipase D1 modulates protein kinase C-epsilon in retinal pigment epithelium cells during inflammatory response.

Inflammation is a key factor in the pathogenesis of several retinal diseases. In view of the essential role of the retinal pigment epithelium in visual function, elucidating the molecular mechanisms elicited by inflammation in this tissue could provide new insights for the treatment of retinal diseases. The aim of the present work was to study protein kinase C signaling and its modulation by phospholipases D in ARPE-19 cells exposed to lipopolysaccharide. This bacterial endotoxin induced protein kinase C-α/ßII phosphorylation and protein kinase-ε translocation to the plasma membrane in ARPE-19 cells. Pre-incubation with selective phospholipase D inhibitors demonstrated that protein kinase C-α phosphorylation depends on phospholipase D1 and 2 while protein kinase C-ε activation depends only on phospholipase D1. The inhibition of α and ß protein kinase C isoforms with Go 6976 did not modify the reduced mitochondrial function induced by lipopolysaccharide. On the contrary, the inhibition of protein kinase C-α, ß and ε with Ro 31-8220 potentiated the decrease in mitochondrial function. Moreover, inhibition of protein kinase C-ε reduced Bcl-2 expression and Akt activation and increased Caspase-3 cleavage in cells treated or not with lipopolysaccharide. Our results demonstrate that through protein kinase C-ε regulation, phospholipase D1 protects retinal pigment epithelium cells from lipopolysaccharide-induced damage.

Age-related changes in retinoic, docosahexaenoic and arachidonic acid modulation in nuclear lipid metabolism.

The aim of this work was to study how age-related changes could modify several enzymatic activities that regulate lipid mediator levels in nuclei from rat cerebellum and how these changes are modulated by all-trans retinoic acid (RA), docosahexaenoic acid (DHA) and arachidonic acid (AA). The higher phosphatidate phosphohydrolase activity and lower diacylglycerol lipase (DAGL) activity observed in aged animals compared with adults could augment diacylglycerol (DAG) availability in the former. Additionally, monoacylglycerol (MAG) availability could be high due to an increase in lysophosphatidate phosphohydrolase (LPAPase) activity and a decrease in monocylglycerol lipase activity. Interestingly, RA, DHA and AA were observed to modulate these enzymatic activities and this modulation was found to change in aged rats. In adult nuclei, whereas RA led to high DAG and MAG production through inhibition of their hydrolytic enzymes, DHA and AA promoted high MAG production by LPAPase and DAGL stimulation. In contrast, in aged nuclei RA caused high MAG generation whereas DHA and AA diminished it through LPAPase activity modulation. These results demonstrate that aging promotes a different nuclear lipid metabolism as well as a different type of non-genomic regulation by RA, DHA and AA, which could be involved in nuclear signaling events.

Early LPS-induced ERK activation in retinal pigment epithelium cells is dependent on PIP 2 -PLC.

This article presents additional data regarding the study "The phospholipase D pathway mediates the inflammatory response of the retinal pigment epithelium" [1]. The new data presented here show that short exposure of RPE cells to lipopolysaccharide (LPS) induces an early and transient activation of the extracellular signal-regulated kinase (ERK1/2). This early ERK1/2 activation is dependent on phosphatidylinositol bisphosphate-phospholipase C (PIP2-PLC). On the contrary, neither the phospholipase D 1 (PLD1) nor the PLD2 inhibition is able to modulate the early ERK1/2 activation induced by LPS in RPE cells.

Insulin-related signaling pathways elicited by light in photoreceptor nuclei from bovine retina.

Retina light stimulation triggers phototransduction events as well as different signaling mechanisms in outer segments (sensorial portion) of photoreceptor cells. We have recently reported a novel light-dependent activation of diacylglycerol kinase (DAGK) and protein kinase C (PKC) at the nuclear level of photoreceptor cells. The aim of the present study was to analyze whether ex-vivo light exposure of bovine retinas also modulates insulin-related signaling pathways in nuclei from photoreceptor cells. To this end, a nuclear fraction enriched in small nuclei from photoreceptor cells (PNF) was obtained using a modified nuclear isolation protocol. In PNF obtained from bovine retinas exposed to light or darkness, the presence of insulin receptor (IR) and phosphorylated insulin receptor (pIR), the activation of Akt, p38 and extracellular signal-regulated kinase (ERK1/2) and the local action of insulin on lipid kinases were studied. Immunofluorescence (IF) and Western blot (WB) studies revealed the presence of IR in photoreceptor nuclei. In PNF a light-dependent increase in IR total content was observed. The presence of activated IR (pIR) was also observed in PNF by WB, being its content higher in PNF from light than in to darkness. Light exposure also produced a significant increase in the content of p-Akt (3 fold) and p-p38 (60%) without changes in total Akt and p38. In addition, an increase in the content of total ERK1/2 (2 fold) was found without changes in p-ERK/total ERK ratio, indicating that light induces translocation of p-ERK to the nucleus. Polyphosphoinositide kinase and diacylglycerol kinase (DAGK) activities were measured in isolated nuclei from light-activated or darkness-adapted retinas through the formation of polyphosphoinositides (PPIs) and phosphatidic acid (PA) using nuclear lipid substrates and [γ-(32)P]ATP as radioactive substrate. A light-dependent increase in PPIs and PA formation was detected when isolated nuclei were exposed to 0.8 µM insulin plus 0.2 mM vanadate. WB studies revealed that retina's exposure to insulin under light condition increased nuclear IR content. In addition, PNF exposure to insulin increased ERK1/2 phosphorylation with no changes in total ERK1/2. Our results demonstrate the presence and the functional state of IR in the nucleus from photoreceptor cells. They also show that molecular signaling components linked to tyrosine kinase receptors and MAPK pathways, such as Akt and ERK1/2, respectively, are present in photoreceptor nuclei and are regulated by insulin and light.

The mouse liver displays daily rhythms in the metabolism of phospholipids and in the activity of lipid synthesizing enzymes.

The circadian system involves central and peripheral oscillators regulating temporally biochemical processes including lipid metabolism; their disruption leads to severe metabolic diseases (obesity, diabetes, etc). Here, we investigated the temporal regulation of glycerophospholipid (GPL) synthesis in mouse liver, a well-known peripheral oscillator. Mice were synchronized to a 12:12 h light-dark (LD) cycle and then released to constant darkness with food ad libitum. Livers collected at different times exhibited a daily rhythmicity in some individual GPL content with highest levels during the subjective day. The activity of GPL-synthesizing/remodeling enzymes: phosphatidate phosphohydrolase 1 (PAP-1/lipin) and lysophospholipid acyltransferases (LPLATs) also displayed significant variations, with higher levels during the subjective day and at dusk. We evaluated the temporal regulation of expression and activity of phosphatidylcholine (PC) synthesizing enzymes. PC is mainly synthesized through the Kennedy pathway with Choline Kinase (ChoK) as a key regulatory enzyme or through the phosphatidylethanolamine (PE) N-methyltransferase (PEMT) pathway. The PC/PE content ratio exhibited a daily variation with lowest levels at night, while ChoKα and PEMT mRNA expression displayed maximal levels at nocturnal phases. Our results demonstrate that mouse liver GPL metabolism oscillates rhythmically with a precise temporal control in the expression and/or activity of specific enzymes.

The phospholipase D pathway mediates the inflammatory response of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) plays an important immunological role in the retina and it is involved in many ocular inflammatory diseases that may end in loss of vision and blindness. In this work the role of phospholipase D (PLD) classical isoforms, PLD1 and PLD2, in the inflammatory response of human RPE cells (ARPE-19) was studied. ARPE-19 cells exposed to lipopolysaccharide (LPS, 10 µg/ml) displayed increased levels of NO production and diminished mitochondrial function after 48 h of incubation. Furthermore, 24h LPS treatment strongly induced cyclooxygenase-2 (COX-2) expression and activation of extracellular signal-regulated kinase (ERK1/2). EGFP-PLDs showed the typical subcellular localization, perinuclear for PLD1 and plasma membrane for PLD2. LPS increased PLD activity by 90% with respect to the control. The presence of PLD1 inhibitor (EVJ 0.15 µM) or PLD2 inhibitor (APV 0.5 µM) reduced LPS-induced COX-2 induction but only PLD2 inhibition reduced ERK1/2 activation. Mitochondrial function was restored after inhibition of PLD2 and ERK1/2. These findings evidence the participation of PLD2 as a promoter of RPE inflammatory response through ERK1/2 and COX-2 regulation. Our results demonstrate for the first time distinctive roles of PLD isoforms in pathological conditions in RPE.

A novel light-dependent activation of DAGK and PKC in bovine photoreceptor nuclei.

In this work, we describe a selective light-dependent distribution of the lipid kinase 1,2-diacylglycerol kinase (EC 2.7.1.107, DAGK) and the phosphorylated protein kinase C alpha (pPKCα) in a nuclear fraction of photoreceptor cells from bovine retinas. A nuclear fraction enriched in small nuclei from photoreceptor cells (PNF), was obtained when a modified nuclear isolation protocol developed by our laboratory was used. We measured and compared DAGK activity as phosphatidic acid (PA) formation in PNF obtained from retinas exposed to light and in retinas kept in darkness using [γ-(32)P]ATP or [(3)H]DAG. In the absence of exogenous substrates and detergents, no changes in DAGK activity were observed. However, when DAGK activity assays were performed in the presence of exogenous substrates, such as stearoyl arachidonoyl glycerol (SAG) or dioleoyl glycerol (DOG), and different detergents (used to make different DAGK isoforms evident), we observed significant light effects on DAGK activity, suggesting the presence of several DAGK isoforms in PNF. Under conditions favoring DAGKζ activity (DOG, Triton X-100, dioleoyl phosphatidylserine and R59022) we observed an increase in PA formation in PNF from retinas exposed to light with respect to those exposed to darkness. In contrast, under conditions favoring DAGKɛ (SAG, octylglucoside and R59022) we observed a decrease in its activity. These results suggest different physiological roles of the above-mentioned DAGK isoforms. Western blot analysis showed that whereas light stimulation of bovine retinas increases DAGKζ nuclear content, it decreases DAGKɛ and DAGKß content in PNF. The role of PIP2-phospholipase C in light-stimulated DAGK activity was demonstrated using U73122. Light was also observed to induce enhanced pPKCα content in PNF. The selective distribution of DAGKζ and ɛ in PNF could be a light-dependent mechanism that in vertebrate retina promotes selective DAG removal and PKC regulation.

Cannabinoid receptor-dependent metabolism of 2-arachidonoylglycerol during aging.

2-Arachidonoylglycerol (2-AG) is one of the principal endocannabinoids involved in the protection against neurodegenerative processes. Cannabinoids primarily interact with the seven-segment transmembrane cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2), both of which are expressed in the central nervous system (CNS). The level of 2-AG is controlled through key enzymes responsible for its synthesis or degradation. We have previously observed a deregulation of 2-AG metabolism in physiological aging. The aim of this study was to analyze how 2-AG metabolism is modulated by CB1/CB2 receptors during aging. To this end, both CB1 and CB2 receptor expression and the enzymatic activities (diacylglycerol lipase (DAGL), lysophosphatidate phosphohydrolase (LPAase) and monoacylglycerol lipase (MAGL)) involved in 2-AG metabolism were analyzed in the presence of cannabinoid receptor (CBR) agonists (WIN and JWH) and/or antagonists (SR1 and SR2) in synaptosomes from adult and aged rat cerebral cortex (CC). Our results demonstrate that: (a) aging decreases the expression of both CBRs; (b) LPAase inhibition, due to the individual action of SR1 or SR2, is reverted in the presence of both antagonists together; (c) LPAase activity is regulated mainly by the CB1 receptor in adult and in aged synaptosomes while the CB2 receptor acquires importance when CB1 is blocked; (d) modulation via CBRs of DAGL and MAGL by both antagonists occurs only in aged synaptosomes, stimulating DAGL and inhibiting MAGL activities; (e) only DAGL stimulation is reverted by WIN. Taken together, the results of the present study show that CB1 and/or CB2 receptor antagonists trigger a significant modulation of 2-AG metabolism, underlining their relevance as therapeutic strategy for controlling endocannabinoid levels in physiological aging.

Daily rhythms of glycerophospholipid synthesis in fibroblast cultures involve differential enzyme contributions.

Circadian clocks regulate the temporal organization of several biochemical processes, including lipid metabolism, and their disruption leads to severe metabolic disorders. Immortalized cell lines acting as circadian clocks display daily variations in [(32)P]phospholipid labeling; however, the regulation of glycerophospholipid (GPL) synthesis by internal clocks remains unknown. Here we found that arrested NIH 3T3 cells synchronized with a 2 h-serum shock exhibited temporal oscillations in a) the labeling of total [(3)H] GPLs, with lowest levels around 28 and 56 h, and b) the activity of GPL-synthesizing and GPL-remodeling enzymes, such as phosphatidate phosphohydrolase 1 (PAP-1) and lysophospholipid acyltransferases (LPLAT), respectively, with antiphase profiles. In addition, we investigated the temporal regulation of phosphatidylcholine (PC) biosynthesis. PC is mainly synthesized through the Kennedy pathway with choline kinase (ChoK) and CTP:phosphocholine cytidylyltranferase (CCT) as key regulatory enzymes. We observed that the PC labeling exhibited daily changes, with the lowest levels every ~28 h, that were accompanied by brief increases in CCT activity and the oscillation in ChoK mRNA expression and activity. Results demonstrate that the metabolisms of GPLs and particularly of PC in synchronized fibroblasts are subject to a complex temporal control involving concerted changes in the expression and/or activities of specific synthesizing enzymes.

Diacylglycerol kinase epsilon in bovine and rat photoreceptor cells. Light-dependent distribution in photoreceptor cells.

The present study shows the selective light-dependent distribution of 1,2-diacylglycerol kinase epsilon (DAGKɛ) in photoreceptor cells from bovine and albino rat retina. Immunofluorescence microscopy in isolated rod outer segments from bleached bovine retinas (BBROS) revealed a higher DAGKɛ signal than that found in rod outer segments from dark-adapted bovine retinas (BDROS). The light-dependent outer segment localization of DAGKɛ was also observed by immunohistochemistry in retinas from albino rats. DAGK activity, measured in terms of phosphatidic acid formation from a) [(3)H]DAG and ATP in the presence of EGTA and R59022, a type I DAGK inhibitor, or b) [γ-(32)P]ATP and 1-stearoyl, 2-arachidonoylglycerol (SAG), was found to be significantly higher in BBROS than in BDROS. Higher light-dependent DAGK activity (condition b) was also found when ROS were isolated from dark-adapted rat retinas exposed to light. Western blot analysis of isolated ROS proteins from bovine and rat retinas confirmed that illumination increases DAGKɛ content in the outer segments of these two species. Light-dependent DAGKɛ localization in the outer segment was not observed when U73122, a phospholipase C inhibitor, was present prior to the exposure of rat eyecups (in situ model) to light. Furthermore, no increased PA synthesis from [(3)H]DAG and ATP was observed in the presence of neomycin prior to the exposure of bovine eyecups to light. Interestingly, when BBROS were pre-phosphorylated with ATP in the presence of 1,2-dioctanoyl sn-glycerol (di-C8) or phorbol dibutyrate (PDBu) as PKC activation conditions, higher DAGK activity was observed than in dephosphorylated controls. Taken together, our findings suggest that the selective distribution of DAGKɛ in photoreceptor cells is a light-dependent mechanism that promotes increased SAG removal and synthesis of 1-stearoyl, 2-arachidonoyl phosphatidic acid in the sensorial portion of this cell, thus demonstrating a novel mechanism of light-regulated DAGK activity in the photoreceptors of two vertebrate species.

Phosphatidic acid metabolism in rat liver cell nuclei.

The aim of the present research was to analyze the pathways for phosphatidic acid metabolism in purified nuclei from liver. Lipid phosphate phosphatase, diacylglycerol lipase, monoacylglycerol lipase and PA-phospholipase type A activities were detected. The presence of lysophosphatidic acid significantly reduced DAG production while sphingosine 1-phoshate and ceramide 1-phosphate reduced MAG formation from PA. Using different enzymatic modulators (detergents and ions) an increase in the PA metabolism by phospholipase type A was observed. Our findings evidence an active PA metabolism in purified liver nuclei which generates important lipid second messengers, and which could thus be involved in nuclear processes such as gene transcription.

Differences in phosphatidic acid signalling and metabolism between ABA and GA treatments of barley aleurone cells.

Phosphatidic acid (PA) is the common lipid product in abscisic acid (ABA) and gibberellic acid (GA) response. In this work we investigated the lipid metabolism in response to both hormones. We could detect an in vivo phospholipase D activity (PLD, EC 3.1.4.4). This PLD produced [(32)P]PA (phosphatidic acid) rapidly (minutes) in the presence of ABA, confirming PA involvement in signal transduction, and transiently, indicating rapid PA removal after generation. The presence of PA removal by phosphatidate phosphatase 1 and 2 isoforms (E.C. 3.1.3.4) was verified in isolated aleurone membranes in vitro, the former but not the latter being specifically responsive to the presence of GA or ABA. The in vitro DGPP phosphatase activity was not modified by short time incubation with GA or ABA while the in vitro PA kinase - that allows the production of 18:2-DGPP from 18:2-PA - is stimulated by ABA. The long term effects (24 h) of ABA or GA on lipid and fatty acid composition of aleurone layer cells were then investigated. An increase in PC and, to a lesser extent, in PE levels is the consequence of both hormone treatments. ABA, in aleurone layer cells, specifically activates a PLD whose product, PA, could be the substrate of PAP1 and/or PAK activities. Neither PLD nor PAK activation can be monitored by GA treatment. The increase in PAP1 activity monitored after ABA or GA treatment might participate in the increase in PC level observed after 24 h hormone incubation.

Aging modifies the enzymatic activities involved in 2-arachidonoylglycerol metabolism.

One of the principal monoacylglycerol (MAG) species in animal tissues is 2-arachidonoylglycerol (2-AG), and the diacylglycerol lipase (DAGL) pathway is the most important 2-AG biosynthetic pathway proposed to date. Lysophosphatidate phosphatase (LPAase) activity is part of another 2-AG-forming pathway in which monoacylglycerol lipase (MAGL) is the major degrading enzyme. The purpose of this study was to analyze the manner in which DAGL, LPAase, and MAGL enzymes are modified in the central nervous system (CNS) during aging. To this end, diacylglycerols (DAGs) and MAGs of different composition were used as substrates of DAGL and MAGL, respectively. All enzymatic activities were evaluated in membrane and soluble fractions as well as in synaptic terminals from the cerebral cortex (CC) of adult and aged rats. Results related to 2-AG metabolism show that aging: (a) decreases DAGL-α expression in the membrane fraction whereas in synaptosomes it increases DAGL-ß and decreases MAGL expression; (b) decreases LPAase activity in both membrane and soluble fractions; (c) decreases DAGL and stimulates LPAase activities in CC synaptic terminals; (d) stimulates membrane-associated MAGL-coupled DAGL activity; and (e) stimulates MAGL activity in CC synaptosomes. Our results also reveal that during aging the net balance between the enzymatic activities involved in 2-AG synthesis and breakdown is low availability of 2-AG in CC membrane fractions and synaptic terminals. Taken together, our results lead us to conclude that these enzymes play crucial roles in the regulation of 2-AG tissue levels during aging.

Distinctive roles of PLD signaling elicited by oxidative stress in synaptic endings from adult and aged rats.

The role of iron in oxidative injury in the nervous system has been extensively described. However, little is known about the role of lipid signal transduction in neurodegeneration processes triggered by iron overload. The purpose of this work was to characterize the regulation and the crosstalk between phosphatidylcholine (PC)-derived diacylglycerol (DAG) and cannonical signaling pathways during iron-induced oxidative stress in cerebral cortex synaptic endings (Syn) obtained from adult (4 months old) and aged (28 months old) rats. DAG production was increased in Syn exposed to iron. This rise in DAG formation was due to phospholipase D1 (PLD1) and PLD2 activations. In adult rats, PKD1, ERK1/2 and PKCα/ßII activations were PLD1 and PLD2 dependent. In contrast, in senile rats, DAG formation catalyzed by PLDs did not participate in PKD1, ERK1/2 and PKCα/ßII regulations, but it was dependent on ERK and PKC activities. Iron-induced oxidative stress promoted an increased localization of PLD1 in membrane rafts, whereas PLD2 was excluded from these domains and appeared to be involved in glutamate transporter function. Our results show a differential regulation and synaptic function of DAG generated by PLDs during iron-induced oxidative stress as a consequence of aging.

Comparative phytohormone profiles, lipid kinase and lipid phosphatase activities in barley aleurone, coleoptile, and root tissues.

We analyzed lipid kinase and lipid phosphatase activities and determined endogenous phytohormone levels by liquid chromatography-tandem mass spectrometry in root and coleoptile tissues following germination of barley (Hordeum vulgare) seeds. The enzymes showing highest activity in aleurone cells were diacylglycerol kinase (DAG-k, EC 2.7.1.107) and phosphatidate kinase (PA-k). The ratio of gibberellins (GAs) to abscisic acid (ABA) was 2-fold higher in aleurone than in coleoptile or root tissues. In coleoptiles, phosphatidylinositol 4-kinase (PI4-k, EC 2.7.1.67) showed the highest enzyme activity, and jasmonic acid (JA) level was higher than in aleurone. In roots, activities of PI4-k, DAG-k, and PA-k were similar, and salicylic acid (SA) showed the highest concentration. In the assays to evaluate the hydrolysis of DGPP (diacylglycerol pyrophosphate) and PA (phosphatidic acid) we observed that PA hydrolysis by LPPs (lipid phosphate phosphatases) was not modified; however, the diacylglycerol pyrophosphate phosphatase (DGPPase) was strikingly higher in coleoptile and root tissues than to aleurone. Relevance of these findings in terms of signaling responses and seedling growth is discussed.

Regulation of phosphatidic acid levels in Trypanosoma cruzi.

Lipid kinases and phosphatases play essential roles in signal transduction processes involved in cytoskeletal rearrangement, membrane trafficking, and cellular differentiation. Phosphatidic acid (PtdOH) is an important mediator lipid in eukaryotic cells, but little is known regarding its regulation in the parasite Trypanosoma cruzi, an agent of Chagas disease. In order to clarify the relationship between PtdOH metabolism and developmental stages of T. cruzi, epimastigotes in culture were subjected to hyperosmotic stress (~1,000 mOsm/L), mimicking the environment in the rectum of vector triatomine bugs. These experimental conditions resulted in differentiation to an intermediate form between epimastigotes and trypomastigotes. Morphological changes of epimastigotes were correlated with an increase in PtdOH mass accomplished by increased enzyme activity of diacylglycerol kinase (DAGK, E.C. 2.7.1.107) and concomitant decreased activity of phosphatidate phosphatases type 1 and type 2 (PAP1, PAP2, E.C. 3.1.3.4). Our results indicate progressive increases of PtdOH levels during the differentiation process, and suggest that the regulation of PtdOH metabolism is an important mechanism in the transition from T. cruzi epimastigote to intermediate form.