Identification of a small RhoA GTPase inhibitor effective in fission yeast and human cells.

The Rho GTPase family proteins are key regulators of cytoskeletal dynamics. Deregulated activity of Rho GTPases is associated with cancers and neurodegenerative diseases, and their potential as drug targets has long been recognized. Using an economically effective drug screening workflow in fission yeast and human cells, we have identified a Rho GTPase inhibitor, O1. By a suppressor mutant screen in fission yeast, we find a point mutation in the rho1 gene that confers resistance to O1. Consistent with the idea that O1 is the direct inhibitor of Rho1, O1 reduced the cellular amount of activated, GTP-bound Rho1 in wild-type cells, but not in the O1-resistant mutant cells, in which the evolutionarily conserved Ala62 residue is mutated to Thr. Similarly, O1 inhibits activity of the human orthologue RhoA GTPase in tissue culture cells. Our studies illustrate the power of yeast phenotypic screens in the identification and characterization of drugs relevant to human cells and have identified a novel GTPase inhibitor for fission yeast and human cells.

Identification of novel microtubule inhibitors effective in fission yeast and human cells and their effects on breast cancer cell lines.

Microtubules are critical for a variety of cellular processes such as chromosome segregation, intracellular transport and cell shape. Drugs against microtubules have been widely used in cancer chemotherapies, though the acquisition of drug resistance has been a significant issue for their use. To identify novel small molecules that inhibit microtubule organization, we conducted sequential phenotypic screening of fission yeast and human cells. From a library of diverse 10 371 chemicals, we identified 11 compounds that inhibit proper mitotic progression both in fission yeast and in HeLa cells. An in vitro assay revealed that five of these compounds are strong inhibitors of tubulin polymerization. These compounds directly bind tubulin and destabilize the structures of tubulin dimers. We showed that one of the compounds, L1, binds to the colchicine-binding site of microtubules and exhibits a preferential potency against a panel of human breast cancer cell lines compared with a control non-cancer cell line. In addition, L1 overcomes cellular drug resistance mediated by ßIII tubulin overexpression and has a strong synergistic effect when combined with the Plk1 inhibitor BI2536. Thus, we have established an economically effective drug screening strategy to target mitosis and microtubules, and have identified a candidate compound for cancer chemotherapy.

Characterization of Chlamydomonas reinhardtii Mutants That Exhibit Strong Positive Phototaxis.

The most motile phototrophic organisms exhibit photo-induced behavioral responses (photobehavior) to inhabit better light conditions for photosynthesis. The unicellular green alga Chlamydomonas reinhardtii is an excellent model organism to study photobehavior. Several years ago, we found that C. reinhardtii cells reverse their phototactic signs (i.e., positive and negative phototaxis) depending on the amount of reactive oxygen species (ROS) accumulated in the cell. However, its molecular mechanism is unclear. In this study, we isolated seven mutants showing positive phototaxis, even after the induction of negative phototaxis (ap1~7: always positive) to understand the ROS-dependent regulatory mechanism for the phototactic sign. We found no common feature in the mutants regarding their growth, high-light tolerance, and photosynthetic phenotypes. Interestingly, five of them grew faster than the wild type. These data suggest that the ROS-dependent regulation of the phototactic sign is not a single pathway and is affected by various cellular factors. Additionally, the isolation and analyses of mutants with defects in phototactic-sign regulation may provide clues for their application to the efficient cultivation of algae.

Reoxygenation with 100% Oxygen Following Hypoxia in Mice Causes Apoptosis.

After hypoxia, reoxygenation with air is the consensus treatment for full-term neonates; however, the effect of hyperoxic reoxygenation of adults is unknown. The present study was designed to investigate the effects of reoxygenation with 100% oxygen after hypoxia on inflammation and apoptosis in mice. Eight-week-old mice were either subjected to hypoxia in 8% oxygen for 30 min or air served as controls. Following hypoxia, mice underwent reoxygenation for 30 min with 21% or 100% oxygen. Tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), caspase-3 and brain derived neurotrophic factor (BDNF) mRNA study and histopathological study were performed. Reoxygenation with 100% oxygen significantly increased TNF-α (2.5 h after hypoxia), IL-1ß (5 h after hypoxia), caspase-3 (8 h after hypoxia) mRNA levels in the whole brain compared with 21% oxygen, and significantly decreased erythropoietin mRNA expression compared with 21% oxygen 9 h after reoxygenation. However, reoxygenation with 100% oxygen and 21% oxygen significantly decreased BDNF mRNA levels compared with control air group. There were no clear abnormal findings showing neuronal death among the three groups. Reoxygenation with 100% oxygen after hypoxia induced inflammation and apoptosis in adult mice. Therefore, these results suggest that the reoxygenation with 100% oxygen after hypoxia has harmful effects on adult brain as well as on neonatal brain.

Effects of sevoflurane exposure during late pregnancy on brain development of offspring mice.

BACKGROUND: Exposure to some anesthetic agents during the fetal period has been shown to induce neurodegeneration or learning deficits in animal models. Sevoflurane is one of the most prevalent general anesthetics; however, the influence of sevoflurane at a clinically relevant concentration on the developing fetal brain remains unknown. OBJECTIVE: We investigated whether a single sevoflurane exposure during the fetal period would affect neuronal development and learning/memory ability in mice. METHODS: Pregnant mice at gestational day 17 were anesthetized with 1.5% sevoflurane in 50% oxygen for 6 h. Mice in the control group were exposed in 50% oxygen without sevoflurane. Pups of some mice in both groups subsequently were delivered early by cesarean section and whole fetal brains were excised. The rest of the pups were delivered naturally at gestational day 20 and were maintained for 8 weeks. The mRNA expression levels of caspase-3, brain derived neurotrophic factor (BDNF), nerve growth factor (NGF), and LIM kinase-1 (LIMK-1) were measured in fetal whole brain and 8-week-old hippocampus sections. Synaptophysin protein in adult hippocampus was assessed immunochemically. In addition, 8-week-old mice were subjected to the radial maze test. RESULTS: No significant difference between sevoflurane and control groups regarding mRNA expression levels of all targets was seen, nor was there an obvious change in synaptophysin protein expression. The results of the maze test revealed that the each-day performance ratios (the rate of errors) of the sevoflurane group were not altered as compared with the control group. CONCLUSIONS: These results suggest that the exposure during late pregnancy to a clinically relevant concentration of sevoflurane does not affect neuronal development and learning/memory ability of offspring mice.

T-5224, a selective inhibitor of c-Fos/activator protein-1, improves survival by inhibiting serum high mobility group box-1 in lethal lipopolysaccharide-induced acute kidney injury model.

BACKGROUND: Sepsis is a potentially fatal syndrome mediated by an early [e.g., tumor necrosis factor-alpha (TNF-α)] and late [high mobility group box-1 (HMGB-1)] proinflammatory cytokine response to infection. Sepsis-induced acute kidney injury (AKI) is associated with a high mortality. C-Fos/activator protein-1 (AP-1) controls the transactivation of proinflammatory cytokines via AP-1 binding in the promoter region. T-5224 is a de novo small molecule inhibitor of c-Fos/AP-1 that controls gene expression of multiple proinflammatory cytokines. We investigated whether T-5224, a selective inhibitor of c-Fos/AP-1, improves survival in lethal lipopolysaccharide (LPS)-induced AKI by inhibiting early (TNF-α) and late (HMGB-1) proinflammatory cytokine response. METHODS: Mice were divided into four groups (control, LPS, LPS + T-5224, and T-5224 only). Control mice were administered polyvinylpyrrolidone (PVP) solution orally, immediately after intraperitoneal (i.p.) saline injection. LPS mice were administered PVP solution orally immediately after i.p. LPS (10 mg/kg) injection. LPS + T-5224 mice were administered T-5224 orally (300 mg/kg) immediately after i.p. LPS injection. T-5224 mice were administered T-5224 orally (300 mg/kg) after i.p. saline injection. Serum concentrations of TNF-α, HMBG-1, and interleukin (IL)-10 were measured by enzyme-linked immunosorbent assay (ELISA). Serum blood urea nitrogen (BUN) and creatinine concentrations were commercially analyzed. Finally, histological examination was performed on the kidney. RESULTS: Treatment with T-5224 decreased serum TNF-α and HMGB-1 levels and increased survival after LPS injection. Furthermore, T-5224 treatment decreased serum BUN and creatinine concentrations but increased serum IL-10 concentration. LPS-induced pathological changes in kidney were attenuated by T-5224 treatment. CONCLUSIONS: These results suggest that T-5224, a selective inhibitor of c-Fos/AP-1, inhibits expression of early and late proinflammatory cytokines, protecting mice from LPS-induced lethality. T-5224 is a potential approach for decreasing lethality in sepsis-induced AKI.

CDK7 regulates the mitochondrial localization of a tail-anchored proapoptotic protein, Hid.

The mitochondrial outer membrane is a major site of apoptosis regulation across phyla. Human and C. elegans Bcl-2 family proteins and Drosophila Hid require the C-terminal tail-anchored (TA) sequence in order to insert into the mitochondrial membrane, but it remains unclear whether cytosolic proteins actively regulate the mitochondrial localization of these proteins. Here, we report that the cdk7 complex regulates the mitochondrial localization of Hid and its ability to induce apoptosis. We identified cdk7 through an in vivo RNAi screen of genes required for cell death. Although CDK7 is best known for its role in transcription and cell-cycle progression, a hypomorphic cdk7 mutant suppressed apoptosis without impairing these other known functions. In this cdk7 mutant background, Hid failed to localize to the mitochondria and failed to bind to recombinant inhibitors of apoptosis (IAPs). These findings indicate that apoptosis is promoted by a newly identified function of CDK7, which couples the mitochondrial localization and IAP binding of Hid.

D-ribose ameliorates cisplatin-induced nephrotoxicity by inhibiting renal inflammation in mice.

Cisplatin is one of the most potent chemotherapeutic anticancer drugs, but it can produce side effects such as nephrotoxicity. Inflammatory cytokines, chemokines and adhesion molecules have important roles in the pathogenesis of cisplatin-induced nephrotoxicity. D-Ribose is a naturally occurring five-carbon monosaccharide that is found in all living cells, and has anti-inflammatory effects in renal ischemia/reperfusion injury. The purpose of this study was to determine the protective effects of D-ribose on cisplatin-induced nephrotoxicity. Forty-eight mice were randomly divided into four groups: control, cisplatin, cisplatin + ribose, and ribose. Mice were given cisplatin (20 mg/kg body weight, intraperitoneally) with or without D-ribose (400 mg/kg body weight, intraperitoneally, immediately after cisplatin injection). At 72 h after cisplatin injection, we measured serum and renal tumor necrosis factor (TNF)-α and renal monocyte chemoattractant protein (MCP)-1 concentrations by enzyme-linked immunosorbent assay; renal expression of intercellular adhesion molecule (ICAM)-1 mRNA by real-time polymerase chain reaction; serum blood urea nitrogen and creatinine; and histological changes. Cisplatin increased serum and renal TNF-α concentrations, renal MCP-1 concentration, and renal ICAM-1 mRNA expression. Treatment with D-ribose attenuated the increase in serum and renal TNF-α concentrations, renal MCP-1 concentration, and renal ICAM-1 mRNA expression. Consequently, cisplatin-induced renal dysfunction and renal tubular necrosis were attenuated by D-ribose treatment. This is believed to be the first time that protective effects of D-ribose on cisplatin-induced nephrotoxicity via inhibition of inflammatory reactions have been investigated. Thus, D-ribose may become a new therapeutic candidate for the treatment of cisplatin-induced nephrotoxicity.

Curcumin ameliorates cisplatin-induced nephrotoxicity by inhibiting renal inflammation in mice.

Inflammatory mechanisms may play an important role in the pathogenesis of cisplatin-induced nephrotoxicity. Curcumin is an orange-yellow polyphenol present in curry spice and has anti-inflammatory and antioxidant effects. The purpose of this study was to determine the protective effects of curcumin on cisplatin-induced nephrotoxicity. Mice were randomly divided into four groups: control, cisplatin, cisplatin + curcumin and curcumin. Mice were given cisplatin (20 mg/kg body weight, intraperitoneally) with or without curcumin treatment (100 mg/kg body weight, intraperitoneally, immediately after cisplatin injection). Serum and renal tumor necrosis factor (TNF)-alpha and renal monocyte chemoattractant protein (MCP)-1 concentrations, intercellular adhesion molecule-1 (ICAM-1) mRNA expression in kidney, renal function and histological changes were determined 72 h after cisplatin injection. Serum TNF-alpha concentration in the cisplatin + curcumin group significantly decreased compared with that in the cisplatin group. Renal TNF-alpha and MCP-1 concentrations and ICAM-1 mRNA expression in kidney in the cisplatin + curcumin group also significantly decreased compared with those in the cisplatin group. Consequently, cisplatin-induced renal dysfunction and renal tubular necrosis scores were attenuated by curcumin treatment. These results indicate that curcumin acts to reduce cisplatin-induced nephrotoxicity through its anti-inflammatory effects. Thus, curcumin may become a new therapeutic candidate for the treatment of cisplatin-induced nephrotoxicity.

The effects of a selective inhibitor of c-Fos/activator protein-1 on endotoxin-induced acute kidney injury in mice.

BACKGROUND: Sepsis has been identified as the most common cause of acute kidney injury (AKI) in intensive care units. Lipopolysaccharide (LPS) induces the production of several proinflammatory cytokines including tumor necrosis factor (TNF)-alpha, a major pathogenetic factor in septic AKI. c-Fos/activator protein (AP)-1 controls the expression of these cytokines by binding directly to AP-1 motifs in the cytokine promoter regions. T-5224 is a new drug developed by computer-aided drug design that selectively inhibits c-Fos/AP-1 binding to DNA. In this study, we tested whether T-5224 has a potential inhibitory effect against LPS-induced AKI, by suppressing the TNF-alpha inflammatory response and other downstream effectors. METHODS: To test this hypothesis, male C57BL/6 mice at 7 weeks old were divided into three groups (control, LPS and T-5224 groups). Mice in the control group received saline intraperitoneally and polyvinylpyrrolidone solution orally. Mice in the LPS group were injected intraperitoneally with a 6 mg/kg dose of LPS and were given polyvinylpyrrolidone solution immediately after LPS injection. In the T-5224 group, mice were administered T-5224 orally at a dose of 300 mg/kg immediately after LPS injection. Serum concentrations of TNF-alpha, interleukin (IL)-1beta, IL-6 and IL-10 were measured by ELISA. Moreover, the expression of intercellular adhesion molecule (ICAM)-1 mRNA in kidney was examined by quantitative real-time RT-PCR. Finally, we evaluated renal histological changes. RESULTS: LPS injection induced high serum levels of TNF-alpha, IL-1beta and IL-6. However, the administration of T-5224 inhibited the LPS-induced increase in these cytokine levels. The serum levels of IL-10 in the LPS group and T-5224 group were markedly elevated compared with the control group. T-5224 also inhibited LPS-induced ICAM-1 mRNA expression. Furthermore histological studies supported an anti-inflammatory role of T-5224. CONCLUSIONS: In endotoxin-induced AKI, T-5224 inhibited the production of TNF-alpha and other downstream effectors. In contrast, T-5224 did not inhibit IL-10, an anti-inflammatory cytokine. These data support that the use of T-5224 is a promising new treatment for septic kidney injury.

Erythropoietin attenuates isoflurane-induced neurodegeneration and learning deficits in the developing mouse brain.

OBJECTIVES: To examine whether recombinant erythropoietin (rEPO) attenuates neurodegeneration and the learning disability induced by isoflurane with the postnatal day 7 (P7) mice. BACKGROUND: Some of general anesthetic agents induce neurodegeneration in developing brain. Several drugs, but not rEPO, were reported as candidates for the prevention of or treatment for neurodegeneration. METHOD AND MATERIALS: We divided P7 mice into three groups at random. One group (IE group) was exposed to 6-h isoflurane (1.0%) after 50,000 IU·kg(-1) rEPO administered subcutaneously. The second group (I) was exposed to isoflurane in the same manner as IE group except saline instead of rEPO. The third group (E) was exposed to air after rEPO administered. The mice were assigned to the radial arm maze on four consecutive days from P56 (day 1) to P59 (day 4). We divided the number of errors each day by that of day 1 to establish each-day performance ratio. After the test, neurodegenerative change in the hilus of dentate gyrus was assessed using Nissl staining. RESULTS: In radial maze test, the performance ratios of day 3 (mean ± sd) were 0.3 ± 0.2 (P < 0.05, vs I group), 0.8 ± 0.5, and 0.6 ± 0.2 in IE, I, and E groups, respectively, while those of day 4 were 0.3 ± 0.1 (P < 0.05), 0.8 ± 0.5, and 0.3 ± 0.2 (P < 0.05), respectively. The histopathological study revealed that in IE group the degenerative neuronal change was attenuated compared with I group. CONCLUSIONS: These results suggested that rEPO attenuated isoflurane-induced neurodegeneration.

[Case of transient paralysis and paresthesia after epidural blood patch].

A 47-year-old woman with postdural puncture headache suffered from transient paralysis and paresthesia immediately after the epidural blood patch. After one and a half hour, these symptoms disappeared spontaneously. We suspect that the spinal cord or spinal nerve root was transiently pressed by the viscous blood mass, because blood sampling maneuver took a few minutes. With passing of time, the viscous blood spread through the epidural space, and neurological symptoms improved. Although the risks of epidural blood patch are relatively low, we should closely pay attention to unexpected side effects.

The tumor suppressor gene H-Rev107 functions as a novel Ca2+-independent cytosolic phospholipase A1/2 of the thiol hydrolase type.

H-Rev107 is a protein that was previously cloned as a negative regulator of proto-oncogene Ras and classified as a class II tumor suppressor. Its structural similarity to lecithin retinol acyltransferase and Ca2+-independent phosphatidylethanolamine (PE) N-acyltransferase led us to analyze H-Rev107 as an enzyme involved in phospholipid metabolism. Here, we show that recombinant H-Rev107s from rat, human, and mouse possess phospholipase (PL) A1 or A2 activity toward phosphatidylcholine (PC). Further examination with purified recombinant protein revealed that H-Rev107 functions as a cytosolic Ca2+-independent PLA(1/2) for PC and PE with higher PLA1 activity than PLA2 activity. Dithiothreitol and iodoacetic acid exhibited stimulatory and inhibitory effects, respectively. Histidine-21 and cysteine-111 of rat H-Rev107 were presumed to form a catalytic dyad based on database analysis, and their single mutants were totally inactive. These results suggested that H-Rev107 is a hydrolase of the thiol type. The N-terminal proline-rich and C-terminal hydrophobic domains of H-Rev107 were earlier reported to be responsible for the regulation of cell proliferation. Analysis of deletion mutants indicated that these domains are also catalytically essential, suggesting relevance of the catalytic activity to the anti-proliferative activity.

Biosynthetic pathways of the endocannabinoid anandamide.

Anandamide (=N-arachidonoylethanolamine) is the first discovered endocannabinoid, and belongs to the class of bioactive, long-chain N-acylethanolamines (NAEs). In animal tissues, anandamide is principally formed together with other NAEs from glycerophospholipid by two successive enzymatic reactions: 1) N-acylation of phosphatidylethanolamine to generate N-acylphosphatidylethanolamine (NAPE) by Ca2+-dependent N-acyltransferase; 2) release of NAE from NAPE by a phosphodiesterase of the phospholipase D type (NAPE-PLD). Although these anandamide-synthesizing enzymes were poorly understood until recently, our cDNA cloning of NAPE-PLD in 2004 enabled molecular-biological approaches to the enzymes. NAPE-PLD is a member of the metallo-beta-lactamase family, which specifically hydrolyzes NAPE among glycerophospholipids, and appears to be constitutively active. Mutagenesis studies suggested that the enzyme functions through a mechanism similar to those of other members of the family. NAPE-PLD is widely expressed in animal tissues, including various regions in rat brain. Its expression level in the brain is very low at birth, and remarkably increases with development. Analysis of NAPE-PLD-deficient mice and other recent studies revealed the presence of NAPE-PLD-independent pathways for the anandamide formation. Furthermore, calcium-independent N-acyltransferase was discovered and characterized. In this article, we will review recent progress in the studies on these enzymes responsible for the biosynthesis of anandamide and other NAEs.

Discovery and characterization of a Ca2+-independent phosphatidylethanolamine N-acyltransferase generating the anandamide precursor and its congeners.

N-Acylphosphatidylethanolamines (NAPEs) are precursors of bioactive N-acylethanolamines, including the endocannabinoid anandamide. In animal tissues, NAPE is formed by transfer of a fatty acyl chain at the sn-1 position of glycerophospholipids to the amino group of phosphatidylethanolamine (PE), and this reaction is believed to be the principal rate-limiting step in N-acylethanolamine synthesis. However, the Ca2+-dependent, membrane-associated N-acyltransferase (NAT) responsible for this reaction has not yet been cloned. In this study, on the basis of the functional similarity of NAT to lecithin-retinol acyltransferase (LRAT), we examined a possible PE N-acylation activity in two rat LRAT homologous proteins. Upon overexpression in COS-7 cells, one protein, named rat LRAT-like protein (RLP)-1, catalyzed transfer of a radioactive acyl group from phosphatidylcholine (PC) to PE, resulting in the formation of radioactive NAPE. However, the RLP-1 activity was detected mainly in the cytosolic rather than membrane fraction and was little stimulated by Ca2+. Moreover, RLP-1 did not show selectivity with respect to the sn-1 and sn-2 positions of PC as an acyl donor and therefore could generate N-arachidonoyl-PE (anandamide precursor) from 2-arachidonoyl-PC and PE. In contrast, under the same assay conditions, partially purified NAT from rat brain was highly Ca2+-dependent, membrane-associated, and specific for the sn-1-acyl group of PC. RLP-1 mRNA was expressed predominantly in testis among various rat tissues, and the testis cytosol exhibited an RLP-1-like activity. These results reveal that RLP-1 can function as a PE N-acyltransferase, catalytically distinguishable from the known Ca2+-dependent NAT.

Functional analysis of the purified anandamide-generating phospholipase D as a member of the metallo-beta-lactamase family.

In animal tissues, bioactive N-acylethanolamines including the endocannabinoid anandamide are formed from their corresponding N-acylphosphatidylethanolamines (NAPEs) by the catalysis of a specific phospholipase D (NAPE-PLD) that belongs to the metallo-beta-lactamase family. Despite its potential physiological importance, NAPE-PLD has not yet been characterized with a purified enzyme preparation. In the present study we expressed a recombinant NAPE-PLD in Escherichia coli and highly purified it. The purified enzyme was remarkably activated in a dose-dependent manner by millimolar concentrations of Mg2+ as well as Ca2+ and, hence, appeared to be constitutively active. The enzyme showed extremely high specificity for NAPEs among various glycerophospholipids but did not reveal obvious selectivity for different long chain or medium chain N-acyl species of NAPEs. These results suggested the ability of NAPE-PLD to degrade different NAPEs without damaging other membrane phospholipids. Metal analysis revealed the presence of catalytically important zinc in NAPE-PLD. In addition, site-directed mutagenesis studies were addressed to several histidine and aspartic acid residues of NAPE-PLD that are highly conserved within the metallo-beta-lactamase family. Single mutations of Asp-147, His-185, His-187, Asp-189, His-190, His-253, Asp-284, and His-321 caused abolishment or remarkable reduction of the catalytic activity. Moreover, when six cysteine residues were individually mutated to serine, only C224S showed a considerably reduced activity. The activities of L207F and H380R found as single nucleotide polymorphisms were also low. Thus, NAPE-PLD appeared to function through a mechanism similar to those of the well characterized members of this family but play a unique role in the lipid metabolism of animal tissues.

Regional distribution and age-dependent expression of N-acylphosphatidylethanolamine-hydrolyzing phospholipase D in rat brain.

The endocannabinoid anandamide (N-arachidonoylethanolamine) and other bioactive long-chain N-acylethanolamines are thought to be formed from their corresponding N-acylphosphatidylethanolamines by a specific phospholipase D (NAPE-PLD) in the brain as well as other tissues. However, regional distribution of NAPE-PLD in the brain has not been examined. In the present study, we investigated the expression levels of NAPE-PLD in nine different regions of rat brain by enzyme assay, western blotting and real-time PCR. The NAPE-PLD activity was detected in all the tested brain regions with the highest activity in thalamus. Similar distribution patterns of NAPE-PLD were observed at protein and mRNA levels. We also found a remarkable increase in the expression levels of protein and mRNA of the brain NAPE-PLD with development, which was in good agreement with the increase in the activity. The age-dependent increase was also seen with several brain regions and other NAPE-PLD-enriched organs (heart and testis). p-Chloromercuribenzoic acid and cetyltrimethylammonium chloride, which inhibited recombinant NAPE-PLD dose-dependently, strongly inhibited the enzyme of all the brain regions. These results demonstrated wide distribution of NAPE-PLD in various brain regions and its age-dependent expression, suggesting the central role of this enzyme in the formation of anandamide and other N-acylethanolamines in the brain.

N-acylphosphatidylethanolamine-hydrolyzing phospholipase D: a novel enzyme of the beta-lactamase fold family releasing anandamide and other N-acylethanolamines.

N-acylethanolamines (NAEs) are a lipid class present in brain and other animal tissues and contains anandamide (an endocannabinoid) and other bioactive substances. NAEs are formed from N-acylphosphatidylethanolamines (NAPEs) by a phospholipase D (PLD)-type enzyme abbreviated to NAPE-PLD. Although this enzyme has been recognized for more than 20 years, its molecular cloning has only recently been achieved by us. We highly purified NAPE-PLD from the particulate fraction of rat heart, and on the basis of peptide sequences with the purified enzyme cloned its cDNA from mouse, rat and human. The deduced primary structures revealed no homology with any PLDs so far reported, but was suggested to belong to the beta-lactamase fold family. When overexpressed in COS-7 cells, the NAPE-PLD activity increased about 1000-fold in comparison with the endogenous activity. The recombinant enzyme generated various long-chain NAEs including anandamide from their corresponding NAPEs at similar rates. However, the enzyme was inactive with phosphatidylethanolamine and phosphatidylcholine and did not catalyze transphosphatidylation, a reaction characteristic of PLD. The enzyme was widely expressed in murine organs with higher levels in brain, testis and kidney. The existence of NAPE-PLD specifically hydrolyzing NAPEs to NAEs emphasizes physiological significance of NAEs including anandamide in brain and other tissues.

Mammalian cells stably overexpressing N-acylphosphatidylethanolamine-hydrolysing phospholipase D exhibit significantly decreased levels of N-acylphosphatidylethanolamines.

In animal tissues, NAEs (N-acylethanolamines), including N-arachidonoylethanolamine (anandamide), are primarily formed from their corresponding NAPEs (N-acylphosphatidylethanolamines) by a phosphodiesterase of the PLD (phospholipase D) type (NAPE-PLD). Recently, we cloned cDNAs of NAPE-PLD from mouse, rat and human [Okamoto, Morishita, Tsuboi, Tonai and Ueda (2004) J. Biol. Chem. 279, 5298-5305]. However, it remained unclear whether NAPE-PLD acts on endogenous NAPEs contained in the membrane of living cells. To address this question, we stably transfected two mammalian cell lines (HEK-293 and CHO-K1) with mouse NAPE-PLD cDNA, and investigated the endogenous levels and compositions of NAPEs and NAEs in these cells, compared with mock-transfected cells, with the aid of GC-MS. The overexpression of NAPE-PLD caused a decrease in the total amount of NAPEs by 50-90% with a 1.5-fold increase in the total amount of NAEs, suggesting that the recombinant NAPE-PLD utilizes endogenous NAPE as a substrate in the cell. Since the compositions of NAEs and NAPEs of NAPE-PLD-overexpressing cells and mock-transfected cells were very similar, the enzyme did not appear to discriminate among the N-acyl groups of endogenous NAPEs. These results confirm that overexpressed NAPE-PLD is capable of forming NAEs, including anandamide, in living cells.