Anandamide (N-arachidonoylethanolamine) is known to be an endogenous ligand of cannabinoid and vanilloid receptors. Its congeners (collectively referred to as Nacylethanolamines) also show a variety of biological activities. These compounds are principally formed from their corresponding N-acyl-phosphatidylethanolamines by a phosphodiesterase of the phospholipase D-type in animal tissues. We purified the enzyme from rat heart, and by the use of the sequences of its internal peptides cloned its complementary DNAs from mouse, rat, and human. The deduced amino acid sequences were composed of 393-396 residues, and showed that the enzyme has no homology with the known phospholipase D enzymes but is classified as a member of the zinc metallohydrolase family of the ␤-lactamase fold. As was overexpressed in COS-7 cells, the recombinant enzyme generated anandamide and other N-acylethanolamines from their corresponding N-acyl-phosphatidylethanolamines at comparable rates. In contrast, the enzyme was inactive with phosphatidylcholine and phosphatidylethanolamine. Assays of the enzyme activity and the messenger RNA and protein levels revealed its wide distribution in murine organs with higher contents in the brain, kidney, and testis. These results confirm that a specific phospholipase D is responsible for the generation of N-acylethanolamines including anandamide, strongly suggesting the physiological importance of lipid molecules of this class.

Bioactive N-acylethanolamines, including anandamide (an endocannabinoid) and N-palmitoylethanolamine (an anti-inflammatory and neuroprotective substance), are hydrolyzed to fatty acids and ethanolamine by fatty acid amide hydrolase. Moreover, we found another amidohydrolase catalyzing the same reaction only at acidic pH, and we purified it from rat lung (Ueda, N., Yamanaka, K., and Yamamoto, S. (2001) J. Biol. Chem. 276, 35552-35557). Here we report complementary DNA cloning and functional expression of the enzyme termed "N-acylethanolamine-hydrolyzing acid amidase (NAAA)" from human, rat, and mouse. The deduced primary structures revealed that NAAA had no homology to fatty acid amide hydrolase but belonged to the choloylglycine hydrolase family. Human NAAA was essentially identical to a gene product that had been noted to resemble acid ceramidase but lacked ceramide hydrolyzing activity. The recombinant human NAAA overexpressed in HEK293 cells hydrolyzed various N-acylethanolamines with N-palmitoylethanolamine as the most reactive substrate. Most interestingly, a very low ceramide hydrolyzing activity was also detected with NAAA, and N-lauroylethanolamine hydrolyzing activity was observed with acid ceramidase. By the use of tunicamycin and endoglycosidase, NAAA was found to be a glycoprotein. Furthermore, the enzyme was proteolytically processed to a shorter form at pH 4.5 but not at pH 7.4. Expression analysis of a green fluorescent protein-NAAA fusion protein showed a lysosome-like distribution in HEK293 cells. The organ distribution of the messenger RNA in rats revealed its wide distribution with the highest expression in lung. These results demonstrated that NAAA is a novel N-acylethanolamine-hydrolyzing enzyme that shows structural and functional similarity to acid ceramidase.

A high throughput screen for neutral, magnesium-dependent sphingomyelinase (SMase) was performed. One inhibitor discovered in the screen, GW4869, functioned as a noncompetitive inhibitor of the enzyme in vitro with an IC 50 of 1 M. It did not inhibit acid SMase at up to at least 150 M. The compound was then evaluated for its ability to inhibit tumor necrosis factor (TNF)-induced activation of neutral SMase (N-SMase) in MCF7 cells. GW4869 (10 M) partially inhibited TNF-induced sphingomyelin (SM) hydrolysis, and 20 M of the compound was protected completely from the loss of SM. The addition of 10 -20 M GW4869 completely inhibited the initial accumulation of ceramide, whereas this effect was partially lost at later time points (24 h). These data therefore support the inhibitory action of GW4869 on N-SMase not only in vitro but also in a cellular model. The addition of GW4869 at both 10 and 20 M did not modify cellular glutathione levels in response to TNF, suggesting that the action of GW4869 occurred downstream of the drop in glutathione, which was shown previously to occur upstream of the activation of N-SMase. Further, whereas TNF treatment also caused a 75% increase of de novo synthesized ceramide after 20 h of incubation, GW4869, at either 10 or 20 M, had no effect on this pathway of ceramide generation. In addition, GW4869 did not significantly impair TNF-induced NF-B translocation to nuclei. Therefore, GW4869 does not interfere with other key TNF-mediated signaling effects. GW4869 was able, in a dose-dependent manner, to significantly protect from cell death as measured by nuclear condensation, caspase activation, PARP degradation, and trypan blue uptake. These protective effects were accompanied by significant inhibition of cytochrome c release from mitochondria and caspase 9 activation, therefore localizing N-SMase activation upstream of mitochondrial dysfunction. In conclusion, our results indicate that NSMase activation is a necessary step for the full development of the cytotoxic program induced by TNF.

Class II α-isoform of phosphatidylinositol 3-kinases (PI3K-C2α) is localized in endosomes, the trans-Golgi network and clathrin-coated vesicles, however, its functional role is little understood. Global or endothelial cell (EC)-specific targeted disruption of PI3K-C2α resulted in embryonic lethality due to defects in sprouting angiogenesis and vascular maturation. PI3K-C2α knockdown in ECs induced decreased phospatidylinositol 3-phosphate-enriched endosomes, impaired endosomal trafficking, and defective delivery of VE-cadherin to EC junctions and its assembly. PI3K-C2α knockdown also impeded cell signaling including vascular endothelial growth factor receptor internalization and endosomal RhoA activation. These together led to defective EC migration, proliferation, tube formation and barrier integrity. Endothelial PI3K-C2α deletion suppressed post-ischemic and tumor angiogenesis, and diminished vascular barrier function, with greatly augmented susceptibility to anaphylaxis and a higher incidence of dissecting aortic aneurysm formation in response to angiotensin II infusion. Thus, PI3K-C2α plays a crucial role in vascular formation and barrier integrity, and represents a new therapeutic target for vascular diseases. 3Formation of the vascular network by vasculogenesis and angiogenesis is essential for embryonic development, repair and remodeling of tissues in adults, as well as tumor growth. The angiogenic response to vascular endothelial growth factor (VEGF) and other factors begins with vascular leakage and dissolution of the subendothelial basement membrane, followed by proliferation and migration of vascular EC 1,2 . Then, formation of the intercellular junctions results in initial sprouts from existing vessels. The newly formed endothelial tubes are associated with mural cells, i.e. smooth muscle cells (SMC) and pericytes, thus becoming mature and stabilized 3 . Tightness of the intercellular junctions, particularly adherens junctions composed of VE-cadherin, controls vascular permeability 4,5 . Quiescent, stabilized vasculature with intact barrier integrity dominates in the healthy condition. In contrast, in pathological conditions, such as tumors, the vasculature is generally inmaturate and leaky. In the case of vascular insult such as excessive angiotensin II (Ang II) activity, increased vascular permeability is asssociated with leukocyte infiltration in the vascular wall and vascular disruption 6,7 . Therefore, stabilization of the vasculature and maintenance of vascular integrity is essential for vascular and tissue homeostasis 8,9 .PI3Ks are an enzyme family that phosphorylates membrane inositol lipids at the 3' position of the inositol ring. The lipid products of PI3Ks serve as important intracellular messengers by interacting with effector proteins, which include protein kinases, guanine nucleotide exchangers for G proteins, and actin cytoskeleton-regulating proteins. Through these actions, PI3Ks regulate a diverse array of cellular processes 10-12 .PI3Ks comprise three classes. Class I PI...

STAT3 regulates glucose homeostasis by suppressing the expression of gluconeogenic genes in the liver. The mechanism by which hepatic STAT3 is regulated by nutritional or hormonal status has remained unknown, however. Here, we show that an increase in the plasma insulin concentration, achieved either by glucose administration or by intravenous insulin infusion, stimulates tyrosine phosphorylation of STAT3 in the liver. This effect of insulin was mediated by the hormone's effects in the brain, and the increase in hepatic IL-6 induced by the brain-insulin action is essential for the activation of STAT3. The inhibition of hepatic glucose production and of expression of gluconeogenic genes induced by intracerebral ventricular insulin infusion was impaired in mice with liver-specific STAT3 deficiency or in mice with IL-6 deficiency. These results thus indicate that IL-6-STAT3 signaling in the liver contributes to insulin action in the brain, leading to the suppression of hepatic glucose production.

No abstract

We investigated the mechanism of endothelin receptor type A (ETA) internalization in Chinese hamster ovary cells using two assays; flow cytometric quantification of cell surface myc-ETA and in situ localization of Cy5-labeled ET-1. In both assays, agonist-dependent internalization of myc-ETA was inhibited by nystatin and filipin, both of which disrupt internalization via caveolae, whereas it was barely affected by chlorpromazine and hypertonic sucrose, both of which disrupt internalization via clathrin-coated pits. In addition to myc-ETA, ET-1 caused intracellular translocation of caveolin-1 and this translocation was also blocked by nystatin but not by chlorpromazine. These results strongly argue that ETA is internalized via caveolae but not clathrincoated pits. Treatment of the cells with cholesterol oxidase reduced cellular cholesterol and caused intracellular translocation of caveolin-1 but did not affect cell surface localization of myc-ETA. In cholesterol oxidasetreated cells, however, both chlorpromazine and hypertonic sucrose effectively blocked ET-1-induced myc-ETA internalization and nystatin was less effective than in untreated cells. Accordingly, expression of a dominant negative form of ␤-arrestin blocked myc-ETA internalization in cholesterol oxidase-treated cells but not in untreated cells. These results suggest that, in Chinese hamster ovary cells, 1) agonist-occupied ETA can be internalized either via caveolae or clathrin-coated pits; 2) of the two, the former is the default pathway; and 3) the oxidative state of cell surface cholesterol is one of the factors involved in the pathway selection.At present, there appear to be at least two distinct internalization pathways for agonist-occupied G protein-coupled receptors (GPCRs) 1 : one via clathrin-coated pits (1, 2) and the other via caveolae (3, 4). The molecular mechanism for sequestration of agonist-occupied GPCRs to clathrin-coated pits has been best studied for ␤ 2 -adrenergic receptors (␤ 2 AR) (2, 5). In brief, upon binding of an agonist, ␤ 2 AR is phosphorylated by a GPCR kinase (GRK) and the phosphorylated receptors bind ␤-arrestin, which then targets the receptors to clathrin-coated pits.Various GPCRs other than ␤ 2 AR have been shown to be phosphorylated by GRK and thought to be targeted to clathrincoated pits in a similar manner (6 -9). Caveolae are small flask-shaped invaginations of the plasma membrane, characterized by high levels of cholesterol and glycosphingolipids and also by the presence of caveolin, a 20 -24-kDa integral membrane protein (2, 3). Although much less is known about the molecular mechanism involved, sequestration of agonist-occupied GPCRs to this microdomain has been demonstrated both by morphological and biochemical methods. For example, electron microscopic studies showed that both agonist-occupied ␤ 2 AR and bradykinin B2 receptors were localized in caveolae (10 -12). For other GPCRs such as angiotensin II type 1 and m2 muscarinic acetylcholine receptors, agonist-dependent sequestration in this microdomain was ...

Mathematical models have become a necessary tool for organizing the rapidly increasing amounts of large-scale data on biochemical pathways and for advanced evaluation of their structure and regulation. Most of these models have addressed specific pathways using either stoichiometric or flux-balance analysis, or fully kinetic Michaelis-Menten representations, metabolic control analysis, or biochemical systems theory. So far, the predictions of kinetic models have rarely been tested using direct experimentation. Here, we validate experimentally a biochemical systems theoretical model of sphingolipid metabolism in yeast. Simulations of metabolic fluxes, enzyme deletion and the effects of inositol (a key regulator of phospholipid metabolism) led to predictions that show significant concordance with experimental results generated post hoc. The model also allowed the simulation of the effects of acute perturbations in fatty-acid precursors of sphingolipids, a situation that is not amenable to direct experimentation. The results demonstrate that modelling now allows testable predictions as well as the design and evaluation of hypothetical 'thought experiments' that may generate new metabolomic approaches.