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.

Anandamide (an endocannabinoid) and other bioactive long-chain NAEs (N-acylethanolamines) are formed by direct release from N-acyl-PE (N-acyl-phosphatidylethanolamine) by a PLD (phospholipase D). However, the possible presence of a two-step pathway from N-acyl-PE has also been suggested previously, which comprises (1) the hydrolysis of N-acyl-PE to N-acyl-lysoPE by PLA1/PLA2 enzyme(s) and (2) the release of NAEs from N-acyllysoPE by lysoPLD (lysophospholipase D) enzyme(s). In the present study we report for the first time the characterization of enzymes responsible for this pathway. The PLA1/PLA2 activity for N-palmitoyl-PE was found in various rat tissues, with the highest activity in the stomach. This stomach enzyme was identified as group IB sPLA2 (secretory PLA2), and its product was determined as N-acyl-1-acyl-lysoPE. Recombinant group IB, IIA and V of sPLA2s were also active with N-palmitoyl-PE, whereas group X sPLA2 and cytosolic PLA2a were inactive. In addition, we found wide distribution of lysoPLD activity generating N-palmitoylethanolamine from N-palmitoyl-lysoPE in rat tissues, with higher activities in the brain and testis. Based on several lines of enzymological evidence, the lysoPLD enzyme could be distinct from the known N-acyl-PE-hydrolysing PLD. sPLA2-IB dose dependently enhanced the production of N-palmitoylethanolamine from N-palmitoyl-PE in the brain homogenate showing the lysoPLD activity. N-Arachidonoyl-PE and N-arachidonoyl-lysoPE as anandamide precursors were also good substrates of sPLA2-IB and the lysoPLD respectively. These results suggest that the sequential actions of PLA2 and lysoPLD may constitute another biosynthetic pathway for NAEs, including anandamide.

BACKGROUND Remote ischemic preconditioning (RIPC) can inhibit recurrent ischemic events effectively in patients with acute or chronic cerebral ischemia. However, it is still unclear whether RIPC can impede ischemic injury after carotid artery stenting (CAS) in patients with severe carotid artery stenosis. METHODS Subjects with severe carotid artery stenosis were recruited in this randomized controlled study, and assigned to RIPC, sham, and no intervention (control) groups. All subjects received standard medical therapy. Subjects in the RIPC and sham groups underwent RIPC and sham RIPC twice daily, respectively, for 2 weeks before CAS. Plasma neuron-specific enolase and S-100B were used to evaluate safety, hypersensitive C-reactive protein, and new ischemic diffusion-weighted imaging lesions were used to determine treatment efficacy. The primary outcomes were the presence of ≥1 newly ischemic brain lesions on diffusion-weighted imaging within 48 hours after stenting and clinical events within 6 months after stenting. RESULTS We randomly assigned 189 subjects in this study (63 subjects in each group). Both RIPC and sham RIPC procedures were well tolerated and completed with high compliance (98.41% and 95.24%, respectively). Neither plasma neuron-specific enolase levels nor S-100B levels changed significantly before and after treatment. No severe adverse event was attributed to RIPC and sham RIPC procedures. The incidence of new diffusion-weighted imaging lesions in the RIPC group (15.87%) was significantly lower than in the sham group (36.51%; relative risk, 0.44; 96% confidence interval, 0.20–0.91; P<0.01) and the control group (41.27%; relative risk, 0.39; 96% confidence interval, 0.21–0.82; P<0.01). The volumes of lesions were smaller in the RIPC group than in the control and sham groups (P<0.01 each). Ischemic events that occurred after CAS were 1 transient ischemic attack in the RIPC group, 2 strokes in the control group, and 2 strokes and 1 transient ischemic attack in the sham group, but these results were not significantly different among the 3 groups (P=0.597). CONCLUSIONS RIPC is safe in patients undergoing CAS, which may be able to decrease ischemic brain injury secondary to CAS. However, the mechanisms and effects of RIPC on clinical outcomes in this cohort of patients need further investigation. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01654666

No abstract

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor expressed in many cell types, including osteoblasts, osteocytes, and osteoclasts. Nrf2 has been considered a master regulator of cytoprotective genes against oxidative and chemical insults. The lack of Nrf2 can induce pathologies in multiple organs. Nrf2 deficiency promotes osteoclast differentiation and osteoclast activity, which leads to an increase in bone resorption. The role of Nrf2 in osteoblast differentiation and osteoblast activity is more complex. Nrf2 mediates anabolic effects within an ideal range. Nrf2 deletion suppresses load induced bone formation and delays fracture healing. Overall, Nrf2 plays an important role in the regulation of bone homeostasis in bone cells.

Bioactive N-acylethanolamines including the endocannabinoid anandamide are known to be hydrolyzed to fatty acids and ethanolamine by fatty acid amide hydrolase (FAAH). In addition, we recently cloned an isozyme termed ''N-acylethanolamine-hydrolyzing acid amidase (NAAA)'', which is active only at acidic pH [Tsuboi, Sun, Okamoto, Araki, Tonai, Ueda, J. Biol. Chem. 285 (2005) 11082 -11092]. However, physiological roles of NAAA remained unclear. Here, we examined a possible contribution of NAAA to the degradation of various Nacylethanolamines in macrophage cells. NAAA mRNA as well as FAAH mRNA was detected in several macrophage-like cells, including RAW264.7, and mouse peritoneal macrophages. The homogenates of RAW264.7 cells showed both the NAAA and FAAH activities which were confirmed with the aid of their respective specific inhibitors, N-cyclohexanecarbonylpentadecylamine (CCP) and URB597. As analyzed with intact cells, RAW264.7 cells and peritoneal macrophages degraded anandamide, N-palmitoylethanolamine, N-oleoylethanolamine, and Nstearoylethanolamine. Pretreatment of the cells with CCP or URB597 partially inhibited the degradation, and a combination of the two compounds caused more profound inhibition. In contrast, the anandamide hydrolysis in mouse brain appeared to be principally attributable to FAAH despite the expression of NAAA in the brain. These results suggested that NAAA and FAAH cooperatively degraded various N-acylethanolamines in macrophages. D

No abstract

No abstract