Harald H.O. Schmid

Occurrence and postmortem generation of anandamide and other long-chain N-acylethanolamines in mammalian brain.

Long‐chain N‐acylethanolamines (NAEs) were assayed in pig, sheep and cow brain by gas chromatography/mass spectrometry of their tert.‐butyldimethylsilyl derivatives in the presence of deuterium‐labeled internal standards. Immediately after death, total NAEs ranged from about 2.7 μg/g wet weight (sheep, cow) to 6.5 μg/g wet weight (pig) and consisted almost exclusively (99%) of saturated and monounsaturated species. Anandamide (20:4n‐6 NAE) comprised about 1% of total NAE in pig and cow brain, but was absent in freshly extracted sheep brain. When pig brain was analysed after 0.5, 1, 3, 4 and 23 h at ambient temperature, NAE levels were found to increase substantially over the entire time period with 20:4n‐6 NAE formation exhibiting a time lag compared to that of saturated and monounsaturated NAEs.

Anandamide, but not 2-arachidonoylglycerol, accumulates during in vivo neurodegeneration

Endogenous cannabinoid receptor ligands (endocannabinoids) may rescue neurons from glutamate excitotoxicity. As these substances also accumulate in cultured immature neurons following neuronal damage, elevated endocannabinoid concentrations may be interpreted as a putative neuroprotective response. However, it is not known how glutamatergic insults affect in vivo endocannabinoid homeostasis, i.e. N‐arachidonoylethanolamine (anandamide) and 2‐arachidonoylglycerol (2‐AG), as well as other constituents of their lipid families, N‐acylethanolamines (NAEs) and 2‐monoacylglycerols (2‐MAGs), respectively. Here we employed three in vivo neonatal rat models characterized by widespread neurodegeneration as a consequence of altered glutamatergic neurotransmission and assessed changes in endocannabinoid homeostasis. A 46‐fold increase of cortical NAE concentrations (anandamide, 13‐fold) was noted 24 h after intracerebral NMDA injection, while less severe insults triggered by mild concussive head trauma or NMDA receptor blockade produced a less pronounced NAE accumulation. By contrast, levels of 2‐AG and other 2‐MAGs were virtually unaffected by the insults employed, rendering it likely that key enzymes in biosynthetic pathways of the two different endocannabinoid structures are not equally associated to intracellular events that cause neuronal damage in vivo. Analysis of cannabinoid CB1 receptor mRNA expression and binding capacity revealed that cortical subfields exhibited an up‐regulation of these parameters following mild concussive head trauma and exposure to NMDA receptor blockade. This may suggest that mild to moderate brain injury may trigger elevated endocannabinoid activity via concomitant increase of anandamide levels, but not 2‐AG, and CB1 receptor density.

Dysregulated Cannabinoid Signaling Disrupts Uterine Receptivity for Embryo Implantation

The mechanisms by which synchronized embryonic development to the blastocyst stage, preparation of the uterus for the receptive state, and reciprocal embryo-uterine interactions for implantation are coordinated are still unclear. We show in this study that preimplantation embryo development became asynchronous in mice that are deficient in brain-type (CB1) and/or spleen-type (CB2) cannabinoid receptor genes. Furthermore, whereas the levels of uterine anandamide (endocannabinoid) and blastocyst CB1 are coordinately down-regulated with the onset of uterine receptivity and blastocyst activation prior to implantation, these levels remained high in the nonreceptive uterus and in dormant blastocysts during delayed implantation and in pregnant, leukemia inhibitory factor (LIF)-deficient mice with implantation failure. These results suggest that a tight regulation of endocannabinoid signaling is important for synchronizing embryo development with uterine receptivity for implantation. Indeed this is consistent with our finding that while an experimentally induced, sustained level of an exogenously administered, natural cannabinoid inhibited implantation in wild-type mice, it failed to do so inCB1 −/− /CB2 −/−double mutant mice. The present study is clinically important because of the widely debated medicinal use of cannabinoids and their reported adverse effects on pregnancy.

Pathways and mechanisms of N-acylethanolamine biosynthesis: can anandamide be generated selectively?

Long-chain N-acylethanolamines (NAEs) and their precursors, N-acylethanolamine phospholipids, are ubiquitous trace constituents of animal and human cells, tissues and body fluids. Their cellular levels appear to be tightly regulated and they accumulate as the result of injury. Saturated and monounsaturated congeners which represent the vast majority of cellular NAEs can have cytoprotective effects while polyunsaturated NAEs, especially 20:4n-6 NAE (anandamide), elicit physiological effects by binding to and activating cannabinoid receptors. It is the purpose of this article to review published data on the pathways and mechanisms of NAE biosynthesis in mammals and to evaluate this information for its physiological significance. The generation and turnover of NAE via N-acyl PE through the transacylation-phosphodiesterase pathway may represent a novel cannabinoid receptor-independent signalling system, analogous to and possibly related to ceramide-mediated cell signalling.

Involvement of the Acid Sphingomyelinase Pathway in UVA-induced Apoptosis

The sphingomyelin-ceramide pathway is an evolutionarily conserved ubiquitous signal transduction system that regulates many cell functions including apoptosis. Sphingomyelin (SM) is hydrolyzed to ceramide by different sphingomyelinases. Ceramide serves as a second messenger in mediating cellular effects of cytokines and stress. In this study, we find that acid sphingomyelinase (SMase) activity was induced by UVA in normal JY lymphoblasts but was not detectable in MS1418 lymphoblasts from Niemann-Pick type D patients who have an inherited deficiency of acid SMase. We also provide evidence that UVA can induce apoptosis by activating acid SMase in normal JY cells. In contrast, UVA-induced apoptosis was inhibited in MS1418 cells. Exogenous SMase and its product, ceramide (10–40 μm), induced apoptosis in JY and MS1418 cells, but the substrate of SMase, SM (20–80 μm), induced apoptosis only in JY cells. These results suggest that UVA-induced apoptosis by SM is dependent on acid SMase activity. We also provide evidence that induction of apoptosis by UVA may occur through activation of JNKs via the acid SMase pathway.

Massive accumulation of N-acylethanolamines after stroke. Cell signalling in acute cerebral ischemia?

We investigated levels and compositions of N‐acylethanolamines (NAEs) and their precursors, N‐acyl phosphatidylethanolamines (N‐acyl PEs), in a rat stroke model applying striatal microdialysis for glutamate assay. Rats (n = 18) were treated with either intravenous saline (control), NMDA receptor antagonist MK801 (1 mg/kg), or CB1 receptor antagonist SR141716A (1 mg/kg) 30 min after permanent middle cerebral artery occlusion (MCAO). MK801 significantly attenuated the release of glutamate in the infarcted striatum (79 ± 22 μmol/L) as compared with controls (322 ± 104 μmol/L). The administration of CB1 antagonist SR141716A had no statistically significant effect on glutamate release (340 ± 89 μmol/L), but reduced infarct volume at 5 h after MCAO significantly by approximately 40%, whereas MK801 treatment resulted in a non‐significant (18%) reduction of infarct volume. In controls, striatal and cortical NAE concentrations were about 30‐fold higher in the infarcted than in the non‐infarcted hemisphere, whereas ipsilateral N‐acyl phosphatidylethanolamine (N‐acyl PE) levels exceeded contralateral levels by only a factor of two to three. Treatment with MK801 or SR141716A, or glutamate release in the infarcted tissue, had no significant effect on these levels. NAE accumulation during acute stroke may be due to increased synthesis as well as decreased degradation, possibly by inhibition of fatty acid amide hydrolase (FAAH).

THE N-ACYLATION-PHOSPHODIESTERASE PATHWAY AND CELL SIGNALLING

Long-chain N-acylethanolamines (NAEs) elicit a variety of biological and pharmacological effects. Anandamide (20:4n-6 NAE) and other polyunsaturated NAEs bind to the cannabinoid receptor and may thus serve as highly specific lipid mediators of cell signalling. NAEs can be formed by phospholipase D-catalyzed hydrolysis of N-acylethanolamine phospholipids or by direct condensation of ethanolamine and fatty acid. So far, most of the latter biosynthetic activity has been shown to be the reverse reaction of the NAE amidohydrolase that catalyzes NAE degradation. Thus, increasing evidence supports the hypothesis that the N-acylation-phosphodiesterase pathway yields not only saturated-monounsaturated NAEs, but polyunsaturated ones, including anandamide, as well.

Accumulation of N-acylethanolamine glycerophospholipids in infarcted myocardium

A new phospholipid was detected in the infarcted areas of canine myocardium 24 h after ligation of the left descending branch of the coronary artery. Both the central and peripheral areas of the infarct contained significant amounts of this lipid (4-6& of lipid phosphorus), but it was not present in the apparently normal portions of the same tissue. The lipid was isolated and characterized by its infrared spectrum, by chemical degradation and by comparison with synthetic and semi-synthetic standards. It was identified as a mixture of N-acylethanolamine glycerophospholipids consisting of diacyl-, alk-1-enylacyl- and alkylacylglycerol species. The N-acyl groups were mainly 16 : 0 and 18 : 0 with smaller proportions of 18 : 1 and 18 : 2. The relative decrease of ethanolamine glycerophospholipids in the infarcated areas and the similarity in their molecular species suggests a percursor-product relationship between these phospholipids.

N-acylethanolamine accumulation in infarcted myocardium

Long-chain N-acylethanolamines were found at levels of 400–500 nmol per g tissue in the infarcted areas of canine myocardium 24 hours after coronary artery ligation. Peripheral infarct areas also contained substantial amounts (200 nmol/g) while apparently normal heart muscle contained very little (< 10 nmol/g). The amide linked fatty acids were mainly 16:0, 18:0, 18:1 and 18:2. Because of its anti-inflammatory activity, N-acylethanolamine may exert beneficial effects in the infarcted area and may be produced as a response to ischemic injury.

Catabolism of N-acylethanolamine phospholipids by dog brain preparations.

Abstract: N‐Acylphosphatidylethanolamine, incubated with dog brain homogenate or microsomes, was hydroyzed to phosphatidic acid and N‐acylethanolamine by a phosphodiesterase of the phospholipase D type. In the absence of F−, phosphatidic acid was further hydrolyzed to diacylglycerol and Pi while N‐acylethanolamine was hydrolyzed by an amidase to fatty acid and ethanolamine. The phosphodiesterase showed an alkaline pH optimum and was also active towards N‐acetylphosphatidyletha‐nolamine, N‐acyl‐lysophosphatidylethanolamine, and glycerophospho(N‐acyl)ethanolamine but showed little activity toward phosphatidylethanolamine and phosphati‐dylcholine. Ca2+ stimulated slightly at low concentrations but inhibited at higher concentrations. Triton X‐100 stim ulated the hydrolysis of N‐acylphosphatidylethanol‐amine, inhibited that of N‐acyl‐lysophosphatidyletha‐nolamine and glycerophospho(N‐acyl)ethanolamine, and had no effect on phosphatidylethanolamine or phospha‐tidylcholine hydrolysis. The N‐acylethanolamine hydrolase (amidase) was also present in the microsomal fraction and exhibited a pH optimum of 10.0. In addition to hydrolysis by the phosphodiesterase, N‐acylphosphati‐dylethanolamine was also catabolized by microsomal phospholipases A1 and/or A2 to N‐acyl‐lysophosphati‐dylethanolamine, some of which was further hydrolyzed to glycerophospho(N‐acyl)ethanolamine.