Impairment of the intestinal microcirculation (IMC) plays a critical role in the pathogenesis of sepsis. Consequently, the protection of the IMC represents a pivotal therapeutic target in severe sepsis. The aim of this study was to examine the effects of endocannabinoid system modulation on the IMC. Experimental animals groups were: control, endotoxemic animals (lipopolysaccharide; LPS), LPS + CB1R agonist, LPS + CB1R antagonist, LPS + CB1R agonist + CB1R antagonist, LPS + CB2R agonist, LPS + CB2R antagonist, LPS + CB2R agonist + CB2R antagonist, LPS + cannabinoid degradation enzyme inhibitor and LPS + enzyme inhibitor + CB2R antagonist. Endotoxemia significantly increased leukocyte adhesion in intestinal submucosal venules, and significantly reduced capillary perfusion of the muscular and mucosal layers of the intestinal wall. In acute experimental endotoxemia, IMC was significantly improved (by reducing leukocyte adhesion and increasing capillary perfusion) with CB1R inhibition or CB2R activation or inhibition of endocannabinoid degradation.

No description available.

N-Acylphosphatidylethanolamines (NAPEs) are precursors of endogenous bioactive lipids, N-acylethanolamines (NAEs). NAPEs, which occur as a minor membrane lipid, are hydrolyzed in a single enzymatic step catalyzed by a type of phospholipase D (PLD) to generate fatty acid ethanolamides. Although, the occurrence of NAPE is widespread in the plant kingdom, the physiological roles remain under appreciated due to the lack of sensitive tools to quantify the pathway metabolites. In Kilaru et al. (2012, Planta, DOI 10.1007/s00425-012-1669-z), comprehensive mass spectrometry (MS)-based methods were developed to gain a clearer understanding of the complex network of metabolites that participate in NAE metabolic pathway. This targeted lipidomics approach allowed insights to be drawn into the implications of altered NAE levels on NAPE content and composition, and the overall regulation of PLD-mediated hydrolysis in Arabidopsis. Based on these results, we point out here the important need for the identification of the precise isoform(s) of PLD in plants that is (are) involved in the regulated hydrolysis of NAPE and formation of NAE lipid mediators in vivo.

Alzheimers sjukdom (AD) är en av de vanligaste former av demenssjukdomar. Sjukdomen karaktäriseras av hjärnatrofi och minnesförlust. Sjukdomen drabbar människor i ålder 65 år och uppåt. 150 000 patienter i Sverige som har demenssjukdom, varvid 90 000 personer har diagnostiserats till AD. Det finns två huvudtyper av Alzheimers sjukdom, den mest vanliga är senil demens vilken utgör 97 % av fallen. Förekomsten av AD karakteriseras av förhöjda beta-amyloid nivåer i hjärnan. Där beta amyloid aggregerar och blir toxiskt samt medför skador på nervceller och dess signaler. Syfte: Syftet med arbetet var att studera rådande forskningsläge gällande endocannabinoider och deras potentiella effekter vid Alzheimers sjukdom. Utifrån syftet ställdes frågeställningar upp, Hur verkar endocannabinoider vid Alzheimers sjukdom? Vilken terapeutisk potential har endocannabinoider vid AD? Metod: Olika databaser bland annat PubMed använts för att hitta information om Endocannabinoider och Alzheimers sjukdom. Resultat: Studierna visade betydelse för närvaro av endocannabinoider och dess skyddande effekt på råttor, cellmodeller samt prover från post mortem patienter med AD. I studie 1 visades olikheter hos människor och råttor i de receptorerna som var viktiga vid fettsyraamid hyrdolas (FAAH) reglering. Det främsta receptorn i FAAH reglering hos råttans hjärna var cannabinoid receptor typ 2, däremot var cannabinoid receptor typ 1 receptor viktig i människans hjärna. Dessa olikheter kan bero på de olika isoformerna av FAAH som uttrycks hos människor till skillnad från det som uttrycks hos råttor. Och det resulterade stimulering av anandamidnedbrytning i humana kontrollprover. Resterande studier visade att hämningen av FAAH ökade anandamidnivåer. Det visade också anandamids möjlighet för skyddande effekter såsom neuroprotektiva, antiinflammatoriska och antioxidativa effekter. Endocannabinoider kan skydda lysosomer från amyloidtoxicitet in-vitro. I studie 2 till skillnad från andra studierna, visades minskningar av amyloid precursor proteinnivåer hos möss som saknade FAAH. Det resulterade minskningar i neuritiska plack som vanligen bildas vid AD. Slutsats: Effekter av endocannabinoider hos råttor, cellkulturer samt hjärnstam från post mortem patienter visade ha neuroprotektiv effekt och kan därmed anses inneha en viktig roll i patogenesen av AD. Effekterna av de utvalda studierna var gjorda på möss, råttor och cellmodell då detta kan försvåra att bevisa och dra en slutsats gällande hur endocannabinoider kan agera hos människor

Mutual cell communication in the human body ensures the proper functioning of the essential mechanisms necessary for the life of the individual and preserving the homeostasis of the whole organism. Such communication is established by various types of signal transmission from the recipient cell to the donor cell, depending on the location and type of communicating cells. One such type is signalization through receptor molecules found on the surface or within the cell receiving the signal. These receptors receive the signal molecule in the form of a ligand and bind it to themselves, while activating the receptor and then triggering the intracellular signaling pathways. The most widely represented receptors in the eukaryotic organism include G-protein-coupled receptors, which represent signaling ensured by activation of the intracellular G-protein complex, and one of the main mechanisms occurring in neuronal signaling and signal transmission in the form of a neurotransmitter. Regulation of the amount of receptors on the surface of the cell and transport of the signal molecule into the intracellular spaces of the cell is ensured by the mechanism of endocytosis, whereby internalization of the ligand- bound receptor in the cytoplasm occurs. One of the most researched mechanisms is clatharin-mediated...

Recently, the occurrence of a mammalian endocannabinoid ligand N-arachidonoylethanolamide (anandamide, AEA, NAE20:4), was reported in early land plants. Unlike seed plants, bryophytes such as Physcomitrella patens possess unique fatty acid composition that includes long-chain fatty acids such as arachidonic acid (AA, 20:4) and eicosapentaenoic acid (EPA, 20:5). We performed targeted lipid profiling to discovere long-chain N-acylethanolamines (NAEs) and their corresponding N-acylphosphatidylethanolamine (NAPE) precursors in Physcomitrella and Selaginella. In protonemal tissues, N-arachidonyl-PE and N-20:5-PE contributed to about 49 % and 30 %, respectively. Matured gametophytes on the other hand showed a 12 % increase in N-20:4-PE and 20 % decline in N-20:5-PE, relative to NAPE content in protonemata. In all haploid developmental stages analyzed, NAE20:4 levels contributed to ~ 23 % of the total NAE while NAE 20:5 remained as a minor component (5 %). Interestingly, in Selaginella moellendorffi, an early vascular plant, N-18:2-PE species was most abundant and 20C-NAEs were present in trace amounts. To understand biological implications of anadamide, we examined the effects of exogenously applied AEA and its corresponding fatty acid (AA) on moss protonemata growth. Both AEA and AA inhibit growth of gametophytes and protonemata in a dose dependent manner, while AEA exclusively affected actin-mediated tip growth. Additionally, we identified moss ortholog for NAPE-hydrolyzing phospholipase D (NAPE-PLD) enzyme that likely generates AEA and a fatty acid amide hydrolase (FAAH) that catabolizes AEA. Both putative PpNAPEPLD and PpFAAH are expressed in E. coli for further characterization. Our data demonstrates the occurrence of evolutionarily conserved NAE metabolic pathway in the moss, with unique composition. Functional and evolutionary implications of this mammalian endocannabinoid in early land plants, however, remains elusive.

N-Acylethanolamines (NAEs) are bioactive acylamides which are involved in diverse biological functions in eukaryotes. Although NAEs are ubiquitous in plants and animals, occurrence of N-arachidonoylethanolamide (anandamide, AEA, NAE20:4) is limited to mammals and early land plants. Metabolism of NAEs and their functional implications in plants are yet to be fully discovered. Unlike seed plants, bryophytes possess unique fatty acid composition that includes abundance of polyunsaturated fatty acids such as arachidonic acid (AA, 20:4) and eicosapentaenoic acid (EPA, 20:5). Moss Physcomitrella patens contains ~18.7 and 15.9 % of AA in gametophores and protonemata, respectively. Therefore, it is hypothesized that P. patens may exhibit a unique NAE metabolite profile. To this extent, we performed lipid profiling and discovered long-chain NAEs and their corresponding N-acyl-phosphatidylethanolamine (NAPE) precursors in Physcomitrella and Selaginella. In protonemal tissues, N-arachidonyl-PE and N-20:5-PE contributed to about 49 % and 30 %, respectively. Matured gametophytes on the other hand showed a 12 % increase in N-20:4-PE and 20 % decline in N-20:5-PE, relative to NAPE content in protonemata. In all haploid developmental stages analyzed, NAE20:4 levels contributed to ~ 23 % of the total NAE while NAE 20:5 remained as a minor component (5 %). Interestingly, in Selaginella moellendorffi, an early vascular plant, N-18:2-PE species was most abundant; minor amounts of N-20:3-PE, N-20:4-PE and N-20:5-PE were also present with only a traceable quantity of NAE20:4. To understand biological implications of AEA, we examined the effects of exogenously applied AEA and its corresponding fatty acid (AA) on moss protonemata growth. Both AEA and AA inhibit growth of gametophytes and protonemata in a dose dependent manner. Additionally, we identified moss ortholog for NAPE-hydrolyzing phospholipase D (NAPE-PLD) enzyme that likely generates AEA. Putative PpNAPE-PLD has been expressed in E. coli for further characterization. Our data demonstrates the occurrence of evolutionarily conserved NAE metabolic pathway in the moss, with an exclusive occurrence of AEA. However, functional and evolutionary implications of this mammalian endocannabinoid in early land plants remain elusive.

Endocannabinoid signaling is well studied in mammals and known to be involved in numerous pathological and physiological processes. Fatty acid amide hydrolase (FAAH) terminates endocannabinoid signaling in mammals. In Physcomitrella patens, we identified nine orthologs of FAAH (PpFAAH1 to PpFAAH9) with the characteristic catalytic triad and amidase signature sequence. Kinetics of PpFAAH1 showed specificity towards anandamide (AEA) at 37°C and pH 8.0. Further biophysical and bioinformatic analyses revealed that, structurally, PpFAAH1 to PpFAAH4 were closely associated to the plant FAAH whereas PpFAAH6 to PpFAAH9 were more closely associated to the animal FAAH. A substrate entry gate or ‘dynamic paddle’ in FAAH is fully formed in vertebrates but absent or not fully developed in non-vertebrates and plants. In planta analysis revealed that PpFAAH responded differently with saturated and unsaturated N-acylethanolamines (NAEs). In vivo amidohydrolase activity showed specificity associated with developmental stages. Additionally, overexpression of PpFAAH1 indicated the need for NAEs in developmental transition. To understand and identify key molecules related to endocannabinoid signaling in P. patens, we used high-throughput RNA sequencing. We analyzed temporal expression of mRNA and long non-coding RNA (lncRNA) in response not only to exogenous anandamide but also its precursor arachidonic acid and abscisic acid (ABA, a stress hormone). From the 40 RNA-seq libraries generated, we identified 4244 novel lncRNAs. The highest number of differentially expressed genes (DEGs) for both mRNA and lncRNA were detected on short-term exposure (1 h) to AEA. Furthermore, gene ontology enrichment analysis showed that 17 genes related to activation of the G protein-coupled receptor signaling pathway were highly expressed along with a number of genes associated with organelle relocation and localization. We identified key signaling components of AEA that showed significant difference when compared with ABA. This study provides a fundamental understanding of novel endocannabinoid signaling in early land plants and a future direction to elucidate its functional role.

Endocannabinoids were known to exist only among Animalia but recent report of their occurrence in early land plants prompted us to study its function and metabolism. In mammals, anandamide, as an endocannabinoid ligand, mediates several neurological and physiological processes, which are terminated by fatty acid amide hydrolase (FAAH). We identified nine orthologs of FAAH in the moss Physcomitrella patens (PpFAAH1 to PpFAAH9) with amidase signature and catalytic triad. The optimal amidase activity for PpFAAH1 was at 37 °C and pH 8.0, with higher specificity to anandamide. Further, the phylogeny and predicted structural analyses of the nine paralogs revealed that PpFAAH1 to PpFAAH4 were closely related to plant FAAH while PpFAAH6 to PpFAAH9 were to the rat FAAH, categorized based on the membrane binding cap, membrane access channel and substrate binding pocket. We also identified that a true ‘dynamic paddle’ that is responsible for tighter regulation of FAAH is recent in vertebrates and absent or not fully emerged in plants and non-vertebrates. These data reveal evolutionary and functional relationship among eukaryotic FAAH orthologs and features that contribute to versatility and tighter regulation of FAAH. Future studies will utilize FAAH mutants of moss to elucidate the role of anandamide in early land plants.

The discovery of a mammalian endocannabinoid, anandamide (N-arachidonylethanolamide; AEA or NAE 20:4) in Physcomitrella patens but not in higher plants prompted our interest in characterizing its metabolism and physiological role in the early land plant. Anandamide acts as an endocannabinoid ligand in the mammalian central and peripheral systems and mediates various physiological responses. Endocannabinoid signaling is terminated by a membrane-bound fatty acid amide hydrolase (FAAH). Based on sequence identity and in silico analyses, we identified nine orthologs of human and Arabidopsis FAAH in P. patens (PpFAAH1 to PpFAAH9). Predicted structural analysis revealed that all the nine PpFAAH contain characteristic amidase signature sequence with a highly conserved catalytic triad and share a number of key features of both plant and animal FAAH. These include a membrane binding cap, membrane access channel, substrate binding pocket and as well as potential for dimerization. Among the nine, gene expression levels for PpFAAH1 and PpFAAH9 were enhanced with exogenous anandamide treatment. Further cloning and heterologous expression, followed by radiolabeled in vitro enzyme assays revealed that PpFAAH1 activity was optimal at 37 °C and pH 8.0. Furthermore, PpFAAH1 showed higher specificity to NAE 20:4 than to other N-acylethanolamines such as NAE 16:0. Highest in planta amide hydrolase activity was noted in microsomes of gametophyte tissues, suggesting the possibility for membrane localization of active FAAH. Interestingly, when FAAH1 was overexpressed, the moss cultures not only showed reduced growth but their transition from protonemal stage to gametophyte was inhibited, which was rescued in part by exogenous AEA. Unlike overexpressors of AtFAAH1, which showed enhanced growth and hypersensitivity to abscisic acid, PpFAAH1 overexpressors showed tolerance to abscisic acid. Together, these data suggest that the occurrence of anandamide and distinct properties of PpFAAH1 in early land plants have physiological implications that are different from that of higher plants.