No abstract

Ceramides serve as bioactive molecules with important roles in cell proliferation and apoptosis. 4 Ceramides (Cer) with different N-acyl side chains (C 14:0-Cer-C 26:0-Cer) possess distinctive roles 5 in cell signaling and are differentially expressed in HCT-116 colon cancer cells. Celecoxib, a 6 selective cyclooxygenase-2 (COX-2) inhibitor, exhibiting antiproliferative effects, activates the 7 sphingolipid pathway. To elucidate the mechanism, HCT-116 cells were treated with 50 µM 8 celecoxib leading to a significant increase of C 16:0-Cer. Interestingly, 50 µM celecoxib resulted in 9 a 2.8 fold increase of ceramide synthase (CerS) activity as measured by a cell-based activity 10 assay. siRNA against several CerSs revealed that CerS6 was predominantly responsible for the 11 increase of C 16:0-Cer in HCT-116 cells. Moreover, the silencing of CerS6 partially protected 12

1 Acetylcholine is synthesized in the majority of non-neuronal cells, for example in human skin. In the present experiments, the in vivo release of acetylcholine was measured by dermal microdialysis. 2 Two microdialysis membranes were inserted intradermally at the medial shank of volunteers. Physiological saline containing 1 mM neostigmine was perfused at a constant rate of 4 ml min À1 and the effluent was collected in six subsequent 20 min periods. Acetylcholine was measured by high-pressure liquid chromatography (HPLC) combined with bioreactors and electrochemical detection. 3 Analysis of the effluent by HPLC showed an acetylcholine peak that disappeared, when the analytical column was packed with acetylcholine-specific esterase, confirming the presence of acetylcholine. 4 In the absence of neostigmine, 71751 pmol acetylcholine (n ¼ 4) was found during a 120 min period. The amount increased to 183743 pmol (n ¼ 34), when the perfusion medium contained 1 mM neostigmine. 5 Injection of 100 MU botulinum toxin subcutaneously blocked sweating completely, but the release of acetylcholine was not affected (botulinum toxin treated skin: 116770 pmol acetylcholine/120 min; untreated skin: 50720 pmol; n ¼ 4). 6 Quinine (1 mM), inhibitor of organic cation transporters, and carnitine (0.1 mM), substrate of the Na þ -dependent carnitine transporter OCTN2, tended to reduce acetylcholine release (by 40%, not significant). 7 Our experiments demonstrate, for the first time, the in vivo release of non-neuronal acetylcholine in human skin. Organic cation transporters are not predominantly involved in the release of non-neuronal acetylcholine from the human skin.

Ceramide synthases (CerS) synthesize chain length specific ceramides (Cer), which mediate cellular processes in a chain length-dependent manner. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), we observed that the genetic deletion of CerS2 suppresses EAE pathology by interaction with granulocyte-colony stimulating factor (G-CSF) signaling and CXC motif chemokine receptor 2 (CXCR2) expression, leading to impaired neutrophil migration. In the present study, we investigated the importance of Cers and their synthesizing/metabolizing enzymes in MS. For this purpose, a longitudinal study with 72 MS patients and 25 healthy volunteers was performed. Blood samples were collected from healthy controls and MS patients over 1- or 3-year periods, respectively. Immune cells were counted using flow cytometry, ceramide levels were determined using liquid chromatography-tandem mass spectrometry, and mRNA expression was analyzed using quantitative PCR. In white blood cells, C16-LacCer and C24-Cer were down-regulated in MS patients in comparison with healthy controls. In plasma, C16-Cer, C24:1-Cer, C16-GluCer, and C24:1-GluCer were up-regulated and C16-LacCer was down-regulated in MS patients in comparison with healthy controls. Blood samples from MS patients were characterized by an increased B-cell number. However, there was no correlation between B-cell number and Cer levels. mRNA expression of Cer metabolizing enzymes and G-CSF signaling enzymes was significantly increased in MS patients. Interestingly, G-CSF receptor (G-CSFR) and CXCR2 mRNA expression correlated with CerS2 and UDP-glucose Cer glucosyltransferase (UGCG) mRNA expression. In conclusion, our results indicate that Cer metabolism is linked to G-CSF signaling in MS.