Ceramides are potent bioactive molecules in cells. for cell senescence, necrosis

Ceramides are potent bioactive molecules in cells. for cell senescence, necrosis and apoptosis [19]C[21]. Some of ceramides effects have been shown to involve activation of different protein phosphatases [22]. However, many other action mechanisms are likely. For instance, ceramide down-regulates the HERG potassium channel [23], and affect the resting membrane potential of thyroid FRTL-cells by processes involving protein kinase C zeta [24]. Ceramides have also been suggested to form pores in bilayer membranes, and more recently also in mitochondrial outer Rabbit Polyclonal to Cytochrome P450 24A1 membranes, thus possibly facilitating apoptosis [25]C[27]. Since so many different ceramide species exists in cells, and because ceramide generation can be ACP-196 enzyme inhibitor highly compartmentalized, it is likely that different signaling pathways are influenced differently, increasing the challenge of understanding the various roles ceramide can have in cell signaling events. Ceramide is a very hydrophobic molecule, and has extremely poor aqueous solubility. This property has greatly hampered studies on the biological ACP-196 enzyme inhibitor activity of ceramides. While ceramides can be generated in cells by activation of endogenous sphingomyelinases [28], [29], or by using bacterial sphingomyelinases, direct addition of natural ceramides to cells has been difficult. This is apparently the main reason why many scientist have used short-chain ceramide analogs (dissolved in organic solvent) to study ceramide effects in cells, since their efficient delivery to cells has been possible. Ceramides do not appear to form similar types of water-soluble complexes with cyclodextrins, as cholesterol does, although in one early study the use of alpha-cyclodextrin facilitated ceramide synthesis, and may have formed some complexes with ceramide [30]. However, liposomal complexes of ceramides and phospholipids can be used instead of solvent to deliver ceramides to cells. As expected, shorter-chain analogs were more potent in affecting cell viability when compared to more physiological ceramides [31], [32]. We have recently shown that ceramides can form fluid bilayers with cholesteryl phosphorylcholine (CholPC C see Figure 1 for structure) [33]. The fluid nature of the bilayers was evident even when palmitoyl ceramide was used with CholPC. Apparently the CholPC/ceramide formulation is stable, since the phosphocholine head group attached to cholesterol can protect both molecules from unfavorable interactions with water. Similar bilayers were previously shown to be formed by CholPC and dimyristoyl glycerol [34]. We have in this study compared the bioactivity of acid. The precipitate was dissolved in 0.1 M NaOH, and mixed with Optiphase Hisafe 3 scintillation liquid (PerkinElmer/Wallac Turku, Finland), and counted ACP-196 enzyme inhibitor for activity. Cell Counting Assay Cell proliferation was also measured by cell counting. Cells were plated on 35-mm plates (100 000 cells/plate) and grown for the indicated times with 0.05 mM of C6-Cer (either as a complex with CholPC or dissolved in DMSO). In control plates, only DMSO was used. At the indicated time, the cells were harvested using PBS containing 0.02% EDTA and 0.1% trypsin, and the cells were counted using trypan blue and a haemocytometer chamber. Cytosolic Calcium Measurements HeLa cells were grown in 35-mm cell culture dishes containing 25-mm poly-L-lysine coated coverslips. At 50% confluence, the cells were exposed for 0.05 mM C6-Cer (either as complex with CholPC, or dissolved in DMSO) for 90 min. Then, the cells were washed three times with HBSS and incubated in HBSS with 2 M Fura 2-AM for 30 min at room temperature. Extracellular Fura 2-AM was then removed by two washes followed by a 15 min incubation in HBSS. The coverslips were transferred to a heated chamber (37C) and placed under an inverted Zeiss Axiovert 35 microscope equipped with a 40 Fluor objective and a SensiCam 12 bit CCD camera (PCO/CD Imaging, Kelheim, Germany). The source for excitation light was a XBO 75W/2 xenon lamp. Correct.