Effects of ceramide-1-phosphate on cultured cells: dependence on dodecane in the vehicle

Abstract

Ceramide-1-phosphate (C1P), the product of ceramide kinase, is a sphingophospholipid with recently recognized signaling properties. In particular, it was reported to be mitogenic and capable of direct stimulation of cytosolic phospholipase A 2a . Much of the present knowledge has relied on the use of C1P of various acyl chain lengths, together with diverse protocols to deliver it to cultured cells. A mixture of ethanol (or methanol) with dodecane, as the vehicle, has become popular. However, the contribution of this solvent to the observed effects of C1P has not been documented. Here, we show that addition of C1P in ethanoldodecane to culture medium leads to irreversible cytotoxic effects. These culminate in mitochondrial swelling, vacuole formation, and cell death. Not only the toxicity of C1P, but also its ability to trigger prostaglandin E 2 release, is fully dependent upon addition of a premade C1P-dodecane mixture. Furthermore, we show that these effects are not restricted to C1P. They result from the capacity of dodecane to interact with phospholipids; hence, they go undetected with a vehicle control. This study should raise awareness about the use of dodecane for phospholipid delivery and, in turn, help in unraveling C1P signaling, which is still poorly

Chain length specificity for activation of cPLA2α by C1P: use of the dodecane delivery system to determine lipid-specific effects

“…C1P at concentrations р 500 nM demonstrated complete lipid specifi city in the induction of AA release from A549 cells. In accord with the recent report by Tauzin et al ( 17 ), lipid specifi city was lost as concentrations of phospholipids increased above 1 M. The loss of lipid specifi city correlated with the loss of cell viability as recently reported by Mitra et al ( 25 ) and Tauzin et al ( 17 ). Thus, our study demonstrates that the contrasting fi ndings between the two laboratories was the difference in concentration of C1P utilized.…”

“…This study also addresses the recent publication by Tauzin et al ( 17 ), which raised doubts as to the lipid specifi city of the activation of cPLA 2 ␣ by C1P when delivered via the well-established EtOH/dodecane system. We specifi cally show that the stimulation of AA release in the macrophages and A549 cells was specifi c for C1P as other related phospholipids such as ceramide and PA failed to do so.…”

“…Tauzin et al ( 17 ) reported that C1P delivered by EtOH/dodecane was rapidly metabolized and its uptake was not enhanced by this delivery system. We hypothesized that this observation on uptake and metabolism may be due to the toxicity induced by using 10 µM C1P delivered with EtOH/dodecane, or the use of the unnatural analog, NBD-C1P.…”

“…The recent report by Tauzin et al ( 17 ) elegantly and comprehensively demonstrated that the biological effects on cells, in particular PGE 2 synthesis and cell death, were not lipid specifi c when: 1 ) high doses of lipids were used (10 M), and 2 ) the lipids were delivered using EtOH/ dodecane. In 2003, our laboratory demonstrated that a lipid-specifi c effect on the activation of cPLA 2 ␣ , induction We then examined the lipid specifi city of cPLA 2 ␣ activation.…”

Suppression of Osteoclastogenesis by N,N-Dimethyl-D-erythro-sphingosine: A Sphingosine Kinase Inhibition-Independent Action

“…Exogenously added S1P (dissolved in methanol) could not rescue the DMS suppression of osteoclast formation even at doses as high as 10 M (data not shown). The methanol/dodecane mixture, which facilitates intracellular delivery of sphingolipids, has been described previously (Tauzin et al, 2007). Therefore, we used a dodecane delivery of S1P into the cells to elucidate whether S1P needs to be transported across the plasma membrane in osteoclastogenesis (Fig.…”