Ceramide 1-phosphate enhances calcium entry through voltage-operated calcium channels by a protein kinase C-dependent mechanism in GH4C1 rat pituitary cells

Abstract

Sphingomyelin derivatives modulate a multitude of cellular processes, including the regulation of [Ca2+]i (the intracellular free calcium concentration). Previous studies have shown that these metabolites often inhibit calcium entry through VOCCs (voltage-operated calcium channels). In the present study, we show that, in pituitary GH4C1 cells, C1P (C2-ceramide 1-phosphate) enhances calcium entry in a dose-dependent manner. The phospholipase C inhibitor U73122 attenuated the response. C1P invoked a small, but significant, increase in the formation of inositol phosphates. Pre-treatment of the cells with pertussis toxin was without an effect on the C1P-evoked increase in [Ca2+]i. The effect of C1P was critically dependent on extracellular calcium, since no increase in [Ca2+]i was observed when cells in a calcium-free buffer were stimulated with C1P. Furthermore, if the cells were retreated with 300 nM of the VOCC inhibitor nimodipine, the effect of C1P was almost totally abolished. In addition, ceramide C8-1-phosphate evoked an increase in [Ca2+]i, but the onset of the response was slow compared with that of C1P. In cells treated with 1 mM thapsigargin for 15 min, C1P still evoked an increase in [Ca2+]i. In patch-clamp experiments in the whole-cell mode, C1P enhanced calcium entry through the VOCCs compared with vehicle-treated cells. Dialysis of the cells with C1P did not enhance the calcium current. On-cell patch-clamp experiments showed an enhanced probability of the VOCCs being open (P(open)) in the presence of C1P. Inhibition of PKC (protein kinase C) with GF109203X and down-regulation of PKC with PMA attenuated the C1P-evoked increase in [Ca2+]i. Furthermore, down-regulation of PKC abolished the effect of C1P on P(open). This is the first report showing that a sphingomyelin derivative enhances calcium entry through VOCCs.

Sphingosine 1-phosphate and ceramide 1-phosphate: expanding roles in cell signaling

“…C1P, an anionic lipid, might therefore interact directly with this domain in a fashion analogous to that by which phosphatidylserine interacts with the C2 domain of PKC. Alternatively, C1P could also indirectly activate cPLA 2 ␣ since exogenous C1P has been reported to induce calcium mobilization (Tornquist et al, 2004). We have recently discovered that C1P is indeed a direct activator of cPLA 2 ␣ that interacts with its C2/CaLB domain (Subramanian et al, 2005).…”

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

“…This led to proposal of the cytosolic phospholipase A 2a (cPLA 2a ) as the first molecular target of C1P (7). Other studies have suggested a role of ceramide kinase/ C1P in mast cell degranulation (8), regulation of acidic sphingomyelinase and control of apoptosis in bone marrowderived macrophages (9), calcium mobilization in GH4C1 pituitary and Jurkat T-cells (10,11), and dopamine release in PC12 cells (12). A role for C1P in fibroblast proliferation was reported a decade ago (13,14).…”

“…Nevertheless, many laboratories, including ours, have tried to elucidate ceramide kinase signaling pathways by adding exogenous C1P to cells in culture. Although reports are still not numerous, there is already complexity attributable to variability in the choice of the acyl chain length of the C1P used, or in the choice of the organic solvent used, such as DMSO (10,11), methanol/chloroform (16), or ethanol (or methanol)/dodecane (5)(6)(7)14); others have prepared fine dispersions of C1P by sonication in aqueous medium (13,15). The ethanol (or methanol)/ dodecane mixture, in particular, used at 98:2, was first reported 10 years ago (14) and proposed to be useful to elucidate the biological functions and mechanisms of action of C1P.…”

Effects of Recombinant Toxin Phospholipase D in Cardiac Muscle of Rats

“…*represent statistical significant difference between groups. In the control and in rLiD1 treated group the dP/dt max and dP/dt min were, respectively (in mmHg/sec) 1536,5±55,7 and 1392,8±37,6 (p = 0.0508); and -819,1±29,0 and -710,3±23,0 (p = 0,010) 5±33,0±11,0±7,7±40,0±39,6±33,6 Discussion Sphingomyelin is the main substrate of phospholipase D. Sphingomyelin products such as sphingosine and ceramide modulate potently cell activity by the activation of a G protein, triggering a cascade of events leading changes in intracellular Ca ++ concentration which affects the activity of enzymes involved in the regulation of Ca ++ and modulating intracellular Ca ++ levels (Tornquist et al, 2004). Ceramide can be phosphorylated by ceramide kinase originating ceramide-1-phosphate.…”