Ceramide inhibits inwardly rectifying K+ currents via a Ras- and Raf-1-dependent pathway in cultured oligodendrocytes

Abstract

Ceramide is a lipid mediator implicated in apoptosis induced by proinflammatory cytokines in many cell types, including oligodendrocytes (OLGs). To determine whether ceramide modulates transmembrane signaling events in OLGs, we studied its effect on intracellular Ca2+ (Cai), resting membrane potential and inwardly rectifying K+ currents (IKir) in cultured neonatal rat OLGs. We report here that (1) exposure to C2-ceramide (cer) rarely increases OLG Cai, whereas sphingosine elicits sustained increase in Cai; (2) cer causes OLG depolarization, an effect mimicked by sphingosine-1-phosphate but not by sphingosine; and (3) cer, but not its inactive analog dihydroceramide, inhibits OLG IKir. The cer effect is attenuated by Ras antibody Y13-259, by protein kinase C inhibitory peptide (19-36), and by suppression of c-Raf-1 expression with antisense raf-1 oligonucleotides. We conclude that cer-induced OLG depolarization is mediated via inhibition of IKir by a Ras- and raf-1-dependent pathway, which results in the phosphorylation of the inward rectifier K+ channel protein.

Fig. 1.

Representative traces illustrating the effect of cer, sph, and SPP on 340:380 fluorescence intensity ratio in fura-2-loaded neonatal OLGs. An increase in 340:380 ratio indicates an increase in Ca_i. A, cer (1–10 μm). B, sph (30 μm), followed by washout with NB and Ca²⁺-free solutions.C, SPP (5 μm). Note that 0.1% BSA and 0.1% DMSO had no effect on OLG Ca_i. D, cer, SPP, and sph in Ca²⁺-free solution (1 EGTA, 0 Ca²⁺).

Fig. 2.

Representative traces illustrating the effect of cer (10 μm), sph (10 μm), and SPP (5 μm) on DiBAC₄ fluorescence in cultured rat OLGs. An increase in fluorescence intensity indicates depolarization.A, Cer-induced depolarization and sph-induced hyperpolarization. Note that 0.1% ethanol (EtOH) and 0.1% DMSO had no effect on OLG RMP. B, SPP-induced depolarization and sph-induced hyperpolarization compared with depolarization induced by high K⁺ (70 mm). C, Effect of sph in the presence of PMA (320 nm). D, Effect of sph and cer in Ca²⁺-free conditions.

Fig. 3.

Cer inhibits I_Kir in cultured rat OLGs. A, Cer (10 μm) induced a decrease in I_Kir amplitudes at 5 min after perfusion. Step pulses ranging from −120 to 80 mV in 20 mV increments were applied for 360 msec from holding potential of −40 mV. The pipette solution contained 140 mm Cs⁺instead of K⁺. Bath solutions contained 5.4 mm K⁺. B, The correspondingI–V plots.

Fig. 4.

Cer-induced I_Kir inhibition involves Ras-dependent pathways. A, Representative traces before (pre) and after (post) perfusion with 10 μm cer. Only currents at −80 mV are shown for simplicity in this and subsequent figures. IgG (+), Inclusion of 0.2 μg/ml control IgG in the intracellular (pipette) solution. RasAb (+), Inclusion of 0.2 μg/ml neutralizing antibody against Ras (Y13-259) in the pipette solution.B, Summarized data showing I_Kirmodulation by cer (10 μm) but not by inactive cer analog dh-cer (10 μm) (*p < 0.0001 for overall ANOVA; p < 0.0001 for ctrl vs cer; p< 0.001 for cer vs dh-cer). Y13-259 prevented the effect of cer onI_Kir (**p < 0.02 for Y13-259 vs ctrl IgG).

Fig. 5.

Examples of autoradiograms illustrating Ras activation by cer in OLGs. Radiolabeled GDP and GTP that bind Ras are detected with thin layer chromatography. Treatment of cer (10 μm) for 5 min increased radiolabeled GTP compared with controls.

Fig. 6.

The role of protein kinases in cer-inducedI_Kir inhibition. A, Representative traces illustrating the effect of PKC and PKA inhibitors on cer action.Left, Inclusion of PKC (19–36) pseudosubstrate (10 μm) in the pipette solution. Right, Inclusion of PKA inhibitor peptide PKI (5–24) (10 μm) in the pipette solution. B, Summarized data showing that pretreatment of OLGs with wortmannin (5 μm), a PI-3-kinase inhibitor, or inclusion of PKI in the pipette solution did not block the effect of cer. In contrast, inhibition of PKC activity by PKCI or by PKC downregulation with 24 hr PMA treatment significantly attenuated cer-induced I_Kir inhibition (*p < 0.05 for cer vs PKCI and for cer vs PMA 24 h).

Fig. 7.

Schematic diagram of possible signaling pathways involved in cer-induced I_Kir inhibition.

Fig. 8.

Cer-induced I_Kir inhibition involves raf-1-dependent pathways. A, Western blot analysis of raf-1 protein expression in cultures treated with 5 μmsense, antisense, or nonsense raf-1 ODNs. Raf-1 protein was detected using a monoclonal antibody against raf-1. Data shown are representative of three experiments. B, Summarized data showing that cer-induced I_Kir inhibition was prevented by antisense raf-1 ODN but not by sense or nonsense raf-1 ODNs (*p < 0.03 for antisense ODNs vs sense and nonsense ODNs). See Materials and Methods for details.

Fig. 9.

PMA-induced I_Kir inhibition in OLGs. A, Representative traces illustrating the effect of PMA in the presence of Ras Ab or in OLGs treated with antisense raf-1 ODN. B, Summarized data illustrating that PMA-inducedI_Kir inhibition was not prevented by Ras Ab or by antisense Raf-1 ODN (p = NS for PMA vs Ras AB + PMA and for PMA vs antisense + PMA).