Exogenous and endogenous ceramides elicit volume-sensitive chloride current in ventricular myocytes

Abstract

Aims: Because ceramide accumulates in several forms of cardiovascular disease and ceramide-induced apoptosis may involve the volume-sensitive Cl(-) current, I(Cl,swell), we assessed whether ceramide activates I(Cl,swell).

Methods and results: I(Cl,swell) was measured in rabbit ventricular myocytes by whole-cell patch clamp after isolating anion currents. Exogenous C(2)-ceramide (C(2)-Cer), a membrane-permeant short-chain ceramide, elicited an outwardly rectifying Cl(-) current in both physiological and symmetrical Cl(-) solutions that was fully inhibited by DCPIB, a specific I(Cl,swell) blocker. In contrast, the metabolically inactive C(2)-Cer analogue C(2)-dihydroceramide (C(2)-H(2)Cer) failed to activate Cl(-) current. Bacterial sphingomyelinase (SMase), which generates endogenous long-chain ceramides as was confirmed by tandem mass spectrometry, also elicited an outwardly rectifying Cl(-) current that was inhibited by DCPIB and tamoxifen, another I(Cl,swell) blocker. Bacterial SMase-induced current was partially reversed by osmotic shrinkage and fully suppressed by ebselen, a scavenger of reactive oxygen species. Outward rectification with physiological and symmetrical Cl(-) gradients, block by DCPIB and tamoxifen, and volume sensitivity are characteristics that identify I(Cl,swell). Insensitivity to C(2)-H(2)Cer and block by ebselen suggest involvement of ceramide signalling rather than direct lipid-channel interaction.

Conclusion: Exogenous and endogenous ceramide elicited I(Cl,swell) in ventricular myocytes. This may contribute to persistent activation of I(Cl,swell) and aspects of altered myocyte function in cardiovascular diseases associated with by ceramide accumulation.

Figure 1

C₂-Cer elicited a Cl⁻ current that resembled I_Cl,swell. (A) Families of currents under control conditions (Ctrl), after C₂-Cer exposure (2 µM, 10 min), and after addition of DCPIB (+DCPIB; 10 μM) in continued presence of C₂-Cer. Holding potential, −60 mV; test potentials, −100 to 60 mV. (B) Current–voltage (I–V) relationships for A. (C) Normalized currents at 60 mV. C₂-Cer increased Cl⁻ current by 0.70 ± 0.09 pA/pF (n = 14, P < 0.001). C₂-Cer-induced current was inhibited by 76 ± 8% (n = 6, P < 0.001) by the I_Cl,swell-specific inhibitor DCPIB; current after DCPIB was not different than control. (D) C₂-Cer-induced currents at 0.2 (n = 3), 0.36 (n = 3), 0.6 (n = 3), 2 (n = 14), and 20 μM (n = 4) and fit (solid line) to EC₅₀ of 0.41 μM and Hill coefficient of 3.6.

Figure 2

C₂-Cer (2 μM, 10 min) activated outwardly rectifying Cl⁻ current in symmetrical Cl⁻. (A) Families of currents and (B) I–V relationships. C₂-Cer-induced current reversed near 0 mV. (C) C₂-Cer-induced current at 60 mV was 1.30 ± 0.35 pA/pF (n = 6, P < 0.01). Outward rectification in symmetrical Cl⁻ and block by DCPIB (Figure 1) indicate C₂-Cer activated I_Cl,swell.

Figure 3

C₂-H₂Cer, a metabolically inactive C₂-Cer analogue, did not alter membrane current, but C₂-Cer elicited I_Cl,swell in the same cell. (A) Typical currents at 60 mV. (B) Cl⁻ current densities in control and with C₂-H₂Cer and C₂-Cer (both: 2 μM, 10 min). C₂-H₂Cer was ineffective (−4 ± 5%, n = 6, NS), whereas C₂-Cer subsequently activated current in all four cells tested (P < 0.01). The data suggest C₂-Cer elicited I_Cl,swell via its normal pathway rather than by non-specific mechanisms.

Figure 4

Bacterial SMase reversibly activated I_Cl,swell. (A) I–V relationships for Cl⁻ current elicited by SMase (0.03 U/mL, 15−18 min) and inhibition by DCPIB (10 μM). (B) SMase increased Cl⁻ current by 1.1 ± 0.1 pA/pF at 60 mV (n = 30), and DCPIB (10 or 30 μM) suppressed 78 ± 6% (n = 7) or 81 ± 6% (n = 4), respectively (P < 0.01 for both). (C) Effect of SMase reversed on washout (18−20 min, n = 3, P < 0.05). (D and E) Exposure to SMase (20 min) generated endogenous long-chain ceramides and depleted a substantial fraction of sarcolemmal sphingomyelins (n = 6; *P < 0.05). For ceramides and sphingomyelins, each lipid species was compared with control using a three-way ANOVA based on two separate experimental data sets, each analysed in triplicate.

Figure 5

Tamoxifen (Tam) inhibited SMase-induced I_Cl,swell. (A) Currents before and after treatment with SMase (0.03 U/mL, 15−18 min) and after the addition of Tam (10 µM). (B) I–V relationships. (C) Tam fully blocks SMase-induced Cl⁻ current (116 ± 16%, n = 5, P < 0.01).

Figure 6

Osmotic shrinkage partially inhibited SMase-induced I_Cl,swell. (A) I–V relationships before (1 T Ctrl) and after (1 T + SMase) exposure to SMase (0.03 U/mL, 18 min) in isosmotic bath solution, and then, after shrinking the same cell in hyperosmotic bath solution containing SMase (1.5 T + SMase; 0.03 U/mL, 15 min). (B) Current densities at 60 mV before and after treatment with SMase in 1 T and 1.5 T bath solutions. Cell shrinkage in 1.5 T partially inhibited the SMase-induced Cl⁻ current (43 ± 8%, n = 6, P < 0.02). This suggested that SMase elicits I_Cl,swell via volume-dependent and volume-independent pathways.

Figure 7

Bacterial SMase-induced Cl⁻ current was inhibited by ebselen. (A) Currents before and after treatment with SMase (0.03 U/mL, 15−18 min) and after the addition of ebselen (20 µM, 5 min). (B) I–V relationships. (C) Ebselen, a glutathione peroxidase mimetic that scavenges H₂O₂, fully blocked SMase-induced Cl⁻ current at 60 mV (n = 5, P < 0.01). These data suggest the SMase-induced Cl⁻ current is elicited by H₂O₂, a downstream mediator of I_Cl,swell.^,

Figure 8

Time course of I_Cl,swell activation by C₂-Cer and bacterial SMase. C₂-Cer data were fit by an exponential function with a time constant of 6.4 ± 1.6 min (R² = 0.98, n = 11), equivalent to a t_1/2 of 4.8 ± 1.2 min. SMase data were fit by a sigmoid function with a t_1/2 = 9.3 ± 0.6 min (R² = 0.99, n = 10). Soluble C₂-Cer may reach the site of activation of I_Cl,swell more quickly than long-chain endogenous ceramides that must first be produced by SMase. Alternatively, ceramides with different chain lengths may activate different sites in the signalling cascade.