Ceramide Channels Increase the Permeability of the Mitochondrial Outer Membrane to Small Proteins

Reagents

The following reagents were purchased from Avanti Polar Lipids (Alabaster, AL): C₂-ceramide, C₂-dihydroceramide, C₁₆-ceramide, C₁₈-dihydroceramide, and asolectin (polar extract of soybean phospholipids). Antimycin A, 2,4-dinitrophenol (DNP), fatty acid-depleted BSA, horse heart cytochrome c, and sodium ascorbate were purchased from Sigma.

Electrophysiological Recordings

Planar membranes were formed by the monolayer method (44), as modified (45), across a 100-µm diameter hole in a Saran partition using 1% (w/v) asolectin (soybean phospholipids), 0.2% (w/v) cholesterol in hexane solution. The aqueous solution contained 1.0 m KCl, 1 mm MgCl₂, and 5 mm HEPES or 5 mm PIPES (pH 7.0). The voltage was clamped (trans-side was ground) and the current was recorded. C₂-ceramide was stirred into the water phase from a Me₂SO solution, whereas C₁₆-ceramide was first dissolved in ethanol at 37 °C prior to addition. In both cases, the final concentration of the vehicle was no more than 0.5%. Fatty acid-depleted BSA was prepared as a stock solution (0.5 mm) in the same aqueous solution bathing the membrane with 0.5% a zide (for storage purposes).

Preparation of Mitochondria

Rat liver mitochondria were isolated by differential centrifugation of tissue homogenate as previously described (46). Briefly, livers from male Sprague-Dawley rats (fasted overnight with water ad labitum) were quickly excised, cut, washed repeatedly in cold isolation medium (70 mm sucrose, 210 mm mannitol, 0.1 mm EGTA, 1.0 mm Tris-Cl, pH 7.4), and minced. Homogenization and differential centrifugation were performed with isolation medium supplemented with 0.5% (w/v) BSA. BSA was removed by a final 9000 × g centrifugation and subsequently mitochondria were suspended in BSA-free medium.

Preparation of Reduced Cytochrome c

1 mm horse heart cytochrome c was reduced by an excess of ascorbate (20 mm), 0.2 mTris-Cl (pH 7.5). The reduced cytochrome c was separated from the ascorbate using a Sephadex G-10 gel filtration column. The concentration of reduced cytochrome c was determined spectrophotometrically (ϵ_{550 nm(Red.-Ox.)} = 18.5 mm⁻¹ cm⁻¹).

Detection of Cytochrome c Permeation through the Outer Membrane

Mitochondria were diluted 20–40-fold in isolation buffer to achieve an appropriate rate of cytochrome c oxidation after hypotonic shock and kept on ice. 20 µl of diluted mitochondria were added to 750 µl of isolation buffer supplemented with 0.5 mm DNP and 5 µm antimycin A (final concentration of 0.3–1.2 mg of mitochondrial protein/ml) and allowed to reach room temperature. Ceramide was added in such a way that the vehicle was no more than 1% of the total volume. Me₂SO was the vehicle for C₂-ceramide and C₂-dihydroceramide. C₁₆-ceramide and C₁₈-ceramide were dissolved in 100% ethanol at 37 °C as described in Ref. 28 and added to mitochondria at room temperature. After the indicated incubation period, 20 µl of reduced cytochrome c (25–35 µm final concentration in the cuvette) was added and the initial rate of oxidation was assayed spectrophotometrically as a decrease in absorbance at 550 nm (Δϵ_Red.-Ox. = 18.5 mm⁻¹ cm⁻¹) or the difference in absorbance at 550 and 536 nm (ϵ_{550 nm} = 27.7 mm⁻¹ cm⁻¹; ϵ_{536 nm} = 7.7 mm⁻¹ cm⁻¹). All initial rates were expressed as nanomoles of cytochrome c oxidized per s/mg of mitochondrial protein. KCN was used to inhibit cytochrome c oxidase and hence stop the reaction. Equal volumes of vehicle and/or dihydroceramide were used as controls.

Assessing the Ability of Ceramides to Release Adenylate Kinase and Fumarase

The intermembrane space enzyme adenylate kinase (47) and the matrix enzyme fumarase (48) were assayed by standard methods. To increase the concentration of any released enzyme in the medium (for detection purposes) following exposure to ceramide, it was necessary to use higher mitochondrial concentrations than those used to measure cytochrome c permeation through the outer membrane. To achieve comparable conditions in both types of experiments, the ceramide to mitochondria ratio was kept constant rather than the total concentration of added ceramide. This can be justified by recalling that ceramide exerts its effect on the membranes and that the critical concentration should be the concentration of ceramide in the membrane; a constant ratio of ceramide to mitochondrial protein should result in a constant level of ceramide in the mitochondrial outer membrane. The literature supports this notion. Muriel et al. (42) found that the concentration of C₂-ceramide that induced the death of 50% of the PC12 cells after 24 h depended on the cell density used. Similarly, Simon and Gear (35) found that C₂-ceramide inhibition of platelet aggregation and its ability to induce 6-carboxyfluorescein release from vesicles was dependent on the ratio of ceramide to total lipid, as opposed to the absolute ceramide concentration. Thus, we scaled the amount of ceramide to the amount of mitochondria used (i.e. moles of ceramide to milligrams of mitochondrial protein).

The mitochondrial preparation was diluted 5-fold in isolation buffer without antimycin A or DNP. A 250-µl aliquot (5 –10 mg of protein/ml) was incubated for 10 min with C₂- or C₁₆-ceramide at either a high or low molar ratio, 5 and 20 µm equivalent ratios of ceramide to milligrams of mitochondrial protein as in the accompanying cytochrome c oxidation experiments. The mitochondria were then pelleted at 12,000 rpm for 5 min and 200 µl of supernatant was then added to 700 µl of either adenylate kinase reaction mixture (50 mm Tris-HCl, pH 7.5, 5 mm MgSO₄, 10 mm glucose, 5 mm ADP, 0.2 mm NADP, 10 units of hexokinase, and 10 units of glucose-6-phosphate dehydrogenase) or fumarase reaction mixture (50 mm sodium phosphate and 50 mm l-malate, pH 7.3). Adenylate kinase was detected as an increase in absorbance at 340 nm. Fumarase was detected as an increase in absorbance at 250 nm. Untreated mitochondria and mitochondria with lysed outer (adenylate kinase) or lysed outer and inner (fumarase) membranes served as negative and positive controls, respectively. In the case of the adenylate kinase assay, the outer mitochondrial membrane was lysed hypotonically. In the fumarase assay, the outer and inner mitochondrial membranes were lysed via sonication under severe hypo-osmotic conditions in the presence of 1 mm EDTA as described in Ref. 49. Equal volumes of vehicle and dihydroceramide were used as controls.

Assessment of Ceramide-induced Protein Release

Five milliliters of mitochondria that was diluted 5-fold (5–10 mg/ml) with isolation buffer (without antimycin A or DNP) was allowed to reach room temperature and then incubated for 10 min with C₂- or C₁₆-ceramide at 20 µm equivalent ratios of moles of ceramide to mitochondrial protein. Untreated mitochondria, dihydroceramide, and vehicles served as controls. The mitochondria were then spun at 35,000 rpm (50Ti rotor) for 30 min at 4 °C. The supernatant was removed and treated with 10% trichloroacetic acid overnight at 4 °C to pellet the proteins. The pellet was washed repeatedly with 1:1 ethanol:ether (v/v) to remove the trichloroacetic acid. The pellets were redissolved by adding Tris-OH, β-mercaptoethanol, and SDS in amounts equivalent to those used in the SDS-PAGE sample buffer and heated to near boiling for 10 min. After returning to room temperature, bromphenol blue was added and then HCl was added just until the color changed. 8 m urea was added to help stabilize the solution and provide for the added density (instead of glycerol). Proteins from the hypotonically lysed mitochondria were diluted 4-, 8-, and 16-fold. Samples were separated on a 15% acrylamide SDS-PAGE supplemented with 4 m urea and the bands stained with GelCode Blue stain (Pierce).

Determination of Protein Concentration

Mitochondrial protein was measured using the BCA method (Pierce). Bovine serum albumin was the standard.

Ceramide Increases the Permeability of the Mitochondrial Outer Membrane to Cytochrome c

It has already been reported that addition of either C₂- or C₁₆-ceramide to isolated mitochondria results in cytochrome c release (27–29). However, from the literature it is not clear how this release might come about. We previously demonstrated that ceramides form large channels in phospholipid membranes (34), whereas the biologically inactive C₂- and C₁₈-dihydroceramides do not (34). The addition of either C₁₆- or C₂-ceramide to the aqueous phase on either one or both sides of a planar phospholipid membrane results in pore formation as indicated by discrete stepwise current increases (Fig. 1). Discontinuous changes in current are diagnostic of channel formation.

Ceramide channels have a wide distribution of discrete conductance increases (34) (Fig. 1), ranging from 1 nanoSiemens to more than 200 nanosiemens. Small channels are seen early on when the total membrane conductance is low. With time, larger discrete conductance increases are evident reflecting the formation of larger structures. Eventually the conductance grows with fluctuations of current overlapping into current noise. Depicted in Fig. 1 are representative examples of some of these small and large channels observed with C₂- and C₁₆-ceramide. According to the bulk properties of water, pores with conductance ranging from 1 to 200 nanosiemens would have estimated diameters ranging from 0.8 to 11 nm, respectively. We hypothesize that similar channels form in the outer mitochondrial membrane, channels large enough to allow cytochrome c to exit. In this hypothesis, ceramide does not trigger a cytochrome c secretion or release mechanism, but simply raises the permeability of the outer mitochondrial membrane, via ceramide channel formation, to include small proteins. The hypothesis predicts the following: 1) cytochrome c should freely permeate and thus both entry to and exit from the intermembrane space should be facilitated; 2) the permeability pathways should be eliminated by removal of ceramide; 3) the release should not be specific to cytochrome c.

To test the first prediction, we did not measure cytochrome c release (already reported in Ref. 20–Ref. 29), but rather the ability of cytochrome c to permeate through the outer membrane. We determined the rate at which cytochrome oxidase of the mitochondrial inner membrane would oxidize exogenously added reduced cytochrome c. Because of the exceedingly small volume of the mitochondrial intermembrane space, cytochrome c would need to cross the outer membrane twice for appreciable oxidation to take place and be detected spectrophotometrically.

Both C₂- and C₁₆-ceramide caused a rapid increase in the rate of cytochrome c oxidation (Fig. 2a). The ceramide-induced permeability increase of the mitochondrial outer membrane occurred in a dose-dependent manner in the concentration range 0.5 to 40 µm. Dihydroceramide and vehicle controls were essentially identical to untreated mitochondria controls (Fig. 2b). To ensure that the decrease in absorbance at 550 nm in mitochondria incubated with ceramide reflected the oxidation of exogenously added reduced cytochrome c and not mitochondrial volume changes (swelling), mitochondria were exposed to C₂- and C₁₆-ceramide without adding exogenously reduced cytochrome c and the absorbance was recorded at 540 and 550 nm. We detected no changes in absorbance at 540 or 550 nm (data not shown). Therefore, the decrease in absorbance at 550 nm in mitochondria incubated with ceramide reflects the oxidation of exogenously added reduced cytochrome c and not volume changes. This does not rule out the possibility that ceramides can cause mitochondrial volume changes as previously reported (50). However, it does exclude the possibility that absorbance changes associated with volume changes were causing part of the recorded change in absorbance.

The measured rates of cytochrome c oxidation were not influenced by direct effects of ceramide on mitochondrial enzymes. Di Paola et al. (28) reported that C₂- and C₁₆-ceramide had stimulatory and inhibitory effects on cytochrome c oxidase, respectively, measured as ascorbate/TMPD oxidase activity, in isolated rat heart mitochondria or isolated respiratory complexes from bovine heart. However, we found that when mitochondria with lysed outer membranes were incubated with C₂- or C₁₆-ceramide, the rate of cytochrome c oxidation was identical to the control (Fig. 2b). Similarly, Gudz et al. (43) found no effect of C₂-ceramide on cytochrome c oxidase activity in isolated rat heart mitochondria, using the ascorbate/TMPD oxidase system in the same range of C₂-ceramide concentrations as utilized here.

In addition, ceramides have been reported to affect complex III (43), other components of the electron transport system (28), and the inner mitochondrial membrane potential (ΔΨ) (20, 27–29). Although important, these effects were not relevant as the medium was supplemented with both the complex III inhibitor antimycin A and the uncoupler DNP. Antimycin A prevents re-reduction of cytochrome c and DNP eliminates the protonmotive force that could slow down cytochrome oxidase activity. Contrary to the findings of Ghafourifar et al. (29) that ceramide-induced cytochrome c release was blocked in the absence of ΔΨ (achieved when they blocked respiration and uncoupled mitochondria), we detect cytochrome c permeation through the outer mitochondrial membrane in both uncoupled (Fig. 2, a and b) and coupled (data not shown) mitochondria incubated with C₂- and C₁₆-ceramide.

The Permeability Increase Induced by C₂-ceramide Can Be Reversed

If the C₂-ceramide-induced increase in permeability of the mitochondrial outer membrane to cytochrome c is because of ceramide channels in dynamic equilibrium with monomers then the normal permeability should be restored when ceramide monomers are removed from solution, resulting in channel disassembly (prediction number 2). Experiments with planar membranes showed that fatty acid-depleted BSA could be used to remove added ceramide. In the representative experiment in Fig. 3a, 10 µm fatty acid-depleted BSA (a 2:1 mol ratio of BSA to C₂-ceramide) was added to the aqueous phase on both sides of a phospholipid membrane containing C₂-ceramide channels. The membrane conductance decreased in a stepwise manner until the total membrane conductance returned to baseline. The time required for a complete disassembly of ceramide channels by BSA varied greatly between experiments. Although the conductance decreased soon after BSA addition, it took between 5 min and 2 h before all the channels disassembled. This may indicate that the structures formed have a varying degree of structural stability. In any case, BSA can be used as a tool to disassemble C₂-ceramide channels.

This approach was applied to intact mitochondria. A 5-min preincubation of isolated mitochondria with 20 µm C₂-ceramide was followed by a 15-min incubation with excess BSA (83 µm; 5:1, BSA:C₂-ceramide mole ratio). This resulted in a marked drop in the cytochrome c oxidation rate indicating an almost complete restoration of outer membrane permeability characteristics (Fig. 3b). If the BSA was simply preventing any further effects of the short-chain ceramide on the outer membrane permeability instead of actually reversing the ceramide channels then the rate of oxidation following the 5-min 20 µm C₂-ceramide and then the 15-min 83 µm BSA incubation would only be lower than a 20-min incubation with 20 µm C₂-ceramide. Instead, the rate of oxidation was substantially lower than the rate seen with a 5-min exposure of the mitochondria to 20 µm C₂-ceramide and was almost as low as the untreated mitochondria. Incubation of the mitochondria with BSA prior to C2-ceramide addition or mixing the BSA and C₂-ceramide prior to their addition also had very low rates of oxidation, showing that BSA can both reverse and inhibit C₂-ceramide channel formation (Fig. 3b).

Complete reversal was not achieved, in contrast with the observations made in the planar membrane experiments, and this was likely because of the inability to incubate the mitochondria at room temperature for a sufficient amount of time to allow for 100% reversal without their degradation. The time required for complete reversal of the C₂-ceramide channels in the planar membrane experiments was highly variable and for the most part required a longer time period than 15 min. Mitochondria with lysed outer membranes exposed to BSA had essentially identical rates of oxidation of the reduced cytochrome c as lysed mitochondria not exposed to BSA, ruling out an effect of the BSA on cytochrome oxidase activity. An inert molecule impermeable to the outer membrane, polyvinyl pyrrolidine (40 kDa), was used as a control for osmotic changes and the possibility that a reduction in the volume of the intermembrane space might somehow affect the rate of cytochrome c oxidation. Addition of 83 µm polyvinyl pyrrolidine had no effect.

Only C₂-ceramide was used for the BSA reversal experiments because of the inability of BSA to reverse or prevent the permeability induced by C₁₆-ceramide, which could be because of a greater affinity of C₁₆-ceramide for membranes as opposed to BSA. C₁₆-ceramide has two long fatty acyl chains like phospholipids, whereas C₂-ceramide has only one as in the free fatty acids that bind to BSA. By doubling the non-polar portion of ceramide one would expect that the hydrophobic component of the interaction energy between ceramide and membranes would also double. This change could not only make ceramide-membrane interactions stronger than ceramide-BSA interactions, but also greatly reduce the free ceramide concentration in solution, favoring the dissociation of ceramide from BSA. In addition, BSA is known to bind associated forms of organic ligands poorly relative to unassociated forms (monomers) (51)

C₂- and C₁₆-ceramides Allow the Release of Low Molecular Weight Proteins from the Intermembrane Space

It has been proposed that cytochrome c release is the prime mitochondrial target of ceramide (29). However, if C₂- and C₁₆-ceramide induced cytochrome c release was because of ceramide channel formation in the outer mitochondrial membrane then these channels should not be specific to the release of cytochrome c, but should also allow the release of other proteins from the mitochondrial intermembrane space (prediction number 3). We therefore assayed for adenylate kinase (26 kDa) (52) release from the intermembrane space of isolated mitochondria by C₂- and C₁₆-ceramide addition. To obtain a sufficient amount of adenylate kinase activity, a higher amount of mitochondria was used and therefore 5 and 20 µm equivalent ceramide to mitochondria ratios (labeled low and high) were used to achieve comparable conditions as in the cytochrome c permeability assays. Mitochondria incubated for 10 min with C₂- and C₁₆- ceramide, but not their dihydroceramide counterparts, released adenylate kinase from the intermembrane space; an increased amount of adenylate kinase was released at the higher ceramide dose (Table I). Vehicles and dihydroceramides were essentially identical to the untreated mitochondrial control. Therefore, the ceramide channels allow the passage of adenylate kinase as well as cytochrome c.

To assess whether the inner membrane was also being permeabilized to proteins, we tested for the release of fumarase (50 kDa) (53), a soluble matrix enzyme, in mitochondrial supernatants. This is a typical test for damage/disruption of the mitochondrial inner membrane (53). No increase in fumarase activity was detected in supernatants from mitochondria incubated with C₂- or C₁₆-ceramide at either low or high ceramide to mitochondrial protein ratios (Table I).

If ceramide channels were forming in the mitochondrial outer membrane that allow the release of relatively small soluble proteins from the intermembrane space, then other proteins of similar molecular weight should also be released. SDS-PAGE results show that C₂- and C₁₆-ceramides favor the release of low molecular weight proteins from mitochondria over high molecular weight ones (Fig. 4, a–c). The bands from the representative gel in Fig. 4a were quantified using densitometry and the individual gel background for each lane in the gel was subtracted out. To more accurately reflect the differences between the ceramides, dihydroceramide, and positive control (mitochondria with hypo-osmotically lysed outer membranes), the data from the untreated mitochondrial control (the background) was also subtracted out. Fig. 4b quantitatively represents the differences in the proteins released by the ceramides and those that were present in the intermembrane space. C₁₈-dihydroceramide was essentially identical to the untreated mitochondria, as its intensity was basically zero when the data from the mitochondrial control was subtracted out (Fig. 4b). Mitochondria with hypo-osmotically lysed outer membranes show proteins both above and below the R_F value of 0.2 (M_r of 60,000). However, mitochondria incubated with C₂- and C₁₆-ceramides show proteins only above the R_F value of 0.2 (Fig. 4b). Therefore, there was a sharp molecular weight cut-off of the proteins released by C₂- and C₁₆-ceramides (Fig. 4b).

Upon initial examination of Fig. 4b, it appears that more of the high molecular weight proteins (less than 60,000, but greater than about 20,000) were released by the ceramides than the lower molecular weight ones (less than about 20,000). However, when the data was expressed as the percent of total proteins in the intermembrane space (released by osmotic shock), on average the same percentage of proteins less than the cut-off of 60,000 was released by the ceramides (Fig. 4c). It appears that at a ceramide dose of 18 (nanomoles of ceramide/mg of mitochondrial protein) of either C₂- or C₁₆-ceramide increases the permeability of the outer membranes of about 10–15% of mitochondria to proteins of molecular weight ≤ 60,000 (Table II).