Ceramide triggers intracellular calcium release via the IP3 receptor in Xenopus laevis oocytes

Evgeny Kobrinsky1, Andrew I. Spielman2, Sophia Rosenzweig2, and Andrew R. Marks1

1 Molecular Cardiology Program, Divisions of Cardiology and Circulatory Physiology, Departments of Medicine and Pharmacology, Columbia University College of Physicians and Surgeons, New York 10032; and 2 New York University College of Dentistry, Basic Science Division, New York, New York 10010


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Ceramide, a product of sphingomyelin turnover, is a lipid second messenger that mediates diverse signaling pathways, including those leading to cell cycle arrest and differentiation. The mechanism(s) by which ceramide signals downstream events have not been fully elucidated. Here we show that, in Xenopus laevis oocytes, ceramide-induced maturation is associated with the release of intracellular calcium stores. Ceramide caused a dose-dependent elevation in the second messenger inositol 1,4,5-trisphosphate (IP3) via activation of Gq/11alpha and phospholipase C-beta X. Elevation of IP3, in turn, activated the IP3 receptor calcium release channel on the endoplasmic reticulum, resulting in a rise in cytoplasmic calcium. Thus our study demonstrates that cross talk between the ceramide and phosphoinositide signaling pathways modulates intracellular calcium homeostasis.

meiotic maturation; calcium signaling; phospholipase C


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SPHINGOLIPID METABOLITES are important second messengers involved in cellular signaling pathways (44). Second messenger metabolites of sphingolipids are generated in response to diverse stimuli, including progesterone (50), tumor necrosis factor-alpha , and interleukin-1 (25). These stimuli trigger intracellular hydrolysis of sphingomyelin via sphingomyelinases, resulting in the generation of ceramide, which can be further metabolized to sphingosine and subsequently phosphorylated to form sphingosine 1-phosphate by ceramidase and sphingosine kinase, respectively (25).

Ceramide mediates signaling leading to apoptosis and the regulation of cell cycle progression (17, 41, 55). Direct targets for ceramide have been identified, including a ceramide-activated protein kinase (29, 56), a ceramide-activated phosphatase (9), the guanine nucleotide exchange factor vav (18), and protein kinase C (30).

The steroid hormone progesterone, the physiological trigger of Xenopus laevis oocyte meiotic maturation, stimulates sphingomyelin hydrolysis in oocytes, resulting in a time- and concentration-dependent increase in ceramide mass and a decrease in sphingomyelin mass (50). Moreover, direct application of C2- and C6-ceramide analogs triggers meiotic cell cycle progression in Xenopus oocytes (8, 50).

Previous studies identified an important role for intracellular calcium release via the inositol 1,4,5-trisphosphate receptor (IP3R) on the endoplasmic reticulum in determining the rate of progesterone-induced maturation in Xenopus oocytes (24). However, the signaling events linking progesterone and 1,4,5-trisphosphate (IP3)-induced intracellular calcium release have not been fully elucidated.

The present study was designed to gain a better understanding of calcium-dependent signaling during meiotic maturation and the possible role of ceramide therein. Toward this end, we examined ceramide-induced maturation in Xenopus laevis oocytes. We showed that ceramide causes a rise in IP3, resulting in the release of intracellular calcium stores that are mediated by Gq/11alpha and phospholipase C (PLC)-beta X.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. Ceramides, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholine (ET-18-OCH3), U-73122, and pertussis toxin (PTX) were purchased from Calbiochem (Palo Alto, CA). Ceramides were dissolved in 98% ethanol and 2% dodecane (Sigma Chemical, St. Louis, MO). Stock solutions of ceramides were stored at -80°C. Ceramide was sonicated in the cell culture medium for 40 min immediately before use. Sphingomyelinases from Staphylococcus auerus and from Streptomyces species were purchased from Sigma. Cells were incubated with PTX as previously described (48). PTX (stock 100 µg/ml) was activated by incubation in 100 mM dithiothreitol (2:1 volume PTX to dithiothreitol) at 37°C for 30 min. Cells injected with PTX mRNA (30 µg/ml) were also stored in PTX-containing medium (2 µg/ml) for 18-24 h before voltage clamp. PTX mRNA and QEHA peptide were kind gifts of Drs. E. Reuveny and D. Logothetis, respectively. Oligonucleotide injection was performed as previously described (24). The sequences of the IP3R and PLC-beta X oligonucleotides were as follows: IP3R antisense, 5'-AACTAGACATCTTGTCTGACATTGCTGCAG-3'; IP3R sense, 5'-CTGCAGCAATGTCAGACAAGATGTCTAGTT-3'; PLC-beta X antisense, 5'-TCATATCATCACTTAGATCAAGGATCTCCGG-3'; PLC-beta X sense, 5'-CCGGAGATCCTTGATCTAAGTGATGATATGA-3'.

Oocytes. Ovarian fragments were surgically removed from adult female Xenopus laevis frogs previously anesthetized by hypothermia. The fragments were washed in NDE solution (5 mM HEPES-NaOH, pH 7.6, 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 2.5 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin). Fully grown stage 6 oocytes (11) were treated with collagenase (2 mg/ml type I collagenase; Sigma). Oocytes were maintained in NDE solution at 18°C until further use. Meiotically mature eggs were induced by incubation in ND-96 solution (96 mM NaCl, 2 mM KCl, 2 mM MgCl2, and 5 mM HEPES, pH 7.5) containing 10 µg/ml progesterone (Sigma). Oocytes were judged to be meiotically mature by the appearance of a white spot on the animal pole, indicating the release of the first polar body (24). For quantitative comparison, the time required for the maturation of 50% of oocytes marked by the appearance of germinal vesicle breakdown was determined (16).

Electrophysiological measurements. Electrophysiological measurements were performed in ND-96 solution (24). Currents were recorded using a double-microelectrode voltage clamp and a TEV 200 amplifier (Dagan). Data are presented as mean values ± SE, where n is the number of oocytes examined. Statistical analysis was performed using Student's t-test, and significance was accepted at P < 0.05. In all cases, similar data were obtained from oocytes derived from two or more animals. The calcium-activated chloride current [ICl(Ca)] had a reversal potential of 21.0 ± 3.0 mV (n = 4) corresponding to the reversal potential of the chloride current (1) and was blocked by 300 µM of niflumic acid (51), a specific inhibitor of ICl(Ca) (n = 3). Experiments were performed in nominally calcium-free media to exclude the possibility that the rise in cytosolic calcium resulted from calcium influx. C16-ceramide injections were performed using a stock solution of 250 µM C16-ceramide such that 20 nl yielded an estimated intracellular concentration of 10 µM, assuming cytoplasmic oocyte volume of 0.5 µl. External application of sphingomyelinase (0.1-8.0 U/ml) induced nonspecific plasma membrane leaks that prevented recording of ICl(Ca) or measurement of IP3 levels in Xenopus oocytes. Oocytes were incubated with the indicated tyrosine kinase inhibitors (Calbiochem) for 3 h. Inhibitors were dissolved in 0.1% DMSO, and controls were treated with 0.1% DMSO alone. The rabbit anti-Gq/11alpha antibody (52) was from NEN Life Sciences (Boston, MA). Oocytes were injected with 50 nl of the undiluted antibody and incubated for 3 h, and controls were injected with 50 nl of boiled antibody.

IP3 measurements. Freshly isolated Xenopus laevis oocytes (150/tube) were stimulated with 150 µl of ceramide at the indicated concentration in ND-96 buffer at 23°C. After 0, 30, or 120 s, 150 µl of ice-cold 9% perchloric acid (PCA) were added to the mixture to quench IP3 formation. Oocytes were immediately homogenized with a glass pestle, and samples were placed on ice. For time 0, oocytes were first quenched with 150 µl of 9% PCA. All subsequent steps were performed at 4°C unless otherwise specified using siliconized tubes, pipettes, and syringes. After quenching with 9% PCA and homogenization, each sample was centrifuged at 17,000 g for 30 min, the volume of the supernatant in each tube was measured, and 330 µl were separated into aliquots and kept at -20°C until use. IP3 was extracted from each tube by precipitation with potassium perchlorate in the presence of KOH and HEPES (38). The supernatant (400 µl) was assayed for IP3 (39, 46) by binding to a radiolabeled, purified IP3-binding protein isolated from bovine adrenal gland (39). Bovine adrenal cortex was dissected from 6 to 10 glands, homogenized in 200 mM NaHCO3 and 1 mM dithiothreitol at 4°C. The homogenate was centrifuged two times at 5,000 g for 20 min. The final pellet was resuspended in 12 ml of homogenization buffer at a protein concentration of 20-40 mg/ml. The binding protein was separated into aliquots at 30 mg/tube and kept at -80°C until use; each tube was thawed only one time. Protein determination was performed using the Bio-Rad (Hercules, CA) protein assay with bovine gamma globulin as the standard.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Ceramide-induced maturation. In the present study, we found that sphingomyelinase and C2- and C16-ceramide induced maturation in Xenopus laevis oocytes (Fig. 1). Progesterone-induced oocyte maturation required 7.2 ± 0.9 h (n = 5). C16-ceramide also induced maturation but with a slower time course requiring 9.8 ± 1.1 h (n = 5, P < 0.01; Fig. 1). Added together, C16-ceramide and progesterone acted synergistically, reducing the time required to achieve maturation to 5.5 ± 0.8 h (n = 5, P < 0.01). Injection of heparin (20 nl, 10 mg/ml) to inhibit IP3R slowed the time courses of progesterone-induced maturation (11.3 ± 0.3 h, n = 5, P < 0.01) and C16-ceramide-induced maturation (12.3 ± 1.3 h, n = 5, P < 0.01). Sphingomyelinase injected into the oocytes to achieve a final concentration of 1 U/ml also induced Xenopus oocyte maturation (6.0 ± 0.7 h, n = 5), and injection of heparin (20 nl, 10 mg/ml) slowed the time course of sphingomyelinase-induced maturation (11.7 ± 0.3 h, n = 5, P < 0.01; Fig. 1). C2-ceramide (final concentration 7.5 µM) induced Xenopus oocyte maturation (8.5 ± 0.3 h, n = 5; Fig. 1), which is consistent with recent findings (33): injection of heparin slowed the time of C2-ceramide-induced maturation (12.5 ± 0.2 h, n = 5, P < 0.01). Injection of solvent had no effect on oocyte maturation. Taken together, our data indicate that both ceramide and the IP3R can modulate the time course of progesterone-induced maturation. Our data further suggest that IP3-mediated intracellular calcium release plays a role in modulating progesterone-induced maturation in agreement with data from earlier studies (20, 24). A possible explanation for the slower time course of ceramide-induced maturation, compared with progesterone-induced maturation, may be that ceramide must diffuse to a specific location within the cell to activate downstream signaling.


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Fig. 1.   Ceramide-induced maturation in Xenopus oocytes. The time required to achieve meiotic maturation was determined in response to progesterone (10 µg/ml); progesterone (10 µg/ml) plus heparin (20 nl, 10 mg/ml) injection; progesterone (10 µg/ml) plus C16-ceramide (2 µM) injection; C16-ceramide (2 µM) injection; injection of C16-ceramide (2 µM) and heparin (20 nl, 10 mg/ml); C2-ceramide (7.5 µM) injection; injection of C2-ceramide (7.5 µM) and heparin (20 nl, 10 mg/ml); sphingomyelinase (1 U/ml) injection; and injection of sphingomyelinase (1 U/ml) and heparin (20 nl, 10 mg/ml). Bars represent the time in hours (mean ± SD) required to achieve germinal vesicle breakdown in 50% of oocytes. Each experiment consisted of 30 oocytes and was repeated 5 times with oocytes from 5 different frogs.

Ceramide-induced calcium release. Application of a cell-permeable analog of C2-ceramide (Fig. 2A) induced the release of intracellular calcium in a dose-dependent manner in Xenopus laevis oocytes. Cytosolic calcium levels were monitored by recording ICl(Ca) in Xenopus oocytes as previously described (24). Maximal release of intracellular calcium was seen with 50 µM C2-ceramide [ICl(Ca) = 959 ± 190 nA, n = 20; Fig. 2A]. A biologically inactive form of ceramide, dihydroceramide (10 µM; see Ref. 2), induced only negligible release of calcium [ICl(Ca) = 12.5 ± 14.0 nA, n = 10; Fig. 2A]. C8-ceramide (100 µM) also induced calcium release in oocytes [ICl(Ca) = 402 ± 95 nA, n = 5].


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Fig. 2.   Ceramide-induced intracellular calcium release in Xenopus oocytes. Traces (top) show representative calcium-activated chloride currents for each condition indicated below. A: external addition of C2-ceramide (arrows) induced intracellular calcium release, whereas the inactive form dihydro C2-ceramide did not. B: injection of C16-ceramide (arrows) induced intracellular calcium release.

Injection of a nonpermeable analog of natural C16-ceramide to achieve an intracellular concentration of 10 µM also induced the release of intracellular calcium [ICl(Ca) = 110.0 ± 19.5 nA, n = 42; Fig. 2B]. The response to C16-ceramide was dose dependent [0.5 µM C16-ceramide-induced ICl(Ca) = 40.5 ± 18.5 nA, n = 9; 2 µM C16-ceramide-induced ICl(Ca) = 71.0 ± 20.0 nA, n = 10; Fig. 2B]. Ethanol, the major component of the vehicle for ceramide, is known to induce calcium release in Xenopus laevis oocytes (21). However, injection of the vehicle alone induced an ICl(Ca) = 21.0 ± 7.0 nA (n = 8, P < 0.01; Fig. 2B), which was significantly smaller than the response to C16-ceramide. The amount of vehicle was equivalent to that required to yield a 10 µM intracellular concentration of C16-ceramide. The cell-permeable ceramide analog C2-ceramide was applied externally in the perfusion bath, whereas the nonpermeable C16-ceramide was injected locally. Therefore, C2-ceramide was acting on the entire oocyte membrane, whereas C16-ceramide acted locally on the plasma membrane near the injection site. This explains why we consistently observed a greater effect in response to C2-ceramide. Sphingomyelinase injected in Xenopus oocytes (estimated final concentration 1 U/ml) induced an ICl(Ca) =179.7 ± 35.0 nA (n = 30; Fig. 3B).


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Fig. 3.   Ceramide-induced release of intracellular calcium via activation of phospholipase C (PLC)-beta X by Gq/11alpha . A: inhibiting PLC-beta X expression using an antisense oligonucleotide blocked the release of intracellular calcium in response to 50 µM C2-ceramide (n = 10). Injection of the corresponding sense oligonucleotide had no inhibitory effect (n = 10). In each oocyte, the response to C2-ceramide (50 µM) was determined before injection of oligonucleotides followed 22 h later by a second application of C2-ceramide (50 µM). B: blocking the activity of Gq/11alpha with an anti-Gq/11alpha antibody (52) inhibits the release of intracellular calcium in response to C16-ceramide (boiled antibody injected, n = 23; antibody injected, n = 29) and to sphingomyelinase (boiled antibody injected, n = 30; antibody injected, n = 10). In each oocyte, the response to C16-ceramide (2 µM) and to sphingomyelinase (1 U/ml) was determined 3 h after injection of anti-Gq/11alpha antibody. Arrows indicate the injection of C16-ceramide and sphingomyelinase.

The ceramide-induced increase in cytosolic calcium was due to intracellular calcium release, since the experiments were performed with negligible extracellular calcium. Xenopus laevis oocytes have IP3R but do not express the other major form of intracellular calcium release channel, the ryanodine receptor (24, 26). On this basis, we hypothesized that the ceramide-induced elevation of cytosolic calcium was mediated through the phosphoinositide pathway, resulting in activation of IP3-induced intracellular calcium release. Therefore, we examined the effects of ceramide on the known components of the phosphoinositide/calcium release pathway: heterotrimeric GTP-binding proteins, nonreceptor protein tyrosine kinases, PLC, IP3, and intracellular calcium stores.

Activaton of IP3R by ceramide. Previous studies have shown that progesterone induces a transient increase in IP3 levels in Xenopus oocytes (49). C2-ceramide (5 µM) induced an approximately threefold increase in IP3 level (from 0.258 ± 0.047 to 0.785 ± 0.090 nmol/oocyte, n = 7, P < 0.001), with the peak occurring within 30 s (Fig. 4A and Table 1).


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Fig. 4.   Ceramide-induced inositol 1,4,5-trisphosphate (IP3) production and activation of intracellular calcium release via the IP3 receptor in Xenopus oocytes. A: IP3 levels increased rapidly and transiently within 30 s after application of 5 µM C2-ceramide (n = 7 oocytes). B: inhibition of IP3 receptor expression using antisense oligonucleotides (24) blocked the release of intracellular calcium in response to C2-ceramide (50 µM, n = 5). Injection of the corresponding sense oligonucleotide had no inhibitory effect (n = 5). The response to C2-ceramide (50 µM) was determined in each oocyte before injection of oligonucleotides (control), followed 22 h later by application of C2-ceramide (50 µM; arrows).


                              
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Table 1.   Ceramide-induced IP3 production in Xenopus oocytes

Injection of heparin (20 nl of a 10-mg/ml solution), an IP3R blocker, inhibited the ceramide-induced release of intracellular calcium. Heparin, a well-established inhibitor of IP3-induced calcium release (13), no doubt has other cellular effects in addition to blocking the IP3R. However, the fact that it inhibits intracellular calcium release in the oocytes in the manner shown in the present study is consistent with its action on the IP3R on the endoplasmic reticulum. With heparin, C2-ceramide (5 µM) induced an ICl(Ca) = 40.5 ± 17.0 nA (n = 14), and C16-ceramide (10 µM) induced ICl(Ca) = 35.5 ± 13.0 nA (n = 14) compared with the responses without heparin for C2-ceramide [5 µM; ICl(Ca) = 118.5 ± 23.0 nA (n = 12, P < 0.01)] and for C16-ceramide [10 µM; ICl(Ca) = 134.5 ± 11.0 nA (n = 12, P < 0.01)]. Injection of IP3R antisense oligonucleotides to inhibit IP3R expression, as previously described (24, 26), also inhibited the C2 (50 µM)-ceramide-induced calcium response, whereas injection of sense oligonucleotides did not inhibit the response (Fig. 4B). Taken together, these data suggest that ceramide triggers the release of intracellular calcium by (directly or indirectly) activating PLC, resulting in IP3 production and activation of the IP3R on the endoplasmic reticulum.

Involvement of PLC-beta X via Gq/11alpha in ceramide-mediated calcium release. Previous reports (3, 54) have shown that PLC-gamma plays a role in oocyte maturation and egg activation. It was also reported (43) that C2- and C6-ceramides were able to increase tyrosine phosphorylation of the focal adhesion kinase p125FAK. C16-ceramide-induced ICl(Ca) was not inhibited by the tyrosine kinase inhibitors genistein (50 µM) or herbimycin A (10 µM). In control oocytes (incubated with 0.1% DMSO), injection of C16-ceramide (2 µM) induced ICl(Ca) = 76 ± 5.4 nA (n = 20) compared with 67 ± 5.2 nA [n = 10, P = statistically not significant (NS)] for oocytes incubated with 10 µM herbimycin A and 106.5 ± 32.7 (n = 10, P = NS) for oocytes incubated with 50 µM genistein. Similar results were obtained when both genistein and herbimycin A were used in combination (data not shown). These data indicate that activation of PLC-gamma through tyrosine phosphorylation is not involved in the calcium-mobilizing action of ceramide in Xenopus oocytes.

It has been reported that PLC-beta X (31) activation via a PTX-insensitive Gqalpha can lead to maturation of Xenopus oocytes (19). PTX, an inhibitor of the Gialpha subunit of the heterotrimeric GTP-binding protein, had no effect on the response to C16-ceramide. Injection of C16-ceramide (to yield an estimated intracellular concentration of 2 µM) induced an ICl(Ca) = 75.0 ± 13.5 nA (n = 15) in control oocytes and ICl(Ca) = 78.5 ± 29.5 nA (n = 15, P = NS) in oocytes incubated with PTX (2 µg/ml) for 24 h. C16-ceramide (2 µM) induced an ICl(Ca) = 58.5 ± 13.0 nA (n = 10) in control oocytes and ICl(Ca) = 61.5 ± 17.0 nA (n = 10, P = NS) in oocytes injected with PTX mRNA. Inhibition of ICl(Ca) evoked by administration of ACh to oocytes expressing M1-muscarinic receptors served as a positive control for both PTX and PTX mRNA, as described previously (31).

PLC-beta X can also be activated via Gbeta gamma (14). Injection of a synthetic peptide, QEHA, derived from the COOH-terminus of the adenylate cyclase 2 isoform that is known to inhibit Gbeta gamma signaling (5) failed to inhibit C16-ceramide (10 µM)-induced calcium release, with ICl(Ca) = 105 ± 25 nA (n = 15) in control oocytes and ICl(Ca) = 109 ± 31 nA (n = 17, P = NS) in injected oocytes.

In contrast, blocking PLC-beta X with either 10 µM ET-18-OCH3 (n = 3) or 10 µM U-73122 (n = 2; see Ref. 47) resulted in 100% inhibition of C2-ceramide (50 µM)-induced intracellular calcium release [ICl(Ca) = 0 nA]. Injection of oocytes with antisense oligonucleotides to PLC-beta X (31) resulted in inhibition of the response to ceramide: 50 µM C2-ceramide induced an ICl(Ca) = 415 ± 115 nA (n = 10) in sense oligonucleotide-injected oocytes and ICl(Ca) = 87 ± 45 nA in antisense oligonucleotide-injected oocytes (n = 10, P < 0.05; Fig. 3A). These data suggested that activation of PLC-beta X via a PTX-insensitive form of Galpha was involved in the ceramide-mediated mobilization of intracellular calcium.

An antibody against the COOH-terminus of Gq/11alpha (52) potently inhibited C16-ceramide (2 µM)- and sphingomyelinase (final concentration 1 U/ml)-induced ICl(Ca) (Fig. 3B). C16-ceramide (2 µM) induced an ICl(Ca) = 91.2 ± 24.3 nA in control (boiled antibody injected) oocytes (n = 23) and 3.3 ± 1.6 nA in anti-Gq/11alpha -injected oocytes (n = 24, P < 0.005). Injection of sphingomyelinase induced an ICl(Ca) = 179.7 ± 35.0 nA in control (boiled antibody injected) oocytes (n = 30) and 29.3 ± 8.5 nA in anti-Gq/11alpha oocytes (n = 10, P < 0.005). These data indicated that Gq/11alpha participates in the activation of PLC-beta X after addition of ceramide in Xenopus oocytes. Interestingly, expression of the Gqalpha family of G protein subunits is increased during Xenopus oocyte maturation (15).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results of the present study demonstrate that ceramide causes a dose-dependent elevation in the second messenger IP3 via activation of Gq/11alpha and PLC-beta X. Elevation of IP3, in turn, activates the IP3R/calcium release channel on the endoplasmic reticulum, resulting in a rise in cytoplasmic calcium. Intracellular calcium release is important for controlling the rate of progesterone (24)- and ceramide-induced maturation in Xenopus laevis oocytes.

Previous reports have demonstrated that C6-ceramide can potentiate calcium release in human platelets (53). In contrast, C2-ceramide did not mobilize calcium in either human T lymphocytes (Jurkat; see Ref. 42) or in a pancreatic duct adenocarcinoma cell line (37). Thus the calcium-mobilizing effects of ceramide appear to be cell-type specific.

The concentrations of ceramide used in the present study (from 2 to 50 µM) are consistent with those used in many previous studies (7, 22, 36). Moreover, because only ~10% of externally applied C2-ceramide is taken up by the cell, the concentration of C2-ceramide inside the cell should be ~10 times less than the concentration of ceramide in our external solution (2).

The effects reported in our studies were observed using both a truncated form of ceramide, C2-ceramide, as well as naturally occurring C16-ceramide and sphingomyelinase. Extensive studies by others (2) have demonstrated that, during the first 30 min after C2-ceramide application, ~1% of C2-ceramide is metabolized and, even after 4 h, ~90% of the C2-ceramide remains intact in cells, demonstrating that C2-ceramide is poorly metabolized. In our own experiments, concentration-dependent effects on calcium signaling were observed within 5 min. Thus it is likely that the effects we report were due to ceramide and not to its metabolites. Interestingly, it has been reported that, in Xenopus oocytes, sphingosine is rapidly converted to ceramide, and short-chain ceramides are converted to long-chain ceramides without accumulation of sphingosine (50).

Inhibition of the ceramide-induced calcium release by heparin and IP3R antisense oligonucleotides suggests that calcium release occurs via the IP3R. It has been reported that sphingosine-induced calcium release occurs via an IP3-independent mechanism (6, 23, 32, 45). In Xenopus oocytes, injection of sphingosine 1-phosphate does not induce calcium release (12). This suggests that ceramide-induced mobilization of intracellular calcium stores is unlikely to be due to the conversion of ceramide to sphingosine 1-phosphate.

The effects of ceramide versus those of sphingosine appear to be somewhat cell specific. For example, in dermal fibroblasts, sphingosine (5 and 30 µM) induced hydrolysis of phosphoinositides within 5 s, resulting in elevated IP3 levels, followed 30-40 s later by increased levels of intracellular calcium (4). Moreover, in these same cells, sphingosine 1-phosphate induced a more rapid intracellular calcium release than sphingosine but did not raise IP3 levels, and C2-ceramide did not affect phosphoinositide turnover or calcium mobilization. PTX partially inhibited the sphingosine-mediated rise in IP3 levels consistent with an effect involving G proteins and PLC.

In considering the effects of sphingosine 1-phosphate on calcium mobilization, it is important to note that sphingosine 1-phosphate acts extracellularly as well as intracellularly. Extracellular sphingosine 1-phosphate binds to a specific receptor, endothelial differentiation gene 1 (EDG1), that activates PLC to mobilize calcium (36, 57). This extracellular effect of sphingosine 1-phosphate does not explain the observed effects of ceramide in our experiments, since the same calcium-mobilizing effects were seen when ceramide was injected intracellularly; therefore, ceramide could not be acting on the sphingosine 1-phosphate receptor.

The delay in the time course of Xenopus oocyte maturation due to heparin injection was reproducible and consistent with our previously published data (24) that showed blocking the expression of the IP3R delays progesterone-induced maturation. Indeed, the magnitude of the heparin-induced delay is similar to that seen when IP3R expression is blocked with antisense oligonucleotides, as we reported previously (24). Nevertheless, the signals governing maturation are complex, and one cannot exclude the possibility that certain metabolites of ceramide play an active role. However, the fact that both C16-ceramide and C2-ceramide had similar effects on calcium signaling argues that ceramide itself is involved in the IP3-dependent calcium signal during maturation, since the metabolism of C2-ceramide is negligible.

A growing list of molecules with diverse structural properties (e.g., neuropeptides, peptide hormones, polyamines, venom peptides, local anesthetics, taste substances) have been shown to directly activate G proteins (34, 35). Ceramide could act in a similar manner, by direct activation of Gq/11alpha protein. Alternatively, G protein-coupled receptors may attain an active conformation in the absence of agonist by spontaneous isomerization and may yield constitutive, agonist-independent activity (27, 40 ). Although we do not know the mechanism by which ceramide may activate Gq/11alpha , it is possible that ceramide can indirectly induce agonist-independent activity of Gq/11alpha -coupled receptors and thus activate the downstream cascade, resulting in the production of IP3.

In summary, we have demonstrated, for the first time, cross talk between the ceramide and phosphoinositide signaling pathways. The link between these two lipid second messenger pathways provides a mechanism for the mobilization of intracellular calcium observed in response to elevated levels of ceramide. Calcium oscillations triggered by ceramide-mediated activation of IP3R may be involved in the regulation of gene expression (10, 28) during meiotic maturation in Xenopus laevis oocytes.


    ACKNOWLEDGEMENTS

We thank I. Tabas and R. Iyengar for helpful comments on the manuscript, D. Logothetis for the PTX mRNA, and E. Reuveny for the QEHA peptide.


    FOOTNOTES

This study was supported by National Institutes of Health (NIH) Grants AI-39794, HL-61503, and HL-56180 to A. R. Marks and NIH Grant DE-10745 and a grant from the Binational Agricultural Research and Development Fund foundation to A. I. Spielman. E. Kobrinsky is on leave from the Institute of Theoretical and Experimental Biophysics, Pushchino, Moscow Region, Russia.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: A. R. Marks, Molecular Cardiology Program, Box 65, Columbia Univ. College of Physicians & Surgeons, Rm. 9-401, 630 West 168th St., New York, NY 10032 (E-mail: arm42{at}columbia.edu).

Received 13 April 1999; accepted in final form 14 June 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 277(4):C665-C672
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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