Bradykinin inhibits ceramide production and activates phospholipase D in rabbit cortical collecting duct cells

Gele Liu1, Leonard Kleine2, Rania Nasrallah1, and Richard L. Hébert1,3

1 Departments of Cellular and Molecular Medicine, 2 Biochemistry, Microbiology and Immunology, and 3 Medicine, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5


    ABSTRACT
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Recent reports suggest that inflammatory cytokines, growth factors, and vasoconstrictor peptides induce sphingomyelinase (SMase) activity. This results in the hydrolysis of sphingomyelin (SM) into ceramide, which is implicated in various cellular functions. Although ceramide regulates phospholipase D (PLD) activity, there is controversy about this relationship. Thus we investigated whether the effect of bradykinin (BK), a proinflammatory factor and vasodilator, was mediated by ceramide signal transduction and by PLD. In rabbit cortical collecting duct (RCCD) cells, BK increased SM levels and decreased ceramide levels in a time-dependent manner. Thus SMase activity was inhibited by BK. Also, the production of ceramide was regulated in a concentration-dependent manner. The BK-B1 antagonist [Lys-des-Arg9,Leu8]BK did not affect ceramide signal transduction but the BK-B2 antagonist (Hoe-140) blocked the effect of BK on SMase, suggesting that the BK-B2 receptor mediates BK-induced inhibition of ceramide generation. Our results show that exogenous SMase significantly hydrolyzed endogenous SM to form ceramide and weakly activated PLD. In contrast, BK induced a significant activation of PLD. However, additive effects of BK and ceramide on PLD activity were not observed. We concluded that in RCCD cells, the BK-induced second messengers ceramide and phosphatidic acid were generated by distinct signal transduction mechanisms, namely the SMase and PLD pathways.

sphingomyelin; sphingomyelinase


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INTRODUCTION
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CERAMIDE, A KEY PRODUCT of sphingolipids, is rapidly emerging as a component of a wide variety of signal transduction pathways, which produce specific biological effects. Sphingomyelin (SM) is composed of a very long chain, saturated or mono-unsaturated fatty acid, a sphingosin base backbone (predominantly sphingosine), and a phosphocholine headgroup (1, 3, 30). The headgroup appears to come from phosphatidylcholine (PC), but other sources have not been ruled out. SM turnover involves removal of the headgroup and amide-linked fatty acid by sphingomyelinases (SMase) and ceramidases, respectively. SMase action is the only known mechanism for SM hydrolysis in mammalian cells. However, most, if not all, mammalian cells appear capable of signaling through the SM pathway (36). The action of certain extracellular agents results in SM hydrolysis by SMase and the concomitant generation of ceramide (23, 31, 36). Indeed, ceramide is emerging as a putative second messenger, mediating the effects of extracellular agents on cell growth, differentiation, and apoptosis (38, 50).

Endogenous SMases can be placed into three classes: acidic SMase (aSMase), neutral SMase (nSMase), and alkaline SMase (23). Apparently, nSMase and aSMase play important roles in ceramide signal transduction. Exogenous SMase, mainly from bacteria, is an important tool used in investigating ceramide signal transduction (6, 51). Addition of exogenous SMase decreases endogenous SM (12). The ceramide that is generated by exogenous SMase induces apoptosis in human myeloid leukemia cells (18). The synthetic, cell-permeable, short-chain ceramides such as C2-(N-acetylsphingosine), C6-(N-hexanoylsphingosine), and C8-(N-octanoylsphingosine) also have been used as tools in investigating and understanding the pathway of ceramide signal transduction (20, 44).

Interestingly, phospholipase D (PLD) promotes the apoptotic DNA fragmentation induced by tumor necrosis factor-alpha (TNF-alpha ) (19). Ceramides can modify cell signaling via PLD (14). Meacci et al. (29) suggests that ceramide can activate PLD in human fibroblasts, an effect that was mimicked by treatment with Staphylococcus aureus SMase. In contrast, ceramide inhibited IgE-mediated activation of PLD in rat basophilic leukemia cells (34). Adding C6-ceramide to undifferentiated cells resulted in the inhibition of PLD activity and reductions in diacylglycerol (DAG) and phosphatidate (PA) levels in the rat neuroblastoma N1E-115 (8). In general, ceramide is considered to be a negative modulator of PLD (3, 53); however, ceramide is also able to activate PLD in specific cell types (29). Therefore, the relationship between ceramide and PLD activity requires further investigation.

Recent results suggest that inflammatory cytokines such as TNF-alpha (9, 26, 27), growth factors such as nerve growth factor (NGF) (11), and vasoconstrictor peptides such as endothelin-1 (ET-1) (5, 46, 48) induce SMase activity, resulting in SM hydrolysis to produce ceramide. Kidney, in particular the collecting duct segment of the nephron, represents a major site of renal sodium and water reabsorption. In the present study, we have used a recently characterized rabbit cortical collecting duct (RCCD) cell line (4) to study the regulation of SMase and PLD by BK in this rabbit nephron segment. These RCCD cells represent a heterogenous population of principal, alpha -intercalated, and beta -intercalated epithelial cell types as assessed by specific monoclonal antibodies (Mab 703 and Mab 503) and Arachis hypogea lectin staining. They have retained both their morphological and hormonal response properties and possess the signaling machinery present downstream of angiotensin signaling (4). However, it is not known whether the effects of BK involve ceramide signal transduction or whether there is cross talk between BK- and ceramide-induced PLD activity. Therefore, we studied the cellular signaling of SM-ceramide and PLD activity induced by BK in RCCD cells.


    MATERIALS AND METHODS
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BK, S. aureus SMase, N-acetyl-D-sphingosine (C2-ceramide), adenosine 5'-trisphosphate, phosphatidylethanol (PEt), PC, 1,2-dioleoyl-rac-glycerol (C18:1[cis]-9), L-alpha -phosphatidic acid, cardiolipin, and all other drugs, standards, and salts were purchased from Sigma. DMEM-F12 media and newborn calf serum were obtained from GIBCO-BRL. Escherichia coli DAG kinase (specific activity: 13 U/mg protein) and D609 (tricyclodecan-9-yl-xanthogenate, potassium salt) were purchased from Calbiochem. [Methyl-14C]choline chloride (specific activity: 55.0 mCi/mmol) and [gamma -32P]ATP (specific activity: >3,000 Ci/mmol) were supplied by Amersham. [14C]myristic acid (specific activity: 40-60 mCi/mmol) was obtained from DuPont-New England Nuclear. Thin-layer chromatography (TLC) plates (0.25 mm thick) were purchased from Whatman. HPLC grade solvents were supplied by British Drug House.

Cell culture. The RCCD cells (4) from passages 3-25 were grown in DMEM-F12 medium containing 10% fetal calf serum, 0.4% penicillin/streptomycin solution, 15 mM HEPES, 50 nM hydrocortisone, 44 mM sodium bicarbonate, and ITS (insulin-transferrin-sodium selenite). Cells were grown to confluence in an atmosphere of 5% CO2 at 37°C and then routinely incubated in standard medium without serum for 24 h before treatment.

Treatment and solvent extraction of cell lipids. The cells were washed three times with DMEM-F12 medium and incubated for 30 min before treatment. The experiments were performed on RCCD cells cultured in 100 × 20-mm plates, and three dishes were pooled per sample. After different treatments, the reaction was terminated by three washings with ice-cold DMEM-F12 media. The lipid extraction method was based on that of Bligh and Dyer (2). After centrifugation at 500 g for 2 min at 4°C, the cells were transferred to an ice-cold ground glass homogenizer containing 0.5 ml chloroform/methanol/HCl (20:40:1, vol/vol/vol), and homogenized on ice. The homogenate was left on ice for 30 min. The mixture was then transferred to a microcentrifuge tube, and the homogenizer was rinsed with 1 ml chloroform. The washings and 0.3 ml 1 M NaCl were added to the microcentrifuge tube, vortexed, and spun at 11,000 g for 5 min at 4°C. The upper aqueous layer was discarded, and the lower lipid-containing layer was transferred to a 1-ml glass Chrompack vial, dried under a stream of O2-free N2 gas, and redissolved in 200 µl chloroform. The samples were stored at -20°C until analyzed. The particulate protein interface was air-dried, dissolved in 0.5 ml 2 M NaOH, and assayed for protein according to Lowry's method (24).

Measurement of radioactivity. [14C]SM, [32P]ceramide, [14C]ceramide, and [14C]PEt were measured using the Molecular Dynamics System with the ImageQuaNT computer software provided by Molecular Dynamics. The TLC plates were exposed to phosphor screens for 48 h (32P) or 72 h (14C) in exposure cassettes and were scanned with the phosphor imager. Results were obtained as volume per spot and expressed as volumes per milligrams protein. Volume calculations include the area and density of the radioactivity-generated spots. Results are expressed as percentage of control.

Measurement of sphingomyelin levels. Because choline is a component of sphingolipids (43), cells were prelabeled with 1 µCi/ml [methyl-14C]choline chloride in DMEM-F12 media 24 h before the treatment. The cells were then stimulated with or without 100 nM BK (for 1, 5, 10 and 15 min) or 10 µM (for 1, 5, 10, 15, 30, 60, 90, and 120 min). Lipids were then extracted, and 20 µl (10 mg/ml in chloroform) of SM + PC were added to each sample as internal standards. The TLC plates were heated to 80°C for 30 min. The samples (25 µl in chloroform) were spotted onto the plates. SM + PC (20 µl of 10 mg/ml in chloroform) were added as external standards. The plates were developed in chloroform/methanol/acetic acid/water (50:30:8:5, vol/vol/vol/vol). TLC plates were dried, and I2 staining was used to mark the areas corresponding to SM and PC.

Quantitation of ceramide by the [gamma -32P]ATP and DAG kinase method. We quantified ceramide using the [gamma -32P]ATP and DAG kinase method, as described by Preiss et al. (37) and modified by Wright et al. (49). Briefly, cells were stimulated with BK (100 nM) for 5, 10, 20, and 30 min or with S. aureus SMase (0.1 U/ml) for 5 min. The lipids were extracted and stored in chloroform at -20°C until used. The chloroform was then evaporated under N2. A blank tube and a standard ceramide tube were always included. For each 10 µl of DAG kinase (20 mU), 50 µl of reaction buffer, 10 µl of 20 mmol/l dithiothreitol, and 10 µl of [gamma -32P]ATP (2.5 × 105 dpm/nmol) were added, and the mixture was incubated at 25°C for 30 min. Reaction buffer contained 25 mmol/l MgCl2 and 2 mmol/l EGTA were dissolved in 50 ml of 100 mmol/l imidazole (pH 6.6), and the pH was readjusted to 6.6. The reaction was terminated by the addition of 0.5 ml ice-cold chloroform/methanol (1:2 vol/vol). The lipids were extracted by the addition of 0.5 ml chloroform and 0.5 ml 1 M NaCl. The mixture was spun at 12,000 g for 3 min, and the upper aqueous phase was discarded. To improve the extraction of ceramide and to remove the excess [gamma -32P]ATP, we modified the procedure as follows: the lower organic phase was sequentially washed with 0.75 ml of 1% perchloric acid with 0.3 ml chloroform/methanol (1:2 vol/vol), 0.2 ml chloroform, and 0.2 ml water. The resultant organic phase containing the radioactive ceramide was dried under a stream of N2 and reconstituted in 25 µl chloroform/methanol (95:5, vol/vol). The reconstituted sample was spotted onto a Silica Gel 60 TLC plate, which had been previously heat activated, and developed in a solvent mixture of chloroform/acetone/methanol/acetic acid/water (10:4:3:2:1, vol/vol/vol/vol/vol).

Direct measurement of ceramide production using [14C]myristic acid. To directly measure ceramide, we have developed a method using [14C]myristic acid-labeled cells and a TLC solvent of ethyl acetate/acetic acid/trimethylpentane. RCCD cells were labeled with [14C]myristic acid for 24 h in the cell culture environment. After the extraction of lipids, the samples were spotted on an oxalate-coated Silica Gel 60 TLC plate. The plate was developed with a solvent of ethyl acetate/acetic acid/trimethylpentane (EAT solvent; 9:2:5, vol/vol/vol). With this method, we were able to separate [14C]ceramide from other [14C]myristate-containing neutral lipids. The external standard ceramide and endogenous ceramide produced by exogenous SMase served to confirm the position of [14C]ceramide.

To investigate the ceramide concentration response to BK, we stimulated [14C]myristic acid-labeled RCCD cells with BK (from 10-9 M to 10-5 M) for 20 min. Lipids were extracted from cells and the 14C-labeled ceramide was separated from other 14C-containing lipids by TLC with the EAT solvent (see Fig. 3). To determine which BK receptor subtype mediates BK-induced ceramide signal transduction, we stimulated [14C]myristic acid-labeled RCCD cells with BK (100 nM) for 20 min with or without the BK-B1 receptor antagonist [Lys-des-Arg9,Leu8]BK (10 µM) or the BK-B2 receptor antagonist Hoe-140 (10 µM). Then lipids were extracted from cells, and the 14C-labeled ceramide was separated from other 14C-containing lipids on TLC plates (see Fig. 4). These experiments were repeated three times.

Measurement of PLD activity. To measure PLD activity, we labeled RCCD cells with 1 µCi/ml [14C]myristic acid (47) in DMEM-F12 media 24 h before the treatment. In the presence of ethanol, the formation of [14C]PEt, a specific product of PLD activity, was determined (3). The cells were treated with 100 nM BK with or without S. aureus SMase (0.1 U/ml) or C2-ceramide (50 µM), or with S. aureus SMase (0.1 U/ml) with or without D609 (10 µM) in the presence of 1% ethanol. D609, a PLC inhibitor (33) specific for phosphatidylcholine-phospholipase C (PC-PLC) (42), does not inhibit phosphatidylinositol-PLC, PLA2, or PLD in U937 cells. D609 has been used to establish the functional coupling of PC-PLC with SMase (42). After extraction of lipids, the samples were spotted on an oxalate-coated Silica Gel 60 TLC plate. The plate was developed with a solvent of ethyl acetate/acetic acid/trimethylpentane (9:2:5, by volume). With this method, we were able to separate [14C]PEt from other [14C]myristate-containing neutral lipids. These experiments were repeated four times.

Statistics. To determine the statistical significance of differences between more than two groups, we used ANOVA and the Bonferroni multiple comparison tests (4, 21). Differences of P < 0.05 were considered statistically significant. Results were presented as means ± SE of three or four independent experiments.


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RESULTS
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BK increases the SM level. SM is hydrolyzed by SMase to produce ceramide. To determine the SM pool response to BK, we stimulated cells with BK (100 nM or 10 µM) for the times indicated in Fig. 1, A-C. For both concentrations of BK, [14C]SM levels increased for 5 min, at which time they were significantly different from the control level (39 ± 7%, n = 3, P < 0.01 for 100 nM; 45 ± 5%, n = 3, P < 0.01 for 10 µM). The levels of SM then reached a plateau and remained at that level for up to 15 min (100 nM) or 120 min (10 µM). The increased [14C]SM levels in the presence of 10 µM BK were always higher than those with 100 nM BK. Thus the [14C]SM levels increased in a time-dependent manner.


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Fig. 1.   Bradykinin (BK) increases sphingomyelin (SM) level. [Methyl-14C]choline chloride-labeled rabbit cortical collecting duct (RCCD) cells were stimulated with 10 µM BK for 30-120 min (A and B) or with 100 nM or 10 µM BK for 1-15 min (C). Lipids were extracted from cells, and labeled SM was separated from other 14C-containing lipids by thin-layer chromatography (TLC). A represents typical image, whereas B and C represent results obtained after scanning. The 2 bands represent 2 isoforms of SM. Results were obtained as volume per spot (vol/mg protein). Volume calculations include area and density of radioactivity-generated spots with phosphor imager. Data are expressed as means ± SE from 3 independent experiments. black-diamond , 10 µM BK; , 100 nM BK. * P < 0.01.

Time-dependent reduction in ceramide production by BK. To further determine whether BK inhibits SMase activity, ceramide production was measured (Fig. 2, A and B) by an indirect method, whereby ceramide generation was phosphorylated in the presence of sn-1,2-diacylglycerol kinase (DGK) and [gamma -32P]ATP to form [32P]ceramide phosphate. Low levels of ceramide were present in the unstimulated control cells. BK (100 nM) decreased the ceramide levels by 25 ± 3%, 48 ± 5%, and 52 ± 4% at 10, 20, and 30 min, respectively (n = 3, P < 0.02) (Fig. 2B). This decrease in ceramide started after 5 min and lasted up to 30 min. These changes reflect the time-dependent increase in [14C]SM levels that were obtained in Fig. 1. The exogenous standard ceramide and [32P]ceramide phosphate produced by exogenous SMase further confirmed that the [32P]ceramide phosphate induced by BK was coming from the SM pool (Fig. 2A).


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Fig. 2.   Time-dependent reduction in ceramide production by BK. RCCD cells were stimulated with 100 nM BK for 5, 10, 20, and 30 min, and lipids were extracted. Lipid samples were treated with reaction solution containing Escherichia coli diacylglycerol (DAG) kinase and [gamma -32P]ATP. Blank and exogenous standard ceramide tubes were similarly treated with reaction solution. As a separate control, cells were treated with exogenous sphingomyelinase (SMase; 0.1 U/ml) for 15 min and extracted lipids were treated with reaction solution. [32P]ceramide-phosphate was separated from other lipids by TLC. A: representative image of [32P]ceramide-phosphate shows blank; standard ceramide; control; 5, 10, 20, and 30 min of BK stimulation; and exogenous SMase stimulation. B: results obtained after scanning. The 2 bands represent 2 isoforms of ceramide. Results were obtained as volume per spot (vol/mg protein). Volume calculations include area and density of radioactivity-generated spots with phosphor imager. Data are expressed as means ± SE from 3 independent experiments. * P < 0.05; ** P < 0.02.

Concentration-dependent reduction in ceramide production by BK. To confirm whether BK inhibits SMase activity and to investigate the concentration response to BK, we directly measured ceramide in [14C]myristic acid-labeled RCCD cells after stimulation with different concentrations of BK. The data (Fig. 3, A and B) suggest that within 20 min, BK causes a concentration-dependent decrease in ceramide levels (39 ± 3%, 47 ± 2%, 55 ± 6%, 60 ± 4%, and 65 ± 5% for the concentrations of BK from 10-9 M to 10-5 M, respectively).


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Fig. 3.   Concentration-response curve for BK-induced reduction of ceramide levels. [14C]myristic acid-labeled RCCD cells were stimulated with BK (between 10-9 and 10-5 M) for 20 min. Lipids were extracted from cells and 14C-labeled ceramide was separated from other 14C-containing lipids by TLC. A: representative image of [14C]ceramide shows control and BK stimulation from 10-9 to 10-5 M. B: results obtained after scanning. The 2 bands represent 2 isoforms of ceramide. Results were obtained as volume per spot (vol/mg protein). Volume calculations include area and density of radioactivity-generated spots with phosphor imager. Data are expressed as means ± SE from 3 independent experiments. * P < 0.01.

BK-B2 receptor mediates effect of BK on SMase. It is known that BK signals through two receptor subtypes, BK-B1 and BK-B2 (38, 49). To determine which receptor subtype is responsible for ceramide signal transduction, we stimulated [14C]myristic acid-labeled RCCD cells with BK (100 nM) for 20 min with or without BK-B1 receptor antagonist or BK-B2 receptor antagonist; [14C]ceramide production was directly measured by TLC using the EAT solvent. Figure 4 demonstrates that BK decreased ceramide levels by 52% and that neither of the receptor antagonists have any significant effect by themselves (Delta 4 ± 0.2% and Delta 8.8 ± 0.3%, respectively) on ceramide levels. The presence of the BK-B1 receptor antagonist did not affect the decrease in ceramide levels induced by BK (Delta 56.7 ± 3%). In contrast, the BK-B2 receptor antagonist completely inhibited the effect of BK (Delta 9.9 ± 0.2%), indicating that BK-B2 receptor mediated the effect of BK on ceramide production.


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Fig. 4.   BK-B2 receptor mediates effect of BK on SMase. [14C]myristic acid-labeled RCCD cells were stimulated with BK (100 nM) for 20 min with or without the BK-B1 receptor antagonist [Lys-des-Arg9,Leu8]BK (10 µM) or the BK-B2 receptor antagonist Hoe-140 (10 µM). Lipids were extracted from cells and 14C-labeled ceramide was separated from other 14C-containing lipids by TLC. A: representative image of [14C]ceramide shows control, BK, BK-B1 receptor antagonist, BK + BK-B1, BK-B2 receptor antagonist, and BK + BK-B2. B: results obtained after scanning. The 2 bands represent 2 isoforms of ceramide. Results were obtained as volume per spot (vol/mg protein). Volume calculations include area and density of radioactivity-generated spots with phosphor imager. Data are expressed as means ± SE from 3 independent experiments. * P < 0.01.

Effect of BK and exogenous SMase on PLD activity. PLD activity was measured using a transphosphatidylation reaction unique to the enzyme, in which the phospholipid headgroup is exchanged for ethanol, producing PEt in preference to PA (47). We have determined the effect of BK, with or without exogenous SMase, on PLD activity (Fig. 5, A and B). In the presence of 1% ethanol, BK (100 nM) stimulation of [14C]myristate-labeled cells resulted in a level of [14C]PEt of 186 ± 57% (n = 4, P < 0.05) above the control levels at 10 min. Also, SMase (0.1 U/ml) increased [14C]PEt production by 45 ± 8% above the control levels (n = 4, P < 0.05). There was a slight increase in [14C]PEt formation (226 ± 32% above controls) when both BK and SMase were present, but this difference was not statistically significant (NS; n = 4), suggesting that exogenous SMase did not enhance BK stimulation of [14C]PEt formation.


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Fig. 5.   Effect of BK and exogenous SMase on phospholipase D (PLD) activity. In presence of 1% ethanol, [14C]myristic acid-labeled RCCD cells were stimulated with BK (100 nM) for 10 min with or without Staphylococcus aureus SMase (0.1 U/ml) for 15 min. PLD activity was measured using a transphosphatidylation reaction unique to the enzyme, in which phospholipid headgroup is exchanged for ethanol, producing phosphatidylethanol (PEt) in preference to phosphatidate (PA). 14C-labeled PEt was extracted from cells and separated from other 14C-containing lipids on TLC plates. A: representative image of [14C]PEt shows control, BK, exogenous SMase, and BK + exogenous SMase. B: results obtained after scanning. Results were obtained as volume per spot (vol/mg protein). Volume calculations include area and density of radioactivity-generated spots with phosphor imager. Data are expressed as means ± SE from 4 independent experiments. * P < 0.05.

Effect of BK on endogenous ceramide induced by exogenous SMase. The effect of BK on ceramide induced by exogenous SMase is shown in Fig. 6, A and B. Exogenous S. aureus SMase (0.1 U/ml) catalyses the hydrolysis of endogenous SM to form ceramide. The [14C]ceramide, which was produced in the presence of exogenous SMase, was significantly higher than the levels present in untreated controls or in BK-treated cells (668 ± 47% over the control level, n = 4, P < 0.01). However, BK (100 nM) did not inhibit the [14C]ceramide formation (BK + SMase, 699 ± 65% over the control levels) that was induced by exogenous SMase (n = 4, P = NS). Also, D609, a PLC antagonist specific for PC-PLC (3, 21), did not block the increase in [14C]ceramide (666 ± 72% over the control levels) induced by exogenous SMase (n = 4, P = NS). These results suggested that BK did not influence the formation of endogenous ceramide induced by exogenous SMase, and that PC-PLC was not involved.


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Fig. 6.   Effect of BK on endogenous ceramide induced by exogenous SMase. [14C]myristic acid-labeled RCCD cells were stimulated with BK (100 nM) for 10 min with or without S. aureus SMase (0.1 U/ml) for 15 min, or with SMase ± D609 (10 µM). Lipids were extracted from cells and 14C-labeled ceramide was separated from other 14C-containing lipids on TLC plates. A: representative image of [14C]ceramide shows control, BK, exogenous SMase, exogenous SMase + D609, and BK + exogenous SMase. B: results obtained after scanning. The 2 bands represent 2 isoforms of ceramide. Results were obtained as volume per spot (vol/mg protein). Volume calculations include area and density of radioactivity-generated spots with phosphor imager. Data are expressed as means ± SE from 4 independent experiments. * P < 0.05; ** P < 0.01.

BK-mediated interaction between endogenous or exogenous ceramide and PLD. To determine whether there is cross talk between the ceramide and PLD pathways, we investigated whether there was an interaction between endogenous or exogenous ceramide and BK-stimulated PLD. Cells were preincubated with 50 µM C2-ceramide for 1 h or with 0.1 U/ml exogenous SMase for 5 min. In the presence of 1% ethanol, cells were either left untreated or treated with 100 nM BK for 10 min. Exogenous C2-ceramide alone did not affect PLD activity and did not influence BK-stimulated [14C]PEt formation (Fig. 7, A and B). We were able to monitor the activity of PLD and SMase in the same sample. Prelabeling the cells with [14C]myristic acid labels both the SM pool and the PC pool. The [14C]ceramide produced by SMase, via hydrolysis of SM, and the [14C]PEt produced from PC, after stimulation of PLD, can be measured on the same TLC plate. Figure 8, A and B, demonstrates that at the same time, BK stimulates PLD activity to produce PEt and inhibits ceramide formation. Similarly, exogenous SMase-stimulated ceramide formation was accompanied by an increase in PLD activity. However, when both BK and exogenous SMase were present, there was no apparent additive effect on PLD activity or on ceramide production (Fig. 8, A and B). Figure 9 illustrates the distinct pathways by which the effects of BK on SMase and PLD are mediated.


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Fig. 7.   Effect of BK and exogenous ceramide on PLD activity. [14C]myristic acid-labeled RCCD cells were preincubated with C2-ceramide (50 µM) for 1 h, and then were stimulated with BK (100 nM) for 10 min in the presence of 1% ethanol. 14C-labeled PEt was extracted from cells and separated from other 14C-containing lipids by TLC. A: representative image of [14C]PEt shows control, BK, exogenous C2-ceramide, and BK + exogenous C2-ceramide. B: results obtained after scanning. Results were obtained as volume per spot (vol/mg protein). Volume calculations include area and density of radioactivity-generated spots with phosphor imager. Data are expressed as means ± SE from 4 independent experiments. * P < 0.05.


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Fig. 8.   Effect of BK and SMase on ceramide production and PLD activity. [14C]myristic acid-labeled RCCD cells were stimulated with BK (100 nM) for 10 min ± S. aureus SMase 0.1 U/ml for 15 min in presence of 1% ethanol. Lipids were extracted and both 14C-labeled ceramide and 14C-labeled PEt were separated by TLC and identified on same plate. A: representative image of [14C]ceramide and of [14C]PEt shows control, BK, exogenous SMase, exogenous SMase + D609, and BK + exogenous SMase. B: results obtained after scanning [14C]PEt and [14C]ceramide bands. The 2 bands for ceramide represent 2 isoforms of ceramide. Results were obtained as volume per spot (vol/mg protein). Volume calculations include area and density of radioactivity-generated spots with phosphor imager. Data are expressed as means ± SE from 4 independent experiments. Open bars, PEt; shaded bars, ceramide. * P < 0.05; ** P < 0.01..


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Fig. 9.   Effects of BK on SMase and on PLD are mediated by distinct pathways. BK inhibited SMase activity and induced PLD activity. Endogenous and exogenous ceramide did not influence PLD activity induced by BK. Cross talk between BK-inhibited SMase activity and BK-induced PLD activity was not observed. In RCCD cells, BK does not activate PLD via production of ceramide. Effect of BK on SMase-ceramide pathway is distinct from its effect on PLD-PA pathway. PC, phosphatidylcholine; solid line, positive response; dotted line, negative response.


    DISCUSSION
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REFERENCES

The interest in ceramide stems from the fact that it is involved in the control of cell differentiation, proliferation, and apoptosis (7, 26, 50). It also seems to be involved in many other cellular activities, making ceramide an important component of signal transduction pathways (3). Furthermore, ceramide is a precursor for ceramide-1-phosphate, sphingosine, and sphingosine-1-phosphate, which are now also recognized as important second messengers.

SMase action is the only known mechanism for SM hydrolysis in mammalian cells (36). However, most, if not all, mammalian cells appear capable of signaling through the SM pathway (36). Our results provide evidence that there is SMase activity in RCCD cells and that this enzyme is inhibited by BK. The possibility that BK exerts some of its effects through modulation of the SM-ceramide signaling pathway was provided by several reports that suggest that inflammatory cytokines such as TNF-alpha (26, 27) and the vasoconstrictor peptide endothelin-1 (ET-1) (5, 46, 48) act via the activation of SMase. Thus we considered the possibility that the proinflammatory and vasodilatory effects of BK could (10, 45) be mediated by the inhibition of SMase. The present study indicates that BK was able to decrease SM hydrolysis and ceramide production by inhibition of SMase activity in RCCD cells. This provides for the possibility that BK might operate, in part, by opposing the SMase-activating effect of other agents. Because the SM-ceramide pathway has been implicated in these events, it is possible that BK is involved in counteracting the ceramide-generating signals initiated by a variety of agents. This suggests that BK may play a role in regulating the overall balance of events such as vasoconstriction and vasodilatation.

Very few studies have investigated the effect of BK on ceramide signaling. Meacci et al. (28, 29) showed that BK is able to activate sphingolipid metabolism in human fibroblasts. BK provoked a rapid and significant increase in ceramide followed by a transient rise in sphingosine. However, BK neither decreased the SM pool nor activated neutral or acidic SMases, thus the possibility remains that glycosphingolipids might be the source of ceramide in human fibroblasts. Actually, ceramide may come from a variety of metabolic pathways such as de novo synthesis (16), dihydroceramide (17), glycerophospholipids (3), and sulfatides (13). However, SM is the main source of ceramide, and this is the pathway being most investigated at the present time. The different effects of BK on RCCD cells and human fibroblasts can probably be attributed to differences in cell or tissue specificity. Our data clearly suggest that the increase in SM levels occurred concomitantly with a decrease in ceramide after BK stimulation.

The measurement of ceramide is usually done using the [gamma -32P]ATP and DAG kinase method, as described by Preiss et al. (37) and modified by Wright et al. (49). However, this is an indirect method for the measurement of ceramide. It requires the phosphorylation of ceramide to [32P]ceramide-phosphate, which is separated and measured on TLC plates. This method requires a long time to complete and is complex and expensive. During the experiments, we have developed a new method for the direct measurement of ceramide production using [14C]myristic acid and a solvent of ethyl acetate/acetic acid/trimethylpentane for TLC development. With this method, we were able to separate [14C]ceramide from other [14C]myristate-containing neutral lipids. The external standard ceramide and endogenous ceramide produced by exogenous SMase were used to confirm the position of [14C]ceramide on the plates. Both methods gave the same results. BK induced time- and concentration-dependent increases in SM levels and a decrease in ceramide production. Although SMase activity was not measured directly, the results point convincingly to the inhibition of SMase by BK.

BK receptors are generally divided into two major subtypes, BK-B1 and BK-B2 (40, 52). BK-B1 mediates the rapid, acute response (smooth muscle contraction or relaxation) as well as some effects occurring more slowly (e.g., collagen synthesis) (40). The BK-B2 receptor is responsible for most of the biological effects of the kinins, including arterial vasodilatation, plasma extravasation, venoconstriction, activation of sensory fibers (e.g., fibers for pain), and stimulation of the release of prostaglandins, endothelium-dependent relaxing factor (from endothelia), norepinephrine (from nerve terminals and adrenals), and other endogenous agents (25, 40, 41). It was reported that in rat medullary collecting duct, the BK-B2 antagonist Hoe-140 increased chloride and water absorption (32), suggesting that BK-B2 is the constitutive subtype in the kidney collecting duct. To determine which BK-receptor subtype affects ceramide signal transduction, we stimulated [14C]myristic acid-labeled RCCD cells with BK in the presence of BK receptor B1 or B2 antagonists. The BK-B1 antagonist [Lys-des-Arg9,Leu8]BK had no direct effect on ceramide production and did not affect the inhibition of ceramide production induced by BK. In contrast, although the BK-B2 antagonist Hoe-140 did not affect ceramide levels by itself, it completely blocked the effect of BK. These results suggest that the BK receptor B2, but not B1, mediates BK-induced inhibition of ceramide signal transduction.

At present there are no specific inhibitors for SMase activity, but exogenous SMase (6, 51) and short-chain ceramides such as C2-ceramide (22) are widely used as tools to investigate ceramide signal transduction. Exogenous SMase is extensively used in the literature to mimic the effect of ceramide generating signals (6, 11, 51). It is not clear how exogenous SMase functions to generate an intercellular signal. Presumably, at least some of the ceramide that is generated on the cell surface enters into the cell. Thus we used both exogenous SMase and C2-ceramide in our experiments. Exogenous SMase induced the production of very high levels of intracellular ceramide. That exogenously added SMase is very effective in hydrolysing endogenous SM has been widely reported in the literature (6, 51). BK did not decrease the amount of ceramide that was elicited by exogenous SMase. This is not unexpected because the concentration of exogenous SMase was very high. These studies using exogenous SMase served mainly as control to generate high levels of ceramide and to study the effect of ceramide on PLD (next series of experiments). We have previously demonstrated that BK activates PC-PLC in Madin-Darby canine kidney cells (21). The DAG that is produced due to action of PC-PLC has been reported to be an activator of SMase (42). Thus we took advantage of the fact that D609 is a potent inhibitor of PC-PLC (42). Our results showed that D609 did not have any effect on the levels of ceramide elicited by exogenous SMase.

Our data shows that BK induced a dramatic activation of PLD. In the same samples, a significant decrease in ceramide levels was observed. In contrast, exogenous SMase, which causes the production of very high levels of ceramide, elicits only a very slight, albeit significant, activation of PLD. The presence of exogenous SMase did not have any effect on the BK-induced PLD activity. It is possible that exogenous SMase would have a direct effect on hydrolysis of PC to PEt. Because we observed production of very high levels of ceramide and activation of PLD simultaneously in the same samples, it is probable that the activation of PLD by exogenous SMase was mediated by ceramide. Whether ceramide activates PLD directly or indirectly is not clear, in light of the fact that sphingosine, the hydrolysis product of ceramide, is a potent activator of PLD (3, 29). The cell-permeable short chain C2 ceramide does not, by itself, activate PLD, and does not blunt the activation of PLD due to BK. These results would tend to support the possibility that exogenous SMase might be acting directly on PLD. However, much evidence indicates that short chain ceramides do not mimic all of the effects of endogenous ceramide (8, 35, 39). Thus it was not surprising that exogenous C2-ceramide did not affect PLD. Because exogenous C2-ceramide, which would also be hydrolysed to sphingosine, did not affect PLD, we suggest that it was ceramide and not sphingosine that directly activates PLD. Our results indicating ceramide is an activator of PLD are in agreement with those of Meacci (28, 29). Having obtained evidence that ceramide activates PLD, we would have expected that the decrease in ceramide levels induced by BK would lead to less activation of PLD. Because PLD is activated by ceramide we suggest that activation of PLD by BK is not mediated via the BK-B2 receptor/SMase/ceramide pathway but involves the activation of PLD by a different signaling pathway, possibly mediated by a different receptor subtype. It is known that, in a number of different cell types, BK activates PLD through G-protein coupled receptors, an effect mediated in part by PKC (21, 28, 29). That BK can activate PLD in kidney cells has been previously shown by us (21) and others (28, 29), however, the relationship between BK's effect on ceramide signaling and PLD activity had not been studied previously. The involvement of ceramide in the regulation of PLD activity is controversial. Ceramide has been previously described as a negative modulator of PLD activity (3). Gomez-Munoz et al. (15) showed that C2- and C6-ceramides blocked the activation of PLD by sphingosine 1-phosphate in rat fibroblasts. However, Meacci et al. (28, 29) suggested that ceramide is an activator of BK-mediated PLD activity. When BK and exogenous SMase were present at the same time, no synergistic or additive effects were observed. The activation of PLD was generally higher than that obtained with BK alone, but this increase was not statistically significant.

In the present study, we have demonstrated that exogenous SMase, whose only known activity is to hydrolyse SM to ceramide and phosphocholine, activated PLD. We have also shown that BK inhibits SMase and activates PLD in RCCD cells. These two observation strongly suggest two separate signaling pathways. The inhibition of SMase was mediated by the BK-B2 receptor subtype. Although both BK and ceramide activate PLD, no synergism or additive effect was noted between the two PLD activators. We speculate that BK function, in part, by counteracting the effects of SMase-activating pathways.


    ACKNOWLEDGEMENTS

This research was supported by the Kidney Foundation of Canada and Medical Research Council of Canada (MT-14103). The BK-B1 and BK-2 receptor antagonists were a generous gift from Dr. Domenico Regoli (Department of Pharmacology, University of Sherbrooke).


    FOOTNOTES

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: R. L. Hébert, Dept. of Cellular and Molecular Medicine, Univ. of Ottawa, 451 Smyth Road, Ottawa, Ontario, K1H 8M5, Canada (E-mail: rhebert{at}uottawa.ca).

Received 30 June 1998; accepted in final form 23 December 1998.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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