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Am J Physiol Heart Circ Physiol 282: H175-H183, 2002. First published September 27, 2001; doi:10.1152/ajpheart.00379.2001
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Vol. 282, Issue 1, H175-H183, January 2002

Bradykinin stimulates ceramide production by activating specific BK-B1 receptor in rat small artery

Leonard Kleine1,*, Gele Liu3,*, Normand Leblanc2, and Richard L. Hébert3

1 Departments of Biochemistry, Microbiology, and Immunology, and 2 Department of Physiology, Montreal Heart Institute, University of Montreal, Montreal, Quebec H3C 3J7; and 3 Department of Cellular and Molecular Medicine, Kidney Research Centre, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Bradykinin (BK), a proinflammatory factor and vasodilator, causes functional change of the small artery. However, it is not clear whether any of these changes induced by BK are mediated by N-acetyl-D-sphingosine (ceramide). Therefore, we investigated whether BK affects the hydrolysis of sphingomyelin and generation of ceramide in the intact rat small artery. Our results suggest that BK induces sphingomyelin hydrolysis and increases ceramide production in a time- and dose-dependent manner. Relative to controls, BK causes a 50% decrease in sphingomyelin levels. Ceramide levels increase in response to BK with the highest level being obtained with 10-8 M BK as well as similar amounts of ceramide are generated when exogenous sphingomyelinase (SMase) is added. We then determined which of the two BK receptors (BK-B1 antagonist Lys-Des-Arg9-Leu8-BK or the BK-B2 antagonist HOE-140) are implicated in the BK-induced generation of ceramide. The BK-B2 antagonist did not alter the effect of BK on ceramide generation, whereas the BK-B1 antagonist blocked the BK-induced production of ceramide. Although ceramide had no effect on KCl-induced constrictions, ceramide dilated preconstricted (phenylephrine) small pressurized rat mesenteric arteries by ~40%. These results suggest that the activation of the BK-B1 receptor mediates the BK-induced activation of SMase and of the production of ceramide. In conclusion, BK-mediated effects on vascular tone may be due, at least in part, to the increased production of ceramide.

phenylephrine; receptor antagonists; sphingomyelin; vascular tone


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE FIELD OF SPHINGOLIPID and N-acetyl-D-sphingosine (ceramide) signal transduction has recently advanced rapidly. Experimental results (18, 19) suggest that ceramide has important roles in many cell types. Sphingomyelin (SM) is composed of long chain saturated or monounsaturated fatty acid, a sphingosine base backbone, and a phosphocholine head group (3, 5, 37). Hydrolysis and synthesis of the sphingolipids that form the SM pool together form a dynamic balanced pathway called the "sphingomyelin pathway." SM turnover involves removal of the head group and amide-linked fatty acid by sphingomyelinases (SMases) and ceramidases, respectively. SMase action is the only known mechanism for SM hydrolysis in mammalian cells. Most, if not all, mammalian cells appear capable of signaling through the SM pathway (29). SM that is present either on the outer or on the inner layer of the plasma membrane can be used to generate ceramide that is involved in signal transduction pathways (11). Exogenous SMases are mainly of bacterial origin and have become important tools for investigating ceramide signal transduction, in particular because few antagonists for endogenous SMases are available. In general, exogenous SMase mimics the effect of ceramide-generating signals and is effective in hydrolyzing endogenous SM (6, 8, 41). It has access to the bulk of cellular SM, which is located on the external portion of the lipid bilayer (36).

Ceramide has been proposed as a novel second messenger that mediates a variety of biological processes (18, 19). Specific stimuli induce SMase activity, which results in SM hydrolysis and ceramide production. Ceramide may activate or inhibit a variety of signals, for example: protein kinases, phosphatases, or phospholipases, which affect multiple downstream targets, including activation of nuclear transcription factors, leading to a variety of distinct cellular effects (19). A large variety of stimuli induce SM hydrolysis and ceramide formation (18, 19). There is evidence for extensive tissue specificity as indicated by the fact that for any given SMase activating stimulus, not all cells or tissues respond to that agent by hydrolyzing sphingomyelin; furthermore, different final effects can be obtained in different cell types in response to the same SMase activating agent (1, 12, 13).

As a novel lipid second messenger, ceramide derived from sphingolipids plays a central role in signal transduction and cell regulation. We (18) have previously shown that the effects of BK, a proinflammatory factor and vasodilator, were mediated by ceramide and by phospholipase D in cultured rabbit cortical collecting duct (RCCD) cells. In RCCD cells, BK increased SM levels and decreased ceramide levels. The BK-B1 receptor antagonist did not affect ceramide but the BK-B2 antagonist blocked the effect of BK on SMase, suggesting that the BK-B2 receptor mediates BK-induced inhibition of ceramide generation in RCCD cells. There is evidence in smooth muscle cells that sphingolipids are involved in regulating cell proliferation. For instance, in mesangial cells and A7R5 smooth muscle cells, platelet-derived growth factor increases sphingosine levels (19) by stimulating SMase and ceramidase activities (7). We do not have much information with regard to contractile responses. Cell-permeable ceramides and SMase treatment caused relaxation of phenylephrine (PE)-contracted endothelium-denuded rat thoracic aortic rings (16). In contrast, cell-permeable ceramide induced contraction of isolated rabbit rectosigmoid smooth muscle cells (35). However, in vascular smooth muscle, one effect of ceramide that may have particular relevance for smooth muscle contraction is the activation of the serine-threonine kinase Raf (15, 42), an upstream regulator of extracellular signal-regulated kinase (ERK) (34). Sphingosine induced vasoconstriction in coronary arteries, an effect that appeared to be mediated by the release of cyclooxygenase-sensitive vasoconstrictor prostanoids from the endothelium (24). Thus the SM pathway may be an important regulator of vascular function.

However, most studies to date have examined the role of ceramide in the context of cytokine signaling from cells in culture, whereas few studies have been done using intact tissues. No studies have been performed exploring the potential role of BK with regard to ceramide and SMase signaling by using intact tissues. Of interest, it is not known whether the effects of BK involve ceramide and SMase signaling nor whether the BK-induced increase in ceramide production may influence vascular tone in the intact pressurized rat mesenteric small artery. Therefore, we studied the effect of BK on ceramide and SMase in rat small artery.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Lipid Measurements

BK, Staphylococcus aureus, SMase, and all other standards, drugs, and salts were purchased from Sigma (St. Louis, MO). Medium 199 (M-199) was obtained from GIBCO-BRL. [Methyl-14C]choline chloride (specific activity 55.0 mCi/mmol) was 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. All solvents, acids used for lipid extraction such as chloroform and methanol, and high-perfomance liquid chromatography grade solvents were supplied by BDH.

Animals and Tissue Preparation

Adult male Sprague-Dawley rats, body wt ~225-275 g, were anesthetized using Pentothal Sodium (Somnotol, MTC Pharmaceuticals). The mesentery was immediately excised and kept in ice-cold physiological salt solution composed of (in mM) 119 NaCl, 4.7 KCl, 25 NaHCO3, 1.17 MgSO4 · 7 H2O, 1.18 KH2PO4, 0.026 K2 EDTA, 5.5 glucose, and 2.5 CaCl2 · 2H2O, pH 7.2, until dissection. Adjoining fat and connective tissues were removed from the vessels with the use of a dissection microscope. Rat mesenteric arteries of ~280-300 µm internal diameter were used for all of the experiments. A collection of five small arteries, each of which was ~2 cm long, was used for each point; each sample contains ~40 µg of protein.

Radiolabeling of small arteries. An appropriate aliquot of radioactive material was dried down under a stream of nitrogen, placed in 200 ml of M-199 solution and redissolved by bath sonication for 5 min. Vessels were incubated in M-199 solution for 3 h at 37°C, washed in 3 ml of M-199 for 15 min, and then transferred to 3 ml of fresh M-199 solution.

Solvent extraction of small artery lipids. The lipid extraction method was based on that of Bligh and Dyer (4) as described by Liu et al. (18). After BK or SMase stimulation, the reaction was terminated by transferring the vessels to an ice-cold ground glass homogenizer containing 0.5 ml chloroform-methanol-HCl (20:40:1, vol/vol/vol), homogenized, and left on ice for 30 min. The mixture was transferred to a microcentrifuge tube and the homogenizer was rinsed with 1 ml of chloroform. These washings were added to the tube along with 0.3 ml 1 M NaCl, vortexed, and spun at 11,000 g for 5 min at 4°C. The upper aqueous layer was discarded, and the lower layer, containing the lipids, was transferred to a 1-ml glass Chrompack vial, dried under a stream of N2, and redissolved in 200 µl of chloroform. 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 the method of Lowry et al. (20).

Measurement of sphingomyelin hydrolysis. Because choline is a component of sphingolipids (10), small arteries were prelabeled with 1 µCi/ml [methyl-14C]choline chloride in M-199 solution for 3 h at 37°C to label the sphingomyelin pool. Small arteries were then stimulated with or without BK (10-8, 10-7, and 10-6 M) for 30 min. Lipids were extracted and internal standards were added to each sample: 20 µl (10 mg/ml in chloroform) of SM + phosphatidylcholine. The TLC plates were preheated to 80°C for 30 min. The samples (25 µl in chloroform) were spotted onto the plates along with external standards: SM + PC (20 µl of 10 mg/ml in chloroform). The plates were developed in chloroform-methanol-acetic acid-water (50:30:8:5, vol/vol/vol/vol), dried, and stained with I2 to mark the areas corresponding to SM and phosphorylcholine.

Measurement of [14C]ceramide production. To investigate the time and concentration-response of ceramide production to BK, [14C]myristic acid-labeled small arteries were stimulated with BK for 1, 5, and 30 min with 10-10-10-6 M BK. Lipids were extracted from small arteries and the 14C-labeled ceramide was separated from other 14C-containing lipids by TLC using the ethyl acetate-acetic acid-trimethylpentane solvent (9:2:5 vol/vol/vol). To determine which BK receptor subtype mediates BK-induced ceramide production, [14C]myristic acid-labeled small arteries were stimulated with BK (10-8 M) for 30 min with or without the BK-B1 receptor antagonist (Lys-Des-Arg9-Leu8-BK, 1 µM) or the BK-B2 receptor antagonist (HOE-140, 1 µM). Lipids were then extracted from arteries, and the 14C-labeled ceramide was separated from other 14C-containing lipids on TLC plates. These experiments were repeated three times.

Measurement of radioactivity. [14C]SM and [14C]ceramide were measured using the Molecular Dynamics System with ImageQuaNT computer software. The developed TLC plates were exposed to phosphor screens for 72 h in exposure cassettes and were scanned with the phosphorimager. Results, obtained as volume per spot, are expressed as volume per milligram of protein. Volume calculations include the area and density of the radioactivity-generated spots. Results are expressed as a percentage of control.

Statistics

To determine the statistical significance of differences between more than two groups, analysis of variance and Bonferroni multiple comparison tests were used (18). Differences of P < 0.05 were considered statistically significant. Results are presented as means ± SE of 3-4 independent experiments.

Functional Assays

Tissue preparation. Functional assays on intact pressurized rat mesenteric arteries were carried out as previously described (31) except that the endothelium was not removed. In brief, third-branch small arteries of ~7.5 mm in length (mean lumen diameter in the absence of transmural pressure was 158 ± 7 µm, n = 13) were carefully dissected from the vascular bed, mounted on borosilicate cannulas in a perfusion chamber, and attached using silk suture threads. Criteria for viability and suitability of the artery included constriction to boluses of 1 M KCl and 10 mM PE, respectively, followed by evidence for a significant endothelium-dependent dilation by exposing the preparation to a bolus of 1 mM acetylcholine, and no pressure leak between the proximal and distal ends of the vessel. Suitable arteries were then allowed to equilibrate at 60 mmHg for 30-60 min at 36 ± 0.5°C before the experimental protocols were initiated.

Diameter measurements. An image of the arteriole was projected onto a video monitor via a microscope-mounted charge-coupled device camera (model KP-113, Hitachi) at a final magnification of ×4. Lumen diameter of the vessel was monitored continuously by edge detection. Pressure transducers were attached at both the proximal and distal ends of the artery. Throughout all experiments, mesenteric vessels were pressurized at 60 mmHg. This pressure was monitored online from the proximal end of the vessel with the use of a pressure monitor (model PM-4, Living Systems Instrumentation; Burlington, VT). Video (diameter) and pressure were recorded simultaneously on a conventional videotape recorder (Vetter; Rebersburg, PA) and on a Pentium-based personal computer using Axoscope software (version 8, Axon Instruments; Foster City, CA) at a sampling rate of 20 Hz/channel. Final analysis was performed using Axoscope and Origin software (version 4.1, Microcal Software; Northampton, MA).

Solutions. The solution used to dissect the arteries contained (in mM) 120 NaCl, 25 NaHCO3, 4.2 KCl, 0.6 KH2PO4, 1.2 MgCl2, 11 glucose, and 1.8 CaCl2. The normal bathing (control) solution was identical to the dissecting solution. Passive diameter changes to pressure were obtained by exposing the arteriole to a Ca2+-free solution of similar composition to the dissecting solution except for the omission of CaCl2 and addition of 2 mM EGTA. The high-K+ (45 mM) solution was obtained by equiosmolar replacement of NaCl by KCl in the control solution. Test solutions were prepared by adding agents to the final concentrations into the control solution. PE and acetylcholine were prepared as 10 mM and 1 mM stock solutions, respectively, in control solutions. A stock solution of ceramide was prepared in dimethyl sulfoxide (DMSO) at concentrations of 50 mM; the final concentration of DMSO never exceeded 0.1%, which by itself produced no effect on PE-induced tone. All drugs were obtained from Sigma. Solutions were bubbled throughout with 5% CO2-12% O2-83% N2 (pH 7.4).

Equation used and statistical analysis. Percent relaxation at 60 mmHg (%dilation) due to ceramide (Fig. 1B) was calculated using the following equation
%Relaxation<IT>={</IT>[Diam<SUB>(ceramide)</SUB><IT>−</IT>Diam<SUB>(PE)</SUB>]

<IT>&cjs0823;  </IT>[Diam<SUB>(0Ca<IT>−</IT>EGTA)</SUB><IT>−</IT>Diam<SUB>(PE)</SUB>]<IT>}×</IT>100
where Diam(ceramide), Diam(PE), and Diam(0Ca-EGTA) are the diameters measured in the presence of 50 µM ceramide + 1 µM PE, PE alone, or Ca2+-free EGTA (passive dilation) solutions, respectively. Results are presented as means ± SE of three or four independent experiments, with n referring to the number of experiments. With the use of Statistica for Windows 98 (version 5.5), statistical significance between individual means was determined using an unpaired Student's t-test or a one-way analysis of variance test with Tukey's honestly significant difference post hoc multiple-range test for repeated measure. P < 0.05 was considered to be statistically significant.


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  		Fig. 1.   Bradykinin (BK) induces sphingomyelin (SM) hydrolysis. [14C]choline chloride-labeled small arteries were stimulated with BK at 10-8-10-6 M for 30 min. Lipids were extracted from arteries, and the labeled SM was separated from other 14C-containing lipids by thin-layer chromatography (TLC). A: representative image. B: results obtained after scanning. The two bands represent two isoforms of SM. Data are means ± SE from 3 independent experiments. *P < 0.01.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

BK Stimulates Sphingomyelin Hydrolysis

SM is hydrolyzed by SMase to produce ceramide and phosphorylcholine. The arteries were prelabeled with [14C]choline chloride; thus a decrease in the radioactivity associated with sphingomyelin represents hydrolysis of sphingomyelin by SMase. To determine the SMase response to BK, arteries were stimulated with 10-8-10-6 M BK for 30 min (Fig. 1). At all of these concentrations of BK, [14C]SM levels decrease by ~50%, which is significantly different from the control levels (P < 0.01, n = 3). Higher concentrations of BK did not cause a further decrease in the levels of SM.

Concentration-Dependent Increase in Ceramide Production by BK

To confirm that BK activates SMase and to investigate the concentration response of ceramide production to BK, rat small arteries were labeled with [14C]myristic acid, and the [14C]ceramide produced was measured after stimulation of rat small arteries with different concentrations of BK (10-10-10-6 M for 30 min). The data in Fig. 2 demonstrate a concentration-dependent increment in ceramide as BK is increased from 10-10 M (n = 3, P < 0.05) to 10-8 M (n = 3, P < 0.01). At 10-8 M BK, the production of [14C]ceramide reached a level that is threefold higher than control levels. The production of ceramide then reached a plateau and remained at that level as the concentration of BK was increased to 10-6 M. 


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  		Fig. 2.   BK increases ceramide production in a concentration-response fashion. [14C]myristic acid-labeled small arteries were stimulated with BK at 10-10-10-6 M for 30 min. Lipids were extracted from arteries and the 14C-labeled ceramide was separated from other 14C-containing lipids with the use of TLC. A: representative image of [14C]ceramide. B: results obtained after scanning. Data are expressed as means ± SE from 3 independent experiments. *P < 0.05, **P < 0.03, and ***P < 0.01.

Time-Dependent Increase of Ceramide Production Induced by BK

To further determine whether BK increases SMase activity, ceramide production was measured at different times after treatment. Small arteries labeled with [14C]myristic acid were then treated with 10-8 M BK for various times. Figure 3 shows that ceramide production increased gradually with time, reaching a threefold increase at 30 min (n = 3, P < 0.01). No further increase was observed if the treatment was continued past 30 min. The exogenous standard ceramide and [32P]ceramide phosphate produced by exogenous SMase confirmed that the [32P]ceramide phosphate induced by BK was coming from the SM pool.


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  		Fig. 3.   Time-dependent increase in ceramide production induced by BK. [14C]myristic acid-labeled small arteries were stimulated with BK for 1, 5, and 30 min at 10-8 M. Lipids were extracted from arteries, and the 14C-labeled ceramide was separated from other 14C-containing lipids by TLC. A: representative image of [14C]ceramide. B: results obtained after scanning. Data are means ± SE from 3 independent experiments. *P < 0.05; **P < 0.01.

Exogenous SMase Induces Ceramide Production

Exogenous SMase is generally used as a tool to study ceramide signal transduction and is often used as a positive control to demonstrate the hydrolysis of SM. Exogenous S. aureus SMase (0.1 U/ml) catalyses the hydrolysis of endogenous SM to form ceramide. The effect of exogenous SMase or BK on ceramide production is shown in Fig. 4. Either exogenous SMase or BK (10-8 M) catalyzed the hydrolysis of endogenous SM to form ceramide. The [14C]ceramide production induced by exogenous SMase or BK was significantly higher (more than twofold) than the levels in the untreated controls (P < 0.01, n = 3).


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  		Fig. 4.   Effect of BK or exogenous sphingomyelinase (SMase) on endogenous ceramide. [14C]myristic acid-labeled small arteries were stimulated with BK (10-8 M) or S. aureus SMase (0.1 U/ml) for 30 min. Lipids were extracted from arteries, and the 14C-labeled ceramide was separated from other 14C-containing lipids on TLC plates. A: representative image of [14C]ceramide. B: results obtained after scanning. Data are means ± SE from 3 independent experiments. *P < 0.01.

BK-B1 Receptor Mediates Effect of BK on SMase

It is known that BK signals through two receptor subtypes, BK-B1 or BK-B2 (38, 49). To determine which receptor subtype is responsible for ceramide signal transduction, [14C]myristic acid-labeled rat small arteries were stimulated with 10-8 M BK for 30 min ±10-6 M BK-B1 receptor antagonist or ±10-6 M BK-B2 receptor antagonist. Figure 5 demonstrates that BK increased ceramide levels and the presence of the BK-B2 receptor antagonist did not block the increase in ceramide levels induced by BK. In contrast, in Fig. 6, the BK-B1 receptor antagonist completely inhibited the effect of BK on ceramide production, indicating that the BK-B1 receptor subtype mediated the effect of BK on ceramide production in the rat small artery.


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  		Fig. 5.   BK-B2 receptor does not mediate the effect of BK on ceramide production. [14C]myristic acid-labeled small arteries were preincubated with BK-B2 receptor antagonist (HOE-140, 10-6 M) for 30 min and then were stimulated with BK (10-8 M for 30 min). Lipids were extracted from cells, and 14C-labeled ceramide was separated from other 14C-containing lipids by the use of TLC. A: representative image of [14C]ceramide for control, BK (10-8 M), BK-B2 antagonist (10-6 M), and BK + BK-B2 antagonist (10-8 + 10-6 M). B: results obtained after scanning. Data are means ± SE from 3 independent experiments. *P < 0.01.



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  		Fig. 6.   BK-B1 receptor mediates the effect of BK on ceramide production. [14C]myristic acid-labeled small arteries were preincubated with BK-B1 receptor antagonist [Lys-des-Arg9, Leu8]-BK (10-6 M for 30 min) and then were stimulated with BK at 10-8 M for 30 min. Lipids were extracted from cells, and the 14C-labeled ceramide was separated from other 14C-containing lipids by TLC. A: representative image of [14C]ceramide for control, BK (10-8 M), BK-B1 antagonist (10-6 M), and BK + BK-B1 antagonist (10-8 + 10-6 M). B: results obtained after scanning. Data are means ± SE from 3 independent experiments. *P < 0.05; **P < 0.01.

Ceramide Effects on Vasomotor Tone

Functional experiments were performed to test the hypothesis that ceramide can influence vasomotor tone in rat mesenteric arteries of similar caliber to those used in biochemical experiments. Figure 7A illustrates a sample experiment where we tested the effects of ceramide on the lumen diameter of a preconstricted mesenteric artery pressurized to 60 mmHg. In the absence of an agonist, lumen diameter was 412 µm. These small arteries exhibited very little (<10% on average) myogenic active tone (in the absence of an agonist), which can be measured by subtracting the lumen diameter versus pressure relationship obtained in normal medium from the one registered in Ca2+-free medium (passive diameter). As shown, application of 1 µM PE, a alpha 1-adrenergic receptor agonist, constricted the artery to a diameter of ~170 µm. Application of 50 µM ceramide in the continued presence of PE slowly dilated the vessel, which eventually reached a diameter ~270 µm after 62 min. Exposure of the preparation to a bolus of ACh (1 mM) led to a rapid transient vasorelaxation of which maximum level approached that measured in control and Ca2+-free conditions, suggesting that the endothelium was still functional in the presence of ceramide. Figure 7B shows mean data from two separate series of experiments. The graph plots the time course of changes in lumen diameter expressed as a percentage of dilation relative to the passive diameter (100% level) estimated in Ca2+-free EGTA medium and maximum constriction induced by 1 µM PE (0% level). Similar to Fig. 7A, 50 µM ceramide induced an ~35% dilation, which took ~10 min to reach a steady state (n = 6). The relaxation mediated by ceramide was not due to a desensitization of the alpha 1-adrenergic receptor because the magnitude of the PE-induced constriction remained stable for >60 min as determined in parallel time control experiments carried out in the absence of ceramide (n = 3, open circles).


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  		Fig. 7.   Effects of ceramide on phenylephrine (PE)- and high-K+-mediated tone in intact pressurized rat mesenteric small arteries. A: sample experiment displaying the changes in lumen diameter of a rat pressurized (60 mmHg) mesenteric artery exposed to the alpha 1-agonist PE and then to ceramide in the presence of PE, as indicated by the thick bars. A bolus of 1 mM acetylcholine (ACh) was applied to the preparation, which led to a potent and persistent vasorelaxation. At the end, all drugs were washed out and the artery was superfused with a Ca2+-free medium containing 2 mM EGTA to determine the passive diameter of this vessel. B: mean time course of changes in the vasoactive properties of intact pressurized (60 mmHg) mesenteric arteries exposed to PE alone (open circle , n = 3) or PE and ceramide (, n = 6), as indicated by the thick bars. Data were expressed as %dilation relative to the maximum constriction induced by PE (0%) and the fully dilated level obtained in Ca2+-free medium (100%). *P < 0.05, significant difference between PE alone and PE + ceramide at identical exposure times. C: bar graph showing the effect of ceramide on the constriction mediated by elevated extracellular K+ concentration ([K+]o). All preparations were pressurized to 60 mmHg. Elevation of [K+]o from 5.4 to 45 mM (15 min) significantly reduced lumen diameter, which was not significantly influenced by a 60-min exposure to 50 µM ceramide (n = 4). dagger P < 0.01, significant difference between groups.

We also examined the possible effects of 50 µM ceramide on high-K+-induced constriction. As shown in Fig. 7C, increasing the concentration of K+ in the bathing medium from 5.4 to 45 mM, 15-min exposure, reduced lumen diameter by 57% on average. A 60-min exposure to ceramide produced no effect on the K+-induced constriction (n = 4). Our data indicate that while ceramide had no influence on the KCl-induced constriction, this compound partially dilated vessels, which were preconstricted with the alpha 1-adrenoceptor agonist PE. These results suggest that BK-induced increased ceramide production also influence vascular tone in resistance arteries.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Many physiological functions have been ascribed to BK, including the promotion of contraction and relaxation of vascular and nonvascular smooth muscles, renal hemodynamics, cell proliferation, inflammation, edema, and pain (9, 10, 22, 28). BK has been shown to induce relaxation of many types of arteries, for example, in coronary arteries (2, 14, 30, 32, 33), pancreaticoduodenal artery (28), and rat mesenteric arteries (3, 5, 37). Contraction has also been observed, for example, in rat tail arteries (45) and in human umbilical and rabbit jugular veins (19, 30); this contraction is sometimes transient and followed and/or preceded by relaxation (30, 32, 33).

Few reports (18, 19, 21) have investigated the effect of BK on ceramide signaling. In human fibroblasts, BK activated sphingolipid metabolism and resulted in a rapid rise in ceramide. We also recently reported (18) that BK acts in part by modifying sphingolipid signal transduction. In contrast to the results obtained with human fibroblasts, BK inhibited the production of ceramide in RCCD cells. We speculated that BK could possibly be involved in counteracting the effects of agents that cause the generation of ceramide and thus have a regulating role in vasoconstriction and vasodilatation. The present study indicates that BK stimulates the rapid hydrolysis of sphingomyelin in rat mesenteric small arteries. The amount of 14C-labeled sphingomyelin obtained from arteries prelabeled with [methyl-14C]choline chloride decreases rapidly after treatment with BK. The effect is significant within 5 min of treatment and is maximal at 30 min. As expected, the decrease in sphingomyelin is accompanied quite closely by an increase in ceramide. The generation of ceramide, as measured by the generation of 14C-labeled ceramide produced after stimulation of [14C]myristic acid-labeled cells with BK, is concentration dependent with the maximal amount being produced in response to 10-8 M BK. These results resemble those obtained in human fibroblasts in response to BK treatment but differ from the effect seen in RCCD cells (18). These results can probably be attributed to differences in cell specificity. We used exogenous SMase as a positive control to demonstrate the generation of ceramide. This treatment resulted in a rapid generation of ceramide. Exogenous SMase is widely used as a tool to investigate ceramide signal transduction. It is used to mimic the effect of ceramide generating signals (18, 19). Although it is not clear how exogenous SMase functions to generate an intracellular signal, some of the ceramide, which is generated on the cell surface, enters into the cell. That exogenously added SMase is very effective in hydrolyzing endogenous SM has been clearly demonstrated (18, 19, 27). Our results show that 10-8 M BK generate a ceramide signal comparable with that of 0.1 U/ml exogenous SMase.

BK receptors are generally divided into two major subtypes, BK-B1 and BK-B2 (18, 30, 32, 33). BK acts primarily by binding to the BK-B2 receptor, which is responsible for most of the following effects of BK: arterial vasodilatation, plasma extravasation, venoconstriction, activation of sensory fibers (pain), release of prostaglandins, and relaxing factors from the endothelium (19, 30, 32, 33). BK binds to the BK-B1 receptor; however, the receptor has a much higher affinity for the metabolite des-[Arg9]-BK. To determine which BK receptor subtype affects ceramide signal transduction, [14C]myristic acid-labeled small arteries were stimulated with BK in the presence of BK receptor B1 or B2 antagonists. The BK-B2 antagonist HOE-140 did not block the effect of BK on ceramide generation. In contrast, the BK-B1 antagonist Lys-Des-Arg9-Leu8-BK, which had no effect of background levels of ceramide, blocked very efficiently the generation of ceramide induced by BK. These results suggest that the BK-B1 receptor, and not the BK-B2 receptor, mediates the ceramide-generating effect of BK.

These results are in contrast to RCCD cells where we demonstrated that the BK-B2 receptor mediates the effect of BK on the inhibition of ceramide generation. In general, it is the BK-B2 receptor that is responsible for the contraction-inducing effect of BK on vessels such as the human umbilical vein (33), the rabbit jugular vein (33), and rat tail arteries (38) or for the BK-induced relaxation of pig coronary arteries (14, 32) and pancreatic arteries (28). More importantly, the relaxation that is induced by BK in rat mesenteric arteries is also mediated by the BK-B2 receptor (33, 37). It has been demonstrated that both types of BK receptors are present on rabbit superior mesenteric artery smooth muscle cells. Stimulation of the BK-B2 receptor by BK or the BK-B1 receptor by des-[Arg9]-BK results in intracellular Ca2+ signals that are transient or sustained, respectively. We have not measured Ca2+ signals; however, it is known that ceramide and other sphingolipid metabolites are implicated in generating these signals including in smooth muscle cells (19, 26, 27).

It seems that the BK-B2 receptor mediates the effect of BK on inhibition of SMase in RCCD cells, whereas activation of the BK-B1 receptor leads to activation of SMase in rat mesenteric small arteries. Whether this is true of the two receptor subtypes in other cell types remains to be determined. Also unknown at the present time are the signaling pathways involved downstream of the BK-B1 or BK-B2 receptors and upstream of sphingomyelinase (Fig. 8). There are presently many potential candidates that could be implicated: the ERK1, ERK2 mitogen-activated protein kinase pathway, the stress-activated protein kinase/c-jun NH2-terminal kinase or p38 pathways, various protein phosphatases, phospholipase A2, phosphatidylinositol or phosphatidylcholine phospholipase C, phospholipase D, protein kinase C, Ca2+, or nitric oxide (19). Also unknown at the present time is the involvement of other sphingolipid metabolites (sphingosine, sphingosine-1-phosphate, and ceramide-1-phosphate) in this model.


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  		Fig. 8.   BK induces SMase activity via the BK-B1 receptor in the rat small artery. Little is known about ceramide signal transduction and SMase activity as modulated by BK in cells and tissues. BK signals through 2 different receptor subtypes (BK-B1 and BK-B2). BK increases ceramide production and the hydrolysis of SMase activity via BK-B1 receptor subtype in rat small artery.

Our functional studies in intact rat mesenteric arteries show that a permeable form of ceramide, at a concentration estimated to be similar to that produced by a dose of BK eliciting maximum production of this second messenger, significantly dilated preconstricted intact rat mesenteric small arteries studied under isobaric condition. At the concentration of 1 µM, the alpha 1-agonist PE is known to produce a contraction in this preparation that is near maximal and is entirely dependent on Ca2+ influx through voltage-gated L-type Ca2+ channels (40). In the presence of PE, ceramide did not attenuate contractility by suppressing smooth muscle Ca2+ channels because it had no effect on the constriction mediated by high-K+-induced membrane depolarization, which is abolished by dihydropyridines (40). The lack of effect of ceramide on high-K+-mediated constriction also rules out a nonspecific effect of this compound on the calmodulin and myosin light chain kinase pathway leading to actomyosin interaction.

Several possibilities could explain the effects of ceramide on PE-induced tone: 1) hyperpolarization of the smooth muscle cells by either enhancement of K+ channel activity (25) or block of Ca2+-activated Cl- (31) or nonselective cation (39) channels would result in reduced Ca2+ influx through voltage-gated L-type Ca2+ channels; 2) alterations in ion channel activity or the signaling pathways controlling the release of endothelium-derived relaxing (nitric oxide, prostaglandin I2, and endothelial-derived hyperpolarizing factor) or constricting (endothelin) factors; 3) modulation of the alpha 1-adrenoceptor signaling pathway in the smooth muscle cells leading to a reduced sensitivity of the contractile machinery to intracellular Ca2+ levels. In contrast with our study, ceramide has been reported (17) to constrict bovine coronary arteries, possibly by inhibiting Ca2+-dependent K+ channels through a redox mechanism.

Interestingly, Ca2+-dependent K+ channels channels appear to play, at most, a minor role in the response of rabbit mesenteric arterial smooth muscle cells to PE (23). Recently, Zhang et al. (43) showed that ceramide constricts bovine small coronary arteries by impairing nitric oxide-mediated vasorelaxation, which would appear to involve an increased production of superoxide anion. Our results demonstrate in rat mesenteric small arteries: 1) the presence of SMase, 2) SM hydrolysis is induced by BK, 3) SMase is activated by BK, 4) ceramide is generated in a time- and concentration-dependent manner, 5) the BK-B1 receptor subtype is implicated in mediating the effect of BK on activation of SMase, and 6) ceramide induces a vasodilatation in mesenteric arteries, suggesting that ceramide bear differential effects on vascular tone in different vascular beds.


    ACKNOWLEDGEMENTS

The authors thank Marie-Andrée Lupien for excellent technical and analytical assistance in the functional studies. The BK-B1 and BK-B2 receptor antagonists were a generous gift from Dr. Domenico Regoli, Department of Pharmacology, Faculty of Medicine, University of Sherbrooke.


    FOOTNOTES

* L. Kleine and G. Liu contributed equally to this work.

This study was supported by the Kidney Foundation of Canada, Canadian Institutes of Health Research Grant MT-14103 (to R. L. Hébert), by the Heart and Stroke Foundation of Québec, the Canadian Institutes of Health Research, and by the Montréal Heart Institute (to N. Leblanc). N. Leblanc holds a Fonds de la Recherche en Santé du Québec scholarship.

Address for reprint requests and other correspondence: R. L. Hébert, Dept. of Cellular and Molecular Medicine, Univ. of Ottawa, 451 Smyth Rd., Rm. 1337, Ottawa, Ontario K1H 8M5, Canada (E-mail: rlhebert{at}uottawa.ca).

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. Section 1734 solely to indicate this fact.

First published September 27, 2001; 10.1152/ajpheart.00379.2001

Received 8 May 2001; accepted in final form 11 September 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 282(1):H175-H183
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