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Am J Physiol Heart Circ Physiol 275: H1592-H1598, 1998;
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Vol. 275, Issue 5, H1592-H1598, November 1998

TNF-alpha -induced endothelium-independent vasodilation: a role for phospholipase A2-dependent ceramide signaling

Douglas G. Johns and R. Clinton Webb

Department of Physiology, University of Michigan, Ann Arbor, Michigan 48109

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Ceramide is a novel second messenger generated by hydrolysis of membrane sphingomyelin by a neutral sphingomyelinase (nSMase). Cytokines such as tumor necrosis factor-alpha (TNF-alpha ) have been shown to increase intracellular ceramide through phospholipase A2 (PLA2)-dependent activation of nSMase. TNF-alpha has been shown to cause endothelium-independent relaxation in isolated blood vessels. We have previously shown that exogenously applied sphingomyelinase and ceramide cause endothelium-independent vasodilation in rat thoracic aortas (D. G. Johns, H. Osborn, and R. C. Webb. Biochem. Biophys. Res. Commun. 237: 95-97, 1997). In the present study, we tested the hypothesis that ceramide mediates TNF-alpha -induced vasodilation. In phenylephrine-contracted rat thoracic aortic rings (no endothelium), TNF-alpha caused concentration-dependent relaxation in the presence of cyclooxygenase and lipoxygenase inhibitors. The phospholipase A2 antagonist 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid (DEDA; 50 µM) and the nonselective PLA2 antagonist quinacrine (30 µM) inhibited TNF-alpha -induced relaxation. In cultured rat aortic vascular smooth muscle cells, TNF-alpha (10-7 g/ml) increased intracellular ceramide 1.5-fold over basal level (0.08 nmol/mg protein), which was blocked by the PLA2 antagonist DEDA (50 µM). We conclude that PLA2 activation and increased ceramide generation play a role in mediating TNF-alpha -induced endothelium-independent vasodilation.

cytokines; smooth muscle relaxation; sphingolipid signaling; sepsis

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

CYTOKINES SUCH AS tumor necrosis factor-alpha (TNF-alpha ) have been shown to have both endothelium-dependent and -independent vasodilator properties (14, 36). Such properties imply an important role for TNF-alpha -induced vascular responses in cases in which plasma TNF-alpha levels are elevated such as sepsis/endotoxemia, vascular damage associated with hypertension, and obesity (5, 8, 39). Indeed, the vasodilator effects of TNF-alpha may be responsible for the marked hypotension seen in septic shock. The endothelium-dependent component has been attributed to activation of endothelial nitric oxide (NO) synthase, generation of NO, and relaxation of vascular smooth muscle (3, 34). The endothelium-independent component of TNF-alpha -induced vasodilation has not been characterized.

In other systems, TNF-alpha has been shown to stimulate a membrane-associated, neutral-optimum pH-acting sphingomyelinase (nSMase) resulting in hydrolysis of sphingomyelin and generation of ceramide and phosphocholine (19). Additionally, evidence demonstrates that arachidonic acid generated by membrane-associated phospholipase A2 (PLA2) may be a mediator of the signal from the TNF-alpha receptor to nSMase (16). Ceramide is a sphingolipid second messenger implicated in transducing the cellular signals initiated by TNF-alpha in other physiological systems, but a role for ceramide in mediating the effects of TNF-alpha in the vasculature has not been investigated.

Ceramide generated through hydrolysis of membrane sphingomyelin is structurally very similar to diacylglycerol, a product of phosphoinositide-phospholipase C activity. Ceramide has been shown to activate a serine/threonine-specific protein phosphatase of the protein phosphatase 2A family (ceramide-activated protein phosphatase), a serine/threonine proline-directed protein kinase (ceramide-activated protein kinase), and inhibit translocation of phorbol ester-sensitive isoforms of the classic protein kinase C (PKC) family (6, 23, 18). The latter function in vascular smooth muscle implies a vasodilator role for ceramide, given the contraction-promoting nature of PKC in vascular smooth muscle. We have previously shown that exogenously applied cell-permeant ceramide and nSMase result in concentration-dependent vasodilation in contracted isolated rat thoracic aortic rings and that nSMase treatment of cultured vascular smooth muscle cells elicits intracellular ceramide generation (17).

We hypothesized that ceramide signaling mediates TNF-alpha -induced vasodilation. To address this hypothesis, we tested the following specific aims: 1) to verify that TNF-alpha causes endothelium-independent vasodilation, 2) to determine whether TNF-alpha -induced vasodilation is PLA2 dependent, 3) to determine whether TNF-alpha causes ceramide generation in cultured vascular smooth muscle cells, and 4) to determine whether TNF-alpha -induced ceramide generation is PLA2 dependent. The hypothesized signaling pathway is illustrated in Fig. 1.


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  		Fig. 1.   Hypothesis for mechanism of endothelium-independent tumor necrosis factor-alpha (TNF-alpha )-induced relaxation in vascular smooth muscle. TNF-alpha binds to its receptor (TNF-alpha -R) in plasma membrane, resulting in activation of membrane-associated phospholipase A2 (PLA2). Arachidonic acid generated by hydrolysis of membrane phospholipids by PLA2 stimulates a membrane-associated neutral sphingomyelinase (SMase), resulting in formation of intracellular ceramide. Ceramide elicits relaxation of vascular smooth muscle through an uncharacterized mechanism. Pharmacological manipulation of PLA2 is accomplished with antagonists 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid (DEDA) and quinacrine.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Isolated vascular segment preparation. Male Sprague-Dawley rats (Charles River) weighing 200-250 g were anesthetized with pentobarbital sodium (50 mg/kg ip), and aortas were removed and placed under a dissecting microscope in cold physiological salt solution (PSS; 130 mM NaCl, 4.5 mM KCl, 1.18 mM KHPO4, 1.17 mM MgSO4, 1.6 mM CaCl2 · 2H2O, 14.9 mM NaHCO3, 5.5 mM dextrose, and 0.03 mM CaNa2 EDTA). The vessels were then cleaned of adherent fat and connective tissue and cut into rings (4 mm long). The endothelium was removed from aortic segments by a gentle rubbing procedure with the tips of a pair of forceps. These rings were mounted in an organ chamber containing PSS aerated with 95% O2-5% CO2 for measurement of isometric force development. The absence of endothelium was functionally evaluated by relaxation to acetylcholine (10-7 M) after contraction in response to phenylephrine (10-8 M). All preparations were allowed to equilibrate for 90 min under a constant passive force (~3 g) before an experiment was begun. This level of passive force was determined to be optimal for maximal force development to 10-6 M phenylephrine. All experiments were conducted in the presence of indomethacin (10-5 M) to inhibit cyclooxygenase. In some experiments, eicosatrienoic acid was added to inhibit lipoxygenase.

Muscle bath experimental protocols. After aortic ring preparation, a concentration-response curve to phenylephrine (10-9 to 10-5 M) was constructed to determine the EC50 contractile response. After the effects of phenylephrine were washed out, vessel segments were contracted with an EC50 concentration of phenylephrine. When the contraction reached a plateau phase, a relaxation response to human recombinant TNF-alpha was conducted in either the presence or absence of drugs to inhibit PLA2. The PLA2 inhibitors tested were 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid (DEDA) and quinacrine. Relaxation to cell-permeant ceramide was also tested by addition of C2-ceramide (10-5 M) or ethanol vehicle to the plateau phase of a contraction to an EC50 concentration of phenylephrine.

Cell culture. Vascular smooth muscle cells were isolated and cultured by explantation. Thoracic aortas from male Sprague-Dawley rats were removed and cleaned of adherent fat and connective tissue. Aortic segments were cut lengthwise, and the endothelium was removed by a gentle rubbing procedure with a cotton swab moistened with PSS. Segments were washed three times in sterile PSS and cut into small pieces that were seated in DMEM supplemented with 30% fetal bovine serum. After a 7- to 10-day period of cell proliferation, vessel segments were removed, and cells were passaged upon confluency. Cells were maintained in 10-cm plates through 10 passages in DMEM supplemented with 10% fetal bovine serum. The presence of alpha -actin was confirmed by staining with a FITC-conjugated anti-alpha -actin antibody and fluorescence microscopy.

Extraction of cellular lipids. Lipids were extracted from vascular smooth muscle cells using the methods of Van Veldoven and Bell (37). Briefly, after treatment with human recombinant TNF-alpha , cells were rinsed three times with cold phosphate-buffered saline and scraped in 4 ml ice-cold methanol. Chloroform and water were added to make the chloroform-methanol-water ratio 1:2:0.8. Samples were sonicated and centrifuged at 3,000 rpm for 30 min. The supernatant was transferred to another tube, and chloroform and 1 M NaCl were added to make the chloroform-methanol-NaCl ratio 2:1:0.8. The pellet was reserved for measurement of cellular protein using a Bio-Rad assay kit. Samples were vortexed and centrifuged at 1,500 rpm for 5 min to partition the aqueous from the organic fraction. The lower layer (organic layer) was isolated and dried under nitrogen.

Measurement of intracellular ceramide. Ceramide levels were determined using modified methods described by Preiss et al. (29). Briefly, dried lipid extracts were solubilized in 20 µl of a solution of 7.5% octyl-beta -D-glucopyranoside and 5 mM cardiolipin in 1 mM diethylenetriaminepentaacetic acid. After a 10-min incubation period at room temperature, 50 µl of reaction mixture (100 mM imidazole HCl, 100 mM NaCl, 25 mM MgCl2, and 2 mM EGTA) and 20 µl dithiothreitol (10 mM) were added to each sample. The enzymatic step was initiated with the addition of 10 µl of a 1:1 solution of Escherichia coli diacylglycerol kinase (13 U/mg protein) reaction mixture and 10 µl of a 1:100 solution of [gamma -32P]ATP (0.5-1.0 µCi/nmol) with 10 mM ATP. After a 45-min incubation period at 25°C, the reaction was stopped with 4 ml of chloroform-methanol (1:1) and 1 ml of 1 M NaCl. Samples were centrifuged for 5 min at 1,500 rpm for partitioning. The lower organic layer was isolated and washed with 2 ml perchloric acid (1%), and samples were centrifuged at 1,500 rpm for 5 min. The perchloric acid wash was repeated, and the organic layer was isolated and dried under nitrogen. Ceramide-1-phosphate was resolved using high-performance thin-layer chromatography (HPTLC) with a solution containing chloroform-acetone-methanol-acetic acid-water (10:4:3:2:1). HPTLC plates were exposed to phosphorimager screens, and ceramide-1-phosphate levels were quantitated with a phosphorimager. Ceramide values were normalized to amount of cellular protein.

Chemicals. Human recombinant TNF-alpha , phenylephrine HCl, acetylcholine, indomethacin, sodium nitroprusside, N-acetylsphingosine (C2-ceramide), quinacrine, dithiothreitol, diethylenetriaminepentaacetic acid, anti-alpha -actin antibody, octyl-beta -D-glucopyranoside, imidazole, and DEDA were purchased from Sigma Chemical (St. Louis, MO). Cardiolipin was purchased from Avanti Polar Lipids (Alabaster, AL). E. coli diacylglycerol kinase and eicosatrienoic acid were purchased from Calbiochem (La Jolla, CA). The protein assay kit was purchased from Bio-Rad Laboratories (Hercules, CA).

Statistics. For muscle bath experiments, data are presented as means ± SE. For ceramide measurements, data are presented as means ± SE of triplicate replicates from one of three experiments with similar results. For two-group comparisons, Student's t-test was used with a P value <0.05 being considered significant. In multiple-testing procedures, the Bonferroni correction was applied.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

TNF-alpha and cell-permeant ceramide induce endothelium-independent vasodilation. Experiments were carried out to evaluate the endothelium-independent effects of TNF-alpha on contracted rat thoracic aortic segments. Figure 2A is a representative tracing from an experiment illustrating the effect of 10-8 g/ml TNF-alpha on a phenylephrine-contracted rat thoracic aortic ring denuded of endothelium. In all experiments, detectable relaxation occurred within 5-15 min after stimulation with TNF-alpha . Figure 2B illustrates the vasodilatory effect of cell-permeant ceramide (C2-ceramide; 10-5 M) in a phenylephrine-contracted rat thoracic aortic ring (no endothelium). The concentration of phenylephrine used was that which elicited a half-maximal (EC50) contraction. All muscle bath experiments were carried out in the presence of indomethacin (10-5 M), a cyclooxygenase antagonist. Incubation of aortic segments with an inhibitor of 5- and 12-lipoxygenase, 5,8,11-eicosatrienoic acid (20 µM), did not affect relaxation to TNF-alpha (data not shown).


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  		Fig. 2.   TNF-alpha and cell-permeant ceramide cause endothelium-independent vasodilation. Representative tracings from tissue bath experiments are shown. A: endothelium-denuded aortic segments were contracted with an EC50 concentration of phenylephrine and treated with TNF-alpha as described in METHODS. Relaxation to TNF-alpha occurred within 15 min of application of cytokine. B: endothelium-denuded aortic segments were contracted with an EC50 concentration of phenylephrine and treated with C2-ceramide as described in METHODS. Thirty minutes after washout, contractile responses to phenylephrine returned to control levels.

Effect of PLA2 inhibition on TNF-alpha -induced vasodilation. If the vasodilator effects of TNF-alpha are dependent on PLA2, then inhibition of PLA2 with pharmacological antagonists should result in inhibition of TNF-alpha -induced vasodilation. Figure 3A illustrates concentration-dependent vasodilation by TNF-alpha in phenylephrine-contracted aortic rings denuded of endothelium. Preincubation of the vessel segments with quinacrine (30 µM) for 30 min resulted in complete inhibition of TNF-alpha -induced vasodilation and a small degree of contraction with increasing concentrations of TNF-alpha . This concentration of quinacrine represents an average IC50 concentration for PLA2 inhibition based on work by other investigators (10, 38). Incubation of aortic segments with DEDA (50 µM) for 30 min resulted in inhibition of TNF-alpha -induced vasodilation at TNF-alpha concentrations of 10-9, 3 × 10-9, and 10-8 g/ml (30, 45, and 50%, respectively). Incubation of aortic segments with DEDA (50 µM) did not affect relaxation to sodium nitroprusside (data not shown). The inhibition of TNF-alpha -induced relaxation by DEDA was concentration dependent (Fig. 3B). A submaximal concentration of TNF-alpha (3 × 10-9 g/ml) was used to relax phenylephrine-contracted (EC50) aortic segments, followed by a concentration-response treatment with DEDA during a plateau phase of relaxation.


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  		Fig. 3.   Inhibitors of PLA2 block relaxation response to TNF-alpha . A: vessel segments were incubated with either DEDA or quinacrine dihydrochloride for 30 min, and TNF-alpha relaxation experiments were carried out as described in METHODS. DEDA (50 µM) inhibited relaxation to TNF-alpha concentrations ranging from 10-9 to 10-8 g/ml. Quinacrine (30 µM) inhibited relaxation to TNF-alpha concentrations ranging from 3 × 10-10 to 10-8 g/ml. * P < 0.05. B: vessel segments were contracted with phenylephrine (PE) and relaxed with a submaximal concentration of TNF-alpha (3 × 10-9 g/ml). Increasing concentrations of DEDA were sequentially added after a plateau relaxation was reached. DEDA caused a concentration-dependent rescue of phenylephrine-induced contraction.

TNF-alpha increases intracellular ceramide levels in cultured vascular smooth muscle cells. Based on the muscle bath results in which TNF-alpha caused endothelium-independent, PLA2-dependent relaxation, we investigated whether TNF-alpha would cause an increase in intracellular ceramide levels. Cultured vascular smooth muscle cells from rat thoracic aorta were treated with TNF-alpha (10-7 g/ml) for 5, 15, and 30 min. Cellular lipids were extracted, and intracellular ceramide levels were measured as described in METHODS. Figure 4A illustrates the increase in intracellular ceramide seen in the phosphorimager scan of the HPTLC analysis. Incubation of vascular smooth muscle cells with TNF-alpha resulted in a 1.5-fold increase in intracellular ceramide at 15 min and a subsequent reduction at 30 min (Fig. 4B). Time points beyond 30 min were not investigated. Under basal conditions, cultured vascular smooth muscle cells contained 0.075 ± 0.003 nmol ceramide/mg protein.


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  		Fig. 4.   TNF-alpha causes time-dependent intracellular ceramide generation in cultured vascular smooth muscle cells. A: image from phosphorimager scan of high-performance thin-layer chromatography (HPTLC) analysis. Dried, radiolabeled lipid extracts from treated cultured vascular smooth muscle cells were resolubilized with chloroform and run on HPTLC plates as described in METHODS. Ceramide standards were included for quantitation purposes. B: cultured vascular smooth muscle cells were treated with TNF-alpha (10-7 g/ml) for time points indicated, and intracellular ceramide was measured as described in METHODS. Ceramide levels increased 1.5-fold over control levels (0.075 nmol/mg protein) after 15 min of exposure to TNF-alpha and returned to near control levels by 30 min. Data are presented as means ± SE of triplicate replicates from 1 of 3 independent experiments with similar results. * P < 0.05.

Inhibition of PLA2 with DEDA blocks TNF-alpha -induced ceramide generation. To investigate the involvement of PLA2 in TNF-alpha -induced ceramide generation, we used an inhibitor of membrane-associated PLA2, DEDA. Cultured vascular smooth muscle cells were treated with vehicle (0.01% ethanol), DEDA alone, or DEDA in combination with 10-7 g/ml TNF-alpha (DEDA concentration, 50 µM) for the intervals indicated. Vehicle alone had no effect on intracellular ceramide levels. Figure 5 illustrates blockade of TNF-alpha -induced ceramide generation by DEDA.


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  		Fig. 5.   PLA2 antagonist DEDA blocks TNF-alpha -induced ceramide generation in cultured vascular smooth muscle cells. Cultured vascular smooth muscle cells were treated with vehicle or appropriate agent for time points indicated. Cellular lipids were extracted, and ceramide was quantitated as described in METHODS. PLA2 antagonist DEDA blocked increase in intracellular ceramide induced by TNF-alpha . open circle , TNF-alpha (10-7 g/ml) alone; , TNF-alpha  + DEDA (50 µM). Data are presented as means of percent change from control of triplicate replicates from 1 of 3 independent experiments with similar results. * P < 0.05 vs. DEDA treated. dagger  P < 0.05 vs. vehicle.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The purpose of this study was to examine a possible signal transduction mechanism mediating the endothelium-independent effects of TNF-alpha in the vasculature. The overall hypothesis was that ceramide signaling mediates endothelium-independent vasodilation to TNF-alpha . The main findings from this study were that TNF-alpha elicits endothelium-independent relaxation, which is dependent on PLA2 and that TNF-alpha elicits PLA2-dependent ceramide generation in cultured vascular smooth muscle cells, implying a role for TNF-alpha -induced ceramide signaling in the vasculature (Fig. 1).

TNF-alpha and vascular reactivity. It has been well documented that TNF-alpha causes vasodilation in a number of vascular preparations (3, 11, 34). Previous work by others was presumably undertaken to examine the vascular role of TNF-alpha during the severe hypotension that accompanies sepsis and endotoxemia, cases in which plasma levels of TNF-alpha are markedly increased (8, 36). More recent studies implicate TNF-alpha as a potential regulator of blood pressure. Ferreri et al. (9) document increased TNF-alpha production in medullary thick ascending limb tubules from angiotensin II-dependent hypertensive rats. In addition, treatment of these rats with anti-TNF antiserum potentiates the increase in mean arterial pressure seen in this model of hypertension, implying a counterregulatory mechanism for TNF-alpha against the pressor effect of angiotensin II (9). The vasodilatory effects of TNF-alpha have been most commonly attributed to endothelium-dependent generation of NO and subsequent relaxation of vascular smooth muscle (3, 34). However, Hollenberg et al. (14) showed that maximal inhibition of endothelial nitric oxide synthase with NG-nitro-L-arginine and blockade of cyclooxygenase with indomethacin did not completely inhibit the relaxation response of rat aortic rings to TNF-alpha (14). Indeed, in the present study, we have verified that both TNF-alpha and cell-permeant ceramide elicit a marked relaxation in blood vessel segments devoid of endothelium in the presence of indomethacin. Therefore, it is clear that another yet uncharacterized mechanism is responsible for endothelium-independent, cyclooxygenase-independent relaxation of vascular smooth muscle in response to TNF-alpha . We have previously demonstrated that ceramide and exogenously applied sphingomyelinase cause relaxation of contracted rat thoracic aortic rings in the absence of endothelium (17). Therefore, if TNF-alpha increases ceramide production in vascular smooth muscle, it is possible that the endothelium-independent mechanism for TNF-alpha -induced relaxation involves ceramide signaling.

TNF-alpha and ceramide generation. Early studies investigating a relationship between cytokines and sphingolipid signaling documented a TNF-alpha -induced increase in intracellular ceramide with a concomitant decrease in membrane sphingomyelin (19). A novel signaling cascade was discovered, in which nSMase was activated in response to stimulation with TNF-alpha and other cytokines such as interferon-gamma and interleukin-1beta (2, 7, 19, 23, 24). Here, we have substantiated this causal relationship by documenting TNF-alpha -induced ceramide generation in vascular smooth muscle cells for the first time. In the present study, the increase in ceramide in cultured vascular smooth muscle cells was transient, with maximum levels detected after 15 min of treatment with TNF-alpha . The time course of the increase in ceramide levels in response to cytokines is variable among various cell types. In HL-60 cells, Kim et al. (19) showed that sphingomyelin turnover reached a maximum 1 h after stimulation with 30 nM TNF-alpha and returned to control levels by 2 h. In RINm5f cells, ceramide levels reach a maximum level at 5 min, which returns to control levels after 20 min of stimulation with interleukin-1beta (40). In the present study, the time course of maximum ceramide generation in the cultured rat aortic smooth muscle cells is within the range of the time course of TNF-alpha -induced relaxation in the muscle bath, where peak ceramide generation in cells and the initiation of relaxation occurred within 15 min of exposure to the cytokine. The increase in ceramide in the cultured smooth muscle cells is transient, and levels return to baseline after 30 min, a time during which relaxation to TNF-alpha is sustained in intact tissue segments.

The transient nature of the ceramide response to TNF-alpha might imply that a transient relaxation response to TNF-alpha should occur, instead of the plateaued relaxation phase we observed. One reason for this apparent incongruity may be that ceramide generated by hydrolysis of membrane sphingomyelin is known to interact with several possible cellular targets. Among these are a ceramide-activated protein phosphatase, ceramide-activated protein kinase, and PKC (6, 18, 23). It is possible, therefore, that ceramide initiates a signaling cascade involving one or more of these targets, resulting in a more sustained effect downstream of ceramide generation. A second possibility is that the TNF-alpha -induced ceramide generation profile in cultured smooth muscle cells may differ from that which may occur in the intact vessel. Studies have shown that vascular smooth muscle cells in culture lose some of the phenotypic characteristics found in those of intact vessel segments (32, 35). Such changes may contribute to the differences between the cell culture and muscle bath data.

The magnitude of the ceramide response to TNF-alpha is in agreement with other studies in which TNF-alpha - and other cytokine-induced ceramide generation was investigated in different cell types. Between various cell types, basal ceramide levels vary. Basal ceramide levels in our cultured rat aorta smooth muscle cells were well within the range of values stated by others regarding other cell types. Kim et al. (19) documented that treatment of HL-60 cells resulted in a 1.4-fold increase in intracellular ceramide after treatment with 30 nM TNF-alpha (10-11 g/ml). In EL4 thyoma cells, interleukin-1beta (40 ng/ml) induced a 1.3-fold increase in ceramide (24). Basal ceramide values also may vary between different groups of cells of the same cell type. Nakamura et al. (26) documented basal ceramide levels ranging from ~1 to 3 nmol/mg protein in the same study. We have previously reported basal ceramide levels of 0.3 nmol/mg protein in cultured rat thoracic aorta smooth muscle cells (17). In this study, basal levels were 0.075 nmol/mg protein. One explanation for this difference is that cells used for each study were cultured from aortic explants from rats of different sources.

Role of PLA2. TNF-alpha has been shown to stimulate PLA2-mediated arachidonic acid generation and subsequent ceramide generation in HL-60 cells and L929 cells (15, 16). Jayadev et al. (16) documented that exogenous melittin, a PLA2 activator, or arachidonic acid stimulates ceramide generation in HL-60 cells. The PLA2 isoform responsible for this effect of arachidonic acid is described as membrane associated and melittin sensitive (22). The PLA2 antagonist inhibitor DEDA was chosen for this experiment because of its specificity for the membrane-associated, melittin-specific isoform of PLA2.

Other groups have shown that TNF-alpha stimulates expression and activity of other isoforms of PLA2, including a cytosolic and secretory isoform (23, 24). Pharmacological inhibitors such as methyl arachidonyl fluorophosphate are specific for the calcium-dependent and -independent cytosolic isoform of PLA2. In the present study, an IC50 concentration of DEDA partially blocked the relaxation response to TNF-alpha . Nonspecific effects of DEDA are unlikely in this study, evidenced by the lack of any effect of DEDA on sodium nitroprusside relaxation. An IC50 concentration of quinacrine, a nonselective PLA2 antagonist, completely blocked and partially reversed the relaxation response to TNF-alpha . The partial reversal of the relaxation response may be because of nonspecific effects of quinacrine on potassium channels (33). We conclude that the melittin-sensitive isoform of PLA2 is likely involved in the endothelium-independent relaxation response to TNF-alpha , but the involvement of other isoforms cannot be ruled out.

Arachidonic acid can be metabolized by 5- and 12-lipoxygenase to form 5-hydroxyeicosatetraenoic acid (5-HETE) and 12-hydroxyeicosatetraenoic acid (12-HETE), respectively (4). Both 5-HETE and 12-HETE are vasoactive (13), and to rule out contribution of these arachidonic acid metabolites to the observed effect of TNF-alpha , we used a selective inhibitor of 5- and 12-lipoxygenase, eicosatrienoic acid. Because inhibition of lipoxygenase had no effect, we can rule out contribution of 5- and 12-HETE to TNF-alpha -induced vasodilation. Cyclooxygenase products were ruled out by the inclusion of indomethacin in all experiments.

In the cultured rat aortic smooth muscle cells, we observed complete blockade of ceramide generation in response to TNF-alpha with an IC50 concentration of DEDA. Another interesting observation is that during treatment with DEDA and TNF-alpha , ceramide levels decreased to 70% of control values at 15 min. A possible explanation is that PLA2 activity in unstimulated cells might contribute to basal ceramide levels. We observed a slight but statistically insignificant reduction in basal ceramide levels in cells treated with DEDA alone for 30 min (data not shown). A more likely explanation is that TNF-alpha stimulates a number of different signaling pathways, including PLA2, which may affect ceramide generation by nSMase. Blockade of the PLA2 component of TNF-alpha signaling, then, might allow a yet unknown inhibitory mechanism to decrease nSMase activity and reduce ceramide levels.

Possible mechanism for TNF-alpha -induced ceramide-mediated relaxation. Although both TNF-alpha and ceramide have been shown to cause endothelium-independent relaxation, a precise mechanism for ceramide-mediated relaxation has not been characterized. The wide range of intracellular targets for ceramide provides a number of possible mechanisms for ceramide-mediated relaxation. Ceramide has been shown to activate a serine/threonine-directed protein phosphatase of the protein phosphatase 2A family of phosphatases (6). Knapp et al. (20) showed that inhibition of protein phosphatases 1A and 2A resulted in an increase in tone of bovine coronary arterial rings, implying a relaxant role for these enzymes.

Also, it has been documented that ceramide inhibits PKC in nonvascular cell types. Specifically, ceramide has been shown to inhibit translocation of PKC-alpha in situ and activity of PKC-alpha in vitro (18, 21). The calcium-dependent isoforms of PKC are important for contraction in vascular smooth muscle in response to adrenergic agonists and other phospholipase C-linked vasoconstrictors (1, 12, 25). The mode of ceramide-mediated inhibition is unknown but may be related to structural similarity between ceramide and diacylglycerol. Ceramide has been shown to interact directly with a zinc fingerlike lipid binding domain of diacylglycerol kinase, an enzyme which shows specificity for phosphorylating both ceramide and diacylglycerol (28). This lipid-binding domain is very similar to the lipid-binding domain of the phorbol ester-sensitive PKC family. Therefore, it is possible that ceramide acts as a partial agonist for PKC, inhibiting the enzyme by competitive antagonism in the presence of diacylglycerol.

In summary, the results from the present study indicate that isolated rat aortic segments exhibit endothelium-independent vasodilation to TNF-alpha that is partially dependent on PLA2. This was demonstrated by concentration-dependent inhibition of the relaxation response to TNF-alpha by the PLA2 inhibitors DEDA and quinacrine in endothelium-denuded rat thoracic aortic rings. TNF-alpha also induced an increase in intracellular ceramide levels in cultured vascular smooth muscle cells that was also PLA2 dependent. From these results, we conclude that ceramide signaling represents a novel mechanism mediating the endothelium-independent vasodilatory effects of TNF-alpha .

    ACKNOWLEDGEMENTS

These studies were supported by National Heart, Lung, and Blood Institute Grant HL-18575.

    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: D. G. Johns, Dept. of Physiology, 7812 Medical Sciences Bldg. II, Univ. of Michigan Medical School, Ann Arbor, MI 48109-0622.

Received 3 April 1998; accepted in final form 14 July 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Heart Circ Physiol 275(5):H1592-H1598
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