Effects of Exogenous Sphinganine, Sphingosine, and Sphingosine-1-Phosphate on Relaxation and Contraction of Porcine Thoracic Aortic and Pulm
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《毒物学科学杂志》
Department of Veterinary Pathobiology College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802, USA and Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802
ABSTRACT
Fumonisin mycotoxicosis in pigs causes a decrease in mean aortic pressure, an increase in mean pulmonary arterial pressure, and increases in serum concentrations of sphinganine (3.2 μM) and sphingosine (1.4 μM). To determine a causal relationship between the hemodynamic changes and sphingolipid alterations, we examined the in vitro effects of sphinganine, sphingosine, and sphingosine-1-phosphate on porcine aortic and pulmonary arterial rings. Both sphinganine and sphingosine relaxed uncontracted and phenylephrine-contracted aortic rings at 10 μM and 1 μM, respectively. Sphingosine (10 μM) relaxed uncontracted and phenylephrine-contracted pulmonary arterial rings, whereas sphingosine-1-phosphate (10 μM) contracted pulmonary arterial rings. Sphingosine (3 μM) also impaired the contractile response of pulmonary artery rings to 60 mM KCl. The results suggested that the systemic hypotension caused by fumonisin is mediated, in part, by increases in serum sphinganine and sphingosine concentrations, and the pulmonary hypertension is mediated, in part, by increased sphingosine-1-phosphate concentrations.
Key Words: fumonisin mycotoxicosis; sphinganine; sphingosine; sphingosine-1-phosphate; aortic and pulmonary arterial rings; pigs.
INTRODUCTION
Fumonisin B1, a mycotoxin produced by the fungus Fusarium verticillioides, is a ubiquitous contaminant of corn and a potent inhibitor of ceramide synthase, a key enzyme in the sphingolipid biosynthetic pathway. Fumonisin ingestion therefore results in dose-dependent accumulation of sphinganine (dihydrosphingosine) and sphingosine (Riley et al., 1993). The ingestion of toxic doses of fumonisin B1 by pigs results in left-sided heart failure, bradycardia, systemic arterial hypotension, pulmonary artery hypertension, pulmonary edema, and death (Constable et al., 2000; Smith et al., 1999, 2000). These cardiovascular changes are accompanied by serum concentrations of sphinganine (3.2 μM) and sphingosine (1.4 μM), which are greatly increased above normal mean values (sphinganine, 0.01 μM; sphingosine, 0.06 μM; Smith et al., 1999).
Recent studies suggest that sphingosine-1-phosphate, and to a lesser extent sphingosine, play important roles in regulating vascular tone (Coussin et al., 2002; Dantas et al., 2003; Salomone et al., 2003). Fumonisin-induced increases in sphinganine and sphingosine concentrations are associated with systemic arterial hypotension and pulmonary artery hypertension in pigs (Constable et al., 2000, 2003; Smith et al., 1999, 2000); however, this does not conclusively demonstrate a direct effect of sphinganine and sphingosine on porcine vascular smooth muscle because affected pigs have left-sided heart failure and increased pulmonary artery wedge pressures, which can produce systemic arterial hypotension and pulmonary artery hypertension, respectively. Because we suspect sphingolipid-mediated vascular dysfunction plays an important role in fumonisin-induced pulmonary edema in pigs (Constable et al., 2003; Smith et al., 1999, 2000), we hypothesized that sphinganine and sphingosine would relax porcine vascular smooth muscle and that sphingosine-1-phosphate would contract porcine vascular smooth muscle. We therefore examined the in vitro effects of these sphingolipids on porcine thoracic aortic and pulmonary arterial rings in order to gain additional insight into the mechanism for fumonisin-induced systemic hypotension and pulmonary hypertension.
MATERIALS AND METHODS
Animals.
The study was approved by our institutional animal care and use committee. Five to six week-old, female and castrated male, cross-bred pigs, obtained from the Veterinary Research Farm of the University of Illinois, were anesthetized by im administration of ketamine (10 mg/kg) and xylazine (2 mg/kg). Sodium heparin (150 IU/kg) was administered intravenously and the pigs were euthanized by sodium pentobarbital (iv; 60 mg/kg). Descending thoracic aortae and second order right and left intralobular pulmonary arteries were quickly excised and immersed in cold (4°C) physiologic salt solution (119 mM NaCl, 3.8 mM KCl, 1.7 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 24 mM NaHCO3 and 11 mM dextrose at a pH of 7.4, adjusted using NaOH, and aerated with 95% O2/5% CO2).
Preparation of vascular rings.
Loose connective tissue was carefully removed, and the isolated aortae and arteries were cut into 3 to 4 mm wide rings. Experiments on the aortic rings were conducted on the day of harvest. Pulmonary arterial rings were stored overnight in the physiologic salt solution with 2% Ficoll at 4°C (Wright et al., 1995), and experiments were conducted on the following day.
Rings were mounted vertically in water-jacketed glass tissue baths (World Precision Instruments Inc., Sarasota, FL) by passing each ring through two parallel, horizontal stainless steel wires. The upper wire was attached to a force displacement transducer (World Precision Instruments Inc., Sarasota, FL) and the lower wire was anchored onto a support foot at the bottom of a micrometer drive, thereby allowing incremental stretch of the vascular rings. Tissue baths contained 30 ml of physiologic salt solution equilibrated at 37°C and were continuously aerated with 95% O2/5% CO2. The isometric tension was recorded and digitized on a computer acquisition system (software WINDAQ v1.32 & DI200AC v1.54, DATAQ Instruments Inc, Akron, OH) for off line analysis.
All rings were equilibrated for 30 min at a tension of 21 g (thoracic aorta) or 6 g (pulmonary artery) that were previously determined to be optimal for maximal force development to 10–6 M phenylephrine (data not shown). Rings were then challenged with K+-depolarizing solution, containing 60 mM of KCl (equimolar substitution of NaCl in the physiologic salt solution by KCl) to test tissue viability. Aortic and pulmonary arterial rings that generated less than 3 g and 1 g of force to 60 mM KCl, respectively, were discarded (16% of aortic rings and 2% of pulmonary arterial rings were discarded). Viable rings were washed 3 times at 5 min intervals with physiologic salt solution, and allowed a second 30 min equilibration period before the test agent was administered.
Response to sphingolipids.
Concentration-response curves to sphinganine, sphingosine, and sphingosine-1-phosphate were constructed from data obtained after cumulative addition of stock solutions (10–8 to 10–4.5 μM) or an equivalent volume of solvent (0.001–3% dimethylsulfoxide in 0.9% NaCl, control) to each tissue bath. At each concentration, rings were allowed 15 min to reach equilibrium tension. In separate studies, rings were precontracted by 6 μM phenylephrine for 15 min before the addition of sphinganine, sphingosine, sphingosine-1-phosphate or solvent (0.3% dimethylsulfoxide in 0.9% NaCl, control). These studies determined the effect of sphingolipid concentration on changing the tension of uncontracted and contracted vascular rings.
The effect of preincubation for 15 min with sphinganine, sphingosine, and sphingosine-1-phosphate (3 μM) on the cumulative concentration-response relationship for phenylephrine (thoracic aorta and pulmonary artery rings) and KCl (pulmonary artery rings) was also determined. For comparison, a vascular ring from the same pig was preincubated with an equivalent volume of solvent (0.3% dimethylsulfoxide in 0.9% NaCl). A sphingolipid concentration of 3 μM was selected for the study based on serum sphinganine and sphingosine concentrations of 3.2 μM and 1.4 μM, respectively, in pigs observed with fumonisin-induced systemic hypotension and pulmonary artery hypertension (Smith et al., 2000).
Phenylephrine was administered because it contracts vascular smooth muscle by binding to the 1 receptor on the cell membrane, stimulating phospholipase C to cleave phosphatidylinositol 4,5 bisphosphate to water soluble inositol triphosphate (IP3) and hydrophobic diacylglycerol; inositol triphosphate causes the immediate release of stored calcium by binding to an IP3 receptor on the membrane side of the intracellular store, whereas diacylglycerol activates protein kinase C, ultimately increasing myofilament sensitivity to calcium (Sato et al., 2001). KCl was administered because it contracts vascular smooth muscle by opening voltage operated calcium channels (L-type calcium channels), thereby increasing the intracellular calcium concentration. Comparing the effect of sphingolipids on the contractile response to phenylephrine and KCl therefore allowed differentiation of the effect of sphingolipids on receptor and non-receptor mediated contraction.
Chemicals.
Sphinganine, sphingosine, and sphingosine-1-phosphate were from Matreya Inc. (Pleasant Gap, PA) and all other reagents were from Sigma-Aldrich (St. Louis, MO). Solutions were made fresh daily by dissolving the chemical agent in dimethylsulfoxide (sphingolipids) or double distilled deionized water (phenylephrine). The volume of solution added was <0.1% of the bath volume.
Data analysis.
Dose response curves for sphinganine, sphingosine, and sphingosine-1-phosphate in uncontracted and 6 μM phenylephrine-contracted rings were plotted with responses expressed as percentage of the tension induced by 60 mM KCl solution. Because sphingolipids induced a small change in active tension relative to that induced by 60 mM KCl, and because there was a small (<10% of 60 mM KCl-induced tension) time-dependent drift in tension in untreated (control) rings, the stated tension represented the difference in active tension from the matched control ring at each concentration.
Phenylephrine and KCl dose response curves for rings pretreated with 3 μM sphinganine, sphingosine, and sphingosine-1-phosphate or untreated (control) were fitted by nonlinear regression (PROC NLIN of SAS/STAT 8.2, SAS Institute Inc., Cary, NC) using the Marquardt iterative method and a three-parameter logistic regression equation:
where T is the tension, C is the concentration of the vasoconstrictor agent (phenylephrine or KCl), Tmax is the maximum response, EC50 is the concentration of the vasoconstrictor agent when T = 50% of Tmax, and S (slope) indicates the gradient of the sigmoidal concentration-tension curve at EC50.
Statistical analysis.
All data are presented as least squares mean ± SE, and a p value <0.05 was considered significant. Statistical analysis was conducted using the PROC MIXED procedure of SAS/STAT 8.2 (SAS Institute Inc., Cary, NC) with pig declared as a random effect. When there was a significant effect of concentration in the dose response curves for sphinganine, sphingosine, and sphingosine-1-phosphate in uncontracted and 6 μM phenylephrine-contracted rings, least squares means were compared to the lowest concentration value for each sphingolipid using Bonferroni adjusted p values. When there was a significant effect of treatment in the phenylephrine and KCl dose response curves for rings pretreated with 3 μM sphingolipid or untreated (control), least square means were compared to control at each concentration using Dunnett's test.
RESULTS
Effects of Sphingolipids on Aortic Rings
Both sphinganine and sphingosine (10 μM) relaxed uncontracted aortic rings in a concentration-related fashion, whereas sphingosine-1-phosphate (10 μM) had no effect on tension (Fig. 1). The relaxation effects of sphinganine and sphingosine were more evident in phenylephrine-contracted rings; both sphinganine and sphingosine (1 μM) caused relaxation. Sphingosine caused a concentration dependent relaxation in phenylephrine contracted rings, whereas sphinganine decreased tension at 1 μM and 3 μM, but not at 10 μM. Sphingosine-1-phosphate slightly decreased the tension of phenylephrine contracted rings, but the maximal decrease seen at 1 μM did not reach statistical significance.
Effects of Sphingolipids on Pulmonary Arterial Rings
Sphinganine (30 μM) and sphingosine (10 μM) relaxed uncontracted pulmonary artery rings in a concentration-related fashion, whereas sphingosine-1-phosphate (10 μM) increased tension (Fig. 2). The relaxant effect of sphingosine was more evident in phenylephrine-contracted rings, whereas sphinganine and sphingosine-1-phosphate did not alter tension in phenylephrine-contracted rings.
Effects of Sphingolipids on the Phenylephrine Dose Response Curve
The mean Tmax value for phenylephrine in control aortic rings (162% of 60 mM KCl contraction) was greater than that in control pulmonary artery rings (90% of 60 mM KCl contraction; Fig. 3), indicating that thoracic aortic rings were comparatively more responsive to 1-mediated contraction than voltage-operated calcium channel mediated contraction.
Three μM sphingosine and sphingosine-1-phosphate shifted the phenylephrine concentration-tension curve of aortic rings to the left, although the change in EC50 was small (Fig. 3). In contrast, sphinganine did not shift the curve to the left. The maximal developed tension (Tmax) was similar for the four groups. Taken together, these results indicated that sphingosine and sphingosine-1-phosphate slightly increased the sensitivity of aortic rings to phenylephrine.
Three μM sphinganine, sphingosine, and sphingosine-1-phosphate had no effect on the EC50 or Tmax values of the phenylephrine concentration-tension curve of pulmonary artery rings (Fig. 3). However, at low phenylephrine concentrations, 3 μM sphingosine decreased tension (indicating relaxation) and 3 μM sphingosine-1-phosphate increased tension (indicating contraction). These results were consistent with those obtained earlier for pulmonary artery rings.
Effects of Sphingolipids on the KCl Dose Response Curve
Three μM sphinganine, sphingosine, and sphingosine-1-phosphate had no effect on the EC50 or Tmax values of the KCl concentration-tension curve of aortic rings (Fig. 4).
Three μM sphinganine and sphingosine-1-phosphate also had no effect on the EC50 or Tmax values of the KCl concentration-tension curve of pulmonary artery rings (Fig. 4). However, 3 μM sphingosine decreased Tmax without changing EC50.
DISCUSSION
We were interested in investigating pathways for sphingolipid-mediated alterations in vascular smooth muscle tone because fumonisin markedly increases the serum concentrations of sphinganine and sphingosine, as well as inducing systemic hypotension and pulmonary hypertension in pigs (Constable et al., 2000, 2003; Smith et al., 1999, 2000). Fumonisin administration is also suspected to increase serum concentrations of sphingosine-1-phosphate and sphinganine-1-phosphate in pigs as a result of the action of sphingosine kinase on sphingosine and sphinganine, respectively. The present study demonstrates that sphinganine (10 μM) and sphingosine (1 μM) relaxed porcine thoracic aortic and pulmonary arterial rings, and sphingosine-1-phosphate (10 μM) contracted pulmonary arterial rings. This study appears to be the first to demonstrate that sphinganine has vasoactive properties and that sphingosine-1-phosphate constricts pulmonary artery rings. Our in vivo and in vitro findings in concert suggest that increases in serum sphinganine or sphingosine concentration may mediate part of the systemic hypotension in pigs with fumonisin toxicity and that increases in serum sphingosine-1-phosphate concentration may mediate part of the pulmonary hypertension. Further studies are needed to elucidate the potential effects of sphinganine-1-phosphate and intracellular sphinganine and sphingosine on vascular smooth muscle tone.
We found that sphinganine and sphingosine (1 to 10 μM) relaxed vascular smooth muscle, and the effect was more pronounced in phenylephrine contracted rings than uncontracted rings. Protein kinase C plays an important role in the maintenance of phenylephrine-induced contraction (Lee et al., 1999). Sphinganine and sphingosine (1 to 10 μM) inhibits protein kinase C (Merrill et al., 1989). Therefore, protein kinase C inhibition was the likely cause for the vasorelaxant effect of sphinganine and sphingosine. Additionally, our finding that sphingosine relaxed vascular smooth muscle was consistent with those of two other studies; 3 μM sphingosine relaxed porcine coronary arterial rings precontracted with prostaglandin F2 (Murohara et al., 1996), and 3 μM sphingosine relaxed uncontracted canine cerebral artery rings (Zheng et al., 2000). In contrast, 0.1 to 10 μM sphingosine had no effect on phenylephrine contracted rat thoracic aortic rings (Johns et al., 1999).
Interestingly, 3 μM sphingosine, but not 3 μM sphinganine, decreased the maximal tension induced by 60 mM KCl in pulmonary artery rings (Fig. 4). This result cannot be attributed to sphingosine induced protein kinase C inhibition because the KCl dose-response curve is unaffected by protein kinase C inhibition (Shimamoto et al., 1993). Sphingosine causes a dose-dependent prolongation of the closed time of the L-type Ca2+ channel in rat (Yasui and Palade, 1996) and rabbit cardiac cells (IC50 = 38 μM; Sharma et al., 2000). Sphingosine also blocks L-type Ca2+ channels (IC50 < 1 μM) in rabbit skeletal muscle (Sabbadini et al., 1992). Therefore, L-type Ca2+ channel blockade may be totally or partly responsible for this observation, and suggests that sphingosine may relax vascular smooth muscle by more than one pathway. The relative contribution of sphingosine-induced protein kinase C inhibition and L-type Ca2+ channel blockade to sphingosine-induced vasorelaxation requires further study.
We found that sphingosine-1-phosphate (10 μM) caused a modest contraction of pig pulmonary arterial rings but did not contract pig aortic rings. Sphingosine-1-phosphate binds to cell membrane receptors named S1P2 and S1P3, which are G-protein-coupled receptors formerly known as endothelial differentiation gene (EDG) receptors. S1P2 is suspected to be specific for cerebral blood vessels, whereas S1P3 is believed to be present in both central and peripheral vessels (Salomone et al., 2003) including aorta and pulmonary artery; S1P3 activation therefore provides the most likely pathway for sphingosine-1-phosphate mediated pulmonary artery contraction in pigs. Additionally, previous studies have determined that a similar concentration of sphingosine-1-phosphate (10 μM) caused a modest contraction of isolated rat mesenteric and intrarenal microvessels (Bischoff et al., 2000) and canine coronary arteries (Sugiyama et al., 2000), but failed to contract rat carotid and femoral artery rings, mouse aortic rings (Salomone et al., 2003), or rat aortic rings (Coussin et al., 2002). In contrast, much lower concentrations (0.1 μM) of sphingosine-1-phosphate contracted dog, rat, mouse, and rabbit basilar and middle cerebral arteries (Tosaka et al., 2001; Salomone et al., 2003) and rat portal veins (Ikeda et al., 2004), and even lower concentrations of sphingosine-1-phosphate (0.01 μM) transiently relaxed rat mesenteric arterioles via endothelium dependent nitric oxide production (Dantas et al., 2003). Taken together, these results indicate marked site specific differences in the effect of sphingosine-1-phosphate on vascular smooth muscle. The absence of sphingosine-1-phosphate-mediated contraction of pig and mouse aortic rings and rat carotid and femoral arteries has been attributed to differences in S1P isoform receptor density in different vascular beds (Coussin et al., 2002; Mazurais et al., 2002).
The EC50 for KCl (22 mM) for second order pulmonary artery rings in this study was similar to that (25 mM) for third and fifth order pulmonary artery rings (Zellers and Vanhoutte, 1989). However, the EC50 for phenylephrine of second order pulmonary artery rings (10–6.7) and aortic rings (10–6.4) was lower than that for third and fifth order pulmonary artery rings (10–5.2 and 10–5.6, respectively; Zellers and Vanhoutte, 1989), first order pulmonary artery rings (10–5.7; Gustin et al., 1993), and 10–5.4 for anterior mesenteric artery rings in pigs (Nielsen et al., 1991).
Our in vitro finding that sphinganine and sphingosine have vasorelaxant effects is consistent with our previous in vivo finding that fumonisin ingestion alters the aortic input impedance spectrum of pigs (Constable et al., 2003). Because fumonisin ingestion results in toxic effects on the heart and vasculature, fumonisin should be considered as a cardiovascular toxin. The relative importance of cardiac and vascular toxicity following fumonisin ingestion may vary from species to species. Cardiac toxicity appears relatively more important in pigs because they die from acute left-sided heart failure (Smith et al., 1999), whereas vascular toxicity may be more important in horses because they die from vasogenic cerebral edema and leukoencephalomalacia that has been attributed to inadequate autoregulation of cerebral blood flow (Foreman et al., 2004; Smith et al., 2002).
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ABSTRACT
Fumonisin mycotoxicosis in pigs causes a decrease in mean aortic pressure, an increase in mean pulmonary arterial pressure, and increases in serum concentrations of sphinganine (3.2 μM) and sphingosine (1.4 μM). To determine a causal relationship between the hemodynamic changes and sphingolipid alterations, we examined the in vitro effects of sphinganine, sphingosine, and sphingosine-1-phosphate on porcine aortic and pulmonary arterial rings. Both sphinganine and sphingosine relaxed uncontracted and phenylephrine-contracted aortic rings at 10 μM and 1 μM, respectively. Sphingosine (10 μM) relaxed uncontracted and phenylephrine-contracted pulmonary arterial rings, whereas sphingosine-1-phosphate (10 μM) contracted pulmonary arterial rings. Sphingosine (3 μM) also impaired the contractile response of pulmonary artery rings to 60 mM KCl. The results suggested that the systemic hypotension caused by fumonisin is mediated, in part, by increases in serum sphinganine and sphingosine concentrations, and the pulmonary hypertension is mediated, in part, by increased sphingosine-1-phosphate concentrations.
Key Words: fumonisin mycotoxicosis; sphinganine; sphingosine; sphingosine-1-phosphate; aortic and pulmonary arterial rings; pigs.
INTRODUCTION
Fumonisin B1, a mycotoxin produced by the fungus Fusarium verticillioides, is a ubiquitous contaminant of corn and a potent inhibitor of ceramide synthase, a key enzyme in the sphingolipid biosynthetic pathway. Fumonisin ingestion therefore results in dose-dependent accumulation of sphinganine (dihydrosphingosine) and sphingosine (Riley et al., 1993). The ingestion of toxic doses of fumonisin B1 by pigs results in left-sided heart failure, bradycardia, systemic arterial hypotension, pulmonary artery hypertension, pulmonary edema, and death (Constable et al., 2000; Smith et al., 1999, 2000). These cardiovascular changes are accompanied by serum concentrations of sphinganine (3.2 μM) and sphingosine (1.4 μM), which are greatly increased above normal mean values (sphinganine, 0.01 μM; sphingosine, 0.06 μM; Smith et al., 1999).
Recent studies suggest that sphingosine-1-phosphate, and to a lesser extent sphingosine, play important roles in regulating vascular tone (Coussin et al., 2002; Dantas et al., 2003; Salomone et al., 2003). Fumonisin-induced increases in sphinganine and sphingosine concentrations are associated with systemic arterial hypotension and pulmonary artery hypertension in pigs (Constable et al., 2000, 2003; Smith et al., 1999, 2000); however, this does not conclusively demonstrate a direct effect of sphinganine and sphingosine on porcine vascular smooth muscle because affected pigs have left-sided heart failure and increased pulmonary artery wedge pressures, which can produce systemic arterial hypotension and pulmonary artery hypertension, respectively. Because we suspect sphingolipid-mediated vascular dysfunction plays an important role in fumonisin-induced pulmonary edema in pigs (Constable et al., 2003; Smith et al., 1999, 2000), we hypothesized that sphinganine and sphingosine would relax porcine vascular smooth muscle and that sphingosine-1-phosphate would contract porcine vascular smooth muscle. We therefore examined the in vitro effects of these sphingolipids on porcine thoracic aortic and pulmonary arterial rings in order to gain additional insight into the mechanism for fumonisin-induced systemic hypotension and pulmonary hypertension.
MATERIALS AND METHODS
Animals.
The study was approved by our institutional animal care and use committee. Five to six week-old, female and castrated male, cross-bred pigs, obtained from the Veterinary Research Farm of the University of Illinois, were anesthetized by im administration of ketamine (10 mg/kg) and xylazine (2 mg/kg). Sodium heparin (150 IU/kg) was administered intravenously and the pigs were euthanized by sodium pentobarbital (iv; 60 mg/kg). Descending thoracic aortae and second order right and left intralobular pulmonary arteries were quickly excised and immersed in cold (4°C) physiologic salt solution (119 mM NaCl, 3.8 mM KCl, 1.7 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 24 mM NaHCO3 and 11 mM dextrose at a pH of 7.4, adjusted using NaOH, and aerated with 95% O2/5% CO2).
Preparation of vascular rings.
Loose connective tissue was carefully removed, and the isolated aortae and arteries were cut into 3 to 4 mm wide rings. Experiments on the aortic rings were conducted on the day of harvest. Pulmonary arterial rings were stored overnight in the physiologic salt solution with 2% Ficoll at 4°C (Wright et al., 1995), and experiments were conducted on the following day.
Rings were mounted vertically in water-jacketed glass tissue baths (World Precision Instruments Inc., Sarasota, FL) by passing each ring through two parallel, horizontal stainless steel wires. The upper wire was attached to a force displacement transducer (World Precision Instruments Inc., Sarasota, FL) and the lower wire was anchored onto a support foot at the bottom of a micrometer drive, thereby allowing incremental stretch of the vascular rings. Tissue baths contained 30 ml of physiologic salt solution equilibrated at 37°C and were continuously aerated with 95% O2/5% CO2. The isometric tension was recorded and digitized on a computer acquisition system (software WINDAQ v1.32 & DI200AC v1.54, DATAQ Instruments Inc, Akron, OH) for off line analysis.
All rings were equilibrated for 30 min at a tension of 21 g (thoracic aorta) or 6 g (pulmonary artery) that were previously determined to be optimal for maximal force development to 10–6 M phenylephrine (data not shown). Rings were then challenged with K+-depolarizing solution, containing 60 mM of KCl (equimolar substitution of NaCl in the physiologic salt solution by KCl) to test tissue viability. Aortic and pulmonary arterial rings that generated less than 3 g and 1 g of force to 60 mM KCl, respectively, were discarded (16% of aortic rings and 2% of pulmonary arterial rings were discarded). Viable rings were washed 3 times at 5 min intervals with physiologic salt solution, and allowed a second 30 min equilibration period before the test agent was administered.
Response to sphingolipids.
Concentration-response curves to sphinganine, sphingosine, and sphingosine-1-phosphate were constructed from data obtained after cumulative addition of stock solutions (10–8 to 10–4.5 μM) or an equivalent volume of solvent (0.001–3% dimethylsulfoxide in 0.9% NaCl, control) to each tissue bath. At each concentration, rings were allowed 15 min to reach equilibrium tension. In separate studies, rings were precontracted by 6 μM phenylephrine for 15 min before the addition of sphinganine, sphingosine, sphingosine-1-phosphate or solvent (0.3% dimethylsulfoxide in 0.9% NaCl, control). These studies determined the effect of sphingolipid concentration on changing the tension of uncontracted and contracted vascular rings.
The effect of preincubation for 15 min with sphinganine, sphingosine, and sphingosine-1-phosphate (3 μM) on the cumulative concentration-response relationship for phenylephrine (thoracic aorta and pulmonary artery rings) and KCl (pulmonary artery rings) was also determined. For comparison, a vascular ring from the same pig was preincubated with an equivalent volume of solvent (0.3% dimethylsulfoxide in 0.9% NaCl). A sphingolipid concentration of 3 μM was selected for the study based on serum sphinganine and sphingosine concentrations of 3.2 μM and 1.4 μM, respectively, in pigs observed with fumonisin-induced systemic hypotension and pulmonary artery hypertension (Smith et al., 2000).
Phenylephrine was administered because it contracts vascular smooth muscle by binding to the 1 receptor on the cell membrane, stimulating phospholipase C to cleave phosphatidylinositol 4,5 bisphosphate to water soluble inositol triphosphate (IP3) and hydrophobic diacylglycerol; inositol triphosphate causes the immediate release of stored calcium by binding to an IP3 receptor on the membrane side of the intracellular store, whereas diacylglycerol activates protein kinase C, ultimately increasing myofilament sensitivity to calcium (Sato et al., 2001). KCl was administered because it contracts vascular smooth muscle by opening voltage operated calcium channels (L-type calcium channels), thereby increasing the intracellular calcium concentration. Comparing the effect of sphingolipids on the contractile response to phenylephrine and KCl therefore allowed differentiation of the effect of sphingolipids on receptor and non-receptor mediated contraction.
Chemicals.
Sphinganine, sphingosine, and sphingosine-1-phosphate were from Matreya Inc. (Pleasant Gap, PA) and all other reagents were from Sigma-Aldrich (St. Louis, MO). Solutions were made fresh daily by dissolving the chemical agent in dimethylsulfoxide (sphingolipids) or double distilled deionized water (phenylephrine). The volume of solution added was <0.1% of the bath volume.
Data analysis.
Dose response curves for sphinganine, sphingosine, and sphingosine-1-phosphate in uncontracted and 6 μM phenylephrine-contracted rings were plotted with responses expressed as percentage of the tension induced by 60 mM KCl solution. Because sphingolipids induced a small change in active tension relative to that induced by 60 mM KCl, and because there was a small (<10% of 60 mM KCl-induced tension) time-dependent drift in tension in untreated (control) rings, the stated tension represented the difference in active tension from the matched control ring at each concentration.
Phenylephrine and KCl dose response curves for rings pretreated with 3 μM sphinganine, sphingosine, and sphingosine-1-phosphate or untreated (control) were fitted by nonlinear regression (PROC NLIN of SAS/STAT 8.2, SAS Institute Inc., Cary, NC) using the Marquardt iterative method and a three-parameter logistic regression equation:
where T is the tension, C is the concentration of the vasoconstrictor agent (phenylephrine or KCl), Tmax is the maximum response, EC50 is the concentration of the vasoconstrictor agent when T = 50% of Tmax, and S (slope) indicates the gradient of the sigmoidal concentration-tension curve at EC50.
Statistical analysis.
All data are presented as least squares mean ± SE, and a p value <0.05 was considered significant. Statistical analysis was conducted using the PROC MIXED procedure of SAS/STAT 8.2 (SAS Institute Inc., Cary, NC) with pig declared as a random effect. When there was a significant effect of concentration in the dose response curves for sphinganine, sphingosine, and sphingosine-1-phosphate in uncontracted and 6 μM phenylephrine-contracted rings, least squares means were compared to the lowest concentration value for each sphingolipid using Bonferroni adjusted p values. When there was a significant effect of treatment in the phenylephrine and KCl dose response curves for rings pretreated with 3 μM sphingolipid or untreated (control), least square means were compared to control at each concentration using Dunnett's test.
RESULTS
Effects of Sphingolipids on Aortic Rings
Both sphinganine and sphingosine (10 μM) relaxed uncontracted aortic rings in a concentration-related fashion, whereas sphingosine-1-phosphate (10 μM) had no effect on tension (Fig. 1). The relaxation effects of sphinganine and sphingosine were more evident in phenylephrine-contracted rings; both sphinganine and sphingosine (1 μM) caused relaxation. Sphingosine caused a concentration dependent relaxation in phenylephrine contracted rings, whereas sphinganine decreased tension at 1 μM and 3 μM, but not at 10 μM. Sphingosine-1-phosphate slightly decreased the tension of phenylephrine contracted rings, but the maximal decrease seen at 1 μM did not reach statistical significance.
Effects of Sphingolipids on Pulmonary Arterial Rings
Sphinganine (30 μM) and sphingosine (10 μM) relaxed uncontracted pulmonary artery rings in a concentration-related fashion, whereas sphingosine-1-phosphate (10 μM) increased tension (Fig. 2). The relaxant effect of sphingosine was more evident in phenylephrine-contracted rings, whereas sphinganine and sphingosine-1-phosphate did not alter tension in phenylephrine-contracted rings.
Effects of Sphingolipids on the Phenylephrine Dose Response Curve
The mean Tmax value for phenylephrine in control aortic rings (162% of 60 mM KCl contraction) was greater than that in control pulmonary artery rings (90% of 60 mM KCl contraction; Fig. 3), indicating that thoracic aortic rings were comparatively more responsive to 1-mediated contraction than voltage-operated calcium channel mediated contraction.
Three μM sphingosine and sphingosine-1-phosphate shifted the phenylephrine concentration-tension curve of aortic rings to the left, although the change in EC50 was small (Fig. 3). In contrast, sphinganine did not shift the curve to the left. The maximal developed tension (Tmax) was similar for the four groups. Taken together, these results indicated that sphingosine and sphingosine-1-phosphate slightly increased the sensitivity of aortic rings to phenylephrine.
Three μM sphinganine, sphingosine, and sphingosine-1-phosphate had no effect on the EC50 or Tmax values of the phenylephrine concentration-tension curve of pulmonary artery rings (Fig. 3). However, at low phenylephrine concentrations, 3 μM sphingosine decreased tension (indicating relaxation) and 3 μM sphingosine-1-phosphate increased tension (indicating contraction). These results were consistent with those obtained earlier for pulmonary artery rings.
Effects of Sphingolipids on the KCl Dose Response Curve
Three μM sphinganine, sphingosine, and sphingosine-1-phosphate had no effect on the EC50 or Tmax values of the KCl concentration-tension curve of aortic rings (Fig. 4).
Three μM sphinganine and sphingosine-1-phosphate also had no effect on the EC50 or Tmax values of the KCl concentration-tension curve of pulmonary artery rings (Fig. 4). However, 3 μM sphingosine decreased Tmax without changing EC50.
DISCUSSION
We were interested in investigating pathways for sphingolipid-mediated alterations in vascular smooth muscle tone because fumonisin markedly increases the serum concentrations of sphinganine and sphingosine, as well as inducing systemic hypotension and pulmonary hypertension in pigs (Constable et al., 2000, 2003; Smith et al., 1999, 2000). Fumonisin administration is also suspected to increase serum concentrations of sphingosine-1-phosphate and sphinganine-1-phosphate in pigs as a result of the action of sphingosine kinase on sphingosine and sphinganine, respectively. The present study demonstrates that sphinganine (10 μM) and sphingosine (1 μM) relaxed porcine thoracic aortic and pulmonary arterial rings, and sphingosine-1-phosphate (10 μM) contracted pulmonary arterial rings. This study appears to be the first to demonstrate that sphinganine has vasoactive properties and that sphingosine-1-phosphate constricts pulmonary artery rings. Our in vivo and in vitro findings in concert suggest that increases in serum sphinganine or sphingosine concentration may mediate part of the systemic hypotension in pigs with fumonisin toxicity and that increases in serum sphingosine-1-phosphate concentration may mediate part of the pulmonary hypertension. Further studies are needed to elucidate the potential effects of sphinganine-1-phosphate and intracellular sphinganine and sphingosine on vascular smooth muscle tone.
We found that sphinganine and sphingosine (1 to 10 μM) relaxed vascular smooth muscle, and the effect was more pronounced in phenylephrine contracted rings than uncontracted rings. Protein kinase C plays an important role in the maintenance of phenylephrine-induced contraction (Lee et al., 1999). Sphinganine and sphingosine (1 to 10 μM) inhibits protein kinase C (Merrill et al., 1989). Therefore, protein kinase C inhibition was the likely cause for the vasorelaxant effect of sphinganine and sphingosine. Additionally, our finding that sphingosine relaxed vascular smooth muscle was consistent with those of two other studies; 3 μM sphingosine relaxed porcine coronary arterial rings precontracted with prostaglandin F2 (Murohara et al., 1996), and 3 μM sphingosine relaxed uncontracted canine cerebral artery rings (Zheng et al., 2000). In contrast, 0.1 to 10 μM sphingosine had no effect on phenylephrine contracted rat thoracic aortic rings (Johns et al., 1999).
Interestingly, 3 μM sphingosine, but not 3 μM sphinganine, decreased the maximal tension induced by 60 mM KCl in pulmonary artery rings (Fig. 4). This result cannot be attributed to sphingosine induced protein kinase C inhibition because the KCl dose-response curve is unaffected by protein kinase C inhibition (Shimamoto et al., 1993). Sphingosine causes a dose-dependent prolongation of the closed time of the L-type Ca2+ channel in rat (Yasui and Palade, 1996) and rabbit cardiac cells (IC50 = 38 μM; Sharma et al., 2000). Sphingosine also blocks L-type Ca2+ channels (IC50 < 1 μM) in rabbit skeletal muscle (Sabbadini et al., 1992). Therefore, L-type Ca2+ channel blockade may be totally or partly responsible for this observation, and suggests that sphingosine may relax vascular smooth muscle by more than one pathway. The relative contribution of sphingosine-induced protein kinase C inhibition and L-type Ca2+ channel blockade to sphingosine-induced vasorelaxation requires further study.
We found that sphingosine-1-phosphate (10 μM) caused a modest contraction of pig pulmonary arterial rings but did not contract pig aortic rings. Sphingosine-1-phosphate binds to cell membrane receptors named S1P2 and S1P3, which are G-protein-coupled receptors formerly known as endothelial differentiation gene (EDG) receptors. S1P2 is suspected to be specific for cerebral blood vessels, whereas S1P3 is believed to be present in both central and peripheral vessels (Salomone et al., 2003) including aorta and pulmonary artery; S1P3 activation therefore provides the most likely pathway for sphingosine-1-phosphate mediated pulmonary artery contraction in pigs. Additionally, previous studies have determined that a similar concentration of sphingosine-1-phosphate (10 μM) caused a modest contraction of isolated rat mesenteric and intrarenal microvessels (Bischoff et al., 2000) and canine coronary arteries (Sugiyama et al., 2000), but failed to contract rat carotid and femoral artery rings, mouse aortic rings (Salomone et al., 2003), or rat aortic rings (Coussin et al., 2002). In contrast, much lower concentrations (0.1 μM) of sphingosine-1-phosphate contracted dog, rat, mouse, and rabbit basilar and middle cerebral arteries (Tosaka et al., 2001; Salomone et al., 2003) and rat portal veins (Ikeda et al., 2004), and even lower concentrations of sphingosine-1-phosphate (0.01 μM) transiently relaxed rat mesenteric arterioles via endothelium dependent nitric oxide production (Dantas et al., 2003). Taken together, these results indicate marked site specific differences in the effect of sphingosine-1-phosphate on vascular smooth muscle. The absence of sphingosine-1-phosphate-mediated contraction of pig and mouse aortic rings and rat carotid and femoral arteries has been attributed to differences in S1P isoform receptor density in different vascular beds (Coussin et al., 2002; Mazurais et al., 2002).
The EC50 for KCl (22 mM) for second order pulmonary artery rings in this study was similar to that (25 mM) for third and fifth order pulmonary artery rings (Zellers and Vanhoutte, 1989). However, the EC50 for phenylephrine of second order pulmonary artery rings (10–6.7) and aortic rings (10–6.4) was lower than that for third and fifth order pulmonary artery rings (10–5.2 and 10–5.6, respectively; Zellers and Vanhoutte, 1989), first order pulmonary artery rings (10–5.7; Gustin et al., 1993), and 10–5.4 for anterior mesenteric artery rings in pigs (Nielsen et al., 1991).
Our in vitro finding that sphinganine and sphingosine have vasorelaxant effects is consistent with our previous in vivo finding that fumonisin ingestion alters the aortic input impedance spectrum of pigs (Constable et al., 2003). Because fumonisin ingestion results in toxic effects on the heart and vasculature, fumonisin should be considered as a cardiovascular toxin. The relative importance of cardiac and vascular toxicity following fumonisin ingestion may vary from species to species. Cardiac toxicity appears relatively more important in pigs because they die from acute left-sided heart failure (Smith et al., 1999), whereas vascular toxicity may be more important in horses because they die from vasogenic cerebral edema and leukoencephalomalacia that has been attributed to inadequate autoregulation of cerebral blood flow (Foreman et al., 2004; Smith et al., 2002).
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