INTRODUCTION

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ACCUMULATING EVIDENCE SUGGESTS that the sphingolipid signaling pathway plays an important role in regulating vascular tone in health and disease (41, 42). Two of the major intermediaries in the sphingolipid signaling pathway, ceramide and sphingosine, are suspected to mediate some of the vascular effects of endotoxemia (17, 22; Fig. 1), and another component of the pathway, sphingosine-1-phosphate, is a vasoactive molecule released by platelets (36). Acute endotoxemia is accompanied by profound systemic arterial hypotension, which results primarily from vascular smooth muscle relaxation rather than decreased cardiac contractility (4). The endothelium-dependent component of endotoxin-induced hypotension is believed to be due to relaxation of vascular smooth muscle in response to activation of endothelial nitric oxide synthase and generation of nitric oxide, as well as endothelial synthesis of prostanoids (9, 31). The mechanism for the endothelium-independent component of endotoxin-induced hypotension is currently uncertain but is probably mediated by tumor necrosis factor- (TNF-) (17), which induces endothelium-independent vasodilation through phospholipase A₂-dependent activation of a plasma membrane-associated, neutral pH-operating, magnesium-dependent form of sphingomyelinase that hydrolyzes membrane sphingomyelin to ceramide (17) and ultimately sphingosine or sphingosine-1-phosphate (Fig. 1). Sphingosine relaxes prostaglandin F₂-contracted pig coronary arteries (22) and phenylephrine- contracted pig thoracic aorta (13). Ceramide relaxes phenylephrine-contracted rat mesenteric arteries (18). A membrane-permeable form of ceramide, C₂-ceramide, relaxes phenylephrine-contracted rat thoracic aorta and decreases phenylephrine-induced elevations in intracellular Ca²⁺ concentration ([Ca²⁺]_i) (15-17, 41); similar results were reported after administration of C₈-ceramide-1-phosphate and sphingosine (42). The vascular smooth muscle response to sphingosine and ceramide is consistent with blockade of L-type Ca²⁺ channels (41).

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Sphingolipid biosynthetic pathway and sphingomyelin pathway. Fumonisin inhibits ceramide synthase (sphinganine/sphingosine-N-acyltransferase) in the sphingolipid biosynthetic pathway, increasing intracellular sphinganine and sphingosine concentrations. The sphingomyelin pathway involves activation of neutral sphingomyelinase. Endotoxin causes the release of tumor necrosis factor- (TNF-), which binds to the TNF- receptor on the cell membrane, activates phospholipase A2, and stimulates the activity of a plasma membrane-associated, neutral pH-operating, magnesium-dependent form of sphingomyelinase. Hydrolysis of membrane sphingomyelin produces ceramide and ultimately sphingosine and sphingosine-1-phosphate. Both ceramide and sphingosine relax vascular smooth muscle, probably by decreasing Ca2+ entry through L-type Ca2+ channels and inhibiting intracellular Ca2+ release from the ryanodine receptor of sarcoplasmic reticulum. The X-and-arrow symbol indicates the enzymatic pathway inhibited by fumonisin.

Fumonisin provides a valuable pharmacological tool for altering the sphingolipid signaling pathway because it is a potent inhibitor of ceramide synthase (sphinganine/sphingosine N-acyltransferase) (38), a key enzyme in the pathway for de novo sphingolipid biosynthesis (Fig. 1). Fumonisins are a group of mycotoxins produced primarily by Fusarium verticillioides, a fungus that commonly contaminates corn. Ingestion of feed contaminated with fumonisin causes leukoencephalomalacia in horses (39) and pulmonary edema in pigs (10, 11, 28, 34, 35). Fumonisin-induced ceramide synthase inhibition results in increased concentrations of sphinganine and sphingosine in serum and tissues (28, 38; Fig. 1), accompanied by decreased cardiac contractility, heart rate, and cardiac output in pigs (5, 32, 34, 35) and horses (33), and systemic arterial hypotension in pigs (35). Fumonisin mycotoxicosis in pigs and horses provides a naturally occurring example of morbidity and mortality due to abnormal sphingolipid metabolism, thereby providing a valuable model for characterizing the cardiovascular functions of the sphingolipid signaling pathway in health and disease.

Alteration of the sphingolipid signaling pathway by administration of fumonisin increases systemic vascular resistance (SVR) in pigs (5, 32, 35) and horses (33). The increase in SVR reflected constriction of systemic resistance vessels or was a mathematical result of its method of calculation, where SVR = [mean arterial pressure  central venous pressure]/cardiac output, because affected animals had decreased cardiac output. We hypothesized that the fumonisin-induced increase in SVR was due to contraction of systemic resistance vessels in response to decreased cardiac contractility, heart rate, cardiac output, and sphinganine or sphingosine-induced relaxation of systemic conduit vessels. Therefore, the objectives of this study were to characterize the in vivo effect of altering the sphingolipid signaling pathway by blocking ceramide synthase on systemic conduit and resistance vessels, and to determine whether the vascular responses to standard pharmacological agents (isoproterenol, nitroprusside, phenylephrine, and Ca²⁺) were similar in fumonisin-fed pigs and control pigs. Isoproterenol, the classic ₁- and ₂-agonist, was used to examine the effect of altered sphingolipid signaling pathway on ₂-mediated vasodilation. Nitroprusside, the direct acting vascular smooth muscle relaxant, was used to examine the effect of altered sphingolipid signaling pathway on systemic arterial vasodilation. Phenylephrine, the classic ₁-agonist, was used to examine the effect of altered sphingolipid signaling pathway on systemic arterial vasoconstriction. Ca²⁺ was administered to examine the effect of increased plasma [Ca²⁺] on hemodynamic values. To achieve our objectives, we measured the aortic input impedance spectrum in -chloralose-anesthetized pigs.

	MATERIALS AND METHODS

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Animals and treatment. This study was approved by our institutional committee on the care and use of laboratory animals. Sixteen healthy castrated male cross-bred pigs weighing 44 ± 4 kg (means ± SD) were housed individually in stalls 5-7 days before treatment. Pigs had free access to water and were fed an 18% protein grower diet that was free of fumonisin B₁ (detection limit <0.1 ppm), fumonisin B₂ (detection limit <0.1 ppm), aflatoxin, vomitoxin, T-2 toxin, ochratoxin, and zearalenone. Culture material containing a high concentration of fumonisin was prepared and analyzed as described previously (35). At the end of the acclimation period, a random number table was used to assign pigs to one of two groups. Treated pigs (n = 7) received culture material containing 20 mg · kg¹ · day¹ fumonisin B₁ mixed into the grower diet, whereas control pigs (n = 9) were fed only the grower diet on the same schedule as treated pigs. Feed consumption was monitored to ensure that the pigs ingested the fumonisin dose. In our laboratory, this fumonisin dose causes pulmonary edema and death within 3-5 days of initial exposure.

Instrumentation. Seventy-two hours after the start of fumonisin administration, the pigs were anesthetized by intramuscular injection of 2 mg/kg xylazine and 10 mg/kg ketamine hydrochloride, followed by mask induction with 3-5% halothane in 100% O₂. Pigs were then orotracheally intubated, placed in dorsal recumbency on a water-circulating heating blanket, and ventilated with supplemental oxygen (4 l/min) on a volume respirator (Harvard Apparatus) at a tidal volume of 10 ml/kg and respiratory frequency of 20 breaths/min. Anesthesia was subsequently maintained by intravenous administration of -chloralose (50 mg/kg initially, then 15 mg · kg¹ · h¹; Sigma; St. Louis, MO) and butorphanol (0.5 mg/kg im, every 3 h). This anesthetic protocol produces minimal cardiovascular depression in pigs (5). Arterial pH, PCO₂, and PO₂ were measured periodically during instrumentation and maintained within normal limits (pH 7.40-7.50; Pa_CO2, 35-45 mmHg; Pa_O2, >100 mmHg) by adjusting tidal volume. Ventilator settings were not altered once instrumentation was completed. Pulmonary artery blood temperature was monitored during the study, and the temperature of the heating blanket was adjusted to maintain normal blood temperature (39.6 ± 0.6°C). Lactated Ringer solution (4 ml · kg¹ · h¹) was administered intravenously for the duration of the study.

Aortic pressure signal calibration. The aortic pressure-flow catheter was presoaked in 0.9% NaCl solution at 37°C for at least 30 min before use and calibrated in vitro with a transducer control unit (Millar Instruments). Zero pressure reference was obtained with the pressure sensor barely submerged in the isotonic saline solution. Pressure transducers had a range of 50-300 mmHg, flat frequency response to 10 kHz, no phase or amplitude error, drift of <6 mmHg in 12 h, and natural frequency of 35 kHz or greater. Because the pressure recording system had no amplitude or phase error at the studied frequencies (<15 Hz), specific adjustments of the pressure signal were not undertaken when characterizing the aortic impedance spectrum and hydraulic power spectrum.

Aortic flow-velocity signal calibration. The electromagnetic flow-velocity signal (cm/s) was calibrated in terms of volumetric flow (cm³/s) during each hemodynamic recording period by in vivo calibration against simultaneously obtained thermodilution stroke volumes (the mean of three determinations at constant heart rate). The area under the aortic flow-velocity curve was integrated to provide electromagnetic stroke volume, and the gain on the computer was altered to ensure equivalence of the two stroke volumes. This calibration technique was required because the electromagnetic transducer signal was proportional to flow velocity instead of volumetric flow, and assumed the last third of the diastolic flow signal represented zero flow, the velocity profile in the region studied was flat, and that the ascending aortic diameter was constant throughout ejection (25).

Experimental protocol. Arterial blood was obtained as soon as an artery was catheterized and serum harvested for subsequent sphinganine and sphingosine assays and serum biochemical analyses. After instrumentation had been completed, pigs were monitored for 15 min to ensure hemodynamic stability before obtaining baseline measurements. The cardiovascular response to sequential intravenous administration of the following pharmacological agents was examined: isoproterenol HCl (0.04 µg · kg¹ · min¹; Isuprel, Sanofi Winthrop Pharmaceuticals, New York, NY); sodium nitroprusside (4 µg · kg¹ · min¹; Nitropress, Abbott; N. Chicago, IL); phenylephrine HCl (4 µg · kg¹ · min¹; United Research Laboratories, Philadelphia, PA); and Ca²⁺ (4 mg · kg¹ · min¹; as Ca²⁺ gluconate). Each pharmacological agent was infused for at least 5 min until stable hemodynamic values were obtained. After each pharmacological agent was infused, hemodynamic parameters were monitored for at least 15 min until they were stable and had returned to preinfusion values. Pigs were euthanized at the end of the study by administration of pentobarbital sodium (60 mg/kg iv), and left ventricular tissue was harvested.

Sphingolipid analyses. Serum derived from arterial blood and left ventricular tissue were stored at 20°C and thawed immediately before determination of free sphinganine and sphingosine concentrations by using previously described methods (35).

Blood gas analysis. Blood gas analyses were performed immediately by the platinum and glass electrode technique, blood hemoglobin concentration was determined with the use of spectrophotometry, and plasma ionized [Ca²⁺] was determined with the use of an ion-specific electrode (model ABL330, Radiometer; Copenhagen, Denmark).

Hemodynamic analyses. Hemodynamic data were analyzed off-line with the use of a personal computer and custom-designed software. End diastole was defined as the point corresponding to the peak of the R wave of the ECG. This reference point was used for signal averaging techniques. The calibrated ascending aortic pressure and flow-velocity waveforms from 10 consecutive beats were signal averaged to minimize noise in the pressure and flow-velocity recording systems, and submitted to Fourier analysis. The noise level of the flow-velocity and pressure signals were determined for each pig during baseline by performing Fourier analysis on the diastolic portion of the aortic flow and pressure signal. Only flow harmonics with moduli greater than the maximum noise level for the flow-velocity signal (typically 8 cm/s or ~2% of peak flow) or pressure (typically 0.2 mmHg or ~0.2% of systolic arterial pressure) were included in subsequent calculations. The phase lag of 1.3/Hz (6 ms) for the flow-velocity signal also was accounted for in subsequent calculations. Postectopic beats and those beats with suboptimal flow-velocity signals were excluded from analysis.

Aortic impedance spectrum. The input impedance modulus and phase angle for each harmonic were calculated as the ratio of the pressure to flow moduli and the difference between the pressure and flow phase angles, respectively, with the convention that a negative-phase angle indicated that flow leads pressure (24). Characteristic impedance (Z_c) was calculated from the average of impedance moduli after the first minimum to the noise limit for the flow recording system. A reflectance index, peripheral resistance (Z₀), was calculated from the ratio of mean pressure to mean flow; Z₀ represents the static (nonpulsatile) component of input impedance (Z_i) and approximates total peripheral resistance when right atrial pressure is low (24), as in this study. The reflection coefficient at the zero, first, and second harmonics (₀, ₁, and ₂) were calculated from the frequency-dependent reflection coefficient spectrum by using the ratio (Z_i  Z_c)/(Z_i + Z_c) (1). Reflectance characteristics were also assessed by determining the frequency of the first minimum impedance modulus and by estimating the frequency for the first zero crossing of phase angle (phase 0), which was obtained by linear interpolation (24).

Aortic hydraulic power spectrum. Mean hydraulic pressure power (W_M), oscillatory hydraulic pressure power (W_o), and total hydraulic pressure power (W_T) were calculated from the hydraulic power spectrum (24). The ratio of oscillatory to total hydraulic pressure power output (W_o/W_T) was also determined to assess the efficiency of energy transfer from the left ventricle to the systemic circulation (24, 40).

Statistical analysis. Data are presented as means ± SD. Two-way analysis of variance (treatment and pharmacological agent) with repeated measures on one factor (pharmacological agent) was used for comparison. Appropriate post tests were conducted whenever the F test was significant (P < 0.05). Multiple pairwise comparisons were conducted between or within treatment groups by using the Bonferroni inequality to keep the experimentwide error rate at P < 0.05 for each family of comparisons. Within-treatment group comparisons were to baseline values. Between-treatment group comparisons for each variable were made for each pharmacological agent. Variables with nonnormal distributions or unequal variances were log transformed or ranked before analysis of variance was performed.

	RESULTS

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Sphingolipid analyses. Fumonisin-treated pigs had increased serum and left ventricular sphinganine and sphingosine concentrations, as well as increased serum and left ventricular sphinganine to sphingosine ratios compared with controls (Table 1).

Blood gas analysis. Fumonisin-treated pigs had an increased blood hemoglobin concentration but similar blood pH, PCO₂, PO₂, bicarbonate concentration, base excess, and plasma ionized [Ca²⁺] to controls (Table 1).

Hemodynamic parameters. Fumonisin-treated pigs had a lower cardiac output and stroke volume than control pigs, but similar heart rate, mean arterial pressure, aortic dP/dt_max, mean pulmonary artery pressure, and mean central venous pressure, peak aortic flow, and aortic dQ/dt_max (Table 2). The time from end diastole to aortic dQ/dt_max and dP/dt_max was longer in fumonisin-treated pigs than in controls. Taken together, these hemodynamic changes indicated decreased left ventricular contractility in fumonisin-treated pigs.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Aortic input impedance spectrum and pressure (P) and flow velocity (Q) waves in the ascending aorta of pigs with ceramide synthase blockade (fumonisin, n = 7) or pigs with a normal sphingolipid biosynthetic pathway (control, n = 9).

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Effect of intravenous administration of isoproterenol HCl on the aortic input impedance spectrum and pressure and flow-velocity waves in the ascending aorta of pigs with ceramide synthase blockade (fumonisin, n = 7) or pigs with a normal sphingolipid biosynthetic pathway (control, n = 9).

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Effect of intravenous administration of sodium nitroprusside on the aortic input impedance spectrum and pressure and flow-velocity waves in the ascending aorta of pigs with ceramide synthase blockade (fumonisin, n = 7) or pigs with a normal sphingolipid biosynthetic pathway (control, n = 9).

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Effect of intravenous administration of phenylephrine HCl on the aortic input impedance spectrum and pressure and flow-velocity waves in the ascending aorta of pigs with ceramide synthase blockade (fumonisin, n = 7) or pigs with a normal sphingolipid biosynthetic pathway (control, n = 9).

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Effect of intravenous administration of Ca2+ gluconate on the aortic input impedance spectrum and pressure and flow-velocity waves in the ascending aorta of pigs with ceramide synthase blockade (fumonisin, n = 7) or pigs with a normal sphingolipid biosynthetic pathway (control, n = 9).

Aortic impedance spectrum. The impedance spectra of fumonisin-treated pigs differed from that of control pigs (Fig. 2 and Table 3), as indicated by a lower value for Z_c, higher values for Z₀, the frequency of the first minimum impedance modulus, and the reflection coefficient at the ₀, ₁, and ₂, and a tendency (P = 0.075) toward an increased value for phase 0. Because mean arterial pressure was similar for both groups (Table 2), these changes in the aortic impedance spectrum indicate relaxation of systemic conduit vessels (decreased Z_c) and contraction of systemic resistance vessels (increased Z₀, ₀, ₁, and ₂, and frequency of first minimum impedance modulus) in fumonisin-treated pigs.

Aortic hydraulic power spectrum. Fumonisin-treated pigs had similar W_M, W_T, W_o/W_T, and W_T/cardiac output (CO), but lower W_o, than control pigs (Table 3). The absence of major changes in the aortic hydraulic power spectrum was indicative that left ventricular aortic coupling was not altered energetically in fumonisin-treated pigs.

	DISCUSSION

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Blocking ceramide synthase by administering fumonisin B₁ increased serum and left ventricular sphinganine and sphingosine concentrations and decreased cardiac output. These changes were consistent with those previously reported in pigs ingesting high daily doses of fumonisin B₁ (>16 mg/kg; see Ref. 11 for review). The major new findings of this study were that altering the sphingolipid signaling system by blocking ceramide synthase caused marked changes in aortic pressure and flow-velocity waves, characterized by decreased vascular tone in systemic conduit vessels and increased vascular tone in systemic resistance vessels. Moreover, ceramide synthase-blocked pigs appeared to have a greater dependence on constriction of systemic resistance vessels for maintaining normal mean arterial pressure, and an increased sensitivity to isoproterenol, nitroprusside, and phenylephrine administration.

The aortic input impedance spectrum provides a quantitative and independent method for characterizing the response of the systemic circulation to an agent that affects the cardiovascular system because it incorporates both steady-state and pulsatile aspects of SVR. The aortic impedance spectrum is determined by the heart rate, mean arterial pressure, the compliance and vascular tone of the proximal aorta, the extent of constriction of systemic resistance vessels, and the distance from the proximal aorta to peripheral reflecting sites (26). The difference in impedance spectra between ceramide synthase-blocked pigs and control pigs (higher values for Z₀, phase 0, and frequency of first minimum impedance modulus, and lower value for Z_c for fumonisin-treated pigs) mirrored those in dogs administered the direct acting vasodilator agent nitroprusside, indicating that fumonisin-treated pigs have relaxation of systemic conduit vessels and contraction (presumably reflex) of systemic resistance vessels (37). Vasodilation typically is accompanied by a decrease in Z₀, loss of the discrete minimal modulus value, and a decrease in phase fluctation (26, 27), as observed in the impedance spectra of fumonisin-treated and control pigs administered isoproterenol and nitroprusside. The increase in the frequency of phase 0 and first minimum accompanied the increase in mean aortic pressure after Ca²⁺ and phenylephrine administration, and the decrease in the frequency of phase 0 and first minimum accompanied the decrease in mean aortic pressure after isoproterenol and nitroprusside administration. These changes were due in part to altered reflection because of a changes in blood pressure and in vascular tone (1, 26).

The results of this study clearly demonstrate that alterations in the sphingolipid biosynthetic pathway affects the systemic vasculature of pigs. This response may have been mediated by increased serum sphinganine concentration (0.004 µM in control pigs; 0.165 µM in treated pigs), increased serum sphingosine concentration (0.017 µM in control pigs; 0.032 µM in treated pigs), or vascular smooth muscle sphinganine and sphingosine concentrations (not measured). We believe that both serum and vascular smooth muscle sphingolipid concentrations were increased in the pigs in this study because we (6) recently determined that daily ingestion of 0.22-0.66 mg/kg body wt fumonisin B₁ for 6 mo increased aortic sphinganine and sphingosine concentrations in Sinclair minipigs; for comparison, the pigs in the study reported here ingested 20 mg/ kg body wt fumonisin B₁ each day. In pigs, relaxation of phenylephrine contracted thoracic aortic rings occurred when sphinganine 0.1 µM and sphingosine 1 µM (13), and relaxation of prostaglandin F₂-contracted pig coronary arteries occurred when sphingosine 3 µM (22). Taken together, these results suggest that sphinganine may be an important vasodilator agent in pigs administered fumonisin; however, a role for sphinganine in vascular reactivity has not been extensively examined. Sphingosine reversibly and potently inhibits L-type Ca²⁺ channels (EC₅₀ = 0.1 µM) and ryanodine receptors (EC₅₀ = 0.5 µM) of rabbit skeletal muscle fibers (29), and the changes in the aortic input impedance spectrum in pigs administered fumonisin were consistent with sphingosine-mediated L-type Ca²⁺ channel blockade. Because arterial smooth muscle tone is believed to be regulated primarily by L-type Ca²⁺ channel activity (23), sphingosine-induced inhibition of systemic arterial smooth muscle L-type Ca²⁺ channels, if present, would induce vasodilation. However, there may be species-specific differences in the vascular effects of sphingolipids, as sphingosine (10 µM) did not relax the phenylephrine-contracted rat thoracic aorta (15), and ceramide did not relax or contract large bovine coronary arteries but did contract small bovine coronary arteries (20).

Ceramide synthase blockade increased the sensitivity of systemic resistance vessels to isoproterenol, nitroprusside, and phenylephrine. The increased sensitivity to the vasodilator agents isoproterenol and nitroprusside was due, in part, to decreased cardiovascular reserve, which we have documented previously in pigs ingesting similar doses of fumonisin (5, 32, 34, 35). The mechanism for the increased sensitivity to the vasoconstrictor agent phenylephrine is probably sphingosine enhancement of 1-receptor signaling because cytosolic sphingosine increases phospholipase C activity, thereby resulting in G protein-coupled enhancement of phosphatidylinositol turnover, increased release of Ca²⁺ from the sarcoplasmic reticulum (3), and vasoconstriction. Another potential pathway by which ceramide synthase blockade increases the sensitivity of systemic resistance vessels to phenylephrine is rapid metabolism of cytosolic sphingosine to sphingosine-1-phosphate by endoplasmic reticulum-bound sphingosine kinase (Fig. 1); sphingosine-1-phosphate then directly induces Ca²⁺ release from intracellular Ca²⁺ stores (7, 8), leading to vasoconstriction.

We did not examine the effect of fumonisin ingestion on the pulmonary circulation. It is possible that ceramide synthase blockade exerts a different physiological response on pulmonary vasculature, as fumonisin B₁ induces pulmonary hypertension in pigs (34, 35) that is accompanied by accumulation of membranous material in the cytocavitary network of the pulmonary capillary endothelium. In contrast, fumonisin B₁ does not appear to cause morphologic changes in the systemic vasculature (10, 11).

The results of this study provide additional information on a potential mechanism for altered vascular responsiveness in endotoxemic shock. Acute endotoxemia causes the release of TNF- (21), and TNF- induces endothelium-dependent and endothelium-independent vasodilation (31). The endothelium-independent TNF- signaling pathway is shown in Fig. 1, which involves an acute signal-transduction mechanism involving phospholipase A₂ activation of neutral sphingomyelinase, hydrolysis of sphingomyelin to ceramide, and conversion of ceramide to sphingosine. Activation of the TNF- signaling pathway will therefore increase ceramide and sphingosine concentrations in vascular smooth muscle; in contrast, ceramide synthase blockade by fumonisin increases sphinganine and sphingosine concentrations in vascular smooth muscle (6) and is widely believed to decrease tissue ceramide concentration. Therefore, the results of this and other studies (13, 22, 41) suggest that in vascular smooth muscle, TNF- induced increased sphingosine concentrations will lead to vasodilation through blockade of L-type Ca²⁺ channels, while increasing the sensitivity to ₁-mediated vasoconstriction. Interestingly, carotid and cranial mesenteric arteries from pigs with acute (3 h) endotoxemia have increased sensitivity to the vasodilator effects of nitroglycerin and the vasoconstrictive effects of norepinephrine (2); these responses are consistent with those expected from TNF- induced increases in vascular smooth muscle sphingosine concentration.

In conclusion, the results of this study in pigs indicate that ceramide synthase blockade caused relaxation of systemic conduit vessels, which was accompanied by contraction of systemic resistance vessels. The vascular effects associated with ceramide synthase blockade were probably due to an increase in plasma and tissue sphinganine or sphingosine concentrations. Because sphingosine is an important physiological mediator in numerous cellular pathways and disease processes, including endotoxic shock, additional studies are warranted to improve our understanding of the mechanism for the vascular effects of altered sphingolipid metabolism.