The sphingolipid derivatives ceramide, sphingosine and sphingosine-1-phosphate (S1P) exert profound effects on cellular homeostasis. Ceramide plays an important role in apoptotic responses to various stress stimuli, whereas S1P is implicated in cell survival and proliferation. Because of these opposing effects and the fact that ceramide can be enzymatically converted to S1P and vice versa, this system is referred to as the ceramide/S1P rheostat. Besides their opposing growth effects, S1P and ceramide have also counteracting effects on vascular contraction and relaxation when applied exogenously. However, it is unknown whether vasoactive compounds actively make use of the endogenous ceramide/S1P rheostat. Therefore, we have investigated if manipulation of the rheostat by inhibition of sphingosine kinase (one of the key enzymes involved in the conversion of ceramide to S1P) influences the vasoconstrictor responses to a variety of vasoactive compounds.

Carotid arteries from male Wistar rats weighing 240-260 g (anaesthetised with 100 mg/kg pentobarbitone i.p.) were isolated and 2mm artery segments were mounted into a wire myograph according to Mulvany et al. (1977). The myograph bath contained a Krebs-Henseleit buffer at 37°C aerated with 5%CO₂/95%O₂, pH=7.4. Isometric force of contraction was measured continuously and is presented as mN/mm segment length. All data are expressed as means ±SE for the number of experiments (n) as indicated. Data are analyzed by Student’s t-test or one-way ANOVA where appropriate. A P value of less than 0.05 is considered significant.

Sphingosine kinase inhibition by 10 µM dimethylsphingosine (DMS) induced a significant leftward shift of the concentration response curves for angiotensin II (pEC₅₀ 9.09±0.08 vs. 8.58±0.04 for control, n=7-8) and endothelin-1 ( pEC₅₀ 9.03±0.10 vs 8.65±0.04, n=7-8) . This was accompanied by a small but significant increase in efficacy of angiotensin II and endothelin-1 (3.47±0.15 mN/mm vs 3.06±0.10 mN/mm and 4.83±0.23 mN/mm vs 4.24±0.12 mN/mm, respectively, n=7-8) . In contrast, DMS had no effect on the concentration response curves for phenylephrine (pEC₅₀ 7.58±0.07 vs 7.60±0.07, E_max 4.15±0.11 mN/mm vs 3.99±0.18 mN/mm, n=5) or KCl (pEC₅₀ 1.97±0.05 vs 1.94±0.03, E_max 3.25±0.16 mN/mm vs 3.34±0.13 mN/mm, n=4-6) . Interestingly, inhibition of nitric oxide synthase by 100 µM N-nitro-L-arginine (L-NNA) mimicked the shift in potency of angiotensin II by DMS (pEC₅₀ 9.07±0.08, n=7), although there was a more pronounced increase in efficacy (E_max 4.46±0.21 mN/mm, n=7, P<0.05 compared to 10 µM DMS). When combined together with 100 m M L-NNA, 10 µM DMS had no additional effects on the concentration response curve for angiotensin II (pEC₅₀ 9,08±0.16, E_max 4.26±0.33 mN/mm, n=7, P>0.05 compared to 100 µM L-NNA alone).

We conclude the ceramide/S1P rheostat is involved in the contractile effects of angiotensin II and endothelin-1, but not in that of phenylephrine or KCl. Angiotensin II and endothelin-1 shift the rheostat into the direction of S1P, which has a vasorelaxant effect in the isolated rat carotid artery segments. This relaxant effect is most likely the result of S1P-mediated NO release.