Sphingosine-1-phosphate and sphingosylphosphorylcholine constrict renal and mesenteric microvessels in vitro

Abstract

Sphingolipids such as sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine (SPPC) can act both intracellularly and at G-protein-coupled receptors, some of which were cloned and designated as Edg-receptors. Sphingolipid-induced vascular effects were determined in isolated rat mesenteric and intrarenal microvessels. Additionally, sphingolipid-induced elevations in intracellular Ca(2+) concentration were measured in cultured rat aortic smooth muscle cells. SPPC and SPP (0.1-100 micromol l(-1)) caused concentration-dependent contraction of mesenteric and intrarenal microvessels (e.g. SPPC in mesenteric microvessels pEC(50) 5.63+/-0.17 and E(max) 49+/-3% of noradrenaline), with other sphingolipids being less active. The vasoconstrictor effect of SPPC in mesenteric microvessels was stereospecific (pEC(50) D-erythro-SPPC 5.69+/-0.08, L-threo-SPPC 5.31+/-0.06) and inhibited by pretreatment with pertussis toxin (E(max) from 44+/-5 to 19+/-4%), by chelation of extracellular Ca(2+) with EGTA and by nitrendipine (E(max) from 40+/-6 to 6+/-1 and 29+/-6%, respectively). Mechanical endothelial denudation or NO synthase inhibition did not alter the SPPC effects, while indomethacin reduced them (E(max) from 87+/-3 to 70+/-4%). SPP and SPPC caused transient increases in intracellular Ca(2+) concentrations in rat aortic smooth muscle cells in a pertussis toxin-sensitive manner. Our data demonstrate that SPP and SPPC cause vasoconstriction of isolated rat microvessels and increase intracellular Ca(2+) concentrations in cultured rat aortic smooth muscle cells. These effects appear to occur via receptors coupled to pertussis toxin-sensitive G-proteins. This is the first demonstration of effects of SPP and SPPC on vascular tone and suggests that sphingolipids may be an hitherto unrecognized class of endogenous regulators of vascular tone.

Figure 1

Effects of sphingolipids on rat mesenteric and renal microvessels in vitro. Data are mean±s.e.mean (n=8–10) and normalized as % of maximal noradrenaline values (≈18 and 6 mN for mesenteric and intrarenal vessels, respectively). SPP, SPPC, SPH, PSY, and GLU concentration-dependently constricted isolated mesenteric (A) and intrarenal (B) microvessels.

Figure 2

Effects of SPPC diastereoisomers on rat mesenteric microvessels in vitro. Data are mean±s.e.mean (n=8–14) and normalized as % of maximal noradrenaline values (≈16 mN). D-erythro-SPPC was significantly more potent and efficient than L-threo-SPPC (P<0.05, see results) with intermediate values for racemic SPPC.

Figure 3

Effect of PTX treatment (10 μg kg⁻¹ 3 days before the experiment) on SPPC-induced vasoconstriction in rat mesenteric microvessels in vitro. Data are mean±s.e.mean (n=16–17) and normalized as % of maximal noradrenaline values (≈20 mN). In microvessels of PTX-treated rats, the SPPC-induced vasoconstriction was significantly reduced compared to vehicle-treated animals (P<0.05, see results).

Figure 4

Effects of EGTA (0.5 mmol l⁻¹ with nominally Ca²⁺-free buffer in the organ bath) and nitrendipine (300 nmol l⁻¹) treatment on SPPC-induced contraction of rat mesenteric microvessels in vitro. Data are mean±s.e.mean (n=11–13) and normalized as % of maximal noradrenaline values (≈18 mN). Nitrendipine significantly attenuated SPPC-induced vasoconstriction (P<0.05 in a two-way analysis of variance) compared to controls, while EGTA abolished it.

Figure 5

Effects of endothelial mediators on SPPC-induced contraction of rat mesenteric microvessels in vitro. Data are mean±s.e.mean (n=8–9) and normalized as % of maximal noradrenaline values (≈13 mN and 19 mN for A and B, respectively). Effects of N^G-nitro-L-arginine (L-NA, 1 mmol l⁻¹) and indomethacin (10 μmol l⁻¹). L-NA alone did not affect SPPC-induced vasoconstriction, while indomethacin alone or in combination with L-NA slightly but significantly decreased the response (P<0.05 in a two-way analysis of variance (A). Effects of SPPC in intact and denuded mesenteric microvessels which did not differ significantly in their responsiveness in a two-way analysis of variance (B).

Figure 6

Original recordings of elevations of intracellular Ca²⁺ concentrations by SPP and SPPC in cultured rat aortic smooth muscle cells. Intracellular Ca²⁺ concentrations were measured in Fura2-loaded cells pretreated with or without 100 ng ml⁻¹ PTX for 16 h. The arrows indicate time points of addition of SPP (1 μmol l⁻¹) or SPPC (10 μmol l⁻¹). SPP (A) and SPPC (C) rapidly and transiently elevated intracellular Ca²⁺ concentrations with a larger increase upon stimulation with SPP compared to SPPC. The effect of SPP and SPPC was strongly attenuated by PTX (B, D).