1 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. 2 Sphingolipid-induced vascular e ects 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. 3 SPPC and SPP (0.1 ± 100 mmol l 71 ) 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 e ect of SPPC in mesenteric microvessels was stereospeci®c (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 e ects, while indomethacin reduced them (E max from 87+3 to 70+4%). 4 SPP and SPPC caused transient increases in intracellular Ca 2+ concentrations in rat aortic smooth muscle cells in a pertussis toxin-sensitive manner. 5 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 e ects appear to occur via receptors coupled to pertussis toxin-sensitive G-proteins. This is the ®rst demonstration of e ects of SPP and SPPC on vascular tone and suggests that sphingolipids may be an hitherto unrecognized class of endogenous regulators of vascular tone.

1 Sphingolipids such as sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine constrict isolated rat intrarenal and mesenteric microvessels in vitro. The present study investigates their e ects on the cardiovascular system in vivo in anaesthetized rats. 2 The animals were given intravenous or intrarenal arterial bolus injections of sphingolipids (0.1 ± 100 mg kg 71 ) with subsequent measurements of mean arterial pressure, heart rate and renal and mesenteric blood¯ows (RBF, MBF) using a pressure transducer and electromagnetic¯ow probes, respectively. 3 Intravenous injection of SPP rapidly (within 30 s), transiently and dose-dependently reduced RBF (maximally 74.0+0.3 ml min 71 ) and MBF (maximally 71.4+0.2 ml min 71 ), without a ecting mean arterial pressure or heart rate. Other sphingolipids had no signi®cant e ect. 4 Intrarenal arterial SPP administration caused greater blood¯ow reductions (maximally 76.4+0.3 ml min 71 ) than systemic administration. Upon intrarenal administration, sphingosylphosphorylcholine also lowered RBF (maximally 72.8+0.6 ml min 71 ), while the other sphingolipids remained without e ect. 5 Pretreatment with pertussis toxin (PTX, 10 mg kg 71 ) 3 days before the acute experiment abolished the SPP-induced reductions of RBF and MBF. 6 These data demonstrate, that SPP is a potent vasoconstrictor in vivo, particularly in the renal vasculature, while the other structurally related sphingolipids had little if any e ects. The PTXsensitivity strongly suggests that the e ects of SPP on renal and mesenteric blood¯ow are mediated by receptors coupled to G i -type G-proteins.

1 We have compared the signalling mechanisms involved in the pertussis toxin-sensitive and -insensitive contraction of rat isolated mesenteric microvessels elicited by sphingosylphosphorylcholine (SPC) and noradrenaline (NA), respectively. 2 The phospholipase D inhibitor butan-1-ol (0.3%), the store-operated Ca 2+ channel inhibitor SK&F 96,365 (10 mM), the tyrosine kinase inhibitor genistein (10 mM), and the src inhibitor PP2 (10 mM) as well as the negative controls (0.3% butan-2-ol and 10 mM diadzein and PP3) had only little e ect against either agonist. 3 Inhibitors of phosphatidylinositol-3-kinase (wortmannin and LY 294,002, 10 mM each) or of mitogen-activated protein kinase kinase (PD 98,059 and U 126, 10 mM each) did not consistently attenuate NA-and SPC-induced contraction as compared to their vehicles or negative controls (LY 303,511 or U 124). 4 The phospholipase C inhibitor U 73,122 (10 mM) markedly inhibited the SPC-and NA-induced contraction (70% and 88% inhibition of the response to the highest NA and SPC concentration, respectively), whereas its negative control U 73,343 (10 mM) caused only less than 30% inhibition. 5 The rho-kinase inhibitors Y 27,632 (10 mM) and fasudil (30 mM) caused a rightward-shift of the NA concentration-response curve by 0.7 ± 0.8 log units and reduced the response to 10 mM SPC by 88% and 83%, respectively. 6 These data suggest that SPC and NA, while acting on di erent receptors coupling to di erent G-protein classes, elicit contraction of rat mesenteric microvessels by similar signalling pathways including phospholipase C and rho-kinase.

Since extracts from the plants Rhois aromatica and Solidaginis virgaurea are being used in the phytotherapy of bladder dysfunction including the overactive bladder syndrome, and since muscarinic receptors are the main pharmacological target in the treatment of bladder dysfunction, we have investigated whether these extracts can inhibit carbachol-induced, muscarinic receptor-mediated contraction of rat and human bladder. In vitro contraction experiments were performed with rat and human bladder strips. Radioligand binding and inositol phosphate accumulation studies were done with cells transfected with human M(2) or M(3) muscarinic receptors. Both extracts concentration-dependently (final concentrations 0.01-0.1%) inhibited carbachol-induced contraction of rat and human bladder with insurmountable antagonism. Radioligand binding experiments and inositol phosphate accumulation studies with cloned receptors demonstrated direct but non-competitive effects on muscarinic receptors. Reductions of KCl-induced bladder contraction demonstrated that inhibition by the higher extract concentrations also involved receptor-independent effects. We conclude that extracts from Rhois aromatica and Solidaginis virgaurea inhibit muscarinic receptor-mediated contraction of rat and human bladder. While this could contribute to the beneficial effects of these extracts in patients with bladder dysfunction, such therapeutic effects remain to be demonstrated in controlled clinical studies.

1 We have investigated the vasodilating eects of D-erythro-C2-ceramide (C2-ceramide) in methoxamine-contracted rat mesenteric microvessels. 2 C2-ceramide (10 ± 100 mM) caused a concentration-dependent, slowly developing relaxation which reached maximum values after &10 min and partially abated thereafter. 3 Endothelium removal or inhibitors of guanylyl cyclase (3 mM ODQ), protein kinase A (10 mM H7, 1 mM H89) and various types of K + channels (10 mM BaCl 2 , 3 mM tetraethylammonium, 30 nM charybdotoxin, 30 nM iberiotoxin, 300 nM apamine, 10 mM glibenclamide) had only small if any inhibitory eects against C2-ceramide-induced vasodilation, but some of them attenuated vasodilation by sodium nitroprusside or isoprenaline. A combination of ODQ and charybdotoxin almost completely abolished C2-ceramide-induced vasodilation. 4 A second administration of C2-ceramide caused a detectable but weaker relaxation. L-threo-C2-ceramide (100 mM), which should not be a substrate to ceramide metabolism, had no biphasic time course. The ceramidase inhibitor (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (100 mM) alone caused some vasodilation, indicating vasodilation by endogenous ceramides, and also hastened relaxation by exogenous C2-ceramide. The late-developing reversal of C2-ceramideinduced vasodilation was absent when a-adrenergic tone was removed by addition of 10 mM phentolamine. 5 We conclude that C2-ceramide relaxes rat resistance vessels in an endothelium-independent manner which is prevented only by combined inhibition of guanylyl cyclase and charybdotoxinsensitive K + channels. The vasodilation abates with time partly due to desensitization of the ceramide response and partly due to metabolism of C2-ceramide to an inactive metabolite.