Sphingosine-1-Phosphate Receptor 1 Regulates Cardiac Function by Modulating Ca2+ Sensitivity and Na+/H+ Exchange and Mediates Protection by Ischemic Preconditioning.

BACKGROUND: Sphingosine-1-phosphate plays vital roles in cardiomyocyte physiology, myocardial ischemia-reperfusion injury, and ischemic preconditioning. The function of the cardiomyocyte sphingosine-1-phosphate receptor 1 (S1P1) in vivo is unknown. METHODS AND RESULTS: Cardiomyocyte-restricted deletion of S1P1 in mice (S1P1 (α) (MHCC) (re)) resulted in progressive cardiomyopathy, compromised response to dobutamine, and premature death. Isolated cardiomyocytes from S1P1 (α) (MHCC) (re) mice revealed reduced diastolic and systolic Ca(2+) concentrations that were secondary to reduced intracellular Na(+) and caused by suppressed activity of the sarcolemmal Na(+)/H(+) exchanger NHE-1 in the absence of S1P1. This scenario was successfully reproduced in wild-type cardiomyocytes by pharmacological inhibition of S1P1 or sphingosine kinases. Furthermore, Sarcomere shortening of S1P1 (α) (MHCC) (re) cardiomyocytes was intact, but sarcomere relaxation was attenuated and Ca(2+) sensitivity increased, respectively. This went along with reduced phosphorylation of regulatory myofilament proteins such as myosin light chain 2, myosin-binding protein C, and troponin I. In addition, S1P1 mediated the inhibitory effect of exogenous sphingosine-1-phosphate on ß-adrenergic-induced cardiomyocyte contractility by inhibiting the adenylate cyclase. Furthermore, ischemic precondtioning was abolished in S1P1 (α) (MHCC) (re) mice and was accompanied by defective Akt activation during preconditioning. CONCLUSIONS: Tonic S1P1 signaling by endogenous sphingosine-1-phosphate contributes to intracellular Ca(2+) homeostasis by maintaining basal NHE-1 activity and controls simultaneously myofibril Ca(2+) sensitivity through its inhibitory effect on adenylate cyclase. Cardioprotection by ischemic precondtioning depends on intact S1P1 signaling. These key findings on S1P1 functions in cardiac physiology may offer novel therapeutic approaches to cardiac diseases.

Sphingosine-1-phosphate receptor 3 mediates sphingosine-1-phosphate induced release of weibel-palade bodies from endothelial cells.

Sphingosine-1-phosphate (S1P) is an agonist for five distinct G-protein coupled receptors, that is released by platelets, mast cells, erythrocytes and endothelial cells. S1P promotes endothelial cell barrier function and induces release of endothelial cell-specific storage-organelles designated Weibel-Palade bodies (WPBs). S1P-mediated enhancement of endothelial cell barrier function is dependent on S1P receptor 1 (S1PR1) mediated signaling events that result in the activation of the small GTPase Rac1. Recently, we have reported that Rac1 regulates epinephrine-induced WPB exocytosis following its activation by phosphatidylinositol-3,4,5-triphosphate-dependent Rac exchange factor 1 (PREX1). S1P has also been described to induce WPB exocytosis. Here, we confirm that S1P induces release of WPBs using von Willebrand factor (VWF) as a marker. Using siRNA mediated knockdown of gene expression we show that S1PR1 is not involved in S1P-mediated release of WPBs. In contrast depletion of the S1PR3 greatly reduced S1P-induced release of VWF. S1P-mediated enhancement of endothelial barrier function was not affected by S1PR3-depletion whereas it was greatly impaired in cells lacking S1PR1. The Rho kinase inhibitor Y27632 completely abrogated S1P-mediated release of VWF. Also, the calcium chelator BAPTA-AM significantly reduced S1P-induced release of VWF. Our findings indicate that S1P-induced release of haemostatic, inflammatory and angiogenic components stored within WPBs depends on the S1PR3.

Dietary fish oil improves endothelial function and lowers blood pressure via suppression of sphingolipid-mediated contractions in spontaneously hypertensive rats.

OBJECTIVE: Long-chain n-3 polyunsaturated fatty acids from oily fish reduce blood pressure (BP) in hypertension. Previously, we demonstrated that hypertension is associated with marked alterations in sphingolipid biology and elevated ceramide-induced vasoconstriction. Here we investigated in spontaneously hypertensive rats (SHRs) whether fish oil improves endothelial function including reduced vascular contraction induced via the sphingolipid cascade, resulting in reduced BP. METHODS: Twelve-week-old SHRs were fed a control or fish oil-enriched diet during 12 weeks, and BP was recorded. Plasma sphingolipid levels were quantified by mass spectrometry and the response of isolated carotid arteries towards different stimuli was measured. Furthermore, erythrocyte membrane fatty acid composition, thromboxane A2 formation and cytokine secretion in ex-vivo lipopolysaccharide-stimulated thoracic aorta segments were determined. RESULTS: The fish oil diet reduced the mean arterial BP (P < 0.001) and improved endothelial function, as indicated by a substantially increased relaxation potential towards ex-vivo methacholine exposure of the carotid arteries (P < 0.001). The long-chain n-3 polyunsaturated fatty acid diet resulted in altered levels of specific (glucosyl)ceramide subspecies (P < 0.05), reduced membrane arachidonic acid content (P < 0.001) and decreased thromboxane concentrations in plasma (P < 0.01). Concomitantly, the fish oil diet largely reduced ceramide-induced contractions (P < 0.01), which are predominantly mediated by thromboxane. Furthermore, thromboxane A2 and interleukin-10 were reduced in supernatants of lipopolysaccharide-stimulated thoracic aorta of SHRs fed the fish oil diet while RANTES (regulated on activation, normal T-cell expressed and secreted) was enhanced. This may contribute to reduced vasoconstriction in vivo. CONCLUSIONS: Dietary fish oil lowers BP in SHRs and improves endothelial function in association with suppression of sphingolipid-dependent vascular contraction.

Sphingosine-1-phosphate receptor-1 controls venous endothelial barrier integrity in zebrafish.

OBJECTIVE: Endothelial sphingosine-1-phosphate (S1P) receptor-1 (S1P(1)) affects different vascular functions, including blood vessel maturation and permeability. Here, we characterized the role of the zS1P(1) ortholog in vascular development in zebrafish. METHODS AND RESULTS: zS1P(1) is expressed in dorsal aorta and posterior cardinal vein of zebrafish embryos at 24 to 30 hours postfertilization. zS1P(1) downregulation by antisense morpholino oligonucleotide injection causes early pericardial edema, lack of blood circulation, alterations of posterior cardinal vein structure, and late generalized edema. Also, zS1P(1) morphants are characterized by downregulation of vascular endothelial cadherin (VE-cadherin) and Eph receptor EphB4a expression and by disorganization of zonula occludens 1 junctions in posterior cardinal vein endothelium, with no alterations of dorsal aorta endothelium. VE-cadherin knockdown results in similar vascular alterations, whereas VE-cadherin overexpression is sufficient to rescue venous vascular integrity defects and EphB4a downregulation in zS1P(1) morphants. Finally, S1P(1) small interfering RNA transfection and the S1P(1) antagonist (R)-3-amino-(3-hexylphenylamino)-4-oxobutylphosphonic acid (W146) cause EPHB4 receptor down-modulation in human umbilical vein endothelial cells and the assembly of zonula occludens 1 intercellular contacts is prevented by the EPHB4 antagonist TNYL-RAW peptide in these cells. CONCLUSIONS: The data demonstrate a nonredundant role of zS1P(1) in the regulation of venous endothelial barrier in zebrafish and identify a S1P(1)/VE-cadherin/EphB4a genetic pathway that controls venous vascular integrity.

FTY720 (fingolimod) increases vascular tone and blood pressure in spontaneously hypertensive rats via inhibition of sphingosine kinase.

BACKGROUND AND PURPOSE: FTY720 (Fingolimod) is a recently approved orally administered drug for the treatment of multiple sclerosis. Phase II and III clinical trials have demonstrated that this drug modestly increases BP. We previously showed that inhibition of sphingosine kinase increases vascular tone and BP in hypertensive, but not normotensive rats. Since FTY720 is reported to have inhibitory effects on sphingosine kinase, we investigated whether FTY720 increases vascular tone and BP only in hypertensive rats via this mechanism. EXPERIMENTAL APPROACH: The contractile and BP modulating effects of FTY720 were studied in vivo and ex vivo (wire myography) in age-matched normotensive Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). KEY RESULTS: Oral administration of FTY720 induced an increase in mean arterial pressure in SHR, whereas a decrease in BP was observed in WKY rats, as measured 24 h after administration. Similar to the sphingosine kinase inhibitor dimethylsphingosine (DMS), FTY720 induced large contractions in isolated carotid arteries from SHR, but not in those from WKY. In contrast, the phosphorylated form of FTY720 did not induce contractions in isolated carotid arteries from SHR. FTY720-induced contractions were inhibited by endothelium denudation, COX and thromboxane synthase inhibitors, and by thromboxane receptor antagonism, indicating that (like DMS-induced contractions) they were endothelium-dependent and mediated by thromboxane A2. CONCLUSIONS AND IMPLICATIONS: These data demonstrate that FTY720 increases vascular tone and BP only in hypertensive rats, most likely as a result of its inhibitory effect on sphingosine kinase.

Antihypertensive treatment differentially affects vascular sphingolipid biology in spontaneously hypertensive rats.

BACKGROUND: We have previously shown that essential hypertension in humans and spontaneously hypertensive rats (SHR), is associated with increased levels of ceramide and marked alterations in sphingolipid biology. Pharmacological elevation of ceramide in isolated carotid arteries of SHR leads to vasoconstriction via a calcium-independent phospholipase A(2), cyclooxygenase-1 and thromboxane synthase-dependent release of thromboxane A(2). This phenomenon is almost absent in vessels from normotensive Wistar Kyoto (WKY) rats. Here we investigated whether lowering of blood pressure can reverse elevated ceramide levels and reduce ceramide-mediated contractions in SHR. METHODS AND FINDINGS: For this purpose SHR were treated for 4 weeks with the angiotensin II type 1 receptor antagonist losartan or the vasodilator hydralazine. Both drugs decreased blood pressure equally (SBP untreated SHR: 191±7 mmHg, losartan: 125±5 mmHg and hydralazine: 113±14 mmHg). The blood pressure lowering was associated with a 20-25% reduction in vascular ceramide levels and improved endothelial function of isolated carotid arteries in both groups. Interestingly, losartan, but not hydralazine treatment, markedly reduced sphingomyelinase-induced contractions. While both drugs lowered cyclooxygenase-1 expression, only losartan and not hydralazine, reduced the endothelial expression of calcium-independent phospholipase A(2). The latter finding may explain the effect of losartan treatment on sphingomyelinase-induced vascular contraction. CONCLUSION: In summary, this study corroborates the importance of sphingolipid biology in blood pressure control and specifically shows that blood pressure lowering reduces vascular ceramide levels in SHR and that losartan treatment, but not blood pressure lowering per se, reduces ceramide-mediated arterial contractions.

Hypertension is associated with marked alterations in sphingolipid biology: a potential role for ceramide.

BACKGROUND: Hypertension is, amongst others, characterized by endothelial dysfunction and vascular remodeling. As sphingolipids have been implicated in both the regulation of vascular contractility and growth, we investigated whether sphingolipid biology is altered in hypertension and whether this is reflected in altered vascular function. METHODS AND FINDINGS: In isolated carotid arteries from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats, shifting the ceramide/S1P ratio towards ceramide dominance by administration of a sphingosine kinase inhibitor (dimethylsphingosine) or exogenous application of sphingomyelinase, induced marked endothelium-dependent contractions in SHR vessels (DMS: 1.4±0.4 and SMase: 2.1±0.1 mN/mm; n = 10), that were virtually absent in WKY vessels (DMS: 0.0±0.0 and SMase: 0.6±0.1 mN/mm; n = 9, p<0.05). Imaging mass spectrometry and immunohistochemistry indicated that these contractions were most likely mediated by ceramide and dependent on iPLA(2), cyclooxygenase-1 and thromboxane synthase. Expression levels of these enzymes were higher in SHR vessels. In concurrence, infusion of dimethylsphingosine caused a marked rise in blood pressure in anesthetized SHR (42±4%; n = 7), but not in WKY (-12±10%; n = 6). Lipidomics analysis by mass spectrometry, revealed elevated levels of ceramide in arterial tissue of SHR compared to WKY (691±42 vs. 419±27 pmol, n = 3-5 respectively, p<0.05). These pronounced alterations in SHR sphingolipid biology are also reflected in increased plasma ceramide levels (513±19 pmol WKY vs. 645±25 pmol SHR, n = 6-12, p<0.05). Interestingly, we observed similar increases in ceramide levels (correlating with hypertension grade) in plasma from humans with essential hypertension (185±8 pmol vs. 252±23 pmol; n = 18 normotensive vs. n = 19 hypertensive patients, p<0.05). CONCLUSIONS: Hypertension is associated with marked alterations in vascular sphingolipid biology such as elevated ceramide levels and signaling, that contribute to increased vascular tone.

Agonist-dependent effects of mutations in the sphingosine-1-phosphate type 1 receptor.

The sphingosine-1-phosphate type 1 (S1P(1)) receptor is a new target in the treatment of auto-immune diseases as evidenced by the recent approval of FTY720 (Fingolimod). The ligand-binding pocket of the S1P(1) receptor has been generally characterised but detailed insight into ligand-specific differences is still lacking. The aim of the current study is to determine differences in ligand-induced S1P(1) receptor activation using an in silico guided site-directed mutagenesis strategy. S1P(1) mutant receptors (modifications of residues Y98(2.57), R120(3.28), F125(3.33)) were probed with a chemically diverse set of S1P(1) agonists (S1P, dihydro-S1P (dhS1P), R-, S- and racemic FTY720-P, VPC24191, SEW2871). Mutation of the R(3.28) residue generally results in a reduction of the potency of all ligands although the synthetic ligands including FTY720-P are less sensitive to these mutations. The Y(2.57)F mutation does not affect the potency of any of the ligands tested, but for all ligands except FTY720-P a significant decrease in potency is observed at the Y(2.57)A mutant. The F(3.33)A mutation significantly decreased the potency of FTY720-P and is detrimental for SEW2871 and VPC24191. The non-aromatic endogenous ligands S1P and dhS1P are less affected by this mutation. Our in silico guided mutagenesis studies identified new molecular determinants of ligand-induced S1P(1) receptor activation: 1) the flexibility of the polar head of the agonist to maintain a tight H-bond network with R(3.28) and 2) the ability of the agonist to make aromatic π-stacking interactions with F(3.33). Interestingly, FTY720-P has both chemical properties and is the only ligand that can efficiently activate the Y(2.57)A mutant.

Sphingosine 1-phosphate receptor 1 and 3 are upregulated in multiple sclerosis lesions.

Sphingolipids are a class of biologically active lipids that have a role in multiple biological processes including inflammation. Sphingolipids exert their functions by direct signaling or through signaling by their specific receptors. Phosphorylated FTY720 (FTY720P) is a sphingosine 1-phosphate (S1P) analogue that is currently in trial for treatment of multiple sclerosis (MS), which targets all S1P receptors but S1P(2). To date, however, it remains unknown whether FTY720P may exert direct anti-inflammatory effects within the central nervous system (CNS), because data concerning S1P receptor expression and regulation under pathological conditions in the human brain are lacking. To investigate potential regulation of S1P receptors in the human brain during MS, we performed immunohistochemical analysis of S1P receptor 1 and 3 expression in well-characterized MS lesions. A strong increase in S1P receptor 1 and 3 expression on reactive astrocytes was detected in active and chronic inactive MS lesions. In addition, we treated primary cultures of human astrocytes with the proinflammatory cytokine tumor necrosis factor-alpha to identify the regulation of S1P(1/3) on astrocytes under pathological conditions. Importantly, we demonstrate that FTY720P exerts an anti-inflammatory action on human astrocytes by limiting secretion of proinflammatory cytokines. Our data demonstrate that reactive astrocytes in MS lesions and cultured under proinflammatory conditions strongly enhance expression of S1P receptors 1 and 3. Results from this study indicate that astrocytes may act as a yet-unknown target within the CNS for the anti-inflammatory effects observed after FTY720P administration in the treatment of MS.

Sphingolipids and the orchestration of endothelium-derived vasoactive factors: when endothelial function demands greasing.

Vasomotor tone is regulated by a complex interplay of a variety of extrinsic neurohumoral and intrinsic factors. It is the endothelium that has a major influence on smooth muscle cell tone via the release of intrinsic vasoactive factors and is therefore an important regulator of vasomotor tone. Sphingolipids are an emerging class of lipid mediators with important physiological properties. In the last two decades it has not only become increasingly clear that sphingolipid signaling plays a pivotal role in immune function, but also its role in the vascular system is now becoming more recognized. In this mini-review we will highlight the possible cross-talk between sphingolipids and intrinsic vasoactive factors released by the endothelium. Via this cross-talk sphingolipids can orchestrate vasomotor tone and may therefore also be involved in the pathophysiology of disease states associated with endothelial dysfunction.

Sphingosine-1-phosphate receptors: zooming in on ligand-induced intracellular trafficking and its functional implications.

Regulatory processes including receptor phosphorylation and intracellular trafficking, also referred to as receptor internalization, are important processes to terminate G protein-coupled receptor (GPCR) signaling. Compelling evidence now indicates that internalization of a receptor is not necessarily the endpoint of signaling, but can also be the beginning of the activation of intracellular signaling pathways. Sphingosine-1-phosphate (S1P) receptors, which are activated by the endogenous phospholipid S1P, belong to the family of GPCRs. Interestingly, there is evidence indicating differential intracellular trafficking of one of the S1P receptor subtypes, the S1P1 receptor, upon agonist activation by either S1P or the synthetic agonist FTY720-P. Moreover, the differential effect of FTY720-P on S1P1 receptor regulation has been suggested to be the mechanism of action of this drug, which is now in Phase III clinical trials for the treatment of multiple sclerosis. It is thus of importance to get a good insight into the regulation of S1P receptors. This review therefore gives a detailed overview about the current state of knowledge on S1P receptor internalization and its functional implications, including some data on nuclear signaling of S1P receptors.

Effects of muscarinic receptor stimulation on Ca2+ transient, cAMP production and pacemaker frequency of rabbit sinoatrial node cells.

We investigated the contribution of the intracellular calcium (Ca (i) (2+) ) transient to acetylcholine (ACh)-mediated reduction of pacemaker frequency and cAMP content in rabbit sinoatrial nodal (SAN) cells. Action potentials (whole cell perforated patch clamp) and Ca (i) (2+) transients (Indo-1 fluorescence) were recorded from single isolated rabbit SAN cells, whereas intracellular cAMP content was measured in SAN cell suspensions using a cAMP assay (LANCE((R))). Our data show that the Ca (i) (2+) transient, like the hyperpolarization-activated "funny current" (I (f)) and the ACh-sensitive potassium current (I (K,ACh)), is an important determinant of ACh-mediated pacemaker slowing. When I (f) and I (K,ACh) were both inhibited, by cesium (2 mM) and tertiapin (100 nM), respectively, 1 micro M ACh was still able to reduce pacemaker frequency by 72%. In these I (f) and I (K,ACh)-inhibited SAN cells, good correlations were found between the ACh-mediated change in interbeat interval and the ACh-mediated change in Ca (i) (2+) transient decay (r (2) = 0.98) and slow diastolic Ca (i) (2+) rise (r (2) = 0.73). Inhibition of the Ca (i) (2+) transient by ryanodine (3 microM) or BAPTA-AM (5 microM) facilitated ACh-mediated pacemaker slowing. Furthermore, ACh depressed the Ca (i) (2+) transient and reduced the sarcoplasmic reticulum (SR) Ca(2+) content, all in a concentration-dependent fashion. At 1 microM ACh, the spontaneous activity and Ca (i) (2+) transient were abolished, but completely recovered when cAMP production was stimulated by forskolin (10 microM) and I (K,ACh) was inhibited by tertiapin (100 nM). Also, inhibition of the Ca (i) (2+) transient by ryanodine (3 microM) or BAPTA-AM (25 microM) exaggerated the ACh-mediated inhibition of cAMP content, indicating that Ca (i) (2+) affects cAMP production in SAN cells. In conclusion, muscarinic receptor stimulation inhibits the Ca (i) (2+) transient via a cAMP-dependent signaling pathway. Inhibition of the Ca (i) (2+) transient contributes to pacemaker slowing and inhibits Ca (i) (2+) -stimulated cAMP production. Thus, we provide functional evidence for the contribution of the Ca (i) (2+) transient to ACh-induced inhibition of pacemaker activity and cAMP content in rabbit SAN cells.

Sphingosine-1-phosphate regulates RGS2 and RGS16 mRNA expression in vascular smooth muscle cells.

Regulator of G protein signalling (RGS) protein expression is altered under growth promoting conditions in vascular smooth muscle cells (VSMCs). Since sphingosine-1-phosphate (S1P) is an important growth stimulatory factor, we investigated whether stimulation of VSMCs with S1P results in alterations in mRNA expression levels of several RGS proteins and which signalling components are involved. VSMCs were stimulated with S1P and mRNA expression levels of RGS2, RGS3, RGS4, RGS5 and RGS16 were measured by real-time polymerase chain reaction. S1P caused a time-dependent up-regulation of RGS2 and RGS16 mRNA expression. FTY720-P, a S1P(1)/S1P(3-5) agonist, did not regulate RGS2 mRNA levels although it did up-regulate RGS16 mRNA expression. Pertussis toxin treatment revealed that the S1P-induced RGS16 expression was G(i/o)-dependent whereas up-regulation of RGS2 mRNA was not. Phosphatidylinositol 3-kinase, protein kinase C and mitogen-activated protein kinase kinase apparently were not involved in the S1P-induced up-regulation of both RGS proteins. The present study demonstrates that S1P induces RGS2 and RGS16 mRNA expression but uses distinct S1P receptor subtypes and signalling pathways to regulate expression of these RGS proteins.

Do gene polymorphisms alone or in combination affect the function of human beta3-adrenoceptors?

BACKGROUND AND PURPOSE: beta(3)-Adrenoceptors mediate many important physiological functions, for example, in the urinary bladder. The corresponding gene is polymorphic, and the W64R (Trp64Arg) single nucleotide polymorphism has been associated with disease states such as obesity, type 2 diabetes and bladder dysfunction. While these clinical data suggest that the 64R variant is hypofunctional, previous in vitro studies in which this variant was generated by site-directed mutagenesis and subsequent transfection have not consistently confirmed this. EXPERIMENTAL APPROACH: We transfected the wild-type human beta(3)-adrenoceptor and the 64R variant and also the more recently discovered 265M and 306F variants as well as 64R/265M and 64R/306F double mutants into human embryonic kidney cells and selected clones expressing the receptors at a density of about 100 fmol mg protein(-1). Receptor activation was measured by cAMP accumulation and ligand affinity by radioligand binding. Desensitisation was assessed as alterations of cAMP responses after prolonged agonist treatment. KEY RESULTS: Neither mutated receptor exhibited alterations in efficacy or potency for cAMP accumulation for any of five agonists (isoprenaline, noradrenaline, YM 178, FK 4664, CGP 12 177). In competition binding studies, the mutations did not affect the ability of any agonist to bind to the receptor. Wild-type receptors and the 64R variant exhibited similar isoprenaline-induced functional desensitization during a 24 h treatment. CONCLUSIONS AND IMPLICATIONS: None of the polymorphisms tested here significantly altered the interaction of isoprenaline, noradrenaline, YM 178, FK 4664 or CGP 12 177 with the human beta(3)-adrenoceptor when expressed at near physiological levels in a human cell line.

Are RGS2 gene polymorphisms associated with high blood pressure in an ethnicity- and gender-specific manner?

BACKGROUND: Polymorphisms in the Regulator of G-protein Signaling 2 (RGS2) gene have been reported to be associated with hypertension (HT) in Japanese women and black Americans of either gender but not in white Americans or Japanese men. We have tested whether these proposed ethnicity- and gender-specific associations between RGS2 gene polymorphisms and HT can be confirmed in an independent population of male and female blacks, whites, and south Asians. METHODS: A population-based sample of 1379 black, white Dutch, and south Asian subjects from the Amsterdam area was genotyped for eight polymorphisms in the RGS2 gene. All analyses were done separately per ethnic group. The phenotype high blood pressure was defined as a dichotomous variable comparing HT vs. normotension (NT) and as a linear variable using systolic blood pressure (SBP) in a multiple regression analysis with concomitant antihypertensive medication, age and body mass index as coexplanatory variables. RESULTS: Ethnic differences in the frequency of polymorphisms and haplotypes (HAPs) derived thereof were in line with previous studies. Our data do not confirm previously reported ethnicity- or gender-specific associations regardless which phenotype definition was used. While the D allele of 1891-1892TC insertion/deletion polymorphism showed association in several groups, they differed from previously reported ones. Haplotype-phenotype analysis was not more sensitive to detect genotype-phenotype associations than individual alleles. CONCLUSIONS: Previously reported ethnicity- and gender-specific associations of RGS2 genotype and hypertensive phenotype are not robust. Nevertheless, the 1891-1892TC insertion/deletion polymorphism warrants further investigation.

The effect of bladder outlet obstruction on alpha1- and beta-adrenoceptor expression and function.

AIMS: To explore possible changes in expression and/or function of alpha(1)- and beta-adrenoceptor subtypes as a cause for bladder dysfunction in a rat model of bladder outlet obstruction (BOO). METHODS: BOO was induced in rats by partial urethral ligature. Contraction and relaxation experiments were performed with isolated bladder strips from BOO, sham-operated and non-operated (control) rats 7 days after BOO induction. mRNA expression of alpha(1)- and beta-adrenoceptor subtypes was assessed by quantitative real-time PCR. RESULTS: Receptor-independent contraction or relaxation did not differ between BOO and sham rats. The alpha(1)-agonists methoxamine and A-61,603 caused only weak contraction without major differences between groups. Against KCl-induced tone, the beta-adrenoceptor agonists noradrenaline and isoprenaline caused similar relaxation in BOO and sham rats, whereas relaxation in response to the beta(3)-selective BRL 37,344 was attenuated. Against passive tension, noradrenaline induced relaxation in sham and control rats; in contrast, noradrenaline induced contraction at low concentrations and relaxation at high concentrations in BOO rats. The contraction component was abolished by the alpha(1)-antagonist prazosin. The mRNA expression of alpha(1D)-adrenoceptors was increased in BOO, whereas none of the other receptor mRNAs were up-regulated. CONCLUSIONS: In a rat BOO model, weak contraction responses to alpha(1)-agonists and relaxation responses to beta-agonists are not altered to a major extent. Nevertheless, relaxation responses to the endogenous agonist noradrenaline are turned into alpha(1)-adrenoceptor-mediated contraction responses in BOO, possibly due to an up-regulation of alpha(1D)-adrenoceptors.

Activation of sphingosine kinase by muscarinic receptors enhances NO-mediated and attenuates EDHF-mediated vasorelaxation.

Local formation of the sphingomyelin metabolite sphingosine-1-phosphate (S1P) within the vascular wall has been shown to modulate vascular reactivity. In this study we investigated whether sphingosine kinase, the enzyme responsible for S1P synthesis, plays a role in muscarinic receptor-mediated NO production and vascular relaxation in different blood vessel types. For this purpose, sphingosine kinase translocation and sphingolipid-dependent NO-production after muscarinic receptor stimulation were assessed in an endothelial cell line. Furthermore, we used the sphingosine kinase inhibitor N,N-dimethylsphingosine (DMS) to investigate the role of sphingosine kinase in the relaxant responses to the muscarinic agonist methacholine (MCh) in isolated rat aorta and mesenteric arteries. Activation of M(3)-receptors in an endothelial cell line induced a fast translocation of YFP-tagged sphingosine kinase-1 from the cytosol to the plasma membrane. Concomitant NO-production in this cell line was partially inhibited by DMS. Accordingly, in rat aorta the relaxant responses to MCh were attenuated in the presence of DMS, while the responses to the NO-donor sodium nitroprusside were unaltered. In contrast, DMS enhanced the relaxant responses to MCh in mesenteric artery preparations. This effect could also be observed in the presence of NO synthase and cyclooxygenase inhibitors, indicating that sphingosine kinase inhibition specifically enhanced endothelium-derived hyperpolarizing factor-mediated (i.e. non-NO and non-prostacyclin-dependent) relaxation. We conclude that sphingosine kinase differentially regulates vascular tone in different vessel types, enhancing NO-dependent vasorelaxation but counteracting EDHF-dependent vasorelaxation. This observation enhances our understanding of the complex mechanisms by which sphingolipids regulate vascular homeostasis. Moreover, a disturbed regulation of sphingolipid metabolism in the vascular wall may therefore play a role in the aetiology/pathology of disease states characterized by endothelial dysfunction.

S1P receptor signalling and RGS proteins; expression and function in vascular smooth muscle cells and transfected CHO cells.

Sphingosine-1-phosphate (S1P) signalling via G protein-coupled receptors is important for the regulation of cell function and differentiation. Specific Regulators of G protein Signalling (RGS) proteins modulate the function of these receptors in many cell types including vascular smooth muscle cells (VSMCs). Therefore, we investigated the role of altered expression levels of RGS proteins in S1P receptor function in VSMCs and transfected CHO cells. The mRNA expression of the S1P(1) receptor, RGS4 and RGS16 were down-regulated in VSMCs during phenotypic modulation induced by culturing, whereas mRNA levels of RGS2, RGS3, S1P(2) and S1P(3) receptors were unchanged. Interestingly, the expression level of RGS5 was transiently up-regulated. Despite major alterations in RGS levels, S1P-induced calcium elevation in VSMCs was not altered. Co-transfection of RGS2, RGS3, RGS4, RGS5 and RGS16 into CHO-Flp-In cells stably expressing the S1P(1) or S1P(3) receptor did not modify S1P-induced inhibition of cAMP accumulation to a major extent. Similar results were obtained with SEW2871, a selective S1P(1) receptor agonist. However, the inhibition of cAMP accumulation by the agonist FTY720-P via the S1P(1) receptor was significantly decreased by co-transfection with RGS5. These results indicate that mRNA of the S1P(1) receptor, RGS4, RGS5 and RGS16 is differentially regulated during phenotypic modulation. However, major alterations in RGS protein expression have only limited effect on S1P receptor function.

Regulation of G protein-coupled receptor signalling: focus on the cardiovascular system and regulator of G protein signalling proteins.

G protein-coupled receptors (GPCRs) are involved in many biological processes. Therefore, GPCR function is tightly controlled both at receptor level and at the level of signalling components. Well-known mechanisms by which GPCR function can be regulated comprise desensitization/resensitization processes and GPCR up- and downregulation. GPCR function can also be regulated by several proteins that directly interact with the receptor and thereby modulate receptor activity. An additional mechanism by which receptor signalling is regulated involves an emerging class of proteins, the so-called regulators of G protein signalling (RGS). In this review we will describe some of these control mechanisms in more detail with some specific examples in the cardiovascular system. In addition, we will provide an overview on RGS proteins and the involvement of RGS proteins in cardiovascular function.

Sphingolipid signalling in the cardiovascular system: good, bad or both?

Sphingolipids are biologically active lipids that play important roles in various cellular processes and the sphingomyelin metabolites ceramide, sphingosine and sphingosine-1-phosphate can act as signalling molecules in most cell types. With the recent development of the immunosuppressant drug FTY720 (Fingolimod) which after phosphorylation in vivo acts as a sphingosine-1-phosphate receptor agonist, research on the role of sphingolipids in the immune and other organ systems was triggered enormously. Since it was reported that FTY720 induced a modest, but significant transient decrease in heart rate in animals and humans, the question was raised which pharmacological properties of drugs targeting sphingolipid signalling will affect cardiovascular function in vivo. The answer to this question will most likely also indicate what type of drug could be used to treat cardiovascular disease. The latter is becoming increasingly important because of the increasing population carrying characteristics of the metabolic syndrome. This syndrome is, amongst others, characterized by obesity, hypertension, atherosclerosis and diabetes. As such, individuals with this syndrome are at increased risk of heart disease. Now numerous studies have investigated sphingolipid effects in the cardiovascular system, can we speculate whether certain sphingolipids under specific conditions are good, bad or maybe both? In this review we will give a brief overview of the pathophysiological role of sphingolipids in cardiovascular disease. In addition, we will try to answer how drugs that target sphingolipid signalling will potentially influence cardiovascular function and whether these drugs would be useful to treat cardiovascular disease.