OKL-1111, A modified cyclodextrin as a potential universal reversal agent for anticoagulants.

BACKGROUND: Antithrombotic therapy is inevitably associated with a risk for bleeding and these bleeding complications can be life-threatening. Recently, specific reversal agents were developed for the direct factor Xa and thrombin inhibitors (DOACs). However, next to the fact that these agents are relatively expensive, the use of selective reversal agents complicates treatment of bleeding patients in practice. In a series of screening experiments, we discovered a class of cyclodextrins with procoagulant properties. In this study we characterize a lead compound, OKL-1111, and demonstrate its potential use as a universal reversal agent. OBJECTIVES: To assess the anticoagulant reversal properties of OKL-1111, in vitro and in vivo. METHODS: The effect of OKL-1111 on coagulation in the absence and presence of DOACs was investigated in a thrombin generation assay. Its reversal effect on a variety of anticoagulants in vivo was investigated in a rat tail cut bleeding model. A possible prothrombotic action of OKL-1111 was assessed in a Wessler model in rabbits. RESULTS: OKL-1111 concentration-dependently reversed the in vitro anticoagulant effects of dabigatran, rivaroxaban, apixaban and edoxaban in the thrombin generation assay. Also in the absence of a DOAC, OKL-1111 concentration-dependently accelerated coagulation in this assay, but did not initiate coagulation. The reversal effect was also seen for all DOACs in the rat tail cut bleeding model. In addition, when tested with other anticoagulants, OKL-1111 also reversed the anticoagulant effect of the vitamin K antagonist warfarin, the low molecular weight heparin enoxaparin, the pentasaccharide fondaparinux and the platelet inhibitor clopidogrel in vivo. OKL-1111 did not have prothrombotic effects in the Wessler model. CONCLUSION: OKL-1111 is a procoagulant cyclodextrin with a currently unknown working mechanism that has potential to become a universal reversal agent for anticoagulants and platelet inhibitors.

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.

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.

Plasma ceramide is increased and associated with proteinuria in women with pre-eclampsia and HELLP syndrome.

OBJECTIVES: Ceramide is a sphingolipid with anti-angiogenic and pro-apoptotic properties that has shown to be increased in plasma of women with pre-eclampsia. We aimed to compare plasma and placental sphingolipid content among normotensive pregnant women and pre-eclamptic women with and without HELLP syndrome and we aimed to assess whether ceramide is related to hypertension and proteinuria in pre-eclampsia. STUDY DESIGN: Case-control study. Participants were recruited from the Department of Obstetrics at the Academic Medical Center in Amsterdam, The Netherlands. In total 48 pregnant women were included: 24 with pre-eclampsia and 24 normotensive controls. Of the 24 pre-eclamptic women, 11 had HELLP syndrome. MAIN OUTCOME MEASURES: Plasma and placental ceramide content and correlation with blood pressure and protein excretion in pre-eclampsia. RESULTS: Total plasma, but not placental, ceramide was higher in pre-eclamptic women with HELLP syndrome (11200 95% CI 9531-12870 nmol/ml, n = 11) compared to pre-eclamptic women without HELLP (7413 95% CI 5928-8898 nmol/ml, n = 13, p < 0.001) and normotensive pregnant women (7404 95% CI 6695-8112 nmol/ml, n = 24, p < 0.001). Maternal circulating ceramide levels were strongly associated with proteinuria (r = 0.621, n = 24, p = 0.001) in pre-eclamptic women and inversely correlated with gestational age at delivery (r = 0.771, p < 0.01) in pre-eclamptic women with HELLP syndrome. Plasma ceramide was not correlated with blood pressure. CONCLUSION: Plasma but not placental ceramide content is increased in women with pre-eclampsia and HELLP syndrome. The strong positive correlation with proteinuria and the inverse correlation with gestational age at delivery indicate that excess plasma ceramide may contribute to the pathophysiology of pre-eclampsia and HELLP.

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.

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.

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.

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.

Signal transduction underlying the control of urinary bladder smooth muscle tone by muscarinic receptors and beta-adrenoceptors.

The normal physiological contraction of the urinary bladder, which is required for voiding, is predominantly mediated by muscarinic receptors, primarily the M3 subtype, with the M2 subtype providing a secondary backup role. Bladder relaxation, which is required for urine storage, is mediated by beta-adrenoceptors, in most species involving a strong beta3-component. An excessive stimulation of contraction or a reduced relaxation of the detrusor smooth muscle during the storage phase of the micturition cycle may contribute to bladder dysfunction known as the overactive bladder. Therefore, interference with the signal transduction of these receptors may be a viable approach to develop drugs for the treatment of overactive bladder. The prototypical signaling pathway of M3 receptors is activation of phospholipase C (PLC), and this pathway is also activated in the bladder. Nevertheless, PLC apparently contributes only in a very minor way to bladder contraction. Rather, muscarinic-receptor-mediated bladder contraction involves voltage-operated Ca2+ channels and Rho kinase. The prototypical signaling pathway of beta-adrenoceptors is an activation of adenylyl cyclase with the subsequent formation of cAMP. Nevertheless, cAMP apparently contributes in a minor way only to beta-adrenoceptor-mediated bladder relaxation. BKCa channels may play a greater role in beta-adrenoceptor-mediated bladder relaxation. We conclude that apart from muscarinic receptor antagonists and beta-adrenoceptor agonists, inhibitors of Rho kinase and activators of BKCa channels may have potential to treat an overactive bladder.

Sphingosine-1-phosphate signaling in the cardiovascular system.

Increasing evidence suggests a key role for the bioactive lipid sphingosine-1-phosphate (S1P) and its G-protein-coupled receptors (S1P(1-5)) in the cardiovascular system. Recent advances in sphingolipid research indicates that cardiomyocyte, vascular smooth muscle and endothelial cell function is greatly influenced by the relative expression and activity both of S1P receptors and of the enzymes involved in sphingolipid metabolism. For instance, the discovery and development of S1P receptor agonists such as FTY720 has clearly indicated the involvement of S1P receptors in the regulation of heart rate. In addition, sphingolipid metabolism induced, for example, by tumour necrosis factor-alpha or angiotensin II plays an important role in vessel structure, function and tone.

Rho kinase: a target for treating urinary bladder dysfunction?

Urinary incontinence and other urinary storage symptoms are frequent in the general population but available treatments have limited efficacy and tolerability. Rho kinase (ROCK) has a central role in the regulation of smooth muscle contraction, including that of the urinary bladder. Recent experimental evidence indicates that this role could be deregulated and exacerbated in local and systemic pathological conditions that affect the bladder. In vitro studies with prototypical ROCK inhibitors such as Y27632 and in vivo data from animal models indicate that such drugs have potential as future treatments for bladder dysfunction.

Validation of a rapid, non-radioactive method to quantify internalisation of G-protein coupled receptors.

Agonist exposure can cause internalisation of G-protein coupled receptors (GPCRs), which may be a part of desensitisation but also of cellular signaling. Previous methods to study internalisation have been tedious or only poorly quantitative. Therefore, we have developed and validated a quantitative method using a sphingosine-1-phosphate (S1P) receptor as a model. Because of a lack of suitable binding studies, it has been difficult to study S1P receptor internalisation. Using a N-terminal HisG-tag, S1P(1) receptors on the cell membrane can be visualised via immunocytochemistry with a specific anti-HisG antibody. S1P-induced internalisation was concentration dependent and was quantified using a microplate reader, detecting either absorbance, a fluorescent or luminescent signal, depending on the antibodies used. Among those, the fluorescence detection method was the most convenient to use. The relative ease of this method makes it suitable to measure a large number of data points, e.g. to compare the potency and efficacy of receptor ligands.

Sphingosine kinase-dependent activation of endothelial nitric oxide synthase by angiotensin II.

OBJECTIVE: In addition to their role in programmed cell death, cell survival, and cell growth, sphingolipid metabolites such as ceramide, sphingosine, and sphingosine-1-phosphate have vasoactive properties. Besides their occurrence in blood, they can also be formed locally in the vascular wall itself in response to external stimuli. This study was performed to investigate whether vasoactive compounds modulate sphingolipid metabolism in the vascular wall and how this might contribute to the vascular responses. METHODS AND RESULTS: In isolated rat carotid arteries, the contractile responses to angiotensin II are enhanced by the sphingosine kinase inhibitor dimethylsphingosine. Endothelium removal or NO synthase inhibition by N(omega)-nitro-L-arginine results in a similar enhancement. Angiotensin II concentration-dependently induces NO production in an endothelial cell line, which can be diminished by dimethylsphingosine. Using immunoblotting and intracellular calcium measurements, we demonstrate that this sphingosine kinase-dependent endothelial NO synthase activation is mediated via both phosphatidylinositol 3-kinase/Akt and calcium-dependent pathways. CONCLUSIONS: Angiotensin II induces a sphingosine kinase-dependent activation of endothelial NO synthase, which partially counteracts the contractile responses in isolated artery preparations. This pathway may be of importance under pathological circumstances with reduced NO bioavailability. Moreover, a disturbed sphingolipid metabolism in the vascular wall may lead to reduced NO bioavailability and endothelial dysfunction.

Vascular effects of sphingolipids.

UNLABELLED: The sphingomyelin metabolites ceramide, sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC) are emerging modulators of vascular tone. While ceramide appears to act primarily intracellularly, S1P and SPC appear to mainly work via specific receptors, although those for SPC have not yet been defined unequivocally. Each of the sphingomyelin metabolites can induce both vasoconstriction and vasodilatation and, in some cases--ceramide on the one hand, and S1P and SPC on the other hand--have opposite effects on vascular tone. The differences in effects between vessels may relate to the relative roles of endothelial and smooth muscle cells in mediating them, as well as to the distinct expression patterns of S1P receptors among vascular beds and among endothelial and smooth muscle cells. Recent evidence suggests that vascular tone is not only modulated by sphingomyelin metabolites which are exogenously added or reach the vessel wall via the bloodstream but also by those formed locally by cells in the vessel wall. Such local formation can be induced by known vasoactive agents such as angiotensin II and may serve a signalling function. CONCLUSION: We conclude that sphingomyelin metabolites are important endogenous modulators of vascular function, which may contribute to the pathophysiology of some diseases and be targets for therapeutic interventions.

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.

Cardiovascular effects of sphingosine-1-phosphate and other sphingomyelin metabolites.

Upon various stimuli, cells metabolize sphingomyelin from the cellular plasma membrane to form sphingosylphosphorylcholine (SPC) or ceramide. The latter can be further metabolized to sphingosine and then sphingosine-1-phosphate (S1P). Apart from local formation, S1P and SPC are major constituents of blood plasma. All four sphingomyelin metabolites (SMM) can act upon intracellular targets, and at least S1P and probably also SPC can additionally act upon G-protein-coupled receptors. While the molecular identity of the SPC receptors remains unclear, several subtypes of S1P receptors have been cloned and their distribution in cardiovascular tissues is described. In the heart SMM can alter intracellular Ca(2+) release, particularly via the ryanodine receptor, and conductance of various ion channels in the plasma membrane, particularly I(K(Ach)). While the various SMM differ somewhat in their effects, the above alterations of ion homeostasis result in reduced cardiac function in most cases, and ceramide and/or sphingosine may be the mediators of the negative inotropic effects of tumour necrosis factor. In the vasculature, SMM mainly act as acute vasoconstrictors in most vessels, but ceramide can be a vasodilator. SMM-induced vasoconstriction involves mobilization of Ca(2+) from intracellular stores, influx of extracellular Ca(2+) via L-type channels and activation of a rho-kinase. Extended exposure to SMM, particularly S1P, promotes several stages of the angiogenic process like endothelial cell activation, migration, proliferation, tube formation and vascular maturation. We propose that SMM are an important class of endogenous modulators of cardiovascular function.

The influence of endogenously generated reactive oxygen species on the inotropic and chronotropic effects of adrenoceptor and ET-receptor stimulation.

Reactive oxygen species (ROS) play a role in cardiovascular diseases such as heart failure and hypertension. Furthermore, increasing evidence has accumulated suggesting that ROS can also be formed subsequent to the stimulation of various receptors, thus functioning as second messengers. The objective of the present study was to elucidate the role of intracellular-generated ROS in the inotropic and chronotropic effects of the alpha1- and beta-adrenoceptor and the ET-receptor stimulation in isolated rat atria. In addition, we investigated whether the MAPKerk pathway is involved in the ROS-provoked rise of contractile force. For this purpose hydrogen peroxide was applied, which is known to serve several endogenous functions as a second messenger. Moreover, hydrogen peroxide readily crosses cell membranes, which thus allows to mimic the intracellular formation. Preincubation of atria with EUK 8 (400 microM), a cell permeable superoxide dismutase- and catalase-mimetic, reduced the positive inotropic effect upon alpha1-adrenoceptor and ET-receptor stimulation. The responsiveness to beta-adrenoceptor stimulation remained unaffected by this pretreatment. The chronotropic effects were not altered by preincubation with EUK 8. In contrast to the MAPK(p38) inhibitor SB203580 (2 and 10 microM), the two MKKmek inhibitors PD98059 (30 and 100 microM) and U0126 (10 microM) significantly attenuated the positive inotropic response to hydrogen peroxide in isolated rat left atria. In addition, inhibition of the Na+/H+ exchange (NHE) by cariporide (1 microM) counteracted ROS-provoked increase of contractile force. From the present study we conclude that the inotropic responses to alpha1-adrenoceptor and ET-receptor stimulation are, at least partially, caused by intracellular-formed ROS, that subsequently may activate the MAPKerk pathway and the NHE.

A mitogen-activated protein kinase is involved in the inotropic but not chronotropic actions of adrenoceptor agonists and endothelin-1.

The activation of mitogen-activated protein kinase (MAPK) pathways in the heart, for instance by alpha(1)-adrenoceptor agonists and endothelin-1, has primarily been associated with cellular growth regulation. Here we have investigated a possible role of MAPK pathways in the inotropic and chronotropic effects of adrenoceptor agonists and endothelin-1 in isolated rat left and right atria. Inotropic and chronotropic responses of the isolated atria to methoxamine, isoprenaline and endothelin-1 were measured in the absence and presence of inhibitors of MAPK pathways. The MAPK kinase (MKK(mek)) inhibitors PD98059 (100 microM) and U0126 (10 microM) significantly inhibited the inotropic responses to the alpha(1)-adrenoceptor agonist methoxamine (300 microM) and endothelin-1 (50 nM), but not the chronotropic responses to these agonists. U0126 but not PD98059 inhibited the inotropic response to 3 microM isoprenaline. None of the aforementioned inotropic and chronotropic effects were inhibited by the MAPKP(p38) inhibitor SB203580 (2 microM). We conclude that activation of the PD98059/U0126-sensitive MAPK pathway is essential for the inotropic but not chronotropic actions of adrenoceptor agonists and endothelin-1.

Vasopressin-induced vasoconstriction is dependent on MAPKerk1/2 phosphorylation.

To investigate the involvement of the mitogen-activated protein kinase (MAPK) family of extracellular signal-regulated kinase (ERK) 1 and 2 (MAPKerk1/2) in the vasopressin-mediated vasoconstriction in the rat aorta. Vasopressin-induced vasoconstriction was measured in isolated rat thoracic aortae in the presence or absence of MAPKerk1/2 kinase (MKKmek1/2) inhibitors. Thereafter the MAPKerk1/2 phosphorylation in the rat aorta was quantified using Western blot analysis. Vasopressin (1-300 nm) induced a concentration-dependent vasoconstriction, which could be inhibited concentration dependently by the selective MKKmek1/2 inhibitors, PD 98059 (10 and 100 microm) and U 0126 (10 and 100 microm). Western blot analysis revealed a 2.7 +/- 0.6-fold increase in the MAPKerk1/2 phosphorylation induced by vasopressin (300 nm). This phosphorylation could be dose dependently prevented by both PD 98059 (100 microm) and U 0126 (10 and 100 microm). These results indicate that vasoconstriction induced by vasopressin is partly regulated by the MAPKerk1/2 pathway.

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.