ET Receptor Stimulation Research Articles

Heparin-binding protein (HBP/CAP37) - a link to endothelin-1 in endotoxemia-induced pulmonary oedema?

Vascular leakage and oedema formation are key components in sepsis. In septic patients, plasma levels of the vasoconstrictive and pro-inflammatory peptide endothelin-1 (ET-1) correlate with mortality. During sepsis, neutrophils release heparin-binding protein (HBP) known to increase vascular permeability and to be a promising biomarker of human sepsis. As disruption of ET-signalling in endotoxemia attenuates formation of oedema, we hypothesized that this effect could be related to decreased levels of HBP. To investigate this, we studied the effects of ET-receptor antagonism on plasma HBP and oedema formation in a porcine model of sepsis. In addition, to further characterize a potential endothelin/HBP interaction, we investigated the effects of graded ET-receptor agonist infusions. Sixteen anesthetized pigs were subjected to 5 h of endotoxemia and were randomized to receive either the ET-receptor antagonist tezosentan or vehicle after 2 h. Haemodynamics, gas-exchange and lung water were monitored. In separate experiments, plasma HBP was measured in eight non-endotoxemic animals exposed to graded infusion of ET-1 or sarafotoxin 6c. Endotoxemia increased plasma ET-1, plasma HBP, and extravascular lung water. Tezosentan-treatment markedly attenuated plasma HBP and extravascular lung water, and these parameters correlated significantly. Tezosentan decreased pulmonary vascular resistance and increased respiratory compliance. In non-endotoxemic pigs graded ET-1 and sarafotoxin 6c infusions caused a dose-dependent increase in plasma HBP. ET-receptor antagonism reduces porcine endotoxin-induced pulmonary oedema and plasma levels of the oedema-promoting protein HBP. Moreover, direct ET-receptor stimulation distinctively increases plasma HBP. Together, these results suggest a novel mechanism by which ET-1 contributes to formation of oedema during experimental sepsis.

Hypertension During Vascular Endothelial Growth Factor Inhibition: Focus on Nitric Oxide, Endothelin-1, and Oxidative Stress

Angiogenesis inhibition with humanized antibodies targeting vascular endothelial growth factor (VEGF) or orally active small tyrosine kinase inhibitors targeting VEGF receptors has become an established treatment modality for various forms of cancer. A common side effect of angiogenesis inhibition is the development of sometimes severe hypertension, which simultaneously appears to be predictive for a favorable antitumor response. Since VEGF increases the expression and activity of endothelial nitric oxide synthase, it has been assumed that the mean blood pressure (MAP) rise during angiogenesis inhibition is caused by a decrease in nitric oxide bioavailability. Yet, the results from experimental and clinical studies exploring this possibility are conflicting. Recent studies provided evidence that the MAP rise during angiogenesis inhibition rather is mediated by activation of the endothelin-1 (ET-1) axis, which, among others, induces oxidative stress. Nevertheless, conclusive evidence for the involvement of reactive oxygen species in the MAP rise could not be obtained so far. The mechanism underlying activation of the ET-1 axis during angiogenesis inhibition is unclear, and this activation was not anticipated in view of studies showing that VEGF stimulates both the expression and production of ET-1 by endothelial cells. In fact, this activation of the ET-1 axis may support the use of ET receptor antagonists for the treatment of angiogenesis inhibition-induced hypertension, especially because ET receptor stimulation in vascular smooth muscle cells results in VEGF production and mitogenesis in a mitogen-activated protein kinase pathway-dependent manner.

Endothelin receptor antagonism and cancer

The endothelin peptides have an important role in the cancer-stromal interactions that promote tumour growth. Endothelin-1 (ET-1), clinically the most investigated endothelin, is a vital agent in the growth and progression of several tumours including prostate, ovarian, colorectal, bladder, breast and lung carcinomas. ET-1 exerts its effects through the activation of two distinct receptors, ET(A) and ET(B). Once activated, these receptors transmit signals via numerous intracellular signalling pathways. The effects of ET receptor stimulation in cancer cells or cancer-associated cells include proliferation, resistance to apoptosis, angiogenesis, migration and subsequent invasion. At present, the manipulation of the endothelin axis within the pre-clinical setting is the subject of intense investigation. Recent studies into ET receptor antagonism have produced interesting results highlighting the fact that these receptors may provide novel targets for a new generation of chemotherapeutic agents in a variety of cancers.

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.

β-Adrenergic and endothelin receptor interaction in dilated human cardiomyopathic myocardium

Background: Although end-stage dilated cardiomyopathy (DCM) is characterized by defects in β-adrenergic receptor (β-AR) activity and increased endothelin-1 (ET-1), possible interactions between these 2 systems remain to be defined. Accordingly, the goal of this study was to determine the effects of ET receptor activation on β-AR signaling through measurement of cyclic adenosine monophosphate (cAMP) in normal and DCM myocardium. Methods and Results: Myocardial sarcolemmal preparations were prepared from normal human (n = 6), dilated cardiomyopathic (n = 10), and ischemic cardiomyopathic (ICM, n = 10) tissue. Basal cAMP production was measured in the presence of ET-1 alone (10−6 to 0−9 mol/L) as well as after (−)isoproterenol (10−6 to 10−2 mol/L) or forskolin (0.05 to 30.0 μmol/L) stimulation. β-AR and ET receptor profiles were determined by radiolabeled ligand assays. ET-1 inhibited basal cAMP production in all preparations in a concentration-dependent manner. However, β-AR-stimulated cAMP production by either isoproterenol or forskolin was not significantly affected by ET-1. β-AR receptor density was reduced, and a selective reduction of the ETB receptor occurred in both forms of DCM. Conclusions: Under basal conditions, ET receptor stimulation reduced cAMP levels, which may influence contractility, particularly with DCM.

Effects of Endothelin-1 on Ca2+Signaling in Guinea-Pig Ventricular Myocytes: Role of Protein Kinase C

The effects of ET-1 on contraction, Ca2+transient andl-type Ca2+current (ICa,L) were studied in single cells isolated from ventricles of guinea-pig hearts. The aim of our study was to elucidate the mechanism of the positive inotropic effect during endothelin receptor stimulation by focusing on the role of PKC. ET-1 at concentrations of 5 and 10 nmproduced a biphasic pattern of inotropism: a first decrease in contraction by 34.4±2.5% of the control followed by a sustained increase in contraction by 66.6±8.4% (mean±SEM,n=9). The Ca2+transient decreased by 13.5±1.0% during the negative inotropic phase, while it increased by 58.1±8.4% (n=10) during the positive inotropic phase. Using the whole-cell voltage-clamp technique with conventional microelectrodes, the application of ET-1 (5 nm) increased theICa,Lby 32.6±5.1% (n=10), which was preceded by a short-lived decrease inICa,L. Incubation of myocytes with pertussis toxin (PTX, at 2μg/ml for >3 h at 35°C) failed to block the ET-1-induced enhancement ofICa,L. The increases in contraction, Ca2+transient, andICa,Lby ET-1 were inhibited by pretreatment with 5-N-methyl-N-isobutyl amiloride (MIA; 10μm), an amiloride analog, and a novel selective Na+/H+exchange inhibitor HOE694 (10μm). To determine whether activation of protein kinase C (PKC) is responsible for the enhancement ofICa,Lby ET-1, we tested a PKC inhibitor, GF109203X, and found that it does exert an inhibitory effect on the ET-1-inducedICa,Lincrease. Our study suggests that during ET receptor stimulation an increase inICa,Ldue to stimulation of Na+/H+exchange via PKC activation causes an increase in Ca2+transients and thereby in the contractile force of the ventricular myocytes.