Long-lasting physiological antagonism of calcitonin gene-related peptide towards endothelin-1 in rat mesenteric arteries and human coronary arteries.

Endothelin-1 causes long-lasting contraction via endothelin type A receptor (ETAR) in isolated rat mesenteric arteries (RMA) that cannot be readily terminated by removing the agonist, or by adding the ETAR antagonist BQ123 or the NO donor sodium nitroprusside. It could be terminated by adding calcitonin-gene related peptide (CGRP), most likely because CGRP causes ET-1/ETAR dissociation. Here we investigated this phenomenon in human coronary microarteries (HCMA). We simultaneously verified the effects of CGRP in RMA and HCMA towards other vasoconstrictors, i.e., the α1-adrenoceptor agonist phenylephrine, the thromboxane A2 analog U46619 (9,11-dideoxy-11α,9α-epoxy-methano-prostaglandin F2α) and KCl. Long-lasting contraction (remaining after washing away the agonist) was observed for ET-1 in RMA, but not HCMA. Constrictions to phenylephrine, U46619 or KCl did not last upon washing. When added on top of ET-1-initiated contraction in RMA, CGRP effectively counteracted vasoconstriction, i.e., it caused full relaxation. Inhibitory effects of CGRP were also observed when briefly exposing RMA and HCMA to CGRP 1h before the addition of ET-1. Similar inhibitory effects of transient CGRP pre-incubation were seen towards phenylephrine, U46619 or KCl in RMA and HCMA. In conclusion, our data imply that CGRP, like ET-1, causes long-lasting effects that remain apparent up to 1h after its removal from the organ bath. Thus, in addition to the reported dissociation of ET-1/ETAR complexes, CGRP causes long-lasting non-selective arterial smooth muscle relaxation that may add to the neuropeptide being a physiological antagonist of arterial effects of ET-1. Long-lasting, washout-resistant ET-1/ETAR interaction does not occur in HCMAs.

Assessment of endothelin-A receptor expression in subcutaneous and orthotopic thyroid carcinoma xenografts in vivo employing optical imaging methods.

Endothelin (ET) receptor dysregulation has been described in a number of pathophysiological processes, including cardiovascular disorders, renal failure, and cancer. The aim of this study was to evaluate the expression of the ET-A receptor (ET(A)R) in murine models of thyroid carcinoma using optical imaging methods. A recently developed near-infrared fluorescent tracer was first assessed in isolated artery preparations for its functional performance in comparison with known ET(A)R antagonists BQ123 and PD156707. Before evaluation of the tracer in vivo, different thyroid carcinoma cell lines were characterized with respect to their ET receptor expression by RT-PCR and autoradiography. In vivo, sc and orthotopic papillary thyroid tumor xenografts were clearly visualized by fluorescence reflectance imaging and fluorescence-mediated tomography up to 48 h after injection of the tracer. Binding specificity of the probe was demonstrated by predosing with PD156707 as a competing inhibitor. In conclusion, optical imaging with a fluorescent ET(A)R tracer allows the noninvasive imaging of tumor-associated ET(A)R expression in vivo. In the future, this technique may help surgeons to evaluate lesion dimensions in intraoperative settings (e.g. thyroidectomy).

Endothelin-1 and -2: two amino acids matter.

AIMS: Endothelin-1 (ET-1) and endothelin-2 (ET-2; Trp(6)Leu(7)ET-1) are expressed by different cell types, but are considered to display identical pharmacological properties on endothelin receptors. We studied agonist-dependent aspects of endothelin(A) (ET(A))-receptor function and the importance of amino acids 6 and 7 of ET-1 and ET-2 in this respect. MAIN METHODS: We used isolated rat mesenteric resistance arteries in wire myographs, in a setting that minimizes influences of endothelium and sensorimotor nerves, to study arterial smooth muscle ET(A)-receptor-mediated vasomotor responses, to ET-1, ET-2 and chimeras thereof (Trp(6)ET-1 and Leu(7)ET-1). KEY FINDINGS: ET-1 and ET-2 cause arterial contractions with comparable sensitivities and maximal responses. BQ123 (ET(A)-antagonist) reduces sensitivity to ET-1 more potently than that to ET-2 (pK(B): 7.1 ± 0.2 versus 5.6 ± 0.4). However, 1 µM BQ123 relaxes maximal contractile responses to ET-2 more markedly than those to ET-1. Leu(7)ET-1 is a contractile agonist with lower potency and similar maximal effect compared to ET-1 and greater sensitivity to BQ123 than ET-2. Up to 256 nM Trp(6)ET-1 did not cause contraction and did not antagonize arterial responses to ET-1. SIGNIFICANCE: Arterial smooth muscle ET(A)-receptor function displays agonist-dependent aspects. This involves roles of amino acids on position 6 and 7 of the endothelin sequence. Agonist-dependent pathologies may benefit from the design of specific, agonist-selective ET-receptor antagonists.

Calcitonin gene-related peptide terminates long-lasting vasopressor responses to endothelin 1 in vivo.

Slow dissociation of endothelin 1 from its endothelin A receptors is responsible for the long-lasting vasoconstrictor effects of the peptide. We showed recently that calcitonin gene-related peptide selectively terminates long-lasting contractile responses to endothelin 1 in isolated rat mesenteric arteries. Here we assessed whether the antiendothelinergic effect of calcitonin gene-related peptide is vascular bed specific and may terminate long-lasting pressor responses to exogenous and locally produced endothelin 1 in vivo. Regional heterogeneity of the calcitonin gene-related peptide/endothelin A receptor cross-talk was explored in arteries isolated from various rat organs. Endothelin A receptor-mediated arterial contractions were terminated by calcitonin gene-related peptide in rat mesenteric, renal, and spermatic arteries but not in basilar, coronary, epigastric, gastric, splenic, and saphenous arteries. Endothelin A receptor antagonism only ended endothelin 1-induced contractions in spermatic arteries. In anesthetized rats, instrumented with Doppler flow probes to record regional blood flows, long-lasting pressor and vasoconstrictor responses to an intravenous bolus injection of endothelin 1 or big endothelin 1 were transiently reduced by sodium nitroprusside (NO donor) but terminated by intravenously administered calcitonin gene-related peptide. In conscious rats, calcitonin gene-related peptide but not sodium nitroprusside terminated prolonged (>60-minute) pressor responses to endothelin 1 but not those to intravenous infusion of phenylephrine. In conclusion, pressor responses to circulating and locally produced endothelin 1 that are resistant to endothelin receptor antagonism and NO can be terminated by a regionally selective effect of calcitonin gene-related peptide. Calcitonin gene related peptide receptor agonism may represent a novel strategy to treat endothelin 1-associated cardiovascular pathologies.

ETA-receptor antagonists or allosteric modulators?

The paracrine signaling peptide endothelin-1 (ET1) is involved in cardiovascular diseases, cancer and chronic pain. It acts on class A G-protein-coupled receptors (GPCRs) but displays atypical pharmacology. It binds tightly to ET receptor type A (ET(A)) and causes long-lasting effects. In resistance arteries, the long-lasting contractile effects can only be partly and reversibly relaxed by low-molecular-weight ET(A) antagonists (ERAs). However, the neuropeptide calcitonin-gene-related peptide selectively terminates binding of ET1 to ET(A). We propose that ET1 binds polyvalently to ET(A) and that ERAs and the physiological antagonist allosterically reduce ET(A) functions. Combining the two-state model and the two-domain model of GPCR function and considering receptor activation beyond agonist binding might lead to better anti-endothelinergic drugs. Future studies could lead to compounds that discriminate between ET(A)-mediated effects of the endogenous isopeptides ET1, ET2 and ET3 and that become more effective when the activity of the endogenous endothelin system is elevated.

Stimuli of sensory-motor nerves terminate arterial contractile effects of endothelin-1 by CGRP and dissociation of ET-1/ET(A)-receptor complexes.

BACKGROUND: Endothelin-1 (ET-1), a long-acting paracrine mediator, is implicated in cardiovascular diseases but clinical trials with ET-receptor antagonists were not successful in some areas. We tested whether the quasi-irreversible receptor-binding of ET-1 (i) limits reversing effects of the antagonists and (ii) can be selectively dissociated by an endogenous counterbalancing mechanism. METHODOLOGY/PRINCIPAL FINDINGS: In isolated rat mesenteric resistance arteries, ET(A)-antagonists, endothelium-derived relaxing factors and synthetic vasodilators transiently reduced contractile effects of ET-1 but did not prevent persistent effects of the peptide. Stimuli of peri-vascular vasodilator sensory-motor nerves such as capsaicin not only reduced but also terminated long-lasting effects of ET-1. This was prevented by CGRP-receptor antagonists and was mimicked by exogenous calcitonin gene-related peptide (CGRP). Using 2-photon laser scanning microscopy in vital intact arteries, capsaicin and CGRP, but not ET(A)-antagonism, were observed to promote dissociation of pre-existing ET-1/ET(A)-receptor complexes. CONCLUSIONS: Irreversible binding and activation of ET(A)-receptors by ET-1 (i) occur at an antagonist-insensitive site of the receptor and (ii) are selectively terminated by endogenously released CGRP. Hence, natural stimuli of sensory-motor nerves that stimulate release of endogenous CGRP can be considered for therapy of diseases involving ET-1.