Urotensin-II signaling mechanism in rat coronary artery: role of STIM1 and Orai1-dependent store operated calcium influx in vasoconstriction.

OBJECTIVE: Human urotensin-II (UII) is considered the most potentendogenous vasoconstrictor discovered to date, although the precise mechanism activated downstream of its receptor UTS2R in blood vessels remains elusive. The aim of this study was to determine the role of the store operated Ca(2+) entry (SOCE) signaling pathway in UII-induced coronary artery vasoconstriction. METHODS AND RESULTS: We used a combination of isometric tension measurement, Ca(2+) imaging, pharmacology, and molecular approaches to study UII-mediated rat coronary artery vasoconstriction and intracellular Ca(2+) mobilization in coronary smooth muscle cells. We found that UII promoted dose-dependent vasoconstriction and elicited Ca(2+) and Mn(2+) influx, which were sensitive to classical SOCE inhibitors. In addition, knockdown of either STIM1 or Orai1 essentially inhibited UII-mediated SOCE and prevented UII but not high-KCL evoked contraction in transfected coronary artery. Moreover, we found that Ca(2+)-independent phospholipase A(2)ß was involved in UII effects and that is colocalized with STIM1 in different submembrane compartments. Importantly, STIM1 but not Orai1 downregulation inhibits significantly independent phospholipase A(2) activation. Furthermore, lysophosphatidylcholine, an independent phospholipase A(2) product, activated Orai1 but not STIM1-dependent contraction and SOCE. CONCLUSIONS: Here, we demonstrated that different critical players of SOCE signaling pathway are required for UII-induced vasoconstriction of rat coronary artery.

Urocortin-2 induces vasorelaxation of coronary arteries isolated from patients with heart failure.

1. Urocortin-2 (Ucn2) is a vasoactive peptide belonging to the corticotrophin-releasing factor (CRF) family that has potent cardiovascular actions. It has been suggested that Ucn2 participates in the pathophysiology of heart failure. However, little is known about the mechanisms underlying the action of Ucn2 in human coronary arteries. The aim of the present study was to assess the effects of Ucn2 on the vascular tone of human coronary arteries dissected from heart failure patients. 2. Human coronary arteries were dissected from the hearts of patients subjected to orthotopic heart transplantation. Coronary arteries were obtained from 17 patients with heart failure due to dilated cardiomyopathy of ischaemic origin in Stage III-IV of the New York Heart Association classification. Changes in tone were measured in arterial rings using force transducers. 3. Application of increasing concentrations of Ucn2 (5-20 nmol/L) to arterial rings precontracted with agonists induced dose-dependent relaxation of the coronary artery, which was independent of endothelial cell activation. Furthermore, the inhibition of the adenylyl cyclase by MDL-12 (100 nmol/L) and protein kinase A (PKA) by H89 (1 µmol/L) prevented Ucn2-mediated relaxation of coronary artery rings. 4. The results of the present study suggest that, in heart failure patients, Ucn2 could be useful in modulating coronary artery circulation independent of endothelial integrity through mechanisms that involve adenylyl cyclase activation and PKA stimulation. The findings warrant further investigation of the role of Ucn2 in circulatory regulation and its potential therapeutic application in heart disease.

Urotensin-II promotes vascular smooth muscle cell proliferation through store-operated calcium entry and EGFR transactivation.

AIMS: Urotensin-II (UII) is a vasoactive peptide that promotes vascular smooth muscle cells (VSMCs) proliferation and is involved in the pathogenesis of atherosclerosis, restenosis, and vascular remodelling. This study aimed to determine the role of calcium (Ca(2+))-dependent signalling and alternative signalling pathways in UII-evoked VSMCs proliferation focusing on store-operated Ca(2+) entry (SOCE) and epithelium growth factor receptor (EGFR) transactivation. METHODS AND RESULTS: We used primary cultures of VSMCs isolated from Wistar rat aorta to investigate the effects of UII on intracellular Ca(2+) mobilization, and proliferation determined by the 5-bromo-2-deoxyuridine (BrdU) assay. We found that UII enhanced intracellular Ca(2+) concentration ([Ca(2+)]i) which was significantly reduced by classical SOCE inhibitors and by knockdown of essential components of the SOCE such as stromal interaction molecule 1 (STIM1), Orai1, or TRPC1. Moreover, UII activated a Gd(3+)-sensitive current with similar features of the Ca(2+) release-activated Ca(2+) current (ICRAC). Additionally, UII stimulated VSMCs proliferation and Ca(2+)/cAMP response element-binding protein (CREB) activation through the SOCE pathway that involved STIM1, Orai1, and TRPC1. Co-immunoprecipitation experiments showed that UII promoted the association between Orai1 and STIM1, and between Orai1 and TRPC1. Moreover, we determined that EGFR transactivation, extracellular signal-regulated kinase (ERK) and Ca(2+)/calmodulin-dependent kinase (CaMK) signalling pathways were involved in both UII-mediated Ca(2+) influx, CREB activation and VSMCs proliferation. CONCLUSION: Our data show for the first time that UII-induced VSMCs proliferation and CREB activation requires a complex signalling pathway that involves on the one hand SOCE mediated by STIM1, Orai1, and TRPC1, and on the other hand EGFR, ERK, and CaMK activation.

Urocortin induces positive inotropic effect in rat heart.

AIMS: The aim of this study is to evaluate the positive inotropic effect of urocortin (Ucn) and to characterize its signalling pathways. METHODS AND RESULTS: Contractility was measured in ex vivo Langendorff-perfused hearts isolated from Wistar rats. Isolated ventricular cardiomyocytes were used to analyse intracellular calcium ([Ca(2+)](i)) transients evoked by electrical stimulation and L-type Ca(2+) current by confocal microscopy and whole-cell patch-clamping, respectively. The application of Ucn to perfused hearts induced progressive, sustained, and potent inotropic and lusitropic effects that were dose-dependent with an EC(50) of approximately 8 nM. Ucn effects were independent of protein kinase A (PKA) activation but were significantly reduced by protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) inhibitors and by brefeldin A, an antagonist of guanine nucleotide exchange factor, suggested to be an inhibitor of exchange protein activated by cAMP (Epac). These whole-organ effects were correlated with the inotropic effects observed in isolated cells: Ucn increased I(CaL) density, [Ca(2+)](i) transients, cell shortening and Ca(2+) content of sarcoplasmic reticulum. CONCLUSION: Our results show that Ucn evokes potent positive inotropic and lusitropic effects mediated, at least in part, by an increase in I(CaL) and [Ca(2+)](i) transient amplitude. These effects may involve the activation of Epac, PKC, and MAPK signalling pathways.