Laboratoire International Associe Samuel Of Champlain

Samuel, Canada

Laboratoire International Associe Samuel Of Champlain

Samuel, Canada
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Doan N.D.,University of Québec | Doan N.D.,Laboratoire International Associe Samuel Of Champlain | Nguyen T.T.M.,University of Québec | Nguyen T.T.M.,Laboratoire International Associe Samuel Of Champlain | And 8 more authors.
British Journal of Pharmacology | Year: 2012

Background and Purpose During the past decade, a few GPCRs have been characterized at the nuclear membrane where they exert complementary physiological functions. In this study, we investigated (1) the presence of a functional urotensin-II (U-II) receptor (UT) in rat heart nuclear extracts and (2) the propensity of U-II and U-II-related peptide (URP) to cross the plasma membrane in a receptor-independent manner. Experimental Approach Biochemical and pharmacological methods including competitive binding assays, photoaffinity labelling, immunoblotting as well as de novo RNA synthesis were used to characterize the presence of functional UT receptors in rat heart nuclei. In addition, confocal microscopy and flow cytometry analysis were used to investigate the cellular uptake of fluorescent U-II and URP derivatives. Key Results The presence of specific U-II binding sites was demonstrated in rat heart nuclear extracts. Moreover, such subcellular localization was also observed in monkey heart extracts. In vitro transcription initiation assays on rat, freshly isolated, heart nuclei suggested that nuclear UT receptors are functional, and that U-II, but not URP, participates in nuclear UT-associated gene expression. Surprisingly, hU-II and URP efficiently crossed the plasma membrane in a receptor-independent mechanism involving endocytosis through caveolin-coated pits; this uptake of hU-II, but not that of URP, was dependent on extracellular pH. Conclusion Our results suggest that (1) U-II and URP can differentially modulate nuclear UT functions such as gene expression, and (2) both ligands can reach the internal cellular space through a receptor-independent mechanism. © 2011 The British Pharmacological Society.

Chatenet D.,University of Québec | Chatenet D.,Laboratoire International Associe Samuel Of Champlain | Letourneau M.,University of Québec | Letourneau M.,Laboratoire International Associe Samuel Of Champlain | And 7 more authors.
British Journal of Pharmacology | Year: 2013

Background and Purpose Recent evidence suggested that urotensin II (UII) and its paralog peptide UII-related peptide (URP) might exert common but also divergent physiological actions. Unfortunately, none of the existing antagonists were designed to discriminate specific UII- or URP-Associated actions, and our understanding, on how these two endogenous peptides can trigger different, but also common responses, is limited. Experimental Approach Ex vivo rat and monkey aortic ring contraction as well as dissociation kinetics studies using transfected CHO cells expressing the human urotensin (UT) receptors were used in this study. Key Results Ex vivo rat and monkey aortic ring contraction studies revealed the propensity of [Pep4]URP to decrease the maximal response of human UII (hUII) without any significant change in potency, whereas no effect was noticeable on the URP-induced vasoconstriction. Dissociation experiments demonstrated the ability of [Pep4]URP to increase the dissociation rate of hUII, but not URP. Surprisingly, URP, an equipotent UII paralog, was also able to accelerate the dissociation rate of membrane-bound 125I-hUII, whereas hUII had no noticeable effect on URP dissociation kinetics. Further experiments suggested that an interaction between the glutamic residue at position 1 of hUII and the UT receptor seems to be critical to induce conformational changes associated with agonistic activation. Finally, we demonstrated that the N-terminal domain of the rat UII isoform was able to act as a specific antagonist of the URP-Associated actions. Conclusion Such compounds, that is [Pep4]URP and rUII(1-7), should prove to be useful as new pharmacological tools to decipher the specific role of UII and URP in vitro but also in vivo. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Tadevosyan A.,University of Montréal | Tadevosyan A.,Montreal Heart Institute | Villeneuve L.R.,Montreal Heart Institute | Fournier A.,University of Québec | And 9 more authors.
Methods | Year: 2016

In addition to cell surface membranes, numerous G protein-coupled receptors (GPCRs) are located on intracellular membranes including the nuclear envelope. Although the role of numerous GPCRs at the cell surface has been well characterized, the physiological function of these same receptors located on intracellular membranes remains to be determined. Here, we employ a novel caged Ang-II analog, cAng-II, to compare the effects of the activation of cell surface versus intracellular angiotensin receptors in intact cardiomyocytes. When added extracellularly to HEK 293 cells, Ang-II and photolysed cAng-II increased ERK1/2 phosphorylation (via AT1R) and cGMP production (AT2R). In contrast unphotolysed cAng-II did not. Cellular uptake of cAng-II was 6-fold greater than that of Ang-II and comparable to the HIV TAT(48-60) peptide. Intracellular photolysis of cAng-II induced an increase in nucleoplasmic Ca2+ ([Ca2+]n) that was greater than that induced by extracellular application of Ang-II. We conclude that cell-permeable ligands that can access intracellular GPCRs may evoke responses distinct from those with access restricted to the same receptor located on the cell surface. © 2015 Elsevier Inc.

Nguyen T.-T.M.,University of Québec | Nguyen T.-T.M.,Laboratoire International Associe Samuel Of Champlain | Letourneau M.,University of Québec | Letourneau M.,Laboratoire International Associe Samuel Of Champlain | And 4 more authors.
International Journal of Biochemistry and Cell Biology | Year: 2012

Urotensin II (UII) and its receptor UT, are widely expressed in the cardiovascular and central nervous system, where they exert regulatory actions under both physiological and pathological conditions. Our study, aimed at investigating the presence of functional nuclear UT in various rat and monkey tissues as well as in human cell lines, demonstrated for the first time by Western blot analysis and confocal immunofluorescence a tissue-specific nuclear expression of this receptor (heart and central nervous system). This nuclear UT was further characterized pharmacologically through radioligand binding studies using specific ligands of the urotensinergic system, as well as somatostatin. In 2D-gel experiments, we observed the presence of different post-translational modifications between membrane and nuclear UT receptors in brain extracts. Transcription initiation assays showed de novo RNA synthesis caused by UII and Urotensin-related peptide (URP) which were inhibited by an UT antagonist urantide. In hypoxic/ischemic conditions, UT receptors were differentially modulated in regard to subcellular localization. Thus, the unique regiospecificity of the nuclear UT receptor along with its particular modulation under hypoxic conditions could indicate a specific and complementary physiological role that could be correlated with pro-angiogenic and/or neuromodulatory actions of UII, both in the cardiovascular and central nervous system. © 2012 Elsevier Ltd. All rights reserved.

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