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Calcimimetic R-568 and Its Enantiomer S-568 Increase Nitric Oxide Release in Human Endothelial Cells

BACKGROUND: Calcimimetics, such as R-568, are thought to activate G protein-linked Ca(2+)-sensing receptor (CaSR) by allosterically increasing the affinity of the receptor for Ca(2+) allowing for efficient control of uremic hyperparathyroidism. Several recent studies suggest they possess additional...

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Autores principales: Bonomini, Mario, Giardinelli, Annalisa, Morabito, Caterina, Di Silvestre, Sara, Di Cesare, Moreno, Di Pietro, Natalia, Sirolli, Vittorio, Formoso, Gloria, Amoroso, Luigi, Mariggiò, Maria Addolorata, Pandolfi, Assunta
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3266284/
https://www.ncbi.nlm.nih.gov/pubmed/22295103
http://dx.doi.org/10.1371/journal.pone.0030682
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author Bonomini, Mario
Giardinelli, Annalisa
Morabito, Caterina
Di Silvestre, Sara
Di Cesare, Moreno
Di Pietro, Natalia
Sirolli, Vittorio
Formoso, Gloria
Amoroso, Luigi
Mariggiò, Maria Addolorata
Pandolfi, Assunta
author_facet Bonomini, Mario
Giardinelli, Annalisa
Morabito, Caterina
Di Silvestre, Sara
Di Cesare, Moreno
Di Pietro, Natalia
Sirolli, Vittorio
Formoso, Gloria
Amoroso, Luigi
Mariggiò, Maria Addolorata
Pandolfi, Assunta
author_sort Bonomini, Mario
collection PubMed
description BACKGROUND: Calcimimetics, such as R-568, are thought to activate G protein-linked Ca(2+)-sensing receptor (CaSR) by allosterically increasing the affinity of the receptor for Ca(2+) allowing for efficient control of uremic hyperparathyroidism. Several recent studies suggest they possess additional vascular actions. Although it has been postulated that calcimimetics may have a direct effect on CaSR in the blood vessels, further studies are needed to elucidate their vascular CaSR-dependent versus CaSR-independent effects. METHODOLOGY/PRINCIPAL FINDINGS: Focusing on human umbilical vein endothelial cells (HUVECs), we studied the CaSR expression and distribution by Immunofluorescence and Western Blot analysis. CaSR function was evaluated by measuring the potential effect of calcimimetic R-568 and its enantiomer S-568 upon the modulation of intracellular Ca(2+) levels (using a single cell approach and FURA-2AM), in the presence or absence of Calhex-231, a negative modulator of CaSR. To address their potential vascular functions, we also evaluated R- and S-568-stimulated enzymatic release of Nitric Oxide (NO) by DAF-2DA, by Nitric Oxide Synthase (NOS) radiometric assay (both in HUVECs and in Human Aortic Endothelial Cells) and by measuring eNOS-ser1177 phosphorylation levels (Immunoblotting). We show that, although the CaSR protein was expressed in HUVECs, it was mainly distributed in cytoplasm while the functional CaSR dimers, usually localized on the plasma membrane, were absent. In addition, regardless of the presence or absence of Calhex-231, both R- and S-568 significantly increased intracellular Ca(2+) levels by mobilization of Ca(2+) from intracellular stores, which in turn augmented NO release by a time- and Ca(2+)-dependent increase in eNOS-ser1177 phosphorylation levels. CONCLUSIONS/SIGNIFICANCE: Taken together, these data indicate that in human endothelium there is no stereoselectivity in the responses to calcimimetics and that CaSR is probably not involved in the action of R- and S-568. This suggests an additional mechanism in support of the CaSR-independent role of calcimimetics as vasculotrope agents.
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spelling pubmed-32662842012-01-31 Calcimimetic R-568 and Its Enantiomer S-568 Increase Nitric Oxide Release in Human Endothelial Cells Bonomini, Mario Giardinelli, Annalisa Morabito, Caterina Di Silvestre, Sara Di Cesare, Moreno Di Pietro, Natalia Sirolli, Vittorio Formoso, Gloria Amoroso, Luigi Mariggiò, Maria Addolorata Pandolfi, Assunta PLoS One Research Article BACKGROUND: Calcimimetics, such as R-568, are thought to activate G protein-linked Ca(2+)-sensing receptor (CaSR) by allosterically increasing the affinity of the receptor for Ca(2+) allowing for efficient control of uremic hyperparathyroidism. Several recent studies suggest they possess additional vascular actions. Although it has been postulated that calcimimetics may have a direct effect on CaSR in the blood vessels, further studies are needed to elucidate their vascular CaSR-dependent versus CaSR-independent effects. METHODOLOGY/PRINCIPAL FINDINGS: Focusing on human umbilical vein endothelial cells (HUVECs), we studied the CaSR expression and distribution by Immunofluorescence and Western Blot analysis. CaSR function was evaluated by measuring the potential effect of calcimimetic R-568 and its enantiomer S-568 upon the modulation of intracellular Ca(2+) levels (using a single cell approach and FURA-2AM), in the presence or absence of Calhex-231, a negative modulator of CaSR. To address their potential vascular functions, we also evaluated R- and S-568-stimulated enzymatic release of Nitric Oxide (NO) by DAF-2DA, by Nitric Oxide Synthase (NOS) radiometric assay (both in HUVECs and in Human Aortic Endothelial Cells) and by measuring eNOS-ser1177 phosphorylation levels (Immunoblotting). We show that, although the CaSR protein was expressed in HUVECs, it was mainly distributed in cytoplasm while the functional CaSR dimers, usually localized on the plasma membrane, were absent. In addition, regardless of the presence or absence of Calhex-231, both R- and S-568 significantly increased intracellular Ca(2+) levels by mobilization of Ca(2+) from intracellular stores, which in turn augmented NO release by a time- and Ca(2+)-dependent increase in eNOS-ser1177 phosphorylation levels. CONCLUSIONS/SIGNIFICANCE: Taken together, these data indicate that in human endothelium there is no stereoselectivity in the responses to calcimimetics and that CaSR is probably not involved in the action of R- and S-568. This suggests an additional mechanism in support of the CaSR-independent role of calcimimetics as vasculotrope agents. Public Library of Science 2012-01-25 /pmc/articles/PMC3266284/ /pubmed/22295103 http://dx.doi.org/10.1371/journal.pone.0030682 Text en Bonomini et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Bonomini, Mario
Giardinelli, Annalisa
Morabito, Caterina
Di Silvestre, Sara
Di Cesare, Moreno
Di Pietro, Natalia
Sirolli, Vittorio
Formoso, Gloria
Amoroso, Luigi
Mariggiò, Maria Addolorata
Pandolfi, Assunta
Calcimimetic R-568 and Its Enantiomer S-568 Increase Nitric Oxide Release in Human Endothelial Cells
title Calcimimetic R-568 and Its Enantiomer S-568 Increase Nitric Oxide Release in Human Endothelial Cells
title_full Calcimimetic R-568 and Its Enantiomer S-568 Increase Nitric Oxide Release in Human Endothelial Cells
title_fullStr Calcimimetic R-568 and Its Enantiomer S-568 Increase Nitric Oxide Release in Human Endothelial Cells
title_full_unstemmed Calcimimetic R-568 and Its Enantiomer S-568 Increase Nitric Oxide Release in Human Endothelial Cells
title_short Calcimimetic R-568 and Its Enantiomer S-568 Increase Nitric Oxide Release in Human Endothelial Cells
title_sort calcimimetic r-568 and its enantiomer s-568 increase nitric oxide release in human endothelial cells
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3266284/
https://www.ncbi.nlm.nih.gov/pubmed/22295103
http://dx.doi.org/10.1371/journal.pone.0030682
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