title: Calcimimetic R-568 and its enantiomer S-568 increase nitric oxide release in human endothelial cells.
abstract: 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.
  
  
  RESULTS
  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
  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.
authors.affiliation: Institute of Nephrology-Department of Medicine, University G. d'Annunzio, Chieti-Pescara, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: Bonomini
authors.forename: Annalisa
authors.initials: A
authors.lastname: Giardinelli
authors.forename: Caterina
authors.initials: C
authors.lastname: Morabito
authors.forename: Sara
authors.initials: S
authors.lastname: Di Silvestre
authors.forename: Moreno
authors.initials: M
authors.lastname: Di Cesare
authors.forename: Natalia
authors.initials: N
authors.lastname: Di Pietro
authors.forename: Vittorio
authors.initials: V
authors.lastname: Sirolli
authors.forename: Gloria
authors.initials: G
authors.lastname: Formoso
authors.forename: Luigi
authors.initials: L
authors.lastname: Amoroso
authors.forename: Maria Addolorata
authors.initials: MA
authors.lastname: Mariggiò
authors.forename: Assunta
authors.initials: A
authors.lastname: Pandolfi
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry* / Q000494:pharmacology*; D001011:Aorta / Q000166:cytology; D057966:Calcimimetic Agents / Q000737:chemistry* / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D010627:Phenethylamines; D011437:Propylamines; D044169:Receptors, Calcium-Sensing / Q000378:metabolism; D013237:Stereoisomerism
chemical_list: D000814:Aniline Compounds; D057966:Calcimimetic Agents; C107873:N-(2-chlorophenylpropyl)-1-(3-methoxyphenyl)ethylamine; D010627:Phenethylamines; D011437:Propylamines; D044169:Receptors, Calcium-Sensing; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D002118:Calcium