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 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.

Plasma protein carbonylation in chronic uremia.

BACKGROUND: Carbonylation is an irreversible modification caused by the introduction into proteins of carbonyl derivatives (aldehydes and ketones), which can alter protein structure and function and lead to cellular dysfunction and tissue damage. Chronic uremia may be associated with an increased carbonyl overload ("carbonyl stress"), though carbonyl formation has been proposed so far for major plasma proteins only. In this study we looked for evidence and for the targets of plasma protein carbonylation in patients on hemodialysis. We also examined the effect of in vitro carbonylated albumin on mRNA levels of endothelial cell adhesion molecules involved in early atherogenesis. METHODS: Carbonylated proteins in uremic plasma were detected by a covalent hydrazine bait strategy and identified by combining electrophoretic separation with mass spectrometry analysis of tryptic digests. Some plasma samples were first depleted of albumin and immunoglobulins to improve detection of lower abundance proteins. The functional impact of carbonylation was assessed in human vein endothelial cells by studying models of modified human serum albumin. RESULTS: Post-dialysis plasma carbonylated protein levels were significantly increased compared to pre-dialysis levels. Susceptibility to carbonyl formation was described on a open-platform investigation for a number of plasma proteins, albumin being the main scavenger of carbonyl reactive species. Incubation of endothelial cells with low doses of carbonylated albumin caused a significant increase in intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 mRNA levels. CONCLUSIONS: Chronic uremia appears as a state of "carbonyl stress" targeting several different plasma proteins. Carbonylated albumin displayed biological effects that may be relevant to uremic atherosclerosis.

Proteomics characterization of protein adsorption onto hemodialysis membranes.

Protein-adsorptive properties are a key feature of membranes used for hemodialysis treatment. Protein adsorption is vital to the biocompatibility of a membrane material and influences membrane's performance. The object of the present study is to investigate membrane biocompatibility by correlating the adsorbed proteome repertoire with structural feature of the membrane surfaces. Minidialyzers of identical structural characteristics composed of either cellulose diacetate or ethylenevinyl alcohol materials were employed to develop an ex vivo apparatus to investigate protein adsorption. Adsorbed proteins were eluted by a strong chaotropic buffer condition and investigated by 2-DE coupled to both MALDI-TOF mass spectrometry (MS) mass fingerprinting and fragmentation analysis on a nanoLC-MS/MS hybrid instrument. Membrane surface characterization included evaluation of roughness (atomic force microscopy), elemental chemical composition (X-ray-photoelectron-spectroscopy), and hydrophilicity (pulsed nuclear magnetic resonance). The present study identifies a number of different proteins as common or characteristic of filter material interaction, showing that proteomic techniques are a promising approach for the investigation of proteins surface-adsorbed onto hemodialysis membrane. Proteomic analysis enables the characterization of protein layers of unknown composition.