Effect of R-568 on Akt activation and NO production in vSMCs from WKY and SHR rats.

<p>(A) vSMCs stimulated with 1μM of R568 (R) at different times (30 seconds, 1 minute, 2 minutes) in the presence or absence of Calhex 231 (C, 1 μM) were subjected to immunoblotting for total and phosphorylated forms of Akt (phospho-Akt^Ser473). Total β-actin levels were used as loading control. A representative set of immunoblots is shown for experiments that were repeated independently 3 times. Each bar represents the mean ± SD of densitometric analysis for phosphorylated proteins normalized to their respective total forms. *p < 0.02 <i>vs</i> respective basal (CTRL), **p < 0.05 R+C <i>vs</i> respective R. (B) NO production determined by conversion of L-[3H]-arginine into L-[3H]-citrulline in vSMCs stimulated with 1μM of R568 in the presence or absence of Calhex 231 (1 μM), data are expressed as pmol/NO/min/mgprottot (picomoles /Nitric Oxide/ minutes/ milligrams protein total). Each bar represents the mean ± SD of 3 independent experiments. *p < 0.02 R568 <i>vs</i> respective basal, **p < 0.01 R568+Calhex <i>vs</i> respective R568.</p

Effect of endothelium removal and CaSR inhibition on vascular response to R-568.

<p>(A) MVB isolated from 8-week old WKY (closed symbols) and SHR (open symbols) were stimulated with increasing concentrations of R-568 in the presence (circles) or in the absence (squares) of endothelium. Results shown are the mean ± SEM of 3 (WKY) and 3 (SHR) independent experiments. Data from each curve were normalized as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0202354#pone.0202354.g001" target="_blank">Fig 1</a>. (B) MVB isolated from 8-week old WKY (closed symbols) and SHR (open symbols) were stimulated with increasing concentrations of R-568 in the absence (circles) or after pretreatment with (diamonds) Calhex-231 (3 μM/20 min). Results are the mean ± SEM of 3 WKY and 3 SHR independent experiments. Data from each curve were normalized as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0202354#pone.0202354.g001" target="_blank">Fig 1</a>. *p < 0.05 <i>vs</i> respective CTRL curve.</p

Evaluation of signaling pathways activated by R-568 in MVB from WKY and SHR.

<p>MVB homogenates from experiments described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0202354#pone.0202354.g001" target="_blank">Fig 1</a> were subjected to immunoblotting for total and phosphorylated forms of Akt (phospho-Akt^Ser473), and eNOS (phospho-eNOS^Ser1177). Total β-actin levels were used as loading control. A representative set of immunoblots is shown for experiments that were repeated independently 3 times. Each bar represents the mean ± SEM of densitometric analysis for phosphorylated proteins normalized to their respective total forms. *p < 0.05 <i>vs</i> respective basal.</p

CaSR expression in WKY- and SHR-vSMVCs.

<p>(A) Representative morphometric aspect of confluent vSMC cultures from normotensive rats (WKY) and spontaneously hypertensive rats (SHR) (magnification x 10; scale bar: 300 μM). The cells were plated and morphologically examined three different times in the same experimental conditions. (B) Representative pattern of electrophoretic bands (170–100, 70–55 and 40–25 kDa) that results from immunoblotting analysis of CaSR expression in WKY- and SHR-vSMCs lysates and its negative (NC, HEK293 empty vector transfected control cell lysate) and positive (PC, HEK293 CaSR transiently transfected cell lysate) controls. (C) Representative images of α-SMA flow cytometry analysis in WKY-and SHR-vSMCs and secondary antibody alone (control).</p

Evaluation of endothelial mediators involved in vascular effects of R-568 in MVB from WKY and SHR rats.

<p>(A) and (B) Representative tracings of vascular response to R-568 (10 nM—30 μM) in MVB from WKY (A) or SHR (B) pretreated with L-NAME, CTX and INDO are shown. Each symbol represents the start of a 4-min perfusion. (C) MVB isolated from 8-week old WKY (closed symbols) were stimulated with increasing concentrations of R-568 in the absence (circles) or in the presence of L-NAME (100 μM/20 min; squares), L-NAME+CTX (300 nM/20 min; triangles), L-NAME+CTX+INDO (10 μM/20 min; diamonds). Results are the mean ± SEM of 4 WKY independent experiments. (D) MVB isolated from 8-week old SHR (open symbols) were stimulated with increasing concentrations of R-568 in the absence (circles) or in the presence of L-NAME (100 μM/20 min; squares), L-NAME+CTX (300 nM/20 min; triangles), L-NAME+CTX+INDO (10 μ M/20 min; diamonds). Results are the mean ± SEM of 4 WKY and 4 SHR independent experiments. *p < 0.04 and ^#p < 0.05 <i>vs</i> respective CTRL curve.</p

Effects of 100 µM R-568 and S-568 with and without extra and intracellular Ca²⁺ on NO production.

<p>Intracellular NO levels (mean value ± SE) in DAF-2DA-loaded HUVECs (A–C). Time effect of R-568 or S-568 100 µM on eNOS-ser1177 phosphorylation (D). Representative immunoblot of eNOS-ser1177 phosphorylation (Upper Panel). Representative data from three experiments (means ± SD, Lower Panel, p<0.003 vs 0′). Phospho-eNOS (p-eNOS) was normalized vs eNOS total.</p

Effects of different doses of R-568 or S-568 on HUVECs [Ca²⁺]_i.

<p>Traces (mean value ± SE) representing [Ca²⁺]i variations in FURA-2AM-loaded HUVECs stimulated with R-568 (A–C) or S-568 (D–F) at 1 µM (A and D), 10 µM (B and E) or 100 µM (C and F) concentrations. Representative traces after stimulation with R-568 100 µM + Calhex 10 µM (inset C) or S-568 100 µM + Calhex 10 µM (inset F).</p

Effects of R-568 and S-568 on HUVEC and HAEC NOS activity.

<p>*p<0.05 (R- and S-568 vs Control).</p

CaSR protein expression in HUVECs by Immunofluorescence Confocal Microscopy and Western Blot.

<p>Immunofluorescent localization of CaSR in HUVECs with specific antibody and negative control after fixation and permeabilization protocol (A and B), or after fixation but not membrane permeabilization (C and D). Representative Western Blot of CaSR protein levels in HAoVSMC, HAEC and HUVEC total lysate, and in HUVEC membrane and cytoplasm extracts (E).</p

Effects of R-568 or S-568 on HUVECs [Ca²⁺]_i without extra or intracellular Ca²⁺.

<p>Representative Ca²⁺-variations in FURA-2AM-loaded HUVECs stimulated with 100 µM R-568 or 100 µM S-568 in Ca²⁺ free medium (A and B) or after depletion of intracellular Ca²⁺ stores by thapsigargin (tg) (C and D).</p