Journal: Cardiovascular Research

Article Title: Endothelial nitric oxide synthase activity is inhibited by the plasma membrane calcium ATPase in human endothelial cells

doi: 10.1093/cvr/cvq077

Figure Lengend Snippet: Endogenous PMCA and eNOS interact in human endothelial cells. (A) eNOS and PMCA co-precipitate in endothelial cells. (Left panels) Protein lysates isolated from primary HUVEC or HDMEC were incubated with an anti-PMCA monoclonal antibody (5F10) and immunoprecipitated proteins analysed by western blot with an anti-eNOS rabbit polyclonal antibody. (Right panels) Endothelial protein lysates were immunoprecipitated with an anti-eNOS rabbit polyclonal antibody (α-eNOS) and immunoprecipitated proteins probed against the 5F10 anti-PMCA monoclonal antibody. (B) PMCA isoforms 1, 2, and 4 are expressed in endothelial cells. Protein lysates isolated from HUVEC or HDMEC were precipitated with an anti-PMCA monoclonal antibody (5F10) and precipitated proteins were probed with rabbit polyclonal antibodies recognizing specifically PMCA isoforms 1, 2, or 4. (C) Protein extracts isolated from HUVEC were immunoprecipitated with polyclonal antibodies recognizing specifically the isoforms 1 (α-PMCA1) or 2 (α-PMCA2) of PMCA. Immunoprecipitated proteins were probed with a mouse anti-eNOS antibody (Zymed). Likewise, protein extracts were precipitated with the JA9 anti-PMCA4 monoclonal antibody (α-PMCA4) and immunoprecipitated proteins subsequently probed with a rabbit anti-eNOS polyclonal antibody. In all cases, eNOS was present within the immunoprecipitated proteins, suggesting that PMCA 1, 2, and 4 isoforms interact with eNOS in endothelial cells. Immunoprecipitation with an irrelevant antibody against firefly luciferase (α-Luc) was included as a negative control in all immunoprecipitation experiments.

Article Snippet: 13 For transfection of primary endothelial cells, 1 × 10 6 HUVEC were transfected with 5 μg of the indicated expression plasmids in 100 μL of HUVEC nucleofector solution from a HUVEC nucleofector kit (Amaxa) following the recommendations of the manufacturer.

Techniques: Isolation, Incubation, Immunoprecipitation, Western Blot, Luciferase, Negative Control

Journal: Cardiovascular Research

Article Title: Endothelial nitric oxide synthase activity is inhibited by the plasma membrane calcium ATPase in human endothelial cells

doi: 10.1093/cvr/cvq077

Figure Lengend Snippet: Endogenous PMCA and calcineurin interact in endothelial cells. Protein lysates from HUVEC were incubated with an anti-PMCA monoclonal antibody (5F10), anti-Luciferase antibody (α-Luc), or an anti-calcineurin antibody (α-Cal) as indicated. Immunoprecipitated proteins were analysed by western blot with antibodies recognizing specifically isoform 1 (α-PMCA1), 2 (α-PMCA2), or 4 (α-PMCA4) of PMCA. The presence of the PMCA isoforms within the immunoprecipitated proteins indicates that endogenous PMCA and calcineurin interact in endothelial cells.

Article Snippet: 13 For transfection of primary endothelial cells, 1 × 10 6 HUVEC were transfected with 5 μg of the indicated expression plasmids in 100 μL of HUVEC nucleofector solution from a HUVEC nucleofector kit (Amaxa) following the recommendations of the manufacturer.

Techniques: Incubation, Luciferase, Immunoprecipitation, Western Blot

Journal: Cardiovascular Research

Article Title: Endothelial nitric oxide synthase activity is inhibited by the plasma membrane calcium ATPase in human endothelial cells

doi: 10.1093/cvr/cvq077

Figure Lengend Snippet: PMCA negatively regulates NO production in endothelial cells. Human PMCA2b or PMCA4b were ectopically expressed in HUVEC by transfection of five μg of the corresponding expression vector (A). NO synthesis was induced by addition of the A23187 ionophore (0.5 μM) 3 min before lyses. NO-dependent cGMP production was calculated in relation to HUVEC transfected with 5 μg of pcDNA3-Empty vector. Mean ± SEM values of six independent experiments are shown. *P < 0.05. (B) Intracellular nitric oxide level in transfected HUVEC was assessed by staining cells with the NO-sensitive dye DAF-FM. NO synthesis was stimulated by addition of acetylcholine (100 μM) for 5 min. Representative microscopy fields of DAF-FM staining in cells are shown in left panels. Mean ± SEM values of fluorescence units measured from 25 different microscopy fields taken from six independent preparations are shown. **P < 0.01.

Article Snippet: 13 For transfection of primary endothelial cells, 1 × 10 6 HUVEC were transfected with 5 μg of the indicated expression plasmids in 100 μL of HUVEC nucleofector solution from a HUVEC nucleofector kit (Amaxa) following the recommendations of the manufacturer.

Techniques: Transfection, Expressing, Plasmid Preparation, Staining, Microscopy, Fluorescence

Journal: Cardiovascular Research

Article Title: Endothelial nitric oxide synthase activity is inhibited by the plasma membrane calcium ATPase in human endothelial cells

doi: 10.1093/cvr/cvq077

Figure Lengend Snippet: PMCA modulates eNOS phosphorylation in regulatory residues Thr-495 and Ser-1177. (A) Ectopic expression of PMCA2b or 4b in endothelial cells increases the phosphorylation status of endogenous eNOS in Thr-495. HUVEC or HDMEC were transfected with 5 μg of plasmids pcDNA3-hPMCA2b, pcDNA-hPMCA4b, or p-cDNA3-Empty (negative control) and proteins analysed by western blot with a mouse antibody recognizing specifically eNOS phosphorylated in the residue Thr-495 (α-eNOS(pT495)). Histogram shows data as mean ± SEM of seven independent experiments performed with four different batches of cells. **P < 0.01. (B) Expression of PMCA2b in endothelial cells leads to a reduction in eNOS Ser-1177 phosphorylation. HUVEC or HDMEC were transfected with 5 μg of plasmids pcDNA3-hPMCA2b or pcDNA3-Empty (negative control). Transfected cells were incubated with VEGF (25 ng/mL) for the times indicated to induce phosphorylation of eNOS in Ser-1177 as described.25 Protein lysates were analysed by western blot with a mouse antibody recognizing specifically eNOS phosphorylated in Ser-1177 (α-eNOS(pS1177). Histogram shows data as mean ± SEM of six independent experiments performed with four different batches of cells. *P < 0.05, **P < 0.01. (C) Disruption of the PMCA–eNOS interaction reverses the PMCA-dependent effect on eNOS Thr-495 phosphorylation. HMVEC (left panels) or HUVEC (right panels) were transfected with either 10 μg of empty vector; 5 μg of empty vector plus 5 μg of a vector encoding PMCA2b or PMCA4b as indicated; or 5 μg of an expression vector for the indicated PMCA protein plus 5 μg of a pFlag vector encoding the corresponding interaction domain. Western blot was performed as described in (A). Histogram shows data as mean ± SEM of five independent experiments performed with three different batches of cells. **P < 0.01. In all experiments, total levels of eNOS protein were determined by analysing lysates from transfected cells using a polyclonal rabbit anti-eNOS antibody (α-eNOS total).

Article Snippet: 13 For transfection of primary endothelial cells, 1 × 10 6 HUVEC were transfected with 5 μg of the indicated expression plasmids in 100 μL of HUVEC nucleofector solution from a HUVEC nucleofector kit (Amaxa) following the recommendations of the manufacturer.

Techniques: Phospho-proteomics, Expressing, Transfection, Negative Control, Western Blot, Residue, Incubation, Disruption, Plasmid Preparation