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Proteomics reveals potential non-neuronal cholinergic receptor-effectors in endothelial cells.

Zhang YY, Shen W, Zhang LC, Pan ZY, Long CL, Cui WY, Zhang YF, Wang H - Acta Pharmacol. Sin. (2014)

Bottom Line: In this study, we used a proteomic approach to study potential downstream receptor-effectors of NNAS that were involved in regulating cellular function in endothelial cells.Western blotting analyses showed that treatment of the cells with cholinergic agonists significantly decreased the expression of destrin, FKBP1A and MIF, and increased the expression of profilin-1.A set of proteins differentially expressed in endothelial cells in response to cholinergic agonists may have important implications for the downstream biological effects of NNAS.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Drug Research Center, Institute of Health and Environmental Medicine, Academy of Military Medical Sciences, Beijing 100850, China.

ABSTRACT

Aim: The non-neuronal acetylcholine system (NNAS) in endothelial cells participates in modulating endothelial function, vascular tone, angiogenesis and inflammation, thus plays a critical role in cardiovascular diseases. In this study, we used a proteomic approach to study potential downstream receptor-effectors of NNAS that were involved in regulating cellular function in endothelial cells.

Methods: Human umbilical vein endothelial cells were incubated in the presence of acetylcholine, oxotremorine, pilocarpine or nicotine at the concentration of 10 μmol/L for 12 h, and the expressed proteins in the cells were separated and identified with two-dimensional electrophoresis (2-DE) and LC-MS. The protein spots with the largest changes were identified by LC-MS. Biowork software was used for database search of the peptide mass fingerprints.

Results: Over 1200 polypeptides were reproducibly detected in 2-DE with a pH range of 3-10. Acetylcholine, oxotremorine, pilocarpine and nicotine treatment caused 16, 9, 8 and 9 protein spots, respectively, expressed differentially. Four protein spots were identified as destrin, FK506 binding protein 1A (FKBP1A), macrophage migration inhibitory factor (MIF) and profilin-1. Western blotting analyses showed that treatment of the cells with cholinergic agonists significantly decreased the expression of destrin, FKBP1A and MIF, and increased the expression of profilin-1.

Conclusion: A set of proteins differentially expressed in endothelial cells in response to cholinergic agonists may have important implications for the downstream biological effects of NNAS.

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Related in: MedlinePlus

Two-dimensional gel showing the proteome of cultured HUVECs at a pH range of 3–10 after stimulation with cholinergic agonists. The cells were incubated with different agonists at pharmacological concentration of 10 μmol/L for 12 h. Positions of the differentially expressed protein spots with corresponding spot numbers are indicated with arrows. These results are representative of at least four independent experiments. A1/A2: before/after acetylcholine treatment, protein maps were stained with silver; A3: differentially expressed spots between the control and acetylcholine group, spots in A3 were visualized with Coomassie Brilliant Blue staining. The expression-changed proteins with spot numbers were identified and they are listed in Table 2. B1/B2: before/after oxotremorine treatment, silver staining; B3: differentially expressed protein spots between control and oxotremorine, Coomassie Brilliant Blue staining. The expression-changed proteins with the spot numbers are listed in Table 3. C1/C2: before/after pilocarpine treatment, silver staining; C3: differentially expressed protein spots between control and pilocarpine, Coomassie Brilliant Blue staining. The protein expression changes with the spot numbers were listed in Table 4. D1/D2: before/after nicotine treatment, silver staining; D3: differentially expressed protein spots between control and nicotine, Coomassie Brilliant Blue staining. The protein expression changes with the spot numbers are listed in Table 5. (E–H) show magnified views of some differentially expressed proteins between the control and each acetylcholine receptor agonist treatment group. (E) differentially expressed proteins between the control and acetylcholine treatment group. (F) between the control and oxotremorine treatment group. (G) between the control and pilocarpine treatment group. (H) between the control and nicotine treatment group.
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fig1A: Two-dimensional gel showing the proteome of cultured HUVECs at a pH range of 3–10 after stimulation with cholinergic agonists. The cells were incubated with different agonists at pharmacological concentration of 10 μmol/L for 12 h. Positions of the differentially expressed protein spots with corresponding spot numbers are indicated with arrows. These results are representative of at least four independent experiments. A1/A2: before/after acetylcholine treatment, protein maps were stained with silver; A3: differentially expressed spots between the control and acetylcholine group, spots in A3 were visualized with Coomassie Brilliant Blue staining. The expression-changed proteins with spot numbers were identified and they are listed in Table 2. B1/B2: before/after oxotremorine treatment, silver staining; B3: differentially expressed protein spots between control and oxotremorine, Coomassie Brilliant Blue staining. The expression-changed proteins with the spot numbers are listed in Table 3. C1/C2: before/after pilocarpine treatment, silver staining; C3: differentially expressed protein spots between control and pilocarpine, Coomassie Brilliant Blue staining. The protein expression changes with the spot numbers were listed in Table 4. D1/D2: before/after nicotine treatment, silver staining; D3: differentially expressed protein spots between control and nicotine, Coomassie Brilliant Blue staining. The protein expression changes with the spot numbers are listed in Table 5. (E–H) show magnified views of some differentially expressed proteins between the control and each acetylcholine receptor agonist treatment group. (E) differentially expressed proteins between the control and acetylcholine treatment group. (F) between the control and oxotremorine treatment group. (G) between the control and pilocarpine treatment group. (H) between the control and nicotine treatment group.

Mentions: In this work, we established the 2-DE protein patterns of the control group and the endothelial cells activated by ACh, Oxo, Pilo, and Nico. More than 1200 protein spots were detected on the 2-DE gels that showed similar protein patterns with silver staining of 300 μg of total proteins. Figure 1 displays the overall 2-DE patterns of protein extracts of control/ACh (Figure 1A), control/Oxo (Figure 1B), control/Pilo (Figure 1C), and control/Nico (Figure 1D). Based on analysis with Image Master 2D Platinum software, among all the detected spots, 16, 8, 8 and 9 spots were separately changed in cells treated with ACh, Oxo, Pilo and Nico, showing detectable changes above 2-fold and the match rate was above 60% (Figure 1E–1H).


Proteomics reveals potential non-neuronal cholinergic receptor-effectors in endothelial cells.

Zhang YY, Shen W, Zhang LC, Pan ZY, Long CL, Cui WY, Zhang YF, Wang H - Acta Pharmacol. Sin. (2014)

Two-dimensional gel showing the proteome of cultured HUVECs at a pH range of 3–10 after stimulation with cholinergic agonists. The cells were incubated with different agonists at pharmacological concentration of 10 μmol/L for 12 h. Positions of the differentially expressed protein spots with corresponding spot numbers are indicated with arrows. These results are representative of at least four independent experiments. A1/A2: before/after acetylcholine treatment, protein maps were stained with silver; A3: differentially expressed spots between the control and acetylcholine group, spots in A3 were visualized with Coomassie Brilliant Blue staining. The expression-changed proteins with spot numbers were identified and they are listed in Table 2. B1/B2: before/after oxotremorine treatment, silver staining; B3: differentially expressed protein spots between control and oxotremorine, Coomassie Brilliant Blue staining. The expression-changed proteins with the spot numbers are listed in Table 3. C1/C2: before/after pilocarpine treatment, silver staining; C3: differentially expressed protein spots between control and pilocarpine, Coomassie Brilliant Blue staining. The protein expression changes with the spot numbers were listed in Table 4. D1/D2: before/after nicotine treatment, silver staining; D3: differentially expressed protein spots between control and nicotine, Coomassie Brilliant Blue staining. The protein expression changes with the spot numbers are listed in Table 5. (E–H) show magnified views of some differentially expressed proteins between the control and each acetylcholine receptor agonist treatment group. (E) differentially expressed proteins between the control and acetylcholine treatment group. (F) between the control and oxotremorine treatment group. (G) between the control and pilocarpine treatment group. (H) between the control and nicotine treatment group.
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fig1A: Two-dimensional gel showing the proteome of cultured HUVECs at a pH range of 3–10 after stimulation with cholinergic agonists. The cells were incubated with different agonists at pharmacological concentration of 10 μmol/L for 12 h. Positions of the differentially expressed protein spots with corresponding spot numbers are indicated with arrows. These results are representative of at least four independent experiments. A1/A2: before/after acetylcholine treatment, protein maps were stained with silver; A3: differentially expressed spots between the control and acetylcholine group, spots in A3 were visualized with Coomassie Brilliant Blue staining. The expression-changed proteins with spot numbers were identified and they are listed in Table 2. B1/B2: before/after oxotremorine treatment, silver staining; B3: differentially expressed protein spots between control and oxotremorine, Coomassie Brilliant Blue staining. The expression-changed proteins with the spot numbers are listed in Table 3. C1/C2: before/after pilocarpine treatment, silver staining; C3: differentially expressed protein spots between control and pilocarpine, Coomassie Brilliant Blue staining. The protein expression changes with the spot numbers were listed in Table 4. D1/D2: before/after nicotine treatment, silver staining; D3: differentially expressed protein spots between control and nicotine, Coomassie Brilliant Blue staining. The protein expression changes with the spot numbers are listed in Table 5. (E–H) show magnified views of some differentially expressed proteins between the control and each acetylcholine receptor agonist treatment group. (E) differentially expressed proteins between the control and acetylcholine treatment group. (F) between the control and oxotremorine treatment group. (G) between the control and pilocarpine treatment group. (H) between the control and nicotine treatment group.
Mentions: In this work, we established the 2-DE protein patterns of the control group and the endothelial cells activated by ACh, Oxo, Pilo, and Nico. More than 1200 protein spots were detected on the 2-DE gels that showed similar protein patterns with silver staining of 300 μg of total proteins. Figure 1 displays the overall 2-DE patterns of protein extracts of control/ACh (Figure 1A), control/Oxo (Figure 1B), control/Pilo (Figure 1C), and control/Nico (Figure 1D). Based on analysis with Image Master 2D Platinum software, among all the detected spots, 16, 8, 8 and 9 spots were separately changed in cells treated with ACh, Oxo, Pilo and Nico, showing detectable changes above 2-fold and the match rate was above 60% (Figure 1E–1H).

Bottom Line: In this study, we used a proteomic approach to study potential downstream receptor-effectors of NNAS that were involved in regulating cellular function in endothelial cells.Western blotting analyses showed that treatment of the cells with cholinergic agonists significantly decreased the expression of destrin, FKBP1A and MIF, and increased the expression of profilin-1.A set of proteins differentially expressed in endothelial cells in response to cholinergic agonists may have important implications for the downstream biological effects of NNAS.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Drug Research Center, Institute of Health and Environmental Medicine, Academy of Military Medical Sciences, Beijing 100850, China.

ABSTRACT

Aim: The non-neuronal acetylcholine system (NNAS) in endothelial cells participates in modulating endothelial function, vascular tone, angiogenesis and inflammation, thus plays a critical role in cardiovascular diseases. In this study, we used a proteomic approach to study potential downstream receptor-effectors of NNAS that were involved in regulating cellular function in endothelial cells.

Methods: Human umbilical vein endothelial cells were incubated in the presence of acetylcholine, oxotremorine, pilocarpine or nicotine at the concentration of 10 μmol/L for 12 h, and the expressed proteins in the cells were separated and identified with two-dimensional electrophoresis (2-DE) and LC-MS. The protein spots with the largest changes were identified by LC-MS. Biowork software was used for database search of the peptide mass fingerprints.

Results: Over 1200 polypeptides were reproducibly detected in 2-DE with a pH range of 3-10. Acetylcholine, oxotremorine, pilocarpine and nicotine treatment caused 16, 9, 8 and 9 protein spots, respectively, expressed differentially. Four protein spots were identified as destrin, FK506 binding protein 1A (FKBP1A), macrophage migration inhibitory factor (MIF) and profilin-1. Western blotting analyses showed that treatment of the cells with cholinergic agonists significantly decreased the expression of destrin, FKBP1A and MIF, and increased the expression of profilin-1.

Conclusion: A set of proteins differentially expressed in endothelial cells in response to cholinergic agonists may have important implications for the downstream biological effects of NNAS.

Show MeSH
Related in: MedlinePlus