Volume 59, Issue 1, Pages 76-86 (January 2001) Dopamine induces ERK activation in renal epithelial cells through H2O2 produced by monoamine oxidase  Cécile Vindis, Marie-Hélène Séguélas, Stephen Lanier, Angelo Parini, Claudie Cambon  Kidney International  Volume 59, Issue 1, Pages 76-86 (January 2001) DOI: 10.1046/j.1523-1755.2001.00468.x Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 1 Effect of dopamine (DA) and tyramine in proximal tubule cell [3H]thymidine incorporation. Subconfluent quiescent proximal tubule cell primary cultures were incubated for 24 hours with or without various concentrations of DA (A) or tyramine (B) in presence of [3H]thymidine, as described in the Methods section. In parallel experiments, cells were preincubated with 1 μmol/L pargyline, 1 mmol/L NAC, 20 μmol/L PD 98059, or 3 μmol/L GBR 12909 for 30 minutes prior to 5 μmol/L DA (C) or 10 μmol/L tyramine (D) addition. Values are mean ± SEM of three separate experiments made in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001. Kidney International 2001 59, 76-86DOI: (10.1046/j.1523-1755.2001.00468.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 2 Dopamine (DA) induces extracellular–regulated kinase (ERK) activation in proximal tubule cells. (A) Proximal tubule cell lysates were prepared following 5 μmol/L DA treatment for the indicated times, as described in the Methods section. Cell lysates were immunoblotted with the antiphosphospecific p42/44 MAPK (anti-pMAPK) or anti-ERK2 antibodies. (B) In parallel experiments, cells were preincubated with or without 1 mmol/L NAC, 1 μmol/L pargyline, 3 μmol/L GBR 12909, 10 μmol/L (-)-sulpiride or SCH 23390 for 30 minutes prior to addition of HBSS (C) or 5 μmol/L DA (D) for five minutes. The blots are representative of three experiments. Kidney International 2001 59, 76-86DOI: (10.1046/j.1523-1755.2001.00468.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 3 Tyramine induces ERK activation in proximal tubule cells. (A) Proximal tubule cell lysates were prepared following 10 μmol/L tyramine treatment for the indicated times, as described in the Methods section. Cell lysates were immunoblotted with the antiphosphospecific p42/44 MAPK (anti-pMAPK) or anti-ERK2 antibodies. (B) In parallel experiments, cells were preincubated with or without 1 mmol/L NAC or 1 μmol/L pargyline for 30 minutes prior to addition of HBSS (C) or 10 μmol/L tyramine (T) for five minutes. The blots are representative of three experiments. Kidney International 2001 59, 76-86DOI: (10.1046/j.1523-1755.2001.00468.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 4 Dopamine (DA) and tyramine induce tyrosine phosphorylation of Shc in proximal tubule cells. (A) Proximal tubule cells were treated with 5 μmol/L DA or 10 μmol/L tyramine for the indicated times. Cell lysates were immunoprecipitated with anti-Shc antibody (IP: Shc) and immunoblotted with antiphosphotyrosine-HRP-conjugated antibody (anti-pTyr) or anti-Shc antibody, as described in the Methods section. (B) In parallel experiments, cells were preincubated with or without 1 mmol/L NAC, 1 μmol/L pargyline, or 3 μmol/L GBR 12909 for 30 minutes prior to the addition of HBSS (C), 5 μmol/L DA (D), or 10 μmol/L tyramine (T) for two minutes. The blots are representative of three experiments. Kidney International 2001 59, 76-86DOI: (10.1046/j.1523-1755.2001.00468.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 5 Monoamine oxidase (MAO)-dependent H2O2 production in human embryonic kidney 293 (HEK 293) cell lines. (A) Generation of chemiluminescence (CL) was monitored in HEK 293-MAO-B (•) and HEK 293-WT cells (▪) for 30 minutes after tyramine (10 μmol/L) addition (at time indicated by the arrow), as described in the Methods section. In parallel experiments, the HEK 293-MAO B cells were preincubated with 1 μmol/L clorgyline (▵) or Ro 19-6327 (○) for 30 minutes prior to tyramine addition. (B) Cell lysates from HEK 293-WT and HEK 293-MAO-B cells were immunoblotted with a rabbit polyclonal antiserum to both MAO isoforms, as described in the Methods section. Each curve and blot are representative of three separate experiments. Kidney International 2001 59, 76-86DOI: (10.1046/j.1523-1755.2001.00468.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 6 DA induce MAO-dependent Shc tyrosine phosphorylation and ERKs activation in HEK 293 cells. 293-WT (left panels) and 293-MAO-B cells (right panels) were incubated with 5 μmol/L DA for the indicated times. Cell lysates were prepared as described in the Methods section. (A) A part of cell lysates was immunoprecipitated with anti-Shc antibody (IP:Shc) and immunoblotted sequentially with antiphosphotyrosine-HRP–conjugated antibody or anti-Shc antibody. (B) Phosphorylated 42 and 44 kD ERKs and ERK-2 were detected sequentially with antiphospho-MAP kinase (anti-pMAPK) and anti-ERK2 antibodies as described in the Methods section. (C) In parallel experiments, 293-MAO-B cells were preincubated with or without 1 mmol/L NAC or 1 μmol/L pargyline for 30 minutes prior to addition of HBSS (C) or 5 μmol/L DA (D) for five minutes, and phosphorylated 42 and 44 kD ERKs and ERK-2 were detected sequentially. Apparent molecular weights (kD) are indicated on the right. The blots are representative of three experiments. Kidney International 2001 59, 76-86DOI: (10.1046/j.1523-1755.2001.00468.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 7 Effect of DA in HEK 293 cell proliferation. Subconfluent quiescent HEK 293 wild-type (293-WT; □) and transfected cells (293-MAO-B; ) were incubated for 24 hours with or without various concentrations of DA. [3H]thymidine incorporation assay (A) and cell number counting (B) were assessed as described in the Methods section. In parallel experiments, HEK 293-MAO-B cells were preincubated with or without 1 μmol/L pargyline, 1 mmol/L NAC, or 20 μmol/L PD 98059 for 30 minutes and incubated for 24 hours with (▪) or without (□) 5 μmol/L DA (DA) prior to [3H]thymidine incorporation assay (C) or cell number counting (D). Values are mean ± SEM of three separate experiments made in triplicate. *P < 0.05; ***P < 0.001. Kidney International 2001 59, 76-86DOI: (10.1046/j.1523-1755.2001.00468.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 7 Effect of DA in HEK 293 cell proliferation. Subconfluent quiescent HEK 293 wild-type (293-WT; □) and transfected cells (293-MAO-B; ) were incubated for 24 hours with or without various concentrations of DA. [3H]thymidine incorporation assay (A) and cell number counting (B) were assessed as described in the Methods section. In parallel experiments, HEK 293-MAO-B cells were preincubated with or without 1 μmol/L pargyline, 1 mmol/L NAC, or 20 μmol/L PD 98059 for 30 minutes and incubated for 24 hours with (▪) or without (□) 5 μmol/L DA (DA) prior to [3H]thymidine incorporation assay (C) or cell number counting (D). Values are mean ± SEM of three separate experiments made in triplicate. *P < 0.05; ***P < 0.001. Kidney International 2001 59, 76-86DOI: (10.1046/j.1523-1755.2001.00468.x) Copyright © 2001 International Society of Nephrology Terms and Conditions