Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Extracellular 3?,5?-cAMP-Adenosine Pathway Inhibits Glomerular Mesangial Cell Growth

doi: 10.1124/jpet.110.166371

Figure Lengend Snippet: Line graphs showing the time-dependent (left) and concentration-dependent (right) metabolism of 3′,5′-cAMP to 5′-AMP (top), adenosine (middle), and inosine (bottom) by cultured human GMCs. For the time course study, cells were treated for 1 to 120 min under standard tissue culture conditions with 30 μM 3′,5′-cAMP (n = 6). For the concentration-dependence study, cells were treated with different concentrations (0.01–30 μM) of 3′,5′-cAMP (n = 6) for 60 min. 5′-AMP, adenosine, and inosine in the medium were analyzed by HPLC. Values are means ± S.E.M. of number of human GMCs cultures. *, p < 0.05 compared with levels at time 0 or levels in medium of GMCs not treated with 3′,5′-cAMP.

Article Snippet: Human GMCs in the 3rd passage were obtained from Lonza Walkersville, Inc. (Walkersville, MD), and human GMCs in the 5th to 6th passages were used for all experiments.

Techniques: Concentration Assay, Cell Culture

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Extracellular 3?,5?-cAMP-Adenosine Pathway Inhibits Glomerular Mesangial Cell Growth

doi: 10.1124/jpet.110.166371

Figure Lengend Snippet: Bar graphs showing the metabolism of 3′,5′-cAMP to 5′-AMP (top), adenosine (middle), and inosine (bottom) by human GMCs in the presence and absence of various inhibitors. Cells were treated for 60 min under standard tissue culture conditions with Dulbecco's PBS (Veh; n = 6) or 3′,5′-cAMP (30 μM; n = 6) in the absence or presence of IBMX (1 mM; n = 6), AMPCP (0.1 mM; n = 6), or DPSPX (0.1 mM; n = 6). 5′-AMP, adenosine, and inosine in the medium were analyzed by HPLC. Values are means ± S.E.M. of number of GMC cultures. *, p < 0.05 compared with corresponding vehicle group in pair; †, p < 0.05 compared with control GMCs treated with 3′,5′-cAMP.

Article Snippet: Human GMCs in the 3rd passage were obtained from Lonza Walkersville, Inc. (Walkersville, MD), and human GMCs in the 5th to 6th passages were used for all experiments.

Techniques:

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Extracellular 3?,5?-cAMP-Adenosine Pathway Inhibits Glomerular Mesangial Cell Growth

doi: 10.1124/jpet.110.166371

Figure Lengend Snippet: Bar graphs comparing the inhibitory effects of 3′,5′-cAMP (10 μM) or Br-cAMP (10 μM) on FCS-induced DNA synthesis ([3H]thymidine incorporation) (top), collagen synthesis ([3H]proline incorporation) (middle), and cell number (bottom) in cultured human GMCs in the presence and absence of KF17837 (KF; 10 nM), DPSPX (10 nM), DPCPX (10 nM), or VUF5574 (VUF; 10 nM). Values for each bar represent means ± S.E.M. from three to four separate experiments, conducted in quadruplicate, using separate cultures. *, p < 0.01 compared with control (2.5% FCS); §, p < 0.01 compared with 3′,5′-cAMP.

Article Snippet: Human GMCs in the 3rd passage were obtained from Lonza Walkersville, Inc. (Walkersville, MD), and human GMCs in the 5th to 6th passages were used for all experiments.

Techniques: DNA Synthesis, Cell Culture

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Extracellular 3?,5?-cAMP-Adenosine Pathway Inhibits Glomerular Mesangial Cell Growth

doi: 10.1124/jpet.110.166371

Figure Lengend Snippet: A, bar graphs showing the inhibitory effects of FOR on 2.5% FCS-induced DNA synthesis ([3H]thymidine incorporation) (top) and collagen synthesis ([3H]proline incorporation) (bottom) in cultured human GMCs, in the presence and absence of DPSPX (10 nM), KF17837 (10 nM; KF), DPCPX (10 nM), VUF5574 (10 nM; VUF), or DDA (10 μM). Data are presented as percentage of control. Values for each bar represent mean ± S.E.M. from three to four separate experiments, conducted in quadruplicate. *, p < 0.01 compared with control (2.5% FCS); §, p < 0.01 compared with 3′,5′-cAMP or forskolin alone. B, bar graphs showing the inhibitory effects of 3′,5′-cAMP (10 μM) and FOR (10 μM) on FCS (2.5%)-induced DNA synthesis (top) and collagen synthesis (bottom) in human GMCs in the presence of EHNA (10 μM) plus ITUB (0.1 μM). Also shown are the effects of DPSPX (10 nM), KF17837 (10 nM; KF), DPCPX (10 nM), VUF5574 (10 nM; VUF), and DDA (10 μM) on responses to EHNA + ITUB + FOR. *, p < 0.05 versus cells treated with vehicle; §, p < 0.01 versus cells treated with FOR + EHNA + ITUB in PBS; †, p < 0.01 compared with 3′,5′-cAMP or forskolin alone. Data are presented as percentage of control. Values for each bar represent mean ± S.E.M. from three to four separate experiments, conducted in quadruplicate.

Article Snippet: Human GMCs in the 3rd passage were obtained from Lonza Walkersville, Inc. (Walkersville, MD), and human GMCs in the 5th to 6th passages were used for all experiments.

Techniques: DNA Synthesis, Cell Culture

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Extracellular 3?,5?-cAMP-Adenosine Pathway Inhibits Glomerular Mesangial Cell Growth

doi: 10.1124/jpet.110.166371

Figure Lengend Snippet: A, bar graph depicting the inhibitory effects of 3′,5′-cAMP (10 μM) and forskolin (10 μM) on PDGF-BB (25 ng/ml)-induced MAPK activity in human GMCs in the presence and absence of DPSPX (10 nM), DPCPX (10 nM), or VUF5574 (10 nM; VUF). *, p < 0.01 compared with control (PDGF-BB). B, bar graph showing the synthesis of 3′,5′-cAMP (cAMP) (top) and adenosine (bottom) by human GMCs in response to FOR (10 μM) in the presence and absence of EHNA (10 μM) plus ITUB (0.1 μM), EHNA + ITUB + low-dose-DPSPX (10 nM), EHNA + ITUB + KF17837 (10 nM), or EHNA + ITUB + DDA (10 μM). Results for 3′,5′-cAMP and adenosine are expressed as nM/106 cells, and each value represents mean ± S.E.M. from six experiments conducted with separate cultures. *, p < 0.01 compared with respective vehicle; †, p < 0.01 versus cells treated with FOR + EHNA + ITUB; §, p < 0.01 compared with vehicle (PBS) only.

Article Snippet: Human GMCs in the 3rd passage were obtained from Lonza Walkersville, Inc. (Walkersville, MD), and human GMCs in the 5th to 6th passages were used for all experiments.

Techniques: Activity Assay