Cardiac Fibroblasts Express the cAMP-Adenosine Pathway by Raghvendra K. Dubey, Delbert G. Gillespie, Zaichuan Mi, and Edwin K. Jackson Hypertension Volume 36(3):337-342 September 1, 2000 Copyright © American Heart Association, Inc. All rights reserved.

Schematic representation of the hypothesized role of the cAMP-adenosine pathway in CFs. Adenosine generated from cAMP by the sequential actions of PDE and 5′-NT provides adenosine to the A2 receptor, which induces antimitogenic effects via activation of adenylyl cyclase–dependent or –independent pathways. Schematic representation of the hypothesized role of the cAMP-adenosine pathway in CFs. Adenosine generated from cAMP by the sequential actions of PDE and 5′-NT provides adenosine to the A2 receptor, which induces antimitogenic effects via activation of adenylyl cyclase–dependent or –independent pathways. Hormone indicates adenylyl cyclase–stimulating hormone; Gs, stimulatory G protein; AC, adenylyl cyclase; ADO, adenosine; Tr, adenosine transporter; and DDA, 2′, 5′-dideoxyadenosine (adenylyl cyclase inhibitor). IBMX is a cell-permeable phosphodiesterase inhibitor; AMPCP is an ecto-5′NT inhibitor; DPSPX is an inhibitor of extracellular PDE at high concentrations and blocks A1/A2 adenosine receptors at low concentrations; and KF17837 is a selective A2 receptor antagonist. Raghvendra K. Dubey et al. Hypertension. 2000;36:337-342 Copyright © American Heart Association, Inc. All rights reserved.

Time-dependent (A) and concentration-dependent (B) metabolism of cAMP to AMP, adenosine, and inosine by ventricular CFs in 3-D culture. Time-dependent (A) and concentration-dependent (B) metabolism of cAMP to AMP, adenosine, and inosine by ventricular CFs in 3-D culture. For the time-course study, cells were treated for 1 to 30 minutes under standard tissue culture conditions with 30 μmol/L cAMP (n=6). For the concentration-dependence study, cells were treated with different concentrations (0.01 to 30 μmol/L) of cAMP (n=6) for 60 minutes. AMP, adenosine, and inosine in the medium were analyzed by high-performance liquid chromatography. Values are mean±SEM of number of rat CF cultures (n). See text for statistical comparisons. Raghvendra K. Dubey et al. Hypertension. 2000;36:337-342 Copyright © American Heart Association, Inc. All rights reserved.

Metabolism of cAMP to AMP, adenosine, and inosine by ventricular CFs in 3-D culture in the presence and absence of various inhibitors. Metabolism of cAMP to AMP, adenosine, and inosine by ventricular CFs in 3-D culture in the presence and absence of various inhibitors. Cells were treated for 60 minutes under standard tissue culture conditions with buffered Dulbecco’s PBS (Veh; n=6) or cAMP (30 μmol/L; n=6) in the absence or presence of IBMX (1 mmol/L; PDE inhibitor; n=6), AMPCP (0.1 mmol/L; ecto-5′-NT inhibitor; n=6), or DPSPX (ecto-PDE inhibitor; 0.1 mmol/L; n=6), and AMP, adenosine, and inosine in the medium were analyzed by high-performance liquid chromatography. Values are mean±SEM of number of rat CF cultures (n). DL indicates detection limit; *P<0.05 compared with corresponding vehicle group in pair; †P<0.05 compared with control CFs treated with cAMP. Raghvendra K. Dubey et al. Hypertension. 2000;36:337-342 Copyright © American Heart Association, Inc. All rights reserved.

Comparison of the inhibitory effects of cAMP (10 μmol/L; A) or 8-bromo-cAMP (Br-cAMP; 10 μmol/L; B) on FCS-induced 3H-thymidine incorporation in ventricular CFs in 3-D culture in the presence and absence of KF17837 (KF; 1 nmol/L; A2 receptor antagonist), DPSPX (1 nmol/L; A1/A2 receptor antagonist), or DPCPX (10 nmol/L; A1 receptor antagonist). Comparison of the inhibitory effects of cAMP (10 μmol/L; A) or 8-bromo-cAMP (Br-cAMP; 10 μmol/L; B) on FCS-induced 3H-thymidine incorporation in ventricular CFs in 3-D culture in the presence and absence of KF17837 (KF; 1 nmol/L; A2 receptor antagonist), DPSPX (1 nmol/L; A1/A2 receptor antagonist), or DPCPX (10 nmol/L; A1 receptor antagonist). Values for each bar represent mean±SEM from 3 to 4 separate experiments, conducted in quadruplicate, using separate cultures. *P<0.01 compared with control (Cont; 2.5% FCS); §P<0.01 compared with DPSPX, DPCPX, and KF; †P<0.01 compared with cAMP. Raghvendra K. Dubey et al. Hypertension. 2000;36:337-342 Copyright © American Heart Association, Inc. All rights reserved.

Comparison of the inhibitory effects of cAMP on FCS-induced 3H-thymidine incorporation (A), cell number (B), and 3H-leucine incorporation (C) in ventricular CFs in 3-D cultures, in the presence and absence of KF17837 (KF; 1 nmol/L; A2 receptor antagonist), DPSPX (1 nmol/L; A1/A2 receptor antagonist), or DPCPX (1 nmol/L; A1 receptor antagonist). Comparison of the inhibitory effects of cAMP on FCS-induced 3H-thymidine incorporation (A), cell number (B), and 3H-leucine incorporation (C) in ventricular CFs in 3-D cultures, in the presence and absence of KF17837 (KF; 1 nmol/L; A2 receptor antagonist), DPSPX (1 nmol/L; A1/A2 receptor antagonist), or DPCPX (1 nmol/L; A1 receptor antagonist). Values represent mean±SEM from 3 to 4 separate experiments, conducted in quadruplicate, using separate cultures. *P<0.01 compared with control (2.5% FCS); §P<0.01 compared with cAMP. Raghvendra K. Dubey et al. Hypertension. 2000;36:337-342 Copyright © American Heart Association, Inc. All rights reserved.

Modulatory effects of EHNA (10 μmol/L; adenosine deaminase inhibitor) plus IDO (0.1 μmol/L; adenosine kinase inhibitor) on the inhibitory effects of cAMP (10 μmol/L) on 2.5% FCS-induced thymidine incorporation (A) and cell proliferation (B) in ventricular CFs in 3-D cultures. Modulatory effects of EHNA (10 μmol/L; adenosine deaminase inhibitor) plus IDO (0.1 μmol/L; adenosine kinase inhibitor) on the inhibitory effects of cAMP (10 μmol/L) on 2.5% FCS-induced thymidine incorporation (A) and cell proliferation (B) in ventricular CFs in 3-D cultures. Results (mean±SEM) are from a representative experiment conducted in quadruplicate. Similar results were obtained in 3 separate experiments conducted in quadruplicate. *P<0.01 compared with control (FCS); †P<0.01 compared with cAMP and EHNA+IDO. Raghvendra K. Dubey et al. Hypertension. 2000;36:337-342 Copyright © American Heart Association, Inc. All rights reserved.