Figure 5.

Acquisition of adenosine A₃ receptor-mediated cyclic AMP and cardioprotective responses. Atrial cells were cultured from chicken embryos 14 days in ovo and transfected with pcDNA3 or with pcDNA3/hA₃R (full-length cDNA encoding human adenosine A₃ receptor subcloned in pcDNA3). (A) The ability of A₃ receptor agonist Cl-IB-MECA to mediate inhibition of isoproterenol-stimulated cyclic AMP accumulation in untransfected atrial cells, in cells transfected with pcDNA3, or in cells transfected with pcDNA3/hA₃R was determined. DPCPX (1 μM) was included to block the A₁ receptor. Data were means and standard errors of triplicate determinations and were typical of five other experiments. ∗, The cyclic AMP level was significantly less than that obtained in cells not exposed to Cl-IB-MECA (control cells). At 10 nM of Cl-IB-MECA, the A₃ receptor-mediated inhibition of the cyclic AMP level, expressed as percentage decrease from control cells, was significantly more in pcDNA3/hA₃R-transfected cells than in pcDNA3-transfected cells or untransfected cells (one-way ANOVA and t test, P < 0.01). (B) The ability of Cl-IB-MECA to cause cardioprotection in untransfected atrial cells, in cells transfected with pcDNA3, or in cells transfected with pcDNA3/hA₃R was determined. Cells were exposed to 10 nM of Cl-IB-MECA and 1 μM of DPCPX for 5 min. Media were replaced with fresh media lacking Cl-IB-MECA or DPCPX. Cells were then incubated under room air for 30 min before being exposed to 90 min of simulated ischemia. The percentage of myocytes killed and the amount of creatine kinase released (data not shown) were determined after the 90-min ischemic period. Data were the means and standard errors of seven experiments. In untransfected cells or in cells transfected with pcDNA3 or pcDNA3/hA₃R, prior A₃ agonist stimulation reduced the percentage of cells killed or the amount of CK released compared with cells not preexposed to A₃ agonist (control cells) (∗, t test, P < 0.05). However, the A₃ receptor-mediated reduction in the number of cells killed or the amount of CK released, expressed as percentage of decrease from those obtained in the control cells, was significantly more in pcDNA3/hA₃R-transfected than in pcDNA3-transfected cells or untransfected cells (∗∗, one-way ANOVA and t test, P < 0.01).