The rat cardiomyoblast cell line H9c2 is widely used as an in-vitro model of cardiac muscle for studies investigating cardioprotection (Katinuma et al. 2005). G-protein coupled receptors including Gi/o-protein linked adenosine A₁/A₃ and δ/κ-opioid receptors play a major role in cardioprotection (Gross, 2003; Mubagwa & Flameng, 2000). However, the functional expression of adenosine A₁/A₃ and δ/κ-opioid receptors on H9c2 cells has not been reported. The aim of this study was to determine whether these receptors are expressed in these cells. Functional coupling to Gi/o-protein was assessed by monitoring the ability of selective agonists to inhibit forskolin-stimulated accumulation of [³H]-cAMP in cells pre-labelled with [³H]-adenine (Scrivens & Dickenson, 2005). The selective adenosine A₁ receptor agonist CPA induced a modest inhibition of 10 μM forskolin-stimulated [³H]-cAMP accumulation in H9c2 cells. Interestingly, the concentration-response curve for CPA-induced inhibition of forskolin-stimulated [³H]-cAMP accumulation was biphasic. Lower concentrations of CPA (maximal inhibition occurring at 100 nM; 22 ± 4%; n=5) inhibited cAMP accumulation induced by 10 μM forskolin, with an approximate pIC50 of 8.8 ± 0.7 (2 nM; n=5). At 1 μM CPA no inhibition of forskolin-stimulated [³H]-cAMP accumulation was observed (114 ± 11% of the forskolin alone response = 100%) which may suggest coupling of the adenosine A₁ receptor to Gs-protein (Baker & Hill, 2007). Pre-treatment with pertussis toxin (Gi/o-protein blocker; 100 ng/ml for 16 h) completely abolished 100 nM CPA-mediated inhibition of forskolin-stimulated [³H]-cAMP accumulation (119 ± 4% of forskolin response in presence of pertussis toxin =100%; n=6). These data suggest functional expression of the adenosine A₁ receptor to Gi/o-protein (and possibly Gs protein) in H9c2 cells. In contrast the adenosine A₃ receptor agonist Cl-IB-MECA (up to 1 μM) had no significant effect on forskolin-stimulated [³H]-cAMP accumulation. However, 10 μM Cl-IB-MECA induced a significant augmentation of forskolin-stimulated [³H]-cAMP accumulation (176 ± 10% compared to forskolin response alone = 100%; n=8). Interestingly, 10 μM Cl-IB-MECA-mediated augmentation of forskolin-stimulated [³H]-cAMP accumulation was reduced by the adenosine A₁ receptor antagonist DPCPX (10 μM; 108 ± 7% compared to forskolin alone = 100%). The selective κ-opioid receptor agonist (-)-U-50488 (100 nM) induced a robust inhibition of 1 μM forskolin-stimulated [³H]-cAMP accumulation (61 ± 16% inhibition; n=3), whereas the δ-opioid receptor agonist SNC 80 (100 nM) had no significant effect.In summary, these data have shown that H9c2 cells express functional adenosine A₁ and κ-opioid receptors.