Fig. 2.
Validation of functional absence of A2BAR signaling in SS-Adora2b mutant rats. a Mutation of Adora2b in SS-Adora2b mutant rats confirmed by PCR. PCR was performed using DNA obtained from tail or ear snips and primers that flank the deleted segment, as described in the “Methods” section. Gel lanes labeled “SS” correspond to DNA from three SS controls (323-bp fragment) and lanes labeled “mut” correspond to DNA from three SS-Adora2b mutant rats (161-bp fragment) confirming a 162-bp deletion. b Loss of BAY-induced hyperglycemia in SS-Adora2b mutant rats. SS control or SS-Adora2b mutant rats (n = 3–6 male rats/group) were injected i.p. with 1 mg/kg BAY or vehicle, and the blood glucose concentration was measured with a One-Touch Ultra Glucometer at the times indicated. (Inset) Dose-response relationship of BAY to induce hyperglycemia in SS rats. n = 3–6 male rats/dose. *p < 0.05 and **p < 0.01 vs. the vehicle-treated SS control group by Student’s t test. c Loss of BAY-induced IL-6 secretion by dermal or pulmonary fibroblasts isolated from SS-Adora2b mutant rats. Following a 24-h serum deprivation, dermal or pulmonary fibroblasts were stimulated with increasing concentrations of BAY for 24 h, after which the media was collected and assayed for IL-6 content by ELISA. n = 3–10; *p < 0.05 and **p < 0.01 vs. the SS control group by Student’s t test. d Loss of BAY- and NECA-induced cAMP accumulation in pulmonary fibroblasts isolated from SS-Adora2b mutant rats. Fibroblasts were treated with BAY (0.3 μM) or NECA (1 μM) for 1 h in the presence of the phosphodiesterase inhibitor Ro-20-1274 (20 μM). Intracellular accumulation of cAMP was assessed using a competition protein binding assay, as described in the “Methods” section. Forskolin (10 M) was used as a positive control. Data are means ± SEM. n = 5; *p < 0.05 and **p < 0.01 vs. the SS control group by Student’s t test