Disruption of the A(3) adenosine receptor gene in mice and its effect on stimulated inflammatory cells

The A(3) adenosine receptor (A3AR) is one of four receptor subtypes for ade nosine and is expressed in a broad spectrum of tissues. In order to study t he function of A3AR, a mouse line carrying a mutant A(3) allele was generat ed. Mice homozygous for targeted disruption of the A3AR gene, A3AR(-/-) are fertile and visually and histologically indistinguishable from wild type m ice. The lack of a functional receptor in the A3AR-/- mice was confirmed by molecular and pharmacological analyses. The absence of A3AR protein expres sion in the A3AR(-/-) mice was demonstrated by lack of N-6-(4-amino-3-[I-12 5]iodobenzyl)adenosine binding to bone marrow-derived mast cell membranes t hat were found to express high levels of A3AR in wild type mice. In A3AR(-/ -) mice, the density of A(1) and A(2A) adenosine receptor subtypes was the same as in A3AR(+/+) mice as determined by radioligand binding to brain mem branes. Additionally, A(2B), receptor transcript expression was not affecte d by ablation of the A3AR gene. A3AR(-/-) mice have basal heart rates and a rterial blood pressures indistinguishable from A3AR(+/+) mice. Functionally , in contrast to wild type mice, adenosine and the A3AR-specific agonist, 2 -chloro-N-6-(3-iodobenzyl)-adenosine-5'-N-methyl-carboxamide (S-Cl-IB-MECA) , elicit no potentiation of antigen-dependent degranulation of bone marrow- derived mast cells from A3AR(-/-) mice as measured by hexosaminidase releas e. Also, the ability of 2Cl-IB-MECA to inhibit lipopolysaccharide-induced t umor necrosis factor-alpha production in vivo was decreased in A3AR(-/-) mi ce in comparison to A3AR(+/+) mice. The A(2A) adenosine receptor agonist, 2 -p-(2-carboxyethyl)phenylamino)-5'-N-ethylcarboxamidoadenosine, produced in hibition of lipopolysaccharide-stimulated tumor necrosis factor-alpha produ ction in both A3AR(-/-) and A3AR(+/+) mice. These results show that the inh ibition in vivo can be mediated by multiple subtypes, specifically the A(3) and A(2A) adenosine receptors, and A3AR activation plays an important role in both pro- and anti-inflammatory responses.