We previously demonstrated a vascular network response initiated by elevated tissue concentrations of adenosine that is distinct from the dilation caused when adenosine is applied directly to the arteriole. The purpose of this study was to elucidate the potential mechanism(s) for the different responses. In the cheek pouch of anesthetized hamster, arteriolar responses were measured when adenosine (10(-4)M) was applied with micropipette into the tissue 500 microm from the arteriole (n=67, baseline diameter 22+/-0.6 microm) or onto the arteriole itself. Application of adenosine to the vessel or into the tissue caused arteriolar dilation with similar concentration profiles. In stark contrast, the concentration profiles were significantly different for vessel and tissue initiated dilation when either sodium nitroprusside or methacholine was tested. Arteriolar dilation was not enhanced when adenosine was simultaneously applied with two pipettes at along a single arteriole; however, the dilation doubled when adenosine was applied simultaneously at arteriole and tissue. Control dilations caused by tissue adenosine (5+/-0.4 microm) were not altered by superfusion of the A(1) receptor antagonist DPCPX (10(-6)M; 4.6+/-0.3 microm), A(2B) receptor antagonist alloxazine (10(-6)M; 6+/-0.8 microm), or A(3) receptor antagonist MRS1220 (5 x 10(-9)M; 6+/-0.8 microm) but were abolished by the selective A(2A) receptor antagonist ZM241385 (10(-7)M; 1+/-0.2 microm), suggesting that activation of A(2A) receptors mediates these network responses. Disruption of arteriolar endothelium and direct arteriolar application of ZM241385 (10(-7)M; 5+/-0.4 microm) did not alter the dilation caused by tissue adenosine. However, local application of ZM241385 into the tissue inhibited adenosine-induced network responses (2+/-0.3 microm). Furthermore, application into the tissue of A(2A) receptor agonist CGS21680 (10(-5)M), but not A(1) (CPA; 10(-4)M), A2b (NECA, 10(-4)M) or A3 (IB-MECA; 10(-4)M) receptor agonists mimicked the adenosine network response. These data demonstrate dual, complimentary, yet distinct pathways for network dilations induced by increases in tissue adenosine.