Multiple receptor subtypes and multiple mechanisms of dilation are involved in vascular network dilation caused by adenosine.

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.

Inducible NO synthase dependent S-nitrosylation and activation of arginase1 contribute to age-related endothelial dysfunction.

Endothelial function is impaired in aging because of a decrease in NO bioavailability. This may be, in part, attributable to increased arginase activity, which reciprocally regulates NO synthase (NOS) by competing for the common substrate, L-arginine. However, the high Km of arginase (>1 mmol/L) compared with NOS (2 to 20 micromol/L) seemingly makes direct competition for substrate unlikely. One of the mechanisms by which NO exerts its effects is by posttranslational modification through S-nitrosylation of protein cysteines. We tested the hypothesis that arginase1 activity is modulated by this mechanism, which serves to alter its substrate affinity, allowing competition with NOS for L-arginine. We demonstrate that arginase1 activity is altered by S-nitrosylation, both in vitro and ex vivo. Furthermore, using site-directed mutagenesis we demonstrate that 2 cysteine residues (C168 and C303) are able to undergo nitrosylation. S-Nitrosylation of C303 stabilizes the arginase1 trimer and reduces its Km value 6-fold. Finally, arginase1 nitrosylation is increased (and thus its Km decreased) in blood vessels from aging rats, likely contributing to impaired NO bioavailability and endothelial dysfunction. This is mediated by inducible NOS, which is expressed in the aging endothelium. These findings suggest that S-nitrosylated arginase1 can compete with NOS for L-arginine and contribute to endothelial dysfunction in the aging cardiovascular system.