Previously, it was reported that red blood cells (RBCs) are required to demonstrate participation of nitric oxide (NO) in the regulation of rabbit pulmonary vascular resistance (PVR). RBCs do not synthesize NO; hence, we postulated that ATP, present in millimolar amounts in RBCs, was the mediator, which evoked NO synthesis in the vascular endothelium. First, we found that deformation of RBCs, as occurs on passage across the pulmonary circulation with increasing flow rate, evoked increments in ATP release. Here, ATP (300 nM), administered to isolated, salt solution-perfused (PSS) rabbit lungs, decreased total and upstream (arterial) PVR, a response inhibited by N G -nitro-L-arginine methyl ester (L-NAME, 100 M). In lungs perfused with PSS containing RBCs, L-NAME increased total and upstream PVR. In lungs perfused with PSS containing glibenclamide-treated RBCs, which inhibits ATP release, L-NAME was without effect. Apyrase grade VII (8 U/ml), which degrades ATP to AMP, was without effect on PVR in PSS-perfused lungs. These results are consistent with the hypothesis that ATP, released from RBCs as they traverse the pulmonary circulation, evokes endogenous NO synthesis.adenosine-5Ј triphosphate; red blood cell EXTRACELLULAR ATP has been suggested to play an important role in the regulation of vascular resistance in a number of vascular beds, including the kidney (24, 26), mesentery (5, 28), heart (17, 20), and lung (8,10,11,12,16,31). The spacial relationship between the cell that is the source of extracellular ATP and vascular smooth muscle is an important determinant of the vascular response to ATP. Thus ATP released from nerve terminals adjacent to vascular smooth muscle would be expected to activate purinergic receptors that produce contraction of that muscle (18,21). In contrast, ATP released from formed elements in the circulation such as red blood cells (RBCs) (6,9,31,33) or ATP released from the endothelium itself (27) would interact with purinergic receptors present on the endothelium. The stimulation of such receptors has been shown to result in the synthesis of endothelium-derived relaxing factors, including nitric oxide (NO) (5,8,12,11,16). Thus extracellular ATP released from nerve terminals would be expected to increase vascular resistance, whereas ATP released into the vascular lumen could be an important mechanism for decreasing vascular resistance.We reported previously that 1) ATP is released from rabbit RBCs as they traverse the pulmonary circulation (29) and 2) RBCs that are capable of releasing ATP were a requisite component in the perfusate of isolated rabbit lungs to demonstrate the participation of NO as a determinant of vascular resistance (31, 33). In the work presented here, we present evidence that ATP, in the absence of RBCs, is capable of promoting NO synthesis in the pulmonary circulation of the rabbit. Thus we determined the effect of ATP infused into the circulation of isolated rabbit lungs on vascular resistance. Moreover, we present evidence that the major mechanism by which ATP ac...