The most important risk factor for the development of glaucoma is elevated intraocular pressure (IOP). Hypotensive drugs decrease IOP, preventing optic nerve damage and further vision loss. The balance between aqueous humor (AH) production and drainage determines IOP, and problems in AH outflow pathways are associated with open-angle glaucoma development. Previous studies have shown the presence of diadenosine tetraphosphate (Ap(4)A) and pentaphosphate (Ap(5)A) in the AH. Topic application of Ap(4)A to the cornea decreased IOP, whereas Ap(5)A increased it. Because dinucleoside polyphosphates stimulate P2Y purinergic receptors, we studied their presence in trabecular meshwork (TM) cells. Additionally, the effects of diadenosine polyphosphates (Ap(n)As; n = 3-5) and Up(4)U (P(1),P(4)-(diuridine 5')-tetraphosphate; INS365) in outflow facility were tested. P2Y(1), P2Y(2), and P2Y(4) receptors were detected in TM cells by Western blot and immunocytochemistry. In TM cells, Ap(3)A, Ap(4)A, and Ap(5)A induced discrete intracellular calcium concentration ([Ca(2+)](i)) mobilizations compared with higher and more sustained [Ca(2+)](i) mobilizations after Up(4)U application. In bovine ocular anterior segments perfused at constant pressure, 1 microM Ap(3)A or Ap(4)A increased outflow facility, whereas Up(4)U or Ap(5)A did not modify it. 2-MeSADP, a selective P2Y(1) agonist, induced outflow facility increases similar to those obtained after Ap(3)A and Ap(4)A, and these were prevented by addition of the selective P2Y(1) receptor antagonist MRS-2179 (2'-deoxy-N(6)-methyladenosine-3',5'-diphosphate). Our results demonstrate that the hypotensive effect of Ap(4)A and other dinucleotides is mediated, at least in part, by increasing trabecular outflow facility through activation of P2Y(1) receptors. The latter would seem to be an interesting target in the development of antiglaucomatous drugs to selectively increase AH outflow.