Trigeminal afferent neurons express ionotropic P2X receptors for extracellular ATP which are known to be sensitive to low interstitial pH. Both conditions - ATP release and tissue acidosis - may occur in the dura following the ischemia phase of migraine attacks. Aim of this study was to investigate whether and how ATP and protons may cooperate in exciting meningeal afferents. After removal of the cerebral hemispheres hemisected scull cavities of adult Wistar rats were used as organ bath of their own lining, the dura mater. The dura was chemically stimulated and the amounts of immunoreactive calcitonin gene-related peptide (iCGRP) and prostaglandin E(2) (PGE(2)) released into incubation fluid were measured using enzyme immunoassays. Stimulation with ATP (10(-4) and 10(-3)M) augmented iPGE(2) release dose-dependently whereas iCGRP secretion was minimally enhanced only if the dura had previously been depleted of extracellular ATP using hexokinase. Acid buffer solutions (pH 5.9 and 5.4) resulted in pH-dependent increase of iCGRP release but reduced iPGE(2) release. Purines (ATP 10(-3)>UTP 10(-4)M>ATP 10(-4)M) and PGE(2) (10(-5)M) were found to facilitate the proton-induced increase in iCGRP release. The proton-reduction of PGE(2) release was overcome by adding ATP (10(-3)M). S(+)-flurbiprofen (10(-6)M) suppressed both the basal and stimulated iPGE(2) release and prevented the ATP(10(-4)M)-induced facilitation of the proton response. The facilitating effect of ATP was also blocked under suramin, a non-selective P2 antagonist, and under reactive blue, an non-selective P2Y-antagonist, but not under pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, a P2X-antagonist. The present results provide evidence that ATP has poor, if at all, direct excitatory effects on CGRP-containing trigeminal nerve endings in the isolated dura and its facilitatory action seems to depend on G-protein coupled P2Y receptors and secondary PGE(2) release. The UTP effect and the antagonist profile is indicative for the P2Y(2) receptor subtype.