To determine the role of 17beta-estradiol and involvement of intracellular phosphatidylinositol-3-kinase signaling in cross-organ sensitization between the descending colon and the urethra, we analyzed urethra reflex activity and protein expressions in lumbosacral (L6-S2) spinal dorsal horn in response to mustard oil instillation into the descending colon in ovariectomized female rats. When compared with vehicle solution, intracolonic mustard oil sensitized the NMDA receptor NR2B subunit-dependent reflex activity and increased expression levels of phosphorylated Akt (pAkt) and phosphorylated NR2B (pNR2B) in dorsal horn. Facilitation of reflex sensitization and increases in protein expressions of pAkt and pNR2B in dorsal horn were induced after pre-treatment with a subcutaneous injection of 17beta-estradiol (5 microg/kg), 6 h ahead of time, when compared with vehicle solution. This phenomenon was reversed both by intrathecal pre-treatment with ICI 182780 (0.25 mg/kg, i.t.) and LY294002 (50 mg/kg, i.t.). Immunoprecipitation of dorsal horn tissue revealed a protein-protein interaction between pAkt and pNR2B increases, 6 h following the subcutaneous 17beta-estradiol when compared with vehicle injections. Results indicate 17beta-estradiol may activate the phosphatidylinositol-3-kinase cascade, which subsequently phosphorylates the NR2B subunit, via spinal estrogen receptors ERalpha/ERbeta, to facilitate NMDA-dependent cross-organ sensitization, which is presumed to underlie pelvic viscero-visceral referred pain.