Estrogen Affects the Colon-Urethra Crosstalk via Spinal Kalirin/PSD-95-NR2B-Dependent NR2B Phosphorylation in Rats

Epidemiological studies have demonstrated patients suffering from irritable bowel syndrome (IBS) have a significantly higher prevalence of the lower urinary tract (LUT) dysfunctions with chronic pelvic pain (CPP). In this project, we will try to investigate the spinal mechanism of the colon-urethra crosstalk (CUC), a form cross-organ sensitization between pelvic viscera that possibly underlies the comorbidity of bowel and urogenital dysfunctions/pain in the pelvic area. In the first year, we try to elucidate the contribution of spinal kalrin, a member of Rho-guanine nucleotide exchange factor implicated in forms of synaptic plasticity, to the CUC. In addition, the possibility that the interaction between post-synaptic density-95 (PSD-95) and NR2B subunit of N-methyl-D-aspartate receptor (NMDAR) which enhances NR2B phosphorylation play a downstream of spinal kalirin to underlying the CUC will also be investigated. Accordingly, the evoked urethra electromyogram in response to intra-colonic mustard oil (MO) or corn oil (CO; a vehicle solution for MO) instillation will be recorded and compared in in vivo rats. The abundance of of spinal kalirin, PSD-95, total NR2B (tRN2B), and phosphorylated NR2B (pNR2B) will be evaluated by Western blot analysis of the lumbosacral dorsal horn samples (L6-S2) dissected at 120 min post-MO/CO instillation, a time point that MO exhibited sustained irritation in the urethra electromyogram in our previous studies. Moreover, the interaction between kalirin, PSD-95, and tRN2B will investigated using coprecipitation and immunohistochemical analysis of the dorsal horn samples. For estrogen has been demonstrated to positively impact CUC, in year 2, the possibility that estrogen-enhanced CUC is mediated by the spinal kalirin/PSD-95-NR2B/pNR2B cascade will be evaluated by comparing and contrasting the CUC in ovariectomized rats with or without estrogen supplement. Moreover, the abundance, the protein-protein interaction, and cellular locations of spinal kalirin, PSD-95, tRN2B, and/or pNR2B will be evaluated by Western blot, coprecipitation, and immunohistochemistry analysises of the lumbosacral dorsal horn samples (L6-S2) dissected from ovarietomized animals with or without estrogen suplement at 120 min post-MO/CO instillation. In addition, ERα and ERβ agonists will be administered to the ovariectomized rat; ERα and ERβ antagonist will be done to the estrodiol-supplement ovariectomized rats to elucidate their roles. For the symptoms and sings of endometriosis are recognized to be caused by an elevated estrogen level. Considering an animal has been established elsewhere to mimic the pathology of endometriosis, we will try to reestablish this animal model and, after checking the plasma estrogen level, evaluate the CUC in endometriosis and sham-operated animals in the year 3. Also the abundance, the protein-protein interaction, and cellular locations of spinal kalirin, PSD-95, tRN2B, and/or pNR2B will analyzed in these groups to elucidate the hypothesis that the high estrogen level in endometriosis animal could lead to enhanced CUC, possibly mediated by the spinal kalirin/PSD-95-NR2B/pNR2B cascade. Similarly, ERα and ERβ antagonist will apply to the endometriosis rats to elucidate their roles. Through this project, we hope we could provide evidences supporting for the crucial role of spinal kalirin not only in neural plasticity underlying the high co-morbidity of IBS and CCP with urogenital disorders but also the exaggerated pelvic pain in patients suffering from endometriosis. Our result could provide a good starting point for developing medical intervention for urogenital pain caused by irritation of other pelvic viscera.