Class of 2009 Abstract / OBJECTIVES: To develop a novel characterization system utilizing immunofluorescent FLAG tagged EP2 and EP4 receptors to assist in the explanation of their unique cell signaling properties for exploitation in future drug development design. METHODS: Plasmids were obtained and isolated that contained cDNAs encoding FLAG-tagged EP2 and EP4 receptors for transient expression in HEK-293 cells. The sequences of these plasmids were confirmed by restriction enzyme analysis and DNA sequencing. Transfected cells were treated with vehicle, PGE2 or forskolin to assess appropriate receptor functionality based on cAMP induction. RESULTS: The two PGE2 receptor subtypes, EP2 and EP4, are similar in their activation of adenylyl cyclase (AC) and subsequent up regulation of cAMP production. These receptors differ, however, in that EP2 more efficiently stimulates cAMP production and EP4 signaling involves the activation of phosphatidylinositol 3-kinase (PI3K) and extracellular signal related kinases (ERKs). The PGE2- treated cells responded as predicted with intracellular production of cAMP, with the EP2 receptor responding more efficiently than the EP4 receptor. CONCLUSIONS: The intent is for these cells to be used as a novel assay system for the development of future selective EP2 and EP4 agonists. This research could potentially benefit in selectively targeting EP2 or EP4 pathways linked to prevalent ailments such as pain, fever, inflammation, possibly cancer or bone growth.

Transition of the cardiopulmonary circulation at birth requires functional closure of the ductus arteriosus (DA). The DA is an arterial shunt that is vital in the fetus for diverting the pulmonary circulation away from the uninflated lungs. Failure of the vessel to functionally close after birth is known as patent DA, which is the second most common congenital heart disease. Patent DA may seriously compromise neonatal health and current pharmacological treatments are often limited by serious complications or a significant failure rate, thereby increasing the necessity for surgical intervention. Recently, we were the first to show that genetic or pharmacological inactivation of cyclooxygenase (COX) -2 produces postnatal patent DA in mice. We also demonstrated that the DA expresses high levels of COX-2 during normal closure after birth, suggesting novel contractile actions of COX-2-dependent prostanoids in the DA. In humans, patent DA is more common in preterm infants than those born at full-term, however, mechanism(s) responsible for the reduced DA closure have not been identified. In the current studies, we examined COX-1 and COX-2 expression in the DA at multiple stages of gestation to determine whether alterations in the expression of these enzymes contribute to patent DA in preterm mice. Using real-time PCR, analysis of the time-course of COX-2 mRNA in the fetal mouse DA indicated that COX-2 expression significantly increased with advancing gestational age. The preterm (day 17.5) neonatal mouse DA showed attenuated COX-2 expression, as compared to the full-term (day 19.5) neonatal DA at 3 hours after birth. Furthermore, the DA of preterm neonatal mice showed incomplete closure after 3 hours of birth, a time-point when the DA of full-term neonates was completely remodeled. These data indicate a correlation between reduced DA closure and attenuated COX-2 expression. Additionally, COX-2 expression was significantly attenuated in the DA of mice deficient in the prostanoid receptor EP4, which also show a patent DA phenotype, suggesting the importance of this receptor for the induction of COX-2 required for DA closure. Overall, these studies suggest that attenuated expression of COX-2 may contribute to increased patent DA at preterm gestation.

Prostaglandins are integral mediators in reproductive processes but their exact role in uterine function is still not clear. In addition, ethical restraints have limited the availability of human tissue to investigate uterine prostanoid receptor populations. The aim of this thesis was to characterise the prostanoid receptors on the human and rat myometrium in order to evaluate the potential of the rat as an animal model of human uterine function and disease. For functional analysis of myometrial prostanoid receptors the immersion technique was utilised. LC-ESI-MS/MS was also used to measure the ex vivo myometrial release of prostanoid metabolites. The results show that both the rat and human uterus displays cyclical changes in uterine motility, with myogenicity greatest in the follicular and oestrus stages. The data also indicate that whilst the human uterus is responsive to EP3, EP2, TP, FP and IP receptor agonists, a functional population of only EP3, EP2 and FP receptors is present on the rat uterus, although the TP receptor appears to be upregulated at gestation and post-partum. The results also show that myometrial prostanoid release in the human uterus is cyclically regulated, with the greatest amount of prostaglandins being released during the late follicular stage. In conclusion, although similarities do exist with regard to the ovarian regulation of uterine motility in both the rat and human uterus, the differences in the apparent functional prostaglandin receptor populations between the two species suggest further work is required before the rat can be used as a model of human uterine function.

Prostaglandins are integral mediators in reproductive processes but their exact role in uterine function is still not clear. In addition, ethical restraints have limited the availability of human tissue to investigate uterine prostanoid receptor populations. The aim of this thesis was to characterise the prostanoid receptors on the human and rat myometrium in order to evaluate the potential of the rat as an animal model of human uterine function and disease. For functional analysis of myometrial prostanoid receptors the immersion technique was utilised. LC-ESI-MS/MS was also used to measure the ex vivo myometrial release of prostanoid metabolites. The results show that both the rat and human uterus displays cyclical changes in uterine motility, with myogenicity greatest in the follicular and oestrus stages. The data also indicate that whilst the human uterus is responsive to EP3, EP2, TP, FP and IP receptor agonists, a functional population of only EP3, EP2 and FP receptors is present on the rat uterus, although the TP receptor appears to be upregulated at gestation and post-partum. The results also show that myometrial prostanoid release in the human uterus is cyclically regulated, with the greatest amount of prostaglandins being released during the late follicular stage. In conclusion, although similarities do exist with regard to the ovarian regulation of uterine motility in both the rat and human uterus, the differences in the apparent functional prostaglandin receptor populations between the two species suggest further work is required before the rat can be used as a model of human uterine function. / Allergan Inc