<p>Since the pioneering work of Pickles et al . (1965), prostanoids have been implicated in the pain and discomfort of primary dysmenorrhea. Accordingly, current pharmacotherapy is based on the inhibition of prostanoid synthesis. However, 10% to 25% of women who suffer from primary dysmenorrhea fail to gain relief from such therapy. The development of alternative therapies to treat these women has been hindered by the fact that the effects of prostanoids on human nonpregnant myometrium have not been quantified in a rigorous way. The prostanoid thromboxane A2 , causes vascular smooth muscle contraction by interacting with specific prostanoid receptors known as TP receptors, the nomenclature follows recent International Union of Pharmacology recommendations where each prostanoid receptor is designated by the letter P, preceded by a letter signifying the most potent natural prostanoid agonist at that receptor. At the time my thesis was undertaken, several observations suggested that the TP receptor may be involved in the physiological and pathophysiological control of myometrial contractility. Therefore, the purpose of this study was to thoroughly characterize the TP receptor population in human nonpregnant myometrium. I evaluated the pharmacological characteristics of the myometrial TP receptor via in vitro functional and radioligand binding studies and employed reverse transcription-polymerase chain reaction assays to evaluate TP receptor mRNA expression. Both U-46,619 and I-BOP produced concentration-dependent contraction of human myometrial strips in vitro (p EC50 = 6.9 ± 0.27; and 7.8 ±0.60, respectively). The contractile activity induced by U-46,619 was attenuated by seven selective TP receptor antagonists. Lastly, the sensitivity of human nonpregnant myometrium was not regulated by anatomical location, tissue orientation or menstrual cycle status of the donor. The binding of [125 I]-BOP to human myometrial membranes was saturable, selective and displaceable. Equilibrium binding of [125 I]-BOP identified one class of sites, Kd = 3.4 nM (pKd = 8.7 ± 0.4) and a maximum binding of 323.1 ± 361.5 fmol/mg protein. The addition of the non-hydrolyzable GTP analog GTPγS (100 μM), to the assay had no effect on [125 I]-BOP binding. The rank order of potency for the seven TP receptor antagonists in displacing [125 I]-Bop from its binding site was correlated (r = 0.75) with the rank order of potency in inhibiting U-46,619-induced contraction of myometrial strips. Ligands selective for other prostanoid receptors were unable to significantly displace [ 125 I]-BOP binding. A novel qualitative RT-PCR methodology was developed and with this technique TP receptor mRNA expression was demonstrated in human nonpregnant myometrium excised from different uterine locations, from donors in both the proliferative and secretory phases of the menstrual cycle. The basis for a semi-quantitative RT-PCR methodology was established and an examination of potential influences on TP receptor mRNA expression, such as tissue excision site and donor menstrual cycle status, was begun. Lastly, the semi-quantitative data describing the amplification of TP receptor mRNA was highly variable, however the factor(s) responsible for such high variability remain to be determined. All taken together, these results suggest that a single homogeneous population of TP receptors, most closely resembling the putative low affinity TP receptor population in human platelets, resides in human nonpregnant myometrium.</p> / Doctor of Philosophy (PhD)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/7445 |
| Date | 09 1900 |
| Creators | Senchyna, Michelle |
| Contributors | Crankshaw, D.J., Health Sciences |
| Source Sets | McMaster University |
| Detected Language | English |
| Type | thesis |
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