NEUROHORMONAL &FREE RADICAL ACTIVATION OF BLADDER CELLS

Mast cells are involved in allergic reactions where they secrete a number of potent mediators, but have also been triggered by neuropeptides. Allergic problems, such as asthma and urticaria, show a sex ratio which changes from a high prevalence in males during childhood to one in females during puberty. Moreover, mast cells have been associated with certain autoimmune syndromes which have a high prevalence in women, such as interstitial cystitis (IC) and irritable bowel disease. Preliminary results have shown that estradiol augment mast cell secretion in response to acetylcholine (Ach), and substance P (SP) which are secreted from neurons in the bladder. Moreover, progesterone causes mast cell secretion directly and rat basophil leukemia (RBL) cells, a mucosal mast cell line kept in culture, show specific progesterone binding with a Kd of 10-(9) M. This proposal will characterize the effect of steroid sex hormones on secretion from RBL cells and purified rat peritoneal mast cells in vitro, as well as from rat and human mast cells in situ stimulated by the stable Ach analog, carbachol, and SP. Estradiol and progesterone binding will be examined by autoradiography using radiolabelled agonists, as well as by receptor affinity using established techniques. In addition, we will identify hormone binding by cell fractionation studies. Free radicals and other reactive oxygen species may participate in mast cell activation and cellular and tissue injury during IC. Experiments will assess direct cellular and tissue effects mediated by reactive oxygen, and indirect effects mediated by initial interaction of these reactants with mast cell granular components, giving rise to secondary cytotoxic species. Direct effects to be assayed include activation of mast cells and RBL cells, cell survival, and injury to rat and human bladder tissue explants. Indirect effects will be assayed by monitoring the chemical production of secondary reactants in a cell-free system, followed by assessing the biological effects of these reactants using the same endpoints employed in the direct damage studies. Results from this study will help us understand the effect of sex hormones on neural activation of mast cells, especially as they pertain to IC, the roles of free radicals on the function of mast cells and involvement in cell and tissue damage, and may permit the construction of an in situ model of IC which would allow testing of possible therapeutic modalities.