Regulation of inducible nitric oxide synthase - Consequences in experimental models of bladder disease

Abstract: Inducible nitric oxide synthase (iNOS) expression is associated with various pathophysiological conditions in the lower urinary tract. The aims of this thesis have been to investigate the regulation of iNOS in bladder smooth muscle cells and the consequences on neuromuscular regulation, cell growth and differentiation. Primary cultures of rat bladder smooth muscle cells (BSMC) expressed iNOS mRNA and protein upon stimulation with the cytokines IL-1? and TNF-? in combination. No constitutive isoforms of NOS were detected at the mRNA level. BSMC were put into different stages of differentiation. The capacity of BSMC to express iNOS was negatively correlated to differentiation status measured as smooth muscle myosin-heavy chain expression. iNOS was shown to be preferentially expressed in immature dedifferentiated BSMC. Actin cytoskeletal dynamics and Rho signalling were involved in regulation of cytokine-induced iNOS expression in BSMC. Phenotypic changes and impairment in actin cytoskeletal formation may potentiate cytokine activation and in turn increase NO production during bladder disease. The consequences of iNOS induction on bladder neuromuscular function were investigated by incubating isolated rat bladder strips with cytokines and recording the mechanical activity in organ baths. iNOS protein was observed in the smooth muscle layer in cytokine stimulated bladder strips. Nerve-mediated, but not acetylcholine-mediated, contraction of the bladder was impaired in bladder strips exposed to cytokines. This impairment was restored by the iNOS inhibitor aminoguanidine and partially by the neurotrophic factor BDNF. iNOS induction affected bladder function and bladder nerves were more sensitive to NO exposure than smooth muscle. The activity and expression of different NOS isoforms were investigated in an in vivo model of bladder hypertrophy induced by urethral obstruction in rats. During obstruction the nNOS activity decreased whereas iNOS activity increased initially. The total NOS activity was decreased. iNOS was expressed in smooth muscle, urothelium and inflammatory cells. Exogenous NO exposure decreased DNA- and protein-synthesis and stimulated differentiation in cultured BSMC. The results indicate that the decreased NO production during obstruction may stimulate a growth response and phenotypic changes in the bladders smooth muscle. The results in this thesis show that the smooth muscle in the bladder should be considered as a source of iNOS expression and NO production during inflammation. Neuronally derived NO is suggested to be involved in bladder smooth muscle physiology by maintaining and stabilizing the smooth muscle phenotype.