Formation and role of nitric oxide in host defense reactions in the urinary bladder

Sammanfattning: Nitric oxide (NO) is an important biological mediator and cell signaling molecule. It is also involved in several host defense reactions. NO is formed in vivo from L-arginine by the enzyme NO synthase (NOS). However, it can also be formed non-enzymatically from acidified nitrite. The main aim of this thesis was to investigate the formation of NO and its role in host defense in the urinary bladder with focus on intravesical instillation of bacillus Calmette-Guérin (BCG) in the treatment of bladder cancer. Furthermore, to establish a method for measurement of NO oxidation products (nitrite and nitrate) in human urine by capillary electrophoresis (CE). T24 and MBT-2 bladder cancer cells showed both calcium-dependent and calciumindependent NOS activity whereas in cultured normal human urothelial cells (NHU) only calciurndependent NOS activity was detected. BCG and cytokines induced calcium-independent NOS activity in bladder cancer cells and NHU cells. Cytokine treatment inhibited the growth of bladder cancer cells partly via induction of calcium-independent NOS activity. In agreement, exogenously administered NO inhibited the growth of bladder cancer cells. On the other hand, the NO substrate L-arginine showed a dose-dependent proliferative effect on bladder cancer cells. In line with the in vitro findings, bladder tumor biopsies showed both calcium-dependent and calcium- independent NOS activities whereas only calcium-dependent NOS activity was detected in bladder mucosal biopsies. BCG treatment induced calcium-dependent and calcium-independent NOS activity in bladder mucosal biopsies. In patients treated with BCG, bladder NO concentrations were markedly increased after the first instillation and over a six month treatment course. NO formation from nitrite containing acidified urine was hugely increased and addition of ascorbic acid augmented the NO formation. Nitrite addition to acidified urine inhibited the growth of E. coli and T24 bladder cancer cells. These results may explain a bacteriostatic effect of ascorbic acid in the treatment of urinary tract infection and may be of importance in the treatment of bladder cancer. A sensitive, simple and rapid technique was developed for measurement of nitrite and nitrate in human urine and cell culture medium by CE using UV detection. The method allows detection of basal nitrite and nitrate concentrations in urine avoiding interference of urine sample matrices and pH with detection. This is of importance to elucidate the role of NO in the urinary tract. In summary, the results indicate that BCG induces long-term local formation of NO in the urinary bladder via induction of NOS activity in urothelial cells. The induction of NOS is likely mediated by endogenous cytokine production following BCG treatment. Endogenous NO formation following NOS induction may inhibit the growth of bladder cancer cells. Non-enzymatically formed NO or other nitrogen oxides from nitrite containing acidified urine may have a role in host defense reactions in the urinary bladder.