ABSTRACT
Purpose. Nitrotyrosine was quantitated in rabbit bladder muscle and mucosa using two analytical systems: Western blotting analyses and a 96-well plate quantitative analysis kit. Materials and Methods. Rabbit bladder muscle and mucosa were obtained from control rabbits. For the Western analysis, the samples were loaded into a SDS page gel and then transferred to a PVDF membrane. The optical density was measured using a Kodak Scanner. Using the 96-well plate, the samples and standards were loaded, incubated with primary and secondary antibody, washed and vacuumed with 10x wash buffer three times between each incubation period. Stop buffer was added to the plate and the results were quantified via the plate reader. Results. For both muscle and mucosa tissue, the optical density readings were linear with tissue concentration; the concentration of nitrotyrosine in the mucosa was significantly higher than in the muscle. However, whereas the Western blot analysis is based on relative optical densities, the 96-well plate kit provides a truly quantitative analysis. Discussion. Mucosa tissue displayed a higher density of nitrotyrosine than did detrusor muscle tissue. This may well be due to the significantly higher metabolic activity of the mucosa compared to the muscle.
ABSTRACT
PURPOSE: Superoxide dismutase (SOD) and catalase are two important antioxidant mechanisms that work together to reduce free radical damage. Intracellular free calcium in smooth muscle can change rapidly and many enzymes can be affected. The sensitivity of SOD and catalase activity to calcium was determined in both rabbit bladder smooth muscle and mucosa. MATERIALS AND METHODS: Calcium sensitivity was analyzed by determining SOD and catalase activity in muscle and mucosa at the following calcium concentrations: 0 (in the presence of 1 mM EGTA), 1 and 5 mM CaCl(2). RESULTS: SOD: EGTA resulted in increased SOD activity of bladder smooth muscle, whereas both 1 and 5 mM calcium significantly decreased SOD activity. EGTA had no effect on SOD activity of the mucosa whereas 1 and 5 mM calcium decreased SOD activity of the muscle. Catalase: 1 mM calcium resulted in decreased catalase activity of the muscle and no change in the activity of the mucosa, whereas 5 mM calcium resulted in increased catalase activity of the mucosa but no change in the activity of the muscle. DISCUSSION: Mucosa showed more SOD and catalase activity than the muscle. Both SOD and catalase showed differing sensitivities to EGTA and calcium.
