Pereira y su proceso de modernización 1920-

For unstable detrusor 5 o f the 10 specimens used for determination of total ATPase activity were also used for determination of the effect o f lOOpM ARL 67156. From Michaelis-Menten curve fits to the mean total ATPase data an estimated pKm of 2.72 ± 0.25 (mean Km = 1.90mM) and Vmax of 1.37 ± 0.46 nmoles min mg (n= 9) was obtained. In paired experiments, the mean velocity o f ATP degradation by detrusor from unstable bladders was lower in the presence of lOOpM ARL 67156 at all concentrations tested (n= 5), see figure 3.22. The inhibition was statistically significant at 100 and lOOOpM ATP (paired r-test, p< 0.05). Analysis of variance showed that the concentration-velocity curves were significantly different in the absence and presence o f ARL 67156 (p<0.005). From Michaelis-Menten curve fits to the mean ARL-resistant data an estimated pKm of 2.50 ± 0.23 (mean Km= 3.16mM) and Vmax o f 1.38 ± 0.59 moles min mg'^ (n= 5) was obtained and were not significantly different from the total ATPase values.

The mean velocity of degradation of ATP in the presence o f the inhibitor was approximately 58% of the control value at lOOpM ATP, 71% o f the control value at lOOOpM ATP, and 76% of the control value at 5000pM ATP.

V elocity (nmol min* Vg'^) T otal 0.8 0.6 A R L 6 7 1 5 6 resistant 0.4 0.2 0.0 0 1000 2000 3000 4000 5000 [ATP]

Figure 3.22; Michaelis-Menton plot o f ATP degradation by ATPase activity o f detrusor samples from unstable bladders in the absence (filled circles) and presence o f lOOpM ARL 67156 (open circles). Paired data. Values are means ± standard deviations. Analysis o f variance showed that the concentration-velocity curves were significantly different in the absence and presence o f ARL 67156 (p<0.0001).

Again, to determine the portion of enzyme activity affected by ARL 67156 inhibition the ARL 67156 sensitive fraction of the total activity was also plotted, see figure 3.23. Analysis of variance showed that the total concentration- velocity and the ARL 67156 sensitive curves were significantly different (p<0.0001). From Michaelis-Menten curve fits to the mean ARL-sensitive data an estimated pKm of 3.14 ± 0.45 (mean Km= 0.72mM) and Vmax of 0.36 ± 0.26 nmoles min mg~^ (n= 5) were obtained. The Vmax value for the ARL-sensitive curve was significantly lower than for the total ATPase activity.

Table R5 shows mean values, standard deviations and n numbers for ATP degradation by human detrusor from unstable bladders.

V elocity (nmol min'%g'^) 1.4 n 1.0- Total 0.8- 0.6- 0 .4 - ARL 67156 sensitive 0.2- 0.0 5000 0 1000 2000 3000 4000 [ATP] |iM

Figure 3.23; Michaelis-Menten plot o f ATP degradation (filled circles) and ARL 67156-sensitive ATP degradation (inhibition calculated as total velocity - velocity in the presence o f lOOpM ARL 67156) by ATPase activity o f detrusor samples from unstable human bladders. Values are means ± standard deviations. Analysis o f variance showed that the concentration-velocity curves were significantly different in the absence and presence o f ARL 67156 (p<0.0001).

Table R5. Velocity o f ATP degradation by detrusor from unstable bladders.

"^denotes values are significantly lower than those in the absence o f lOOjuMARL 67156 (unpaired t-test). § denotes difference in the calculated ARL 67156 sensitive values and total activity. Values are means ±s.d. and n= number o f experiments.

[ATPJpM Velocity (nmoles min_"/Mg";

Total activity ARL 67156 resistant ARL 67156 sensitive

Mean SD (n) Mean SD (n) Mean SD (n)

100 0.09 0.05 10 0.07* 0.05 5 0.054§ 0.025 5 200 0.14 0.04 8 500 0.25 0.08 9 0.22 0.03 5 0.05§ 0.06 5 1000 0.42 0.13 10 0.31* 0.06 5 0.12§ 0.06 5 2000 0.80 0.28 9 0.50 0.10 5 0.15§ 0.22 5 5000 1.30 0.40 9 0.77 0.19 5 0.24§ 0.32 5 Vmax 1.37 0.46 9 1.38 0.59 5 0.36§ 0.26 5 (nmoles min'^mg'^) pK-m 2.72 0.25 9 2.50 0.23 5 3.14 0.45 5

3.3. s Comparison o f A TP degradation velocities and A R L 67156 sensitivity in stable and unstable detrusor samples

T h e m e a n v e l o c i t y o f A T P d e g r a d a t io n w a s lo w e r in d e tr u s o r str ip s o b ta in e d fr o m u n s t a b le b la d d e r s ( n = 1 0 ) c o m p a r e d w it h str ip s fr o m s t a b le b la d d e r s ( n = 1 1 ) at a ll A T P c o n c e n t r a tio n s t e s t e d ( s e e f ig u r e 3 .2 4 ) . T h is d if f e r e n c e w a s s ig n if ic a n t at c o n c e n t r a t io n s o f 1 0 0 , 2 0 0 ( p < 0 .0 5 ) , 5 0 0 ( p < 0 .0 0 5 ) , 1 0 0 0 an d 2 0 0 0 p M ( p < 0 .0 5 ) . T h e Vmax o b ta in e d fr o m u n s t a b le b la d d e r s 1 .3 7 ± 0 .4 6 ( n = 9 ) c o m p a r e d w it h str ip s f r o m s t a b le b la d d e r s 2 . 5 4 ± 1 .5 0 n m o le s m in m g ~ ' ( n = 1 0 ) w a s a ls o s ig n if ic a n t ly l o w e r ( p < 0 .0 5 ) . C o m p a r is o n o f th e e n tir e d a ta s e ts u s in g a n a ly s is o f v a r ia n c e s h o w e d th a t th e c o n c e n t r a t io n - v e lo c it y c u r v e s w e r e s ig n if ic a n t ly d iff e r e n t ( p < 0 .0 5 ) . Velocity (nmol m in 'V g ‘*) 2 .5 - 2.0- 1.0 - 0 .5 - 0.0 1000 2000 3000 4000 5000 0 [ATP] |liM

Figure 3.24; M ichaelis-M enton plot o f ATP degradation by ATPase activity o f detrusor samples from stable bladders (filled circles) and from unstable bladders (open circles). Values are means ± standard deviations. Analysis o f variance showed that the concentration-velocity curves were not significantly different.

100|iM ARL 67156 had a greater inhibitory effect on ATP degradation in detrusor strips obtained from stable bladders (n= 8) than in detrusor strips obtained from unstable bladders (n= 5) at all concentrations tested (see figure 3.25). This difference was not significant at individual concentrations of ATP but when the entire ARL 67156 sensitive data sets were compared using analysis of variance the two curves were significantly different (p<0.005). For the unstable group the ARL sensitive curve had a Vmax value of 0.36 ± 0.26 nmoles min mg“* (n= 5) significantly lower than that obtained for the stable group, 0.94 ± 0.41 nmoles min mg'* (n= 8, p< 0.05). Again, the pKm values of the two groups were not significantly different from one another.

Velocity (nmol m in 'V g ’*) 0.8- 0.6- 0 .4 - 0.2- 0.0 0 1000 2000 3000 4000 5000 [ATP] |iM

Figure 3.25; Michaelis-Menton plot o f the ARL 67156-sensitive ATP degradation (inhibition calculated as total velocity - velocity in the presence o f lOOpM ARL 67156) by ATPase activity o f detrusor samples from stable bladders (filled circles) and from unstable bladders (open circles). Values are means ± standard deviations. Analysis o f variance showed that the ARL sensitive concentration-velocity curves were significantly different from each other.

The ARL 67156 resistant ATPase activity of detrusor from stable bladders and detrusor from unstable bladders was similar and not significantly different at any of the individual ATP concentrations (see figure 3.26). However, comparison of the entire data sets over all concentrations tested using analysis of variance showed that the ARL 67156 resistant concentration-velocity curves were significantly different from one another (p< 0.05). Since the proportion of ATPase activity blocked by the ectoATPase inhibitor ARL 67156 was greater in detrusor strips obtained from stable bladders than in detrusor strips from unstable bladders yet the residual ARL 67156 resistant ATPase activity of the two groups was similar the lower sensitivity of detrusor from unstable bladders to ARL 67156 inhibition suggests that ARL 67156 sensitive ecto-ATPases are less abundant and/or less active in unstable bladder samples than in samples from stable bladders.

Velocity (nmol min’*mg*')

1 .4 - 1 .2 - 1 .0 - 0 .8 - 0.6- 0 .4 - 0.2- 0.0 0 1000 2000 3000 4000 5000 [ATP] pM

Figure 3.26; Michaelis-Menton plot o f the ARL 67156 resistant proportion o f ATP degradation by ATPase activity o f detrusor samples from stable bladders (filled circles) and from unstable bladders (open circles). Values are means ± standard deviations. Analysis o f variance showed that the ARL 67156 concentration-velocity curves were significantly different from each other (p< 0.05).

When EDTA (5mM) was added to a Ca^^-free buffer the ATPase activity of detrusor from stable bladders was less at 0.18 nmoles /mg/ml compared with the control value of 0.64 nmoles /mg/ml, demonstrating that most (i.e. 72%) of the ATPase activity of the tissue was due to a divalent cation dependent ATPase (n= 6, p<0.001). A cocktail of ouabain (ImM), a specific inhibitor of transport Na^, K^-ATPase, soduim azide (ImM), an inhibitor of ATP-ADP diphosphohydrolase and oligomycin (50 pg/ml), an inhibitor of mitochondrial ATPase, had no significant effect on ATP hydrolysis at lOOOpM ATP (n= 6). See figure 3.27.

Velocity at lOOOpM ATP (nmol min mg*")

0.8 0.6- 0.4 0.2 H 0.0 EDTA ARL Control Inhibitors

Figure 3.27; Comparison o f ATP degradation by ectoATPase activity o f detrusor samples from stable bladders in the absence and presence o f inhibitors {ouabain (Im M ), soduim azide (Im M ), and oligom ycin (50 pg/m l)}, a calcium free EDTA (5mM) containing buffer and lOOpM ARL 67156. Values are means ± standard deviations.

3 .4 P I - R

S