Experimental procedure

Strips of detrusor muscle were prepared as described above and placed into incubation vials and equilibriated in 3.5 ml of gassed high calcium Tyrode’s maintained at 37°C on the Driblock sample concentrator for 20 minutes. The pre­ wash buffer was then exchanged for 1.75 ml of fresh buffer alone, or buffer containing one o f the potential inhibitors, and pre-incubated for 5 minutes prior to the addition of ATP. For each assay run a control vial with no muscle strip added

was set up to correct for changes to sample concentration due to evaporation. Aliquots (7.5-30 pi) o f ATP stock solutions of 10 mM, 100 mM and 500 mM ATP were then added to the incubations vials for final concentrations o f 100 - 5000 pM. Assays were performed in order o f sample concentration (from low to high) to minimise subsequent assays firom ATP contamination.

10 pi aliquot samples o f the incubations vials were taken at time intervals of 2 ,5 , 10, 15 and 25 minutes fi*om the addition of ATP. Each 10 pi sample was immediately diluted 100-fold in ice-cold Ca^^ free Tyrode’s containing EDTA (5mM) solution to terminate breakdown.

A clear bottomed, opaque sided 96 well plate (Packard) was previously prepared by aliquoting lOOpl of Tris-Acetate Buffer into each well. 50pl aliquots o f the diluted samples/ ATP standards were transferred to wells on the plate. 40pl of the luciferin-luciferase ATP monitoring reagent (Labsystems, Helsinki, Finland) was then added to the wells and the plate immediately transferred to the Victor luminometer. The luminometer was set to collect the total light emitted fi"om each well over a 5 second period. The plate reading chamber thermostat was set at 20°C.

The effect o f 100 pM ectoATPase inhibitor, ARL 67156, was tested for its ability to inhibit degradation of ATP by detrusor samples at concentrations o f 100-5000 pM. ARL 67156 was added firom a 30 mM stock to a final concentration of 100 pM and incubated for 5 minutes prior to the addition of ATP. In a study of ectoATPase activity in human blood cells ARL 67156 caused inhibition o f ATP degradation by approximately 70-80% (Crack et a l, 1995). ARL 67156 at a concentration of lOOpM also caused significant enhancement o f neurogenic and

exogenous ATP evoked contractions without significantly affecting contractions evoked by exogenously applied a,p-me-ATP in guinea-pig bladder muscle strips (Westfall, e ta l, 1997).

To determine the ectoATPase component of total ATPase activity several other ATPase inhibitors, such as ouabain, oligomycin and Na azide, were added to the assay medium. The dependence of enzyme activity on Ca^^ and Mg^"^, and its insensitivity to the above agents, all inhibitors of intracellular ATPases, are characteristics for ectoATPases (Pearson, 1985). The effect o f removing calcium on the rate of degradation of 1000 pM ATP was tested by using a Ca^^-fi-ee Tyrode’s solution containing EDTA (5mM). Each strip was pre-incubated in the Ca^^-ffee EDTA Tyrode’s solution for 10 minutes prior to the addition of ATP.

The effect of ouabain (ImM), a specific inhibitor o f the Na^/K^ -ATPase transporter, sodium azide (ImM), an inhibitor of ATP-ADP diphosphohydrolase and oligomycin (SOpg/ml), an inhibitor of mitochondrial ATPase on the rate o f degradation of lOOOpM ATP was tested by adding each drug firom stocks to the required final concentration. After these inhibitors had been added the detrusor strip was incubated for 5 minutes prior to the addition o f ATP.

2,3,4 Calibration,

Calibration of the system to establish the relationship between the number of relative light units (RLU’s) counted by the luminometer and the concentration of ATP present in the well was performed prior to each assay run. ATP stocks were diluted in Tyrode’s containing EDTA (5mM) solution to provide standards over the concentration range ICT* - 10"^ M. This procedure defined a curve described by a rectangular hyperbola which was then used to calculate the concentration o f ATP present in the test samples (figure 2.3). The lower limit of detection of ATP claimed by the manufacturers (assuming use of a Labsystems luminometer) was 10" M. Despite following the manufacturers guidelines, calibration of the reagent determined a lower limit of detection of ATP of approximately 10‘* M. The reason for this discrepancy is unknown but was not considered a methodological constraint as calibration of the system was reproducible. Test samples were diluted for luciferin-luciferase luminometric determination o f ATP to a concentration of between 0.1 and lOpM ATP.

Relative Light Units 5000000 4000000 3000000 2000000 1000000 - [ATP] M

Figure 2.3; C a lib r a tio n o f lu c if e r in - lu c if e r a s e A T P m o n it o r in g r e a g e n t s h o w i n g th e r e la t io n s h ip b e t w e e n t h e c o n c e n t r a tio n o f A T P p r e s e n t an d th e R e la t iv e L ig h t U n it s ( R L U ) r e c o r d e d b y th e lu m in o m e t e r . V a lu e s p lo tt e d are m e a n s ± sta n d a rd d e v ia t io n s , n = 10. E s tim a te d m a x im u m = 4 4 6 0 0 0 0 R L U , h a lf - m a x im a l su b str a te c o n c e n t r a tio n = 1 .3 8 p M A T P , s lo p e c o e f f i c i e n t = 1 .2 , r v a lu e = 0 .9 9 9 4 .

2 .3 .5 D a t a P r e s e n t a t i o n AND A n a l y s i s

From the calibration curve the concentration of ATP present in the well was determined. This value was then corrected for dilution and used to calculate the number of moles of ATP present in the incubation vial. The initial velocity was then calculated by linear regression of a graph of the number o f moles of ATP degraded against the time from addition o f ATP at which the sample was taken. Kinetic reaction curves were first plotted to check the linearity of the decline in [ATP] with time. From these graphs it was determined that an approximate measure o f the initial rate vq (obtained from the slope of the tangent to the reaction curve at zero time) could be derived from a linear regression to the number of moles o f ATP remaining at time points 0, 2, 5 and 10 minutes for initial [ATP] of 100, 200 and 500pM and by linear regression to time points 0, 2 ,5 , 10, 15 and 25 minutes for initial [ATP] o f 1000, 2000 and 5000 pM. Each velocity was then normalised to the samples weight and Michaelis-Menton plots o f nanomoles ATP min* mg'^ consumed at each initial [ATP] produced. A more detailed explanation of this data treatment is given below.