solutions. The following interventions were used, made up to the required concentrations in Tyrode’s solution.
a) Carbachol; from a 10 mM stock solution kept at 4 °C. The solution was made using 0.913 g carbachol (Sigma St Louis, Mo, USA) dissolved in 500 ml R /0 water. Experimental concentrations (range 0.01 - 100 pM) were made by dilution in Tyrode’s solution.
b) ATP: from solid just prior to use. 0.0294 g Na2ATP (Sigma St Louis, Mo, USA)
was dissolved in 5 ml of R /0 water to give a 10 mM solution. Experimental concentrations (range 0.001 - 30 pM) were made by dilution in Tyrode’s solution.
c) KCI: - from solid by dissolving 0.567 g KCI (AnalaR® (BDH Poole) in 100 ml of Tyrode’s solution to produce an final concentration of 80 mM. No correction for osmotic strength or hypertonicity was made for this solution.
d) Caffeine: - from 0.1942 g caffeine (Sigma St Louis, Mo, USA) in 100 ml Tyrode’s solution to give a 10 mM solution.
e) Forskolin: - a 1 pM stock solution was made by dissolving of 2.1 mg of forskolin (Sigma St Louis, Mo, USA) in 5 ml of dimethyl sulphoxide (DMSO; BDH Poole). This stock was aliquoted and stored in the dark at -2 0 °C (stable for 1 month). It was then diluted to 0.1 ml in 100 ml for experiments, giving a final concentration of 1 nM.
f) Cholera toxin (ctx. Sigma St Louis, Mo, USA ( C-8052)): - a stock solution of 10 pg/ml was made by dissolving 0.5 mg of ctx solid in 50 ml of R /0 water. The stock was then diluted in Tyrode’s solution to a dilution of 0.3 ml per 100ml, i.e. a concentration of 30 ng/ml.
g) Dibutyryl cyclic-AMP (Sigma): - from solid at the time of the experiment. A stock solution concentration of 1 pM was made by dissolving 4.914 mg of dessicated solid in 10 ml of R /0 water. I ml aliquots were stored frozen and diluted at 0.1 ml per 100 ml for experiments, i.e. a concentration of 1 nM.
2.4.5 Epifluorescence microscopy - experimental protocols.
Experiments were undertaken on single cells, prepared as described above (Sections 2.1.2 and 2.2). Once the cells were loaded with Fura-2 and allowed to adhere to the glass coverslip base of the perfusion dish, the flow of oxygenated
Tyrode’s was œmmenced and cell selection was performed. This was initially carried out under phase contrast at a magnification of x100 (x10 eyepiece and x10 objective). The cells were examined to find one that was as close to the centre of the dish as possible. As can be seen from figure 2.3 the flow was across the dish on a diagonal axis and therefore the presumption was made that, this way, a cell was in an ideal position to receive rapid and continuous exposure to the perfusing solution. The morphology of the cell was also examined to ensure that it had the characteristic spindle shape. Once a cell had been identified the objective was changed to a x40 quartz oil interface objective (Olympus Uapo 340 nm, numerical aperture 1.3, WD 0.1 mm) to give a total magnification of x400. The fluorescence of the cell was then checked by visual inspection under 340 nm illumination. If there was visible green fluorescence then the cell was selected and used for the experiment.
The rotating filter wheel was set to sychronise with the sample and hold amplifiers, the microscope covered to prevent all external light from entering the system and the cell was viewed on the CCD camera monitor under red (<580 nm) illumination. The absolute zero levels for the two channels (340 and 380 nm) were recorded with no excitation. A shutter between the filter wheel and the cell was then opened and the PMT tube voltage was increased to -800 to 1100 V to give two sample- and-hold excitation outputs (one each from 340 and 380 nm excitation) as well as the ratio trace. Once the PMT voltage was set the cell was removed from the field of view using the microscope stage adjusters. This then gave a reading of the
background fluorescence, which was used as a baseline for the fluorescence signals with the cell in place. The cell was returned to the field of view and the position of the 340, 380 nm and ratio signals were checked to ensure that the traces returned to their original values.
Interventions evoking a change of the intracellular [Ca^^ - a Ca-transient - were preceded and followed by control exposure to standard Tyrode’s solution. Thus to establish dose-response curves, cumulative additions of agonist were not carried out to avoid uncertainties from gradual desensitisation by the agonist. The magnitude of the response was recorded from the average of the steady-state base-line levels. Agonists were added until a response was seen and then flow was switched back to control Tyrode’s solution. Between interventions a standard time of five minutes was allowed to ensure wash-off of any agonist and recovery of the cell; this time was chosen after initial experiments showed that consistent, repeated responses could be evoked with the interventions used in this thesis. This protocol was adhered to in all cases except for restitution experiments (Results, chapter 5) variable intervals between pairs of exposures were used and
the magnitude of the second expressed as a percentage of the first. To
investigate the effect of an intervention on the magnitude of a Ca-transient, control responses were obtained before and after introduction of the agent and the average used as a 100% control response.
At the beginning of each experiment the effect of 10 pM carbachol was tested. If there was no Ca-transient after one minute the cell was rejected - it was assumed that the cell was either not a detrusor myocyte (ie. a fibroblast) or had been badly damaged by the dissociation process.
2.4.6 Data analysis
Data are presented as mean ± s.d., except where indicated when median (25%, 75% interquartiles) are also quoted. Skewness of data sets of values x, was tested by calculating the skewness coefficient, Vb = rc\z/(n\2.^n\2),
where m3 is the third moment of the mean (=S(xrXf/n - X is the sample mean