ENZIMÁTICA, A PARTIR DE AISLADO DE CHOCHO

Patch-clamp protocols were designed and run using the pulsed sampling module of the CED software package. The sampling frequency was 4KHz and the signals were filtered at 2KHz using a Bessel filter on the Axopatch-lC. All protocols were of a squarewave form and run from an initial holding potential of -60mV as it has previously been found that 80% of the current is available from this potential (Montgomery and Fry, 1992).

The gain settings of the Axopatch-lC were: Voltage IV / mV and Current 0.01 V / pA. The following voltage-clamp protocols were used during the course of the experiments:

1. Current-voltage relationship (CVR). 2. Steady-state inactivation (INACT). 3. Peak inward current (PIC).

2.7.1 CVR Protocol

Current-voltage relationships were determined using a series of twelve clamp steps increasing by lOmV each time from a holding potential of -60mV. Each step was 100ms long and ica was sampled 5ms before the start of each step for 120ms. The protocol is summarised in Figure M4 and the steps were repeated at 5s intervals.

^ ( m V ) .60 60 40 20 0 -20 -40 lOOms Sampling Window 12 0m s

2.7.2 INACT Protocol

The voltage dependence of inactivation involved a two pulse protocol. The first preconditioning pulse depolarised the membrane from -60mV to 0 mV for 2s and was immediately followed by a 100 ms depolarising pulse to +10mV. The latter step has previously been shown to elicit the peak ica (See Results). This sequence was repeated at 5s intervals for seven preconditioning steps decreasing each time by 10 mV and is summarised in Figure M5. (mV) 0 ■ -20 : -40 : -60' 10 100ms 10 2s Sampling Window 120ms Figure M5. Steady-state inactivation protocol.

The time constant of the reactivation process, Treact, was determined by varying the interval between two clamp steps from -60mV to +10mV as shown in Figure M6. Treact was found to be 138ms and it can be seen from Figure M7 that under these conditions the reactivation process is complete after an interval of Is. For other clamp steps at smaller voltages Xreact will be shorter and so the preconditioning pulse was maintained for 2s in order to ensure that the time dependent processes were complete in all cases. This was important because if ica was still inactivating then it would not be possible to elicit another maximal response.

2.7.3 PIC Protocol

The peak inward current protocol was designed as a series of 100ms depolariations from -60 to +10mV at 15s intervals and a 120ms sampling window was used for each step.

10 V (mV) m -60 100ms

— II —

Figure M6. Two pulse protocol for determining the time constant of reactivation. At short time intervals a single sampling window was used to encompass both pulses, at longer intervals two separate windows were used, each 120ms long.

0.8 - 00 0.6 - 0.4 - 0 200 400 600 800 1000 1200 Time (ms)

Figure M7. Time interval between test and prepulse depolarisations to lOmV, plotted against test pulse ica±SD expressed as a percentage of the prepulse value, 4<n<7. The curve was fitted using the equation y= 1 - e where t is the time interval and Xreact the time constant of reactivation.

2.8 Controls

Patch-clamp experiments were performed with each individual cell acting as its own control, ica in isolated human detrusor myocytes exibited the phenomenon of rundown, that is the peak ica became progressively smaller in any one cell with

succeeding patch-clamp protocols. This effect is illustrated in Figure M8 and is discussed further in the results chapter.

1 min

%

Figure M8. Segment o f calcium current rundown elicited using the PIC protocol.

This rundown however was noted to be extremely variable between different cells and in order to assess the effect of any given intervention the CVR protocol was run in groups of seven, as shown in Figure M9, at two minute intervals. Peak ica for the intervention (CVR 4) was then com pared to the mean of the values obtained in Tyrode's namely CVR's 3 and 5. If this mean value was an accurate estim ate of the value that would have been obtained had there not been an intervention then this should also hold true for the CVR's performed before and afterwards. The mean value of peak iCa in CVR's 1 and 3 was therefore compared to CVR 2 and similarly for CVR's 5, 6 and 7. This assumed a linear relationship in each segment of the rundow n process examined and only those experiments in which the rundown could be predicted in this way are included in the results section. All control values referred to in the text represent the mean of two results as ju st described unless otherw ise stated. This method was also used to compare all the other values that were subsequently derived from the i-v relationship.

Intervention Peaki Ca

□

D

d

Figure M9. Graphical representation on an exaggerated scale of the method for running intervention protocols based on a series of seven CVR's.