ESTACIÓN DE CLASIFICACIÓN Y APROVECHAMIENTO ECA

The data below show that the sensitivity of cultured detrusor myocytes to carbachol is unchanged by the process of culture as shown by the similarity of the ApCa dose response curves and exemplified by their pECso values. The cells used were only taken from passages 0 to 3 (Table 4.3) after which their ability to respond to carbachol became less predictable (see below). The resting [Ca^‘"]i for cultured detrusor myocytes was 121 nM (101-144 nM (pCa 6.91±0.08 mean data ± s.e.m.) n=35 cells). The mean value was not significantly different from that of freshly isolated myocytes 107 nM (84-138 nM).

Table 4.3 cells used in the derivation of dose-response relationships to carbachol, using cultured detursor myocytes.

Passage Number Number of cells used

PO 1

PI 0

P2 4

P3 2

Cells at passage numbers greater than 3 were more likely to generate long augmented responses and not a characteristic transient as demonstrated by a

rapid rise and fall of [Ca^^], as seen in fresh cells and cells from early passages (figure 4.1). Furthermore there was a greater chance of spontaneous activity, as demonstrated in the example of Figure 4.6. This could occur before or after carbachol interventions and therefore data from these experiments was impossible to reliably interpret. In one cell cultured after passage 8 it was possible however to elicit a carbachol Ca^'"-transient demonstrating that even after multiple cultures cells would still respond to agonists.

1 min 1380

450

Figure 4.6 an example of the spontaneous activity seen in a passage 8 cultured detrusor myocyte. The resting [Ca^^], for this cell was 550 nM.

Figure 4.7 shows the dose-response relationship to carbachol in the cultured detrusor myocytes with a mean pECso of 5.56 ± 0.34. This is equivalent to a mean

ApCa % maximum response

120.0

100.0

80.0

60.0

40.0

20.0

0.0

100

Figure 4.7 mean ApCa as a function of the carbachol concentration for cultured detrusor myocytes. The ordinate is expressed as the percentage of the estimated maximum responses from each experiment The absolute magnitude of responses in different cell types is given in section 4.7. Mean data ± s.d. n=6.

Whilst the sensitivity of these cells was similar to their freshly isolated counterparts there was evidence of some desensitization. In four of the experiments used to generate dose-response relationships (below, figure 4.7) the A[Ca^'"]i was lower in 100 pM carbachol than that seen at 30 pM. This was a phenomenon not seen in the freshly isolated myocytes. However, the set of ApCa values for the response to 30 pM and 100 pM carbachol were not significantly different (p>0.05, see table

4.4). Hence there was no statistical evidence of desensitisation of the cultured detrusor myocytes to high concentrations of carbachol.

Table 4.4 values of ApCa in response to 30 p M and 100 pM carbachol.

[carbachol] pM Mean ApCa ± s.d n=6

30 0.33 ± 0.20

100 0.35 ± 0.26

However, in individual experiments where the 100 pM carbachol response was reduced this may have affected the calculated mean value of ECso This was tested by recalculating the ECso for each experiment when omitting the datum point obtained at that concentration. The pECso was 5.55 ± 0 .1 8 (mean ECso 2.81 pM, 1.86 - 4.27 pM) and should be compared to that when the 100 pM data were included (pECso 5.56 ± 0.34, above). The two values were not significantly different which corroborates the conclusion that any apparent desensitisation to high carbachol concentrations in individual experiments did not affect the mean estimated pECso-

Figure 4.8 shows the carbachol dose-response curve with the 100 pM data omitted.

ApCa % maximum response

120.0

100.0

20.0

0.0

Figure 4.8 mean ApCa as a function of the carbachol concentration for cultured detrusor myocytes The 100 p M data points have been removed. Values plotted as a percentage of the maximum estimated response. Mean data ± s.d. n=6

The Hill coefficient, r\, gives an indication of co-operativity within any system. A value of unity would mean one-to-one relationship between agonist activation and generation of the end-response (i.e a rise of Ca^""). If the value were greater than unity this would imply co-operativity within the system that, requiring more than one agonist unit to generate the end-response or downstream amplification. The

Hill coefficient calculated for the action of carbachol on generating a Ca^'^-transient was 1.93 ± 0.84 for freshly isolated cells and 1.45 ± 0.50 for the cultured myocytes The mean values of ri were significantly greater than unity in both cell groups, but

were not different between the two groups. The reason why is greater than unity

cannot be determined by this experimental approach and requires determination of the binding kinetics of carbachol to the cell membrane or the gain of intracellular second messenger systems in generating a rise of the intracellular [Ca^'^].

Table 4.5 lists the pECso values and Hill coefficients, q, from freshly isolated and cultured detrusor myocytes for the A[Ca^^]i in response to carbachol. There was no significant difference, for the respective parameters, in the two sets (p>0.1) Table 4.5 - Mean pECso values (± s.d.) and Hill coefficient values for freshly isolated and cultured detrusor myocytes in response to carbachol.

Freshly isolated myocytes Cultured myocytes

n=6 n=6

pECso ± s.d. 5.68 ± 0.42 5.56 ± 0.34

T| (Hill coefficient) ± s.d. 1.93 ± 0 .8 4 1.45 ± 0 .5 0