McCarthy, C.; (2007) Extracellular ATP signalling pathways in detrusor smooth muscle. Doctoral thesis , University of London.

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Abstract

Two neurotransmitters, ACh and ATP, initiate nerve-mediated contractions in detrusor smooth muscle from guinea-pigs and humans with bladder over-activity whereas only ACh has a role in muscle from stable human bladders. Both Acetylcholine and ATP are rapidly broken down, by acetylcholinesterases and ectonucleotidases respectively. The activity of ectonucleotidase has been shown to be reduced in the unstable human detrusor compared to stable. The reduced activity may contribute to the development of the purinergic component of the unstable human detrusor contraction. In vitro muscle strip experiments showed that apyrase significantly reduced the force of EFS contractions in guinea pig and over active human detrusor. Tension in guinea-pig was reduced to 81.5 1 4.5 % of control and by 87.5 7.5 % in overactive human. Apyrase had no effect in stable human detrusor. In guinea-pig detrusor the ectonucleotidase inhibitor ARL 67156 significantly increased the force of EFS contractions. The ectonucleotidase activity was investigated by measuring the degradation of a number of concentrations of ATP by detrusor samples. The ATP degradation rate in guinea pig and human detrusor was significantly reduced by the presence of ARL 67156. The ARL 67156 sensitive fraction which represented the ectonucleotidase activity solely was not significantly different between the stable and overactive detrusor groups. RNA extracted from human detrusor samples was used in gene expression assays to investigate the presence of four members of the ectonuleotidase family. ENTPDase 1, 2, 3, and 5 was present in all 18 human detrusor samples. There was significantly less ENTPDase 1 and 3 in over active detrusor samples compared to those that were stable. There was no difference in the levels of ENTPDase 2 and 5 between the two detrusor groups. Novel ATP sensitive biosensors were used to measure the real time release of ATP from guinea pig detrusor over the frequency range 1-20 Hz. The ATP signal reached a maximal response at a lower frequency compared to tension. The ATP signal kl/2, 4.41 3.02 HZ, was significantly lower than that of the tension generated, 12.0 2.59 Hz. In the presence of 1 iM atropine the frequency dependence of the ATP signal and tension is comparable. There was no significant difference between the kl/2 of the ATP signal, 7.29 3.5 Hz, and that of tension, 3.12 1.6 Hz. The apyrase experiments show a correlation between the effect of apyrase on nerve-mediated contractions in human and guinea-pig detrusor and the proportion of atropine resistance. This is consistent with the hypothesis that ATP contributes to nerve-mediated contractions in guinea-pig and over-active human bladders by incomplete breakdown prior to its activation of detrusor smooth muscle. This was corroborated by an enhancement of contraction strength in guinea-pig tissue by ARL 67156, which would have reduced further ATP breakdown. The reduced ATP breakdown may be linked to the difference in the levels of ENTPDase 1 and 3 between stable and overactive detrusor. The use of ATP sensitive biosensors show it is possible to measure the real time release of ATP from detrusor. The ATP release is frequency dependent and there is evidence to suggest that there is preferential release at lower stimulation frequencies.

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