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. 2019 Dec;176(24):4720-4730.
doi: 10.1111/bph.14840. Epub 2019 Nov 3.

Characterisation of nerve-mediated ATP release from bladder detrusor muscle and its pathological implications

Carly J McCarthy  1   2 , Youko Ikeda  3   2 , Deborah Skennerton  2 , Basu Chakrabarty  4 , Anthony J Kanai  3 , Rita I Jabr  5 , Christopher H Fry  2   4
Affiliations

Characterisation of nerve-mediated ATP release from bladder detrusor muscle and its pathological implications

Carly J McCarthy et al. Br J Pharmacol. 2019 Dec.

Abstract

Background and purpose: This study aims to characterise the molecular mechanisms that determine variability of atropine resistance of nerve-mediated contractions in human and guinea pig detrusor smooth muscle.

Experimental approach: Atropine resistance of nerve-mediated contractions and the role of P2X1 receptors, were assessed in isolated preparations from guinea pigs and also humans with or without overactive bladder syndrome, from which the mucosa was removed. Nerve-mediated ATP release was measured directly with amperometric ATP-sensitive electrodes. Ecto-ATPase activity of guinea pig and human detrusor samples was measured in vitro by measuring the concentration-dependent rate of ATP breakdown. The transcription of ecto-ATPase subtypes in human samples was measured by qPCR.

Key results: Atropine resistance was greatest in guinea pig detrusor, absent in human tissue from normally functioning bladders, and intermediate in human overactive bladder. Greater atropine resistance correlated with reduction of contractions by the ATP-diphosphohydrolase apyrase, directly implicating ATP in their generation. E-NTPDase-1 was the most abundantly transcribed ecto-ATPase of those tested, and transcription was reduced in tissue from human overactive, compared to normal, bladders. E-NTPDase-1 enzymic activity was inversely related to the magnitude of atropine resistance. Nerve-mediated ATP release was continually measured and varied with stimulation frequency over the range of 1-16 Hz.

Conclusion and implications: Atropine resistance in nerve-mediated detrusor contractions is due to ATP release and its magnitude is inversely related to E-NTPDase-1 activity. ATP is released under different stimulation conditions compared with ACh, implying different routes for their release.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
ATP transients in guinea pig detrusor muscle preparations. (a) Recordings of isometric tension (upper blue trace) and outputs from ATP‐selective electrode and null electrodes (lower black traces), the difference recording (ATP–null, red trace) is also shown. Stimulation was a 3‐s train at 2 Hz. Arrows above the tension and ATP–null traces show the respective times of peak values. The inset shows a calibration curve of an ATP electrode with a linear fit, as well as a sample calibration trace. (b) Tension (blue) and ATP–null (red) traces in the presence of 10‐μM carbachol. (c) Tension (lower) and ATP–null (upper) traces in the absence and presence of 1‐μM tetrodotoxin (TTX), in this example at 8‐Hz stimulation. (d) Tension (lower) and ATP–null (upper) traces in the absence and presence of 1‐μM atropine, in this example at 4‐Hz stimulation
Figure 2
Figure 2
ecto‐ATPase activity in detrusor smooth muscle. (a) Sample experiment from human‐stable detrusor tissue of the initial rate of ATP hydrolysis as a function of the starting ATP concentration, in the absence (total) and presence (ARL independent) of 100‐μM ARL‐67156. The difference between the two (ARL dependent) is also plotted as a measure of ecto‐ATPase activity. The V max and K M values of the ARL‐dependent fraction are shown. (b) The relationship between ecto‐ATPase V max and the percentage purinergic component of the contraction (8‐Hz stimulation) as determined by (a) the percentage residual contraction with atropine (closed circles) or percentage reduction of the contraction by apyrase (open circles). Numbers of preparations in the three cohorts of tissue, contributing to the contractile data (ordinate) and ecto‐ATPase data (abscissa), are shown in Table 1 and Figure S1b,d,g
Figure 3
Figure 3
ATP‐transients and nerve‐mediated contractions. Data from guinea pig preparations. (a) Frequency dependence of tension (lower, blue traces) and ATP–null (upper, red traces) traces. (b) Dependence of the peak ATP transient (closed, red circles) and tension (black, closed squares) on stimulation frequency. Values of the half‐maximal frequency (f 1/2) for tension (T, f 1/2,T) are shown. The mean value for the ATP‐transient magnitude at 24 Hz was not used for the curve fit. Also shown is the frequency dependence of tension in the presence of 1‐μM atropine (closed, blue squares). Data are mean ± SD, n = 18 for tension values and n = 6 for ATP data. See Figure S1i for f 1/2,T and f 1/2,ATP values in individual preparations
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