Frequency-dependent characteristics of nerve-mediated ATP and acetylcholine release from detrusor smooth muscle

Abstract

New findings: What is the central question of this study? Is the frequency dependence of co-transmitter release from postganglionic nerve fibres different for each transmitter? What is the main finding and its importance? Release of co-transmitters from the parasympathetic supply to detrusor smooth muscle can be independently regulated. This offers a targeted drug model to reduce selectively the release of transmitter associated with human pathologies (ATP) and may also be applicable to other smooth muscle-based disorders of visceral tissues.

Abstract: Nerve-mediated contractions of detrusor smooth muscle are mediated by acetylcholine (ACh) and ATP release in most animals. However, with the normal human bladder, only ACh is a functional transmitter, but in benign pathologies such as overactive bladder (OAB), ATP re-emerges as a secondary transmitter. The selective regulation of ATP release offers a therapeutic approach to manage OAB, in contrast to current primary strategies that target ACh actions. However, the release characteristics of nerve-mediated ACh and ATP are poorly defined and this study aimed to measure the frequency dependence of ACh and ATP release and determine if selective regulation of ATP or ACh was possible. Experiments were carried out in vitro with mouse detrusor with nerve-mediated ATP and ACh release measured simultaneously with tension recording. ATP was released in two frequency-dependent components, both at lower frequencies (mid-range 0.4 and 5.5 Hz stimulation) compared to a single compartment release of ACh at 14 Hz. Intervention with the phosphodiesterase type-5 inhibitor sildenafil attenuated ATP release, equally from both components, but had no effect on ACh release. These data demonstrate that nerve-mediated ACh and ATP release characteristics are distinct and may be separately manipulated. This offers a potential targeted drug model to manage benign lower urinary tract conditions such as OAB.

Keywords: ATP; acetylcholine; bladder; detrusor smooth muscle; neurotransmitter.

Figure 1.. Frequency-dependence of nerve-mediated contraction amplitude; mouse detrusor.

Lines are best-fits of a one- (1, dotted line) or a two (2, solid line) component model: see Methods for details. The two-component model shows a significantly better fit (F(_2,5)=175.8; p<0.0001). Data are mean±SD, n=12.

Figure 2.. Frequency-dependence of nerve-mediated ATP release; mouse detrusor.

Lines are best-fits of a one- (1, dotted line) or a two (2, solid line) component model. The two-component model shows a significantly better fit (F(_2,5)=87.1; p<0.0001). Data are mean±SD, n=12.

Figure 3.. Frequency-dependence of nerve-mediated ACh release; mouse detrusor.

The line is a best-fit of a one-component model. A two-component model fit showed no improvement in the goodness-of-fit (F(_2,5)=2.47; p=0.179). Data are mean ±SD, n=12 preparations.

Figure 4.. Real-time recording of nerve-mediated tension and ATP release; guinea-pig detrusor.

A: ATP and tension recorded with a 3-s train of pulses at 1 Hz frequency. Black arrowheads mark spontaneous ATP and tension transients. B: ATP and tension recorded with a 3-s train of pulses at 12 Hz frequency. C: Superimposition of the ATP transients in parts A and B normalised to their respective maximum values: note the different time courses of the 1 and 12 Hz transients.

Figure 5.. Effect of sildenafil (20 μM) on tension and ATP, ACh release: mouse detrusor.

A: Frequency-dependent reduction of nerve-mediated contractions; the line is a fit of eqn 3 (Methods). The dotted line shows the 0% reduction. B: bar chart of tension values in control and with sildenafil during stimulation at 4 Hz (left; **p=0.00283, n=7) and 40 Hz (right; p=0.259, n=7). C: Bar chart of nerve-mediated ATP release in control and with sildenafil for the low frequency (left, **p=0.000526, n=7) and high frequency (right; **p=0.00286, n=7) components. D: Bar chart of nerve-mediated ACh release in control and with sildenafil (*p=0.925, n=5). All data mean±SD.

Figure 5.. Effect of sildenafil (20 μM) on tension and ATP, ACh release: mouse detrusor.

A: Frequency-dependent reduction of nerve-mediated contractions; the line is a fit of eqn 3 (Methods). The dotted line shows the 0% reduction. B: bar chart of tension values in control and with sildenafil during stimulation at 4 Hz (left; **p=0.00283, n=7) and 40 Hz (right; p=0.259, n=7). C: Bar chart of nerve-mediated ATP release in control and with sildenafil for the low frequency (left, **p=0.000526, n=7) and high frequency (right; **p=0.00286, n=7) components. D: Bar chart of nerve-mediated ACh release in control and with sildenafil (*p=0.925, n=5). All data mean±SD.