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

Basu Chakrabarty, Katie Aitchison, Paul White, Carly J McCarthy, Anthony J Kanai, Chris H Fry*

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)peer-review

5 Citations (Scopus)
22 Downloads (Pure)

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
Original languageEnglish
Pages (from-to)350-358
Number of pages9
JournalExperimental Physiology
Volume107
Issue number4
Early online date14 Feb 2022
DOIs
Publication statusE-pub ahead of print - 14 Feb 2022

Bibliographical note

Funding Information:
All animal care and experimental procedures were in compliance with University of Bristol Ethics Committee approvals (UB/18/010; UB/21/064) and carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and Amendment Regulations (SI 2012/3039). The study also complies with ethical principles under which Experimental Physiology operates and the principles of United States National Institutes of Health. Animal studies are reported in compliance with ARRIVE guidelines (Percie du Sert et al., 2020). Animals were housed on a 12 h on/off light cycle with free access to food and water. All animal care and experimental procedures were in compliance with University of Bristol Ethics Committee approvals (UB/18/010; UB/21/064) and carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and Amendment Regulations (SI 2012/3039). The study also complies with ethical principles under which Experimental Physiology operates and the principles of United States National Institutes of Health. Animal studies are reported in compliance with ARRIVE guidelines (Percie du Sert et al., 2020). Animals were housed on a 12 h on/off light cycle with free access to food and water. C56BL/6 female mice (12 weeks, Harlan UK Ltd) or Dunkin–Hartley female guinea-pigs (250–350 g, sourced from designated suppliers by the university animal service unit) were euthanised by cervical dislocation. The bladder was removed after a midline laparotomy, the neck and trigone region cut away, and the dome laid out as a sheet after an anterior wall incision. Strips (mice with intact mucosa: 4–5 mm length, 1 mm diameter; guinea-pigs, mucosa removed: 5 mm length, 2 mm cross-section) were dissected and tied in a horizontal trough between a fixed hook and an isometric force transducer. Preparations were superfused (3 ml.min−1) at 36°C with Tyrode's solution. Contractions were generated by electrical field stimulation (EFS; 0.1 ms pulses, 0.5–40 Hz, 3 s trains every 90 s). Agents were added to the superfusate and their effects on nerve-mediated contractions and transmitter release were measured. Tension amplitude was normalised to preparation weight (mN.mg−1). Superfusate samples (50 μl) were taken from a fixed location relative to the preparation, 3 mm along the preparation and 1 mm lateral, and immediately stored on ice. Samples were taken before EFS and 2 s after its initiation, and nerve-mediated release was the difference between these two values. ACh concentration was measured using a choline/acetylcholine fluorometric assay (MAK056, Sigma-Aldrich, St Louis, MO, USA; 535/587 nm, excitation/emission) as per the manufacturer's instructions. Briefly, the superfusate sample was gently mixed at room temperature with 50 μl assay mix, to convert acetylcholine to choline, and an aliquot added to a well of a 96-well plate. The plate was covered with aluminium foil and placed on a horizontal shaker for 30 min. Choline standards (0–250 pmol.l−1 in five equal steps) were included and produced linear calibration curves (r2 = 0.9994 ± 0.0002, n = 15). Superfusate samples (100 μl) were taken as for ACh measurements and stored on ice before analysis with a luciferin–luciferase luminometry assay (FLAAM, Sigma-Aldrich, UK), per the manufacturer's instructions. Luminescence was recorded from a luminometer (Glomax 20/20, Promega, Madison, WI, USA), calibrated with ATP standards (0.1–1000 pmol.l−1); calibrations were linear on a log–log scale across this range, using a blank of Tyrode's solution. Electrodes (50 μm diameter, 2 mm active tip; Sarissa Biomedical Ltd, Coventry, UK) were placed on the preparation surface, parallel to the longitudinal axis. A similar null electrode, lacking the sensing layer, was placed about 200 μm away and both were polarised to 0.65 V by carbon fibre potentiostats (MicroC, WPI, Hitchin, UK). Both outputs formed differential inputs to a low common mode rejection amplifier to reduce EFS artefacts and the output was digitised (1 kHz) for recording. Glycerol (2 mM) was added to all superfusates, required as an intermediate for the electrode detection of ATP. Prior to recordings, electrodes were calibrated in situ by adding 0.2–50 μmol.l−1 Na2ATP to the superfusate – electrodes showed a linear response over this range. Tyrode's solution contained (mM): NaCl, 118; NaHCO3, 24; KCl, 4.0; NaH2PO4, 0.4; MgCl2, 1.0; CaCl2, 1.8; glucose, 6.1; sodium pyruvate, 5.0; 5% CO2, 95% O2, pH 7.4. Sildenafil stock samples (10 mmol.l−1 in dimethyl sulfoxide) were added to Tyrode's solution to obtain a final concentration of 20 μM, a half-maximal concentration to reduce agonist-induced detrusor contractions (Chakrabarty et al., 2019). All chemicals were from Sigma-Aldrich (UK). Data are means ± SD and differences between multiple data sets were tested with repeated measures two-way ANOVA and Tukey's post hoc tests; the null hypothesis was rejected at P < 0.05; n-values refer to the number of animals. Differences between paired data sets were tested with paired Student's t-test. Statistical and curve-fitting analyses were undertaken with KaleidaGraph (Synergy Software, Reading, PA, USA). Frequency–response data were fitted to a one-component (Equation 1) or a linear two-component (Equation 2) function (equivalent to the Hill–Langmuir equation) by a non-linear least-squares Levenberg–Marquardt algorithm (Marquardt, 1963): 1 Yf=(Ymax×fm)/f1/2m+fm\begin{equation}Y\left(f \right) = ({Y_{{\rm{max}}}} \times {f^m})/\left({f_{1/2}^m + {f^m}} \right)\end{equation}2 Yf=(YA,max×fm)/fA,1/2m+fm+(YB,max×fm)/fB,1/2m+fm\begin{equation}Y\left(f \right) = ({Y_{{\rm{A}},{\rm{max}}}} \times {f^m})/\left({f_{{\rm{A}},1/2}^m + {f^m}} \right) + ({Y_{{\rm{B}},{\rm{max}}}} \times {f^m})/\left({f_{{\rm{B}},1/2}^m + {f^m}} \right)\end{equation} In Equation (1), Ymax is the estimated value of Y (tension (T), ATP or ACh) at the highest frequencies and f1/2 the frequency required to achieve Ymax/2. m is a constant; with tension and ATP data, a best fit was obtained with m = 2; with ACh data with m = 3. In Equation (2) the two components each had a magnitude YA,max (low frequencies) and YB,max (high frequencies), each with respective f1/2 values (fA,1/2 and fB,1/2). A two-stage hierarchical regression assessed if the improvement of fit with Equation (2) over Equation (1) was statistically significant at α = 0.05 or 0.01. This was tested using tables of the upper significance of the F-distribution, with two and five degrees of freedom, corresponding to the increase in the number of parameters using Equation (2) and the degrees of freedom of the residual mean square error from 10 data values. Reduction of tension values, Tredn, with sildenafil in Figure 5a was fitted by: 3 Trednf=Tf0−Tf0−f∞×fm/f1/2m+fm\begin{equation}{T_{{\rm{redn}}}}\ \left(f \right) = {T_{f0}}\ - \left[{\left({{T_{f0 - f\infty }} \times {f^m}} \right)/\left({f_{1/2}^m + {f^m}} \right)} \right]\end{equation}where Tf0$ {T}_{f0}$ is the estimated maximum reduction at low frequencies; Tf0−f∞${T_{f0 - f\infty }}\ $is the difference of reduction at low and high frequencies; m = 2; and f1/2 is that where frequency-dependent decline was half-maximal.

Publisher Copyright:
© 2022 The Authors. Experimental Physiology © 2022 The Physiological Society.

Keywords

  • acetylcholine
  • ATP
  • bladder
  • detrusor smooth muscle
  • Neurotransmitter

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