The effects of P2X receptor antagonists on non-cholinergic neurotransmission in guinea-pig isolated urinary bladder

Charles Kennedy, Paul Tasker and Timothy Westfall, Department of Physiology & Pharmacology, University of Strathclyde, SIBS, 27 Taylor Street, Glasgow G4 0NR.

Parasympathetic nerves provide the major excitatory innervation to the detrusor muscle of the urinary bladder, but it was shown as long ago as the 19 th century that a substantial component of the neurotransmission is atropine-resistant (Langley & Anderson, 1895). Subsequently, desensitisation of P2X1 receptors by α,β-meATP was found to abolish the non-cholinergic component, identifying ATP as a cotransmitter with acetylcholine (Kasakov & Burnstock, 1983). The aim of this study was to characterise the effects of P2X receptor antagonists on the non-cholinergic component of neurotransmission.

Longitudinal strips ( ~ 12 mm x 3 mm) of adult, male Dunkin Hartley guinea-pig urinary bladder were mounted under isometric conditions in 2 ml baths at 35 °C. Tension was recorded by Grass FT03 transducers and a Powerlab/4e (AD Instruments) system. Intramural nerves were stimulated by electrical field stimulation (EFS) at 4 Hz, 0.15 ms pulse width, supramax. voltage, for 20 s at 10 min intervals, via two parallel, platinum wire electrodes. Drugs were added directly to the bathing solution and washed out by replacement with drug-free solution. Atropine (1 µM) and prazosin (100 nM) were present throughout. Data are expressed as mean ± sem or geometric mean with 95% confidence limits (95% cl), and were compared by Student’s t-test, or one-way ANOVA and Tukey’s comparison as appropriate. Concentration-inhibition response curves were fitted to the data by logistic (Hill equation), non-linear regression analysis.

Atropine (1 µM) abolished contractions evoked by exogenous acetylcholine (10 µM) (n=4), but reduced the peak amplitude of responses to 4 Hz EFS by only 27.7 ± 7.9% (n=6). PPADS (0.1-100 µM) and suramin (1-300 µM) inhibited the atropine-resistant contractions with IC50 (95% cl) values of 6.9 µM (0.7-78.9 µM, n=5) and 13.8 µM (7.9-24.1 µM, n=6) respectively, but 30-40% of the response remained at the highest concentrations used. Tetrodotoxin (1 µM) abolished the remaining responses. PPADS and suramin also inhibited contractions elicited by exogenous ATP (300 µM) and α,β-meATP (1 µM), with potencies similar to those against 4Hz EFS (n=4-8). Whilst the responses to α,β-meATP were abolished, 30-40% of the peak response to ATP was not. The atropine-resistant neurogenic contractions were also only partially inhibited by 100 µM of the P2X antagonists NF279 (31.9 ± 3.7% decrease, n=7), MRS2159 (57.0 ± 4.4% decrease, n=5) and reactive blue 2 (32.8 ± 5.2% decrease, n=6), all of which abolished contractions evoked by α,β-meATP (1 µM) . In contrast, desensitisation of P2X1 receptors by administration of a,b -meATP (50 µM) abolished responses to 4 Hz EFS (n=12), α,β-meATP (1 µM, n=4) and ATP (300 m M, n=5).

Thus, whilst the non-cholinergic component of neurotransmission is abolished by desensitisation of the P2X1 receptor by α,β-meATP, it is only partially inhibited by P2X1 antagonists. At present the mechanism underlying the atropine- and P2X1 receptor antagonist-resistant component of parasympathetic neurotransmission is unclear.

Langley, KN & Anderson, HK (1895). J. Physiol., 19, 71-139.
Kasakov, L & Burnstock, G (1983). Eur. J. Pharmacol., 86, 291-294.