Spontaneous release of acetylcholine from autonomic nerves in the bladder

Abstract

Background and purpose: Bladder contractility is regulated by intrinsic myogenic mechanisms interacting with autonomic nerves. In this study, we have investigated the physiological role of spontaneous release of acetylcholine in guinea pig and rat bladders.

Experimental approach: Conventional isotonic or pressure transducers were used to record contractile activity of guinea pig and rat bladders.

Key results: Hyoscine (3 micromol x L(-1)), but not tetrodotoxin (TTX, 1 micromol x L(-1)), reduced basal tension, distension-evoked contractile activity and physostigmine (1 micromol x L(-1))-evoked contractions of the whole guinea pig bladder and muscle strips in vitro. omega-Conotoxin GVIA (0.3 micromol x L(-1)) did not affect physostigmine-induced contractions when given either alone or in combination with omega-agatoxin IVA (0.1 micromol x L(-1)) and SNX 482 (0.3 micromol x L(-1)). After 5 days in organotypic culture, when extrinsic nerves had significantly degenerated, the ability of physostigmine to induce contractions was reduced in the dorso-medial strips, but not in lateral strips (which have around 15 times more intramural neurones). Most muscle strips from adult rats lacked intramural neurones. After 5 days in culture, physostigmine-induced or electrical field stimulation-induced contractions of the rat bladder strips were greatly reduced. In anaesthetized rats, topical application of physostigmine (5-500 nmol) on the bladder produced a TTX-resistant tonic contraction that was abolished by atropine (4.4 micromol x kg(-1) i.v.).

Conclusions and implications: The data indicate that there is spontaneous TTX-resistant release of acetylcholine from autonomic cholinergic extrinsic and intrinsic nerves, which significantly affects bladder contractility. This release is resistant to blockade of N, P/Q and R type Ca(2+) channels.

Figure 2

Effect of hyoscine and tetrodotoxin (TTX) on distension-induced contractile activity in the whole guinea pig bladder, in vitro. (A) Typical recording of 4th distension (0.5 mL·min⁻¹ for 6 min)-evoked contractile activity and (B) 5th distension after 15 min of application of hyoscine (3 µmol·L⁻¹). (C) Decay of distension-evoked contractile activity in control experiments (N= 5). Note that 4th–6th distensions evoked similar contractile activity of the bladder. (D) Averaged data of the effects of hyoscine (3 µmol·L⁻¹, N= 5) and TTX (1 µmol·L⁻¹, N= 5) on 3 mL distension-induced bladder contractions. *, significant difference from control, P < 0.01.

Figure 5

Intramural neurones in the whole guinea pig bladder and in isolated muscle strips. (A) Intramural neurones density map, indicating the number of intramural neurones in the whole bladder. Each square pixel on the map corresponds to 4 mm²; total number of neurones = 1805. (B) Averaged data of the density of intramural nerve cell bodies (number of neurones divided by area of the tissue) in fresh and 5 day cultured muscle strips from lateral (strips 1 & 2) and dorso-medial (strips 3) regions of the bladder. (C) Choline acetyltransferase-immunoreactive intramural neurones in a ganglion in the bladder wall. (D) Single intramural neurone located outside a ganglion shows multipolar morphology by tubulin immunoreactivity. (E) Clearly fragmented tubulin immunoreactive nerve fibres (marked by arrows) in the nerve trunk of the dorsal medial muscle strip after 5 days in culture. Such fragmented axons were not seen in freshly fixed preparations. Each value in (B) is the mean from 6–10 guinea pigs. * and ^#, significant difference from strips 1 & 2, P < 0.001.

Figure 1

Effect of hyoscine on spontaneous contractile activity in the whole guinea pig bladder and isolated muscle strips in vitro. (A and B) Application of hyoscine (3 µmol·L⁻¹) in the presence of tetrodotoxin (TTX) (1 µmol·L⁻¹) inhibited spontaneous contractions of the whole bladder in vitro (A) and of muscle strips (B). (C and D) Averaged data of the effects of hyoscine (3 µmol·L⁻¹) in the presence of TTX (1 µmol·L⁻¹) on spontaneous contractions of the whole bladder (C, N= 4) and of isolated muscle strips (D, n= 8, N= 7). * and ^#, significant difference from TTX, P < 0.01 and P < 0.05 respectively.

Figure 3

Typical tracings indicating the effects of tetrodotoxin (TTX) and hyoscine on the action of physostigmine (Physo) in the whole guinea pig bladder and isolated muscle strips in vitro. (A) Physostigmine (1 µmol·L⁻¹) evoked powerful phasic contractions of the isolated whole guinea pig bladder in vitro; insert below shows physostigmine-induced contractions on faster time scale. TTX (1 µmol·L⁻¹) did not affect, but hyoscine (3 µmol·L⁻¹) nearly blocked phasic contractions evoked by physostigmine. (B) Physostigmine (1 µmol·L⁻¹) evoked phasic contractions of isolated muscle strips; insert below shows physostigmine-induced contractions on faster time scale. TTX (1 µmol·L⁻¹) did not affect contractions, but hyoscine (3 µmol·L⁻¹) nearly abolished them. (C and D) Averaged data of the effects of TTX (1 µmol·L⁻¹) and hyoscine (3 µmol·L⁻¹) on physostigmine-induced contractions in the whole bladder in vitro (C) and in isolated muscle strips (D). Each value in (C) and (D) is the mean from four (C) and six (D) guinea pigs. *, significant difference from control, P < 0.001 in (C) and P < 0.01 in (D); ^#, significant difference from TTX, P < 0.001 in (C) and P < 0.05 in (D).

Figure 4

Effects of CTX alone and of a cocktail of toxins (CTX, ATX and SNX 482) on physostigmine (Physo)- and EFS (10 Hz)-induced contractions in isolated muscle strips of the guinea pig bladder. (A) Averaged data from six guinea pigs shows the lack of effect of CTX (0.3 µmol·L⁻¹) on physostigmine (1 µmol·L⁻¹)-induced contractions in the presence of TTX (1 µmol·L⁻¹). (B) Averaged data (N= 7) of the effect of CTX (0.3 µmol·L⁻¹) on EFS (10 Hz)-induced contractions. (C) Averaged data from five guinea pigs of the effects of the cocktail of toxins [CTX (0.3 µmol·L⁻¹), ATX (0.1 µmol·L⁻¹) and SNX 482 (0.3 µmol·L⁻¹)] on physostigmine (1 µmol·L⁻¹)-induced contractions, in the presence of TTX (1 µmol·L⁻¹). (D) Averaged data (N= 5) of the effect of the same cocktail of toxins on EFS (10 Hz)-induced contractions revealing significant, but incomplete blockade. (E) Averaged data (N= 6) of the corresponding time control for the effect of 1 µmol·L⁻¹ physostigmine (physo 1 = 25–30 min and physo 2 = 45–50 min) alone. *, significant difference from TTX, P < 0.01; ^#, significant difference from control, P < 0.001. ATX, ω-agatoxin IVA; CTX, ω-conotoxin GVIA; EFS, electrical field stimulation; TTX, tetrodotoxin.

Figure 6

Effects of physostigmine and electrical field stimulation (EFS) (10 Hz) on 5 day cultured dorso-medial and lateral muscle strips in the guinea pig bladder. In contrast to high KCl, physostigmine (1 µmol·L⁻¹) and EFS (10 Hz) evoked significantly smaller contractile responses in dorso-medial strips (A) than in lateral strips after 5 days in culture (B). An application of 80 mmol·L⁻¹ KCl is shown by arrowhead in (A) and (B). In (A), arrows show spontaneous contractions while filled dots mark EFS-induced contractions (in A and B). (C and D) Averaged data of the effects of physostigmine (1 µmol·L⁻¹) in fresh and 5 day cultured strips from dorso-medial (C) and lateral (D) regions of the bladder. Note that the effects of physostigmine in (A–D) are in the presence of tetrodotoxin (1 µmol·L⁻¹). Each value in (C) and (D) is the mean from four to seven guinea pigs. *, significant difference from fresh control tissue, P < 0.05.

Figure 7

Effects of physostigmine and density of intramural neurones in the fresh and cultured muscle strips of the rat bladder. (A) Typical traces demonstrate the effects of physostigmine (3 µmol·L⁻¹) on a fresh muscle strip of the rat bladder in the presence of tetrodotoxin (TTX) (1 µmol·L⁻¹). Note that TTX blocked EFS-induced contractions (evoked by 10 Hz, marked by filled dots). (B) Averaged data on the density of intramural neurones (number of neurones divided by area of the tissue) in fresh and 5 day cultured muscle strips from the rat bladder. Note the very different vertical scale from Figure 5B. (C) Physostigmine (3 µmol·L⁻¹)-induced contractile effect was significantly reduced after 5 days in organ culture compared with control fresh strips. Each value in (B) and (C) is the mean from four to five rats. *, significant difference from fresh tissue, P < 0.01.

Figure 8

Effects of physostigmine on the rat bladder in vivo. Typical tracings showing the effects of physostigmine (Physo; 500 nmol, 50 µL applied topically, indicated by arrow) in the rat bladder, after topical application of tetrodotoxin (TTX) (5 nmol, 50 µL). Note that TTX blocked the distension-induced reflex contractions. Atropine (4.4 µmole·kg⁻¹, i.v.) abolished the effect of physostigmine.