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. 2022 Mar;179(6):1187-1200.
doi: 10.1111/bph.15685. Epub 2021 Oct 27.

Evidence for tetrodotoxin-resistant spontaneous myogenic contractions of mouse isolated stomach that are dependent on acetylcholine

Weigang Cai  1 , Raj Makwana  1 , Marilisa Straface  1 , Armen Gharibans  2 , Paul L R Andrews  3 , Gareth J Sanger  1
Affiliations

Evidence for tetrodotoxin-resistant spontaneous myogenic contractions of mouse isolated stomach that are dependent on acetylcholine

Weigang Cai et al. Br J Pharmacol. 2022 Mar.

Abstract

Background and purpose: Gastric pacemaker cells, interstitial cells of Cajal (ICC), are believed to initiate myogenic (non-neuronal) contractions. These become damaged in gastroparesis, associated with dysrhythmic electrical activity and nausea. We utilised mouse isolated stomach to model myogenic contractions and investigate their origin and actions of interstitial cells of Cajal modulators.

Experimental approach: Intraluminal pressure was recorded following distension with a physiological volume; tone, contraction amplitude and frequency were quantified. Compounds were bath applied.

Key results: The stomach exhibited regular large amplitude contractions (median amplitude 9.0 [4.7-14.8] cmH2 O, frequency 2.9 [2.5-3.4] c.p.m; n = 20), appearing to progress aborally. Tetrodotoxin (TTX, 10-6 M) had no effect on tone, frequency or amplitude but blocked responses to nerve stimulation. ω-conotoxin GVIA (10-7 M) ± TTX was without effect on baseline motility. In the presence of TTX, (1) atropine (10-10 -10-6 M) reduced contraction amplitude and frequency in a concentration-related manner (pIC50 7.5 ± 0.3 M for amplitude), (2) CaCC channel (previously ANO1) inhibitors MONNA and CaCCinh-A01 reduced contraction amplitude (significant at 10-5 , 10-4 M respectively) and frequency (significant at 10-5 M), and (3), neostigmine (10-5 M) evoked a large, variable, increase in contraction amplitude, reduced by atropine (10-8 -10-6 M) but unaffected (exploratory study) by the H1 receptor antagonist mepyramine (10-6 M).

Conclusions and implications: The distended mouse stomach exhibited myogenic contractions, resistant to blockade of neural activity by TTX. In the presence of TTX, these contractions were prevented or reduced by compounds blocking interstitial cells of Cajal activity or by atropine and enhanced by neostigmine (antagonised by atropine), suggesting involvement of non-neuronal ACh in their regulation.

Keywords: ACh; ANO1; CaCC; interstitial cells of Cajal; myogenic contractions; nausea; stomach.

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Conflict of interest statement

WC, MS, RM, AG and PLRA declare no potential conflict of interest. GJS receives a research grant from Takeda Pharmaceuticals.

Figures

FIGURE 1
FIGURE 1
Baseline analysis of the mouse isolated stomach spontaneous contractions pre‐ and post‐tetrodotoxin (TTX). (a) The spontaneous contractions pre‐ and post‐TTX (10−6 M) administration. Black arrows show the small amplitude, high frequency (SAHF) contractions, between and superimposed upon the high amplitude, low frequency (LALF) contractions. (b) Analysis (see Section 2.3.1) of the spontaneous contraction amplitude (LALF) and frequency from 20 preparations pre‐TTX and post‐TTX (10−6 M) administration (see Section 2 for details) and 7 time‐matched (periods A and B) preparations not treated with TTX. The median value and interquartile ranges are shown together with the individual data points. (c) An example of the pressure record averaged (see Section 2.3.2) over 10 min before TTX and between 20 and 30 min after TTX (10−6 M). The solid line and the shadow represent the average and the SEM value, respectively. Time 0 corresponds to the peak of each contraction. (d) Representative recording showing block of electrical stimulation (circumoesophageal electrodes 10 Hz, 0.5 ms, 20 V, 1 min, horizontal bar) induced reduction in tone and abolition of contractions by TTX (10−6 M)
FIGURE 2
FIGURE 2
Antagonism of mouse stomach myogenic contractions by atropine. (a) Examples of the pressure record averaged following incremental concentrations of atropine in the presence of TTX (10−6 M; see Section 2.3.2 for analysis). The solid line represents the average, and the shadow illustrates the SEM. Time 0 corresponds to the peak of each contraction. (b) Examples of 5‐min segments of original pressure recordings showing the effect of incremental concentrations of atropine in the presence of tetrodotoxin (TTX; 10−6 M). (c) Concentration–response curves for the effect of atropine on the contraction amplitude, frequency and tone relative to the baseline values in the presence of TTX (10−6 M). Individual values are plotted together with the best fit curve (n = 5). Friedman's test/Dunn's comparison of each atropine concentration with TTX; *P < 0.05
FIGURE 3
FIGURE 3
Antagonism of mouse stomach myogenic contractions by the calcium activated chloride channel (CaCC) inhibitor (CaCCinh‐A01). (a) Examples of the pressure record averaged following incremental concentrations of CaCCinh‐A01 in the presence of tetrodotoxin (TTX; 10−6 M; see Section 2.3.2 for analysis). The solid line represents the average, and the shadow illustrates the SEM. Time 0 corresponds to the peak of each contraction. (b) Examples of 5‐min segments of original pressure recordings showing the effect of incremental concentrations of CaCCinh‐A01 in the presence of TTX (10−6 M). (c) Concentration–response curves for the effect of CaCCinh‐A01 on the contraction amplitude, frequency and tone relative to the baseline values in the presence of TTX (10−6 M). Values are median with all individual values plotted (n = 5). Friedman's test/Dunn's comparison of all concentrations of CaCCinh‐A01 with TTX (see Section 2.7); *P < 0.05
FIGURE 4
FIGURE 4
Antagonism of mouse stomach myogenic contractions by the calcium activated chloride channel (CaCC) inhibitor (MONNA). (a) Examples of the pressure record averaged following incremental concentrations of MONNA in the presence of tetrodotoxin (TTX; 10−6 M; see Section 2.3.2 for analysis). The solid line represents the average, and the shadow illustrates the SEM. Time 0 corresponds to the peak of each contraction. (b) Examples of 5‐min segments of original pressure recordings showing the effect of incremental concentrations of MONNA in the presence of TTX (10−6 M). (c) Concentration–response curves for the effect of MONNA on the contraction amplitude, frequency and tone relative to the baseline values in the presence of TTX (10−6 M). Values are median with all individual values plotted (n = 5). Friedman's test/Dunn's comparison of all concentrations of MONNA with TTX (see Section 2.7); *P < 0.05
FIGURE 5
FIGURE 5
Antagonism of neostigmine enhanced myogenic contractile activity by atropine. (a) An original recording selected to illustrate the protracted stimulation of contraction amplitude by neostigmine (10−5 M) in the presence of tetrodotoxin (TTX; 10−6 M). In this experiment, the effects of atropine (10−9–10−6 M) were investigated 30 min after neostigmine. (b) Quantification of the effect of incremental concentrations of atropine (10−9–10−6 M) on the response to neostigmine (10−5 M) in the presence of TTX (10−6 M) with addition of atropine commencing 15 min after neostigmine (see Section 2). The effect of atropine on contraction amplitude, frequency and tone is plotted in relation to the TTX + neostigmine response. Responses are plotted as median with individual values plotted. Friedman's test/Dunn's test comparing responses to incremental concentrations of atropine to the response to neostigmine in the presence of TTX; *P < 0.05
FIGURE 6
FIGURE 6
Preliminary experiments showing the effect of mepyramine on the response to neostigmine in the presence of tetrodotoxin (TTX). (a) Examples of 5‐min segments of original pressure recordings showing the effect of incremental concentrations of mepyramine (10−6–10−4 M) on the response to neostigmine (10−5 M) in the presence of TTX (10−6 M). (b) Concentration–response curves for the effect of mepyramine on the contraction amplitude, frequency and tone relative to values in the presence of neostigmine (10−5 M) and TTX (10−6 M). Responses are plotted as median with all individual values shown (n = 3)
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