Whether or not M3R was involved in the intestinal epithelial barrier function has been unclear, as receptor subtype-selective compounds are not currently available and mice lacking the M3R seemed to retain cholinergic intestinal ion transport, probably due to compensatory mechanisms [21]. In the present study using a novel highly selective M3 PAM (PAM-369), we showed for the first time that M3R does help maintain the intestinal epithelial barrier function via induction of intestinal epithelial Cl− secretion in a similar manner to EP4R, and potentiation of M3R by M3 PAM ameliorated indomethacin-induced small intestinal injury.
This study demonstrated that both EP4R and M3R contribute to the intestinal epithelial Cl− secretion in a similar manner, using the application of receptor antagonists and an extracellular Cl−-free condition. Intestinal epithelial Cl− secretion through apical Cl− channels, mainly cystic fibrosis transmembrane conductance regulator (CFTR), is known to be indispensable for the formation of water secretion and the mucin layer, which plays a crucial role in the intestinal mucosal barrier function [8, 10, 22, 23]. Cl− secretion is accompanied by secretion of Na+ in a paracellular pathway, which is followed by water secretion [24]. One important point of the present study is that the involvement of M3R in intestinal epithelial Cl− secretion has been definitely determined using PAM-369 in combination with complete blockade of the Isc increase via 4-DAMP, an M3R antagonist. It is considered that activation of EP4R enhances apical Cl− conductance via CFTR through an increase in cyclic adenosine monophosphate (cAMP). In contrast, it is reported that activation of M3R reinforces basolateral K+ efflux in a Ca2+-dependent manner, leading to an increased uptake of Cl− into epithelial cells via Na/K/2Cl cotransporter (NKCC1), which results in an increased driving force for intestinal epithelial Cl− secretion [24].
Use of PAM-369 also led us to discover the important fact that a small amount of endogenous acetylcholine continues to be produced basally in intestinal tissue. Application of PAM-369 at a high concentration slowly but significantly increased the basal Isc in the absence of carbachol, even though PAM-369 exerts no effects on the gastrointestinal tract in the absence of an orthosteric ligand for M3R. This was further confirmed by the finding that the PAM-369-induced increase in basal Isc was significantly potentiated by neostigmine. Given that 4-DAMP had no effects on the basal Isc, the amount of basally produced acetylcholine is not sufficient to cause a basal increase in the Isc itself; however, the basal response to acetylcholine was unmasked by allosteric changes of M3R by 10 µM PAM-369.
We were unable to clarify in our study why a small amount of endogenous acetylcholine continued to be produced basally. It has been reported that a basal level of acetylcholine release was also observed in unstimulated bladder tissue [25]. One speculation is that basal stimulation of M3R with acetylcholine at a very low level that cannot induce any response itself might contribute to the conduct of an appropriate reaction or avoid an unexpectedly strong reaction by adjusting the M3R sensitivity to orthosteric ligands. Further studies will be required to clarify this point in the future.
The present study successfully demonstrated for the first time that M3R was a viable therapeutic target for NSAID-induced enteropathy. Impairment of the intestinal epithelial barrier function by inhibition of the PGE-EP4 signal transduction pathway is a primary cause of NSAID-induced enteropathy. An EP4R agonist reportedly alleviated indomethacin-induced small intestinal injury in rodents [12]. Consistent with previous studies, we confirmed that activation of EP4R by misoprostol increased intestinal epithelial Cl− secretion ex vivo, and misoprostol was indeed effective in preventing indomethacin-induced small intestinal injury in our in vivo mice model. Although misoprostol is currently clinically available, common side effects, such as abdominal pain and diarrhea, are not usually tolerated by patients with NSAID-induced enteropathy in clinical practice [14, 15]. Since M3R activation can induce intestinal epithelial Cl− secretion in a similar manner to EP4R activation, we hypothesized that M3R would be a viable substitute for EP4R in preventing NSAID-induced enteropathy. As expected, potentiation of M3R ameliorated indomethacin-induced small intestinal injury in a similar manner to misoprostol.
This study provided further evidence that M3R is a viable therapeutic target for NSAID-induced enteropathy. It should be noted in the present study that the PAM-369-induced increase in the basal Isc was significantly enhanced after indomethacin treatment, indicating that M3R can be enhanced by induction of NSAID-induced enteropathy. Indeed, the cholinergic system, including M3R and ChAT, can reportedly be enhanced in acute small intestinal inflammation to prevent further progression of organ damage [26]. Consistent with the results concerning the Isc in the Ussing chamber, we confirmed that the expression of M3R (but not M1R or M2R) was significantly up-regulated by indomethacin treatment, while the ChAT expression was unchanged. Given that 4-DAMP had no effect on the basal Isc but the PAM-369-induced increase in basal Isc was potentiated by indomethacin treatment, not only the increased expression of M3R itself but also the increased sensitivity of the orthostatic site and/or allosteric site to M3R may be present during indomethacin treatment. It is unlikely that functional M3R is newly synthesized in a short time [27], but its up-regulation by indomethacin treatment is possibly brought by endocytic trafficking [28]. Further investigations will be needed to clarify the mechanism underlying how M3R signaling was enhanced under conditions where the production of prostaglandin is suppressed. More importantly, combined treatment of misoprostol and PAM-369 had no additional effects (Supplemental Fig. 2), suggesting that both agents may exert their effects through the same final pathway, such as CFTR. In NSAID-induced enteropathy, up-regulation of M3R signaling might be induced in order to compensate for the down-regulated PGE-EP4 pathway in terms of the intestinal epithelial barrier function (Fig. 7). In this regard, M3R may indeed be a therapeutic target for NSAID-induced enteropathy.
In conclusion, M3R plays a role in intestinal epithelial Cl− secretion in a similar manner to EP4R in terms of the intestinal mucosal barrier function. Suppression of prostaglandin production by indomethacin induced an increased expression of M3R and enhanced M3R signaling, possibly via a compensatory mechanism. The further potentiation of M3 receptor by PAM-369 ameliorated indomethacin-induced small intestinal injury. M3R is a promising target for the treatment or prevention of NSAID-induced enteropathy.