The role of Ano1 in mediating cholinergic neurotransmission in the murine gastric fundus Article Swipe

Gastrointestinal (GI) motility relies on coordinated enteric motor neurotransmission. Excitatory and inhibitory enteric neurons innervate the cells of the SIP syncytium, an electrical syncytium comprising smooth muscle cells (SMCs), interstitial cells of Cajal (ICC) and platelet derived growth factor receptor α-immunopositive (PDGFRα+) cells. The integrated output of these electrically coupled cells determines the contraction and relaxation patterns of GI muscles (Sung et al. 2018). The traditional concept of cholinergic neurotransmission in the GI musculature is that excitatory neurotransmitters act exclusively on SMCs (Goyal & Chaudhury, 2010). An alternative hypothesis involving an intermediary role for ICC in the process of excitatory neurotransmission has since been explored (Fig. 1). Intramuscular ICC (ICC-IM) have been shown to form close synapse-like contacts with enteric neurons, and studies have indicated that neurotransmitters released from nerve varicosities act primarily on receptors on ICC-IM, and resulting electrical signals are conducted to SMCs via gap junctions (Ward et al. 2000). The cells and conductances responsible for mediating post-junctional neuroeffector responses have been subject to controversy. The development of W/WV mice, which lack ICC-IM in the gastric fundus, allowed this controversy to be further investigated. Some studies have shown that post-junctional responses were significantly attenuated or absent in W/WV mice, supporting the idea that ICC-IM are involved in enteric neurotransmission (Ward et al. 2000). However, other studies on WS/WS rats (which also lack ICC-IM) showed that post-junctional responses were unchanged, concluding that ICC-IM are not required for normal enteric neurotransmission (Zhang et al. 2011). It is now well established that ICC exclusively express the calcium-activated chloride channel (CaCC) Ano1 in the GI tract. Activation of Ano1 causes spontaneous transient inward currents (STICs) as a result of chloride efflux. This depolarization is then passively conducted to SMCs via gap junctions, resulting in SMC depolarization, calcium entry via L-type calcium channels and initiation of excitation–contraction coupling. In a recent study published in The Journal of Physiology, Sung & colleagues 2018 investigated whether Ano1 channels on ICC-IM, rather than non-selective cation channels (NSCCs) on SMCs, are the primary conductance mediating cholinergic neurotransmission in the mouse fundus. Fundus muscle was chosen for this study because it has no basal pacemaker activity that could obscure post-junctional responses. Previous studies exploring the role of Ano1 in pacemaker activity used global Ano1 knockout (Ano1−/−) models. Sung et al. 2018 were the first group to examine enteric neuroeffector transmission in the Ano1−/− model. Importantly, Sung et al. 2018 also utilized the Cre-loxP system to generate mice with an inducible conditional knockout (KO) of Ano1 exclusively in ICC (iAno1−/−). The global KO model has limitations, including possible compensatory mechanisms and neonatal lethality. It was difficult to fully explore the role of Ano1 in enteric neurotransmission in neonates, so the inducible model provided a better physiological representation of the role of Ano1 in ICC in adult animals.The authors used antibodies against Kit and Ano1 to label for ICC in the mouse fundus. In adult (Ano1+/+) animals, Ano1 immunoreactivity was resolved in ICC-IM but not in SMCs. Importantly, cellular co-localization of Kit and Ano1 was also observed in ICC-IM of postnatal day (P)3–P5 (Ano1+/+) animals, but only Kit+ ICC-IM were found at a similar density in Ano1−/− littermates; as expected Ano1 was absent in ICC-IM. These results revealed the presence of Ano1 in ICC-IM of the fundus of adult and neonatal mice. Morphological relationships between cholinergic nerve fibres and ICC-IM were studied using antibodies against vesicular acetylcholine transporter (vAChT), to identify cholinergic motor nerves, and Ano1. The results showed vAChT+ nerve fibres were closely apposed to ICC-IM in the fundus of adult and P5 (Ano1+/+) animals. This suggested that cholinergic neurotransmission acts primarily through ICC-IM. Cholinergic neurotransmission is the main excitatory input in fundus muscles, causing transient depolarization of post-junctional cell membrane potentials, known as excitatory junction potentials (EJPs). Whole-cell patch clamp was performed to investigate the carbachol (CCh)-activated conductance in ICC-IM and SMCs from the fundus. KitcopGFP/+ mice allowed the unequivocal identification of isolated ICC-IM. SMCs were identified by their characteristic spindle shaped morphology, and enhanced green fluorescent protein (eGFP) was used to sort smooth muscle cells via Myh11eGFP/+ mice. CCh (10 μM) evoked a CaCC conductance in ICC-IM and a NSCC conductance in SMCs. These responses were abolished by atropine (1 μM). Since the CaCC Ano1 is known to be expressed by ICC, the authors tested the effects of Ano1 inhibitors on inward currents in ICC-IM. The Ano1 blocker benzbromarone (1 μM) inhibited STICs in ICC-IM, and blocked the effect of CCh. Inward currents were reduced by another Ano1 blocker, T16Ainh-A01 (10 μM), but subsequently increased after addition of CCh (10 μM). To inhibit CCh-evoked inward currents, a higher concentration of T16Ainh-A01 (30 μM) was necessary. Although Ano1 was not resolved in SMCs of the murine fundus, the effect of T16Ainh-A01 on inward currents was tested as a negative control. T16Ainh-A01 did not inhibit the CCh-evoked NSCC currents in SMCs, suggesting that Ano1 is present and active in ICC-IM but not in SMCs. To investigate whether the inward current activated by CCh in ICC-IM is partially mediated by a NSCC, the effects of a NSCC inhibitor (La3+) were studied. La3+ (10 μM) had no effects on STICs in ICC-IM and CCh still evoked a large inward current. In contrast, La3+ reduced the small noisy currents and inhibited the effect of CCh in SMCs, indicating that a NSCC mediated the conductance in SMCs rather than Ano1. These results suggested that cholinergic neurotransmission activates different conductances in murine fundus ICC-IM and SMCs. To uncover whether ICC-IM are the cells primarily responsible for post-junctional electrical responses in fundus tissues, electrical field stimulation (EFS) was performed on P5 Ano1+/+ and global KO Ano1−/− mice. Ano1+/+ mice displayed spontaneous transient depolarizations (STDs) under control conditions; these were greatly diminished or absent in Ano1−/− mice. This indicated an important role for Ano1 channels in the excitatory response. EFS evoked a biphasic response in Ano1+/+ mice, but the excitatory component was absent in Ano1−/− mutants. Addition of the nitric oxide synthase blocker NG-nitro-l-arginine (l-NNA; 100 μM) abolished the inhibitory junction potential (IJP) component and increased the amplitude of the EJP in Ano1+/+ mice. Neostigmine (1 μM), a cholinesterase inhibitor, induced slower developing responses to EFS in Ano1+/+ mice. Neostigmine was an important positive control because it also evoked depolarization of Ano1−/− mice, which showed that ACh breakdown was not the reason for the lack of excitatory response in SMCs after EFS. These results demonstrated that cholinergic responses in intact fundus muscles require Ano1. Corresponding mechanical recordings of fundus contractions also confirmed these findings. Previous studies have reported that Ano1 is necessary for the development and proliferation of ICC (Stanich et al. 2011). It was therefore vital to check if the ICC population was reduced in Ano1−/− mutants, as this could be the reason for compromised post-junctional responses to cholinergic neurotransmission. Kit immunohistochemistry revealed no difference in the number of ICC-IM in gastric fundus between Ano1+/+ and Ano1−/− mice. To ensure that the Ano1−/− model had no phenotypic characteristics responsible for altering the cholinergic response, an ICC specific inducible conditional KO of Ano1 (iAno1−/−) was used. Immunohistochemistry showed that Ano1 expression was significantly reduced in the fundus of iAno1−/− mice. EFS evoked the same effects on the iAno1−/− model as seen in the P5 Ano1−/− model, providing further evidence that the lack of cholinergic excitatory responses in both young and adult mice was due to ICC-IM lacking Ano1. To further highlight the importance of Ano1 in cholinergic responses, the authors showed that benzbromarone (5 μM) blocked EJPs in Ano1+/+ fundus muscle. The mechanical responses after addition of benzbromarone displayed a significant reduction in EFS-evoked contractions. To confirm that benzbromarone was not having significant off-target effects, exogenous ACh (1 μM) was added, which caused muscle contraction. Overall, these results support the hypothesis that Ano1 channels in ICC-IM are the primary conductance for cholinergic neurotransmission in murine gastric fundus muscle. ICC involvement in neurotransmission is controversial. Goyal & Chaudhury 2010 argued that the study carried out by Ward et al. 2000 did not consider the effects of cholinergic stimulation on SMCs directly. Another study by Zhang et al. 2011 concluded that cholinergic activity was still present and normal in WS/WS rat stomachs. They suggested that although the presence of cholinergic activity was not due to upregulation of muscarinic receptors in ICC-IM, it could have been due to upregulation of an unstudied subtype in SMCs. Zhang et al. 2011 also argued that the lack of cholinergic response shown in previous W/WV studies (Ward et al. 2000) was due to ACh breakdown before it reached the receptors on SMCs. Sung et al. 2018 challenged this hypothesis through the use of neostigmine controls. The current study suggests cholinergic neurotransmitters act primarily on ICC-IM through muscarinic receptors, leading to cell depolarization via Ano1 activation. This depolarization is then transduced to SMCs via gap junctions, culminating in depolarization, opening of L-type calcium channels and muscle contraction. This study provides compelling evidence that ICC-IM have an intermediary role in cholinergic neurotransmission in murine proximal stomach. The authors are currently on a year's undergraduate research sabbatical at the University of Nevada, Reno, School of Medicine (the institution affiliated with the authors of the focus article) as part of an ongoing International Undergraduate Research Program between Queen's University Belfast, The University of Manchester and The University of Nevada, Reno. T.L.M. presented the work at a journal club presentation. T.L.M., E.P.N.B. and R.T.S.K. drafted and edited the manuscript. All authors approved the final version of the manuscript before submission and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. The authors would like to thank Dr Bernard Drumm and Dr Caroline Cobine for their guidance and help with this review.