Reply from Kenton M. Sanders, Bhupal P. Bhetwal and Brian A. Perrino

Contractile tone is a requisite factor in normal filling and emptying of the proximal stomach. Neural inputs are key regulators of tone (thus gastric volume and compliance). During eating there is net inhibitory drive from nitrergic/purinergic neurons, and as the proximal stomach delivers food to the distal stomach, cholinergic influences gradually increase tone, empty the fundus and restore the resting volume of the stomach. For many years investigators have attempted to determine what cells and mechanisms mediate the post-junctional responses to neurotransmitters in the fundus. Knowing the answers to these questions might reveal useful therapeutics for conditions, such as dyspepsia and gastroparesis, that affect many patients worldwide. Our recent study (Bhetwal et al. 2013) was designed to determine whether the same pathways are activated by neurotransmitters released from enteric motor neurons as those activated by neurotransmitter substances added to a solution bathing a muscle strip. We reasoned that a relatively tiny mass of neurotransmitter released in a punctate manner from nerve varicosities might achieve different concentration profiles and bind to different populations of receptors compared to high-mass amounts of bath-applied transmitter diffusing freely through the extracellular spaces in muscles. Our findings indicate that the post-junctional receptors activated during cholinergic neurotransmission must be different from those activated when muscles are bathed in cholinergic agonists. Use of W/WV mice allowed us to further refine the role of interstitial cells of Cajal (ICC) in cholinergic neurotransmission, and we do not believe our previous findings were contradicted by the new data, as Professor Goyal suggests. We found that when ACh is released from neurons, there is an increase in the phosphorylation of CPI-17, a protein known to increase Ca2+ sensitivity in smooth muscle contraction. We did not resolve phosphorylation of MYPT1, another mediator of enhanced Ca2+ sensitivity, when ACh was released from neurons, but MYPT1 was clearly phosphorylated when carbachol was added to bath solutions. Thus, our conclusion that neurally released ACh binds mainly to receptors on cells other than smooth muscle cells (SMCs) is based on these data. Phosphorylation of CPI-17 appeared to result from a Ca2+-dependent protein kinase C. Then we attempted to perturb the system by inhibiting the metabolism of ACh released from neurons or by using W/WV muscles missing most of the ICC-IM that form close associations with the terminals of motor neurons. In both conditions ACh released from neurons caused phosphorylation of MYPT1 as a post-junctional mechanism activated by cholinergic nerve stimulation. An explanation for these data is that ACh released from motor neurons binds primarily to receptors on ICC, and this results in depolarization of these cells by activation of Ca2+-activated Cl− channels. Depolarization of ICC conducts to SMCs via gap junctions that exist between ICC and SMCs in the stomach (Komuro et al. 1999; Horiguchi et al. 2003). Depolarization of SMCs enhances Ca2+ entry via voltage-dependent Ca2+ channels. Removing ICC may increase the access of the neurotransmitter to receptors on SMCs that are coupled through G proteins to Rho kinase and phosphorylation of MYPT1. Removing the tiny volumes formed between motor nerve terminals and ICC may reduce the rate of metabolism of ACh because post-junctional concentrations of neurotransmitter may not reach levels as high when released generally into the interstitium. Therefore, the ‘barrier’ against overflow of ACh onto SMC receptors produced by junctions of ICC and enteric nerve terminals does not physically restrict the diffusion of neurotransmitter but rather the amount of available transmitter is reduced by the robust enzymatic activity of acetylcholine esterase. Professor Goyal seems to favour the traditional view that neurotransmitters course freely through the interstitial volume, and responses in gastrointestinal (GI) muscles are the sole result of neurotransmitter binding to SMC receptors. However, morphology shows that the receptive field in GI muscles is populated by at least three types of electrically coupled cells (SMCs, ICC and another interstitial cell that is selectively labelled with antibodies against PDGFRα– PDGFRα+ cells), and we have referred to the receptive field in these muscles as the SIP syncytium (Sanders et al. 2012). When a neurotransmitter is released from a nerve varicosity the response is determined by multiple factors, as discussed in more depth elsewhere (Sanders et al. 2010): (i) the concentration profile of the transmitter as it diffuses within the interstitium, (ii) the expression of receptors with affinity for the neurotransmitter by cells near the site of release, (iii) efficacious coupling of receptors to effector mechanisms, and (iv) the rate of metabolism, uptake of the transmitter, or other means of reducing transmitter concentration. Since all SIP cells express receptors for enteric motor neurotransmitters, it is likely that the post-junctional response is highly integrated and not as simple as transduction by SMCs. Redundant receptors and pathways may provide a ‘safety factor’ when normal response mechanisms are lost or damaged. Our findings have shown ICC to be a first-line mediator of cholinergic electrophysiological responses (Ward et al. 2000), but our recent study (Bhetwal et al. 2013) demonstrates that contractile responses are preserved when ICC are reduced in numbers via recruitment of SMC Ca2+ sensitization pathways. Professor Goyal seems to suggest that if contractile responses to motor nerve stimulation are preserved in the absence of ICC, then these cells are irrelevant to GI motility. This view may be too simplistic, and of greater importance is the likelihood that the causes for GI motility disorders may not be as black and white as traditional concepts might imply. The question of whether cholinergic responses in animals lacking ICC are normal might be the most cogent point for future investigation. Loss of electrophysiological responsiveness and increasing the gain on Ca2+ sensitization mechanisms by recruiting MYPT1 phosphorylation, as occur in fundus muscles lacking ICC, may distort responses to other agonists, such as prostaglandins, hormones, or nitric oxide, and alter compliance responses during gastric filling and/or the rate at which the proximal stomach empties into the distal stomach. Abnormal responses to otherwise normal signalling by the many bioactive substances at play in the postprandial stomach may be at the heart of functional dyspepsia and adversely affect the rate of gastric emptying. We would suggest that normal responses are likely to result from the functional apparatus (e.g. the SIP syncytium) developed through evolution in wild-type animals. Since evolution is primarily concerned with function, and not so much with form, it is imprudent to expect that synaptic or post-junctional structures mediating motor responses in visceral smooth muscles must be identical to synaptic structures in the CNS or neuromuscular junctions in skeletal muscles. Therefore, it may be premature to discount ICC and label them as ‘expendable’ when as yet, we have an incomplete understanding of the mechanisms regulating gastric compliance and little or no certainty about the pathophysiological basis for gastric motility disorders.