CrossTalk proposal: Interstitial cells are involved and physiologically important in neuromuscular transmission in the gut

Traditional thinking has held that enteric motor neurons communicate with smooth muscle cells (SMCs) exclusively by volume transmission (Sarna, 2008). However, the neuromuscular apparatus of the gastrointestinal (GI) tract is complicated by the presence of interstitial cells of Cajal (ICC; KIT⁺ cells) and PDGFRα⁺ cells (cells labelled by antibodies against platelet‐derived growth factor receptor α), which cluster along varicose fibres of motor neurons and form gap junctions with SMCs. The three types of cells form an electrical syncytium known (from the first letter of each of the three cell types) as the SIP syncytium (Sanders et al. 2012). SIP cells express receptors for motor neurotransmitters and respond to neurotransmitters. These observations suggest that interstitial cells are innervated by motor neurons and physiological responses of GI muscles are the integrated product of the SIP syncytium.

ICC and PDGFRα⁺ cells lie in close proximity to varicosities of motor neurons in GI muscles. In the case of ICC, very close junctions (<20 nm) are common. Junctions of this sort have also been reported between varicosities and SMCs, but these appear less common (Daniel & Posey‐Daniel, 1984). Close contacts between varicosities and interstitial cells suggest exposure to high concentrations of neurotransmitters during neurotransmission. Nerve varicosities and ICC express pre‐ and post‐junctional proteins associated with synaptic contacts (Beckett et al. 2005). The nature of synapse‐like specializations between neurons and ICC are still under investigation, but such junctions suggest that neurotransmitters are released in close proximity to ICC.

There are differences in post‐junctional responses in muscles lacking ICC. Nitrergic inhibitory junction potentials (IJPs) were lost in gastric muscles of W/W^V mice with decreased intramuscular ICC (Burns et al. 1996). Cholinergic responses were also reduced in W/W^V muscles (Ward et al. 2000), leading to the suggestion that ICC have a role in mediating excitatory and inhibitory neuromuscular responses in GI muscles.

PDGFRα⁺ cells have receptors for purinergic neurotransmitters. PDGFRα⁺ cells express both P2Y1 receptors and SK3 channels, fundamental components of the inhibitory responses to purines in the gut. Purines (P2Y1 agonists) activate Ca²⁺ transients and SK currents in PDGFRα⁺ cells, and purinergic neurotransmitter responses are activated in PDGFRα⁺ cells prior to responses in SMCs (Kurahashi et al. 2011; Baker et al. 2013, 2015). P2Y1 receptor agonists hyperpolarize PDGFRα⁺ cells to the equilibrium potential for K⁺ ions, but inward currents and depolarization constitute responses to P2Y1 agonists in SMCs held at physiological potentials (Kurahashi et al. 2014). Thus, it appears unlikely that SMCs mediate the signature fast IJPs elicited by purinergic nerve stimulation in the gut. Stimulation of enteric neurons elicits Ca²⁺ transients in PDGFRα⁺ cells in situ with kinetics consistent with direct innervation of these cells (Baker et al. 2015). Purinergic responses in PDGFRα⁺ cells are inhibited by P2Y1 receptor antagonists and in muscles from P2ry1 ^−/− mice (Kurahashi et al. 2011; Baker et al. 2015).

SMCs clearly have receptors and effectors for motor neurotransmission, but ICC and PDGFRα⁺ cells also express suitable receptors and effectors. The ionic conductances mediating post‐junctional responses are also expressed by ICC or PDGFRα⁺ cells, but display greatly reduced expression or absence in SMCs (Hwang et al. 2009; Zhu et al. 2011; Kurahashi et al. 2011, 2014). So even if neurotransmitters reach SMCs in effective concentrations, non‐physiological responses might be elicited if these are the main pathways activated during neurotransmission (e.g. activation of a non‐selective cation conductance in response to muscarinic agonists; activation of an inward current in response to purines).

Mechanisms controlling Ca²⁺ sensitivity in SMCs also couple to neurotransmitter receptors: muscarinic receptor coupling through Gα_q/11 activates protein kinase C and phosphorylation of CPI‐17; coupling through Gα_12/13 activates Rho kinase (Somlyo & Somlyo, 2003). Both pathways lead to inhibition of myosin phosphatase and enhanced contraction. Other pathways mediate cyclic nucleotide‐dependent desensitization. Application of muscarinic agonists to solutions bathing GI muscles causes phosphorylation of CPI‐17, activation of Rho kinase and phosphorylation of an inhibitory subunit of myosin phosphatase. We tested whether these pathways are activated by cholinergic neurotransmission. CPI‐17 was phosphorylated in response to ACh released from motor neurons, but Rho kinase was not activated (Bhetwal et al. 2013). Thus, ACh released from neurons appears to activate different types of receptors, possibly on different cells, from muscarinic agonists added to solutions bathing the muscles. When ACh hydrolysis was blocked by inhibiting cholinesterases, SMC receptors coupled to Rho kinase were activated. The same thing occurred in muscles of W/W^V mice where there were few ICC. These results suggest that ACh released from neurons is rapidly metabolized by acetylcholinesterase expressed by motor neurons (Worth et al. 2015). Metabolism of ACh, possibly in the restricted volumes between varicosities and ICC, reduces the availability of transmitter for diffusion to more distant receptors. Inhibition of acetylcholinesterase or absence of ICC appears to extend the effective volume of neurotransmission such that effective concentrations of ACh reach SMC receptors and recruit new pathways for Ca²⁺ sensitization. An enhanced sphere of influence for ACh was also found in electophysiological experiments (Ward et al. 2000). Similar observations have been made with nitrergic neurotransmission. Telokin, a protein mediating cyclic nucleotide‐dependent Ca²⁺ desensitization in GI muscles (Khromov et al. 2006), was phosphorylated by addition of NO donors, but was not phosphorylated when NO was released from motor neurons (An et al. 2015).

Recent studies using cell‐specific gene deactivation techniques have suggested that SMCs and ICC both participate in nitrergic responses. Mice with cell‐specific reduction in soluble guanylyl cyclase (sGC) were used to evaluate which cells mediate nitrergic relaxation in lower oesophageal sphincter, gastric fundus and colon. In each organ portions of nitrergic responses were attributable to SMCs, ICC or integrated responses of both cell types (Groneberg et al. 2013, 2015; Lies et al. 2014 a,b, 2015). Nitrergic IJPs were reduced when sGC was knocked‐out in either ICC or SMC in colon or fundus (Lies et al. 2014 a).

Some investigators have been resistant to the idea that interstitial cells are involved in motor neurotransmission (Goyal & Chaudhury, 2010), but it should be noted that evidence for exclusive innervation of SMCs is largely assumptive. The fact that nerve stimulation causes relaxation or contraction is not strong evidence that SMCs are the only cells responsible for mediating neural responses. Arguments against a role for interstitial cells in neurotransmission are that SMCs have receptors and effectors and motor responses are retained in mutant mice with reduced populations of ICC (Goyal & Chaudhury, 2010). Observing motor responses in mutants lacking ICC does not mean that interstitial cells have no role in physiological enteric neurotransmission (Huizinga et al. 2008; Zhang et al. 2010; Zhang et al. 2011), because as detailed above, loss of these cells can be compensated for in some circumstances. Recent gene deactivation experiments are consistent with the idea that nitrergic responses are partially mediated by pathways in SMCs and ICC (Lies et al. 2014 b). Reponses to cholinergic, purinergic and peptidergic neurotransmitters result from receptors and pathways distributed throughout cells of the SIP. The breadth of current evidence suggests that all three cell types of the SIP syncytium participate in transduction and generation of responses to motor neurotransmission. Thus, the conclusion that interstitial cells are involved and physiologically important in motor neurotransmission in GI muscles and organs seems inescapable.

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Additional information

Competing interests

None declared.

Author contributions

All authors have approved the final version of the manuscript 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.

Funding

Work on nitrergic regulation of GI muscles by K.M.S. and S.M.W. has been supported by a Program Project Grant from the NIDDK: P01 DK41315. A.F. was supported by Deutsche Forschungsgemeinschaft (FR 1725/1‐5).

Biography

Kenton Sanders and Sean Ward are Professors of Physiology and Cell Biology at the University of Nevada School of Medicine. They have worked on isolating and understanding the role of interstitial cells in visceral smooth muscles for nearly three decades. They were the first to isolate these cells and to identify their functions as pacemaker cells and as mediators of enteric motor neurotransmission. Andreas Friebe is a Professor of Physiology in the Physiologisches Institut at the Universität Würzburg. He is an expert on the functions of soluble guanylyl cyclase and has used inducible genetic deletion of this signalling molecule to better understand the cellular targets for nitric oxide signalling in a number of tissues.