In the gut, smooth muscle bundles are separated by intramuscular spaces containing nerve fibres and interstitial cells including intramuscular interstitial cells of Cajal (ICC-IM), a subset of a family of ICC (Komuro 2006). Traditionally, smooth muscle cells (SMCs) are believed to transduce the action of neurotransmitters that cause muscle contraction or relaxation by a process called direct neuromuscular transmission (NMT). Neuro-ICC-IM and neuro-smooth muscle (SM) transmissions regulate functions of ICC and SMCs, respectively. Initially proposed by Imaizumi & Hanna (1969), Sanders and colleagues championed the idea that the smooth muscle responses to nerve stimulation required mandatory transduction by the ICC-IM (indirect NMT) (Burns et al. 1996; Ward et al. 2000). The role of the smooth muscles was considered simply to mount mechanical contraction or relaxation in response to electrical signals transduced in ICC-IM. It has also been suggested that both ICC-IM and SMCs transduced neural signals to the smooth muscle. According to one view, ICC-IM is involved only in certain situations (direct or indirect NMT) (Bhetwal et al. 2013; Klein et al. 2013). According to another, ICC-IM is involved in parallel with smooth muscles (direct and indirect NMT) (Groneberg et al. 2013). However, most of the available data continue to support the traditional view of direct NMT, not requiring ICC-IM (Goyal & Chaudhury 2010) (see Table 1).
|Involvement of ICC-IM in NMT|
|Type of NMT||Direct||Indirect||Direct or indirect (situational)||Direct and indirect (dual)|
|Proposers||Traditional||Burn et al. 1996; Ward et al. 2000||Klein et al. 2013; Bhetwal et al. 2013||Groneberg et al. 2013|
|Neurotransmitters involved||All transmitters||Nitrergic and cholinergic||Nitrergic and cholinergic||Nitrergic only|
|Lack of evidence for true synaptic innervation of the ICC-IM*||Consistent||Against||Against||Consistent|
|Evidence for direct innervation of SM*||Consistent||Against||Against||Consistent|
|Evidence for the presence of functional signalling molecules in SM*||Consistent||Against||Consistent||Consistent|
|Lack of evidence for effective conduction from ICC-IM to SM via gap junctions*||Consistent||Against||Against||Against|
|Origin of nitrergic inhibitory junction potential in SM*||Consistent||Against||Against||Consistent|
|Preserved nitrergic NMT in ICC deficiency*||Consistent||Against||Consistent||Consistent|
|Loss of nitrergic inhibition with presumed deletion of NO-GC from ICC*||Against?||Consistent||Against||Consistent|
|Preserved cholinergic NMT in ICC deficiency*||Consistent||Against||Against||Consistent|
The traditional model: direct NMT
The direct NMT is supported by the following: (1) close synapse-like contacts of motor nerve varicosities with smooth muscles (Komuro 2012), (2) the presence of nitrergic and cholinergic signalling molecules in the smooth muscles (Bhetwal et al. 2013; Cobine et al. 2014; Lies et al. 2014a), (3) the presence of nitrergic and cholinergic signalling in the smooth muscles (Wang et al. 1996; Zhang et al. 1998), (4) expected effects of agonists of neurotransmitters in isolated smooth muscle (Wang et al. 1996; Zhang et al. 1998), and (5) demonstration of active generation of nitrergic inhibitory junction potential in the SMC (He & Goyal 2012).
Mandatory role of ICC-IM: indirect NMT
An assumed lack of direct NMT led to the conclusion of a mandatory role of ICC-IM in nitrergic NMT. This conclusion was supported by the following: (1) an assumed lack of direct innervation of smooth muscle and presence of exclusive close synaptic contacts of nerve varicosities with ICC-IM (Sanders et al. 2014a), (2) the abundance of the nitric oxide sensitive-guanylate cyclase (NO-GC) in ICC-IM but not in smooth muscle (Lies et al. 2014a), and (3) the reported loss of nitrergic NMT in ICC-IM-deficient W/Wv mice (Burns et al. 1996). The mandatory role of cholinergic transmission was based on the reported loss of cholinergic NMT in ICC-IM deficiency (Ward et al. 2000; Klein et al. 2013).
However, as summarized below, there is substantial evidence against the mandatory role of the ICC in nitrergic NMT.
First, close synapse-like junctions between the nerves and ICC are also present between the nerves and the smooth muscles (Mitsui & Komuro 2002). However, true synapses with a synaptic cavity are present at neither neuro-ICC nor neuro-smooth muscle junctions (Komuro 2012).
Second, a key assumption in the model of the role of ICC-IM in NMT is that the gap junctions conduct electrical potentials from ICC-IM to smooth muscles (Sanders et al. 2014a). Although gap junctions have been identified, their functional efficiency in conducting electrical signals has been shown to be poor (Sibave et al. 2006; Daniel et al. 2007).
Third, NO-GC is present in the smooth muscle, ICC and PDGFRα+ fibroblast-like cells and abundance of NO-GC does not correlate with greater function. PDGFRα+ fibroblast-like cells have the highest concentration of NO-GC, yet they do not participate in NMT (Cobine et al. 2014; Lies et al. 2014a).
Forth, several studies have reported that nitrergic inhibitory junction potential (IJP) and smooth muscle relaxation are preserved in ICC-IM-deficient W/Wv mice and Ws/Ws rats (Sivarao et al., 2001, 2008; Huizinga et al. 2008; Zhang et al. 2010). Most recently, Sanders and colleagues (2014b) suggested that the discrepant results may be due to an incomplete loss of ICC in the interrogated tissues. They reported that complete absence of ICC-IM was required for a loss of nitrergic relaxation. However, in the absence of ICC-IM, there is no barrier between neurally released neurotransmitter and SMCs, leaving direct NMT intact (Bhetwal et al. 2013; Klein et al. 2013). Therefore, the report of loss of nitrergic neurotransmission in the absence of ICC in W/Wv mice (Sanders 2014b) is difficult to reconcile.
Evidence against a mandatory role of the ICC cholinergic NMT is the failure to reproduce the reported loss of cholinergic NMT in W/Wv mice (Ward et al. 2000; Zhang et al. 2011; Bhetwal 2013; Goyal 2013).
Optional role of ICC-IM: direct or indirect NMT
Klein et al. (2013) reported that ‘ICC-specific’ deletion of protein kinase G1 (Prkg1) abolished nitrergic NMT, but deletion of ICC left nitrergic neurotransmission intact. To explain these paradoxical findings the authors made an intriguing proposal, that when present ICC-IM are involved in NMT, but when ICC-IM is absent, direct NMT takes place. This model was based on the presence of assumed true neuro-ICC-IM synapses that can restrict the accessibility of the released nitric oxide (NO) to the smooth muscle (Beckett et al. 2005). However, no true synapses have been identified between ICC and smooth muscles (Komuro 2012; Goyal & Chaudhury 2013). Moreover, NO is a highly diffusible gas (diffusion constant 3300 μ2 s−1) (Lancaster 1997). Therefore, ICC-IM cannot restrict it to having effects on adjacent smooth muscles. A similar unsubstantiated model was used to explain the persistence of cholinergic responses in W/Wv mice (Bhetwal et al. 2013; Goyal 2013).
Complementary role of ICC-IM: direct and indirect NMT
For nitrergic NMT, Groneberg and colleagues (2013) reported that cell-specific knockdown of NO-GC in SMCs or ICC did not affect nitrergic relaxation. However, double knockdown of NO-GC in both SMCs and ICC abolished nitrergic NMT (Groneberg et al. 2013). Other studies have reported that incomplete deletion of NO-GC in ICC results in a dominant loss of nitrergic relaxation (Lies et al. 2014b; Groneberg et al. 2015). Moreover, Klein et al. 2013 has reported that partial deletion of Prkg1 abolishes nitrergic NMT, but partial deletion of ICC does not (Klein et al. 2013). It is not clear why incomplete deletion of NO-GC has a more profound effect on nitrergic NMT than incomplete deletion of ICC. It is possible that cKIT-CreERT2 mutants may also affect NO-GC in smooth muscles. Validation studies using immunohistochemistry are not sensitive enough to reveal changes in NO-GC in smooth muscles (Groneberg et al. 2015). Finally, a complementary role of ICC in NMT is difficult to comprehend in the absence of a functional gap junction that can transmit signals from the ICC to SMC (Sibave et al. 2006; Daniel et al. 2007). A complementary role of cholinergic NMT is also speculated but without any experimental data to support it (Groneberg et al. 2015).
Overall, available evidence is most consistent with the traditional model of direct NMT without the involvement of ICC-IM. Parallel neuro-ICC and neuro-SM transmissions may independently regulate the functions of the ICC-IM and SMCs, respectively. There is little evidence for an obligatory or optional involvement of ICC-IM in NMT. A complementary role and physiological importance of ICC-IM in nitrergic NMT remain speculative. Moreover, it is unlikely that ICC-IM would only be involved in nitrergic NMT while a variety of other neurotransmitters will use direct NMT.