Year: 2015
Type: article
Abstract: Gastrointestinal muscles receive both excitatory and inhibitory innervation, and both positive and negative influences impart important regulatory control of motility and transit. Twenty-five years after nitric oxide (NO) was identified as a key neurotransmitter of enteric inhibitory motor neurons (Bult et al. 1990), the cells mediating nitrergic motor effects and the cellular mechanisms responsible for relaxation remain controversial. Three cell types found within GI muscles, i.e. smooth muscle cells (SMCs), interstitial cells of Cajal (ICC) and PDGFRα+ cells, form an electrical syncytium (the SIP syncytium) that integrates intrinsic and regulatory inputs (Sanders et al. 2014). A recent paper in The Journal of Physiology by Lies and coworkers addresses the question of which cell types mediate nitrergic motor responses in the longitudinal muscle layer of the murine colon (Lies et al. 2015). The authors used cell-specific, tamoxifen-inducible expression of Cre recombinase (iCre) in SMCs or ICC bred to mice with floxed Gucy1b3 (Gucy1b3flox/flox) to control expression of β1 subunits of soluble guanylate cyclase (GC), an essential component of NO receptors. The rationale was that elimination of functional receptors in ICC or SMCs would reveal the relative importance of these cells in nitrergic responses. Activation of iCre in ICC resulted in slowing of the frequency and enhancement in the regularity of spontaneous contractions. This response was mimicked by pharmacological blockade of nitrergic transmission in wild-type muscles with tetrodotoxin, NG-nitro-l-arginine methyl ester (l-NAME) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). In contrast, activation of iCre in SMCs did not alter the basal contractile pattern. These data suggest that ongoing NO production and release from neurons inhibits the ability of ICC to coordinate contractile activity. This could possibly be accomplished by blocking the ability of ICC to propagate pacemaker activity. This observation is curious, however, because NO normally reduces contractile frequency and/or abolishes contractions in GI muscles (e.g. Gallego et al. 2008). The difference may relate to the fact that circular muscles were used for most previous studies of nitrergic regulation. In fact, one wonders why Lies and coworkers focused on longitudinal muscles exclusively in their study. This muscle, while capable of shortening colonic segments, as noted by the authors, has an ambiguous role in colonic motility. Another important observation was that nitrergic responses elicited by electrical field stimulation (EFS) appeared to be mediated mainly by SMCs, because responses to repetitive EFS in colonic muscles of mice with iCre activated in SMCs were similar to the responses of colonic muscles with global GC knockout. Thus, nitrergic responses appear to be more complicated than thought previously, and different cells may mediate different aspects of the integrated responses. Cell specific activation of iCre and excision of key alleles in targeted cells (i.e. turning off expression of selected genes) seems like the ideal experiment for physiologists. However, some studies have noted either patchy activation of iCre or incomplete deactivation of the targeted alleles with this approach (Bao et al. 2013). Thus, a question remaining in the study by Lies and coworkers is the degree of knockdown accomplished by activation of iCre in SMCs and ICC. The authors used the term GC knockout based on immunohistochemical evaluation of GCβ1 protein in specific cell-types after tamoxifen injections. While immunohistochemistry (IHC) is useful for identifying the presence of protein X-like immunoreactivity in cells, it is not reliable as a means of determining whether a protein is not present and/or not functional. Expression of GCβ1 in cells of the SIP syncytium provides an excellent example of this point. GCβ1 is either not resolved or poorly resolved by IHC in SMC of GI muscles (Iino et al. 2008), but this protein is clearly functional in SMCs, as demonstrated in the experiments of Lies and coworkers. GCβ1 is resolved in ICC by IHC (Iino et al. 2008), but it fell below the level of resolution in the majority of cells after treatment with tamoxifen (Lies et al. 2015). The question begged by such an observation is whether GCβ1 was knocked out or whether it was still present and functional, but at levels below resolution by IHC. The problem arises from the fact that iCre may not be highly efficient in excising both floxed alleles in mice homozygous for the floxed gene (i.e. geneflox/flox), and heterozygotes frequently fail to show an altered phenotype. If cells expressing Gucy1b3flox/flox had only a single allele deactivated, it is possible that functional levels of protein could remain. Cellular genotyping and quantification of transcripts in targeted cells before and after tamoxifen treatments should become a standard for studies of this type. Lastly, our understanding of neural regulation in the GI tract is growing, but there is a cell missing from the analysis by Lies and coworkers. PDGFRα+ cells have already been shown to have function in purinergic responses in GI muscles (Kurahashi et al. 2011), and they also express GCα1 and GCβ1 (Iino et al. 2008). Thus, functional receptors for NO are likely to be present in these cells. A role for PDGFRα+ cells in nitrergic responses has not been discovered, and at present, mice with selective expression of iCre in PDGFRα+ cells have not been utilized for studies of the GI tract. As the study by Lies and coworkers demonstrates, different SIP cells may participate in nitrergic regulation in specialized ways. Thus, complete understanding of nitrergic mechanisms may not be accomplished until the nitrergic responses of each cell type in the SIP syncytium are clarified. None declared. This work was supported by a program project grant: P01 DK 41315 (KMS and KDK) and an R01 project: DK078736 (KDK) from the NIDDK. The authors also acknowledge helpful discussions with Drs. Wei Yan, Caroline Cobine and Sean Ward.
Source: The Journal of Physiology
DOI: 10.1113/jp271320
Authors: Kenton M. Sanders, Kathleen D. Keef
Institution: University of Nevada, Reno
Language: English
Subfield: Gastroenterology
Field: Medicine
Domain: Health Sciences
Open Access status: hybrid
Estimated APC paid: $5,400
OpenAlex ID: W2269047102