Caroline A. Cobine

Relationship between interstitial cells of Cajal, fibroblast-like cells and inhibitory motor nerves in the internal anal sphincter

Interstitial cells of Cajal (ICC) have been shown to participate in nitrergic neurotransmission in various regions of the gastrointestinal (GI) tract. Recently, fibroblast-like cells, which are positive for platelet-derived growth factor receptor α (PDGFRα+), have been suggested to participate additionally in inhibitory neurotransmission in the GI tract. The distribution of ICC and PDGFRα+ cell populations and their relationship to inhibitory nerves within the mouse internal anal sphincter (IAS) are unknown. Immunohistochemical techniques and confocal microscopy were therefore used to examine the density and arrangement of ICC, PDGFRα+ cells and neuronal nitric-oxide-synthase-positive (nNOS+) nerve fibers in the IAS of wild-type (WT) and W/Wv mice. Of the total tissue volume within the IAS circular muscle layer, 18% consisted in highly branched PDGFRα+ cells (PDGFRα+-IM). Other populations of PDGFRα+ cells were observed within the submucosa and along the serosal and myenteric surfaces. Spindle-shaped intramuscular ICC (ICC-IM) were present in the WT mouse IAS but were largely absent from the W/Wv IAS. The ICC-IM volume (5% of tissue volume) in the WT mouse IAS was significantly smaller than that of PDGFRα+-IM. Stellate-shaped submucosal ICC (ICC-SM) were observed in the WT and W/Wv IAS. Minimum surface distance analysis revealed that nNOS+ nerve fibers were closely aligned with both ICC-IM and PDGFRα+-IM. An even closer association was seen between ICC-IM and PDGFRα+-IM. Thus, a close morphological arrangement exists between inhibitory motor neurons, ICC-IM and PDGFRα+-IM suggesting that some functional interaction occurs between them contributing to inhibitory neurotransmission in the IAS.

Changes in neuromuscular transmission in the W/Wv mouse internal anal sphincter

Background  Intramuscular interstitial cells of Cajal (ICC‐IM) have been shown to participate in nitrergic neuromuscular transmission (NMT) in various regions of the gastrointestinal (GI) tract, but their role in the internal anal sphincter (IAS) is still uncertain. Contractile studies of the IAS in the W/Wv mouse (a model in which ICC‐IM numbers are markedly reduced) have reported that nitrergic NMT persists and that ICC‐IM are not required. However, neither the changes in electrical events underlying NMT nor the contributions of other non‐nitrergic neural pathways have been examined in this model.

Functional role of vasoactive intestinal polypeptide in inhibitory motor innervation in the mouse internal anal sphincter

Vasoactive intestinal polypeptide (VIP)‐ergic neuromuscular transmission (NMT) was examined in the internal anal sphincter of wild‐type mice and compared with that of VIP−/− mice. Relaxation and hyperpolarization during brief trains of electrical field stimulation (EFS; 4 s) were mediated by purinergic and nitrergic NMT. During longer stimulus trains, a non‐purinergic, non‐nitrergic (NNNP) relaxation and hyperpolarization slowly developed and persisted for several minutes beyond the end of the stimulus train. The NNNP NMT was abolished by VIP receptor antagonists, absent in the VIP−/− mouse internal anal sphincter and mimicked by exogenous VIP. These data suggest that NNNP NMT gives rise to ultraslow relaxation and hyperpolarization that is mediated by VIP. In vivo, this pathway may be activated with larger rectal distensions, leading to a more prolonged anal relaxation.

Nitrergic neuromuscular transmission in the mouse internal anal sphincter is accomplished by multiple pathways and postjunctional effector cells

The effector cells and second messengers participating in nitrergic neuromuscular transmission (NMT) were investigated in the mouse internal anal sphincter (IAS). Protein expression of guanylate cyclase (GCα, GCβ) and cyclic GMP-dependent protein kinase I (cGKI) were examined in cryostat sections with dual-labeling immunohistochemical techniques in PDGFRα(+) cells, interstitial cells of Cajal (ICC), and smooth muscle cells (SMC). Gene expression levels were determined with quantitative PCR of dispersed cells from Pdgfrα(egfp/+), Kit(copGFP/+), and smMHC(Cre-egfp) mice sorted with FACS. The relative gene and protein expression levels of GCα and GCβ were PDGFRα(+) cells > ICC ≫ SMC. In contrast, cGKI gene expression sequence was SMC = ICC > PDGFRα(+) cells whereas cGKI protein expression sequence was neurons > SMC ≫ ICC = PDGFRα(+) cells. The functional role of cGKI was investigated in cGKI(-/-) mice. Relaxation with 8-bromo (8-Br)-cGMP was greatly reduced in cGKI(-/-) mice whereas responses to sodium nitroprusside (SNP) were partially reduced and forskolin responses were unchanged. A nitrergic relaxation occurred with nerve stimulation (NS, 5 Hz, 60 s) in cGKI(+/+) and cGKI(-/-) mice although there was a small reduction in the cGKI(-/-) mouse. N(ω)-nitro-l-arginine (l-NNA) abolished responses during the first 20-30 s of NS in both animals. The GC inhibitor ODQ greatly reduced or abolished SNP and nitrergic NS responses in both animals. These data confirm an essential role for GC in NO-induced relaxation in the IAS. However, the expression of GC and cGKI by all three cell types suggests that each may participate in coordinating muscular responses to NO. The persistence of nitrergic NMT in the cGKI(-/-) mouse suggests the presence of a significant GC-dependent, cGKI-independent pathway.

Spatial organization and coordination of slow waves in the mouse anorectum

The internal anal sphincter (IAS) develops tone important for maintaining high anal pressure and continence whereas tone in the rectum is less. To investigate tone generation, the electrical properties [membrane potential (Em) and slow waves (SWs)] and morphology of the mouse IAS and distal rectum were compared. SWs were greatest in amplitude and frequency at the distal end of the IAS and declined toward the rectum. SWs were also coordinated to a greater degree in the circumferential than the oral direction. The circular muscle was divided into ‘minibundles’ in the IAS but not rectum. Intramuscular interstitial cells of Cajal and platelet‐derived growth factor receptor alpha‐positive cells were present in each minibundle making each a possible candidate for SW generation. The features that distinguish the IAS from rectum (i.e. depolarized Em, larger and higher frequency SWs and multiunit configuration) are all properties that are predicted to result in greater tone generation.

Inhibitory Neural Regulation of the Ca2+ Transients in Intramuscular Interstitial Cells of Cajal in the Small Intestine

Gastrointestinal motility is coordinated by enteric neurons. Both inhibitory and excitatory motor neurons innervate the syncytium consisting of smooth muscle cells (SMCs) interstitial cells of Cajal (ICC) and PDGFRα+ cells (SIP syncytium). Confocal imaging of mouse small intestines from animals expressing GCaMP3 in ICC were used to investigate inhibitory neural regulation of ICC in the deep muscular plexus (ICC-DMP). We hypothesized that Ca2+ signaling in ICC-DMP can be modulated by inhibitory enteric neural input. ICC-DMP lie in close proximity to the varicosities of motor neurons and generate ongoing Ca2+ transients that underlie activation of Ca2+-dependent Cl− channels and regulate the excitability of SMCs in the SIP syncytium. Electrical field stimulation (EFS) caused inhibition of Ca2+ for the first 2–3 s of stimulation, and then Ca2+ transients escaped from inhibition. The NO donor (DEA-NONOate) inhibited Ca2+ transients and Nω-Nitro-L-arginine (L-NNA) or a guanylate cyclase inhibitor (ODQ) blocked inhibition induced by EFS. Purinergic neurotransmission did not affect Ca2+ transients in ICC-DMP. Purinergic neurotransmission elicits hyperpolarization of the SIP syncytium by activation of K+ channels in PDGFRα+ cells. Generalized hyperpolarization of SIP cells by pinacidil (KATP agonist) or MRS2365 (P2Y1 agonist) also had no effect on Ca2+ transients in ICC-DMP. Peptidergic transmitter receptors (VIP and PACAP) are expressed in ICC and can modulate ICC-DMP Ca2+ transients. In summary Ca2+ transients in ICC-DMP are blocked by enteric inhibitory neurotransmission. ICC-DMP lack a voltage-dependent mechanism for regulating Ca2+ release, and this protects Ca2+ handling in ICC-DMP from membrane potential changes in other SIP cells.

AB285. SPR-12 Evidence supporting a pivotal role for intramuscular interstitial cells of Cajal in the generation of pacemaker activity, phasic contractions and tone in the internal anal sphincter

Objective A fundamental property of the internal anal sphincter (IAS) is its ability to generate tone. However, the mechanism underlying tone generation is still controversial. In other GI regions, interstitial cells of Cajal (ICC) have been demonstrated to be the pacemaker cells that generate slow waves (SWs). We have shown that SWs are also present in the IAS along with a population of intramuscular ICC (ICC-IM). However, the identity of the cell that generates SWs in the IAS is still uncertain. We hypothesize that SWs are generated by ICC-IM via a rise in intracellular calcium, activation of calcium-activated chloride channels (ANO1) and voltage-dependent L-type calcium channels (CavL). We further propose that SWs play a critical role in tone development in the IAS through the generation and summation of phasic contractile activity. Methods Cell specific protein expression was examined with immunohistochemistry while gene expression was determined with qPCR on whole muscles and cells isolated with FACS. An inducible Cre/loxP technique was used to express a genetically-encoded Ca2+ biosensor (GCaMP3) in a cell-specific manner. Calcium transients were imaged from the IAS of mice expressing GCaMP3 in ICC (Kit-GCaMP3) and smooth muscle cells (SMC; smMHC-GCaMP3) with an Olympus DSU. Results Dual labeling immunohistochemistry revealed ANO1 expression in ICC but not SMC while gene expression of ANO1 (Ano1) was 26× greater in FACS-sorted ICC than in sorted SMC. In contrast, gene expression of CavL (Cacna1c) was only 2× greater in SMC than in ICC. Calcium transients were visualized in Kit-GCaMP3+ cells in the circular muscle layer of the IAS. Kit-GCaMP3+ cells exhibited rhythmic whole cell Ca2+ transients that occurred at the same frequency as the SWs we have previously recorded from this muscle. The activity of adjacent Kit-GCaMP3+ cells (<200 µm) was synchronized suggesting coupling between ICC-IM. Rhythmic calcium transients in Kit-GCaMP3+ cells were abolished by removal of extracellular calcium, by the CavL blocker nicardipine (1 µM) and the ANO1 blocker CaCCinh-A01 (3–10 µM). SWs and phasic contractions were also abolished by these blockers. Like SWs, Ca2+ transients in both smMHC-GCaMP3+ and Kit-GCaMP3+ cells were greatest in frequency at the distal extremity of the IAS and declined in the proximal direction. However, when the distal edge of the IAS was removed, both SWs and Ca2+ transients persisted in the remaining muscle although there was a reduction in frequency. Conclusions This study provides the first direct evidence that ICC-IM are the pacemaker cells of the IAS. The properties of Ca2+ transients in Kit-GCaMP3+ and smMHC-GCaMP3+ cells are commensurate with the electrical and contractile properties of this muscle. The ability of nicardipine and CaCCinh-A01 to abolish tone, SWs and rhythmic calcium transients in Kit-GCaMP3+ cells suggests a causal relationship between these events. The importance of the distal edge of the IAS in regulating phasic contractions in this muscle is underscored by the decline in both SW and Ca2+ transient frequency and amplitude in the proximal direction indicating that like the heart there is a dominant site of pacemaker activity in the IAS. The observation that the greatest frequency of these events is at the distal edge suggests an important function in ensuring fecal continence. Funding Source(s) NIH DK078736

65 Role of ANO1 in Slow Wave and Tone Generation in the Internal Anal Sphincter

test) with aging. To determine whether these pro-inflammatory cytokines affected ENSC survival, TUNEL assays were performed on ENSCs cultured in conditioned media from young (CM-young) and old (CM-old) LMMP. CM-old caused a over a 2-fold increase in apoptosis compared to CM-young, an effect completely reversed by anti-IL6 neutralizing antibody (11.1±0.6% for CM-old, 4.9±1.2% for CM-young, 4.9±1.4% for CM-old+anti-IL6, p<.05 by ANOVA). Conclusion: This data suggests that a shift in macrophage polarization from M2 to M1 with aging is associated with an inflammatory state characterized by increased pro-inflammatory cytokines. These pro-inflammatory cytokines, particularly IL-6, cause increased apoptosis of ENSCs contributing to age-related degeneration of the ENS.