Saur, Dieter

Myenteric plexus interstitial cells of Cajal (ICC-MY) in the small intestine are Kit+electrical pacemakers that express the Ano1/TMEM16A Ca2+-activated Cl–channel, whose functions in the gastrointestinal tract remain incompletely understood. In this study, an inducible Cre-LoxP-based approach was used to advance the understanding of Ano1 in ICC-MY of adult mouse small intestine. KitCreERT2/+;Ano1Fl/Flmice were treated with tamoxifen or vehicle, and small intestines (mucosa free) were examined. Quantitative RT-PCR demonstrated ~50% reduction in Ano1 mRNA in intestines of conditional knockouts (cKOs) compared with vehicle-treated controls. Whole mount immunohistochemistry showed a mosaic/patchy pattern loss of Ano1 protein in ICC networks. Ca2+transients in ICC-MY network of cKOs displayed reduced duration compared with highly synchronized controls and showed synchronized and desynchronized profiles. When matched, the rank order for Ano1 expression in Ca2+signal imaged fields of view was as follows: vehicle controls>>>cKO(synchronized)>cKO(desynchronized). Maintenance of Ca2+transients’ synchronicity despite high loss of Ano1 indicates a large functional reserve of Ano1 in the ICC-MY network. Slow waves in cKOs displayed reduced duration and increased inter-slow-wave interval and occurred in regular- and irregular-amplitude oscillating patterns. The latter activity suggested ongoing interaction by independent interacting oscillators. Lack of slow waves and depolarization, previously reported for neonatal constitutive knockouts, were also seen. In summary, Ano1 in adults regulates gastrointestinal function by determining Ca2+transients and electrical activity depending on the level of Ano1 expression. Partial Ano1 loss results in Ca2+transients and slow waves displaying reduced duration, while complete and widespread absence of Ano1 in ICC-MY causes lack of slow wave and desynchronized Ca2+transients.NEW & NOTEWORTHY The Ca2+-activated Cl−channel, Ano1, in interstitial cells of Cajal (ICC) is necessary for normal gastrointestinal motility. We knocked out Ano1 to varying degrees in ICC of adult mice. Partial knockout of Ano1 shortened the widths of electrical slow waves and Ca2+transients in myenteric ICC but Ca2+transient synchronicity was preserved. Near-complete knockout was necessary for transient desynchronization and loss of slow waves, indicating a large functional reserve of Ano1 in ICC.View this article's corresponding video summary at https://youtu.be/cyPtDP0KLY4 .

Abstract Background Delayed gastric emptying in diabetic mice and humans is associated with changes in macrophage phenotype and loss of interstitial cells of Cajal ( ICC ) in the gastric muscle layers. In diabetic mice, classically activated M1 macrophages are associated with delayed gastric emptying, whereas alternatively activated M2 macrophages are associated with normal gastric emptying. This study aimed to determine if secreted factors from M1 macrophages could injure mouse ICC in primary culture. Methods Cultures of gastric ICC were treated with conditioned medium ( CM ) from activated bone marrow‐derived macrophages ( BMDM s) and the effect of CM was quantified by counting ICC per high‐powered field. Key Results Bone marrow‐derived macrophages were activated to a M1 or M2 phenotype confirmed by qRT ‐ PCR . Conditioned medium from M1 macrophages reduced ICC numbers by 41.1%, whereas M2‐ CM had no effect as compared to unconditioned, control media. Immunoblot analysis of 40 chemokines/cytokines found 12 that were significantly increased in M1‐ CM , including tumor necrosis factor alpha ( TNF ‐α). ELISA detected 0.697±0.03 ng mL −1 TNF ‐α in M1‐ CM . Recombinant mouse TNF ‐α reduced Kit expression and ICC numbers in a concentration‐dependent manner ( EC 50 = 0.817 ng mL −1 ). Blocking M1‐ CM TNF ‐α with a neutralizing antibody preserved ICC numbers. The caspase inhibitor Z‐ VAD .fmk partly preserved ICC numbers (cells/field; 6.63±1.04, 9.82±1.80 w/Z‐ VAD .fmk, n=6, P <.05). Conclusions & Inferences This work demonstrates that TNF ‐α secreted from M1 macrophages can result in Kit loss and directly injure ICC in vitro partly through caspase‐dependent apoptosis and may play an important role in ICC depletion in diabetic gastroparesis.