Seth T. Eisenman

Inhibition of Cell Proliferation by a Selective Inhibitor of the Ca2+-activated Cl− Channel, Ano1

BACKGROUND Ion channels play important roles in regulation of cellular proliferation. Ano1 (TMEM16A) is a Ca(2+)-activated Cl(-) channel expressed in several tumors and cell types. In the muscle layers of the gastrointestinal tract Ano1 is selectively expressed in interstitial cells of Cajal (ICC) and appears to be required for normal gastrointestinal slow wave electrical activity. However, Ano1 is expressed in all classes of ICC, including those that do not generate slow waves suggesting that Ano1 may have other functions. Indeed, a role for Ano1 in regulating proliferation of tumors and ICC has been recently suggested. Recently, a high-throughput screen identified a small molecule, T16A(inh)-A01 as a specific inhibitor of Ano1. AIM To investigate the effect of the T16A(inh)-A01 inhibitor on proliferation in ICC and in the Ano1-expressing human pancreatic cancer cell line CFPAC-1. METHODS Inhibition of Ano1 was demonstrated by whole cell voltage clamp recordings of currents in cells transfected with full-length human Ano1. The effect of T16A(inh)-A01 on ICC proliferation was examined in situ in organotypic cultures of intact mouse small intestinal smooth muscle strips and in primary cell cultures prepared from these tissues. ICC were identified by Kit immunoreactivity. Proliferating ICC and CFPAC-1 cells were identified by immunoreactivity for the nuclear antigen Ki67 or EdU incorporation, respectively. RESULTS T16A(inh)-A01 inhibited Ca(2+)-activated Cl(-) currents by 60% at 10μM in a voltage-independent fashion. Proliferation of ICC was significantly reduced in primary cultures from BALB/c mice following treatment with T16A(inh)-A01. Proliferation of the CFPAC-1 human cell-line was also reduced by T16A(inh)-A01. In organotypic cultures of smooth muscle strips from mouse jejunum, the proliferation of ICC was reduced but the total number of proliferating cells/confocal stack was not affected, suggesting that the inhibitory effect was specific for ICC. CONCLUSIONS The selective Ano1 inhibitor T16A(inh)-A01 inhibited Ca(2+)-activated Cl(-) currents, reduced the number of proliferating ICC in culture and inhibited proliferation in the pancreatic cancer cell line CFPAC-1. These data support the notion that chloride channels in general and Ano1 in particular are involved in the regulation of proliferation.

Ano1, a Ca2+‐activated Cl− channel, coordinates contractility in mouse intestine by Ca2+ transient coordination between interstitial cells of Cajal

Ano1, a Ca2+‐activated Cl− channel, is expressed in interstitial cells of Cajal (ICC) throughout the gut. We report here that it is required to maintain coordinated Ca2+ transients within myenteric ICC of mouse small intestine. Ca2+ transients in Ano1 WT mice were rhythmic and coordinated whereas uncoordinated Ca2+ transients were seen in knockout mice. Ca2+ transients were un‐coordinated following pharmacological block of Ano1 in WT mice using niflumic acid, 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid and 4,4′‐diisothiocyanato‐2,2′‐stilbenedisulfonic acid disodium salt. Transient knockdown of Ano1 in organotypic cultures with short hairpin RNA to Ano1 in WT tissues also caused loss of coordinated Ca2+ transients. Contractility of Ano1 knockout mouse intestinal segments in organ bath experiments was significantly decreased, less coordinated and non‐rhythmic. Spatiotemporal maps from knockout mouse small intestine also showed loss of phasic contractile activity. This study provides important information on the basic mechanisms driving coordinated contractile activity in the gastrointestinal tract.

Diabetic Csf1op/op Mice Lacking Macrophages Are Protected Against the Development of Delayed Gastric Emptying

Background & Aims Diabetic gastroparesis is associated with changes in interstitial cells of Cajal (ICC), neurons, and smooth muscle cells in both animal models and humans. Macrophages appear to be critical to the development of cellular damage that leads to delayed gastric emptying (GE), but the mechanisms involved are not well understood. Csf1op/op (Op/Op) mice lack biologically active Csf1 (macrophage colony stimulating factor), resulting in the absence of Csf1-dependent tissue macrophages. We used Csf1op/op mice to determine the role of macrophages in the development of delayed GE. Methods Animals were injected with streptozotocin to make them diabetic. GE was determined weekly. Immunohistochemistry was used to identify macrophages and ICC networks in the gastric muscular layers. Oxidative stress was measured by serum malondialdehyde (MDA) levels. Quantitative reverse-transcription polymerase chain reaction was used to measure levels of mRNA. Results Csf1op/op mice had normal ICC. With onset of diabetes both Csf1op/op and wild-type Csf1+/+ mice developed increased levels of oxidative stress (75.8 ± 9.1 and 41.2 ± 13.6 nmol/mL MDA, respectively). Wild-type Csf1+/+ mice developed delayed GE after the onset of diabetes (4 of 13) whereas no diabetic Csf1op/op mouse developed delayed GE (0 of 15, P = .035). The ICC were disrupted in diabetic wild-type Csf1+/+ mice with delayed GE but remained normal in diabetic Csf1op/op mice. Conclusions Cellular injury and development of delayed GE in diabetes requires the presence of muscle layer macrophages. Targeting macrophages may be an effective therapeutic option to prevent cellular damage and development of delayed GE in diabetes.

Tumor necrosis factor alpha derived from classically activated "M1" macrophages reduces interstitial cell of Cajal numbers

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.

Expression and function of the Scn5a‐encoded voltage‐gated sodium channel NaV1.5 in the rat jejunum

The SCN5A‐encoded voltage‐gated sodium channel NaV1.5 is expressed in human jejunum and colon. Mutations in NaV1.5 are associated with gastrointestinal motility disorders. The rat gastrointestinal tract expresses voltage‐gated sodium channels, but their molecular identity and role in rat gastrointestinal electrophysiology are unknown.

163 Hyperglycemia-Induced Hyperplasia of Interstitial Cells of Cajal (ICC) and ICC Stem Cells (ICC-SC) Is Associated With Accelerated Gastric Emptying in Obese Diabetic LeprDb/Db Mice

Background & Aims: ICC depletion is the most common cellular change in diabetic gastroparesis. However, in up to 22% of diabetic patients with gastric symptoms, gastric emptying (GE) is accelerated, rather than delayed. The fate of ICC in these patients is unclear. Previously we reported ICC and ICC-SC hyperplasia in hyperinsulinemic, diabetic Lepr mice. Here, we studied the relationship between GE of solids and ICC/ICC-SC numbers, and investigated the contribution of insulin/IGF1-dependent Kit ligand (Kitl) expression and hyperglycemia-induced ERK MAPK activation to ICC/ICC-SC hyperplasia. Methods: Lepr (n=32) and agesexand strain-matched controls (n=30) were studied 13-65 weeks after the onset of diabetes. ICC and ICC-SC were quantified by flow cytometry. Serum insulin was measured by enzyme immunoassay. Oxidative stress was determined by measuring serum malondialdehyde (MDA) and gastric 8OHdG. GE of solids was analyzed by 13C breath test. Gene expression was studied by qRT-PCR and WB. Effects of high glucose were investigated in murine gastric ICC primary cultures, a conditionally immortalized cell line derived from murine gastric ICC (ICL2A), and in an ICC-SC line isolated from the mouse stomach (2XSCS2F10). ERKMAPK signaling was evaluated byWB and pharmacological inhibition of MAPKK with PD98059. Cell proliferation and apoptosis were determined byMTS assay andCaspase Glo-3/7 assay, respectively. Results: Leprmice had significantly higher blood glucose (median[IQR]: 515[462;577] vs. 124[115;134] mg/dL; P,0.001) and serum insulin levels. Serum MDA and gastric 8OHdG were moderately increased (2.4and 2.2-fold, respectively, P,0.001). In Lepr mice, ICC and ICC-SC increased 2.0±0.1-fold (P,0.001) and 1.5±0.3-fold (P,0.05), respectively, and GE was accelerated (T1/2: 67±10 vs. 100±11 min; P,0.05). GE was similarly accelerated in Kit mice with generalized ICC hyperplasia due to an activating Kit mutation. In Lepr mice, total/soluble Kitl mRNA and Kitl protein were reduced. High glucose (500 mg/dL) significantly increased the numbers of primary ICC and stimulated proliferation and ERK MAPK phosphorylation in ICL2A and 2XSCS2F10 cells. PD98059 (20 μM) inhibited cell proliferation induced by high glucose without affecting basal proliferation. In contrast, osmotic stress induced by mannitol had no effect on ICC numbers, cell proliferation and ERK MAPK phosphorylation. ICC and ICC-SC were resistant to apoptosis induced by hyperglycemia at levels seen in Lepr mice. Conclusions: In the absence of major oxidative stress, hyperglycemia induces ICCSC and ICC hyperplasia via the ERK MAPK pathway even in the presence of reduced Kitl signaling. ICC hyperplasia, in turn, leads to accelerated GE and may contribute to gastric symptoms in a subset of diabetic patients. Grant support: NIH DK58185, DK68055.

Expression of RAD21 immunoreactivity in myenteric neurons of the human and mouse small intestine

RAD21 is a double‐strand‐break repair protein and component of the cohesin complex with key roles in cellular functions. A RAD21 loss‐of‐function mutation was found in cases of chronic intestinal pseudo‐obstruction (CIPO) with associated enteric neuronal loss. Analysis of RAD21 expression in the enteric nervous system is lacking, thus we aimed to characterize RAD21 immunoreactivity (IR) in myenteric ganglia.

Change in Populations of Macrophages Promotes Development of Delayed Gastric Emptying in Mice.

BACKGROUND & AIMS Muscularis propria macrophages lie close to cells that regulate gastrointestinal motor function, including interstitial cells of Cajal (ICC) and myenteric neurons. In animal models of diabetic gastroparesis, development of delayed gastric emptying has been associated with loss of macrophages that express cytoprotective markers and reduced networks of ICC. Mice with long-term diabetes and normal gastric emptying have macrophages that express anti-inflammatory markers and have normal gastric ICC. Mice homozygous for the osteopetrosis spontaneous mutation in the colony-stimulating factor 1 gene (Csf1op/op) do not have macrophages; when they are given streptozotocin to induce diabetes, they do not develop delayed gastric emptying. We investigated whether population of the gastric muscularis propria of diabetic Csf1op/op mice with macrophages is necessary to change gastric emptying, ICC, and myenteric neurons and investigated the macrophage-derived factors that determine whether diabetic mice do or do not develop delayed gastric emptying. METHODS Wild-type and Csf1op/op mice were given streptozotocin to induce diabetes. Some Csf1op/op mice were given daily intraperitoneal injections of CSF1 for 7 weeks; gastric tissues were collected and cellular distributions were analyzed by immunohistochemistry. CD45+, CD11b+, F4/80+ macrophages were dissociated from gastric muscularis propria, isolated by flow cytometry and analyzed by quantitative real-time polymerase chain reaction. Cultured gastric muscularis propria from Csf1op/op mice was exposed to medium that was conditioned by culture with bone marrow-derived macrophages from wild-type mice. RESULTS Gastric muscularis propria from Csf1op/op mice given CSF1 contained macrophages; 11 of 15 diabetic mice given CSF1 developed delayed gastric emptying and had damaged ICC. In non-diabetic Csf1op/op mice, administration of CSF1 reduced numbers of gastric myenteric neurons but did not affect the proportion of nitrergic neurons or ICC. In diabetic Csf1op/op mice given CSF1 that developed delayed gastric emptying, the proportion of nitrergic neurons was the same as in non-diabetic wild-type controls. Medium conditioned by macrophages previously exposed to oxidative injury caused damage to ICC in cultured gastric muscularis propria from Csf1op/op mice; neutralizing antibodies against IL6R or TNF prevented this damage to ICC. CD45+, CD11b+, and F4/80+ macrophages isolated from diabetic wild-type mice with delayed gastric emptying expressed higher levels of messenger RNAs encoding inflammatory markers (IL6 and inducible nitric oxide synthase) and lower levels of messenger RNAs encoding markers of anti-inflammatory cells (heme oxygenase 1, arginase 1, and FIZZ1) than macrophages isolated from diabetic mice with normal gastric emptying. CONCLUSIONS In studies of Csf1op/op and wild-type mice with diabetes, we found delayed gastric emptying to be associated with increased production of inflammatory factors, and reduced production of anti-inflammatory factors, by macrophages, leading to loss of ICC.

385 Conditional Genomic Deletion of Ano1 in Kit-Expressing Cells of Adult Mice Results in Loss of Slow Waves and Reduced Coordination of Ca2+ Transients in Myenteric Interstitial Cells of Cajal of the Small Intestine

In the enteric nervous system (ENS), glia outnumber neurons by 4 to 6-fold and form an extensive network throughout the gastrointestinal tract. Enteric glia are essential for normal gastrointestinal function and play roles in regulating epithelial barrier integrity, epithelial cell proliferation and neuronal support. While glial subtypes can be clearly distinguished in the central and peripheral nervous systems (CNS and PNS), it remains unknown whether similar glial diversity exists in the ENS. Because of their morphology and expression of Glial Fibrillary Acidic Protein (GFAP), until recently, enteric glia were thought to resemble astrocytes. We tested the hypothesis that enteric glia instead constitute a unique and heterogeneous group of glial cells. To define the level of heterogeneity, we first analyzed expression of the markers S100β, Sox10, GFAP, and proteolipid protein 1 (PLP1) in the small and large intestine of adult mice. Sox10 and S100β are widely expressed by enteric glia throughout the intestine. GFAP expression, however, is more restricted. Marker expression in combination with cellular location reproducibly distinguished subpopulations of enteric glia, suggesting that functional subtypes are likely to exist. Unexpectedly, we found that PLP1 is widely expressed by enteric glia, although they do not myelinate axons. We then performed RNA sequencing analysis (RNA-Seq) on PLP1-expressing cells in the mouse intestine and compared their gene expression to that of other types of glia in the CNS and PNS. This showed that enteric glia are transcriptionally distinct from other glial classes, and share the greatest similarity to myelinating glia. The gene expression database generated by this study will facilitate future studies of glial function in gastrointestinal physiology.

Su2029 Medium Conditioned With Conventionally-Activated M1 Macrophages Inhibits Survival of Mouse Interstitial Cells of Cajal in Primary Culture

Background: Application of the surfactant benzylkonium chloride (BAC) to the serosal surface of the intestine causes ablative neurodegeneration within two days. Modest neurogenesis within 4-8 weeks of ablation has been observed in this model with neurons derived from glial precursors. We hypothesized that non-ablative concentrations of BAC would spare potential neural precursors and allow a more robust neuroregeneration. Aims: To determine whether low concentration BAC causes neurodegeneration and subsequent neurogenesis and whether markers of neural progenitors are expressed following BAC treatment. Methods: In C57Bl/6 mice, following laparotomy, a 1x0.2cm piece of absorbent paper soaked in 0.0001% BAC was applied to the serosal surface of the jejunum for 5 min. Following various periods of recovery, fixed segments of the external muscle layers were immunostained for HuC/D (mature enteric neurons), S100β (enteric glia), doublecortin (DCX; immature neurons), and/or SOX2 (neural pluripotency transcription factor). Immunoreactive (IR) cells were enumerated as the number of cells per ganglion. In some mice, EdU (100mg/kg IP) was administered daily between 12-16d post-BAC before animals were euthanized 21d postBAC. Results: In mice two days after BAC treatment there was a significant reduction in the number of HuC/D-IR cells per ganglion in the region of treatment (27 ± 8) compared to an orally-adjacent region (61 ± 7; P<0.05). In mice 21 days after BAC treatment, the number of neurons per ganglion in the treated region (45 ± 5) were no longer significantly less than the orally-adjacent region (61 ± 6; P<0.05). There was no evidence for EdU incorporation in HuC/D-IR cells. Although DCX-IR was detected in the rostral migratory stream of the CNS, DCX-IR was rarely detected in the myenteric plexus. SOX2-IR was increased in HuC/ D-IR neurons at 21d post-BAC. SOX2-IR was detected in S100β-IR cells as early as 2d postBAC and S100β-IR was detected in a proportion of HuC/D-IR cells in the BAC-treated region between 2-14d post-BAC. Conclusion: Low concentration BAC caused moderate neurodegeneration in the myenteric plexus of the mouse jejunum. The number of myenteric neurons increased by 21d post-BAC. The pluripotency transcription factor SOX2, which in the CNS is capable of reprogramming astrocytes to mature neurons, is expressed in enteric glial cells early following BAC treatment and in a proportion of mature neurons by 21d post-BAC. These data support the concept of neuroregeneration of the enteric nervous system, and suggest that SOX2 expression in enteric glia may drive adult neurogenesis without cell proliferation.