Anion exchanger 2 (AE2), the principal bicarbonate secretor in the human biliary tree, is down‐regulated in primary biliary cholangitis. AE2 creates a “bicarbonate umbrella” that protects cholangiocytes from the proapoptotic effects of bile salts by maintaining them deprotonated. We observed that knockdown of AE2 sensitized immortalized H69 human cholangiocytes to not only bile salt‐induced apoptosis (BSIA) but also etoposide‐induced apoptosis. Because the toxicity of etoposide is pH‐independent, there could be a more general mechanism for sensitization of AE2‐depleted cholangiocytes to apoptotic stimuli. We found that AE2 deficiency led to intracellular bicarbonate accumulation and increased expression and activity of soluble adenylyl cyclase (sAC), an evolutionarily conserved bicarbonate sensor. Thus, we hypothesized that sAC regulates BSIA. H69 cholangiocytes and primary mouse cholangiocytes were used as models. The sAC‐specific inhibitor KH7 not only reversed sensitization to BSIA in AE2‐depleted H69 cholangiocytes but even completely prevented BSIA. sAC knockdown by tetracycline‐inducible short hairpin RNA also prevented BSIA. In addition, sAC inhibition reversed BSIA membrane blebbing, nuclear condensation, and DNA fragmentation. Furthermore, sAC inhibition also prevented BSIA in primary mouse cholangiocytes. Mechanistically, sAC inhibition prevented Bax phosphorylation at Thr167 and mitochondrial translocation of Bax and cytochrome c release but not c‐Jun N‐terminal kinase activation during BSIA. Finally, BSIA in H69 cholangiocytes was inhibited by intracellular Ca2+ chelation, aggravated by thapsigargin, and unaffected by removal of extracellular calcium. Conclusions: BSIA is regulated by sAC, depends on intracellular Ca2+ stores, and is mediated by the intrinsic apoptotic pathway; down‐regulation of AE2 in primary biliary cholangitis sensitizes cholangiocytes to apoptotic insults by activating sAC, which may play a crucial role in disease pathogenesis. (Hepatology 2016;64:522‐534)

Background & Aims To better understand the pathogenesis of primary sclerosing cholangitis, anti‐ and pro‐inflammatory factors were studied in bile. Methods Ductal bile of PSC patients (n = 36) and controls (n = 20) was collected by endoscopic retrograde cholangiography. Gallbladder bile was collected at liver transplantation. Bile samples were analysed for cytokines, FGF19 and biliary lipids. Hepatobiliary tissues of PSC and non‐PSC patients (n = 8–11 per patient group) were collected at transplantation and were analysed for IL8 and FGF19 mRNA expression and IL8 localization. The effect of IL8 on proliferation of primary human cholangiocytes and expression of pro‐fibrotic genes was studied. Results In PSC patients, median IL8 in ductal bile was 6.6 ng/ml vs. 0.24 ng/ml in controls. Median IL8 in gallbladder bile was 7.6 ng/ml in PSC vs. 2.2 and 0.3 ng/ml in two control groups. IL8 mRNA in PSC gallbladder was increased and bile ducts stained positive for IL8. In vitro, IL8 induced proliferation of primary human cholangiocytes and increased the expression of pro‐fibrotic genes. Conclusion Elevation of IL8 in bile of PSC patients, collected at different stages of disease, indicates an ongoing inflammatory stimulus that drives IL8 production. This challenges the idea that advanced PSC is a burned‐out disease, and calls for reconsideration of anti‐inflammatory therapy in PSC.

The evolutionarily conserved soluble adenylyl cyclase (sAC, adcy10) was recently identified as a unique source of cAMP in the cytoplasm and the nucleus. Its activity is regulated by bicarbonate and fine-tuned by calcium. As such, and in conjunction with carbonic anhydrase (CA), sAC constitutes an HCO−3/CO−2/pH sensor. In both alpha-intercalated cells of the collecting duct and the clear cells of the epididymis, sAC is expressed at significant level and involved in pH homeostasis via apical recruitment of vacuolar H+-ATPase (VHA) in a PKA-dependent manner. In addition to maintenance of pH homeostasis, sAC is also involved in metabolic regulation such as coupling of Krebs cycle to oxidative phosphorylation via bicarbonate/CO2 sensing. Additionally, sAC also regulates CFTR channel and plays an important role in regulation of barrier function and apoptosis. These observations suggest that sAC, via bicarbonate-sensing, plays an important role in maintaining homeostatic status of cells against fluctuations in their microenvironment.

The in vitro generation of terminally differentiated hepatocytes is an unmet need. We investigated the contribution of oxygen concentration to differentiation in human liver cell lines HepaRG and C3A. HepaRG cells were cultured under hypoxia (5%O2), normoxia (21%O2) or hyperoxia (40%O2). Cultures were analysed for hepatic functions, gene transcript levels, and protein expression of albumin, hepatic transcription factor CEBPα, hepatic progenitor marker SOX9, and hypoxia inducible factor (HIF)1α. C3A cells were analysed after exposure to normoxia or hyperoxia. In hyperoxic HepaRG cultures, urea cycle activity, bile acid synthesis, CytochromeP450 3A4 (CYP3A4) activity and ammonia elimination were 165–266% increased. These effects were reproduced in C3A cells. Whole transcriptome analysis of HepaRG cells revealed that 240 (of 23.223) probes were differentially expressed under hyperoxia, with an overrepresentation of genes involved in hepatic differentiation, metabolism and extracellular signalling. Under hypoxia, CYP3A4 activity and ammonia elimination were inhibited almost completely and 5/5 tested hepatic genes and 2/3 tested hepatic transcription factor genes were downregulated. Protein expression of SOX9 and HIF1α was strongly positive in hypoxic cultures, variable in normoxic cultures and predominantly negative in hyperoxic cultures. Conversely, albumin and CEBPα expression were highest in hyperoxic cultures. HepaRG cells that were serially passaged under hypoxia maintained their capacity to differentiate under normoxia, in contrast to cells passaged under normoxia. Hyperoxia increases hepatocyte differentiation in HepaRG and C3A cells. In contrast, hypoxia maintains stem cell characteristics and inhibits hepatic differentiation of HepaRG cells, possibly through the activity of HIF1α.Electronic supplementary materialThe online version of this article (10.1007/s12079-018-0456-4) contains supplementary material, which is available to authorized users.

Following high summer temperatures in Taiwan, erratic fruit production and yellowed cladode have been observed in red-fleshed ‘Da Hong’ pitaya (Hylocereus polyrhizus). However, the specific environmental influences that result in the yield loss and cladode damage are unknown. The aim of this study was to evaluate how high temperature affects fruit production and cladode yellowing of ‘Da Hong’ pitaya under controlled conditions. One-year-old field-grown potted plants with moderately yellow-colored cladodes were placed in the phytotron at either 40/30 °C ± 1 °C [day/night, high-temperature treatment (HT)] or 30/20 °C ± 1 °C [day/night, control (CK)] during the natural long-day reproductive period. Floral bud development duration, flower opening behavior, fruit set and development, as well as fruit characteristics and seed setting (which was expressed as the estimated number of seeds), and the weight per fruit at harvest were investigated. In addition, the percentage of dry matter and color change (regreening) of cladodes were examined. We found that floral bud development was completed 8 days earlier than the control, but the time of blooming was 2 to 3 hours later within a day; and fruit set, fruit size, seed weight, and peel color were strongly suppressed in HT-treated plants compared with the control. Furthermore, both the estimated seed number and seed weight were positively correlated with fruit weight, suggesting that reduced seed setting and weight arising from incomplete fertilization in the HT plants may have resulted in fruit drop and smaller fruit. Although the color on the sunny (sun-exposed) side of the cladode remained yellow, the percentage of dry matter in the HT cladodes was not significantly different from the control, indicating that the yellow-colored cladodes did not reduce their carbon supply potential. The results indicate that HT during bloom led to poorer fruit set and lower fruit weight, presumably due to lower seed setting/weight per fruit arising from incomplete fertilization. The HT treatment also caused less regreening of cladodes, but this did not seem to impact fruit production. Further study is required to ascertain whether disrupted stamens or pistils resulting from HT treatment lead to incomplete fertilization.

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