bluuebunny/crossref_metadata_2025 huggingface dataset:
CD133 expression is modulated at a post‐translational level during sodium butyrate‐induced differentiation of HT29 human colon cancer cells
Authors: Sgambato Alessandro , Errico Federica , Puglisi Maria Ausiliatrice , Corbi Maddalena , Boninsegna Alma , Rettino Alessandro , Genovese Giannicola , Coco Claudio , Gasbarrini Antonio , Cittadini Achille
Published: June 2021

Human CD133 is a cell surface glycoprotein whose expression has been shown to characterize normal as well as cancer stem cells in several tissues. Despite the enormous interest for this molecule, its functions and ligands and its involvement in essential properties of stem cells such as self‐renewal and differentiation remain unknown. In the present study, expression of the CD133 protein was analyzed during the sodium butyrate‐induced differentiation of the HT29 colon cancer cell line. Treatment induced a progressive decrease of CD133 expression, as assessed by flow cytometry using the AC133 monoclonal antibody, in a time‐ and dose‐dependent fashion. No relationship was observed between CD133 protein evaluated by flow cytometry and mRNA expression level and no changes were detected in the methylation status of the CD133 gene promoter. Moreover, the expression of the CD133 protein, evaluated by western blot analysis using an antibody directed against the C‐terminal intracytoplasmic region of human CD133 protein, did not correlate with flow cytometry results. Different results were also obtained using the two antibodies to analyze the expression of the CD133 molecule in human colon cancers. These findings demonstrate that CD133 expression might undergo a complex regulation during differentiation of colon cells which likely occurs at a post‐transcriptional level.
WNK4 inhibits NCC protein expression through MAPK ERK1/2 signaling pathway
Authors: Zhou Bo , Zhang Yiqian , Wang Dexuan , Zhuang Jieqiu , Zhang Xuemei , Gu Dingying , Cai Hui
Published: June 2021

WNK ((with no lysine (k)) kinase is a subfamily of serine/threonine kinases. Mutations in two members of this kinase family (WNK1 and WNK4) cause pseudohypoaldosteronism type II featuring hypertension, hyperkalemia and metabolic acidosis. WNK1 and WNK4 were shown to modulate NCC activity through SPAK and OSR1. Activation of PKC by a phorbol ester inhibits NCC function via activation of ERK1/2 kinase. To investigate whether WNK4 affects NCC via MAPK signaling pathway besides SPAK and OSR1, we first determine whether WNK4 phosphorylates ERK1/2 kinase. We found that WNK4 increases the phosphorylation of ERK1/2 in a dose‐dependent manner in mouse distal convoluted tubule (mDCT) cells, whereas WNK4 mutants with the PHA II mutations (E562K and R1185C) do not increase the ERK1/2 phosphorylation. We next showed that hypertonic stimulation significantly increased ERK1/2 phosphorylation in mDCT cells. The ERK1/2 activation by hypertonic stimulation was significantly attenuated by WNK4 siRNA knock‐down expression (23.19 ± 8.21 % in siWNK4 group vs 100 % in control group, p <0.01, n=3). We further showed that WNK4 knock‐down significantly increases total NCC expression (100 ± 0 % vs 120.7 ± 6.2 %, p <0.05, n=4) as well as NCC surface expression (100 ± 0 % vs 256.8 ± 50.3 %, p <0.05, n=4). We conclude that the inhibitory effect of WNK4 on NCC is mediated by MAPK ERK 1/2 signaling pathway.

Note: BZ and YZ contributed equally to this work.
Dopamine inhibits the Na+/H+ Exchanger NHE3 via Protein Phosphatase 2A
Authors: Bobulescu Ion Alexandru , Quiñones Henry , Gisler Serge M , Di Sole Francesca , Shi Mingjun , Hu Ming‐Chang , Zhang Jianning , Fuster Daniel , Mumby Marc , Moe Orson
Published: June 2021

Nephrogenic dopamine is a potent natriuretic paracrine/autocrine hormone that is central for mammalian sodium homeostasis. In the renal proximal tubule, dopamine induces natriuresis partly by inhibiting the sodium/proton exchanger NHE3. The signaling pathways involved in this regulation are incompletely understood. We describe the role of the serine/threonine protein phosphatase 2A (PP2A) in the regulation of NHE3 by dopamine. The PP2A regulatory subunit B56 delta isoform directly associates with the carboxy‐terminal hydrophilic putative cytoplasmic domain of NHE3 (NHE3‐cyto), as demonstrated by yeast‐two‐hybrid, co‐immunoprecipitation, blot overlay and in vitro pull‐down assays. This interaction involves more than one region of NHE3‐cyto. NHE3‐cyto is a substrate for purified PP2A in an in vitro dephosphorylation reaction. In cultured renal cells, inhibition of PP2A by either okadaic acid or by overexpression of the simian virus 40 (SV40) small t antigen blocks the ability of dopamine to inhibit NHE3 activity and to reduce surface NHE3 protein. Dopamine‐induced NHE3 internalization is also blocked by okadaic acid ex vivo in rat kidney cortical slices. These studies demonstrate that PP2A is an integral and critical part of the signal transduction pathway between dopamine receptor activation and NHE3 inhibition.
Alternative Splicing of the murine Tmem16a Transcript in Heart
Authors: O'Driscoll Kate Eileen , Hatton William J , Britton Fiona C
Published: June 2021

TMEM16A and TMEM16B, members of a new family of proteins, have been shown to function as calcium‐activated chloride channels (ClCa). Alternative exons were identified in the human TMEM16A transcript, which confer differences in the voltage‐ and calcium‐dependence of the channel. Calcium‐activated chloride currents (IClCa) are widely distributed in cardiac tissues and play important roles in cardiac excitability, yet the gene(s) encoding cardiac ClCa is unknown. We investigated the expression of Tmem16 paralogs in mouse heart and demonstrate that several family members, including Tmem16a, but not Tmem16b are expressed. We hypothesize that Tmem16a in its role as a ClCa, may contribute to this conductance in cardiac tissues. Quantitative RT‐PCR revealed that Tmem16a is more abundant in atria than ventricle. Three alternative exons of 66 bp, 12 bp and 78 bp were identified in the mouse Tmem16a transcript, which encode for additional channel regions. We performed exon‐specific RT‐PCR to identify the inclusion or exclusion of alternative exons in Tmem16a. Multiple Tmem16a channel isoforms consisting of various combinations of alternatively spliced exons were identified in atria and ventricle. The presence of Tmem16a variants and their different regional expression in heart raises the possibility that molecular diversity of cardiac ClCa may be generated by alternative Tmem16a channel structures.
Vasomotion in rat small mesenteric arteries is bestrophin‐3 dependent.
Authors: Aalkjær Christian , Brøgger Torbjørn , Bødtkjer Donna Briggs , Matchkov Vladimir
Published: June 2021

We have hypothesized the importance of a cGMP‐dependent Ca‐activated Cl current for synchronization of smooth muscle cells (SMCs) in the vascular wall and have shown that cGMP‐dependent Ca‐activated Cl current dependens on bestrophin‐3 (Best‐3) expression.

To study the role of Best‐3 for vascular tone, we transfected rat small mesenteric arteries in vivo with siRNA against Best‐3 and with non‐functional mutated Best‐3 siRNA, as a control. The arteries were tested 3 days after in vitro for isometric force development and for [Ca2+]i.

Best‐3 protein expression was downregulated with specific siRNA by 65.0±5.8 % versus control arteries transfected with mutated siRNA. The transfection did not affect either maximal contractile response or sensitivity to noradrenalin (NA). Relaxation to acetylcholine was not affected either. The amplitude of NA‐induced vasomotion in arteries downregulated for Best‐3 was significantly lower than in the controls, while frequency was not different. The downregulation of Best‐3 partly desynchronized the global [Ca2+]i oscillations in SMCs and asynchronous Ca waves appeared.

Our study demonstrates the importance of Best‐3 for synchronization of SMCs and, thus, generation of vasomotion.
Mineralocorticoids stimulate the activity and expression of renal H,K‐ATPases
Authors: Greenlee Megan Michelle , Lynch I. Jeanette , Gumz Michelle L. , Cain Brian D. , Wingo Charles S.
Published: June 2021

Mineralocorticoids such as deoxycorticosterone activate Na+ reabsorption and H+ secretion in the renal collecting duct. In this study, the effect of deoxycorticosterone pivalate (DOCP) on renal H+,K+‐ATPase activity and expression was investigated. Eight days after female wild type (WT) mice received DOCP, collecting ducts were perfused in vitro to measure H+,K+‐ATPase‐dependent H+ secretion after an acute intracellular acid load. DOCP stimulated H+,K+‐ATPase mediated H+ secretion in A type intercalated cells of inner cortical collecting ducts by 70% whereas no effect of DOCP was observed in B type intercalated cells compared to control. DOCP dramatically increased HKα2 mRNA expression in outer and inner medulla (246% and 1040%, respectively) and did not affect HKα1 mRNA expression. DOCP treatment caused significant body weight gain in WT mice and had little effect on body weight in mice lacking both α subunits (HKα1,2−/−). DOCP caused a similar degree of hypokalemia in WT and HKα1,2−/− mice. Importantly, WT developed metabolic alkalosis whereas HKα1,2−/− mice did not. In summary, these data clearly demonstrate a physiological role for renal H+,K+‐ATPases in mineralocorticoid stimulated H+ secretion and suggest a role for these enzymes in Na+ reabsorption.

Studies were funded by NIH RO1‐DK‐049750 to C.S.W and 5T32DK007518‐22 to M.M.G.
Vasopressin‐mediated signaling in the distal convoluted tubule regulates phosphorylation of the thiazide‐sensitive Na+‐Cl− cotransporter (NCC) at two conserved threonine residues
Authors: Pedersen Nis Borbye , Hofmeister Marlene Vind , Nielsen Jakob , Fenton Robert Andrew
Published: June 2021

The Na+‐Cl− cotransporter (NCC) is important for renal electrolyte balance. Phosphorylation of NCC has been proposed to play a major role in its transport activity and cellular localization. Using recently generated phospho‐specific antibodies against two conserved N‐terminal phosphorylation sites (Thr53, Thr58 and Thr53/Thr58) we assessed the role of vasopressin (AVP) in regulating NCC in vivo. In AVP‐deficient Brattleboro rats, acute dDAVP exposure for 15 min significantly increased pNCC abundance at the apical plasma membrane approximately 300%. Acute dDAVP did not alter the abundance of the STE20/SPS1‐related proline/alanine‐rich kinase (SPAK) previously implicated in NCC phosphorylation. Early and late DCTs increased their intracellular Ca2+ levels in response to 1 min of superfusion of dDAVP (2 × 10−10 M), confirming that the DCT is an AVP responsive segment. In rats fed a high salt diet alongside AT1‐R blockade by candesartan, similar increases in pNCC abundance were observed following chronic or acute dDAVP. Candesartan treatment reduced the abundances of total SPAK and pSPAK, suggesting that this kinase can be regulated via the AT1‐R in vivo. Taken together our results indicate that AVP is a potent regulator of NCC activity.

Funding is provided by the Danish Medical Research Council, the Novo Nordisk Fond and the Carlsberg Foundation.
Betaine Supplementation Improved Adipose Tissue Function in Mice Fed High‐Fat Diet: A Mechanism for Hepatoprotective Effect of Betaine in Non‐alcoholic Fatty Liver Disease
Authors: Wang Zhigang , Song Zhenyuan
Published: June 2021

Adipose tissue dysfunction plays a central role in the progression of nonalcoholic fatty liver disease (NAFLD). Promising beneficial effects of betainesupplementation on NAFLD have been reported in bothclinical investigations and experimental studies; however, underlying mechanisms are still elusive. In this study, we examined the effects ofbetaine supplementation on hepatic fat accumulation and injury in mice fed high‐fatdiet and evaluated mechanisms underlying its hepatoprotective effects. Male C57BL/6 mice were divided into four groups and started on one of fourtreatments: control diet (Con), control diet supplemented with betaine(BT), high‐fat diet (HF), and high‐fat diet supplemented with betaine (HB). Betaine was supplemented in the drinking water (1%: wt/vol). Our results showed betaine alleviated hepatic steatosis and liver injury in this animal model, which were associated with attenuation of insulin resistance (GTT) and recoveries of abnormal adiponectin, leptin, and resistin productions. Further investigation revealed that alleviation of endoplasmic reticulum (ER) stress response (manifested by increased GRP78/CHOP protein abundance and JNK activation) in adipose tissue may play a mechanistic role in betaine's hepatoprotective effects in NAFLD. Our findings suggest that betaine might serve as a safe and efficacious therapeutic tool for NAFLD by improving adipose tissue function.
Rosiglitazone improves characteristics of Non‐Alcoholic Steatohepatitis (NASH) in a mouse model of metabolic syndrome
Authors: Gupte Anisha A. , Liu Joey Z. , Minze Laurie J. , Ren Yuelan , Wiles Jessica R. , Collins Alan R. , Lyon Christopher J. , Hsueh Willa A.
Published: June 2021

NASH, which is closely associated with metabolic syndrome (MS), is the most common cause of chronic liver disease in the Western world, but has no approved treatment. Middle‐aged (MA, 12‐month) male LDLR−/− mice fed 3 months of high‐fat diet (HFD) develop MS (obesity, elevated fasting glucose and triglycerides, and decreased HDL), and reveal a classical NASH phenotype of liver steatosis, inflammation and fibrosis. We hypothesized that treatment with the insulin‐sensitizer rosiglitazone (RSG) could prevent NASH by attenuating MS development and age‐related oxidative stress. RSG treatment dramatically reduced, but did not completely prevent, steatosis in HFD‐fed MA LDLR−/− mice. RSG reversed HFD‐mediated decreases in liver fatty acid oxidation genes (CPT1, ACADm, HADHa), and attenuated HFD‐induced decreases in antioxidant genes (DJ‐1, Nrf2, NQO1, SOD2, GPX‐1 and ‐4). But RSG had no effect on HFD‐generated increases in fibrosis genes (Coll1α2, Coll4α1 and TGF‐β1) or inflammation genes (MCP‐1, TNFα, CD68). RSG‐treatment of palmitate‐treated primary hepatocytes showed that RSG increased expression of both fatty acid oxidation and synthesis genes. RSG induced β‐oxidation in liver mitochondria, as seen by increased respiratory control ratios and ADP to ATP conversion with palmitoyl‐carnitine. RSG may thus prevent steatosis, at least partially, through direct effects to promote hepatocyte β‐oxidation.
Casein kinase 2 (CK2) phosphorylates occludin at Ser408 to increase intra‐tight junction diffusion and reduce paracellular barrier function
Authors: Raleigh David Ronan , Marchiando Amanda , Boe Devin , Wang Yingmin , Shen Le , Turner Jerrold R
Published: June 2021

CK2 phosphorylates the carboxy‐terminal cytoplasmic tail of the tight junction protein occludin. We sought to determine the consequences of CK2‐mediated occludin phosphorylation and identify critical target residues(s). The CK2 inhibitors TBB and DMAT each elevated transepithelial resistance (TER) of Caco‐2 monolayers. This regulation was CK2‐ and occludin‐dependent, as it did not occur after siRNA knockdown (KD) of either protein. To assess the functional relevance of previously identified CK2 targets Thr404 and Ser408, wild type or T408D/S408D occludin was expressed in occludin KD cell lines. Both constructs restored TER, which was reduced by occludin KD, but only wild type occludin re‐established the TER response to CK2 inhibition. Fluorescence recovery after photobleaching (FRAP) showed a reduced occludin mobile fraction after either chemical‐ or siRNA‐mediated CK2 inhibition, which was associated with increased occludin density at the tight junction. DMAT similarly reduced occludin FRAP in vivo. In vitro, T404A, T404D, and S408D occludin mutants had FRAP behavior indistinguishable from wild type occludin, but S408D was unaffected by CK2 inhibition. Conversely, S408A occludin FRAP at the tight junction was reduced, relative to wild type, and was unaffected by CK2 inhibition. Thus, CK2‐mediated phosphorylation of occludin S408 accelerates tight junction remodeling and increases permeability.
Morphometrical analysis of ultrastructural transformation in gastric parietal cells reverting from the active to the resting state processed by high‐pressure freezing.
Authors: Sawaguchi Akira , Toyoshima Fumiyo
Published: June 2021

To elucidate a functional transformation of gastric parietal cells, we have newly developed an isolated rat gastric mucosa model whose parietal cells exhibited a reverting process from the active to resting state of acid secretion. Briefly, the parietal cells were treated with H2‐receptor blocker following prior stimulation of acid secretion in the model, and cryofixed by high‐pressure freezing for electron microscopy. As a result, immunohistochemistry of H+/K+‐ATPase demonstrated a progressive translocation of H+/K+‐ATPase from the apical to the cytoplasmic region. The ultrastructure of parietal cells at 5 min in the reverting phase was quite similar to that of maximally stimulated one. The apical membranes were subsequently internalized into the cytoplasm forming unique penta‐laminar membranes. Interestingly, at 90 min in the reverting phase, the penta‐laminar membranes formed a number of multilamellar autophagosomes that were intensely labeled for H+/K+‐ATPase. Then, the parietal cells exhibited well‐developed Golgi apparatus and lysosomal compartments, and mostly reverted to their resting conformation at 120 min in the reverting phase. Morphometrical analysis indicated a remarkable decrease in the apical membrane/tubulovesicular membrane ratio and increase in the number of both lysosomes and multivesicular bodies as the reverting process.
Sonic Hedgehog signaling mediates bone marrow‐derived mesenchymal stem cell proliferation and recruitment
Authors: Donnelly Jessica M , Chawla Ambreesh , Houghton JeanMarie , Zavros Yana
Published: June 2021

Objective

Bone marrow‐derived mesenchymal stem cells (MSCs) are recruited to the site of chronic inflammation during gastric cancer progression. Given that increased interferon‐gamma (IFNγ) and Sonic Hedgehog (Shh) expression contribute to cancer development, the role of the Shh signaling pathway as a mediator of IFNγ induced proliferation and recruitment of MSCs was studied.

Methods

MSCs transduced with lentiviral particles expressing shRNA for Shh (MSCsShhKO) or empty vector (MSCsVect) were analyzed for cell cycle changes by flow cytometry. In vivo, MSCs tagged with RFP were transferred (via tail vein injection) into C57BL/6 mice and treated with either PBS or IFNγ (1μg/mouse/day) for 14 days. To identify the mechanism of recruitment, chemokine SDF‐1α was used in a chemotaxis assay with MSCsShhKO and MSCsVect.

Results

IFNγ significantly increased MSC proliferation, a response mediated by the secretion of Shh from cultured cells and blocked by Hedgehog signaling inhibitor cyclopamine. MSCsShhKO were arrested in G0/G1 phase compared to MSCsVect. Mice injected with IFNγ showed significant recruitment of RFP‐tagged MSCs to the gastric mucosa. Compared to MSCsVect, MSCsShhKO failed to migrate toward the SDF‐1α reflected by decreased CXCR4 expression.

Conclusion

IFNγ induces proliferation and initiates recruitment of MSCs, a mechanism mediated by Shh signaling.

(Supported by start‐up funds, Zavros)
Edema‐induced alterations in the regulation of myosin light chain phosphorylation in intestinal smooth muscle.
Authors: Uray Karen , Shah Shinil , Cox Charles S
Published: June 2021

We have shown that edema decreases intestinal contractile activity via decreased intestinal smooth muscle myosin light chain (MLC) phosphorylation. The objective of this study was to determine the mechanism by which edema decreases MLC phosphorylation. Thus, regulation of MLC phosphorylation was studied in a rodent model of intestinal edema. Edema was induced by a combination of resuscitative fluid administration and induction of mesenteric venous hypertension. Edema‐induced changes in MLC kinase (MLCK) activity and phosphorylation of MYPT1 subunit of MLC phosphatase (MLCP) were measured in intestinal smooth muscle. Phosphorylation of the MYPT1 subunit in the presence of rho kinase (ROK), zipper interacting protein kinase (ZIPK), and p21 activated kinase (PAK) inhibitors, Y‐27632, SM‐1 peptide, and IPA‐3 respectively, was also measured. No significant changes in MLCK activity were observed in the edematous intestinal smooth muscle. In contrast, phosphorylation of the MYPT1 subunit of MLCP was significantly decreased in edematous intestinal smooth muscle compared to controls. Data suggest that both ROK and ZIPK were involved in edema‐induced decreases in MYPT1 phosphorylation. We conclude from these data that edema‐induced decreases in the inhibitory phosphorylation of MYPT1 subunit of MLCP via regulation of ROK and ZIPK result in decreased MLC phosphorylation in edematous intestinal smooth muscle.
Minihepcidins: Small Peptides Involved in Disulfide Exchange with Ferroportin Act as Agonists
Authors: Preza Gloria Cuevas , Ruchala Piotr , Nemeth Elizabeta , Ganz Tomas
Published: June 2021

The hepcidin‐ferroportin interaction is the central regulatory event in systemic iron homeostasis. To facilitate the understanding of hepcidin structure and to aid the development of useful hepcidin agonists we searched for the minimal structure that would display hepcidin agonist activity. The bioactivity of candidate peptides was assayed by flow cytometry measuring the degradation of GFP‐ferroportin in a cellular model. We previously showed that the five N‐terminal residues, DTHFP, were essential but not sufficient for bioactivity. Our ferroportin studies identified C326 cysteine thiol as an essential feature of the binding site for hepcidin suggesting that disulfide exchange may be necessary for hepcidin agonist activity. We therefore synthesized a series of minihepcidins that contained the N‐terminus but also included the first or second cysteine in a thiol form. These peptides exerted substantial bioactivity. Substitution of the cysteine with isosteric serine not capable of disulfide exchange ablated minihepcidin activity, as did blocking of the cysteine thiol. Substitution with unnatural amino acids capable of disulfide exchange retained activity and confirmed the importance of ligand‐receptor disulfide formation. Additional substitutions were devised to increase the resistance of hepcidin to proteolysis and its chemical stability. Small hepcidin agonists should provide the benefit of decreased cost of production, favorable pharmacokinetics and possibly oral bioavailability, which should make them useful as therapeutics for iron overload disorders.
Triclosan may alter the composition of the intestinal microbiota and increase the susceptibility to experimental murine colitis
Authors: Pasch Erica , Walton Kristen LW
Published: June 2021

Evidence suggests that alterations in the intestinal microbiota may play a role in the pathophysiology of inflammatory bowel disease. Triclosan, an antibacterial agent found in consumer products, is detectable in plasma and urine from sample human populations. The current study determined whether low doses of triclosan, mimicking environmental exposure, alter the intestinal microbiota and susceptibility to experimental colitis. Mice were given triclosan in the drinking water (10 μg/ml) for 6 days, followed by induction of colitis by 3.5% dextran sulfate sodium (DSS). Fecal bacteria were cultured for quantification of lactobacilli and coliforms. Fecal DNA was analyzed by PCR using bacterial genus‐specific 16S rRNA primers. Colitis was assessed by disease activity index (DAI) score, histological scoring, and western blot for cyclooxygenase‐2 (COX‐2). The day 4 DAI for the DSS + triclosan (DSS+T) group was higher than the DSS treated mice, but there was no difference at day 5 or 6. Triclosan did not alter induction of COX‐2 expression by DSS. Total fecal lactobacilli were greater in the control and triclosan versus DSS and DSS+T groups. Coliforms were increased by DSS and increased the most in DSS+T group. We conclude that low‐dose exposure to triclosan may increase susceptibility to DSS colitis may influence the composition of the intestinal microbiota. Supported by the MWSU College of Liberal Arts and Sciences.
Role of a non‐CFTR chloride channel in intestinal epithelial chloride secretion
Authors: Kazi Mirajul Hoque , Woodward Owen M , Guggino Sandra E , Tse Chung‐Ming
Published: June 2021

The cystic fibrosis transmembrane regulator (CFTR) is the major Cl channel found in the apical membrane of intestinal epithelial cells. Protein kinase A (PKA) phosphorylates and opens CFTR. Exchange protein directly activated by cAMP (Epac) upon binding of cAMP, activates PKA‐independent signaling via small G proteins. We tested the hypothesis that Epac are involved in cAMP‐stimulated Cl secretion. Human intestinal T84 cells and mouse intestine were used for short circuit current (Isc) measurement in response to agonist stimulated Cl secretion. Epac activator, 8‐pCPT‐2′‐O‐Me‐cAMP, elevated [Ca2+]i, activated Rap2 protein and induced Cl secretion in intact and basolateral membrane permeabilized T84 cells and mouse ileum. These effects of 8‐pCPT‐2′‐O‐Me‐cAMP were completely abolished by BAPTA‐AM, but not by H89. In contrast, T84 cells with silenced Epac1 protein had reduced Isc response to forskolin which was completely inhibited by H89 but not by the phospholipase C inhibitor, U73122. The lack of effect of 8‐pCPT‐2′‐O‐Me‐cAMP either to CFTRinhi‐172, glibenclamide or on whole cell patch clamp recording of Cl current in CHO cells transiently expressing the human CFTR, implicates that CFTR channels are not involved. Biophysical characterization of the Epac1‐dependent Cl conductance in Ussing chambers suggests that Epac mediated Cl conductance was hyperpolarization‐activated, inward rectifying, and displays a Cl−>Br−>I− permeability sequence. These results lead us to conclude that the Epac‐Rap‐PLC‐[Ca2+]i signaling pathway is involved in cAMP‐stimulated Cl secretion through non‐CFTR Cl channel.
MK571‐sensitive extracellular ATP release in intestinal epithelia
Authors: Nakano Takanari , Akiba Yasutada , Engel Eli , Guth Paul H , Kaunitz Jonathan D
Published: June 2021

Epithelial ATP release is an essential component of purinergic secretory regulation. Although the mechanism of extracellular ATP release from epithelial cells is unknown, the involvement of ATP channels, such as pannexin hemichannels and the ATP‐binding cassette (ABC) transporters has been hypothesized. We measured extracellular ATP release from confluent Caco‐2 cell monolayers in order to further study this mechanism. Ecto‐ATPase activity of alkaline phosphatase, CD39, 5′‐nucleotidase, and other brush border ecto‐enzymes was inhibited by non‐cytotoxic concentrations of EDTA, enabling the measurement of supernatant ATP using the luciferin‐luciferase assay. In the presence of EDTA, [ATP] increased linearly, consistent with constitutive cellular ATP release. Among the pharmacological reagents reported as inhibitors for ATP release, MK571, a specific multidrug resistant‐associated protein (MRP) inhibitor dose‐dependently inhibited the ATP release, with no inhibition due to stilbene anion transport inhibitors, pannexin inhibitors, or multidrug resistance protein (MDR) 1 substrates. These results suggest MRP‐associated ATP release in intestinal epithelia, a novel mechanism for transmembrane ATP transport.

NIH‐NIDDK R01 DK54221 & R21 DK081199
Isolated Cells of the Sheep Rumen and Omasum Express Maxi‐anion Channels Permeable to Acetate, Propionate and Butyrate
Authors: Stumpff Friederike , Georgi Maria Ifigenia , Martens Holger
Published: June 2021

Large quantities of short chain fatty acids (SCFA) are absorbed by the gut, representing the major source of energy in the ruminant. Both salivary buffering of SCFA in the forestomachs and intraepithelial mechanisms are necessary to prevent clinical complications. A recent study suggests that basolateral efflux of acetate from the rumen of sheep may occur through an anion channel with p(Cl−) ~350 pS and p(acetate−) ~140 pS. Using the patch clamp technique, we investigated the permeability of this channel for other SCFA and demonstrate its expression by cells isolated from the omasum. Whole cell currents were sensitive to DIDS and p‐chloromercuribenzoate with p(Cl−) > p(acetate−) > p(propionate−) > p(butyrate−) > p(gluconate−). Inside‐out data in symmetrical NMDG‐propionate solution showed a conductance of 114 ± 10 pS for propionate−. The size of channel events at positive pipette potential changed with the dominant anion in the bath, confirming the permeability sequence obtained. Data from asymmetric solutions could be fitted with a model assuming independence, with marginal negative interaction. We propose that protons taken up with SCFA are extruded apically via Na+/H+ exchange. While butyrate is mostly metabolized, the anions of acetate‐ and propionate‐ leave basolaterally via the channel described, driven by the potential generated by the Na+‐K+‐ATPase. German Sci. Found. STU 258/4‐1; Schaumann Stiftung
Microbiota and dietary nutrients regulate digestive enzyme activity in the zebrafish intestine
Authors: Semova Ivana , Rawls John Franklin
Published: June 2021

The microbial community residing in the intestine (microbiota) is an important environmental factor regulating nutrient metabolism and energy storage. An improved understanding of the dynamic relationships between host, microbiota and diet could lead to novel therapies for metabolic diseases. The objective of this study is to define the role of the gut microbiota and diet on zebrafish nutrient digestion. We compared in vivo digestive physiology of gnotobiotic zebrafish larvae exposed to different microbial and dietary stimuli, by feeding fluorescent reporters of phospholipase A2 (PED6) and protease activity (EnzChek). Colonization of germ‐free zebrafish at 3 days post‐fertilization (dpf) with a normal microbiota resulted in increased PED6 and EnzChek digestion within 24 hours independent of diet. In vitro assays revealed that gut microbes can contribute directly to PED6 and EnzChek processing. We find that microbial stimulation of PED6 and EnzChek digestion in vivo is not associated with altered feeding behavior, but is dependent on exogenous nutrients by 6dpf. Our results indicate that microbial and nutritional signals interact to stimulate digestive enzyme activity in the zebrafish gut. Future experiments will determine if microbial and nutritional status influences production or release of pancreatic enzymes, and test the role of specific nutrients and bacterial genes. NIH DK073695 and DK081426
The G‐protein coupled receptor GPR40 mediates long chain fatty acid induced cholecystokinin secretion
Authors: Liou Alice Peiyu , Lu Xinping , Zhao Xilin , Pechhold Susanne , Raybould Helen , Wank Steve
Published: June 2021

INTRODUCTION

Long chain fatty acid receptors (LCFARs) have been implicated in the chemosensation of dietary fats, however direct studies on native “I” cells have been difficult due to their rare and isolated distribution. The objective of this study was to determine whether dietary fat‐induced cholecystokinin (CCK) secretion is directly mediated by GPR40 expressed on I cells isolated from duodenal mucosa of CCK‐eGFP BAC transgenic mice by fluorescence activated cell sorting.

METHODS

Isolated CCK‐eGFP cells were evaluated for LCFAR gene expression by Taqman RT‐PCR. GPR40−/− mice were crossed with CCK‐eGFP mice to evaluate the functional relevance of GPR40 on linolenic acid‐stimulated CCK secretion, measured by radioimmunoassay, in isolated CCK‐eGFP cells. Also, plasma CCK secretion in response to olive oil gavage was compared between GPR40+/+ and GPR40−/− mice.

RESULTS

Isolated CCK‐eGFP cells display a 100‐fold greater expression of GPR40 compared to non‐eGFP cells. In vitro, linolenic acid increased CCK secretion by 53% in GPR40+/+ CCK‐eGFP cells compared to no significant secretion in GPR40−/− CCK‐eGFP cells. In vivo, an olive oil gavage significantly increased plasma CCK levels by 5.7‐fold in GPR40+/+ mice compared to an increase of 3.1‐fold in GPR40−/− mice.

CONCLUSION

This study is the first to demonstrate a direct chemosensory role of GPR40 in dietary fat induced CCK secretion.
Effect of chronic alcohol consumption on vitamin B1 (thiamin) uptake and metabolism by rat pancreatic acinar cells
Authors: Veerapazham Thillai Sekar , Said Hamid M
Published: June 2021

Thiamin plays a vital role in normal cellular metabolism and it deficiency leads to impairment in the function of pancreatic acinar cells. Little is known about thiamin uptake and metabolism by pancreatic acinar cells and the effect of chronic alcohol consumption on these events. We addressed these issues using cultured rat pancreatic acinar AR42J cells and primary acinar cells isolated from rat pancreas. The results showed thiamin uptake by pancreatic acinar cells to be via a specific carrier‐mediated process. Both thiamin transporters 1 & 2 (THTR‐1 and THTR‐2) were expressed in pancreatic acinar cells at the protein and mRNA levels. Chronic alcohol feeding for 4 weeks (36% calories) was found to lead to a significant reduction in carrier‐mediated thiamin uptake compared to uptake by cells from pair‐fed controls. This was associated with a marked reduction in mRNA level of THTR‐1 and THTR‐2. Similarly, chronic alcohol feeding lead to a significant reduction in mRNA level of the thiamin metabolizing enzymes thiamin pyrophophokinase, thiamin pyrophosphatase, and thiamin monophosphatase compared to pair‐fed controls. These results show for the first time that thiamin uptake by pancreatic acinar cells is carrier‐mediated. Further, chronic alcohol feeding leads to inhibition in thiamin uptake and metabolism in these cells [Supported by NIH grants DK56061‐6 and AA018071 thiamin intestine and pancreas]
Therapeutic targeting of mitochondrial superoxide in hypertension
Authors: Dikalov Sergey , Bikineyeva Alfiya , Budzyn Klaudia , Nazarewicz Rafal , Lewis William , Harrison David G , Dikalova Anna
Published: June 2021

INTRODUCTION

Superoxide (O2•) has been implicated in the pathogenesis of many human diseases including hypertension, however commonly employed antioxidants have proven ineffective in clinical trials. It is possible that these agents are not adequately delivered to the subcellular sites of O2• production.

HYPOTHESIS

Because the mitochondria are important source of O2•, we postulated that mitochondrial targeting of O2• scavenging would have therapeutic benefit.

RESULTS

In this study, we found that the hormone angiotensin II increased endothelial mitochondrial O2•. Treatment with the mitochondria‐targeted SOD mimetic mitoTEMPO decreased mitochondrial O2•, inhibited the total cellular O2•, reduced cellular NADPH oxidase activity and restored endothelial NO, while a similar dose of non‐targeted TEMPOL was not effective. These effects were mimicked by overexpressing the mitochondrial MnSOD (SOD2), while SOD2 depletion with siRNA increased both basal and angiotensin II‐stimulated cellular O2•. Treatment of mice in vivo with mitoTEMPO after the onset of angiotensin II‐ and DOCA‐salt induced hypertension decreased blood pressure by 30 mm Hg, decreased vascular O2•, increased NO• production and improved endothelium‐dependent relaxation. Transgenic mice overexpressing mitochondrial SOD2 demonstrated attenuated angiotensin II‐induced hypertension and vascular oxidative stress similar to mice treated with mitoTEMPO.

CONCLUSION

These studies show that mitochondrial O2• is important for the development of hypertension and that mitochondria‐targeted SOD mimetic could have therapeutic benefits in this and possibly other diseases.

Supported by AHA and NIH grant No 09GRNT2220128, HL058000 and HL075209.
Mitochondrial Complex I Deficiency is One of the Major Causes of Mitochondrial Oxidative Stess in Zucker Obese Fatty Rat
Authors: Pung Yuh Fen , Hafemeister Jennifer L , Chilian William M
Published: June 2021

Mitochondria dysfunction has been implicated in a wide variety of diseases involving tissue with high energy demand such as the myocardium. We have recently observed an elevated level of cardiac mitochondrial ROS in a rat model with metabolic syndrome (MS), the Zucker obese fatty rat (ZOF). We thus hypothesized that cardiac mitochondrial oxidative stress in ZOF rats is due to mitochondrial dysfunction. Because complex I (CI) is one of the major “sites” of e− leakage, we evaluated its activity. CI activity was ~20% decreased in ZOF as compared to lean rats (n=3). Concomitant with the reduced CI activity, there was also a ~25% reduction in the expression level of NDUFS4 (ZOF<Lean), a 18‐kDa nuclear‐encoded mitochondrial protein, involved in electron transfer and stability of CI assembly. To evaluate if these biochemical meausures of mitochondrial dysfunction were also evident in mitochondrial structure, we studied mitochondrial ultrastructure of ZOF and lean rats using transmission electron microscopy. In ZOF vs Lean, we observed an increase in the numbers of mitochondria per field and larger subsarcolemmal mitochondrial clusters. We speculate that e− leakage from CI might be one of the major causes of mitochondrial oxidative stress in ZOF. Our study will provide insights into the role of CI in relation to mitochondrial oxidative stress, with the ultimate aim to understand heart failure in patients with MS.
Elevated angiotensin II attenuates activation of voltage‐gated sodium channels in heart failure rats: involvement of mitochondria‐derived superoxide
Authors: Zhang Libin , Zheng Hong , Tu Huiyin , Tran Thai Paul , Muelleman Robert L. , Zimmerman Matthew C. , Li Yu‐Long
Published: June 2021

Our previous study has shown that chronic heart failure (CHF) reduces expression and activation of voltage‐gated sodium (Nav) channels in baroreceptor neurons, which are involved in the blunted baroreceptor neuron excitability and contribute to the impairment of baroreflex in CHF state. Here we examined whether angiotensin II (Ang II) mediates CHF‐induced hypoactivation of Nav channels via mitochondria‐derived superoxide. Ang II concentration, expression of angiotensin II type I (AT1) receptors, and superoxide production were higher in nodose neurons from CHF rats than that from sham rats. Activity of manganese superoxide dismutase (MnSOD) was reduced in nodose neurons of CHF rats. Whole cell patch clamp data showed that Nav current density in baroreceptor neurons from CHF rats was lower than that from sham rats. L158,809 (AT1 receptor antagonist, 1 μM) and adenovirus MnSOD gene transfer (1 × 1010 pfu/ml) significantly increased Nav current density in baroreceptor neurons from CHF rats but not from sham rats. These results indicate that endogenous Ang II‐mitochondrial superoxide contributes to the Nav channel dysfunction of the baroreceptor neurons in CHF.
Oral intake of rosiglitazone promotes central antihypertensive effect via upregulation of mitochondrial uncoupling protein and alleviation of oxidative stress in rostral ventrolateral medulla of spontaneously hypertensive rats
Authors: Chan Samuel H.H. , Chang Alice Y.W. , Chan Julie Y.H.
Published: June 2021

Rosiglitazone, a synthetic ligand of transcription factor peroxisome proliferator‐activated receptor‐γ (PPARγ), possess blood pressure‐lowering effect beyond insulin sensitizing. We test the hypothesis that oral intake of rosiglitazone exerts central antihypertensive effect by amelioration of oxidative stress in rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons reside, via transcriptional upregulation of mitochondrial uncoupling protein 2 (UCP2). Oral intake of rosiglitazone resulted in vasodepression in adult spontaneously hypertensive rats (SHR) but not normotensive WKY rats. This antihypertensive effect of rosiglitazone was abrogated by microinjection bilaterally into RVLM of PPARγ small interfering RNA (siRNA). Oral intake of rosiglitazone also upregulated the UCP2 and ameliorated the heightened superoxide level in RVLM of SHR. Protection against oxidative stress in RVLM by rosiglitazone was abrogated by PPARγ siRNA or by knocking down ucp2 mRNA. The same treatments also reversed the antihypertensive effect of rosiglitazone. These results suggest that oral intake of rosiglitazone promotes central antihypertensive effect through a PPARγ‐dependent protection against oxidative stress in RVLM via transcriptional upregulation of UCP2. Supported by research grants NSC98‐2321‐B‐182A‐004 and NSC 97‐2321‐B075B‐001 from National Science Council, Taiwan.
Sinoaortic Denervation (SAD) Prevents Training‐Induced Plasticity in Preautonomic Oxytocinergic and Vasopressinergic Pathways
Authors: Cavalleri Marina T. , Burgi Katia , Jordao Maria T. , Ceroni Alexandre , Cruz Josiane C. , Michelini Lisete C.
Published: June 2021

Baroreceptor signaling plays a crucial role in driving training‐induced beneficial adjustments mediated by central autonomic pathways (Exp Physiol 2009;94:630–640; 947–960). We sought now to identify these pathways in WKY and SHR. Rats submitted to SAD or SHAM surgery were trained (55% VO2 max, for 3 month) or kept sedentary (S). After hemodynamic measurements at rest, rats were anesthetized for transcardiac perfusion (PBS). Brains were removed, frozen and sliced to isolate dorsal and ventral brain stem (comprising the solitary‐vagal complex and RVLM) and hypothalamic paraventricular nucleus (PVN); tissue samples were processed for oxytocin (OT), vasopressin (VP) and their receptors’ expression (Real Time PCR, GAPDH as the reporter gene). T‐induced pressure fall (−8% in the SHR) and resting bradycardia (−9% in both groups) were absent after SAD. There were no changes on receptors’ expression in target areas but SAD completely blocked T‐induced OT mRNA (4.3‐fold and 20% increases in SHAM WKYT and SHRT, respectively) and VP mRNA expression (+2.4‐fold in SHAM SHRT vs. S controls) within the PVN. OT and VP immunoreactivities within the PVN confirmed these responses. Baroreceptor inputs are decisive to drive plastic changes in preautonomic neurons. T‐induced PVN OTergic expression is present in both normotensive and hypertensive, while T‐induced VPergic expression is only observed in hypertensive individuals
The hypothalamic paraventricular nucleus is required for insulin's action to increase baroreflex gain of lumbar sympathetic nerve activity
Authors: Cassaglia Priscila Andrea , Brooks Virginia L
Published: June 2021

During pregnancy, low brain insulin levels are associated with impaired baroreflex control of heart rate (HR) and sympathetic nerve activity (SNA), and intracerebral insulin infusion normalizes baroreflex gain (BRG). The specific site of insulin action is unknown, though a likely candidate is the paraventricular nucleus (PVN). To test this hypothesis, BRG of lumbar SNA (LSNA) and HR were obtained in chloralose anesthetized female rats before and at 30min intervals during iv infusion of insulin (15mU/kg/min, n=5) or saline (n=5). Insulin increased LSNA (P<0.05; in % baseline) to 166±18 (30min), 224±43 (60min), 219±46 (90min), and 227±49 (120min). Similarly, insulin increased LSNA BRG (P<0.05; in %LSNA/mmHg) from 2.6±0.8 to 2.9±0.7 (30min), 4.5±0.9 (60min), 4.3±0.5 (90min), and 4.7±0.6 (120min). Insulin did not affect basal HR or HR BRG. No changes in LSNA, HR or BRG were observed with saline infusion. In another group (n=4), the effect of insulin to increase LSNA and LSNA gain was reversed by blockade of PVN via bilateral microinjection of muscimol (M; 60nL of 1mM). LSNA (% baseline) increased to 223±37 after insulin and fell to 126±11 after M (P<0.05). LSNA gain (%LSNA/mmHg) increased from 2.8±1.3 to 4.4±1.2, and decreased to 2.4±0.7 after M (P<0.05). In summary, iv insulin increased LSNA and LSNA gain. This action was reversed by PVN blockade, suggesting that insulin acts via the PVN to improve BRG. Support: the EMBL/GenBank Data Libraries under Accession No. HL088552
Enteral feeding induces profound early intestinal adaptation in a neonatal piglet model of short bowel syndrome
Authors: Dodge M Elaine , Bertolo Robert , Brunton Janet A
Published: June 2021

Our objective was to characterize small intestinal (SI) adaptation in a novel piglet model. Piglets (n = 32, 12–13 d old) were randomized to 1) 80% SI resection with total parenteral nutrition (TPN) feeding (R‐TPN), 2) 80% SI resection with TPN + enteral feeding (R‐EN) or 3) sham SI transection with TPN + enteral feeding (Sham‐EN). In R pigs, the distal 100 cm of ileum (residual SI) and 30 cm of proximal SI was left intact. All pigs received TPN after surgery. R‐EN and Sham‐EN piglets also received continuous gastric infusions of elemental diet starting on d 3 (40:60 TPN:EN). Piglets were killed 4, 6 or 10 d post‐surgery. Cell proliferation in residual SI, assessed by incorporation of 5‐bromo‐2′‐deoxyuridine (BrdU) was significantly greater in R‐EN animals (29.5 ± 4.7 %) compared to Sham‐EN (13.63 ± 5.7 %) and R‐TPN (12.97 ± 8.9 %) animals at d 4 (P<0.05). R‐EN at d 4 also had higher rates of mucosal protein synthesis than Sham‐EN (P<0.001) and R‐TPN animals (P<0.05), (87.2 ± 3.2 vs 53.2 ± 2.1 and 59.7 ± 9.7 %/d, respectively). Villous height and crypt depth were greater in R‐EN animals compared to Sham‐EN animals at d 6 and 10 (p<0.05). Less than 24 h of enteral feeding increased rates of cell proliferation and protein synthesis; this translated into greater villous height and crypt depth only 2 d later. This piglet model will be very useful in assessing the efficacy of nutritional interventions targeting SI adaptation. (SickKids Foundation)
Cardiac autonomic regulation in hyperthyroid subjects during sleep
Authors: Tobaldini Eleonora , Pecis Marica , Bulgheroni Mara , Colombo Silvia , Muratori Milena , Bevilacqua Maurizio , Porta Alberto , Montano Nicola
Published: June 2021

Hyperthyroidism, a model of reversible hyperadrenergic state, is characterized by an altered autonomic regulation and by a reduced sleep quality, but no data are available on sleep characteristics and autonomic control during sleep in hyperthyroid (HYPERTH) compared to euthyroid (EUTH) state.

The aim of our study was to assess autonomic control during sleep in Hyperth and Euth using symbolic analysis (SA) of heart rate variability (HRV). A polysomnographic study was performed in six women with diagnosed HYPERTH and when recovered to EUTH, after suppression therapy. The time series of RR intervals were constructed for each sleep stage and analyzed using SA, a method that is based on the conversion of time series into symbols, the construction of patterns and the identification of four families: 0V (no variation), marker of sympathetic modulation, 1V, 2LV and 2UV (two unlike variations), marker of parasympathetic modulation.

Sleep quality parameters were similar in HYPERTH and EUTH. As to HRV, in Hyperth REM sleep was characterized by an increased 0V% and a decreased 2UV%. Comparing to Hypert, Euthyr showed a decrease of heart rate and sympathetic modulation and an increase of vagal modulation in all sleep stages. In conclusion, in HYPERTH patients, REM is characterized by a predominant sympathetic control and the alterations of autonomic cardiac control during sleep seem to be reverted after treatment.