bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Anopheles gambiae Slc26a5 and potential role in malaria
Authors: Brin Lauren R , Hirata Taku , Scileppi Elyse M , Holmes Heather L , Linser Paul J , Romero Michael F
Published: June 2021

Malaria transmission requires Plasmodium invasion of the Anopheles mosquito midgut. This critical first step involves adhesion to chondroitin sulfate (ChSO4)[Dinglasan et al, PNAS 2007], a midgut epithelial ligand. Sulfate (SO42−) for ChSO4 biosynthesis is supplied by midgut SO42− uptake. Slc26 proteins transport many anions: Cl−, HCO3−, oxalate (ox2−), and SO42−. Recently, we found that Drosophila Slc26a5 is an electrogenic Cl−/SO42−, Cl−/ox2− and Cl−/nHCO3− exchanger [Hirata et al, JASN 2009], and SO42− transport exceeded ox2− transport. Therefore we searched EST and genomic databases to identify two Slc26a5 orthologues (AgSlc26a5A and ‐B). We expressed AgSlc26a5A and B in Xenopus oocytes to evaluate transport using microelectrodes. AgSlc26a5A is found in the salivary glands and gastric caeca. However, AgSlc26a5A did not obviously transport Cl−, HCO3−, SO42− or ox2−. On the other hand, AgSlc26a5B has transport properties similar to mouse Slc26a6 and Drosophila Slc26a5. That is, AgSlc26a5B mediates electrogenic Cl−/SO42−, Cl−/ox2− and Cl−/nHCO3− exchange. And, AgSlc26a5B is enriched in the hindgut (including Malpighian tubules) and the posterior midgut. These results suggest that AgSlc26a5B can alkalinize the Anopheles midgut and is also a candidate for SO42− supplier to midgut cell. Thus, we hypothesize that control of AgSlc26a5B activity could control parasite (Plasmodium) invasion.
FK506 binding protein (FKBP12) may contribute to 4‐chloro‐m‐cresol induced adenosine in human B cells
Authors: Bina Saiid , Capacchione John , Bayarsaikhan Munkhuu , Muldoon Sheila , Bunger Rolf
Published: June 2021

Introduction

Ryanodine receptor (RyR1) agonist 4‐chloro‐m‐cresol (4CmC) stimulates normoxic adenosine (ADO) production in B cells, especially from malignant hyperthermia susceptible individuals. In an attempt to identify a pathway for this action of 4CmC, FK506, an immunosuppressant drug that disrupts the interaction of FK506‐binding protein (FKBP12) with RyR1, and Xestospongin C (XeC), a specific inhibitor of IP3R were tested. FKBP12 forms a complex with the RyR and IP3R to regulate their physiological release of intracellular Ca2+.

Methods

B cells were suspended in HBSS + ADO deaminase inhibitor (EHNA) followed by addition of FK506 or XeC and incubated at 37°C for 10 min. In a 2nd experiment; 10min after addition of FK506 or XeC, 4CmC (2mM) was applied and incubation continued for 30 min. After cellular reactions termination, supernatant [ADO] was assayed using HPLC.

Results

(means ± SEM for 106 cells): ADO basal level was 0.422 ± 0.097μM (n=7). FK506 (2, 20, 200μM) increased ADO levels by 59 ± 4, 207 ± 21, and 240 ± 24% respectively. FK506 (2, 20, 200μM) increased the sensitivity of B cells to 4CmC by 4±5, 13±4, and 49±10%. XeC did not alter basal or 4CmC‐induced ADO in B cells.

Conclusion

FK506 likely displaces FKBP12 from the RyR1 receptor in B cells resulting in leaky Ca2+ channels and to cytosolic Ca2+ accumulation. This stimulates Ca2+ pumping, generating ADP and AMP leading to formation of ADO in the presence of EHNA.
c‐Src Directly Interacts with the C‐terminal Domain of Occludin by involving SH2 and SH3 domains
Authors: Seth Ankur , Samak Geetha , Zola Courtney E , Rao R.K.
Published: June 2021

The effect of H2O2 on c‐Src translocation to tight junction (TJ) and the direct binding to occludin were investigated. Caco‐2 cell monolayers were exposed to H2O2. Src activation was measured by FRET‐based Src kinase assay and immunoblot analysis for c‐Src(pY418). Association of c‐Src with TJ proteins was determined by co‐immunoprecipitation and confocal microscopy. Direct binding of c‐Src with C‐terminal domain of occludin determined by pair‐wise binding using c‐Src and GST‐Occludin‐C (C‐terminal domain). GST‐Occludin‐C(Ocldn)Δ 378–385 and GST‐OcldnY379/383D were used to determine whether interaction is an Enzyme‐Substrate binding. Recombinant SH2 and SH3 domains of c‐Src were used to determine their role in occludin binding. H2O2 activated c‐Src and increased co‐immunoprecipitation of occludin with c‐Src(pY418). H2O2 increased localization of c‐Src(pY418) at the TJ. c‐Src binds to GST‐Ocldn‐C as well as GST‐OcldnΔ378–385 and GST‐OcldnY379/383D indicating that the interaction is not just an enzyme‐substrate binding. SH2 and SH3 domains dose‐dependently attenuated c‐Src binding to GST‐OcldnWT and GST‐OcldnΔ378–385 indicating that these domains are involved in c‐Src binding to occludin. Incubation of GST‐SH2 and GST‐SH3 with Caco‐2 cell extract pulled down occludin. Results indicate that H2O2 induces translocation of c‐Src to TJ by direct binding to occludin, which involves SH2 and SH3 domains.
Vitamin D regulates MUC17 expression in Caco‐2 cells.
Authors: Tabikh Sara Q , Tang Eva YK , Resta Silvia
Published: June 2021

Mucins are components of the gastrointestinal barrier and their downregulation contributes to chronic gut inflammation. Calcium plays an important role in stabilizing negatively charged mucins expressed by intestinal cells. Vitamin D regulates intestinal calcium absorption, through the nuclear vitamin D receptor (VDR). MUC17 gene regulator contains a VDR responsive element. We hypothesized that lack of VDR expression would disrupt mucin expression in intestinal epithelial cells. Caco‐2 cells treated with siVDR or untreated control were tested for MUC17 mRNA expression by PCR and by immuno‐histochemistry. Treatment with low calcium concentration by calcium switch and vitamin D3 was also studied. siVDR Caco‐2 cells had lower levels of MUC17 vs controls (p<0.01). MUC17 mRNA expression was decreased by 2.5 in siVDR cells. In cell exposed to low calcium no MUC17 was detectable. Restoration of calcium levels in the siVDR Caco‐2 evoked a rapid short‐lived recovery of MUC17 mRNA expression, albeit the mucin expression remained unchanged. Vitamin D3 treatment had no effect on siVDR Caco‐2. However, untreated controls, expressing normal levels of VDR showed an increased MUC17 mRNA and protein expression. Our data support the hypothesis that vitamin D has a protective effect on the gut epithelial barrier by promoting mucin expression and homeostasis.

NIH‐DK062096 (SR).
βArrestin2/βArrestin1 (βA2/βA1) ratio determines cell death or cell proliferation in response to platelet‐activating factor (PAF)
Authors: Issac Marla , Pazin Marina , Caplan Michael , Jilling Tamas
Published: June 2021

PAF, a potent inflammatory mediator, induces apoptosis in intestinal epithelial cells (IEC) and is involved in gastrointestinal disorders, yet it causes proliferation and cell survival in breast and prostate cancer cells. We hypothesized that these contrasting effects on cell death and survival are mediated by differential expression of βA2 and βA1. There is a significantly higher βA2/βA1 mRNA ratio in MDA‐MB‐231 (MDA) breast cancer cells (1.286±0.477) compared to IEC (0.015±0.004; p=0.028), without a difference in PAFR expression as determined by quantitative real time PCR. PAF induces a dose dependent increase of caspase activity, but not proliferation in IEC. Conversely, PAF stimulates proliferation but not caspase activity in MDA. pERK & pAkt increased in response to cPAF in MDA, but pERK and pAkt decreased in IEC. Over expression of βA2 in IEC abrogated cPAF's effect on caspase activation and shRNA‐mediated knock down of βA2 in MDA negated PAF‐induced proliferation. In summary, higher βA2/βA1 prevents cPAF‐induced apoptosis and supports pro‐proliferative signaling by cPAF, perhaps by modulating signaling via the Akt and ERK pathways.
TGF β Supports, Platelet‐activating Factor (PAF) Inhibits Intestinal Epithelial Cell (IEC) Migration In Vitro via Effects on Phoshatidylinositol 3 Kinase (PI3K)
Authors: Pazin Marina Viktoria , Rudnicki Jean , Yang Yirong , Caplan Michael S , Jilling Tamas
Published: June 2021

We aimed to identify mechanisms involved in the regulation of IEC migration by growth factors that are known to be beneficial to prevent mucosal injury and by (PAF), a mediator implicated in inflammatory bowel diseases. In 5% FBS, IEC6 migrate, on fibronectin, for two hours at an average linear rate of V=0.38±0.04 ìm/min, while PIP3 concentration oscillates in the leading edge of lamellipodia at 83mHz, as assessed by live cell imaging and using AKT‐PH domain‐GFP fusion protein as a PIP3 sensor. FBS withdrawal aborts PIP3 oscillations and migration in the second hour (V=0.03±0.02 ìm/min), correlating with steady state dephosphorylation of AKT on S473. Activation of the PAF receptor by 20ìM carbamyl‐PAF or inhibition of PI3K using 10 ìM LY294002 in the presence of FBS leads to the same outcome. TGFâ (1ng/ml) partly restores migration (V=0.22±0.04 ìm/min) and steady state AKT phosphorylation in the absence of FBS. AKT phosphorylation, but not migration, is restored by either EGF and/or IGF (10ng/ml each). Though all three growth factors compensate for the loss of steady state Akt phosphorylation upon FBS withdrawal, only TGFâ is able to restore migration. Further studies are aimed to determine whether divergent effects by TGFβ and EGF/IGF on PIP3 oscillations and/or focal adhesions explain the discrepancy between effects by these growth factors on steady state Akt phosphorylation and migration. Supported by DK062960
Effects of Aminoguanidine (AG) in a Rat Pancreatic Tumor Cell line
Authors: Chowdhury Parimal
Published: June 2021

Aminoguanidine is a compound that reportedly exerts both pro‐ and antioxidant effects. In animal models and in humans it was shown as an antioxidant against lipid peroxidation. The current study evaluated the direct effect of AG on rat pancreatic tumor cell.

Methods

AR42J cells were grown in F‐12 nutrient medium at 370C and 5% CO2. At log phase, they were separated, sub cultured for 3 days at specified dose and time with hydrogen peroxide and aminoguanidine. The cellular extracts were subjected to Western blotting employing antibodies to ERK ½ and analyzed further by immunohistochemistry. Cell proliferation lipid peroxidation assay was also conducted. Cell functional assay was conducted with CCK‐8 (10–10 M) incubated for 30 min at 370C and was assessed by the amount of amylase released in the media.

Results

Incubation of AR42J cells with aminoguanidine induced 3–5 fold activation of phospho ERK‐1/2. This was confirmed by immunohistochemistry with FITC conjugated antibody to ERK1/2. CCK‐stimulated amylase release was enhanced in the presence of aminoguanidine.

Conclusion

These data suggest that ERK‐1/2, a proliferative signal of MAPK pathway, is induced and enhanced in AR42J cells by aminoguanidine. This is coupled with enhanced cell function and proliferation suggesting that aminoguanidine acts as a pro‐oxidant in this cell line.
Testosterone down‐regulates sodium‐hydrogen exchanger isoform 8 (NHE8) expression
Authors: Xu Hua , Zhang Bo , Li Jing , Tong Weiqun , Chen Huacong , King Nolan , Ghishan Fayez K
Published: June 2021

NHE8 is the newest member of the sodium‐hydrogen exchanger family. It is expressed in intestinal and renal epithelial cells. We have shown that NHE8 is important for intestinal sodium absorption early in life and that its expression is regulated by many physiological factors. Since studies have shown that certain genes are regulated differently by gender, we investigated if NHE8 is one of these genes. Therefore, we compared NHE8 expression in male and female mice. RNA and brush‐border membrane protein were isolated from the intestinal mucosa of mice. Real‐time PCR was used to determine NHE8 mRNA levels, and Western blot was used to determine NHE8 protein levels. We performed the same experiments on Caco2 cells to study the effect of sex hormones on NHE8 expression. Our results showed that at four weeks of age, RNA and protein expression of NHE8 was similar between male and female mice, but at eight weeks of age, RNA and protein expression of NHE8 was significantly reduced by ~50% in male mice compared to female mice. In Caco2 cells, estrogen (100nM, 18 hours) had no effect whereas testosterone (100nM, 18 hours) inhibited NHE8 expression at RNA and protein levels that are comparable with the in vivo observations. In conclusion, our results suggest that NHE8 expression differs between male mice and female mice. The difference is likely due to testosterone's inhibitory effect. Our study was supported by NIH grant R01DK073638.
Expression of PDE5 and its stimulation by cGMP‐dependent protein kinase (PKG) determine the magnitude of smooth muscle relaxation in different regions of the stomach
Authors: Al‐Shboul Othman , Mahavadi Sunila , Grider John R , Murthy Karnam S.
Published: June 2021

The contractile phenotypes of proximal and distal gastric smooth muscle are distinct with proximal smooth muscle exhibiting tonic characteristics and distal smooth muscle phasic characteristics. In smooth muscle, the main cGMP hydrolyzing enzyme phosphodiesterase 5 (PDE5) plays an important role in determining the strength and duration of cGMP signaling, and thus muscle relaxation. The activity of PDE5 is stimulated by phosphorylation via PKG.

Aim

To determine the expression and regulation of PDE5 activity in proximal versus distal regions of the stomach.

Methods

Expression of PDE5 and PKG‐mediated phosphorylation of PDE5 at Ser 92 were measured by western blot. PDE5 activity was measured using [3H]cGMP and cGMP‐mediated relaxation was measured by scanning micrometry.

Results

Muscle cells from proximal stomach contained 2 times more PDE5 than muscle cells from distal stomach. Activation of PKG by the NO‐donor caused an increase in PDE5 phosphorylation and activity, but the responses were higher in muscle cells from proximal stomach. In contrast, muscle relaxation was greater in muscle cells from distal stomach consistent with the lower amounts of PDE5 in muscle cells from this region.

Conclusion

. Higher expression of PDE5 and greater PKG‐mediated augmentation of PDE5 activity in muscle cells from proximal stomach correlate with the tonic phenotype of muscle in proximal stomach relative to distal stomach.
Effect of ezetimibe on the response of incretin secretion to intestine lipid ingestion
Authors: Yang Li , Li Xiaoming , Ji Yong , Wang David Q.‐H. , Howles Philip N. , Hui David Y. , Tso Patrick
Published: June 2021

Background

Ezetimibe (EZ) is a potent inhibitor of cholesterol absorption by enterocytes. Although EZ minimally affects fat absorption, it is unknown if EZ affects the secretion of incretins glucose‐dependent insulinotropic polypeptide (GIP) and glucagon‐like peptide‐1 (GLP‐1) during triglyceride (TG) absorption.

Aim

We determined if EZ influences the lymphatic secretion of incretins stimulated by the ingestion of Ensure.

Methods

In SD rats, the mesenteric lymph duct was cannulated and a duodenal infusion tube was installed. EZ (0.67 mg/kg) or saline was infused duodenally 2 h prior to the feeding of a 3 ml bolus dose of Ensure (4.43 kcal). To determine glucose, TG, cholesterol, GIP, and GLP‐1 concentrations, lymph samples were collected 2 h before and 2 h after infusion of Ensure.

Results

EZ did not affect lymph flow, glucose or TG output stimulated by Ensure compared with the control rats. As expected, EZ did significantly reduce lymphatic cholesterol output. Interestingly, EZ significantly reduced the stimulation of lymphatic secretion of GIP and GLP‐1 induced by Ensure.

Conclusions

Reduced secretion of GIP and GLP‐1 by the enteroendocrine cells is associated with decreased cholesterol secretion in EZ‐treated rats. Moreover, this response of intestinal incretin secretion is independent from the uptake of dietary TG. The mechanism involved remains to be elucidated. This work was supported by NIH.
FUNCTIONAL ROLES OF CALCIUM‐SENSING RECEPTOR
Authors: Nakamura Eiji , Hasumura Mai , Gabriel Ana San , Uneyama Hisayuki , Eto Yuzuru , Torii Kunio
Published: June 2021

Calcium sensing‐receptor (CaSR) is known as one of the family C GPCRs, which chemosense L‐amino acids, including metabotropic glutamate receptors (mGluRs), taste 1 receptor T1R1/T1R3 and GPRC6A. To clarify the functional roles of CaSR in stomach, we fractionized different kinds of cells (mucus secreting cells, parietal cells, chief cells, smaller endocrine cells and larger endocrine cells) from the dispersed rat gastric mucosal cells using a counterflow elutriation and density gradient centrifugation, and then characterized the expressions of L‐amino acid receptors in these cell fractions. We found that D cells were concentrated in the smaller endocrine fraction using immunocytochemistry. Real‐time RT‐PCR analysis revealed that the expressions of L‐amino acid receptors were diverse among these cell fractions, however, D cell‐rich fraction showed comparatively specific expressions for CaSR and some Gi‐coupling mGluR subtypes. Interestingly, CaSR agonists such as Cinacalcet, L‐phenylalanine and L‐histidine, potently stimulated somatostatin release from cultured D cells. From these results, we propose that CaSR would be an interesting target to regulate the gastric functions via the induction of somatostatin release from D cells.
The combined administration of multiple soluble factors in the repair of chronically infarcted rat myocardium
Authors: Hwang Hyosook , Kloner Robert A.
Published: June 2021

The purpose of this study was to investigate a potential benefit of simultaneous administration of multiple soluble factors (SF) in the repair of chronically infarcted rat myocardium. Rats subjected to a permanent coronary artery occlusion (MI) received a cocktail of SF or a phosphate buffer. Four soluble factors, fibroblast growth factor‐2 (2 μg), insulin‐like growth factor‐1 (1 μg), hepatocyte growth factor (2 μg), and stromal cell‐derived factor‐1α (0.6 μg) were injected directly into the ischemic myocardium at the onset of occlusion and subsequently at 3, 7, 14, and 21 days of surgery (ip). Cardiac contractile function, infarct size and remodeling, and blood vessel density were studied at 4 weeks post surgery. Infarct size, left ventricular circumference and cavity volume, thinning ratio, and expansion index were not statistically different between groups. Treatment of SF did not alter ejection fraction, compared to control. No statistical difference in total blood vessel density in the infarct zone was observed in SF vs. control. In conclusion, our results showed that there were no enhancements in cardiac function, reductions in infarct size, improvements in remodeling, nor increases in vasculature density in SF vs. control. These results do not support the study hypothesis that the combined use of SF benefits the hearts with myocardial infarct.
Amino acids regulate expression of Antizyme‐1 and modulate Ornithine Decarboxylase Activation
Authors: Ray Ramesh M , Viar Mary Jane , Johnson Leonard R.
Published: June 2021

In a glucose‐salt solution (EBSS), asparagine (ASN) stimulates ornithine decarboxylase (ODC) activity in a dose‐dependent manner. We determined the mechanism by which ASN induces ODC activity. In the absence of amino acids (AA), an antizyme 20‐kDa isoform (AZ‐1‐20), which inhibits ODC activity, increased in a time‐dependent fashion. ASN prevented the accumulation of AZ‐1‐20 and rapidly induced ODC activity, as did glutamine and α‐amino iso butyric acid. In contrast, lysine and valine induced AZ‐1‐20 expression and decreased ASN‐induced ODC activity. ODC induction by ASN was lower in DMEM containing AA as compared to that in DMEM without AA, suggesting that AZ‐1‐20‐inducing AA may partially neutralize the effect of ASN. Incubation in EBSS for 6 hrs increased AZ‐1‐20 protein levels, and addition of ASN decreased accumulated AZ‐1‐20. Cycloheximide similarly caused elimination of accumulated AZ‐1‐20 but did not alter the rate of decrease due to ASN. Actinomycin‐D failed to prevent AZ‐1‐20 expression in EBSS. These results suggest that ASN modulates AZ‐1‐20 levels by regulating synthesis. ASN prevented the phosphorylation and caused accumulation of eukaryotic initiation factor binding protein (4EBP1). Thus, by preventing the phosphorylation of 4EBP1, ASN causes it to accumulate and bind to eIF4E resulting in the inhibition of the synthesis of AZ‐1‐20, allowing the activation of ODC.
Growth factors EGF and HGF suppress hepcidin by direct interference with bone morphogenetic protein signaling
Authors: Goodnough Julia B , Ganz Tomas
Published: June 2021

The iron‐regulatory hormone hepcidin is a 25‐amino‐acid peptide synthesized in the liver. Hepcidin inhibits iron export into plasma and is in turn regulated by iron through a BMP6‐activated pathway. Erythropoietic activity and certain liver diseases decrease hepcidin synthesis through unknown mechanisms.

We found that hepatocyte growth factor (HGF) was a potent suppressor of hepcidin in hepatocytes and in hepatic cell lines. At physiologic concentrations, HGF dose‐dependently suppressed hepcidin mRNA and hepcidin promoter‐luciferase. HGF also opposed hepcidin induction by BMP6 in both systems. HGF was likely acting directly on BMP signaling because HGF prevented the induction of an unrelated BMP‐sensitive reporter by BMP6, as well as ‐2, ‐4, and ‐9. In primary mouse hepatocytes, BMP‐driven Smad1/5/8 nuclear translocation was suppressed by HGF. The hepatocytes displayed increased activation of the MAP kinase Erk and related phosphorylation of a regulatory motif in R‐Smads known to result in nuclear exclusion. Thus, decreased nuclear concentration of activated Smad1/5/8 is a likely mechanism of the HGF suppression of hepcidin response to BMP. EGF also suppressed the induction of hepcidin mRNA and the hepcidin‐luciferase reporter by BMPs, likely by a similar mechanism. EGF, HGF or other growth factors using MAPKs could mediate the suppression of hepcidin in chronic liver diseases.

This research is supported by National Institutes of Health grant #1F30DK082151.
Hepcidin independent anemia of inflammation
Authors: Rivera Seth , Shprung Dana , Limbrick Donald , Gabayan Victoria , Ganz Tomas
Published: June 2021

Because hepcidin overproduction mimics anemia of inflammation and absence of hepcidin protects against the acute hypoferremia seen in response to inflammation, hepcidin is presumed to be necessary for the development of anemia of inflammation (AI, anemia of chronic disease). However no independent studies have tested this hypothesis. Using two mouse models of anemia of inflammation and hepcidin deficient mice, we sought to determine whether hepcidin is necessary for the development of anemia of inflammation. Hepcidin knockout mice maintained on a standard iron diet were largely protected against AI. However, these mice have severe iron overload prior to the onset of inflammation, which may protect them against AI. We therefore sought to prevent the development of iron overload prior to the onset of inflammation by restricting dietary iron immediately after weaning. Hepcidin knockout mice without baseline iron overload developed anemia as severe as wild type mice and this anemia was iron restricted. Therefore, hepcidin is not necessary for the development of anemia in some inflammatory diseases. Hepcidin antagonists may not be effective in treating some patients with AI.
Comparative analysis of innate immune related transcriptional responses to pathogens in Caenorhabditis elegans
Authors: Balamurugan Krishnaswamy , Karuthapandian Shanmugiahthevar
Published: June 2021

Caenorhabditis elegans responds to infection with a simple innate immune system, allows the nematode to be used as a tractable model host for infectious disease and for host‐pathogen interactions related innate immunity studies. Salmonella typhi, Vibrio parahaemolyticus, Shigella flexneri and Pseudomonas aeruginosa are gram‐negative pathogenic bacteria which causes several infections to humans. The feeding based pathogenicity liquid‐assay facilitates comparative physiological/transcriptional analysis. Kinetic studies revealed the LT50 varied significantly for each pathogens‐mediated infection to the animals. CFU assay revealed the multiplication pathogens inside C. elegans after infections. Less than 6 hr of pathogens exposed animals indicated the impact of food source after infections. Solid assays showed pathogen avoidance behavior. Total RNA isolated from pathogens exposed and control animals showed the initial burst at the level of expression of few putative antimicrobial genes. RT‐PCR was done for different time periods. The 4–24 hr pathogen exposed C. elegans were analyzed for the expression of many genes including Clec‐60, Lys‐7 and Scl‐2. Some of the putative antimicrobial genes such as lysozymes and C‐type lectins appear to be upregulated in C. elegans infected with pathogens during the initial hours of exposure. The kinetic studies are currently underway.
Oxidative stress inhibits Klotho activation of TRPV5 and TRPV6 in intestinal epithelial cells
Authors: Maruggi Marco , Nguyen Amanda Tu , Resta Silvia
Published: June 2021

Calcium is an essential ion in all organisms. We have shown that calcium metabolism is disregulated during chronic inflammation and aging. Klotho is a major player in cell senescence and is associated with calcium metabolism. We hypothesized that oxidative stress in intestinal epithelial cells may cause disregulated calcium balance in the gastrointestinal tract via Klotho disfunction. Caco‐2 and HT29/cl 19A cells were exposed to oxidative stress (H2O2, 100 nM) for 24–72 hr. The cells were then tested for Klotho, TRPV5, TRPV6 mRNA and protein expression, and for calcium transport. H2O2 treatment significantly decreased expression and activity of Klotho vs controls in a time‐dependent manner (1‐, 3‐, 1‐fold decrease at 24, 48, 72 hr, respectively). In parallel, TRPV5 and TRPV6 activity was decreased. Calcium transport was significantly reduced at 24–48 hr, and completely inhibited at 72 hr. H2O2 treatment reversed the co‐immunprecipitation of Klotho and the calcium channels by western blot, and co‐localization by confocal microscopy. We conclude that oxidative stress is in part responsible for the altered calcium metabolism observed in chronic inflammatory diseases through inhibition of Klotho activation of the major intestinal epithelial cell calcium channels. (NIH‐DK062096, SR).
Enterocyte fatty acid oxidation may influence eating through vagal afferent signals
Authors: Langhans Wolfgang , Egle Claudia , Schober Gudrun , Arnold Myrtha
Published: June 2021

Mercaptoacetate (MA) and other fatty acid oxidation (FAO) inhibitors supposedly stimulate eating by inhibiting hepatic FAO, thus activating a hepatic vagal afferent signal to eat. This concept has been challenged because hepatic parenchymal vagal afferent innervation is scarce and because experimentally induced changes in hepatic FAO often fail to affect eating. Nevertheless, IP MA must act in the abdomen to stimulate eating because its effect was blocked by subdiaphragmatic vagal deafferentation (SDA) (Brandt et al, Neurosci Letters 411:104, 2007). This suggests a role of the small intestine in the effects of IP MA on eating. To examine this idea, we equipped adult male rats with jejunal catheters and tested the effects on eating of intrajejunal infusions (30 sec, 1 ml/kg, start at 3 h into the light phase) of MA (100, 200, and 400 μmol/kg BW). MA (200 and 400 μmol/kg) increased (P < 0.05) 30 and 60 min food intake vs. control, this effect was absent in rats after SDA, and MA infused into the superior mesenteric artery (SMA) increased (within 10 min) the activity of 34 (out of 37) serotonin‐sensitive single units isolated from celiac vagal fascicles originating in the proximal small intestine. Because the SMA supplies most of the small intestine, these data support the hypothesis that small intestinal enterocytes may serve as FAO sensors and influence eating through changes in intestinal vagal afferent activity.
JAK/STAT activation induced by Angiotensin II in rats isolated pancreatic islets.
Authors: Alves Eduardo Silva , Haidar André Abou , Quadors Caren DalMoura , Carvalho Diego Soares , Morgan Daniela , Rocha Marlene Santos , Carpinelli Angelo Rafael , Hirata Aparecida Emiko
Published: June 2021

Angiotensin II (Ang II) controls blood pressure, electrolyte balance, cell growth and vascular remodeling. In addition Ang II activates NAD(P)H oxidase in several tissues and has an important participation in the control of insulin secretion. There is a growing body of evidence demonstrating the occurrence of hypertension, insulin resistance and impairment of pancreatic beta cell function in the development of type II diabetes. In several tissues, Ang II induces JAK/STAT activation and phosphorylation which involve superoxide generation from NAD(P)H oxidase. The aim of the present study was to study the JAK/STAT association stimulated by Ang II. For this purpose, isolated pancreatic islets were incubated in presence or absence of ANG II (10− 8M). Samples were immunoprecitipated with anti‐JAK2 antibody and immublotted with anti‐STAT1 or STAT3 antibodies. Western blotting analysis showed that Ang II induced an increase JAK2/STAT1 (42%) and STAT3 (38%) association when compared with control group (glucose 2.8mM). In addition Ang II was also able to induce a three‐fold increase in protein content of others kinases such as ERK1/2.

Financial Support: Fapesp
Erythropoietin regulates intestinal metal transporter expression
Authors: Pourvali Katayoun , Chung Bomee , Sharp Paul
Published: June 2021

Levels of the hormone erythropoietin (Epo) are elevated in iron deficiency anaemia (IDA). In IDA, intestinal iron absorption is up‐regulated reflecting the increased erythroid iron demand. The absorption of copper is also increased in models of iron deficiency. Epo is thought to influence iron absorption indirectly via inhibition of hepatic hepcidin production; however, we have hypothesized that Epo also acts directly on enterocytes to regulate absorption. Here we have examined the effects of Epo on iron transporter expression in human intestinal Caco‐2 cells. We have also determined whether Epo may play a role in the increased intestinal copper transport observed in iron deficiency. Cells were treated with Epo (1U/ml) for 24h and transporter expression was measured by Real‐Time PCR. Following Epo treatment, expression of the iron transporters DMT1 and ferroportin; the copper transporters hCTR1 and ATP7b; and the Cu‐dependent ferrioxidase hephaestin were increased significantly. Transepithelial iron transport was also significantly increased in Epo‐treated cells. Our data demonstrate that Epo can act directly on intestinal epithelial cells to increase iron absorption. Increased expression of copper transporters and hephaestin in Epo‐treated cells is consistent with the essential role of copper in maintaining iron availability for metabolism. KP is funded by Shaheed Beheshti Medical University, Iran.
Antioxidant intravenous supplementation improves cardiac output during dynamic exercise in college soccer athletes
Authors: Chun Jong‐Mok , Kwak Jung‐Ja , Choi Hyun‐Min , Park Siyoung , Lee Joon‐Hee , Kim Jong‐Kyung , Jung Jae Woo , Nho Hosung
Published: June 2021

Antioxidant supplements are used by athletes as a means to counteract the oxidative stress of exercise. However, the effect of chronic antioxidant supplements on cardiovascular responses to dynamic exercise is not well understood. Therefore, we investigated the hemodynamic responses during cycle exercise after intravenous supplements of antioxidant vitamins in college soccer athletes. Eleven healthy subjects were instrumented to measure heart rate (HR), stroke volume (SV), cardiac output (CO) and mean arterial pressure (MAP) before and after 700 ml intravenous supplementation with antioxidant compound (B‐com, vitamin C, gluthation, vitamin B12, vitamin B5, vitamin B6, L‐ carnitine, magnesium sulphate, glycchyrizin, vitamin B1, thiot acid in isotonic sodium chloride solution), 14 times for one month. Total vascular conductance (127.1±8.7 vs. 137.2±9.3 ml/min/Hg), SV (123.7±7.8 vs. 137.8±7.7 ml), and CO (11.8±0.8 vs. 13±0.9 L) were significantly increased after the antioxidant supplements, where there were no differences in MAP and HR. The results indicate that antioxidant compound‐induced increases in SV and CO can increase oxygen delivery to exercising skeletal muscles and improve exercise performance. Effects may be due to attenuation of sympathetic‐induced vasoconstriction in skeletal muscle during dynamic exercise. Supported by Kyung Nam Pharm CO.
Ionotropic glutamate receptors in the paraventricular nucleus (PVN) are required for sympathoexcitation due to angiotensin II (A II) in the subfornical organ (SFO) of nonpregnant and pregnant rats
Authors: Kvochina Lyudmyla I. , Hasser Eileen M. , Heesch Cheryl M.
Published: June 2021

Circulating A II is increased and CNS actions of A II may support sympathetic nerve activity (SNA) in pregnancy. We previously reported greater increases in mean arterial pressure (MAP) and lumbar SNA (LSNA) in response to AT1 receptor activation in the SFO in term pregnant (P) compared to nonpregnant (NP) rats. The pathway from the SFO requires AT1 receptors in the PVN. Ionotropic glutamate receptors (iGluRs) in the PVN also contribute to sympathoexcitation from the SFO and current experiments evaluated the role of iGluRs in the PVN. Responses to microinjection of A II into the SFO were tested before and after iGluR blockade (kynurenate, KYN) in the PVN. To eliminate compensatory arterial baroreflex responses, sinoaortic denervation was performed in 5 nonpregnant (NP) and 4 term pregnant (P) inactin anesthetized rats. Baseline MAP was lower in P (84±13 mmHg) compared to NP (124±11 mmHg) rats. Increases in MAP (ΔMAP: P = +16.5±3 mmHg; NP = +10±1 mmHg) and LSNA (ΔLSNA: P= 15±3%; NP= 8±1%) in response to A II (20 μM, 50 nl) in the SFO were greater in P rats. Bilateral microinjection of KYN (27 mM, 100 nl) into the PVN had minimal effects alone, but eliminated responses to activation of the SFO in both groups (ΔMAP: P= −1±1 mmHg; NP= −1±2 mmHg; ΔLSNA: P= 0±1%; NP= 1.5±1%) Thus, both iGluRs and AT1 receptors in the PVN are required for sympathoexcitation mediated by SFO A II in NP and P rats. (NIH HL 091164)
Role of CK2 and calcineurin in increased NMDA receptor activity of PVN presympathetic neurons in hypertension
Authors: Ye Zengyou , Li De‐Pei , Pan Hui‐Lin
Published: June 2021

Increased glutamatergic input to presympathetic neurons in the paraventricular nucleus (PVN) plays a critical role in increased sympathetic outflow in hypertension. However, the molecular mechanism underlying increased NMDA receptor activity in the PVN in hypertension is not known. Here we determined the role of protein kinase CK2 and protein phosphatase calcineurin in regulating NMDA receptor activity in spinally projecting PVN neurons in brain slices from spontaneously hypertensive rats (SHR) and Wistar‐Kyoto rats (WKY). The NMDA current (0.68 ± 0.05 vs. 0.32 ± 0.06 pA/pF, P < 0.05) and the ratio (0.72 ± 0.07 vs. 0.35 ± 0.02, P < 0.05) of NMDA/AMPA currents of retrogradely labeled PVN neurons were significantly larger in SHR than in WKY. Inhibition of CK2 with DRB or TBB normalized the increase in NMDA currents and had no effect on AMPA currents in SHR. Inhibition of calcineurin with FK506 significantly increased NMDA currents in WKY but not in SHR. Furthermore, in the presence of FK506, DRB had little effect on NMDA currents in SHR and WKY. Our study provides important new information that the phosphorylation and dephosphorylation process tonically regulates NMDA receptor function. The imbalance between CK2 and calcineurin activity in the PVN likely contributes to augmented NMDA receptor activity in presympathetic neurons and increased sympathetic outflow in hypertension.
Cardiac chronotropic and inotropic responses evoked from right or left sides of dorsomedial hypothalamus
Authors: Xavier Carlos Henrique , Beig Mirza Irfan , Ianzer Danielle , Fontes Marco Peliky , Nalivaiko Eugene
Published: June 2021

We previously reported a functional asymmetry in the descending cardiovascular pathways from dorsomedial hypothalamus (DMH). Right DMH is critical for expressing full tachycardic responses to stress, and its activation evokes more pronounced tachycardia compared to left DMH. We next evaluated the cardiac chronotropic and inotropic responses evoked by right (R) or left (L) sides of DMH. In anesthetized adult male Wistar rats (n=7) we compared changes in LVP, LVdP/dt, ECG, and AP evoked by a microinjection of a GABAA antagonist bicuculline (40pmol/100nl) into L‐ or R‐DMH. R‐DMH activation evoked greater increases in HR (Δ: R 108±12 vs. L 63±9bpm), LVP (Δ: R 43±10 vs. L 22±5mmHg) and LVdP/dtpeak (Δ: R 106±17 vs. L 77±15%) compared to the L‐DMH activation (P<0.05 for all), whereas pressor responses were similar for both sides. R‐DMH activation provoked greater number of ectopic beats (Δ: R 16±10 vs. L 7±3 ectopic beats/10min, P<0.05). No differences were found in ECG intervals after L‐ or R‐DMH activation. Atenolol (2mg/kg i.v.) blocked cardiac chronotropic and inotropic responses evoked by unilateral L‐ or R‐DMH activation. We conclude that right DMH has dominant effects on cardiac inotropic and chronotropic changes, and that it may be involved in the genesis of cardiac arrhythmias. Support: FAPEMIG‐PPM and CNPq.
Altered expression of heme oxygenase‐1 in the hypothalamic paraventricular nucleus of heart failure rats
Authors: Biancardi Vinicia Campana , Son Sook Jin , Stern Javier E.
Published: June 2021

The paraventricular nucleus of the hypothalamus (PVN) is known to be involved in increased neurohumoral drive in heart failure (HF). While abundant evidence supports blunted nitric oxide (NO) function as an underlying mechanism, the potential role of carbon monoxide (CO), another gas molecule highly related to NO, has not yet been explored. Thus, we aimed in this study to determine whether the expression of heme ‐ oxygenase 1 (HO1), a CO‐synthesizing enzyme was altered in the PVN during heart failure. HO1 immunohistochemical studies were performed in sham and ischemic congestive heart failure rats. Both neuronal and glial‐like staining patterns were observed in the PVN. Quantification of immunofluorescence intensity within individual PVN neurons demonstrated a significant increase (~20%) of HO1 in the PVN of heart failure rats. No differences however were observed in the supraoptic nucleus. Ongoing studies aim to determine whether changes in HO1 levels affect both neurosecretory and autonomic‐related neurons, as well as their electrophysiological activity. Based on our recent observation supporting CO as an excitatory gas molecule, our results suggest that enhanced CO levels within the PVN of heart failure rats may contribute to enhanced neurohumoral activation in this disease. Supported by NIH HL68725.
Reduced reactive oxygen species generation in rostral ventrolateral medulla suppresses the pressor response induced by the excitation of the paraventricular nucleus of the hypothalamus in spontaneously hypertensive rats
Authors: Nishihara Masaaki , Hirooka Yoshitaka , Matsukawa Ryuuichi , Ito Koji , Kishi Takuya , Sunagawa Kenji
Published: June 2021

Background

Increased generation of reactive oxygen species (ROS) in the rostral ventrolateral medulla (RVLM) contribute to the neural mechanism of hypertension. The RVLM receives excitatory synaptic inputs from the paraventricular nucleus (PVN). It is also suggested that the angiotensin II type 1 receptors in the RVLM are activated by the PVN stimulation. Therefore, we examined whether the excitatory synaptic inputs derived from the PVN mediate the ROS in the RVLM or not.

Methods and Results

We used 14 to 18‐week‐old male Wistar‐Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). We transfected adenovirus vectors encoding manganese superoxide dismutase gene (AdMnSOD) into the bilateral RVLM. Then, we microinjected bicuculline (BIC) (100pmol) into the unilateral PVN on day 7 after gene transfer. Microinjection of BIC into the PVN increased mean arterial blood pressure (MAP) in both SHR and MnSOD‐transfected SHR, however, the pressor response was significantly attenuated in MnSOD‐transfected SHR (SHR: 33±2 mmHg; MnSOD‐transfected SHR: 20±1 mmHg, n=5, P<0.05). Overexpression of MnSOD in the RVLM did not suppress BIC‐elicited MAP elevation in WKY rats (WKY rats: 14±1 mmHg; MnSOD‐transfected WKY rats: 13±1 mmHg, n=3).

Conclusion

Excitatory synaptic inputs arising from activation of the PVN to the RVLM are mediated, at least in part, by ROS generation in SHR, but not in WKY.
Short chain fatty acid supplementation increases MUC2 expression and mucin production in a short bowel piglet receiving TPN
Authors: Nehrling Erica , Bartholome Anne , Albin Dave , Tappenden Kelly Anne
Published: June 2021

Total parenteral nutrition (TPN) is associated with goblet cell atrophy increasing the risk of bacterial translocation. Butyrate has been shown to enhance mucin production and may be an effective clinical treatment for TPN associated intestinal atrophy. This study assessed the effect of butyrate and short chain fatty acid (SCFA) mixtures on MUC2 expression at transcriptional and translational levels in a short bowel syndrome piglet receiving TPN. Piglets underwent massive small bowel resection and received saline, 9mM butyrate, 60mM butyrate or SCFA mixture parenterally for 12 or 72 hours. Quantitative real time PCR showed an increase in MUC2 expression in 9mM compared to other treatments (p=.04). High iron diamine alcian blue stain revealed a trend increase (p=.102) of sulfated mucins in jejunal villi in 9mM over other groups. MUC2 expression tended to increase over time in the ileum regardless of treatment (p=.069). Augmentation of MUC2 and sulfomucins in the proximal bowel by butyrate supplemented TPN enhances barrier function and decreases the risk of bacterial translocation thereby lending support to the use of butyrate as an efficacious treatment of TPN associated intestinal atrophy.

Grant Funding Source: NIDDK 5R01DK057682
Adrenergic tone does not tonically restrain limb venous compliance in healthy humans
Authors: Sielatycki John A. , Spilk Samson J. , Monahan Kevin D.
Published: June 2021

Previously we reported that sympathetic nervous system activation, induced via baroreceptor unloading, acutely decreased calf venous compliance in men. To test the hypothesis that sympathetic nervous system outflow tonically restrains limb venous compliance in healthy humans we studied 7 men (30±4 years old, mean±SE) before and during systemic alpha‐adrenergic (0.1428 mg/kg phentolamine priming dose over 5 min followed by 0.01428 mg/kg/min maintenance dose) and beta‐adrenergic blockade (0.25 mg/kg propranolol priming dose over 15 min followed by 0.004 mg/kg/min maintenance dose). Venous compliance was determined in supine subjects by inflating a thigh venous collecting cuff to 60 mmHg for 8 min, then decreasing pressure at a rate of 1 mmHg/s while measuring calf volume (strain gauge plethysmography). The slope of the pressure‐compliance relation (compliance=β1+2*β2*cuff pressure), which is the first derivative of the quadratic pressure‐volume relation [(Δ limb volume)=β0+β1*(cuff pressure)+β2*(cuff pressure)2] during reductions in collecting cuff pressure, was used to quantify venous compliance. Calf venous compliance was not increased by adrenergic blockade (−2.11±0.43 and −1.49±0.18 before and during adrenergic blockade, respectively). These data suggest that adrenergic tone does not tonically restrain calf venous compliance in healthy men.

Support: NIH HL92309, AG24420 and M01 RR10732
Autonomic regulation in patients with diabetic neuropathy
Authors: Wang Siqi , Randall David C , Knapp Charles F , Nelson Kevin R , Patwardhan Abhijit R , Kilfoil Peter J , Evans Joyce M
Published: June 2021

Cardiac and vascular damage resulting from autonomic neuropathy is a complication of diabetes that often goes undiagnosed. The aims of this study were 1) to quantify damage to cardiac and vascular function in patients diagnosed with peripheral neuropathy, and 2) to develop noninvasive measures to diagnose autonomic neuropathy.

METHODS

Continuous blood pressure, ECG, blood flow and respiration were monitored in 7 able‐bodied subjects (AB), 9 diabetics without (D0), and 7 diabetics with (D1), neuropathy during supine rest and 70° head up tilt (HUT). Mean values, spectral power and partial coherence analyses were used to discriminate groups.

RESULTS

In response to HUT, diabetics increased heart rate to higher levels, but were unable to maintain BP as well as did AB. Low frequency spectral power of SBP and high frequency power of RRI were higher in AB than diabetics during rest and HUT. AB had the highest, and D1 the lowest, slope of baroreflex sequences during rest and HUT.

CONCLUSIONS

Our results support the concept that diabetes diminishes both sympathetic and parasympathetic influence on cardiac and vascular function and suggest that noninvasive indices may help discriminate patients with autonomic neuropathy. Supported by NIH RO1 NS39774, Kentucky NASA EPSCOR WKU 52611, and UK GCRC USPHS grant #M01RR02602. We acknowledge contributions of A Hartman, RN, L Krompak, L Mohney, D Silcox and R Schneider.
Relationship between breathing and cardiovascular function at rest: Sex related differences
Authors: Wehrwein Erica A , Hart E C , Charkoudian N , Joyner M J , Widmer RJ , Eisenach J H , Wallin BG
Published: June 2021

Young men display a positive association between breathing rate and muscle sympathetic nerve activity (MSNA). We compared breathing rate, peroneal MSNA and cardiovascular variables at rest in 17 men (age 27 ± 2 yrs) and 15 women (age 25 ± 1 yrs). Breathing rates and MSNA were similar in men and women but women had significantly lower systolic and mean blood pressures, cardiac output and stroke volume. In men breathing rate correlated positively to MSNA (r = 0.58, p<0.05) but not in women (r = 0.12, p> 0.05).. In men respiratory rate correlated positively to total peripheral resistance (r = 0.65, p<0.05) and inversely to cardiac output (r = − 0.84, p<0.05) and heart rate (r = − 0.60, p<0.05) but there were no such relationships in women (p> 0.05 for all). In another group of subjects, linear regression was performed on respiratory rate and brachial arterial plasma norepinephrine. There was no relationship in men (r=0.0847, n=38) or women (r=0.0367, n=74). Importantly, MSNA does not always predict plasma NE levels at rest. The positive relationship between breathing and MSNA in men, and the inverse coupling of breathing to cardiac output and heart rate suggest that influences of respiration may be important, not only for dynamic, but also for “tonic” cardiovascular function. The lack of relationships among these variables in women shows that there are fundamental differences in basic blood pressure regulation between the sexes.
Molecular effect pathways involved in crustacean molting and in its disruption
Authors: TUMBURU LAXMINATH , Gao Yongping , Wheatly Michele G.
Published: June 2021

In crustaceans, calcium (Ca2+) is not only involved in the role as an intracellular second messenger, but also in the cyclical cycle of mineralization/demineralization knowns as molting cycle. Molting is an endocrine‐mediated process, controlled by ecysteroids. Numerous endocrine disrupting chemicals (EDCs) have been found to interfere with edcysteroid signaling, thereby disrupting the natural molting process. We hypothesize that the hormonal regulation of Ca2+ flux in crustacean molting occurs through a pathway involving cyclic nucleotides. We have successfully cloned and sequenced the ligand‐binding cDNA fragment of ecdysteroid receptor (EcR) and retinoid‐x‐receptor (RXR) from the hepatopancreas of the crayfish, Procambarus clarkii. Using real‐time PCR, we have determined in our observations so far, that molting is a function of ecdysteroid signalling, and can temporally regulate Ca2+ transporters. We have selected two EDCs, a synthetic estrogen 17α‐ethinyl‐estradiol (EE2), and a natural estrogen 17β‐estradiol (E2) to address the hypothesis of this study. Studies are underway to determine the effect of EE2 and E2 on the levels cyclic nucleotides (cAMP and cGMP) and the expression of nuclear receptors (EcR and RXR) and Ca2+ transporters (SERCA: Sarco/Endoplasmic Reticulum Ca2+‐ATPase; PMCA: Plasma Membrane Ca2+‐ATPase; CaM: Calmodulin). (Supported by Grants IBN 0445202 to MGW, YG and CMG).