bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effects of Renin‐Angiotensin System on Endoplasmic Reticulum Stress in Pancreatic Beta Cells
Authors: Sopontammarak Boontharick , Menikdiwela Kalhara , Ramalingam Latha , Moustaid‐Moussa Naima
Published: June 2021

Type 2 diabetes is an emerging epidemic and affects more than 25 million people in the United States. The Renin Angiotensin System (RAS) is classically known to regulate blood pressure and fluid balance. Interestingly, components of RAS, including the product angiotensin II (Ang II), which is the active hormone of this system, are expressed in both adipose tissues and pancreas. Studies conducted in our laboratory have shown that mice overexpressing RAS specifically in adipose tissue had insulin resistance, higher levels of inflammation, and endoplasmic reticulum (ER) stress. However, no studies have investigated the relationship between RAS and ER stress in beta cells. Hence, we hypothesized that activation of RAS in beta cells leads to increased ER stress and reduced insulin secretion. To test this hypothesis, we treated clonal beta cells with Ang II, and found that Ang II significantly increased the expression of ER stress genes such as CHOP and ATF4. In addition, Ang II treatments also reduced insulin secretion. Furthermore, inhibition of Ang II production in beta cells using an angiotensin converting enzyme inhibitor (ACEi) significantly reduced ER stress. Additionally, pharmacological inhibition of angiotensin type I and II receptors significantly reduced ER stress. These results indicate involvement of the RAS pathway in ER stress in pancreatic beta cells. We are currently determining effects of these RAS inhibitors on insulin secretion. In conclusion, this research will help better understand the role of RAS activation via ER stress on diabetes progression and may help develop new anti‐diabetes therapeutic targets.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Protein Phosphatase PHLPP1 Suppresses Insulin Signaling and Inflammation in Mouse Model
Authors: Lorden Gema , Skovsø Søs , Riopel Matthew , Cohen‐Katsenelson Ksenya , Johnson James D. , Newton Alexandra C.
Published: June 2021

The PH domain Leucine Rich Repeat Protein Phosphatase 1 (PHLPP1) directly dephosphorylates and inactivates Akt2, the Akt isozyme that is a key transducer of insulin signaling. Elevated levels of PHLPP1 protein have been associated with obesity and insulin resistance in humans, suggesting PHLPP1 may be a novel therapeutic target to combat insulin resistance in diabetes and metabolic syndrome. Here we examine how loss of PHLPP1 affects insulin signaling using the Phlpp1−/− mice. Insulin tolerance tests of mice on a normal chow diet reveal a modest but statistically‐significant increase in the insulin sensitivity of mice lacking PHLPP1 compared to littermate Phlpp1+/+ controls. Analyses of liver from these mice reveal elevated Akt signaling in the mice lacking PHLPP1, as assessed by increased phosphorylation of Akt itself and downstream substrates such as GSK‐3 and MDM2. In addition, consistent with previous studies, the steady state levels of EGF receptor are elevated. We also note that there was an approximately 15% increase in the steady state levels of PHLPP2. Consistent with in vivo results, deletion of PHLPP1 in HepG2 liver cells also increases insulin‐dependent signaling, assessed by increased phosphorylation of the insulin receptor and Akt. Thus, both in vivo and in vitro studies are consistent with PHLPP1 suppressing insulin signaling. In contrast to results from normal chow diet, Phlpp1−/− and Phlpp1+/+ mice subjected to a high fat diet had the same insulin sensitivity. Furthermore, biochemical analysis of liver from these mice revealed a striking decrease in phosphorylation of Akt and downstream substrates such as S6kinase, and a decrease in PKCα. PHLPP2 levels remained elevated. Additionally, we also found that in livers, high fat diet promotes an up‐regulation of pro‐inflammatory genes such as Il6 and Tnfa, which is significantly enhanced in PHLPP1 deficient mice. Our data are consistent with a model in which loss of PHLPP1 enhances both insulin sensitivity and inflammation. The pro‐inflammatory effects of high fat diet may be exacerbated in the Phlpp1−/− cells, masking an insulin sensitivity phenotype.

Support or Funding Information

This work was funded by NIH P01DK054441

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Proteomic Changes After Surgical Repair in Congenital Heart Diseases
Authors: He Guo‐Wei , Hou Hai‐Tao , Xuan Chao , Wang Jun , Liu Li‐Xin , Zhang Jian‐Feng , Yang Qin
Published: June 2021

Background

The final goal for treatment of congenital heart diseases (CHD) is to resume not only the normal heart structure but also physiology. The present study evaluates the proteomics changes before and after surgery in CHD with tetralogy of Fallot (TOF) or ventricular septal defect (VSD).

Methods

Differential plasma proteins by using two‐dimensional gel electrophoresis and mass spectrometry followed by ELISA validation in new cohort were analyzed in patients with TOF (n=82) or VSD (n=82) compared to normal controls (n=97) preoperatively and 6‐month postoperatively.

Results

A total of 473 protein spots preoperatively and 515 postoperatively including those significantly (p<0.01) downregulated or upregulated were identified. The ELISA validation for chosen proteins demonstrated that in VSD patients, postoperative alpha‐1‐acid glycoprotein 2 and serum amyloid p‐component were completely recovered (p>0.05, compared to control) and complement component C3c was partially recovered (p<0.05, compared to control) towards normal after surgery. In TOF patients, postoperative fibrinogen gamma chain was completely and gelsolin was partially recovered.

Conclusions

We have for the first time demonstrated that CHD repair surgery not only corrects the structure malformation but also resumes the normality of altered proteins that may be developed as biomarkers. Identification of the recovered or unchanged proteins may be used for evaluation of the surgical results and for facilitating the personalized management for CHD.

Support or Funding Information

Supported by grants from the National Natural Science Foundation of China (81170148 & 81641017), Zhejiang Provincial Natural Science Foundation (LY15H020008), & Tianjin Binhai Key Platform for Creative Research Program (2012‐BK110004), Binhai New Area Health Bureau (2012BWKZ008, 2016BWKY007, 2016BWKZ003).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The relationship between anemia and sudden cardiac death in severe aortic stenosis
Authors: Ducharme‐Smith Allison , Chahal Anwar A. , Gersh Bernard J. , Somers Virend K. , Sawatari Hiro , Brady Peter A. , Nkomo Vuyi T. , Pellikka Patricia A.
Published: June 2021

Background

Aortic stenosis (AS) affects, and is associated with significant comorbidity and mortality, including sudden cardiac death (SCD) from unknown mechanisms. However, it is difficult to predict which patients with AS will suffer a SCD event. Anemia and low iron are known risk factors for mortality in heart failure populations and AS. We hypothesized that anemia in patients with AS is associated with increased risk of SCD.

Purpose

Understand the relationship and prognostic implications between anemia and SCD in patients with severe AS.

Methods and Results

We selected 8,144 of 10,850 patients with severe AS (mean transvalvular gradient ≥40 mmHg or peak velocity ≥4 m/s or calculated valve area < 1 cm2) from the Mayo Clinic Aortic Stenosis Registry, which includes all consecutive patients with severe AS over the time period January 1st, 1983 to September 30th, 2015. Subjects in this analysis (median age 76 years and 57% male) were diagnosed starting in 1995 and had hemoglobin (Hb) measurements within one year of diagnosis and prior to intervention. The mean Hb level was 12.8 (± 1.9) g/dL. The prevalence of anemia (Hb<13.0g/dL for men, <12.0g/dL for women) was 40%, 14% microcytic, 20% macrocytic, with the remainder normocytic. Of the 8,144 patients, a total of 4,440 died (10‐year survival 39%; and 240 were confirmed as SCD events [10 year cumulative incidence 4%]). Anemia was associated with increased all‐cause mortality (HR 1.87, 95% CI 1.76–1.99, P=<0.001) and increased SCD events (HR 1.58, 95% CI 1.22–2.06, P=<0.001) in patients with severe AS. Every SD decrease in Hb (1.9 g/dL) was associated with an increased risk of SCD (HR 1.42, 95% CI 1.24–1.62, P<0.001).

Conclusions

Anemia is highly prevalent in patients with severe aortic stenosis and is independently associated with increased all‐cause mortality, and more specifically with increased SCD risk. Possible mechanisms include anemia aggrevating myocardial hypoxia, increasing propensity for arrhythmia and abnormal Bezold‐Jarisch reflex mismatch. Anemia may be a useful prognostic marker and a potential therapeutic target in patients with severe AS.

Support or Funding Information

CAAC is supported by AHA17POST33400211 and NIH HL134885. This study was supported by a grant from the Department of Cardiovascular Medicine, Mayo Clinic Rochester.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A comparative study on phenotypic and molecular characteristics of ischemic stroke resistant animal model
Authors: Kang hyunbon , Won Jinyoung , Hong Yonggeun
Published: June 2021

The use of animal stroke models has substantially increased to develop the therapeutic agents in preclinical studies according to the high mortality rate of cardiovascular disease. Animal ischemic stroke model is generally classified into three types, global ischemia, focal ischemia, and hypoxic ischemia. These animal models have been extensively used in ischemic stroke studies. However, ischemia induced injury models reveal several limitations to identify underlying pathogenesis of stroke. The purpose of this study was to develop the stroke resistant model using ischemia induced cell death signal targeted gene, DAPK1/Pin1 deletion. In addition, stroke resistant model was screened the major phenotypes, physiological functions, and molecular characteristics in organ system. DAPK1/Pin1 single gene knockout (KO) and double gene KO (DKO) mice were generated to determine whether DAPK1 and Pin1 contribute to neuronal death in ischemic injury. The genotype was confirmed by PCR using genomic DNA. DAPK1/Pin1 DKO mice were produced by crossing female DAPK1/Pin1 heterozygous (hetero) with identical genotype male mice. The offspring from DAPK1/Pin1 hetero mice showed significant difference compared with wild type (WT). Body weight and body length of Pin1 KO and DAPK1/Pin1 DKO mice were less than WT and DAPK1 KO mice. DAPK1/Pin1 DKO mice exhibited restricted growth rate. To examine the therapeutic effects of DAPK1/Pin1 deficiency, KO mice were subjected to MCAo. DAPK1/Pin1 DKO mice exhibited smaller infarct size and reduced neurologic deficits compared with single gene KO. Taken together, ischemic stroke resistant model induced by DAPK1/Pin1 genetic suppression could be potential targets for treating stroke.

Support or Funding Information

Funds: NRF‐2013R1A2A2A01067169, NRF‐2017R1A2A2A01067169, KRIBB‐KGM4611714

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CXCR4 inhibitor, MSX‐122 suppresses AOM‐induced colon cancer in Apc+/Min mouse
Authors: Bissonnette Bruce Marc , Dougherty Urszula , Mustafi Reba , Haider Haider I. , Joseph Loren , Souris Jeffrey , Hart John A. , Pewkow Joel R. , LI Yan Chun
Published: June 2021

Introduction

In prior studies, we showed that EGFR is required for tumor promotion by Western diet in the azoxymethane (AOM) model of sporadic colon cancer and the AOM/DSS model of inflammation‐associated colon cancer. We also demonstrated that ADAM17, the metalloprotease responsible for shedding membrane‐bound EGFR ligands, controls tumor development in both the AOM and the Apc+/mutant multiple intestinal neoplasia (Min) mouse model. Furthermore, we established that Sdf1a‐CXCR4 signals activated EGFR by an ADAM17‐dependent mechanism in colon cancer cells; and that Western diet up‐regulated Sdf1a‐CXCR4 and ADAM17 in colonic mucosa. We hypothesized that CXCR4 blockade might inhibit colon cancer development. The aim of this study was to test the ability of a novel CXCR4 antagonist, MSX‐122 (substituted benzylamine) to inhibit AOM‐promoted colon cancer in Apc+/Min mice. Min mice without AOM treatment mostly develop small intestinal tumors, whereas AOM‐treated Apc+/Min mice develop colon cancers (Suzui et al 2002).

Methods

In studies in cell culture, we examined the ability of MSX‐122 to block Sdf1a‐induced EGFR transactivation. In vivo, Apc+/Min mice were given AOM (10 mg i.p./kg body weight) and two weeks later mice were started on a Western diet (20% fat). Mice received MSX‐122 i.p. (2.5 mg/kg body weight, n=12) or vehicle (DMSO, n=13) three times weekly and were killed after 10 wks.

Results

MSX‐122 blocked EGFR transactivation by Sdf1a in HT29 colon cancer cells as assessed by reductions in pErbB2, pAKT and pERK. In vivo, MSX‐122 was well tolerated with mice showing comparable weight gain to vehicle‐treated mice. As expected, AOM induced colon tumors in Apc+/Min mice. MSX‐122 significantly reduced colon tumor incidence (92.3% vs. 50.0%, p=0.025) and cancer incidence (69.2% vs. 16.7%, p=0.015). MSX‐122 also reduced tumor burden as assessed by total tumor multiplicity (1.8 vs. 1.0 tumor per TBM; p=0.02), tumor volume (169.5 mm3 vs. 61.2 mm3, p<0.023) and tumor proliferation as assessed by Ki67 staining (37.2±3.6% vs. 25.3±5.8%, p<0.005). Cancer multiplicity also appeared to be decreased (1.6 vs. 1.0 tumor/TBM, p=0.17).

Conclusions

MSX‐122 blocked EGFR transactivation by CXCR4 agonist Sdf1a in colon cancer cells. MSX‐122 also inhibited colon tumor development and progression in the AOM‐promoted Apc+/Min mouse model. We speculate that MSX‐122, which was well tolerated in a phase 1b clinical trial (Painter, W. 2015), could serve as a chemopreventive agent in individuals at increased risk for colon cancer.

Support or Funding Information

NIH R01CA164124

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cannabinoid Type‐1 Receptors can Mediate the Antinociceptive Effects of Heroin in Nonhuman Primates
Authors: Nilges Mark R. , Bondy Zachary , Grace Joseph Allie , Winsauer Peter
Published: June 2021

Opioid analgesics can provide adequate pain relief under some conditions; however, adverse effects such as dependence, abuse liability, and respiratory depression are major clinical complaints. Cannabinoids such as THC (the psychoactive constituent in marijuana) can produce analgesic effects under some conditions, but generally at higher doses that produce behaviorally disruptive effects. Combinations of opioids and THC produce thermal antinociceptive effects in a synergistic manner, such that ineffective doses of each drug can produce antinociception when given in combination. In this experiment, male nonhuman primates (rhesus macaques) were administered THC in combination with cumulative doses of heroin to determine if THC enhances the antinociceptive effects of heroin. Furthermore, we tested the effects of rimonabant, a selective cannabinoid type‐1 receptor (CB1R) antagonist, to determine the antinociceptive contribution of CB1R. Antinociception was assessed using a tail‐dip procedure. Briefly, the animals were secured in standard primate chairs, tails were shaved, and dipped in a mixed order in three different water temperatures (40°, 50°, and 55°C) until the tail was withdrawn or 20 seconds elapsed. Latency (sec) to remove the tail was converted to percent maximum effect (%MPE). The initial tail dips of a session served as the baseline latency for each temperature, and the subsequent injections were either vehicle (control), THC (0.1 – 1 mg/kg), and/or rimonabant (0.32 mg/kg). These injections were then followed by cumulative doses of either heroin (0.032 – 0.32 mg/kg) or vehicle. Each set of tail dips was separated by a 15‐min period that also served as an inter‐injection interval for the cumulative doses of heroin. When THC was administered prior to heroin, there was a dose‐dependent leftward shift in the heroin dose‐effect curve at both 50° and 55°C temperatures. When rimonabant (0.32 mg/kg) was administered prior to heroin, there was a rightward shift in the heroin dose‐effect curve at the 50°C water temperature, but no change was detected at 55°C. When rimonabant (0.32 mg/kg) preceded THC (1 mg/kg), the enhancement of heroin‐induced antinociception was partially antagonized at the 50°C water temperature, but not at 55°C. Overall, rimonabant attenuated the antinociceptive effects of heroin alone and partially antagonized the enhanced antinociceptive effects of the THC‐heroin combinations at the lower stimulus temperature. These data are consistent with the notion that THC can potentiate the antinociceptive effects of an opioid agonist in a CB1‐dependent manner.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biophysical identification of the 5‐HT2A receptor:5‐HT2C receptor interaction in vitro
Authors: Felsing Daniel Eric , Anastasio Noelle C. , Miszkiel Joanna M. , Gilbertson Scott , Moeller Frederick Gerrard , Allen John A. , Cunningham Kathryn A.
Published: June 2021

Disruption of serotonin 5‐HT2A receptor (5‐HT2AR) and 5‐HT2CR function is implicated in a range of neuropsychiatric disorders with these receptors being essential sites of action for anti‐psychotic and anti‐obesity medications. Based on evidence that the 5‐HT2AR and 5‐HT2CR co‐localize and co‐immunoprecipitate from neurons in the medial prefrontal cortex of the rat (Anastasio et al, ACS Chem. Neurosci. 15:1248, 2015), we hypothesize that these two receptors form a heteromeric complex in living cells. Here, we utilize three distinct biophysical and immunocytochemistry‐based approaches to identify and study this receptor complex in living cells. First, we generated a split luciferase complementation assay to test the hypothesis that the 5‐HT2AR and 5‐HT2CR are in close proximity (< 50 nm). We predicted that inactive luciferase fragments fused to each receptor would be brought into close proximity when receptors form a complex, thereby reconstituting the enzymatic activity of luciferase. A robust complementation (i.e., increased luminescence) was observed upon addition of the substrate D‐luciferin, an outcome that was dependent on protein levels assessed in radioligand binding studies (n=3, p<0.001). Co‐transfection with either the wildtype 5‐HT2AR or 5‐HT2CR inhibited the complementation (n=3, p<0.01). Using a proximity ligation assay, we demonstrate expression of the existence of 5‐HT2AR:5‐HT2CR heteroreceptor complexes in vitro, indicating the protomers are within 40 nm of each other (n=5, p<0.05). To further delineate the receptors are within very close proximity (< 10 nm) in living cells, we generated 5‐HT2AR and 5‐HT2CR constructs for use in a bioluminescence resonance energy transfer (BRET) assay. Preliminary results indicate that the 5‐HT2AR and 5‐HT2CR form a robust BRET interaction creating a maximal signal of 230 mBRET units and a BRET50 of 2.1, which was more than double the signal generated in control studies with the β2‐adrenergic receptor. Taken together, we have identified a very close 5‐HT2AR:5‐HT2CR interaction in living cells. Ongoing research will employ these technologies to further investigate the formation and function of the receptor complex.

Support or Funding Information

NIDA T32 DA07287 “Neural and Pharmacological Mechanisms of Abused Drugs”

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Blood‐brain Barrier Penetrant and Orally Bioavailable Antidotes to Organophosphate Poisoning
Authors: Shyong Yan‐Jye , Garcia Arnold , Sepulveda Yadira , Radić Zoran , Momper Jeremiah , Taylor Palmer
Published: June 2021

We have developed a series of antidotes to organophosphate poisoning, hydroxyiminoacetamido alkylamines, that ionize as cations, anions, zwitterions and form an appreciable neutral species at physiologic pH values. As such they cross the blood‐brain barrier and are orally bioavailable. They appear to be effective in mice and macaques in reversing organophosphate toxicity from accumulating sarin and paraoxon (Rosenberg et al., 2017, Chem‐Biol Interact 274, 50–57; Toxicol Lett. https//doi.org/10.1016/jtoxlet.2017.10.025). We examine here the blood and tissue disposition of these agents in the absence of organophosphate to ascertain pharmacokinetic profiles and achieve optimal loading and maintenance dosing of this family of antidotes. Our data show comparable absorption and tissue distribution after tail vein and retro‐orbital injection with only a slight delay when the antidote is administered intramuscularly. Hence, systemic distribution can be rapidly achieved to reverse acute toxicity. Initial studies of tissue disposition show both rapid transfer into the brain, along with rapid clearance from tissues and blood through likely transport mechanisms. Such kinetics are consistent with post‐administration Protection Ratios of the organophosphate after acute exposure and in vitro profiles of reactivation of the organophosphate inhibited enzyme. As with the quaternary oximes such as 2‐PAM and HI‐6, clearance is rapid requiring a parenteral loading dose and then parenteral or oral maintenance dosing to reverse or avert toxicity and fatality. Such studies in mice enable one to consider effective antidote treatment schemes for longer acting pesticides and acute exposure to the volatile sarin and related nerve agents through respiratory and dermal absorption routes.

Support or Funding Information

Supported in part by NINDS, UO1‐058046

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biasing μ Opioid Receptors with G Protein Inhibitors to Improve Opioid Analgesics
Authors: Campbell Adrian Phillip , Smrcka Alan V.
Published: June 2021

The development of “safer” opioids is difficult owing to the fact that the undesirable effects of opioid analgesics are all on‐target effects at the μ opioid receptor (MOR). A new exciting approach to avoiding side effects has been development of “biased” MOR agonists that alter receptor conformation to preferentially activate analgesic pathways downstream, and away from pathways that promote undesirable side effects [1]. One such drug, Oliceridine, developed by Trevena pharmaceuticals has shown analgesic efficacy in acute pain with limited side effects in Phase IIb clinical trials. Our laboratory has pioneered an alternate approach to biasing GPCR signalling with small molecules that bind to the Gβγ subunits downstream of receptor activation to selectively modify signals downstream of GPCRs. Two prototype molecules, gallein and M119 potentiate the analgesic potency of morphine, without potentiating side effects typically associated with opioid use including acute tolerance, respiratory depression, constipation, hyperlocomotion, reward preference or physical dependence [2–4].

To identify novel small molecule inhibitors of Gβγ with more drug‐like potential we screened a 140, 000 small molecule library comprised of diversity sets from Maybridge and Chemdiv. 36 compounds passed primary testing, triplicate retesting, dose response analysis, counterscreening for assay interfering compounds and triaging to exclude predicted aggregators, PAINS, toxic or reactive compounds and structures with limited drug‐like potential. The molecules have potencies of 2–100 μM in our primary Alphascreen assay (compared to 4 μM for the control SIGK peptide) and multiple compounds inhibit Gβγ activation of PLCβ in in vitro assays of PLC activity with potency equal to or better than the small molecule gallein. One compound has also shown potentiation of opioid analgesia similar to morphine in mice. The hits possess a variety of novel scaffolds and represent good starting points for further medicinal chemistry optimization and represent possible novel potentiators of opioid analgesia.

Support or Funding Information

5 R01GM0817720‐08

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel Approach to Safer Analgesics: Mu Opioid Receptor Agonist & Delta Opioid Receptor Antagonist Peptidomimetics
Authors: Fernández Thomas Joaquín , Griggs Nicholas , Nastase Anthony , Anand Jessica , Jutkiewicz Emily J. , Mosberg Henry , Traynor John
Published: June 2021

Opioid analgesics exert their therapeutic and unwanted effects by activation of the mu‐opioid receptor (MOR). Repeated administration of opioid analgesics and subsequent chronic MOR activation promotes opioid tolerance and physical dependence. It has been proposed that activation of MOR with concurrent antagonism of the delta‐opioid receptor (DOR) can attenuate the development of opioid tolerance and dependence. To further investigate the utilization of concurrent MOR agonism and DOR antagonism we designed, synthesized, and characterized a library of “peptidomimetics” that have elements of opioid peptides vital for activity but retain small molecule‐like features necessary for blood‐brain‐barrier penetration. Compounds were evaluated in membrane preparations of cloned cell lines expressing either MOR, DOR, or kappa‐opioid receptors (KOR). [35S]‐GTPγS binding to measure G protein‐coupled activation was used. Full agonists at each opioid receptor (DAMGO, DPDPE, and U69,593, respectively) were employed as standards. Maximum binding and potency (EC50) values were determined from concentration‐effect curves. To measure affinity (Ki) values, competition binding using [3H]‐diprenorphine was performed. Peptidomimetics that demonstrated promising bi‐functional activity were tested for antinociceptive activity in vivo. Compared to repeated morphine‐treated mice, repeated peptidomimetic treated mice developed less analgesic tolerance or physical dependence. Analysis of in vitro and in vivo data has permitted structure‐activity relationships to guide further chemical synthesis and the discovery of opioid analgesics that may have improved clinical utility.

Support or Funding Information

Supported by DA‐03910

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulatory Effect of Fruit‐derived Nanoparticle on Intestinal Transporters
Authors: Fujita Daichi , Arai Toshiki , Shirasaki Yuma , Iwamoto Yui , Komori Hisakazu , Nakanishi Takeo , Tamai Ikumi
Published: June 2021

Drug food interaction is an important issue, since intake of food often alters pharmacokinetics and subsequently reduces efficacy and/or adverse effect of concurrently administered drugs. Although it has been reported that diet or beverage affects drug transporters and drug metabolizing enzymes, they were explained only by small molecules in foods. Recently, several studies have shown that plant‐derived nanoparticles (NPs) taken up by the intestinal cells affect intestinal function. NP should contain large molecules such as protein, lipid and nucleic acid derived plant cell stably. Although large molecules are not stable by digestion and impermeable into the cells, those in NP could be overcome those matters. Thus, we investigated that edible plant‐derived NP on intestinal transporter as factor involved in drug‐food interaction. Apple and grapefruit were squeezed and NP fraction was collected by ultra‐centrifugal separation. The fraction contained 100 to 400 nm particles and small molecules such as flavonoid and furanocoumarins that affect intestinal transporter were mostly excluded. When lipid membrane of NP fraction is labeled by PKH26 and incubated with Caco‐2 cells, the signals of fluorescence of labeled NP were observed in the cells, showing internalization of the NP. When mRNA expression and transport activity in Caco‐2 cells were evaluated after exposure with those NP fraction, several transporters such as ASBT, OCTN2 and OATP2B1 out of examined nine transporters showed decreased expressions and activity. Moreover, when NPs were disrupted by sonication or boiling, NPs were significantly decreased and those changes in expression of transporter genes were mostly disappeared. When mRNA expression in HEK293 cells transfected with cDNA of OATP2B1 was evaluated by those NP fraction, mRNA of OATP2B1 was only decreased in HEK293 cells transfected with plasmid containing 3′ untranslated region (UTR) of OATP2B1. These results suggested that edible plant‐derived NPs, which reduce mRNA expression of intestinal transporters, could be one of possible mechanisms of drug‐food interaction. Furthermore, 3′UTR was involved in decreased mRNA of OATP2B1, suggesting that plant microRNA contained affected mRNA expression. In conclusion, fruit‐derived NP reduced mRNA expression and activity of intestinal transporter, suggesting that high molecules in NP are involved in drug food interaction. Moreover, it is possible that plant‐derived NPs contribute to regulation of transporters for drugs and nutrients absorption by expression modulation of transporters.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Wnt and TLR4 Pathway modulators as a Promising Therapeutic Strategy in the Treatment of Glioblastoma
Authors: Esposito Emanuela , Casili Giovanna , Campolo Michela , Lanza Marika , Filippone Alessia , Cuzzocrea Salvatore
Published: June 2021

Glioblastomas (GMB) are highly aggressive brain tumors. Despite recent improvements of care in the fields of surgical resection, radiotherapy and alkylating chemotherapy, the 5‐year survival rate for patients with GBM remains low and additional therapies are needed. Various pathways are involved in glioma tumorigenesis, among which the Wingless (Wnt) signaling. Dickkopf protein‐related protein 3 (Dkk‐3) interacts with proteins of Wnt pathway as inhibitor. The Wnt signaling contributes to activity of the claudins, important components of Tight junctions (TJ), implicated in blood‐brain barrier (BBB) formation, whose expression was altered selectively in cerebral microvessels of human GBM. The aim of this study was to determine the role of Wnt pathways to directly regulate tumor grown, apoptosis process by targeting Dkk‐3, TJ alteration involving claudin‐5 and to suggest possible therapeutic interactions involving Wnt/Toll‐like receptors (TLRs) pathways. In the present study we investigated the expression of Dkk‐3, Claudin‐5, apoptosis markers and TLR‐4 receptor protein levels in in vitro studies on U‐138MG and A‐172 human glioma cell lines and in GBM human patient's tissues. We showed a significant Dkk‐3 and claudin‐5 down regulation, with the apoptosis process involvement and with an interesting TLR‐4/Wnt modulation. We concluded that Wnt and TLR4 pathway modulators might shift the balance from tumor growth stasis to cytotoxic responses, representing a promising approach in the treatment of glioblastoma.

Support or Funding Information

No founding

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The broad‐spectrum antiemetic efficacy of the ryanodine receptor antagonist, dantrolene, in the least shrew (Cryptotis parva)
Authors: Zhong Weixia , Darmani Nissar A.
Published: June 2021

Dantrolene, a muscle relaxant, is clinically used for treatment of muscle spasticity as well as malignant hyperthermia since it inhibits Ca2+ ion release from intracellular sarcoplasmic reticulum stores by antagonizing ryanodine receptors (RyRs). We have recently demonstrated that intracellular Ca2+ mobilization is involved in emesis, and the dihydropyridines, L‐type Ca2+ channels blockers such as nifedipine and amlodipine, have broad spectrum antiemetic activity. Substantial evidence indicates that a physical and functional connection between LTCC and RyR mediates Ca2+‐induced Ca2+ release. The aim of this study was to evaluate the antiemetic efficacy of dantrolene. Fully effective emetic doses of diverse emetogens such as selective and/or nonselective agonists of serotonergic 5‐HT3 (e.g. 5‐HT or 2‐Me‐5‐HT)‐, tachykinin NK1 (e.g. GR73632)‐, dopamine D2 (e.g. quinpirole)‐, cholinergic M1 (e.g. McN‐A343 or pilocarpine)‐receptors, the Ca2+ LTCC agonist (FPL 64176), the inhibitor of the endoplasmic reticulum Ca2+ pump (thapsigargin), or the chemotherapeutic agent cisplatin, were administered intraperitoneally in the least shrew to induce vomiting. The antiemetic potential of dantrolene was evaluated against the discussed emetogens. Dantrolene pre‐treatment (0.1–10 mg/kg, i.p.) in a dose‐dependent and potent manner attenuated both the vomit frequency and percentage of shrews vomiting in response to administration of 5‐HT (5 mg/kg), 2‐Me‐5‐HT (5 mg/kg), GR73632 (5 mg/kg), quinpirole (2 mg/kg), McN‐A343 (2 mg/kg), pilocarpine (2 mg/kg), FPL 64176 (10 mg/kg) or thapsigargin (0.5 mg/kg). Though dantrolene alone failed to affect cisplatin‐induced vomiting, a combination of non‐effective doses of dantrolene (2 mg/kg) and the 5‐HT3 receptor antagonist palonosetron (0.05 mg/kg) were more effective against chemotherapeutic agent cisplatin‐induced vomiting during both acute and delayed phase than their corresponding doses tested alone. In conclusion our data supports the notion that: i) Ca2+‐mobilization signals play an important role in emesis; and ii) the RyRs can be a potential therapeutic target for the treatment of emesis.

Support or Funding Information

NIH‐NCI RO1CA207287

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The effects of cardiac contraction on cerebral oxygenation during supraventricular and ventricular tachycardia
Authors: Komine Hidehiko , Machino Takeshi , Yamagami Fumi , Nogami Akihiko , Aonuma Kazutaka , Tsurushima Hideo , Ishii Kei , Hatori Yasuhiro , Gwak Jongseong , Takahashi Naoki , Kitazaki Satoshi , Akamatsu Motoyuki
Published: June 2021

Cerebral vasoconstriction during hypotension with ventricular tachycardia has been reported, but the underling mechanism was unclear. We tested a hypothesis that the cardiac contraction, i.e. heart rate and/or contractility, causes cerebral vasoconstriction and thereby a decrease in cerebral oxygenation during supraventricular tachycardia (SVT) and ventricular tachycardia (VT). Heart rate (HR), arterial blood pressure (AP), and the prefrontal tissue oxygen index (TOI) with near‐infrared spectroscopy were measured during electrophysiological study in 8 patients. We selected similar hypotension trials ranging from −30 to −50 mmHg in SVT (10 trials, 4 patients) and VT (9 trials, 4 patients), and then compared HR and TOI responses. Although the decreases in AP were similar in SVT and VT (−37 ± 2 vs. −38 ± 2 mmHg), the prefrontal TOI decreased greater in SVT than VT (−5.2 ± 0.4 vs. −3.3 ± 0.6%, P < 0.05). HR increased greater in SVT than VT (85 ± 3 vs. 74 ± 8 beats/min, P < 0.05), and the increase in HR correlated with the TOI fall (r = −0.80, P < 0.05). The ejection fraction in SVT and VT were 70 ± 2 and 44 ± 14%. These results suggest that a higher cardiac contraction with tachycardia may lead to decrease in cerebral oxygenation, which is irrespective of hypotension.

Support or Funding Information

This study is supported by Automotive and Medical Concert Consortium (AMECC)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Duty Cycle Impairs Functional Sympatholysis During Moderate Intensity Hand‐grip Exercise
Authors: Caldwell Jacob T. , Hammer Shane M. , Post Hunter K. , Alexander Andrew M. , Didier Kaylin K. , Lovoy Garrett M. , Ade Carl J.
Published: June 2021

Functional sympatholysis is a well‐known mechanism that blunts the sympathetic vasoconstrictor signal in working muscle, enabling blood flow to be maintained during exercise in the moderate intensity domain. Recent work has demonstrated that exercise intensity and contraction frequency effects functional sympatholysis via changes in metabolic rate. However, less is known about the effects duty cycle (DC) on functional sympatholysis, which alters the time that intramuscular pressure is developed throughout a contraction cycle independent of changes in frequency and intensity. Given that a higher duty cycle (i.e., 50% DC) attenuates muscle blood flow to the working limb, potentially leading to a greater metabolite buildup, we hypothesized that a 20% DC would show greater percent change in forearm vascular conductance (%Δ FVC) and forearm blood flow (%Δ FBF) when compared to 50% duty cycle.

METHODS

Eight healthy male subjects (age: 25 ± 1 yrs; height: 176.8 ± 1.5 cm; weight: 84 ± 5 kg) agreed to participate in the current investigation. Subjects began with a four‐minute resting protocol, two‐minutes of supine blood pressure and blood flow and two‐minutes utilizing lower‐body negative pressure (LBNP; ‐30 mmHg) to increase sympathetic outflow. Subjects then underwent two exercise conditions in a randomized order consisting of 20% and 50% DC (20%:50% or 50%:20%) with ~10 min between trials to allow blood pressure and blood flow to return to baseline. All hand‐grip exercise was performed at 20 contractions per minute at a workload of 20% of subject's maximal voluntary contraction.

RESULTS

Absolute FVC during the 20% duty cycle condition was significantly higher than the 50% duty cycle (20%: 398 ± 34; 50%: 327 ± 37 ml min−1 mmHg−1; P < 0.01). FBF was also significantly higher in the 20% duty cycle condition (20%: 392 ± 21; 50%: 330 ± 29 ml min−1; P < 0.01). During steady state hand‐grip exercise coupled with LBNP, %Δ FVC, an indicator of sympathetically‐mediated vasoconstriction, showed a larger percent reduction during 20% duty cycle (20%: −12.6 ± 1.5%; 50%: 1.4 ± 3.9%; P < 0.01).

CONCLUSION

The results of the current investigation suggest that the 20% DC had a greater sympathetically‐mediated vasoconstrictor response compared to the 50% duty cycle.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dynamic Renal Blood Flow Autoregulation During Recovery From Acute Kidney Injury (AKI)
Authors: Thomson Scott Culver , Pham Hai , Singh Prabhleen
Published: June 2021

Background

If efficiency of RBF autoregulation is impaired during recovery from AKI, then the recovering kidney would be susceptible to recurrent injury from fluctuating blood pressure or nerve traffic. Hence, it is worthwhile to know about RBF autoregulation in the injured kidney for someone prescribing treatments to patients with AKI that impinge on systemic hemodynamics, such as hemodialysis.

Methods

Wistar rats were subjected to 60 minutes of bilateral renal ischemia and studied again after 1, 7, or 28 days (4–7 animals per group). GFR was measured by inulin clearance and while RBF (Transonics flow probe) and BP (arterial catheter) were recorded for 2 hours at 1Hz. Time series were used to calculate admittance gain from short‐term Fourier transforms, and autoregulatory step responses in the time domain by a method of moving averages.

Results

RBF returned to baseline within 1 day of ischemia‐reperfusion, while GFR normalized some time between the 1 and 4 week time points. Step responses reveal the RBF autoregulation was less efficient at the 1 and 7 day time points when GFR was low but was nearly normal at 4 weeks into renal recovery. Tubuloglomerular feedback (TGF) signatures (resonance in the admittance gain around 0.03 Hz and transient overshoot in the step response at ~ 30 seconds) were missing while the GFR was low, but were restored at 4 weeks when GFR had returned to normal. Admittance gain shown in figure where TGF and myogenic resonance peaks denoted. Step responses shown without error bars for clarity, but the major apparent intergroup differences were significant.

Conclusion

The findings are consistent with prior prediction that RBF autoregulation should be less efficient in the absence of a varying TGF signal, which is necessarily the case when GFR is low. A caveat is that, while the spontaneous BP power was good enough to yield robust admittance functions and step responses, these studies don't address the response to large BP disturbance, where the deficit in autoregulation could be more profound. Additional experiments with BP forcings would be required for that.

Support or Funding Information

Department of Veterans Affairs
Denervation of Peripheral Chemoreceptors Decreases Heart Rate During Bilateral Carotid Occlusion in Unanesthetized Rats
Authors: Brognara Fernanda , Castania Jaci Airton , Dias Daniel Penteado Martins , Kanashiro Alexandre , Ulloa Luis , Fazan Rubens , Salgado Helio Cesar
Published: June 2021

Bilateral carotid occlusion (BCO) has been used as a maneuver to cause global reflex activation of the sympathetic nervous system in both unanesthetized and anesthetized animals. It is known that the cardiovascular effects of BCO involve not only the baroreceptors but also the chemoreceptors. However, to our knowledge, the cardiovascular responses produced during BCO in the absence of the peripheral chemoreceptors has not been studied in unanesthetized rats. Therefore, the present study aimed to examine the responses of arterial pressure and heart rate to BCO in unanesthetized rats with selective denervation of peripheral chemoreceptors. Under ketamine/xylazine anesthesia Wistar Hannover rats were implanted with pneumatic cuffs around the common carotid arteries for BCO. Catheters were also inserted into the femoral artery and vein for blood pressure recording and drug administration (to test the effectiveness of denervation), respectively. Rats were divided into two groups: INTACT (n = 7) and chemoreceptors removed (CHEMO‐X; n = 8). In the next day, BCO was performed for 20s in unanesthetized rats. As compared to baseline condition BCO promoted, in both groups, an increase in systolic (INTACT: 133 ± 5 vs. 211 ± 8 mmHg; CHEMO‐X: 142 ± 3 vs. 210 ± 4 mmHg), diastolic (INTACT: 92 ± 5 vs. 145 ± 7 mmHg; CHEMO‐X: 100 ± 2 vs. 140 ± 3 mmHg) and mean arterial pressure (INTACT: 111 ± 5 vs. 173 ± 7 mmHg; CHEMO‐X: 119 ± 2 vs. 168 ± 3 mmHg). Of note, the increase in arterial pressure was similar in both groups; however, while BCO did not promote a change of the heart rate in the INTACT group (321 ± 9 vs. 318 ± 7 bpm), the rats from the QUIMIO‐X group displayed significant bradycardia (352 ± 7 vs. 315 ± 14 bpm). These findings indicate that the presence of the peripheral chemoreceptors did not affect the hypertensive response elicited by BCO, but their absence determined a reflex bradycardia during this occlusive maneuver. This mechanism deserves further investigation.

Support or Funding Information

FAPESP (Proc. 2017/05163‐6 and 2013/20549‐7).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Association of Bilateral Pulse Wave Velocity and Inter‐Arm Systolic Blood Pressure Difference in Apparently Healthy Individuals
Authors: Jensen Brock T. , Walsh Maureen A. , Clarke Melanie M. , Allen Sarah R. , Wikert Tianna N. , Brush Amanda C. , Holmstrup Michael E.
Published: June 2021

Bilateral, either simultaneous or sequentially measured, blood pressure (BP) is used for the screening and diagnosis of inter‐arm systolic blood pressure difference (ISBPD). ISBPD, wherein a difference of ≥10 mmHg in systolic BP between arms is noted, is associated with peripheral vascular disease, type 2 diabetes, and arterial stiffness. Similarly, arterial stiffness is a strong determinant of cardiovascular risk. Pulse wave velocity (PWV) is widely used as the gold‐standard measurement of arterial stiffness. However, bilateral PWV is not commonly performed, and procedural measurement of PWV has yet to achieve clear standardization. Further, existing evidence is very limited regarding the relationship of bilateral carotid‐radial PWV measurement and ISBPD.

PURPOSE

To determine the association between bilateral BP and carotid‐radial PWV.

METHODS

To identify the relationship between bilateral carotid‐radial PWV measurement and BP, we studied 53 subjects (26±7.4 years, 57% female) with no known cardiovascular disease. Bilateral BP was acquired both sequentially (BPSEQ) and simultaneously (BPSIM) following a 10‐minute rest period in the supine position. Right and left carotid‐radial distances were measured using an Infantometer. Bilateral carotid‐radial PWV was obtained using a Complior Analyse™ simultaneously. T‐tests were performed to determine differences in BPSEQ and BPSIM and right and left carotid‐radial pulse wave velocity (PWVR; PWVL), and correlation coefficients determined the relationship between bilateral BP and PWV differences (PWVD).

RESULTS

Bilateral BPSEQ and BPSIM measurement was similar (1.5±7.2 mmHg and 1.2±4.6 mmHg, respectively; P > 0.05) and BP was not different between arms regardless of method (P > 0.05). Interestingly, bilateral carotid‐radial PWV was different (PWVR = 7.8±1.3 m/s and PWVL = 7.3±1.1 m/s; P < 0.05). BPSEQ and BPSIM is poorly correlated to PWVD (R = −.09 and R = .009, respectively; P > 0.05). However, PWVR was strongly correlated to PWVD (R = .63; P < 0.05).

CONCLUSIONS

Greater PWV is associated with a greater bilateral PWV difference. The consideration for bilateral measurement of PWV deserves further investigation. Bilateral BP and PWV differences may be valuable additions to a cardiovascular risk profile.

Support or Funding Information

Pennsylvania State System of Higher Education, Faculty Professional Development Council

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.