bluuebunny/crossref_metadata_2025 huggingface dataset:
Elucidating the Role of DEPTOR in Cultured Glioblastoma Multiforme Cancer Cells
Authors: Ryan Patrick J , Deaver J William , Koo Sunwoo , Touissaint L Gerard , Fluckey James D
Published: June 2021

Glioblastoma multiforme (GBM) is the primary malignancy of the brain, representing 45% of all central nervous system tumors. Aggressive and incurable, GBM is generally fatal within two years of diagnosis, despite the best treatment. The cause and progression of these tumors remains a mystery; however, what is known about GBM pathology indicates a role for the anabolic AKT/mTOR pathway in tumorigenesis and growth. Our group and others recently identified an endogenously produced direct inhibitor of mTOR, DEPTOR, which binds mTOR and potently inhibits its activity. As previous attempts to prevent cell growth in GBM have only been marginally successful, manipulation of an effective innate inhibitor of mTOR could provide a viable avenue toward new therapeutics for this deadly cancer. With this in mind, we set out to characterize the role of DEPTOR in cultured glioblastoma cells. Immortalized U87 glioblastoma cells were grown and harvested in cell culture, along with U87 cells selected for invasive potential to model a more aggressive subset of tumors (designated IM3), and cultured astrocytes to provide an adjacent tissue comparison. Western blot methodology was used to investigate the status of several key proteins in the AKT/mTOR axis (including DEPTOR), as well as the relative abundance and phosphorylation status of downstream targets of mTOR (S6K1 and 4EBP1). We determined that key measures of anabolic signaling were elevated in the cultured GBM cells, with double the ratio of phosphorylated to total S6K1 observed in both GBM lines versus the astrocyte, along with reductions in total 4EBP1, indicating upregulation of cap‐dependent protein synthesis. Further, DEPTOR expression was reduced five‐fold in cultured GBM cells, indicating that this “brake” on mTOR has been removed, resulting in rampant and out‐of‐control anabolism. Taken together, these findings hint that lack of DEPTOR may play a role in GBM pathology, and suggest that further investigation into the interplay between DEPTOR, mTOR, and cancer cell growth in GBM is warranted.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Fatty Acid Oxidation and Glucose Oxidation in Endothelial Cell Inflammation and Permeability Associated with Acute Lung Injury
Authors: Ruiz Vania Lopez , Leonard Antony , Rahman Arshad , Fazal Fabeha
Published: June 2021

Acute lung injury (ALI) is a common cause of respiratory failure in critically ill patients with a mortality rate of 38.5%. ALI can be precipitated by either direct insults such as pneumonia, aspiration or via indirect insults such as sepsis and multiple trauma, to the lungs. The vascular endothelium forming the innermost lining of all pulmonary blood vessels is the major barrier that protects air spaces against vascular fluid entry. Because of their localization, endothelial cells (EC) are constantly exposed to oxygen and nutrients that fuel cell metabolism. However, very little is known about the role of EC metabolism in the regulation of EC inflammation and permeability, the two major pathogenic features of ALI. Recent studies have highlighted that fatty acid oxidation (FAO) is central to the tight regulation of EC permeability required to maintain vessel functionality. The purpose of the present study is to understand the mechanistic link between cell metabolism and thrombin‐induced EC inflammation and permeability and to understand the relevance of this linkage in the pathophysiology of ALI. Human Pulmonary Artery Endothelial cells (HPAECs) were stimulated with proinflammatory mediator thrombin in the presence and absence of Etomoxir, an inhibitor of FAO or dichloroacetate (DCA), an inhibitor of pyruvate hydrogenase kinase (PHK). Our results indicate that both Etomoxir and DCA inhibited thrombin‐induced inflammatory responses as observed by decrease in the secretion of cytokines IL‐6 and IL‐8. In addition real time changes in transendothelial electrical resistance (TER) were measured to monitor endothelial permeability after exposure of HPAECs to Etomoxir or DCA, followed by thrombin treatment. Our data indicate that Etomoxir had no effect on thrombin‐induced decrease in TER however it delayed the recovery after 1 hour of thrombin challenge. DCA in contrast showed no effect on thrombin‐induced EC permeability. Together our data suggest that metabolic pathways are critical to EC inflammation and permeability induced by thrombin.

Support or Funding Information

Supported by: NIH (HL130870)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cardiovascular Responses to the Exercise Pressor Reflex in Pediatric Heart Transplant Recipients
Authors: Blushke Corey R , Boyes Natasha G , Lahti Dana S , Kosokowsky Kylee , Marciniuk Darcy , Butcher Scotty J , Erlandson Marta C , Wright Kristi D , Pharis Scott , Pockett Charissa , Tomczak Corey R
Published: June 2021

Pediatric heart transplant recipients (HTR) have reduced exercise tolerance despite normal systolic ventricular function. A slower heart rate adaptation to exercise in HTR due to cardiac denervation is commonly observed in HTR patients, which explains, in part, the exercise intolerance. We tested the hypothesis that the cardiovascular response to exercise pressor reflex activation from handgrip exercise would be blunted in HTR.

Methods

Six pediatric HTR (3f/3m; mean±SD, 10±3 yrs) and five healthy‐matched controls (CTL; 3f/2m; 10±2 yrs) were studied. After 3 min of resting baseline, subjects performed 2 min of isometric handgrip (HG) exercise at 40% maximal voluntary contraction, followed by 3 min post‐exercise circulatory occlusion (PECO). Beat‐by‐beat mean arterial pressure, heart rate, and modelflow stroke volume and cardiac output were recorded. Data were analysed as the change (Δ) from rest using two‐way repeated measures ANOVA (2 × 3; group × condition) and Holm‐Sidak multiple comparisons analysis. Significance was accepted at P<0.05.

Results

Mean arterial pressure increased in HTR (Δ17±15 mmHg) and CTL (Δ13±16 mL) during HG (P<0.001) and remained elevated above baseline during PECO (HTR: Δ10±10 mmHg; CTL: Δ11±9 mmHg; P<0.05), with no between group differences. Heart rate did not significantly increase during HG in HTR (Δ4±5 bpm, P=0.235) but did in CTL (Δ14±11 bpm, P<0.001), and was 9 bpm lower in HTR vs CTL (P=0.005). Heart rate was not different during PECO from rest in HTR and CTL. Stroke volume increased in HTR (Δ3±1 mL) and CTL (Δ3±3 mL) during HG (P<0.001) and remained elevated above baseline during PECO (HTR: Δ3±2 mL; CTL: Δ4±3 mL; P<0.001), with no between group differences. Cardiac output did not significantly increase during HG in HTR (Δ0.34±0.21 L/min, P=0.074) but did in CTL (Δ0.71±0.54 L/min, P<0.001), and was 0.37 L/min lower in HTR vs CTL (P=0.035). Cardiac output was not different during PECO from rest in HTR and CTL.

Conclusions

Owing to the impaired heart rate response during handgrip exercise, cardiac output was lower in pediatric HTR compared to healthy‐matched controls in response to exercise pressor reflex activation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Paracrine and Intracellular Angiotensin II on Protein Kinase C, MAP kinase ERK1/2, and Nuclear Factor‐κB Signaling Responses in Mouse Glomerular Mesangial cells
Authors: Leite Ana Paula Oliveira , Li Xiao C. , Zhuo Jia L.
Published: June 2021

Angiotensin II (ANG II) acts not only as a vasoconstrictor, but also as a growth‐promoting cytokine and chemokine, proinflammatory and profibrotic factor in the kidney. Glomerular mesangial cells (MC) are a key target for endocrine, paracrine, autocrine, and intracrine ANG II. Augmentation of local paracrine and intracrine ANG II generation in MCs may induce glomerular injury by promoting MC cell growth, hypertrophy and fibrosis. In the present study, we performed phosphoproteomic characterization of ANG II‐induced protein kinase C (p‐PKCα), MAP kinase ERK1/2 (p‐ERK1/2), p38 MAPK, and nuclear factor‐κB (p65 and p50 subunits) signaling responses in murine glomerular MCs. Murine mesangial cells (MES‐13, ATCC) were grown to ~80% confluence in 6‐well plates in DMEM and Ham's F‐12 medium, containing 5% fetal bovine serum, and 1% penicillin‐streptomycin, maintained at 37°C and 95% O2/5% CO2. After starved at a serum‐free medium for 24 h, MCs were then treated with low to high levels of ANG II (0.1 to 100 nM) for 5 to 20 min, or transfected with a mitochondria‐targeting intracellular ANG II for 48 h (mito‐ECFP/ANG II, 4 μg/well), with or without pretreatment with the AT1 receptor blocker losartan (1 μM), AT2 receptor blocker PD123319 (1 μM) or both blockers. Proteins were extracted for Western blot analysis of p‐PKCα, p‐ERK1/2, p38 MAPK, and p65 and p50 subunits of NF‐κB, respectively. ANG II significantly increased p‐ERK1/2 and p‐PKCα in both dose‐ and time‐dependent manners, with peak responses to ANG II occurred at 0.1 and 1.0 nM, 5–10 min (p<0.05), respectively. ANG II also increased p38 MAPK response, but it peaked at 1 nM, 20 min (p<0.05). By contrast, ANG II appeared to decrease the NF‐κB responses at 1 nM, 10 min, which were reversed by losartan and PD123319. The expression of mito‐ECFP/ANG II selectively in the MC mitochondria significantly increased mitochondrial complex proteins (CI‐CV), which were blocked by losartan (p<0.01). Our results suggest that both paracrine and intracellular ANG II may activate AT1 receptors to induce important intracellular PKC, MAP kinase ERK1/2, p38 MAPK, and NF‐κB signaling responses in MCs, which may contribute to ANG II‐induced glomerular injury in hypertension and kidney diseases.

Support or Funding Information

Supported by National Institute of Health grants, 2R01DK067299‐10A1 and 2R01DK102924‐03A1 to Zhuo, and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior ‐ CAPES to Leite.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Influence of GATOR1 Sub Complex NPRL2 on Anabolic Signaling During Insulin Resistance in Rats
Authors: OReilly Colleen , Greene Nicholas , Wiggs Michael , Fluckey James
Published: June 2021

Skeletal muscle metabolism plays a significant role in dysregulation of protein synthesis signaling pathways in skeletal muscle is a contributing factor in multiple disease states including Type 2 Diabetes (T2D). In insulin resistant rats, there is a well‐documented increase in mammalian target of Rapamycin (mTOR) activity, in contrast to observed muscle mass losses. In several types of cancer, the same mTOR increases were attributed to a deficiency in at least one of the GATOR1 sub complexes (DEPDC5, NPRL2, NPRL3). GATOR1 is a GTPase activating protein, which when active, is an mTOR inhibitor. When one or more of these sub complexes are deficient, GATOR1 will not inhibit mTOR. NPRL2 is specifically associated with the Gap activity observed with Gator1 and it's mTOR inhibition. It has been shown that specific knockout of NPRL2 in mouse skeletal muscle resulted in increased mTORC1 activity. While this deficiency and its relationship to downstream targets of mTOR are documented in multiple cancer models, the relationship of GATOR1 sub complexes with insulin resistance is less clear. The purpose of this study was to determine the relationship between NPRL2 and the observed mTOR response to insulin resistance as well as this relationship with or without exercise.

METHODS

Male obese and lean zucker rats were given twelve days of acclimation and then assigned to control or exercise groups blocked for lean mass. The exercise groups were put through 4 bouts of resistance exercise over an eight day period using the “squat” resistance model.

RESULTS

Our results indicate NPRL2 content is lower in the obese rats with or without prior exercise. This corresponds to higher mTOR activity, demonstrated by phosphorylation of p70S6k, being higher in the obese rats than in the lean rats.

CONCLUSION

These results contribute to the understanding of insulin resistance and muscle's metabolic role in that resistance. NPRL2 and the GATOR1 complex may be another key player in the understanding of the mTOR dysregulation with metabolic disease.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Skeletal Muscle Anabolic Response Following Incubation with Protein Kinase C Activator OAG
Authors: Deaver J William , O'Reilly Colleen L , Cross Kristina M , Fluckey James D
Published: June 2021

Purpose

For years, there has been a growing interest in the role of lipids in the progression of type 2 diabetes. Specifically, molecules such as diacylglycerol have garnered attention due to its concomitant increase with the progression of insulin resistance and type 2 diabetes. These experiments aim to further characterize the role of diacylglycerol in cellular metabolism by using the diacylglycerol mimetic, 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) alongside mTOR inhibitors rapamycin or Torin 1.

Methods

C2C12 myoblasts were grown and maintained in 10 cm plates with high glucose DMEM growth media until reaching ~80% confluence, at which point they were transitioned to a high glucose differentiation media to encourage myotube formation. Six days following differentiation, plates were randomly assigned to treatment groups (n=3) and treated in the presence of 4% deuterium oxide for 24 hours. Myotubes were harvested with cell scrapers in the presence of Norris lysis buffer, and cellular contents were fractionated for measures of both protein synthesis rates and anabolic signaling via western blot analysis.

Results

Presence of OAG (20 μM) did not significantly alter 24 hour rates of protein synthesis (1.728%/hr) compared to controls (1.630%/hr) (p = 1.000). Rapamycin (1.333%/hr) and Torin 1 (1.187%/hr) treatments significantly reduced protein synthesis rates compared to controls (p = 0.003 and p < 0.001 respectively). However, OAG did not have any statistically significant effect on mitigating the suppressive effects of rapamycin (OAG+Rapamycin 1.380%/hr) (p=1.000) or Torin 1 (OAG+Torin 1 1.158%/hr) (p=1.000).

Conclusions

OAG is known to be a potent activator of Protein Kinase C as a cell permeable analog of diacylglycerol. These data indicate that a 24‐hour incubation with OAG is not sufficient to significantly perturb fractional rates of protein synthesis in murine skeletal muscle myotubes.

Support or Funding Information

Work by this author was partially supported by the Sydney & J.L. Huffines Institute for Sports Medicine & Human Performance, and a Texas A&M College of Education and Human Development Graduate Research award.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Attenuation of Ovalbumin‐induced Airway Inflammation by Compound of Portulaca oleracea L. Extract through Regulating Mast Cell‐Mediated Inflammatory Responses
Authors: Lee Soyoung
Published: June 2021

Asthma is a chronic airway inflammatory condition characterized by breathing difficulty due to bronchial and mucosal inflammation, which reduces the patient's life quality, and needs continuous pharmacological treatments. Therefore, drug development for the treatment of asthma contributes to healthy life. It has been previously reported that Portulaca oleracea L. have beneficial effects such as anti‐cancer, anti‐oxidant. The compound of Portulaca oleracea L. extracts (CPL) is derivatives of compound isolated from Portulaca oleracea L. extract. The aim of present study was to elucidate whether CPL modulates the asthma and to study its possible mechanisms of action using ovalbumin (OVA)‐induced airway inflammatory mouse model, and cell‐based in vitro models. In mouse model, CPL treated groups were reduced airway constriction and immune cell infiltration in the lung. CPL treated groups were inhibited the release of β‐hexosaminidase, and the level of pro‐inflammatory cytokines in bronchoalveolar lavage fluid. Moreover, CPL treated groups were alleviated the level of immunoglobulin (Ig) such as IgE, OVA‐specific IgE, IgG1 and IgG2a in serum, and the level of pro‐inflammatory cytokines in the lung. These symptoms are associated with the immune response of mast cell, RBL‐2H3 cells were used to evaluate for the effecting mechanism of CPL. In RBL‐2H3 cells, pre‐treatment of CPL suppressed the release of histamine and β‐hexosaminidase, and the expression of pro‐inflammatory cytokines. Taken together, our findings suggest that CPL could facilitate the prevention or treatment of asthma‐like allergic inflammatory diseases.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biphenylindanone A, a prototypical mGlu2 positive allosteric modulator, alleviates dyskinesia and psychosis in the parkinsonian marmoset
Authors: Hamadjida Adjia , Nuara Stephen , Gourdon Jim , Huot Philippe
Published: June 2021

Objective

To determine the effect of the prototypical and selective metabotropic glutamate 2 (mGlu2) receptor positive allosteric modulator (PAM) biphenylindanone A (BINA) on L‐3,4‐dihydroxyphenylalanine (L‐DOPA)‐induced psychosis‐like behaviours (PLBs) and dyskinesia in the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐lesioned marmoset model of Parkinson's disease (PD).

Background

Psychosis and dyskinesia undermine the quality of life of as many as 50–95% of patients with advanced PD. Available therapies are few, and they may elicit important side effects. We have recently demonstrated that activation of mGlu2 receptors with another prototypical PAM, LY‐487,379, reduces both PLBs and dyskinesia in the MPTP‐lesioned primate. Here, we seek to determine the effects of PAM BINA on these 2 treatment‐related complications, in the MPTP‐lesioned marmoset.

Methods

Six common marmosets (Callithrix jacchus) were rendered parkinsonian by MPTP injection. Following repeated administration of L‐DOPA/benserazide (L‐DOPA) to elicit stable PLBs and dyskinesia, they were administered acute challenges of BINA (0.1, 1 and 10 mg/kg) or vehicle, in combinaison with L‐DOPA after which the severity of PLBs, dyskinesia and parkinsonian disability was rated.

Results

BINA (0.1, 1 and 10 mg/kg) significantly reduced the severity of peak dose PLBs, by ≈33.3%, ≈ 55.6 and ≈ 61.1% (P < 0.05, P < 0.01 and P < 0.01), respectively when compared to L‐DOPA/vehicle. Severity of peak dose dyskinesia was also reduced, by ≈ 28.9%, ≈ 52.3 and ≈57.8% (P < 0.05, P < 0.01 and P < 0.01), respectively, when BINA (0.1, 1 and 10 mg/kg) was added to L‐DOPA, compared to L‐DOPA/vehicle. The anti‐psychotic and anti‐dyskinetic effects of BINA were achieved without interfering with the therapeutic effect of L‐DOPA on parkinsonian disability.

Conclusions

BINA is the second mGlu2 PAM that was effective at alleviating PLBs and dyskinesia in the gold‐standard animal model of PD, the parkinsonian primate. The results presented here further add to the increasing data suggesting that mGlu2 positive allosteric modulation may be effective to diminish both PD psychosis and L‐DOPA‐induced dyskinesia.

Support or Funding Information

Fonds de Recherche Québec ‐ Santé, Parkinson Canada, Natural Sciences and Engineering Research Council of Canada, Weston Brain Institute, Michael J Fox Foundation for Parkinson's Research, Healthy Brains for Healthy Lives.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Electroacupuncture Inhibits Opioid‐induced Hyperalgesia in a Mouse Model of Thermal Nociception
Authors: Lee Ming Tatt , Chen Yi‐Hung , Chiou Lih‐Chu
Published: June 2021

Opioids remain the gold standard for the treatment of moderate to severe acute pain, however, repeated administration of opioids would inflict pain sensitization to the patients, a condition termed opioid‐induced hyperalgesia (OIH). One of the strategies to prevent OIH in clinical practice is by co‐administration of non‐opioid analgesics. We have recently revealed an unprecedented non‐opioid‐mediated analgesic effect induced by electroacupuncture at PC6 (Neiguan) acupoint (EA‐PC6), which overlays the median nerve, in mice.1 Briefly, EA‐PC6 at low frequency (2 Hz, 2 mA, 0.15 ms) can trigger orexin release from the lateral hypothalamus into the ventrolateral periaqueductal gray (vlPAG), a midbrain region for initiating descending pain inhibition. Orexin then activates postsynaptic orexin 1 receptors (OX1Rs), a Gq proteincoupled receptor family, in the vlPAG, resulting in synthesis of 2‐arachydonoylglycerol (2‐AG), an endocannabinoid that produces retrograde inhibition of GABA release by activating presynaptic cannabinoid 1 receptors (CB1Rs), leading to disinhibition of the vlPAG and ultimately inducing analgesia. Importantly, this EA‐PC6‐induced analgesic effect (EA‐PC6‐IA) is not blocked by naloxone, a non‐selective opioid receptor antagonist. Given the opioid‐independent nature of EA‐PC6‐IA, we further examined whether EA‐PC6 can prevent the development of OIH in male mice on thermal nociception test. OIH in mice were induced via repeated injection of morphine (twice/day, 10 mg/kg each, i.p.) over the course of 3 days, and a final dose (10mg/kg, i.p.) on the 4th day. Immediately after the final morphine injection, EA‐PC6 was given for 20 min in OIH‐induced mice, The mice were subsequently subjected to hotplate test. The control groups included the negative control group which received isoflurane anesthesia only, the sham group with needle insertion at PC6 without electrostimulation and the non‐acupoint group (electrostimulation at the deltoid muscle where there are very few acupoints). The results showed that EA‐PC6 inhibited repeated morphine injection‐induced thermal hyperalgesia. Importantly, this analgesic effect was blocked by a pre‐treatment of either SB334867 (an OX1R antagonist, 15 mg/kg, i.p.) or AM251 (an CB1R antagonist, 1.1 mg/kg, i.p.). These findings suggest that EA‐PC6 is a potential novel pain management strategy in OIH‐inflicted patients, such as terminal cancer patients.

Support or Funding Information

This study was supported by Ministry of Science and Technology, Taiwan (MOST107‐2321‐B‐002‐010, MOST107‐2811‐B‐002‐008), National Health Research Institutes, Taiwan (NHRI‐EX107‐10733NI) and Ministry of Education, Taiwan.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel Drug Target for Migraine: The GABAA Receptor α6 Subtype in Trigeminal Ganglia
Authors: Chiou Lih‐Chu , Tzeng Hung‐Ruei , Fan Pi‐Chuan , Sieghart Werner , Ernst Margot , Knutson Daniel E , Cook James
Published: June 2021

Migraine, an unmet medical need, is due to an overactive trigeminovascular system (TGVS). The trigeminal ganglion (TG), a hub of the TGVS, transmits the pain signals arising from neurogenic inflammation in the meninges to the brainstem. Previously, we have revealed an unprecedented role of the α6 subunit‐containing GABAA receptor (the GABAA receptor α6 subtype, α6GABAAR) on TG in the neuropathogenesis of migraine using the α6GABAAR‐specific positive allosteric modulators (PAMs)1 we developed. In a rat model of migraine induced by intracisternal injection of capsaicin, we showed that Compound 6, a potent α6GABAAR‐selective PAM via acting on TG, attenuated TGVS activation‐induced peripheral and central responses, using immunohistochemical approaches.2 Here, we further investigated whether positive modulating TG α6GABAARs would attenuate migraine‐related nociceptive behaviors induced by repetitive injections of nitroglycerin (NTG), which induces headache in human due to dural vasodilatation. We gave NTG (10 mg/kg, i.p.) to mice (6–8 weeks, male ICR) once every two days for 5 times. The mouse grimace scale (MGS) was used to score, from 0–2, the headache‐like behaviors, including orbital tightening, nose bulge, cheek bulge, and the changes of ear position and whisker. The severity of migraine was assessed by the MGS on day 9 (the 5th session). To evaluate the abortive treatment efficacy of α6GABAAR PAMs, drug were administered 20 min after NTG injection on day 9. For their prevention efficacy, drugs were given 30 min before NTG injection in each session. Repeated NTG injections significantly increased MGS. Compound 6 and its deuterated derivative that has a longer half‐life, DK‐56‐I, significantly attenuated the elevated MGS induced by NTG. Importantly, the effect of the α6GABAAR‐selective PAM was antagonized by i.p. injection of furosemide (20 mg/kg), an α6GABAA R‐selective antagonist that is blood‐brain barrier‐impermeable. It was mimicked by sumatriptan, an effective drug for migraine abortion. Besides, we found that DK‐56‐I was effective in preventing repeated NTG‐induced elevation of MGS, like topiramate, an effective drug for migraine prevention. We also found that α6GABAARs are expressed in TG neurons and satellite glial cells of mice. These results suggest that the TG α6GABAAR is a novel therapeutic target for migraine and that the α6GABAAR‐selective PAM is a novel treatment for migraine abortion and prevention.

Support or Funding Information

Supported grants: MOST 107‐2321‐B‐002‐010 and NHRI‐EX107‐10733NI.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Reversal of the Development of Antinociceptive Effects to Chronic Morphine in Mice by Fecal Microbiota Transplantation (FMT)
Authors: Kang James Minho , Dewey William L , Akbarali Hamid I
Published: June 2021

Morphine remains one of the most frequently prescribed drugs for the treatment of moderate to severe pain, including pain due to cancer or surgery. However, the long‐term use of this excellent pain reliever in man is limited by side‐effects that include analgesic tolerance, dependence and constipation. We have previously shown that depletion of gut bacteria by oral vancomycin results in the loss of antinociceptive tolerance, decreases epithelial permeability and reduces colonic inflammation by chronic morphine. The aim of this study was to investigate the effect of colonizing the gut with non‐opioid commensal microbiome on reversal of morphine‐induced antinociceptive tolerance. Gut bacteria were collected from 5 day placebo and morphine pelleted mice from fecal pellets designated as donor mice groups (PP‐FMT and MP‐FMT). The microbiome was then orally gavaged (1×109 cells/dose) daily for 5 days to placebo (PP) and morphine (MP) pelleted (75 mg pellet, 5 days) recipient mice groups, and the presence of antinociceptive tolerance was determined by morphine challenge (10 mg/kg, s.c.) in the tail immersion and hot plate tests. Daily gavage of the microbiome from morphine pelleted mice to the morphine‐pelleted recipient mice (MP+MP‐FMT) resulted in antinociceptive tolerance to morphine challenge. However, gavage of the microbiome from placebo pelleted donor mice to the morphine pelleted recipient mice (PP+MP‐FMT), prevented the development of antinociceptive tolerance. We further investigated whether an exogenous microbiome could reverse chronic morphine induced antinociceptive tolerance development. In these studies, oral gavage of gut microbiome was commenced after development of tolerance to morphine. Similar to the induction of tolerance, oral gavage of placebo microbiome to morphine tolerant mice (MP+PP‐FMT) reversed antinociceptive tolerance. In order to determine if transplantation of the gut microbiome also prevents the loss of epithelial integrity by chronic morphine treatment, intestinal permeability was measured by oral administration of FITC (Fluorescein isothiocyanate)‐dextran (44 mg/kg, p.o.) in placebo or morphine pelleted mice for 5 days. FITC levels were significantly higher in morphine pelleted mice (MP) treated with the microbiome from morphine donors (MP‐FMT) than placebo pelleted controls (PP, p<0.0001). However, the epithelial integrity was maintained in morphine pelleted mice (MP) treated with the commensal microbiome from placebo donors (PP‐FMT). These studies define a novel microbiome‐gut‐brain interaction in the development of opioid tolerance.

Support or Funding Information

NIH R01 DA036975 and NIH T32 DA007027

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Order of Fixed Ratio Presentation on Demand for Self‐administered Remifentanil in Rats
Authors: Maguire David R , Minervini Vanessa , France Charles P
Published: June 2021

Behavioral economics has been used extensively to study factors that control operant behavior, including quantification of reinforcing effectiveness of drugs. Consumption of a commodity decreases with increasing price, and the rate of decrease reflects demand elasticity, which is inversely related to reinforcing effectiveness; drugs with low elasticity have greater effectiveness than those with higher elasticity. Price is often manipulated by varying the number of responses required to obtain an infusion (e.g., fixed ratio [FR] schedule); however, many studies present price using only one order, which could introduce sequence effects that influence estimates of demand. This study examined the impact of the order of FR presentation on demand for the mu opioid receptor agonist remifentanil (0.0032 mg/kg/infusion) using a within‐subjects, reversal design. Seven male rats lever pressed for iv infusions. The FR varied across 3‐session blocks, yielding a demand curve. During the first and third phases of the study, FR values were presented in a strictly ascending order; during the second phase, FR values were presented in a mixed order. Rats obtained more than 60 infusions on average per session under baseline conditions (i.e., FR1) during each of the three phases, and the number of infusions decreased systematically with increasing FR values. Estimates of elasticity (i.e., reinforcing effectiveness) were not statistically different across the three phases indicating that the order of ratio presentation did not alter estimates of demand and further demonstrating the marked robustness of price as a source of control over operant behavior, including behavior maintained by drug reinforcers.

Support or Funding Information

This study was supported, in part, by the Welch Foundation (AQ‐0039).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CHARACTERIZATION OF EXPRESSION PATTERN OF SOMATOSTATIN 4 RECEPTOR IN THE MOUSE BRAIN
Authors: Pohóczky Krisztina , Kecskés Angéla , Bakai Rita , Kormos Viktória , Szentes Nikolett , Szőke Éva , Bölcskei Kata , Udvarácz Ildikó , Ábrahám István , Varga Zoltán , Gaszner Balázs , Helyes Zsuzsanna
Published: June 2021

Somatostatin is a cyclic neuropeptide which is expressed in brain regions related to pain‐ and mood regulation. It has 5 inhibitory G‐protein coupled receptors, and subtype 4 (sst4) mediates analgesic, anti‐inflammatory and antidepressant effects without endocrine actions. It was suggested to be a novel drug target for chronic neuropathic pain. However, its central nervous system distribution and mechanism of the inhibitory actions are not known due to the lack of specific antibody.

We mapped sst4 expression using β‐galactosidase immunohistochemistry in the brain of Sst4 knockout (KO) mice where sst4 was replaced by the lacZ gene. Since KO mice are not appropriate for determining functional changes and co‐localization, we also performed ultrasensitive RNAscope‐based in situ hybridization and neurochemical characterization on formalin‐fixed, paraffin‐embedded coronal sections of wild type mice. Sst4 mRNA was also quantified by qPCR in the positive regions.

Strong sst4‐related β‐galactosidase immunopositivity was detected in the hippocampus, moderate in the medial septum, amygdala, habenula in both sexes. Remarkably more intensive signal was seen in the primary somatosensory cortex of male mice compared to females. Sst4 mRNA was abundant in the hippocampal CA1 region, amygdala, spinal cord, sensory and motor cortices (layer 5). It was co‐localized with vesicular glutamate transporter 1 in most regions and choline‐acetyl transferase in the habenula. Sst4 mRNA was significantly higher in the dorsal root ganglia compared the spinal cord. These are the first data for sst4 expression in glutamatergic and cholinergic excitatory neurons of the nociceptive pathway. This might explain its unique value to simultaneously inhibit chronic pain and depression.

Support or Funding Information

ÚNKP17‐3‐III‐PTE‐166, GINOP‐2.3.3.‐15‐2016‐00050, GINOP‐2.3.3.‐15‐2016‐00048, EFOP‐3.6.2‐16‐2017‐00008, KTIA_NAP_13‐2‐2014‐0022, KTIA_NAP_13‐1‐2013‐0001, 207653/2018/FEKUTSTRAT

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the role of endothelial cell‐specific p110β isoform of PI3K as a potential target for anti‐angiogenic therapy
Authors: Azad Abul Kalam , Zhabyeyev Pavel , Oudit Gavin , Moore Ronald , Murray Allan
Published: June 2021

Introduction

Angiogenesis‐inhibitor drugs targeting Vascular Endothelial Growth Factor (VEGF) signalling to the endothelial cell (EC) are used to treat various cancers. However, tumors become resistant to this therapy due to the recruitment of alternative growth factors, acting via cognate EC receptor tyrosine kinases (RTK) or g‐protein coupled receptors (GPCR), to cue neo‐angiogenesis. In ECs, the PI3 kinase p110β isoform is uniquely coupled to both RTKs and GPCRs. Endothelial‐specific p110β inactivation impairs angiogenic sprouting and tip cell marker gene expression in vitro. These data indicate that p110β mediates pro‐angiogenic signals. We hypothesize that EC PI3 kinase‐β activity mediates tumor angiogenesis escape from sunitinib therapy.

Methods

Mouse Lewis lung carcinoma (LLC1) or B16F10 melanoma cells were implanted subcutaneously in EC‐specific p110β knockout (ECβKO) or control mice. Sunitinib (40mg/kg/day) treatment was initiated when the tumors reached an average volume of 200 mm3, then tumor growth was monitored, and all mice were euthanized when the average tumor volume reached 1500 mm3. Second, to model metastasis, B16F10 cells were injected intravenously in ECβKO or control mice, then treated with sunitinib for 20 days. Pimonidazole was administrated 1 hour before euthanasia. Immunohistochemical analyses were performed for CD31‐positive vessels and pimonidazole‐positive hypoxic areas.

Results

EC‐specific p110β loss with sunitinib treatment decreases the growth of subcutaneous LLC1 and B16F10 melanoma cells among syngeneic mice vs the sunitinib alone‐treated control mice. Similarly, EC p110β loss with sunitinib decreases B16F10 metastases in the lung, and the overall tumor area in lung cross‐section, vs sunitinib‐treated control mice. Further, subcutaneous primary and metastatic tumors had a marked decrease in CD31‐positive microvessels in ECβKO vs control mice, accompanied by a significant reduction in tip cell marker gene expression. Surprisingly, pimonidazole‐positive hypoxic area in the tumors was normalized in ECβKO vs control mice. We found that inactivation of EC‐p110β increased NG2‐positive pericyte coverage of tumor microvessels and arterioles.

Conclusions

These findings demonstrate that EC‐specific inactivation of p110β in combination with sunitinib decreases primary tumor growth and tumor metastasis vs sunitinib treatment alone. The density of the tumor vasculature and tip cell gene expression is reduced, but tumor oxygen delivery is normalized. Inhibition of endothelial p110β may be useful as adjuvant therapy with sunitinib, and may facilitate delivery and/or response of the tumor to conventional chemotherapy agents.

Support or Funding Information

Canadian Cancer Society

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chemotherapeutic Potential of Novel Selenium (Se) Analog of Histone Deacetylase (HDAC) Inhibitor, Suberoylanilide Hydroxamic Acid (SAHA) in Experimental Colorectal Cancer
Authors: Kaushal Naveen , Ghanghas Preety , Desai Dhimant , Raza Kaisar , Bhalla Aman
Published: June 2021

Colorectal cancer (CRC), one of the leading causes of cancer related deaths worldwide is an outcome of both genetic and epigenetic changes in colonic epithelia leading to the formation of adenocarcinomas. Histone deacetylases (HDACs) through their key roles in regulation of cellular processes, are associated with the development and progression of cancer. Thus, in this regard, HDAC inhibitors have become promising therapeutic targets. These inhibitors induce cell cycle arrest, differentiation, autophagy and apoptotic cell death by inhibiting multiple signaling pathways. Suberoylanilide hydroxamic acid (SAHA), a second generation HDAC inhibitor has been approved by US‐FDA for the treatment of cutaneous T‐Cell lymphoma, however its anti‐cancerous effects against colorectal cancer is still unknown. Also, essential dietary element Selenium (Se) has been found to have an important role in CRC prevention via modulating redox sensitive molecular pathways. Therefore, the present study aimed to explore the anti‐tumorigenic role of SAHA and its Se analog namely, SelSA‐1 (25 mg/kg) in DMH induced experimental colon cancer model in Balb/c mice. The toxicity studies and pharmacokinetic characterization of SelSA‐1 exhibited better tolerance, absorption and elimination rate than SAHA. Further, histological and morphological analyses showed that SelSA‐1 has better/comparable chemotherapeutic potential compared to SAHA at lower doses than SAHA. Further, the positive redox modulatory effects of SelSA‐1 and SAHA in CRC associated oxidative stress were also noteworthy, projecting the plausible use of similar analogs as compounds of choice for cancer treatments based on their safety profiles, efficacy and specificity in terms of chemotherapeutic potential.

Support or Funding Information

Department of Science and Technology (DST), Govt. of India, New Delhi; University Grant Commission (UGC), Govt. of India, New Delhi

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High salt diet reduced cardiac angiotensin‐(1‐7) but not ACE2 activity in SHR
Authors: Varagic Jasmina , Brosnihan K. Bridget , Chappell Mark C. , Ferrario Carlos M.
Published: June 2021

Despite numerous studies suggesting that angiotensin‐(1‐7) [Ang‐(1‐7)] counterbalances the biological actions of angiotensin II (Ang II), little is known about its response in salt‐induced left ventricular (LV) remodeling. Thus, we examined whether an inadequate balance between these peptides might contribute to the previously shown LV hypertrophic and proliferative response to high dietary salt intake in spontaneously hypertensive rats (SHR) (Varagic, AJP 2008). Eight week‐old male SHR were given an 8% salt diet for 5 weeks (Salt; n=8) and age‐matched controls received standard chow (Control; n=6).

Increased salt intake exaggerated hypertension (221 ± 10 mm Hg) compared to rats given a normal salt diet (188 ± 9 mmHg) and aggravated cardiac hypertrophy and fibrosis. Although LV tissue Ang II content was not changed, high dietary salt intake reduced the cardiac content of Ang‐(1‐7) as well as Ang‐(1‐7)/Ang II ratio (Figure). Despite the reduction in Ang‐(1‐7), salt excess did not influence cardiac ACE2 activity (Salt: 21.01 ± 2.05 vs. Control: 22.25 ± 1.55 fmol/mg/min). In conclusion, the significant reduction in the cardiac content of Ang‐(1‐7) in response to a high salt diet suggests a critical role for the peptide in the development of salt‐related cardiac remodeling. The inability to increase ACE2 suggests that other enzymes may affect Ang‐(1‐7) metabolism under the conditions of dietary salt excess.
Respiratory quotient and energy expenditure after consumption of low vs. high carbohydrate meals
Authors: Smith Julie Katherine , Kumagai Melissa , Bergman Sarah , Stadler Diane
Published: June 2021

Macronutrient consumption influences respiratory quotient (RQ) and energy expenditure (EE) in vivo. EE may be higher after LC than high carbohydrate (HC) meals which may confer a metabolic advantage that contributes to greater weight loss. We used a randomized crossover trial to test the hypothesis that LC meals result in higher postprandial EE and lower RQ than HC meals. Eight healthy, normal weight adults consumed a run‐in diet for 3 days and were admitted into the Clinical Research Center on the 4th day. Resting EE was measured for 45 minutes by indirect calorimetry, then subjects ate a HC or LC meal and postprandial EE was measured for 45 minutes every hour for 4 hr. A 2nd meal of the same composition was consumed and postprandial EE was measured for an additional 4 hr. This procedure was repeated after a >3 day washout period when subjects ate meals of the alternate diet. Postprandial AUC was calculated for EE, RQ, CO2 production (VCO2) and oxygen consumption (VO2) after the entire 9 hr period. Differences between diets in AUC for each variable were measured by one‐tailed paired t‐tests. After consumption of the LC meals, AUC of VO2 was not different but AUC of VCO2 was lower (2.17±1.94 vs 2.04±1.50, p<0.01) resulting in a lower AUC of RQ (4.10±0.63 vs 4.65±0.58; p<0.01). There was no difference in AUC EE over the 9 hr studied; however, the patterns of change of EE suggest that there may be differences if studied for longer periods of time.
CARPAL MOTION DURING MOBILIZATION
Authors: ZAKHARY RIZKALLA , BURKS ROBIN , SALEM GEORGE
Published: June 2021

Introduction

Therapists treating hand and wrist problems frequently perform mobilizations of the radiocarpal and intercarpal joints. The purpose of this investigation was to use prosection, digital photography (2D & 3D), and radiographic techniques to quantify carpal movement during wrist flexion and extension.

Methods

The soft tissue of the antebrachium was dissected sufficiently to allow placement of marker wires into the dorsal surfaces of the carpals, radius, and 3rd metacarpal. The tendons for the wrist motors were freed and used to flex, extend, radially and ulnarly deviate the wrist. The topography of the joint surfaces of the capitohamate and triquetrohamate joints were examined stereoscopically.

Results

Absolute and relative motion of the carpals, as identified using radiographic analysis, is presented in Table 1. Total wrist motion was 61° for flexion and 33° for extension. The hamate demonstrated the greatest absolute and relative motion during flexion; whereas the capitate demonstrated the greatest motion in extension. Examination of the joint surfaces of the isolated carpals suggested that the motions of flexion and extension at the "midcarpal" joint occur around an axis that passes through the scaphoid, capitate, hamate and triquetrum, with the scaphoid and the medial portion of the triquetrum convex distally and the capitate and hamate convex proximally.
Possible functional consequences of structural adaptation along single arterioles
Authors: Jacobsen Jens Christian Brings , Hornbech Morten , Henriksen Freja , Jensen Lars Jørn , Holstein‐Rathlou Niels‐Henrik
Published: June 2021

Arterioles are normally thought of as having a constant structural diameter between branch points. In unbranched rat cremaster arterioles, however, structural diameter increases downstream along the vessel (upstream tapering) without change in cross‐sectional area of the vascular wall (1). We addressed the possible functional consequence of this configuration by flow‐simulations comparing myogenically active vessels of uniform structural radius with those having an upstream tapering shape. In our model cross‐sectional area of the vascular wall remains invariant along the vessel and the flow is the same in all cases.

Results

The vascular wall material can be arranged in an upstream tapering configuration such that circumferential wall stress is uniform along the vessel. The stress remains uniform during flow‐autoregulation when perfusion pressure is changed. In contrast, when the structural radius is uniform along the vessel wall stress is much higher upstream than downstream, and this difference is maintained under variations in perfusion pressure.

Conclusion

In a myogenically active vessel upstream tapering of the inner radius allows the circumferential wall stress, and hence possibly the contractile state of the wall smooth muscle cells, to remain constant along the vessel at a given pressure. This is not the case for a vessel with a uniform radius. The upstream tapering shape is consistent with the vascular wall cells having a uniform point of operation as regards stress‐induced myogenic contraction.
Contribution of the arterial baroreflex to the differential responses of renal and lumbar sympathetic nerve activity and heart rate to freezing behavior in conscious rats
Authors: Miki Kenju , Kondo Naomi , Shibasaki Manabu
Published: June 2021

The present study examined the potential contribution of the arterial baroreflex to the differential responses of renal (RSNA) and lumbar sympathetic nerve activity (LSNA) and heart rate (HR) observed during freezing behavior, which was reported previously (FASEB J. 21:6 A882, 2007). Male Wistar rats were chronically instrumented with electrodes for measurements of RSNA, LSNA, and electrocardiogram, and with arterial and venous catheters. At least three days after the surgery, rats were exposed to white noise with 90dB over 10 min following a 60 min control period. The baroreflex curve for RSNA, LSNA and HR was determined by changing systemic arterial pressure using rapid intravenous infusions of vasoactive drugs and then fitted to an inverse sigmoid function curve. During the freezing behavior, the baroreflex curve for RSNA was shifted upward with a significant (P<0.05) increase in the response range of 115% compared with control level; while the baroreflex curve for LSNA remained unchanged during freezing behavior. The baroreflex curve for HR was shifted leftward with a significant (P<0.05) decrease in the midpoint pressure of 11mmHg relative to the control level. These data suggest that the differential responses of RSNA, LSNA and HR observed during freezing behavior may be explained, in part, by differential shifts in baroreflex curves for RSNA, LSNA and HR in rats.