bluuebunny/crossref_metadata_2025 huggingface dataset:
Urinary Biomarkers of Oxidative Stress and Insulin Resistance in Childhood Obesity
Authors: Ramachandra Nayana , Rodriguez Luisa , Devaraj Sridevi
Published: June 2021

Obesity is a worldwide epidemic and is increasing in children. Obesity can lead to cardiovascular complications and diabetes and is associated with increased oxidative stress. However, characterization of urinary biomarkers of oxidative stress and its correlation to biomarkers of insulin resistance has not been studied in children. Thus, the aim of this study was to correlate biomarkers of oxidative stress to biomarkers of insulin resistance in obese children compared to lean children.

Following informed consent, 52 children, 28 lean and 24 obese, were recruited to this study at Texas Children's Hospital. Out of the obese subjects, 48% had metabolic syndrome as defined by the National Cholesterol Education Program.

Measures of oxidative stress included 8‐hydroxydeoxyguanosine (8OHdG), 8‐Isoprostane, protein carbonyl, nitrotyrosine, and thiobarbituric acid reactive substances. Assays were performed using enzyme‐linked immunosorbent assays and the coefficient of variation for all assays were <10%. Paired T‐tests were used to compute statistical significance of parametric data and Wilcoxon tests were used for non‐parametric data. Among all the biomarkers of oxidative stress, 8‐OHdG was significantly increased in the obese subjects compared to the control (P‐value <0.01) even when adjusted for BMI. There was a significant correlation of 8‐OHdG with features of the Metabolic syndrome and HOMA‐IR index.

Thus, urinary 8‐hydroxyguanosine is a reliable indicator of oxidative stress in obese children and also correlates with insulin sensitivity. Since this marker is measured in urine, it affords a good non‐invasive method to detect children who are at increased cardiovascular risk.
Human and Canine Codon Optimization of Measles and Canine Distemper Viral Nucleic Acid Sequences Increases Sequence Identity and Protein Expression Compared to Wild Type and Human Codon Sub‐Optimized Constructs
Authors: Uhl Elizabeth , Osborn Michelle , Michel Frank , Hogan Robert
Published: June 2021

The role of codon usage bias (CUB) in the host adaptation and pathogenicity of human measles virus (HMV) and canine distemper virus (CDV) is being investigated and compared. Analysis of nucleic acid sequences for HMV and CDV nucleoprotein (N) revealed that human and canine CUB optimized constructs, as compared to wild type (WT) and human CUB sub‐optimized constructs, were characterized by increased nucleic acid and codon identities, and increased protein expression when transfected into human (293 HEK) cells. The purpose of this study was to characterize human and canine codon optimized nucleic acid sequences of the MHV and CDV F, M, H,L and P/C proteins. All human and canine CUB optimized HMV and CDV nucleic acid sequences had increased nucleic acid and codon identity as compared to WT sequences. Protein expression in human cells transfected with human and canine CUB optimized constructs of the F, M, H and P/C proteins was assessed using florescence, Cellomic and where possible western analysis. Higher protein expression was present in cells transfected with the human and canine codon optimized constructs compared to cells transfected with HMV and CDV WT or HMV suboptimized constructs. These results suggest that further adaptation of HMV and CDV to the mammalian CUB could enhance viral protein expression and pathogenesis.
Resistin promotes angiogenesis in endothelial progenitor cells through inhibition of microRNA‐206
Authors: Su Chen‐Ming , Chang Weng‐Cheng , Tang Chih‐Hsin
Published: June 2021

Resistin has a proinflammatory function in human that contributes to the link between inflammation and cardiovascular disease and is also associated with rheumatoid arthritis (RA).The aim of this study was to examine the potential function ofresistin in human circulating endothelial progenitor cells (EPCs) during angiogenesis in RA.EPCs isolated from healthy donors were used to investigate the mechanisms underlying EPCs migration and tube formation. In addition, potential microRNA‐206 (miR‐206) targets were validated using real‐time quantitative polymerase chain reaction, western blotting, and a luciferase reporter assay. Resistin is highly expressed in synovial fluids and tissues in RA patients and mice with collagen‐induced arthritis and promotes both EPC homing to synovia and angiogenesis. We show that resistin directly induces significant upregulation of vascular endothelial growth factor (VEGF) expression in EPCs and promotes expression of the VEGF 3ʹ untranslated region in the absence of a miR‐206 binding site. Our study is the first to demonstrate that a signaling pathway involving miR‐206 is involved in resistin‐induced VEGF expression and that EPCs migrate into RA joints in vitro and in vivo; these results provide support for resistin as a therapeutic target of RA.
Effects of extrasynaptic GABA‐A signaling in the PAG on morphine antinociception.
Authors: Tonsfeldt Karen , Suchland Katherine , Ingram Susan
Published: June 2021

Mu‐opioid receptors (MOPrs) reduce GABA‐mediated inhibition of ventrolateral periaqueductal gray (vlPAG) output neurons to activate the descending pain modulatory system and induce antinociception. Both tonic and phasic GABAA‐mediated currents are observed in the vlPAG. Tonic GABAA currents are reduced in vlPAG neurons from female rats pretreated with Complete Freud's Adjuvant (CFA) for 5‐7 days to induce chronic inflammatory pain (2‐way ANOVA; F (1, 45) = 15.24, p = 0.0003). The mRNA and protein levels of the GABAA delta subunit, known to mediate tonic currents in other brain areas, are increased in the vlPAG of female, but not male rats, pretreated with CFA. The decrease in current in females may be due to the rapid clearance of GABA because of upregulation of GABA transporters. Neuronal GAT1 transcripts were increased in female vlPAG pretreated with CFA (Two‐way ANOVA, F (1, 16) = 25.63, p < 0.0001). Glial GAT3 transcripts were decreased in males and increased in female vlPAG pretreated with CFA (Two way ANOVA, F (1, 16) = 32.93). Finally, we explored the role of the GABAA delta subunit in morphine‐induced antinociception with the novel selective allosteric agonist (DS2). DS2 (3 µg) was injected in to the vlPAG of male and female rats prior to cumulative morphine doses and testing of thermal nociceptive thresholds using the hot plate test. DS2 significantly decreased the ED50 of morphine in male rats (vehicle = 4.8 µg vs. DS2 = 10.8 µg, F(1,61) = 6.57, p = 0.013). Interestingly, female rats exhibited a leftward shift of the morphine dose response following DS2 microinjection (vehicle = 15.5 µg vs. DS2 = 5.1 µg, p < 0.05). These data support a role for the GABAA receptors containing the delta subunit in pain modulation in vivo.
Endomorphins and DAMGO Utilize Different Mechanisms to Produce Analgesia
Authors: Marrone Gina , Grinnell Steven , Rossi Grace , Pintar John , Majumdar Susruta , Pan Yingxian , Pasternak Gavril
Published: June 2021

There is significant variability among animals in their response to mu opioid receptor (MOR1) analgesics. The mu opioid receptor gene (OPRM1) undergoes extensive alternative splicing, producing several splice variants with 1, 6, and 7 transmembrane (TM) domains. Variants containing 6 TMs lack TM1 and are associated with the exon 11 promoter. We previously showed that mice lacking exon 11 variants respond normally to methadone and morphine, but fentanyl, heroin, and morphine‐6‐glucuronide potency is significantly reduced (Pan et al, 2009). Here, we report that in exon 11 KO mice the DAMGO response is intact both supraspinally and spinally but endomorphin analgesiais significantly reduced. [3H]DAMGO binding in exon 11 KO brain and spinal cord was not significantly altered, but binding of the endomorphin 2 analog 125I‐DAPP (dmt(125I)‐D‐Ala‐Phe‐Phe‐NH2) was reduced.No binding of [3H]DAMGO or 125I‐DAPP was detected in mice lacking both exon 1 7TM MOR variants and exon 11 6TM variants. In [35S]GTPγS studies of brain and spinal cord binding, max stimulation by DAMGO was equal in wild type and exon 11 KO mice whereas IDAPP stimulation was reduced in exon 11 KO mice. Overall, our results indicate that DAMGO does not recruit 6TM exon 11 variants for binding and analgesia, whereas endomorphins require a mechanism including 6TM exon 11 variants for analgesia and a significant portion of binding.
The Effects of RGS4 on Delta Opioid Receptor‐mediated Behaviors in Mice
Authors: Dripps Isaac , Jutkiewicz Emily
Published: June 2021

The delta opioid receptor (DOPr) is member of the opioid receptor family of G protein‐coupled receptors. Activation of DOPr induces antinociception and antidepressant‐like effects in animal models without the constipation, respiratory depression, and abuse liability associated with mu opioid receptor agonists such as morphine. In addition, some DOPr agonists cause convulsions, hindering their development as therapeutics in humans. Regulator of G protein signaling 4 (RGS4) proteins have been shown to act as negative modulators of DOPr‐mediated G protein signaling in vitro. However, the role of RGS4 in modulating DOPr‐mediated behaviors in vivo has yet to be determined. To this end, we compared the ability of the DOPr agonist SNC80 to induce antinociception, antidepressant‐like effects, and convulsions in wildtype and RGS4 knockout mice. Loss of RGS4 potentiated SNC80‐induced peripherally‐mediated antinociception in the acetic acid stretch assay and centrally‐mediated antihyperalgesia in a nitroglycerin‐induced thermal hyperalgesia assay in a DOPr‐dependent manner. There was no change in the dose of SNC80 needed to induce a convulsion. Furthermore, in the tail suspension test, RGS4 knockout mice showed no significant difference in SNC80‐induced antidepressant‐like effects. Taken together, these data suggest that RGS4 negatively modulates some, but not all DOPr‐mediated behaviors. Because RGS4 modulates DOPr‐mediated G protein signaling, these data are consistent with G protein‐independent signaling mechanisms generating DOPr‐mediated antidepressant‐like effects and convulsions. Future studies will focus on determining the mechanisms underlying differential modulation of DOPr‐mediated behaviors by RGS4. Research support was provided by start‐up funds to EMJ.
Effect of an Anti‐Cocaine Monoclonal Antibody on Body Weights in Self‐Administering Rats
Authors: Sparks Emily , Wetzel Hanna , Norman Andrew
Published: June 2021

A recombinant humanized anti‐cocaine monoclonal antibody (mAb), h2E2 is intended as an immunotherapy for cocaine abuse. This mAb sequesters cocaine in the plasma and inhibits cocaine entry into the brain in rats. h2E2 also increased the cocaine priming threshold in rats indicating that it could reduce the probability of cocaine‐induced relapse in cocaine addicts. However, once cocaine self‐administration was reinstated, h2E2 caused an increase in cocaine consumption in rats. It is possible that the increase in cocaine consumption and the increase in peripheral cocaine concentration in the presence of a foreign protein may be sufficient to induce cardiovascular or other peripherally‐mediated toxicity. Furthermore, multiple treatments with h2E2 will be required clinically and this could provoke toxic immune responses. As the loss of body weight of rats represents a standard indicator of animal health and an FDA‐sanctioned measure of drug toxicity, the effects of a repeated treatment with h2E2 on rat's body weights during cocaine self‐administration were investigated. Rats trained to self‐administer cocaine were infused with h2E2 (120 mg/kg) i.v. Body weights were measured 6 days a week over a 3‐week baseline period, 5 weeks after the first h2E2 infusion and over 5 weeks after the second. No significant weight loss was observed despite an initial moderate increase in cocaine consumption after each h2E2 treatment. There was no evidence that the increase in cocaine consumption in the case of relapse or repeated treatments with h2E2 would represent a safety concern during the treatment of cocaine abusers. Supported by NIH DP1DA031386.
The Protein Kinase C Inhibitor Tamoxifen Inhibits Neurochemical and Reinforcing Behavioral Effects of Amphetamine
Authors: Mikelman Sarah , Guptaroy Bipasha , Jutkiewicz Emily , Gnegy Margaret
Published: June 2021

The amphetamine (AMPH) class of drugs exerts its reinforcing effects primarily by reversing the normal function of the dopamine transporter (DAT) and increasing synaptic dopamine in the striatum and nucleus accumbens. We found that inhibition of protein kinase C (PKC) decreases AMPH‐stimulated efflux without affecting normal dopamine uptake by the dopamine transporter. Here, we investigate the estrogen receptor modulator tamoxifen, the only known brain permeable PKC inhibitor, as a potential therapeutic scaffold for the treatment of AMPH abuse. We tested the effect of tamoxifen in vitro on AMPH‐stimulated dopamine efflux in rat striatal synaptosomes and DAT‐containing neuroblastoma (N2A) cells, and in vivo on AMPH‐stimulated locomotor behavior and self‐administration of AMPH in rats. Tamoxifen and its metabolite 4‐hydroxytamoxifen (at 3 μM) decrease AMPH‐stimulated dopamine efflux in vitro in rat striatal synaptosomes by approximately 30% and in hDAT‐N2A cells by 40%. The inhibitory activity of tamoxifen on AMPH‐stimulated dopamine efflux was unaffected by knockdown of estrogen receptor alpha, supporting our hypothesis that tamoxifen inhibits AMPH action independently of its ER modulating effects. Most strikingly, pretreatment of rats with tamoxifen blocks AMPH‐stimulated hyperactivity and dose‐dependently (3‐9 mg/kg) decreases responding for AMPH in a self‐administration paradigm. We conclude that tamoxifen shows potential as a pharmaceutical lead in the development of a therapeutic for the treatment of amphetamine abuse. Supported by DA11697, DA007267, GM07767
Low Dose Mephedrone Depicts a Combined Dopaminergic and Serotonergic Discriminative Stimulus Effect
Authors: Saber Iman , Ward Sara Jane , Rawls Scott , Walker Ellen
Published: June 2021

Mephedrone (MEPH) is a recreational synthetic cathinone and a major component of psychoactive 'bath salts.' In previous studies, MEPH increases dopamine (DA) and serotonin (5‐HT) release and inhibits DA and 5‐HT synaptosomal uptake. A previous assay used a high dose MEPH to establish a discriminative stimulus and found 5‐HT stimulants to fully substitute for MEPH (Varner et al., Psychopharmacology 225:675, 2013). The purpose of this experiment was to pharmacologically profile MEPH using a very low training dose in a drug discrimination procedure. Sprague‐Dawley rats were trained using a two‐choice drug discrimination assay to examine the properties of 0.5 mg/kg MEPH. We hypothesized that MEPH would produce discriminative stimulus effects based upon multiple component cues of DA and/or 5‐HT, and therefore these cues would be blocked by DA and/or 5‐HT antagonists. When tested, the DA agonists cocaine, methamphetamine, and d‐amphetamine produced 23, 99, and 77 % MEPH‐like responding while 5‐HT agonists meta‐chlorophenylpiperazine (mCPP) and 3, 4‐methylenedioxymethamphetamine (MDMA) produced 64 and 97 % MEPH‐like responding, respectively. The DA1 receptor antagonist SCH23390 produced a reduction in MEPH discriminative stimulus effects while the 5‐HT2C receptor antagonist SB242084 completely reversed the effects of the training dose of MEPH. The DA2 antagonist sulpiride produced increased rate‐decreasing effects in combination with MEPH. In conclusion, a very low dose of MEPH was established as a discriminative stimulus and it appears the composite pharmacological profile is a unique composition of DA and 5‐HT activation.

R21 DA032718
Measuring the Potencies of RTI‐55 and Bupropion Using Self‐Administration in Rats as a Bioassay
Authors: Wetzel Hanna , Tsibulsky Vladimir , Norman Andrew
Published: June 2021

Self‐administration of dopamine agonists in rats can be used as an in vivo bioassay to determine their half‐lives and pharmacodynamic (PD) potency. During maintained self‐administration, the intervals between self‐administrations (T) is described by the logarithmic equation T=ln(1+Du/Dst)/(t1/2/ln2) (Eq.1), where Du = unit dose, Dst = satiety threshold and t1/ 2 = agonist half‐life. Self‐administration is a quantal response induced at an equiactive agonist concentration (Dst), making Dst a measure of PD potency. Non‐linear regression analysis of T as a function of Du can be used to calculate t1/2. According to this model t1/2 = T when Du = Dst. Therefore, the range of Du must exceed Dst to provide an accurate estimate of Dst and t1/2. Long half‐life drugs require long sessions in order to have sufficient number of intervals to reliably measure T. Eq.1 was used to estimate Dst and t1/2 of the dopamine transporter inhibitors cocaine, WIN35428, bupropion and RTI‐55, and the direct agonist apomorphine. Bupropion and RTI‐55 have the lowest and highest affinities but relatively similar t1/2. Self‐administration of both drugs was regular and T increased proportionally to Du, consistent with Eq.1. Bupropion and RTI‐55 had Dst of 35 µmol/kg and 0.3 µmol/kg and t1/2 of 39 and 129 min respectively. These t1/2 estimates are consistent with literature values. Despite the >100‐fold difference in PD potencies, the difference in intervals at Du = Dst (38 min for bupropion, 119 min for RTI‐55) was only 3‐fold, the same as the difference t1/2 . This illustrates the importance of selecting appropriate Du and session duration (12‐24 h) when using self‐administration as a pharmacological bioassay of agonist PK and potency. Supported by NIH grant DP1DA031386.
Effects of 3,4‐Methylenedioxypyrovalerone (MDPV) and 3,4‐Methylenedioxymethamphetamine (MDMA) on Place Conditioning in Mice
Authors: Gannon Brenda , Tobacyk Julia , Fantegrossi William
Published: June 2021

Recently, there has been an increase in abuse of new synthetic psychostimulant drugs derived from the cathinone structure. These products are typically referred to as “bath salts”, and one of the most common psychoactive constituents of these preparations is 3,4‐methylenedioxypyrovalerone (MDPV). In these studies, we used the conditioned place preference (CPP) paradigm to explore the apparent appetitive effects of MDPV in mice, as compared to those of the structurally similar psychostimulant 3,4‐methylenedioxymethamphetamine (MDMA). The primary aims of this study were to investigate whether MDPV and MDMA induce CPP in mice, to determine the dose‐responsiveness of the CPPs elicited by these compounds, to compare the magnitude of CPP between drugs, and explore whether binge exposure to either MDPV or MDMA one week before the first CPP pairing increased preference scores for either drug, perhaps consistent with the concept of “sensitization” to abuse‐related effects of these compounds. Adult male NIH Swiss mice were conditioned with MDPV and MDMA at doses of 1, 3, or 10 mg/kg (i.p.), with mice receiving two pairing sessions (one saline‐one drug) per day for 3 days. Binge exposure animals were treated with 3 mg/kg MDPV or MDMA every two hours over an eight hour period, and then conditioned for CPP one week later. Both MDPV and MDMA elicited significant place preference across the doses studied, with MDPV producing CPP equal to or higher than that of MDMA. The effects of binge exposure on CPP will be discussed. Given the public health concerns related to “bath salts” use, future studies of both the acute and chronic effects of these compounds are necessary to fully understand the effects of these drugs and their potential for abuse. Studies supported by T32 DA022981.
Modified dextromethorphan displays antidepressant‐like effects in the forced swim test
Authors: Scandinaro Anna , Nguyen Linda , Matsumoto Rae
Published: June 2021

Dextromethorphan (DM) may serve as a safer alternative to ketamine as a fast acting antidepressant due to overlapping pharmacology. Our lab has previously shown DM produces antidepressant‐like effects through sigma‐1 (σ1) receptors in the forced swim test (FST) in male Swiss, Webster mice. However DM's rapid metabolism poses a challenge in its use as a routine clinical drug. Two approaches to inhibit its metabolism are 1) addition of the CYP2D6 inhibitor, quinidine, and 2) chemically modifying DM. The present study aimed to determine if modified DM (MDM) by itself and in the presence of quinidine elicits antidepressant‐like effects and if σ1 receptors are involved. Our results showed MDM (3‐30 mg/kg, i.p.) produces a dose‐dependent decrease in immobility time in the FST, indicative of antidepressant‐like effects. MDM also had stimulatory effects, but they cannot account for its antidepressant‐like effects because there was no correlation between the two measures. Pretreatment with BD1063 (σ1 receptor antagonist) to examine the potential role of σ1 receptors failed to block MDM's antidepressant‐like effects. Co‐administration of quinidine to slow the metabolism of MDM produced a similar dose‐dependent decrease in immobility time in the FST compared to MDM alone. Quinidine with MDM, however, had no stimulatory effects. In conclusion, MDM has antidepressant‐like effects independent of σ1 receptors in the FST. Addition of quinidine does not alter the antidepressant‐like effects of MDM, but reduces its stimulant effects, suggesting a possible way to reduce the abuse potential and increase the safety of MDM.
Hallucinogens Activate a Specific Population of Neurons in the Cortex
Authors: Martin David , Porretta Connie , Nichols Charles
Published: June 2021

Serotonin hallucinogens produce a range of cognitive, sensory, and emotional effects in man. Recently, a resurgence of clinical interest in these compounds has developed because they can produce pronounced and long‐lasting psychological changes. While the structural backbones and receptor pharmacology of hallucinogens vary, their primary effects arise from agonism of the 5‐HT2A receptor. However, our understanding of how these drugs produce changes in brain function at a neuronal level and network level is incomplete. The studies reported here aim to uncover properties of cells which are activated by the hallucinogens lysergic acid diethylamide (LSD) and 2,5‐dimethoxy‐4‐iodoamphetamine (DOI). Adult rats are treated with LSD, DOI, or saline, and the somatosensory and medial prefrontal cortices are collected and dissociated into single cell suspensions. We then use fluorescence‐activated flow cytometry (FACS) to separate neurons from glia and activated neurons from non‐activated neurons. Isolated populations of neurons are examined for changes in mRNA levels using qPCR analysis. We find an enrichment of a broad collection of immediate early genes (including c‐fos, fosb, arc, krox‐20/egr‐2) in a small population of activated neurons in the cortex. Additionally, the abundance of a variety of other genes (htr2a, slc17a7, gad67) in activated versus non‐activated cells provides evidence for which specific cell types are being directly activated by hallucinogens. Further research is required to determine the role the transcriptional program in activated cells plays in the development of long‐term effects initiated by hallucinogens.
Association Rate Constants as Determinants of Ligand Selectivity:Lessons from The Dopamine And The Serotonin Transporter
Authors: Freissmuth Michael , Hasenhuetl Peter , Sucic Sonja , Sitte Harald , Sandtner Walter
Published: June 2021

Cocaine and methylphenidate (MPH) act as competitive inhibitors at the dopamine transporter (DAT). Both drugs bind DAT with similar affinities, but they differ fundamentally with respect to their action on the closely related serotonin transporter (SERT): cocaine inhibits SERT and DAT in a comparable concentration range, but MPH is highly selective for DAT. Conversely, other inhibitors exist, e.g. desipramine, which has a profile opposite to MPH: desipramine blocks SERT with much higher affinity than DAT. The kinetic determinants of this ligand selectivity have remained enigmatic due to methodological limitations. The prevailing view is to ‐ explicitly or implicitly – assume that – by analogy with affinity – selectivity is mainly determined by the dissociation rate of a ligand. We developed an electrophysiological approach to measure the binding of SERT and DAT inhibitors with high temporal precision. The method explored allows for measuring association (kon) and dissociation rates (koff) of drugs over a wide affinity range. We exemplify our approach by comparing the rates of cocaine‐, MPH‐ and desipramine‐binding to SERT and DAT. Cocaine has a two‐fold higher affinity for DAT than for SERT, which is accounted for by a twofold slower koff. In contrast the low affinity of MPH for SERT was due to a 20‐fold slower kon than that for binding of MPH to DAT. Conversely, the association of desipramine to SERT occurred at a rate that was 10 times faster than the rate observed for DAT. These findings highlight an important contribution of the forward binding reaction to drug selectivity.
Pharmacological and Functional Characterization of Adenosine A1 and A2A Receptors in Caudate Nucleus of Neonatal Lambs
Authors: Ibe Basil , McBride Shaemion , Mier Mark , Bernal Gilberto , Guerra Catalina , Rajaee Arezoo , Koos Brian
Published: June 2021

The basal ganglia including the caudate nucleus (CN) are critically involved in motor function with connections that include the sensorimotor sector of the thalamus. The CN, which has a high density of A2AR, has been implicated in the plasticity of the developing brain. We set out to identify intracellular signaling pathways in CN cells that are linked to A2AR activation that may be involved in neonatal brain plasticity. The hypothesis is that downstream effects of A2AR‐activation included modulation of cAMP/PKA signaling in CN cells. Culture of primary CN cells of neonatal lambs 4‐8d old were stimulated with different concentrations (uM, 10‐0.1) of CGS 21680 (CGS, A2AR agonist) compared to NECA. cAMP release and expression of A2AR, A1R, and PKA proteins were measured. Stimulation of cell proliferation by these agonists was also studied. CGS release of cAMP was dose‐dependent. A2AR antagonist SCH 58261(SCH) inhibited cAMP release. Also CGS increased while NECA decreased A2A receptor expression without affecting PKA expression. Both CGS and NECA stimulated caudate cell proliferation, PKA antagonist Rp‐cAMPS inhibited CGS stimulation of cell proliferation, A2AR, and PKA expression. These data show that cells from the caudate nucleus of neonatal lambs express functional A2AR and A1R that are linked to cAMP‐PKA signaling. These data may explain the greater plasticity of the neonatal brain attributed to the caudate nucleus. Supported by: Grant 20842: UCLA/LABiomed
Radiation Nephropathy is Mitigated by Epoxyeicosatrienoic acid Analog
Authors: Vo Bao Vi , Khan Md. Abdul , Neckar Jan , Sharma Amit , Fish Brian , Cohen Eric , Imig John
Published: June 2021

We investigated the kidney protective effect of a novel epoxyeicosatrienoic acid analog EET‐A in experimental radiation‐induced nephropathy caused by total body irradiation (TBI, 11Gy). Male WAG/RijCmcr rats were divided into three groups: normal rats (control), and TBI rats treated with either vehicle (TBI‐V) or EET‐A (10 mg/kg/d, TBI‐EET‐A) in drinking water started from day‐2 post TBI. Urine and plasma samples were collected after 12‐wk treatment to assess kidney protective effect of EET‐A by determining blood urea nitrogen (BUN), albuminuria, nephrinuria, and renal histopathological examination. TBI resulted in elevated BUN levels in TBI‐V compared to control group (44±5 vs. 18±1 mg/dL, P<0.05). In TBI rats, EET‐A treatment reduced BUN levels (27±3 mg/dL, P<0.05). TBI‐V group had elevated urinary albumin (310±50 mg/d) compared to control (0.9±0.1 mg/d), and EET‐A treatment reduced urinary albumin levels by 65% (P<0.05). TBI‐V group also had elevated urinary nephrin levels (5.7±0.9 mg/d) compared to control (0.07±0.01 mg/d). EET‐A treatment in TBI rats reduced urinary nephrin levels by 80% (P<0.05). Moreover, TBI resulted in renal histopathological changes that included glomerular and tubular injury, and EET‐A treatment reduced this injury by 50%. In conclusion, we demonstrate that EET‐A treatment mitigates radiation‐induced nephropathy, and that an EET analog could be a new therapeutic target to treat radiation‐induced nephropathy.
Substrate‐Dependent Inhibition of Human Renal Organic Cation Transporter 2 (OCT2) by Cimetidine
Authors: YIN JIA , Duan Haichuan , Isoherranen Nina , Wang Joanne
Published: June 2021

Inhibition of transporter‐mediated renal drug secretion is a significant concern for clinical drug‐drug interactions (DDIs). In the recent draft FDA guidance for drug interaction studies, metformin and cimetidine were respectively suggested as the model substrate and inhibitor for in vivo DDI assessment for OCT2, a major renal transporter responsible for tubular secretion of cationic drugs. Emerging evidence suggests that the inhibition potency of a specific compound towards OCT2 is substrate‐dependent, but the available data was mostly obtained with experimental compounds that are not used clinically. The aim of this study was to determine whether cimetidine inhibition of OCT2 differs towards metformin and atenolol, two clinically used drugs predominantly eliminated by the kidney. The IC50 values and mechanism of inhibition were determined by transport assays in Flp‐In 293 cells stably expressing human OCT2. The IC50 values of cimetidine towards OCT2‐mediated metformin and atenolol transport were 93.5 ± 1.1 μM, and 9.9 ± 1.1 μM respectively. Detailed kinetics analysis further revealed a mixed inhibition model for both substrate drugs. The Ki values based on mixed inhibition model differed more than 70 fold for metformin and atenolol. The data suggest that with a Cmax value of 8 ~ 12 μM, cimetidine will significantly inhibit OCT2‐mediated atenolol renal secretion in vivo without affecting OCT2‐mediated metformin transport. Therefore, data obtained from one probe substrate may not be applicable to other drug substrates in assessing DDI potentials for OCT2.

This study was supported by the NIH grants R01GM066233 and TL1 TR000422.
IL‐33/ST2 signaling is anti‐cardiac hypertrophic and spatially restricted in cardiomyocytes after pressure overload
Authors: Chen Wei‐Yu , Lee Richard
Published: June 2021

ST2, a member of the interleukin (IL)‐1 receptor family, plays a major role in immune and inflammatory responses. At least two types of ST2 are identified including membrane‐bound ST2L and a soluble form (sST2). IL‐33 binds to the ST2L activates downstream signaling, whereas the sST2 acts as a decoy receptor by sequestering IL‐33. To investigate how IL‐33/ST2 signaling mediates anti‐cardiac hypertrophy in vivo, we performed adenoviral gene transfer of ST2L and sST2 genes into left ventricular wall and analyzed cardiac hypertrophy after transverse aortic constriction (TAC)‐induced pressure overload. Adenovirus carry GFP, ST2L, or sST2 were injected into left ventricular wall of wild type (WT) or ST2‐deficient mice. We then performed TAC surgery and analyzed the outcomes. ST2‐deficiency exacerbates TAC‐induced cardiac hypertrophy, which is rescued by ST2L transfer. Gene transfer of sST2 into WT mice, but not the ST2‐deficient mice, exacerbates cardiac hypertrophy after TAC. Cross‐sectional analysis showed that ST2L‐expressing cells were lesser hypertrophic, whereas sST2‐expressing cells are more hypertrophic. Furthermore, ST2L‐mediated anti‐hypertrophic effect was constrained in ST2L‐expressing cells. In WT mice, sST2‐expressing cells affect only in immediately adjacent cells but not in cells farther away. Collectively, IL‐33/ST2 signaling mediated anti‐hypertrophic effect is highly constrained in ST2L‐expressing cardiomyocytes.
Regulation of Middle Meningeal Artery (MMA) Diameter by ATP‐sensitive potassium channels
Authors: Syed Arsalan , Koide Masayo , Brayden Joseph , Wellman George
Published: June 2021

The cellular mechanisms contributing to migraine headache are poorly understood, but may involve prolonged dilation of cranial arteries, specifically the MMA. Agonist‐induced activation of ATP‐sensitive potassium (KATP) channels is known to play an important role in the modulation of arterial diameter in a number of vascular beds. The objective of the current study was to determine if a tonic vasodilator influence resulting from basal KATP channel activity is present in isolated, pressurized rat MMA. At an intravascular pressure of 40 mmHg, arteries developed pressure‐induced constriction representing an approximate 40 % decrease in diameter. Treatment of arteries with either of the KATP channel inhibitors glibenclamide (10 µM) or PNU37883 (10 µM) induced a further decrease in diameter of ∼ 20 %. Also consistent with basal KATP activity, glibenclamide induced a membrane potential depolarization of ∼ 14 mV in MMA segments at an intravascular pressure of 40 mmHg. Further, in MMA loaded with the ratiometric Ca2+ indicator, Fura‐2‐AM, glibenclamide‐induced MMA constriction was correlated with a simultaneous increase in the ratio of 340 nm/380 nm excited fura‐2 fluorescence, consistent with an increase in intracellular Ca2+. Interestingly, the PKA inhibitor, H89 (1 µM), abolished glibenclamide‐induced MMA constriction suggesting that PKA activity may underlie tonic KATP channel activation. Glibenclamide or PNU37883 did not alter the diameter of isolated cerebral arteries. Together these results suggest that tonic KATP channel activity plays a key role in regulation of MMA, but not cerebral artery, diameter and may represent a potential target in the development of treatments for migraine headache.Supported by NIH P01 HL095488, Totman Trust and the Peter Martin Fund.
TRPV1‐mediated Ca2+ Influx and Middle Meningeal Artery Constriction
Authors: Manuelyan I. , Syed A , Koide M. , Shui B , Sonkusare S. , Kotlikoff M. , Nelson M. , Wellman G.
Published: June 2021

The causes of migraine and cluster headaches are not clear, but may involve dilation of the meningeal vasculature. Recent evidence indicates that transient receptor potential channel V1 (TRPV1) agonists may benefit patients with migraine or cluster headaches. Here, we test the hypothesis that capsaicin, a TRPV1 agonist, constricts middle meningeal artery (MMA) via activation of smooth muscle (SM) TRPV1 channels. Consistent with this hypothesis, capsaicin induced constriction of isolated pressurized rat MMA (EC50 ∼ 100 nM) that was blocked by the TRPV1‐selective antagonist, capsazepine (1 µM). Interestingly, capsaicin (10 µM) did not constrict cerebral arteries. Capsaicin‐induced MMA constriction was reduced ∼ 34% by the voltage‐dependent calcium channel (VDCC) blocker diltiazem (100 µM) and abolished by removal of extracellular Ca2+. To assess the direct impact of capsaicin on Ca2+ influx, MMA myocytes were freshly isolated from transgenic (acta2‐GCaMP5‐mCherry) mice with SM‐specific expression of Ca2+‐sensitive GCaMP5 and Ca2+‐independent mCherry proteins. In these cells, 1 µM capsaicin increased the fluorescence ratio of GCaMP5/mCherry by 4‐fold. Further, optical imaging of unitary Ca2+ events (sparklets) in intact MMA using Fluo‐4 revealed elementary capsaicin‐induced signals with a quantal fractional fluorescence change consistent with TRPV1 channels. In sum, these findings suggest capsaicin‐induced MMA constriction results from activation of TRPV1 channels on vascular myocytes. Capsaicin‐induced Ca2+ influx in MMA SM likely reflects both direct Ca2+ entry via TRPV1 channels and VDCC activation via TRPV1‐mediated Na+ influx. Supported by: Totman Trust, Peter Martin Fund, AHA and NIH.
Protective Effects ofEuterpe oleracea Mart. (An Amazon Plant) on Experimental Metabolic Syndrome in C57BL/6 Mice
Authors: da Costa Cristiane , da Rocha Ana Paula , de Bem Graziele , Santos Izabelle , Daleprane Julio , de Moura Roberto , Resende Angela
Published: June 2021

The study was designed to determine the protective effects of Euterpe oleracea (açaí) stone extract (ASE, 300 mg/kg/day), in C57BL/6J mice fed a high‐fat diet (HFD) that delineate components of metabolic syndrome (MS). All experiments on animals were approved by the Animal Care and Use Committee of the Biology Institute Rio de Janeiro State University. C57BL/6 mice at 6 weeks of age were fed a control diet and received water (CD) or ASE (CD+ASE) and a HFD (60% fat) that received water (HFD) or ASE (HFD+ASE) for 12 weeks.Body weight, plasma and liver lipids, glucose and insulin plasma levels were determined and the insulin resistance measured by HOMA index. Oxidative damage and antioxidant activities were evaluated in liver homogenate by spectrophotometry. Expression of insulin receptor (IR), phosphorylated AKT (pAkt), HMGCoa‐R, pAMPK, ABCG 8 and tubulin protein were evaluated in liver by western blotting. Hepatic steatosis was evaluated in liver by histology. Increased body weight, plasma and liver lipids, glucose levels and insulin resistance, oxidative damage and expression of HMGCoa‐R and SREBP‐1 were observed in HFD and reduced by ASE. Antioxidant activities and expression of IR, pAKT, pAMPK and ABCG8 were reduced in HFD and increased by ASE.Hepatic steatosis was greater in HFD and the treatment with ASE reduced this change. The results demonstrate a protective effect of an extract obtained from açaí stone in metabolic syndrome, since the obesity, hypercholesterolemia, hyperglicemia, insulin resistance and hepatic steatosis induced by HFD were improved by oral treatment with the extract. Financial Support: CNPq and FAPERJ.
Identification of Type I Interferon as a Novel Therapeutic Approach for Targeting Alveolar Rhabdomyosarcoma
Authors: Nieves Kiomaris , Henrich Ian , Young Robert , Quick Laura , Bledsoe Krista , Chou Margaret
Published: June 2021

Bone and soft tissue tumors (BSTT) are a group of neoplasms most commonly found in children. The TRE17/ ubiquitin‐specific protease 6 (USP6) oncogene is intimately involved in several BSTTs, including alveolar rhabdomyosarcoma (ARMS). The molecular functions of TRE17 in ARMS pathogenesis are unknown. However, recent unpublished data from our lab has identified the Jak1‐STAT1/3 pathway as a key effector of TRE17.

Type I Interferon (IFN) has been used previously to treat sarcomas. IFN functions through Jak1/STAT1. Interestingly, we found that TRE17 renders ARMS hypersensitive to IFN‐induced apoptosis in vitro. TRE17‐expressing ARMS, but not control ARMS, underwent apoptosis after 24h of IFN treatment. In addition to inducing apoptosis, IFN also resulted in the degradation of TRE17. The exact mechanisms behind the sensitization and subsequent downregulation of TRE17 in ARMS remain to be uncovered. The degradation of TRE17 appears to require its ubiquitin‐specific protease activity. Currently, work is being done to find the mechanism of TRE17 degradation upon exposure to IFN; preliminary data suggest that the proteosomal pathway is responsible. These studies may identify methods to sustain or induce TRE17 expression, which would render ARMS susceptible to killing by IFN, and ultimately allow exploration of IFN as a therapeutic agent for ARMS treatment.
Stress Responses Initiated in Cardiomyocytes during Hypothermia‐Induced Rewarming Shock
Authors: Schaible Niccole , Han Young Soo , Zaidan Louai , Arteaga Grace , Tveita Torkjel , Sieck Gary
Published: June 2021

Rewarming shock following accidental hypothermia is a potentially fatal scenario involving cardiac dysfunction. Previously our lab demonstrated that rewarming shock results in a decrease in Ca2+ sensitivity of contraction in cardiac muscle due to an increase in cTnI phosphorylation. In this study, we hypothesized that rewarming shock initiates a cascade of intracellular events including reactive oxygen species (ROS) formation, endoplasmic reticulum (ER) stress, and mitochondrial disruption. Isolated cardiomyocytes from Sprague‐Dawley rats were exposed to a hypothermia/rewarming protocol (cooled for 1 h, held at 17oC for 30 min, rewarmed for 1 h) that results in contractile dysfunction indicated by an increase in cytosolic Ca2+ and reduced cell shortening measured using an IonOptix system. Rewarming shock increased ROS levels measured by an HPLC‐based dihydroethidium assay and by MitoSox fluorescence. Hypothermia/rewarming was also associated with ER stress indicated by increased expression of ER stress markers (Grp78, PERK, and CHOP) measured by Western blot. Finally, rewarming shock was associated with mitochondrial disruption evidenced by mitochondrial membrane depolarization (decreased in TMRM fluorescence) and by an increase in cytosolic cytochrome c (measured by Western blot). Our results support our overall hypothesis and suggest that these ER and mitochondrial intracellular events may underlie myocardial contractile dysfunction following hypothermia/rewarming.

Supported by NIH Grant 5T32HL105355‐02 (GCS)
The Effect of Doxorubicin Administration on Intramuscular BCAA Concentrations in the Rat
Authors: Fabris Sergio , MacLean David
Published: June 2021

The purpose of the present study was to examine the effect of Doxorubicin (DOX) administration on total intramuscular branch chained amino acids (BCAA) in the soleus (SOL) and white gastrocnemius (WG) of the rat following a single IP administration of 1.5 or 4.5 mg/kg DOX after 24, 48, 72, 96, 120, 144, 168 and 192 hrs. The administration of 1.5 mg/kg DOX did not cause significant changes in BCAA concentrations in the SOL or the WG. However the 4.5 mg/kg dose resulted in a decrease (P<0.05) of BCAA in the SOL 24, 168 and 192 hours (105±8, 120±10 and 155±15 µmol/kg dw, respectively) post injection compared to control (304±34 µmol/kg dw). Similarly, BCAA significantly decreased in the WG 24, 144, 168 and 192 hours (93±8, 123±10, 107±11 and 154±19 µmol/kg dw, respectively) after the administration of the 4.5 mg/kg dose compared to control (317±44 µmol/kg dw). BCAA concentrations in the SOL were decreased (P<0.05) following the 4.5 mg/kg dose, when compared to 1.5 mg/kg, after 24 (224±3 %), 72 (138±3 %), 144 (154±2 %), 168 (183±5 %) and 192 hours (149±6 %). Likewise, BCAA concentrations in the WG were decreased (P<0.05) following the administration of 4.5 mg/kg DOX after 24 (288±8 %), 144 (224±1 %), 168 (239±8 %) and 192 hours (151±11 %) when compared to the 1.5 mg/kg dose. There were no consistent differences when comparing between muscle types. These data represent, for the first time, alterations in BCAA in the skeletal muscle as a function of DOX administration. This presents a significant clinical implication as a single administration of DOX effectively reduces BCAA which promotes muscle wasting and a poor clinical outcome. Supported by NSERC.
Mitochondria‐Targeted Catalase Does Not Enhance Myogenesis following Cardiotoxin Muscle Injury and Radiation Exposure
Authors: Cotter Joshua , Baker Michael , Akers Lewis , Acharya Munjal , Limoli Charles , Caiozzo Vincent
Published: June 2021

Radiation inhibits myogenesis following muscle damage from cardiotoxin (CTX) injection. Cellular oxidative stress may contribute to this inhibition. The objective of the current study was to investigate the effects of human mitochondrial‐targeted catalase (MCAT) following muscle damage induced by cardiotoxin with and without exposure to radiation. Adult male mice (C57BL/J6) were assigned to one of eight groups: CTX wild‐type or CTX+MCAT; irradiation (IRR) or no IRR; and 4 or 14 days. Following CTX injection into the tibialis anterior (TA), mice assigned to the irradiation groups were exposed to a dose of 7 Gy of gamma irradiation. The TA muscles of both legs were harvested and stained by hematoxylin and eosin for histological analysis. Cross sectional area (CSA) of regenerating fibers (centralized nuclei) was analyzed. No significant differences in CSA were found between wild‐type and MCAT mice at days 4 or 14. Irradiation resulted in significant damage at day 4 in both wild‐type and MCAT mice with no centralized nuclei discernable. Irradiation caused a significant blunting of regeneration at day 14 compared to the non‐irradiated groups (p < 0.001) with no significant difference between CTX+IRR and CTX+MCAT+IRR. These results indicate that a single dose of clinically relevant radiation resulted in the inhibition of myogenesis 14 days following CTX injection that was not affected by catalase targeted to the mitochondria.
Targeted Deletion of all Caldesmon Splice Variants in Mice Results in Abdominal Wall Closure Defects
Authors: Schroeter Mechthild , Frohn Marina , Pütz Sandra , Koch Manuel , Peche Vivek , Metzler Doris , Noegel Angelika , Neiss Wolfram , Pfitzer Gabriele
Published: June 2021

Ample evidence obtained in vitro indicates that the thin filament linked protein, caldesmon (CaD) is involved in regulation of smooth muscle contraction. Homozygous h‐CaD deficient mice were reported to be viable and exhibit a but small slowing of relaxation (Guo et al., 2013). However, the results are confounded by upregulation of non‐muscle l‐CaD in smooth muscle. Thus, the in vivo function of CaD is still not clear. Here we report that deletion of all splice variants of Cald1 results in perinatal lethality whereas the life span of heterozygotes (hets) is normal. Gene targeting was achieved by deleting exons 2‐13 in the murine Cald1 gene. Homologous recombination was confirmed on DNA level by Southern blots and PCR, and on protein level by Western Blotting. Homozygous offspring followed Mendelian rules at E15.5 but was abrogated at E19.5. The bodyweight of the ‐/‐ fetuses at E18.5 was reduced by ~20%. The most prominent feature of the ‐/‐ fetuses is the persistence of the physiological umbilical hernia whereby the size of the abdominal wall defect is variable. Bladder contractility of adult hets was not significantly affected. In conclusion, our results indicate that deletion of CaD does not result in early embryonic lethality but it may be essential for organ maturation. Moreover, lack of h‐CaD but not lack of all splice variants including non‐muscle l‐CaD is compatible with life.
Reduced Relaxation Responses to Heme‐dependent and –independent Soluble Guanylyl Cyclase Activation in Mesenteric Arteries of a Rat Model of Type 2 Diabetes
Authors: Goulopoulou Styliani , Hannan Johanna , Matsumoto Takayuki , Ergul Adviye , Webb R Clinton
Published: June 2021

Endothelial dysfunction is a main feature of type 2 diabetes. Impairment in vascular smooth muscle cell function may also contribute to diabetes‐associated microvascular and macrovascular complications. We hypothesized that mesenteric resistance arteries (MA) from non‐obese rats with type 2 diabetes (Goto‐Kakizaki, GK) would have reduced responses to soluble guanylyl cyclase (sGC) activation as compared to controls (Wistar). Endothelium‐dependent and –independent relaxation was assessed in isolated arterial segments mounted in a wire myograph. GK MA had reduced responses to acetylcholine (ACh; ‐logEC50: 7.92 ± 0.08 vs. 7.65 ± 0.06, p<0.05) and sodium nitroprusside (SNP; ‐logEC50: 8.43 ± 0.08 vs. 7.84 ± 0.06, p<0.05), but there were no group differences in 8‐Br‐cGMP‐induced relaxation and PKG‐1 and phosphorylated VASPSer239 expression (p>0.05). GK MA had attenuated responses to BAY41‐2272 (heme‐dependent sGC stimulator; ‐logEC50: 7.55 ± 0.07 vs. 6.88 ± 0.08, p<0.05) and BAY58‐2667 (heme‐independent sGC stimulator; ‐logEC50: 10.82 ± 0.07 vs. 10.27 ± 0.08, p<0.05) compared to control values but pharmacological inhibition of sGC abolished these differences. A reduction in sGC activity may account for impaired vascular smooth muscle responsiveness to dilatory stimuli in type 2 diabetes.