bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of opioids and cannabinoids during chronic morphine treatment in rhesus monkeys (658.3)
Authors: Zanettini Claudio , Gerak Lisa , France Charles
Published: June 2021

Opioids remain the drugs of choice for treating moderate‐severe pain, although concerns about abuse and dependence limit their use. Therapeutic utility might be improved by combining opioids with other drugs to enhance analgesic effects, but only if other (adverse) effects of opioids are not similarly enhanced. This study examined changes in the antinociceptive (warm water tail withdrawal) effects of morphine and the cannabinoid receptor agonist WIN‐55,212 resulting from twice daily treatment with 3.2 mg/kg morphine in 4 monkeys. Before treatment, morphine and WIN‐55,212 increased tail withdrawal latency with similar potency (ED50= 1.2 and 0.8 mg/kg, respectively); during treatment dose‐effect curves of both drugs were significantly shifted rightward (12.9‐ and 3.4‐fold, respectively). Acute discontinuation of treatment increased heart rate and observable signs that can be indicative of withdrawal (yawning, vocalization, and twitching); these effects were reversed when morphine treatment resumed. These results demonstrate robust tolerance to the antinociceptive effects of morphine under conditions that generate modest physical dependence (withdrawal). That chronic morphine treatment might confer reduced potency of a cannabinoid receptor agonist raises concerns regarding the therapeutic utility of combining opioids and cannabinoids to treat pain.

Grant Funding Source: Supported by R01DA005018 and K05DA17918
VTA GABA(A) receptors mediate maladaptive disinhibition of dopamine release in the NAc ő a potential mechanism for increased risk taking behavior following adolescent voluntary alcohol intake (658.9)
Authors: Schindler Abigail , Tsusui Kimberly , Clark Jeremy
Published: June 2021

Alcohol is the most commonly abused drug among adolescents and shows the highest liability of all abused drugs. We have previously demonstrated that voluntary alcohol consumption by adolescent rats results in maladaptive risk‐taking behavior and phasic dopamine release in adulthood, as assessed by a probability discounting task and fast scan cyclic voltammetry (FSCV). These findings suggest that adolescent alcohol exposure‐induced changes in striatal dopamine release could bias choice by assigning greater value to the risky option, but the underlying mechanisms remain unknown. GABAA receptors located in the VTA are a potential candidate for mediating these effects. Using electrical stimulation of PPT glutamatergic afferents to VTA DA neurons, here we demonstrate that adult rats previously exposed to alcohol during adolescence show increased phasic dopamine release in the NAc, as compared to controls. PPT stimulation elicits GABAA receptor dependent current in VTA DA neurons, raising the possibility that adolescent alcohol intake produces this increased phasic dopamine release through downregulation of GABAA receptors on VTA DA neurons. Systemic administration of L‐838,417, a GABAA receptor α2, α3, and α5 subunit allosteric agonist, causes a dose‐dependent decrease in phasic DA release elicited by PPT stimulation in control rats, and this decrease is enhanced in rats exposed to alcohol during adolescence. Additionally, imaging flow cytometry was used to measure expression of GABAA receptor subunits on neurons of the ventral midbrain. Preliminary results suggest that GABAA receptor α3, but not α1, subunit surface expression is decreased following adolescent alcohol intake. Taken together, these results provide unique insight into the potential role that GABAergic modulation of dopamine neurons plays in the maladaptive risk taking behavior seen following adolescent alcohol exposure and highlights new potential therapeutic targets.

Grant Funding Source: R01AA021121 and T32AA07455
Endomorphin analogs with analgesic actions mediated through exon 11 variants of the mu opioid receptor (MOR‐1) (659.10)
Authors: Marrone Gina , Grinnell Stephen , Pan Ying‐Xian , Pintar John , Majumdar Susruta , Pasternak Gavril
Published: June 2021

Multiple mu opioid receptors (MOR‐1) have been confirmed with the cloning of several mu splice variants. These variants are generated by distinct promoters associated with exon 1 and exon 11. Previously, our lab (Pan et al., 2009, PNAS) observed that the potency of morphine‐6β‐glucuronide, heroin, and fentanyl analgesia was significantly reduced in mice lacking truncated 6 transmembrane (6TM) exon 11 variants of MOR‐1. In contrast, morphine analgesia was intact. We now present evidence that the mu selective peptides endomorphin‐1 and endomorphin‐2 also require exon 11 variants for their analgesic actions supraspinally. An endomorphin‐2 analog, DAPP (dmt‐DAla‐Phe‐Phe‐NH2), produces analgesia via exon 11 variants of MOR‐1 as well. In vitro, approximately 50% of [125I]DAPP binding was lost in the brains of exon 11 knockout mice, whereas almost no specific binding was detected in triple knockout mice lacking MOR‐1 exon 1 variants, delta (DOR‐1), and kappa (KOR‐1). Consistent with our previous behavioral findings, morphine‐6β‐glucuronide, 6‐acetyl‐morphine, and fentanyl were less potent competitors of [125I]DAPP in the brains of exon 11 knockout mice compared to C57/B6 wild type mice. Overall, our findings provide further evidence for the functional role of truncated 6TM variants of MOR‐1. These results also suggest that mu analgesics, including peptides, can be subclassified based on their affinities to exon 11 associated splice variants.

Grant Funding Source: NIDA DA02615, DA06142 and DA07242
Abundant sigma‐1 receptor expression in the rat dorsal root ganglion (659.14)
Authors: Kim HungTae , Duellman Tyler , Mavlyutov Tim , Ruoho Arnold , Yang Jay
Published: June 2021

Intrathecal administration of Sigma‐1 receptor (S1R) antagonists attenuate the pain behavior of rodents in both inflammatory and neuropathic pain models. However, the S1R localization in the spinal cord shows a selective ventral horn motor neuron distribution suggesting the high likelihood of S1R in the dorsal root ganglion (DRG) mediating the pain relief by intrathecally administered drugs. Here, we demonstrate the presence of a strong S1R immuno‐fluorescence in all rat DRG neurons co‐localizing with the NSE marker but not in the GFAP‐positive satellite cells At the protein level, Western blots confirmed that the DRG demonstrated a significantly greater S1R expression per ug of total protein compared to liver, heart, spinal cord or cortex. However, qPCR demonstrated greater expression of the S1R mRNA in the liver and heart compared to the DRG indicating the presence of a potential organ‐specific translational or post‐translational regulation of S1R expression. We hope to exploit this and other mechanisms for down regulating S1R expression in the DRG as potential anatomically‐targeted therapeutic strategy for the treatment of inflammatory and neuropathic pain.

Grant Funding Source: Supported by NIH R01 Grant
The role of GABA in hyperbaric oxygen‐induced acute antinociception in mice (659.16)
Authors: Liu Shulin , Shirachi Donald , Quock Raymond
Published: June 2021

Earlier we reported that mice responded with an acute antinociceptive effect during an 11‐min treatment with HBO2 at 3.5 atmospheres absolute (ATA) (Ohgami et al., NeuroReport 20:1325, 2009). We endeavored to explore the possible role of GABA in this HBO2‐induced effect by pretreating different groups of male NIH Swiss mice, 20‐30 g, intracerebroventricularly (i.c.v.) or intrathecally (i.t.) with various drugs that influenced GABA neurotransmission. The pretreatment drugs were the GABAA antagonist SR 95531, the GABAB antagonist CGP 35348 and the GABA neuronal uptake‐inhibitor (±)‐nipecotic acid. Antinociceptive responsiveness to HBO2 was assessed using the acetic acid‐induced abdominal constriction test conducted in a small animal hyperbaric chamber. HBO2 at 3.5 ATA evoked a strong antinociceptive effect that was antagonized in dose‐related manner by i.c.v.‐ or i.t.‐administered SR 95531. I.c.v. and i.t. pretreatment with CGP 35348 or nipecotic acid potentiated HBO2‐induced antinociception at higher doses. These data strongly suggest that GABA is involved in the acute antinociceptive response of mice to HBO2.

Grant Funding Source: Supported by NIH Grant AT‐007222 and the Allen I. White Distinguished Professorship.
B2 bradykinin receptor‐expressing nociceptors are differentially sensitive to the microtubule‐targeting agent, paclitaxel (659.3)
Authors: LoCoco Peter , Scofield Harrison , Chavera Teresa , Mooberry Susan , Berg Kelly , Clarke William
Published: June 2021

Paclitaxel (TAX) produces debilitating peripheral neuropathy accompanied by pain. Interestingly, patients complain more frequently of increased sensitivity to cold but not hot temperatures. Thus, we hypothesize that TAX differentially damages functional subpopulations of peripheral pain‐sensing neurons (i.e. nociceptors). Here, we evaluated TAX effects on bradykinin (BK) receptor‐expressing nociceptors. Paw withdrawal latency (PWL) to a heat or cold stimulus was determined following TAX treatment. PWL of TAX rats to cold was decreased, whereas PWL to heat was increased, suggesting that TAX differentially affected cold‐ and heat‐responsive nociceptors. We then evaluated PWL to cold and heat following i.pl. BK injections. As expected, BK produced a transient cold and heat allodynia in vehicle rats. By contrast, TAX treatment reduced heat, but prolonged cold allodynia. Treatment of primary nociceptor cultures with TAX produced a time‐dependent, tri‐phasic effect on BK‐stimulated phospholipase C activity that was blocked by a B2, but not B1, antagonist. Taken together, these data suggest that nociceptor subpopulations are differentially sensitive to TAX. Understanding why some nociceptors are resistant to effects by TAX could reveal novel treatment/preventative strategies for chemotherapy‐induced peripheral neuropathy.

Grant Funding Source: Supported by USPS grant 8ULITR000149, Greehey Fellowship, and Translational Science Training Program
Chronic morphine enhances nicotine responses in single mouse enteric neurons (659.9)
Authors: Gade Aravind , Dewey William , Akbarali Hamid
Published: June 2021

Opioids are excellent pain relievers. A major side‐effect of chronic opioid treatment is constipation whereas withdrawal following chronic exposure leads to diarrhea and increased gastrointestinal motility. These effects of chronic opioids are mediated by mu opioid receptors expressed on enteric neurons. Previous in‐vitro studies have shown that chronic opioids enhance sensitivity to nicotinic receptor agonists. In this study we examined the sensitivity to nicotine following short (10 minutes) and long‐term exposure (16‐20 hours) to morphine in isolated mouse single enteric neurons using patch‐clamp techniques. Nicotine (1mM) induced inward currents from holding potential of ‐60 mV that were significantly increased in neurons exposed for long‐term but not short‐term to morphine. However a significant shift was not seen in the EC (50) values in all neurons. The maximal current densities induced by 1mM nicotine were 168 ± 29 pA/pF, 135 ± 31 and 296 ± 51 pA/pF in control, short‐term and long‐term morphine treated cells respectively, and the EC (50) values were 44 ± 17 µM (n = 9), 42 ± 14 (n = 5) and 26 ± 7 µM (n = 11) in the same order. Shift in the maximal currents but not in the EC (50) values suggests a shift in the efficacy but not potency of nicotine after long term exposure to morphine. Studying the mechanisms involved in this enhancement is hypothesized to give a better insight into methods for treating opioid dependent and withdrawal symptoms. Supported by NIH grants DK046367 and DA024009.
Inhalation of multi‐walled carbon nanotubes affects lung resistance and compliance and evokes airway hyperreactivity to methacholine in rats (660.1)
Authors: Fedan Jeffrey , Thompson Janet , Frazer David , Porter Dale , Tsuruoka Shuji , Endo Morinobu , McKinney Walter
Published: June 2021

The incorporation of carbon nanotubes into new materials has raised concerns about their potential hazards to workers exposed during manufacturing. In animals, following pharyngeal aspiration, i.t. instillation and inhalation exposures to MWCNT, airway inflammation and lung fibrosis have been reported. However, the effects of inhalation of MWCNT on pulmonary function and reactivity to inhaled methacholine (MCh), a bronchoconstrictor, have not been studied. Therefore, we investigated whether inhaled MWCNT affect lung resistance (RL) and dynamic compliance (CDyn), and/or reactivity to inhaled MCh aerosol. Male rats were exposed for 6 h to filtered air or 5 mg/m3 of aerosolized MWCNT (MWNT‐7; Hodogaya Chemical Co.; 20 ‐ 50 walls; median length, 3.86 μm; mean width, 49 nm; mass median aerodynamic diameter, 1.5 μm; particle count aerodynamic diameter mode, ~0.4 μm). Basal RL and CDyn were measured 18 h and 7 d after the end of exposure, and responses to increasing concentrations of MCh were obtained. Eighteen h after exposure basal RL was increased and basal CDyn was decreased; 7 d after exposure basal RL and CDyn were not changed. Reactivity to MCh (RL) was increased and CDyn responses were decreased at 18 h but not 7 d after exposure. The results indicate that lung function and airway reactivity changes occur acutely, but that these changes subside over time following a single MWCNT inhalation exposure.

Grant Funding Source: Supported by NIOSH
Nerve growth factor reduces amiloride‐sensitive Na+ transport in human airway epithelial cells through a trkA‐Erk1/2 mediated pathway (660.10)
Authors: Shimko Michael , Zaccone Eric , Thompson Janet , Fedan Jeffrey
Published: June 2021

Nerve growth factor (NGF) is over‐expressed in airways of patients with inflammatory lung diseases. Ion transport by the epithelial cells of the lung is responsible for controlling the periciliary liquid layer, and changes in ion transport can accompany disease. To investigate NGF’s effects on airway epithelium, human primary cultured epithelial cells were placed in an Ussing chamber to measure short‐circuit current (Isc). NGF (1 ng/ml), applied apically, reduced Isc (4.5%) and reduced amiloride (apical, 3.5×10‐5 M) ‐sensitive Na+ transport (control, 52%; NGF‐treated, 38%). To investigate whether the NGF receptor, trkA, is responsible for mediating the responses to NGF, cells were incubated with either the tyrosine kinase inhibitor K‐252a (10 nM, apical) or DMSO (0.004 %) in the absence or presence of NGF. K‐252a did not affect Na+transport, but attenuated the NGF‐induced reduction in Na+ transport. To investigate whether trkA activates the Erk1/2 signaling cascade, which results in the down‐regulation of ENaC, cells were incubated with PD‐98059 (30 µM, apical and basolateral) or DMSO (0.06 %) in the absence or presence of NGF. PD‐98059 did not affect Na+ transport, but attenuated the NGF‐induced reduction in Na+ transport. These results indicate that NGF reduces Na+ transport through a trkA‐Erk1/2 mediated signaling pathway.

Grant Funding Source: Supported by NIOSH
Butter flavoring‐elicited relaxation and bioelectric responses of rat airway are independent of bitter taste receptors (TAS2R) (660.14)
Authors: Zaccone Eric , Shimko Mike , Thompson Janet , Fedan Jeffrey
Published: June 2021

“Popcorn workers’ lung” is a fixed obstructive pulmonary disease caused by inhalation of artificial butter flavoring vapor. In the isolated, perfused rat trachea preparation, the butter flavorings, diacetyl and 2,3‐pentanedione elicit airway smooth muscle (ASM) relaxation. TAS2Rs mediate relaxation of ASM by increasing the concentration of intracellular Ca2+ via large‐conductance Ca2+‐activated K+ (BKCa) channels. This study investigated the potential involvement of TAS2Rs in relaxant responses of tracheal ASM and bioelectric responses of tracheal epithelial cells in response to denatonium (1 mM), a TAS2R agonist, diacetyl (3 mM) and 2,3‐pentanedione (3 mM). In rat tracheal strips contracted with methacholine (3×10−5 M), denatonium‐induced relaxation responses were inhibited by iberiotoxin (IbTx; 100 nM), a BKCa channel blocker, suggesting that denatonium‐induced relaxation is mediated by TAS2R. However, relaxation responses to the butter flavorings were not antagonized by IbTx. Whether TAS2R mediate bioelectric responses to denatonium and flavorings in the epithelium was investigated. The decrease in short‐circuit current (Isc) in response to denatonium and flavorings were not blocked by apical IbTx, suggesting that the responses in the epithelium were not mediated by TAS2R. Our findings indicate that BKCa channels are not involved in flavoring‐induced bronchodilation or Isc responses in rat airways.
The efficacy of apocynin in mitigating LPS‐induced acute lung injury when given prior to, but not after LPS insult (660.3)
Authors: Lee Intae , Dodia Chandra , Chatterjee Shampa , Feinstein Sheldon , Fisher Aron
Published: June 2021

Apocynin (4‐hydroxy‐3‐methoxyacetophenone; APO) is an inhibitor of NADPH oxidase (NOX2) activation and a possible scavenger of reactive O2 species (ROS). Regulating NOX2 may play a crucial role in LPS (lipopolysacharide)‐induced ROS production and the pathogenesis of ALI. To evaluate the effect of APO on lung injury in mice following intra‐tracheal infusion of LPS (0111:B4 from E. coli) at 1 mg/kg, mice received iv APO at 2.5 mg/kg at 0.5h prior to LPS or 2h post LPS. Measurements were performed 24h post LPS. LPS significantly increased lung infiltration with inflammatory cells, lung alveolar‐capillary permeability (lung wet to dry ratio, leakage of protein and FITC‐dextran into the bronchoalveolar lavage fluid), expression of pro‐inflammatory cytokines (IL‐6, TNF‐α) and a chemokine (MIP‐2). APO dramatically reduced all of these parameters when administered at 0.5h prior to LPS but had no significant effect when administered at 2h post LPS. Thus, APO when administered prophylactically prevented lung inflammation, excessive ROS production, and cell injury but was not effective when administered during the early stages of LPS‐induced inflammation. These findings contrast with the recently reported effectiveness of MJ33, also an inhibitor of NOX2 activation, when administered 2 h post LPS. The mechanism for the difference between the 2 inhibitors requires further investigation.

Grant Funding Source: NHLBI‐R01‐HL105509 (PI: Dr. Aron B. Fisher)
A novel approach to bronchodilation: combining the Kv7 channel activator retigabine with the β2‐adrenergic agonist formoterol (660.9)
Authors: Haick Jennifer , Brueggemann Lioubov , Byron Kenneth
Published: June 2021

Kv7 potassium channels are expressed in airway smooth muscle cells (ASMCs) and bronchoconstrictor agonists, such as methacholine and histamine, inhibit Kv7 currents. Inhibition of Kv7 channels in rat and human airways is sufficient to induce bronchoconstriction. Long‐acting β2‐adrenergic agonists (LABAs) are commonly used in the treatment of asthma. However, long term use of these drugs may lead to tolerance. Here we provide evidence that the Kv7 channel activator retigabine can oppose agonist‐induced suppression of Kv7 currents and enhance bronchodilation by the LABA formoterol. Dose‐dependent bronchodilation by retigabine and formoterol was measured using rat precision cut lung slices (PCLS). A submaximal dose of formoterol (10nM) induced relaxation of methacholine‐constricted airways, but this relaxation was transient despite continued treatment. The approximate EC50 dose of retigabine (10µM) enhanced formoterol‐induced relaxation and eliminated the apparent desensitization. Similar results were obtained in human PCLS pre‐constricted with histamine, suggesting a beneficial effect of combining a Kv7 channel activator with standard bronchodilator therapy.

Grant Funding Source: Supported by Loyola University Chicago
Effects of self‐administered methamphetamine on learning‐to‐learn in the squirrel monkey (661.1)
Authors: Kangas Brian , Bergman Jack
Published: June 2021

Long‐term chronic exposure to methamphetamine (MA) has been related to profound neural impact including, but not limited to, damage to monoamine nerve terminals. In turn, neural changes following repeated MA administration may be related to adverse effects on learning and other cognitive performance. However, the characterization of such neurobehavioral deficits remains incomplete. The present study assessed the effects of ongoing MA self‐administration on touchscreen‐based discrimination learning in nonhuman primates. Prior to daily sessions of MA self‐administration (i.e., 20‐hr after the previous day’s session), monkeys engaged in a repeated acquisition task in which changes in the rate of discrimination learning for a stimulus pair were assessed over the successive presentation of novel stimulus pairs. Results thus far indicate that ongoing MA self‐administration produces markedly deleterious effects on the development of learning sets in discrimination learning. Importantly, the magnitude of adverse effects on learning appears to be correlated with the level of daily MA intake among individual subjects.

Grant Funding Source: Supported by grant K01‐DA035974 from NIDA
Reinforcing effects of abused “bath salts” constituent 3,4‐methylenedioxypyrovalerone in mice and rats (661.7)
Authors: Gannon Brenda , Norwood Andrew , Tai Sherrica , Bailey Jessica , Fantegrossi William
Published: June 2021

Recently, synthetic analogues of naturally‐occurring cathinone in commercial “bath salt” preparations have emerged as psychostimulant‐like drugs of abuse. 3,4‐Methylenedioxypyrovalerone (MDPV) is a common constituent of these illicit products, and recent studies implicate monoamine transporters as mediators of its pharmacological effects. In these studies, adult male NIH Swiss mice and Sprague Dawley rats were used to assess reinforcing effects of MDPV in assays of two‐bottle choice oral self‐administration (mice), responding maintained by a drug‐paired stimulus complex (mice), and intravenous self‐administration (rats). In mice, MDPV concentrations sufficient to produce locomotor stimulation (0.1 and 0.3 mg/ml) and lethality (1 mg/ml) were consumed when the alternative fluid was 0.1 mg/ml quinine. However, when water was available as an alternative to MDPV, very little MDPV consumption occurred at the concentrations which previously elicited biological effects. In subsequent studies, mice were implanted with radiotelemetry probes to simultaneously measure core temperature and locomotor activity, and provided with MDPV solutions to drink (0.03, 0.1, 0.3, and 1 mg/ml). Separate groups of mice were trained to respond under an FR5 schedule reinforced by milk presentations. In pairing sessions, MDPV (0, 1 or 10 mg/kg) was administered IP in the presence of a novel light+tone stimulus complex. The light+tone stimulus complex was contingently available under a PR schedule after 0, 3 or 6 pairings with MDPV, but no apparent reinforcing effects were observed. In contrast, MDPV was an effective intravenous reinforcer across a wide range of unit doses (0.003, 0.01, 0.03, 0.1, and 0.3 mg/kg/infusion) in rats previously trained to self‐administer phencyclidine (PCP) and its analogue methoxetamine (MXE). Possible explanations for the discrepancy in apparent reinforcing effects of MDPV may be related to route of administration or species differences and will be discussed.

Grant Funding Source: Supported by RR020146 and RR029884
The insecticides carbaryl and carbofuran show high affinity for hMT2 melatonin receptors (662.12)
Authors: Popovska‐Gorevski Marina , Dubocovich Margarita , Rajnarayanan Rajendram
Published: June 2021

Carbaryl (1‐naphthyl methylcarbamate) and carbofuran (2,3‐dihydro‐2,2‐dimethyl‐7‐benzofuranyl methylcarbamate) are among the most toxic insecticides, known to increase diabetes risk. Using an integrated pharmacoinformatics‐molecular docking approach, we found carbaryl and carbofuran to be structurally similar to melatonin. These compounds docked into our predictive 3D model of the MT2 melatonin receptor with relatively high binding affinity scores. Carbaryl and carbofuran then were tested for competition with 2‐[125I]‐iodomelatonin (300nM) binding to hMT1 or hMT2 receptors stably expressed in CHO cells. Biphasic competition curves suggest that carbaryl binds to two affinity states of the hMT1 (IC50Hi=6.8nM, IC50Lo=22µM) and hMT2 (IC50Hi=16nM, IC50Lo= 0.9μM) receptors while carbofuran binds two sites on the hMT2 (IC50Hi=27nM, IC50Lo=11μM) receptor. Compared to carbaryl, carbofuran affinity for hMT2 was lower, but it showed selectivity, as it did not compete for hMT1 binding. GTP (100 mM) did not affect binding affinity of carbaryl for either hMT1 or hMT2 receptors. However, GTP significantly decreased the affinity of carbofuran for hMT2 receptors suggesting potential agonist efficacy. These data support the utility of molecular modeling for screening potential melatonin ligands and suggest that carbaryl and carbofuran could interact with pancreatic MT2 receptors, known to affect glucose homeostasis and insulin secretion.

Grant Funding Source: Supported by UB Funds
Probing the structural determinants of the central region of third intracellular loop of human dopamine D1‐class receptor subtypes (662.9)
Authors: Albaker Awatif , Yang Xiaodi , Liang Binhui , Charette Andrew , Tiberi Mario
Published: June 2021

Dopamine D1‐class receptors (D1, D5) are seven transmembrane receptors and play a major role in regulating important physiological effects in the brain and in the periphery. The first and the second intracellular loops are highly conserved between D1R and D5R while the third intracellular loop is the most divergent. The alpha‐helical of the amino and the carboxyl ends of the third intracellular loop (IL3) of GPCRs play a pivotal role in receptor activation and G proteins coupling. Meanwhile, it remains unclear whether the central region of IL3 linking the cytoplasmic extension of TM5 and TM6 significantly contributes to GPCR activation and G protein coupling. Here, we address this issue using either deletions of the conserved N‐terminal moiety or divergent C‐terminal moiety of the central region of IL3 of human D1‐class receptors (D1ΔN and D5ΔN; D1ΔC and D5ΔC). Also, we test the effect of agonist‐induced desensitization on these mutants. The agonist affinities were significantly increased and decreased at D1ΔN and D5ΔN, respectively. However, D1ΔC and D5ΔC displayed drastically increased agonist affinity. We also observed full abolition of constitutive activity of D1ΔN and D5ΔN, and a severe blunting in agonist‐induced cAMP formation. In contrast, constitutive and agonist‐dependent activity of D1ΔC and D5ΔC were considerably increased. Overall, our study strongly suggests a critical role of the central region of IL3 in subtype‐specific activation of D1‐class dopaminergic signaling. Potentially, this may be achieved through distinct motifs located in the N‐ and C‐terminal moiety of the central region of IL3

Grant Funding Source: supported by a Graduate Scholarship from King Saud University (to AB) and OMHF grant (to MT)
Mitochondrial reactive oxygen species induce hyperexcitability in nociceptive airway sensory nerves via PKC activation (663.5)
Authors: Taylor‐Clark Thomas , Hadley Stephen , Bahia Parmvir
Published: June 2021

Activation of airway nociceptive C‐fibers evokes cough and parasympathetic reflexes. Nociceptor excitability is increased by inflammation, worsening symptoms in asthma. Airway C‐fiber terminals are packed with mitochondria. Inflammatory signaling modulates mitochondrial function, causing reactive oxygen species (ROS) production. We hypothesize that these ROS increase nociceptor excitability. We recorded action potentials in single C‐fibers using an ex vivo mouse lung‐ganglia preparation. “Nociceptors” are slow fibers, sensitive to capsaicin (via TRPV1). Antimycin A, a mitochondrial complex III inhibitor that evokes ROS, had no effect on the excitability of non‐nociceptive nerves. Ten minutes after antimycin A, nociceptive nerves developed hyperexcitability to chemical and mechanical stimuli. Antimycin‐induced hyperexcitability was blocked by N‐acetyl cysteine and dithiothreitol but not by TRPV1 knockout. ROS can activate protein kinase C independently of Ca2+. Here, the PKC inhibitor bisindolylmaleimide I (BIM I), but not the inactive analogue BIM V, abolished antimycin‐induced hyperexcitability. Antimycin A also caused PKC translocation via ROS. Lastly, exogenous H2O2 induced nociceptor hyperexcitability, which was blocked by BIM I. We conclude that acute modulation of nerve terminal mitochondria can induce airway nociceptor hyperexcitability via ROS‐activated PKC pathways.

Grant Funding Source: Supported by R01HL119802‐01
In humans with type 2 diabetes, prostacyclin receptor expression increases as hemoglobin A1c levels increase (664.6)
Authors: Stephenson Alan , Knebel Stephanie , Sprague Randy
Published: June 2021

In healthy humans (HH), exposure of erythrocytes to reduced O2 tension stimulates the release of O2 as well as the potent vasodilator adenosine triphosphate (ATP). Erythrocytes of humans with type 2 diabetes (DM2) fail to release ATP when exposed to reduced O2 tension. However, iloprost (1 µM)‐induced stimulation of the prostacyclin receptor (IPR) activates a signaling pathway distinct from the low O2 pathway resulting in increased cAMP and ATP release in erythrocytes of HH and humans with DM2. In fact, as glycemic control worsens (indicated by increases in HbA1c) IPR‐mediated ATP release increases. We hypothesized that with DM2, the increased ATP release may result from increased IPR expression or decreased phosphodiesterase (PDE) 3 activity. Previously, we reported that when PDE3 activity was inhibited with cilostazol, a selective PDE3 inhibitor, iloprost (1 µM)‐induced ATP release was not different between HH and DM2, indicating that PDE3 activity is decreased in erythrocytes from humans with DM2. Here, using Western blot analysis, we found that expression of IPR monomeric (40, 50, and 65 kD, n=8, P<0.05) and dimeric isoforms (MW greater than 65 kD, n=8, P<0.02) was directly correlated with levels of HbA1c. Taken together with the PDE studies, when compared to HH erythrocytes, decreased PDE3 activity and increased expression of the IPR as glycemic control worsens may explain the increased iloprost‐induced ATP release in erythrocytes from humans with DM2.

Grant Funding Source: ADA and NIH
Ionizing radiation promotes the acquisition of a senescence‐associated secretory phenotype and impairs angiogenic capacity in cerebromicrovascular endothelial cells: role of increased DNA damage and decreased DNA repair capacity in microvascular radiosens (665.3)
Authors: Ungvari Zoltan , Podlutsky Andrej , Sosnowska Danuta , Tucsek Zsuzsanna , Toth Peter , Deak Ferenc , Gautam Tripti , Csiszar Anna , Sonntag William
Published: June 2021

Cerebromicrovascular rarefaction is believed to play a central role in cognitive impairment in patients receiving whole‐brain irradiation therapy. To elucidate the mechanism underlying the deleterious effects of γ‐irradiation on the cerebral microcirculation, rat primary cerebromicrovascular endothelial cells (CMVECs) were irradiated in vitro. We found that in CMVECs, γ‐irradiation (2‐8 Gy) elicited increased DNA damage, which was repaired less efficiently in CMVECs compared with neurons, microglia, and astrocytes. Increased genomic injury in CMVECs associated with increased apoptotic cell death. In the surviving cells, γ‐irradiation promotes premature senescence (indicated by SA‐β‐galactosidase positivity and upregulation of p16 (INK4a)), which was associated with impaired angiogenic capacity (decreased proliferation and tube‐forming capacity). γ‐Irradiated CMVECs acquired a senescence‐associated secretory phenotype, characterized by upregulation of proinflammatory cytokines and chemokines (including IL‐6, IL‐1α, and MCP‐1). Collectively, increased vulnerability of γ‐irradiated CMVECs and their impaired angiogenic capacity likely contribute to cerebromicrovascular rarefaction and prevent regeneration of the microvasculature postirradiation. The acquisition of a senescence‐associated secretory phenotype in irradiated CMVECs is biologically highly significant as changes in the cytokine microenvironment in the hippocampus may affect diverse biological processes relevant for normal neuronal function (including regulation of neurogenesis and the maintenance of the blood brain barrier).

Grant Funding Source: Supported by NIH and the Ellison Medical Foundation
Functional consequences of chronic mast cell activation by aged mesenteric lymphatic vessels (666.1)
Authors: Gashev Anatoliy , Chatterjee Victor , Tobin Richard , Newell Rogers M. Karen
Published: June 2021

Here we present novel data related to interactions between lymphatic vessels, mast cells (MCs), eosinophils, and immune cells. MCs are known to inhabit in greater density the tissue near mesenteric lymphatic vessels (MLVs). Acute activation induces MC degranulation with subsequent recruitment of eosinophils and of MHC class II+ cells towards MLVs. Aging permanently influences mesenteric MCs by inducing chronic basal activation, thereby influencing contractility of aged MLVs. At the same time, the number of intact MCs available to react acutely to inflammatory stimuli decreases with age. The diminished number and activity of MCs in aged tissues also diminishes traffic and activation of eosinophils and other immune cells that are attracted to aged MLVs. Concurrently, MCs can function as helper cells for B cells that can produce IgE. IgE has a relatively short half‐life in the absence of chronic MC activation. We propose that a key component of aging‐associated alterations of “mesenteric lymphatic system‐mast cells/eosinophils‐inflammatory cells” system is the increased basal activation of resident MCs and eosinophils in aged mesentery that promote chronic inflammation while being insufficient to recruit enough anti‐inflammatory MHC class II+ cells and eosinophils, resulting in the inability to prevent a smooth transition from acute inflammation to specific immune cell activation. NIH R01 AG030578.

Grant Funding Source: NIH R01 AG030578
Pump function in isolated juvenile rodent lymphatics (666.2)
Authors: Moor Andrea , Breslin Jerome
Published: June 2021

Primary lymphedema typically afflicts children, however, studies of lymphatic physiology generally examine lymphatic vessels from adult animals. The goal of this study was to evaluate responses of isolated lymphatic vessels obtained from juvenile (5‐7 week old) Sprague‐Dawley rats to 1) changes in luminal pressure, or 2) substance P, an endogenous lymphatic constricting agent. Isolated lymphatics were mounted onto resistance‐matched glass micropipettes bathed in 37°C albumin physiological salt solution (APSS) at 2 cm H2O luminal pressure. Pressure was stepped to 4, 6, and 8 cm H2O. In other experiments, the response to 10 nM substance P was assessed. Video recordings were used to obtain contraction frequency (CF), end diastolic diameter (EDD), end systolic diameter (ESD), and contraction amplitude (AMP = EDD ‐ ESD). Ca2+‐free APSS was used to obtain the maximal passive diameter (MaxD), and the ability of lymphatics to reestablish tone and pumping upon switching back to normal APSS was also evaluated. Tone was calculated as 100*(MaxD‐EDD)/MaxD. In response to elevations in luminal pressure, juvenile lymphatics displayed increases in EDD, ESD, and CF. In contrast, the amplitude of the phasic contractions decreased. Lymphatic tone was also diminished. After measuring passive diameter in Ca2+‐free solution, upon changing back to Ca2+‐containing APSS lymphatics reestablished tone and pumping respectively. The addition of substance P to the bath elicited an immediate constriction of lymphatics and increased CF over a 5 min period. Substance P also caused constriction even in the presence of 10‐8‐10‐7M nifedipine, an L‐type calcium channel blocker, however lymphatic pumping did not resume. These experiments demonstrate similarities in isolated juvenile rat lymphatics with previous studies using lymphatic vessels isolated from adult rats.
Bilirubin loading of the heart: a novel treatment for ischemia‐reperfusion injury (667.5)
Authors: Bakrania Bhavisha , Du Toit Eugene , Headrick John , Bulmer Andrew
Published: June 2021

Cardiovascular Disease (CVD) is the world’s greatest killer, claiming the lives of around 17.2 million men and women per year. Bilirubin is a novel antioxidant, which has been cited to reduce ischemia‐reperfusion injury in various tissues; however research into cardiac ischemia‐reperfusion injury is yet to be completed. The aim of this research was to determine whether exogenous bilirubin is capable of reducing ischemia‐reperfusion injury, in the heart. Young male (12 weeks old) Wistar isolated rat hearts were treated with bilirubin (50mM) for a period of 30 minutes before (Pre) or after (Post) a 30 minute no flow ischemic period. As well as functional recovery, tissue damage was measured by collecting effluent samples and quantifying infarct size. The left atria was removed from each heart after their respective treatment periods to allow analysis, through Pearson's correlation, of recovery and bilirubin concentration in the tissue. Diastolic pressure in both Pre and Post treated hearts was most significantly reduced after 90 minutes of reperfusion (Control, 81.01±14.73 mmHg, Pre, 58.92±14.23 mmHg, Post, 64.74±13.21 mmHg). The pre‐treated hearts revealed a significant recovery in left ventricular developed pressure (LVDP) in comparison to controls (Control, 34.72±12.67 %, Pre, 54.32±12.87 %). In addition, biomarkers of tissue damage, as well as infarct area, were significantly reduced in both pre‐treated and post‐treated hearts, in comparison to controls. Although the mechanisms are still unclear, this data suggests that bilirubin loading on the heart, either before or after ischemic insult, can preserve heart tissue and function.
Gp91ds‐tat, a selective NADPH oxidase peptide inhibitor, increases blood nitric oxide bioavailability in hind limb ischemia and reperfusion (667.7)
Authors: Walker Sydney , Galbreath Tyler , Chen Qian , Barsotti Robert , Patel Harsh , Chau William , Young Lindon
Published: June 2021

I/R injury induces cell death and organ dysfunction in part due to a burst of reactive oxygen species that occurs upon the reintroduction of oxygen into the ischemic area, leading to endothelial dysfunction: decreased blood NO and increased hydrogen peroxide (H2O2 ) levels. We’ve previously shown in isolated rat hearts subjected to I/R injury, gp91ds‐tat attenuated cardiac contractile dysfunction and reduced infarct size compared to controls presumably by the inhibition of NADPH oxidase induced superoxide release. Superoxide can quench NO via the formation of peroxynitrite and also be converted to H2O2 in blood. We attempted to confirm this hypothesis using a rat hind limb I/R model that permitted real time measurements of changes in blood NO and H2O2. NO or H2O2 microsensors were inserted into both femoral veins in anesthetized male rats. One limb’s femoral artery/vein is subjected to I(30min)/R(45min) while the other served as a non‐ischemic sham. Preliminary results show blood NO release significantly increased by the end of reperfusion in gp91ds‐tat treated rats (1.2 mg/kg, MW 2452g/mol, n=5) compared to saline treated rats (n=3;p<0.05). The preliminary data suggest that gp91ds‐tat attenuates I/R induced endothelial dysfunction. The results of gp91ds‐tat on blood H2O2 levels are forthcoming.
Visualizing the endothelium from inside intact arteries at physiological pressures reveals pulsatile linear, circular and spiral Ca2+ waves (668.1)
Authors: Wilson Calum , Saunter Christopher , Zagnoni Michele , Girkin John , McCarron John
Published: June 2021

Ca2+ signalling in the endothelium is central to the control of vascular function and to the changes that occur in vascular disease. However, visualizing Ca2+ signals in endothelial cells in intact, pressurised arteries is difficult (particularly in the larger arteries where atherosclerosis and re‐stenosis occurs), due to medial and adventitial thickness. A novel wide‐field, epi‐fluorescent microscope with subcellular resolution, that enables endothelium imaging from the inside of large pressurised arteries, has been developed. A side‐viewing Gradient Index (GRIN) relay lens, inserted into the lumen of pressurised arteries, permits the endothelium to be imaged with a 350 µm diameter field of view ‐ an area encompassing approximately 200 cells. Acetylcholine‐activation of the endothelium evoked pulsatile, repetitive Ca2+ waves that initiated at several sites (measured by fluorescent indicators). Waves progressed through the endothelium linearly, in expanding circles and in spiral forms. The Ca2+ waves show properties of excitable systems i.e. regenerative propagation and annihilation of colliding wavefronts. The GRIN lens approach offers a new level of understanding in the study of endothelial Ca2+ signalling and we propose the endothelium, via Ca2+ signals, behaves as an excitable medium in physiological conditions.

Grant Funding Source: Wellcome Trust; British Heart Foundation; EPSRC
Role of endoplasmic reticulum chaperone protein BiP in endotoxin‐induced acute lung injury (669.10)
Authors: Fazal Fabeha , Schwarzenbart Amanda , Leonard Antony
Published: June 2021

Endoplasmic reticulum (ER) makes up approximately 10% of the cell volume and plays an important role in multiple cellular processes such as calcium homeostasis, protein synthesis, folding and secretion, and lipid biosynthesis. Disturbances in ER homeostasis result in ER stress, which is implicated in a wide range of pathological conditions such as ischemic injury, neurodegenerative disorders, metabolic diseases and more recently in inflammatory conditions. A key step in the pathogenesis of acute lung injury (ALI), an inflammatory disease, involves alveolarcapillary membrane injury. This leads to infiltration of inflammatory cells into the alveolar airspaces and increased permeability, resulting in pulmonary edema. In this study, we report a critical role for ER chaperone and signaling regulator BiP in LPS‐induced ALI. Our data shows that AB5 cytotoxin family member subtilase cytotoxin (SubAB), which selectively cleaves and inactivates BiP, triggers ER stress and protects mice from endotoxin‐induced lung injury. In WT mice, LPS inhalation induced significant polymorphonuclear leukocyte (PMN) recruitment into bronchoalveolar lavage (BAL); however, mice pretreated to intraperitoneal injection of SubAB were protected from PMN influx into the alveolar airspaces. Consistently, LPS‐induced level of adhesion protein ICAM‐1, which plays a critical role in PMN adhesion and endothelial transmigration, was also reduced in SubAB treated mice. In addition, LPS‐induced microvascular permeability was restored upon SubAB pretreatment, as evidenced by a marked decrease in the level of albumin in the BAL. In contrast, SubAA272B, a SubAB active site mutant that cannot induce ER stress, failed to show the protective effect. These findings suggest that preconditioning the mice with ER stress ameliorates ALI caused by LPS.

Grant Funding Source: NIH
Differentional effect of black raspberry extract on two primary human microvascular endothelial cells isolated from intestine and esophagus (669.2)
Authors: Medda Rituparna , Nie Linghui , Link Benjamin , Lyros Orestis , Otterson Mary , Rafiee Parvaneh
Published: June 2021

BACKGROUND: Polyphenolic compounds (anthocyanins, flavonoid glycosides) in berries have been shown to prevent the carcinogenesis and tumorigenesis in esophagus and digestive tract. Angiogenesis has been implicated in the pathogenesis of inflammatory bowel disease (IBD). In this study, we aimed to determine the effects of black raspberry extract (BRE) on human primary microvascular endothelial cells isolated from intestine (HIMEC) and esophagus (HEMEC).

METHODS: HIMEC and HEMEC monolayer were activated with either with VEGF or TNF‐α/IL‐1β with or without BRE. Effects of BRE on cells survival, apoptosis, proliferation, migration and tube formation were determined. COX‐2, ICAM‐1 and VCAM‐1 genes and proteins expression were assessed by qPCR and immunobloting and nuclear localization of p65 was observed by immunofluorescence staining.

RESULTS: VEGF increased cell migration, proliferation and in vitro tube formation and BRE treatment of cells suppressed these effects in both HIMEC and HEMEC. TNF‐α/IL‐1β treatment translocated the p65 subunit of NFκB to nucleus, increased COX‐2, ICAM‐1 and VCAM‐1 gene and protein expression and the PGE2 activity in HIMEC and HEMEC. However, BRE treatment only was effective in inhibiting the COX2, NFκB and ICAM‐1 and VCAM‐1 in HEMEC, but exerted no inhibitory effect on HIMEC.

CONCLUSIONS: Taken together, BRE is a potent anti‐inflammatory agent for HEMEC but not HIMEC.

Grant Funding Source: MCW Digestive Disease
Soluble JAM‐C induces monocyte migration via Erk (669.5)
Authors: Tuma Elizabeth , Imhof Beat , Koch Alisa , Rabquer Bradley
Published: June 2021

Purpose: The migration of leukocytes, including monocytes (MNs), is a major contributor to inflammation in autoimmune diseases such as rheumatoid arthritis (RA). In RA, MN migration to the joint synovial tissue is aided by junctional adhesion molecules (JAMs) expressed by endothelial cells (ECs). The EC JAMs bind integrins found on leukocytes, promoting their ingress. JAM‐C is the ligand for the integrin Mac‐1 (Cd11b/CD18). Like some adhesion molecules, JAM‐C has been found in soluble form, and our recent work has shown that soluble JAM‐C (sJAM‐C) is upregulated in RA synovial fluid. Some soluble adhesion molecules have also previously been shown to induce leukocyte migration. Based on this, we hypothesized that sJAM‐C promotes THP‐1 MN migration by binding Mac‐1 and inducing the phosphorylation of Erk.

Methods & Results: To test this hypothesis, we cultured THP‐1 cells to model MN function. Using qPCR (n=2) we found that THP‐1 MNs express the Cd11b portion of Mac‐1. Next, in vitro MN chemotaxis assays allowed us to determine that sJAM‐C is chemotactic for MNs in a concentration dependent fashion (n=3, p<0.05). In addition, our Western blotting showed that sJAM‐C induces the phosphorylation of Erk in a time dependent fashion (n=3).

Conclusions: Our results indicate that THP‐1 MNs express Mac‐1, and suggests that sJAM‐C binding to Mac‐1 induces THP‐1 migration via the Erk pathway.

Grant Funding Source: Supported by Albion College
Smooth muscle cell organization in the stem region of the gracilis collateral circulation (670.1)
Authors: Krall Amanda , Nehzati Ashkon , Bynum Alexander , Laiho Lily , Cardinal Trevor
Published: June 2021

Many patients who suffer from the ischemic Peripheral Arterial Occlusive Disease (PAOD) experience intermittent claudication, pain during exercise, due to the build up of atherosclerotic plaques in the arteries of the lower extremities. Impaired vasodilation is a possible explanation for intermittent claudication. Collateral vessels function as natural bypasses around the occluded arteries and the increase in flow into these vessels causes them to outwardly remodel, which will improve their efficiency. The capacity to vasodilate is imperative for maximal effectiveness and function in collaterals. Functional vasodilation in the stem region collateral vessels is impaired at day 7 following femoral ligation, which can be attributed to smooth muscle cell malfunction. The increase in vessel diameter in outwardly remodeled collaterals is not due to cell proliferation. One possible explanation for diameter increase in collateral vessels is that the vascular smooth muscle cells undergo mechanoadaptation to acclimate to the increase in blood flow. To investigate whether mechanoadaptation is the cause of outward remodeling in the profunda femoris artery (collateral stem), we ligated the femoral artery. At day 7 following surgery, we evaluated maximal vasodilation using sodium nitroprusside and papaverine before perfusion fixation. The profunda was then resected and immunostained with α‐smooth muscle actin before measuring smooth muscle cell length and overlap, using confocal microscopy.