bluuebunny/crossref_metadata_2025 huggingface dataset:
Antidotes for Acute Cannabinoid Intoxication
Authors: Vemuri Kiran , Raghav Jimit , Makriyannis Alexandros
Published: June 2021

The cannabis constituent Δ9‐tetrahydrocannabinol (THC), now being legalized for recreational and medical use, exerts its psychotropic effects (marijuana “high”) via CB1 receptor activation. Synthetic psychoactive cannabinoids (SPCs) such as JWH‐018, illegally sold as “designer drugs”, mimic the effects of THC, a CB1 partial agonist, with higher potency, acting via CB1 receptors. Although acute toxicity is benign for consuming high doses of THC, patients admitted to the ER may exhibit symptoms of hyperemesis, irritation of conjunctival and bronchial tissue, along with impaired cognition and motor skills. SPCs act as full CB1R agonists wherein clinical manifestations associated with their consumption include, neurotoxicity represented by several negative behavioral outcomes, and even death. While such adverse events may differ among patients, current adjunctive treatments include intubation, use of benzodiazepines, antipsychotics, antihistamines, adenosine, and hypnotics, along with i.v. saline, and supportive care. Currently, there are no effective treatments available for directly blocking and reversing the effects of cannabinoid poisoning. To address this critical public‐health issue, we have initiated a drug discovery program to develop antidotes that can counteract the intoxicating/adverse effects of CB1 activation and be therapeutically useful in an emergency setting. Although CB1 antagonists such as rimonabant appear to be promising pharmacotherapeutics, this class of drugs are known to produce nausea and unacceptable pro‐depressant effects. We have now discovered a novel class of potent, selective and brain penetrant CB1 antagonists, displaying better safety profiles as compared to rimonabant. In male CD‐1 mice, these novel CB1 antagonists represented by AM11503 (10 mg/kg, i.v), antagonized the hypothermic effect induced by THC (30 mg/kg, i.p.) and was equipotent to rimonabant (10 mg/kg, i.v.) in rapidly normalizing (≤ 60 min, post‐antagonist) the hypothermia induced by an acute dose of JWH‐018 (6 mg/kg, i.p). To mimic an ER‐like situation, antagonists were given i.v., 60 min post‐ JWH‐018, where the agonist effect is at its maximum. AM11503 (10 mg/kg, i.v or 18 mg/kg, i.p) displayed no side‐effects when compared to rimonabant and AM251, which, at similar doses, induced significant stereotypic behavior (pruritic responses). Remarkably, when tested for their ability to block the neurotoxic effects of SPCs, AM11503 (10 mg/kg, i.v.) fully blocked tremors and convulsions induced by “suprapharmacological” doses of JWH‐018 (18 mg/kg, i.p). Lead optimization efforts along with safety and efficacy studies are further underway. Here, we established proof‐of‐concept and present preliminary data towards developing the first specific antidote for cannabinoid intoxication.

Support or Funding Information

Supported by NIDA/NIH grant DA045882 (to KV)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanism of Prenatal Cannabinoid Exposure Mediated Memory Loss in Adolescent Offspring: Opportunities for Identifying Therapeutic Target
Authors: Pinky Priyanka Das , Bloemer Jenna , Setti Sharay E , Heslin Ryan T , Smith Warren D , Du Yifeng , Dityatev Alexander , Reed Miranda N , Suppiramaniam Vishnu
Published: June 2021

Cannabis use during pregnancy has increased by 62% from 2002 through 2014 and is now the most commonly used illicit drug during pregnancy, in part due to its ability to reduce morning sickness during pregnancy. Several studies have demonstrated that neural network activity which underlies typical cognitive and behavioral processes can be altered by prenatal cannabinoid exposure (PCE) leading to a long lasting effects on adult behavior. Here, we have investigated the impact of PCE in adolescent offspring in hippocampus dependent learning and memory via performing a series of behavioral, electrophysiological and immunochemical studies. An osmotic pump filled with either vehicle or the cannabinoid receptor full agonist WIN55, 212–2 (2 mg/kg body weight/day) was implanted subcutaneously in gestational day 4, which delivered the drug at a constant rate until the pups were born. Contextual Fear Conditioning and Morris Water Maze test were performed to investigate the hippocampus based memory revealing significant learning deficits in the PCE group. To investigate the mechanisms behind observed behavioral deficits, electrophysiological experiments were performed on acute hippocampal slices. Reduced long‐term potentiation and enhanced and long term depression were observed in the PCE group demonstrating impaired synaptic plasticity. To identify alterations in the downstream signaling cascade involved in glutamatergic neurotransmission, immunoblotting experiment was performed. Immunoblotting data showed increased Cannabinoid Receptor Type 1(CB1) expression along with reduced Neural Cell Adhesion Molecule (NCAM) expression. NCAM is important for maintaining proper neuronal connections, and it modulates NMDA mediated glutamatergic neurotransmission. Based on our data, we hypothesize that a reduction in NCAM may be responsible for the learning and memory deficits observed in PCE animals and can be used as a therapeutic target to ameliorate the cognitive deficits in PCE offspring.

Support or Funding Information

No Available Funding Source

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cocaine increases the stimulation of dopamine release, at variance with atypical dopamine uptake inhibitors. New clues for the abuse liability of psychostimulants?
Authors: Keighron Jacqueline D , Bonaventura Jordi , Li Yang , Cao Jian Jing , DeMarco Emily M , Sandtner Walter , Michaelides Michael , Sitte Harald H. , Newman Amy H. , Tanda Gianluigi
Published: June 2021

Dopamine uptake inhibitors (DUIs), such as cocaine and methylphenidate, are characterized by their ability to bind to the dopamine transporter (DAT) and prevent dopamine (DA) from being transported from the extracellular to the intracellular side of a neuronal terminal. It was originally posited that all DUIs would have the same effect on behavior, neurochemistry, and abuse liability in animal models as well as humans. However, a subset of these drugs, atypical DUIs (e.g. JHW007), have shown dissimilar profiles. While these DUIs have the same target and similar binding affinities to typical DUIs, their effects on behavior and neurochemistry are markedly different and predict little to no abuse liability in animal models.

The goal of the present study was to determine how the neurochemistry of this atypical subset of DUIs differs from typical DUIs in mice. A pharmacological approach was taken utilizing Fast Scan Cyclic Voltammetry (FSCV), for real time monitoring of DA release and reuptake from the terminal; Fiber Photometry (FP), for real time monitoring of Ca2+ fluctuations in the terminal; and Electrophysiological (Ephys) ligand displacement experiments, to characterize the interactions between DUIs and the DAT.

FSCV data indicate that cocaine and other abused DUIs have effects on both DA reuptake (by binding to the DAT and blocking DA transport) and stimulated release (causing an increase in mobilization of DA vesicles). In contrast the atypical DUIs, such as JHW007, have similar effects on DA reuptake, but do not cause a significant increase in stimulated DA release. FP experiments show how this difference influences Ca2+ fluctuations. JHW007 has a greater dose‐dependent impact on the influx of Ca2+ following stimulation than cocaine. Further Ephys experiments indicate a correlation between DA release and the off‐rate for each DUI, while no significant correlation was found for DA release and DAT affinity. Our data suggest 1) the dopaminergic effects of cocaine and other typical DUIs are not solely related to their ability to block the DAT; 2) DAT affinity alone is not an indicator for abuse liability; 3) the effect of the DUIs on the quaternary structure of the DAT and its downstream interactions may play an important role in the effects of typical DUIs related to abuse. Our preclinical results suggest that atypical DUIs can mitigate the effects of cocaine, leading to possible treatment candidates for psychostimulant use disorders in humans.

Support or Funding Information

This research was funded in part by the Medication Development Program, National Institute on Drug Abuse, IRP, NIH/DHHS

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
AAV‐7F9‐Fc Protects Against METH‐Induced Hyperthermia and Decreases in Blood Brain Barrier Tight Junction Expression in a Rodent Model
Authors: Bolden Chris , McLeroy‐Charles Kennede , Hay Charles E , Margaritas Paris , Peterson Eric
Published: June 2021

Methamphetamine (METH) is a potent psychostimulant, with no FDA‐approved pharmacological treatment options. METH can adversely affect brain homeostasis by initiating dysfunction in the Blood Brain Barrier (BBB) and hyperthermia. The BBB is a semipermeable barrier composed of endothelial cells and pericytes along blood capillaries. These cells are held together by tight junction (TJ) proteins (e.g., claudin‐5, occludin, etc) which limit paracellular transport and help regulate body temperature. METH can decrease the expression of these TJ proteins and induce hyperthermia leading to BBB dysfunction. Thus, a novel therapy that could mitigate these effects is needed.

A novel treatment under evaluation in our laboratory is an anti‐METH single chain variable antibody‐fusion antibody (7F9‐Fc) packaged into adeno‐associated virus (AAV8) particles. AAV‐7F9‐Fc works as a pharmacokinetic antagonist to slow and reduce entry of METH into the brain. AAV‐7F9‐Fc has achieved long‐term serum concentrations in mice (>7 months from a single dose) and has been shown to alter locomotor effects of METH in mice. This treatment could have clinical potential to attenuate the effects of METH during drug use and aid addiction recovery.

We hypothesized that an AAV delivered anti‐METH antibody fragment (AAV‐7F9‐Fc) will mitigate METH‐induced hyperthermia and BBB dysfunction. To begin to test this hypothesis, we recorded baseline temperatures of mice 1 h before administration of either saline or METH (10 mg/kg). Male BALB/c mice were randomized into one of five groups (n=5/group); 1) Saline + Saline, 2) Saline + METH, 3) AAV‐7F9‐Fc + Saline, 4) AAV‐7F9‐Fc + METH, 5) AAV‐empty vector + saline. After administration of either Saline or METH, rectal temperatures were probed for 1 h, and brains were collected at the 3 h time point. Brains were dissected on dry ice into key dopaminergic regions such as the Frontal Cortex and Hippocampus. After dissection, proteins were extracted via RIPA buffer and analyzed for their expression by Western Blot. In Saline+METH treated mice, a 40% decrease in expression of Claudin‐5 and a 45% decrease in expression of Occludin (p < 0.05) were observed in the Frontal Cortex. These results were mitigated in AAV‐7F9‐Fc + METH mice in the Frontal Cortex and Hippocampus. These results indicate two important findings in an acute setting. This indicates that AAV‐7F9‐Fc has the potential to mitigate in an acute administration of METH, 1). the ability of METH to induce Hyperthermia, and 2). cause a reduction of critical tight junction protein expression in the CNS.

Support or Funding Information

This research was funded by NIH/NIDA:R01‐DA036600 & R01 DA036600‐03S1, NIH/NIGMS:T32‐GM106999, and R25GM083297.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Reward deficits in an animal model of compulsive eating
Authors: Cottone Pietro , Moore Catherine F , Leonard Michael , Miczek Klaus , Sabino Valentina
Published: June 2021

Background

Compulsive eating, which affects an estimated 15 million adults in the US, can be conceptualized as overeating to relieve a negative emotional state. One hypothesized underlying mechanism is diminished reward. Overeating of palatable diets alter mesolimbic dopamine neurotransmission, likely contributing to the persistence of excessive intake. However, it is not yet known if overeating of palatable food alters reward sensitivity, and whether reward is differentially altered between phases of palatable food access and withdrawal.

Methods

In this study, we investigated sensitivity to d‐Amphetamine, a dopamine releaser, in rats undergoing ad libitum palatable diet cycling, a model of compulsive eating. In this model, animals receive 2 days of access to a highly palatable, chocolate flavored, high‐sucrose diet (‘P Phase’) followed by 5 days of access to a control chow diet (‘C Phase’). In all experiments, we investigated d‐ Amphetamine sensitivity during access to the palatable diet (‘P Phase’) as well as when animals were withdrawn from palatable food (‘C Phase’). To test behavioral and molecular response to d‐ Amphetamine, we used a locomotor response assay, conditioned place preference test, intra‐cranial self‐stimulation paradigm, and in vivo microdialysis. Finally, we tested oral self‐administration of d‐Amphetamine in the home cage. To examine dopaminergic signaling targets, gene expression of dopamine transporter (DAT) and tyrosine hydroxylase (TH) in the ventral tegmental area (VTA) were determined using quantitative polymerase chain reaction (qPCR).

Results

During the P phase, diet alternated rats showed reduced sensitivity to d‐Amphetamine's stimulating (locomotor assay), rewarding (CPP), and reward‐enhancing effects (ICSS). In diet cycled animals, d‐Amphetamine‐evoked dopamine efflux was blunted. Chow/Palatable rats had higher self‐administration of d‐Amphetamine during the P Phase. Levels of DAT mRNA were increased in the VTA in Chow/Palatable rats during the P Phase.

Conclusions

These results indicate that a history of overconsumption of palatable food causes reward deficits and associated alterations in the mesolimbic pathway that led to increased self‐administration of d‐ Amphetamine.

Support or Funding Information

R01‐030425, F31‐DA044664

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Icariin improves the cognitive function of APP/PS1 mice via suppressing ER stress
Authors: Li Fei , Zhang Yangyang , Shi Jingshan , Gong Qihai
Published: June 2021

Alzheimer's disease (AD) is a progressive neurodegenerative disorder including many cognitive processes decline and neurons loss. Massive misfolded or unfolded proteins gathered in endoplasmic reticulum (ER) can disturb ER function and induce ER stress, which results in neuronal death in AD. Icariin (ICA), extracted from Chinese herb, has a wide range of neuron protection and has been researched in AD treatment. The current study was focusing on the effect of ICA on ER stress in AD animal model. We found that ICA improved 12‐month aged APP/PS1 mice behavior performance, reduced Aβ1‐40/42 deposition in hippocampal region. We also observed the APP‐ Aβ pathway, and found ICA decreased the protein expression of APP and BACE1, as well as increased the level of ADAM10, indicating that ICA inhibited aberrant APP‐Aβ metabolism by reducing APP and β‐secretase. In order to observe the effect of ICA on ER stress in AD condition, we tested the ER stress marker GRP78 and the results showed that ICA decreased the protein and mRNA level of GRP78 in APP/PS1 mice hippocampus. We further observed the main three branches of ER stress, the phosphorylation level PERK and eIF2α has been decreased in APP/PS1 mice after ICA treatment, the downstream factors of eIF2α (ATF4 and CHOP) were also decreased, but IRE1 phosphorylation and ATF6 were not affected in ICA treated mice. This suggested that ICA maybe inhibit ER stress through altering the PERK/eIF2α pathway. Furthermore, we found the neuron apoptosis in APP/PS1 mice were ameliorated after ICA treatment, the level of cleaved‐casepase‐3, ‐9, ‐12, as well as Bax/Bcl‐2 ratio were dramatically decreased in ICA treated APP/PS1 mice. These results demonstrated that ICA could serve as a potent ER stress inhibitor in AD research.

Support or Funding Information

This work was supported by the National Natural Science Foundation of China (Grant No. 81560594); Guizhou Province International Science and Technology cooperation project (Qiankehewai G [2014]7011).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Fear Extinction and Myocardial Infarction on Corticotropin Releasing Hormone Receptor 1 mRNA in Rostral Locus Coeruleus of Rats
Authors: Rose Deborah , Kuckertz Anne , Scrogin Karie
Published: June 2021

We found that 8 weeks after myocardial infarction (MI) female, but not male rats showed deficits in extinction of conditioned fear (P<0.05). Small hairpin RNA‐mediated knockdown of corticotropin releasing hormone receptor 1 (CRF1) around the rostral locus coeruleus (rLC) reversed deficits in fear extinction. Female and male rats subjected to MI and sham knockdown showed reduced CRF1 mRNA levels 24 hours after extinction training compared to sham MI/sham knockdown controls (P<0.01). CRF1 mRNA levels in the rLC region were directly related to the extent of freezing observed during extinction trials in rats subjected to sham knockdown (P<0.05). These findings suggest either that exposure to feared cues during extinction contributed to acute reductions in CRF1 mRNA in the rLC region, or that chronic reductions in CRF1 mRNA levels resulted from MI. Here, we determined if CRF1 mRNA levels in the rLC region were reduced after MI in male rats. Fresh frozen brains of rats exposed to coronary artery ligation (n=4) or sham surgery (n=3) 8 weeks prior to euthanasia were processed for fluorescent in situ hybridization. Brain sections containing the rLC were processed for detection of CRF1‐, vesicular glutamate transporter 2‐ (VGLUT2, a glutamatergic cell marker) and glutamate decarboxylase 2 mRNA (GAD2, a GABAergic cell marker). Assessments from the medial parabrachial nucleus to the lateral border of the posterodorsal tegmental nucleus and ventral border of Barrington's nucleus were performed. Cells were counted bilaterally and averaged within rat. No differences in CRF1 mRNA were observed between groups. A small number of CRF1 mRNA positive cells (39±6) close to the rLC did not show VGLUT2 or GAD2 mRNA label. Numbers of GAD2‐ (90±15) and VGLUT2 mRNA‐positive cells (365±30) did not differ between groups. CRF1‐ and VGLUT2 mRNA‐positive cells were found in high density in the medial parabrachial nucleus and in the lateral dorsal tegmental nucleus. CRF1‐ and GAD2 mRNA‐positive cells were found medially, near the posterodorsal tegmental nucleus. The percent of VGLUT2 mRNA‐positive cells (72±4) that co‐expressed CRF1 mRNA was greater than the percent of GAD2 mRNA‐positive cells that co‐expressed CRF1 mRNA (60±5, p<0.001). The integrated density of CRF1 mRNA fluorescence was greater in VGLUT2 mRNA‐positive cells than in GAD2 mRNA‐positive cells (P<0.05). The data indicate that CRF1 mRNA is expressed primarily by glutamatergic cells in the rLC region of the male rat and that reductions in CRF1 mRNA levels observed in the rLC region 24 hours after extinction in MI rats are likely due to stress responses during fear extinction rather than to chronic effects of MI.

Support or Funding Information

Cardiovascular Research Institute, Loyola University Chicago

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Role of Serotonin Transporter, Plasma Membrane Monoamine Transporter, and Organic Cation Transporter 3 in the Antidepressant‐like Effects of Ketamine
Authors: Bowman Melodi Ann , Vitela Melissa , Owens W. Anthony , Koek Wouter , Daws Lynette C.
Published: June 2021

Twenty percent of adults are diagnosed with major depressive disorder, which is typically treated with selective serotonin reuptake inhibitors (SSRIs). However, SSRIs take approximately six weeks to produce therapeutic effects. Recently, low doses of ketamine, a noncompetitive N‐methyl‐ D‐aspartate (NMDA) receptor antagonist, have been shown to produce rapid and long‐lasting antidepressant effects. Studies that have begun to examine the mechanisms underlying these effects have focused on intracellular changes mediated by NMDA and/or AMPA receptors. However, the traditional view of the etiology of depression involves the need for an increase in extracellular serotonin to regulate mood. Surprisingly, there appears to be little research into the effects of ketamine on serotonin. One study found an increase in extracellular serotonin following ketamine, consistent with results from another study showing that ketamine inhibited serotonin uptake. Together, these findings suggest a role for serotonin in the antidepressant‐like effects of ketamine, and putatively one involving transporters capable of serotonin uptake. The serotonin transporter (SERT) is an obvious candidate, but recent evidence points to the plasma membrane monoamine transporter (PMAT) and organic cation transporter (OCT3) as important players in serotonin uptake as well. In order to investigate a role for these transporters in the action of ketamine to inhibit serotonin uptake, we used in vivo chronoamperometry to measure serotonin clearance from extracellular fluid of CA3 region of the hippocampus. In wildtype mice, ketamine robustly inhibited serotonin clearance. Ongoing chronoamperometry studies are using knockout lines of SERT, PMAT and OCT3 mice to parse apart their roles in the action of ketamine to inhibit serotonin clearance. Parallel studies using these knockout (KO) lines in the forced swim test support the involvement of all three transporters in the antidepressant‐like effect of ketamine. As expected, ketamine (32 mg/kg) produced robust antidepressant‐like effects in wildtype mice, but in marked contrast, was without effect in SERT KO, PMAT KO, and OCT3 KO mice. Ketamine did not influence locomotor activity in SERT KO and wildtype mice, ruling out differences in the action of ketamine on general activity accounting for the contrasting effect of ketamine in the FST between these genotypes. Experiments assessing the locomotor actions of ketamine in PMAT KO and OCT3 KO mice are ongoing. Together, these data suggest that blockade of SERT, PMAT, and/or OCT3 may be important for the antidepressant‐like effects of ketamine.

Support or Funding Information

R01 MH093320

R01 MH106978

T32 NS082145

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Oxycodone‐induced gene expression adaptations in the brain reward center in a murine model of neuropathic pain
Authors: Pryce Kerri D. , Kronman Hope , Ramakrishnan Aarthi , Gaspari Sevasti , Pena Catherine , Shen Li , Nestler Eric , Zachariou Venetia
Published: June 2021

Chronic neuropathic pain is a complex condition with complications beyond physical symptoms that include depression, anxiety, and cognitive issues. One in five patients suffering from chronic pain is prescribed an opiate drug such as oxycodone for pain relief. Long‐term use of oxycodone and other opiates for the treatment of chronic pain can lead to physical dependence and addiction. Recent studies from our group revealed that chronic neuropathic pain promotes several adaptations in the brain reward regions responsible for both the sensory and affective components associated with chronic pain (Descalzi et al., 2017, Sci. Signaling). Here, we are using Next Generation RNA‐sequencing to gain insight on the gene expression adaptations triggered by persistent exposure to oxycodone and to determine the impact of neuropathic pain in maladaptive plasticity to oxycodone. We employed the spared nerve injury model (SNI) of neuropathic pain in adult C57Bl/6 mice and assessed peripheral neuropathy symptoms (thermal hyperalgesia, mechanical allodynia) for ~9 weeks. Mice were then treated daily with oxycodone, for two weeks and gene expression was monitored three weeks after oxycodone withdrawal. Oxycodone treatment induced thermal hyperalgesia regardless of pain states, while withdrawal produced significant mechanical allodynia. We next monitored changes in gene expression for three brain regions implicated in the symptoms of chronic neuropathic pain: medial prefrontal cortex (mPFC), nucleus accumbens (NAc) and the ventral tegmental area (VTA). Although oxycodone treatment promotes mostly unique transcriptome profile across brain regions, we observed similar downstream effectors and transcription factors to be affected in pain states when compared with non‐pain states. We are currently validating these genes using transgenic mouse lines to investigate their roles in potentiating oxycodone induced analgesia as well as physical dependence and addiction‐like behaviors. Understanding the gene adaptions in the brain after oxycodone treatment provides novel insight into the cellular machinery involved in maladaptive responses to drugs of abuse in the presence and absence of pain.

Support or Funding Information

5T32DA007135‐34, NIDA, R01 NS086444‐01, NINDS, and P01 DA008227, NIDA

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Combinations of haloperidol and morphine produce maximal levels of anti‐hyperalgesic or potentiation effects in rats with neuropathic pain
Authors: MENA‐VALDES LICET CARIDAD , ESPINOSA‐JUAREZ JOSUE VIDAL , BLANCO‐HERNANDEZ YISEL , LOPEZ‐MUÑOZ FRANCISCO JAVIER
Published: June 2021

Neuropathic pain (NP) is a chronic disorder that result from an injury or dysfunction at central or peripheral nervous system, promoting alterations in control and modulation of nociceptive signalling pathways. The haloperidol is a butyrophenone prototype, that recently some authors have described as anti‐nociceptive drug mediated by sigma‐1 receptors antagonism. Besides, morphine is a prototype analgesic opioid drug currently used in the NP treatment.

Objective

To detect the optimal combinations generated by therapy of haloperidol with morphine in NP induced by chronic constriction injury (CCI).

Methods

The anti‐hyperalgesic effects of haloperidol (0.017–0.100 mg/Kg, s.c.) and morphine (0.01–3.16 mg/Kg, s.c.) were determined after single‐doses, both in monotherapy and combined, using the von Frey test. Evaluations were done until 10 days post‐surgery at 30, 60, 90, 120 and 180 minutes after drugs administration and data were processed using Surface of Synergic Interaction analysis (SSI).

Results

Haloperidol and morphine showed a dose‐dependent anti‐hyperalgesic effects. From 16 combinations studied, six were potentiation, while remaining 10 combinations resulted in an additive effect or limited by the maximum effect of the individual doses. The combination morphine (0.0316 mg/Kg) and haloperidol (0.01778 mg/Kg) showed the maximum potentiation effect (p<0.001). In turn, the combination of maximum anti‐hyperalgesic efficacy (p<0.01) was morphine (1.0 mg/Kg) and haloperidol (0.1 mg/Kg). Interestingly, was detected that is required a haloperidol single dose tenfold higher than haloperidol dose employed by the combination of maximum potentiation for reach similar antinociceptive effect.

Conclusions

This study showed that haloperidol and morphine in combination can produce addition and potentiation effects in neuropathic pain, and the SSI analysis allowed to detect the optimal combinations to produce maximum efficacy or maximum potentiation of anti‐hyperalgesic effects.

Support or Funding Information

Department of Pharmacobiology, Cinvestav‐Unidad Coapa, C.P. 14330, Tlálpan, Mexico City, Mexico.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chronic and Intermittent Ethanol Treatment Differentially Regulates Membrane Compartmentalization of G‐protein Subunits in Rat Brain
Authors: Miller James , Wiley Aidan , Luessen Deborah , McCool Brian , Chen Rong
Published: June 2021

Introduction

Ethanol exposure produces profound changes in the trafficking and activity of many G‐protein coupled receptors (GPCRs) which are associated with alcohol preference, tolerance, and dependence. However, the molecular mechanism involved in ethanol‐induced changes in receptor function remains unclear. Our lab previously found that ethanol treatment increases cholesterol content in rat brain, altering membrane lipid composition. Cholesterol, enriched in lipid rafts of membranes, plays an important role in membrane integrity and promotes the recruitment of signaling molecules. Ethanol‐induced disruption of brain cholesterol homeostasis would influence the signaling of many membrane‐bound GPCRs and channels. This study aimed to examine whether CIE also causes changes in the membrane localization of G‐protein subunits which are tightly associated with many GPCRs and channels.

Methods

Male Sprague Dawley rats were exposed to 7 cycles of 12 hours of ethanol vapor inhalation followed by 12 hours of normal air. Rats were sacrificed 24 hours after the final ethanol exposure and the prefrontal cortex (PFC) tissue was dissected. Samples were homogenized followed by discontinuous sucrose density (5%–40%) ultracentrifugation and separated by density into 15 fractions. Western blotting was performed to determine the localization of G‐protein subunits on in lipid raft and non‐lipid raft fractions.

Results

CIE selectively altered membrane compartmentalization of certain subunits of G‐protein. There was an increase in localization of Gαo subunit from lipid rafts to non‐lipid rafts in CIE‐treated rat PFC. In contrast, CIE caused Gβ subunit to translocate from non‐lipid rafts to lipid rafts. Moreover, CIE had no effect on membrane compartmentalization of Gαi3 and Gαq. These results indicate selective changes in location of certain G‐protein subunits, altering proximal microdomain interaction with membrane channels and receptors and thus downstream signaling. This provides a possible mechanism underlying cholesterol‐mediated changes in GPCR signaling in rat brain tissue.

Support or Funding Information

This project is supported by NIH R01DA042862, P50DA006634, F31AA025532.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phospholipase Cɛ Regulation of Cardiac Fibroblasts
Authors: Brown Loren M. , Rodriguez Soneida S. , Smrcka Alan V.
Published: June 2021

Heart disease is one of the leading causes of death in the United States. Cardiac fibrosis is commonly associated with heart diseases, such as cardiac hypertrophy and myocardial infarctions. This is characterized as an excess deposition of extracellular matrix that results in a stiffening of the heart. Cardiac fibrosis can lead to arrhythmias, decreased diastolic function and eventually heart failure. Upon injury to the heart, cardiac fibroblasts transition to an activated myofibroblast state, characterized by the expression of α‐smooth muscle actin (αSMA). While the activation of fibroblasts is initially beneficial to the heart, chronic activation of fibroblasts leads to excessive deposition of collagen resulting in the development of fibrosis. Phospholipase Cɛ (PLCɛ) is an important signaling enzyme that is activated by small GTPases downstream of G‐protein coupled receptors (GPCRs) and receptor tyrosine kinases and is important in the cardiac myocyte for the development of cardiac hypertrophy. In this study we sought to elucidate the role of PLCɛ in the transition of cardiac fibroblasts to activated myofibroblasts. Using neonatal rat cardiac fibroblasts, we demonstrate that PLCɛ is highly expressed in fibroblasts compared to myocytes at the mRNA and protein level. Adenovirus encoding for a PLCɛ‐shRNA was used to knockdown PLCɛ in fibroblasts and the ability of the cells to transition to a myofibroblast was examined. Knocking down PLCɛ decreases the mRNA expression of pro‐fibrotic genes such as αSMA, collagen 1, and TGFβ indicating that PLCɛ can regulate pro‐fibrotic gene expression in cardiac fibroblasts. The transition to myofibroblasts induced by transforming growth factor‐β (TGFβ) is reduced in cells treated with PLCɛ‐shRNA compared to cells treated with a control shRNA as assessed by staining of αSMA. In addition, knocking down PLCɛ reduces the ability of the cardiac fibroblasts to respond to pro‐fibrotic Gq‐coupled GPCRs such as the angiotensin receptor and the protease‐activated receptor 1. These data indicate that PLCɛ has an important role in pro‐fibrotic signaling in cardiac fibroblasts and can regulate the myofibroblast transition. Future studies will focus on elucidating the molecular mechanism by which PLCɛ is involved in the pro‐fibrotic signaling response.

Support or Funding Information

This project is supported by grant number T32‐HL007853 from the NIH

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of the Ca2+‐sensing capacity of STIM1 by the G protein‐coupled estrogen receptor
Authors: Pekas Devon , Gebert‐Oberle Briana , Giles Jennifer , Underwood Timothy , VerMeer Mark , Tran Kim
Published: June 2021

The stromal interaction molecule 1 (STIM1) is essential for activation of store‐operated Ca2+entry (SOCE). STIM1 contains in its intra‐endoplasmic reticular (ER) domain a canonical EF hand (cEF) that houses a Ca2+‐binding loop. This loop serves as a Ca2+sensor; Ca2+leaving its binding loop upon ER Ca2+depletion initiates conformational changes leading to STIM1 oligomerization and SOCE activation. We recently reported that activation of the G protein‐coupled estrogen receptor (GPER) clamps intracellular Ca2+signals via multifaceted inhibitory actions on both Ca2+efflux and SOCE; the latter results partly from alteration in phosphorylation at serines 575, 608 and 621 of STIM1. Here, we identified a dynamic interaction between GPER and STIM1 that directly controls the Ca2+‐sensing capacity of STIM1. GPER coimmunoprecipitates with STIM1 in resting primary endothelial cells, an association enhanced by treatment with either GPER agonist G‐1 or activation of SOCE using thapsigargin. This effect is not altered by combined S575/608/621A substitutions. Coimmunoprecipitation analysis using truncated versions of STIM1 showed that GPER associates with the cEF domain. Confocal microscopy identified a GPER's submembrane domain that localizes GPER to the ER. To assess the role of GPER‐STIM1 interaction in controlling STIM1's Ca2+sensing capacity, we generated a series of biosensors that incorporated STIM1's Ca2+‐binding loop in singlet, doublet or triplet. These biosensors faithfully report Ca2+binding to the loop, with tripled dynamic range by the triple‐loop biosensor and loss of Ca2+binding by charge substitutions targeting strategic elements for Ca2+binding. Using peptides corresponding to submembrane domains of GPER and these STIM1 biosensors, we identified a robust Ca2+‐independent interaction between a submembrane domain and the Ca2+‐binding loop. The charge substitutions that abolish Ca2+binding only reduce but do not negate the interaction, indicating that Ca2+and the GPER domain bind to different regions of the loop. Consistently, we have observed that GPER overexpression decreases SOCE and GPER knockdown substantially increases SOCE. These data reveal a new interaction between GPER and STIM1 that directly controls STIM1's Ca2+‐sensing capacity and SOCE activation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CCK1 ‐ a Very Peculiar G Protein‐Coupled Receptor
Authors: Cui Zong Jie
Published: June 2021

The class A G protein‐coupled receptor cholecystokinin 1 (CCK1) plays a predominant role in regulating exocrine pancreatic secretion both in mammals and in birds. CCK1R supra‐maximal stimulation leads to one of the most commonly used murine models of acute pancreatitis. The CCK‐CCK1R system is a well conserved feeding regulator in evolution from worms, insects, other invertebrates, vertebrates, to primates. In the murine brain, CCK1R is mainly expressed in the hippocampus and a few highly defined extracortical regions. These expression patterns determine that CCK1R modulation is important in both biology and medicine. Indeed some special characteristics revealed recently have made CCK1 stand out among all the class A GPCR. Although CCK1 is not susceptible to superoxide or hydrogen peroxide oxidative regulation, it is permanently activated by singlet oxygen (1O2) which can be generated conveniently in a Type II photodynamic action. We have used Fura‐2 fluorescent calcium imaging to show that CCK1R is activated permanently by photodynamic action with photosensitiser sulphonated aluminium phthalocyanine (SALPC) in the freshly isolated rat pancreatic acini. Such permanent photodynamic activation of CCK1R with SALPC could be reproduced in rat pancreatic acinar tumor cell AR4‐2J, and in cell lines ectopically expressing human hCCK1R. Both CCK1R in AR4‐2J and ectopically expressed in other cell lines (CHO, HEK) could be permanently activated by photodynamic action with the genetically encoded protein photosensitisers KillerRed and miniSOG. Other than permanent activation by 1O2, CCK1R activation by CCK in the freshly isolated rat pancreatic acini is quenched by mixed reactive oxygen species generated by neutrophil respiratory burst, and by neutrophil extracellular trap component histones. Further, CCK1R activation by CCK or by 1O2 in AR4‐2J is sensitized by histones. Our ongoing work is examining the potential for CCK1R analogues from invertebrates to be permanently activated by photodynamic action with SALPC, KillerRed, miniSOG or their variants. We are also looking at the possibility to extend the permanent activation by 1O2 (pAb1O2) property of CCK1R to other GPCR by the generation of chimeric receptors. The outcomes of such experiments will provide useful toolkits for remote photon‐driven activations of the CCK1R system in vivo from invertebrates to vertebrates, birds, mammals and non‐human primates.

Support or Funding Information

This work was supported by The Natural Science Foundation of China (31670856).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synthesis, Characterization and Evaluation of Cadmium Sulfide Nanoparticles Capped with Dextrin and Functionalized with Temozolomide
Authors: Leo‐Campos Frida , Rodriguez Patricia , Reyes‐Esparza Jorge , Rodriguez‐Fragoso Lourdes
Published: June 2021

Nanoparticles are currently used as drug carriers. Temozolomide is an antineoplastic drug for tumor in central nervous system. It presents a half‐life of 1.8 hours. For this reason, requires high doses that are limited by side effects. Studies about the use of nanoparticles as drugs transporters have reported an increase the drug solubility, increase in half‐life and sustained release of the drug because it is protected from degradation and loss of therapeutic effect. This group has development and studied CdS nanoparticles capped with polymers and have showed being biocompatible and effective as drug carriers. To synthesize and characterize cadmium sulfide nanoparticles capped with dextrin functionalized with temozolomide and observe cell uptake. By means of the chemical method, the precursors were allowed to react to obtain the synthesis of CdS/dextrin nanoparticles. Subsequently, the nanoparticles were functionalized with temozolomide through a chemical method in which the OH groups of the dextrin were bounded to the NH2 Through the technique of Fourier‐transform infrared, the characteristic bonds were observed: at 840 cm−1 the α‐D glucopyranose bond, at 927 cm−1 the α(1–4) bonds; 1159 cm−1 glucopyranosyl units and at 1079 cm−1 the C‐O‐C, at 1025 cm−1 the C‐O bond, at 3399 cm−1 the OH group characteristic of dextrin. At 2001 cm−1 the C=N=N of diazomethane was found which is a toxic compound resulting from the formation of nanoparticles. Also at 3400 cm−1 there is the link N=H and 1610 cm−1 C = C or C = N of the temozolomide. According to the X‐ray results, the widening of 3 peaks corresponding to the crystalline planes characteristic of the cubic phase of the CdS is observed which are: (111), (220) and (311). Using the Scherrer formula d = 0.8λ/βcos θ was calculated that the average size of the nanoparticles is 3 nm. Due to the size of the semiconductor nanoparticles of CdS‐dextrin, they emit green fluorescence characteristic. Therefore, tumor cells treated with nanoparticles functionalized with temozolomide in epifluorescence microscope were observed and it was observed that the nanoparticles reached the cytoplasm and core of cells and produces the effect of temozolomide arresting cell cycle in phase G2 + M. The synthesis and characterization of CdS/dextrin nanoparticles functionalized with temozolomide resulted in 5nm, which are uptake by the cells and are distributed on the cytoplasm and core exerting the effect of the drug. The CdS/dextrin has the potential for being used as drug carriers and facilitating the pharmacological because cells up take them.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Sub‐Chronic Inhalation of Crude Oil Vapor (COV) on Reactivity to Methacholine and Neural Regulation of Rat Airways in vitro
Authors: Fedan Jeffrey S , Russ Kristen A , Thompson Janet A , Jackson Mark C , McKinney Walter G
Published: June 2021

Inhalation of crude oil vapor (COV), a mixture of hydrocarbon vapors and volatile organic compounds (VOCs), by oil and gas industry workers has led to a number of deaths. Workers are potentially exposed to COV during sampling, gauging and filling crude oil storage tanks. Inasmuch as the effects of COV inhalation on various functions of the lung is undescribed, we have been investigating the effects of COV inhalation in a rat model. Earlier, we found increases in transepithelial resistance 1 d post‐exposure to COV (300ppm total VOCs, 6 h/d×1 d; whole body chambers). Lung resistance was increased 90 dpost‐exposure, but no changes occurred in lung compliance, airway reactivity in vivo, or epithelial ion transport in vitro, 1, 28 and 90 d after this acute exposure. Here, rats were exposed to COV sub‐chronically (300 ppm total VOCs, 6 h/d×28 d), and airway reactivity tomethacholine (MCh) in vitro and neurogenic responses of airway smooth muscle in vitro were investigated 1, 28 and 90 d post‐exposure. Reactivity to MCh and the modulatory role of the airway epithelium was assessed by comparing contractile responses of the isolated, perfused trachea to extraluminally (EL)‐and intraluminally (IL)‐applied MCh. Reactivity to EL‐applied MCh was unaffected, but reactivity to IL‐applied MCh was decreased at 90 d post‐exposure, indicative of an enhanced inhibitory effect of the epithelium. To investigate the effect of sub‐chronic COV exposure on neural regulation of airway smooth muscle, tracheal strips were placed in an organchamber between two platinum ring electrodes to record isometric tension. Electric field stimulation (EFS) was delivered across the electrodes to stimulate frequency‐dependent contractile responses of the muscle. Exposure of rats to COV had no effect on EFS‐evoked contractions at any post‐exposure time point, indicating that neurons releasing acetylcholine in the airway wall were not a target of COV. The results indicate that the airway epithelium is affected by inhalation of COV, and that cholinergic motor nerves are not. Physiological systems in the lung have different, exposure time‐ and post‐exposure time‐dependent susceptibilities to inhaled COV's effects.

Support or Funding Information

NIOSH 7939ZYAC

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Oncogenic Gαq and Gα11 Inhibition by FR900359 in Uveal Melanoma
Authors: Lapadula Dominic , Farias Eduardo , Randolph Clinita E. , Purwin Timothy J. , McGrath Dougan , Charpentier Thomas H. , Zhang Lihong , Wu Shihua , Terai Mizue , Sato Takami , Tall Gregory G. , Zhou Naiming , Wedegaertner Philip B. , Aplin Andrew E. , Aguirre‐Ghiso Julio , Benovic Jeffrey L.
Published: June 2021

Uveal melanoma is the most common intraocular tumor in adults and often metastasizes to the liver, leaving patients with few options. Recurrent activating mutations in the G proteins, Gαq and Gα11, are observed in approximately 93% of all uveal melanomas. While therapeutic intervention of downstream Gαq/11 targets has been unsuccessful in treating uveal melanoma, using in vitro [35S]GTPγS binding and cell signaling assays we have found that the Gαq/11 inhibitor, FR900359 (FR), effectively inhibits oncogenic Gαq/11 signaling in uveal melanoma cells expressing either mutant Gαq or Gα11. Inhibition of oncogenic Gαq/11 by FR results in cell cycle arrest and induction of apoptosis. Furthermore, colony formation is prevented by FR treatment of uveal melanoma cells in 3D‐cell culture, providing promise for future in vivo studies. This suggests direct inhibition of activating Gαq/11 mutants may be a potential means of treating uveal melanoma.

Support or Funding Information

Dr. Ralph and Marian Falk Medical Research Trust and NIH award F31 CA225064

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.