bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inflammation Produced by Alcohol Synergizes with Methamphetamine to Cause Dopaminergic Deficits
Authors: Blaker Amanda L. , Yamamoto Bryan K. 
Published: June 2021

Alcohol and methamphetamine (Meth) are often co‐abused, in that ~77% of Meth addicts also display an alcohol use disorder. However, the neurochemical consequences of this comorbid drug taking behavior are unknown. We tested the hypothesis that serial exposure to ethanol (EtOH) and Meth produces greater neurotoxicity compared to either drug alone. Sprague Dawley rats were allowed drinking access to a 2 bottle choice of 10% EtOH and water, every other day for 4 weeks, followed by one day of binge Meth (10 mg/kg × 4 injections). Over 4 weeks, both EtOH intake (g/kg) and preference (% total volume) increased (p<0.05). EtOH drinking alone caused inflammation, evidenced by significant increases in lipopolysaccharide (LPS) in serum and increased LPS and cyclooxygenase‐2 (COX‐2) in the striatum 24 hours after the last day of EtOH compared to water drinking (*p<0.05 vs. Water). 7 days after Meth or saline injections, striatal dopamine (DA) was depleted by 50% in Meth‐treated rats and by 95% in EtOH+Meth treated rats (*p<0.05 vs. Water+Saline). The DA transporter, DAT, followed a similar trend. Importantly, EtOH alone did not affect DA or DAT concentrations in the brain. Additionally, the amount of EtOH imbibed significantly correlated with the extent of Meth‐induced DA depletions (r2=0.89; p<0.001). The significantly enhanced DA depletions produced by the serial exposure to EtOH drinking and Meth (Meth alone vs. EtOH+Meth; p<0.05) were blocked by the COX inhibitor, ketoprofen (5 mg/kg) administered twice daily on the intermittent withdrawal days during the EtOH drinking period. To determine whether the loss of DA was paralleled by changes in the substantia nigra pars compacta (SNpc), cell bodies were stained for tyrosine hydroxylase (TH). Immunofluorescence showed a significant loss of TH‐positive cells in the SNpc. To ascertain if the loss of DA at the cell body and terminal levels resulted in Parkinsonian‐like motor deficits, motor function was tested 7 days after the last drug exposure. Rotarod test results showed that neither EtOH alone nor Meth alone affected motor behavior, but the serial exposure to the drugs significantly decreased the latency to fall off the rod. These results show the inflammation during EtOH drinking increases the sensitivity to Meth neurotoxicity in the striatum. Future studies will examine if inflammation mediates the loss of TH and motor function, and the extent to which glutamate‐mediated excitotoxicity plays a role in this DAergic loss.Support or Funding InformationNIH DA007606This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Adenosine G Protein‐Coupled Receptor Biased Agonism to Treat Ischemic Heart Disease
Authors: May Lauren Therese , Baltos Jo‐Anne , Chuo Chung H. , Vecchio Elizabeth A. , Wang Bing H. , Kompa Andrew R. , Christopoulos Arthur , Scammells Peter J. , White Paul J. 
Published: June 2021

Ischemic heart disease and secondary heart failure places an immense burden on society. Stimulation of adenosine G protein‐coupled receptors (ARs) represents a powerful protective mechanism to decrease cardiac ischemia‐reperfusion injury (IRI). Despite this, therapeutic targeting of ARs has been largely unsuccessful due to on‐target adverse effects, including pronounced bradycardia, atrioventricular block and hypotension (1). Biased agonism has the potential to overcome these limitations by enabling the separation of therapeutic from adverse effects (2).AimTo profile prototypical and biased AR agonism in isolated cardiac cells. Subsequent studies investigated the in vivo profile of AR biased agonism in acute and longer‐term rat models of cardiac ischemia‐reperfusion injury.ResultsIn isolated rat cardiomyocytes, prototypical and biased AR agonists stimulated reperfusion injury salvage kinase (RISK) signalling pathways, including Akt and ERK1/2, and promoted cardioprotection. Exposure to in vitro simulated ischemia‐reperfusion conditions differentially influenced the RISK signalling stimulated by prototypical and biased AR agonists. Within an in vivo model of acute myocardial ischemia‐reperfusion injury, both biased and prototypical agonists stimulated an A1AR‐mediated reduction in infarct size and an improvement in cardiac function post‐IRI. However, in contrast to prototypical agonists, our biased agonist had no significant adverse hemodynamic effects. Furthermore, in a longer‐term in vivo model of myocardial IRI, an AR biased agonist improved cardiac function and decreased cardiac hypertrophy and fibrosis.DiscussionInsights into AR signalling in cardiomyocytes under normoxic and simulated ischemia‐reperfusion conditions suggests that context‐specific biased agonism stimulates a preferential signalling profile. Furthermore, AR biased agonism can promote cardioprotection and decrease pathological cardiac remodelling in the absence of bradycardia, a profile suggestive of ligand bias. Collectively, our findings demonstrate that AR biased agonism can stimulate potent cardioprotection in the absence of adverse hemodynamic effects and therefore represents an attractive therapeutic approach for the treatment of ischemic heart disease.Support or Funding InformationThis work was supported by Project Grant APP1084487 of the National Health and Medical Research Council of Australia (NHMRC). J‐A.B., CHC and E.A.V are recipients of the Australian Government Research Training Program Scholarship. A.C. is a Senior Principal Research Fellow of the NHMRC. L.T.M. is an Australian Heart Foundation Future Leaders Fellow.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Role of Distal Helix 5 as a Determinant of GPCR‐G protein Coupling Selectivity
Authors: Okashah Najeah , Wan Qingwen , Inoue Asuka , Lambert Nevin 
Published: June 2021

G protein‐coupled receptors (GPCRs) interact with heterotrimeric G proteins to mediate important physiological responses, and compounds that bind to GPCRs represent approximately one third of FDA‐approved drugs. In humans, hundreds of GPCRs converge onto 16 G alpha subunits that are grouped into four families, and individual receptors select between these subtypes. Prior work suggests that the distal half of helix 5 (H5), the extreme C‐terminus of the G alpha subunit, is a key determinant of GPCR‐G protein coupling selectivity. The objective of this study was to determine the relative contributions of the extreme C‐terminus (H5.17‐H5.26) and the rest of the G alpha subunit (HN.1‐H5.16) to GPCR‐G protein coupling selectivity.We constructed a panel of G alpha subunits that included a wild‐type G protein from each of the four families (Gs‐long, Gi1, Gq, and G12), and all twelve possible chimeras where the last 10 amino acids (H5.17‐H5.26) of each G alpha subunit is replaced by those of another (e.g. Gsi‐10). These formed heterotrimers with venus‐labeled betagamma subunits, and coupling to Rluc8‐labeled GPCRs was measured using bioluminescence resonance energy transfer (BRET). BRET was measured in the presence and absence of both agonists and nucleotides in digitonin‐permeabilized HEK293 Gs/q/12 knockout cells which, together with pertussis toxin, were used to minimize interference from endogenous G proteins.Our results suggest that distal H5 influences GPCR‐G protein coupling, but by itself is not sufficient to determine selectivity. Furthermore, for some receptors, exchanging distal H5 had little effect on G protein coupling. For example, M3 muscarinic acetylcholine receptors coupled to Gqs‐10 more strongly than to Gsq‐10, suggesting that for this receptor the key selectivity determinants reside proximal to distal H5. In addition, the distal H5 of G12 was generally permissive, e.g. A1 adenosine receptors coupled to Gi12‐10 nearly as well as wild‐type Gi1. Taken together our results suggest that GPCR‐G protein selectivity determinants are likely to be distributed throughout the G protein alpha subunit, and distal H5 is only one such determinant. Moreover, G protein specificity determinants are likely to vary for different GPCRs. Future experiments will specifically address proposed specificity‐determining sites outside of distal H5.Support or Funding InformationSupported by NIH grants GM078319 and GM109879.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Distinct regulatory domains on the N‐ and C‐termini of Kv7.4 and Kv7.5 contribute to both their voltage‐dependence of activation and their responses to PKA and PKC.
Authors: Byron Kenneth L. , Brueggemann Lyubov I. , Cribbs Leanne L. 
Published: June 2021

We previously determined that homomeric Kv7.4 potassium channels expressed in smooth muscle cells are insensitive to regulation by protein kinases A and C (PKA or PKC), whereas homomeric Kv7.5 channels are robustly enhanced by PKA and robustly inhibited by PKC activation. To determine the molecular basis for these different responses of Kv7.4 and Kv7.5 channels, using A7r5 vascular smooth muscle cells as an expression system, we examined the regulation of chimeric channels, constructed by inserting subdomains of Kv7.5 into Kv7.4, to determine whether particular parts of Kv7.5 can confer sensitivity onto Kv7.4. A putative PKA phosphorylation site, S53, is located on the N‐terminus of Kv7.5, but is missing from Kv7.4. We found that replacing the Kv7.4 N‐terminus with the Kv7.5 N‐terminus (Q5N‐Q4) rendered Kv7.4 significantly more responsive to a PKA‐activating stimulus than was wild‐type Kv7.4, but this Q5N‐Q4 chimera was still insensitive to AVP (a PKC‐activating stimulus). PKC is thought to target sites on the C‐terminus of Kv7.5, so we switched the C‐terminus of Kv7.4 with that from Kv7.5 (Q5C‐Q4). Preliminary results suggest that this Q5C‐Q4 chimera is sensitive to PKC‐dependent current suppression. The Q5N‐Q4 chimera had voltage‐dependence of activation similar to WT Kv7.4 (V0.5=−31 ± 3 mV for Q5N‐Q4 and −34 ± 2 mV for WT Kv7.4), whereas the Q5C‐Q4 chimera activated at voltages similar to WT Kv7.5 (V0.5=−44 ± 1 mV for Q5C‐Q4 and −45 ± 2 mV for WT Kv7.5). Our findings suggest that distinct regulatory domains on the N‐ and C‐termini of Kv7.5 and Kv7.4 contribute to both the voltage‐dependence of activation and the differential regulation of the channels by protein kinases.Support or Funding InformationThe project described was supported by Award Number I01BX007080 from the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The G Protein Gamma Subunit, Ste18, is a Phosphorylation‐Dependent pH Sensor that Controls MAPK Activation in Yeast
Authors: Toosi Zahra Nassiri , Torres Matthew P. 
Published: June 2021

As essential transducers of extracellular stimuli such as hormones and neurotransmitters, G protein signaling (GPS) systems – comprised of seven transmembrane receptors (GPCRs), heterotrimeric G‐proteins (Gαβγ), and regulators of G‐protein signaling (RGS) proteins – are important regulators of cell/environment homeostasis in all eukaryotes. Yeast harbor a single canonical GPS system that regulates a pheromone‐dependent mating response, which has long served as a tool for discovering regulators fundamental for all eukaryotic GPS systems. In wild type yeast, the activation rate and amplitude of pheromone signaling is severely dampened by negative feedback phosphorylation of the canonical Gγ subunit, Ste18, and the MAPK scaffold, Ste5. Here, we show that Ste18 is a pH sensor that undergoes combinatorial phosphorylation/dephosphorylation in response to fluctuations in intracellular pH (pHi). Using a site‐specific phosphorylation‐dependent electrophoretic mobility assay and a pHi probe (pHlourin), we show that Ste18S3 is rapidly phosphorylated in response to intracellular acidification, while Ste18S7 is rapidly phosphorylated in response to mating pheromone. When pHi is lowered, basal phosphorylation on Ste18S7 decreases concomitantly with increasing phosphorylation on Ste18S3. In contrast, pheromone stimulation activates phosphorylation on Ste18S7, but not Ste18S3. We show that phosphomimic mutations on either phosphosite function to prohibit phosphorylation of the other non‐mutated site, demonstrating that phosphorylation is mutually exclusive. Furthermore, phosphorylation at either site, mimicked by glutamate mutations, is sufficient to inhibit ultra‐fast and intense output from the pheromone pathway. Thus, our findings suggest that Ste18Nt acts as a phosphorylation‐dependent pH‐sensor that is capable of restricting pheromone pathway output under acid stress conditions when yeast mating is sub‐optimal. Considering that dysregulation of pHi is linked to chronic and life‐threatening diseases such as cancer, stroke, and Alzheimer's disease, our data reveal the possibility that Gγ subunits serve as pH sensors important for regulating G protein signaling in such diseases.Support or Funding InformationNational Institutes of Health (NIH)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Estrogen‐dependent epigenetic regulation of soluble epoxide hydrolase via DNA methylation
Authors: Yang Yang‐Ming , Sun Dong , Kandhi Sharath , Froogh Ghezal , Hammock Bruce D. , Huang An 
Published: June 2021

We have demonstrated that female‐specific downregulation of soluble epoxide hydrolase (sEH) protein expression in cardiac, pulmonary, and vascular tissues favors a greater contribution of epoxyeicosatrienoic acids (EETs) in females. Thus, to elucidate molecular mechanism(s) responsible for the sexually dimorphic phenotype of sEH expression, we tested the hypothesis that female‐specific downregulation of sEH expression is driven by estrogen‐dependent methylation of the Ephx2 gene.MethodsMesenteric arteries isolated from male, female, ovariectomized female (OV) and OV with estrogen replacement (OVE) mice, as well as human cell line (HEK293T) were used.ResultsMethylation‐specific PCR (MSP) and bisulfite genomic sequencing (BGS) analysis indicate significant increases in DNA/CG methylation in vessels of female and OVE compared to those of male and OV mice. The same increase in CG methylation was also observed in male vessels incubated with a physiological concentration of 17b‐estradiol (17b‐E2) for 48 hours. All vessels that displayed increases in CG methylation were concomitantly associated with decreases in their Ephx2 mRNA and protein, suggesting a methylation‐induced gene silencing. Transient transfection assays indicate that the activity of Ephx2 promoter‐coding luciferase was significantly attenuated in HEK293T cells treated with 17b‐E2, which was prevented by additional treatment with an estrogen receptor (ER) antagonist (ICI). Chromatin immunoprecipitation (ChIP) analysis indicates significantly reduced binding activities of transcription factors (including SP1, AP‐1 and NFκB with their binding elements located in the Ephx2 promoter) in vessels of female mice and human cells treated with 17b‐E2, responses that were prevented by ICI and Decitabine (DNA methyltransferase inhibitor), respectively. In conclusion, estrogen/ER‐dependent methylation of the promoter of Ephx2 gene silences sEH expression, which is involved in specific transcription factor‐directed regulatory pathways.Support or Funding Information(This work was supported by grants NIH HL070653 and HL129797, and also partially supported by NIEHS grant R01 ES02710 and NIEHS Superfund Research Program P42 ES04699).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibitor of Soluble Epoxide Hydrolase Attenuates Decline in Learning and Memory of Diabetic Rats
Authors: Goswami Sumanta Kumar , Minaz Nathani , Razdan Rema , Hammock Bruce D. 
Published: June 2021

Epoxyeicosatrienoic acids (EETs) are epoxide derivatives formed from arachidonic acid and are neuroprotectant. These bioactive lipids are degraded by soluble epoxide hydrolase (sEH). Pharmacological inhibition of sEH enhances the synaptic function in the central nervous system and has a protective role in the age‐related cognitive decline. We have evaluated the hypothesis that the sEH inhibitor TPPU might prevent a diabetes‐induced decline in learning and memory which is associated with alteration in the level of neurotransmitter and oxidative stress. Type 1 diabetes (streptozotocin 52 mg/kg, i.p.) was induced in rats and the animals were treated with TPPU (0.1 and 0.3 mg/kg body weight; p.o.) for 8 weeks. The learning and memory functions were assessed by Barnes maze and step down test. Level of oxidative stress indicators (reduced glutathione and malondialdehyde) and neurotransmitters (γ‐Aminobutyric acid, norepinephrine, and dopamine), and activity of acetylcholinesterase and were measured in the discrete regions of the brain. Our results revealed that treatment with TPPU which penetrates the blood‐brain barrier significantly improved learning and memory performance in diabetic rats with a parallel decrease in the level of blood sugar. Moreover, treatment with TPPU significantly minimized the diabetes‐induced alteration in levels of neurotransmitters and activity of acetylcholinesterase. In addition, TPPU protected anti‐oxidant defense system. Based on our experimental findings, it can be concluded that the inhibition of sEH is a viable strategy for retrieving diabetes‐induced decline in learning and memory.Support or Funding InformationThis study was partially funded by the National Institute of Environmental Health Sciences (NIEHS) [Grant R01 ES002710], NIEHS Superfund Research Program [Grant P42 ES004699], West Coast Central Comprehensive Metabolomics Center [Grant U24 DK097154] to BDH.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of Soluble Epoxide Hydrolase Protects the Brain from Permanent Middle Cerebral Artery Occlusion
Authors: Koehler Raymond C. , Tu Ranran , Zhang Xiaofan 
Published: June 2021

Inhibition of soluble epoxide hydrolase (sEH) has been shown to reduce infarct volume after transient middle cerebral artery occlusion (MCAO), but to the best of our knowledge, has not been tested in a model of permanent MCAO. Here, we produced permanent MCAO in male C57Bl6 mice by coagulation of the distal MCA and administered vehicle or the sEH inhibitor TPPU [1‐trifluoromethoxyphenyl‐3‐(1‐propionylpiperidin‐4‐yl)urea] at 90 min after MCAO and daily over the subsequent 6 days. Protein expression of sEH was measured at 1, 3, 7, 14, and 28 days after MCAO and was found to nearly double at 3 and 7 days after MCAO before decreasing at 14 days and returning to the surgical sham control levels at 28 days. Tissue activity of sEH tripled 3 days after MCAO and was attenuated by 2.7, 64.5, and 62.6% with ip administration of 0.3, 1, and 3 mg/kg/day TPPU. Subsequent experiments used a dose of 1 mg/kg/day. Infarct volume was significantly decreased from 23.9±8.2% (±SD) in the vehicle group to 16.6±4.1% in the TPPU group (P<0.01, n=13). Likewise, sensorimotor function, as assessed by the time to recognize adhesive tape on the affected paw, was significantly improved at 1, 3, 7, and 14 days after MCAO in the TPPU‐treated group. Tissue cytokine mRNA was measured at 1, 3, and 7 days after MCAO. Relative to vehicle treatment, TPPU attenuated the increase in IL‐1β and TNFα mRNA at 3 days after MCAO and augmented the increase in IL‐10 mRNA at 7 days after MCAO. These results indicate that ischemic neuroprotection by administration of a sEH inhibitor does not require reperfusion and that TPPU limits expression of pro‐inflammatory cytokine expression and augments the delayed increase in reparative IL‐10 expression after permanent focal cerebral ischemia.Support or Funding InformationSupported by NIH NS060703This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of Potent Soluble Epoxide Hydrolase Inhibitors for Veterinarian Usage
Authors: Morisseau Christophe , Shihadih Diyala S. , Harris Todd R. , Kodani Sean D. , Hwang Sung‐Hee , Lee Kin Sing S. , Hamamoto Briana , Guedes Alonso , Hammock Bruce D. 
Published: June 2021

The veterinary pharmacopeia to treat pain and inflammation in companion animals is very limited. Currently, cyclooxygenase inhibitors (both NSAIDs and COXIBs) are the main class of drugs available to reduce pain and inflammation in cats, dogs and horses. However, these drugs often have insufficient efficacy and a great number of known adverse effects, underlying the need for novel pain medication for companion animals. Soluble epoxide hydrolase (sEH) inhibitors are a new class of anti‐inflammatory and analgesic drugs being tested in humans. They block the hydrolysis, and thus increase endogenous concentrations, of signaling molecules called epoxy‐fatty acids (EpFAs) that modulate inflammation and nociception. In addition to their use in human, sEH inhibitors may be a suitable alternative to cyclooxygenase inhibitors in companion animals. However, the compounds optimized for usage in primates and rodents are typically not sufficiently potent inhibitors of other animals' sEH for in vivo usage. Here, we screened a library of 2,300 inhibitors developed for the human enzyme with partially purified feline, canine and equine hepatic sEH to identify inhibitors that are broadly potent among species. Six candidate sEH inhibitors that are very potent across species (IC50 < 1nM for each enzyme tested) were identified. Their microsomal stability was then measured in hepatic extracts from cat, dog and horse, as well as their solubility in drug‐able solvents. Based on this work, trans‐4‐{4‐[3‐(4‐trifluoromethoxy‐phenyl)‐ureido]‐cyclohexyloxy}‐benzoic acid was selected for further testing in veterinary clinical trials of pain and inflammation in companion animals.Support or Funding InformationThis work was supported by the National Institute of Environmental Health Sciences grant numbers ES002710 and ES004699.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.