bluuebunny/crossref_metadata_2025 huggingface dataset:
Lysophosphatidylethanolamine acts on type 1 lysophosphatidic acid receptor in MDA‐MB‐231 breast cancer cells
Authors: IM DONG‐SOON , PARK SOO‐JIN , LEE KYOUNG‐PIL
Published: June 2021

Lysophosphatidylethanolamine (LPE) is a lyso‐type metabolite of phosphatidylethanolamine, a plasma membrane component. Actions of LPE have been implicated to be mediated through G‐protein‐ coupled receptor (GPCR). GPCRs for lysophosphatidic acid, a structurally‐similar representative lipid mediator, have not been implicated in LPE‐mediated actions in SK‐OV3 and OVCAR‐3 ovarian cancer cells and in the receptor over‐expression systems. In the present study, LPE‐induced intracellular Ca2+ increase was observed in MDA‐MB‐231 cells but not other breast cancer cell lines. LPE‐ and LPA‐induced responses showed homologous and heterologous desensitization. Furthermore, antagonists for LPA1 and LPA3, VPC32183 and Ki16425, inhibited LPE‐induced Ca2+ increase and Knockdown of LPA1 by transfection of LPA1 siRNA also completely inhibited LPE‐induced Ca2+ rise in the cells. Pertussis toxin, a specific inhibitor of Gi/o proteins, edelfosin, an inhibitor of phospholipase C, 2‐APB, an inhibitor of IP3 receptor, completely inhibited LPE‐induced response. However, HA130, an inhibitor of autotoxin/lysophospholipase D, did not inhibit the response. Therefore, LPE is supposed to act on LPA1/Gi/o proteins/phospholipase C/IP3/Ca2+ rise in MDA‐MB‐231 breast cancer cells.

This work was supported by MRC program (2009–0083538) and by Basic Science Research Program (2011–0021158) of the NRF of Korea.
Modulation of the cAMP pathway by Pseudomonas aeruginosa quorum sensing molecules
Authors: Stoddart Leigh , Coward William , Williams Paul , Hill Steve
Published: June 2021

Pseudomonas aeruginosa (PA) uses quorum sensing molecules (QSMs) to coordinate the production of its virulence factors. These QSMs have also been shown to induce responses within the host mammalian cells. We have investigated the ability of two QSMs, the P. aeruginosa quinolone signal (PQS) and N‐3‐(oxododecanoyl)‐L‐homoserine lactone (OdDHL), to stimulate a variety of second messenger pathways within HEK293 cells. Both QSMs had little effect upon calcium release or ERK1/2 phosphorylation. In contrast, when examining basal cAMP levels it was found that PQS had minimal effects but OdDHL caused a marked decrease in the cAMP levels in a concentration dependent manner. Both PQS and OdDHL stimulated a decrease in forskolin stimulated cAMP, with PQS having effects at lower concentrations than OdDHL. This was also true when stimulating cAMP production via adenosine or β‐adrenergic receptors endogenously expressed in these cells. Pertussis toxin treatment was unable to alter the effects on cAMP modulated by PQS and OdDHL suggesting that Gαi/o proteins are not directly involved. Interestingly the phosphodiesterase 4 (PDE4) inhibitor rolipram caused a moderate reversal in the QSM simulated inhibition of cAMP levels. Understanding the effects of QSMs upon the cAMP pathway may uncover a novel target for the treatment of PA infections.

This work was supported by a University of Nottingham Bridging the Gaps award.
Intersectin enhances Cbl ubiquitylation of epidermal growth factor receptor through regulation of Sprouty2‐Cbl interaction
Authors: Okur Mustafa Nazir , Zhu Jolene Ooi Yu , Guy Graeme , O'Bryan John P
Published: June 2021

Ubiquitylation of receptor tyrosine kinases plays a critical role in regulating the trafficking and lysosomal degradation of these important signaling molecules. We identified the multi‐domain scaffolding protein intersectin (ITSN) as an important regulator of this process. ITSN stimulates ubiquitylation of the epidermal growth factor receptor (EGFR) through enhancing the activity of the Cbl E3 ubiquitin ligase. However, the precise mechanism through which ITSN enhanced Cbl activity was unclear. In this study, we find that ITSN enhances Cbl activity through disrupting the interaction of Cbl with the Sprouty2 (Spry2) inhibitory protein. We demonstrate that ITSN binds Pro‐rich regions in both Cbl and Spry2 and that interaction of ITSN1 with Spry2 disrupts Spry2‐Cbl interaction resulting in enhanced ubiquitylation of the EGFR. Disruption of ITSN1 binding to Spry2, ITSN1 binding site in Spry2 results in enhanced Cbl‐Spry2 interaction and inhibition of receptor ubiquitylation. This study demonstrates that ITSN enhances Cbl activity by modulating the interaction of Cbl with Spry2. In addition, our results reveal a new level of complexity in the regulation of Cbl through the interaction with ITSN and Spry2.
Cholinergic manipulations modulate the discriminative stimulus effects of methamphetamine in C57BL/6J mice
Authors: Grant Kathleen A , Eastwood Emily , Davis Natalie L , McCracken Aubrey D , Ford Matthew M
Published: June 2021

Cholinergic transmission modulates several aspects of stimulant abuse, including initial exposure, transition to dependence, and relapse risk. The goals of the current work were to 1) establish methamphetamine (MA) as a discriminative stimulus (SD) in mice, 2) assess the ability of M1‐ or M1/M4‐active compounds to generalize from or block the SD effects of MA, and 3) evaluate the nACh receptor subtypes underlying the MA‐like SD effects of nicotine. Sixteen male C57BL/6J mice were trained to discriminate 1 mg/kg MA (i.p.) from saline on a FR‐12 schedule. Following acquisition (41 ± 3 sessions), substitution testing revealed that the M1/M4‐preferrring agonist xanomeline and the M1‐preferring agonist n‐desmethylclozapine were perceived as saline‐like whereas the non‐selective antagonist scopolamine and the M1‐preferring antagonists trihexyphenidyl and dicyclomine exhibited low partial substitution for MA. Pretreatments were administered 10‐min prior to MA during blocking tests, and it was observed that dicyclomine producing a leftward shift in the MA dose‐response curve whereas xanomeline resulted in a non‐significant rightward shift. Nicotine partially generalized (50%) from MA in mice, and pretreatment with the nACh antagonists mecamylamine and DHβE as well as the partial agonist varenicline completely blocked the MA‐like SD effects of nicotine. Supported by DA018165, AA016849 and OD011092.
Interactions of dopamine (DA) D2‐like receptor availability and DA transporters (DAT) on cocaine self‐administration in female cynomolgus monkeys
Authors: Nader Michael A. , Duke Angela N. , Gage Donald , Nader Susan H. , Bell Michelle , Voll Ronald , Howell Leonard L.
Published: June 2021

Using PET imaging, we recently reported that female monkeys who become dominant showed a significant increase in dopamine (DA) D2‐like receptor availability compared to subordinate animals who showed significant decreases in DA transporter (DAT) availability (Nader et al. 2012). In contrast to males, it appears that higher D2‐like availability was associated with greater vulnerability to cocaine reinforcement. However, it is possible that DAT availability was the more important vulnerability trait. The goal of this study was to further examine the role of D2‐like receptor and DAT availability on cocaine self‐administration (SA) in female cynomolgus monkeys (n=10). Individually housed female monkeys were scanned twice, once with the D2‐like receptor ligand [11C]raclopride and once with the DAT ligand [18F]FECNT. All monkeys were trained to respond under a multiple fixed‐interval 3‐min schedule of food and cocaine reinforcement. Food was available in components 1 and 3 and drug (or saline) in components 2 and 4. Although still preliminary, there appears to be no relationship between food‐maintained responding and DAT availability. Cocaine will be made available for SA and responding will be correlated with D2‐like and DAT availability. These studies will provide important information related to DA receptor function and vulnerability in a female monkey model of cocaine abuse. DA017763
Increased Go activity in C57Bl/6J mice enhances sensitivity to a model of epilepsy
Authors: Kehrl Jason , Dalton Hans , Kohut Kevin , Stern Matt , Neubig Richard
Published: June 2021

Many patients suffering from temporal lope epilepsy (TLE) are refractory. This work focused on understanding two potential new therapeutic targets, the hetero‐trimeric G‐proteins Go and Gi2. We hypothesized that increasing activity of these proteins would be protective from seizures as inhibition of these proteins enhances seizure susceptibility. Mouse lines were created with increased activity of Go (Gαo RGSi) or Gi2 (Gαi2 RGSi) through the introduction of a point mutation in the α subunit. Both mouse lines were exposed to a chemical kindling model with relevance to TLE. C57Bl/6J Gαo+/RGSi mice have a faster onset to seizure while Gαi2+/RGSi mice have no change compared to controls. 129SvImJ Gαo+/RGSi mice however, kindle no faster than controls. Furthermore, a 129 modifier region on Chromosome 17 reduces the rate at which B6 Gαo+/RGSi mice kindle. Here we have demonstrated an in vivo role for Go and a novel modifier region in regulating susceptibility to a kindling model of epilepsy however we have yet to identify if these effects are through developmental or acute signaling changes. Supported by NIH grant RO1‐GM39561 (RRN), 5T32GM008322–19 (JMK) and PhRMA Foundation Fellowship to JMK
Enhanced Efficacy and Reduced Side Effects of Standard Antiepileptics by New Kava Combinations
Authors: Nassar Noha N , Tawfiq Rasha Tawfiq A. , El‐Eraky Wafaa I. , El‐Denshary Ezzeldein S. M.
Published: June 2021

The long‐term use of anticonvulsant drugs possess many unwanted effects, thus new safe combinations are urgently mandated. Hence, the present study aimed to investigate the anticonvulsant effect of kava or its combination with a synthetic anticonvulsant drug, carbamazepine (CBZ) or diazepam (DZ). Animals were divided into two subsets, each comprising 9 groups as follows: GP I received 1% tween80 orally and served as control, while GPs (II) and (III) received kava at two dose levels (100 and 200mg/kg, p.o.). The remaining six groups received (IV) CBZ alone (30 mg/kg, p.o) or kava in combination with CBZ (V) (15 mg/kg, p.o) or (VI) (30 mg/kg, p.o), (VII) DZ alone (10 mg/kg, p.o) or kava in combination with DZ (VIII) (5 mg/kg, p.o) or (IX) (10 mg/kg, p.o). Results of the present study revealed that kava increased the maximal electroshock threshold (MEST) and potentiated the anticonvulsant effect of both, CBZ and DZ following both acute and chronic treatment. Moreover, neither kava nor its combination with CBZ or DZ impaired motor coordination acutely or chronically. Furthermore, kava ameliorated both the reduction in locomotor activity as well as the changes in liver enzymes induced by chronic administration of CBZ or DZ. The biochemical parameters were further confirmed with histopathological findings. In conclusion, the present study suggests the possibility of combing a low dose of CBZ or DZ with kava to reduce harmful effects and improve the quality of life in patients chronically treated with these synthetic anticonvulsant drugs.
Minocycline down‐regulated MMP‐9 and PARP in male and female mice in an embolic model of ischemic stroke
Authors: Sazonova Irina Y , Hoda Md Nasrul , Zemskova Marina A , Hess David C
Published: June 2021

Background and Purpose

Acute ischemic stroke is one of the leading causes of morbidity and mortality in the U.S. Recently, minocycline has been shown to provide neurovascular protection reducing acute cerebral injury by means of its efficiency to inhibit MMP‐9 and PARP. However, it is unclear whether minocycline is effective in females. Here, our aim was to determine if there is a sex‐specific change in MMP‐9 and PARP levels in response to minocycline treatment. We analyzed the neuroprotective mechanisms of minocycline in both sexes using an embolic stroke model in mice that closely mimics acute thromboembolic stroke in humans.

Methods

Embolic stroke was initiated by injection of a thrombus to the right middle cerebral artery. Minocycline was tested in adult males and adult ovariectomized females (23±3 weeks old; n=9–23 animals/group). Behavioral outcomes, infarct volumes, and expression of MMP‐9, PARP, and caspase‐3 were assessed.

Results

Without minocycline treatment, cerebral blood flow, infarct size, and neurological scores were comparable in both males and ovariectomized females. Stroke up‐regulated MMP‐9 and PARP levels in the brain in both genders, and caspase‐3 level in females. Minocycline significantly reduced the infarct volume (P<0.0001), improved neurologic score (P<0.0001) and reduced expression of MMP‐9 (p<0.05) and PARP (p<0.05) in all animals. However, minocycline did not affect caspase‐3 levels.

Conclusion

In a thromboembolic stroke model minocycline is neuroprotective inhibiting MMP‐9 and PARP irrespective of mouse sex.

Research support: NIH‐R21 award and GHSU start‐up to I.Y.S
Manganese treatments decrease immunofluorescence emissions of post‐synaptic dopamine D2 receptors
Authors: Opoku Rachael , Chekayev Yelena , Carroll Margaret A. , Catapane Edward J.
Published: June 2021

Manganese (Mn) a neurotoxin causing Manganism, a Parkinsons‐like disease, disrupts dopamine (DA) neurotransmission. Gill lateral cell cilia of Crassostrea virginica are controlled by DA innervations. DA is cilio‐inhibition. Our lab showed post‐synaptic DA receptors in gill cells are D2 type and Mn blocks cilio‐inhibitory effects of DA. Questions exist in the literature if Mn decreases the number of D2 receptors in brain. To test this we used histoimmunofluorescence techniques to visualize DA D2 receptors in gill and ganglia of C. virginica. Animals were treated with 500 FM of Mn for 5 days. Paraffin embedded sections were viewed with a phase contrast fluorescence microscope with a ProgRes C3 Peltier cooled camera. Antibody treated sections showed bright FITC fluorescence in lateral ciliated cells and other areas of gill and ganglia. Sections lacking 1E antibody treatment did not display similar fluorescence. We analyzed fluorescence intensity of the cells using ImageJ software. Intensity of Mn treated cells was 70% less than controls. The study identifies DA D2 receptors in gill cells and cerebral and visceral ganglia and shows a negative correlation between fluorescence intensity of the receptors in Mn treated animals. The question if the decrease in intensity is due to decrease in actual number of receptors or if Mn alters protein conformation of D2 receptor and D2‐ligand binding sites needs to be further explored.
Optical imaging of molecular targets of phytoagent deoxyelephantopin against mammary adenocarcinoma cell activities
Authors: Shyur Lie‐Fen , Lee Wai‐Leng , Shiau Jeng‐Yuan , Ying Laiqiang , Gervay‐Hague Jacquelyn
Published: June 2021

Deoxyelephantopin (DET), a major germacranolide sesquiterpene lactone isolated from the medicinal herb Elephantopus scaber L. exhibits significant bioactivities in suppressing breast cancer cell growth and metastasis as demonstrated in our recent studies. Optical imaging has become an emerging technique in non‐invasive, real time and high resolution modalities for drug delivery and cancer targeting. In this study, we designed and synthesized a DET‐near infrared (NIR) fluorescent dye conjugate, aiming to investigate and identify the direct or interacting molecular targets of DET in breast cancer cells. Confocal immunofluorescence microscopy study showed that DET‐NIR co‐localized with exosomes in mesenchymal breast cancer cells, murine TS/A and human MDA‐MB‐231, suggesting that DET‐induced oxidative stress led to centrosomal protein aggregation in cancer cells also involved exosome accumulation. Proteomic study using 2‐D DIGE showed that 11 exosomal proteins matched with ExoCarta database were responsive to DET treatment. Tumor exosomes are shown to play a role in immuno‐suppression and cancer progression. Results of further competition binding assay and proteomic study showed that annexin A2 is directly interacting or binding to DET‐NIR, implying the phytoagent can regulate signaling transduction in breast cancer cells through modulating its membrane trafficking. (NSC 100–2325‐B‐001–008, Taiwan)
Gene and protein expression in a rodent model of social stress: Implications for depression‐cardiovascular disease comorbidity
Authors: Wood Susan K , Lee Catherine S , Zhang Xiao‐Yan , Rappaport Eric F , Valentino Rita J
Published: June 2021

Psychosocial stress is linked to psychiatric disorders (eg, depression) and comorbid cardiovascular disease, but underlying mechanisms remain unknown. We previously identified two distinct phenotypic responses to social stress in rats (resident‐intruder model). One population exhibited passive behaviors, assuming a defeat posture within a short latency (SL, <300s). The other exhibited proactive behaviors and a longer latency (LL, >;300s). The SL phenotype was associated with endocrine and behavioral characteristics resembling depression and heart rate variability alterations indicating greater cardiovascular disease risk. As the locus coeruleus (LC) and dorsal raphe (DR) are implicated in the pathogenesis of depression, this study hybridized RNA from the LC or DR (n=4–6/group) to RT2 Profiler PCR arrays (SABiosciences) to identify expression of 88 genes involved in GPCR signaling. 22 genes (13 up, 9 down) in LC and 20 genes (11 up, 9 down) in DR were differentially expressed in SL vs LL rats (>;1.5 fold), many involved in inflammation. Elisa analysis confirmed stress‐induced suppression of brain Il1β and MCP‐1 in LL rats and increased Il1β in SL rats. These data suggest distinct stress‐induced adaptations in inflammatory markers may underlie susceptibility to a depressive phenotype and offers targets that may impact depression‐cardiovascular disease comorbidity. MH058250, MH040008, NARSAD‐17830
Novel high‐throughput DNase I assay
Authors: Jang Dae Song , Apostolov Eugene O , Penthala Narsimha , Zheng Guangrong , Crooks Peter , Basnakian Alexei G
Published: June 2021

DNase I is the most active and abundant endonuclease in the body, mediates toxic injury of kidney cells induced by cisplatin. This presents a major obstacle for cancer chemotherapy. However, neither inhibitors of DNase I nor high‐throughput screening methods for screening of high‐volume chemical libraries for DNase I inhibitors are available. To overcome this problem, we have developed a high‐throughput DNase I assay. The assay is based on the increase of fluorescence intensity when fluorophore‐labeled oligonucleotides are degraded by a DNase. It is optimized for a 96‐well plate format. The assay is highly sensitive to DNase I compared to other endonucleases, reliable (Z′≥0.6), operationally simple, and has low intra‐ and inter‐assay variability. The assay was used to screen a chemical library and several potential DNase I inhibitors were identified. After comparison, one best inhibitor was selected and shown to protect against cisplatin‐induced kidney cell death in vitro. This assay will be suitable for identifying potential inhibitors of DNase I and other endonucleases. NIH R01 DK078908 and VA Merit Review grants to AGB.
Allele‐Dependent miRNA Regulation of MMP9 and the Importance of Coding Region Polymorphisms in Gene Expression
Authors: Duellman Tyler , Warren Chris , Yang Jay
Published: June 2021

During the course of a study investigating the functional consequences of Matrix Metalloproteinase 9 (MMP9) single nucleotide polymorphisms (SNPs), as part of a case: control genotype correlation analysis with abdominal aortic aneurysm, we identified a SNP, rs41427445 (N38S), which resulted in a profound decrease in the amount of secreted MMP9 protein. Bioinformatic analysis of miRNAs targeting polymorphic sites within the coding region of MMP9 indicated a number of miRNAs that targeted polymorphic sites in an allele‐dependent fashion potentially altering post‐transcriptional regulation of the polymorphic mRNA. The analysis indicated the presence of two miRNAs, hsa‐miR‐671–3p and hsa‐miR‐657 that preferentially targeted the N38S mRNA with higher affinity than wild type MMP9 mRNA. miR inhibitors (antisense oligomers) and “silent mutagenesis” (disruption of miRNA affinity but maintenance of amino acid sequence) validated these dual miRNAs as mediating the suppression of N38S‐MMP9 expression in an allelic‐dependent fashion revealing miRNA targeting the coding region as novel regulators of MMP9 expression.
Genome‐wide binding and transcriptome analysis of human farnesoid X receptor in the liver
Authors: Zhan Le , Liu Hui‐xin , He Yuqi , Fang Yaping , Wan Yu‐Jui Yvonne , Fang Jianwen , Guo Grace Liejun
Published: June 2021

Farnesoid X receptor (FXR, NR1H4) is a ligand‐activated transcription factor, belonging to the nuclear receptor superfamily. FXR is highly expressed in the liver and functions as the bile acid sensor. FXR deficiency is implicated in numerous liver diseases, and modulation of FXR in mice has been used as animal models to study human liver physiology and pathology. By using chromatin immunoprecipitation followed by deep sequencing (ChIP‐seq), genome‐wide binding of FXR in mice indicates that FXR may be involved in regulating diverse pathways in the liver. However, limited information exists in terms of human FXR function and the suitability of using murine models to study it. To compare and contrast the genome‐wide FXR binding in the liver of mice and humans, ChIP‐seq were performed in frozen human livers of healthy male and female donors (FHLs) and livers of mice treated with FXR agonist (GW4064), as well as in primary human hepatocytes (PHHs) and a hepatoma cell line, HepG2 cells, with or without FXR activation. In addition, RNA‐seq was performed to determine the transcriptome with FXR activation in PHHs. The results showed similarities and differences of genome‐wide FXR binding in the liver between humans and mice in terms of motif analysis and pathway prediction. In addition, differences in FXR binding between male and female humans were not significant. In summary, we have established genome‐wide FXR binding and transcriptome in human livers. These results will aid in determining the human FXR functions, as well as judging the suitability of using mouse models to study human FXR function.
CYP2D6 expression is regulated by polymorphisms that affect splicing and trancription: new biomarkers for CYP2D6 activity
Authors: Wang Danxin , Poi Ming J , Sun Xiaochun , Gaedigk Andrea , Leeder J. Steven , Sadee Wolfgang
Published: June 2021

Cytochrome P450 2D6 (CYP2D6) metabolizes ~25% of clinical used drugs. CYP2D6 variants are currently used as biomarkers to predict CYP2D6 activity, but the relationship between genotype and phenotype is still ambiguous in most individuals except in poor metabolizers. In this study, we showed common SNP located in exon 6 (rs16947, *2) affects exon 6 splicing, resulting in 2 fold reduction in total CYP2D6 mRNA expression via nonsense‐mediated RNA decay. In addition we identified a novel SNP (E‐SNP) located in an enhancer region over 100 kb downstream of CYP2D6 that increases CYP2D6 transcription 2 fold. Testing in a pediatric cohort of 160 individuals with metabolic activity measured as the ratio of dextromethorphan/metabolites (DM/DX), SNP rs16947 alone is associated with reduced metabolic activity, while haplotype containing both rs16947 and E‐SNP has similar metabolic activity as reference haplotype, indicating gain of function E‐SNP can compensate for the loss of function effect of rs16947. Our data also indicate that the key SNP in *41 rs28371725 does not have independent effect on CYP2D6 mRNA level, the in vivo reduced activity effects associated with *41 was resulted from the lack of E‐SNP in *41 haplotype which also contain rs16947. These results suggest that the combination of rs16947 and E‐SNP may become a new biomarker for predicting CYP2D6 activity.

Supported by NIH grant U01 GM092655.
Metabolism‐Directed Structure Optimization of Benzimidazole‐Based F. Tularensis Enoyl‐Reductase (FabI) Inhibitors
Authors: Zhang Yan‐Yan , Johnson Michael E. , Ghosh Arun K. , Jeong Hyunyoung
Published: June 2021

FabI is an attractive antibiotic target against F. Tularensis, a Category A biowarfare agent of high risk to public health. Our previous study identified a novel class of benzimidazole analogs possessing excellent FabI inhibitory activity but unknown metabolic stability. The objective of this study was to characterize structure‐metabolism relationships (SMR) for this series to guide chemical modification. To this end, hepatic microsomal stability and bioactivity data were obtained for 22 lead compounds. Methyl, methoxy, or methylenedioxy substitution at the core structure was found unfavorable to the metabolic stability. Metabolite identification studies on 4 model compounds revealed hydroxylation of the benzimidazole ring as the major metabolic route. Introduction of methyl groups on this moiety provided additional metabolic soft spots. Cyclic modification strategy was then proposed, and 9 cyclopentane and oxacyclopentane derivatives were newly synthesized, which showed 4–13‐fold increases in t1/2 without impaired bioactivity. Subsequently, quantitative SMR models were successfully constructed using genetic algorithm variable selection, multi‐linear regression and artificial neural network techniques. Collectively, SMR for the FabI inhibitors were systematically analyzed, and the results should help product optimization for optimal pharmacokinetic properties.
Surgery‐induced inflammation reduces morphine distribution into cerebrospinal fluid
Authors: Wang Yan , Landry Kathryn , Issa Mark , Sleno Lekha , Julian Lisa , Hall Richard , Goralski Kerry
Published: June 2021

Objective

To determine the effects of surgery‐induced inflammation on morphine distribution across blood‐brain barrier (BBB)

Methods

Blood and cerebrospinal fluid (CSF) concentrations of morphine were measured in adult patients undergoing aortic aneurism surgery with (n=12) and without (n=13) cardiopulmonary bypass (CPB). Morphine concentrations were determined by mass‐spectrometry. A Q‐plex cytokine ELISA was used to measure plasma and CSF cytokines.

Results

The surgical inflammatory response was characterized by increased plasma concentrations IL‐6, a known regulator of BBB drug transporters. The plasma area under the curve from 0 to 24 h (AUC0–24h) of interleukin‐6 (IL‐6) was significantly higher (p=0.02) in the CBP (7900±8180 pg/ml h) versus the non‐CPB (1690±1680 pg/ml h) group, indicating that individuals receiving CPB had a more robust inflammatory response. The CSF to plasma AUC0–24h ratio of morphine in the CPB versus non‐CPB groups was 1.06±0.57 and 2.51±2.08 (p=0.03), respectively, indicating that less morphine penetrated into the brain following CPB when a larger inflammatory response was present.

Conclusion

CSF morphine exposure is lower in individuals undergoing aortic aneurism with CPB. This effect may be due to IL‐6‐mediated changes in blood‐brain barrier drug uptake or efflux transporter function.

The HSFC, PEF and CAS funded the project.
Investigating novel mechanisms of chemerin signalling in mature adipocytes
Authors: Dranse Helen J , Muruganandan Shanmugam , Sinal Christopher J
Published: June 2021

BACKGROUND

Chemerin is a recently identified adipocyte‐secreted signalling molecule (adipokine) that plays local and systemic roles in the regulation of adipocyte differentiation, metabolism, and inflammation. Chemerin levels are increased in obese patients, however little is known regarding the signal transduction pathways coupled to chemerin signalling and how altered chemerin levels in obesity affect adipose function.

OBJECTIVE

To identify key signalling pathways and molecular targets influenced by chemerin signalling.

RESULTS

Microarray analysis revealed that a number of genes with important roles in adipose tissue remodeling were up‐regulated following treatment of murine adipocytes with a neutralizing chemerin antibody. Functionally, this resulted in increased matrix metalloproteinase activity and recruitment of macrophages towards adipocyte‐conditioned medium. Treatment with a NFkB inhibitor was able to reverse these effects, suggesting a NFkB‐dependent mechanism.

CONCLUSIONS

Chemerin mediates the expression of genes involved in adipose tissue remodeling and inflammation through a NFkB‐dependent pathway. This novel role for chemerin signalling likely has important effects on adipose tissue when chemerin levels are modified in obesity.

Supported by CIHR, NSERC, and Killam Trusts.
Selectivity of synthetic CXCR4 agonists
Authors: Lefrancois Marilou , St‐Louis Étienne , Boulais Philip E , Lefebvre Marie‐Reine , Mona Christine , Leduc Richard , Lavigne Pierre , Heveker Nikolaus , Escher Emanuel
Published: June 2021

CXCR4, a widely distributed chemokine receptor, is known for its major role in cancer metastasis and AIDS pathogenesis. Pharmacotherapy with antagonistic ligands is greatly hindered since it blocks the many essential physiological roles of CXCR4, causing serious undesired effects. CXCR4 has only one known endogenous ligand, CXCL12, a 68 amino acid peptide that only binds to CXCR4 and CXCR7. To improve this situation we have discovered CXCR4 agonists from in silico homology modeling and the crystal structure of CXCR4: we hypothesized that peptide chimera of T140 (a potent inverse agonist of CXCR4) and the N‐terminal fragments of CXCL12 could act as agonists on CXCR4; such compounds proved to be the first potent synthetic CXCR4 agonists. We have shown that these synthetic peptides were able to induce chemotaxis both in vitro and in vivo, and to activate the typical Gi signaling pathway of CXCR4 in nanomolar concentrations, similarly to CXCL12. This study aims to assess the CXCR4 selectivity of these synthetic agonists. Binding assays on CXCR7 and CXCR4 were performed and showed affinities nanomolar for CXCR4 but micromolar for CXCR7, indicating up to a thousand‐fold selectivity. Functional selectivity was assessed on β‐arrestin‐2 recruitment to CXCR4 and to CXCR7. The surprising result is that our synthetic CXCR4 agonists appear to induce β‐arrestin recruitment on CXCR7 only and not on CXCR4.
Characterization of the anticancer pharmacology of novel jadomycin molecules
Authors: Hall Steven Robert , Robertson Andrew , Jakeman David L. , Goralski Kerry B.
Published: June 2021

Introduction

Jadomycins are natural anticancer products produced by the soil bacteria S. Venezuelae. The object was to examine the cytotoxicity and mechanisms of action of jadomycins in control and taxol, etoposide, and mitoxantrone‐resistant MCF7 breast cancer cells that respectively overexpress the ATP binding cassette (ABC) transporters ABCB1, ABCC1, or ABCG2.

Methods and Results

Lactate dehydrogenase (LDH) assays were used to determine the cytotoxicity of jadomycins DNV, L, B, SPhG, F, W, S, and T. All jadomycins were 100% efficacious at killing control MCF7 cells with EC50 values of 4.3 to 42.3 μM. The jadomycins effectively killed the ABCB1, ABCC1, and ABCG2 overexpressing MCF7 cells with a slight 1.5–3.5‐fold reduction in potency relative to the control MCF7 cells. Using PCR cancer gene arrays and quantitative PCR assays, the expression of 15 cancer‐related genes was found to be altered by jadomycin B or S treatment. This included up to a 23.7‐fold increase in the expression of the antioxidant encoding thioredoxin reductase 1 (TXNRD1). Reactive oxygen species (ROS) assays indicated ROS levels are elevated after jadomycin B, S, or SPhG treatment.

Conclusions

Jadomycin cytotoxicity in MCF7 breast cancer cells is not dependent of ABCB1, ABCC1, or ABCG2 function. The mechanisms of jadomycin action may include the production of ROS and subsequent cellular oxidative damage.

This project is funded by NSERC, CIHR, NSHRF, and BHCRI.
Tricyclic compounds inhibit the OATP1A2 transporter
Authors: Lu Jennifer , Moya Liliam Gabriela Guilarte , Leung Yat Hei , Gaudette Fleur , Turgeon Jacques
Published: June 2021

Background

OATP1A2 is a membrane transporter potentially involved in the absorption of various drugs. Previous data demonstrated that the uptake of rosuvastatin through OATP1A2 can be inhibited by several β‐blockers, where carvedilol is the most potent inhibitor. Carvedilol structurally differs from the other β‐blockers tested by its tricyclic moiety. The goal of this study was to determine whether the tricyclic structure of carvedilol is responsible for its strong inhibitory effect on OATP1A2.

Methods

A HEK293 cell line overexpressing OATP1A2 was used as model. They were co‐incubated in the presence of rosuvastatin and increasing concentrations of different tricyclic compounds. The amount of rosuvastatin transported in the cells was measured by HPLC.

Results

Most tricyclic compounds evaluated inhibited rosuvastatin uptake through OATP1A2 with different IC50. Our data show that the inhibition is competitive.

Compound
IC50 (μM)
Compound
IC50 (μM)

carazolol
4.0
nortriptyline
14.8

amitriptyline
5.0
chlorpromazine
29.6

imipramine
12.6
desipramine
77.9

trimipramine
13.6
carbamazepine
>;100

doxepin
14.0
carbazole
No effect

clomipramine
14.7
phenothiazine
No effect

Conclusions

The inhibitory component is made up of the tricyclic ring with a short aliphatic chain. Consequently, drugs with a similar structure may strongly modulate the transport of OATP1A2 substrates.

Funding: CIHR, FRSQ, Foundation CHUM
Examination of gender bias in fatty acid ethyl ester and ethyl glucuronide hair analysis for determination of excessive alcohol use
Authors: Gareri Joey , Rao Chitra , Koren Gideon
Published: June 2021

Fatty acid ethyl esters (FAEE) and ethyl glucuronide (EtG) in hair are validated biomarkers of chronic excessive alcohol use. Clinical studies examining dose‐response performance have been primarily conducted in male populations and other studies have shown an impact of hair cosmetics in producing both false‐negative EtG results and false‐positive FAEE results; suggesting a possible bias in using these biomarkers in female populations. n=199 female and n=73 male hair samples that tested positive for FAEE (>; 0.50 ng/mg) by GCMS following headspace solid‐phase micro‐extraction were tested for EtG by LC‐MSMS. A cut‐off value of 27 pg/mg‐EtG was applied. Clinical histories obtained via social worker or hair donor were reviewed against results where available. A higher rate of correspondence was found between FAEE and EtG levels amongst male (74.0%) over female (36.7%) subjects overall. Amongst female samples, there was a decrease in FAEE/EtG agreement for cosmetically‐coloured samples (20.8%) over untreated samples (42.5%). Twenty‐six percent of discordant female samples demonstrated reliable alternative evidence of alcohol abuse suggesting false‐negative EtG results. The findings demonstrate that these two biomarkers are best used in tandem when assessing alcohol consumption in a female population.
Characterization of the Vascular Phenotype of the Equilibrative Nucleoside Transporter 1 Knockout Mouse
Authors: Best Keisha Arielle , Bone Derek , Gros Robert , Hammond James
Published: June 2021

Adenosine is important in the regulation of vascular tone and the Equilibrative Nucleoside Transporter 1 (ENT1) is a regulator of adenosine concentrations. Thus, changes in ENT1 expression and activity may alter vascular tone regulation. We therefore examined the cardiovascular phenotype of ENT1‐knockout (KO) as compared to wild type (WT). Heart rates (HRs) and blood pressures (BPs) of KO and WT littermate male mice were obtained. Vascular reactivity was assessed in aortic ring segments obtained from 3 and 6 month old KO and WT mice. Morphological assessment of vascularized tissues was performed using H&E staining. BPs under awake and anesthetised conditions were not significantly different between KO and WT. However, awake HRs were significantly different (KO:570±22 vs. WT:674±22 beats; p<0.01). However, anesthetised HRs were not. KCl‐mediated significantly greater constriction (>;30%) in KO rings compared to WT. However, no significant differences between the two genotypes were observed for the different receptor agonists. H&E staining yielded evidence of a decreased luminal area as well as thinner aortic wall in KO mice at the age of 3 months. The enhanced effect of KCl in KO aortic rings suggests a physiological difference in the regulation of the vasoconstriction response. Decreased lumen and aortic wall thickness suggests a compensatory mechanism due to reduced HR in KO mice. These studies suggest a vascular phenotype associated with a loss of ENT1. This model will aid in unraveling the role of adenosine in vascular regulation.
Preserved PAR2 calcium signalling in endothelial cells of hypertensive mice
Authors: Hennessey John Charles , Stuyvers Bruno D , McGuire John J
Published: June 2021

In hypertension where other signal transduction pathways are attenuated, protease‐activated receptor 2 (PAR2)‐mediated vascular relaxation is preserved. Despite this difference, PAR2 calcium events in vascular endothelium are not understood. We hypothesize that PAR2‐mediated calcium events are preserved in isolated mesenteric endothelial cells from hypertensive mice. Calcium activity was measured by spinning‐disk confocal microscopy in PAR2 wild type (PAR2‐WT) and PAR2 deficient (PAR2‐KO) mice treated subcutaneously with saline or angiotensin II (hypertensive). Isolated endothelial cells were incubated with fluorescent calcium indicator, fluo‐4 AM, and then exposed to a PAR2‐activating peptide 2‐furoyl LIGRLO‐amide (2fly) or muscarinic M3 agonist acetylcholine. Our results show the number of calcium release sites and event rate are preserved in hypertensive PAR2‐WT exposed to 2fly. Acetylcholine‐induced calcium release sites and event rate were attenuated by 21% and 24% respectively. Similar results were found in hypertensive PAR2‐KO. Two types of calcium event were identified; small peripheral and repetitive, large central amplitude events. We conclude that PAR2‐mediated calcium signaling in isolated resistance endothelial cells is preserved in hypertension. Research funded by Canadian Institutes of Health Research and Research & Development Corporation Newfoundland and Labrador.
Role of the lipid peroxidation product, 4‐hydroxynonenal, in the in the development of nitrate tolerance
Authors: D'souza Yohan , Bennett Brian M.
Published: June 2021

Aldehyde dehydrogenase 2 (ALDH2) is important for the detoxification of 4‐hydroxy‐2‐nonenal (HNE), a toxic aldehyde produced by oxidative stress‐induced lipid peroxidation, which can form protein adducts and alter cell function. Tolerance to nitrates such as nitroglycerin (GTN) is associated with oxidative stress, inactivation of ALDH2, and decreased GTN‐induced cGMP accumulation and vasodilation. We hypothesized that GTN‐induced inactivation of ALDH2 results in increased HNE adduct formation of key proteins, and consequently an altered vasodilator response to GTN. This was assessed in a cell culture model (PK1 cells), and in aortae from GTN‐tolerant rats and ALDH2 null mice. Immunoblot analysis indicated a marked increase in HNE adduct formation in all three preparations. Preincubation of PK1 cells with HNE resulted in a dose‐dependent decrease in GTN‐induced cGMP accumulation, and pretreatment of isolated rat aorta with HNE resulted in dose‐dependent decreases in the vasodilator response to GTN, thus mimicking GTN‐tolerance. Pretreatment of aortae from ALDH2 null mice with 10 μM HNE also resulted in a desensitized vasodilator response, and mimicked the desensitized response observed in GTN tolerance. The data are consistent with the notion of a primary role of HNE protein adduct formation in the development of GTN tolerance. This work was supported by grant from the Canadian Institutes of Health Research (MOP 81175)
Association of two polymorphisms in the Cx40 promoter with hypertension and leftventricular hypertrophy
Authors: Schmidt Kjestine , Kaiser Frank J , Schunkert Heribert , Erdmann Jeanette , de Wit Cor
Published: June 2021

Signalling between cells is enabled by gap junctions composed of connexins (Cx). The isoform Cx40 is encoded by GJA5 and expressed in the endothelium, renin‐producing cells, atrial cardiomyocytes, and the cardiac conduction system. The functional relevance of Cx40 is emphasized by the phenotype of Cx40‐deficient mice, namely a renin‐dependent hypertension, an arteriolar communication deficit, and a cardiac hypertrophy. A genome‐wide association study identified a single nucleotide polymorphism (SNP) in the intron of GJA5 being associated with left ventricular hypertrophy (LVH). Since LVH often results from hypertension we analysed GJA5 in probands carrying the intron SNP. Sequencing of the Cx40 coding region did not reveal any alterations in 167 probands. In the putative promoter region of the Cx40 transcript 1A we identified two linked SNPs (−44G>;A, +71A>;G) at a significantly increased frequency in this cohort (40% vs. 5–7% in controls). Assuming a recessive model these SNPs were associated with hypertension and LVH. Surprisingly, this association was only found in men. However, analysing the promoter activity of this region at various lengths in a reporter gene assay using HeLa or HMEC‐1 cells did not reveal any modulation by the SNPs. In conclusion, we identified two coupled SNPs associated with LVH/hypertension in a preselected cohort, but the underlying pathomechanisms remain to be explored.
Computational model of NO and NO oxidative species transport in the microcirculation
Authors: Madrasi Kumpal Jagdish , Tsoukias Nikolaos
Published: June 2021

Sufficient availability of nitric oxide (NO) in the smooth muscles of blood vessels is important for its function as the proposed endothelium derived relaxing factor. NO may be preserved through the formation of NO2− anions or nitrosothiols which have a longer half‐life than NO in the circulation and thus may be transporting NO. A 2 D axisymmetric diffusion model was used to observe NO and NO oxidative species transport and to test the potential contribution of NO2− and nitrosothiols to smooth muscle NO levels according to data derived in previous research. With physiological concentrations of NO2− (10 μM – 20 μM) in the smooth muscle tissue, sufficient NO is generated to control blood vessel tone. NO2− reduction was more efficient in the lumen as compared to the tissue. Mitochondrial NO oxidase activity in the tissue regions was found to be an important factor for generating smooth muscle levels of NO2−. It was observed that the major nitrosating mechanism in the circulation is the •NO2 radical provided NO oxidation is accelerated in the hydrophobic regions of tissue layers and nitrosothiols may deliver minor amounts of NO through the Cu+ dependent enzyme activity. This study was supported by the NIH grant SC1HL095101
Impaired Ca2+ signaling following acutely elevated glucose in mouse endothelial cell tubes
Authors: Socha Matthew J. , Domeier Timothy L. , Sowers James R. , Segal Steven S.
Published: June 2021

Calcium signaling is integral to endothelial function and may be altered by hyperglycemia, a hallmark of diabetes. We tested the hypothesis that acute exposure to elevated glucose impairs Ca2+ signaling of native endothelium from resistance arteries of skeletal muscle. Intact endothelial cell tubes were isolated from superior epigastric arteries of male C57BL/6 mice (3–4 mos). Monitoring Ca2+ levels during exposure to control (10 mM) or elevated (20 mM) glucose for 30 min had no effect on resting Ca2+ (Δ fura‐2 ratio: 0.13±0.03 vs. 0.14±0.01, respectively; n=3). After loading with fluo‐4 dye, Ca2+ responses were evoked with 1 μM acetylcholine (ACh) in physiological solutions with normal (10 mM) and elevated (20 or 30 mM) glucose (with mannitol as an osmotic control) and imaged using spinning‐disc confocal microscopy. Exposure to 20 mM glucose for 30 min diminished peak Ca2+ responses (F/F0) to ACh (from 3.14±0.27 to 2.11±0.12; n=3, P<0.05) that were otherwise maintained in 10 mM glucose. These reductions in F/F0 with 20 mM glucose persisted for 1 hr following restoration of 10 mM glucose. Elevating glucose to 30 mM effectively abolished Ca2+ responses to ACh, which did not recover within 1 hr. Acute exposure to elevated glucose can impair endothelial Ca2+ signaling in the endothelium of resistance arteries and thereby contribute to endothelial dysfunction in diabetes. (NIH R37HL041026, R01HL086483, F32HL107050)
N‐cadherins Serve as a Mechanotransducer in Mid‐Cerebral Artery Smooth Muscle
Authors: Sun zhe , Li Min , Li zhaohui , Hill Michael A. , Meininger Gerald A.
Published: June 2021

Similar to the extracellular matrix‐integrin‐cytoskeleton axis, cell‐to‐cell N‐cadherin‐adhesion sites are linked to the cytoskeleton. We investigated the hypothesis that these sites also act as sites of mechanotransduction in vascular smooth muscle cells (VSMC). Consistent with our previous observations that N‐cadherin‐directed antibodies inhibited the myogenic response in rat skeletal muscle arterioles, knock‐down of N‐cadherin expression with specific si‐RNA also abolished myogenic tone in rat mid‐cerebral arteries (MCA). Immunolabeling of N‐cadherin demonstrated it to be distributed along the borders between adjacent VSMCs. In VSMCs isolated from MCA and transfected with N‐cadherin‐GFP, clustering of N‐cadherin‐GFP formed around an N‐cadherin‐coated microsphere (10 μm) brought into contact with the cell using an atomic force microscope (AFM). After 20 min of contact with the cell surface, application of a pulling force (1 nN; via the AFM) to the microsphere resulted in a VSMC contractile response. The contractile response was characterized by the VSMC generating a force to oppose the pulling force, and by recruitment of actin‐RFP and RhoA‐GFP to the N‐cadherin adhesion sites. Collectively, these data strongly support an important role for cell‐to‐cell N‐cadherin adhesion sites in mediating VSM mechanotransduction. (1P01HL095486 to G.A.M. and AHA 0835676N to Z.S.)
Aging increases the amplitude of spontaneous transient outward currents in murine resistance artery smooth muscle cells
Authors: Hayoz Sebastien , Segal Steven S. , Jackson William F.
Published: June 2021

Large conductance Ca2+‐activated K+ channels (BKCa) contribute to negative feedback regulation of microvascular tone. However, the effects of aging on BKCa function are unclear. The purpose of this study was to determine: 1) if enzymatically‐isolated smooth muscle cells (SMCs) from skeletal muscle resistance arteries display BKCa‐ and ryanodine receptor (RyR)‐dependent spontaneous transient outward currents (STOCs); and 2) whether aging affects STOC properties. Using perforated patch clamp recording, individual SMCs from Young (≥ 3 months) and Old (≥ 24 months) male C57BL/6 mice displayed STOCs at membrane potentials ≥ −30 mV, with the amplitude and frequency of STOCs increasing with membrane depolarization (n = 19–24; p < 0.05). STOCs were inhibited by BKCa channel blockers (1 μM paxilline, 100 nM iberiotoxin) and by a RyR antagonist (100 μM tetracaine) (n ≥ 5; p < 0.05 each). At +30 mV holding potential, STOC amplitude = 3.2 ± 0.32 pA/pF and frequency = 3.88 ± 0.41 Hz (n = 67) in SMCs from Young. STOC amplitude increased to 4.97 ± 0.5 pA/pF (n = 61; p < 0.05) in SMCs from Old, with no significant difference in frequency (3.15 ± 0.31 Hz; p >; 0.05). SMCs from murine resistance arteries display BKCa‐ and RyR‐dependent STOCs and aging increases STOC amplitude. Age‐related change in BKCa activity may impact the regulation of SMC function and contribute to age‐induced alterations in muscle blood flow regulation. (NIH R01HL086483).