bluuebunny/crossref_metadata_2025 huggingface dataset:
The Mu‐Delta Opioid Receptor Heterodimer Promotes Acute and Chronic Morphine Induced Dependence/Withdrawal in Mice
Authors: Nguyen Paul , Keresztes Attila , Olson Keith , Hruby Victor , Streicher John M.
Published: June 2021

Opioid drugs like morphine are the gold standard for treating acute and chronic pain, but induce detrimental side effects such as tolerance and dependence. It has been suggested that the mu‐delta opioid receptor heterodimer (MDOR) transduces some of these side effects and that heterodimer targeted drugs could be a solution to weaken these side effects. We have thus created an MDOR selective antagonist called D24M. We evaluated D24M in vitro, and found a ~100 fold selectivity for the MDOR over the monomers. Similarly, we performed hot water tail‐flick experiments in mice in the presence of various doses of D24M against CYM51010 and Deltorphin‐2 (MDOR selective agonist), and DAMGO (MOR monomer selective agonist), and found that D24M potently (A50=2–7.8 nmol) blocked MDOR activity with no activity against the MOR monomer up to 10 nmol. To test the ability of D24M to lessen acute or chronic morphine‐induced dependence and withdrawal, we established acute (4 hr) and chronic (4 day) morphine dependence models, with 1 nmol D24M or vehicle injected 5 minutes prior to naloxone precipitation of withdrawal. We found that D24M strongly reduced jumping behavior in dependent mice in both the acutely and chronically models; further confirming that the MDOR promotes withdrawal behavior, and that D24M could possibly be a promising drug candidate for opioid dependence and withdrawal. These discoveries will further determine the role of the MDOR in vivo, and provide a novel tool that could greatly impact opioid heterodimer research.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of Swimming Exercise in Combination with non‐Steroidal Anti‐Inflammatory Drugs on Inflammatory Nociception in Rats
Authors: Alsalem Mohammad , Altarifi Ahmad , Kalha Zain , Alzoubi Abdallah , Alfaqih Mahmoud , Kana'an Saddam
Published: June 2021

Pain is a growing health problem with increasing prevalence and burden worldwide. Major approaches for pain control include pharmacological and non‐pharmacological interventions. Pharmacological treatment is achieved by the use of analgesics, such as non‐steroidal anti‐inflammatory drugs (NSAIDs) and opioids. However, these medications are usually accompanied with many side effects upon prolonged use. Physical exercise is an example of non‐pharmacological interventions for pain control. In this study, we examined the effect of one of the most commonly prescribed NSAIDs, ibuprofen, on nociception threshold, with or without swimming exercise in rats. Two swimming protocols were used; Prophylactic group and treatment group. Nociception was induced by the intraplantar injection of Complete Freund's Adjuvant (CFA). Nociception threshold was measured using von Frey filaments in an up‐down protocol. The goal of this study was to determine the effect of swimming exercise on nociception threshold and ibuprofen‐induced antinociception in inflammatory pain rat model. We hypothesized that swimming exercise will enhance the ibuprofen‐induced antinociception in rats. Ibuprofen produced an antinociception effect only at the highest dose (32 mg/kg), with a duration that lasted for 17 days. A dose of 32 mg/kg ibuprofen produced significant increase in pain threshold in swimming treatment and prophylactic swimming groups when compared to ibuprofen alone group. Moreover, Ibuprofen produced significant antinociception after 10 mg/kg drug dose compared to vehicle in the prophylactic swimming group only. The duration of mechanical allodynia after CA injection was decreased in both swimming groups to 7 days only. In conclusion, the combination of ibuprofen and swimming exercise was shown to be effective in controlling nociception in a rat inflammatory pain model. The combination of exercise and ibuprofen could be suggested as an effective intervention to help control chronic pain. In addition, prophylactic swimming can increase the potency of analgesic medications, by lowering the administered drug dose, and thus, lowering the risk for ibuprofen‐induced side effects.

Support or Funding Information

JUST 130/2016

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Conformational Dynamics Contribute to Phospholipase Cβ Activity
Authors: Van Camp Michelle Marie , Garland‐Kuntz Elisabeth , Lyon Angeline
Published: June 2021

Phospholipase Cβ (PLCβ) enzymes hydrolyze the lipid phosphatidylinositol‐4,5‐bisphosphate (PIP2) to produce the second messengers inositol‐1,4,5‐triphosphate (IP3) and diacylglycerol (DAG). Production of these second messengers leads to many diverse physiological responses, including vascular smooth muscle contraction and inflammation. Previous structural and functional studies of PLCβ have revealed a highly conserved catalytic core that adopts a compact, globular structure and forms the minimal fragment that retains lipase activity. However, a growing body of evidence suggests that the PLCβ PH domain, which interacts with EF hands and TIM barrel of the catalytic core in crystal structures, may be flexible in solution. Using a combination of small angle x‐ray scattering (SAXS), cross‐linking studies, and biochemical assays, we provide the first structural data demonstrating that the PH is in fact conformationally flexible in solution. The PH domain adopts two major states: an open state wherein the PH domain is extended away from the core, and a closed state consistent with the crystal structures. We also provide evidence that these conformational states are functionally significant. These findings provide new evidence of conformational heterogeneity of PLC enzymes in solution and reveal new insights into their roles in cell signaling and cardiovascular disease.

Support or Funding Information

American Heart Association Scientist Development Grant 16SDG29920017; Purdue Center for Cancer Research; Showalter Foundation; all to A.M.L

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Amino‐terminal region plays an important role in transport function of human organic anion transporting polypeptide 1B1
Authors: Hong Mei , Chen Jie , Xiang Zhaojian , Fang Zihui , Ni Chunxu
Published: June 2021

Objective

The family of organic anion transporting polypeptides (OATPs, gene symbol SLCO) mediates sodium‐independent transport of a wide variety of endogenous and exogenous compounds. OATPs are considered as key players in drug absorption, distribution and excretion due to their broad substrate specificity, wide tissue distribution and involvement in drug‐drug interactions. OATP1B1 is specifically located at the basolateral membrane of human hepatocytes and serves a crucial role in drug clearance from the body. Previous studies have shown that transmembrane domains (TMs) of OATP1B1 are essential structural features for proper function of the transporter.

Methods

Alanine‐scanning of the putative transmembrane domain 1 and truncation of the N‐terminus of OATP1B1 were carried out, and the uptake function and protein expression of mutants were analyzed.

Results

Two positively charged amino acid residues, K41 and K49, were identified to be important for uptake function of the transporter protein. Alanine substitution of K41 altered substrate binding at the high affinity component; while K49A exhibited increased Km value at the low affinity site. In addition, our study of the N‐terminal residues of OATP1B1 found out that residues ranging from 19–27 are essential for protein stability and uptake function of the transporter protein.

Conclusions

The N‐terminus region of OATP1B1 is important for maintaining proper function and protein expression level of the transporter.

Support or Funding Information

This work was supported by the National Natural Science Foundation of China Grants [81373473] to Mei Hong.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rgs8 and Rgs16 protect against pancreatitis and PDA cancer progression
Authors: Wilkie Tom , Choudhuri Shreoshi Pal , Zolghadri Yalda
Published: June 2021

Pancreatic ductal adenocarcinoma (PDA) has the highest death rate among major cancers, and new treatments are desperately needed. KCR8‐16 mice are an excellent model for identification, characterization, and in vivo validation of novel PDA therapeutics. Kras oncogenic mutations (e.g. KrasG12D) are found in over 90% of human PDA. Kras can be activated by protein kinase and G‐Protein Coupled Receptor (GPCR) signaling. Regulator of G protein signaling (Rgs) proteins regulate GPCR signaling by accelerating the GTPase activity of Gq/11‐ and Gi‐class alpha subunits. Activating alleles of Gq/11 that are resistant to Rgs inhibition are found in benign precursors of PDA in humans. We previously reported that Rgs8 and Rgs16 are in vivo reporters of Kras activity in pancreatic intraepithelial neoplasia (PanIN), intraductal papillary mucinous neoplasm (IPMN), and PDA progression in KIC;Rgs16::GFP mice (p48::Cre/+; KrasG12D/+; Cdkn2af/f; Rgs16GFP ) (DMM 8, 2015). To identify the role Rgs8 and Rgs16 in PDA, we crossed the Rgs8‐16 double knockout into pancreas specific KrasG12D (KC) mutant mice (termed KCR8‐16). We found that deletion of Rgs8 and Rgs16 dramatically accelerated PDA progression in the KC backgrond. Additional pancreatic stress evoked by caerulein treatment caused immediate and pancreas‐wide progression to PDA in KCR8‐16 mice. Our study suggest Rgs8 and Rgs16 act as tumor suppressor genes in PDA initiation and progression. Moreover, KCR8‐16 and KIC;Rgs16::GFP mice can be used as excellent models for identification and rapid in vivo validation of PDA therapeutics.

Support or Funding Information

NCI R01 CA192381

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Promiscuous Biotin Ligase as a Global Strategy to Interrogate the Protein Interactome of Human ABCC4
Authors: Looi‐Somoye Remi , Wijaya Juwina , Peng Junmin , Schuetz John
Published: June 2021

ATP‐Binding Cassette transporter sub‐family C member 4, ABCC4, is a plasma membrane transporter with the ability to export prostaglandins and cancer chemotherapeutics. In acute myeloid leukemia (AML), ABCC4 interacts with a binding protein through its PDZ motif1. This interaction with a highly expressed PDZ protein, a protein which correlated with worse overall survival for AML patients, affects ABCC4 plasma membrane localization and retention, leading to profound effects on its function and drug resistance. The intense focus on ABCC4's PDZ motif as a locus for protein interaction has left other potential protein interactions that might regulate ABCC4 function largely unexplored. Little is actually known about how ABCC4 traffics to and from the plasma membrane. In the present study, we developed a global approach to not just identify potential interacting proteins, but also determine the proteins required for ABCC4 trafficking and internalization. We used a promiscuous biotin ligase, BioID2, which covalently attaches a biotin moiety to nearby proteins in the presence of biotin. Fusing BioID2 to the N‐terminus of ABCC4 did not alter its glycosylation, plasma membrane localization, or function. Upon addition of biotin, the BioID2‐ABCC4 fusion protein was capable of biotinylating itself as well as ABCC4 proximal and interacting proteins. We then developed an immunoprecipitation and elution protocol and strategy to specifically identify proteins biotinylated by the BioID2‐ABCC4 fusion protein. With successful immunoprecipitation of biotinylated proteins and identification by mass spectrometry, proteins within the vicinity of ABCC4, from its genesis at the endoplasmic reticulum to its final destination at the plasma membrane, will be identified. At the same time, proteins involved in ABCC4 recycling to degradation will also be discovered. Overall, this study will provide unique insights into the mechanisms of ABCC4 trafficking and identify proteins that will allow for modulation of ABCC4 level. This will be beneficial for malignancies associated with high ABCC4 expression, including neuroblastoma, prostate, non‐small cell lung, and endometrial cancers.

Support or Funding Information

This work was supported by NIH and ALSAC.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Polyamine pathway as a player in breast cancer cell proliferation in diabetic conditions
Authors: Capellen Caleb , Ortega Jose , Morwitzer M. Jane , Dunworth Matthew , Casero Robert A. , Chandra Surabhi
Published: June 2021

Diabetes/hyperglycemia has been known to increase cancer cell proliferation, leading to metastasis and even mortality. High blood sugar is also known to reduce the effects of cancer therapeutics. Polyamines (spermine, spermidine, and putrescine) which are involved in several cellular processes such as cell growth, replication, and transcription, have been shown to be elevated in certain cancers including colon cancer and skin cancer. We hypothesized that polyamines are involved with proliferation of breast cancer cells in diabetic conditions. Cell lines used in this study included MCF‐7 (early stage breast cancer), MDA‐MB‐231 (late stage breast cancer), and MCF‐10A (normal breast epithelial cells). Cells were treated with normal glucose (NG, 5mM) or high glucose (HG, 25 mM) in the presence/absence of polyamine pathway inhibitors. Both MDA‐MB‐231 and MCF‐10A cells showed significant proliferation with HG treatment after 72 hr, which was not observed in MCF‐7 cells. Simultaneous treatment with an ornithine decarboxylase (polyamine synthesis enzyme) inhibitor, difluoromethylornithine (DFMO) could prevent this effect. Interestingly, while spermine levels were not altered with HG treatment in breast cancer cells (MCF‐7 and MDA‐MB‐231), they were significantly increased in MCF‐10A cells. This is possibly due to the fact that cancer cells have endogenously high levels of polyamines, such that further increase is prevented to avoid toxicity from polyamines. Nevertheless, DFMO was able to decrease spermine and spermidine levels induced with glucose treatments, suggesting a role of polyamine pathway in regulating growth. Putrescine levels were undetectable in all samples. In conclusion, polyamine pathway is involved with HG mediated normal breast epithelial cell and breast cancer cell proliferation. Future studies will involve further analysis of the mediators which regulate polyamine production.

Support or Funding Information

UNK Faculty Start‐up Funds (SC); Undergraduate Research Fellowship Grant (CC and JO)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Down Regulation of Add3 in Astrocytes Disrupts the Actin Cytoskeleton in Association with Decreasing Small Molecule Uptake and May Contribute to Cognitive Deficits in FHH rats
Authors: Travis Olivia Kristell , Wang Shaoxun , He Xiaochen , Roman Richard J. , Fan Fan
Published: June 2021

We previously reported that the expression of Add3 is decreased in various tissues, including brain and cerebral vessels, and further decreases with age in FHH rats. More recently, we demonstrated that Add3 dysfunction contributes to cerebral vascular impairments in FHH rats, and they exhibit BBB leakage and neurodegeneration after the development of hypertension. In addition, Aβ protein expression in the brain in FHH rats is increased as early as 8 weeks of age. Reactive astrogliosis is a common finding in neurodegenerative diseases and activated astrocytes promote neurodegeneration. Moreover, astrocyte dysfunction affects Aβ clearance and Aβ accumulation is a characteristic biomarker of Alzheimer's disease. To further explore whether astrocytes contribute to neurodegeneration and cognitive impairments in FHH rats, we knocked down Add3 expression using Add3 Dicer‐substrate RNAi (DsiRNA) in human astrocytes and found that GFAP expression was markedly enhanced in comparison to scrambled siRNA treated cells. F‐actin distribution was significantly reduced in the Add3 DsiRNA treated cells (19.3 ± 6.7%, n = 6) when compared to the control cells (34.9 ± 3.0%, n = 6). In addition, the Add3 DsiRNA treated cells took up less fluorescence‐labeled dextran (10 kD) than the control cells suggesting the capability of uptake of small molecule, such as Aβ may be diminished. We found that DOCA/salt induced hypertensive FHH rats exhibit a reduction in neuronal density and size in the hippocampus. This was associated with loss of capillary density (22.7 ± 0.001% in FHH and 9.6 ± 0.014% in control rats, n = 7 and 9, respectively), and greater expression of GFAP positive astrocytes and loss of neurons in affected areas. Hypertensive FHH rats took 2.5 times longer to escape from an eight‐arm water maze as compared to control strains. These results suggest that down regulation of Add3 in astrocytes disrupts the actin cytoskeleton to induce reactive astrogliosis and may affect Aβ clearance, all of which may contribute to neurodegeneration and cognitive deficit associated with aging and hypertension in FHH rats.

Support or Funding Information

This study was supported by grants HL36279 (RJR) and DK104184 (RJR), AG050049 (FF), P20GM104357 (RJR, FF ) from the National Institutes of Health; 16GRNT31200036 (FF) from the American Heart Association. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ang II‐Mediated Regulation of AGT Protein Synthesis in Rat Brainstem Astrocytes is βarrestin1 Dependent
Authors: Negussie Shmuel , Lymperopoulos Anastasios , Clark Michelle Alexa
Published: June 2021

Objective

Study the role of βarrestins in Angiotensin (Ang) II‐mediated regulation of angiotensinogen (AGT) protein synthesis in astrocytes isolated from the brainstem of spontaneously hypertensive rats (SHRs) or normotensive rats.

Background

The βarrestins (βarrestin1 and‐2) terminate G protein signaling by G protein‐coupled receptors (GPCRs) and, at the same time, they initiate their own “second wave” of signaling from GPCRs, independently of G proteins. Ang II, through its cognate GPCR, Angiotensin II type1 receptor (AT1R), signals via both G proteins and βarrestins in various cell types. In our previous study, we reported that SII (an Ang II peptide analog which is a βarrestin‐biased agonist), similarly to Ang II, upregulates AGT synthesis in a time‐dependent manner. Here, we sought to delineate whether Ang II‐mediated regulation of AGT protein synthesis in brainstem‐derived astrocytes is G protein or βarrestin or both G protein and βarrestin dependent.

Methods

Primary cultures of brainstem astrocytes were isolated from the brains of 2–3 days old pups. Double‐stranded siRNAs (Dharmacon) targeting rat βarrestin1 or βarrestin2 were used to knockdown βarrestins. Non‐silencing siRNA (Dharmacon) was used as a control. RNiMAX (Invitrogen) was used as a transfection reagent. Ang II (100 nM) and 10 μM SII (the βarrestin‐biased agonist) were used to study AT1R‐mediated AGT protein expression. Protein analysis was done using Western blotting and mRNA measurement was done using quantitative real‐time PCR. The results in SHR astrocytes were compared to normotensive rat astrocytes.

Results

Our results indicate that βarrestin1 is the major βarrestin isoform in astrocytes isolated from both SHR and normotensive rat brainstem astrocytes. Ang II upregulated AGT protein synthesis in both SHR and normotensive rat astrocytes. This effect was higher in SHR as compared to normotensive rats. siRNA mediated knockdown of βarrestin1 abolished Ang II‐mediated AGT protein synthesis. However, βarrestin2 knockdown did not inhibit Ang II‐mediated AGT protein synthesis.

Conclusion

In this study, we showed that βarrestin1 plays a major role in the Ang II‐AT1R‐mediated regulation of AGT protein synthesis in rat brainstem astrocytes in both SHR and normotensive rat models, which could be an important therapeutic target for the treatment of hypertension and other cardiovascular diseases.

Support or Funding Information

Funding for this project was provided through a Nova Southeastern University President's Faculty Research and Development Grant (PFRDG)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Toll‐Like Receptor 9 (TLR9) Is Associated with Vascular Dysfunction in 2‐Kydney 1‐Clip (2K1C) Hipertensive Mice.
Authors: Rodrigues Fernanda Luciano , da Costa Rafael Menezes , Silva Carlos Alberto Aguiar , Bomfim Gisele Facholi , Carneiro Fernando Silva , Tostes Rita , Junior Rubens Fazan , Salgado Helio Cesar
Published: June 2021

Introduction

Renovascular hypertension is the leading cause of secondary hypertension and commonly resistant to antihypertensive therapy. The involvement of toll‐like receptors 9 (TLR9) has already been demonstrated in spontaneously hypertensive rats (SHR) and their activation seems to contribute to the increase in arterial pressure (AP). Therefore, we hypothesized that TLR9 are activated in two‐kidney one‐ clip (2K1C) hypertension, eliciting inflammation and vascular dysfunction, playing a role in the increase of the AP.

Methods and Results

Wild type C57BL (WT) and TLR9 knockout (TLR9_KO) mice were anesthetized with isoflurane and were implanted with a silver clip (0.12 mm) around the left renal artery (SHAM operated mice were not implanted with the clip). After 4 weeks, the mice were also anesthetized with isoflurane for direct AP measurement. Afterwards the third‐order mesenteric resistance arteries (MRA) were removed for vascular function evaluation. AP was higher in 2K1C WT mice (133 ± 2 vs 93 ± 4 mmHg in SHAM WT mice), while this increase was partially prevented by the absence of TLR9 (114 ± 5 mmHg in 2K1C TLR9_KO mice). Vascular dysfunction, characterized by increased contractile responses to phenylephrine (Emax: 167 ± 2.2 vs 126 ± 3.5 KCl % in SHAM WT mice) and reduced relaxation to acetylcholine (Emax: 69 ± 1.6 vs 88 ± 1.4 PE % in SHAM WT mice) and sodium nitroprusside (pD2: 7.31 ± 0.07 vs 7.85 ± 0.05 in SHAM WT mice) was found in 2K1C WT mice but not in 2K1C TLR9_KO mice (Emax: 128 ± 2.9 KCl %, 89 ± 2.0 PE % and pD2: 7.78 ± 0.06, when compared to SHAM WT mice, to phenylephrine, acetylcholine and sodium nitroprusside, respectively).

Conclusion

TLR9 play an important role in vascular dysfunction and AP increase in 2K1C hypertension.

Support or Funding Information

Financial support: FAPESP (Proc. 2015/21976‐1, 2013/20549‐7 and 2016/11988‐5).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modulation of Gastrointestinal Smooth Muscle Motility by Losartan in Rats
Authors: Aigbe Flora Ruth , Tobechukwu Mercy Ezinwa , Akindere Olanrewaju Kingsley , Chijioke Micah Chimeremeze , Jaiyesimi Temitope Jonathan , Shonubi Fatima Ayodeji , Adeyemi Oluwafunmilayo Olaide
Published: June 2021

A drug‐induced gastrointestinal disturbance is an important adverse drug reaction. It has been reported to occasionally present with inflammatory bowel disease‐like manifestations. The mechanism by which this occurs for several drugs is yet to be fully understood. Time and money are sometimes wasted in the diagnosis and treatment (owing to inappropriate strategies) of gastroenterological cases that are simply drug‐induced. Proper assessment of the gastrointestinal actions of drugs would contribute to the knowledge required to significantly address this situation. Losartan is an antihypertensive implicated in diarrhoea, constipation and abdominal cramps, mechanisms of which are yet to be well understood. This preclinical study was carried out to examine the action of losartan on gastrointestinal smooth muscles using rat tissues. Albino rats' caecal and duodenal tissues (4–5 cm) were excised, suspended in tissue baths containing aerated Tyrode physiological solution and connected to Powerlab data acquisition system via isometric force transducers. The direct effect of losartan (0.332–990 μg/ml) and its influence on contractile responses to acetylcholine (1.83×10−5–5.45×10−3 μM), carbachol (1.82×10−4–5.42×10−2 μM) and potassium chloride (2.28×10 – 6.64×10 μM) were determined. Concentration‐dependent contraction of the rats' caecum and duodenum tissues by losartan was observed; with median effective concentration (EC50) values of 8.30 μg/ml and 8.41 μg/ml for both tissues respectively. Losartan significantly (p<0.01) reduced acetylcholine‐induced contraction of the rats' caecum. A significant (p<0.0001) reversal of carbachol‐induced contraction by losartan at 990 μg/ml was observed in the caecum and duodenum tissues. Significant (p<0.0001) reduction in potassium chloride‐induced contraction of the duodenum was also noted, and a similar trend was observed with the caecum. These findings demonstrate that losartan modulates intestinal smooth muscle tone by interfering with one or more of the several pathways involved in gastrointestinal motility. One pathway that seems likely from the observations made in this study is the cholinomimetic pathway. Additional studies to further elucidate the involvement of this pathway and other mechanisms through which losartan mediates its action would enhance understanding of its pharmacology in this regard. The knowledge thus gained would allow for possible optimization of its therapeutic benefit to mankind, as a more rational approach to its use would become possible, due to the availability of additional relevant information.

Support or Funding Information

This work was carried out without external funding.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Snake Venom‐Derived Hemocoagulase Modified with Factor Xa Restores Hemostasis in Heparin‐Treated, Hypocoagulant Mice
Authors: Jackson Madeline , Adili Reheman , Dai Xiangrong , Li Benjamin Xiaoyi , Holinstat Michael
Published: June 2021

Background

Excessive, post‐traumatic bleeding is the leading cause of potentially preventable death in the United States. Nearly one‐third of trauma patients present with coagulopathy upon hospital admission, which significantly increases the incidence of multiple organ failure and death when compared with healthy patients with similar injuries. A hemocoagulase, such as batroxobin, is a thrombin‐like serine protease that is derived from the venom of either the Bothrops Jararaca or Bothops atrox snake and has the ability to convert fibrinogen to fibrin, thus enhancing clot formation and ultimately reducing blood loss in hemorrhagic injuries. Slounase is a modified hemocoagulase combined with Factor Xa (FXa) which may have better hemostatic efficacy in hypocoagulant conditions compared to batroxobin alone. However, the effects of this modified hemocoagulase in the presence of a coagulopathy has not been well characterized in vivo due to lack of reliable hemostasis models.

Aim

To determine the hemostatic efficacy of slounase compared to both batroxobin and FXa in hypocoagulant murine models using real‐time intravital microscopy.

Methods

The effects of slounase, batroxobin, and Fxa, at doses of both 1 U and 0.1 U, were assessed in vivo by laser ablation of arterioles in the cremaster muscle under real‐time microscopy. Prior to injury, mice were intravenously injected with 25 U heparin to induce hypocoagulant conditions.

Results

Intravenous injection of 25 U heparin inhibited clot formation, significantly increasing bleeding time in untreated, hypocoagulant mice. Injection of slounase, FXa, and Batroxobin did not cause platelet activation in absence of injury. After arteriole injury, FXa did not have a significant effect on clot formation, as FXa alone is inhibited by heparin. Batroxobin‐treated mice had some fibrin formation, however, there was no noticeable change in platelet recruitment. Slounase, at both 0.1 U and 1 U, resulted in a significant enhancement of platelet‐fibrin content, restored clot formation, and significantly shortened bleeding time.

Conclusions

This in vivo study clearly demonstrated that slounase restores hemostasis in hypocoagulant mice by enhancing both platelet activation and fibrin formation, while batroxobin alone only enhances formation of fibrin. Our data strongly suggests that slounase has a better hemostatic efficacy in heparin‐induced hypocoagulant conditions than hemocoagulase alone.

Support or Funding Information

This work was funded by LEE'S PHARM and in part through the National Institutes of Health grants: R01 GM105671 (MH), R01 HL114405 (MH), and R01 MD007880 (MH)

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 Marine Pharmacology and Pharmaceuticals Pipeline in 2017
Authors: Trieu Jefferson , Newman David J. , Glaser Keith B. , Mayer Alejandro M.
Published: June 2021

The ocean's organisms continue to deliver a chemical biodiversity that contributes to both the global preclinical and clinical pharmaceutical pipelines. Thus, in November 2017, the clinical marine pharmaceutical pipeline consisted of six marine‐derived drugs approved by the United States Food and Drug Administration (FDA): for cancer, cytarabine (Cytosar‐U®, Depocyt®, FDA‐approved 1969); for pain, ziconotide (Prialt®, FDA‐approved 2004); for hypertriglyceridemia, omega‐3‐acid ethyl esters (Lovaza®, FDA‐approved 2004); for cancer, eribulin mesylate (Halaven®, FDA‐approved 2010), brentuximab vedotin (Adcetris®, FDA‐approved 2011), and trabectedin (Yondelis®, FDA‐approved 2015). Furthermore, there were 23 marine‐derived compounds in clinical trials: 6 marine‐derived compounds in Phase III, 8 compounds in Phase II, and at least 9 compounds in Phase I, many of them auristatin‐containing antibody drug conjugates. Regular updates on the clinical marine pharmaceutical pipeline are posted at http://marinepharmacology.midwestern.edu/clinPipeline.htm. Furthermore, the global preclinical marine pharmacology pipeline continues to generate considerable data on multiple pharmacological classes, as highlighted in a recent review: A. M. S. Mayer, A. D. Rodríguez, O. Taglialatela‐Scafati and N. Fusetani. Marine Pharmacology in 2012–2013: Marine Compounds with Antibacterial, Antidiabetic, Antifungal, Anti‐Inflammatory, Antiprotozoal, Antituberculosis, and Antiviral Activities; Affecting the Immune and Nervous Systems, and other Miscellaneous Mechanisms of Action. Marine Drugs 15 (9): 273, 2017. We thus conclude that in November 2017, both the marine pharmacology preclinical and clinical pharmaceutical pipelines continued to remain very active.

Support or Funding Information

Supported by Midwestern University.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anti‐allergic effect of elecampane camphor from Saussurea lappa in ovalbumin‐induced asthma model
Authors: Nam So‐Yeon , Lee Bo‐Kyung , Im Dong‐Soon , Park Soo‐Jin , Kang Saeromi
Published: June 2021

Background

In Oriental countries, the root of Saussurea lappa is used to treat asthma, rheumatism, and other conditions. Extracts of Saussurea lappa were reported to alleviate house dust mite‐induced atopic‐like dermatitis in Nc/Nga mice, and sesquiterpene lactones were isolated.

METHODS

To elucidate whether elecampane camphor, a constuent of Saussurea lappa extract has beneficial effects on allergic asthma, anti‐asthma effects were studied using female Balb/c mice and rat RBL‐2H3 mast cells. Antigen‐induced degranulation was measured in vitro by measuring β‐hexosaminidase activity. A murine ovalbumin‐induced allergic asthma model was used to test the in vivo efficacy of the elecampane camphor.

RESULTS

Antigen‐induced degranulation was inhibited by the elecampane camphor. Sensitization and challenge of ovalbumin induced allergic asthma responses. Administration of elecampane camphor decreased numbers of inflammatory cells, especially eosinophils, and reduced the expression and secretion of Th2 cytokines, including IL‐4 and IL‐13, in lung tissues and bronchoalveolar lavage fluid. Histologic studies showed administration of elecampane camphor reduced inflammatory signs and mucin production in lungs.

CONCLUSION

The findings provide evidence that elecampane camphor has a potential use as anti‐allergic therapeutics.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ameliorative potentials of Annona muricata against Potassium dichromate‐induced hypertension through antioxidant defence system
Authors: Ola‐Davies Olufunke Eunice , Oyagbemi Ademola Adetokunbo , Omobowale Temidayo Olutayo , Akande Isreal Adetokunbo
Published: June 2021

Potassium dichromate is an environmental and industrial pollutant. It has already been reported that the accumulation of K2Cr2O7 can alter the activities of some enzymes involved in the free‐radical metabolism and also decrease the activities of some enzymes involved in the antioxidant defense system. This study investigated the antihypertensive effect of Annona muricata extract against K2Cr2O7 induced hypertension. Fifty rats were used for this study and were divided into ten rats per group. Rats in Group A received normal saline, Groups B (Annona muricata extract alone at 250 mg/kg, Group C K2Cr2O7 at 30 mg/kg, Groups D and E were pre‐treated with extract at 250 mg/kg and Gallic acid at 60 mg/kg for fourteen days respectively and thereafter administered with single intra‐peritoneal injection of K2Cr2O7 (30 mg/kg). Administration of K2Cr2O7 significantly increased systolic, diastolic and mean arterial pressure. Further, pre‐treatment with extract at 250 mg/kg and Gallic acid at 60 mg/kg significantly reduced high blood pressure to near normal. Also, K2Cr2O7 led to significant increase in hydrogen peroxide (H2O2) generated, malondialdehyde contents, protein carbonyl together with significant reductions in reduced glutathione (GSH), glutathione‐S‐transferase (GST), glutathione peroxidase (GPx) and superoxide dismutase (SOD). Serum nitric oxide (NO) also reduced significantly whereas serum advanced oxidative protein product (AOPP), myeloperoxidase (MPO) and xanthine oxidase (XO) significantly increased in K2Cr2O7 alone‐administered rats. Immunohistochemistry of renal Kidney injury molecule (Kim−1) and p38 MAPK showed increased expressions following administration of K2Cr2O7 together with down‐regulation of nuclear factor erythroid 2‐related factor 2 (nrf2). Pre‐treatment with extract at 250 mg/kg and Gallic acid at 60 mg/kg also increased the expressions of nrf2 and down‐regulated Kim−1 and p38. Together, pre‐treatment with extract at 250 mg/kg and Gallic acid at 60 mg/kg normalize blood pressure, reduced markers of oxidative stress, improved antioxidant defense system and serum NO bioavailability

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Increases of capillary blood pressure in the supine position in persons with cervical spinal cord injuries
Authors: Kouda Ken , Nakamura Takeshi , Umemoto Yasunori , Kamijo Yoshi‐ichiro , Tajima Fumihiro
Published: June 2021

Background

Cervical spinal cord injuries (CSCI) subjects have hypotension and most of physicians have believed that capillary pressure in CSCI would be decreased and easily formed pressure ulcers. Systemic blood pressure (BP) decreases are mainly caused by attenuated total peripheral resistance in CSCI. However, BP in capillary was not measured in CSCI. We hypothesized that the measurement of peripheral pressure such as arteriole pressure and capillary pressure could be a simple and efficient means of expecting to occur with lasting tissue damage for CSCI. The purpose of the present study was to investigate capillary BP in CSCI.

Methods

Eleven male CSCI individuals with a lesion between C6 and C7 (ASIA A, age, 32.5 +/− 2.8 y) and 13 healthy subjects (age, 40.1 +/− 3.5 y) participated in the present study. Measurements were performed in the supine position with the legs at heart level. To measure capillary BP, a pressure transducer with probe of Laser Tissue Blood Flowmeter was attached to the pretibial skin and the probe was gradually and continually compressed for five seconds until the output signal of the flowmetry showed constant values. When the output of the velocimeter showed constant values, the pressure of the probe compression should mean the Capillary BP. Because the termination of capillary blood flow demonstrated constant signals of velocimeter and at the time the compression pressure should be the capillary pressure. Simultaneously, systolic and diastolic BP was non‐invasively measured during the measurement on the contralateral arm of the blood flow measuring leg.

Results

A significant lower MBP was observed in CSCI (76.7 +/−3.9 mm Hg) than in healthy subjects (91.0 +/− 4.0 mm Hg). Capillary BP in CSCI (55.7 +/− 2.0 mm Hg) was significantly greater than in control subjects (45.9 +/− 2.3 mm Hg).

Conclusion

We demonstrated here that transection of sympathetic nervous system from medulla to peripheral nerves in CSCI resulted in the increase of capillary pressure. We suggested that the cervical spinal transection diminished tonic impulses of sympathetic nerves to resistant vessels in CSCI.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Phenylephrine and Norepinephrine on the Diameter of the Epineurial Vessels Supplying the Sciatic Nerve
Authors: Cleary Shane R. , Orr Julie I. , Killey Chelsa , Jackson Dwayne N. , Frisbee Jefferson C. , Twynstra Jasna
Published: June 2021

Background

Phenylephrine (PE) and norepinephrine (NE) are known vasoconstrictors; however, little is known about their role in blood flow regulation at the sciatic nerve. The purpose of this study was to determine the effects of PE and NE on the epineurial vessels supplying the sciatic nerve.

Methods

Adult male Sprague‐Dawley rats (323.1 ± 5.2 g) were anaesthetized using urethane, α‐chloralose, then were tracheotomized, catheterized, and had the sciatic nerve exposed. Alexa Fluor 488 Isolectin GS‐IB4 conjugate [178.6 μm/ml] was used to label the endothelial cells of the vessel wall allowing for visualization of the epineurial vessels using intravital video microscopy. PE (10−5 M) or NE (10−5 M) was topically applied to the sciatic region for 4 minutes, separately. Images were obtained at baseline, immediately prior to drug application, and every minute for 4 minutes during application. Data are expressed at the time point that maximum change in diameter occurred. Data are expressed as mean ± standard error.

Results

Application of PE (n=11) resulted in a vasoconstriction (baseline: 22.0 ± 2.1 μm, PE: 19.3 ± 2.0 μm; P<0.05). Similarly, topical application of NE (n=8) induced a vasoconstriction (baseline: 31.2 ± 4.5 μm, NE: 26.5 ± 4.6 μm; P<0.05). Mean arteriolar pressure was unaffected during either PE (baseline: 99.1 ± 5.6 mmHg, PE: 97.4 ± 5.4 mmHg) or NE (baseline: 104.0 ± 4.5 mmHg, NE: 107.3 ± 7.7 mmHg) application.

Conclusion

Both PE and NE caused vasoconstriction at the sciatic nerve, which is consistent with previous studies in other tissues observing vasoconstriction through activation of α‐1 adrenergic receptors. As PE and NE are both α‐1 receptor agonists, these findings suggest that α‐1 receptors may be involved in blood flow regulation at the peripheral nerves.

Support or Funding Information

This study was supported by the SEED Research Grant from The University of Western Ontario.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of aging on Rho‐kinase activity and vascular smooth muscle contractility in skeletal muscle resistance arteries
Authors: Shin Song Yi , Padgham Samuel , Trache Andreea , Woodman Christopher
Published: June 2021

Introduction

Vasoconstrictor responses decline with age in resistance arteries. The mechanism for the decrement in constrictor function is not fully understood but has been proposed to involve signaling through the RhoA pathway.

Objective

We tested whether aging affects Rho kinase (ROCK) activity in aged resistance arteries.

Methods

Soleus feed arteries (SFA) were isolated from young (4 mo) and old (24 mo) male Fischer 344 rats. Smooth muscle cells isolated from one group of SFA were lysed and ELISA assay was used for assessment of both ROCK isoforms, total ROCK 1 and 2, and their activity, phospho ROCK 1 and 2. A separate group of young and old SFA was cannulated with glass micropipettes for assessment of vasoconstrictor function. To isolate the role of smooth muscle in constrictor responses, endothelial cells were removed from cannulated arteries (denuded) by passing 5 ml of air through the lumen of the artery. Vasoconstrictor responses were assessed using increasing whole log doses of norepinephrine (NE; 10−9–10−4 M), angiotensin II (Ang II; 10−11–10−7 M), and phenylephrine (PE; 10−9–10−4 M).

Results

Total ROCK 1 and ROCK 2 was similar in cells isolated from young and old SFA, whereas phospho‐ROCK 1 and phospho‐ROCK 2 were higher in cells isolated from old SFA relative to young arteries. Constrictor responses of denuded SFA to PE and Ang II were significantly impaired in old SFA relative to young SFA whereas constrictor responses to NE were preserved. In addition, presence of a ROCK inhibitor, Y 27632, reduced further the constrictor response to agonists.

Conclusion

The results of this study indicate that smooth muscle contractile function declines with age in SFA. Contractility regulation involves the ROCK pathway, but upregulation of ROCK activity in old arteries is not able to rescue the reduced contractility state in aging.

Support or Funding Information

AHA grant 0765043Y, AHA grant 4150031, and Sydney and J.L. Huffines Institute of Sports Medicine and Human Performance.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Thrombin Cleavage of Endothelial Glycocalyx Syndecan‐4 Ectodomain in Human Lung Vasculature
Authors: Jannaway Melanie , Yang Xiaoyuan (Sherry) , Meegan Jamie E. , Ospina Santiago Rodriguez , Coleman Danielle C. , Yuan Sarah Y.
Published: June 2021

The endothelial glycocalyx (eGCX) is a protective barrier on the luminal surface of endothelial cells. The syndecan family of proteoglycans constitute integral components of the eGCX and can act as receptors for many cell signaling pathways. Under inflammatory conditions, the eGCX can be cleaved and shed by proteases such as matrix metalloproteinases and thrombin. We analyzed the ability of thrombin to cleave syndecan core proteins from the eGCX of microvessels in human lung samples using transmission electron microscopy (TEM) and immunogold staining for syndecan‐4 ectodomain (SDC4ecto). The concentration of SDC4ecto in supernatant from human lung slices treated with thrombin was assessed via enzyme‐linked immunosorbent assay and western blotting. The effect of thrombin‐cleaved recombinant SDC4ecto fragments on endothelial barrier resistance was investigated using electric cell‐substrate impedance sensing assay. TEM immunogold staining of human lung demonstrated decreased expression of SDC4ecto within the eGCX following thrombin treatment. Similarly, the concentration of SDC4ecto in human lung slice supernatant was reduced following thrombin treatment, consistent with western blot analysis showing thrombin‐induced cleavage of both recombinant and endogenous full‐length SDC4ecto. In human umbilical vein endothelial cell monolayers, recombinant SDC4ecto treated with thrombin generated a significant decrease in barrier resistance, and this effect was sensitive to a pharmacological inhibitor of Rho kinase, suggesting the involvement of the RhoA pathway in SDC4ecto signaling. In conclusion, these findings suggest that thrombin causes endothelial barrier injury by cleaving SDC4ecto from the eGCX in human lung vessels. Furthermore, an autocrine mechanism may exist whereby thrombin‐cleaved SDC4ecto can act as a signaling molecule to promote endothelial hyperpermeability.

Support or Funding Information

NIH Grant HL070752 & GM097270

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evidence for modulation of heat induced changes in erythrocytes by cytochalasin D: potential role for oxygen nanobubbles in aggregation
Authors: Weber‐Fishkin Samantha D. , Blasco Joey F. , Gallegos Eva M. , Frame Mary D.
Published: June 2021

Thermal burn injury is associated with an increase in erythrocyte (RBC) aggregation (rouleaux) prior to fibrin clot formation; unresolved rouleaux occlude vessels and contribute to thermal burn injury progression (Clark, 2013). RBC aggregation is reversible, yet the mechanism is unknown. Current models do not include all biophysical phenomena, including the role of the cytoskeleton, and sudden oxygen release from hemoglobin. We previously proposed that thermally induced membrane deformation creates nucleation sites for excess oxygen to coalesce, i.e., form nanobubbles, which may contribute to surface tension induced aggregation. To begin to address the role of the cytoskeleton in membrane deformation through changes in the band 3‐ankyrin/adducin bridge and spectrin‐actin interactions, we have disrupted actin polymerization with cytochalasin D (cytoD). We hypothesized that disrupting the cytoskeleton would alter the apparent presence of oxygen nanobubbles on singlet RBCs. Our experimental model examines both size and greyscale appearance of RBCs with sudden changes in temperature. Temperatures of 41–49 C cause shifts in brighter greyscale and larger apparent RBC size, consistent with light diffraction through concentrated gas bubbles that dissipates with time or return to 37 C (Blasco, 2017). Washed murine RBCs (obtained through a tissue sharing program) were suspended in saline (PBS) containing 1% EtOH, 0.5 uM CytoD (in 0.1% EtOH) or control, and incubated at 37°C prior to use. RBCs settled to a monolayer within an enclosed microchannel at 37°C (heated stage). The stage temperature rapidly heated from 37°C to test temperature (37, 39, 41, 43, 45, 47, or 49°C) and we imaged at 2 Hz for 70 seconds (60X, SWI). Singlet RBCs were followed over time, measuring greyscale and apparent size of the cell (outer rim) and core (central biconcave) (ImageJ). The initial changes were seen during the temperature shift and included increased core greyscale and apparent size of 20–40% between 41–47°C, with corresponding changes in cell size. Over the next several seconds there was temperature dependent fluctuations in size and greyscale of up to 50%. Cells in EtOH were similar overall to controls. CytoD prevented changes in greyscale and in size, and suppressed further fluctuations until higher temperatures where those treated cells had formed echinocytes. The data are consistent with gas bubbles forming at the RBC membrane, causing diffraction of the light, in a temperature dependent fashion. As destabilization of the cytoskeleton prevented these changes, we conclude that the cytoskeleton contributes to membrane shape change necessary for oxygen nanobubble formation with heat.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.