bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a novel interaction between α‐synuclein and VMAT2
Authors: Dean E. Danielle , Shepherd Kennie Raviie , Li Yingjie , Torres Gonzalo E. , Miller Gary W.
Published: June 2021

Several familial forms of Parkinson disease are linked to mutations in α‐synuclein (α‐syn), a major component of Lewy bodies. One proposed role for α‐syn in dopaminergic neurons is the maintenance of dopamine (DA) homeostasis. We have identified an interaction with α‐syn and the vesicular monoamine transporter 2 (VMAT2). VMAT2 sequesters cytosolic DA into vesicles for release at synaptic terminals. Here, we show an interaction between α‐syn and the C‐terminus of VMAT2 by yeast two‐hybrid assay. This interaction is confirmed by coimmunoprecipitation of α‐syn with VMAT2 in SHSY5Y cells. Immunofluorescence confocal microscopy shows both perinuclear and punctate neuritic‐like colocalization of VMAT2 and α‐syn in heterologously coexpressing SHSY5Y cells and natively‐expressing primary mouse nigral neurons. This staining is consistent with vesicle colocalization and a perinuclear compartment such as golgi or endoplasmic reticulum. Finally, coexpression of α‐syn increases tetrabenazine sensitive‐VMAT2 sequestration of 3H‐DA in SHSY5Y cell lysates. This increase in transporter activity is reflective of a 2‐fold increase in Vmax. These data suggest that one function of α‐syn may be to maintain proper storage of DA via regulation of VMAT2. Since the storage of DA also protects DA from oxidation, α‐syn may maintain high VMAT2 activity to protect dopaminergic neurons from cell death. Supported by U54ES‐012068.
Dopamine D1 receptor activation blocks phencyclidine ‐ induced neurotoxicity by enhancing synaptic strength
Authors: Johnson Kenneth M. , Lei Gang
Published: June 2021

PCP‐induced neurotoxicity has been used to model both cellular and behavioral aspects of schizophrenia. This study investigates the mechanism underlying the protective effects of dihydrexidine (DHX), a D1R agonist in cultured cortical neurons. DHX (10 uM) significantly blocks the toxic effects of 1 uM PCP as measured by apoptosis and MTT assays. DHX protection against PCP is PKA dependent, as selective inhibitors of PKA block neuroprotection. However, inhibition of either PKC with bisindolyl‐maleimide I or CaMKII with KN93 had no effect. DHX also significantly increased NR1 phosphorylation at serine 897, which is known to increase NMDAR function. Treatment of neurons with DHX increased the density of NR1 on the cell surface, an effect prevented by lavendustin A, a tyrosine kinase inhibitor or PP2, a SRC kinase family inhibitor, but not by PKI14–22, implying that NMDAR trafficking initiated by D1 receptor activation may be regulated by the tyrosine kinase pathway. Both LvA and PP2 partially blocked the protection by DHX. Moreover, NR2B subunits associated with PSD95 were robustly increased by DHX, suggesting that activation of D1 receptors increases NMDAR synaptic insertion. Thus, we suggest that dopamine D1 activation enhances synaptic strength via increasing NMDAR phosphorylation and trafficking, thereby providing neuroprotection against toxicity caused by blocking NMDAR activity. Supported by DA‐02073.
UCP4 is a key regulator of mitochondrial antioxidant defense in rat PC12 cells
Authors: Kamga Christelle K. , Zhang Shelley X.L. , Fentress Emily S , Wang Yang
Published: June 2021

Reactive oxygen species (ROS)‐induced cellular injury contributes to the pathogenesis of neuronal disorders. Uncoupling proteins (UCPs) facilitate electron flows on the mitochondrial membrane and are expected to decrease ROS production. We previously showed that silencing of UCP4, a central nervous system‐specific UCP, led to apoptosis in PC12 cells. The purpose of the current study was to define the effects of UCP4 on ROS homeostasis and on mitochondrial antioxidants by contrasting the effects of UCP4‐silencing and overexpression. ROS levels were significantly increased in UCP4‐silencing (S2) cells (251.4±1.4% of. controls) but decreased in UCP4‐overexpressing (hUCP4) cells (73.3±1.4% of. controls). MnSOD was upregulated in S2 cells but downregulated in hUCP4 cells. Peroxiredoxin III was significantly downregulated in S2 cells but maintained in hUCP4 cells. Furthermore, mitochondrial levels of GSH were significantly decreased in S2 cells (51.9±8.4% of controls), which was associated with reduced expression of dicarboxylate carrier, a mitochondrial GSH transporter, but increased in hUCP4 cells (155.6±23.1% of. controls). These findings suggest that UCP4 may regulate ROS levels by modifying mitochondrial antioxidants, especially those that are important in reducing H2O2 and preventing the formation of hydroxyl radicals. UCP4 may thus be a therapeutic target for ROS‐induced neuronal disorders.
Brevetoxin sensitizes immature cerebrocortical neurons to NMDA receptor signaling through activation of voltage‐gated sodium channels
Authors: George Joju , Cao Zhengyu , Murray Thomas F
Published: June 2021

Brevetoxins (PbTx) are potent polyether neurotoxins that activate voltage‐gated sodium channels. Inasmuch as [Na+]i has been shown to act as a positive regulator of NMDA receptor currents, we used immature cerebrocortical cultures to assess the influence PbTx‐2 on NMDA receptor mediated calcium influx, excitotoxicity and neurite outgrowth. We first confirmed that PbTx‐2 elevates [Na+]i in cerebrocortical neurons. PbTx‐2 produced a concentration‐dependent elevation of [Na+]i in DIV 2, 4, 6 and 9 cerebrocortical cultures. PbTx‐2 (30 nM) leftward shifted the NMDA‐induced Ca2+ influx concentration‐response curves in cortical cultures. NMDA‐induced excitotoxicity in DIV 4, 6, and 9, but not DIV 2, cerebrocortical cultures was also enhanced by PbTx‐2. The most striking PbTx‐2 potentiation of NMDA‐induced [Ca2+]i elevation occurred in DIV 2 cultures. We used the specific Src kinase inhibitor PP2 to demonstrate that PbTx‐2 potentiation of NMDA‐induced elevation of [Ca2+]i was dependent on Src. Pharmacological studies showed that the PbTx‐2 enhanced neurite outgrowth involves elevation of [Na+]i, enhancement of NMDA receptor signaling and engagement of the CaMKK pathway.
Analysis of secondary structures of odorant receptors
Authors: Nguy May , Qiu Rongde , Feng Ying‐Hong
Published: June 2021

The structure‐function relationship of rhodopsin family GPCRs is well studied whereas little is known about the family of G protein‐coupled odorant receptors (ORs). Here the amino acid sequences and the functional domains of 385 human ORs were analyzed using AlignX, transmembrane (TM) helix prediction, and helix wheel projection. Most importantly, the critical residues, motifs and domains of ORs were examined by comparison with the corresponding ones of the rhodopsin family, especially the bioamine subfamily GPCRs that bind ligands with small size. Our result shows that as many as 59 residues in the TM domains and 33 residues in the non‐TM domains are conserved in >90% of ORs, an increase of 36.1% over 36 residues in the TM domains of the rhodopsin family GPCRs. Interestingly, the distributions of the highly conserved residues are largely distinct between the two family GPCRs, especially, in TM2, TM3, TM4, TM6 and TM7. The TM3 of ORs bearing highly conserved DRY motif for activation of Gα proteins was found to be one residue longer than that of the rhodopsin family GPCRs. These results suggest that ORs may have unique, but rather conserved global conformation and utilize structural mechanisms distinct from GPCRs of the rhodopsin family for ligand binding and activation.

Supported by ASPET SURF award to MN and NIH grant (HL065492) to YHF
MAPPING THE LIGAND INTERACTION OF THE AT1 –RECEPTOR WITH PHOTOPROBES IN ALL POSITIONS OF ANGIOTENSIN II
Authors: Fillion Dany , Beaulieu Marie‐Eve , Lavigne Pierre , Guillemette Gaetan , Leduc Richard , Escher Emanuel
Published: June 2021

We have combined a photoaffinity labelling approach to study the molecular structure of the liganded human angiotensin II (AngII) type 1 receptor (hAT1). Systematic incorporation of the photoprobes p‐benzoyl‐L‐phenylalanine (Bpa) or p–[3–(trifluoromethyl)–3H–diazirin–3–yl]–L–phenylalanine (Tdf) into each of the eight positions of the AngII sequence was carried out. Since the constitutively active mutant [N111G]‐hAT1 (hAT1‐CAM) had a more permissive structure‐activity relationship, photoaffinity labelling studies were carried out on this CAM‐receptor. The AngII‐photoprobe analogues substituted in position 1, 2, 3 and 5 displayed AngII‐like properties on hAT1‐CAM. Analogues substituted in position 7 and 8 were shown to be low nM affinity neutral antagonists whereas analogues substituted in positions 4 and 6 had too low affinities. Receptor labelling experiments evidence localisation of the peptide N‐terminus in the extracellular space of the receptor through contact with most extracellular elements of hAT1 A single contact with ECL2 was found at position 3 of AngII. The middle sequence elements are still ill‐defined but the AngII C‐terminal is in close contact with the trans‐membrane bundle formed by TMD3, 5, 6 and 7, in accord with previous results (Clement, JBC. 2005; 280:27121). This study again confirms the extended ligand structure in the hAT1 receptor.
Ligand‐directed signaling of the serotonin 2A receptor depends on βarrestin‐2
Authors: Bohn Laura M , Raehal Kirsten M. , Schmid Cullen L.
Published: June 2021

The serotonin 2A receptor (5‐HT2AR) mediates the effects of hallucinogenic drugs such as DOI and LSD, as well as the neurotransmitter serotonin. Using mouse embryonic fibroblasts (MEFs) that lack βarrestins and mice that lack βarr2 (βarr2), we show that there is difference in signaling and trafficking of the 5‐HT2AR that diverges at point of βarrestin interactions. In MEFs, DOI induces internalization and activates ERK1/2 in a βarrestin‐independent manner, while serotonin requires βarrestins to internalize the receptor and to mediate most of the ERK1/2 activation. In WT frontal cortical neurons, the 5‐HT2AR is intracellularly distributed; however in βarr2‐KO neurons, the receptor is expressed on the cell surface. WT mice display a head twitch response when treated with hallucinogenic serotonergic agonists and the same response can be observed following high doses of the serotonin precursor, 5‐hydroxytryptophan (5‐HTP). βArr2‐KO mice display head twitches in response to DOI but not to 5‐HTP. Both 5‐HTP and DOI treatments induce ERK1/2 activation in WT frontal cortex; however, only DOI activates ERK in βarr2‐KO mice. Our findings demonstrate that 5‐HT2AR activation by two distinct agonists diverges at the point of βarrestin interactions; in the absence of βarr2, differences between the behavioral responses to these drugs are revealed. NIDA funding: K01 DA14460 (LMB); F31 DA219532 (KMR).
Evolutionarily conserved trafficking signal within the alpha2c adrenergic receptor restricts plasma membrane expression
Authors: Angelotti Timothy , Daunt David , Kobilka Brian , Hurt Carl
Published: June 2021

Plasma membrane (PM) expression of polytopic cargo proteins such as, G‐protein coupled receptors (GPCRs) is required for their activation by extracellular ligands. For some GPCRs such as, the alpha2c‐adrenergic receptor (α2c‐AR), their heterologous expression results in limited PM expression and intracellular (IC) retention in the endoplasmic reticulum (ER). To date, the structural determinants within the α2c‐AR that dictate its trafficking have yet to be elucidated. Utilizing a chimeric α2a‐/α2c‐AR strategy, we were able to localize the region responsible for α2c‐AR peculiar trafficking to an evolutionary conserved hydrophobic sequence within the extracellular amino terminus. Deletion of this trafficking signal in the α2c‐AR resulted in improved PM expression and a reduction in IC retention in HEK 293 and Rat1 fibroblast cell lines. Conversely, transplantation of the α2c‐AR trafficking signal into the α2a‐AR resulted in restricted plasma membrane expression and IC retention. The presence of an evolutionary conserved trafficking signal within the α2c‐AR suggests a yet to be identified retention protein restricting its plasma membrane expression.
Epidermal growth factor receptor transactivation and decreased binding in response to lysophosphatidic acid treatment of airway epithelial cells
Authors: Kassel Karen M. , Toews Myron L.
Published: June 2021

Exposure of BEAS‐2B airway epithelial cells to lysophosphatidic acid (LPA) induces biphasic decreases in epidermal growth factor receptor (EGFR) binding measured with intact cells on ice. A rapid decrease occurs within 15 min and is blocked by the MEK inhibitor U0126. The decrease is sustained to 18 hr, and the protein kinase C (PKC) inhibitor BisI prevents this sustained decrease. We hypothesized that EGFR transactivation by LPA mediates the decreases in EGFR binding. LPA rapidly induced a 2‐fold increase in EGFR tyrosine phosphorylation. The EGFR tyrosine kinase inhibitor AG1478 and the MMP inhibitor GM6001 completely inhibited EGFR phosphorylation but only partially inhibited ERK1/2 phosphorylation and the rapid decrease in EGFR binding, and they had no effect on the sustained decrease. The HB‐EGF inhibitor CRM197 had no effect on the LPA‐induced EGFR phosphorylation or the decrease in binding. U0126 partially inhibited LPA‐induced EGFR phosphorylation but BisI had no effect. These results indicate that the rapid decrease in EGFR binding induced by LPA is partially due to MMP‐mediated EGFR transactivation but that mechanisms other than transactivation also contribute. PKC does not contribute to EGFR phosphorylation, and PKC‐mediated mechanisms other than EGFR transactivation mediate the sustained phase of decreased binding. Supported by funds from GlaxoSmithKline and Nebraska DHHS.
The tyrosine kinase inhibitor genistein inhibits mu‐opioid mediated adenylyl cyclase sensitization in C6 cells expressing a mu‐opioid receptor
Authors: Divin Mary F. , Traynor John R.
Published: June 2021

Acute mu‐opioid treatment of cells leads to inhibition of adenylyl cyclase through Gi/o proteins. Opioid withdrawal is characterized by adenylyl cyclase sensitization and cAMP overshoot, an increase in forskolin‐stimulated cAMP accumulation upon removal of chronic agonist. Kinase enzymes have been implicated in the development of sensitization. Kinase inhibitors with specificity for different enzymes were evaluated for their ability to inhibit sensitization following chronic opioid exposure in C6 rat glioma cells expressing the mu‐receptor (C6mu). Notably, treatment with the tyrosine kinase inhibitor genistein (100 μM) caused a 5‐fold rightward shift in the ability of DAMGO to induce adenylyl cyclase sensitization following 1 h agonist treatment. This effect was surmountable at high concentrations of DAMGO suggesting alternative pathway(s); however, the ability of genistein to inhibit sensitization was not significantly enhanced by combinations of kinase inhibitors. Genistein did not significantly alter basal cAMP levels, acute DAMGO‐mediated inhibition of forskolin‐stimulated cAMP production, or the development of tolerance. Results show that the development of adenylyl cyclase sensitization in C6mu cells is mediated in part by phosphorylation by tyrosine kinase enzymes but alternative unidentified pathways are available in the presence of genistein. Supported by DA04087 and DA19276.
A Luminescent cAMP Assay for determining cellular and tissue cAMP and monitoring the Modulation of Gs and Gi Protein Coupled Receptors
Authors: Goueli Said A , Vidugiriene Jolanta , Zdanovskaia Marina , Hsiao Kevin
Published: June 2021

G Protein Coupled Receptors represent a large validated target for Drug discovery research. The Gs is coupled to activation of adenylate cyclase and Gi is coupled to inhibition of adenylate cyclase while Gq is coupled to activation of phospholipase ƒÒ. Cellular concentrations of second messengers such as cAMP and cGMP have been known to play major roles in smooth muscle relaxation, contractile activity of cardiovascular system, and central nervous system related pathologies. We report here on a new assay for determining cAMP concentrations in cellular and tissue extract and for monitoring modulation of GPCR that are linked to activation or inhibition of adenylate cyclase (Gs or Gi). The assay is homogenous, fast, HTS formatted with high Z¡¦, and adaptable to multi plate formats. We have successfully generated EC50 values for agonists and IC50 values for antagonists of Gs coupled receptors that are similar to those reported in the literature. The assay is easy to use, and can be carried out in less than 60 minutes and does not require antibodies or expensive instrumentation for signal detection and the signal output is relatively stable for few hours.
Differential expression and signaling of adenosine receptors in mouse bone marrow neutrophils
Authors: van der Hoeven Dharini , Auchampach John A.
Published: June 2021

Adenosine suppresses neutrophil activity at sites of inflammation. Most inhibitory effects of adenosine on neutrophil function have been attributed to the A2AAR, although recently we discovered that the A3AR also plays a role. Goals of this study were to determine if exposure to inflammatory stimuli regulates A3AR expression in mouse bone marrow neutrophils (BMNs), and to explore the signaling mechanisms by which the A3AR inhibits neutrophil function. Real time RT‐PCR and radioligand binding analyses revealed that expression of the A2AAR but not the A3AR was increased ~10‐fold in BMNs obtained from LPS‐treated mice compared to controls. Despite selective induction of the A2AAR in BMNs from LPS‐treated mice, potencies of both the A2AAR‐selective agonist CGS 21680 and the A3AR‐selective agonist CP‐532,903 to inhibit fMLP‐induced superoxide production were increased compared to naïve BMNs. Mechanistic studies using naïve BMNs determined that activation of the A3AR inhibits BMN functions neither by increasing intracellular cAMP, nor by cross‐desensitizing chemoattractant or ATP receptors. We conclude that, unlike the A2AAR, A3AR expression is not induced during inflammation. In addition, we conclude that A3AR activation inhibits BMN function by a signaling mechanism that is distinctly different from the A2AAR.

Supported by NIH Grants RO1 HL 077707, RO1 HL 60051 and by AHA Predoctoral Fellowship 0615533Z
Real time measurement of hydrogen peroxide (H2O2)and nitric oxide (NO) release in femoral vein ischemia and reperfusion (I/R): The effects of tetrahydrobiopterin (BH4)/dihydrobiopterin (BH2) and the effects of Protein Kinase C (PKC) epsilon activation (ε+)/inhibition (ε−)
Authors: Zambrano Christopher John , Krass Samuel , Chen Qian , Young Lindon Howard
Published: June 2021

Endothelial nitric oxide synthase (eNOS) is a possible source of oxidative stress (i.e., superoxide (SO) and H2O2) during reperfusion in which BH4 is oxidized to BH2. Moreover, PKC ε positively regulates NO and SO release from eNOS. The effects of BH4/BH2 or PKC ε +/ε− on NO and H2O2 release have not been directly estimated by real‐time measurement in blood vessels during I/R in vivo. NO and H2O2 were directly measured by inserting either NO or H2O2 microsensors (100 μm diameter) into both femoral veins in the anesthetized rat. One femoral vein was subjected to I/R, which was induced by clamping the femoral artery and vein for 20 min ischemia followed by removing the clamp for 45 min reperfusion. The other non‐ischemic femoral vein served as a sham control in the same animal. H2O2 release increased throughout the first 20 min of reperfusion compared to the sham control. PKC ε− (0.4 mg/kg) or BH4 (6.5 mg/kg) significantly reduced H2O2 release while PKC ε+ (0.9mg/kg) or BH2 (2mg/kg) showed a trend to increase H2O2 release throughout reperfusion. BH4 significantly increased NO release while BH2 showed a trend to decrease NO release. The preliminary data thus far support our hypothesis that BH4 or PKC ε− decrease H2O2 release during reperfusion whereas the opposite effect is observed with BH2 or PKC ε+.

This study was supported by NHLBI Grant 1R15HL‐76235‐01 and Center For Chronic Disorders of Aging at PCOM
Cardioprotective effects of epicatechin in ischemia‐reperfusion (IR) injury
Authors: Yamazaki Katrina Go , Romero‐Perez Diego , Rivas Maria , Cruz Michelle , Cortez‐Gomez Brenda , Villarreal Francisco
Published: June 2021

Epidemiological studies have shown a correlation between flavonoid‐rich diets and improved cardiovascular prognosis. Cocoa contains large amounts of flavonoids, in particular flavanols (mainly catechins and epicatechins). Flavonoids possess various properties that may confer protective effects to tissues in the setting of injury. We examined the abiliy of epicatechin to reduce short term IR myocardial injury. For this purpose, epicatechin (1 mg/kg/day) pre‐treatment (Tx) was administered via oral gavage to 250 g male rats for either 2 or 10 days. Control groups received water. IR was induced via a 45 min coronary occlusion. Reperfusion was allowed to occur for 48 h while Tx continued. We measured infarct (MI) size (%), hemodynamics, myeloperoxidase (MPO) activiy, tissue oxidative stress, matrix metalloproteinase‐9 (MMP‐9) and NFκB activities. With 2 days of Tx, no reductions in MI size occurred. After 10 days, a significant (p <0.01) ∼50% reduction in MI size was observed. Effects were independent of changes in blood pressure since epicatechin animas demonstrated sustained hemodynamics. MPO activity and MMP‐9 activity was significantly reduced (∼50%) in the infarct region compared to non‐infarct regions. Tissue oxidative stress and NFκB activity demonstrated trends towards reductions (∼30%) in the infarct region. Results indicate that cocoa flavanols confer significant cardioprotection via anti‐inflammatory actions in the setting of IR injury and that these effects are independent of changes in hemodynamics. Supported by HL‐43617 and 67922.
The Effects of Microcirculatory Responses to Hypovolemic Shock Following Resuscitation with Colloid Solutions
Authors: Mielniczuk Mariusz Jozef , Krokowicz Lukasz , Grykien Christopher , Kusza Krzysztof , Siemionow Maria
Published: June 2021

INTRODUCTION: Aim of the study was comparison of volume replacement with: 6% Hetastarch (HET), 5% Albumin (ALB), and 10% Pentastarch (PENT) following induction of hypovolemic shock (HS). METHODS: Rat cremaster model was used to evaluate microcirculatory changes after HS induction. Rats were randomized into groups: 1) (n=10) had parameters recorded for 4 hrs; 2) (n=10) blood withdrawn, no replacement; 3) (n=6) replaced with HET; 4) (n=6) replaced with ALB; 5) (n=6) replaced with PENT. During 4 hrs measurements of RBC velocity, vessel diameter, functional capillary perfusion (FCP) and leukocyte‐endothelial interactions, endothelial micropermeability (EMP) were recorded. RESULTS: At 4 hrs after resuscitation with HET, ALB, and PENT respectively 70%, 84%, and 85% return of FCP was recorded compared to 33 % in Group II. ALB and PENT resuscitation reduced level of sticking and transmigrated PMN's. Groups: III, IV, V presented higher tissue oxygenation than Group II (HET – 10.1 ± 0.25, ALB – 12.5 ± 0.28 and PENT 11.2 ± 0.25 vs. Group II 8.1 ± 0.15 mmHg). At 5 hrs after HS, EMP index was: Group I – 0.42; Group II – 0.86; Group III – 0.73; Group IV – 0.82; Group V – 0.7.

CONCLUSIONS: After HS resuscitation with Colloid Solutions improved microcirculatory hemodynamics. ALB, and PENT administration decreased level of sticking and transmigrating PMN's. ALB and PENT have beneficial effect on vascular permeability.
ANTECEDENT HYDROGEN SULFIDE ELICITS AN ANTI‐INFLAMMATORY PHENOTYPE IN POSTISCHEMIC MURINE SMALL INTESTINE: ROLE OF BKCa CHANNEL
Authors: Yusof Mozow , Yang Yan , Davis Michael J. , Korthuis Ronald J.
Published: June 2021

We recently demonstrated that preconditioning with an exogenous hydrogen sulfide (H2S) donor (NaHS‐PC) 24 h prior to ischemia and reperfusion (I/R) causes postcapillary venules to shift to an anti‐inflammatory phenotype in C57Bl/6 wild‐type mice such that these vessels fail to support leukocyte rolling (LR) and leukocyte adhesion (LA) during reperfusion. The objective of the present study was to determine whether the calcium‐sensitive large conductance K channel (BKCa channel) is also involved as an initiator of the anti‐inflammatory phenotype elicited by H2S. The postischemic anti‐inflammatory effects of NaHS‐PC were abolished by BKCa channel inhibitor treatment (paxilline) when administered coincident with NaHS, 24 hrs prior to I/R. Moreover, preconditioning with the BKCa channel activator, NS‐1619, was as effective as NaHS‐PC in preventing I/R‐induced leukocyte rolling and adhesion. Our data is consistent with the concept that H2S induces the development of an anti‐adhesive state in I/R by a BKCa channel‐dependent mechanism. (Patch‐clamp studies are presented separately). Supported by AA14945, DK43785 and HL71796
Effects of CO liberated from carbon monoxide‐releasing molecule (CORM)‐2 on modulation of inflammatory response in ischemia/reperfusion (I/R)‐challenged small intestine
Authors: Katada Kazuhiro , Bihari Aurelia , Stephen Jancy , Badhwar Amit , Potter Richard F. , Cepinskas Gediminas
Published: June 2021

CORM‐released CO has been shown to modulate inflammatory response. In this study, we assessed the effects and potential mechanisms of CORM‐2‐released CO on modulation of inflammatory response in mice small intestine following I/R. To this end mice (C57Bl/6) small intestine were challenged with ischemia by occluding SMA for 45 min. CORM‐2 (8 mg/kg; i.v.) was administered: 1) immediately before SMA occlusion or 2) immediately upon reperfusion. Sham operated mice were injected with vehicle (DMSO).

Inflammatory response in the small intestine was assessed by measuring tissue levels of TNF‐[alpha] (ELISA), iNOS, ICAM‐1, and E‐selectin (Western blot) protein, accumulation of PMN (MPO assay) and the number of rolling/adherent leukocytes in the microcirculation of jejunum mucosa (Intravital microscopy) 4 hr following reperfusion.

The obtained results indicate that tissue levels of TNF‐[alpha], iNOS, ICAM‐1 and E‐selectin protein expression and subsequent PMN accumulation were elevated in I/R‐challenged jejunum. The above changes were significantly attenuated in CORM‐2‐treated mice. The most potent inhibition was observed in mice, which were injected with CORM‐2 immediately before induction of ischemia.

Taken together these findings indicate that CORM‐2‐released CO confers anti‐inflammatory effects in I/R‐challenged small intestine (HSFO‐NA5580 and MOP‐68848).
Activation of Endothelial Cell BK Channels by the Gasotransmitter Hydrogen Sulfide
Authors: Davis Michael John , Yang Yan , Yusof Mozow , Korthuis Ronald J
Published: June 2021

Hydrogen sulfide can function as a preconditioning stimulus by preventing leukocyte‐endothelial cell (EC) interactions following ischemia‐reperfusion injury in the intestinal wall. The actions of H2S are prevented by paxilline, an inhibitor of large‐conductance, Ca2+‐activated K+ (BK) channels. Using whole‐cell and single‐channel patch clamp methods, we tested the hypothesis that H2S directly activates BK channels in ECs. Human microvascular endothelial cells (HMECs) were plated on glass coverslips and voltage‐clamped under conditions favorable for recording K+ current. In whole‐cell mode, depolarizing steps from a holding potential of −70 mV resulted in robust, Ca2+‐sensitive K+ currents that were inhibited by IBTX (100 nM). Likewise, excised, inside‐out membrane patches exhibited outward, Ca2+‐ and IBTX‐sensitive K+ currents at positive potentials in the presence of IK/SK channel blockers. Bath application of NaHS (100 μM) increased whole‐cell K+ current by 1.5–2.0 fold and significantly increased the N•Po of single channel K+ currents; these NaHS actions were prevented by IBTX. These results are consistent with a role for EC BK channels in the preconditioning effect of H2S.
A Cannabinoid CB2 Receptor Agonist Attenuated Adhesion Molecules Expression In a Mouse Ischemic/Reperfusion Injury Model
Authors: Zhang Ming , Martin Billy R , Adler Martin W , Razdan Raj K , Ganea Doina , Tuma Ronald
Published: June 2021

It has been shown that leukocyte extravasation, which is mediated by adhesion molecules on both endothelial cells and leukocytes, plays an essential role in cerebral ischemic/reperfusion injury (I/R injury). The CB2 receptor is primarily expressed by cells of immune system and its activation has been shown to attenuate cerebral I/R injury and decrease leukocyte/endothelial interactions in our lab. The purpose of this study was to determine if the protection through CB2 receptor in cerebral I/R injury is associated with changes in adhesion molecules expression. Transient middle cerebral artery occlusion was performed in male C56BL/6 mice using an intraluminal filament method. Mice were treated with a CB2 agonist (0–1966) at 1mg/kg i.v. or equal volume of vehicle at 1 hour before, 1 hour after or 3 hours after ischemia. Cerebral infarct volume was measured by triphenyltetrazolium chloride staining while adhesion molecules ICAM‐1 and Mac‐1 expressions were tested by immunostaining at 24 hours after ischemia. The results demonstrate that CB2 agonist administration reduced infarct volume at all the treatment points and was associated with decreased ICAM‐1 and Mac‐1 expression in ischemic brain region.
Neutrophil‐induced increase in vascular permeability involves activation of the contact system
Authors: Kenne Ellinor , Soehnlein Oliver , Lindbom Lennart
Published: June 2021

Adhesion of polymorphonuclear leukocytes (PMN) to the endothelial cell (EC) lining leads to increased vascular permeability. Plasma prekallikrein (PK), factor XII (FXII) and H‐kininogen are principal components of the contact system assembling on the EC surface. Activated PK cleaves H‐kininogen, liberating bradykinin that triggers EC contraction and paracellular gap formation. Here, we investigated the role of the contact system in PMN‐evoked plasma extravasation. Thioglycollate‐induced pleurisy in the mouse was used to study PMN‐dependent plasma leakage. FITC‐dextran served as plasma marker. The fluorescence intensity and PMN content in the exudate was markedly increased 4 h after thioglycollate injection, whereas in neutropenic animals, the plasma leakage was abolished. Treatment with antagonists to bradykinin B1 and B2 receptors, or an inhibitor to PK/FXII largely reduced fluid extravasation by 79% and 74% respectively. Intravital microscopy of the hamster cheek pouch microcirculation revealed that treatment with the B1/B2 antagonists reduced plasma leakage without reducing PMN adhesion. These results demonstrate a critical role of contact system activation in PMN‐evoked increase in vascular permeability. Our data also indicate that PMN‐dependent permeability changes and PMN extravasation may be regulated independent of each other.
Characterization of the hepatic endothelial glycocalyx of sialidase deficient mice in response to TNFα
Authors: Kowalewska Paulina Monika , Igdoura Suleiman A , Fox‐Robichaud Alison E
Published: June 2021

Evidence exists that components of the venular glycocalyx are shed and sialic acid is cleaved during inflammation. The objective of this study was to examine the role of sialidase in altering the hepatic glycocalyx. Female C57Bl/6 (WT) or sialidase deficient (B6Sm−/−) mice were injected with 500 ng of TNFα or saline and the hepatic microcirculation examined by intravital confocal microscopy at 4h. Sugars present on the vasculature were detected by IV injection of FITC‐labeled Bandeiraea simplicifolia (BS‐1) (detects α‐N‐acetylgalactosamine and α‐galactose residues) or Texas‐Red‐labeled Lycopersicon esculentum (TL) (detects N‐acetylglucosamine residues). Both lectins labeled the sinusoidal endothelium with no significant difference between the groups in the mean fluorescence intensity of the BS‐1 or TLlabeled glycocalyx. However, the saline treated WT mice had a thicker glycocalyx as compared to the saline treated B6Sm−/− mice (876 ±37 nm and 777 ± 18 nm, respectively) and TNFα stimulation resulted in a decrease of the glycocalyx thickness (772 ± 45 nm and 658 ± 23 nm, respectively). Our data supports the idea that components of the glycocalyx are shed during inflammation resulting in loss of glycocalyx height. Surprisingly, these sugars were expressed primarily on the sinusoids rather than venules and in contrast to other vascular beds, were not removed during TNFα‐induced hepatic inflammation.
Increased fibrinogen content induces release of endothelin‐1 from endothelial cells
Authors: Sen Utpal , Tyagi Neetu , Moshal Karni S. , Dean William L. , Roberts Andrew M. , Tyagi Suresh C. , Lominadze David
Published: June 2021

We previously showed that fibrinogen (Fg) increases microvascular constriction in vivo. In the present study we tested the hypothesis that Fg could cause vasoconstriction by affecting release and formation of endothlin‐1 (ET‐1) from vascular endothelial cells (EC). Fg‐induced formation of ET‐1 was studied using rat cardiac microvascular ECs. Confluent ECs were given one of the following treatments: Fg (2 or 4 mg/ml), Fg (4 mg/ml) with extracellular signal‐regulated kinase (ERK) kinase inhibitors (PD98059, or U0126), Fg (4 mg/ml) with an antibody against intercellular adhesion molecule‐1 (ICAM‐1), or medium alone for 45 min. The amount of formed ET‐1 in the cell growth medium was measured by ELISA. Fg caused a dose‐dependent increase in ET‐1 production from ECs. Fg‐induced ET‐1 production was inhibited by ERK kinase inhibitors and by the antibody to ICAM‐1. These data suggest that Fg‐induced vasoconstriction may be mediated through increased release and formation of ET‐1. This mechanism may help to explain a role of Fg in contributing to increased microvascular constriction during diseases such as hypertension, stroke, and diabetes that are accompanied by an increased blood content of Fg.

Supported by NIH grant# HL080394 to DL.
Impaired myogenic tone in overweight rats exposed to simulated sleep apnea is due to elevated iNOS‐derived nitric oxide
Authors: Sweazea Karen L. , Kanagy Nancy L. , Walker Benjimen R.
Published: June 2021

Exposure of rats to eucapnic intermittent hypoxia (E‐IH) results in vascular abnormalities similar to those seen in humans with sleep apnea. However, sleep apnea is often associated with increased adiposity. Therefore, we examined the potential interaction between these factors. Since increased adiposity may be associated with elevated expression of inducible nitric oxide synthase (iNOS), we hypothesized that overweight rats would not exhibit enhanced myogenic tone following exposure to simulated sleep apnea. Rats were fed either chow or a high fat diet (HFD; 60% kcal from fat) for 6 weeks. During the final 14 days of each diet, animals were exposed to either air or E‐IH. Both Chow E‐IH and HFD Air rats developed increased systolic blood pressure, whereas HFD E‐IH rats were less hypertensive and had elevated plasma nitric oxides. Consistent with our hypothesis, myogenic tone in response to increasing intraluminal pressure was attenuated in isolated mesenteric arterioles from both HFD Air and HFD E‐IH rats compared to Chow Air controls. Inhibition of iNOS with L‐N6‐(1‐Iminoethyl) lysine hydrochloride (L‐NIL, 10 μM) restored myogenic reactivity in these arteries to the level of Chow Air rats. Despite normalization of myogenic reactivity, L‐NIL did not restore the diminished pressure‐induced increase in vascular smooth muscle (VSM) calcium to Chow Air levels. These results suggest that HFD‐induced elevation in iNOS‐derived NO impairs myogenic tone in both HFD Air and HFD E‐IH arteries by diminishing VSM calcium sensitivity.
Using micro‐PIV to assess the role of insulin and free radical scavenger therapies in treating the effects of chronic hyperglycemia on the microvascular glycocalyx of NOD mice
Authors: Savery Michele D , Jiang John , Damiano Edward R
Published: June 2021

The existence of a hydrodynamically relevant endothelial surface layer (ESL), ~ 0.5 μm thick in microvessels of wild‐type mouse, is well established under normal physiological conditions. Since nearly all chronic complications of diabetes are derived from vascular disease arising from hyperglycemia, and since the ESL is situated as the first line of defense against vascular damage, we seek to establish the effects of hyperglycemia on the ESL. We applied microviscometric analysis to data obtained using micro‐particle image velocimetry in cremaster‐muscle venules (20–60 μm diameter) of chronically hyperglycemic NOD mice. In mice without treatment, we observed that the ESL thickness is degraded to ~ 0.15 μm (n=20), compared with ~ 0.53 μm (n=23) in healthy wild‐type mice. This represents a significant degradation of the ESL (p<0.05), comparable to that seen after free‐radical damage due to light‐dye treatment. In mice treated with insulin therapy (0.3 u/d LinBit implant) the ESL thickness is degraded to ~ 0.18 μm (n=5). This result is not significantly different from the untreated group. In mice treated with synthetic catalytic free radical scavengers (EUK‐207, Proteome Systems, Ltd.) in addition to insulin therapy, the ESL thickness is ~ 0.49 μm (n=5). This is significantly thicker than that found in either of the chronically hyperglycemic groups (p<0.05) and not significantly different from the control group.
The enhanced protective effect of postconditioning by 17β‐estradiol after transient focal cerebral ischemia in mice
Authors: Shen Jia , Sun Lina , Lin Shupeng , Bai Xiaoyin , Liao Yuanjin , Xia Qiang
Published: June 2021

The aim of this study was to explore the enhancement of 17β‐estradiol (E2) in the neuroprotective effect of ischemic postconditioning (PostC) following transient focal cerebral ischemia in mice. Adult female mice were ovariectomied two weeks before 30 min transient focal cerebral ischemia induced by right middle cerebral artery occlusion. E2 or vehicle (sesame oil) was administered following the reperfusion immediately. PostC consisted of 3 cycles of 15 s reperfusion/15 s occlusion on right common carotid artery. Brains were obtained 24 h, 2 d, 3 d and 5 d after permanent reperfusion. Nissl staining was used to measure the infarct sizes and neurological scores were evaluated. Levels of TNF‐α, IL‐1β and IL‐6, and expressions of caspase 3 and Bcl‐2 in brain homogenates were analyzed. We found that E2 enhanced the infarct‐limiting effect and improvement of neurological score by PostC. Levels of TNF‐α, IL‐1β and IL‐6 in brain homogenates from E2 treated postconditioned mice were significantly lower than those in vehicle treated group. Expression of caspase 3 and Bcl‐2 in brains of postconditioned mice with E2 treatment showed lower levels than that of vehicle treated group. These results provide first evidence that E2 enhances the neuroprotective effect of ischemic PostC at least via antagonizing inflammation and apoptosis induced by cerebral ischemia and reperfusion.
The geranylgeranyl‐transferase inhibitor GGTI‐286 does not reproduce rosuvastatin‐induced delayed preconditioning against oxygen‐glucose deprivation (OGD) in cultured rat cortical neurons
Authors: Domoki Ferenc , Kis Béla , Gáspár Tamás , Bari Ferenc , Busija David W
Published: June 2021

Rosuvastatin (RST) has been recently shown to induce resistance against OGD via depletion of geranylgeranyl pyrophosphate (GGPP) rather than cholesterol levels in cultured neurons. RST also induces GGPP‐sensitive, reversible mitochondrial depolarization, decreased NADH/NADPH and ATP levels, and these alterations may contribute to the OGD‐resistant phenotype. Our purpose was to determine if pharmacological inhibition of protein geranylgeranylation with GGTI‐286 would reproduce the neuroprotective and metabolic effects of RST preconditioning.

Primary cortical neuronal cultures were obtained from E18 Sprague‐Dawley rat fetuses, and were treated with GGTI‐286 (1–10μM) for 3 days before exposure to 3h OGD. GGTI‐286 did not provide protection against OGD. Cell viability determined 4 days after OGD (untreated vs. 5μM GGTI‐286, mean±SEM, n=8–32) was 34±1% vs. 39±1% of normoxic controls. Interestingly, GGTI‐286 (5μM) reduced mitochondrial membrane potential to 80±1%*, and neuronal NADH/NADPH to 79±3%* of control levels (*p<0.05, n=8–32) similarly to RST, but unlike RST, did not affect neuronal ATP levels. In summary, GGTI‐286 is able to replicate some but not all of the GGPP‐sensitive metabolic effects of RST and does not yield OGD resistance. Reduction in ATP levels appears to be crucial in RST‐induced preconditioning against OGD. Supported by OTKA K68976, K63401 and by NIH HL30260, HL65380, HL77731.
Role of whole body autoregulation in the control of blood pressure and sodium balance: insights from computer simulations with Guyton's model
Authors: Montani Jean‐Pierre , Malpas Simon C. , Van Vliet Bruce N.
Published: June 2021

The concept of whole body blood flow autoregulation (WBFAR) has often been used to explain the increase in blood pressure (BP) in various models of salt‐loading hypertension. Computer simulations offer a unique way to test the role of WBFAR in achieving salt balance and BP control during salt‐loading since it is not possible to block WBFAR experimentally. Using Guyton's large circulatory model, we tested, in the presence or absence of WBFAR, the hemodynamic and fluid volume changes that occur after a 10‐fold increase in salt intake in three different situations: (A) normal neurohormonal modulation; (B) fixed circulating angiotensin II at a normal level; (C) full neurohormonal blockade (fixed angiotensin, aldosterone and ANP, and fixed autonomic output). In the presence of WBFAR, BP after salt‐loading remained stable in situation A, but increased by ~10 mmHg in situation B, and by ~30 mmHg in situation C. Even then, changes in cardiac output (CO) and blood volume (BV) were minimal (less than 4% and 2%, respectively). In the absence of WBFAR, the same simulations led to similar changes in BP. However, a nearly twofold increase in CO and an increase in BV by more than 20% were required to achieve salt‐balance in situation C. From the simulations one may conclude that autoregulation limits the amount of fluid retention required to increase BP in order to achieve salt balance during salt‐loading, but does not affect the final level of BP.
Sympathetic activation in the development of DOCA‐salt hypertension
Authors: Kandlikar Sachin , King Andrew J , Novotny Martin , Fink Gregory
Published: June 2021

Indirect evidence indicates that sympathetic activity (SNA) is increased in DOCA‐salt hypertension. Total body norepinephrine (NE) spillover provides an accurate estimate of whole‐body SNA. The purpose of this study was to assess SNA during the development of DOCA‐salt hypertension. Catheters were implanted in male rats for pressure measurements and blood sampling. Rats drank water containing 1% NaCl and 0.2% KCl. After a 7 day recovery and 3 day control period, a DOCA pellet (50 mg/kg, sc) was implanted in some rats, while others had sham surgery. NE spillover was measured following a 90 minute 3H‐NE infusion on control day 2, and days 7 and 14 after DOCA administration or sham surgery. During the control period, mean arterial pressure (MAP) was similar in sham (106 ± 4.2 mmHg) and DOCA (107 ± 4.5 mmHg) rats. MAP was higher in DOCA rats on day 7 and 14 (113 ± 7.5 and 124 ± 5.8 mmHg) than in sham rats (104 ± 2.0 and 108 ± 15.6 mmHg). However, plasma NE concentration, clearance and spillover were not different at any time in DOCA versus sham rats. To examine possible regional sympathetic activation, celiac ganglionectomy (CGX) was performed to selectively denervate the splanchnic organs. MAP was lower in CGX than in sham‐operated rats during DOCA‐salt treatment. In conclusion, global SNA is not increased during development of DOCA‐salt hypertension, but sympathetic activation may occur specifically in the splanchnic region.
The spleen mediates beneficial effects of 5HT‐1A receptor agonists in hypovolemic shock
Authors: Tiniakov Ruslan L , Scrogin Karie E
Published: June 2021

We showed that the 5HT‐1A agonist, 8‐OH‐DPAT, increases cardiac output during hypovolemic shock by a sympathetic‐dependent increase in mean circulatory filling pressure (MCFP), an index of venous tone. To determine if increased MCFP was partially due to splenic constriction and mobilization of stored blood, we examined effects of splenectomy on 8‐OH‐DPAT‐induced changes in MAP, central venous pressure (CVP), MCFP and blood gases in rats during unresuscitated hemorrhagic shock. Splenectomized and intact rats were hemorrhaged to 50 mmHg and maintained there for 25 min, then treated with either 30 nmol/kg of 8‐OH‐DPAT or 200 uL of saline, iv, and monitored for additional 35 min. 8‐OH‐DPAT rapidly increased MAP in both intact and splenectomized rats (+32 ± 5 and +24 ± 4 mmHg; P<0.01). In intact animals, this effect coincided with persistent increases in MCFP (+1.86 ± 0.33 mmHg; P<0.01) and CVP (+0.75 ± 0.4 mmHg; P<0.01) up to 35 min post treatment. In splenectomized rats, the pressor effect of 8‐OH‐DPAT was transient (~20 min) and was not accompanied by increased MCFP. By 35 min after injection, intact 8‐OH‐DPAT‐treated rats had higher venous blood oxygen saturation (33 ± 2 vs. 23 ± 3, 22 ± 3, 24 ± 2 mmHg; P<0.01), as well as higher total bicarbonates (26 ± 1 vs. 17 ± 3, 16 ± 3, 15 ± 3 mmol/L; P<0.01) and lower lactate (4.5 ± 0.4 vs. 12 ± 1.9, 13.1 ± 2.1, 13.3 ± 2.3; P<0.01), compared to all other groups. Intact 8‐OH‐DPAT‐treated rats had a lower spleen‐to‐body weight ratio than intact animals treated with saline (0.20 ± 0.01 vs. 0.25 ± 0.01; P<0.05). Our data indicate that splenic contraction is involved in the beneficial hemodynamic effect of 8‐OH‐DPAT in hypovolemic shock. Supported by HL 076162 and 072354.
A new implantable transmitter for simultaneous recording of sympathetic nerve activity and blood pressure
Authors: Lim Matthew , McCormick Daniel , Kirton Robert , Budgett David , Kondo Masahiro , Pallas Wayne , Guild Sarah‐Jane , Barrett Carolyn , Malpas Simon
Published: June 2021

The development of implantable telemeters for measurement of arterial blood pressure (BP) and sympathetic nerve activity (SNA) in conscious rabbits and rats is presented. These devices incorporate a high gain amplifier for SNA recording. The SNA signal is digitized with 12‐bit resolution and sampled at 8 kHz. Arterial BP is measured (sampled at 500 Hz) by inserting a fluid filled catheter, that is coupled to a pressure sensor, into the aorta. The pressure signal is corrected for atmospheric pressure variations in the wireless receiver situated up to 5 m away. The total implant weight is 14 gm and suitable for implantation in animals over 200 gm. Digital transmission of the signals permits co‐housing of animals without crosstalk. The transmitter is recharged whilst implanted using novel inductive power transfer technology. This effectively allows continuous lifetime monitoring without need for battery refurbishment. The device was implanted into a group of 5 rats and 5 rabbits and provided good quality recordings of SNA and BP during a range of physiological stimuli (baroreflex, chemoreflex, nasopharyngeal). We propose that this technology provides new avenues for exploring the role of the sympathetic nervous system in the development of cardiovascular diseases.