bluuebunny/crossref_metadata_2025 huggingface dataset:
Serine Protease Generation by Oversulfated Chondroitin Sulfate and Contaminants in Heparin: Clinical Implications
Authors: Hoppensteadt Debra , Champlain Jacqueline , Cunanan Josephine , Khan Hussein , Jeske Walter , Kennedy Richard , Fareed Jawed
Published: June 2021

We hypothesized that Oversulfated Chondroitin Sulfate (OSCS) and the other contaminants present in contaminated heparin may mediate the generation of several serine proteases. To address this hypothesis, seven batches of recalled heparins linked to adverse patient outcomes were supplemented to freshly drawn, normal human plasma anti‐coagulated with hirudin. The generation of markers of an immune reaction, as well as rate of formation of these enzymes, was measured in biochemically defined assay conditions using chromophore‐conjugated substrates for Kallikrein, Plasma Kininogenase, C1 esterase, Thrombin, Xa and Plasmin. Assays performed with substrates for Kallikrein, Plasma Kininogenase, and C1 esterase showed a time‐dependent generation of these proteases that was strongest in the samples containing purified contaminant OSCS. Of the six contaminated heparins, four preparations showed a strong activation of these proteases which were significantly different (p<0.050) than the control non‐contaminated heparins. Our studies suggest that heparin contaminants are capable of activating multiple serine proteases, which potentially contribute to the pathogenesis of observed adverse clinical events. Classical complement pathway activation is not suggested by this data as a significant increase in C1 esterase activity is noted with all heparins, including purified OSCS.
The role of histamine H1, H2 and H3 receptors in the stimulation of NT‐3 synthesis in astrocytes
Authors: Carman‐Krzan Marija , Mele Tina , Juric Damijana Mojca
Published: June 2021

Neurotrophin‐3 (NT‐3) is produced by astrocytes, in addition to neurons, and monoamine neurotransmitters play a role in controlling NT‐3 synthesis. We evaluated the active involvement of histamine receptors and intracellular mechanisms in the regulation of NT‐3 production by HA.

HA (1 μM) significantly and transiently elevated NT‐3 mRNA levels by 2.2‐fold after 30 min of incubation following by 2.1‐fold increase in NT‐3 intracellular levels after 6 h. Its stimulation was partly inhibited by selective H1, H2 and H3 antagonists. NT‐3 levels in astrocytes were increased by selective H1, H2 and H3 agonists as well as by adenylyl cyclase activation, PKA activation, PKC activation and mobilization of intracellular Ca2+. HA‐induced increase in NT‐3 cellular levels were significantly reduced by H‐89, staurosporin, KN‐62 and desensitizing pretreatment with TPA. MAP kinase cascade inhibitor PD98059 completely blocked the stimulatory action of HA and all selective agonists. Using quantitative RT‐PCR we confirmed that cultured rat cortical astrocytes express not only H1 and H2 receptors already identified by radioligand binding studies but also H3 receptors which were sensitive to pertussis toxin. In conclusion, the synthesis of astrocytic NT‐3 stimulated by HA is an adaptable process using several parallel histaminergic pathways using H1‐, H2‐, and H3‐receptor pathways. The observed H1, H2 and H3 receptor crosstalk in histaminergic regulation of NT‐3 leads to the convergence of these pathways at the level of MAP kinase activity which in the next step triggers the increased expression and subsequently the synthesis of NT‐3 in astrocytes.
Methamphetamine self‐administration alters gene expression in the rat striatum
Authors: Krasnova Irina N. , Chiflikyan Margarit , Justinova Zuzana , Ladenheim Bruce , McCoy Michael T. , Wood William H. , Becker Kevin G. , Goldberg Steven R. , Cadet Jean Lud
Published: June 2021

Striatum plays a critical role in the development of METH addiction but the molecular adaptations in the striatal neurons caused by METH exposure remain poorly understood. In the present study, we used an extended METH self‐administration model to study changes in gene expression in the rat striatum. Animals self‐administered METH (0.1 mg/kg/injection, i.v.) during 15‐hour sessions for 8 consecutive days. A yoked control group received passive injections of saline. The drug intake for the METH group reached 14.8 mg/kg/day. Rats were euthanized at 2 and 24 hours after the cessation of self‐administration. Transcriptional changes caused by METH were examined in the striatum using Illumina 22K Rat microarrays and validated by qRT‐PCR. METH self‐administration caused upregulation in the expression of transcription factors c‐fos, c‐jun, Nurr1, Nptx1 and neuropeptides cholecystokinin, neurotensin and neuromedin U. METH also induced increases in the expression of BDNF, synaptophysin, synaptotagmin II, syntaxin, cholinergic, glutamate and GABA receptors involved in the regulation of synaptic plasticity. Our results show that METH self‐administration causes changes in the expression of genes known to trigger persistent neuroadaptations in the mammalian brain. Some of these changes might contribute to METH‐related habit learning and development of METH addiction.
Novel GRK and β‐arrestin isotype regulation of Toll‐like receptor 4 signaling
Authors: Grisanti Laurel , Porter James E
Published: June 2021

Adrenergic receptors (ARs) are expressed on immune cell populations, but their precise function is not known. We have shown that α1BAR stimulation synergistically increases IL‐1β release from lipopolysaccharide (LPS)‐challenged human monocytes through an NFκB‐independent, but p38/AP‐1‐dependent mechanism. RNA interference (RNAi) for β‐arrestin (βARR) 1 prevented α1BAR mediated increases in LPS‐induced IL‐1β production. We hypothesized that concurrent α1BAR and TLR4 activation leads to increased IL‐1β levels through βARR1‐dependent activation of p38. We demonstrate using RNAi that βARR2 but not βARR1 is necessary for p38 phosphorylation in monocytes concomitantly treated with phenylephrine (PE) and LPS. Co‐immunoprecipitation and confocal microscopy techniques, showed that MEK3/6 interacts with βARR2, promoting activation of p38. Electrophoretic mobility shift assays (EMSA) demonstrated the importance of βARR2 for AP‐1 activation and inhibition of the NFκB pathway after PE and LPS treatment. Using RNAi we showed the necessity of GRK2 for α1BAR modulation of LPS‐mediated IL‐1β generation, while GRK5 inhibited production of IL‐1β in response to both PE plus LPS and LPS only treatments. This study demonstrates that βARR1 and 2 serve unique functions and provides direct evidence for GRK2/βARR2‐dependent activation of p38, as well as a novel βARR2‐dependent inhibition of NFκB in human monocytes.
Potentiation of Delta Opioid Receptor Inhibition of Adenyly Cyclase Activity By 5‐HT3 Receptor Stimulation in Intact NG108‐15 Cells
Authors: Paul Dennis , Bailey Evangeline
Published: June 2021

Affinity of ligands for delta opioid receptors (DORs) is regulated by concentration of divalent cations, whereas affinity for alpha2 adrenoceptors is not. In intact NG108‐15 cells, we have demonstrated a 4‐fold increase in affinity of DOR ligands by stimulation of 5‐HT3 receptors, a ligand‐gated cation channel. However, whether this regulation of affinity translates to a regulation of function has not been determined. Accordingly, we assessed the effect of the calcium/magnesium ionphore, A23187 and the 5‐HT3 receptor agonist, 2‐methyl‐5‐HT on delta opioid receptor cell signaling. In intact NG108‐15 cells, the effect of DPDPE (0.1nM – 30 nM) or clonidine (1nM ‐ 1μM) on forskolin‐stimulated cAMP production was determined in the absence or presence of A23187 or 3μM 2‐methyl‐5‐HT. As expected, DPDPE inhibited cAMP production dose‐dependently.2M5HT (3μM) produced a biphasic shift in the concentration‐response curve for DPDPE. At low concentrations of DPDPE, 2M5HT shifted the curve 10‐fold leftward, whereas at high concentrations of DPDPE, the curve was not shifted. This effect was attenuated by the 5‐HT3 antagonist, tropisetron. In contrast, 2‐methyl‐5‐HT had no effect on inhibition of forskolin‐stimulated cAMP produced by the alpha2 adrenoceptor agonist, clonidine. These results are evidence that the increase in affinity produced by cation influx translates to a functional increase in potency of agonists for delta opioid receptors. Supported by the Dept. of Pharmacology, LSUHSC.
Gαi binding sites on Calnuc and NUCB2 define a new family of structurally‐related Gαi‐regulatory motifs
Authors: Garcia‐Marcos Mikel , Kietrsunthorn Patrick , Wang HongHui , Ghosh Pradipta , Farquhar Marilyn G
Published: June 2021

Heterotrimeric G proteins are molecular switches modulated by families of structurally and functionally related regulators. GIV is the first non‐receptor GEF that activates Gαi‐subunits via a defined, evolutionarily conserved motif. Here we found that Calnuc and its closely related homolog NUCB2, two calcium‐binding proteins, share a common motif with GIV for Gαi binding and activation. Bioinformatic searches and structural analysis revealed that Calnuc and NUCB2 possess an evolutionarily conserved motif with sequence and structural similarity to GIV's GEF sequence. Using in vitro pulldown and competition assays we demonstrate that this motif binds preferentially to the inactive conformation of Gαi1 and Gαi3 over other Gα‐subunits and docks onto the same site as GIV, i.e., the α3/Switch II cleft. Calnuc binding was impaired when K248 in the α3 helix of Gαi3 was replaced by the corresponding M residue in Gαo, which does not bind to Calnuc. Moreover, mutation of hydrophobic residues from the conserved motif predicted to dock on the α3/Switch II cleft of Gαi3 abolished Calnuc's binding to Gαi3 and impaired its GEF activity in vitro. We also demonstrate that calcium binding to Calnuc and NUCB2 abolishes their interaction with Gαi3, probably via a conformational change that renders their Gαi‐binding residues unexposed. Taken together our results define a new class of structurally‐related Gαi regulatory motifs and provide the structural basis for the properties of Calnuc and NUCB2 binding to Gα‐subunits and its regulation by calcium ions.

Support: NIH grants DKI7780 and CA100768 to M.G.F. Susan G. Komen fellowship KG080079 to M.G‐M CAMS (BWF) and RSA (AGA) to P.G.
Regulation and Activation of Phospholipase Cβ
Authors: Lyon Angeline M. , Tesmer Valerie M. , Dhamsania Vishan , Gutierrez Joanne , Chowdhury Shoaib , Suddala Krishna , Northup John K. , Tesmer John J.G.
Published: June 2021

Phospholipase Cβ (PLCβ), which catalyzes the hydrolysis of phosphatidylinositol‐4,5‐bisphosphate (PIP2) to the second messengers diacylglycerol (DAG) and inositol‐1,4,5‐triphosphate (IP3), is activated in response to specific extracellular stimuli through interactions with the heterotrimeric G protein Gαq. However, the structural and functional mechanisms underlying the regulation of PLCβ activity is poorly understood. In this work we present two new structures of PLCβ homologs purified from endogenous sources that include a highly conserved element from the C‐terminal regulatory domain. This conserved element forms a microdomain that interacts with a conserved surface of the catalytic core. Deletion of or mutation of residues within this microdomain in PLCβ3 results in increased PLCβ activity. Based on the recent structure of the Gαq‐PLCβ3 complex, this microdomain is likely displaced from the catalytic core upon Gαq binding. Thus, the C‐terminal microdomain appears to represent an autoinhibitory element that modulates the basal activity of PLCβ and is involved in allosteric changes mediated by Gαq.

Support was provided by NIH grants HL086865 and HL071818.
Specific association of β4γ5 to adenosine A1 and A2A receptors determined by stable isotope labeling with heavy amino acids in cell culture and mass spectrometry
Authors: McIntire William E , Wang Dora Bigler , Sherman Nicholas , Shannon John , Leonhardt Susan , Yeager Mark , Mayeenuddin Linnia H , Garrison James C
Published: June 2021

Quantitative mass spectrometry was used to characterize native βγ dimers selectively associated with expressed adenosine A1:αi1 and A2A:αS receptor fusion proteins. An A1:αi1 fusion protein was expressed in HEK‐293 cells cultured in media containing [13C6] Arg and [13C6] Lys, and the βγ dimers which incorporated heavy isotopes purified. Heavy βγ was combined with either recombinant βγ purified from Sf9 cells, βγ purified from A2A:αS expressed in HEK‐293 cells cultured in standard media, or βγ enriched from HEK‐293 cells. Samples were separated by SDS‐PAGE, and protein bands containing β and γ were excised, digested with trypsin, separated by HPLC and isotope ratios analyzed by mass spectrometry. Three β isoforms, β1, β2 and β4, and seven γ isoforms, γ2, γ4, γ5, γ7, γ10, γ11 and γ12 were identified in the analysis. Compared to cellular HEK‐293 βγ levels, both fusion proteins had increased levels of β4 and γ5. Also, more β4 associated with A2A:αS fusion protein than the A1:αi1 version. No differences in γ isoforms were observed between the two fusion proteins; however, both contained less γ2, γ10 and γ12 than the enriched HEK‐293 βγ. These results suggest that preferences for particular βγ isoforms are driven in part by structural motifs common to adenosine receptor family members. Supported by AHA Grant 0535350N, a Pilot Award from the UVa Silvio O. Conte Digestive Disease Research Center and NIH Grant R01‐DK‐19952.
Modulation of CGRP‐receptor function by G‐protein βγ subunits
Authors: Meens Merlijn JPMT , Mattheij Nadine JA , Spijkers Léon JA , Alewijnse Astrid E , De Mey Jo GR
Published: June 2021

Activated CGRP–receptors (CGRP‐R) might relax vascular smooth muscle (VSM) via activation of Gsα and by inducing dissociation of endothelin‐1 (ET‐1) from ETA‐receptors (Meens et al. PLos ONE 2010). The latter “terminator effect” is not mimicked by other stimuli of Gsα. Thus, we analyzed roles of G‐protein βγ subunits (Gβγ) in CGRP‐R function. Organ chamber studies with rat arteries and radioligand binding studies on membranes from CHO cells expressing human CGRP‐R were performed. In several types of arteries, isoproterenol (ISO), forskolin and CGRP caused relaxation during K+‐induced contractions. The effects of CGRP did not correlate with those of ISO or forskolin. In mesenteric arteries, contracted with K+‐ or ET‐1, IBMX increased relaxations induced by sodium nitroprusside (SNP) or ISO, but not by CGRP. Gallein and M119 (Gβγ inhibitors), but not fluorescein (negative control), inhibited the relaxing effect and the “terminator effect” of CGRP while not altering arterial effects of i) K+, ii) ET‐1, iii) SNP or iv) ISO. Gallein did not displace [125I]‐CGRP from CGRP‐R but caused an acute fivefold increase of the Bmax for [125I]‐CGRP. In conclusion, activated CGRP‐R cause cyclic nucleotide‐independent relaxation of VSM and terminate arterial effects of ET‐1 via Gβγ. Also, Gβγ seems to restrict agonist‐binding to CGRP‐R.

Performed within TIPharma. M119 was obtained from the National Cancer Institute (US).
Analysis of β‐Adrenergic Receptor Signaling in Gravin (AKAP12) Knockout Mice after Chronic Isoproterenol Stimulation
Authors: Guillory Ashley Nicole , Wijaya Cori S. , Rababa'h Abeer , Yin Xing , McConnell Bradley K.
Published: June 2021

The purpose of this study was to investigate the effect of knockout of gravin, an A kinase anchoring protein, on the beta‐adrenergic receptor (β‐AR) signaling pathway. We measured heart‐to‐body weight ratio (HW/BW), β‐AR density and affinity and cAMP production in wild‐type (WT) and gravin knockout (KO) mice stimulated with isoproterenol (ISO; 30 mg/kg/day x 14 days). Our results show that the WT ISO group had a significantly higher HW/BW ratio than the WT and gravin KO controls (p=0.02) as well as the gravin KO ISO group (WT control: 4.81 mg/g; WT ISO: 6.05 mg/g; KO control: 5.11 mg/g; KO ISO: 5.54 mg/g; p=0.012)The WT treatment group had significantly higher total β‐AR density (WT control: 103.53 fmol/mg; WT ISO: 209.52 fmol/mg; KO control: 118.62 fmol/mg; KO ISO: 146.25 fmol/mg; p=0.011) but significantly lower affinity both compared to the WT and KO controls as well as the gravin KO treatment group (WT control: 0.30; WT ISO: 0.66; KO control: 0.34; KO ISO: 0.23; p=0.002). However, both the WT and the gravin KO treatment group had significantly lower cAMP production when compared to their respective controls (WT control: 0.68 pmol/mg; WT ISO: 0.32 pmol/mg; KO control: 0.66 pmol/mg; KO ISO: 0.24 pmol/mg; p=0.0001). These data indicate that the absence of gravin attempts to maintain normal conditions of the β‐AR signaling pathway even after chronic β‐AR stimulation. This research was supported by NIH R01HL085487.
MicroRNAs 103 and 107 regulate the expression of Human Cytochrome P450 2C8
Authors: Zhang Shuyun , Surapureddi Sailesh , Goldstein Joyce A
Published: June 2021

CYP2C8, which is a highly inducible member of the CYP2C family is involved in the metabolism of therapeutics and endogenous compounds. CYP2C8 is regulated by a variety of nuclear receptors such as GR, CAR and HNF4α. The expression of CYP2C8 has been consistently observed to be low in tumor cell lines. We hypothesized that miRNAs might be involved in the regulation of CYP2C8. Microarray analysis showed several miRNA to be upregulated in HepG2 cell line. A potential recognition element was identified in the 3′ untranslated region of CYP2C8, which matches with miR 103 and miR 107. We investigated if these are involved in CYP2C8 expression. Reporter assays in human hepatocytes with miRNAs revealed that both could identify the recognition element in the 3′ UTR of CYP2C8 mRNA. We found that overexpression of miRNAs in human primary hepatocytes decreases the transactivation of luciferase reporter, whereas the luciferase activities are increased by inhibition of miRNAs. CYP2C8 protein expression in human primary hepatocytes is decreased by overexpression of miRNAs. Conversely, inhibition of miRNAs results in an increase of CYP2C8 protein level. However, neither overexpression nor inhibition of miRNAs affect s the CYP2C8 mRNA levels. We conclude that miR103 and 107 post‐transcriptionally regulate human CYP2C8. The increased expression of miRNAs might be a responsible for low expression of CYP2C8 protein in HepG2 cells.
Progesterone and Estrogen Modulate the Expression of Xenobiotic Transporters in Cultured Mammary Epithelial Cells
Authors: Silver Kristopher , Malreddy Pradeep , Lorch Amy , Schultz Bruce D. , Gehring Ronette
Published: June 2021

Milk is an important source of nutrition, but it can become contaminated when mothers (or cows) ingest drugs or are exposed to toxins in the environment. Some toxins are reportedly concentrated in milk although the underlying mechanisms are not well‐defined, especially across species. Importantly, in vitro models to assess potential exposure through milk are lacking. The goal for these studies is to determine whether a bovine mammary epithelial cell line, BME‐UV, expresses putative drug transporters and to test for hormone‐induced changes in transporter expression profile or activity. Real time PCR was employed to evaluate the expression of xenobiotic transporters in polarized BME‐UV cell monolayers cultured in the presence or absence of progesterone and/or estradiol. P‐glycoprotein (P‐gp) mRNA expression was reduced following exposure to β‐estradiol or progesterone alone, but significantly decreased when the two hormones were used in combination. In contrast, expression of organic anion transporter 3 increased with hormone exposure. Since expression of P‐gp is reduced in lactating mammary epithelia, our results showing a similar decrease in P‐gp expression with hormone exposure in BME‐UV cells suggests that this cell line recapitulates observations in native tissues and thus will provide a powerful tool to quantify transport of distinct drugs or environmental contaminants. [NIH P20‐RR017686 Core C]
Cadmium regulates angiogenesis through AKT, ERK1/2, and HIF‐1 pathways in human lung epithelial cells
Authors: Jing Yi , Liu Ling‐Zhi , Zhu Yingxue , Jiang Yue , Guo Nancy Lan , Barnett John , Rojanasakul Yon , Jiang Bing‐Hua
Published: June 2021

Cadmium is classified as a human carcinogen especially involved in human lung cancer. The mechanism underlying cadmium in inducing carcinogenesis is unclear yet. Angiogenesis has been considered as a fundamental requirement for tumor initiation, proliferation and metastasis. By using in vitro and in vivo models, we showed that cadmium can transform human lung epithelial cells, induce tumor growth and angiogenesis. To understand the molecular mechanism of cadmium in inducing carcinogenesis, we demonstrated that cadmium activated PI3K/AKT and ERK1/2 signaling pathways in a time‐ and concentration‐dependent manner, and increased generation of reactive oxygen species (ROS). Cadmium activated AKT and ERK1/2 through ROS production. Cadmium stimulated angiogenesis by activating AKT and ERK1/2, and by increasing HIF‐1 and VEGF expression. These findings reveal the role of cadmium in inducing tumor growth and angiogenesis, and provide potential underlying molecular mechanism.
Serum biomarkers in young adult and aged Brown Norway (BN) rats following episodic (weekly) ozone exposure
Authors: Gordon Christopher J , Schmid Judy , Lehmann James R , Ledbetter Allen , Ward William , Schladweiler Mette , Jarema Kimberly , Kodavanti Urmila , MacPhail Robert C
Published: June 2021

Ozone (O3) is an air pollutant that is associated with cardiovascular and respiratory diseases. Older adults are considered to be particularly susceptible to oxidant air pollutants such as O3. Serum biomarkers are being sought that would lead to better predictions of susceptibility in the aged to air pollutants. We exposed young adult (4 month) and aged (20 month) male BN rats (N=7) to air (control) or 0.8 ppm O3 for 6 hr/d, 1 d/wk for 17 wks. Respiratory parameters assessed by unrestrained plethysmography the day after exposure revealed that O3 led to airway‐flow limitation. Rats were terminated 24 hr after the last exposure. Bronchoalveolar fluid macrophages were reduced in aged rats and neutrophils increased in young rats exposed to O3. A panel of 58 protein analytes was quantified in serum, including cytokines, chemokines, and tissue factors (Rules Based Medicine). Age affected 20 analytes, including C‐reactive protein, interferon, factor VII, and macrophage inflammatory protein‐2 (MIP‐2). O3 affected 6 analytes, including fibroblast growth factor (basic), MIP‐2, leukemia inhibitor factor, and haptoglobin. Age by O3 interactions were obtained for MIP‐2 and myoglobin. The data illustrate that serum analytes may be beneficial as biomarkers to assess aging and susceptibility to criteria air pollutants. This is an abstract of a proposed presentation and does not reflect US EPA policy.
Mercaptoethylguanidine Attenuates Caustic Esophageal Injury in Rats; A Role for Scavenging of Peroxynitrite
Authors: Guven Ahmet , UYSAL Bulent , CALISKAN Bahadir , OZTAS Emin , KORKMAZ Ahmet , OZTURK Haluk
Published: June 2021

Introduction

After ingestion of caustic material, tissue damage is caused by reactive oxygen species and reactive nitrogen species. Mercaptoethylguanidine (MEG) is a scavenger of peroxynitrite. Thus, this study was designed whether MEG has a beneficial effect on caustic esophageal injury.

Methods

45 rats were allocated into 3 groups; sham‐operated, untreatment and treatment groups. Caustic esophageal burn was created by instilling 15% NaOH in the distal esophagus. The treatment group treated with 10 mg/kg/day MEG i.p. for 5 days. All rats were killed at 28 days. Efficacy of the treatment was assessed by histopathologically and biochemically.

Results

The stenosis index and the histopathologic damage score were significantly lower in the MEG‐treatment group which showed a correlation with tissue hydroxyproline level. In the untreatment group, tissue oxidative stres parameters (malondialdehyde and protein carbonyl content) were significantly higher, antioxidant enzymes activities (SOD and GSH‐Px) were significantly lower than trreatment group. Urinary Nitrate and Nitrite (NOx) levels increased in the treatment and un‐treatment group at the first three days.

Conclusion

Peroxynitrites play an important role in the healing process of caustic esophagitis. MEG might be used a potential adjuvant agent in treatment of esophageal caustic burn by modulating antioxidant defense mechanism.
Lipopolysaccharide enhances TGFβ1 induction of PDGF‐B in bile duct epithelial cells: role of NF‐κB
Authors: Kassel Karen M , Sullivan Bradley P , Luyendyk James P
Published: June 2021

Platelet‐derived growth factor‐B (PDGF‐B) is a potent fibrogenic mediator in the liver. The PDGF‐BB homodimer is expressed by bile duct epithelial cells (BDECs) and contributes to liver fibrosis after bile duct ligation. The mechanism of PDGF‐B induction in BDECs is not known. Transforming growth factor β (TGFβ) and lipopolysaccharide (LPS) also contribute to the fibrogenic response after bile duct ligation. We tested the hypothesis that LPS enhances TGFβ1 induction of PDGF‐B in BDECs. Stimulation of MMNK‐1 cells or primary rat BDECs with LPS did not induce PDGF‐B mRNA expression, but LPS stimulation enhanced TGFβ1 induction of PDGF‐B mRNA. LPS did not affect TGFβ1 activation of a SMAD‐responsive luciferase construct. Rather, stimulation of MMNK‐1 cells with LPS, but not TGFβ1, caused IκBα degradation and activation of a NF‐κB‐responsive luciferase construct. Expression of an IκBα super‐repressor or dominant negative IKK2 inhibited activation of the NF‐κB‐responsive luciferase construct and attenuated the LPS potentiation of TGFβ‐induced PDGF‐B mRNA. The results indicate that LPS activation of a NF‐κB pathway enhances TGFβ1‐induced PDGF‐B expression in BDECs. Financial support: NIH R01 ES017537.
Characterization of a Long Acting Smooth Muscle Myosin Inhibitor, CK‐2125927, as a Novel Therapeutic Mechanism for Bronchodilation
Authors: Pannirselvam Malar , Jia Zhiheng , Durham Nickie , Zavodovskaya Marianna , Lenzi David , Clancy Sheila , Hartman James J , Morgans David J , Morgan Bradley P , Qian Xiangping , Malik Fady I
Published: June 2021

Smooth muscle myosin is a mechanochemical enzyme that hydrolyzes ATP to generate mechanical force; ultimately all signaling pathways that modulate smooth muscle tone converge on the regulation of this motor protein. We previously showed that a novel smooth muscle myosin inhibitor, CK‐2019165, inhibited the methacholine‐induced bronchoconstriction in rat models with a short duration of action (~ 2 hours). Here we describe the discovery and characterization of a long acting smooth muscle myosin inhibitor for bronchodilation. Using high throughput screening, we identified and subsequently optimized a class of selective inhibitors of smooth muscle myosin. These compounds potently inhibit the smooth muscle myosin ATPase (IC50≤ 30 nM). An optimized member of this series, CK‐2125927,, inhibited calcium‐induced contraction of skinned rat tail artery in a concentration dependent manner with an IC50 of ~ 1 μM. CK‐2125927 induced concentration‐dependent relaxation of methacholine‐pre‐constricted rat tracheal rings with an EC50 of ~ 1 μM. Further, CK‐2125927 (10 μM) completely inhibited electrical field stimulation‐induced constriction of guinea pig tracheal rings with a 50% recovery time of > 6 hours, a duration comparable to the long‐acting beta‐adrenergic agonist salmeterol. Together these data suggest CK‐2125927 is a long‐acting smooth muscle myosin inhibitor that may provide a novel therapeutic approach for the treatment of diseases where bronchoconstriction plays a role, such as asthma and COPD.
Interaction of Human Urotensin II and Vasodilator Agents in Human Internal Mammary Artery
Authors: HE Guo‐Wei , BAI Xiao‐Yan , LIU Xiao‐Cheng , YANG Qin
Published: June 2021

Spasm of the internal mammary artery (IMA) may occur in coronary grafting (CABG). We studied the interaction of human urotensin II (hU‐II) and vasodilators (calcium antagonists or glyceryl trinitrate (GTN)) in human IMA segments (n=102, from 46 CABG patients) in myograph (n=6 in each group). hU‐II contracted all IMA. In KCL‐contraction, full (nifedipine:98.9±3.9%) or nearly full (diltiazem:92.7±6.0%) relaxation with 18.6‐fold‐higher potency to nifedipine vs. diltiazem (EC50 −8.01±0.20 vs. −6.74±0.22 logM, p=0.002) and in hU‐II‐contraction, nearly full relaxation (nifedipine: 90.6 ±4.6%; diltiazem: 95.1±2.1%) with 6.2‐fold‐higher potency to nifedipine vs. diltiazem (EC50 −7.25±0.25 vs. −6.46±0.18 logM, p=0.014) were observed. GTN caused nearly full relaxation (95.4±5.8%) but its pretreatment failed to alter, whereas diltiazem and nifedipine pretreatment reduced contraction to hU‐II. Thus, hU‐II is a potent vasoconstrictor in human IMA. Calcium antagonists and GTN relax the contraction caused by hU‐II with different potencies. However, calcium antagonists are more effective in preventing the contraction induced by hU‐II than GTN.

Supported by China Nat'l Ministry of Sci. & Tech. 2009DFB30560 & 2010CB529502(973)åTianjin Municipal Sci. & Tech. Commission 09ZCZDSF04200 &10JCYBJC26400å Hong Kong RGC (CUHK4651/07M & CUHK4789/09M); CUHK2041561, and Providence SV Med. Foundation, Portland, OR.
Acute high glucose concentration increases myogenic tone in bovine retinal arteries through a protein kinase c mechanism
Authors: Owen Mary Pruitt , Andrews Jaiye A. , Zhu Shu , Barman Scott A. , White Richard E.
Published: June 2021

A reduction in retinal blood flow is one of the early features of diabetic retinopathy. Hyperglycemia is an important risk factor for vascular complications in diabetes and is believed to modulate protein kinase C (PKC) activation in the retina. The object of this study was to evaluate the effect of acute exposure to high glucose (25 mM) on myogenic tone in the retinal circulation and to determine whether that myogenic tone can be modified by inhibition of PKC. Branches of the retinal artery were removed from the bovine retinal sheath and pressurized using an arteriograph system. At 60, 80 and 100 mm Hg, significantly more tone (4.2 ± 1.4%, 3.5 ± 0.8% and 3.7 ± 1.1%, respectively) developed in high glucose buffer than in normal buffer (5.5 mM). Chelerythrine, a nonselective PKC inhibitor, blocked the effect of high glucose on myogenic tone and reduced the level of myogenic tone in normal glucose. In addition, western blot results showed that isoforms alpha, beta, delta, epsilon, iota and lambda of PKC are expressed in bovine retinal arterial vascular tissue. To our knowledge, this is the first study to show that acute high glucose concentration increases myogenic tone in isolated retinal arteries through a PKC mechanism. Exploitation of a possible PKC isoform specificity for this effect has potential for delaying, stopping or even reversing diabetic retinopathy. (CCDA and HL‐68026)
Fundamental Change in Neurovascular Coupling after Subarachnoid Hemorrhage
Authors: Koide Masayo , Bonev Adrian D , Nelson Mark T , Wellman George C
Published: June 2021

Subarachnoid hemorrhage (SAH) following cerebral aneurysm rupture is associated with substantial morbidity and mortality against which existing therapeutic options have limited efficacy. The impact of SAH on neurovascular coupling, reflecting the coordinated communication between neurons, astrocytes and parenchymal arterioles, is unknown. Using a combination of two‐photon and infrared‐differential interference contrast microscopy, arteriolar diameter and astrocyte endfoot Ca2+ were simultaneously measured in brain slices from un‐operated, sham‐operated and SAH model of rats. Increased neuronal activity caused by electrical field stimulation (EFS) elicited the predicted elevation in endfoot Ca2+ and vasodilation in brain slices from control animals (Filosa JA et al, 2006). EFS induced a similar increase in endfoot Ca2+, but in marked contrast caused parenchymal arteriolar constriction in brain slices from SAH model animals. Further, elevating endfoot Ca2+ via two‐photon photolysis of caged Ca2+ in the range of 200–500 nM caused parenchymal arteriolar dilation in brain slices from control animals and constriction in brain slices from SAH animals. These data demonstrate that SAH causes a fundamental switch from vasodilation to constriction in response to moderate increases in astrocyte endfoot Ca2+. (Supported by NIH P01 HL095488, R01 HL078983, R01 HL44455, & Totman Medical Research Trust).
Nicotinamide phosphoribosyl transferase coordinates stabilization of plasminogen activator inhibitor‐1 mRNA at the myoendothelial junction
Authors: Heberlein Katherine , Straub Adam C , Best Angela K , Wojta Johann , Isakson Brant E
Published: June 2021

We previously demonstrated that plasminogen activator inhibitor‐1 (PAI‐1) regulates MEJ formation in response to obese conditions. Using TNF‐α to mimic obese conditions, we have shown that PAI‐1 mRNA is stabilized at the myoendothelial junction (MEJ), resulting in increased PAI‐1 protein. How PAI‐1 mRNA is stabilized at the MEJ is unknown. To elucidate this mechanism we show that serpine binding protein‐1 (SERBP1), which stabilizes PAI‐1 mRNA, and nicotinamide phosphoribosyl transferase (NAMPT), which contains a microtubule binding domain (MBD), both increase at the MEJ in response to TNF‐α. We then demonstrate via co‐immunoprecitation an interaction between these two proteins that is enhanced by TNF‐α. Next, using a microtubule binding assay we show that NAMPT can bind microtubules in a dose‐dependent manner, independent of microtubule length. This suggests that SERPB1 and NAMPT may act as a PAI‐1 RNA binding protein (RBP) complex at the MEJ. To determine the importance of the NAMPT MBD for increases in PAI‐1 at the MEJ, we designed a peptide with a TAT‐tag, targeted to the MBD of NAMPT. Treatment with the MBD peptide negated the effects of TNF‐α on PAI‐1 at the MEJ, while a scrambled peptide had no effect. Together, these data indicate that during obesity, stabilization of PAI‐1 mRNA occurs via a PAI‐1 RBP complex that is anchored by NAMPT to microtubules at the MEJ, promoting local increases in PAI‐1 protein.
Activation of the Epac1/Rap1 pathway attenuates fMLP‐induced microvascular hyperpermeability in vivo
Authors: Kurtz Kristine M , Belaidi Marisa B , Breslin Jerome W
Published: June 2021

Elevation of intracellular cAMP levels has been shown to enhance microvascular barrier integrity. Recent studies have shown a novel mechanism of cAMP‐mediated endothelial barrier regulation via the cAMP‐dependent nucleotide exchange factor Epac1 and its downstream effector Rap1 GTPase. We tested the hypothesis that the Epac/Rap1 activator 8‐pCPT‐2′‐O‐Me‐cAMP (8CPT) will inhibit inflammation‐induced hyperpermeability, using 10 μM fMLP as an inflammatory stimulus. Male Sprague Dawley rats (275–325 g) rats were anesthetized, catheterized (artery and vein), and a midline laparotomy was performed. The mesentery was exteriorized for intravital fluorescence microscopic viewing of the mesenteric microcirculation. Rats received an i.v. bolus (100 mg/kg) followed by continuous infusion (0.15 mg/kg/min) of FITC‐albumin. The integrated optical intensity (IOI) of multiple extravascular regions in the mesentery, and arteriolar diameter were measured every 5 min. for a 20‐min. baseline and 1 h with fMLP. Our results show that fMLP caused a marked increase in IOI, that was significantly attenuated by pretreatment with 200 μM 8CPT. Treatment with fMLP also caused constriction of arterioles that was not changed by 8CPT pretreatment. Our results suggest that Epac1/Rap1 activation can attenuate acute inflammation‐induced microvascular leakage. Supported by the American Heart Association and NIH P20RR018766.
Conducted vasodilatation in human coronary arterioles
Authors: Bagi Zsolt , Feher Attila , Garland Chris , Dora Kim
Published: June 2021

Conducted vasomotor responses in the coronary microcirculation match blood flow to metabolic requirements. Here we established whether conducted vasodilatation occurs in human coronary arterioles. Coronary arterioles were dissected from atrial appendage of patients (n=15). In isolated, pressurized arterioles (~ 100 μm) conducted dilatation was initiated by locally administering bradykinin (BK, 100 μM) ejected from a micropipette, positioned in close proximity to the vessel wall. Diameter changes were measured at local and upstream sites (500 and 1,000 μm from the stimulus) with videomicroscopy. Local administration of BK elicited vasodilatation (90±2%), which remained substantial at the upstream sites (500 μm, 53±7%; 1,000 μm, 46±9%). Administration of the gap junction uncoupler carbenoxolone (10 μM) did not affect local responses, but diminished conducted dilatation. The NO synthase inhibitor, L‐NAME had no affect on local responses (82±5%), but significantly reduced dilatation at upstream sites (500 μm, 17±3%; 1,000 μm, 24±6%). Both local and upstream dilatations to BK were diminished by Ca‐activated K‐channel blockers, TRAM34 and apamin and also with 30 mM K+. Thus, BK‐induced dilatation is propagated along the isolated human coronary arteriole. The conducted response is mediated by gap junctional hyperpolarisation spread, which is facilitated by NO. (NIH HL104126, BHF RE/08/004)
Enzymatic glycocalyx treatment impairs insulin‐mediated recruitment of microvascular blood volume and decreases insulin sensitivity in rats
Authors: Eskens Bart , Cobelens Hanneke , Vink Hans , van Teeffelen Jurgen
Published: June 2021

Agonist‐induced impairment of the blood‐exclusion properties of the glycocalyx has been shown to effectively increase microvascular blood volume (MBV) (VanTeeffelen, CVR 2010). Since the rapid insulin‐mediated increase in MBV has been suggested to contribute to insulin's metabolic actions (Vincent, Diabetes 2004), we hypothesize that loss of glycocalyx impairs the ability of insulin to increase MBV and to subsequently dispose blood glucose.

In rats, loss of glycocalyx was induced by a bolus of hyaluronidase; saline served as control. During anesthesia the microcirculation of hindlimb muscle was visualized with a Side Stream Dark field camera before and during intravenous insulin infusion. The RBC column width distribution of visible microvessels was determined in time. In control, median RBC column width did not change during insulin infusion but the P75 and P90 increased rapidly (<10 min) with 0.3 ± 0.1 μm and 0.8 ± 0.4 μm respectively. In the hyaluronidase treated group insulin had no effect on median, P75 and P90 of RBC column width. To measure insulin sensitivity an intravenous insulin tolerance test was performed in conscious rats. The insulin‐mediated decline in blood glucose after 15 minutes was found to be decreased after hyaluronidase treatment from 62 ± 1 to 53 ± 2% (P<0.05).

It is concluded that glycocalyx degradation is associated with an impaired ability for insulin to increase MBV and to dispose glucose.

Supported by DFN 2006.00.027 and NHF 2005T037
The Phosphodiesterase 3 (PDE3) Inhibitor, Cilostazol, Rescues Low O2‐induced ATP Release from Erythrocytes (RBCs) of Humans with Type 2 Diabetes (DM2) Restoring Their Ability to Dilate Skeletal Muscle Arterioles
Authors: Sprague Randy S , Stephenson Alan H , Bowles Elizabeth A , Achilleus David , Ellsworth Mary L
Published: June 2021

Healthy human (HH) RBCs exposed to low O2 release ATP via a pathway requiring increases in cAMP levels which are regulated by PDE3. Isolated skeletal muscle arterioles perfused with these cells dilate when exposed to similar decreases in extraluminal O2. In contrast, DM2 RBCs neither release ATP nor stimulate dilation of arterioles under the same conditions. We investigated the hypothesis that inhibition of PDE3 would rescue low O2‐induced ATP release from DM2 RBCs and permit these cells to stimulate vasodilation. RBCs were exposed to low PO2 (~10 mmHg) and ATP release was determined in the absence or presence of the PDE3 inhibitor, cilostazol (CZ, 100 μM). In the absence of CZ, ATP release from HH (n=6) and DM2 (n=7) RBCs increased by 126±54 (p<0.05) and 10±17% (NS), respectively. Pretreatment of DM2 RBCs with CZ (n=7) augmented low O2‐induced ATP release (105±46%, P<0.05) and restored the ability of DM2 RBCs to stimulate dilation of perfused arterioles exposed to reduced extraluminal O2. These findings are consistent with the hypothesis that decreased ATP release from DM2 RBCs may contribute to peripheral vascular disease due to failure of a control mechanism for the regulation of perfusion distribution to meet tissue O2 needs. Importantly, these studies suggest that PDE3 inhibitors could improve this defect via a heretofore unrecognized effect on RBCs. [Supported by NIH grant HL‐89094 and a Presidential Grant from SLU].
BKCa channels modulate cortical astrocytic CO production in newborn pigs
Authors: Xi Qi , Parfenova Helena , Jaggar Jonathan H. , Leffler Charles W.
Published: June 2021

In newborn pigs, we have previously demonstrated that glutamate‐induced cortical astrocytic carbon monoxide (CO) regulates neurovascular coupling by activating large‐conductance calcium‐activated potassium (BKCa) channels in arteriolar smooth muscle cells. Recently, BKCa channels have been also recorded in cortical astrocytes. It is not known whether astrocytic BKCa channels regulate CO production. Here, we explored the hypothesis that astrocytic CO is fine tuned by BKCa channels in cortical astrocytes of newborn pigs. Major findings from newborn pig freshly isolated cortical astrocytes and astrocytes in primary culture were: (1) paxilline, a BKCa channel blocker, increased intracellular calcium concentration ([Ca2+]i) and elevated CO production; (2) in cortical astrocytes permeabilized with ionomycin, elevation of [Ca2+]i concentration‐dependently increased CO production; and (3) nimodipine, a L‐type calcium channel blocker, blocked glutamate–induced elevations in CO. In summary, this study indicates that [Ca2+]i elevation increases astrocytic CO production. CO activates astrocytic BKCa channels that inhibit voltage‐dependent Ca2+ channel activity, causing [Ca2+]i reduction, thereby decreasing CO production.
Prostanoids mediate functional hyperaemia in healthy young and older men in an oxygen‐dependent manner
Authors: Davies Christopher Steven , Townend Jonathan N , Marshall Janice M
Published: June 2021

We have previously shown in young subjects that the hyperaemia following static exercise is reduced by both hyperoxia and cyclooxygenase inhibition with aspirin. We therefore hypothesised that functional hyperaemia is at least partly mediated by vasodilator prostanoids in an oxygen (O2) dependent manner. In the present study, 12 young (18–24) and 12 older (50–70) healthy, recreationally active male subjects performed static and rhythmic handgrip exercise at 50% of their maximum force for 2 minutes, separated by 30 minutes rest. Experiments were performed on 4 separate days in random order: following either 600mg soluble aspirin in orange squash or placebo, and with either room air or 40%O2 breathed through a face mask during the period of exercise. Forearm blood flow was measured using venous occlusion plethysmography and arterial pressure was recorded by finapress. Venous blood samples were taken from the exercising arm for analysis of venous prostaglandins. Administration of interventions (O2, aspirin and both combined) in young subjects caused a reduction in functional hyperaemia relative to control, whereas in older subjects the interventions caused the hyperaemia to increase. Thus prostanoids are released during exercise in an O2‐dependent manner and production potentially shifts from vasodilator to vasoconstrictor prostanoids with ageing. Research was funded by the British Heart Foundation.
Influence of sex and menstrual phase on the middle cerebral artery blood flow velocity responses to dynamic exercise in humans
Authors: Hartwich Doreen , Kim Areum , Griffin Harry S , Balanos George M , Aldred Sarah , Fadel Paul J , Fisher James P
Published: June 2021

Emerging evidence suggests that resting cerebral blood flow is typically higher in women until menopause. However, it is presently unknown how sex and menstrual phase influence exercise‐induced increases in cerebral perfusion. Middle cerebral artery blood flow velocity (MCA Vmean; transcranial Doppler), heart rate (HR; ECG) and mean blood pressure (MBP; SunTech) were measured at rest and during leg cycling at 50% of HR reserve in young women (21±2 yr; n=8) and men (20±1yr; n=9). Women not taking oral contraceptives were tested during the early (EF, days 1–5) and late (LF, days 10–12) follicular phases of the menstrual cycle. Cerebral vascular conductance index (CVCi) was calculated (MCA Vmean/MBP). At rest HR and MBP were similar between women at EF and LF, and men. However, MCA Vmean was significantly higher in women compared to men (73±3 EF, 75±4 LF and 56±3 men, cm/s). Exercise‐induced increases in MBP were significantly lower in women (Δ+10±1 EF, Δ+10±2 LF) than men (Δ+17±2 mmHg), while increases in HR were similar between groups. The increase in MCA Vmean during exercise was attenuated in women during LF phase (Δ+4±3 cm/s) compared with EF (Δ+10±3 cm/s) and men (Δ+16±5 cm/s). Resting CVCi was higher in men and unchanged from rest to exercise in all groups. These preliminary data suggest that exercise‐induced increases in cerebral perfusion are attenuated during LF phase of the menstrual cycle in women.
Enhanced BK (IK(ca)) channel activity contributes to lack of myogenic response in the cerebral circulation of Fawn Hooded Hypertensive (FHH) Rats
Authors: Pabbidi Mallikarjuna , Juncos Julio , Burke Marilyn , Farley Jerry , Harder David , Roman Richard
Published: June 2021

The present study examined the role of alterations in Iberiotoxin (IBTX) sensitive IK(ca) channel activity in impaired myogenic response in the cerebral circulation of Fawn Hooded Hypertensive (FHH) rats. Isolated perfused middle cerebral arterioles from FHH rats exhibited no myogenic response when pressure was increased from 40 to 140 mmHg. In contrast, vascular diameter decreased by about 50% in an FHH.1BN AR+ congenic strain in which a 2.6 Mb region of BN chromosome 1 was introgressed into the FHH genetic background. Whole‐cell patch‐clamp (1μM free calcium; +40mV) of cerebral vascular smooth muscle cells revealed amplified IK(ca) current densities (pA/pF) in FHH rats compared to AR+ and SD rats (23.2±6;6.9±1.9 and 7.1±1.3 respectively). Additionally, in inside‐out patch clamping, IK(ca) exhibited similar unitary conductance (235 and 245 pS) in both strains. However, open probability of IK(ca) were significantly higher in FHH rats compared to AR+ rats (0.82±0.04 and 0.3±0.09 respectively). These results suggest that enhanced activity of the IK(ca) channel contributes to loss of myogenic response in the cerebral circulation of FHH rats. NIH HL36279, HL59996, DK79306. Roman.
L‐ and T‐ Type Calcium Channels in Cerebral Arteries
Authors: El‐Rahman Rasha Abd , Welsh Suzanne E Brett , Anfinogenova Yana , Harraz Osama , Turner Raymond , Welsh Donald G
Published: June 2021

This study examined the expression, localization, and functional properties of voltage gated Ca2+channels in cerebral arterial smooth muscle cells. An initial PCR and western blot analysis, performed on whole arteries and isolated smooth muscle cells, revealed that the α1 subunits of L‐type (Cav1.2) and T‐type (Cav3.1 and Cav3.2) Ca2+ channels were present in these tissues. Immunohistochemistry confirmed the presence of all 3 Ca2+ channel subunits in specified cellular regions. Cav1.2 labeling was ribbon‐like along the length of the smooth muscle cell whereas T‐type labeling was punctate and localized to periodically spaced regions. Whole cell patch clamp electrophysiology confirmed the presence of a Ca2+current that was only partly blocked by nifedipine, an L‐type Ca2+ channel inhibitor. Characteristic of T‐type Ca2+ channels, the nifedipine‐insensitive component displayed fast inactivation kinetics and was abolished by mibefradil, efondipine and kurtoxin. Pressure myography experiments subsequently revealed that arterial tone was differential affected by L‐ and T‐type Ca2+ channel blockade depending on whether the vessel was pressurized to 20 or 80 mmHg. This differential effect was consistent with the voltage‐dependent properties of the two channel subtypes. In closing, this study found that L‐ and T‐type Ca2+ channels are expressed in cerebral arterial smooth muscle and that both contribute to tone development in accordance within their defined electrophysiological properties. Supported by CIHR.