bluuebunny/crossref_metadata_2025 huggingface dataset:
Cannabinoid CB1 and serotonin 5‐HT1A agonists mediate lower lip retraction by independent mechanisms
Authors: Chopda Girish Rajmal , Anderson Joseph , Makriyannis Alexandros , Nikas Spyridon P , Paronis Carol A
Published: June 2021

Lower lip retraction (LLR) is a behavioral endpoint produced by activation of 5‐HT1A receptors and used to evaluate 5‐HT1A agonists. LLR is also produced by cannabinoid (CB) agonists and we sought to investigate the roles of CB1 and 5‐HT1A receptors in CB‐mediated LLR. Female SD rats (n= 6–8) were used and LLR was scored on a 3‐point scale. The CB agonists, Δ9 tetrahydrocannabinol (THC), 1–10 mg/kg, and AM2389, 0.01–0.1 mg/kg, dose‐dependently produced LLR at 3hr after injection, with ED50 values of 5.6 and 0.022 mg/kg, respectively. The 5‐HT1A agonist, 8‐OHDPAT injected cumulatively at 15min intervals, also dose‐dependently increased LLR, with an ED50 of 0.026 mg/kg. The 8‐OHDPAT dose response curve was shifted 2–5‐fold to the left after 3hr pretreatment with either 0.003–0.01 mg/kg AM2389 or 1 mg/kg THC. These results suggest commonality in the mechanisms by which CB1 and 5‐HT1A agonists produce LLR. However, the CB1 antagonist rimonabant administered as a 30 min pretreatment failed to antagonize LLR effects of 8‐OHDPAT and, similarly, the 5‐HT1A antagonist WAY100635 did not antagonize LLR effects of AM2389. Hence, this study for the first time reports that CB agonists produce LLR in rats by a CB1 dependent, 5‐HT1A independent mechanism. Ongoing studies are examining interactions of THC and 8‐OHDPAT in other in vivo measures, specifically CB1‐mediated diuresis and 5‐HT1A‐mediated increases in punished responding.
Modulation of Nrf2 and NF‐κB Counteracts Multiple Manifestations of Experimental Diabetic Neuropathy: Potential of Sulforaphane and Melatonin
Authors: Negi Geeta , Sharma Shyam S
Published: June 2021

In the recent years Nrf2 and NF‐κB have surfaced as two transcription factors orchestrating antioxidant defense and inflammation, respectively. Since oxidative stress and inflammation play a pivotal role in nerve dysfunction and damage in diabetic neuropathy, Nrf2 and NF‐κB could be major players here too. For in vitro experiments neuro2a cells were incubated with sulforaphane and melatonin (both possess dual antioxidant and anti‐inflammatory activities) in the presence of normal (5.5 mM) and high glucose (30 mM). For in vivo studies, sulforaphane and melatonin were administered to rats 6 weeks post diabetes induction for 14 days. Motor nerve conduction velocity, nerve blood flow and pain behavior were improved and malondialdehyde level was reduced by treatment. Antioxidant effect of both agents was derived from Nrf2 as demonstrated by increased expression of Nrf2 and downstream targets HO‐1 and NQO‐1 in neuro2a cells and sciatic nerve of diabetic animals. NF‐κB inhibition may be responsible for their antiinflammatory activity as there was reduction in its expression and IκB kinase (IKK) phosphorylation along with abrogation of proinflammatory mediators. This study supports the defensive role of Nrf2 under conditions of oxidative stress and also suggests that NF‐κB is an important modulator of inflammatory damage in diabetic neuropathy. Funded by Ministry of Chemicals and Fertilizers, Government of India.
Effects of chronic stress on nitrous oxide (N2O)‐induced antinociceptive and anxiolytic effects in mice
Authors: Emmanouil Dimitris , Yeon Jiwoong , Quock Raymond M.
Published: June 2021

Subanesthetic concentrations of N2O are routinely used in clinical dentistry to produce conscious sedation. The aim of this study was to assess the influence of chronic stress on the antinociceptive and anxiolytic effects of N2O. To induce chronic stress, male NIH Swiss mice (20–25 g) were subjected to a modified forced‐swim test four times over 48 min. Thirty min later, they were exposed to 30%, 50% or 70% N2O in O2, and antinociceptive or anxiolytic responsiveness was assessed using the hot plate test or the light/dark exploration box. Non‐stressed control mice responded to N2O with a dose‐dependent antinociceptive effect, but chronically‐stressed, N2O‐exposed mice showed a marked hyperalgesic response. Exposure of non‐stressed control mice to N2O increased both the time spent in the light compartment and the number of intercompartmental transitions in a generally dose‐dependent manner. Chronic stress did not influence either parameter, but chronically‐stressed, N2O‐exposed mice showed increases in the number of transitions but not in time spent in the light compartment. These findings may be of clinical significance in the use of N2O in patients with chronic stress. Further investigation in the influence of stress on the antinociceptive and anxiolytic actions of N2O is warranted. (Supported in part by NIH Grant GM‐77153 and the Allen I. White Distinguished Professorship.)
Interaction of Protein Kinase C‐related Kinase (PRK) 1 with the TPα and TPβ isoforms of the human Thromboxane A2 receptor: Implications for Prostate Cancer
Authors: Kinsella B Therese , Turner Elizebeth C , Mulvaney Eamon P , Kavanagh David J , Reid Helen M
Published: June 2021

In humans, thromboxane (TX)A2 signals through the TPα and TPβ isoforms of the TXA2 receptor (TP). Here, the RhoA effector protein kinase C‐related kinase (PRK) 1 was identified as an interactant of both TPα and TPβ involving common and unique sequences within their respective carboxyl‐termini and the kinase domain of PRK1. While the interaction with PRK1 is constitutive, agonist‐activation of TPα/TPβ did not regulate the complex but enhanced PRK1 activation leading to phosphorylation of histone H1 in vitro. Altered PRK1/TP expression & signaling are increasingly implicated in certain neoplasms, particularly in androgen‐associated prostate carcinomas. Agonist‐activation of TPα/TPβ led to phosphorylation of histone H3 at Thr11 (H3Thr11), a recognized marker of androgen induced‐chromatin remodeling, in the prostate LNCaP and PC‐3 cell lines but not in primary vascular smooth muscle or endothelial cells. Moreover, this effect was augmented by dihydrotestosterone in androgen‐responsive LNCaP but not in non‐responsive PC‐3 cells. Disruption of PRK1 impaired TPα/TPβ‐mediated H3Thr11 phosphorylation in, and cell migration of, both cell types. Given the role of TXA2 as a potent mediator of RhoA signaling, the identification of PRK1 as a bone fide interactant of TPα/TPβ has broad functional significance such as within the vasculature and in neoplasms in which both PRK1 and the TPs are increasingly implicated. Funded by SFI and HRB.
The α1A‐adrenergic receptor mediates cardiac hypertrophy through the Gαq‐PI3K‐Rac1 signaling pathway
Authors: Mohl Marion , Xiao Xiao-Hui , Balaji Poornima , Issmaa Siiri , Feneley Michael , Graham Robert
Published: June 2021

Using a mouse model of cardiac‐specific over‐expression of the α1A‐adrenergic receptor (α1A‐AR; α1A‐M 66‐fold; α1A‐H 166‐fold) we have recently shown that acute activation of the α1A‐AR results in enhanced contraction through activation of α1A‐AR‐operated Ca2+ entry via Snapin and TRPC6.

We have now defined a hypertrophic signalling pathway involving α1A‐AR‐mediated recruitment of Gαq‐PI3K‐Rac1 that is distinct from the contractile pathway. Cardiomyocytes from adult α1A‐M, α1A‐H and WT mice stimulated with the α1A‐AR‐specific agonist A61603 exhibited dose‐dependent increases in hypertrophic gene expression, protein synthesis, and cell surface area that were proportional to the level of receptor expression and were Gαq‐PI3K‐Rac1‐dependent. Inhibition of the α1A‐AR contractile pathway by blockade of TRPC6 did not inhibit α1A‐AR‐mediated induction of cardiac hypertrophy.

The elucidation of two distinct α1A‐AR‐mediated signalling pathways, one increasing cardiac contraction upon acute activation and the other inducing cardiac hypertrophy upon chronic activation, identifies the α1A‐AR as a potential target for the development of pathway‐specific α1A‐AR‐agonist drugs to treat heart failure.

Sponsor: NHMRC, NHF
Serotonin 5‐HT2C receptor homodimers identified by Fluorescence Correlation Spectroscopy
Authors: Herrick-Davis Katharine , Grinde Ellinor , Mazurkiewicz Joseph E
Published: June 2021

Fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) were applied to determine diffusion rate and oligomeric size of G‐protein‐coupled receptors at the single molecule level. FCS was used to monitor fluctuations in fluorescence intensity arising from fluorescent‐tagged serotonin 5‐ HT2C receptors freely diffusing within the plasma membrane of HEK293 cells and primary hippocampal neurons. FCS reported diffusion rates of 0.9μm2/s and 1.3μm2/s for GFP‐ and YFP‐tagged receptors, respectively. Since the molecular brightness of a fluorescent protein is directly proportional to the number of fluorescent proteins within a cluster it can be used to determine the oligomer state of a protein complex. PCH analysis of fluorescent‐tagged 5‐HT2C receptors provided molecular brightness values that were approximately twice that of GFP and YFP monomeric controls, similar to a dimeric GFP control, and unaltered by 5‐HT. Bimolecular fluorescence complementation (BiFC) of the N‐ and C‐terminal halves of YFP, attached to 5‐HT2C receptors, was observed on the plasma membrane with a brightness equal to the monomeric YFP control. Co‐expression of GFP‐tagged 5‐HT2C receptors with a large excess of non‐tagged 5‐HT2C receptors decreased the brightness by half. These results indicate that the mobile fraction of 5‐HT2C receptors in the plasma membrane is dimeric. Supported by NIH‐ MH86796.
The Nurr1 transcription factor is upregulated by the EP1 prostanoid receptor in monomethylarsonous acid treated UROtsa cells
Authors: Sanchez Christopher M. , Gandolfi A. Jay , Regan John W.
Published: June 2021

Monomethylarsonous acid (MMA(III)) is a toxic metabolite of arsenic that has been used to study the urothelial effects of arsenicals. A previous study has shown that acute treatment of immortalized UROtsa bladder cells with MMA(III) activate cyclooxygenase 2, which along with cyclooxygenase 1, is required for prostaglandin synthesis. Prostaglandins act on receptors that are members of the G‐protein coupled receptors family responsible for regulating pain, fever, and inflammation and are involved in such cellular processes as proliferation, migration, and apoptosis. Recently, we have shown that stimulation of the EP1 prostanoid receptor by prostaglandin E2 upregulates expression of the orphan nuclear receptor Nurr1. Nurr1 is a transcription factor that is essential for the development and maintenance of midbrain dopaminergic neurons and has been shown to regulate both cell survival and death. In the current study, we examined the potential role of prostanoid receptors in the regulation of Nurr1 expression in UROtsa cells. Treatment of UROtsa cells with MMA(III) resulted in a 3‐fold upregulation of Nurr1 protein expression after 1 hour that was sustained for up to 12 hours. Pretreatment of UROtsa cells with SC‐51322, a selective EP1 receptor antagonist, inhibited the upregulation of Nurr1 expression by MMA(III), suggesting involvement of the EP1 prostanoid receptor with the upregulation of Nurr1 by MMA(III).
Palmitoylation of PAR1 regulates the accessibility of C‐tail YXX φ motifs to AP‐2 and internalization
Authors: Canto Isabel , Trejo JoAnn
Published: June 2021

Protease‐activated receptor‐1 (PAR1), a G‐protein coupled receptor (GPCR) for thrombin, is irreversibly and proteolytically activated. Thus, desensitization and intracellular trafficking of PAR1 are critical for proper cellular responses. Palmitoylation of GPCRs regulates various aspects of signaling and trafficking, however the function of palmitoylation in PAR1 signal regulation is not known. We examined palmitoylation in PAR1 wildtype and in a mutant where C‐tail cysteines were converted to alanines. Here, we show that PAR1 is palmitoylated at these C‐tail cysteine residues. Moreover, the PAR1 palmitoylation mutant displayed enhanced constitutive internalization, whereas agonist‐induced internalization was comparable to wildtype receptor. Constitutive internalization of the PAR1 palmitoylation mutant was dependent on the clathrin adaptor protein complex AP‐2, like wildtype receptor. However, unlike wildtype PAR1, AP‐2‐mediated internalization required the proximal rather than the distal YXXφ motif in this mutant. The PAR1 palmitoylation mutant also displayed defects in phosphorylation and ubiquitination. Thus, palmitoylation stabilizes a conformation of the PAR1 C‐tail that is important for trafficking and other post‐translational modifications. Supported by RO1 HL073328 & UC TRDRP.
Regulation of protease‐activated receptor‐1 signaling and trafficking by extracellular loop 2 N‐linked glycosylation
Authors: Soto Antonio Gregorio , Trejo JoAnn
Published: June 2021

Thrombin binds to and cleaves the N‐terminus of the G protein‐coupled receptor protease‐activated receptor‐1 (PAR1) unmasking a new N‐terminus that functions as a tethered ligand. To study the role of PAR1 extracellular loop 2 (ECL2) N‐linked glycosylation in signaling and trafficking we generated asparagine to alanine mutants. Here, we report that the PAR1 ECL2 domain is highly modified by N‐linked glycosylation. Moreover, the PAR1 ECL2 glycosylation mutant displayed an enhanced efficacy in thrombin signaling. The increase in PAR1 ECL2 mutant signaling was not due to defects in desensitization, receptor cleavage kinetics, or recycling back to the cell surface after activation. Interestingly, the PAR1 ECL2 mutant also displayed a delay in agonist‐induced endocytosis. We employed BRET to study possible differences in receptor dimerization and interactions with adaptor proteins. Our BRET data revealed differences in PAR1 wildtype versus ECL2 mutant titration curves as well as differences in beta‐arrestin 2 association. Thus, these findings suggest that PAR1 N‐linked glycosylation at ECL2 affects the conformation of the receptor that results in differential coupling to signaling effectors and components of the endocytic machinery leading to the altered signaling and trafficking. Supported by NIGMS T32 Pharmacological Sciences Training Grant and NHLBI Diversity Supplement Award.
Synthetic tethering increases the potency of PAR2 agonists
Authors: Flynn Andrea , Tillu Dipti V. , Hoffman Justin , Zhang Zhenyu , Vagner Josef , Price Theodore J. , Boitano Scott
Published: June 2021

Protease‐activated receptor‐2 (PAR2) is G protein‐coupled receptor important in a number of protease‐associated pathologies including allergic asthma, cancer and pain. Peptides (SLIGRL‐NH2) that mimic the receptor's native tethered ligand can activate PAR2 without the off‐target effects of proteases. A better understanding of the pathological role of PAR2 is limited by the need for potent, specific agonists. We and others have developed potent PAR2 peptidomimetics (2‐at‐LIGRL‐NH2) based on the trypsin cleavage site and serine substitutions. We hypothesized mimicking the native tethered ligand would increase PAR2 ligand potency. We used palmitate to mimic the natural membrane anchor and a polyethylene glycol (Peg) linker between the anchor and peptidomimetic to make a series of synthetic tethered ligands (STLs; 2‐at‐LIGRLO‐PegN‐Palm; N=1–5). We found 3 Pegs conferred maximal potency; 2‐at‐LIGRL‐Peg3‐Palm induced Ca2+ and MAPK signaling in vitro (EC50 0.4 nM, 12.2 nM) and thermal hyperalgesia in vivo (0.36 nMol), and was specific for PAR2. 2‐at‐LIGRLO‐Peg3‐Palm is 30–1000 times more potent across assays than the best described agonist and specific for PAR2. STLs are a powerful approach to increase ligand potency without sacrificing specificity and may allow for the development of subnanomolar ligands important to understanding the pathologic roles of PAR2. NIH grants NS073664 and T32‐HL007249.
The antipsychotic aripiprazole is a non‐competitive antagonist of dopamine‐stimulated D2 receptor interactions with β‐arrestin‐2
Authors: Free R Benjamin , Dalefield Martin , Miller Brittney N , Doyle Trevor B , Conroy Jennie L , Vangveravong Suwanna , Duan Lihua , Javitch Jonathan A , Mach Robert H , Sibley David R
Published: June 2021

Aripiprazole is a recently approved drug for treating schizophrenia. It is often referred to as a 3rd generation antipsychotic with unique clinical and biological properties. Its mechanism(s) of action has been attributed to either functionally selective properties or low partial agonist activity for D2 receptor signaling. We have now evaluated the ability of aripiprazole, and 12 structurally related analogs (Vangveravong et al., 2011), to regulate D2 receptor‐β‐arrestin‐2 interactions, an important signaling arm of the D2 receptor. Two assays were employed to detect receptor‐arrestin interactions: 1) a bioluminescent resonance energy transfer (BRET) assay; and 2) a β‐galactosidsase complementation assay. In each assay, dopamine (DA) stimulates D2 receptor‐β‐arrestin interactions in a dose‐dependently. None of the aripiprazole derivatives exhibited agonist activity, although each fully antagonized the DA response. Interestingly, when the DA dose‐response curves were performed with increasing concentrations of test ligand (Schild experiments), aripiprazole and four derivatives were found to decrease the ECMAX without affecting the EC50 of DA (non‐competitive inhibition). In contrast, eight derivatives of aripiprazole decreased the EC50 of DA without affecting the ECMAX (competitive inhibition). The structure‐activity relationships for these effects are currently under investigation.
GPCR‐mediated Modulation Of Synaptic Transmission
Authors: Betke Katherine , Rose Kristie , Friedman David , Chen Yunjia , Wang Qin , Gilsbach Ralf , Hein Lutz , Caprioli Richard , Hamm Heidi
Published: June 2021

Synaptic transmission is characterized by exocytotic events which mediate the release of chemical transmitters to facilitate neuronal communication. GPCRs such as the α2A adrenergic receptor play an important role in controlling this process as they act as feedback regulators for transmitter release from presynaptic terminals via their G protein βγ subunits. Many current therapies targeting these receptors exhibit significant side effects, and as such, dissection of the downstream mechanisms by which they modulate exocytosis may reveal more disease‐selective targets for therapeutic intervention.

Gβγ subunits have been shown to bind directly to SNARE proteins to regulate fusion events. Studies suggest unique Gβγ combinations may differentially interact with SNARE proteins to regulate exocytosis; however, no study has been able to describe the subcellular localization of isoforms to presynaptic terminals, nor link their activation to specific receptors. Utilizing synaptosome preparations and targeted mass spectrometric approaches, the current study evaluates the distribution of Gβγ subunits at presynaptic terminals expressing α2A adrenergic receptors and the physiological relevance of the Gβγ‐SNARE interaction. This work was supported by the NIH (EY010291).
Finding novel intragenic suppressors of a constitutively active allele of Gs alpha
Authors: Janevska Daniela , Din Eraj , Tobar-Rubin Raquel , Alvarez Rebecca , Chaudhry Faiza
Published: June 2021

McCune‐Albright Syndrome (MAS) is a genetic disorder caused by mutations that inhibit GTP hydrolysis in Gsα, permanently activating the protein. We have developed a yeast model system for MAS in which mutations in the yeast Gα subunit homologous to those seen in MAS (R297H) prevent colony formation when the mutant allele is the only Gα gene present in the cells. We then constructed of a library of 32,000 secondary mutations in the constitutively active yeast Gα gene with the goal of identifying intragenic suppressors of the MAS mutation. Nine novel suppressor alleles have been identified and have been mapped to the crystal structure of the Gsαƒnprotein. Suppressor mutations have been identified in the GTP‐binding site of the protein, near the βγ‐interaction surface, on a potential effector interaction surface, in switch III, and also on regions of the α subunit with no currently characterized function. The construction of a detailed map of sites on the surface of Gα which can inactivate the MAS allele can drive rational drug discovery of better treatments for MAS patients.

Funded by NIH grant 1R15ED020190‐01
How Gαq Regulates PIP2 Hydrolysis: Molecular Mechanisms and Prospects for Drug Development
Authors: Lyon Angeline Marie , Tesmer Valerie M. , Boguth Cassandra A. , Dhamsania Vishan D. , Thal David M. , Guiterrez Joanne , Chowdhury Shoaib , Northup John K. , Tesmer John J. G.
Published: June 2021

Phospholipase Cβ (PLCβ) is activated in response to specific extracellular stimuli by direct interactions with the heterotrimeric G protein Gαq. However, the molecular mechanisms underlying this regulation are poorly understood. In this work we present four new structures of PLCβ that capture the enzyme in its basal state and in complex with Gαq. These structures not only provide the first high‐resolution models of the C‐terminal regulatory domain in the context of full‐length PLCβ, but also identify regions important for PLCβ regulation and Gαq binding and activation. Deletion or mutation of these regions has profound consequences in the basal activity of PLCβ, the magnitude of Gαq‐stimulated activation, and the affinity for activated Gαq. Taken together, these structures reveal an allosteric mechanism of PLCβ activation by Gαq and define the role of the C‐terminal regulatory domain in this process. Support for this work was provided by NIH grants HL086865 and HL071818 to J.J.G.T. and an American Heart Association postdoctoral fellowship to A.M.L.
Angiotensin (1–7) Antagonizes Angiotensin II Action in SHR Hypothalamic Neurons via Stimulation of PTEN Expression
Authors: Modgil Amit , O'Rourke Stephen T. , Sun Chengwen
Published: June 2021

It was demonstrated previously that Ang (1–7) counter‐regulates Ang II action in neuronal activity of SHR neurons. However, the underlying molecular mechanisms are still not fully known. Our objective in the present study was to characterize the signal transduction mechanism of phosphatidylinositol 3‐kinase (PI3K) and phosphatase and tensin homologue deleted on chromosome ten (PTEN) involvement in the counter‐regulatory effect of Ang (1–7) on Ang II action in neurons cultured from hypothalamus of WKY vs. SH rats using current clamp recording.

Application of Ang II produced a 44% greater increase in neuronal firing in SHR vs. WKY neurons. Chronic treatment with Ang (1–7) (100nM, 24 hrs) and inhibition of PI3K using lentiviral vector (LV‐DNp85α) significantly abolished Ang II mediated increase in neuronal firing rate in SHR vs. WKY neurons. Pretreatment of Ang (1–7) attenuated Ang II‐induced increase in the phosphorylation of Akt by 83% in SHR vs WKY neurons. Treatment with Ang (1–7) (100nM, 24 hrs) significantly enhanced PTEN expression in both WKY and SHR neuronal cultures using western blot and real‐time PCR. These effects of Ang (1–7) were blocked by A‐779 (10 μM), a Mas‐R antagonist. The data demonstrate that Ang (1–7) induces PTEN expression via Mas‐R, and depresses PI3K signaling via PKB/Akt pathway, which leads to a counter‐regulatory effect of Ang (1–7) on Ang II action in neuronal activity of SHR neurons.
Temporal lipid mediator profiles differ between plasma and bronchoalveolar lavage in mice exposed to tobacco smoke
Authors: Nording Malin Linder , Yang Jun , Hoang Laura , Zamora Vanessa , Uyeminami Dale , Espiritu Imelda , Pinkerton Kent E. , Hammock Bruce D. , Luria Ayala
Published: June 2021

Patients with chronic obstructive pulmonary disease exhibit inflammation in the respiratory tract. In order to investigate temporal trends of the inflammatory response, a murine model of short‐term tobacco smoke (TS) exposure was used. Focus was on lipid mediators in the oxylipin family, i.e. oxidized fatty acids including eicosanoids of the cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) metabolic pathways. Mice (C57Bl/6) were exposed to TS for 3, 9, and 15 hours, or filtered air (FA). Bronchoalveolar lavage fluid (BALF) and plasma samples underwent solid phase extraction followed by analysis with liquid chromatography coupled to tandem mass spectrometry. The resulting oxylipin levels were higher in plasma than in BALF, and a larger number of oxylipins was detected in plasma than in BALF (28 vs 21). Furthermore, the fraction of LOX‐derived oxylipins was the same, while the fraction of COX‐ and CYP‐derived oxylipins differed between BALF and plasma. Six of the LOX‐oxylipins in BALF, and none in plasma, were significantly increased after 9h of TS exposure correlating to a significant increase in total number of infiltrated cells (p=0.05). In conclusion, temporal oxylipin profiles may be used to assess the progression of pulmonary inflammation in mice exposed to TS.

TRDRP 16KT‐0037, NIEHS Grants ES02710, P42 ES04699, and the Swedish Research Councils VR, Formas, and Vinnova supported this work.
Inhibition of soluble epoxide hydrolase in mice exposed to tobacco smoke
Authors: Nording Malin Linder , Yang Jun , Hoang Laura , Zamora Vanessa , Uyeminami Dale , Espiritu Imelda , Pinkerton Kent E. , Hammock Bruce D. , Luria Ayala
Published: June 2021

Patients with chronic obstructive pulmonary disease exhibit inflammation in the respiratory tract. Epoxyeicosatrienoic acids (EETs) possess promising anti‐inflammatory properties. Hence, stabilization of EETs through inhibition of the enzyme soluble epoxide hydrolase (sEH) by small molecule inhibitors (sEHI) was investigated in a murine model of short‐term tobacco smoke (TS) exposure. Wild‐type (WT), and sEH knock out (KO) mice were exposed to filtered air (FA) or TS for 9 hours. Bronchoalveolar lavage fluid samples underwent solid phase extraction followed by liquid chromatography coupled to tandem mass spectrometry to facilitate quantitation of EETs, and their corresponding diols (DHETs) derived from arachidonic acid, as well as linoleic acid epoxides (EpOMEs), and diols (DiHOMEs). The total number of infiltrated cells was decreased in TS exposed KO mice, as well as in sEHI‐treated WT mice, compared to non‐treated mice exposed to TS. Both the EET/DHET ratio, and the EpOME/DiHOME ratio were increased in TS‐exposed WT mice treated with sEHI, compared to non‐treated mice exposed to TS. The ratios were further increased in KO mice, both exposed to TS and FA. Our results suggest that inhibition of sEH stabilizes EETs and EpOMEs, with potential beneficial effects in TS exposed mice.

TRDRP 16KT‐0037, NIEHS Grants ES02710, P42 ES04699, and the Swedish Research Councils VR, Formas, and Vinnova supported this work.
SIRT1 enhances endothelium‐dependent relaxation through an eNOS‐independent mechanism
Authors: XU Cheng , Vanhoutte Paul M , Wang Yu
Published: June 2021

SIRT1 is a class III NAD+‐dependent deacetylase which involved in a wide range of cellular processes and possesses vascular protective effects. SIRT1 maintains vascular homeostasis by reducing endothelial activation, preventing endothelial senescence, and promoting endothelium‐dependent relaxations of vascular smooth muscle. To elucidate the molecular mechanisms underlying the SIRT1‐mediated vascular protection, an endothelium‐specific SIRT1 transgenic mouse model was engineered. The vascular responsiveness of the thoracic aorta was determined using a Mulvany‐Halpern wire myograph for the recording of isometric force. The results showed that selective over‐expression of SIRT1 in endothelial cells significantly enhanced acetylcholine‐evoked aortic relaxation in mice of different ages [8, 12, 16 and 20 weeks] and prevented high fat diet‐induced endothelial dysfunction. The enhanced endothelium‐dependent relaxation in the aorta of these SIRT1 transgenic mice persisted in the presence of L‐NAME, an inhibitor of NO synthesis. Moreover, the eNOS activity was not significantly different from that in preparations of wild type mice. These findings suggest that an eNOS/NO independent mechanism is involved in the vascular regulation by SIRT1.
miR‐150 prevents pulmonary inflammation by regulating Interleukin‐1R associated kinase‐2 downstream of focal adhesion kinase
Authors: Rajput Charu , Tauseef Mohammad , Yazbeck Pascal , Thennes Tracy , Mehta Dolly
Published: June 2021

Pulmonary inflammation and severe edema developing post‐sepsis are the main hallmarks of Acute Lung Injury (ALI). We have shown that tamoxifen‐induced endothelial‐specific deletion of FAK led to disruption of lung vascular barrier and generation of pro‐inflammatory cytokines mimicking early phase of ALI. In this study, we addressed the role of microRNA (miR)‐150 in regulating lung inflammation downstream of FAK based on the tenet that the plasma level of mir‐150 were found to be decreased in septic patients. FAK deleted mice lungs showed markedly suppressed miR‐150 expression in miR‐microarray. We confirmed these findings in EC‐FAK−/− lungs and FAK‐depleted human endothelial cells (ECs) using mir‐150 specific primers. Furthermore, we show that in WT mice, miR‐150 expression decreased following LPS‐induced lung injury and subsequently was restored during resolution of lung inflammation. However, in miR‐150−/− mice, LPS persistently induced edema formation. These findings were recapitulated in WT mice where alteration of miR‐150 activity modulated edema formation and inflammation. We identified interleukin‐1R‐associated kinase‐2 (IRAK2), a critical mediator LPS‐induced inflammation, as a downstream effector of mir‐150. We showed augmented IRAK2 levels in EC‐FAK−/− and miR‐150−/− mice lungs as well as FAK knock down ECs. The IRAK2 3′UTR luciferase reporter assay confirmed it as a miR150 target. Our results demonstrate a critical role of miR‐150 downstream of FAK in dampening pulmonary inflammation by targeting IRAK2.
A phosphodiesterase 3B‐based signaling complex integrates exchange protein activated by cAMP 1 and phosphatidylinositol 3‐kinase signals in human arterial endothelial cells
Authors: Maurice Donald H
Published: June 2021

Enzymes of the phosphodiesterase 3 (PDE3) and PDE4 families each regulate the activities of both protein kinases A (PKAs) and exchange proteins activated by cAMP (EPACs) in cells of the cardiovascular system. At present, the mechanisms that allow selected PDEs to individually regulate the activities of these two effectors are ill understood. The objective of this study was to determine how a specific PDE3 variant, namely PDE3B, interacts with and regulates EPAC1‐based signaling in human arterial endothelial cells (HAECs). Using several biochemical approaches, we show that PDE3B and EPAC1 bind directly through protein‐protein interactions. By knocking down PDE3B expression or by antagonizing EPAC1 binding with PDE3B, we show that PDE3B regulates cAMP binding by its tethered EPAC1. Interestingly, we also show that PDE3B binds directly to p84, a PI3Kγ regulatory subunit, and that this interaction allows PI3Kγ recruitment to the PDE3B‐EPAC1 complex. Of potential cardiovascular importance, we demonstrate that PDE3B‐tethered EPAC1 regulates HAEC PI3Kγ activity and that this allows dynamic cAMP‐dependent regulation of HAEC adhesion, spreading, and tubule formation. We identify and molecularly characterize a PDE3B‐based “signalosome” that integrates cAMP‐ and PI3Kγ‐encoded signals and show how this signal integration regulates HAEC functions of importance in angiogenesis.
Anti‐oxidant and anti‐apoptotic effects of fisetin in MPTP‐induced PC12 cells
Authors: Benzeroual Kenza , Patel Mital
Published: June 2021

Several studies mentioned the association of oxidative stress with Parkinson's disease (PD). This study, investigated the anti‐oxidant and anti‐apoptotic mechanisms of two anti‐inflammatory drugs, Fisetin and Ibuprofen in the MPTP‐injured PC12 cells, a rat model of PD. Previously, we demonstrated that MPTP treatment decreased cell viability, increased NO levels and caspase‐3 activity while pretreatment with fisetin reversed all these effects. To further understand the mechanisms involved, we measured NFkB, pro‐apoptotic Bax, and anti‐apoptotic Bcl‐xL protein expression by western blot analysis, and cytochrome c levels using ELISA. Results showed that fisetin dose‐dependently decreased NFkB expression probably due to decreased NO levels. The study also reported that fisetin decreased Bax and increased Bcl‐XL proteins expression, as well decreased cytochrome c levels. This anti‐apoptotic effect was further confirmed with fluorescence microscopy using a fluorescent PSVue480 probe against apoptotic cells. Ibuprofen effects were more related to anti‐oxidant rather than anti‐apoptotic mechanisms. The present study indicates that fisetin may provide a promising approach for the treatment of neurodegenerative diseases such as PD.
p38 MAPK signaling determines CAR‐mediated activation of the CYP2B genes by xenobiotics
Authors: Saito Kosuke , Moore Rick , Negishi Masahiko
Published: June 2021

The current concept for how liver eliminates xenobiotics and therapeutics is: these chemicals activate nuclear receptors CAR and/or PXR which in turn activate genes that encode for enzymes and transporters that metabolize and excrete them, such as the cytochrome P450s (CYPs). Here, we have demonstrated that p38 MAPK signaling is required for CAR to effectively and selectively activate a set of CYP genes and have investigated its molecular mechanism. TCPOBOP‐activated mouse CAR was first found to induce CYP2B6 mRNA only when HepG2 cells were treated with a p38 MAPK activator anisomycin (ANI). siRNA knock down of p38 MAPK strongly repressed the induction in HepG2‐derived Ym17 cells, which stably express mouse CAR. Therefore, p38 MAPK signaling can determine CAR‐mediated activation of the CYP2B6 gene. p38 MAPK appeared to target HNF4α in order to determine CYP2B6 promoter activation, since siRNA knockdown of HNF4α profoundly repressed the increase of CYP2B6 mRNA by co‐treatment with ANI and TCPOBOP in Ym17 cells. In addition to CYP2B6 mRNA, ANI co‐treatment with TCPOBOP increased CYP2C9, but not CYP3A4 mRNA, in Ym17 cells. p38 MAPK is substantially activated in mouse primary hepatocytes and an inhibition of p38 MAPK by SB239063 suppressed Cyp2b10 induction by TCPOBOP in the hepatocytes. p38 MAPK may be the cell signaling gate through which CAR selectively determines its target genes and subsequent metabolic consequences.
The African clawed frog (Xenopus laevis) is sensitive to pyrene exposure during early development
Authors: Snoke Deena B. , Rosato Kaitlyn E. , Richardson Elizabeth M. , Whittemore Susan L.
Published: June 2021

Toxicity of pyrene, a tetracyclic polyaromatic hydrocarbon (PAH), is poorly understood. Pyrene deposition in air, soil, and water results primarily from fossil fuel combustion. Pyrene is also abundant in cigarette smoke. PAHs cross the placenta; urinary 1‐hydroxypyrene (1‐HP) levels, a measure of PAH (and pyrene) exposure, are higher in pregnant smokers and their newborns than in nonsmokers and their newborns. Children in urban and industrial settings excrete more 1‐HP than those from rural areas. It is clear that developing humans are exposed to PAHs, including pyrene, both in utero and postnatally. To better understand how this abundant contaminant may affect development, we exposed the embryos of X. laevis, an important model of vertebrate development, to pyrene. Exposure to 5–10 ppm (25–49 μM) pyrene from stage 10.5–42 (~96 hrs) resulted in 90–100% mortality 48 hrs after their return to control conditions, while exposure to 0.1–1 ppm had no effect. Pyrene‐induced mortality may be stage‐specific. We observed 30% mortality in embryos exposed to 5 ppm until stage 18, a rate in contrast to the 90–100% mortality of embryos exposed to stage 24 or beyond. In addition, pyrene exposure induced edema at all doses (0.1–10 ppm). Based on these findings, further characterization of the toxicity of this common pollutant is warranted and potential health risks to humans assessed.

(Supported by NH‐INBRE grant 1P20RR030360‐01)
Dichloroacetonitrile Induces Protein Modifications in Cortical Astrocyte Cell Line CTX‐TNA2 and Alterations in Fetal Mouse Brain
Authors: Esmat Ahmed , Khalifa Amani E , El-Demerdash Ebtehal , Abdel-naim Ashraf B , El-Mahdy Mohamed A , Zweier Jay L
Published: June 2021

Epidemiological studies have shown an association between disinfection by‐products (DBP) of drinking water chlorination (DWC) and adverse pregnancy outcomes (APO) and indicated the involvement of DBP‐induced developmental toxicity on fetal brains. However, the precise mechanisms involved are not known. We report that dichloroacetonitrile (DCAN), a DBP of DWC, induces protein carbonylation in immortalized rat cortical astrocyte cells CTX‐TNA2 and pathological changes in the brains of fetuses of DCAN‐exposed pregnant albino mice. Exposure of cells to DCAN for 48 hr resulted in increased superoxide generation, decreased GSH/GSSG ratio, protein carbonylation, and inhibition of proteasomal activity. Oral administration of DCAN to pregnant mice resulted in low fetal body weight, neurodegeneration in the fetal cerebral cortex, redox imbalance and induction of apoptosis in the cerebral cortex and the cerebellum of the fetuses. Our data suggest that DCAN induces oxidative protein modifications and proteasomal inhibition which leads to accumulation of altered proteins in astrocytes, resulting in developmental pathological changes in fetal brains all of which result in APO. The findings provide insights toward understanding of the molecular basis of DCAN‐induced developmental pathologies and identify cellular pathways that can serve as tools for their prevention. (Supported by the Egyptian government)
Organophosphate pesticide exposure affects zebrafish developmental physiology
Authors: Garcia Hena , Hole Natalie , Radoniqi Flaka , Schmidt Hayden , Orona Gabriella , Fradinger Erica A
Published: June 2021

Organophosphate pesticides are known to inhibit acetylcholine esterase, an enzyme that degrades acetylcholine at the cholinergic synapse. In this study zebrafish were used to understand the impact chlorpyrifos, dichlorvos, and diazanon on vertebrate developmental physiology. Pre‐epiboly zebrafish embryos were exposed pesticides at concentrations ranging from 1μM to 1mM. Survival, spontaneous movements, heart rate, swimming behavior, and physical abnormalities were examined. All three pesticides were found to be toxic to developing zebrafish embryos at concentrations at or above than 100 μM. However, disruption of physiological processes controlled by the cholinergic neurons was only seen in embryos exposed to chlorpyrifos and dichlorvos. Chlorpyrifos and dichlorvos exposed embryos exhibited increased spontaneous movements, decreased embryonic heart rate, and a transient decrease in larval swimming ability. Although diazanon was the most toxic, it had no effect on these physiological systems. The effects of organophosphate pesticide exposure on vertebrate development are diverse and may be mediated through pathways other than the inhibition of acetylcholine esterase.
The effects of circulating angiotensin II levels on vascular gene expression in normotensive rats
Authors: Priestley Jessica , Fredrich Katherine , Beyer Andreas , Lombard Julian
Published: June 2021

Expression of mRNA for 84 genes implicated in hypertension was evaluated using pathway‐focused PCR arrays (SA Biosciences). Comparisons were made between salt‐sensitive (SS) vs. SS.13BN rats ± angiotensin II (ANG II) infusion (5 ng/kg/min) and in Sprague‐Dawley (SD) rats fed low salt diet (LS), high salt diet (HS), or HS + ANG II. In SS vs. SS.13BN, significant changes (p < 0.05) were observed for 21 genes with tendency for up‐ or down‐regulation (0.05 < p < 0.10) in 15 others. In SD, expression changes occurred in 16 genes, with trends towards up‐ or down‐regulation in 19 others. Angiotensin converting enzyme (ACE) expression was higher with elevated ANG II levels. Angiotensinogen was down‐regulated in SD rats fed HS but not in SS vs. SS.13BN. Several genes important to vasoreactivity showed altered expression with variations in circulating ANG II, including those important in the endothelin (ET) system, e.g., the ETB receptor was down‐regulated in HS vs. LS SD and in SS.13BN vs. SS. In SS.13BN, ET1 and endothelin converting enzyme 1 were down‐regulated vs. SS. The PGI2 receptor was down‐regulated in SS vs. SS.13BN and unchanged in SD. The B2 bradykinin receptor was up‐regulated in HS + ANG II vs. HS SD. These results indicate that plasma ANG II levels affect the expression of genes in cerebral arteries, but there are significant differences in the effect of ANG II on gene expression among strains. [NIH #HL65289 and HL92026].
Cilostazol, a PDE3 (phosphodiesterase 3) inhibitor, enhances prostacyclin receptor‐mediated cAMP accumulation and ATP release in erythrocytes (RBCs) of healthy humans (HH) and humans with type 2 diabetes (DM2)
Authors: Knebel Stephanie , Bowles Elizabeth , Sprague Randy , Stephenson Alan
Published: June 2021

Erythrocytes (RBCs) release ATP when exposed to low oxygen tension (O2), thus matching O2 supply with O2 need in skeletal muscle. Activation of a different pathway, the Gs‐coupled prostacyclin receptor (IPR), results in cAMP accumulation and ATP release from RBCs of HH and DM2. DM2 RBCs, however, demonstrate higher levels of cAMP accumulation and ATP release upon incubation with prostacyclin analog, iloprost (ILO 1μM), when compared to HH RBCs. Previously, we established that PDE3 hydrolyzes cAMP in the IPR‐mediated pathway, limiting cAMP accumulation, and therefore, ATP release from RBCs. Here we investigate the hypothesis that PDE3 activity is decreased in DM2 RBCs, which would explain the increased ILO‐induced cAMP accumulation and ATP release. Pretreatment of RBCs with the selective PDE3 inhibitor cilostazol eliminated differences in ILO‐induced cAMP accumulation and ATP release between DM2 and HH RBCs. Similar results were seen using the prostacyclin analog UT15C. These data suggest that decreased PDE3 activity in DM2 RBCs is responsible for increased cAMP levels and ATP release upon IPR activation. Supported by grants from United Therapeutics and American Diabetes Association.
The role of protease‐activated receptor‐4 in vascular smooth muscle cell migration and proliferation
Authors: Epperson Tenille , Weekes Danielle , Tillery Lakeisha , Mantey Jane , Austin Anita , Motley-Johnson Evangeline
Published: June 2021

Recent studies have shown that thrombin stimulates the activation of extracellular regulated kinase (ERK1/2) and Rho/Rho kinase, which are associated with cellular proliferation and migration in vascular diseases such as hypertrophy and atherosclerosis. Thrombin activates these kinases through protease‐activated receptors (PARs), which are a family of G‐protein coupled receptors activated by the enzymatic cleavage of the extracellular NH2 terminus. After verifying that PAR‐4 was expressed in vascular smooth muscle cells (VSMCs), we used a PAR‐4 activating peptide (AYPGKF) to stimulate the cells in a time‐ and concentration‐dependent manner. Using Western blot analysis, we observed that 25uM of the PAR‐4 agonist maximally phosphorylated ERK1/2 at a time point of 15 minutes. To determine if PAR‐4‐induced phosphorylation of ERK1/2 was dependent on Rho kinase, we pretreated VSMCs with 25uM of the Rho kinase inhibitor (Y27632) for one hour and then stimulated the cells with 25uM of AYPGKF for 15 minutes or 5U/ml of thrombin for 5 minutes. ERK1/2 phosphorylation was significantly attenuated in the presence of the Rho kinase inhibitor when stimulated with PAR‐4. In addition, pretreatment with the rho kinase inhibitor significantly decreased thrombin‐induced ERK1/2 phosphorylation. We demonstrated that PAR‐4 signals via Rho kinase in the phosphorylation of ERK1/2. We conclude that PAR‐4 may be a key mediator in the cellular migration and/or proliferation of VSMCs.

NIH‐NIGMS‐SCORE
Antagonism between PKA and Epac signaling is involved in the regulation of PGE2‐induced ICAM‐1 expression in bEnd.3 cells
Authors: Park Tae Yeop , Lee Kwang Min , Kim Jae Mi , Jung Yi-Sook , Baik Eun Joo , Moon Chang-Hyun , Lee Soo Hwan
Published: June 2021

Increased leukocyte adhesion and infiltration under various pathological conditions is accompanied by increased expression of endothelial ICAM‐1 and the blockade of ICAM‐1 ligation limits leukocyte infiltration and brain damage. PGE2 is known to be the principal pro‐inflammatory prostanoid and play important roles in brain diseases through binding to EP1‐4 receptors. However, there have been contradictory reports on its actions during inflammation processes, especially in vascular endothelial cells. In this study, we investigated the roles of PGE2 in the expression of ICAM‐1 in bEnd.3 cells and therein involved signaling pathways. We investigated that elevation of cAMP is necessary for PGE2‐induced ICAM‐1 expression in bEnd.3 cells. We determined effect of PGE2 on Epac activation using Rap1‐GTP pull down assay and effect of Epac on PI3K/Akt activation as well as ICAM‐1 expression. Next, we showed that loss of Akt activity results in inhibition of PGE2‐induced NF‐κB activation. On the other hand, interestingly enough, PKA inhibitor stimulated Akt phosphorylation and ICAM‐1 expression and specific activator inhibited PGE2‐induced ICAM‐1 expression and Akt phosphorylation. Taken together, these data suggest that this antagonism between PKA and Epac signaling is importantly involved in the regulation of PI3K/Akt signaling axis, and thereby ICAM‐1 expression in cerebrovascular endothelial cells.

This study was supported by grants from Ajou University School of Medicine and Gyunggi‐do through CCRB‐GRRC
S‐nitrosylation of β‐catenin and p‐120 catenin: a novel regulatory mechanism in endothelial hyperpermeability
Authors: Zamorano Patricia , Marin N , Carrasco R , Boric Mauricio , Durán Walter N. , Sánchez Fabiola A.
Published: June 2021

Platelet‐activating factor (PAF) increases endothelial permeability (hyperpermeability) via endothelial NOS (eNOS), which stimulates cGMP production to enhance permeability. We explored the complementary hypothesis that PAF‐stimulated hyperpermeability involves eNOS‐derived NO induced S‐nitrosylation of adherens junction proteins. We measured PAF‐stimulated S‐nitrosylation of β‐catenin and p120‐catenin (p120) in 3 cell lines: ECV‐eNOSGFP, EAhy926 and CVEC. S‐nitrosylation reduced expression of β‐catenin and p120 at the adherens junction and enhanced permeability. To ascertain the significance of eNOS location, we used ECV‐304 cells transfected with constructs that target eNOS to the cytosol (G2AeNOSGFP) and plasma membrane (CAAXeNOSGFP). We detected S‐nitrosylation of β‐catenin and p120 only in ECV‐GFPeNOS‐G2A, i.e., in cells with cytosolic eNOS. PAF‐stimulated S‐nitrosylation decreased the association between β‐catenin and p‐120. PAF significantly decreased the association between β‐catenin and p‐120 only in ECV‐GFPeNOS‐G2A. Inhibitors of NO production and of S‐nitrosylation blocked PAF‐induced S‐nitrosylation and hyperpermeability. Our results demonstrate that agonist‐induced S‐nitrosylation contributes to junctional membrane protein changes that enhance endothelial permeability.