bluuebunny/crossref_metadata_2025 huggingface dataset:
VOLTAGE‐GATED SODIUM CHANNEL ACTIVATION TRIGGERS CALCIUM INFLUX AND HIPPOCAMPAL NEURON COMPLEXITY
Authors: Gomez Dina Akasheh , Murray Thomas F.
Published: June 2021

Background and significance

After stroke, the molecular mechanisms underlying recovery are similar to the development of the nervous system. During development, neuronal activity regulates both the morphology and connectivity of neurons. Neuronal activity primarily involves voltage‐gated sodium channel (VGNaC) activation. Calcium signaling through the N‐methyl‐D‐aspartate receptor (NMDAR) drives morphological changes such as, dendritic arborization, synaptogenesis, and spinogenesis. VGNaC activation influences NMDAR function making this pathway an attractive therapeutic target for stroke recovery.

Hypothesis

Activation of VGNaCs by PbTx‐2 augments NMDAR signaling resulting in and increased Ca2+ influx leading to dendritic arborization and spinogenesis in hippocampal neurons.

Experimental design and Methods

We used the VGNaC gating modifier, brevetoxin (PbTx‐2), to assess the effects of VGNaC activation on neuronal morphology. We defined the calcium‐signaling pathway triggered by PbTx‐2 in a population of hippocampal neurons (HNs), and evaluated PbTx‐2‐induced effects on dendritic arborization and spinogenesis in organotypic hippocampal slice culture (OHSC).

HNs

cells were prepared from E18 Swiss Webster mouse embryos and plated in 96‐well plates. All experiments were run on (10 Days in vitro). A FlexStation2 was used to monitor PbTx‐2‐induced Ca2+ influx.

OHSC

Slices were prepared from P5, Thy‐1 YFP transgenic, mouse pups. Slices were treated with Pbtx‐2 18H after plating. A Leica SP8 confocal microscope was used to generate Z‐stack images of neurons or dendrites. Imaris‐XT software was used to reconstruct 3D‐images of the dendritic arbor, run Sholl analysis, and measure spine density.

Results and Conclusion

In HNs, the EC50 for PbTx‐2‐induced Ca2+ influx was 376 nM (95% CI, 281–504 nM). The PbTx‐2 triggered Ca2+ influx in HNs was through the reverse mode of operation of Na+/Ca2+ exchanger, the NMDAR and the L‐type Ca2+ channel pathways. In OHSC, four‐day treatment with PbTx‐2 (100 nM) produced significant increases in dendritic arbor complexity as determined by Sholl analysis (p<0.005, ANOVA). PbTx‐2 treatment (300 nM) produced significant increases in spine density (p<0.001, ANOVA). Modifying the gating properties of VGNaC using PbTx‐2 enhanced the morphology of hippocampal neurons. Overall, these studies are consistent with the hypothesis that VGNaC activation may represent a novel pharmacological strategy to regulate NMDAR function and aid in recovery post‐stroke.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparison of agonist occupancy of M2 muscarinic receptor‐G protein complexes in myocardial homogenates with functional estimates of active receptor‐state affinity in isolated atria
Authors: Ehlert Frederick J.
Published: June 2021

We have recently developed methods for estimating the active (Kact) and inactive (Kinact) receptor‐state affinity constants of ligands in functional studies on G protein‐coupled receptors. Our approach is valid for in vitro responses measured downstream from receptor activation, such as second messenger signaling in cell lines and responses in isolated tissues. The data required for our analysis include agonist‐mediated responses measured in the absence and presence of reduced receptor expression and modulation with an allosteric agonist (J Pharmacol Toxicol Methods 69:253–279, 2014). We also described an analogous technique that employs a constitutively active receptor mutant (J Pharmacol Toxicol Methods 83:94–106, 2017) instead of an allosteric agonist. In this study, we sought to validate our functional determination of Kact by using an independent biophysical method for estimating Kact. It is well known that muscarinic agonists exhibit high affinity for a subset of M2 muscarinic receptors in myocardial homogenates as measured in competitive binding experiments with [3H]N‐methylscopolamine. This subset of muscarinic binding sites is thought to represent a receptor‐Gi/o protein complex. We measured agonist binding to this site directly using the agonist radioligand, [3H]oxotremorine‐M, and completed agonist/[3H]oxotremorine‐M competitive binding experiments to estimate ligand affinity for this site in the guinea pig myocardium. We estimated a log affinity constant of approximately 8.2 for oxotremorine‐M in competitive binding experiments with [3H]oxotremorine‐M in the presence of 140 mM Na+. The allosteric agonist, LY211,9620 caused about a twofold increase in the affinity of oxotremorine‐for this site. In the presence of low sodium, LY211,9620 had little effect on oxotremorine‐M affinity, suggesting that in presence of both LY211,9620 and low sodium, the observed affinity of oxotremorine‐M is nearly the same as the active state affinity. Under these conditions, the log affinity constant of oxotremorine‐M was approximately 8.8. This value was similar to that estimated in functional studies on the atria (log Kact, 9.1 ± 0.10). These functional studies also provided estimates of the log Kact and Kinact values of the allosteric agonist LY211,9620 (6.3 and 5.1, respectively), the log isomerization constant of the unoccupied M2 muscarinic receptor (Kq, −3.18 ± 0.11), and the log sensitivity constant of the signaling pathway (log KE, 1.87 ± 0.27). Receptor‐state affinity constants provide a more powerful approach to structure activity relationships and to the measurement of agonist bias.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Mechanistic Role of Metal Ions, Ca2+ and Mg2+, in RGS: G‐Protein Interactions
Authors: O'Brien Joseph , Sieng Monita , Hayes Michael , Fowler Andrew , Lyon Angeline , Roman David
Published: June 2021

Regulator of G protein signaling (RGS) proteins are negative regulators of G protein‐coupled receptor (GPCR) signaling through their ability to act as GTPase activating proteins (GAPs) for activated Gα subunits. The RZ subfamily, of which RGS17 is a member, binds to activated Gαo, Gαz, and Gαi1–3 proteins to modulate downstream pathways, including those involved in formation of cyclic AMP and Ca2+‐dependent signaling. In contrast to other RGS proteins, less is known about the regulation of RZ family members. Both crystallization and 1H‐15N 2D HSQC NMR experiments revealed a putative interaction of the metal ion Ca2+ with RGS17 at a defined binding site. Subsequent protein‐protein interaction experiments, using AlphaScreen were used to assess the impact on the ions Ca2+ and Mg2+ on the RGS17 interaction with activated Gαo. The results indicate that both calcium and magnesium have an effect of promoting the RGS17 ‐ Gαo interaction. These studies will extend to examining the selectivity and affinity of RGS17 for other physiologically relevant divalent metal cations, such as Zn2+, Cu2+, and Mn2+. In addition, the residues of RGS17 that bind Ca2+ are conserved in multiple RGS proteins. The functional impact of metal ion binding is likely not limited to RGS17 and a more in‐depth evaluation of these proteins for metal binding deserves further attention.

Support or Funding Information

This work was supported by The University of Iowa Center for Biocatalysis and Bioprocessing and the NIH‐sponsored Predoctoral Training Program in Biotechnology (2 T32 GM008365).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dynamin internalizes tyrosine phosphorylated sphingosine 1 phosphate receptor 1 and impair downstream signaling
Authors: Anwar Mumtaz , Amin Md Ruhul , Mehta Dolly
Published: June 2021

We recently showed that S1P induces S1PR1 phosphorylation at tyrosine 143 residue impairing S1PR1 cell‐surface expression. Here we determined if phosphorylation alters receptor trafficking to the cell‐surface and if so what is the mechanism. The GTPase dynamin is essential for both clathrin‐dependent and clathrin‐independent‐endocytosis of receptors and thereby regulates the abundance of the receptor on the plasma membrane and downstream signaling. Thus, we inhibited dynamin using dynasore, which blocks dynamin GTPase activity in HEK cells transducing native S1PR1, phosphodefective (Y143F)‐S1PR1 and phosphomimicking (Y143D)‐S1PR1 and assessed S1PR1 spatial localization using confocal imaging. Interestingly, we found that inhibition of dynamin function blocked the internalization of Y143D‐S1PR1 leading to cell surface retention of the receptor. Cell‐surface retained S1PR1 induces signaling events by binding Gi heterotrimeric protein which includes calcium mobilization, cAMP generation, activation of MAP kinases. Thus, we assessed if phosphorylation altered S1PR1 downstream signaling. We observed that S1P‐induced enhanced Ca2+ entry in HEK cells expressing Y143D‐S1PR1 in Gi‐dependent manner indicating phosphorylated receptor binds Gi and dynamin and the internalized complex is functional. Further studies are under way to characterize the role of dynamin interaction with Y143 phosphorylated receptor in regulating S1PR1 signaling and endothelial barrier repair function.

Support or Funding Information

NIH

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Crystal Structure of the C‐terminal Guanine Exchange Factor Module of Trio Reveals its Oncogenic Potential
Authors: Bandekar Sumit
Published: June 2021

The C‐terminal guanine exchange factor module of Trio (TrioC) serves as a link between the heterotrimeric G proteins Gαq/11 and the small G‐protein RhoA, joining Gαq/11‐coupled GPCRs to downstream events of cell motility and gene transcription. This ancient signal transduction axis has been identified as crucial to the rise and spread of the fatal malignancy uveal melanoma. Previous studies have shown that TrioC is likely regulated via an autoinhibitory constraint that is released upon binding of Gαq/11. However, the structural determinants of this autoinhibition remain unclear. We have determined the crystal structure of TrioC in its basal state, revealing high‐resolution detail of autoinhibition mediated by the Pleckstrin Homology (PH) domain. We show that truncation of the PH domain activates the module in vitro, showcasing the importance of the αN helix found at the DH/PH junction. We then show using two orthogonal biochemical assays that the disruption of the autoinhibited conformation by point mutations destabilizes and activates the module. Finally, we show that mutations in αN found in cancer patients hyperactivate TrioC. These point mutations likely increase mitogenic signaling through the RhoA axis and may thus represent the first known cancer drivers identified in Trio, operating in an analogous, yet Gαq/11 independent manner. We are in the process of confirming this hypothesis using cell‐based assays.

Support or Funding Information

Rackham Graduate Student Research Grant, University of Michigan Horace H. Rackham Graduate School

Pharmacological Sciences Training Program Fellow, 5T32GM007767–38

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulator of G‐protein Signaling (RGS10) regulates the expression of COX‐2 and TNF‐α in microglia via G‐protein independent mechanism
Authors: Wendimu Menbere Y. , Alqinyah Mohammed , Hooks Shelley
Published: June 2021

Regulators of G‐protein signaling (RGS) are a family of proteins that classically act to inhibit signaling downstream of heterotrimeric G‐proteins. RGS10, a member of the R12 subfamily, has been shown to selectively target G alpha‐i and is enriched in immune cells, such as peripheral macrophages and microglia. Previous reports have characterized RGS10 as an anti‐inflammatory protein, playing a protective role in the nervous and immune system. Specifically, loss of RGS10 in microglia amplifies inflammatory cytokine production such as tumor necrosis factor alpha (TNF‐α) and leads to microglia‐induced neurotoxicity. However, the mechanism by which RGS10 mediates these effects is unknown and has not been linked to a GPCR pathway. In addition to inflammatory cytokines, inflammation and neurotoxicity are enhanced and maintained by the induction of the inflammatory enzyme cyclooxygenase‐2 (COX‐2) which is essential for the production of inflammatory molecules such as prostaglandin E2 (PGE2). However, it is not known if RGS10 influences COX‐2 and PGE2 production. The goal of this study was to directly investigate the role of RGS10 on the expression and function of COX‐2 in microglia, and further determine if RGS10 acts in a canonical G‐protein dependent or G‐protein independent mechanism to regulate inflammatory signaling. Our data revealed that RGS10 deficiency significantly elevates COX‐2 mRNA and protein levels, and enhances the release of PGE2 from microglia. Furthermore, the elevated TNF‐α and COX‐2 signal resulting from RGS10 loss was not affected by G‐protein inhibition, and a GAP‐deficient RGS10 mutant was as effective in inhibiting TNF‐alpha and COX‐2 expression as wild type RGS10, suggesting that the anti‐inflammatory functions of RGS10 are not mediated by its classic action on G‐proteins.

Support or Funding Information

National Institute of Health (NINDS)

National Multiple Sclerosis Society

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Protein Dynamics in Selectivity of Thiadiazolidinone Inhibition of RGS Proteins
Authors: Shaw Vincent , Mohammadiarani Hossein , Vashisth Harish , Neubig Richard
Published: June 2021

Background

Regulator of G‐protein Signaling (RGS) proteins play a critical role in GPCR (G‐protein Coupled Receptor) signaling by binding to active, GTP‐bound Gα subunits and accelerating GTP hydrolysis, thus terminating signaling. There are multiple RGS isoforms with signature tissue distributions, so by targeting RGS proteins in an isoform‐specific manner, better tissue specificity of pharmacological effects may be achieved than by global application of a GPCR agonist. Thiadiazolidinones (TDZDs) are a series of compounds that inhibit RGS proteins by covalent modification of cysteine residues. They are very potent against RGS4, but also act less potently on several other cysteine‐containing RGS proteins. RGS4, RGS8, and RGS19, three proteins that are inhibited by TDZDs, share a cysteine on the α4 helix of the RGS homology domain. RGS19 has only this cysteine, while RGS4 and RGS8 have additional cysteines, including a shared one on the α6‐α7 interhelical loop. Interestingly, these shared cysteines are not exposed to solvent in crystal structures.

Hypothesis

Differences in potency due to covalent modification of the shared cysteine at the α4 helix of RGS proteins is driven by differences in flexibility.

Approach

The rate of covalent modification at individual cysteines can be determined by treating proteins with iodoacetamide, digestion, and detection of adduct by mass spectrometry. In addition, the effect of modification at different cysteines on protein function can be evaluated by measuring TDZD inhibition of binding between Gα and cysteine mutants of RGS proteins.

Results

Deuterium exchange and molecular dynamics studies have shown differential flexibility in key regions of these RGS proteins, which may be necessary for TDZD access to buried cysteines. RGS4 is more potently inhibited by CCG‐50014 (IC50: 81 nM) than RSG19 (IC50: 1.1 μM) or RGS8 (IC50: 1.8 μM). However, when RGS4 or RGS8 are mutated to contain only the α4 helical cysteine, the potency of TDZD inhibition is drastically reduced to below that of RGS19 (RGS4 IC50: 8.5 μM, RGS8 IC50 >100 μM). In accordance with previous molecular simulation data showing this α4 cysteine to be least solvent accessible in RGS4, it had a lower degree of covalent adduct formation by iodoacetamide than other cysteines.

Conclusion

These results indicate that while additional cysteines in RGS4 may be responsible for its high potency of inhibition by TDZDs, differences in the rate of covalent modification and subsequent potency of inhibition at the conserved α4 cysteine is driven by differences in RGS protein flexibility.

Support or Funding Information

NIH T32 GM092715NSF 1507588

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Silencing TRPC6 Gene Attenuates Hypertension‐induced Renal Injury in 5/6 Ablation/Infarction Model
Authors: Wang Weili , Lyu Bingqing , Li Pin‐Lan , Li Ningjun
Published: June 2021

The transient receptor potential canonical 6 (TRPC6) channel is a member of the TRPC family. A “gain of function” mutation of TRPC6 causes familial focal segmental glomerulosclerosis (FSGS) and that overexpression of TRPC6 in podocytes causes a kidney disease consistent with FSGS in animals. Moreover, TRPC6 participates in the mechanical or pressure sensation in various cell types, including in the podocyte slit diaphragm. Therefore, TRPC6 may be the upstream mediator that transmits the pressure stress into downstream molecular pathways to cause hypertension‐induced renal injury. The present study tested the hypothesis that up‐regulated TRPC6 in the kidney mediates the hypertension‐induced renal injury in rats with 5/6 renal ablation/infarction (A/I), a model in which the renal injury is closely associated with blood pressure. Adults male Sprague Dawley rats were divided into shame group, 5/6 A/I control group and 5/6 A/I shRNA group. The left kidneys of shame and 5/6 A/I control groups were treated with intra‐renal transfection of control plasmids and the 5/6 A/I shRNA group TRPC6 shRNA plasmids. The results showed that the levels of renal TRPC6 was increased in 5/6 A/I control group compared with shame group and that the increase of TRPC6 was blocked in 5/6 A/I shRNA groups, the relative protein levels of TRPC6 were 1.0±0.26, 3.9±0.16 and 1.4±0.17 in shame, 5/6 A/I control and 5/6 A/I shRNA groups, respectively. TRPC6 shRNA had no effect on hypertension in 5/6 A/I rats (MAP: 153.9 ± 8.1 mmHg in control groups vs. 154.7 ± 8.9 mmHg in shRNA groups). However, the albuminuria was attenuated in 5/6 A/I shRNA group compared with 5/6 A/I control group. The levels of urinary albumin were 0.8±0.34 mg/Kg/24h in shame, 20.82± 3.7 mg/Kg/24h in 5/6 A/I control and 11.9±1.5 mg/Kg/24h in 5/6 A/I shRNA group, respectively (P=0.037 for control vs. shRNA groups). The regression slop between the blood pressure and the % of glomerulosclerosis was significantly steeper in 5/6 A/I control group than in 5/6 A/I shRNA group (P=0.028 by ANCOVA), indicating a significant attenuation of hypertension‐induced glomerular injuries. These data suggested that hypertension‐induced activation of TRPC6 may be the molecular sensor of pressure stress to produce kidney damage associated with hypertension. (Supported by NIH grants DK107991 to NL and DK54927 to PL).

Support or Funding Information

NIH grants DK107991 to NL and DK54927 to PL

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NFE2L2 (Nrf2) attenuates Tgf‐β1 signaling by elevation of Smad7 in mouse mesangial MES‐13 cells
Authors: Song Min‐Kyun , Ryoo In‐Geun , Ryu Dayoung , Lee Sang‐Hwan , Kwak Mi‐Kyoung
Published: June 2021

Renal fibrosis is the principal process underlying the progression of kidney diseases. Accumulating evidence indicates that oxidative stress plays a crucial role in renal fibrotic diseases. Transforming growth factor‐β1 (Tgf‐β1) is a potent stimulator of profibrotic responses, including expression of fibrosis markers such as extracellular matrix (ECM) and α‐smooth muscle actin (α‐SMA) through a Smad‐dependent pathway. Therefore, Smad‐dependent Tgf‐β1 signaling is known to play pivotal roles in renal fibrosis. In this regard, we investigated the potential involvement of nuclear factor (erythroid‐derived 2)‐like 2 (NFE2L2; Nrf2) in renal fibrosis with Tgf‐β1–Smad signaling. We previously reported that Smad7, a negative regulator of Smad signaling, may be a linker molecule between the Nrf2 pathway and Tgf‐β1 signaling in human renal tubular epithelial HK2 cell. In this study, we confirmed that elevated protein level of Smad7 attenuates Tgf‐β1 signaling by degradation of Tgf‐β1 Receptor 1 (TβR1) in Kelch‐like ECH‐associated protein 1 (Keap1) knockdown mouse mesangial MES‐13 cell, a cellular model of Nrf2 overexpression. As one of potential underlying molecular mechanisms of Smad7 regulation by Nrf2, reduced level of Smad‐ubiquitination‐regulatory factor 1 (Smurf1), an E3 ubiquitin ligase for Smad7, was remarkable. Further, we demonstrated that a Nrf2 inducer bardoxolone methyl ester (BARD), which is on clinical trials for kidney diseases, also elevated Smad7 level and attenuated Tgf‐β1 signaling in MES‐13. Collectively, these results indicate that Nrf2 can be an effective inhibitor of Smad‐dependent Tgf‐β1 signaling, and imply the beneficial role of Nrf2 in renal fibrosis.

Support or Funding Information

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (NRF‐2015R1A2A1A10054384)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sex‐Unrelated Counteraction by Nicotine of the Endotoxemia‐Evoked Facilitation of Renal Vasodilator Capacity in Rats: Roles of α7/α4β2 nAChRs and HSP70
Authors: Wedn Abdalla M. , El‐Gowilly Sahar M. , El‐Mas Mahmoud M.
Published: June 2021

Nicotine has been shown to alleviate renal inflammation and injury induced by endotoxemia. The current study investigated (i) the sex specificity of the nicotine modulation of hemodynamic and renal vasodilatory responses to endotoxemia in rats, and (ii) the roles of α7 or α4β2 nAChRs and related HSP70/TNFα/iNOS signaling in the interaction. Endotoxemia was induced by i.p. administration of lipopolysaccharide (LPS, 5 mg/kg/day.) for 2 consecutive days and changes in systolic blood pressure (SBP, tail‐cuff plethysmography) and vasodilator responsiveness of isolated perfused kidney to acetylcholine (ACh) or 5′‐N‐ethylcarboxamidoadenosine (NECA, adenosine receptor agonist) were evaluated. Compared with saline values, LPS decreased SBP and increased renal vasodilations induced by cumulative bolus injections of ACh (0.01–7.29 nmol) or NECA (1.6–100 nmol). No changes in serum creatinine or urea were observed. These effects were not sexually differentiated as they were similarly observed in the two rat genders. The LPS facilitation of renal vasodilations were attenuated after concurrent administration of aminoguanidine (iNOS inhibitor, 50 mg/kg), pentoxifylline (TNFα inhibitor, 3 mg/kg), or nicotine (0.5, 1, or 2 mg/kg). We also report that the selective blockade of α7‐nAChRs (methyllycaconitine citrate, MLA) or α4β2‐nAChRs (dihydro‐β‐erythroidine, DHβE) (i) potentiated the enhancing action of LPS on ACh or NECA vasodilations, and (ii) abolished the depressant effect of nicotine on LPS facilitation of renal vasodilatory capacity. A similar inhibition of the nicotine effect was seen after inhibition of HSP70 by quercetin. Alternatively, LPS hypotension was eliminated in rats treated with the DHβE/nicotine or quercetin/nicotine regimen in contrast to no effect for nicotine alone or combined with MLA. These findings establish that nicotine offsets the LPS facilitation of the renal vasodilator propensity, an effect that is sex independent and possibly involves a crosstalk between HSP70 and nAChRs of the α7 and α4β2 types.

Support or Funding Information

Supported by the Science and Technology Development Fund, Egypt (STDF Grant No. 14895)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring Structural Conformations of Human P450 1A1 with Diverse Ligands
Authors: Bart Aaron G. , Scott Emily E.
Published: June 2021

The human P450 enzyme 1A1 (CYP1A1) is an extrahepatic P450 often expressed in tumor tissues where it may be clinically significant in procarcinogens and toxin bioactivation. Prototypical CYP1A1 ligands are planar and polycyclic aromatic compounds, such as the procarcinogen benzo[a]pyrene found in environmental combustion and tobacco smoke. The single crystal structure of CYP1A1 is with the polycyclic, planar molecule alpha‐napthoflavone (αNF) and reveals a narrow active site that is consistent with planar CYP1A1 ligands. However, a number of known CYP1A1 substrates and inhibitors do not adhere to these typical ligand features, including promising pro‐drugs in cancer therapy based on a duocarmycin scaffold and clinically used tyrosine kinase inhibitors. Such CYP1A1 ligands could not be accommodated in the active site of the CYP1A1/αNF structure. This study aims to determine how CYP1A1 binds atypical CYP1A1 substrates and inhibitors using co‐crystallization.

Potential ligands for co‐crystallization trials with CYP1A1 were selected based on structural diversity, focusing on those that are not compatible with the 1A1/αNF crystal structure active site dimensions. Refinement of these ligands was performed by ranking based on their ability to inhibit CYP1A1 metabolism of a pro‐luciferin substrate. Anticancer tyrosine kinase inhibitors (erlotinib, imatinib, and ponatinib) displayed nanomolar to low micromolar IC50 values. A series of antifungal azoles, which are generally potent P450 inhibitors, resulted in a range of inhibitory responses. Compact, lower MW clotrimazole, miconazole, and tioconazole exhibited sub‐nanomolar potency, but the much larger extended azoles ketoconazole and posaconazole were also potent nanomolar inhibitors. The azoles fluconazole and letrozole were significantly less potent inhibitors with micromolar IC50 values. The pro‐duocarmycin drug ICT2700 and antitumor benzothiazole GW 610 inhibited CYP1A1 in the sub‐micromolar to nanomolar range. Compounds displaying potent inhibition are being employed in cocrystallization and structure determination. The structural results with atypical CYP1A1 ligands should provide a more robust framework for both prediction of procarcinogen and toxin activation and the development of novel CYP1A1‐activated cancer therapies.

Support or Funding Information

This work was supported by National Institutes of Health Grants F37 GM076343 (to EES).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation of Non‐Caspase Mediated Mechanisms of Death in Response to Treatment with Novel Chalcone in Glioblastoma Cell Lines
Authors: Inbody Laura R. , Stang Christopher R.T. , Eichel Amanda M. , Orahood Cassandra B. , Dick Matt J. , Mohamed Ahmed H. , Hickey Mikaela C. , Khupse Rahul S. , Schneider Ryan A.
Published: June 2021

Despite recent advances in cancer treatments, there is still a paucity of therapeutic options for patients with glioblastoma. We previously reported the efficacy of the synthetic chalcone, RK6, against A‐172, T98G, and U87‐MG human glioblastoma cell lines (IC50 values: 10–20 μM). Despite the moderately potent anticancer activity of RK6, the mechanism of death and putative drug target has yet to be elucidated. Previous efforts to identify potential mechanisms of death included evaluation of executioner caspase activation and production of reactive oxygen species. However, these pathways were found to be not largely involved. As such, further investigation of non‐caspase mediated forms of cell death were performed.

To rule out necrosis as a mechanism of death for RK6, a trypan blue exclusion assay was conducted to examine membrane destabilization. Cellular membranes remained intact following treatment with RK6 at concentrations greater than the IC50 for six hours, suggesting necrosis is not the mechanism of death.

We then sought to determine if cysteine proteases such as calpains or cathepsins were involved in the observed cell death following RK6 exposure. For this, cells were co‐treated with RK6 along with selective and non‐selective calpain and cathepsin inhibitors followed by measurement of cell viability using the XTT cell viability assay. Sublethal concentrations of the calpain and cathepsin inhibitor, N‐Acetyl‐Leu‐Leu‐Norleucinal (ALLN), selective calpain inhibitor, calpeptin, and selective cathepsin B inhibitor, CA‐074‐Me, were used to interrogate these signaling pathways. Inhibition of calpains and cathepsins concomitantly using ALLN had no effect on the efficacy of RK6 in the A‐172, T98G, and U87‐MG glioblastoma cell lines. Similarly, inhibition of cathepsin B with CA‐074‐Me did not affect the efficacy of RK6. Interestingly, inhibition of calpains with calpeptin resulted in a synergistic cytotoxic effect in all three cell lines. The results of these studies suggest that inhibition of calpains further sensitizes the cells to RK6, however concomitant inhibition of calpain and cathepsins does not provide the same effect. Further investigations are ongoing to investigate the nature of the synergy between RK6 and calpeptin and the potential role of other forms of mediated cell death, such as necroptosis, caused by this synthetic chalcone.

Support or Funding Information

This project was supported in part by an American Foundation for Pharmaceutical Education Gateway to Research Award for the second author.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Local regulation of L‐type CaV1.2 channel and vascular reactivity by adenylyl cyclase 5 during diabetic hyperglycemia
Authors: Syed Arsalan U. , Gosh Debapriya , Prada Maria Paz , Reddy Gopireddy , Nystoriak Matthew A. , Chiamvimonvat Nipavan , Santana Luis F. , Xiang Yang Kevin , Nieves‐Cintrón Madeline , Navedo Manuel F.
Published: June 2021

Diabetes mellitus is a debilitating metabolic disorder affecting millions of people worldwide. One of the hallmark symptoms of diabetes is hyperglycemia (elevated blood glucose levels; HG). At least 60% of the diabetic patient develop hypertension and become susceptible to both macro and microvascular complications, including stroke. HG is associated with excessive vascular tone and dysfunction of the blood vessels. Recently, we demonstrated that activation of protein kinase A (PKA) leads to phosphorylation and potentiation of L‐type CaV1.2 channels in vascular smooth muscle resulting is increased vasoconstriction during HG. A fundamental gap in knowledge that remains unexplored is the upstream mechanisms underlying PKA activation. Here, we describe for the first time a critical role for local cAMP production by adenylyl cyclase (AC) isoform 5, upstream of PKA, in regulation and potentiation of CaV1.2 channels leading to vasoconstriction during HG. Consistent with this, membrane‐targeted fluorescence resonance energy transfer (FRET) biosensor for cAMP revealed a small but significant increase in cAMP production by HG, in agreement with local cAMP production. Treatment with forskolin (global cAMP activator) resulted in a significantly larger cAMP signal, aligned with cell wide global cAMP increase. In the presence of the specific AC5 and AC6 inhibitor 2,5,DDA or in vascular smooth muscle from AC5−/− mice, the HG‐induced cAMP increase was abolished. Yet, the forskolin‐induced global increase in cAMP production remained intact. Electrophysiology and pressure‐myography experiments demonstrated that the 2,5 DDA compound and genetic ablation of AC5, but not AC6, abrogated the increased in L‐type CaV1.2 channel activity and vasoconstriction in response to HG. Super‐resolution microscopy and proximity ligation assay confirmed close association between AC5 and L‐type CaV1.2 channels. Taken together, these finding demonstrate a novel role of local AC5 signaling in HG‐induced vasoconstriction due to increased CaV1.2 activity leading to vasoconstriction during diabetic hyperglycemia.

Support or Funding Information

NIH R01HL098200, R01HL121059 and NIH T32‐HL086350

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Developing and characterizing human biomimetic arteriole for studying pulmonary hypertension
Authors: Bhatta Anil , Pagaduan Jayson V. , Chen Xing , West‐Foyle Hoku , Hou Annie , Liu Jiayu , Jin Qianru , Nguyen Thao D. , Kuo Scot , Berkowitz Dan , Gracias David H. , Romer Lewis H.
Published: June 2021

Arteriolar resistance vessels control flow to vascular beds. The structure, mechanics, and function of these blood vessels are critically important to our understanding of the mechanisms of cardiovascular disease pathogenesis. Additionally, in vitro arteriolar mimetic approaches are needed to improve the study of cardiovascular disease, because models based upon rodents vascular function and cells in standard culture containers do not accurately recapitulate the native multicellular architecture of a human vessel wall. Here, we describe novel, highly parallel, mass biofabrication of biomimetic tubular vessel constructs composed of biocompatible scaffold materials, pulmonary microvascular endothelial cells (HPMEC), extracellular matrix components including fibronectin and laminin 3, and human pulmonary artery smooth muscle cells (HPASMC). These constructs had a diameter of ~250 microns, and a length of 1–3 mm. Cells were incorporated into these constructs in patterns that mimic the layering and relative alignment observed in human pulmonary arterioles. Finite Element Analysis showed that the ultra‐thin cylindrical scaffold materials used here could be displaced by the cells in these biomimetic vascular walls to an extent that was similar to displacements of thicker tube composed of softer materials. HPMEC in these biomimetic vessel constructs produced more nitric oxide, and demonstrated higher phosophorylation levels of key nitric oxide signaling proteins (eNOS and Akt) than equal numbers of cells grown on equal surface areas that were flat. Additionally, HPASMC were aligned at tunable angles that mimicked in‐vivo organization using biomolecular micropatterning. Thus, biomimetic arterioles produced in this study exhibited improved endothelial functionality together with multi‐cellular layering and anatomically accurate cellular alignment, and provide a platform for investigation of microvascular function.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modulation of mean arterial pressure and diuresis by renomedullary infusion of selective inhibitors of fatty acid amide hydrolase
Authors: Ahmad Ashfaq , Dempsey Sara K. , Daneva Zdravka , Poklis Justin L. , Li Ningjun , Li Pin Lan , Ritter Joseph K.
Published: June 2021

The kidneys are involved in the long term regulation of blood pressure and diuresis, which may involve various systems along with the endocannabinoid (EC) system. The EC system consists of N‐arachidonoylethanolamine (anandamide, AEA) and 2‐arachidonoylglycerol (2‐AG), cannabinoid receptors (CB‐1 and CB‐2) and hydrolyzing enzymes like fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). Previously we showed that dual inhibition of FAAH and MAGL in the renal medulla increased urinary excretion (UV) and salt excretion without affecting mean arterial pressure (MAP) and these responses are dependent on cyclooxygenase‐2 (COX‐2). The present study hypothesized that selective inhibition of FAAH in the kidney medulla of C57BL/6 mice can modulate MAP, UV, and salt excretory functions of the kidney and that these effects are COX‐2 and CB‐1 dependent. Renal medullary infusion of the selective FAAH inhibitor, PF‐3845(N‐3‐pyridinyl‐4‐[[3‐[[5‐(trifluoromethyl)‐2‐pyridinyl]oxy]phenyl]methyl]‐1‐piperidinecarboxamide), produced a significant drop in MAP only during the post treatment infusion phases but significant increases in UV and salt excretions (all P<0.05) at 15 and 30 nmol/min/kg and 30 nmol/min/kg, respectively, compared to the control pretreatment infusion phase (C1). The MAP‐lowering and UV‐stimulating effects of PF‐3845 were blocked by pretreatment of the mice with the CB‐1 antagonist, rimonabant (3 mg/kg, i.p.) which indicates the involvement of the CB‐1 receptor in both activities and these responses to PF‐3845 were not affected by pretreatment with celecoxib (15 mg/kg, i.v.). Intravenous administration of PF‐3845 significantly reduced MAP at 7.5, 15 and 30 nmol/min/kg (all P<0.05) and increased UV and salt excretion at 15 and 30 nmol/min/kg and 30 nmol/min/kg, respectively. Intramedullary administration of PF‐3845 in homozygous FAAH knock out (FAAH−/−) mice showed no change in MAP at any dose of PF‐3845 but, surprisingly, UV was increased at 7.5, 15 and 30 nmol/min/kg (P<0.05), indicating that the blood pressure‐lowering but not UV‐increasing effect of PF‐3845 was FAAH‐dependent. Analytical data showed significant increases (P<0.05) in AEA in kidneys of wild‐type mice treated with PF‐3845 versus sham‐treated controls. In summary, PF‐3845 administration into the renal medulla produces AEA which lowers MAP, increase urinary and salt excretion and these effects are dependent on CB‐1 receptors but independent of COX‐2 in C57BL/6.

Support or Funding Information

This work was supported by National Institute of Diabetes and Digestive and Kidney Disorders grant, DK102539

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
12‐LOX Oxylipins Derived from Omega‐3 Supplementation with Docosahexanoic Acid Potently Regulate Human Blood Platelets
Authors: Shami Ryan , Hawley Megan , West Joshua , Holman Ted , Holinstat Michael
Published: June 2021

Cardiovascular disease (CVD) is the leading cause of mortality in the United States in both men and women. Thrombotic‐associated ischemic injuries due to excessive platelet activation are largely responsible for the high level of death associated with CVD. Therapies that seek to pharmacologically inhibit platelet activation, aggregation, or clot stabilization, have the potential to reduce the high mortality rates associated with CVD but are also associated with an increased risk of bleeding. The oxygenated products of polyunsaturated fatty acids—oxylipins—have been shown to be critical regulators of platelet function. Previously, it has been shown that Docosahexanoic Acid (DHA) has antiplatelet activity; therefore, we hypothesize that DHA regulates platelet function at least in part through its 12‐lipoxygenase (12‐LOX) metabolites, 11‐ and 14‐ hydroxydocosahexaenoic acid (11‐ and 14‐HDHA). Our study sought to investigate the role that 12‐LOX derived products of DHA, 11‐ and 14‐ HDHA, have on human blood platelet aggregation and activation. Platelet aggregation, granule secretion, and integrin αIIbβ3 activation were measured following agonist stimulation in the presence of DHA, 11‐ or 14‐ HDHA. Both 11‐ and 14‐ HDHA were observed to dose‐dependently attenuate aggregation in response to sub‐maximal concentrations of either thrombin or collagen. Additionally, both 11‐ and 14‐ HDHA were observed to attenuate intracellular signaling as demonstrated by a decrease in granule secretion in platelets treated with DHA compared to vehicle control treated platelets. To further the study, the effects of DHA supplementation in vivo were investigated in wild‐type mice as well as mice lacking 12‐LOX. DHA was found to play an important role in regulation of platelet function in vivo. Future studies will help to elucidate the mechanism by which DHA and its metabolites inhibit platelet function and play a role in supplementation‐dependent regulation of blood clotting. Once completed this will be the first study to clearly define a role for DHA and its 12‐LOX metabolites in the regulation of platelet aggregation and give important insight into the benefits and risks of fish oil supplementation on the cardiovascular system.

Support or Funding Information

This work was funded in part through the National Institutes of Health grants R01 GM105671 (MH), R01 HL114405 (MH), R01 MD007880 (MH)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ovarian Cancer Cell Exosome Interactions with Platelets: A role for the Novel Transmembrane Protein SUSD2
Authors: Rodezno Tania G. , Stein Sarah J. , Graber Katelyn N. , Hora Kirby A. , Madeo Marianna , Roetman Jess J. , Gubbels Jennifer AA. , Larson Mark K.
Published: June 2021

Ovarian cancer (OvCa) is the deadliest gynecologic cancer and ranks as the seventh‐leading cause of cancer death among women. OvCa can metastasize without the aid of the circulatory or lymphatic system by traveling through the peritoneal fluid (ascites), adhering to the mesothelial lining, and spreading to neighboring organs. Furthermore, OvCa tumors can hijack platelets as a means to evade immune responses and medicinal therapies. Recently, a novel tumor cell surface protein, Sushi Domain Containing 2 (SUSD2), was correlated with increased OvCa survival rates, as well as lowered platelet adhesion. Given that OvCa tumor cells are bathed in relatively minimal levels of platelets in the ascites, we hypothesized that OvCa cells can interact with platelets distally via released exosomes, and that exosome‐platelet interaction can be inhibited by abundant SUSD2 presence on the tumor cells. To test this hypothesis, exosomes were harvested from OvCa‐derived cell line media supernatant, isolated via differential ultracentrifugation, and verified via Western blotting for exosome‐specific markers and flow cytometric gating on exosome‐sized cell particles. Exosomes derived from SUSD2‐knockdown cell lines were secreted at significantly higher amounts compared to cells with higher levels of SUSD2 expression. Similarly, exosomes from SUSD2‐knockdown cells had a more pronounced positive effect on agonist‐induced platelet activation as measured by GPIIb/IIIa integrin conformational activation, as well as increased platelet spreading on fibrinogen. The results suggest a mechanistic role of SUSD2 in OvCa exosome production and exosome‐platelet communication, and a link between cancer cell and platelet interaction that does not require proximal contact.

Support or Funding Information

Research was supported in part by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health (P20GM103443), as well as funding from the National Science Foundation/EPSCoR program [IIA‐1355423], the state of South Dakota, the Cynthia Ballentine‐Roesler Memorial Fund, and an undergraduate fellowship from the American Physiological Society.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Non‐invasive assessment of arterial stiffness correlates with age‐related vessel structure: a new Popmètre Device for Rats
Authors: Amaral Sandra Lia , Fabricio Mayara F. , Vidal Mateus R. , Ruiz Thalles F.R. , Zago Anderson S. , Vicentini Carlos A. , Jordão Maria T. , Michelini Lisete C.
Published: June 2021

Arterial stiffness measurement, evaluated by pulse wave velocity (PWV), is a predictor of cardiovascular events. Among the main factors that affect arterial stiffness are cross‐sectional area, medial wall thickening and wall‐to‐lumen ratio. Recently, a device using finger‐toe pulse wave velocity was validated for humans study, but to better understand the mechanisms that change arterial stiffness, it is necessary to use the arterial vessel for genic, proteic and histological analysis. Therefore, a new device – popmètre® – was designed for measurements of PWV in animals and it was not validated yet. The aim of this study was to correlate the PWV using a new device for animal studies with vessel structure in rats of different ages. Forty‐two Wistar rats were allocated into 3 groups of different ages: Young: 1 month; Adult: 4 months and Older: 12 months. Two photodiodes were positioned on the right thoracic and pelvic members for PWV measurement, on anesthetized rats. Then, aorta, carotid, femoral and renal arteries were extracted and prepared for histological analysis (cross sectional area – CSA and wall thickness). Results of PWV and morphometric analysis were then correlated using Pearson correlation test. As expected, PWV was 46% higher in adult rats compared with young rats and 20% higher in aged rats compared with adult ones. Cross sectional area was significantly higher in aged rats compared with young rats in aorta (+151%), carotid (+94%), femoral (+62%) and renal (251%) arteries. Wall thickness was not altered among groups. A significant correlation was observed between CSA and PWV in all arteries: aorta (r=0.5642; p<0.001); carotid (r=0.6854 p<0.001); femoral (r=0.6642 p<0.001) and renal ( r=0.7817; p<0.0001). Wall thickness did not correlate with PWV. The results of this present study allow us to suggest that PWV measurement, obtained by a new device pOpmètri® for small animals, was able to detect age‐related vessel structure in rats.

Support or Funding Information

São Paulo Research Foundation (FAPESP) and Coordination for the Improvement of Higher Education Personnel (CAPES).

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