bluuebunny/crossref_metadata_2025 huggingface dataset:
Neurochemical and Cardiovascular Effects of β‐Methylphenethylamine (BMPEA) Analogs Found in Dietary Supplements
Authors: Baumann Michael H. , Thorndike Eric B. , Rice Kenner C. , Schindler Charles W.
Published: June 2021

Dietary supplements often contain additives not listed on the label. β‐Methylphenethylamine (BMPEA, 2‐phenylpropan‐1‐amine), a structural isomer of the stimulant drug amphetamine, is one such additive. The objective of this study was to determine the neurochemical and cardiovascular effects of BMPEA and its analogs, N‐methyl‐2‐phenylpropan‐1‐amine (MPPA) and N,N‐dimethyl‐2‐phenylpropan‐1‐amine (DMPPA). Synaptosomes were prepared from rat caudate tissue for dopamine transporter (DAT) assays or from whole rat brain minus caudate and cerebellum for norepinephrine transporter (NET) assays. Five male Sprague‐Dawley rats received surgically‐implanted telemetry transmitters for the measurement of blood pressure (BP), heart rate (HR) and locomotor activity. Rats were placed into acoustical cubicles for 3 h each weekday, and drug or vehicle was administered on Tuesdays and Fridays. As expected, amphetamine was a potent substrate‐type releasing agent at DAT (EC50=5 nM) and NET (EC50=8 nM). Like amphetamine, BMPEA was a releaser at DAT (EC50=627 nM) and NET (EC50=125 nM), and MPPA had similar effects. DMPPA was a weak substrate only at NET (EC50=1337 nM). In general, the transmitter releasing actions of BMPEA analogs were more potent at NET than DAT. Amphetamine (0.3–3.0 mg/kg, sc) produced significant dose‐related increases in BP, HR and activity (all F[4,34] > 8.9, p < 0.05). By contrast, BMPEA, MPPA and DMPPA (3.0 and 30 mg/kg, sc) produced significant increases in BP (all F[3,27] > 4.1, p < 0.05) but failed to clearly affect HR or activity. The hypertensive effect of BMPEA was reversed by pretreatment with the α‐adrenergic antagonist prazosin. Our results show that BMPEA and its analogs are biologically active. Although these compounds are unlikely to be abused due to weak effects at DAT, they could produce adverse cardiovascular complications due to prominent transmitter releasing activity at NET.

Support or Funding Information

Supported by IRP, NIDA, NIH, DHHS.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dopaminergic Perturbations from Food Restriction and Exercise are Sex‐Dependently Amplified During Adolescence
Authors: Gilman T Lee , Owens W Anthony , George Christina M. , Metzel Lauren , Daws Lynette C.
Published: June 2021

Eating disorders, which are 2.5‐fold more prevalent in females than males, typically emerge during adolescence. Afflicting at least 3 percent of teenagers, eating disorders such as anorexia nervosa, bulimia nervosa, and binge eating disorder entail severe health consequences in addition to their psychological toll. Yet no effective treatments for eating disorders exist. An established regulator of both eating behaviors and physical activity, the dopaminergic system undergoes a sensitive maturation period during adolescence. However, studies into the role of the dopaminergic system in ontogeny of eating disorders are lacking, as are investigations into dopaminergic system maturation in adolescent females. Here we measured function of the dopamine transporter (DAT), a critical regulator of dopaminergic signaling, using both in vivo high‐speed chronoamperometry and locomotor assays of acute cocaine response. Employing an activity‐based anorexia paradigm for 4–5 days, we investigated how food restriction, free exercise on a running wheel, or the combination thereof impacted DAT function in adult (postnatal day 90) and adolescent (postnatal day 30) Sprague‐Dawley rats of both sexes. Food restriction alone or with exercise in adolescent rats of both sexes produced leftward shifts in the dose‐response to the locomotor‐promoting effects of cocaine, though the maximal response to cocaine was blunted in females within these two groups. In adults, only food restricted females exhibited a significant increase in maximal cocaine‐induced locomotor response. Clearance of dopamine in dorsal striatum was not significantly altered by food restriction and/or exercise in adults. In contrast, food restriction plus exercise significantly attenuated dopamine clearance in adolescent males, and food restriction alone or combined with exercise elicited pronounced dopamine clearance reductions in adolescent females. Together, these findings suggest that adolescent plasticity of the dopaminergic system confers – particularly in females – vulnerability to eating disorders and persistence of associated unhealthy behaviors (e.g., compulsive exercise). Therefore, drugs that enhance dopamine uptake or otherwise reduce dopamine signaling duration may prove efficacious in the treatment of emerging adolescent eating disorders. Ongoing experiments are evaluating striatal DAT expression in these animals, and future experiments will focus on optimizing longer term food restriction and exercise conditions in adolescents to facilitate investigation of pharmacologic interventions.

Support or Funding Information

This research was supported by R21 DA038504 to LCD. TLG is supported by T32 DA031115 to Charles P. France.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Behavioral Effects of α2/α3 Subtype‐Selective GABAA Receptor Positive Allosteric Modulators
Authors: Lewter Lakeisha , Cook James M. , Li Jun‐Xu
Published: June 2021

Millions of individuals suffer from chronic pain. Opioids are effective for treating chronic pain but their use is limited due to undesirable side‐effects. Previous studies have shown that different subunits of the GABAA receptor are associated with different actions of the benzodiazepine site. While α2/α3 subunit‐containing GABAA receptors mediate antinociceptive effects, α1 subunit‐containing GABAA receptors mediate sedative and abuse‐related effects. This study sought to examine the behavioral effects of two α2/α3‐subtype selective PAMs: KRM‐II‐81 and NS16085. In a rat model of inflammatory pain (complete Freund's adjuvant) and a rat model of neuropathic pain (chronic constriction injury), both PAMs and the classical benzodiazepine midazolam attenuated mechanical hyperalgesia (as measured by von Frey test). In the procedure of food‐maintained operant responding, KRM‐II‐81 and NS16085 did not significantly decrease the response rate at doses that produced maximal antinociception. Contrastingly, midazolam significantly reduced the response rate at doses that attenuated mechanical hyperalgesia. In a horizontal wire test aimed to measure muscle relaxation, within the dose range that produced antinociception, only midazolam dose‐dependently increased the percentage of rats unable to grasp the wire, indicating muscle‐relaxant activity. These behavioral effects can be attenuated by benzodiazepine receptor antagonist flumazenil, confirming that the behavioral effects of these subtype‐selective GABAA PAMs are mediated through the benzodiazepine site of GABAA receptors. Taken together, while midazolam produced antinociceptive, rate‐suppressing, and muscle‐relaxant activity at similar doses, both subtype‐selective GABAA receptor PAMs selectively produced antinocicpetive effects. Collectively, these data support the notion of α2/α3‐subtype selective GABAA PAMs as novel analgesics.

Support or Funding Information

R01DA034806

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 Neuropeptide Receptor System, BigLEN‐GPR171, Interacts with the Opioid System to Relieve Pain
Authors: Afrose Leela , Gomes Ivone , Devi Lakshmi A. , Bobeck Erin N.
Published: June 2021

ProSAAS derived peptides, such as BigLEN, are abundantly expressed throughout the brain. We recently identified BigLEN as the endogenous peptide for the orphan receptor GPR171. Immunohistochemical analysis shows that both BigLEN and GPR171 are highly expressed in areas of the brain involved in pain modulation, including the periaqueductal gray (PAG). In addition, we find that GPR171 is co‐localized with mu opioid receptors (MOPr) in the ventrolateral PAG. Also, using immunohistochemistry we have identified that GPR171 is expressed in a portion of vGLut2 and GAD67 positive neurons. This brain region is especially known to contribute to opioid antinociception and tolerance. Given the expression pattern of GPR171, we hypothesized that GPR171 contributes to antinociception and tolerance. To test this, mice were injected with a GPR171 antagonist (MS21570; 5 mg/kg, i.p.) followed by the tail flick pain test which produced an increase in antinociception compared to controls. However, this compound is not as efficacious as morphine and pretreatment with MS21570 10 min prior to morphine (10 mg/kg, s.c.) did not alter morphine‐induced antinociception. To assess the role of GPR171 in morphine tolerance, mice were injected with morphine twice daily for 5 days. A subset of mice were pretreated with MS21570 10 min prior to each morphine injection. Those mice pretreated with MS21570 showed a decrease in the development of morphine tolerance. To understand how these systems may be working together, GPR171 was knocked down in Neuro2A cells which led to a decrease in MOPr‐mediated GTPγS signaling by DAMGO, but had no effect on GTPγS signaling by the delta opioid receptor agonist, deltorphin. This data suggests that the MOPr agonist, DAMGO, requires GPR171 expression in order to signal despite not directly binding to GPR171. Studies investigating GPR171 expression and signaling changes within the PAG following chronic morphine are ongoing. Taken together, this data shows that GPR171 and MOPr interact with one another and GPR171 antagonist may be a novel target to treat pain or opioid tolerance.

Support or Funding Information

Supported by NIH grants, DA019521 and NS026880 (to LAD) and NIDA postdoctoral training grant DA007135 and a Young Investigator Grant from the Brain and Behavior Research Foundation (to ENB).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Heat Shock Protein 90α, Assisted by Co‐Chaperones p23 and Cdc37, Promotes Opioid Anti‐Nociception in the Brain via Promoting ERK MAPK Signaling
Authors: Lei Wei , Duron David I. , Stine Carrie A. , Blagg Brian , Streicher John M.
Published: June 2021

Reducing opioid side effects like constipation and addiction has been a strong focus for improving opioid therapy in treating acute and chronic pain. One such approach includes manipulation of mu opioid receptor (MOR) signaling cascades, such as βarrestin2. However, the MOR signaling complex and mechanisms of signaling regulation are poorly understood. In our earlier work, we identified Heat shock protein 90 (Hsp90) as a novel MOR signaling regulator, which could be a target for improving the therapeutic profile of opioids. We found that intracerebroventricular (icv) 17‐AAG, a well‐established Hsp90 inhibitor, strongly blocked or abolished morphine anti‐nociception in paw incision and HIV neuropathic pain in mice. The current study was designed to identify molecular mechanisms of this regulation, including which isoform(s) and/or co‐chaperone(s) are involved in the regulatory effect of Hsp90 on opioid signaling. Male and female CD‐1 mice were treated with selective inhibitors for different Hsp90 isoforms (KU‐A, KUNB106, KUNG65) and co‐chaperones (KU177, gedunin, celastrol) via icv injection. We found that an Hsp90α selective inhibitor blocked morphine anti‐nociception by ~ 80% in the paw incision model, however, inhibitors for Hsp90β and Grp94 had no impact on morphine anti‐nociception. Knockdown of Hsp90α in the brain by CRISPR‐Cas9 also showed strong inhibition of morphine anti‐nociception in paw incision, confirming the results. Further, inhibition of the co‐chaperones p23 and Cdc37, but not Aha1, showed similar effects as Hsp90α inhibition in the paw incision model. To determine the molecular mechanism of these activities, we found that Hsp90α inhibitor treatment blocked ERK and JNK MAPK activation induced by the selective MOR agonist DAMGO without changing the expression of Hsp70 in mouse periaqueductal grey (PAG). Treatment with the ERK MAPK inhibitor U0126 recapitulated our results of Hsp90 inhibition on paw incision, HIV neuropathic, and tail flick pain, suggesting that ERK MAPK regulation is the means by which Hsp90α regulates morphine anti‐nociception. Identification of the isoforms and co‐chaperones involved in Hsp90 regulation of ERK and opioid anti‐nociception may permit more selective manipulation of the MOR signaling machinery to improve opioid efficacy and reduce side effects.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Glial Glutamate Transporter Activator, LDN‐212320, Attenuates Formalin‐Induced Pain Behavior in Mice
Authors: Alotaibi Ghallab , Rahman Shafiqur
Published: June 2021

Previous studies have shown that glial glutamate transporter‐1 (GLT‐1) in the hippocampus and anterior cingulate cortex (ACC) are critically involved in pain processing and modulation in the central nervous system. The objective of the present study was to investigate the role of GLT‐1 activator, 3‐[[(2‐Methylphenyl) methyl]thio]‐6‐(2‐pyridinyl)‐pyridazine (LDN‐212320), in formalin‐induced pain behavior in mice. Animal received 10 μl injection of formalin (2.5%) into the planter surface of the hind paw and the duration of licking/biting responses was measured. In addition, GLT‐1 expression and extracellular signal‐regulated kinase phosphorylation (pERK) were measured in the hippocampus and ACC using Western blot analysis. The LDN‐212320 (10 or 20 mg/kg, i.p) significantly attenuated formalin‐induced nociceptive behavior. These antinociceptive effects of LDN‐212320 were reversed by systemic administration of dihydrokianic acid (10 mg/kg, i.p), a GLT‐1 antagonist. Moreover, the LDN‐212320 (10 or 20 mg/kg, i.p) increased GLT‐1 expressions in the hippocampus and ACC. Formalin induced‐ERK phosphorylation, a molecular marker of nociception, was significantly reduced by LDN‐212320 (20 mg/kg, i.p) in the hippocampus and ACC. Overall, these results suggest that LDN‐212320 plays a critical role as antinociceptive agent by upregulating GLT‐1 expression in the hippocampus and ACC. Therefore, LDN‐212320 could be a novel therapeutic drug candidate for nociceptive pain.

Support or Funding Information

Supported in part by grant from Saudi Arabian Cultural Mission, USA

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of Antinociception Produced by Positive Allosteric Modulators of the Mu‐Opioid Receptor
Authors: Kandasamy Ram , Traynor John R.
Published: June 2021

Traditional opioids (e.g., morphine) provide analgesia by acting at the orthosteric site on the mu‐opioid receptor (MOR); however, G protein‐coupled receptor function can also be controlled by compounds acting at a separate (allosteric) site on the receptor. We have identified two compounds, BMS‐986122 and BMS‐986187, that bind to the allosteric site on the MOR and enhance the affinity and potency of MOR agonists in vitro. Thus, we hypothesized that BMS‐986122 and BMS‐986187 will produce antinociception by enhancing endogenous and exogenous opioid activity in vivo. Male and female mice were administered BMS‐986122, BMS‐986187, or vehicle via intraperitoneal injections (10 mg/kg; i.p.). Antinociception was assessed using the hot plate and warm water tail withdrawal tests. D,L‐methadone (10 mg/kg) produced antinociception on the tail withdrawal and hot plate tests. Co‐administration of BMS‐986187 did not enhance D,L‐methadone (3.2 mg/kg) antinociception on either test. However, a 30 min pretreatment of BMS‐986187 (10 mg/kg, i.p.) enhanced antinociception produced by an active dose of D,L‐methadone (10 mg/kg), but not an inactive dose (3.2 mg/kg), on the hot plate test. Intraperitoneal injection of both BMS‐986122 and BMS‐986187 alone produced near‐maximal antinociception on the tail withdrawal test for 2 hours that is likely caused by enhancement of endogenous opioid activity. Our data suggest complex interactions underlying antinociception produced by allosteric modulators of MOR that depend on several factors including dose, timing, and orthosteric agonist (methadone vs. endogenous opioid). Further, these data suggest that PAM‐mediated opioid antinociception affect pain processing at spinal and supraspinal levels.

Support or Funding Information

RK was supported by T32 DA007268. Funding provided by R37 DA039997 (to JRT).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ligand‐specific patterns of PTH1R and arrestin3 internalization and trafficking define a novel form of ligand “bias”
Authors: Lee Mi‐Hye , Strungs Erik G. , Luttrell Louis M.
Published: June 2021

The type 1 parathyroid hormone receptor (PTH1R) couples to Gs, Gq/11, and Gi/o family heterotrimeric G proteins and binds arrestins, which mediate receptor desensitization and arrestin‐dependent signaling. It is known that ligand structure can “bias” PTH1R signaling by changing the efficiency with which it couples to these downstream effectors. Here, we characterize the effect of ligand structure on the signaling, internalization, and intracellular trafficking of human PTH1R in a common HEK293 cell background using a panel of four structurally distinct ligands. Two ligands, hPTH(1‐34) and hPTH(1‐31), behave as conventional PTH1R agonists, activating both Gs‐cAMP and Gq/11‐Ca2+ signaling; while [Trp1]‐PTHrP(1‐36) is a Gs‐cAMP selective agonist; and [D‐Trp12 Tyr34]‐bPTH(7‐34) is a Gs‐cAMP selective inverse agonist. All four promote arrestin‐dependent ERK1/2 activation, with [Trp1]‐PTHrP(1‐36) and [D‐Trp12 Tyr34]‐bPTH(7‐34) exhibiting the greatest degree of arrestin‐dependence. To investigate the effect of ligand structure on PTH1R internalization and trafficking, we employed a “bystander BRET” approach in which the BRET pair consisted of a Renilla luciferase‐tagged Ras‐like small GTPase (Rab) marking a specific intracellular membrane compartment (Rab 1, Rab2, Rab5, Rab7, Rab9 or Rab11) and PTH1R‐YFP or arrestin3‐YFP. Three ligands, hPTH(1‐34), hPTH(1‐31), and [Trp1]‐PTHrP(1‐36) promoted PTH1R‐arrestin3 binding and clathrin‐dependent internalization, while [D‐Trp12 Tyr34]‐bPTH(7‐34), which fails to stabilize a PTH1R‐arrestin3 complex, did not promote internalization. In response to hPTH(1‐34) or hPTH(1‐31), PTH1R transited sequentially through early (Rab5) and late (Rab7/9) endosomes, before distributing between the slow recycling (Rab1/2) trans‐Golgi/ER network and rapid recycling (Rab11) endosome pool. Arrestin3 trafficking paralleled the receptor, consistent with the known role of arrestins in promoting slow PTH1R recycling and degradation. In contrast, PTH1R stimulated with [Trp1]‐PTHrP(1‐36) did not accumulate in Rab5 or Rab7/9 positive endosomes, instead transiting rapidly into the Rab11 and Rab2 positive pools. Notably, arrestin3 did not accompany the PTH1R, indicating that rapid dissociation of the PTH1R‐arrestin complex changed the pattern of PTH1R sorting in favor of receptor recycling. These results suggest that ligand structure, in particular the rate of ligand‐receptor‐arrestin complex dissociation, can “bias” not only the effector coupling profile of a G protein‐coupled receptor, but also it subsequent intracellular trafficking and rate of degradation/resensitization.

Support or Funding Information

NIH grants R01 DK055524 (LML), GM095497 (LML); the MUSC FLIPRTETRA Core S10 RR027777 (LML); the Research Service of the Ralph H. Johnson VA Medical Center, Charleston, SC.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Homologous Regulation of Mu Opioid Receptor Recycling by G Beta‐gamma
Authors: Kunselman Jennifer , Zajac Amanda , Weinberg Zara , Puthenveedu Manojkumar
Published: June 2021

The mu opioid receptor (mOR) is the primary target for opioid analgesics that are widely used and abused. Opioid receptors are removed from the cell surface after activation, causing down regulation of responses. Receptor recycling to the surface, which allows recovery of cellular responses, determines acute tolerance to opioids, and might have long‐term effects on signaling. Whether and how mOR recycling is acutely regulated by homologous signals is not clear. Here we used total internal reflection fluorescence microscopy (TIR‐FM) to directly visualize and quantify individual receptor recycling events, to test whether mOR recycling is regulated by opioid signaling downstream of the receptor itself. Attenuation of continued signaling, by removing the agonist, reduced the frequency of recycling events, indicating that opioid signaling increases the rate of mOR recycling. This homologous regulation was reduced in cells treated with the G protein inhibitor pertussis toxin, but not a Protein Kinase A inhibitor. Further, inhibition of G beta‐gamma (Gbg), Phospholipase C, or Protein Kinase C (PKC) abolished the decrease in recycling caused by agonist removal, while activation of Gbg or PKC caused an increase in recycling even in the absence of agonist. This suggests that mOR recycling is regulated by PKC activation through Gbg. Importantly, the homologous regulation was abolished by mutating the serine 363 residue on mOR, a PKC phosphorylation site. Our results support a model where Gbg activation of PLC/PKC and phosphorylation of mOR site S363 is required for the homeostatic regulation of mOR recycling. This represents a novel mechanism for homologous regulation of receptor recycling, which could provide control points for regulation by different opioid drugs.

Support or Funding Information

Supported by the NIH Cellular and Molecular Biology Training Grant T32‐GM007315

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel paradigms governing β1‐adrenergic receptor trafficking in primary adult rat cardiac myocytes
Authors: Nooh Mohammed Mostafa , Mancarella Salvatore M. , Bahouth Suleiman W.
Published: June 2021

Background and Purpose

The β1‐adrenergic receptor (β1‐AR) is a major cardiac G protein‐coupled receptor (GPCR), which mediates cardiac actions of catecholamines and is involved in genesis and treatment of numerous cardiovascular disorders. Catecholamines induce the internalization of the β1‐AR into endosomes and their removal promotes the recycling of endosomal β1‐AR back to the plasma membrane (PM).

Experimental Approach

Trafficking of the β1‐AR and its regulation was determined by confocal microscopy in primary adult rat ventricular myocytes (ARVM), which are terminally differentiated myocytes with unique structures such as t‐tubules and contractile sarcomeres.

Key Results

In unstimulated ARVM, the fluorescently labeled wild type (WT) β1‐AR was expressed on the external membranes (the sarcolemma) of cardiomyocytes. Exposing ARVM to isoprenaline promoted the translocation of surface β1‐AR into small (~225–250 nm) regularly spaced internal punctate structures that overlapped with puncta stained by Di‐8‐ANEPPS, a t‐tubule‐specific dye. Replacing the β‐agonist with a β‐blocker such as alprenolol, induced the recycling of the WT β1‐AR from t‐tubules back to the PM. This step was dependent on two previously identified β1‐AR barcodes, namely the type‐1 PDZ binding motif (PBM) and Ser312 of the β1‐AR, which is phosphorylated by the PBM anchored PKA‐AKAP5‐SAP97 complex.

Conclusions and Implications

We present data to show that translocation of the β1‐AR from t‐tubules back to the PM in ARVM was mediated by a novel sorting mechanism. These findings might explain unique aspects of cardiac β1‐AR signaling under normal or pathological conditions.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Insights into Phospholipase Cɛ Activity
Authors: Garland‐Kuntz Elisabeth Erin , Vago Frank S. , Van Camp Michelle M. , Sieng Monita , Blaine Arryn T. , Jiang Wen , Lyon Angeline M.
Published: June 2021

Phospholipase C (PLC) enzymes hydrolyze phosphatidylinositol lipids to produce second messengers, including inositol‐1,4,5‐triphosphate (IP3) and diacylgycerol (DAG), which increase intracellular calcium and activate protein kinase C (PKC), respectively. PLCɛ contributes to cardiac hypertrophy and contractility, as well as to oncogenic and inflammatory signaling pathways downstream of receptor tyrosine kinases and G protein‐coupled receptors. PLCɛ shares a conserved core with other PLC enzymes, but the roles of individual domains in activity and membrane binding have not been established. Using biochemical assays, small‐angle X‐ray scattering (SAXS), and electron microscopy (EM), we have found that the PLCɛ PH domain is necessary for basal lipase activity, but is dispensable for stability. Importantly, we provide the first structural insights into domain organization of PLCɛ, and reveal that the PH domain is conformationally heterogeneous in solution. Comparisons of the PLCɛ solution structure to that of the closely related PLCβ enzyme reveal that, in contrast to its crystal structure, the PLCβ PH domain is also mobile in solution. We also show the dynamic nature of the PH domain in these enzymes is functionally important, and may contribute to their regulation. These findings provide critical new insights into the solution structures of these signaling enzymes and their roles in cardiovascular disease and cancer.

Support or Funding Information

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

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cellular Compensation for Chronic Decreases in Cyclic AMP Concentration: Gαs‐Deficient Dendritic Cells as a Model and Implications for Allergic Disorder Therapeutics
Authors: Chinn Amy M. , Lee Jihyung , Raz Eyal , Insel Paul A.
Published: June 2021

Dendritic cells (DCs), the main antigen‐presenting cells, induce type II helper T cell (Th2) immune responses, but the mechanism by which this occurs is not fully elucidated. Mice that have a CD11c (DC)‐specific deletion of the Gnas gene (CD11cΔGnas), which encodes the α subunit of the heterotrimeric (αβγ) G protein Gαs, display very low basal cyclic AMP (cAMP) concentrations and an allergic phenotype; the decrease in cAMP and protein kinase A (PKA) activation biases DCs to provoke a Th2 response ex vivo and in vivo (Lee et al., PNAS 112: 1529–34 [2015]). In order to assess how cells compensate for a chronic decrease in cAMP/PKA signaling, we compared the mRNA expression of cAMP pathway components in WT and CD11cΔGnas bone marrow‐derived DCs (BMDCs). Using quantitative real‐time PCR (qPCR), we found that BMDCs from CD11cΔGnas mice have decreased expression of numerous cyclic nucleotide phosphodiesterases (PDEs) that degrade cAMP including PDE4B and 4D, but no overall increase in Gαi proteins or the cAMP transporter MRP4/ABCC4. Treatment of the mouse dendritic cell line DC2.4 with a cell‐permeable PKA‐specific cAMP analog and the Gαs agonist prostaglandin E2 increased PDE4B and 4D expression >2‐fold, indicating the dynamic changes in PDE4B and 4D expression are in response to cellular cAMP concentration. We also assessed the expression of G protein‐coupled receptors (GPCRs; assayed by qPCR‐based Taqman GPCR arrays), including those that regulate cAMP formation. We found a greater number and larger increase in expression for GPCRs that couple to Gαi (rather than other Gα proteins) in CD11cΔGnas BMDCs compared to BMDCs from WT mice. Additionally, expression of multiple PKA subunits (RIIα, RIIβ) decreased >50% in CD11cΔGnas BMDCs. A‐kinase anchor protein (AKAP)6 and AKAP3 have an increase and decrease, respectively, in those BMDCs. Together, these data indicate that DCs with lower cAMP levels have altered expression of both “upstream” cAMP signaling components (GPCRs) and “downstream” components that regulate cAMP levels (PDE isoforms) and actions (PKA subunits, AKAPs). Such changes in expression of GPCRs and post‐G protein components likely reflect withdrawal of “basal”, tonic effects of cAMP, may contribute to the greater Th2 responses and allergic asthma of CD11cΔGnas mice, and may be novel therapeutic targets for allergic asthma.

Support or Funding Information

Supported by NIH grants R56AI110505 and T32GM007752.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Mechanism of Rap1A‐Dependent Activation of PLCɛ
Authors: Sieng Monita , Garland‐Kuntz Elisabeth E. , Lyon Angeline M.
Published: June 2021

Phospholipase C (PLC) enzymes hydrolyze phosphatidylinositol lipids to produce diacylglycerol (DAG) and inositol phosphates, to activate protein kinase C (PKC) and downstream signaling pathways, including cell growth and survival. The PLCɛ subfamily has emerged as a key player in cardiovascular health, where it is required for maximum contractility. However, prolonged activation of PLCɛ results in cardiac hypertrophy and heart failure through its ability to regulate hypertrophic gene expression. This process is regulated by the small GTPase Rap1A, which is activated downstream of β‐adrenergic receptors. Rap1A binds to the C‐terminal Ras association (RA) domain of PLCɛ, simultaneously translocating the complex to the perinuclear region and activating PLCɛ. However, the molecular mechanism of this process is not known. We seek to characterize the interactions between Rap1A and PLCɛ using structural and functional studies to map the Rap1A binding site on PLCɛ and determine whether activation results in conformational changes that release autoinhibition and/or increase membrane association. These studies provide the first molecular details of the Rap1A‐dependent activation of PLCɛ and opens the door to the development of new therapeutic strategies for treating cardiac hypertrophy.

Support or Funding Information

American Heart Association Scientist Development Grant (16SDG29920017) to A.M.L.; Purdue Center for Cancer Research

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanistic Study of a Novel Thienyl Napthalenesulfonate Molecule that Selectively Inhibits Melanoma Cells with Constitutively‐Active ERK1/2
Authors: Martinez Ramon , Huang Weiliang , Centola Garrick , Samadani Ramin , Chen Lijia , Scheenstra Jacob , Fletcher Steven , Kane Maureen , Mackerell Alexander , Shapiro Paul
Published: June 2021

The uncontrolled proliferation of cancer cells is often a result of constitutive activation of the extracellular signal‐regulated kinase (ERK 1/2) signaling pathway. Current trends in the design of kinase inhibitors for the ERK1/2 pathway employ the use of ATP‐competitive scaffolds, targeting the catalytic core of the proteins. As an alternative, we used computer‐aided drug design (CADD) to identify a novel thienyl napthalenesulfonate compound that selectively inhibits ERK2 at a binding site used by F‐site containing substrates, such as members of the Fos family of proteins. This compound has been shown to selectively inhibit the growth of melanoma cells that have constitutively active ERK1/2 via mutations in upstream activators BRaf or NRas. Proteomic and transcriptomic analysis of drug‐treated lysates showed inhibition in the expression of important downstream F‐site containing substrates of ERK 1/2, such as c‐Fos, Fra‐1, and c‐Myc. In addition, increased expression of the protein Nrf2, which is a regulator of pathways generating reactive oxygen species (ROS), suggests that this compound may be involved with impeding cell growth in melanoma cells via upregulation of oxidative stress pathways. When treating cells with known inhibitors of ROS, such as α‐tocopherol or N‐acetyl cysteine, partial to complete cellular proliferation is restored, further supporting implication of oxidative stress in the compound function. In this work, we attempt to further characterize and validate the regulatory mechanisms and apoptotic effect this novel compound is having on cancer cells, and how it can serve as a promising alternate line of therapy to traditional ATP‐competitive kinase inhibitors.

Support or Funding Information

This work was supported in part by an NIGMS Initiative for Maximizing Student Development Grant (2‐R25‐GM55036) and by an NIGMS Institutional Training Grant (T32 GM066706).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phosphatidylethanolamine‐Binding Protein Promotes Opioid Anti‐Nociception in the Brain and Spinal Cord by Reducing βarrestin2 Recruitment to the Mu Opioid Receptor
Authors: Kim Caleb Ya Ill , LaVigne Justin , Edwards Katie , Streicher John M.
Published: June 2021

Opioids have been widely accepted as an effective treatment for chronic pain at a cost of serious side effects, including tolerance and addiction. Side effects are caused in part by desensitization and downregulation of mu opioid receptors (MOR) through recruitment of βarrestin2. Phosphatidylethanolamine‐Binding Protein (PEBP) is known to regulate either Raf Kinase or G Protein Receptor Kinase‐2 (GRK2) through phosphorylation by Protein Kinase C (PKC), and GRK2 is a major player in the recruitment of βarrestin2. However, PEBP has not been investigated for its role in MOR signaling and opioid response. We first tested this using an in vitro model of βarrestin2 recruitment in MOR expressing U2OS cells. We found that by inhibiting PEBP through siRNA knockdown or the small molecule inhibitor locostatin, we could increase the recruitment of βarrestin2 to the MOR in 2 separate assays. Conversely, we found that activating PKC via phorbol myristate acetate (PMA) led to PEBP phosphorylation/activation, and a reduction in βarrestin2 recruitment to the MOR, which could be blocked by PEBP knockdown. We then tested the role of PEBP in vivo by injection of locostatin into the brain or spinal cord, or CRISPR editing of PEBP in the spinal cord. We found that blocking PEBP in vivo led to a strong reduction in morphine‐induced tail flick anti‐nociception in both the brain and spinal cord. Together, these results support the hypothesis that PEBP acts to sequester GRK2 and block βarrestin2 recruitment downstream of the MOR both in vivo and in vitro. These results also suggest that enhanced PKC/PEBP activation by opioid drugs could further reduce opioid side effects and enhance analgesic efficacy.

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This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Reactive Skin Decontamination Lotion (RSDL) Maintains Its Effectiveness against Cutaneous Applications of VX Nerve Agent for at Least 5 Years Past Its Expiration Date
Authors: Clarkson Edward D. , Morgan Julia E. , Chen Paul , Schultz Susan M. , Soni Sunil D. , Capacio Benedict R. , Koplovitz Irwin
Published: June 2021

Reactive Skin Decontamination Lotion (RSDL) is the primary product the US military has fielded to decontaminate cutaneous exposures to chemical warfare agents. RSDL is extremely effective at protecting guinea pigs against cutaneous applications of the nerve agent VX. The 24‐hour cutaneous LD50 of VX in un‐anesthetized haired clipped guinea pigs is 140 µg/kg. When RSDL is used to remove VX two minutes after application the LD50 is 7,310 µg/kg. But RSDL is expensive to field and has a limited operational life. In the summer of 2017 we addressed the question of reducing costs by comparing the effectiveness of two expired (2012, 2014) lots of RSDL, to a lot that expires in April 2020. We applied the LD30 dose of 5,500 µg/kg to groups of n=12 and used the different batches of RSDL to remove the VX. There was no statistical difference in survival (n=10, 8 and 9) between the groups. The RSDL packets did change color as they aged, with the non‐expired RSDL having a bright yellow color, the RSDL that expired in January 2014 having a gray color and the RSDL that expired in April 2012 having a black color. Analysis of the various RSDL packets by proton, carbon and phosphorus NMR spectroscopy indicated that the difference in the component composition was not greater than 1%. To determine if protection was due to physical removal of VX we exposed two groups of n=4 to 5,500 µg/kg and 2,250 µg/kg of VX and used RSDL trainers to remove the VX. In the 5,500 µg/kg group all 4 animals died, and in the 2,250 µg/kg group 2 animals died. These results argue that RSDL can be effective past its expiration date and that the protection provided is not due entirely to physical removal.

The views expressed in this abstract are those of the author(s) and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government. The experimental protocol was approved by the Animal Care and Use Committee at the United States Army Medical Research Institute of Chemical Defense and all procedures were conducted in accordance with the principles stated in the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act of 1966 (P.L. 89‐544), as amended.

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This research was supported by the Defense Threat Reduction Agency – Joint Science and Technology Office, Medical S&T Division.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Quantitative Assessment of Trimethyltin, Kainic Acid and Pyrithiamine Neurotoxicity in Rat Brain and its Correlation with MR Imaging
Authors: Srivastava Anshul , Liachenko Serguei , Paule Merle G. , Sarkar Sumit , Pandey Jai , Negi Geeta , Hanig Joseph
Published: June 2021

Toxicagents such as trimethlytin (TMT), kainic acid (KA) and pyrithiamine (PT) induce neurotoxicity. Previous studies have reported degeneration in cortex, hippocampus, olfactory bulb, thalamus a swell as various other brain areas following exposure. Quantitative assessment of the extent of the degeneration is essential for understanding the functional changes due to such toxicity. In the current study, quantitative analyses of the pathological changes associated with single i.p. injections of TMT(12 mg/kg), KA (10 mg/kg) and PT (.25 mg/kg) were performed using tissue classification and area quantification algorithms based on stain color and OD available in HALO™ image analysis software. Sections containing brain areas to be compared to MRI images were chosen from 81 coronal sections of the whole rat brain stained with amino‐cupric silver and subsequently digitalized. Rats treated with TMT showed degeneration in amygdaloid nucleus (11.79%), cortex (7.71%), hippocampus(6.28%), hypothalamus (5.81%), septal region(4.92%), olfactory bulb (4.72%), thalamus (2.82%), nucleus accumbens (2.34%), caudateputamen (2.11%) and substantia nigra (0.40%). Cortical and hippocampaldegeneration in KA treated animals were 5.78% and 4.47%, respectively. Thalamusand olfactory degeneration in PT treated rats were 3.99% and 1.56% respectively. Saline treated animals served as controls and showed <0.3% changes in these brain areas. Quantification results of histopathology using image analysis were supported by the pathology score where the affected area was scored based on a severity scale. MRI T2 responses in hippocampus and cortex were compared to the toxicity in TMT treated animals as determined via histopathology quantification. Receiver Operating Characteristic (ROC) curves were generated to correlate histopathology and MRI responses. Correlation of MR imaging with quantitative histopathology analyses showed that MRI signals were in concordance with the histopathology and could effectively estimate loci and extent of neurodegeneration. Results suggest that MRI can be used as non‐invasive biomarker for the detection of drug‐induced neuropathology.

Support or Funding Information

Center for Drug Evaluation and Research ‐ Critical Path Award and National Center for Toxicological Research, Food and Drug Administration

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interaction of Respirable Fracking Sand Dust (FSD) with Pulmonary Tissues In vivo and In vitro
Authors: Fedan Jeffrey S. , Thompson Janet A. , Russ Kristen A. , Roberts Jenny R. , Schwegler‐Berry Diane , Mercer Robert R.
Published: June 2021

Sand added to fracking fluid that is pumped into the well bore is used as a proppant to maintain fissures in fractured shale to facilitate gas flow during unconventional gas drilling. Mechanical manipulation of the sand during preparation of fracking fluid gives rise to respirable dust (fracking sand dust; FSD) and inhalation exposures of workers to crystalline silica at levels in excess of regulatory standards. We characterized properties of FSD and some of its biological effects. Analysis of worksite‐derived FSD particles (≤2 μm major axis; >95% crystalline silica) usingenergy‐dispersive X‐ray spectroscopy (EDS) revealed eight categories of differing compositions based on the presence of Si, O, Al, Mg, K, Ca, Mn, Fe, and Co; elemental heterogeneity was present in individual particles. Inhalation of FSD by rats (6 h/d, 4 d, 10 or 30 mg/m3) led to dose‐dependent lung burdens at 1, 7, and 27 dpost‐exposure, with clearance noted following 1 d post‐exposure. In enhanced dark field (EDF) images of trachea, particles associated with epithelium were observed 1 d but not 7 or 27d post‐exposure. EDF studies of clearance revealed that particles in the alveolar region of the lungs were reduced to ~50% and ~30% by 7 and 27 d post‐exposure; none were present at 90 dpost‐exposure. Clearance of FSD appears to be more rapid than that reported for pure crystalline silica (i.e., MIN‐U‐SIL). Previously, inhalation of 30 mg/m3 FSD was observed to inhibit tracheal epithelial active Na+ transport, suggesting that the epithelium is a target of FSD effects. We, therefore, examined the effects of 18 hincubation with apical FSD (0.0001 – 1 mg) on cytokine and lactatedehydrogenase (LDH) release into apical and basolateral media from normal humanbronchial epithelium in air‐interface culture. Changes in cytokine levels in a 65‐cytokine panel of growth factors and inflammatory mediators were modest (0.1 – 1 mg). No LDH was released into the basolateral medium; LDH released into the apical medium was unaffected by FSD. The results indicate that FSD is fairly rapidly cleared from the lungs following inhalation and is without appreciable direct toxicity to airway epithelial cells.

Support or Funding Information

NIOSH 8927ZLDC

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Graphene interactions with human endothelium
Authors: Szukiewicz Dariusz , Skoda Marta , Dudek Ilona
Published: June 2021

The discovery of graphene boosted an interdisciplinary research based on its unique properties and possible applications in biomedicine. Multiple scientific reports concerning graphene interactions with adherent cells showed the potential use of graphene as a biomaterial in tissue engineering.

In this study we examined the influence of pristine graphene, as a monolayer coating or flake suspension, on human endothelial cells (HUVEC) isolated from umbilical veins. Umbilical cords were obtained from healthy gravidas after the cesarean section procedures. Cytotoxicity, proliferation and cell morphology study were performed using various methods, including colorimetric and luminescent cell assays, optical and fluorescence microscopy.

The cytotoxicity assay confirmed dose and time dependent cytotoxicity of tested graphene flakes (G1APS~5000nm and G2APS~450nm). Even the lowest concentration of graphene (10μg/ml) significantly inhibited cell proliferation. The difference between the two tested flake sizes was more distinct during cell mortality study, indicating the greater cytotoxic effect of the smaller graphene particles. Graphene suspended in the cell culture medium created aggregates. Cell imaging revealed efficient accumulation of nanoflakes inside the cells, especially those of the smaller size.

The morphology of the cells seeded on graphene‐coated SiO2 substrates was recorded using fluorescence microscopy. Endothelial cells were immunostained for actin cytoskeleton to characterize its organization, stress fibers creation, cell adhesion and spreading. After a 72h incubation, both the cells incubated on graphene SiO2 substrates and on the reference material for cell plating, reached confluence. No difference in cell shape and morphology was detected. Graphene was shown to be a suitable surface for endothelial cell growth.

Our preliminary investigation confirmed cytotoxic effect of pristine graphene flakes on adherent cells, however demonstrated that CVD grown graphene is not toxic for human endothelial cells. These data show promise for using graphene monolayers in tissue engineering or as a substrate material for implant devices.

Support or Funding Information

WUM grant: 2M2‐W2‐16

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