bluuebunny/crossref_metadata_2025_split huggingface dataset:
PKD inhibitor treatment results in increased PKD activation loop phosphorylation (1055.1)
Authors: Kunkel Maya , Newton Alexandra 
Published: June 2021

Protein kinase D (PKD) transduces an abundance of signals downstream of diacylglycerol (DAG) production and plays a role in many cellular functions, including regulation of Golgi sorting, apoptosis, cell growth, and cell survival. PKD1, originally described as a protein kinase C (PKC) family member and named PKCμ, is now known to be a member of a distinct family of kinases that includes, PKD1, PKD2, and PKD3. The PKD family is related to members of the calcium‐calmodulin kinase (CAMK) superfamily of kinases rather than the PKCs, which belong to the AGC kinase group. PKD activity is acutely regulated via binding to membrane DAG via its C1 domains. Subsequent to DAG binding, PKD is activated via phosphorylation by the upstream kinases, the novel PKCs, at the activation loop followed by autophosphorylation. Thus, this phosphorylated species is often used as a measure of kinase activity. Here we describe that PKD inhibitors promote activation loop phosphorylation downstream of G protein‐coupled receptors. This is reminiscent of similar observations for PKC and Akt, wherein active site inhibitors trap phosphates on the kinase; a phenomenon we have attributed to stabilization of a phosphatase‐resistant conformation of the kinase domain. For PKD, this effect occurred in the presence of active site inhibitors as well as in the presence of non‐competitive inhibitors, a distinction from PKC. These findings underscore the importance of using caution when interpreting kinase activity from kinase phosphorylation status in the presence of small molecule inhibitors.Grant Funding Source: Supported by NIH P01 DK54441
Involvement of KATP channels in hydrogen sulfide‐induced increase in aqueous humor outflow (1060.3)
Authors: Robinson Jenaye , Ezeudu Chinoso , Mitchell Leah , Chitnis Madhura , Opere Catherine , Ohia Sunny , Njie‐Mbye Ya Fatou 
Published: June 2021

Involvement of Katp channels in hydrogen sulfide‐induced increase in aqueous humor outflow Jenaye Robinson1, Chinoso Ezeudu1, Leah Mitchell1, Madhura Chitnis1, Catherine Opere2, Sunny E. Ohia1,Ya Fatou Njie‐Mbye1.1Department of Pharmaceutical Sciences, College of Pharmacy and Health sciences, Texas Southern University, Houston TX 77004,2Department of Pharmacy Sciences, School of Pharmacy and Health Professions, Creighton University, Omaha, NE 68178 We have evidence that hydrogen sulfide (H2S), a novel gasotransmitter can increase aqueous humor (AH) outflow in porcine trabecular meshwork tissues (TM). Purpose: To investigate the underlying mechanism of H2S‐induced increase in AH outflow. Methods: Porcine ocular anterior segment explants were perfused with DMEM maintained at 37ºC, 5% CO2 and constant pressure of 7.35 mmHg. Stabilized explants were exposed to the H2S‐releasing compound, sodium hydrosulfide (NaHS) and its substrate, L‐cysteine. Explants were also treated with the KATP channel antagonist glibenclamide and H2S biosynthetic enzyme inhibitors, aminooxyacetic acid (AOA), or proparglyglycine (PAG). Results: L‐cysteine (1 nM ‐ 1μM) caused a dose‐dependent increase in outflow, reaching a maximal effect at 100 nM [153 ± 7.2% of basal (mean ± SE)]. The effect of L‐cysteine (100 nM) on AH outflow was completely attenuated by AOA (30 μM) and PAG (1 mM). Interestingly, NaHS (100 nM ‐ 10 µM) also produced a concentration‐dependent increase in AH outflow, reaching a maximal effect at 10 μM. Furthermore, the enhancement of outflow caused by NaHS (10 μM) was inhibited significantly (p < 0.01) by glibenclamide (100 µM). Conclusions: We conclude that H2S‐induced increase AH outflow in porcine TM is dependent upon its intramural biosynthesis from L‐cysteine and, is mediated by KATP channels.Grant Funding Source: Supported by NIH/NEI Grant R15EY022215
Characterization of a repurposed agent as a potent inhibitor of DGKa (1062.1)
Authors: Boroda Salome , Harris Thurl , Purow Benjamin , Floyd Desiree 
Published: June 2021

Diacylglycerol Kinases (DGKs) catalyze the conversion of diacylglycerol (DAG) to phosphatidic acid (PA). DGK‐mediated increases in PA concentrations have been found to affect pro‐cancer growth and survival pathways such as the mTORC1 pathway and the HIF ‐1α pathway. As a consequence, the DGKs are promising therapeutic targets for a large range of cancers. Specifically, the attenuation of DGKα by a small molecule inhibitor, R59022, has shown to dramatically decrease cell survival in glioblastoma models (Dominguez et al, 2013). R59022 has only been used in laboratories and completion of clinical trials could take years before its approval for use in humans. Our database searches have revealed a compound similar in structure to R59022 which we sought to repurpose for the inhibition of DGKα. We purified DGKα from mammalian 293 T cells using affinity chromatography and employed both liposome and micelle assays to measure the effects of this compound (which we term compound R) on DGKα activity. We showed that compound R is a strong uncompetitive inhibitor of DGKα with substantially greater potency than R59022. Taken together, our data suggest that compound R could serve as a viable therapeutic agent in targeting glioblastoma.Grant Funding Source: Supported by: National Institute of Health and University of Virginia
Selective interactions of HIV protease inhibitors with human plasma membrane monoamine transporter and organic cation transporters (1064.13)
Authors: Duan Haichuan , Pan Yongmei , Swaan Peter , Unadkat Jashvant , Wang Joanne 
Published: June 2021

The plasma membrane monoamine transporter (PMAT, SLC29A4) is a novel polyspecific organic cation transporter that shares significant substrate and inhibitor overlaps with organic cation transporters (OCTs) in the SLC22 family. Currently, there are no PMAT‐selective inhibitors available to distinguish PMAT activities from those of OCTs. In this study, we first identified HIV protease inhibitors (HIV PIs) as potential PMAT inhibitors through computer‐aided virtual screening using previously developed pharmacophore models. We then developed nonradioactive fluorescent substrate‐based uptake assays for PMAT and OCT1‐3. These assays demonstrated excellent accuracy and robustness for inhibitor screening and characterization. Using these assays, we characterized the interaction of human PMAT and OCT1‐3 with a panel of HIV PIs. Our results showed that PMAT was broadly and strongly inhibited by most HIV PIs whereas OCT2 and OCT3 were relatively resistant to PIs. OCT1 selectively interacted with PIs and was most strongly inhibited by ritonavir and saquinavir. Uptake experiments with [3H]ritonavir further suggested that it is likely a transportable substrate for PMAT. Our results revealed that PMAT and OCTs possess differential sensitivity towards HIV PIs which could have important implications for understanding clinical drug interactions with HIV PIs.Grant Funding Source: Supported by NIH Grants GM066233 and GM066233‐07S1.
Role of the blood‐brain barrier in limiting CNS uptake of pyrethroid insecticides: findings with hCMEC cells as a proxy (1064.14)
Authors: Bruckner James 
Published: June 2021

Pyrethroids, the most widely‐used insecticides in the U.S., generally exhibit low neurotoxicity in humans and other mammals. Kinetic studies of the pyrethroid deltamethrin (DLM) have revealed that levels of the hydrophobic, neurotoxic parent compound are <20% of blood levels in rats. Experiments with hCMEC cells, a human brain endothelial cell line, were undertaken as part of an effort to understand the BBB’s role in limited CNS deposition of DLM. hCMEC cells were grown in EGM‐2 medium, seeded in T‐75 flasks, and allowed to grow to confluency within 3‐4 days. DLM uptake by the cells was concentration‐dependent, indicating a saturable process. Cyclosporin inhibited uptake, suggesting an active influx transport process. Increasing the amount of human serum albumin reduced the free/unbound fraction of DLM, which correlated with decreased cellular uptake. In conclusion: DLM’s pronounced plasma protein/lipoprotein binding (~90+%) appears to limit its availability for BBB passage; and a BBB influx transporter of low capacity and low affinity contributes to hCMEC uptake.Grant Funding Source: Council for the Advancement of Pyrethroid human Risk Assessment (CAPHRA)
Organic anion transporting polypeptide 1B3 can form homodimers (1064.17)
Authors: Zhang Yuchen , Hagenbuch Bruno 
Published: June 2021

Organic Anion Transporting Polypeptide 1B3 (OATP1B3) is under normal physiological conditions mainly expressed at basolateral membrane of human hepatocytes where it is thought to mediate uptake of numerous drugs into the liver. Preliminary data from western blots indicated that OATP1B3 could form multimers. Therefore, we investigated if OATP1B3 indeed forms multimers and whether such multimer formation has any effect on transport function. OATP1B3 constructs with a His‐, a Flag‐ or an HA‐tag at the C‐terminal end were constructed and transiently transfected in HEK293 cells. Uptake of the OATP1B3 model substrates estradiol‐17β‐glucuronide (E17βG) and estrone‐3‐sulfate (E3S) was measured, and immunoprecipitation experiments were performed. Transport of E17βG and E3S into HEK293 cells transiently transfected with OATP1B3‐His, OATP1B3‐Flag or OATP1B3‐HA was comparable to HEK293 cells expressing wild‐type OATP1B3. Chemical cross‐linking experiment in HEK293 cells transiently transfected with OATP1B3‐His suggested the formation of OATP1B3 dimers. However, interactions with other proteins could not be excluded. Thus, HEK293 cells were transfected with a mixture of OATP1B3‐Flag and OATP1B3‐HA and immunoprecipitation with an anti‐HA antibody was performed. Anti‐HA antibodies were able to pull down OATP1B3‐HA as well as OATP1B3‐Flag, demonstrating that OATP1B3 indeed can form homodimers. Co‐transfection of wild‐type OATP1B3 with the nonfunctional OATP1B3 mutant K41C resulted in reduced but still detectable transport, suggesting that the dimerized OATP proteins function individually. In conclusion, OATP1B3 can form homodimers and the functional unit is the protein and not the dimer.Grant Funding Source: Supported by NIH Grant GM077336
5‐Hydroxytryptamine mediated vasocontraction and Ca2+/Calmodulin and Src‐kinase dependent Epidermal Growth Factor Receptor Transactivation in Rat aorta (1065.7)
Authors: Guner Sahika , Ozakca Isil , Kandilci H. Burak , Zoto‐Mustafayeva Teuta , Duman‐Dalkilic Basak , Gurdal Hakan 
Published: June 2021

5‐Hydroxytryptamine (5‐HT) receptors induce vasocontraction and play significant roles in vascular systems. We have previously shown that transactivation of EGFR contributes to α1‐adrenergic receptor mediated vasocontraction. Here, we examined EGFR transactivation in 5‐HT induced vascular contraction and extracellular‐regulated kinase (ERK1/2) activation. Aortic contraction and intracellular [Ca2+] response to 5‐HT in aorta and A7R5 cells, respectively, were obtained in the absence and presence of EGFR inhibitor (AG1478). To characterize EGFR transactivation, 5‐HT stimulated‐EGFR phosphorylation was determined in the absence and presence of AG1478, Ca2+/Calmodulin inhibitor (W7), Src kinase inhibitor (PP2).AG1478 attenuated 5‐HT induced vasocontraction and intracellular [Ca2+] response. 5‐HT mediated EGFR‐transactivation was inhibited by AG1478, W7 and PP2. 5‐HT also led to phosphorylation of myosin light chain kinase (MLCK) and ERK1/2 which were partially inhibited by AG1478, but completely by W7 and PP2.These data suggest that 5‐HT mediated EGFR‐transactivation depends on Ca2+/Calmodulin and Src‐kinase and is partially involved in both vascular contractility and ERK1/2 phosphorylation in rat aorta.Grant Funding Source: This study is supported by TUBITAK 111S131
SIRT1 overexpression protects against high fat diet‐induced cerebral artery endothelial dysfunction (1070.10)
Authors: Walker Ashley , Henson Grant , Reihl Kelly , Lesniewski Lisa , Donato Anthony 
Published: June 2021

High fat (HF) diet is associated with increased cerebrovascular disease risk, but interventional targets to protect cerebral artery function with HF diet have not been elucidated. Sirtuins control cellular energy metabolism and SIRT1 transgenic overexpressing (SIRT‐TG) mice are protected against some of the negative consequences of HF diet, such as glucose intolerance. However, the effect of SIRT1‐TG on cerebral artery function after HF diet is unknown. We measured middle cerebral artery (MCA) endothelium‐dependent dilation to acetylcholine (ACh) in normal chow (NC) and HF diet fed SIRT‐TG and wildtype (WT) mice (6 mo, n=6‐8/group). In NC mice, MCA dilation to ACh did not differ between SIRT1‐TG and WT (max: 66±13% vs. 67±14%, P=0.90). HF WT mice had impaired MCA dilation to ACh (46±11%, P<0.01 vs. NC WT). However, MCA dilation to ACh for HF SIRT‐TG mice was greater than HF WT mice (63±10%, P=0.01) and not different from NC groups (P>0.05). There were no group differences after nitric oxide (NO) synthase inhibition with L‐NAME (P>0.05), indicating that the impaired dilation to ACh in HF WT mice resulted from reduced NO bioavailability. Endothelium‐independent dilation did not differ between groups (P>0.05). In conclusion, HF diet induced cerebral artery endothelial dysfunction by reducing NO bioavailability in WT mice. SIRT1‐TG mice are protected against cerebral artery endothelial dysfunction induced by HF diet.Grant Funding Source: AG029337, AG040297, AG033196, HL007576
Phospholipase C isoforms expression in mouse endothelium (1075.2)
Authors: Béziau Delphine , Toussaint Fanny , Charbel Chimène , Daya Nour , Blanchette Alexandre , Ledoux Jonathan 
Published: June 2021

Phospholipase C (PLC) is a major signal transduction component through the regulation of various intracellular pathways, including PKC and intracellular Ca2+ channels. Up to 13 mammalian PLC isoforms have been identified and are classified in 6 families: PLC‐β, γ, δ, ε, ζ and η. Although expression of PLCs is tissue‐specific, combined expression of different PLCs has been reported. However, endothelial PLC expression remains to be established. Therefore, we sought to determine the expression pattern of PLC in mouse resistance arteries. We first studied the expression of PLC isoforms in mesenteric arteries. We found mRNA encoding for most PLCs with the exception of ζ1, η1 and η2 in whole mesenteric arteries. Similar results were obtained in middle cerebral, coronary and pulmonary arteries. We then elucidated the intracellular distribution of PLCs β3, γ1 and δ1, the main mammalian isoforms, in endothelial cells with in situ immunocytochemistry. We observed a diffuse staining in endothelium with no clearly defined heterogeneous distribution of all three PLCs. Further investigation is necessary to determine the specific PLC isoforms involved in endothelial localized Ca2+ dynamics. Taken together, these studies might strengthen our understanding of endothelial Ca2+ homeostasis mechanisms and therefore endothelial function.Grant Funding Source: Supported by FRQS, HSFC, SQHA and FICM.
Caffeine increases basal eNOS phosphorylation and stimulates endothelial cell growth (1075.6)
Authors: Calleo Adrianna , Harris M. Brennan 
Published: June 2021

Caffeine is one of the most widely used neural stimulants and has both vasodilator and vasoconstrictor effects. In this study, we examined the effects of caffeine on eNOS phosphorylation and on endothelial cell growth. eNOS phosphorylation was examined in human umbilical vein cells (HUVECs) in the absence and presence of the eNOS agonist VEGF using Western blot. Endothelial cell growth was determined using a scratch wound assay in HUVECs. eNOS phosphorylation was increased in HUVECs treated with 100 µM caffeine and 1 mM caffeine for 1 h or 20 ng/ml VEGF for 30 min. No further increases in phosphorylation were observed in cells treated with both caffeine and VEGF. Endothelial cell growth was increased in cells exposed to 100 µM and 1 mM caffeine. Higher concentrations of caffeine impeded cell growth. In conclusion, these data suggest that caffeine acts as an eNOS agonist during short exposures which can augment endothelial cell growth.Grant Funding Source: Supported by NIH/NHLBI 1R15HL108428
Partial genetic disruption of mTOR complex 1 signaling does not improve vascular endothelial function in hypercholesterolemic mice (1076.6)
Authors: Jbeli Aiham , Zhang Bin , Roos Carolyn , Fujimoto Hirokazo , Miller Jordan 
Published: June 2021

Endothelial cell senescence promotes inflammation and impairs endothelium‐dependent vasodilation. While aging, hypercholesterolemia, and caloric excess are known to drive cellular senescence through activation of mTOR, it is unknown whether reduction of the mTORC1 adapter protein RapTOR can improve vasomotor function. Thus, we used hypercholesterolemic mice (Ldlr‐/‐/apoB100/100, or LA) carrying intact copies of Raptor (LA‐Raptor+/+) or were deficient in one copy of Raptor (LA‐ Raptor+/‐). Mice were fed Western Diet for 6 or 12 months, and vasomotor function in aorta was evaluated using isolated organ chamber baths. Maximum relaxation to acetylcholine (MRACH) was progressively impaired from 6 months to 12 months in LA‐Raptor+/+ mice (69±4% and 42±7%, respectively). In contrast to our hypothesis, MRACH was not significantly improved in LA‐Raptor+/‐ mice at either time point (72±3% and 50±8%, respectively). Relaxation to acetylcholine was abrogated by L‐NAME in both strains at both time points and relaxation to nitroprusside was identical between strains at each time point, suggesting that changes in endothelial function were not masked by compensatory mechanisms (e.g., endothelium‐derived hyperpolarizing factors). Furthermore, reduction of RapTOR did not elicit changes in maximal tension generated in response to Prostaglandin F2α at either time point. Collectively, despite strong previous evidence from in vitro model systems, genetic reduction of mTORC1 signaling in vivo does not appear to be sufficient to improve vasomotor function in aging hypercholesterolemic mice. Grant Funding Source: Supported by NIH/NHLBI
Modulation of pressure‐dependent cerebral arterial myogenic tone by a redox‐sensitive signaling event (1079.8)
Authors: Gebremedhin Debebe , Harder David 
Published: June 2021

The objective of this study is to investigate the pressure‐induced redox‐sensitive signaling pathway in the regulation of pressure‐dependent myogenic rat cerebral arterial constriction.Methods. We have investigated effects of step increases in intramural pressure on the generation of ROS and the role of inactivation of the phosphatase PTEN and subsequent activation of the PI3K/Akt signaling pathway in pressure‐induced myogenic constriction of cerebral arteries using a battery of experimental methods.Results. Step increase in intraluminal pressure induced myogenic constriction associated with increased formation of superoxide (O2.‐) and its dismutation product hydrogen peroxide (H2O2). The increase in intraluminal pressure also induced oxidative inactivation of the lipid dual‐phosphatase PTEN and activation of Akt. Pharmacological inhibition of endogenous PTEN activity potentiated pressure‐dependent myogenic constriction, and caused a reduction in NPo of a 238 pS arterial KCa channel current and an increase in [Ca2+]i level in freshly isolated cerebral arterial muscle cells (CAMCs), responses that were attenuated by Inhibition of the PI3K/Akt pathway.Conclusions. We report that an increase in intraluminal pressure‐induced elevated O2.‐production that resulted in oxidative inactivation of PTEN and activation of the PI3K/Akt signaling pathway, which involved in modulation of the development of pressure‐dependent myogenic cerebral arterial constriction.Grant Funding Source: This work is supported by grants from NIH (R01 NIH/NHLBI, HL033833, HL092105 and HL105997)
Mechanisms that regulate PKCδ‐dependent phosphorylation of cardiac troponin I: the role of the C2 domain and ATP‐binding loop phosphorylation S357 (1081.2)
Authors: Steinberg Susan , Gong Jianli , Yao Yongneng , Zhang Pingbo , Van Eyk Jennifer 
Published: June 2021

PKCδ's diverse roles in cell growth, survival, and injury have been attributed to stimulus‐specific differences in PKCδ‐dependent signaling responses. PKCδ exerts membrane‐delimited actions following activation by agonists that stimulate phosphoinositide hydrolysis. PKCδ is released from membranes as a Y311‐phosphorylated enzyme that displays a high level of lipid‐independent activity and altered substrate specificity during oxidative stress. We previously showed that PKCδ phosphorylates cardiac troponin I (cTnI) at S23/S24 when it is allosterically‐activated by lipid‐cofactors and that PKCδ acquires activity toward an additional site on cTnI (T144) during oxidative stress when it is Y311‐phosphorylated by Src. This study identifies an interaction between PKCδ’s phosphotyrosine‐binding C2 domain and the Y311‐phosphorylated hinge region (which resides in a C2 domain consensus‐binding motif) that controls PKCδ's enzymology indirectly by regulating ATP‐positioning loop phosphorylation at S357. Mutagenesis studies show that PKCδ‐S357A is a lipid‐independent enzyme that displays high levels of cTnI‐S23/S24 and T141 kinase activity. In contrast, phosphomimetic or bulky substitutions at S357 prevent cTnI phosphorylation at T141 (but not S23/S24) due to an effect on the enzyme’s phosphoacceptor site selectivity. S‐A substitutions at cognate sites in the highly‐conserved ATP‐binding loops of PKCα and PKA increase catalytic activity, but do not alter phosphoacceptor site selectivity. The C2 domain‐pY311 docking interaction that controls ATP‐binding loop phosphorylation represents a novel structural determinant of PKCδ catalytic activity.Grant Funding Source: Supported by HL77860
Functional neprilysin‐angiotensin‐(1‐7) system within mitochondria of the sheep kidney (1088.9)
Authors: Wilson Bryan , Rose James , Chappell Mark 
Published: June 2021

An intracellular‐based renin‐angiotensin system (RAS) is evident within the kidney, heart and other tissues. AT1 and AT2 receptors, functionally linked to reactive oxygen species and nitric oxide (NO), respectively, are present on both nuclei and mitochondria (Mito). The current study determined whether components of an Ang‐(1‐7) system are also expressed in renal Mito. Mito were isolated from sheep renal cortex by differential centrifugation and discontinuous Percoll gradients. Immunoblots of the Mito fraction revealed protein bands for angiotensinogen (Aogen), renin, Ang‐(1‐7)/Mas receptor, and endothelial nitric oxide synthase (eNOS). Using an Ang I‐directed Aogen antibody, renin activity was demonstrated by the time‐dependent disappearance of intact Aogen which was abolished by renin inhibitor aliskerin. Ang I was predominantly converted to Ang‐(1‐7) by the endopeptidase neprilysin (EC3.4.24.11) and a 90 kDa band was visualized with a monoclonal neprilysin antibody. To assess the functional aspects of the peptide, isolated Mito fractions were loaded with the NO‐sensitive dye diaminofluorescein (DAF, 1ug/ul) for 30 minutes, exposed to various concentrations of Ang‐(1‐7) (0.1 to 100nM). Ang‐(1‐7) (10 nM) significantly increased NO production (50 ± 16%, n=3) over control. We conclude that renal Mito express a functional NEP/Ang‐(1‐7)/Mas receptor system coupled to the NO generation.Grant Funding Source: Supported by NIH grants HL56973, HL52972, HL112237, and HD047584