bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of the high hydrostatic pressure extract of Korean ginseng on hepatic lipid metabolism and AMP‐activated protein kinase activation in HepG2 cells (1045.25)
Authors: Lee Mak‐Soon , Shin Yoonjin , Kim Yangha
Published: June 2021

Ginseng is a medicinal plant widely used for the treatment of various diseases in Asian countries, including Korea, China, and Japan. In this study, we investigated the effects of the high hydrostatic pressure extract of Korean ginseng (Panax Ginseng C.A. Meyer) on hepatic lipid metabolism and AMP‐activated protein kinase (AMPK) activation in HepG2 cells. First, we investigated the potential effects of high hydrostatic pressure extract of fresh ginseng (HFG) on the cytotoxicity and apoptosis in HepG2 cells. Cytotoxicity and apoptosis were unaffected in the concentrations of 1‐50 μg/ml of HFG. Intracellular lipid accumulation as assessed by the quantification of intracellular triglyceride and cholesterol contents. HFG significantly reduced hepatic cholesterol and triglyceride contents in a time‐ or dose‐dependent manner. Furthermore, HFG inhibited expression of fatty acid synthase (FAS), a key enzyme in fatty acid synthesis, and 3‐hydroxy‐3‐methyl glutaryl coenzyme A (HMG‐CoA) reductase, a rate‐limiting enzyme in hepatic cholesterol synthesis. In contrast, HFG increased activity of AMPK, a major regulator of lipid metabolism. These results suggest that HFG reduces hepatic lipid accumulation with inhibition of FAS and HMG‐CoA reductase expression and stimulation of AMPK activity in HepG2 cells. Therefore, HFG may be beneficial for the treatment of fatty liver and dyslipidemia.

Grant Funding Source: This work was supported by the Brain Korea 21 plus project funding (No. 22A20130012143).
Inhibition of osteoclast actin ring formation and activation by phloretin through disturbing integrin αvβ3‐PYK2 pathway (1045.29)
Authors: Lee Eun‐Jung , Kim Yun‐Ho , Kang Young‐Hee
Published: June 2021

Osteoclast activation is important for bone remodeling and is altered in multiple bone disorders such as osteoporosis. This process requires cell adhesion and extensive actin cytoskeletal reorganization. Phloretin, a dihydrochalcone and a type of natural phenols, can be found in apple tree leaves. The goal of this study was to define the process and mechanism of bone resorption. RAW 264.7 macrophages were incubated with 1‐20 μM phloretin for 5 days in the presence of RANKL. RANKL stimulated Akt activation and c‐Src expression, which was blunted by phloretin. The integrin αvβ3, heterodimer of adhesion receptors, is crucial in osteoclast activation mediating cell‐matrix and cell‐cell interactions. Phloretin suppressed integrin β3 induction and PYK2 activation elevated by RANKL, indicating its inhibition of osteoclastic fusion. The activation of PYK2, a major cell adhesion‐activated tyrosine kinase in osteoclasts, is required for the formation of actin rings. Taken together, these results suggest that phloretin was an inhibitor of actin ring formation, disrupting integrin‐PYK2 pathway for the regulation of actin cytoskeletal organization in osteoclasts.

Grant Funding Source: Supported by Brain Korea 21 plus
Effect of high hydrostatic pressure extract of Korean ginseng on adipogenesis in 3T3‐L1 adipocytes (1045.4)
Authors: Oh Soojung , Lee Mak‐Soon , Jung Sunyoon , Kim Chong‐Tai , Kim Yangha
Published: June 2021

Ginseng, especially steam‐and‐dry processed red ginseng, has been shown to decrease adipogenesis in adipose tissue. In the food processing industry, high hydrostatic pressure extraction technique has become an alternative to heat processing. The aim of this study was to comparatively investigate the anti‐adipogenic effects of water extract of red ginseng (WRG) and high hydrostatic pressure extract of fresh ginseng (HPG) in 3T3‐L1 adipocytes. Intracellular lipid and triglyceride were decreased in both WRG and HPG treated cells, while the cell viability remained unaffected. This observed decrease was consistent with the reduction of GPDH activity, a key enzyme in triglyceride biosynthesis. The mRNA expressions of adipogenic genes, including peroxisome proliferator‐activated receptor‐gamma, CCAAT/enhancer‐binding protein‐α, sterol regulatory element‐binding protein‐1c, and adipocyte fatty acid binding protein, were significantly decreased in both WEG and HPG treated cells. The activity of AMPK, a key regulator of energy metabolism, was significantly increased in the HPG treated cells compared to the WRG. From these results, it is assumed that anti‐adipogenic mechanisms of the WRG and HPG in adipocytes might be associated with inhibition of GPDH activity and adipogenic gene expression as well as AMPK activation. Moreover, efficiency of the HPG might be comparable to that of the WRG.

Grant Funding Source: This work was supported by the Brain Korea 21 plus project funding (No. 22A20130012143)
Maternal vasculature changes produced by experimental gestational diabetes mellitus by obesity (1051.10)
Authors: Tufiño Cecilia , Rosas Paula , Bobadilla Rosa
Published: June 2021

Gestational diabetes mellitus is an increasing incidence disease whose short‐term consequences in the maternal vasculature are poorly understood. The purpose of this study was to determine whether an experimental model of obesity induced gestational diabetes mellitus can modify the vasoconstrictor response of isolated arteries and if so, to explore the possible causes. Wistar rats were subjected to a high calorie diet for 7 weeks, of which the last three coincided with the period of gestation. GDM was defined by an abnormal glucose tolerance test. GDM group was compared with a control, obese, and healthy pregnant group. Dose response curves to phenylephrine and angiotensin II were done in isolated aortic rings. Also, plasma levels of angiotensin II and expression of the COX‐1, COX‐2, iNOS and AT1 receptors for angiotensin II were measured. Results showed that GDM group presented impaired glucose tolerance and an increase in the vasoconstrictor response in the presence of losartan or prazosin associated with an increase in the protein expression of AT‐1 receptor, COX‐2 and iNOS. These results provide evidence that obesity occurs DMG deleterious changes in the short‐term maternal vasculature.
Investigation of sorbitan niosomal gel for sustained release ocular delivery of piroxicam (1053.3)
Authors: Abdul Rasool Bazigha , Alsawad Oday , Lootah Hamda , Abusharbain Iman , Abu‐Alhaj Hiba
Published: June 2021

Objective: In this study, niosomal hydrogel formulation was investigated as a promising carrier for piroxicam sustained release ocular delivery.

Methods: Piroxicam loaded niosomes were prepared by thin film hydration technique using various proportions of span and cholesterol. Optimized formulation was selected depending on the highest percent drug entrapment. Morphology, mean size, encapsulation efficiency (EE) and in vitro drug release from niosomes in phosphate buffer pH 7.4, were evaluated. Drug‐cholesterol interaction was studied by FTIR spectroscopy. Drug retention in niosomes was performed at refrigerated temperature and room temperature for the period of two months. The optimized niosomal formulation was incorporated into 1% carbopol gel and evaluated for the ex vivo transocular drug permeation comparing to a plain drug gel through excised albino rabbit cornea using Franz diffusion cell.

Results: Maximum drug EE was obtained with Span 80: cholesterol niosomes of µmolar ratio (300:200). Also they were stable during the storage time. No significant interaction between the drug and cholesterol was detected. The optimized niosomal gel formulation showed prolonged drug release and enhanced drug ocular bioavailability comparing to the plain gel.

Conclusion: piroxicam niosomal formulation was prepared successfully and is useful as a sustained release preparation.
Elasticity measurements predict no membrane toxicity for the cardiolipin‐targeted mitochondrial therapeutic SS‐31 (1054.3)
Authors: Chao Wesley , Bracken Clay , Anderson Olaf , Birk Alexander , Szeto Hazel
Published: June 2021

SS‐31 is a mitochondria‐targeted therapeutic, currently in Phase 2 clinical trials, which protects cristae structure, prevents swelling of mitochondria and improves cellular respiration. We previously identified the target of SS‐31 as cardiolipin (CL), a phospholipid found only in the inner mitochondrial membrane. Here we utilize DOSY NMR to show that the diffusion rate of SS‐31 is reduced in the presence of CL‐containing bicelles, which is the first kinetic information on the interaction of SS‐31 with CL‐containing membranes. Because mitochondrial swelling involves solute transfer across the membrane, in addition to the changes in membrane morphology, it became important to test for membrane‐perturbing effects of SS‐31, particularly In light of evidence that membrane‐perturbation predicts toxicity for small molecules. We utilize a gramicidin‐based fluorescence assay using CL‐containing liposomes to assess drug‐induced membrane perturbations. We find that SS‐31 has no effect on synthetic membrane systems, even at several log orders above therapeutic concentration. The lack of membrane effects suggest that SS‐31 has no toxicity on the membrane, which is consistent with safety data from Phase 1 trials.
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
Reenergizing aged mitochondria to combat retinal degeneration (1060.1)
Authors: Mills William , Alam Nazia , Szeto Hazel
Published: June 2021

The major risk factors for Age‐Related Macular Degeneration (AMD) are age and oxidative stress. Aging is associated with bioenergetic failure, especially of the metabolically active retinal pigment epithelium (RPE), which is required for phagocytic function of photoreceptor outer segments. A2E and lipid peroxides in lipofuscin can increase the feed‐forward loop of oxidative damage to mitochondria in the RPE. Here, we propose a novel approach for resolving feed‐forward mitochondrial damage with the mitochondria‐targeted peptide SS‐31. With its strong antioxidant properties and ability to promote electron transport in aging mitochondria, we show optimization of ATP synthesis and minimization of oxidative stress. We created an age‐relevant RPE model using complex I inhibitors and A2E or lipid peroxide (tBHP) and demonstrated relevant energetic failure. SS‐31 treatment restored ATP synthesis, antioxidant capacity and phagocytic function of the RPE. Moreover, SS‐31 treatment in aged mice and rats improved visual acuity as measured by optokinetic tracking. Thus, our novel mitochondria‐targeted approach will preserve RPE mitochondrial function and will potentially prevent or delay progression of early AMD.
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
Pharmacokinetic study of two docetaxel preparations in Beagle dogs by a LC‐MS/MS method (1064.5)
Authors: Huang Xin , Liu Guanlan , Jiang Zhenzhou , Zhang Luyong
Published: June 2021

Pharmacokinetic study of two docetaxel preparations in Beagle dogs by a LC‐MS/MS method Xin Huang, Guanlan Liu, Zhenzhou Jiang and Luyong Zhang Jiangsu Center for Drug Screening, China Pharmaceutical University, Nanjing 210009, China Objective: To establish a LC‐MS/MS method for determination of docetaxel in dog plasma and compare the PK profiles of two preparations in Beagle dogs. Methods: 12 dogs were divided into two groups and i.v. administrated test and reference preparations, respectively. Plasma samples were collected at different time points. The analytes were separated through a C18 column with a mobile phase of methanol‐0.1% acetic acid water solution (85:15, v/v), and analyzed by ESI in SRM mode. The ions were m/z 830.4→303.9 and 876.4→591.3 for docetaxel and paclitaxel(IS), respectively. The pharmacokinetic parameters were calculated by DAS 2.0 software. Results: The calibration curves were linear over the ranged 3‐2000 ng/mL (r=0.9980) with the LLOQ 3 ng/mL. The within‐ and between‐batch precisions were within 8%, and the accuracy ranged from 94 to 110%. The overall recovery for docetaxel was about 91.3%. The main PK parameters t1/2, Cmax, MRT0‐24 and AUC0‐24 for test and reference preparations were: 7.3±2.0 h and 7.2±2.5 h, 796.1±321.8 ng/mL and 1248.0±178.6 ng/mL, 6.2±1.1 h and 5.3±0.8 h,948.4±100.2 ng·h/mL and 969.7±322.1 ng·h/mL. Conclusion: The method developed is sensitive, specific, rapid, and reproducible. It is suitable for PK studies of docetaxel in dogs. The results above shows no significant differences (P>0.05) between the two preparation in PK parameters in dogs.
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
Organizing signal transduction in the postsynaptic density (107.2)
Authors: Kennedy Mary
Published: June 2021

Excitatory neurons in the CNS receive thousands of synaptic contacts from other excitatory neurons. An increase in the strength of a relatively small number of these synapses can store a memory by binding connected neurons into a circuit in which all of the neurons fire each time the specific memory is evoked. An increase in synaptic strength involves addition of new glutamate receptors to the synaptic membrane, and re‐arrangement of the cytoskeleton to support a larger synapse. Biochemical signaling enzymes that control this process, which is called “activity‐dependent synaptic plasticity,” are organized near the postsynaptic membrane. The enzymes include several protein kinases, adenylyl cyclase, and regulators of Ras and Rap. Many of them are attached to a multiprotein scaffold called the “postsynaptic density.” At least five classes of scaffold proteins contribute to the precise orchestration of changes in synaptic strength: PSD‐95 and other MAGUKS, SHANKs, Homer, AKAPs, and Densin. What we have learned thus far about the dynamic properties of the postsynaptic density reveals new ways to rapidly regulate the number and sensitivity of membrane receptors.
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
Plasmalemmal lipid rafts are required for signal transduction by AGTR1 in neonatal mesangial cells (1088.11)
Authors: Adebiyi Adebowale , Soni Hitesh , John Theresa , Yang Fen
Published: June 2021

Angiotensin II (ANG‐II) receptors (AGTRs) contribute to renal glomerular physiology and pathophysiology in newborns, but the underlying mechanisms that regulate AGTR function in newborn glomeruli are poorly understood. Here, we investigated AGTR expression in primary mesangial cells cultured from newborn pig glomeruli. We also tested the hypothesis that plasmalemmal lipid rafts are essential for ANG‐II‐induced intracellular Ca2+ ([Ca2+]i) elevation and proliferation in the cells. RT‐PCR revealed that newborn pig glomerular mesangial cells (GMCs) express AGTR1, but not AGTR2. ANG‐II‐induced [Ca2+]i elevation in the cells was essentially abolished by EMD 66684, a selective AGTR1 antagonist, but not PD 123319, a selective AGTR2 antagonist. Cyclodextrin (CDX)‐mediated cholesterol depletion did not alter total AGTR1 protein, but reduced cell surface AGTR1 protein expression by ~ 50%. CDX also inhibited ANG‐II‐induced [Ca2+]i elevation by ~ 65%. The COOH‐terminus of porcine AGTR1 contains a caveolin (CAV)‐binding motif. However, newborn pig GMCs express CAV‐1, but not CAV‐2 and CAV‐3. In situ proximity ligation assay detected an association between AGTR1 and CAV‐1 in the cells. A synthetic CAV‐1 scaffolding domain (CSD) peptide attenuated ANG‐II‐induced [Ca2+]i elevation in the cells by ~ 69%. Real‐time imaging of cell growth kinetics revealed that ANG‐II stimulates newborn GMC proliferation. Moreover, ANG‐II‐induced GMC growth was reduced by EMD 66684, CDX, and CSD peptide, but not PD 123319. These findings indicate that AGTR1 is the functional AGTR subtype in newborn pig GMCs. Our data also suggest that lipid raft microdomains and CSD regulate ANG‐II‐induced [Ca2+]i elevation and proliferation in the cells.
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
Anti‐oxidative and anti‐inflammatory role of adipose stem cells in reversing pulmonary hypertension and associated cardiac remodeling (1090.9)
Authors: Rathinasabapathy Anandharajan , Shenoy Vinayak , Bruce Erin , Espejo Andrew , Nair Anand , Francis Joseph , Raizada Mohan , Katovich Michael
Published: June 2021

Pulmonary Hypertension (PH) is a debilitating cardiopulmonary disorder, often resulting in right heart failure. Existing therapy does not curtail the rate of mortality. Here, the protective role of adipose stem cell (ADSC) in attenuating PH was studied. Monocrotaline (MCT) was injected into 8‐weeks old male SD rats to induce PH. Post 2 weeks of MCT challenge, when the RVSP (Right Ventricular Systolic Pressure) was significantly elevated, ADSC were administered. After 4 weeks of MCT induction, RVSP (30.09±1.51 vs 86.71±4.21 mmHg), & RV Hypertrophy (RVH; 0.23±0.01 vs 0.60±0.03) were elevated. ADSC therapy significantly reduced RVSP (55.94±7.35) and RVH (0.34±0.04). Video of echocardiography demonstrated that RV end‐diastolic area, (0.12±0.01 vs 0.33±0.03 cm2) and RV ejection fraction (61.62±0.93 vs 29.47±3.76 %) was significantly dysregulated in PH. Further, the pulmonary blood flow (0.88±0.04 vs 0.58±0.06 m/s) and AT/ET (0.37±0.01 vs 0.18±0.02) was altered by MCT treatment. ADSC reverted all of these cardiac remodeling events. RT‐PCR demonstrated that the markers of fibrosis (TGFβ), pro‐inflammatory cytokines (TLR4, TNFα, IL1β) and oxidative stress (NOX2) were up regulated 4 to 5 fold and anti‐inflammatory cytokine (IL10) was down regulated 6 fold in PH animals. ADSC therapy normalized all of these parameters. Collectively, our data demonstrate that the anti‐oxidant and anti‐inflammatory role of ADSC arrested PH and associated cardiac remodeling and suggest that ADSC intervention could be a potential therapeutic alternative for the treatment of PH.

Grant Funding Source: HL056921 and HL102033
Proteomic profiling of quiescent and activated airway epithelial NKCC1 (1095.9)
Authors: Liedtke Carole , Litman Paul , Gokulrangan Giridharan
Published: June 2021

Protein scaffolds at the amino‐ or carboxyl termini have specific roles in NKCC1 activation and/or membrane expression: Activation of epithelial NKCC1 transport activity also requires posttranslational modification (PTM). The goal of this study is to identify cargo proteins associated with the protein scaffolds and to assess PTM of NKCC1 using proteomic/nano LC‐MS/MS methodology for more accurate modeling of NKCC1 regulation. NKCC1 was immunoprecipitated from Calu‐3 epithelial cells after treatment to modify NKCC1 activity and subjected to 1D gel electrophoresis. Protein bands were excised for in‐gel trypsin digestion followed by LS‐MS/MS analysis. Phosphoenrichment of in‐gel digests was used to identify sites of phosphorylation of NKCC1. We identified proteins associated with plasma membrane, endocytosis, exocytosis, intracellular signaling, structural proteins, endoplasmic reticulum, cytosol, and ubiquitination. We found that activated (okadaic acid treatment) NKCC1 mass decreased and inactive (siSPAK treatment) NKCC1 mass increased. Two unique sites of ubiquitination of NKCC1 were identified. Treatment with hyperosmotic sucrose for 4 min induced robust phosphorylation of NKCC1 in the C‐terminus that decreased after 15 min. We conclude that targeted proteomic analysis using LS‐MS/MS will generate more detailed information about NKCC1 and its associated protein scaffolds.

Grant Funding Source: Supported by a grant from the NIH, RO1‐HL‐58598, and by the Clinical and Translational Science Colla
Calcium signaling in the choroid plexus epithelium (1097.11)
Authors: Longden Thomas , Bonev Adrian , Nelson Mark
Published: June 2021

Choroid plexus epithelial cells (ECs) express a diverse array of pumps and ion channels that facilitate the production of cerebrospinal fluid in the brain ventricles. Our understanding of the molecular players involved in EC function is advancing, but to date little is known about EC calcium (Ca2+) signaling. We thus sought to determine the nature of Ca2+ signaling in native preparations of rat choroid plexus loaded with the Ca2+ indicator Fluo‐4, using high speed confocal imaging of intracellular Ca2+ events.

We observed three major forms of Ca2+ events in ECs: 1) Uni‐ or multicellular propagating waves. 2) Brief, localized, high amplitude events reminiscent of IP3‐mediated Ca2+ release in endothelial cells, which we term Ca2+ plexars.1 3) Low amplitude quantal events reminiscent of TRPV4 sparklets in endothelial cells.2Ca2+ activity in ECs was dependent on extracellular Ca2+, as its removal abolished all signals. Similarly, blockade of the sarco/endoplasmic reticulum ATPase terminated EC Ca2+ signaling. At some sites, Ca2+ plexars initiated sparklet activity.

We conclude that Ca2+ signaling in choroid plexus ECs is dependent on extracellular Ca2+ entry, which causes the release of Ca2+ from intracellular stores. Stores‐mediated plexar events also couple to sparklet events and may lead to regenerative Ca2+ release.

1. Ledoux et al. (2008) PNAS 9: 1397‐1403 2. Sonkusare et al. (2012) Science 336 6081: 597‐601

Grant Funding Source: Supported by National Institutes of Health and the American Heart Association.
Effect of resveratrol on the erectile machinery is due to the underlying mechanisms of the alpha 1‐adrenoceptor activation (1097.2)
Authors: Restini Carolina , Domingos Aline , Pereira Bruna , Vieira David
Published: June 2021

The anococcygeus muscle is part of the erectile machinery in male rodents whose innervation is mainly due the autonomic system. Activation of alpha1‐adreneceptor triggers the production of reactive oxygen species (ROS). Resveratrol (RESV) is an antioxidant compound. Hypothesis: RESV may reduce the contractile responses stimulated by alpha‐1‐adrenergic agonist, phenylephrine (PE). It was evaluated the effect of RESV on the contractile response to PE on the rat anococcygeus muscle. Male Wistar rats (n=5‐7) were killed by cervical dislocation (Ethics Committee Approval: 019/2012), the anococcygeus muscle was isolated and suspended in an organ chamber bath to measure isometric contractions. Preparations were stimulated with PE (1 nmol/L to 100 mmol/L) before and after 20 min incubation with RESV or RESV plus different drugs: prazosin (Pz: 10‐8 mol/L); 2‐APB (10‐4 mol/L); GF109203X (0.5x10‐5mol/L) or atorvastatin (ATV: 10‐4mol/L). Analysis: Student paired t‐test or one‐way ANOVA, Newman‐Keuls post‐test. RESV (10‐4 mol/L) reduced maximum contraction (MC) stimulated with PE (to 69.3±1.1%). Pz (alpha1‐adrenoceptor antagonist) + RESV reduced this MC (to 55.8±4.7%, p<0.0001). 2‐APB (IP3 receptor antagonist) was effective on reducing MC (to 28.8±1.19, p<0,001). RESV+2‐APB also reduced ME (to 5.3±1.4%, p<0.0001). pD2 values were reduced under RESV+2‐APB incubation. Incubation with GF109203X (PKC inhibitor) was effective on reducing MC (to 40.5±7.3%, p<0.0001). pD2 values were also altered after GF109203X incubation. ATV (NADPH oxidase inhibitor) +RESV reduced MC (56.9±7.7%, p<0.0001). The signaling triggered through stimulation of PE on the alpha 1‐adrenoceptors are involved in the production of free radicals, which were sensitive to RESV.

Grant Funding Source: Supported by CNPq/PIBIC, UNAERP
Aldosterone activates colonic K secretion via BK channels (KCa1.1) and membrane trafficking (1097.9)
Authors: Halm Susan , Halm Dan
Published: June 2021

Aldosterone (aldo) activates electrogenic K+ secretion in guinea pig distal colon via a mechanism requiring protein synthesis. Channel blocker sensitivity supported conductive apical K+ exit and basolateral Cl‐ exit during aldo stimulation, similar to adrenergic activation. Paxilline [1 μM], BK channel (KCa1.1, Kcnma1) blocker, inhibited ~50% of short‐circuit current (Isc) and transepithelial conductance (Gt) activated by aldo; and, CaCCinh‐A01 [30 μM], Ca++‐activated Cl‐ channel blocker, eliminated aldoIsc and decreased aldoGt. Signaling for K+ secretion by aldo involved cAMP and serum‐glucocorticoid protein kinase (sgk) as indicated by elimination of aldoIsc with inhibition of soluble adenylyl cyclase by KH7 [30 μM] or sgk by GSK‐650394 [10 μM]. Elimination of aldoIsc by inhibiting dynamin (dyngo‐4a [30 μM]) or clathrin (pitstop‐2 [20 μM]) indicated a requirement for clathrin dependent endocytosis during aldo signaling. Manipulating the ubiquitylation cycle by inhibiting ubiquitin‐E1 ligase (UBEI‐41 [50 μM]) also inhibited aldoIsc; and, disrupting membrane localization of small GTPases such as ras, rho, or rab with farnesyltiosalicylate [10 μM] abolished aldoIsc. Dependence on microtubular transport was supported by loss of aldoIsc with inhibiting dynein (ciliobrevin‐D [10 μM]). Together these results support an aldosterone signaling mechanism for activating electrogenic K+ secretion involving cAMP, sgk, endocytosis, and microtubular transport. [NIH DK65845]