bluuebunny/crossref_metadata_2025 huggingface dataset:
Ang‐(1–7) Induced MAS1 Receptor‐Mediated Angiogenesis in the Rat Microvasculature
Authors: Hoffmann Brian Robert , Stodola Timothy J. , Wagner Jordan R. , Greene Andrew S.
Published: June 2021

The renin‐angiotensin system influences hypertension and angiogenesis. Specifically, angiotensin II (AngII) has been shown to regulate angiogenesis and cause vasoconstriction, fibrosis, cellular growth, and migration through the AT1 receptor. Angiotensin 1–7 (Ang(1–7)), whose actions are mediated by the Mas1 receptor, can act antagonistically to AngII by inducing vasodilation, anti‐fibrotic, anti‐hypertrophic, and anti‐proliferative effects. However, Ang‐(1–7) induced MAS1 receptor signaling is much less defined than AngII signaling, especially during angiogenesis. Here we show that Ang‐(1–7) promotes angiogenesis in the microvasculature of the rat hind limb through the Mas1 receptor independently of AngII and the AT1 receptor. Additionally, we show in an ex vivo tube formation assay that Ang‐(1–7) induces an increased angiogenic response among rat microvascular endothelial cells. Utilizing advanced proteomic methodology, downstream components of the Mas1 receptor signaling pathway were also identified to determine possible signaling pathways in the angiogenic response. Signaling pathway components identified included proteins involved in nascent protein synthesis, exocytosis, and cellular reorganization, along with regulators of nitric oxide, G‐protein, and NF‐kappaB signaling.
Alternaria alternata aeroallergens evoke ATP release and IL‐33 secretion from normal and asthmatic bronchial epithelial cells.
Authors: O'Grady Scott M. , Maniak Peter J. , Lancto Cheryl , Shivappa Nandadevi , Kita Hirohito
Published: June 2021

Exposure of human bronchial epithelial (HBE) cells from normal and asthmatic subjects to Alternaria alternata extract evoked rapid and sustained ATP release with greater efficacy observed in cells from asthmatic subjects. Previously, Alternaria aeroallergens were shown to elicit sustained increases in intracellular [Ca2+]i that depended on coordinated activation of P2Y2 and P2X7 receptors. In this study, pretreatment with BAPTA‐AM inhibited ATP release, indicating dependency on increases in [Ca2+]i. Alternaria‐evoked ATP release exhibited a greater peak response and higher EC50 value in cells obtained from asthmatic subjects compared to normal controls. Furthermore, the maximum increase in [Ca2+]i was greater in cells from asthmatic subjects. The vesicle transport inhibitor brefeldin A blocked Alternaria‐stimulated incorporation of fluorescent lipid (FM1–43) labeled vesicles into the plasma membrane and ATP release. Additionally, inhibiting vesicular ATP uptake with bafilomycin also blocked ATP release similar to brefeldin A. These results indicated that a primary mechanism for Alternaria‐induced ATP release involved Ca2+‐dependent vesicular transport. Serine and cysteine protease inhibitors also reduced Alternaria‐induced ATP release. This result was consistent with our previous data showing that Alternaria‐evoked increases in [Ca2+]i depend on purinoceptor signaling.
Antinociceptive effect of p‐cymene in mice – evidence of involvement of periaqueductal gray area
Authors: de Souza Siqueira Quintans Jullyana , de Santana Michele Fraga , Brito Renan Guedes , de Lucca Júnior Waldecy , Júnior Lucindo Quintans
Published: June 2021

p‐Cymene (PC) is a monoterpene present in the essential oil of many aromatic medicinal plants. We investigated the anti‐hypernociceptive effect of PC and your action in the central nervous system (CNS) in rodents. Male mice were pretreated with PC (25, 50 and 100 mg/kg, i.p.), morphine (5 mg/kg, i.p.) or vehicle (distilled water + one drop of Tween 80 0.2%), 1h before of mechanic hypernociception induction by carrageenan (CG), TNF‐α, prostaglandin (PGE2) and dopamine (DA). Experimental protocols were approved by the Animal Care and Use Committee at the Federal University of Sergipe (CEPA#19/11). The data were analyzed by ANOVA (two‐way) followed by Tukey's test (p<0.05). Our results revealed that acute treatment with PC, all doses, reduced significantly the hypernociception (p<0.01 or p<0.001) caused by CG, TNF‐α, PGE2 and DA. Such results were unlikely to be provoked by motor abnormality. The immunofluorescence assay demonstrated a significant increase in the number of Fos positive cells (p<0.05 or p<0.01) in the periaqueductal gray. Together, our results provide evidence to show that PC attenuates nociception at least through an activation of CNS area, mainly periaqueductal gray.
Acute toxicity evaluation of Genipa americana L. (Rubiaceae)
Authors: de Souza Cavalcanti Karen Pena , Jimenez George Chaves , Silva Elisângela C. B. , Anjos Falba Bernadete Ramos , Souza Ivone Antonia , Cabrita Antonio Silvério
Published: June 2021

;The specie G. americana L. (Rubiaceae) is an exotic plant with edible fruit, aphrodisiac, antianaemic and useful in diseases of the spleen and liver. The objective of this study was to determine the acute toxicity of crude ethanol extract of the fruit of G. americana parameters by evaluating general toxicity. For the tests were selected Swiss albino mice of the species Mus musculus females, weighing 25–30g were divided into groups of six. The animals were kept in polypropylene cages under controlled lighting (cycle 12 hours light/dark) and temperature (25 ± 2 °C), receiving food and water ad libtum. The doses of 1000, 2000 and 3000 mg/kg administered intraperitoneally (VIP) and 1500, 3000 and 4500 mg/kg orally. The 50% lethal dose (LD50) was not determined due to lack of deaths of the animals at the doses employed, being regarded maximal for the route. Among the signs of toxicity observed highlight the stimulating effect on the CNS. The results indicate that the ethanol extract of G. americana has low acute toxicity in mice when administered by oral and intraperitoneal administration.
In vivo and in vitro citotoxic activity of Hylocereus undatus fruit extract in cancer cells
Authors: Calvillo Tannia Uribe , Soriano Alejandro , Rosales Arnoldo Michel , Estrada Manuel Jimenez , Beltrán Elpidio Peña , Enciso Iván Delgado
Published: June 2021

Cancer is the leading cause of death in the world. Transformation from one normal cell into a cancer cell results from several modifications and damage to a genetic level, becoming into a tumor. Antitumorigenic capacity of antioxidants to diminish DNA damage is mediated by the elimination of reactive oxygen species (ROS). Cactaceae family plants have a great diversity of pigments, which present antioxidant activity like betacyanins and vitamin C. Alamar Blue assay was used to study the antiproliferative activity of Hylocereus extract on cervix cancer cell line HeLa (PVH‐18). The highest citotoxic effect in vitro showed by Hylocereus extract was with an initial concentration of 0.02 gr/mL, resulting on 50% mortality. In vivo experiments showed an inhibitory effect on tumor's volume with a decrease in the tumoral growing speed up to 50% trough a period of 35 days of treatment with a daily administration of Hylocereus extract (100 μL). Greater inhibitory effect was observed with the highest concentration of the extract that we used. So, this results demonstrate the capaticy of Hylocereus undatus compounds to diminished cell survival on cervix cancer in vitro e in vivo.
Pro‐Resolving Mediator Production by Human Phagocytes and their Transcellular Biosynthesis with Microparticles in Inflammation‐Resolution
Authors: Dalli Jesmond , Serhan Charles
Published: June 2021

Phagocytes are central in orchestrating the acute host response during inflammation and its timely resolution. Using lipid mediator (LM) metabololipidomcs, we assessed LM levels in neutrophils (PMN), apoptotic PMN and macrophages. During efferocytosis, macrophages upregulate the biosynthesis of specialized pro‐resolving mediators (SPM), including resolvin (Rv) D1 and RvD2, that were further elevated by PMN microparticles (MP). Using deuterium labeled precursors we found that both apoptotic PMN and MP donated precursors to macrophages that were utilized for transcellular SPM biosynthesis. Fresh PMN from peripheral blood with zymosan gave predominantly leukotriene B4, whereas apoptotic PMN displayed both elevated prostaglandin E2 and lipoxin (LX) B4. Lineage differentiated macrophages showed distinct LM profiles where M2 macrophages gave higher SPM including maresin 1 and lower prostaglandins then M1. These profiles were regulated during efferocytosis where M1 cells gave increased SPM including LXA4 while overall LM biosynthesis was reduced in M2. Together these findings establish the LM signatures of human phagocytes and their regulation during distinct stages of acute inflammation‐resolution.

This work was supported by the NIH (grants GM038765 and GM095467).
AMP‐ACTIVATED PROTEIN KINASE AS REGULATOR OF P2Y6 RECEPTOR‐INDUCED INSULIN SECRETION IN MOUSE PANCREATIC BETA CELLS
Authors: Balasubramanian Ramachandran , Maruoka Hiroshi , Jayasekara Suresh MP , Gao Zhanguo , Jacobson Kenneth A
Published: June 2021

5′‐AMP‐activated protein kinase (AMPK) and its pharmacological modulators have been targeted for treating type 2 diabetes. Extracellular UDP activates P2Y6 receptors (P2Y6Rs) in pancreatic β cells to release insulin and reduce apoptosis, which would benefit diabetes. Here, we studied the role of P2Y6R in activation of AMPK and insulin secretion in MIN6 mouse pancreatic beta cells. Treatment with a potent P2Y6R dinucleotide agonist MRS2957 activated AMPK, which was blocked by P2Y6R‐selective antagonist. Also, MRS2957 induced phosphorylation of acetyl‐coenzyme A carboxylase (ACC), a marker of AMPK activity. Calcium chelator BAPTA‐AM, calmodulin‐dependent protein kinase kinase (CaMKK) inhibitor STO‐069 and IP3 receptor antagonist 2‐APB attenuated P2Y6R‐mediated AMPK phosphorylation revealing involvement of intracellular calcium signaling pathways. P2Y6R agonist induced insulin secretion only at high glucose, which was reduced by AMPK gene silencing suggesting an involvement of AMPK in P2Y6 receptor‐mediated insulin secretion. Thus, P2Y6R has a crucial role in β cell function, suggesting its potential as a therapeutic target in diabetes.
Characterization of MT1 melatonin receptor expressing neurons in the medial habenula, habenula commissure and periaqueductal grey of the C3H/HeN mouse brain
Authors: Evely Katherine Michelle , Adamah‐Biassi Ekue Bright , Hudson Randall L. , Dubocovich Margarita L.
Published: June 2021

Melatonin (MLT) is rhythmically secreted from the pineal gland and acts on two G protein‐coupled receptors, termed MT1 and MT2. Brain tissue from a transgenic mouse line expressing red fluorescence protein (RFP) at the MT1 receptor promoter provides a method of localizing the receptor. RFP‐MT1 fluorescence and immunoreactivity was localized to the medial habenula (MHb), habenula commissure (HbC) and the midbrain dorsal medial periaqueductal grey (DMPAG) area. The habenula acts as a relay station from forebrain to midbrain. The downstream PAG plays a prominent role in pain transmission (Behbehani, Prog Neurobiol 1995;46:575–605). The goal of our research is to investigate the distribution of the MT1 receptor in these cholinergic, dopaminergic and glutamatergic neuronal systems. Immunofluorescence co‐staining for RFP along with choline acetyl transferase (ChAT), tyrosine hydroxylase (TH) or vesicular glutamate transporter (VGLUT2) is used to investigate the distribution of the MT1 receptor. Results show RFP‐MT1 expression in the dorsal MHb, clearly separated from ChAT staining in the ventral MHb. TH colocalized with RFP‐MT1 in the HbC, PAG, and the ependymal lining of the aqueduct. VGLUT2 and RFP‐MT1 positive cells are present in dorsal MHb neurons. These results indicate a possible role for the MT1 receptor in the modulation of glutamatergic and dopaminergic neurotransmission. Supported by DA 021870 MLD.
Mapping the Binding of G Protein–Coupled Receptor Kinases 4γ (GRK4γ) with Gαs
Authors: Nguyen Jenny H , Lacuran Alecander E , Oelschlaeger Peter , Andresen Bradley T
Published: June 2021

GRK4γ appears to be constitutively active and two polymorphisms in the RH domain (R65L and A142V) and one in the kinase domain (KD) (A486V) are associated with hypertension. Currently our model is that GRK4γ interacts with inactive Gαs through part of the Gαs helical domain. Since GRK2's RH domain interacts with Gαq, we hypothesized that GRK4γ interacts with inactive Gαs through its RH domain. Since GRK4γ, but not GRK5, interacts with inactive Gαs, we utilized a chimeric approach and co‐immunoprecipitation to identify the region that GRK4γ binds to Gαs. Initially we constructed chimeras of GRK4 & 5 examining multiple helices in the RH domain as well as the point where the RH domain polymorphisms lie. In contrast with the hypothesis, our data indicate that GRK4γ interacts with either the KD or distal portion of the RH domain which is largely absent in GRK4γ. GRK4γ was modeled from GRK6 (PBD:3NYO), and Autodock 4.2 was used to predict other potential Gαs‐helical domain binding sites on GRK4γ. Further chimeric based analysis indicates that the likely mode of interaction is through a region at the distal end of GRK4γ, which is distinct from GRK5. Our current model indicates that GRK4γ is poised next to the receptor allowing for it to efficiently phosphorylate the receptor after Gαs dissociates and binds to adenylyl cyclase, but fails to explain the mechanistic consequence of the R65L and A142V polymorphisms.
G Protein‐Coupled Receptor Kinase 2 (GRK2) is Localized to Centrosomes and Mediates Epidermal Growth Factor‐Promoted Separation of Duplicated Centrosomes
Authors: So Christopher , Michal Allison , Benovic Jeffrey
Published: June 2021

G protein‐coupled receptor kinases (GRKs) play a central role in regulating receptor signaling. Recent studies have suggested a broader role for these enzymes in regulating normal cellular functions. For example, GRK5 has been shown to localize in centrosomes and modulate cell cycle progression. In this study, we demonstrate that GRK2 is also localized to centrosomes. Similar to GRK5, the centrosomal localization of GRK2 is microtubule‐independent and it does not affect normal centrosome duplication. However, in contrast to GRK5, GRK2 is not involved in centrosome nucleation. Interestingly, knockdown of GRK2 inhibits epidermal growth factor (EGF)‐initiated separation of duplicated centrosomes during S phase in both HeLa cells and retinal pigment epithelial 1 (RPE1) cells. This EGFR‐GRK2 mediated process is dependent on the protein kinases MST‐2 and Nek2a but does not involve Polo‐like kinase 1 (PLK1). This separation during S phase results in enhanced centrosome amplification, resulting in a rise in the number of cells demonstrating aberrant duplicated centrosomes. Collectively, these findings demonstrate that GRK2 is localized in centrosomes and plays a central role in mitogen‐promoted centrosome separation.
Sphingosine‐1 phosphate receptor‐1 desensitization mediated by isoforms of protein kinase C: receptor phosphorylation
Authors: Morquecho‐León Marco Antonio , Bazúa‐Valenti Silvana , Romero‐Ávila María Teresa , García‐Sáinz Jesús Adolfo
Published: June 2021

Sphingosine‐1 phosphate (S1P) is important in the regulation of angiogenesis, vascular permeability and stability, development of new blood vessels, vascular maturation, cardiac development, immunity and cellular mobility routing. A deficiency in the regulation of sphingosine phosphate receptor type 1 (S1PR1) can cause health disorders, such as development of autoimmune diseases or formation of new vasculature in solid tumors.

Although it is known that PKC phosphorylates and possibly regulates S1PR1, we don't know which PKC isoforms are involved, as well as target sites. In the present investigation we have found that S1PR1 is phosphorylated by the action of 12‐O‐tetradecanoylphorbol‐13‐acetate, a direct activator of PKCs, and that this phosphorylation does not occur when using a general inhibitor of PKCs bisindolylmaleimide and decreases by approximately 90% when using a specific inhibitor for PKCα (Gö6976).

Using dominant negative isoforms of PKC α and βII we found that receptor phosphorylation mediated by TPA was prevented in approximately 50% and 40% respectively. Site directed mutagenesis showed that the mutant receptor ‐changes serines 351, 353, 355, 358 and 359 to alanines‐(S1PR1–5A) phosphorylation decrease nearly to basal levels in both S1P and TPA stimulations.

Research sponsored by DGAPA IN200812 and CONACyT 177556
PDZ ligand‐dependent phosphorylation of Ser336 in the C‐terminal tail of the P2Y1 receptor blocks agonist‐promoted internalization in HEK293 cells
Authors: Nicholas Robert A , Harden Ken , Qi Ai‐Dong
Published: June 2021

We have shown previously that phosphorylation of Ser352 and Ser354 in the C‐terminus of the ADP‐activated P2Y1 receptor is necessary for agonist‐promoted internalization in MDCK cells. Unexpectedly, ADP addition did not promote internalization of the P2Y1 receptor in HEK293 and 1321N1 cells. Mutational analysis of Ser and Thr residues in the C‐tail of the P2Y1 receptor revealed that a S336A mutation within a highly conserved SRAT motif conferred agonist‐dependent internalization in HEK293 and 1321N1 cells, while it markedly increased the rate and extent of receptor internalization in MDCK cells. Phosphorylation studies showed that Ser336, Ser352 and Ser354 were phosphorylated in response to agonist in HEK293 and 1321N1 cells, while only Ser352 and Ser354 were phosphorylated in MDCK cells. Additionally, mutation of SRAT to DRAD to mimic Ser336 phosphorylation abolished agonist‐promoted internalization of the P2Y1 receptor in MDCK cells. Deletion of the PDZ ligand at the C‐terminus of the receptor conferred agonist‐promoted internalization and prevented phosphorylation of Ser336, suggesting recruitment of a PDZ protein‐dependent kinase that regulates internalization. This study reveals a critical role for Ser336 in the regulation of agonist‐promoted internalization of the P2Y1 receptor that depends on the cell type in which it is expressed. (Supported by NIH grant HL54889).
Selective allosteric modulation of the 5‐HT3A receptor by halogenated indole derivates
Authors: Barnes Nicholas , Chiarenza Alessandra , Gambassi Silvia , Powell Andrew , Grafton Gillian
Published: June 2021

The human 5‐HT3A receptor is a member of the cys‐loop ligand‐gated ion channel family. Allosteric modulators of this receptor have potential roles in the treatment of irritable bowel syndrome. In the present study we have used radioligand binding assays, fluorescence indicators and patch clamp techniques to explore the allosteric modulation of human 5‐HT3A stably expressed in HEK293 cells. We examined a range of halogenated indole derivatives and discovered seven novel positive allosteric modulators (PAMs) of the 5‐HT3 receptor, of which two were examined in more detail. Radioligand binding assays showed that neither 5‐chloroindole nor 5‐iodoindole (up to 100 μM) competed with [3H]‐granisetron for the 5‐HT3A receptor orthosteric site or affected the affinity of ondansetron for the receptor. In contrast both molecules provoked an increase in the affinity of several agonists/partial agonists for the receptor. Importantly, there was no effect on human nicotinic alpha 7 receptor mediated responses, unlike previously described PAMs at the 5‐HT3A receptor, allowing the use of 5‐HT3A/nicotinic alpha‐7 chimeras to map the binding site(s) for these molecules. Such studies will aid rational design of useful therapeutic agents that modulate 5‐HT3 receptor function.
Repurposing anti‐hypertensive drugs for Alzheimer's disease
Authors: Pasinetti Giulio Maria
Published: June 2021

Growing evidence suggests cognitive deterioration in Alzheimer's disease (AD) is directly linked to accumulation of extracellular soluble oligomeric Aβ species in the brain. Oligomeric Aβ induces synapse degeneration and synaptic plasticity disruption, contributing to mechanisms underlying onset and progression of AD dementia. Carvedilol is a brain bioavailable and bioactive small antihypertensive drug that has significantly reduced brain contents of oligomeric Aβ and attenuated cognitive deterioration in transgenic mouse models of AD by directly interfering with Aβ oligomerization. Our study was designed to use a structure activity relationship (SAR) approach to identify novel carvedilol analogs lacking α1/β2 adrenergic receptor activity, which mediate cardiovascular effects while enhancing structural features essential for anti‐Aβ oligomerization activity. In vitro testing confirmed the majority of carvedilol analogs tested retained anti‐oligomerization activity. In vivo efficacy testing showed select carvedilol analogs can reduce Aβ oligomerization in the brain without eliciting any hypotensive side effect. Our study demonstrated that carvedilol analogs can be repositioned for treatment of AD.
Effect of Hydrogen Sulfide on Isolated Bovine Ocular Smooth Muscle
Authors: Okpobiri Anita Chinwe , Njie‐Mbye Ya Fatou , Robinson Jenaye , Chitnis Madhura , Opere Catherine , Ohia Sunny
Published: June 2021

Purpose

To investigate the pharmacological effects of the amino acid, L‐cysteine, a substrate for hydrogen sulfide (H2S) production, on isolated pre‐contracted bovine iris‐ciliary muscle. Furthermore, we examined the mechanism of action of L‐cysteine in eliciting relaxations of this tissue.

Methods

Bovine iris‐ciliary muscle strips were mounted in organ baths containing oxygenated Krebs buffer (pH 7.4, 37□ C). Changes in isometric tension were recorded and analyzed via Force‐displacement transducers. Effects of L‐cysteine on carbachol‐induced tone were studied in the presence of enzyme inhibitors for H2S biosynthetic pathway (AOA; cystathionine β‐synthase inhibitor) and prostanoids (flurbiprofen; cyclo‐oxygenase (COX) inhibitor).

Results

L‐cysteine (0.01 nM–100 μM) evoked a dose‐related relaxation of carbachol‐induced tone in bovine iris‐ciliary muscle, reaching a maximum inhibition of 88% at 10 μM (with an IC50 of 2 nM). Relaxations elicited by L‐cysteine were unaffected by pretreatment of tissues with flurbiprofen (3 μM). The inhibitor of cystathionine β‐synthase, AOA (10 μM) blocked only relaxations caused by high concentrations of L‐cysteine (>; 500 nM).

Conclusions

L‐cysteine can relax pre‐contracted bovine iris‐ciliary muscle, and this effect is dependent on endogenous production of H2S. Furthermore, prostanoids are not involved in the inhibitory action of L‐cysteine in this smooth muscle.
Angiotensin II and mitochondrial‐derived oxidative stress play a role in coronary arteriole remodeling in type 2 diabetes
Authors: Halleck Kathryn Elizabeth , Katz Paige S , Dos Santos Karen Tiago , Trask Aaron J. , Lucchesi Pamela A.
Published: June 2021

Angiotensin II (Ang II) and oxidative stress play a key role in type 2 diabetic (T2DM) vasculopathies. Our previous studies indicated that coronary arterioles (CA) in T2DM (db/db) mice undergo inward hypertrophic remodeling leading to reduced coronary flow reserve. In this study we examined the impact of Ang II Type 1 receptor (AT1R) blockade in CA remodeling by treating 12wk old control and db/db mice with losartan (3mg/kg/day) for 4 wks. CA structure and passive mechanical properties were measured by pressure myography. In losartan‐treated db/db mice (n=10), CA wall thickness and medial‐to‐lumen ratio were significantly reduced vs. untreated db/db. Losartan significantly decreased CA VSMC number in db/db mice measured by DAPI staining. Given the role of the NADPH oxidase in Ang II signaling, we next treated control and db/db mice for 4 weeks with apocynin (5mM). Apocynin had no effect on T2DM‐induced CA remodeling. Conversely, a mitochondrially targeted anti‐oxidant, mito‐tempol (2mmoles/kg/day, for 6 weeks), significantly attenuated T2DM‐inward CA remodeling. In primary cultured VSMC, pretreatment with 2μM mito‐Tempol decreased Ang II (100nM)‐induced ROS generation as measured by DHE staining. These data suggest Ang II‐induced mitochondrial oxidative stress plays a key role in coronary microvascular remodeling in T2DM. Support: R01HL056046 to PAL
A role for Nrf2 in the prevention of salt‐induced vascular dysfunction
Authors: Priestley Jessica R. C. , Dahlgren Allison F. , Weinberg Brian D. , McCord Joe M. , Lombard Julian H.
Published: June 2021

Consumption of a high salt (HS) diet results in vascular oxidant stress and impairs endothelium‐dependent relaxation in a variety of human and experimental models. Nuclear factor erythroid‐2‐related factor 2 (Nrf2) is a transcription factor that induces the expression of many critical antioxidant genes. We measured protein expression of Nrf2 and its cytosolic inhibitor, kelch‐like ECH‐associated protein 1 (Keap1), in mesenteric and cerebral arterial homogenates from Sprague‐Dawley (SD) rats fed HS (4% NaCl), low salt (LS), or HS with low dose angiotensin II (ANG II) infusion to restore normal plasma ANG II levels. While Nrf2 expression was similar between groups, Keap1 expression was significantly lower in ANG II‐infused rats (p < 0.05). The Nrf2:Keap1 ratio normalized to LS was significantly increased in both mesenteric (LS = 100.00% ± 19.76; HS = 176.34% ± 29.98; HS + ANG II = 561.44% ± 210.39) and cerebral (LS = 100.00% ± 16.21; HS = 181.67% ± 69.31; HS + ANG II = 677.55% ± 172.38) arterial beds. The dietary supplement Protandim, shown to induce Nrf2 expression, improved endothelium‐dependent relaxation to acetylcholine in salt‐fed SD rats (HS = 0.50 μm ± 0.96; HS + Protandim = 6.67 μm ± 1.28; p = 0.008) and hamsters (HS = 1.33 μm ± 0.52; HS + Protandim = 5.60 μm ± 0.68; p < 0.001 ). Together, these data suggest a functional role for the Nrf2 antioxidant defense system in preventing salt‐induced vascular dysfunction.
Retinoic acid‐induced regulation of guanylyl cyclase/natriuretic peptide receptor‐A gene expression in gene‐targeted mice via histone H3 modifications
Authors: Kumar Prerna , Periyasamy Ramu , Das Subhankar , Pandey Kailash N
Published: June 2021

Guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) is the biological receptor of cardiac hormones atrial and brain natriuretic peptides (ANP and BNP), which plays an important role in maintaining renal and cardiovascular homeostasis. Present study was designed to uncover the regulatory mechanism involved in Npr1 gene (coding for GC‐A/NPRA) transcription by all‐trans retinoic acid (ATRA) and histone deacetylase (HDAC) inhibitor sodium butyrate (NaBu) in Npr1 gene‐targeted mice. Mice (16‐week old) were injected intraperitonealy with ATRA (0.5 mg/Kg/day), NaBu (0.5 mg/Kg/day), and ATRA‐NaBu (1.0 mg/Kg/day) for 2‐week. The results showed that renal HDAC activity was increased and acetylated histone levels were decreased in gene‐knockout heterozygous (1‐copy; Npr1+/−) mice compared with wild‐type (2‐copy; Npr1+/+) mice; however, HDAC activity was decreased in gene‐duplicated (3‐copy; Npr1++/+) mice. A significant increase in acetylation of histone H3 and H4 and reduction in HDAC activity was observed in ATRA‐ and NaBu‐treated animals. ATRA and NaBu cooperatively activated renal Npr1 mRNA levels by 3‐fold in 1‐copy and 3.5‐fold in 2‐copy mice. The present results indicate that ATRA epigenetically regulates Npr1 gene in vivo via histone modifications and should have important implications in renal and cardiovascular events in disease states. This work was supported by the NIH grant (HL62147).
Potential proarrhythmic alterations in ventricular cardiomyocytes expressing CREM‐IbΔC‐X, a repressory human cardiac isoform of cAMP response element modulator
Authors: Schulte Jan Sebastian , Fehrmann Edda , Tekook Marcel , Heinick Alexander , Seidl Matthias Dodo , Schmitz Wilhelm , Müller Frank Ulrich
Published: June 2021

Transgenic mice with heart directed expression of transcription factor CREM‐IbΔC‐X (TG) develop atrial alterations including dilatation, impaired electrical conduction and impaired regulation of intracellular Ca2+ preceding spontaneous‐onset atrial fibrillation. Here, we studied whether CREM‐IbΔC‐X is linked to proarrhythmic alterations in ventricular cardiomyocytes (CMs). Action potentials were prolonged in TG CMs along with reduced mRNA of the Ito underlying channel subunit Kv4.2 as compared to wild‐type (WT) CMs. In TG vs. WT CMs Ca2+ transient amplitude was unaltered under basal conditions but increased under stimulation with isoproterenol. Under both conditions Ca2+ release was retarded but the Ca2+ decay was accelerated in TG vs. WT CMs. The frequency of Ca2+ sparks was enhanced while spark amplitude was reduced in TG vs. WT CMs. Stress stimulation protocols revealed an enhanced rate of spontaneous Ca2+ releases in TG vs. WT CMs. Hence, human cardiac isoform CREM‐IbΔC‐X is linked to an increased susceptibility to arrhythmia possibly by regulating the transcription of genes modulating AP shape and duration and intracellular Ca2+ cycling. Supported by IZKF Münster
Heterogeneity of cardiac myocyte contractile properties during long‐term stimulation; substrate effects
Authors: Barton Gregory , Diffee Gary
Published: June 2021

Substrate utilization in the heart is well documented and favors fatty acid oxidation in normal healthy hearts. Most studies looking at metabolic effects on contractile properties have been done in the whole heart and little information is known about the cellular contractile response to different substrates. To determine potential substrate effects on contractile properties in isolated myocytes, we measured percent shortening and rate of shortening at time 0 (T0) and after 10 minutes (T10) of continuous stimulation at 1 Hz in myocytes isolated from healthy hearts and bathed in either 11mM glucose (G) or 0.1mM palmitate (P) during contractile measurements. We determined not only mean shortening but the distribution of shortening properties among cells in order to determine how much heterogeneity there was among myocytes. Mean shortening was significantly greater in P (12.2 %) than in G (9.8%). Although the mean shortening significantly decreased in both groups at T10 compared to T0, analysis of the distribution showed nearly all cells decreased % shortening at T10 in P, while the cells in G demonstrated a more heterogeneous response at T10, with one‐third of the cells showing an increased shortening. These results suggest substrate does influence shortening properties, and distributional analysis reveals significant heterogeneity among cells in their response to different substrates.

Supported by NIH AG 030423
NOX2 DEFICIENCY LIMITS THE DEVELOPMENT OF INSULIN RESISTANCE PRODUCED BY A HIGH‐FAT DIET
Authors: Didion Sean P
Published: June 2021

The goal of the present study was to test the hypothesis that Nox2‐derived superoxide contributes to the development of insulin resistance produced by a high fat diet (HFD). C57Bl/6 (wild‐type, WT) and Nox2‐deficient (Nox2−/−) mice were fed a control (10% kcal) or a 60% (kcal) HFD for 12 wks. A HFD produced a similar degree (P>;0.05) of obesity and visceral adiposity in WT and Nox2−/− mice fed a HFD (eg, body weight, 46±1 and 42±4 g, respectively). Fasting glucose levels were significantly higher (P<0.05) in WT mice fed a HFD compared to HFD Nox2−/− mice. A HFD was associated with the development of insulin resistance in WT mice (eg, following a 1 mg/kg ip glucose load, blood glucose levels at 120 min were 362±63 and 170±10 mg/dl in WT mice fed a HFD or a control diet, respectively). However, despite being equally obese as HFD WT mice, HFD Nox2−/− mice were able to clear glucose at a rate similar to that observed in WT mice fed a control diet (eg, at 120 min, blood glucose levels were 207±56 vs. 170±10 mg/dl, respectively). Molecular analysis suggests that that the ability of HFD Nox2−/− mice to clear glucose normally is not reflective of alterations in GLUT4 expression. These genetic data strongly implicate a role for Nox2‐derived superoxide in the development of insulin resistance. Supported by NIH HL‐089884 & HL‐107632.
The Effects of Palmitic Acid and Oleic Acid on Macrophage Gene Expression
Authors: Reyes Ariel Delos , Camell Christina , Smith C. Wayne , Rumbaut Rolando E.
Published: June 2021

Macrophages are critical in regulating inflammation and can be polarized to promote or inhibit inflammation. They have crucial roles in the clearance of excess fatty‐acids; however, their response to individual fatty‐acids are unclear. We hypothesized that bone marrow derived macrophages (BMDMs) treated with oleic acid will have increased anti‐inflammatory gene expression, and BMDM treated with palmitic acid will have increased pro‐inflammatory gene expression. We tested this hypothesis by culturing murine BMDM in the presence of oleic or palmitic acid and analyzing gene expression of markers for pro‐inflammatory (TNFα, CD11c and iNOS) and anti‐inflammatory macrophages (Arg1, MGL1, CD206, Chi3L3 and RETNLA). Palmitic acid increased TNFα expression though decreased CD11c and iNOS expression, whereas oleic acid increased Arg1 and MGL1 expression in BMDMs. We found no change in other genes examined. Additional macrophage markers and samples would need to be analyzed to clearly identify if fatty‐acids can polarize BMDMs; however, our data suggests that individual fatty‐acids can regulate expression of some pro‐ and anti‐inflammatory macrophage markers.

Support: APS Frontiers in Physiology Fellowship; NRSA F‐31‐DK084841‐04; USDA/ARS CHRC 6250–51000‐055
Acute Effects of a Mixed Meal on Arterial Stiffness and Central Hemodynamics in Healthy Adults
Authors: Taylor Jennifer Lynne , Curry Timothy B , Matzek Luke J , Joyner Michael J , Casey Darren P
Published: June 2021

Elevated central hemodynamics and arterial stiffness are associated with increased peripheral resistance and higher sympathetic nervous system activity. Additionally, consumption of a meal is known to be sympathoexcitatory. However, the acute effects of a meal on aortic wave reflection and stiffness are unknown. Therefore, we tested the hypothesis that aortic wave reflection and stiffness would increase following a meal. We examined these effects using high‐fidelity radial arterial pressure waveforms and carotid‐femoral pulse wave velocity (PWV), measured non‐invasively via applanation tonometry, before and 60 and 180 min after ingestion of a liquid mixed meal (Ensure, 40% of daily energy expenditure) in 15 healthy adults (25 ± 2 y; 8M/7F). Both peripheral and central pressures were reduced 180 min from baseline. Yet contrary to our hypothesis, augmentation index (15 ± 4 vs. 2 ± 3 vs. 8 ± 3%), augmentation index normalized for heart rate (9 ± 4 vs. −2 ± 3 vs. 3 ± 3%), augmented pressure (6 ± 2 vs. 1 ± 1 vs. 3 ± 1 mmHg) as well as carotid‐femoral PWV (7.3 ± 0.2 vs. 6.7 ± 0.2 vs. 6.7 ± 0.1 m/s) were substantially reduced at 60 and 180 min following the meal (P < 0.05 for all). Taken together, our results suggest that a liquid mixed meal acutely decreases central hemodynamics and arterial stiffness in healthy adults. NIH K23 DK82424 (TC) P30 DK50456 (MNOC) UL1 RR024150 (MCCTSA) HL105467 (DPC)
Deltorphin‐Dvariant Attenuates Anaerobic Metabolism in a Pig Model of Shock
Authors: Oeltgen Peter , Dickson Eric W.
Published: June 2021

We assessed the ability of a hibernation‐like delta2 specific opioid, Deltorphin‐Dvariant (Delt‐Dvar) to provide ischemic protection in a pig model of cardiopulmonary bypass (CPB). Cardiogenic shock is induced by decreasing CPB flow 50% from baseline levels of 75–80 ml/kg/min to 37–40 ml/kg/min for 30 min and measuring the amount of lactic acid (LA). The CP circulation of a control and test pig weighing 75–85 kg was surgically diverted to an extracorporeal CPB circuit consisting of: Medtronic Affinity Oxygenator and integral hard shell venous reservoir, and a Medtronic affinity arterial blood filter. The test animal received Delt‐Dvar at a conc of 1.43 mg/kg in 30 ml physiological saline while the control received 30 ml physiological saline following 15 min of baseline CPB. This was followed by a 50% reduction in CPB flow for 30 min to 37–40 ml/kg/min, a flow rate mimicking cardiogenic shock. LA were measured as indicators of a switch to compensatory anaerobic metabolism. In Delt‐Dvar animal arterial LA dropped from 5.6 to 4.9 after 30 min of CPB flow at 50% and LA remained at 4.8 (a 30% reduction in LA) at 30 min after resumption of 100% CPB flow. In the control animal baseline LA of 2.8 increased to 6.0 at 30 min of 50% CPB flow and was 8.2 (a 292% increase in LA) 30 min after resumption of 100% CPB flow. This study indicates that Delt‐Dvar can markedly attenuate the onset of anaerobic metabolism in the face of profound ischemia.
Human endothelial colony forming cells and mesenchymal progenitor cells form functional blood vessels and improve rat cardiac function after ischemia/reperfusion injury
Authors: Kang Kyu‐Tae , Coggins Matthew , Xiao Chunyang , Rosenzweig Anthony , Bischoff Joyce
Published: June 2021

We showed previously that human endothelial colony forming cells (ECFC) combined with mesenchymal progenitor cells (MPC) form functional blood vessels in vivo. Here, we investigated whether ECFC+MPC could form functional vascular networks within ischemic myocardium, and whether this would improve cardiac function. Ischemia/reperfusion injury was induced in immune‐deficient rats by ligation of the left anterior descending coronary artery for 40 min. ECFC+MPC (2×106cells, 2:3 ratio) or PBS were injected into the ischemic myocardium after reperfusion. Luciferase‐labeled ECFC showed that 1,600 ECFC were present at day 14, indicating a quantifiable level of cellular retention. Perfused human ECFC‐lined vessels in myocardium were visualized at time points up to three months by femoral vein injection of fluorescently‐tagged human‐specific lectin. Left ventricular (LV) dimensions and heart weight to tibia length ratios were decreased in ECFC+MPC compared to PBS‐injected hearts at 4 months, suggesting that ECFC+MPC injection reduced LV hypertrophy. Pressure‐volume loop measurements showed that cardiac function was at least partially recovered in ECFC+MPC compared to PBS‐injection. Fibrosis area was not different between groups at 4 months. These data suggest that delivery of ECFC+MPC into ischemic myocardium to enhance neovascularization could be a therapeutic approach to restore cardiac function.
PGC‐1α deletion accelerates angiotensin II induced endothelial dysfunction by increasing mitochondrial oxidative stress and vascular aging
Authors: Schulz Eberhard , Kröller‐Schön Swenja , Jansen Thomas , Schüler Andrea , Oelze Matthias , Wenzel Philip , Hausding Michael , Kerakrodi Jasmin Ghaemi , Daiber Andreas , Münzel Thomas
Published: June 2021

Objective

Peroxisome proliferator coactivator 1α (PGC‐1α) is an important mediator of mitochondrial biogenesis and function. Since dysfunctional mitochondria might be involved in the pathogenesis of vascular disease, the current study was designed to investigate the effects of in vivo PGC‐1α deficiency during chronic angiotensin II (ATII) treatment.

Methods and Results

ATII infusion at subpressor dose (0.1mg/kg/d) resulted in mild endothelial dysfunction in wildtype mice, which was significantly aggravated in PGC‐1α knockout mice. In parallel, mitochondrial oxidative stress was increased in aortic rings from ATII‐treated PGC‐1α knockout mice while no change in NO production was observed. The increase in mitochondrial ROS was associated with premature vascular aging (p16INK4 and Chk‐2 expression) and enhanced activity of the pro‐apoptotic JNK signaling pathway. In vivo treatment with the mitochondrial targeted antioxidant mitoTEMPO corrected endothelial dysfunction in ATII‐treated PGC‐1α knockout but not in wildytpe mice, suggesting a crucial role of mitochondrial ROS in this process.

Conclusions

PGC‐1α deletion impairs endothelial function during chronic angiotensin II infusion by increasing mitochondrial ROS production, which results in premature vascular aging and apoptosis.
Role of the scaffold protein striatin in regulating the excitation‐contraction coupling in cardiomyocytes
Authors: Nader Moni , Khalil Bariaa , Abu‐Zaid Ahmed , Inaya A'man , Bakheet Dana , Dzimiri Nduna
Published: June 2021

Striatin (STRN) is a multivalent protein with dynamic domains including caveolin (Cav) and calmodulin (CaM) binding sites; both of which are key regulators of the L‐type calcium channel (LCC). We have previously reported that the formation of the STRN/Cav/CaM complex was calcium sensitive and that overexpression of striatin resulted in a sustained elevation of KCl‐induced intracellular calcium increase. We aimed, in this study, to show whether STRN interacts with LCC and PP2A to regulate cardiomyocyte contractility. Herein we report that both protein phosphatase 2A (PP2A) and the LCC were associated with STRN/Cav/CaM complex and that the interaction between all these components was calcium sensitive (abolished by calcium chelator EGTA). In addition, we also show that overexpression of striatin (adenoviral delivery) in cultured cardiomyocytes increased the rate of contraction by ~ 3 fold when compared to control cells (non‐target adenovirus). Preliminary data showed that knockdown of striatin (shRNA targeting STRN) enhanced the interaction of CaM and Caveolin‐3 suggesting a central role for STRN in articulating the STRN/Cav/CaM/LCC/PP2A complex. Collectively, our data suggest that striatin may emerge as a novel regulator of the L‐type calcium channel in cardiomyocytes and consequently regulate their contractility.

This work was supported by an operating grant from KFSHRC.
Thermoreceptors in the upper gastrointestinal tract independently influence sudomotor activity
Authors: Morris Nathan B , Bain Anthony R , Jay Ollie
Published: June 2021

Warm and cool fluid ingestion during exercise has been previously shown to alter local sweat rate independently of core and skin temperatures. This study strove to identify the location of the thermoreceptors responsible. Six males cycled at 50% VO2max for 75 min in a 24.8 ± 0.7°C and 33.4 ± 3.5% RH environment, while either mouth‐swilling (SW) 1.5°C or 50°C water; or ingesting 1.5°C or 50°C water through a nasogastric (NG) tube in three 3.2 mL·kg−1 boluses after 15, 30, and 45 min of exercise. Mean skin temperature (Tsk), mean local sweat rate (MLSR) and rectal temperature (Tre) were measured continuously. For all trials, Tre and Tskwere similar throughout. In the NG trials, MLSR with 1.5°C water was lower than with 50°C water, after 30, 45, and 60 min (P<0.05), but not after 15 or 75 min (P>;0.05) of exercise. In contrast, during the SW trials, MLSR with 1.5°C and 50°C water was almost identical throughout exercise (P>;0.05). In conclusion, thermoreceptors in the stomach region, rather than thermoreceptors in the mouth, are responsible for the transient alterations of sudomotor activity previously observed following the ingestion of warm and cool water. Supported by NSERC Discovery Grant #386143–2010.
LPS induces the same prostaglandins (PGs) in warm and cold environments, but thermal responses to PGs strongly depend on ambient temperature (Ta)
Authors: de Oliveira Cristiane , Nucci Tatiane B , Eales Justin , Dragic Alexander S , Pan Jennifer , Patel Shreya , Pakai Eszter , Brose Stephen A , Golovko Michail Y , Steiner Alexander A , Romanovsky Andrej A
Published: June 2021

Rats respond to iv LPS with a complex thermoregulatory response, which is represented mostly by fever at a neutral Ta, but mostly by hypothermia at a subneutral Ta. Both the febrile and hypothermic components of this response are mediated by PGs, but the exact mechanism of the fever‐hypothermia switch is unknown. Wistar rats were injected with LPS (10, 100 or 1000 μg/kg iv) at a neutral (30°C) or subneutral (20°C) Ta. At 30°C, the febrile components of the response to each LPS dose were more pronounced; at 20°C, the hypothermic components were predominant. At either Ta, LPS caused a dose‐depended increase in plasma PGs within all five cascades (PGE2, D2, F2á, I2 and TXB2), whether measured by ELISA or LC/MS/MS. Irrespective of the method, we found no difference in the plasma response of any individual PG to any dose of LPS at 30°C vs 20°C. We then studied thermoregulatory responses of rats to iv PGs (0.8 μmol/kg, administered as albumin complexes) at 30 and 20°C. Only PGE2 caused a statistically significant fever, and this response was unaffected by Ta. Several PGs, including E2, D2 and I2, caused hypothermia, and this response was restricted to the subneutral conditions. We conclude that, regardless of Ta, LPS causes the same systemic PG response. The fever‐hypothermia switch involves LPS‐induced PGE2, D2 and I2 causing hypothermia in a subneutral (but not neutral) environment.

Support: NS041233, NS064480, 11SDG4880051
Short‐term training alters the control of mitochondrial respiration rate before maximal oxidative ATP synthesis
Authors: Layec Gwenael , Haseler Luke J , Hoff Jan , Hart Corey R , Liu Xin , Jeong Eun‐Kee
Published: June 2021

Background

There has not been a study that has directly assessed temporal changes in mitochondrial oxidative capacity and metabolic control as a consequence of short‐term exercise training in vivo.

Methods

We used 31P‐magnetic resonance spectroscopy (31P‐MRS) to examine the effect of short‐term plantar flexion exercise training on phosphocreatine (PCr) recovery kinetics and the control of respiration rate. To this aim, we investigated 12 healthy men experienced with this exercise modality (TRA) and 8 time‐control subjects (TC).

Results

After 5 days of training, maximum work rate during incremental plantar flexion exercise was significantly improved (P < 0.01). During the recovery period, the maximal rate of oxidative ATP synthesis (PRE: 28 ± 13 mM.min−1; POST: 26 ± 15 mM.min−1) and the PCr recovery time constant (PRE: 31 ± 19 s; POST: 29 ± 16) were not significantly altered. In contrast, the Hill coefficient (nH) describing the cooperativity between respiration rate and ADP was significantly increased in TRA (PRE:nH =2.7 ± 1.4; POST: nH =3.4 ± 1.9, P < 0.05). Meanwhile, we did not observe any systematic variation in any of these variables in the TC.

Conclusion

This study reveals that 5 days of training induces rapid adaptation in the allosteric control of respiration rate by ADP before any substantial improvement in muscle oxidative capacity.
The pattern of change in skeletal muscle oxidative injury and non‐heme iron during aging differs between fast and slow muscles
Authors: Receno Candace N , Cowley Patrick M , Cromer Alray , Liang Chen , DeRuisseau Keith C
Published: June 2021

The aging‐related elevation in skeletal muscle non‐heme iron (NHI) is thought to be a contributing factor to the development of oxidative stress and sarcopenia. However, the iron status of aging slow twitch muscle has not been reported. This study examined the association between NHI and oxidative injury among two muscles that show a divergent pattern of aging‐related muscle atrophy; the fast twitch tibialis anterior (TA) and slow twitch soleus (Sol). Male F344BN rats were studied at 8, 18, 28, and 36 months of age. The animals were anesthetized and muscles were harvested for analysis of NHI and oxidative injury measures. The TA mass was greater than the Sol (p<0.05) and showed atrophy beginning at 28 months compared to 18 month values (p<0.05). The Sol showed atrophy only at 36 months (p<0.05). NHI concentration (nmol/gww) of the Sol was increased at 36 months (95.4±9.3; p<0.05) compared to the other age groups and coincided with the decline in muscle mass. The TA NHI was also increased at 36 months (46.3±8.2; p<0.05); after the onset of muscle atrophy and to a lesser extent than that of the soleus (p<0.05). Lipid peroxidation was unchanged in the TA (p>;0.05) but increased (p<0.05) in the Sol at 36 months compared to 8 and 18 month groups. In summary, elevations in NHI concentration and oxidative injury were better aligned with the Sol pattern of atrophy than the TA in response to aging.

Supported by NIH 5R03AG032033 (KCD)