bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dietary supplements influence activity of acid‐sensing ion channels (1061.8)
Authors: Agharkar Amruta , Gonzales Eric 
Published: June 2021

Dietary supplements and nutraceuticals have been the focus of research to identify novel therapeutics for a variety of pathologies, including the prevention of long‐term consequences of stroke and reducing pain. Ion channels offer a growing group of molecular targets for treatment, which include the acid‐sensing ion channels (ASICs). Acid‐sensing ion channels (ASICs) are sodium channels that are sensitive to changes in extracellular pH, specifically those changes following injury and ischemia. These channels are expressed most prominently in peripheral and central nervous system. Their role in physiology is yet to be fully understood, but these channels have been implicated in pain sensation and centrally in the neurodegeneration following ischemic stroke. We identified an over‐the‐counter dietary supplement (DS) that shares similarity to guanidine compounds that selectively modulate acid‐sensing ion channels. Thus, we hypothesize that this dietary supplement inhibits channel activity in acid‐sensing ion channels. Our preliminary data show that DS decreased the ASIC1a pH sensitivity by shifting the observed proton activation profile to the right. Furthermore, we observed a change in the Hill coefficient of the DS influenced ASIC1a steady‐state desensitization profile. Future experiments will focus on determining the influence of DS on other acid‐sensing ion channel subtypes and identifying the DS binding site with the protein structure.Grant Funding Source: American Heart Association 12BGIA8820001
Claudin‐1 as a target for modulation of epidermal barriers (1062.6)
Authors: Nakajima Misaki , Yamashita Mayo , Watari Akihiro , Yagi Kiyohito , Fukasawa Masayoshi , Kondoh Masuo 
Published: June 2021

Epithelium plays pivotal roles in barriers that separate the inside of the body from the outside environment. Passage across epithelial barriers is the first step in drug absorption. Epidermal absorption is an ideal route for drug administration because it is non‐invasive and easily controlled. Claudin‐1 (CLDN1) seals tight junctions in the stratum granulosum, but whether modulation of CLDN1 enhances epidermal permeability is unclear. We investigated the effect of an anti‐CLDN1 antibody on the epidermal epithelial barrier by using human epidermal keratinocytes (NHEK), a popular in vitro model of human epithelial cell sheets. Immunoblot and FACS analyses showed that CLDN1 was expressed on NHEK cell membranes. Treatment of NHEK monolayers with anti‐CLDN1 antibody decreased the integrity of tight junctions between adjacent cells and enhanced the permeation of solutes across the cell sheets. Furthermore, treatment of Caco‐2 cell monolayers, a popular model of the human intestinal barrier that expresses CLDN1 and CLDN4, with anti‐CLDN4, but not treatment with anti‐CLDN1, decreased the integrity of tight junctions. These findings suggest that CLND1 may be a target for specific modulation of epidermal barrier.Grant Funding Source: supported by the Ministry of Health, Labor, and Welfare of Japan
Anemia in adenine‐induced chronic renal failure and the influence of treatment with gum acacia thereon (1063.4)
Authors: Ali Badreldin , Al Z'aabi Mohammed , Ramkumar A. , Nemmar A. 
Published: June 2021

Anemia is an early complication of human chronic kidney disease (CKD) and chronic renal failure (CRF) in animals. We investigated the effect of induction of CRF in rats by feeding them adenine (0.75%, w/w, 4 weeks) on erythrocyte count (EC), hematocrit (PCV) and hemoglobin (Hb) concentration, as well as on the activity of L‐γ‐glutamyl transferase (GGT) and iron (Fe), transferrin (Tf), ferritin (F), total iron binding capacity (TIBC) / unsaturated iron binding capacity (UIBC) and hepcidin (Hp) in serum and erythropoietin (Epo) in renal tissue. Renal damage was assessed histopathologically, and also by measuring the serum concentrations of the uremic toxin indoxyl sulfate (IS), creatinine, and urea, and by creatinine clearance. We also assessed the influence of concomitant treatment with gum acacia (GA) in the drinking water (15%, w/v, 4 weeks) on the above analytes. Adenine ‐ induced CRF, accompanied by significant decreases (P < 0.05) in EC, PCV, and Hb, and in the serum concentrations of Fe, Tf, TIBC, UIBC and Epo. It also increased Hp and F levels. GA significantly ameliorated these changes in the adenine‐treated rats. A general improvement in the renal status of rats with CRF after GA is shown due to its anti‐inflammatory and anti‐oxidant actions, and reduction of the uremic toxin IS, which is known to suppress Epo production, and this may be the basis of its ameliorative actions on the indices of anemia studied.Grant Funding Source: Supported by the Research Council of Oman
The human testis as a model of retinoic acid formation (1064.10)
Authors: Arnold Samuel , Kent Travis , Schlatt Stefan , Prasad Bhagwat Prasad , Haenisch Michael , Muller Chip , Hogarth Cathryn , Griswold Michael , Paik Jisun , Walsh Thomas , Amory John , Isoherranen Nina 
Published: June 2021

Retinoic acid (RA), the active form of vitamin A (retinol), is indispensable for maintaining many essential biological processes. RA formation depends on a complex network of enzymes. The rate limiting step is irreversible formation of RA by aldehyde dehydrogenase1A (ALDH1A1‐1A3). However, the specific roles, tissue expression, and localization of these enzymes are not well understood.While RA is necessary for spermatogenesis in animal models, the source of intratesticular RA is unknown. Sertoli cell specific ALDH1A knockouts have demonstrated intratesticular RA formation is required for male fertility. Due to the requirement for intratesticular RA formation and presence of mRNA for all three ALDH1A enzymes in the human testis, the human testis was chosen as a model of the biochemistry of RA formation.In order to model RA formation, a novel LC‐MS/MS based peptide quantification method was developed to quantify ALDH1A in a cohort of 18 men. Each ALDH1A concentration was used to predict the velocity of RA formation in each donor. The accuracy of our predictions was confirmed by measuring intrinsic RA formation and RA concentrations in the same samples. Additionally, weidentified a distinct localization pattern for ALDH1A enzymes in the human testis using immunohistochemistry.In conclusion, these novel techniques were successfully used to model RA formation in human testicular tissue. In the future, RA formation in other tissues can be investigated using the assays developed.Grant Funding Source: Supported by NIH/NICHD grant U54 HD042454 and TL1TR000422
Activation of vascular Toll‐like receptor 3 induces phosphorylation of caldesmon via ERK1/2 pathway (1065.12)
Authors: Carrillo‐Sepulveda Maria Alicia , Hardigan Trevor , Webb R. Clinton 
Published: June 2021

TLR3, a pattern‐recognition receptor of the innate immune system, is constitutively expressed in vascular smooth muscle cells (VSMC) and has been showed to be functional in the vascular system; however its role in contraction remains unknown. Caldesmon (CaD) is a potent and versatile regulatory protein that play crucial role in contractility of smooth muscle. We hypothesized that TLR3 contributes to contractile events in VSMC by phosphorylation of CaD in Ser789. Phosphorylation of myosin light chain (pMLC), marker of contraction, increased by 65% in mouse C57bl/6 aortic smooth muscle cells (ASMC) stimulated with TLR3 agonist polyinosine polycytidylic acid (Poly I:C) (10 μg/mL, 60 min). This effect was accompanied by increased phosphorylation of CaD on Ser789 (3 fold versus control unstimulated ASMC) as well as significantly activation of mitogen‐activated protein/extracellular signal‐regulated kinase (ERK1/2). Pre‐treatment with PD98059 (50μM, 30 min), ERK1/2 inhibitor, abolished Poly I:C‐mediated phosphorylation of CaD. Interestingly, chronic treatment with Poly I:C for 24 hours downregulated by 40% TLR3 expression in ASMC. The present results showed that activation of TLR3 in VSMC causes ERK1/2‐mediated phosphorylation of CaD, thereby contributing to augmented contraction and consequently to development of vascular dysfunction. Our data provide the first functional assessment of the role of TLR3 in vascular contractile events, suggesting TLR3 as a potential new therapeutic target in vascular dysfunction.Grant Funding Source: AHA 13POST14690026
Contribution of nuclear factor E2‐related factor 2 to the atherogenic phenotype transition in coronary arterial smooth muscle cells lacking CD38 gene (1065.16)
Authors: Xu Ming , Li Xiaoxue , Wang Mi , Zhang Yang , Li Pin‐lan 
Published: June 2021

Recent studies have reported that CD38 gene deficiency results in dedifferentiation or transdifferentiation of arterial smooth muscle (SMCs) upon atherogenic stimulations. However, the molecular mechanisms mediating this SMC phenotypic change remain unknown. In the present study, we first characterized the phenotypic change in the primary cultures of coronary arterial myocytes (CAMs) from CD38‐/‐ mice. It was shown that CD38 deficiency decreased the expression of contractile marker calponin and α‐SMA and increased the expression of SMC dedifferentiation marker vimentin, which was accompanied by collagen I accumulation and cell proliferation. This phenotypic change was enhanced by 7‐ketocholesterol (7‐Ket), an atherogenic stimulus in CAMs from CD38‐/‐ mice. We further found that the CD38 deficiency decreased the protein level of nuclear factor E2‐related factor 2 (Nrf2) and inhibited its activity. Given that Nrf2 is a transcription factor that responds to oxidative stress, we determined the role of redox signaling in the regulation of Nrf2 expression and activity. It was demonstrated that in CD38‐/‐ CAMs 7‐Ket failed to produce O2‐., while in CD38+/+ CAMs 7‐Ket‐induced O2‐. production was substantially attenuated by Nox4 gene silencing. In addition, 7‐Ket‐induced increase in Nrf2 activity in CD38+/+ CAMs was significantly inhibited by silencing Nox4 gene. Finally, we demonstrated that both Nrf2 gene silencing and CD38 deletion had similar effect to increase CAM dedifferentiation upon 7‐Ket stimulation. In contrast, the overexpression of Nrf2 gene markedly abolished 7‐Ket‐induced dedifferentiation in CD38‐/‐ CAMs. These results suggest that CD38 activity is required for 7‐Ket‐induced O2‐. production, which increases Nrf2 activity maintaining CAMs in a differentiated status. When CD38 gene expression and function are deficient, the Nrf2 activity is suppressed thereby leading to dedifferentiation of CAMs.Grant Funding Source: HL057244, HL091464 and HL075316
Chronic baclofen treatment influences GABAB activation of G‐proteins and phosphorylation of FAK, GSK3β, and DARPP32 in mesocorticolimbic rat brain regions (1066.3)
Authors: Keegan Bradley , Beveridge Tom , Pezor Jeffrey , Xiao Ruoyu , Sexton Tammy , Childers Steven , Howlett Allyn 
Published: June 2021

The GABAB receptor agonist baclofen is being explored as a potential treatment for cocaine addiction, as therapeutic activation of the GABAergic system is theorized to counterbalance over‐stimulation of the dopaminergic system in brain regions involved in reward. Rats acutely administered i.p. baclofen (5 mg/kg, single dose 15 min. prior to sacking) showed no immediate changes in GABAB‐stimulated GTPγS binding in any brain region but displayed significantly greater phosphorylation of focal adhesion kinase (FAK) in key mesocorticolimbic regions (caudate putamen, cortex, hippocampus, thalamus, and prefrontal cortex) compared to controls, as well as elevated levels of glycogen synthase kinase 3‐beta (GSK3β) phosphorylation in these and other regions (nucleus accumbens, septum, and globus pallidus). In contrast, GABAB‐stimulated GTPγS binding in rats administered chronic (5 mg/kg, t.i.d. for five days) baclofen was significantly lower in the prefrontal cortex, septum, amygdala, and parabrachial nucleus than in the controls. While chronically‐treated rats exhibited similar increases in FAK phosphorylation, they displayed attenuated levels of GSK3β phosphorylation and wide‐spread elevations in levels of DARPP32 phosphorylation compared to those treated acutely or with vehicle. These findings indicate a desensitization of GABAB‐mediated signal transduction in rats following chronic administration of baclofen.Grant Funding Source: Supported by the National Institute on Drug Abuse: R01‐DA03690 and P50‐DA006634.
Pre‐exposure of the urotensin II receptor to urotensin II or urotensin II‐related peptide differentially reduces the response to subsequent additions (1066.8)
Authors: Warren Taylor , Clark Stewart 
Published: June 2021

The Urotensin II receptor (UIIR) is a G protein‐coupled receptor which is activated by urotensin II (UII) and urotensin‐related peptide (URP). Studies show equivalent Kd values and a similar EC50 in Ca2+assays in vitro. The co‐expression of the peptides, spatially and temporally, in vivo leads us to hypothesize that the ligands mediate different post‐activation events. Ca2+ mobilization was used to monitor receptor activation. CHO‐rUIIR cells pre‐treated with 1pM UII have a 40% reduction in response to a second addition of 10nM UII. The same experiment was carried out with equimolar URP; no significant decrease was seen. The same phenomenon was seen when cells were pre‐treated for 6 or 19 minutes, washed, and a second addition added one hour later. UII and URP have identical cyclic regions, but UII has an additional short n‐terminal tail containing a charged residue. The Ca2+ assay was repeated using peptide truncations, and these truncations resulted in differential desensitization. Desensitization mechanisms may lead to proliferative pathways. Therefore, cells were treated with UII or URP overnight. UII dose‐dependently increased MTS reduction (130%; MTS assay) compared to control; URP showed no significant increase. Data suggests low concentrations of UII and URP induce different post‐activation events. A charged residue at the n‐terminal of the cyclic region may alter receptor conformation upon binding.Grant Funding Source: Supported By NIH Grant# R00DA024754‐05S1
Transient receptor potential vanilloid type 4 channel as a putative target molecule for anti‐hypertensive therapy (1067.10)
Authors: Walas Dawid , Paton Julian 
Published: June 2021

TRPV4 is a non‐selective cation channel causing vasodilatation (Willette et al. 2007) and cerebral arterial re‐modelling in response to shear stress (Schierling et al., 2011). Our hypothesis is that TRPV4 is an intracranial sensor that maintains cerebral blood flow by sympathetically mediated modulation of arterial pressure.Using qPCR, we demonstrated that TRPV4 mRNA levels from common carotid arteries of the Spontaneous Hypertensive Rat (SHR) were lower compared to a normotensive Wistar (9 weeks old animals, p=0.002, n=3). mRNA levels in the SHR brain vasculature were age related with older rats showing higher levels (e.g. at 9 weeks old mRNA was 2 times lower than 13 weeks old SHR, p=0.06, N=3). This difference was not detected in Wistar rats (p=0.997, n=3).We next examined the effect of TRPV4 agonist (GSK1016790A) on arterial pressure in anesthetised SHRs fitted with radio‐telemeters. Bolus intravenous injections showed a dose‐dependent reduction in arterial pressure with maximal response achieved at 166 µg/kg and the lowest effective dose being 42 µg/kg (n=3); the latter dose has been reported to cause circulatory collapse in normotensive rats (Willette et al. 2007). Thus, SHR showed a higher tolerance to the agonist consistent with lower mRNA expression.Presence of TRPV4 in the cerebral vessels and its vasodilatory effect are both consistent with it being an exploitable target for improving cerebral blood flow and for controlling hypertension.
MCA‐APK(Dnp) is not a selective substrate of angiotensin‐converting enzyme‐2 (1067.6)
Authors: Carrera Eduardo , Linares Andrea , Joachim Arline , Jean‐Baptiste Malaika , Speth Robert 
Published: June 2021

MCA‐APK(Dnp) is a fluorogenic substrate used in assays of angiotensin‐converting enzyme‐2 (ACE‐2) metabolism. Cleavage of the Pro‐Lys bond removes the quenching Dnp group and induces fluorescence. To ascertain the specificity of MCA‐APK(Dnp) for ACE‐2, we measured metabolism of the substrate in rat kidney in the presence or absence of the ACE‐2 inhibitor MLN‐4760. Frozen rat tissues were homogenized in 19 volumes of 50 mM sodium phosphate at pH 7 with 0.05% Triton X‐100 detergent and centrifuged at 48,000 x g. The supernatant was then diluted 20‐fold in assay buffer for a final concentration of 50 mM sodium phosphate at pH 6, 6.5, 7, or 7.5 and 100 mM NaCl. 20 µL of diluted supernatant, 10 µl of 50 µM MLN‐4760, and 20 µl of 125 µM substrate were added to each well for a reaction volume of 50 µL. Metabolism of MCA‐APK(Dnp) was recorded at 37oC at a wavelength of 393 nm with excitation at 328 nm. Breakdown of MCA‐APK(Dnp) attributable to ACE‐2 represented only 21%, 28%, 44%, and 56% of total enzymatic activity at pH 6, 6.5, 7, and 7.5, respectively. Total activity also increased with decreasing pH. Our results suggest that MCA‐APK(Dnp) is not selective for ACE‐2. When using MCA‐APK(Dnp) as a surrogate substrate for determination of ACE‐2 activity, it is critical to use a selective inhibitor or ACE‐2 activity such as MLN‐4760 to differentiate ACE‐2 versus non‐ACE‐2 mediated metabolism of MCA‐APK(Dnp).Grant Funding Source: NIH‐HL113905
Major role of ryanodine type 2 receptors in global and local intracellular calcium release in arterial smooth muscle (1067.7)
Authors: Kassmann Mario , Schleifenbaum Johanna , Szijártó István , Anistan Yoland‐Marie , Gollasch Maik 
Published: June 2021

Ryanodine receptors (RyR) are calcium‐sensitive ion channels in the sarcoplasmic reticulum (SR) membrane, and are important effectors in arterial smooth muscle excitation‐contraction (EC) coupling. While three different RyR isoforms are expressed in arterial smooth muscle cells (SMCs), the relative contribution of each RyR isoform to vascular function remains unresolved. We generated tamoxifen‐inducible SMC specific knock‐out mice of RyR2 to test the hypothesis of RyR2 contributing in arterial EC coupling, vasoregulation, and systemic blood pressure. Myography and videomicroscopy of isolated arteries, calcium imaging and patch clamp current recordings on isolated SMCs, and radiotelemetric blood pressure measurements were used. Targeted deletion of the gene for RyR2 resulted in a loss in functional coupling of calcium sparks to associated BK potassium channel activation, and increased arterial tone and systemic blood pressure. Aortas, femoral, cerebral and mesenteric arteries from RyR2‐deficient mice showed no contraction in response to RyR activation by 10 mmol/L caffeine. Our data demonstrate that RyR2 are key elements in Cav1.2 channel‐induced EC coupling in large and small arteries. Furthermore, RyR2, by activating BK channels, are important molecular SR calcium release components in translating local calcium signals (Ca2+‐sparks) to vasoregulation and arterial blood pressure.Grant Funding Source: Supported by the Deutsche Forschungsgemeinschaft (DFG)
Stretch‐activation of angiotensin II type 1a receptors contributes to the myogenic response of mouse mesenteric and renal arteries (1067.8)
Authors: Schleifenbaum Johanna , Kassmann Mario , Szijártó István , Hercule Hantz , Weinert Stefanie , Heidenreich Matthias , Pathan Asif , Anistan Yoland‐Marie , Alenina Natalia , Rusch Nancy , Bader Michael , Jentsch Thomas , Gollasch Maik 
Published: June 2021

Wall stretch is a major stimulus for the myogenic response of small arteries to pressure. Recent findings suggest that G protein‐coupled receptors can elicit a stretch response. Our aim was to determine if angiotensin II type 1 receptors (AT1R) in vascular smooth muscle cells (VSMC) exert mechanosensitivity and identify the downstream ion channel mediators of myogenic vasoconstriction. We used mice deficient in AT1R signaling molecules and putative ion channel targets, namely AT1R, angiotensinogen, TRPC6 channels or subtypes of the KCNQ (Kv7) gene family (KCNQ3, 4 or 5). We identified a mechano‐sensing mechanism in mesenteric arteries and the renal circulation that relies on coupling of the AT1R subtype a (AT1aR) to a Gq/11‐protein as a critical event to accomplish the myogenic response. The mechano‐activation occurs after block of AT1R, and in the absence of angiotensinogen or TRPC6. Activation of AT1aR suppresses XE991‐sensitive Kv channel currents in VSMCs, blocking these channels enhances mesenteric and renal myogenic tone. Although KCNQ3, 4 and 5 are expressed in VSMCs, XE991‐sensitive K+ current and myogenic contractions persist in arteries deficient in these channels. Our results provide evidence that myogenic responses of mouse mesenteric and renal arteries rely on ligand‐independent mechano‐activation of AT1aR. This signal relies on an ion channel distinct from TRPC6 or KCNQ3, 4 or 5.Grant Funding Source: Supported by Deutsche Forschungsgemeinschaft (DFG)
Contribution of voltage‐gated potassium channels in cerebrovascular dysfunction associated with a genetic model of ischemic small vessel disease (1068.1)
Authors: Dabertrand Fabrice , Krøigaard Christel , Bonev Adrian , Brayden Joseph , Joutel Anne , Nelson Mark 
Published: June 2021

Dominant mutations in the NOTCH3 gene induce the most common heritable cause of stroke and vascular dementia, referred to as Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL). Using a recently developed mouse model of CADASIL (Joutel, JCI, 2010), we examined the consequences of this mutation on the function of intracerebral arterioles. Elevation of intravascular pressure to 20 mm Hg constricted isolated arterioles from control and CADASIL mice to a similar extent. However, above 30 mm Hg, CADASIL arterioles displayed impaired vascular reactivity. At 40 mm Hg, CADASIL arterioles were 38% less constricted, and their smooth muscle membrane potential was 10 mV more hyperpolarized than control. A pharmacologic approach revealed an unchanged activity of small‐, intermediate‐, large‐conductance calcium‐sensitive potassium channels. However the voltage‐gated potassium (Kv) channel blocker 4‐AP enhanced pressure‐induced constriction to a greater extent in both parenchymal arterioles and pial arteries from CADASIL mice. Conversely, mesenteric resistance arteries displayed similar 4‐AP‐induced constriction and similar myogenic response in both groups. In isolated cerebral myocytes, Kv current density was 30% greater in CADASIL than in control, whereas gating properties were similar. These results support the concept that the CADASIL mutation decreases pressure‐induced vasoconstriction through upregulation of smooth muscle Kv channels in cerebral arteries and arterioles.Grant Funding Source: NIH PO1HL095488, RO1HL44455, the Totman Trust and the Fondation Leducq