bluuebunny/crossref_metadata_2025 huggingface dataset:
Metabolic Specialization of Mouse Embryonic Stem Cells
Authors: McKnight Steven Lanier
Published: June 2021

The cell doubling time of mouse embryonic stem (ES) cells is shorter than that of even the most aggressively growing cancer cells. To investigate whether mouse ES cells might exist in a unique metabolic state supportive of rapid growth, organic extracts were prepared and analyzed by mass spectrometry. Upon comparison of mouse ES cells with differentiating embryoid bodies, such assays revealed profound differences in the abundance of metabolites associated with one‐carbon metabolism. It was also noted that the abundance of both threonine and acetyl‐CoA changed markedly as ES cells differentiate into embryoid bodies. These differences in metabolite abundance were traced to the copious, ES cell‐specific expression of the gene encoding threonine dehydrogenase (TDH). TDH gene expression was observed to be 1,000‐fold higher in ES cells than any of 7 tissues assayed from adult mice. The TDH enzyme catalyzes the mitochondrial breakdown of threonine into glycine and acetyl‐CoA. Glycine is further catabolized by mitochondrial enzymes associated with the glycine cleavage system to foster the charging (methylation) of tetrahydrofolate, a key intermediate in purine synthesis. The acetyl‐CoA produced from TDH‐mediated breakdown of threonine can be fed into the TCA cycle, used as a building block for the synthesis of lipids and sterols, or deployed as a substrate for protein acetylation. This TDH‐dependent metabolic pathway is prototypic of rapidly growing microbial organisms, and is a component of what has been termed the high flux backbone of microbial metabolism. Nutritional, pharmacologic and genetic approaches have been used to confirm the unique dependency of mouse ES cells on threonine consumption as a metabolic fuel. This research was supported by a program project grant funded by The National Cancer Institute and philanthropic contributions from an anonymous donor.
A Chronic Method for Measuring Real‐time Pulse Wave Velocity in Conscious Rodents
Authors: Grenwis Jessica , Bogie Heather , Main Bradley
Published: June 2021

Pulse wave velocity (PWV) is a measure of arterial stiffness and predictor of cardiovascular (CV) risk. PWV is calculated by recording the time it takes for an arterial pressure “pulse wave” to transit between two sites at a known distance apart. Prior to the advent of a small, dual pressure telemetry device (HD‐S21, DSI); PWV data were only attainable using acute measurement techniques in rodents. To investigate chronic, PWV measurement, two male rats (Sprague Dawley® Rat, Charles River Laboratory) were surgically implanted with the HD‐S21. The upstream catheter was placed in the left carotid artery and the downstream catheter was placed approximately 7.25cm away in the descending aorta. Following post‐surgical recovery, 24 hours of baseline arterial pressure and PWV data were collected from each animal. The next day, the rats were administered 30mg/kg of vasoconstrictor Nω‐Nitro‐L‐arginine methyl ester hydrochloride (L‐NAME) via IP injection and data were recorded for 24 hours. Using 10 minute averaging intervals, 24 hours of baseline and post‐dose PWV data were compared. Average baseline PWV measurements of 286 and 357 cm/s increased sevenfold post L‐NAME dosing, resulting in PWV values as high as 1959 and 2561 cm/s due to increased vascular stiffness. This evaluation demonstrated that the HD‐S21 is a feasible tool for investigating chronic PWV measurements in small animal models.
Protein kinase A‐mediated inhibition of T‐type Ca2+ channels in the cerebral circulation
Authors: Harraz Osama F , Welsh Donald G
Published: June 2021

Previous work from our laboratory documented the expression of L‐type (CaV1.2) and T‐type (CaV3.1/CaV3.2) Ca2+ channels in rat cerebral arterial smooth muscle. In this study, we examine, in greater detail, the electrophysiological and regulatory properties of these voltage‐gated Ca2+ channels. Using whole‐cell patch clamp electrophysiology and Ba2+ as charge carrier, whole‐cell Ba2+ current was pharmacologically subdivided into nifedipine‐sensitive and ‐insensitive components. The nifedipine‐sensitive component displayed L‐type characteristics such as slow activation/inactivation at depolarized voltages. In contrast, the nifedipine‐insensitive current displayed T‐type Ca2+ channel properties such as fast activation/inactivation; this current could be further subdivided into CaV3.1 and CaV3.2 components based on Ni2+ sensitivity. Modulators of Protein kinase A (PKA) were found to affect T‐type, but not the L‐type Ca2+ channel activity in cerebral arterial smooth muscle. In greater detail, PKA activators (isoproterenol, forskolin and db‐cAMP) were observed to inhibit peak T‐type Ca2+ current and evoke a leftward shift in the activation/inactivation kinetics. While PKA inhibitors (KT5720, PKI 14–22) did not alter the magnitude of the T‐type Ca2+ channel current, they did prevent forskolin‐mediated inhibition. Forskolin's inhibitory effect on the T‐type Ca2+ channel was similarly abolished by stHt31, a peptide inhibitor of A‐kinase anchoring protein. In summary, the study provides the first detailed electrophysiological delineation of L‐ and T‐type Ca2+ channels in cerebral arterial smooth muscle. Intriguingly, it shows that T‐type Ca2+ channels are also selectively targeted by signaling pathways that mediate arterial vasodilation.
Modulation of Angiotensin II Effects in Vascular Smooth Muscle Cells by microRNAs
Authors: jin wen , Reddy Marpadga A , Chen Zhuo , Putta Sumanth , Lanting Linda , Kato Mitsuo , Park Jung Tak , Chandra Manasa , Wang Charles , Tangirala Rajendra , Natarajan Rama
Published: June 2021

Angiotensin II (AngII)‐mediated vascular smooth muscle cell (VSMC) dysfunction plays a key role in cardiovascular diseases. However, the role of microRNAs (miRNAs) in this process is unclear. We used small RNA deep sequencing to profile AngII‐regulated miRNAs and evaluated their role in rat VSMC. Bioinformatics analysis showed that the predicted targets of AngII‐responsive miRNAs can modulate biological processes relevant to cardiovascular diseases. Further studies showed time‐ and dose‐dependent upregulation of miR‐32 and miR‐212 cluster (miR‐132/212) by AngII. We identified phosphatase and tensin homolog (PTEN) as a novel target of miR‐132 and showed miR‐132 mediates monocyte chemoattractant protein‐1 (MCP‐1) induction in VSMC partly via PTEN repression. miR‐132 overexpression also enhanced cyclic AMP‐response element binding protein (CREB) phosphorylation whereas its inhibition attenuated AngII‐induced CREB activation. Furthermore, miR‐132 induction by AngII required CREB activation, demonstrating a positive feedback loop. Notably, aortas from AngII‐infused mice showed higher levels of miR‐132/212 and MCP‐1. In addition, microarray analysis combined with RT‐QPCR revealed that a subset of genes downregulated by AngII was miR‐132 predicted targets, and they are associated with AngII functions. These results suggest that miR132/212 fine‐tunes and amplifies AngII actions in VSMC.
Researcher Beware! Decreased TG2 and OCT3 Expression in Vascular Smooth Muscle Cells Upon Culture
Authors: Johnson Kyle B , Petersen-Jones Humphrey , Watts Stephanie W
Published: June 2021

Recently, a protein modification has been described, norepinephrine (NE)‐amidation, in which NE is covalently attached to the γ‐glutamyl moiety of a protein‐bound glutamine by members of the transglutaminase (TG) family. While TG activity is necessary for vascular contraction to NE, the full physiological relevance of NE‐amidation in the vasculature is not known. We hypothesized that vascular smooth muscle cells (VSMCs) maintain a phenotype conducive to characterizing the physiological relevance that the NE‐amidation of proteins serves in NE‐elicited growth and migration. The intensity of immunocytochemical staining for the vascular TGs (TG1, TG2, and TG4) was reduced in cultured compared to freshly dissociated rat aorta and vena cava VSMCs. The organic cation transporter 3 (OCT3), a non‐neuronal NE transporter, localized to the cell surface of freshly dissociated VSMCs, while staining was sparse in cultured VMSCs. While freshly dissociated VSMCs took up a NE‐biotin conjugate, cultured VSMCs lost this ability. Western analysis detected less protein for TG2 and OCT3 in cultured VSMCs versus whole tissue (protein in aortic VSMCs: TG2 = 2.5 ± 0.7, OCT3 = 1.3 ± 0.3 % of protein in whole aorta, p≤0.05). These results demonstrate that VSMCs undergo a phenotypic transformation in culture that is not conducive to the characterization of the physiological responses elicited by NE‐amdiation of proteins.
Decreased protein expression and activity of transglutaminases 1 and 2 in arteries from DOCA‐salt rats
Authors: Petersen-Jones Humphrey , Johnson Kyle B. , Thompson Janice M. , Watts Staphanie W.
Published: June 2021

Transglutaminases (TGs) catalyze a variety of protein modification reactions in the vasculature, and TG‐mediated protein remodeling has been implicated in hypertension. We hypothesized that there would be a higher expression of TGs in hypertensive vs normotensive vasculature due to protein cross‐linking catalyzed by the TGs. We used aorta (RA) from deoxycorticosterone acetate (DOCA)‐salt (hypertensive) and sham (normotensive) control rats. Methods included immunohistochemistry (IHC), Western blot analysis, and in situ fluorescent detection of active TGs using the K5 (TG1) and T26 (TG2) peptides. IHC showed a decreased expression of TG1 and TG2 in DOCA‐salt rat aorta (TG1: DOCA 1.22, sham 1.44; TG2: DOCA 0.61, sham 1.61 arbitrary rank units) and in situ detection showed decreased activity of TG1 and TG2 in DOCA‐salt rat aorta (TG1: DOCA 2.0, sham 2.72; TG2: DOCA 1.79, sham 2.56 arbitrary rank units). Similarly, protein expressions of TG1 and TG2 in DOCA‐salt rat aorta were diminished compared to the control (TG1: DOCA 27.8 ± 2.52, sham 51.9 ± 13.0; TG2: DOCA 83.2 ± 3.85, sham 108 ± 5.83 arbitrary densitometry units/alpha actin; p<0.05). We conclude that there is less activity and decreased levels of TG1 and TG2 in hypertensive rat aorta compared to sham. This contradicts our hypothesis and supports the idea that lack of compensatory TGs may a role in the pathology of hypertension.

POIHC70687
Physical association of Angiotensin‐II type 1 Receptor (AT1R) and NOX‐1 mediates NF‐κB and AP‐1‐dependent Interleukin‐ 18 induction and Aortic SMC Migration and Proliferation
Authors: Valente Anthony J , Yoshida Tadashi , Wekerle Ryan , Katsuyama Masato , Chandrasekar Bysani
Published: June 2021

Persistent elevation of angiotensin (Ang)‐II promotes atherosclerosis via oxidative stress and induction of inflammatory cytokines. Since NF‐κB and AP‐1 are important mediators in Ang‐ II signaling pathways, and since the pro‐mitogenic and pro‐migratory factor interleukin (IL)‐18 is an NF‐κB and AP‐1 responsive gene, we investigated whether Ang‐II‐mediated SMC migration and proliferation are IL‐18 dependent. Ang‐II stimulated the migration and proliferation of SMC isolated from rat thoracic aorta. Ang‐II induced O2− generation, IKKβ and JNK activation, p65 and c‐Jun phosphorylation, NF‐κB and AP‐1 activation, and IL‐18 transcription, effects that were attenuated by losartan, DPI, and NOX‐1 knockdown. Both αIL‐18 Ab and IL‐18BP attenuated Ang‐II induced SMC migration and proliferation. Importantly, AT1R binds NOX‐1 in vitro and in vivo. Ang‐II also induced Nox1 transcription via AP‐1. Ang‐II infusion (7 days) increased NOX‐1 mRNA expression, DPI‐inhibitable O2− generation, p65 and c‐Jun phosphorylation, and IL‐18 induction in endothelium‐denuded thoracic aorta in an AT1R‐dependent manner.

Conclusions

Ang‐II stimulates SMC migration and proliferation via AT1R/NOX‐1‐ mediated IKK/NF‐κB and JNK/AP‐1‐dependent IL‐18 induction, and this process may involve AT1R/NOX‐1 physical association. The AT1R/NOX‐1/IL‐18 pathway may be a critical mechanism in atherogenesis and restenosis.
Aquaporin 1(AQP1) Mediates Migration and Growth in Pulmonary Arterial Smooth Muscle Cells (PASMCs)
Authors: Lai Ning , Maylor Julie , Leggett Kyle , Undem Clark , Crow Michael , Shimoda Larissa
Published: June 2021

PASMC proliferation and migration is important for many pathological processes, including the development of pulmonary hypertension; however, the mechanism governing PASMC movement and growth is not fully understood. AQP1, a water channel protein, was shown to aid in migration of endothelial cells. Since AQP1 is abundant in PASMCs, we hypothesized that AQP1 might be important for proliferation and migration in these cells. Rat PASMCs were isolated from intrapulmonary arteries, cultured in SMGM complete media and placed into basal media 24–48 h before experiments. Migration and proliferation were measured using transwell assay and BrdU incorporation, respectively. To over‐express AQP1, PASMCs were infected with 50 ifu/cell for 4 h with an adenoviral construct containing wild‐type AQP1 (AdAQP1), or green fluorescent protein (AdGFP), the control for infection. AdAQP1 infection increased AQP1 protein 2 fold, and was associated with increased proliferation and migration under basal conditions. To deplete AQP1, PASMCs were transfected with AQP1 siRNA (siAQP1) or non‐targeting siRNA. Exposure to siAQP1 decreased AQP1, but not AQP4, protein levels, suggesting specific knockdown of AQP1 and reduced basal proliferation and migration in PASMCs. The results from these gain‐ and loss‐of‐function experiments suggest that AQP1 is a key protein involved in the initiation of PASMC proliferation and migration.
ET‐1‐induced Superoxide‐dependent NFATc3 Activation
Authors: Diaz Juan Manuel Ramiro , Nitta Carlos H , Sherpa Mingma L , Bosc Laura V Gonzalez
Published: June 2021

Endothelin‐1 (ET‐1)‐induced activation of Nuclear Factor of Activated T‐cells isoform c3 (NFATc3) is necessary for the progression of chronic hypoxia (CH)‐induced pulmonary hypertension. Reactive oxygen species (ROS) levels are elevated in CH. ET‐1 increases ROS in pulmonary artery smooth muscle cells (PASMC). ROS participates in NFAT activation in cancer cells. However, it is unknown whether ROS mediates ET‐1‐induced NFATc3 activation in PASMC. Using human PASMC electroporated with a NFATc3‐GFP plasmid, we tested the hypothesis that increased ROS participates in ET‐1‐induced NFATc3 activation. Cells were pretreated with vehicle, a superoxide dismutase mimetic (tempol), a superoxide dismutase inhibitor (diethyldithiocarbamate, DDC) or H2O2. Nuclear fluorescence was measured before and during ET‐1 treatment. To prevent NFATc3 nuclear export, the CRM1 exportin inhibitor leptomycin B was present in all experiments.

Our results show that pretreatment with both tempol and H2O2 inhibited ET‐1‐induced NFATc3 nuclear import. On the other hand, DDC enhanced ET‐1‐induced NFATc3 nuclear import.

To determine the intracellular source of ROS, cells were pretreated with a xanthine oxidase inhibitor, allopurinol (Allo), or a NADPH oxidase inhibitor, apocynin (Apo). Allo but not Apo inhibited ET‐1‐induced NFATc3 nuclear import.

Our results suggest that in human PASMC, ET‐1‐induced NFATc3 nuclear import is dependent on xanthine oxidase‐derived superoxide and/or a reduction in H2O2 levels. Therefore, it is possible that elevated superoxide levels in CH contribute to NFATc3 activation.

Funding source: R01 HL088151
Characterization of NAADP‐Induced Global and Local Calcium Signals in Rat Pulmonary Arterial Smooth Muscle Cells
Authors: Jiang Yong-Liang , Lin Amanda H.Y. , Lee Suengwon , Xia Yang , Sham James S.K.
Published: June 2021

Ca2+ signaling in vascular smooth muscle cells involves global and local interactions between multiple Ca2+ influx and release pathways. Ca2+ release pathways include IP3 receptor‐, ryanodine receptor‐, and nicotinic acid adenine dinucleotide phosphate (NAADP) receptor‐mediated mechanisms. Recent studies demonstrated that the two‐pore channels (TPC1 and TPC2) are NAADP receptors located in the endo‐/lysosomes. Here we characterized the NAADP‐mediated Ca2+ signals in rat pulmonary arterial smooth muscle cells (PASMCs). Application of the membrane permeable NAADP‐AM (0.25–1 μM) to PASMCs elicited concentration‐dependent increase in global [Ca2+]i. It was blocked by the NAADP antagonist NED‐19 or the acidic vacuolar H+‐ATPase bafilomycin A, suggesting Ca2+ release from the lysosomal Ca2+ stores. The Ca2+ response was independent of extracellular Ca2+ influx, and was unaffected by the IP3 receptor blocker xestospongin C; but was partially inhibited by ryanodine or thapsigargin. Moreover, NAADP caused dramatic increase in local Ca2+ release events, which was inhibited by ryanodine or NED‐19. The temporal and spatial properties of the NAADP‐induced local release events were comparable to the spontaneous Ca2+ sparks, the elementary Ca2+ events mediated via the ryanodine receptors. Our results, hence, show that the NAADP‐sensitive release channels are functionally expressed and Ca2+ signals from the NAADP channel‐gated lysosomal stores cross‐activate ryanodine receptors to amplify Ca2+ release in PASMCs.
The adenosine system in skeletal muscle of individuals with essential hypertension and the effect of physical training
Authors: Jensen Lasse Gliemann , Nyberg Michael , Thaning Pia , Hellsten Ylva , Mortensen Stefan Peter
Published: June 2021

The study examined vascular function and the adenosine (ADO) system in skeletal muscle of individuals diagnosed with essential hypertension (EH; N=10) and normotensive (N; N=11) individuals, before and after a period of aerobic training. Before and after the training period, the subjects participated in two experiments in which a) leg blood flow was determined during arterial infusion of ADO, acetylcholine (ACh) or during exercise, and b) interstitial fluid was sampled from v. lateralis muscle during exercise and ADO infusion. Leg vascular conductance (LVC) in response to arterial ADO infusion was similar in the EH and N group. Interstitial ADO formation during exercise was lower in the EH than the N group. In EH, the period of training did not affect vasodilator responsiveness to arterially infused ADO but improved the formation of interstitial ADO and prostacyclin. In the N group, training resulted in a lower LVC in response to arterial ADO infusion, which was paralleled by a lower leg blood flow during submaximal exercise. In conclusion, whereas the responsiveness to circulating ADO in individuals with essential hypertension remained unaltered with training, the capacity to form ADO and prostacyclin at the microcirculatory level was enhanced, suggesting an improvement in microvascular blood flow regulation. In normotensive individuals, aerobic training reduced the vasodilatory response to intraluminal ADO.
Role of mitochondrial reactive oxygen species and actin polymerization in PKC‐dependent constriction of small pulmonary but not mesenteric arteries
Authors: Snow Jessica B. , Sands Michelle A. , Gonzalez Bosc Laura V. , Walker Benjimen R. , Resta Thomas C.
Published: June 2021

PKC plays a central role in the regulation of vascular smooth muscle (VSM) tone. However, little is known regarding mechanisms by which PKC mediates contraction in pulmonary VSM. We hypothesized that PKCβ elicits pulmonary vasoconstriction through a unique signaling axis involving VSM mitochondrial reactive oxygen species (ROS) and actin polymerization not observed in systemic arteries. The PKC activator phorbol 12‐myristate 13‐acetate (PMA) caused constriction in both endothelium‐disrupted, pressurized rat pulmonary (~150 μm diameter) and mesenteric (~165 μm diameter) arterioles that was independent of an increase in VSM Ca2+ (measured by fura‐2 fluorescence). Consistent with our hypothesis, the PKCβ inhibitor LY‐333,531 (10 nM), the O2− scavenger tiron (10 mM), the mitochondrial‐targeted antioxidant Mito‐CP (0.5 μM), and the actin polymerization inhibitor cytochalasin B (10 μM), blunted reactivity to PMA in pulmonary arteries while having no effect in mesenteric arterioles. Furthermore, PMA increased mitochondrial ROS (measured by Mito‐SOX fluorescence) and caused actin polymerization (measured by Alexa Fluor 488 phalloidin/Alexa Fluor 594 DNase staining) in cultured human pulmonary artery smooth muscle cells. We conclude that PKCβ uniquely enhances mitochondrial ROS production and actin polymerization in the pulmonary circulation to mediate vasoconstriction.
Identification of Rtel1, Edn3, and Dnajc5 as candidate genes for rat chromosome 3 blood pressure Quantitative Trait Loci (QTLs) by gene expression profiling under multiple salt‐loading conditions
Authors: Cicila George Thomas , McSweeny Andrew , Pettee Krista M. , Yang Siming , Khuder Sadik A. , Lee Soon Jin
Published: June 2021

Previous work, comparing congenic rat strains carrying Dahl salt‐resistant (R) rat chromosome 3 (RNO3) alleles on a Dahl salt‐sensitive (S) rat background with the hypertensive S rats defined two blood pressure (BP) QTL‐containing intervals. Renal gene expression profiling of S.R(ET3×1) and S.R(ET3×2) congenic substrain rats, compared to S rats, was conducted in two microarray experiments using Affymetrix Rat RAE230 GeneChip sets. The first examined renal expression after 1) maintenance on a low dietary (0.4% NaCl) salt intake or 2) following acute (24 hour) exposure to an elevated dietary salt (4% NaCl) intake, identifying differentially expressed genes by two‐way analysis of variance. The second examined renal expression following chronic (28 day) exposure to an elevated dietary salt (2% NaCl) intake, identifying differential expression by Significance Analysis of Microarrays thresholding. Ingenuity Pathways Analysis (IPA) was used to highlight cellular molecular networks containing differentially expressed probesets likely altered among the three strains. Candidate genes were defined as mapping to RNO3 congenic regions and being 1) differentially‐expressed and/or 2) interacting with one or more genes in an IPA‐identified molecular network. Differential expression of Rtel1, Edn3, and Dnajc5 was found in both the acute and chronic NaCl‐loading experiments by qRT‐PCR.
Epistatic effects of multiple congenic regions on blood pressure and heart weight in Dahl rats under salt‐loading conditions
Authors: Cicila George Thomas , Yerga-Woolwine Shane , Farms Phyllis , Lee Soon Jin , Joe Bina
Published: June 2021

Mammalian complex traits are little studied for combinatorial gene‐gene interactions. To study epistasis among blood pressure (BP) quantitative trait loci (QTLs) we developed bi‐and tri‐congenic strains that introgress R‐rat congenic regions containing RNO3, RNO7, and RNO9 low BP alleles into S rats. S rats, RNO3+RNO7+RNO9 tri‐congenic, and RNO3+RNO7, RNO3+RNO9, and RNO7+RNO9 bi–congenic rats (n=16) were concomitantly raised on a low (0.4% NaCl) salt diet until 6 weeks‐old, fed a higher (2% NaCl) salt diet for 24 days, after which tail‐cuff BP, body weight (BW), and heart weight (HW) were measured. While all bi‐ and tri‐congenic strains had lower BP (P<0.0001) compared to the parental, S, strain, BP differences were not observed among the bi‐ and tri‐congenic strains. Thus, low BP alleles in the congenic regions were non‐additive under this salt‐loading condition. While bi‐ and tri‐congenic strains had significantly lower HW (P<0.0001) compared to S rats, only RNO3+RNO7+RNO9 and RNO3+RNO7 rats had lower BW‐adjusted HW (1201.8 and 1128.0 mg, respectively) compared to S rats (1267.2 mg; P=0.004 and P<0.0001, respectively). Thus, low BP QTL‐containing alleles showed non‐additive effects, while RNO3+RNO7 bicongenic rats having lower BW‐adjusted HW compared with RNO3+RNO7+RNO9 rats (P<0.002). These results provide evidence for strong interactions between previously identified BP QTLs.
Mapping a novel blood pressure quantitative trait locus within a congenic strain spanning a single annotated gene containing segment on rat chromosome 10
Authors: Pillai Resmi , Kumarasamy Sivarajan , Nie Ying , Yerga-Woolwine Shane , Farms Phyllis , Gopalakrishnan Kathirvel , Joe Bina
Published: June 2021

By high resolution substitution mapping using alleles of the LEW rat introgressed onto the Dahl Salt‐sensitive (S) rat, a 320kb congenic segment was previously identified on rat chromosome 10 (RNO10:70750008‐71070581bp) that further increased the blood pressure (BP) of the S rat. A newer iteration of congenic substrains with introgressed segments ranging from 83kb (70750008‐70832849bp) to 134kb (70750008‐70884058bp) were constructed to further fine‐map this region. Out of these, the systolic BP of the congenic strain spanning a 134kb region was significantly lower compared to that of the S rat by −28 mm Hg (p<0.0001). Bioinformatics analysis of this critical 134kb congenic interval on the rat genome and comparative mapping using mouse and human genomes suggested a single protein coding gene, LOC688668 as the sole candidate gene for this locus. Detailed sequence and expression analyses of this gene will be presented. The significance of this work is that it represents a unique high resolution mapping project wherein the BP lowering effect of the 134kb congenic segment is independently observed when isolated from the longer 320kb congenic segment, which had an overall BP increasing effect.
Orthopedic trauma reduces renal hemodynamics in the STZ‐induced diabetic rat
Authors: Manigrasso Michaele Beth , Davis Deborah D , Lu Silu , Xiang Lusha , Hester Robert L
Published: June 2021

Diabetes is an independent risk factor for the development of acute renal dysfunction following orthopedic trauma (OT). Whether OT affects renal hemodynamics has yet to be investigated. The hypothesis tested in this study was that OT reduces renal hemodynamics in type 1 diabetes.

Male lean Zucker rats (12 wks) were divided into 4 treatment groups (n=4/gp): non‐diabetic without trauma (ND‐NT), non‐diabetic with trauma (ND‐T), STZ‐induced diabetic without trauma (D‐NT) and STZ‐induced diabetic with trauma (D‐T). Four wks after induction of diabetes, animals were implanted with catheters and OT was induced by soft tissue injury and local injection of bone components in both hind limbs. Animals were placed in metabolic cages for 12hrs to collect urine and urine albumin excretion (UAE) was measured by ELISA. 24 hrs post‐trauma, glomerular filtration rate (GFR) was measured in conscious rats. Our results show that diabetes after 4 wks causes a reduction in GFR compared to controls. However, OT caused a greater reduction in GFR in diabetics than controls. Our data suggest that OT compromises renal function in the setting of type 1 diabetes.

Group
Body Weight (g)
Glucose (mg/dL)
UAE (mg/day)
GFR/KW (ml/min/g)

ND‐NT
396±25
81±6
0.5±0.1
1.37±0.09

ND‐T
415±20
85±4
1.2±0.7
1.52±0.08

D‐NT
298±12*#
358±27*#
3.3±0.6*#
1.17±0.08#

D‐T
330±14*#
366±15*#
4.6±0.3*#
1.06±0.07*#

Avg±SEM;

P<0.05 vs. ND‐NT;

P<0.05 vs. ND‐T
Combined effect of advanced glycation end products and physiologically relevant dynamic shear stress on endothelial cell functions
Authors: Maria Zahra , Yin Wei , Rubenstein David Alan
Published: June 2021

Advanced glycation end products (AGEs) and disturbed shear stress are hallmarks of cardiovascular diseases. Our goal was to evaluate the combined effect of AGEs and physiologically relevant dynamic shear stress on endothelial cell (EC) functions. ECs were exposed to glycated or non‐glycated albumin for up to 5 days. ECs were then subjected to shear stress (SS) waveforms mimicking cardiac flow, stenosis flow and recirculation flow for 1hr. EC morphology, metabolic activity and shear induced actin alignment was observed. Also, the expression of ICAM, tissue factor and thrombomodulin was measured to quantify inflammatory and thrombogenic potential. We hypothesized that the combination of disturbed SS and AGEs would enhance EC responses to mimic endothelial dysfunction. Results indicate that ECs incubated with AGEs and exposed to stenosis SS had an altered actin alignment and structure. EC metabolic activity in the presence of glycated albumin, decreased when exposed to disturbed flow. Inflammatory and thrombogenic potential was also enhanced. Thus, the results indicate that the presence of AGEs severely alters EC function under various pathological SS conditions. Thanks to OCAST for supporting this work.
A Stress Repair Mechanism That Maintains Vertebrate Structure During Stress
Authors: Coleman Lewis Stanton
Published: June 2021

This presentation describes the “Stress Repair Mechanism”(SRM) that enables the “Universal Theory of Medicine” predicted by Hans Selye. It explains the nature of embryology, physiology, pathology, and stress. SRM function comprises the autonomic nervous system, the vascular endothelium, and the dynamic enzymatic interaction of blood‐borne hepatic Factors VII, VIIIC, IX and X that produces thrombin, soluble fibrin and insoluble fibrin, whose combined effects account for all SRM manifestations. The vascular endothelium is a diaphanous neuroendocrine organ that lines all blood vessels and is the sole constituent of capillary walls. It secretes tissue factor into extravascular tissues, and insulates those tissues from the hepatic enzymes. Tissue disruption exposes tissue factor to the enzymatic interaction and activates tissue repair. The vascular endothelium also releases nitric oxide and von Willebrand Factor into blood in accord with autonomic balance to regulate the enzymatic interaction to govern tissue perfusion and organ function. Therefore, continuously fluctuating combinations of nervous stimuli that affect autonomic balance and forces that disrupt tissues determine SRM activity.
Hyper‐caloric diet enhances aortic endothelial function via increased NOS3 activity and expression in Dahl S rats
Authors: Spradley Frank T , Musall Jacqueline B , Pollock Jennifer S
Published: June 2021

Previously we reported that a 4‐week hyper‐caloric diet (HCD) elicits blunted angiotensinII‐induced aortic constriction in Dahl salt‐sensitive (Dahl S) rats (16 weeks old), but not age‐matched genetic control SS‐13BN rats. We tested the hypothesis that HCD promotes increased NOS3 activity and/or expression as well as endothelial‐dependent relaxation when compared to normal diet (ND) Dahl S rats. Aortic NOS activity was examined by the conversion of L‐arginine to L‐citrulline while NOS3 expression was determined by Western blotting. HCD increased NOS3 activity (22±2 vs. 13±1 cpm/mg protein/30min; P<0.05), abolished NOS2 activity (undetectable vs. 5±1 cpm/mg protein/30min), with no change in NOS1 activity compared to ND Dahl S rats. HCD also increased aortic NOS3 protein expression (0.44±0.04 vs. 0.34±0.04 RDU; P<0.05). Using wire myography, acetylcholine‐induced vasorelaxation was greater in aortae from Dahl S rats on HCD compared to ND (82±3 % vs. 71±3 % max relaxation; P<0.05); NOS inhibition (L‐NAME, 100μm) abolished this response in both diet groups. Sodium nitroprusside mediated aortic relaxation was similar in Dahl S rats on both diets. We conclude that a short‐term HCD in Dahl S rats promotes enhanced endothelial‐dependent vasorelaxation and blunted angiotensinII‐induced vasoconstriction via increased NOS3 activity and expression. Funding: PO1 HL69999 and AHA 10PRE4010001.
Bidirectional regulation of adrenal catecholamine release by prostaglandin E2
Authors: Jewell Mark , Breyer Richard M. , Currie Kevin P.M.
Published: June 2021

Catecholamine secretion from adrenal chromaffin cells is elevated in response to adverse physiological conditions. Similarly, systemic immune challenge leads to intra‐adrenal inflammatory signaling and a resulting induction of synthetic enzymes for prostaglandin E2 (PGE2). In the present study we investigated the functional impact of PGE2 on calcium signaling and catecholamine release from mouse adrenal chromaffin cells. PGE2 potently (EC50 = 5.5 nM) inhibited Cav2 voltage‐gated calcium currents (ICa), while having no effect on resting intracellular [Ca2+] or the peak amplitude of nicotinic acetylcholine receptor currents. The inhibition of ICa is mediated by pertussis toxin‐sensitive G proteins, and reversed by a strongly depolarizing voltage step, characteristic of direct G protein βγ subunit binding to the channel. PGE2 acts primarily through a family of cognate GPCRs (EP1‐EP4). We detected mRNA for all four EP receptors in mouse adrenal gland, but using selective pharmacological tools and EP receptor knockout mice we show the inhibition of ICa is mediated by EP3 receptors. Monitoring changes in membrane capacitance in response to brief depolarizations, we show Ca2+‐ dependent exocytosis is reduced in parallel with ICa in wild‐type mice. Conversely, PGE2 potentiates exocytosis in chromaffin cells isolated from EP3 receptor knockout mice, revealing a signaling modality distinct from the inhibitory EP3 pathway. Using carbon fiber amperometry, we find the potentiation of exocytosis is also apparent in wild type cells during sustained depolarizations mimicking acute stress. Our data support a model in which distinct EP receptor subtypes mediate activity‐dependent, bidirectional modulation of catecholamine release by PGE2. This work is supported by the American Heart Association [11GRNT7890031] and the National Institutes of Health [R01 NS052446].
The fragile X mental retardation protein FMRP plays a role in the DNA damage response
Authors: Shi Yang , Alpatov Roman , Wagner Ulrich , Nakamoto-Kinoshita Mika , Ye Zhen , Luu Ying , Armache Karim J. , Simon Matthew D. , Stuetzer Alexandra , Greer Eric L. , Wang Zhibin , Hu Gang-Qing , Wu Feizhen , Xu Chao , Beavers William N. , Guo Yahong , Bian Chuanbing , Morrison Paul T. , Vakoc Christopher R. , Min Jinrong , Fischle Wolfgang , Kingston Robert E. , Zhao Keji , Ren Bing , Warren Stephen T.
Published: June 2021

In humans, loss of the fragile X mental retardation protein, FMRP, leads to the most common inherited form of intellectual disability, the fragile X syndrome. FMRP is predominantly present in the cytoplasm where it regulates translation of proteins important for synaptic function. However a small percentage of FMRP is localized in the nucleus where its function has remained unclear. We demonstrate that FMRP associates with chromatin and participates in the replication stress‐dependent DNA damage response (DDR), possibly by recognizing specific histone modifications. Taken together, our findings uncover an unexpected role of FMRP in the DDR and suggest that FMRP‐dependent maintenance of genomic stability may be a potential contributing factor in the development of the fragile X syndrome.
Dietary sodium alters beta‐adrenergic receptor mediated vasodilation in men but not women
Authors: Gullixson Leah R , Kost Susan L , Penheiter Alan R , Turner Stephen T , Nicholson Wayne T , Joyner Michael J , Eisenach John H
Published: June 2021

Dietary sodium loading decreases β‐adrenergic receptor mediated vasodilation in hypertensive men, and paradoxically increases this dilation in normotensive men; no information exists in women. The purpose of this investigation was to test the hypothesis that dietary sodium loading and restriction influences β‐receptor mediated vasodilation in a sex‐dependent manner. Healthy normotensive subjects consumed a high (400 mmol/day, n=36, mean age±SEM 23±1 yr, 23 women) or low (10 mmol/day, n=30, 30±1 yr, 14 women) sodium diet for 5 days prior to study. Forearm blood flow (FBF, plethysmography) responses to brachial artery administration of isoproterenol (ISO: 1, 3, 6, 12 ng/100 ml tissue/min) were measured before and after endothelial NO synthase inhibition with NG‐monomethyl‐L‐arginine (L‐NMMA). Consistent with prior reports, the FBF response to ISO was greater in men on high sodium compared to men on low sodium (p=0.03, ISO‐by‐sodium interaction). The highest response to ISO was 16.6±1.7 and 11.0±1.1 ml/100 ml tissue/min (high vs. low, p=0.01). After LNMMA the men's response to ISO did not differ based on sodium. In contrast, in women dietary sodium did not affect the response to ISO before or after LNMMA. We conclude that dietary sodium alters β‐adrenergic receptor mediated vasodilation in men, which appears to be dependent on NO. This is the first study to suggest that these mechanisms of vasodilation are resistant to dietary sodium in women. Support: HL‐089331, CTSA RR‐024150
Na+‐dependent transport of taurine is found only on the abluminal membrane of the blood‐brain barrier
Authors: Rasgado-Flores Hector , Mokashi Ashwini , Hawkins Richard
Published: June 2021

Luminal and abluminal plasma membranes, isolated from bovine brain microvessels, were used to characterize Na+‐dependent and facilitative taurine transport. Initial transmembrane potential was − 59 mV 0, external Na+ was 126 mM, and T= 25°C. Apparent affinity constants of the taurine transporters were determined over a range of taurine concentrations from 0.24 μM to 11.4 μM. Abluminal membranes had Na+‐dependent taurine transport as well as facilitative transport; luminal membranes only had facilitative transport. Apparent Km's for facilitative and Na+‐dependent taurine transports were 0.06 ± 0.02 μM and 0.7 ± 0.1 μM, respectively. Na+‐dependent transport of taurine was voltage dependent between −25 to −101 mV. Transport was over 5 times greater at −101 mV compared with Vm was −25 mV. Sensitivity to external osmolality of Na+‐dependent transport was studied over a range of osmolalities (229 to 398 mOsm/kg H2O) using mannitol as the osmotic agent to adjust osmolality. For these experiments the concentration of Na+wasmaintained constant at 50 mM; the calculated transmembrane potential was −59 mV. Na+‐dependent transport was sensitive to osmolality with the greatest rate observed at 229 mOsm/kg H2O.
Role of Aquaporin2 (Aqp2) in the increased urinary flow of K‐adapted mice
Authors: Contreras Jacob I. , Hatcher Lori I , Cornelius Ryan J. , Sansom Steven C.
Published: June 2021

Mice fed a high K diet maximize K excretion partly by increasing distal flow, which activates large, Ca‐activated K channels in connecting tubules (CNT) and cortical collecting ducts (CCD). Flow is increased by medullary K recycling and inhibition of Cl absorption in the thick ascending limb; however, other events may increase flow. We hypothesized that diuresis of a high K diet is partly due to less Aqp2 in the apical membranes of CNT cells and principal cells (PC) of the CCD. We found that flow increased from 1.3 ±0.5 ml/day in mice fed a control diet (Na =0.3%, K = 0.6%) to 6.7 ±0.5 ml/day in high K fed mice (HK; Na = 0.3%, K =5%). The urine osmolality of control was 2296 ±201 mosm/L and of HK was 1487 ±46 mosm/L. HK consumed three‐fold more water; however, the hematocrits (control =41.5 ±0.3%; HK = 42.3 ±0.6%) were not different, indicating that the diuresis was not the result of a primary increase in water consumption. Estimated urea excretions for controls (1831 ±191 μmoles/day) and HK (1909 ±224 μmoles/day) were not different. Using immunohistochemical staining with anti‐Aqp2, we found that Aqp2, which was intensely stained (26.4±1.6 AU) on the apical membranes of CNT and PC of control, exhibited significantly less apical staining (20.2±1.3 AU) in HK.

Conclusion

The increased flow of HK is not the result primary drinking or an osmotic urea diuresis, but may be partly the result of reduced Aqp2‐mediated water reabsorption.
The N‐terminus of ClC‐3 determines membrane localization
Authors: Lamb Fred S , Collins Malia M
Published: June 2021

CLC family Cl−/H+ anti‐porters are primarily intracellular (IC) proteins that support endosome acidification. Heterologous ClC‐5 current is readily expressed while ClC‐3 is not. We hypothesized that this difference is due to differential membrane localization directed by N‐terminal signal sequences. ClC‐3 and ‐5 C‐terminal GFP and YFP fusion proteins were localized by confocal microscopy. WT ClC‐3‐GFP (both long or short isoforms) localized to enlarged IC vesicles which were much smaller in H+ transport deficient mutants (E224A). Disruption (D15A, D18A) of a putative clathrin binding motif (LLDLLD) in short ClC‐3 yielded isolated bright dots in the plasma membrane. These dots were dissociated into a smooth membrane signal by methyl‐beta‐cyclodextrin, suggesting localization to lipid rafts. WT ClC‐5‐CFP also localized to bright spots in the membrane. A novel, long‐ClC‐5 isoform (69 aa longer) localized to both diffusely to the plasma membrane and to enlarged IC vesicles. When the N‐terminus of ClC‐5 was replaced by that of long‐ClC‐3 (first 70aa) membrane expression was lost and only enlarged IC vesicles were observed. Thus, ClC‐3 appears to traffic through plasma membrane lipid rafts and the N‐terminus is critical for localization. The first 70aa of long ClC‐3 are sufficient to move ClC‐5 into an exclusively IC localization that is indistinguishable from ClC‐3.
The Effects of Synthetic ASIC1a Peptide on Glial Proliferation
Authors: Guercio Jennifer C. , Petroff Elena
Published: June 2021

Increased activity of large conductance calcium and voltage‐activated potassium (BK) channels has been shown to accelerate glial proliferation. Our previous studies indicate that Acid Sensing Ion channels (ASICs) inhibit BK at physiological pH levels, and that this inhibition can be relieved at acidic pH. Both normal glia and glioma express BK and ASIC channels. We hypothesized that ASIC‐BK interaction at normal pH would inhibit glial growth, and that a drop in extracellular pH would accelerate the cell proliferation through endogenous pH‐dependent regulation of BK channel activity by ASICs. Mouse glia from wild type (wt) and ASIC1 knockout (ko) mice were grown at normal pH 7.4 and in more acidic pH 7.0. Lowering extracellular pH increased glial proliferation in wt. However, the pH‐dependent regulation of glial growth is lost in ASIC1a knockout glia. A BK channel blocker charybdotoxin (CTX) inhibited glial growth at both pH 7.4 and 7.0, in wt and ko. To further examine ASIC1a and glial proliferation, a wild type peptide (with a similar amino acid sequence to ASIC1a's toxin‐like domain) and a mutant peptide (arginine and lysine important for BK inhibition were mutated to alanine) were designed. Cultured wild type glia were grown at pH 7.4 and 7.0, and in both conditions with either the wild type peptide or the mutant peptide. The synthetic wild type peptide treatment showed no significant change in proliferation. However, the glial growth in the presence of the mutant peptide doubled when compared to the control conditions. These results are consistent with the expected competition of the peptides with ASIC for interacting with BK. Since the mutant peptide does has no inhibit BK but presumably interacts with it at the same site as ASIC, it leads to increased BK activity and cell proliferation. This study is a step towards understanding the regulation of glial cell proliferation by ASIC‐BK channel interaction and our long term goal of developing novel approaches to treating gliomas. This work is supported by the R15 NIH grant to E.P.
A Deg/ENaC cation conductance regulates migration and cell cycle progression in gliomas
Authors: Rooj Arun Kumar , McNicholas Carmel , Fuller Catherine M
Published: June 2021

Glioma cells express a specific amiloride‐sensitive cation channel composed of ASIC1, α‐ and γENaC. As these tumors are highly invasive, we hypothesized that channel activity was engaging an intracellular signaling pathway regulating migration and proliferation. We used a truncated construct (ASIC1Y67X) and shRNA to knockdown ASIC1 and integrin β1 in D54MG glioma cells, and scratch assays, FACS, patch‐clamp, and immunoblotting, to determine knockdown effects. Cells were synchronized by removal of FBS for 48h prior to the experiments. ASIC1 knockdown reduced scratch area by 39 ± 16% (n≥4 ±SD P<0.001), and significantly increased the percentage of cells in the cell cycle G0/G1 phase (by 19 ± 5% (SD) n≥3 P<0.001) as compared to controls at 24h. Expression of two cell cycle inhibitors, p21Cip1 and p27Kip1 increased by 2.5 fold (n≤5) and 3 fold (n≤6), respectively. Phosphorylation of ERK1/2, a key mediator of proliferation and migration was also reduced (by 47 ± 20% (SD) n≥6 P<0.001) as compared to control at 24h. Knockdown of δENaC (not part of the glioma channel complex), had no effect on any measured parameter. Knockout of integrin β1 inhibited the amiloride‐sensitive current by 89±24% (n≥9; ±SD P<0.001). Our data reveal novel interactions between Deg/ENaC channels, the MAPK pathway and integrins that may form a mechanism by which Na+ current regulates cell proliferation and migration. Supported by NIH Grant # DK037206.
Chemosensitive signaling of locus coeruleus neurons in the Bullfrog, Lithobates catesbeianus
Authors: Santin Joseph , Hartzler Lynn
Published: June 2021

Bullfrogs, Lithobates catesbeianus, experience broad changes in pH inversely proportional to temperature while retaining whole body CO2 stores. Ventilatory patterns to maintain constant CO2 are controlled through negative feedback with chemosensory neurons detecting CO2/pH. We hypothesize that neurons of the locus coeruleus (LC) will respond to hypercapnic acidosis (HA). Whole‐cell patch‐clamp electrophysiological techniques were used to measure firing rates in excitatory (chemosensitivity index (CI)=260±35%), inhibitory (CI=24±18%), and non‐chemosensitive (CI=105±3%) LC neurons in bullfrog brainstem slice preparations. LC neurons have resting membrane potentials of −42±0.6mV, and when stimulated by HA membrane potentials are depolarized to −39±0.7mV (p<0.001). 81% of neurons increased and 11% decreased firing rates when exposed to HA, though increasing ΔCO2 to acidify the superfusate by >2‐fold (0.13–0.33pH units) did not produce different firing rates (p> 0.5). Greater than 80% of LC neurons are excited by HA which corroborate percentages typically reported for neonatal rat LC neurons; however, bullfrog excitatory output is doubled and appears to be maximally activated with lower CO2/H+ changes compared with neonatal rats. Our data begin to provide insight into a neurological mechanism for α‐stat pH regulation in bullfrogs based on chemosensitivity to CO2/pH.
Mitochondrial sensitivity to regulatory signals in muscle energy balance: is it constant during exercise?
Authors: Jeneson Jeroen A.L. , Dash Ranjan , Vinnakota Kalyan C , Wu Fan , Beard Dan A , Wiseman Robert W
Published: June 2021

The sensitivity of mitochondrial ATP synthesis to regulatory signals elevated by muscle exercise was investigated in a rat model using an integrative approach of physiologic experiments and computer simulation. Time courses of hindlimb muscle phosphocreatine (PCr) and Pi and pH during serial contractions and recovery were recorded using in vivo phosphorus NMR spectroscopy. Muscle contractions were elicited using sciatic nerve pacing over a tenfold range of frequencies (0.25–2 Hz) with concurrent force recording from the Achilles tendon. At each contraction frequency, the mitochondrial ATP synthesis rate was determined from PCr and pH dynamics and correlated with the concentrations of the regulatory metabolites ADP and Pi. Data were analyzed using a computational model of mitochondrial ATP synthesis and accounting for cytosolic fluxes of phosphate buffering and glycolytic pathways. The integrated model was compared to empirical data and used to explore the feasibility of competing hypotheses regarding the control of mitochondrial dehydrogenases and respiratory fluxes.