bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of eukaryotic translation initiation factor 5A (eIF5A) in skeletal muscle stem cell differentiation
Authors: Luchessi Augusto Ducati , Hirabara Sandro Massao , Lambertucci Rafael Herling , Cambiaghi Tavane David , Neto Claudio Miguel Costa , Curi Rui
Published: June 2021

The protein eIF5A is involved in different steps of mRNA metabolism. This factor contains a special amino acid residue named hypusine. Hypusine is required for the eIF5A activity being produced by a post‐translational modification involving spermidine as substrate. Stem cells (satellite cells) from skeletal muscle rats were submitted to differentiation and an increase of eIF5A gene expression was observed. An increased content of eIF5A protein was observed in satellite cells on differentiation in comparison to non‐differentiated satellite cells and muscle tissue. The treatment with GC7, a hypusination inhibitor, abolished the differentiation process. In association with the differentiation blockage, an increase in glucose consumption and lactate production and a decrease in glucose and palmitate decarboxylation were observed. Also, there was a reduction in cell proliferation and protein synthesis. L‐arginine, a partial suppressor of muscle dystrophic phenotype and spermidine precursor, partially abolished the GC7 effects. Our results reveal a new physiological role for eIF5A and contribute to elucidate the molecular mechanisms involved in muscle differentiation.

Supported by: FAPESP, CNPq and CAPES.
Src family Protein tyrosine kinase( PTK) modulates the effect of SGK1 and WNK4 on ROMK channels.
Authors: Yue Peng , Leng Qiang , Lin Daohong , Pan Chunyang , Wang Wenhui
Published: June 2021

We used the whole cell patch clamp technique to study the role of c‐Src in modulating the effect of WNK4 and SGK1 on ROMK channels. Expression of c‐Src decreased K current from 1080 pA to 520 pA in HEK293T cells transfected with GFP‐ROMK1+c‐Src. The inhibitory effect of PTK was abolished either by treatment of cells with herbimycin A or in cells transfected with R1Y337A, in which tyrosine residue 337, a PTK phosphorylation site of ROMK1, was mutated to alanine. Coexpression of WNK4 decreased K current by 50% in cells transfected with ROMK1 (from 1050 to 560 pA) or R1Y337A (from 1790 to 800 pA). The inhibitory effect of WNK4 on ROMK1 or ROMK mutant was abolished when SGK1 is coexpressed (R1+WNK4+SGK1, 1040 pA; R1Y337A+WNK4+SGK1, 1740 pA), suggesting that SGK1 antagonizing the WNK4‐induced inhibition of ROMK. However, expression of c‐Src significantly decreased K current again in cells transfected with R1+WNK4+SGK1 (650 pA) or with R1Y337A+WNK4+SGK1 (870 pA). In contrast, expression of c‐Src had no effect on K current in cells transfected with R1337A or R1Y337A+SGK1. We conclude that c‐Src inhibits ROMK channels by phosphorylating ROMK channels and by antagonizing the SGK1‐mediated inhibition of WNK4.
Receptor Specificity in AKAP79/150 Actions on M‐type K+ Channels
Authors: Zhang Jie , Bal Manjot , Zaika Oleg , Shapiro Mark S.
Published: June 2021

Protein kinase C (PKC) phosphorylation of M‐type (KCNQ2‐5) K+ channels facilitated by A‐kinase‐anchoring protein (AKAP)79/150 is involved in M current (IM) suppression by stimulation of Gq/11‐coupled muscarinic M1, but not bradykinin B2, receptors in cultured superior cervical ganglion (SCG) neurons (Hoshi et al., Nat. Cell Biol. 7:1066‐73). Purinergic P2Y receptors also depress IM in SCG cells via a similar mechanism to that of bradykinin involving IP3‐mediated Ca2+ signals. We asked if purinergic modulation also involves AKAP79/150. Transfection of δA‐AKAP79, which lacks the "A domain" necessary for PKC binding, did not affect IM suppression by the purinergic agonist UTP (2 μM), nor by bradykinin (100 nM), but did reduce IM suppression by the muscarinic agonist oxotremorine (oxo‐M, 1 μM). For cells transfected with δA‐AKAP79, IM suppression by UTP, bradykinin or oxo‐M was 43.5 ± 5.9% (n=7), 36.9 ± 2.7% (n=5) and 25.6 ± 2.1% (n=14), respectively. For cells transfected with GFP only, the suppressions were 45.0 ± 3.6% (n=10), 33.7 ± 2.6% (n=4) and 49.6 ± 1.2% (n=9, p<0.01), respectively. We also tested association of AKAP79 with M1, B2 and P2Y6 receptors via FRET experiments on CHO cells under TIRF microscopy. Indeed, FRET between AKAP79 and P2Y6 or B2 receptors was much less than for M1 receptors. Our data suggest that AKAP79/150 is not involved in purinergic suppression of M current. Supported by NIH grant RO1 NS043394.
Genetic loss of KCa3.1 causes subtle erythrocyte macrocytosis and progressive splenomegaly
Authors: Kaistha Brajesh Pratap , Grgic Ivica , Kaistha Anuradha , Hoyer Joachim , Köhler Ralf
Published: June 2021

KCa3.1, also known as Gardos channel in erythrocytes, is considered to be a major regulator of red blood cell (RBC) volume by mediating K+ efflux. However, the functional importance of KCa3.1 in RBC in vivo is still incompletely understood. Here we used KCa3.1‐/‐ mice to investigate the consequences of KCa3.1 deficiency for RBC metrics, functions and sequestration. RBCs of KCa3.1‐/‐ mice of all ages were mildly macrocytic but their biconcave appearance preserved. RBCs number, total hemoglobin and hematocrit were unchanged in the adult KCa3.1‐/‐ mice and increased in the premature KCa3.1‐/‐ mice. Filterability, Ca2+ dependent volume decrease and osmotic tolerance of RBCs lacking KCa3.1 were noticeably reduced when compared to RBC of wt‐mice. Deformability to increasing shear stress was unchanged. Strikingly, KCa3.1‐/‐ mice developed progressive splenomegaly which was considerable in the >6‐month‐old mice and was paralleled by increased iron deposition in the aged‐mice presumably as a consequence of enhanced RBC sequestration. Injections of the KCa3.1‐blocker TRAM‐34 also produced mild splenomegaly in wt‐mice. We conclude that genetic deficit of erythroid KCa3.1 causes mild RBC macrocytosis, presumably leading to reduced filterability, and impairs volume regulation. These RBC defects result in mild but progressive splenomegaly.
Role of miRNAs in post‐transcriptional gene regulation by high NaCl
Authors: Bravo Ignacio J , Ferraris Joan D , Williams Chester K , Burg Maurice B
Published: June 2021

High NaCl increases mRNA and protein abundance of numerous gene products, including the osmoprotective transcription factor TonEBP/OREBP. High NaCl increases TonEBP/OREBP abundance by stabilizing its mRNA. miRNAs regulate gene expression post‐transcriptionally by destabilizing target mRNAs and inhibiting translation. The purposes of the present studies were 1) to find whether miRNAs regulate TonEBP/OREBP abundance and 2) to identify miRNAs that are regulated by the level of NaCl. DICER and Ago2 are necessary for maturation and activity of miRNAs. We knocked down DICER and Ago2 in HEK293 cells by conditional expression of specific sh_miRNAs. Knocking down either DICER or Ago2 increases TonEBP/OREBP protein abundance, suggesting that miRNAs are involved in its regulation. In order to begin investigating the role of miRNAs in high NaCl‐induced changes of expression of proteins, including TonEBP/OREBP, we conducted a microarray screen of miRNAs affected by high NaCl. We adapted HEK293 cells to 500 mosmol/kg (NaCl added) through many passages or kept them at 300 mosmol/kg. We isolated miRNAs from them with a mirVana Kit, then screened with microarrays containing 470 probes (LC Sciences). Levels of 11 miRNAs are significantly greater at 500 mosmol/kg and 9 are less. Several of these miRNAs have likely target sites in the 3′‐UTRs of TonEBP/OREBP and other tonicity regulated genes. We conclude that the level of NaCl regulates several miRNAs and that these miRNAs may be involved in post‐transcriptional regulation of TonEBP/OREBP and other proteins.
Renal Collecting Duct GSK3ß Deletion Affects Response To AVP By Reducing Adenylate Cyclase Activity
Authors: Rao Reena , Patel Satish , Woodgett Jim , Matthew Breyer , Raymond Harris
Published: June 2021

Glycogen synthase kinase 3 (GSK3) is potently inhibited by lithium, a common treatment for bipolar disorders, which impairs urinary concentrating ability in 20‐30% of patients. To examine if GSK3ß is crucial for regulating vasopressin (AVP) dependent water reabsorption in the renal collecting duct (CD), we developed CD specific GSK3ß knockout mice (GSK3ß‐CD null). These mice had lower urine osmolality, renal aquaporin 2 (AQP2) mRNA and protein levels following water deprivation or dDAVP treatment, compared to wild type (WT) mice. dDAVP and forskolin induced cAMP generation was lower in the GSK3ß‐CD null mice and GSK3ß inhibitor treated WT mice or IMCD cells. We measured the renal AC activity in membrane preparation from WT and GSK3ß ‐CD null mice. dDAVP or forskolin induced AC activity in GSK3ß‐CD null group was reduced by up to 50% and 40% respectively compared to the WT group. Over 68% suppression of AC activity was observed in WT membranes treated with SB216763. These studies show for the first time that GSK3ß inhibition or deletion leads to lower cAMP generation due to reduced AC activity, which could be responsible for low AQP2 expression and impaired response to AVP in the GSK3ß‐CD null mice.
Techniques in microfluidics: creation of microspheres for cellular encapsulation
Authors: Tesche Leora Jeanne , Gavigan Thomas , Samuelson Lisa , Gerber David
Published: June 2021

Background

With new innovations in stem cell research, cell therapy as a treatment modality has broad potential. Current pursuits range from treatment of diabetes and cardiovascular disorders to wound healing and aesthetic medicine. For stem cell therapy to be realized, a reliable method for transplanting cells is critical. Currently, technical difficulties including mechanical shearing and failure of a cell to engraft limit the long‐term survival of transplanted cells. Microencapsulation of cells within a protective biosphere allows cells to be safely injected into the host where they can proliferate and engraft providing therapeutic benefit.

Research Proposal

This abstract describes a novel technique of creating a two layered microsphere. In this model, cells suspended within a nutrient rich collagen matrix are encapsulated by a protective alginate coating.

Methods

Specially patterned microfluidic molds are created using lithography. T‐junction geometry within the mold creates 200μ spheres which contain select cell populations. Phase contrast imaging is used to track cell growth within the microspheres.

Results

Hepatic cells survive and proliferate within this microenvironment for extended periods in culture, up to 2 weeks.

Discussion

Perfecting this technique has implications in studying 3‐dimesional cell culture as well as cellular transplantation.
Characterization of bacterial species associated with dental disparities of the appalachian community
Authors: Owen Sarah K , Lumkomska Ewa , Olson Joan
Published: June 2021

Periodontal disease in West Virginian families is the highest in the nation. The recognized link between periodontal disease and other negative health outcomes makes understanding the origin of this disparity an important medical problem. Studies have shown that over 500 bacterial species can make up the complex biofilm communities of dental plaques. The purpose of this study was to determine if there were differences in the bacterial biofilm community in subgingival plaques of the Appalachian community that can be linked to their increased periodontal disease. Dental plaques from high and low periodontal disease participants were collected in association with the COHRA (Center for Oral Health Research in Appalachia) project, the DNA extracted, the 16S RNA gene amplified and sequenced, and the sequences were analyzed using a BLAST comparison. Our studies found Selenomonas sp. to be prominent bacteria in an individual with high periodontal disease, while a protective species, Veillonella, found in low disease participants was decreased. (Supported by NIH Grant 5P20RR016477 to the West Virginia IDeA Network for Biomedical Research Excellence)
ROS generated with Ischemia are a Redox Signal for Angiogenesis
Authors: Chatterjee Shampa , Hong NanKang , Yu Kevin , Fisher Aron
Published: June 2021

We have reported earlier that ischemia in the pulmonary vasculature results in reactive oxygen species (ROS) generation due to closure of a KATP channel leading to endothelial NADPH oxidase activation. Here, we investigated the effect of ischemic ROS on the angiogenic potential of endothelial cells in vitro and in vivo. Flow adapted ischemic mouse pulmonary microvascular endothelial cells (PMVEC) (72 h flow adaptation, 1 h ischemia) injected into a matrigel matrix showed ~4 fold higher tube formation than static cells. Tube formation was not detected in PMVEC derived from mice with knockout of KATP channels or gp91phox subunit of NADPH oxidase. (These null cells do not generate ROS with ischemia). Tube formation in static cells could be increased by the addition of exogenous ROS but flow adapted cells were unaffected. Using an in vivo model, ischemia (femoral artery ligation) resulted in neovascularization as ascertained by microscopic evaluation in wild type mice but was significantly decreased in mice with knockout of KATP channel or the gp91phox subunit of NADPH oxidase. Increased VEGF expression was observed in ischemic tissue of wild type mice and was abolished in KATP channel and gp91phox null mice. We conclude that ROS with ischemia serves as a signal for angiogenesis and that the ROS triggered VEGF formation represents an attempt at neovascularization to restore the impeded blood flow.
Changes in neuropeptide expression in hypothalamic neurons of rats with chronic heart failure
Authors: Llewellyn‐Smith Ida J , Smith Cameron M
Published: June 2021

Pre‐autonomic and neuroendocrine neurons in the hypothalamic paraventricular nucleus (PVN) are important for regulating blood pressure and fluid balance. In chronic heart failure (CHF), changes occur in the anatomy and physiology of the PVN to compensate for reduced heart function. We investigated the effect of CHF on hypothalamic neurons immunoreactive for corticotropin releasing factor (CRF), vasopressin (VP), enkephalin (ENK), pro‐ENK or nitric oxide synthase (NOS). To induce myocardial infarctions that occupied >40% of the left ventricle, we ligated left coronary arteries in male rats and perfused them 6‐8 weeks later. Hypothalamic sections from CHF rats and control rats without ligations were stained with immunoperoxidase and immunoreactive neurons were counted. We found more CRF‐ and VP‐immunoreactive neurons in the PVN of CHF than control rats (p<0.05, n=5‐6 rats/group). There was also a trend towards a decreased number of NOS‐immunoreactive neurons in the PVN in CHF rats (p=0.07, n=5/group). ENK neurons were not found in the PVN itself but in the anterior hypothalamic area. The number of ENK‐ (p=0.34, n=4/group) and pro‐ENK‐immunoreactive (p=0.57, n=3/group) neurons did not differ between CHF and control rats. These results suggest that CHF causes widespread changes in the neurochemistry of hypothalamic neurons involved in the regulation of autonomic function.

Support: NH&MRC of Australia
The sympathetic chemoreflex response to hypoxia in humans is sensitised by prior exposure to 8 h of isocapnic hypoxia
Authors: Croft Quentin , Sander Mikael , MacDonald Ian , Simpson Liz , Dorrington Keith , Robbins Peter
Published: June 2021

In healthy humans the ventilatory sensitivity to acute hypoxia is increased by prior exposure to hypoxia for 8 h. Here we test the hypothesis that the sympathetic chemoreflex is similarly sensitised by a prior short‐term exposure to hypoxia. Healthy humans (n=9) were exposed to eucapnic hypoxia (controlled end‐expiratory PO2 = 45 mmHg) for 10 minutes before and after 8 h in an hypoxic chamber (controlled end‐expiratory PO2 = 50 mmHg). Muscle sympathetic nervous activity (MSNA) was determined by peroneal microneurography (morning recording in one leg and evening recording from the contralateral leg). Acute hypoxic exposure before vs after the chamber resulted in oxygen desaturation (arterial saturations 82±1 vs 81±1 %) and increases in heart rate (+15±2 vs +20±1 b·min‐1), ventilation (+8±1 vs +20±1 l·min‐1), and MSNA (+61±14 vs +154±34 % total activity), all p<0.05. These results demonstrate robust sensitization of sympathetic as well as ventilatory chemoreflexes in healthy humans following an 8‐h exposure to mild hypoxia.

This work was supported by The Wellcome Trust and University College, Oxford.
Mechanisms underpinning the hypertension in juvenile rats submitted to chronic intermittent hypoxia
Authors: Zoccal Daniel B , Bonagamba Leni GH. , Antunes‐Rodrigues José , Paton Julian FR , Machado Benedito H
Published: June 2021

Neural and/or hormonal mechanisms underlying the hypertension of juvenile male Wistar rats (20‐21 days) exposed to chronic intermittent hypoxia (CIH, 6% O2 for 40 s every 9 min, 8 h/day) for 10 days were evaluated. Prior to CIH, baseline systolic arterial pressure (SAP), measured indirectly, was similar between CIH and control rats. After CIH, SAP recorded directly was higher in CIH (n=28) than in control group [n=29, (131±3 vs 115±2 mmHg, P<0.05)]. This higher SAP of CIH rats was associated with increases in the power of oscillatory components at low and high (respiratory‐related) frequencies, but not with changes in spontaneous cardiac baroreflex, in comparison to controls. Sequential i.v. injections of losartan (AT1 receptor antagonist) and AVPx (Vasopressin1a receptor antagonist) did not affect baseline mean arterial pressure (MAP) in either CIH (n=8) or control groups (n=9), while hexamethonium (ganglionic blocker), injected after losartan and AVPx, produced a larger depressor response in CIH than in control group (‐53±2 vs ‐42±2 mmHg, P<0.05). Fifteen days after the cessation of CIH exposure MAP of CIH rats returned to normal levels. The data indicate that the hypertension observed in CIH‐treated juvenile rats is mainly dependent on sympathetic overactivity and is under a higher respiratory modulation, suggesting augmented respiratory‐sympathetic coupling in CIH‐induced hypertension. Supported by FAPESP and CNPq.
Increased vasopressin transmission from the hypothalamic paraventricular nucleus (PVN) to the rostroventrolateral medulla (RVLM) augments cardiorespiratory outflow in CIH‐conditioned rats
Authors: Kc Prabha , Balan Kannan V , Tjoe Steven S , Martin Richard J , LaManna Joseph C , Dick Thomas E
Published: June 2021

Conditioning with chronic intermittent hypoxia (CIH) causes hypertension associated with elevated sympathetic nerve activity and increased cardiorespiratory sensitivity to hypoxia. Hypoxia sensed by the carotid bodies drives cardiorespiratory regulating sites, including the PVN via the nucleus tractus solitarius. Our hypothesis is that CIH‐conditioning alters the balance of excitatory and inhibitory influences on CR control via the PVN. Male SD rats were exposed to CIH for 8 h/day for 10 days. Blood pressure (BP), heart rate (HR), diaphragm (DEMG) and genioglossus (GGEMG) activity were recorded in anesthetized, ventilated and vagotomized rats. The PVN was disinhibited by injecting bicuculline (BIC, a GABAA receptor antagonist, 100 pmol) before and after blocking AVP receptors in the RVLM with SR49059 (200 pmol/side, bilateral). In control rats, BIC increased BP, HR, minute DEMG and GGEMG. Following RVLM blockade, these variables were significantly decreased (p < 0.05). Although more pronounced, similar responses were observed in CIH‐conditioned rats; however, it required a higher dose of blocker (400 pmol/side, bilateral) to blunt the effect (p < 0.05). These finding suggests that AVP modulates cardiorespiratory output via the RVLM and CIH‐conditioned rats show augmented AVP transmission, suggesting AVP as a candidate neuropeptide that controls sympathetic tone and respiratory output during CIH.

Supported by: HL 87620.
5HT1A Receptor Mediated Inhibition of Glutamatergic Neurotransmission to Cardiac Vagal Neurons Following Hypoxia and Hypercapnia
Authors: Mendelowitz David , Kamendi Harriet W
Published: June 2021

Cardiac vagal neurons (CVNs) are excited by serotonergic and purinergic pathways recruited post hypoxia / hypercapnia (H/H). Surprisingly, excitatory glutamatergic inputs to CVNs are inhibited post H/H. This study examines the mechanisms of H/H induced inhibition of glutamatergic neurotransmission to CVNs. Spontaneous and inspiratory‐related EPSCs were recorded from identified CVNs before, during and post H/H. WAY 100635 (100 microM), a specific antagonist for 5HT1A receptors, restored a glutamatergic pathway to CVNs in the recovery period following H/H. The purinergic and serotonergic pathways to CVNs active post H/H were not altered by blocking 5HT1A receptors and WAY 100635 had no effect before, and during H/H. This study indicates endogenous activation of serotonergic 5HT1A receptors decreases glutamatergic neurotransmission to CVNs following H/H, likely via a presynaptic site of action.
Systematic comparison of intrinsic membrane properties of locus coeruleus neurons in Mecp2‐null and wild‐type mice
Authors: Zhang Xiaoli , Cui Ningren , Wu Zhongying , Jiang Chun
Published: June 2021

Rett syndrome caused by mutations in MECP2 (Methyl‐CpG binding protein 2) gene shows autonomic abnormalities, in which brainstem norepinephrinergic (NE) systems are involved. To determine if the Mecp2 defect affects intrinsic membrane properties of locus coeruleus (LC) NE neurons, whole‐cell current clamp was performed in brain slices of 3‐4 weeks old Mecp2‐/y and wild‐type mice. Of 102 neurons studied, no statistical differences were found in passive membrane properties except the time constant that was significantly smaller in Mecp2‐/y neurons. Most LC neurons in both groups displayed spontaneous firing activity. The action potential duration was prolonged in Mecp2‐/y mice due to an extended rising phase. Voltage clamp in acutely dissociated LC neurons confirmed that the density of voltage‐activated Na+ currents was reduced in Mecp2‐/y mice. Mecp2 knockout produced a negative shift of half inactivation potential of delayed excitation, and had rather small effect on spike frequency adaption. The overall amplitude of afterhyperpolarization (AHP) was lower in Mecp2‐/y neurons with a clear manifestation of the fast AHP. No evident bursting activity and post‐inhibition rebound was observed in both groups. The electrophysiological abnormalities of LC NE neurons involving likely defects in Na+ and K+ channels seem to contribute to the autonomic dysfunction in Mecp2‐/y mice. (supported by NIH R01‐HL067890)
Effects of high salt diet on NF‐KB and CREB DNA binding activity in heart, kidney and hypothalamus of SHR
Authors: Shang Qianhui , Wang HongWei , Stewart Alexandre FR , Leenen Frans H. H.
Published: June 2021

In Dahl S or SHR on high salt diet, activation of NF‐κB contributes to cardiac hypertrophy and renal damage, which may be offset by activation of CREB. The brain RAAS mediates the salt‐induced hypertension, possibly by NF‐κB in the PVN. We assessed effects of high salt diet on DNA‐binding activity of NF‐κB and CREB in the heart, kidney and PVN of WKY rats and SHR on high (8%) or reg (0.6%) salt diet for 2 or 4 weeks (n=6). NF‐κB and CREB DNA binding activity were studied by electrophoretic mobility shift assay.

In WKY, high salt only increased CREB DNA binding activity in the LV and NFκB in the PVN. In SHR, high salt increased NF‐κB DNA‐binding activity in the heart and kidney, but decreased CREB binding activity in the heart, kidney, and the PVN.

Conclusion

High salt causes an inverse relationship between NF‐κB and CREB in the LV, kidney and PVN which may contribute to salt induced sympathetic hyperactivity, LV and renal hypertrophy in SHR.
Inhibition of Matrix Metalloproteinase‐9 Reverses Changes in Vascular Wall Structure and Function of Thoracic Aorta of Dahl Salt‐Sensitive Rats
Authors: Rodriguez‐Alvarez Walter E , Tyagi Neetu , Sen Utpal , Kumar Munish , Ngo Tuan , Adeagbo Ayotunde S , Joshua Irving G , Tyagi Suresh C
Published: June 2021

Left ventricular hypertrophy (LVH) and cardiac fibrosis in DSS hypertensive rats were associated with matrix proteins alteration. DSS hypertension was characterized by a cardiac dysfunction. Present study determines if salt sensitive hypertension induced changes in vascular structure and function are attenuated by inhibition of MMP‐9. To test the hypothesis, vascular function and levels of MMP‐9 and TIM‐4 were determined in thoracic aorta segments of 16 week male DSS (D), and Lewis (L) rats. Groups were fed high salt diet for 6‐8 weeks. Rats were regrouped: DSS plus MMP‐9 inhibitor (SB‐3CT) treatment (D+I) and DSS without inhibitor (D). Aortic function was assessed on myobath. MMP‐9 and TIMP‐4 levels were measured by Western blot, reverse zymography and confocal staining. Acetylcholine‐induced relaxation response of the aortic rings was decreased in D as compared to L group, but normalized in D+I group. Sodium nitroprusside‐induced relaxation response was not altered. MMP‐9 levels were significantly high in aortic segments of D as compared to L group, and were significantly reduced in D+I group. There was significant decrease in TIMP‐4 levels in the D as compared to L group. The result suggests that increase in MMP‐9 instigates vascular hypertrophy and dysfunction in Dahl salt‐sensitive rats.
Hemolysis‐induced pulmonary hypertension and treatment with infused or inhaled nitrite, or inhaled NO in the newborn lamb
Authors: Vrancken K , Schroeder H , Terry M H , Merrill‐Henry J , Power G G , Blood A B
Published: June 2021

Background

NO is a tonic vasodilator in the lung. Plasma‐free hemoglobin scavenges NO, resulting in pulmonary vasoconstriction. Oxidized hemoglobin, produced by the reaction of NO with oxyhemoglobin, does not scavenge NO. We hypothesized that NO, administered as a gas (iNO) or as nitrite that is endogeneously converted to NO, would attenuate pulmonary hypertension caused by hemolysis.

Methods

Intravenous hemolyzed blood was administered to newborn lambs for 180 min while measuring pulmonary arterial pressure and flow. Changes in pulmonary and systemic pressures and resistances were measured. During the final 90 min of hemoglobin infusion, intravenous nitrite, nebulized nitrite, or iNO (20ppm) were administered.

Results

Hemolyzed blood resulted in a significant increase in pulmonary (p<0.01) but not systemic vascular resistance that was sustained throughout the first 90 min of hemolysate infusion. iNO resulted in a rapid return of pulmonary vascular resistance to baseline levels, associated with nearly complete oxidation of plasma hemoglobin (p<0.01). Inhaled nitrite resulted in a significant increase in exhaled NO gas, and a significant decrease in pulmonary vascular resistance. Intravenous nitrite did not alter pulmonary vascular resistance significantly.

Conclusions

Hemolyzed blood causes selective pulmonary hypertension. Inhaled nitrite produces vasoactive amounts of NO. The benefits of iNO therapy for treatment of hemolysis‐induced pulmonary hypertension may result from both direct vasodilation as well as oxidation of the plasma free hemoglobin to methemoglobin, which does not scavenge NO.
Beta‐arrestin and vasomotor control in the spontaneously hypertensive rat
Authors: Mattar Louis , Jackson Dwayne N , Ellis Chris G , Noble Earl , Shoemaker J Kevin
Published: June 2021

We tested the hypothesis that spontaneously hypertensive rats (SHR) exhibit reduced desensitization of the neuropeptide Y (NPY) Y1 receptor (Y1R) leading to altered hindlimb vasomotor control. ?‐arrestin 2 ( ?‐arr) is a key regulatory protein involved in the desensitization of the Y1R (as well as many other G‐protein coupled receptors (GPCRs)). Reduced ?‐arr would lead to less desensitization, and prolonged signal transduction. In vivo measurements of arterial pressure and femoral blood flow (Transonic) were measured in 7‐week old anaesthesized male SHR animals (n=5) and their genetic control (WKY; n=6) rats. Red vastus lateralis from the animals were analyzed for ?‐arr content by western blotting techniques. SHR content of ?‐arr was 1412 ± 827 units while ?‐arr content in the WKY animals was 3546 ± 898 units (p = 0.003; mean ± SD). These data are consistent with the 55% (p = 0.07) and 60% (p = 0.06) reductions in both hindlimb blood flow (ml/min) and conductance (ml/min/mmHg) to exogenous NPY (r = 0.77, p = 0.02 and r = 0.66, p = 0.05 respectively). β‐arr was also correlated with reductions in hindlimb flow to exogenous phenylephrine (r =0.76, p = 0.02). These data implicate a role for β‐arr in Y1R and alpha adrenergic receptor sensitization in hypertension. This work was supported by CIHR.
Extracellular acidosis activates ASIC‐like channels in freshly isolated cerebral artery smooth muscle cells
Authors: Chung Wen‐Shuo , Farley Jerry M. , Drummond Heather A.
Published: June 2021

Our laboratory has shown that certain Acid Sensing Ion Channels (ASIC) mediate pressure‐induced constriction and migration of vascular smooth muscle cells (VSMC). However, electrophysiological evidence of ASIC channels in VSMC is lacking. The purpose of this study was to test the hypothesis that isolated cerebral artery VSMC express ASIC‐like channels. Conventional whole‐cell patch‐clamp technique was used. The criteria of Na+ selectivity and gating by extracellular acidosis, features of most ASIC channels, were used to confirm the presence of ASIC‐like channels. We found extracellular acid induced inward currents in 46% of cells tested (n=126, pH 6.5 ‐5.0). At pH = 6.0, the mean amplitude of the current normalized to whole cell capacitance was 7.6±0.9 pA/pF (n=22). While initial and sustained responses to acidosis varied among cells, the ASIC blocker amiloride (100 mM) consistently inhibited the initial response (n = 18, pH 6.0‐6.5). Extracellular acidosis (pH = 6.0) caused depolarization of 7.2±2.4 mV from the resting membrane potential of 25.4±4.1 mV (n = 8) and shifted the whole‐cell reversal potential toward the Nernst potential of Na+ (n = 6). Substitution of extracellular Na+ by N‐methyl‐d‐glucamine abolished the inward current (n = 8), indicating Na+ is the major charge carrier. These findings suggest that functional ASIC‐like currents are present in cerebral VSMCs. Supported by the‐NHLBI.
Modulation of the vascular KATP channel by the reactive carbonyl methylglyoxal (MGO)
Authors: Yang Yang , Cui Ningren , Jiang Chun
Published: June 2021

The vascular KATP channel made of Kir6.1 and SUR2B plays an important role in systemic response and susceptibility to diabetes and sepsis in which the channel may be modulated by reactive oxygen and carbonyl species. MGO is a carbonyl glycolytic intermediate that is highly reactive. Here we show evidence for the channel modulation by MGO. Acute exposure to MGO activated the Kir6.1/SUR2B channel expressed in HEK293 cells with EC50 ~1.5 mM. A pretreatment with a KATP channel inhibitor completely blocked the MGO effect. In excised patches, MGO augmented Popen without changing single‐channel conductance. In contrast, a prolonged treatment with MGO produced marked suppression of the KATP currents with the time constant 3 hrs. This inhibition was associated with a reduction in Kir6.1/SUR2B mRNAs revealed by RT‐PCR and qPCR in isolated aortic rings and cultured A10 aortic smooth muscle cells. The effect of MGO did not seem to result from an inhibition of Kir6.1 mRNA synthesis, as the MGO effect remained after transcriptional inhibition with actinomycin‐D. MGO did not affect the mRNA level in cell‐free condition. In conclusion, our results suggest that MGO is a strong KATP channel regulator, activating the channel with acute exposure and inhibiting the channel with a prolonged treatment (supported by NIH‐HL067890).
Dynamic PKCδ‐Mediated Trafficking of TRPM4 Regulates Smooth Muscle Excitability
Authors: Crnich Rachael , Amberg Gregory C. , Gonzales Albert L. , Tamkun Michael M. , Earley Scott
Published: June 2021

The melastatin Transient Receptor Potential (TRP) channel TRPM4 is vital for pressure‐induced smooth muscle cell (SMC) depolarization and constriction of cerebral arteries. Protein kinase C (PKC) activity contributes to SMC excitability by a number of mechanisms, including TRPM4 activation. Although strong evidence shows a correlation between PKC, TRPM4 activity, and subsequent depolarization, the mechanisms underlying regulation of TRPM4 are unclear. Several TRP channels are regulated by dynamic, directed movement of channel protein into/out of the plasma membrane. Thus, we hypothesized that rapid insertion of TRPM4 into the plasma membrane in response to PKC activity regulates channel dynamics and controls SMC excitability. Consistent with this hypothesis, live‐cell fluorescence recovery after photobleaching (FRAP) analysis of GFP‐tagged TRPM4 indicate that the channel is mobile. Cell surface biotinylation and total internal reflection (TIRF) microscopy reveal that TRPM4 is rapidly inserted into the plasma membrane in response to PKCδ activity. Pressure‐myograph studies of intact cerebral arteries demonstrate that TRPM4 and PKCδ are involved in PKC‐induced constriction. We conclude that stimulation of PKCδ activity leads to membrane depolarization and vasoconstriction by increasing the number of TRPM4 channels in the sarcolemma. AHA0535226N
The effects of corticosterone on stress‐induced weight loss and corticotrophin releasing factor (CRF) mRNA expression in the paraventricular nucleus of the hypothalamus (PVN)
Authors: Scherer Isabell Juliane , Holmes Philip V , Harris Ruth BS
Published: June 2021

Rats subjected to 3 hours of restraint on 3 consecutive days (repeated restraint, RR) lose weight and do not return to the weight of non‐stressed controls (CTRL). This study tested the effect of corticosterone (CORT) on sustained weight loss by subjecting intact, adrenalectomized (ADX) and ADX with CORT replacement (ADX CORT) rats to RR. Both CTRL ADX CORT and RR ADX CORT groups were injected with 2.0 mg/kg CORT before each stress. All other rats were injected with 2.0 mL/kg saline. CORT measurements on day 2 of RR confirmed the effect of ADX and replication of stress‐induced cort in the cort injected rats. RR ADX CORT rats had the same initial and sustained weight loss as RR Intact rats. CTRL ADX CORT and RR ADX rats lost half as much weight as RR Intact rats on the days of stress and maintained the weight loss for a shorter period of time than RR Intact or RR ADX CORT rats. Thus both stress‐induced CORT and a second, undefined aspect of RR are required for the pattern of weight loss found in Intact RR rats. In a second set of rats, PVN CRF mRNA expression was increased in all ADX rats and in RR Intact rats compared with CTRL Intact rats at the end of 3 hours of restraint on the second day of restraint. CORT injections did not inhibit expression in ADX rats. This suggests that CORT contributes to initial and sustained weight loss in RR rats through a mechanism independent of PVN CRF mRNA. This study was funded by NIH grant MH06828101.
Anoretic effects of neuropeptide VF are mediated via central mu and kappa subtypes of opioid receptors
Authors: Sliwa Lindsay Nicole , Cline Mark
Published: June 2021

Recently, we demonstrated that neuropeptide VF (NPVF) causes hypothalamus associated satiety using chicks as models. The present study was designed to better understand some of the central mechanisms that mediate these effects. In experiment 1, co‐injection of NPVF and beta‐funaltrexamine (FNA, a mu opioid receptor antagonist) did not cause a greater suppression of food intake than when NPVF and FNA were injected alone. In experiment 2, co‐injection of NPVF and ICI‐174,864 (ICI, a delta opioid receptor antagonist) caused a more potent reduction in food intake than when NPVF and ICI were injected alone. In experiment 3, co‐injection of NPVF and nor‐binaltorphimine (BNI, a kappa opioid receptor antagonist) did not cause a greater suppression of food intake than when NPVF and BNI were injected alone. The orexigenic effects of neuropeptide Y and beta‐endorphin (both ligands of opioid receptors) were suppressed by NPVF co‐injection. These results suggest that NPVF‐induced satiety has a relationship with mu and kappa but not delta subtypes of opioid receptors, and likely causes suppression of the chick's innate orexigenic system.
NAD(P)H:quinone oxidoreductase 1 (NQO1) activity threshold in intact pulmonary arterial endothelial cells (PAEC)
Authors: Bongard Robert , Krenz Gary , Lindemer Brian , Merker Marilyn
Published: June 2021

One pulmonary endothelial metabolic function is reduction of blood borne redox compounds via NQO1, wherein phase II enzyme induction is a potential means to optimize this function. However, a threshold above which increases in NQO1 activity do not support increases in NQO1 substrate toxicity is seen in other cell types. The goal of this study was to use a kinetic approach to evaluate whether PAEC also exhibit an NQO1 activity threshold. PAEC NQO1 activity in cell cytosol fraction was induced ~6 fold (46.2±3.4 vs 268.1±28.5 nmol/min/mg protein) by exposure to sulforaphane (5 μM;24 hr). However, when intact cell NQO1 activity was measured by addition of duroquinone (DQ; 0‐50 μM) to the extracellular medium, the maximal reduction rate was only ~3 fold higher in sulforaphane than control cells (10.0±0.6 vs 32.2±1.3 nmol/min/mg protein). The possibility that NADPH availability was limiting NQO1 activity in sulforaphane exposed cells was suggested by the observation that glucose‐6‐phosphate dehydrogenase was induced only 1.2 fold (19.6 ± 1.2 vs 23.9 ± 1.6 nmol/min/mg protein). The study demonstrates a threshold effect for intact PAEC NQO1 activity and implies that the pulmonary endothelial NQO1 capacity to activate blood borne anticancer drugs or detoxify xenobiotics is limited by factors other than enzyme activity measured in cytosol fractions. Support: HL‐65537 and Dept. of Veterans Affairs.
Manipulation of lipid bioaccessibility influences postprandial vitamin E concentrations in healthy human subjects: implications for inflammation, oxidative stress and vascular responses.
Authors: Berry Sarah E , Lapsley Karen , Tydeman Elizabeth A , Lewis Hannah B , Phalora Ravneet , Rosborough Jennifer , Picout David R , Ellis Peter R
Published: June 2021

Postprandial lipemia is associated with an acute elevation in oxidative stress, inflammation and impaired vascular function, which is attenuated by the presence of antioxidant vitamins. We previously reported that slow release (low bioaccessibility) of lipid from whole almonds (WA) during digestion, compared with free oil from almond (AO) or sunflower (SO) seeds, reduced postprandial lipemia (over 8h), but no effects on oxidative stress and vascular function were found1. Subjects (n=20, aged 18‐40y) were fed test meals containing 50g fat provided as WA, AO or SO in a randomised cross‐over design, with 1‐wk washout. Additional analysis to investigate the effects of differences in lipid bioaccessibility on changes in plasma vitamin E and interleukin 6 (IL‐6; inflammatory marker) concentrations are presented. Plasma vitamin E concentrations were lower (mean incremental area under curve with 95% CI) following the WA meal (69 (25, 113)) vs. the AO (150 (211, 88)) and CO (137 (190, 84)) meals; P=0.003. IL‐6 concentrations increased significantly from fasting following the test meals (P=0.023), but did not differ between meals. The concomitant increase in plasma vitamin E with postprandial lipemia from the free oils may counterbalance the deleterious effects of exaggerated lipemia on oxidative stress, inflammation and vascular function.

1Berry et al. Am J Clin Nutr 2008;88:922‐9
α‐Smooth muscle actin differential regulation in hESC derived mesenchymal cells
Authors: Boyd Nolan Lee , Stice Steven L.
Published: June 2021

We have derived mesenchymal cell lines from H9 hESC in a monolayer culture. The derived cells are positive for multiple mesenchymal markers (CD73, CD90, CD105 and CD166) while being negative for markers of hematopoietic and endothelial precursors (CD31, CD34, CD45, CD133 and CD146). Seeding the cells into a collagen I lattice (24‐well plate, 1ml of rat tail collagen I [1mg/ml] and 1.25x105cells/ml) for 7d demonstrated they are highly contractile reducing the construct to ~25% of its original size. We have also demonstrated the derived cells can differentiate toward osteogenic and chondrogenic cells, but not adipogenic suggesting they are a mesenchymal progenitor cell. It is suggested that MSC are capable of also differentiating into smooth muscle cells (SMC), therefore we examined their ability to express αSMA when exposed to various growth factors (TGF‐β1 and PDGF‐B) and serum. Cells grown in serum‐free media showed varying amounts of αSMA by Western blot, but when exposed to TGFβ1 (1 ng/ml) there was a consistent up‐regulation of αSMA protein. In contrast, both PDGF‐B (10 ng/ml) and serum (10%) did not produce any expression of αSMA. This suggests the derived mesenchymal cells may have differential regulation of αSMA expression and could be a model for SMC differentiation from precursor cells. This research was supported by grants and fellowships from NIH and GTEC.
Increasing concentrations of central leptin treatment enhances bone marrow cell differentiation in ob/ob mice
Authors: Bartell Shoshana M , Gaddam Dhanunjaya , Ambati Suresh , Rayalam Srujana , Hartzell Diane L , Hamrick Mark , She Jin‐Xiong , Della‐Fera Mary Anne , Baile Clifton
Published: June 2021

There are contradictory findings when assessing central leptin treatment on bone mass in rodents. The objective of this study was to determine the effect of central leptin administration on bone marrow gene expression in leptin deficient ob/ob mice (15‐wk old, init BW=60.6g). Leptin (1.5 μg/d or 0.38 μg/d) or control was continuously delivered intracerebroventricularly (ICV) via osmotic pumps for 12 days (n=6). Leptin decreased BW (41.2 vs 53.1 vs 63.7, p<0.01; mean of 1.5 leptin vs 0.38 leptin vs control) & body fat (22.3 vs 30.6 vs 35.0g, p<0.01) & increased distal tibial (.10 vs .07 vs .07g/cm2, p<0.01) & lumbar (.10 vs .07 vs .07g/cm2, p<0.01) BMD. mRNA expression levels of genes involved in adipogenesis & cell survival (eg., PPARγ, Bcl‐2) were decreased, demonstrating an enhancement in the sensitivity to leptin‐stimulated adipocyte apoptosis in the bone marrow. Expression levels of genes associated with osteogenesis (Runx2) were increased, while those associated with osteoclastogenesis (RANK) were decreased, which is consistent with the observed leptin‐stimulated bone growth & inclination of cells to differentiate into osteoblasts. These results indicate that increasing concentrations of leptin promote expression of pro‐osteogenic factors in the bone marrow & enhanced bone formation in ob/ob mice. Supported by the GA Research Alliance (GRA) Eminent Scholar endowment (CAB) & GRA Challenge Grant (CAB & JXS).
Intergeniculate leaflet c‐Fos induction is attenuated in mice lacking normal vestibular input
Authors: Ringgold Kristyn Michelle , Fuller Charles A.
Published: June 2021

The circadian pacemaker can be phase shifted by appropriately timed locomotor activity, however the mechanism(s) involved remain poorly understood. Investigations suggest the intergeniculate leaflet of the thalamus is an integral part of the pathway utilized by locomotor stimuli in modulation of the circadian timing system (CTS). We have demonstrated a role for the neurovestibular system in phase shifts of the pacemaker in mice exposed to the gravitoinertial input associated with locomotion. This study tested the hypothesis that timed access to a running wheel would induce IGL c‐Fos in only those animals with functional input from the vestibular macular organs. This study used head tilt (het) mice lacking vestibular otoconia, chemically labyrinthectomized (Labx) mice and wildtype (WT) littermates. After 10 days in constant darkness, animals were assigned to either a running‐wheel pulse (WP) or cage control (CC) group. At circadian time (CT) 4, WP mice were moved to a running wheel and CC mice to a wheel‐free cage. At CT 8, animals were sacrificed and brain tissue processed for histology. We found significantly more IGL c‐fos in WP WT mice than in CC WT or het or Labx mice in either condition. The increase in IGL c‐fos induction among only WT mice supports the role of macular gravity receptive cells in modulation of the CTS while also providing a functional mechanism by which locomotor activity may affect the pacemaker.
Rat Pancreatic Response to Simulated Microgravity by Hindlimb Suspension
Authors: Chowdhury Parimal , Soulsby Michael E. , Ali Nawab , Mehta Rahul
Published: June 2021

Hindlimb suspension (HLS) of rats is a validated model of simulated microgravity representing weightlessness. This study examines the stress response induced by HLS on the structural integrity of endocrine and exocrine pancreas in rats. The influence of a dietary soyprotein meal was also examined. Four groups of animals were used: two unsuspended control having each group fed with either Purina chow or a meal containing 100% custom made soyprotein meal and two suspended groups with the same feeding schedule but suspended for one week by their tail. After one week, the animals were sacrificed to collect pancreatic tissues for measurements of malondialdyde (MDA) and histopathological changes. The results show that HLS significantly increased MDA levels in addition to marked structural changes in the exocrine and endocrine pancreas as determined by vacuolation, vacuolar fusion, pyknosis and increased size and number of islets. These effects by HLS have been significantly reversed with dietary soyprotein meal. The results suggest that pancreatic response to one week suspension by HLS is induced by oxidative stress and soyprotein appeared to have the protective effect on this organ (Supported by a grant from Arkansas Space Grant Consortium).
Contrasting effects of intermittent and sustained hypoxia on HIF‐2α expression
Authors: Nanduri Jayasri , Wang Ning , Yuan Guoxiang , Khan Shakil
Published: June 2021

Intermittent hypoxia (IH) occurs in many pathological conditions including recurrent apneas. Hypoxia‐Inducible Factor‐1 and 2 (HIF‐1 and 2) mediate transcriptional responses to low O2. A previous study showed that HIF‐1 mediates some of the IH‐evoked physiological responses. Because HIF‐2α, the O2 regulated subunit, is an orthologue of HIF‐1α, we examined the effects of IH on HIF‐2α expression, in PC12 cells. In contrast to the up‐regulation of HIF‐1α, IH down regulated HIF‐2α in a stimulus‐dependent and reversible manner. Sustained hypoxia, up‐regulated both HIF‐1 and 2α. Neither prolyl hydroxylases / proteosome nor changes in HIF‐2α mRNA could account for the IH‐induced HIF‐2α degradation. Calcium chelator (BAPTA) and calpain protease inhibitors (ALLM, ALLN) prevented IH‐evoked degradation of HIF‐2α. IH activated calpains and down‐regulated the endogenous calpain inhibitor calpastatin. IH‐evoked HIF‐2α degradation led to decreased SOD2 enzyme activity due to transcriptional down‐regulation, resulting in oxidative stress. Over expression of transcriptionally active HIF‐2α prevented IH‐evoked oxidative stress and restored SOD2 activity. These observations demonstrate that, unlike continuous hypoxia, IH leads to down‐regulation of HIF‐2α via a novel signaling pathway involving calpains resulting in oxidative stress. Supported by NIH‐HL‐90554 and HL‐76537.