bluuebunny/crossref_metadata_2025 huggingface dataset:
Acute alcohol ingestion blunts arterial pressure but not sympathetic neural responses to orthostatic stress in humans
Authors: Stream Sarah F , Schwartz Christopher E , Klein Jenna C , Carter Jason R
Published: June 2021

Acute alcohol consumption has been reported to decrease mean arterial pressure (MAP) during orthostatic challenge, a response that may contribute to alcohol‐mediated syncope. Muscle sympathetic nerve activity (MSNA) increases during orthostatic stress to help maintain MAP, yet the influence of alcohol on MSNA during orthostatic stress has not been determined. We hypothesize that alcohol ingestion will blunt MSNA responses to lower body negative pressure (LBNP). MAP, MSNA, and heart rate (HR) were recorded during progressive LBNP (−5, −10, −15, −20, −30, and −40 mmHg; 3 min/stage) in 22 subjects (age 25 ± 1 yrs). After a baseline progressive LBNP (Trial 1), subjects were randomly assigned to either the placebo (n=11) or alcohol (n=11) group and repeated the progressive LBNP (Trial 2). Resting MAP increased (P<0.05) similarly with placebo (83 ± 2 to 88 ± 2 mmHg) and alcohol (82 ± 2 to 87 ± 3 mmHg). Resting HR (60 ± 2 to 64 ± 2 beats/min) and MSNA (15 ± 4 to 24 ± 4 bursts/min) increased (P<0.05) with alcohol, but not placebo. A significant time × condition × group interaction (P<0.05) was observed for MAP, demonstrating a more pronounced decrease in MAP during progressive LBNP with alcohol. MSNA and HR increased during all LBNP protocols, but there were no differences between trials or groups. In conclusion, alcohol decreases MAP responses to orthostatic stress, but these decreases do not appear to be mediated by MSNA. Supported by NIH.
Combined Heat and Mental Stress Alters Neurovascular Control in Humans
Authors: Klein Jenna C , Crandall Craig G , Brothers R Matthew , Carter Jason R
Published: June 2021

This study examined the effect of combined heat and mental stress on neurovascular control. We hypothesized that muscle sympathetic nerve activity (MSNA) and forearm vascular conductance (FVC) responses to mental stress (MS) would be augmented during heat stress (HS). Twelve subjects performed 5 min of MS during normothermia (NT: Tcore; 37 ± 0 °C) and HS (38 ± 0 °C). Heart rate (HR), mean arterial pressure (MAP), MSNA, forearm skin vascular conductance (SkVCf), and FVC were recorded. HS increased HR, MSNA, SkVCf, and FVC at rest, but did not change MAP. MS increased MAP during both conditions, but increases during HS (Δ10 ± 2 mmHg) were blunted compared to NT (Δ15 ± 1 mmHg; interaction, P<0.01). MS increased total MSNA during both conditions, but increases during HS were greater (Δ4668 ± 1224 units) relative to NT (Δ1267 ± 558 units; interaction, P<0.01). MS decreased SkVCf during HS (Δ−19 ± 5 units; P<0.001), but not during NT (Δ−1 ± 0 units). MS elicited similar increases in MSNA burst frequency, HR, and FVC during both conditions. In conclusion, these findings indicate that MS elicits a blunted increase of MAP during HS, despite greater increases in total MSNA and cutaneous vasoconstriction. These findings could be of particular interest to those exposed to mentally demanding tasks in hyperthermic environmental conditions (i.e. soldiers, firefighters, and athletes). Supported by APS and NIH.
Toward comprehensive quantitative proteomics using subcellular fractionation and LC‐MS/MS
Authors: Luginbuhl Kelli , Pisitkun Trairak , Rinschen Markus , Hoffert Jason , Knepper Mark A
Published: June 2021

Our previous LC‐MS/MS profiling in cultured renal mpkCCD cells has detected 2999 unique proteins. This compares with 7922 transcripts found by Affymetrix microarrays in these cells (http://dir.nhlbi.nih.gov/papers/lkem/mpkccdtr/Default.aspx).

Comprehensive quantification requires, as a pre‐requisite, that we can detect virtually all of the proteins that correspond to the expressed transcripts. With a goal of comprehensive proteomic profiling, we asked if subcellular fractionation prior to LC‐MS/MS could increase the number of proteins detected. Gene ontology analysis of the transcriptomic data revealed that approximately 29% of the detected transcripts code for nuclear proteins, a much larger percentage than previously found by LC‐MS/MS. To increase protein identifications in mpkCCD cells, we used a standard procedure for subcellular fractionation into nuclear extract, nuclear pellet and cytoplasmic fractions followed by 1D SDS‐PAGE and in‐gel trysinization. Using high sensitivity LC‐MS/MS (LTQ‐Orbitrap) and multiple spectral search algorithms, while limiting false positive identifications to <1% (target/decoy), we identified a total of 6188 unique proteins including 2521 that were in the two nuclear fractions but not in the cytoplasmic fraction. With further improvements in mass spectrometry, comprehensive identification (and quantification) of proteins should soon be feasible.
Genetic Variation of the Alpha Subunit of the Epithelial Na+ Channel Influences Exhaled Na+ in Response to Albuterol in Humans
Authors: Foxx‐Lupo William T. , Wheatley Courtney M. , Cassuto Nicholas A. , Wong Eric C. , Patanwala Asad E. , Delamere Nicholas A. , Snyder Eric M.
Published: June 2021

Epithelial Na+ Channels (ENaC) are located on alveolar cells and are important in lung fluid clearance through the removal of Na+ from the alveolar airspace. Previous work has demonstrated genetic variation of the alpha subunit of ENaC at amino acid 663 is important in channel function with the wild‐type (A663A) cells demonstrating attenuated function when compared to A663T cells. We sought to determine the influence of genetic variation at position 663 of ENaC on exhaled Na+ in healthy humans. To determine this we compared exhaled Na+ in 14 wild type (WT, AA at amino acid 663) and 13 non‐wild type (NWT, AT or TT at amino acid 663) subjects (age=28±8 vs. 29±10yrs., ht.=175±12 vs. 171±10cm., wt=69±12 vs. 73±14kg., BMI=23±3 vs. 25±4kg/m2, VO2peak=107±25% vs. 100±31% predicted, mean±SD, for WT and NWT, respectively) at baseline and at 30, 60 and 90 minutes following the administration of a nebulized β‐agonist (albuterol sulfate, 2.5mg diluted in 3ml normal saline). We found that the WT group had a higher baseline level of exhaled Na+ (p<0.05) and a greater response to a β‐agonist (baseline= 3.1±1.8 vs. 2.3±1.5mmol/l; 30minpost= 2.1±0.7 vs. 2.2±0.8mmol/l; 60minpost= 2.0±0.5 vs. 2.3±1.0mmol/l; 90minpost= 1.8±0.8 vs. 2.6±1.5mmol/l, mean±SD, for WT and NWT, respectively, p<0.05). These results suggest genetic variation of ENaC influences β‐adrenergic receptor stimulated Na+ clearance in the lungs.
Proteasome inhibition suppresses injury‐induced myelin ovoid formation and Schwann cell dedifferentiation in the peripheral nerves
Authors: Park Hwan Tae , Lee Hyun Kyoung , Shin Yoon Kyung , Jung Junyang , Suh Duk Joon , Lee Dae‐Hee , Baek Sun Yong
Published: June 2021

The ubiquitin‐proteasome system (UPS), lysosomes and autophagy are essential protein degradation systems for the regulation of a variety of cellular physiological events including the cellular response to injury. It was recently reported that axonal degeneration by traumatic insults or retrieval of nerve growth factors is mediated by the UPS and autophagy. In the peripheral nerves, axonal degeneration after injury is accompanied by myelin degradation which is tightly related to the reactive changes of Schwann cells called dedifferentiation. In this study, we examined the role of the UPS, lysosomal proteases and autophagy in the early phase of Wallerian degeneration of injured peripheral nerves. We found that nerve injury induced an increase in ubiquitin conjugation and lysosomal‐associated membrane protein‐1 expression within one day without any biochemical evidence for autophagy activation. Using an ex vivo explant culture of the sciatic nerve, we observed that inhibiting proteasomes or lysosomal serine proteases prevented myelin degradation, whereas this was not observed when inhibiting autophagy. Interestingly, proteasome inhibition, but not leupeptin, prevented Schwann cells from inducing dedifferentiation markers such as p75 nerve growth factor receptor and glial fibrillary acidic protein in vitro and in vivo. In addition, proteasome inhibitors induced cell cycle arrest and cellular process formation in cultured Schwann cells. Taken together, these findings indicate that the UPS plays a role in phenotype changes of Schwann cells in response to nerve injury.
Effect of AT1 receptor blockade on intermittent hypoxia‐induced endothelial dysfunction
Authors: Marcus Noah J , Philippi Nathan R , Bird Cynthia E , Schultz Harold D , Li Yulong , Morgan Barbara J
Published: June 2021

Exposure of rats to chronic intermittent hypoxia (CIH) impairs vasodilator responsiveness and raises arterial pressure. CIH‐induced increases in arterial pressure are prevented by blockade of the angiotensin type 1 receptor (AT1R); however, it is unknown if renin‐angiotensin system (RAS) activity plays a role in CIH‐induced vascular dysfunction. Sprague Dawley rats were exposed to CIH, while control rats (NORM) were housed under normoxic conditions. A subset of animals from each group was treated with an AT1R blocker, losartan (Los), during the exposure period. Arterial pressure was monitored by telemetry. After 4 weeks, gracilis arteries (GA) were isolated and cannulated with glass micropipettes, and pressurized to 80 mmHg. Diameters were measured using video microscopy before and after exposure to acetylcholine (10−6 M) or sodium nitroprusside (10−4 M) in the superfusate. CIH caused elevations in arterial pressure when intermittent hypoxia cycles were administered and also when the animals were unperturbed. Los attenuated both increases. Acetylcholine‐induced vasodilation was blunted in CIH vs. NORM, and this was prevented by Los treatment. Responses to nitroprusside were not different between CIH‐exposed and control animals. These results suggest that vascular dysfunction associated with CIH is dependent in part on RAS signaling through the AT1R. Funded by #HL074072.
PDGF‐BB signaling in adventitial fibroblasts during hypoxia‐induced pulmonary hypertension
Authors: Panzhinskiy Evgeniy , Shields Kristin , Zawada Michael , Stenmark Kurt , Das Mita
Published: June 2021

PDGF‐BB contributes to vascular remodeling during the development of hypoxia‐induced pulmonary hypertension (HPH). Adventitial fibroblasts (AdFibs) are the first cells activated in HPH. We tested the hypothesis that PDGF‐BB‐induced activation of AdFibs is a key regulator of HPH development. Newborn calves were exposed to high altitude (4750 m) (HA) for 2 weeks to induce HPH. Phosphorylated PDGFβ receptor (PDGFβ‐R) levels were markedly elevated in the thickened adventitia of the vessels in HA lungs compared to that of control. PDGFβ‐R expression was similarly activated in AdFib isolated from the main pulmonary artery of HA animals as evaluated by immunoblot analysis. PDGF‐BB stimulated greater proliferation rate and reactive oxygen species (ROS) production in HA cells compared to the AdFibs of control animals. ROS scavengers, N‐Acetyl Cysteine (NAC), and superoxide dismutase mimetic, TEMPOL, selectively inhibited PDGF‐BB‐induced proliferation and ROS production in HA cells. PDGF‐BB induced activation of ERK1/2 and JNK1/2, but not p38 MAP kinase in both cell types. However, both NAC and TEMPOL attenuated PDGF‐BB‐stimulated heightened phosphorylation levels of ERK1/2 and JNK1/2 in HA cells only. Our results suggest that dramatic adventitial remodeling during the progression of HPH depends on PDGF‐BB‐induced AdFib proliferation mediated through ROS production and ERK/JNK MAP kinase activation.

Funded by HL64917; P20RR016474
Protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac) in human pulmonary artery smooth muscle cells: expression, function and downstream signaling
Authors: Kwon Ohmin , Murray Fiona , Insel Paul A.
Published: June 2021

The second messenger cAMP inhibits human pulmonary artery smooth muscle (PASMC) proliferation, although the mechanisms involved have not been fully elucidated. The actions of cAMP are mediated primarily by protein kinase A (PKA) and Exchange protein activated by cAMP (Epac), thus we sought to investigate their expression, function and downstream signaling in PASMC. Real‐time PCR revealed that PKARIα, PKARIIβ, Epac‐1 and Epac‐2 are all expressed in PASMC. cAMP analogs that selectively activate PKARIα [8‐Cl‐cAMP (8Cl)], PKARIIβ [N6‐ Pheny‐cAMP, (N6)] and Epac [8‐pCPT‐2‐O‐Me‐cAMP, (8Me)] all dose‐dependently (100 nM‐100 μM, 24 hr) inhibited PASMC proliferation. Both 8Cl and N6, unlike 8Me, increased CREB and VASP activation. In contrast 8Me, but not 8Cl or N6, increased Rap‐1 and decreased ERK and AKT activation (50 μM, 10 min). In PASMC the PKA inhibitor [(PKI), 5μM, 24hr] blunted the anti‐proliferative effect of 8Cl and N6, but not that of 8Me. Inhibition of the Epac effector Rap‐1 [by GGTI‐298, 10μM, 24hr] did not inhibit the anti‐proliferative effect of 8Me in PASMC, suggesting a Rap‐1 independent mechanism. These results indicate that PASMC express both PKA and Epac and that activation of both decreases PASMC proliferation. Targeting both PKA and Epac‐dependent pathways may thus be beneficial in diseases such as pulmonary hypertension that are characterized by increased PASMC. Supported by NIH grants.
Reciprocal regulation of Na+/H+ exchanger isoform 1 (NHE1) and Na+/H+ exchange regulatory factor 1 (NHERF1) in Hypoxic Pulmonary Arterial Smooth Muscle Cells (PASMCs)
Authors: Maylor Julie Anne , Lu Wenju , Pisarcik Sarah , Walker Jasmine , Undem Clark , Myers Allen C. , Shimoda Larissa A.
Published: June 2021

Increased PASMC contraction and growth during chronic hypoxia (CH) may be due to cytoskeletal rearrangement involving transmembrane and cytosolic proteins. We found that the ion transporter, NHE1, is upregulated in chronically hypoxic rats (10% O2; 3 wk) and is required for development of hypoxic pulmonary hypertension. In addition to regulating pH, NHE1 was recently found to bind phosphorylated ezrin (p‐ezrin), an actin filament binding protein. NHERF1 also binds p‐ezrin, at the same site as NHE1. Since the transmembrane spanning NHE1 can act as an anchor, but cytosolic NHERF1 cannot, we hypothesized that changes in NHE1/ezrin interactions during hypoxia enables actin filaments to be tethered to the cell membrane, promoting PASMC contraction and growth. Confocal images of immunofluorescent stained chronically hypoxic mouse lung sections confirmed co‐localization of p‐ezrin and smooth muscle cell‐specific α‐actin (SM‐actin). Immunoblots showed increased NHE1, increased p‐ezrin and decreased NHERF1 levels in response to hypoxia. Co‐immunoprecipitation studies showed increased NHE1 binding to p‐ezrin and SM‐actin with hypoxia (4% O2; 24 hr), and decreased NHERF1 binding. These results suggest reciprocal regulation of NHERF1 and NHE1 expression in PASMC during hypoxia, with both proteins competing to bind p‐ezrin. Increased NHE1/ezrin interactions may contribute to hypoxic pulmonary hypertension.
The role and regulation of Inhibitor of Differentiation (Id) family members in hypoxic pulmonary hypertension
Authors: Lowery Jonathan Wayne , Anderson Lynda , Jones Mark , de Caestecker Mark Peter
Published: June 2021

Aberrations in BMP signaling have been implicated in the etiology of Pulmonary Arterial Hypertension. However, the specific role of BMP signaling in the normal pulmonary vasculature is not known. Here, we evaluate the necessity of the downstream BMP target Id1 in the vascular remodeling of murine hypoxic pulmonary hypertension (PH). Id1, which is strongly induced by 1 and 3 weeks hypoxia, is a dominant negative member of the basic helix‐loop‐helix family of transcription factors and promotes cell de‐differentiation, proliferation, and migration. However, we show that wild type, Id1+/−, and Id1−/− mutant mice do not differ in the development of hypoxic PH, as determined by increased right ventricular (RV) systolic pressure and RV hypertrophy. Furthermore, loss of Id1 does not lead to defects in hypoxia‐induced proliferation or muscularization of peripheral pulmonary vessels. Upon assessing compensation for loss of Id1 by other Id family members, we found that both Id2 and Id3 are induced by hypoxia and display a partially overlapping expression pattern with Id1. Moreover, expression of Id2 and Id3 are selectively elevated in peripheral vessel vascular smooth muscle cells of Id1−/− mutant mice. Collectively, our data suggest that, in response to chronic hypoxia, Id1 may promote vascular remodeling, but functional compensation for the loss of Id1 might occur through the upregulation of other Id family members.
Effect of chronic alcohol feeding on vitamin B1 (thiamine) transport in rat kidneys
Authors: Subramanian Veedamali S , Subramanya Sandeep B , Said Hamid M
Published: June 2021

Renal uptake of thiamine plays an important role in regulating the vitamin body homeostasis and is mediated by thiamine transporters1 and 2 (THTR1 and THTR2). Little is known about the effect of chronic alcohol consumption on renal thiamine transport. We addressed this issue in this investigation using purified renal brush border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) isolated from rats fed alcohol liquid diet (36% calories) for 2, 4 and 6 weeks and compared the findings to those of pair‐fed controls (alcohol free liquid diet). Results showed that chronic ethanol feeding significantly inhibits carrier mediated thiamine uptake in renal BBMV and the effect was evident as early as 2 weeks after the start of alcohol feeding. Uptake of thiamine by renal BLMV was similarly inhibited. This decrease in thiamine transport was associated with parallel decrease in mRNA level of THTR1 and 2 (detected by real‐time PCR) as well as in level of transcription of their respective genes [established from level of expression of heterogeneous nuclear RNA (hnRNA)]. These studies show for the first time that chronic alcohol consumption leads to inhibition in renal thiamine uptake and the effect appears to be mediated (at least in part) via transcriptional mechanism(s). (Supported by grants from DVA and NIH).
Ethnic differences in renal water handling: a “thirsty” phenotype?
Authors: Bankir Lise , Pratt John H , Weder Alan B
Published: June 2021

Because African Americans (AA) tend to concentrate urine more than do Caucasians (CA) [Bankir, CJASN 2007] and to reabsorb Na more avidly, which could contribute to high blood pressure, 2 studies addressed ethnic differences in thick ascending limb (TAL) function. One, based on furosemide effects, concluded that TAL activity is greater in AA [Chan, Hypertension 2008]. Another showed that AA excrete a water load more slowly than CA, suggesting lesser TAL activity in AA [Sachdeva, Hypertension 2009]. This apparent contradiction actually reflects different functions of the medullary (M) and cortical (C) TAL subsegments (urine concentration and dilution, respectively) as illustrated by functional adaptations in mammals. Marine mammals, which never need to excrete solute‐free water, exhibit a proportionately much thinner cortex than other mammals [Beuchat, AJP 1996] suggesting low CTAL transport. In rats, chronic infusion of the V2 receptor agonist dDAVP resulted in large hypertrophy of MTAL and atrophy of CTAL [Bankir, Pflugers Arch. 1988]. Humans evolved in a hot, arid climate in which water conservation enhanced survival. Increased MTAL and decreased CTAL transport in AA have complementary effects, both favoring water conservation, which could be less critical for CA residing in cooler, wetter climates. Racial differences in TAL function could reflect adaptation by natural selection to local environments.
Na/Bicarbonate Cotransporter NBCn1 is Upregulated under Acidic Conditions and Enhances Ammonium Transport in the Kidney Medullary Thick Ascending Limb Cell Line
Authors: LEE SOOJUNG , LEE HYE JEONG , PARK HAE JEONG , YANG HAN SOO , CUCORANU DELIA , RAJBHANDARI IRA , COOPER DEBORAH S. , CHOI INYEONG
Published: June 2021

The sodium/bicarbonate cotransporter NBCn1 regulates intracellular pH (pHi) and transepithelial HCO–3 movement. We examined NBCn1 expression and activity under acidic conditions and its contribution to ammonium movement in the mouse medullary thick ascending limb (MTAL) cell line ST‐1. Incubating cells at pH 6.8 for 24 h lowered the mean steady‐state pHi from 7.10 to 6.56. Immunoblot showed NBCn1 upregulation after acidic incubation. The Cl/HCO3 exchanger AE2 was present but its expression remained unaffected by acidic incubation. Acidic incubation decreased 14C‐methylammonium/ammonia (MA) accumulation by 22%. The 14C‐MA accumulation after acidic incubation was unaffected by 0.5 mM DIDS, whereas the accumulation in controls was reduced by DIDS. In CO2/HCO–3‐free solution, cells at normal pH accumulated 14C‐MA over 7 min and reached a plateau. In CO2/HCO–3 solution, however, cells markedly accumulated 14C‐MA over 30 min. This CO2/HCO–3‐dependent accumulation was reduced by DIDS. Cl– removal reduced the accumulation and this reduction was more substantial with DIDS. Expressed in Xenopus oocytes, NBCn1 increased CO2/HCO–3‐dependent 14C‐MA accumulation, which was abolished by replacing Na+. Two‐electrode voltage clamp exhibited negligible current upon NH4Cl application. We conclude that NBCn1 in the MTAL is upregulated under acidic conditions and the upregulation enhances ammonium uptake.
Improved Renal Tubular Function Detected by Magnetic Resonance Imaging after Revascularization of Experimental Renal Artery Stenosis
Authors: Warner Lizette , Woollard John R , Glockner James D , Krier James D , Zhu Xiang Y , Textor Steve , Lerman Lilach O
Published: June 2021

Renal artery stenosis (RAS) may lead to hypertension and renal injury. Reversibility of renal tubular dysfunction in RAS upon revascularization is unclear. We tested the hypothesis that percutaneous transluminal renal angioplasty (PTRA) would restore tubular function assessed as medullary oxygenation response to furosemide, a diuretic that inhibits medullary tubular transport. This was studied in 16 pigs after 6 wks of RAS, using blood oxygen level dependent magnetic resonance imaging (BOLD MRI) before and after furosemide. Renal blood flow (RBF) and glomerular filtration rate (GFR) were determined with multidetector computed tomography. Pigs were then randomized for PTRA or sham (n=8 each) and studied again 4 wks later; 8 normal pigs served as controls.

Results

Stenotic kidney RBF, GFR, and BOLD MRI responses were reduced in RAS shams (Figure). PTRA decreased the degree of stenosis (11±7 vs. 71±5% in untreated RAS) and blood pressure (108±3 vs. 130±9 mmHg, p<0.05). Renal and tubular function were improved in RAS+PTRA, although not to normal (Figure). Non‐stenotic kidneys showed no change in tubular function. Therefore, tubular solute transport‐related oxygen consumption can be partially restored and detected by BOLD MRI 4 wks after PTRA in post stenotic swine kidneys. This noninvasive method may be useful to assess the functional status of kidneys in RAS patients. Supported by NIH, HL085307.
Brain‐derived neutrotrophic factor (BDNF) binding is required for its receptor TrkB activation in the supraoptic nuclei (SON) following dehydration in the rat
Authors: Carreno Flavia Regina , Cunningham J Thomas
Published: June 2021

Osmotic stimulation increases BDNF release and mRNA expression in the SON. Our previous studies indicate that the BDNF receptor TrkB is activated (indicated by phosphorylation status) in the SON following 48h water deprivation (WD). Phosphorylation of TrkB receptor can occur by either BDNF binding or transactivation. We tested the role of BDNF in phosphorylation of TrkB following WD by directly infusing the SON with a TrkB‐Fc fusion protein which prevents BDNF from binding to TrkB. Rats were instrumented with right SON cannula coupled to mini‐osmotic pumps (Alzet model 2004) prefilled with vehicle (0.9% saline) or TrkB‐Fc fusion protein (50ug/ul). Brain punches that contained the right (cannulated) and left (uncannulated) SON were collected separately from euhydrated controls and WD rats. Punches were subjected to Western Blot analysis for total TrkB full length and its phosphorylated TrkB levels. Densitometric measurements of the immunoreactive bands were normalized using GAPDH. In the left SON contralateral to the cannula, TrkB phosphorylation was significantly enhanced followed 48hWD (P<0.01; n=4). Analyis of the right SON receiving TrkB‐Fc showed that the TrkB receptor phosphorylation following WD was significantly attenuated (P<0.05; n=4). Our results show that following WD, TrkB phosphorylation in the SON is likely BDNF‐mediated. NIH R01 HL62579
Fyn kinase‐TrkB receptor‐NMDAR2B glutamate receptor subunit (NR2B) physical interaction is increased in the supraoptic nuclei (SON) following dehydration in the rat
Authors: McGovern Adam Andrew , Carreno Flavia Regina , Cunningham J Thomas
Published: June 2021

In the SON, dehydration activates brain‐derived neurotrophic factor receptor (TrkB) and downstream src‐kinase pathways such as Fyn. Fyn can mediate tyrosine phosphorylation of NR2B subunits. We tested the hypothesis that TrkB receptor‐dependent Fyn kinase activation phosphorylates NR2B subunits following dehydration in the SON. Brain punches that contained the SON were collected from euhydrated controls, 48 h water deprived rats (WD), and rats water deprived for 46 h followed by access to water for 4 h (4hR). Punches were homogenized in NP‐40 buffer and the total lysate was subjected to Western Blot analysisof NR2B and tyrosine‐phosphorylated forms of NR2B (pNR2BY). Densitometric measurements of the immunoreactive bands were normalized using GAPDH. Co‐Immunoprecipitations (Co‐IP) were conducted to evaluate physical interaction between Fyn kinase and NR2 and TrkB. Although no changes were observed for total NR2B levels, pNR2BY levels were significantly increased (P<0.01) in the SON of WD animals compared to controls. In 4hR rats, pNR2BY decreased to control levels. Physical interaction of Fyn kinase and NR2B and TrkB respectively were increased followed dehydration (P<0.01). Our data suggest that increased NR2B tyrosine phosphorylation in the SON following dehydration may be due to TrkB receptor dependent Fyn activation that regulates glutamate receptor function. NIH R01 HL62579
In vivo Inhibition of Renal Heme Oxygenase‐1 with an Imidazole‐Dioxolane Inhibitor, QC‐13.
Authors: Csongradi Eva , Vera Trinity , Stec David E.
Published: June 2021

Recent studies have indentified imidazole‐dioxolane based compounds as novel heme oxyenase inhibitors. While these compounds have been demonstrated to be specific HO inhibitors in vitro, they have yet to be used to inhibit renal HO activity in vivo. The goal of this study was to determine the effectiveness of the imidazole‐dioxolane HO‐1 inhibitor, QC‐13, in the inhibition of renal HO activity in vivo. HO‐1 was induced in mice by treatment with cobalt protoporphyrin (COPP, 50 mg/kg, sc.). After 5 days, QC‐13 was delivered either by continuous intrarenal medullay interstitial (IRMI) infusion (25 μM) for 72 hours or by two i.p injections (52 mg/kg) over a 48 hour period. IRMI infusion of QC‐13 resulted in a 46 ± 14 % decrease in medullary HO activity as compared to control CoPP treated medulla (n=5). IRMI infusion of QC‐13 had no significant effect on cortical HO activity which averaged 107 ± 17 % of COPP treated cortex (n=5). In contrast, the systemic administration of QC‐13 resulted in significant decrease in renal cortical HO activity but had no significant effect on medullary HO activity with HO activity averaging 48 ± 8 %, and 79 ± 12 % of control CoPP treated mice in cortex and medulla, respectively. In contrast to classical porphyrin based HO inhibitors, IRMI infusion of QC‐13 did not induce HO‐1 protein levels as determined by Western blot analysis of medullary protein samples. Our results demonstrated that imidazole‐dioxolane inhibitors are selective HO‐1 inhibitors in vivo which can inhibit HO‐1 activity independent of HO‐1 induction. These inhibitors may be useful tools to elucidate the role of renal HO‐1 in numerous physiologic and pathophysiologic conditions. This study was supported by NIH grants HL‐088421 and HL‐088421‐S1.
The voltage‐operated Potassium channel Kv1.1 as new player in renal Magnesium handling
Authors: Glaudemans Bob , van der Wijst Jenny , Scola Rosana , Lorenzoni Paulo , Heister Angeline , van der Kemp Annemiete , Knoers Nine , Hoenderop Joost , Bindels Rene
Published: June 2021

Genetic studies in families with hereditary forms of renal Mg2+ wasting revealed new genes involved in Mg2+ homeostasis such as Mg2+ channel TRPM6. Likewise, this study aims to find the affected gene in a Brazilian family with isolated dominant hypomagnesemia. SNP‐based linkage analysis was used to identify a 11.6 cM critical region on chromosome 12. We sequenced the KCNA1 gene and identified a heterozygous A763G mutation that co‐segregated with the disorder. KCNA1 encodes voltage‐gated K+ channel Kv.1.1 in which the mutation causes substitution of a conserved asparagine at position 255 for an aspartic acid (N255D). Kv.1.1 is expressed in kidney, where it co‐localizes with TRPM6 at the distal convoluted tubule (DCT) luminal membrane as shown by immunohistochemistry. Functional analysis showed that the N255D mutation results in a non‐functional channel with a dominant negative effect on the wild‐type channel. Next, we revealed that the N255D channel fails to hyperpolarize the cell membrane. The N255 residue was further characterized by substitution with six amino acids (E, Q, A, V, T, H). The Kv1.1 N255E and N255Q mutants were not functional, while the other mutants were similar to wild‐type. Yet, their channel kinetics significantly changed, suggesting a role for Kv1.1 N255 in channel gating. Kv1.1 likely establishes the membrane potential across the DCT luminal membrane to drive TRPM6‐mediated Mg2+ reabsorption.
Changes on respiratory chemosensitivity after vagotomy in rats
Authors: Moreira Thiago S , Takakura Ana C
Published: June 2021

Breathing relies on a rhythmically active network of brainstem neurons that is driven by central chemoreceptors and other sources of tonic excitatory drive. The retrotrapezoid nucleus (RTN) contains central respiratory chemoreceptors that are activated by carotid body and inhibited by lung inflation. In the present study, we examined whether the vagus nerve interfere with the activity of RTN chemosensitive neurons in rats. Mean arterial pressure (MAP), pO2, pCO2, pH, phrenic nerve activity (PNA) and RTN CO2‐activated neurons were recorded in urethane‐anesthetized intact or vagotomized male Wistar rats (250–280 g, n = 7/group). In vagotomized rats, without artificial ventilation, pCO2 increased (48 ± 3, vs. intact rats: 44 ± 6%) and pH is reduced (7.27 ± 0.05, vs. intact rats: 7.45 ± 0.05), without change MAP and pO2. The CO2 threshold of RTN neurons (3.2 ± 0.2%, vs. intact rats: 4.5 ± 0.4%) and PNA (4.1 ± 0.2%, vs. intact rats: 5.6 ± 0.3%) was lower in vagotomized rats. After the connection of the vagotomized rats in artificial ventilation, pCO2 (43 ± 4%), pH (7.48 ±0.16) and CO2 threshold of RTN (4.8 ± 0.9%) and PNA (5.7 ± 0.3%) returned to levels observed in intact anesthetized preparations. The present results support the idea of the importance of the vagus nerve on the control of the respiratory chemosensitivity in rats.

Financial support: FAPESP (06/60174‐9 to TSM).
Further evidence of redox modulation of neurons in a CO2‐chemosensitive area: normobaric hyperoxia (95%O2) stimulates CO2‐chemosensitive and –insensitive neurons in the solitary complex (SC)
Authors: Matott Michael Patrick , Landon Carol S , Putnam Robert W , Dean Jay B
Published: June 2021

We previously reported that CO2‐excited neurons in SC, in medullary slices maintained in 95%O2, are stimulated by chemical oxidants and hyperbaric O2 (JAP 95:910‐921, 2003; AJP 286:C940–951, 2004). Here we test the hypothesis that SC neurons, maintained in 40%O2 rather than 95%O2 (to reduce oxidative stress), are viable and stimulated by normobaric hyperoxia (95%O2). Slices (P1–21) were harvested in chilled ACSF gassed with 95%O2 and immediately transferred to ACSF (22–24°C) equilibrated with 40%O2‐5%CO2 in N2. Whole cell recordings (35–37°C) were established in 40%O2 (n=44) and tested using hyperoxia (40→95%O2) and hypercapnia (5→10 or 15%CO2). Hyperoxia stimulated 8/44 neurons and hypercapnia stimulated 5/44 neurons; of these 4/8 were stimulated by both O2 and CO2. Hyperoxia usually increased firing rate and decreased input resistance whereas hypercapnia increased firing rate and input resistance. Most neurons tested were insensitive to hyperoxia and hypercapnia (31/44). Compared to previous slice studies that used 95%O2 control, there is a smaller proportion of CO2‐excited SC neurons in slices maintained in 40%O2. These data indicate that medullary slices are viable in 40%O2 and that hyperoxic stress stimulates SC neurons, including CO2‐excited neurons. These data also suggest that oxidative stimuli increases the incidence of CO2‐chemosensitivity in SC neurons (ONR N000140110179, NIH R01HL56683)
Oxygen transport from air to cell: The impact of age
Authors: Amann Markus , Wray Walter , Wary Claire , Monnet Aurelien , Carlier Pierre G. , Richardson Russell S.
Published: June 2021

To evaluate potential age‐related differences in the O2 transport from air to skeletal muscle at rest, we studied 14 young (25 ± 2) and 13 old (71 ± 3) healthy males. Femoral venous and arterial catheterization was combined with the measurement of femoral blood flow (QL) (ultrasound Doppler) to quantify single leg O2 delivery and O2 consumption (VO2). Proton nuclear magnetic resonance spectroscopy was used to determine basal myoglobin desaturation (deoxy‐Mb) and intracellular O2 pressure (PiO2) was calculated using an O2/Mb half‐saturation of 3.2 mmHg. Utilizing these data, a Bohr integration technique was used to calculate muscle O2 diffusion capacity (DmO2). Despite a similar leg muscle mass (~9.5 l) and basal leg VO2 (~24 ml/min), QL and O2 delivery were significantly lower in the old vs young subjects (270 vs 324 ml/min and 52 vs 63 ml/min, respectively). However, neither myoglobin concentration (~0.3 mM) nor the average basal deoxy‐Mb signal (~11%) were different between the two age groups. Consequently, the calculated PiO2 was nearly identical in the old and young subjects (~32 mmHg) and calculated resting DmO2 was 28% greater in the elderly. Our data confirm previous reports of attenuated QL and O2 delivery with age, but suggest that, as leg VO2 and PiO2 were similar between young and old, aging improves O2 diffusion from capillary to myocyte at rest.
Murine Carotid Body Responses to Hypoxia: In vivo Carotid Sinus Nerve Recordings in the DBA/2J and A/J Strains.
Authors: Pichard Luis , Sgambati Francis , Kostuk Eric , Bashai Pejmon , Fitzgerald Robert , Shirahata Machiko
Published: June 2021

The role of the carotid body (CB) as a primary chemosensor is well established in large mammals. Due to our ability to better modify the genome the mouse has been used for hypoxic chemosensing research. The only method by which one can study the global and unified CB response to hypoxia is via recording the carotid sinus nerve (CSN) activity. Our laboratory has been using the two inbred strains of mice, DBA/2J and A/J. In general, the DBA/2J is more sensitive, with an elevated cardiorespiratory response to hypoxia compared to the A/J strain. Thus, the purpose of this study is to establish a stable in vivo model for recording CSN activity in these two strains of mice. CSN activity was recorded from the central end just prior to its junction to the glossopharyngeal nerve using a glass suction pipette in six DBA/2J and A/J mice. Baroreceptor activity was mechanically disrupted. Following a baseline recording, the inspired gases were changed from hyperoxia to FIO2=0.21, 0.15 and 0.10. CSN activity was then analyzed using Fast Fourier Transform (FFT) based time‐frequency spectral analysis. The data showed a hyperbolic relationship between PO2 and CSN activity in the DBA/2J mice. The A/J mice showed a significant attenuation in their carotid body neural output when compared to the DBA/2J mouse at all comparable levels of normoxia and hypoxia. Thus, the model established methods to examine carotid body responses to hypoxia in mice and showed a clear difference in the carotid body generated neural output in the DBA/2J mouse when compared to the A/J. This model can now be used to study how the different genetic make‐up, by using other strains and knockouts, can alter the overall in‐vivo response to hypoxia. Support: F31HL096450, HL081345, AHA 09GRNT2080158, HL50712 and HL61596.
Leptin deficiency impairs hypercapnic ventilatory responses independently of changes in body weight
Authors: Bassi Mirian , do Carmo Jussara Marcia , Hamza Sherren , Hall John Edward
Published: June 2021

Leptin‐deficient (ob/ob) mice exhibit ventilatory depression during hypercapnia which can be normalized by intracerebroventricular injections of leptin, suggesting a central role of leptin on ventilatory control. However, it is still unclear whether leptin's effect on ventilatory function occurs independently of the weight loss that occurs during leptin treatment. In this study we investigated ventilatory responses to hypercapnia (7% CO2) in 15‐week‐old obese ob/ob mice fed ad‐lib (n=5, 62±1 g) and in non‐obese ob/ob mice (n=5, 26±1 g) that were fed fixed amounts of food since weaning to match the body weight of wild‐type (WT) control mice (n=3, 27±1 g). Pulmonary ventilation (VE), tidal volume (VT) and frequency (FR) were analyzed by the plethysmography method. Our results demonstrate that hypercarpnic ventilatory responses were significantly (P≤0.05) lower in obese ob/ob mice and lean pair‐weighted ob/ob mice compared to WT controls (138±18, 107±25 and 173±40 ml.min‐1, respectively). The reduction in VE appears to be caused by a significantly smaller VT in the ob/ob groups (0.42±0.04 and 0.37±0.03 ml, respectively, for obese and lean ob/ob mice) compared to WT mice (0.54±0.04 ml). Baseline VE and FR during hypercapnia did not differ among the 3 groups. These results suggest that impaired ventilatory responses to hypercapnia in leptin‐deficient mice are independent of changes in body weight.
Chronic Administration of Fluoxetine Markedly Attenuates the Hypercapnic Ventilatory Response in Urethane‐anesthetized Female Mice
Authors: Bale Tejus A. , Solomon Irene C.
Published: June 2021

Selective serotonin reuptake inhibitors (SSRIs) are commonly prescribed to treat anxiety disorders in humans, and these agents have been reported to reduce hyperventilation as well as hypersensitivity to increased levels of CO2. In rodent models, conflicting observations regarding the effects of SSRIs have been reported. For example, in unanesthetized male rats, administration of the SSRI fluoxetine hydrochloride (FHCL) is ineffective in altering normocpanic and hypercapnic ventilatory activity while in urethane‐anesthetized male mice, FHCL increases basal breathing frequency. In humans, FHCL has also been shown to differentially affect behavioral and neural function in males vs females. Thus, the current study was undertaken to evaluate the effects of FHCL on ventilatory activity in female mice. To accomplish this, diaphragm EMG activity was recorded in spontaneously breathing urethane‐anesthetized female C57BL/6 mice following 28‐d continuous infusion of FHCL (~2 mg/kg/day, n=5) or vehicle (50% DMSO:50% saline, n=5) under baseline conditions and in response to 7% CO2. We found that 7% CO2 elicited an increase in EMG burst frequency (P<0.05, as expected) in vehicle‐treated mice; however, this increase in burst frequency was markedly attenuated or abolished (P<0.05) in FHCL‐treated mice. These findings suggest that SSRIs may alter the sensitivity to hypercapnia in female mice. Supportedby NS045321
Mammary Epithelial Cells Modulate TRPM6 Expression in Response to Variations of Magnesium Availability
Authors: Wolf Federica I , Trapani Valentina , Mastrototaro Lucia , Cittadini Achille
Published: June 2021

Mammary epithelial cells (HC11) chronically adapted to grow in a low‐magnesium (0.05 mM vs. 0.5 mM) or in a high‐magnesium (40 mM) medium were used to investigate on the mechanisms of cell magnesium transport under conditions of non‐physiological magnesium availability. Magnesium influx was higher in low‐magnesium cells compared to control or high‐magnesium cells, this influx was abolished by Co(III)hexaammine, an inhibitor of magnesium channels. Among the Mg‐transporting channels recently characterized, e.g. MagT1, SLC41A1, transient receptor potential meastatin (TRPM7) was shown to mediate magnesium uptake ubiquitously at cell/tissue level, whereas TRPM6 seems to be responsible for transcellular magnesium absorption or re‐absorption in the intestine and kidney, respectively. We found that HC11 cells grown in low magnesium upregulated mRNA for TRPM6 and not for TRPM7 or MagT1. Consistent with these results, TRPM6 protein level as assessed by western blot, was inversely related to extracellular magnesium, namely low‐magnesium cells > control cells > high‐magnesium cells and was also rapidly up‐ or down‐modulated in HC11 cells acutely deprived of magnesium or in low‐magnesium cells re‐added with magnesium, respectively. These results suggest, for the first time, that TRPM6 contributes to regulating magnesium influx in mammary epithelial cells.

Supported by the Italian MIUR‐PRIN 2007ZT39FN.
The Eleven‐nineteen Lysine‐rich Leukemia Gene (ELL) Couples Transcription Elongation with Epigenetic Signaling
Authors: Fufa Temesgen D , Wakano Clay , Byun Jung S , Haggerty Cynthia M , Elenitoba‐Johnson Kojo , Thirman Michael J , Raffeld Mark , Hewitt Stephen , Gardner Kevin
Published: June 2021

The human Eleven‐nineteen Lysine‐rich Leukemia gene (ELL) was first identified as a chromosomal translocation partner for the histone‐methyltransferase mixed lineage leukemia protein (MLL) and was subsequently found to be a RNA polymerase II elongation factor. Though studies in several eukaryotic models including yeast and Drosophila are beginning to shed light on ELL function, just how ELL and its oncogenic fusion with MLL influences gene regulation in mammalian systems remains to be defined. We have profiled the interaction partners of ELL in mammalian cells by sedimentation analysis, immunoprecipitation, mass spectrometry and genome location analysis. These analyses revealed that in addition to the positive transcription elongation factor b (P‐TEFb), ELL associates dynamically with various readers, writers, and erasers of the histone code. These include, the histone acetyltransferase p300, members of the histone deacetylase complex and components of the SET1 methyltransferase complex. Surprisingly, ELL undergoes a dynamic subcellular redistribution in response to mitogen stimulation, forming compartment specific complexes in the nucleus and cytosol. These findings provide new insights into the mechanisms that couple the crosstalk between transcriptional elongation and epigenetic regulation in mammalian cells.

Funding: Intramural Research Program of the NCI/NIH, USA
Genetic Expression Analysis of Immunostimulatory Botanicals on Human Peripheral Blood Mononuclear Cells Associated with Inflammatory Processes
Authors: Lasku Arben , Denzler Karen , Jacobs Bertram , Rochon Yvan , Langland Jeffrey , Waters Robert , Brown Kurtis , Chrystal Joshua , Eang Sana , Leek Dan , Singleton Vera
Published: June 2021

Many hundreds of botanicals have a place in complementary and alternative medicine for therapeutic use in antimicrobial, immunostimulatory, and “wound healing” activity. While there exists many centuries of anecdotal evidence on the activity and efficacy of these botanicals, limited scientific evidence exists on how these botanicals may be modulating the immune and inflammatory responses. Using microarray analysis, this study provides novel insight into putative genes which may be involved in the physiological responses following administration of botanical extracts. Using extracts from immunostimulatory herbs (Astragalus membranaceus, Sambucus cerulea, Andrographis paniculata) and an immunosuppressive herb (Urtica dioica), the data presented supports previous cytokine studies on these herbs as well as identifying additional genes which may be involved in immune cell activation and migration and various inflammatory responses, including wound healing, angiogenesis, and blood pressure modulation. The data presented provides a more extensive picture on how these herbs may be instigating their biological effects on the immune and inflammatory responses.
Modulation of Neuropeptide‐Y receptor expression by IGF‐1 and atheroma‐associated cytokines in carotid plaques of symptomatic and asymptomatic patients
Authors: Pankajakshan Divya , Jia Guanghong , Pipinos Iraklis , Agrawal Devendra
Published: June 2021

Neuropeptide‐Y (NPY) is involved in the induction of immune‐mediated inflammation. But, its role in atherosclerotic plaques is unknown. Here, we examined NPY‐receptor(R) expression in the presence of IGF‐1 and TNF‐α in plaque smooth muscle cells (pSMCs) of symptomatic(S) and asymptomatic(AS) patients with carotid stenosis. IGF‐1 increased NPY‐Y1 and NPY‐Y5 expression in S (1.5 and 1.2‐fold) and AS pSMCs (2 and 1.5‐fold, respectively). TNF‐α significantly decreased NPY‐Y1R (10‐fold) in S with no change in AS pSMCs. NPY‐Y5R expression decreased significantly in both S and AS pSMCs by TNF‐α. The combined stimulation of the cells with IGF‐1 and TNF‐α increased NPY‐Y1 expression by 1.2‐fold in AS pSMCs but no effects in S pSMCs. In both S and AS pSMCs, IGF‐1 increased NPY‐Y5 expression, but the effect of IGF‐1 in the presence TNF‐α was abolished. IGF‐1 increased (80%) NPY‐Y2 receptor expression in AS pSMCs, but had no significant effect in S pSMCs. NPY‐Y2 expression was higher in AS (2.5‐fold) and S pSMCs (3.5‐fold) upon TNF‐α stimulation than respective controls. Thus, IGF‐1 and TNF‐α differentially modulate NPY‐Y1, ‐Y2 and ‐Y5 receptor expression in AS and S pSMCs. A balance between the inflammatory cytokines and IGF‐1 level at the plaque milieu is critical in determining SMC proliferation or apoptosis at the site of fibrous cap leading to the plaque stability/instability in symptomatic patients with carotid stenosis.

(Supported by NIH R01HL073349)
Rapid and consistent porcine model for chronic total occlusions in the peripheral vasculature
Authors: Tunev Stefan , Caceres Alejandra , Melder Robert , Wilcox Josiah
Published: June 2021

Development and testing of treatment options for chronic total occlusions (CTO) still remains one of the last frontiers of vascular interventions. The lack of a consistent CTO animal model is one of the hurdles precluding efficient treatment advances in this field.

We describe here a porcine model of a calcified CTO achieved by an intravascular delivery of de‐mineralized bone matrix (DBM) products in the femoral arteries. DBM is essentially de‐mineralized allograft bone particles with osteoinductive properties. In this study, the DBM material was delivered to each vessel via a guide catheter based approach. At 28 days, each treated vessel was evaluated angiographically, the animals were sacrificed and the treated vessels segments were assessed histopathologically. CTO was detected in 73% of the treated vessels. Morphologically, the CTO lesions were characterized by a mixed tissue growth composed of well vascularized collagenous matrix, mineralized and un‐mineralized bone fragments and variable mononuclear inflammation. This tissue reaction appeared well adhered to the vascular tunica media. Vascular channels lined by endothelium were present in vessels with incomplete chronic occlusion.

This animal model can provide a platform for 1) development and testing of new CTO crossing devices, 2) evaluation of stents in calcified vessels 3) identification and evaluation of pharmacological entities in context of calcification and 4) development and evaluation of decalcification treatments.