bluuebunny/crossref_metadata_2025 huggingface dataset:
Zinc mediated vasoconstriction in the isolated perfused rat lung
Authors: Bauer Eileen Maria , Pitt Bruce , St. Croix Claudette Marie
Published: June 2021

Hypoxic pulmonary vasoconstriction (HPV) is a complex multifactorial physiological response resulting in the shunting of blood flow to oxygen rich areas of the lung. Recent data from our laboratory suggest that zinc signaling plays a role in the effects of acute hypoxia mediated nitric oxide biosynthesis, and this pathway contributes to HPV. The objective of this study was to assess the ability of the pulmonary vasculature to constrict in response to exogenous zinc. We used the isolated perfused rat lung to determine the effect of exogenous zinc on pulmonary arterial pressure (PaP). Dose‐dependent constriction was observed in response to zinc supplied in the perfusate. The response to zinc was independent of the oxygen tension of the inspirate, as vasoconstriction was observed under both hypoxic and normoxic conditions. In addition, removing Ca2+ from the perfusate had no effect on the ability of Zn to induce changes in PaP; suggesting that Zn‐mediated constriction may be Ca2+ independent. These data provide evidence that Zn induces pulmonary vasoconstriction and suggest that this response may be via a novel mechanism independent of oxygen tension or Ca2+. Funded by RHL081421‐01.
AQP1 and AQP4 gene polymorphisms and endurance performance status in hispanics
Authors: Matos Carla , Nieves Juan J , Castro Paola , Morgado Andres , Salgado Jeanette , Martinez Juan L , Lopez‐Taylor Juan , Rivera Miguel A
Published: June 2021

Associations between single nucleotide polymorphisms (SNPs) in the AQP1 and AQP4 genes with endurance performance status were recently reported (MSSE 37(5):S164, 2005 and MSSE 39(5):S279, 2007, respectively). This study attempted to characterize the association between the AQP1 and AQP4 genes SNPs and endurance performance status in Hispanics. In a case‐control study, genomic DNA was successfully genotyped from 460 Hispanic subjects. Cases (n = 115) were long distance runners finishers in the highest 3 percentile, by age and gender, during their participation in an international marathon (42 km) race, while healthy controls (n=168) were those in the lowest 3 percentile for their respective age and gender. Controls (n = 64) were subjects with severely compromised cardiorespiratory functions. The AQP1 and AQP4 genes SNPs were detected by the tetra‐primer ARMS‐PCR procedure. Genotypes were determined by PCR product size. The observed allelic and genotypic frequencies were compared by endurance performance status using Chi‐square test statistic. The three expected AQP1 and AQP4 genotypes were observed in each of the three groups. No significant (P>0.05) associations were observed between genotypes or SNPs carrier status and endurance performance classification. CONCLUSIONS: We found no support for the hypothesis of association between the AQP1 and AQP4 genes SNPs and endurance performance status in Hispanics.

Supported by CRC‐RCMI UPR‐MSC Grant #1P20RR11126, RCMI Basic Sciences #G12RR03051.
Hypothalamic dopaminergic activity improves aerobic performance in rats.
Authors: Balthazar Cláudio Heitor , Coimbra Cândido Celso
Published: June 2021

Male Wistar rats (250–300g) were used in all experiments. Under anesthesia with an association of ketamine and xylazine, the animals received an implant of guide cannulae into the right lateral cerebral ventricle. After recovery from surgery, the rats were familiarized to run on a motor‐driven treadmill during 5 consecutive days. On the day of the experiments, 2μl of dopamine (Dp) 5x10−3M or SCH23390 5x10−3M (D1 antagonist) or Eticlopride 5x10−3M (D2 antagonist) or saline, at random, was intracerebroventricularly (ICV) injected and then the rats were submitted to a progressive exercise protocol on a treadmill until fatigue. The ICV injection of Dp 5x10−3M improved AP in rats, as seen by increased time to fatigue: Saline: 16.8±3.0 min vs Dp: 26.3±5.0 min (p<0.03; n=6) and increased VO2 max. ΔVO2 max: Saline: 18.2±2.2 ml.Kg.min−1 vs Dp: 26.5±5.5 ml.Kg.min−1 (p<0.03; n=6). In contrast, the ICV injection of D1 and D2 antagonists reduced the time to fatigue in rats about 75% and 50% respectively (p<0.03 vs Saline; n=6) and reduced VO2 max. ΔVO2 max: SCH23390: 4.4±2.2 ml.Kg.min−1; Eticlopride: 11.4±1.2 ml.Kg.min−1 (p<0.03 vs Saline: 18.2±2.2 ml.Kg.min−1; n=6).We conclude that central dopaminergic tonus has an ergogenic influence on running performance and VO2 max. Supported by: CNPq; FAPEMIG.
Glucose restriction inhibits glycolytic flux and limits proliferation in lung microvascular endothelial cells
Authors: Parra‐Bonilla Glenda , Alvarez Diego F. , Cioffi Donna L. , Stevens Troy
Published: June 2021

Pulmonary microvascular endothelial cells (PMVECs) exhibit increased glycolytic flux, which may contribute to their highly proliferative nature. We therefore sought to determine whether PMVECs rely on extracellular glucose to sustain glycolysis and growth under normoxic conditions. We performed growth curves and pH measurements using PMVECs and pulmonary artery endothelial cells (PAECs) in different glucose concentrations: High (22 mM), normal (11 mM) and low (5 mM). PMVEC proliferation was greatest in high glucose, and decreased progressively with lower glucose concentrations. In contrast, glucose concentration did not affect PAEC growth. Whereas PMVECs acidified the media in high glucose, they did not acidify the media at low glucose concentrations; PAECs did not acidify the media at any glucose concentration. QRT‐PCR analysis for glucose transporter‐1 (GLUT‐1), hexokinase‐2 (HK‐2), phosphofructokinase (PFK), and lactate dehydrogenase (LDH) revealed that media glucose did not influence upon the expression of these key glycolytic enzymes, suggesting genes responsible for increased glycolysis are constitutively upregulated in PMVECs. The constitutive upregulation of GLUT‐1, HK‐2, PFK, and LDH in PMVECs illustrate the intrinsic preference of these cells to use aerobic glycolysis during proliferation. Thus, glucose availability is a key determinant of growth and acidosis in PMVECs. HL66299.
Microtubule‐Dependent Apical Translocation of Mitochondria in Pulmonary Endothelial Cells Under Shear Stress
Authors: Reed Darla , Fadjoh Serge , Al‐Mehdi Abu‐Bakr , Gillespie Mark
Published: June 2021

Mitochondria are motile organelles that play key roles in numerous cellular pathways including calcium regulation, apoptosis, and ATP production. Mitochondrial apical translocation may be an essential response to the changing requirements under shear stress since shear stress directly affects the apex of polarized pulmonary endothelial cells. We hypothesize therefore that flow induces mitochondrial translocation to the apex of pulmonary endothelial cells. To study this idea, rat pulmonary microvascular endothelial cells (PMVEC) were subjected to shear stress at 14 dynes/cm2 over a period of three to six hours. MitoTracker Red 580 (MTR) was utilized to image and track mitochondrial movement. The area of the MTR signal at the apex, corresponding to localization of the mitochondria mass, was quantified in 3D‐reconstructed individual cells at different time points and plotted as a percent of total apical area. Our results show that the apical MTR signal increased significantly at 3 and 6 h of shear‐stress compared with 0 h of flow, indicating flow‐induced mitochondrial translocation to the apex. Treatment with 50 nM nocodozole abolished the mitochondrial translocation response with shear stress. We conclude that mitochondria translocate to the cellular apex under shears stress and that microtubule integrity is essential for this translocation.
Expression of gonadotropin receptors in the rat hypothalamo‐pituitary axis
Authors: Hull Kerry Lynn , Nutley Mark , Render Caroline , Harvey Steve
Published: June 2021

The gonadotropins luteinizing hormone (LH) and follicle stimulating hormone (FSH) control numerous aspects of neural function. They also inhibit gonadotropin secretion at hypothalamic and possibly pituitary sites. Surprisingly, little attention has been focused on the possible involvement of neural LH and FSH receptors in these effects. In partial support of this hypothesis, Lei et al. detected LH receptor transcripts in brain but not pituitary tissue. The possibility that FSH and LH receptor transcripts may be present in the brain and pituitary gland of the rat was therefore investigated, using the polymerase chain reaction. LH receptor cDNAs of expected size (600 bp) were generated from ovarian or testicular reverse‐transcribed RNA. Conversely, a smaller cDNA moiety of 520 bp was amplified from reverse‐transcribed RNA isolated from the hypothalamus, pituitary gland, and extrahypothalamic brain regions. Similarly, several FSH receptor cDNA moieties were amplified in reverse‐transcribed RNA from the testis and ovary, including the 990 bp moiety predicted by the full‐length FSH receptor sequence and a 600 bp cDNA. Only the 600 bp cDNA was detected in the brain and pituitary gland. These results therefore suggest that the FSH and LH receptor genes are expressed in neural and pituitary tissue. However, these receptor transcripts may not encode full‐length receptors. This work was supported by NSERC.
The cigarette smoke component cadmium inhibits the expression and function of the CFTR chloride channel in lung epithelial cells
Authors: Cormet‐Boyaka Estelle , Rennolds Jessica , Maloney Kevin
Published: June 2021

The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel that is mostly expressed in epithelial cells. Mutations in the cftr gene, that affect CFTR trafficking and/or function, lead to Cystic Fibrosis. Cystic Fibrosis is an inherited chronic disease affecting primarily the lungs. Therefore CFTR is necessary for the normal function of this organ. It was recently reported that cigarette smoke reduces the expression of CFTR (Cantin et al., 2006). However, the mechanisms underlying this inhibitory effect were not elucidated. We found that the suppressor effects of cigarette smoke could be specifically reproduced by cadmium, but not other cigarette smoke components such as nicotine (an alkaloid) or acrolein (an aldehyde). The cigarette smoke component cadmium (50 μM) decreases the expression of the CFTR protein by 61.5 ± 10.6 % in the bronchial epithelial cells Calu‐3. Furthermore, Calu‐3 cells treated for 5 days with 50 μM cadmium resulted in 38.9 ± 6.3% inhibition of CFTR currents indicating that CFTR function was also affected by cadmium in polarized cells. Cadmium is a toxic heavy metal that is present in cigarette smoke as well as contaminated air, food and water, and is believed to interfere with several physiological processes. Our findings identify cadmium as a key component present in cigarette smoke that is responsible for the inhibition of CFTR observed in lung epithelial cells.
Highly Ca2+ ‐selective TRPM channels regulate IP3‐dependent oscillatory Ca2+ signaling in the C. elegans intestine
Authors: Xing Juan , Yan Xiaohui , Estevez Ana , Strange Kevin
Published: June 2021

Posterior body wall muscle contraction (pBoc) in C. elegans occurs rhythmically and mediates defecation. pBoc is controlled by IP3‐dependent Ca2+ oscillations in the intestine. The intestinal epithelium can be studied by patch clamp electrophysiology, Ca2+ imaging, reverse/forward genetics analysis, molecular biology and thus provides a powerful model to develop an integrated understanding of a non‐excitable cell oscillatory Ca2+ signaling pathway. Intestinal cells express a TRPM‐like outwardly rectifying Ca2+ (ORCa) current. Two TRPM homologs, GON‐2 and GTL‐1, are expressed in the intestine. Using deletion and severe loss‐of‐function alleles of the gtl‐1 and gon‐2 genes, we demonstrate that GON‐2 and GTL‐1 are both required for maintaining rhythmic pBoc and intestinal Ca2+ oscillations. Loss of GTL‐1 and GON‐2 function inhibits IORCa ∼70% and ∼90%, respectively. IORCa is undetectable in gon‐2; gtl‐1 double mutant cells. Our results demonstrate that 1) both gon‐2 and gtl‐1 are required for ORCa channel function, and 2) GON‐2 and GTL‐1 function interdependently to mediate IORCa. Epistasis analysis suggests that GON‐2 and GTL‐1 function in the IP3 signaling pathway to regulate intestinal Ca2+ oscillations. We postulate that GON‐2 and GTL‐1 form heteromeric ORCa channels and function to regulate IP3 receptor activity and possibly to refill ER Ca2+ stores.

Source of research support: NIH R01 grant GM74229
Molecular mechanism in protein kinase C‐induced inhibition of recombinant vascular KATP channels
Authors: Jiao Jundong , Garg vivek , Yang Baofeng , Hu Keli
Published: June 2021

It is known that activation of protein kinase C (PKC) inhibits KATP channels in vascular smooth muscles. This signal transduction pathway has been implicated in the regulation of blood flow by endogenous vasoactive transmitters. However, the mechanism by which PKC down‐regulates KATP channels remains elusive. To test the hypothesis that caveolae‐dependent internalization of KATP channels is involved in PKC‐mediated inhibition of the channel, we employed HEK293 cells transfected with Kir6.1/SUR2B, a potential cloned equivalent of vascular smooth muscles. Phorbol 12‐myristate 13‐acetate (PMA, 1 μM) treatment at 37°C for 15 minutes caused substantial redistribution of KATP channels from cell membrane to intracellular vesicles, whereas distrupting caveolae by depletion of cholesterol with methyl‐betacyclodextrin significantly abolished the actions of PMA. Whole‐cell voltage clamp experiments revealed that a dominant negative mutant of dynamin (K44E) significantly reduced PMA‐induced inhibition of KATP currents in comparison with wild‐type dynamin (26.5±2.9% vs 49.9±4.8%, p<0.01, n=5). Consistently, immunofluorescence microscopy further demonstrated that dynamin K44E largely prevented the internalization of KATP channels induced by PMA. We concluded that activation of PKC by PMA inhibits recombinant vascular KATP channels via a dynamin‐dependent, caveolar‐related mechanism.
UTP regulations of short‐circuit current in alveolar epithelial cells involve distinct mechanisms
Authors: Yang Chuanxiu , Liu Lin
Published: June 2021

UTP was reported to inhibit alveolar fluid clearance. However, the contribution of alveolar type I and type II cells to this process are unknown. In this study, we investigated the effects of UTP on short‐circuit current (Isc) in alveolar type II‐like (AT2) and type I‐like cell (AT1) monolayer. AT2 and AT1 were obtained by culturing primary type II cells on air‐liquid and liquid‐liquid models, respectively. Real‐time PCR and immunostaining of cell markers confirmed the cell phenotypes. Luminal treatment with UTP (10 μM) increased Isc value of AT2 (ΔIsc = 1.66 ± 0.25 μA/cm2) but decreased Isc value of AT1 (ΔIsc = 2.81 ± 0.26 μA/cm2). Pretreatment with amiloride, the inhibitor of epithelium Na+ channel, abolished the UTP‐induced ΔIsc on AT1 and had no effect on that of AT2. Pretreatment with Cl− channel blockers, DIDS, CFTRinhi‐172, NPPB greatly inhibited the UTP‐induced ΔIsc in both cell types. The general blocker of K+ channel, BaCl2, eliminated the UTP‐induced ΔIsc only in AT2. The latter was also blocked by clotrimazole and clofilium, indicating the role of IKCa and KvLQT1. Our results suggested that UTP induced opposite changes in Isc in AT2 and AT1. Epithelium Na+ channel and Cl− channel contribute to the regulations of UTP in AT1 while K+ channel and Cl− channel contribute to that of AT2. (Supported by NIH HL083188)
Shear Stress Induces Synthetic‐to‐contractile Phenotypic Change of Smooth Muscle Cells via Paracrine Effect of Prostacyclin from Endothelial Cells and the PPAR‐α/δ Pathways
Authors: Tsai Min‐Chien , Hsu Tzu‐Fang , Chien Shu , Chiu Jeng‐Jiann
Published: June 2021

Endothelial cells (ECs) exposed to shear stress may exert paracrine effects to modulate the phenotype of smooth muscle cells (SMCs). We investigated the role of shear stress on ECs in SMC phenotypic modulation, using our EC/SMC co‐culture system in which these 2 types of cells were separated by a porous membrane. Application of shear stress (12 dynes/cm2) to EC/SMC induced SMC phenotypic change to a contractile state. This phenotypic change of SMCs was also observed by treating SMCs with media collected from ECs. For SMCs transfected with ligand‐binding domains of peroxisome proliferator activated receptor (PPAR)‐α, −δ, or −γ, treatment with media from ECs induced the ligand binding activities of these PPARs, suggesting that ECs exposed to shear stress release ligands of these PPARs. The shear‐induced SM22 expression in SMCs was inhibited by treating SMCs with antagonists of PPAR‐α (MK886) and −δ (sulindac sulfide), suggesting that PPAR‐α/δ pathways play a role in SMC phenotypic change. Shear stress to ECs induced their release of prostacyclin, which contributes to phenotypic modulation of SMCs. Our findings provide new insights into the role of shear stress on ECs in phenotypic modulation of SMCs.
Deficiency of MAP Kinase Phosphatase‐1 Induces Pulmonary Hypertension in Mice.
Authors: Das Mita , Shields Kristin M , Burns Nana , Harral Julie , Homma Noriyuki , Oka Masahiko , West James
Published: June 2021

MAP Kinase Phosphatase‐1 (MKP‐1), deactivator of MAP kinases, is a critical inflammatory regulator. We tested the hypothesis that MKP‐1 will control pulmonary hypertension development by dephosphorylating MAP kinases using MKP‐1 wild type (MKP‐1+/+), heterozygous (MKP‐1+/−), and null (MKP‐1−/−) mice. Animals were exposed to sea level (SL), Denver altitude (DA; 5280 feet) and severe altitude (HYP; 17,000 feet) for 6 weeks. Marked increase in right ventricular systolic pressure was found in MKP‐1+/− and MKP‐1−/− mice which were raised in SL and DA. Intensity of αSMA staining was increased in both MKP‐1+/− and MKP‐1−/−HYP lungs. Vessel to alveoli ratio was decreased in HYP lungs of MKP‐1+/− and MKP‐1−/−. Vessel (<50μm) wall thickness to diameter ratio was not affected by hypoxia in MKP‐1+/+ lungs (DA: 0.14 vs. HYP: 0.12), but increased in MKP‐1+/− (DA: 0.12 vs. HYP: 0.18) and MKP‐1−/− (DA: 0.12 vs. HYP: 0.2) lungs. Activation levels of ERK1/2 and JNK1/2 were similar in all groups. However, phosphop38 levels were greatest in the MKP‐1−/− lungs with positive reaction in vessels, respiratory epithelium, and interstitial cells. COX‐2, an inflammatory marker, also had strongest immunoreactivity in the MKP‐1+/− and MKP‐1−/− lungs. Therefore, lack of MKP‐1 induces sustained p38 activation and subsequent inflammation which might be a major contributor in the development of pulmonary hypertension. Funded by NHLBI HL 06491
Role of Copper and Homocysteine in Pressure Overload Heart Failure
Authors: Hughes William Michael , Rodriguez Walter E , Rosenberger Dorothea S , Moshal Karni S. , Sen Utpal , Chen Jing , Kang Y. James , Tyagi Suresh C.
Published: June 2021

Elevated levels of homocysteine (Hcy) known as hyperhomocysteinemia (HHcy) causes dilated cardiomyopathy (DCM). Hcy chelates copper and impairs copper‐dependent enzymes. To test the hypothesis that copper supplement regresses left ventricle hypertrophy (LVH), fibrosis and endothelial dysfunction in pressure overload DCM hearts (Figure 1), we created aortic constriction (AC) in mice and treated with or without 20 ppm copper supplements in diet for 16 weeks. The mice were grouped as sham; AC, sham+Cu; and AC+Cu. The cardiac function was assessed by ECG and Echocardiography. The levels of fibrosis was assessed by measuring matrix metalloproteinase (MMP), tissue inhibitor of metalloproteinase (TIMP) and lysl oxidase (LOX) by zymography and Western blot analyses. The results suggest that cardiac function was restored with copper supplement. Fractional shortening was 51.3±0.9% in AC; 62.7±3.7% in sham; 60.9±4.7% in sham+Cu; and 62.8±3.1% in AC+Cu. The QRS complexes from ECG were: 13.0±1.4 ms in sham; 14.0±2.8 ms in AC; and 13.0±2.1 ms in AC+Cu. Western blot analyses show that MMP‐13, TIMP‐3, and TIMP‐4 activity decreases in AC and then is improved in AC+Cu. TIMP‐1 and LOX activity increases in AC and returns to sham value in AC+Cu. The data suggests that copper supplement restores cardiac function in a pressure overload DCM heart.
Diameter and Compliance of the greater Saphenous Vein‐The Effect of Age and Glyceryl Trinitrates.
Authors: Zachrisson Helene , Lindenberger Marcus , Länne Toste
Published: June 2021

OBJECTIVES: The greater saphenous vein (GSV) is commonly used in autologous vein graft surgery in the lower limb. GSV diameter (D) has proven to influence graft patency and venous compliance (C) might be of importance. The aim of the study was to evaluate the effect of age on GSV D and C, and the effect of glyceryl trinitrate (GTN).

METHODS : The D and C of the GSV, with and without GTN, were examined with B‐mode ultrasound in 14 young (YM, 25±1 y) and 12 old (OM, 70±2 y) males. The GSV D was measured at rest, and after 6 min of venous stasis (60 mmHg). Pressure‐diameter curves during a linear venous pressure decrease were produced. C with was calculated using the quadratic regression equation (?diameter) = β0 + β1·(cuff pressure) + β2·(cuff pressure)2

RESULTS: The GSV D at rest was 3.7±0,2 mm and 2.8±0.2 mm in YM and OM respectively, P<0.01. Venous congestion increased GSV D significantly in YM and OM respectively, (P<0.001). Mean GSV C (β1) was 0,017±0.0035 and 0.0085±0,0039 in YM and OM respectively, (P=0.1). GTN increased resting D by 13% in OM , (P<0.01), but not in YM. After GTN, compliance was significant greater in YM, (p<0.05).

CONCLUSIONS: The diameter, and compliance of the GSV decreases with age, indicating defective venous wall function. The effect of venous compliance on graft patency deserves further study.
14,15‐Epoxyeicosatrienoic acid increases endothelial permeability via TRPV4 and KCa channel activation in mouse lung
Authors: Jian Ming‐Yuan , Liedtke Wolfgang , Townsley Mary I.
Published: June 2021

We have shown that the increased permeability response to 14,15‐epoxyeicosatrienoic acid (EET) in rat lung requires Ca2+ entry via the vanilloid transient receptor potential channel TRPV4. The current study tested the hypothesis that the permeability response to 14,15‐EET requires both TRPV4 and Ca2+‐activated K+ channels

(KCa). In lungs isolated from anesthetized TRPV4+/+ (WT) and TRPV4−/− (KO) mice, endothelial permeability was measured via the filtration coefficient (Kf) at baseline and after treatment with 14,15‐EET (3 μM). Baseline Kf was no different in WT and KO groups (0.011±0.001 vs 0.010±0.001 ml/min/cmH2O/g dry wt, mean±SEM). In WT lungs perfused with physiological buffer (n=6), 14,15‐EET increased Kf 2.9‐fold (p<0.001), a response blocked in lungs pretreated with the TRPV antagonist ruthenium red (3 μM, n=5), and absent in KO lungs (n=5). The permeability response to 14,15‐EET in WT lungs was attenuated in low Ca2+ (0.02 mM) but returned with Ca2+ addback (2.2 mM), in the absence (n=4) or presence of iberiotoxin (100 nM, n=4). Permeability did not increase with Ca2+ addback in groups treated with ruthenium red (n=4), the combination (n=4) of charybdotoxin (100 nM)/apamin (300 nM) or apamin alone (n=3). We conclude that intermediate (IK) and/or small (SK) conductance KCa channels contribute to the TRPV4‐mediated Ca2+ entry‐dependent endothelial permeability response to 14,15‐EET.

Supported by HL066299.
Increased AQP2 and AQP3 expression in renal cortex in COX‐2 deficient mice
Authors: Norregaard Rikke , Topcu Sukru Oguzkan , Madsen Kirsten , Nielsen Soren , Jensen Boye L. , Frokiaer Jorgen
Published: June 2021

Previously we demonstrated that COX‐2 activity contributes to downregulation of aquaporin‐2 (AQP2) in rats subjected to 24h of bilateral ureteral obstruction (BUO). In this study we hypothesized that COX‐2 exerts tonic suppression of renal AQPs. We investigated the expression of AQPs and cyclooxygenase‐1 (COX‐1) in cortex (COR) and inner medulla (IM) tissue from COX‐2−/− mice. Kidneys from 4 week old COX‐2−/− (C57Bl6, 1. generation) mice and wild type (WT) littermates were prepared for semiquantitative immunoblotting. A subset of mice was perfusion fixed for immunohistochemistry (IHC). Plasma creatinine, osmolality and urea levels were increased in the COX‐2−/− mice compared to the WT. COX‐2 protein was not detectable in COX‐2−/− kidneys. COX‐1 abundance was increased in both COR and IM in COX‐2 KO mice compared to the WT. This was confirmed by IHC showing labeling of collecting ducts. AQP1 expression was unchanged in COR. AQP2 protein level was increased in COR of COX‐2−/− compared to WT but was not different in IM. The expression of AQP3 was increased in COR of COX‐2−/− mice and IHC showed intense labeling of the basolateral membranes of the cortical collecting ducts in the KO mice.

The data indicate that COX‐2 is involved in regulation of the expression of AQP2 and AQP3. Further studies are required but our preliminary results indicate that COX‐2 may contribute to segmental regulation of water reabsorption.
Studies of Cold Injury in Organotypic Hippocampal Slice Cultures
Authors: Warren Daniel E. , McKleroy William , Martinez Zachariah , Bickler Philip
Published: June 2021

Hypothermia is used as a therapeutic agent to minimize the damaging effects of ischemia associated with trauma, myocardial infarction, and cardiopulmonary bypass. Its utility, however, is limited by its injurious effects, especially in brain. Hippocampal slice cultures prepared from nine or ten day old rats were acutely cooled to 4°C from 37°C for 6 hours followed by acute rewarming to 37°C. Slice death was quantified using propidium iodide staining after 30 minutes of rewarming when cooled under the following conditions: with the non‐specific Na+ and K+ channel blocker bupivacaine, 2% CO2, University of Wisconsin (UW) organ preservation solution, and the inhalation anesthetic isoflurane. Six hours of acute cooling followed by rapid rewarming resulted in 25–40% slice death. Compared to untreated slices, bupivacaine and hypocapnia did not affect survival. UW solution increased slice death while isoflurane prevented it entirely. These results demonstrate the utility of this in vitro system for identifying injurious and protective agents during deep cooling in hippocampi of cold‐sensitive mammals. The system also lends itself to exploring the physiological and molecular changes responsible for differential survival during hypothermia, such as the mechanisms underlying isoflurane protection observed in the present study.
Inhalation of Dry, Normobaric Hypoxic Air Impairs Thermal Conditioning of Nasal Air in Humans: Potential Role of Nitric Oxide
Authors: Peebles Evan Richard , Roberts MF , Giraud GD , Holden WE
Published: June 2021

Hypoxic, dry, cold air is a stress to nasal air temperature conditioning (NATC) at high altitude, where humans often complain of nasal irritation. The vasodilator nitric oxide (NO) may modulate NATC (J Appl Physiol 87:1260, 1999). Hypoxic, dry (but not cold) air also reduces NO release into nasal air (Resp Physiol 114:285, 1998). Thus, we asked if simulated high altitude (normobaric hypoxic, dry air) would impair NATC. We infused (12 L/min) normobaric, hypoxic (100% N2), dry (0% humidity) air (21° C) or dry air (21° C, 21% O2, balance N2) into the nasal passages of seven adult humans. During breath holding, air entered the nose, passed around the soft palate, exited from the mouth and was collected for analysis of nasal NO release by chemiluminescence. NO output (nL/min) was calculated as the product of NO concentration and flow rate (timed collection measured in a Tissot gasometer). Nasal air temperatures were measured at one cm intervals from the nasal verge to a depth of 7 cm by thermocouples. Exponential curve fitting generated a half‐point value—the depth where nasal air reached half the difference between ambient and maximum cavity temperature. Half point depth increased during normobaric hypoxic, dry air infusion compared with dry air indicating impediment of NATC (see table). Nitric oxide release decreased during normobaric hypoxic, dry air infusion compared with compressed air. We confirm that nasal NO release is impaired by hypoxic, dry air and further suggest that, hypoxic air impedes NATC. Our findings are consistent with a role of NO in NATC and may help explain nasal irritation in humans at high altitude.

NO output (nL/min)

Half‐point (cm)

(Supported by the Murdock Foundation).
Does chronic stimulation of both nicotinic and opioid receptors contribute to the loss of O2 and CO2 sensitivity in perinatal adrenomedullary chromaffin cells?
Authors: Buttigieg Josef , Brown Stephen T , Zhang Min , Nurse Colin A
Published: June 2021

Adaptation of the neonate to extrauterine life depends critically on catecholamine secretion (CAT) from adrenomedullary chromaffin cells (AMC). This CAT secretion is triggered by asphyxial stressors (e.g. hypoxia and hypercapnia) associated with the birthing process and these act directly on AMC, prior to functional splanchnic innervation. These sensing mechanisms are lost postnatally along a time course correlating roughly with innervation of AMC. Thus, the majority of juvenile AMC (>14 days) are unable to sense directly hypoxia and hypercapnia, although there is evidence that these properties may return following denervation. The splanchnic nerve releases various neurotransmitters including acetylcholine (ACh) which activate nicotinic ACh receptors (nAChR) and enkephalins which activate opioid receptors on AMC. Here, we test the hypothesis that activation of these receptors may initiate a signaling cascade leading to the loss of O2 and/or CO2 sensitivity. Recently, we showed that chronic stimulation of nAChR on AMC with nicotine, results in the loss of hypoxic, but not hypercapnic, sensitivity (Buttigieg et al. FASEB J 2007, in press). This loss of hypoxic sensitivity appears to be mediated by up‐regulation of KATP channels, which are activated by low PO2. Preliminary studies based on exposure of AMC to chronic opioid receptor stimulation suggest that this treatment leads to loss of both hypercapnic and hypoxic sensitivity. Thus, synaptic stimulation of opioid and nACh receptors on AMC may contribute to the normal developmental loss of O2 and CO2 sensitivity. Funded by: The Heart and Stroke Foundation (HSF) of Ontario. JB and SB were funded by a Focus on Stroke award from HSF of Canada.
Modulation of BK channel expression in mouse carotid body
Authors: Lieu TinaMarie , Fujii Koichi , Sham James S.K. , Shirahata Machiko
Published: June 2021

The carotid body (CB) is a major arterial chemoreceptor that senses low arterial PO2. Inhibition of large conductance calcium‐activated K+ (BK) channels have been hypothesized to be one of the initial key steps in hypoxic chemotransduction pathways of the glomus cell (GC). Our previous study indicates that the iberiotoxin‐sensitive BK current was more robust in GC of DBA/2J mice (high responder to hypoxia) than that of A/J mice (low responder to hypoxia). Further, glial‐derived neurotrophic factor (GDNF) appears to modulate the expression of BK channels. Since GDNF is induced by oxidative stress, we have hypothesized that oxidative stress and GDNF influences gene expression and function of BK channels, resulting in different phenotype of the GCs between the two strains of mice. We have developed a culturing system for undissociated whole CB. The carotid bifurcations with the CBs were harvested and cultured in defined media with different PO2 levels (21–95% O2) at various durations (4–24 hours). Real‐time PCR analysis revealed that GDNF levels increased in all cultured conditions tested in both strains, but the levels returned toward the control after 24 hours of culture. BKβ4 expression was similar to GDNF expression, while BKα1 and BKβ2 expression did not show consistent changes. Exposure of the DBA/2J's CB to anti‐GDNF for 12 hours decreased BKα1, β2, β4 mRNA expressions. These results are complimentary to our previous study showing that supplementation of GDNF in the A/J's CB increased functional expression of BK channels. The results further suggest that BKβ4 subunit expression may be influenced by environmental O2.

Supported by HL72293 & HL 081345
Indices of orthostatic intolerance following 10 days of bedrest in healthy, older men and women
Authors: Hays Nicholas P. , Lombeida Juan , Ronsen Ola , Symons T. Brock , Kortebein Patrick , Kajkenova Oumitana , Ferrando Arny A. , Paddon‐Jones Douglas , Wolfe Robert R. , Evans William J.
Published: June 2021

Moving from a supine to standing position (orthostasis) requires multiple cardiovascular and autonomic adjustments to maintain blood pressure and cerebral perfusion. We examined cardiovascular and catecholamine responses to a standardized orthostatic challenge before and after 10 d of bedrest in healthy men and women (n = 24) aged 55 – 80 y. The orthostatic test involved measurements of heart rate, blood pressure, and serum catecholamines during 10 min of supine rest followed by 10 min of standing. A test was terminated if the subject experienced any orthostatic symptoms (e.g. lightheadedness). Two subjects were symptomatic during pre‐bedrest testing, while 5 subjects were symptomatic post‐bedrest. Heart rate during the standing portion of the test was higher than the supine value; the difference between standing and supine values was significantly greater at post‐ vs. pre‐bedrest (22.3 ± 9.7 vs. 14.1 ± 15.8 bpm, respectively, p = 0.01). The difference between standing and supine serum norepinephrine values was also higher at post‐ vs. pre‐bedrest (282.6 ± 167.0 vs. 162.8 ± 271.0 pg/mL, p = 0.05). No significant differences in blood pressure or epinephrine concentration were observed. These data indicate that 10 d of bedrest can result in greater indices of orthostatic intolerance in previously asymptomatic healthy older adults. NIH P01AG023591, M01RR14288, and M01RR00073.
Impaired generation of NO and mitochondrial superoxide underlie abnormal diazoxide induced vasodilation in insulin resistance
Authors: Katakam Prasad V.G. , Snipes James A. , Busija David W.
Published: June 2021

Insulin resistance (IR) is a risk factor for cerebrovascular disease. We evaluated responses to diazoxide, a putative mitochondrial KATP channel opener, in cerebral arteries from Zucker obese rats (ZO) with IR and lean rats (ZL). Diameter changes to diazoxide were determined in the presence and absence of endothelium and inhibitors of eNOS (L‐NAME) and NADPH oxidase (apocynin). Changes in mitochondrial membrane potential (TMRE) and superoxide (HEt) in response to diazoxide were also determined. Diazoxide induced vasodilation was reduced in ZO compared to ZL (‰ maximal dilation: 37±3 in ZL vs. 21±4 in ZO, p<0.05). Vasodilation was reduced by endothelial denudation or L‐NAME but unaffected by apocynin in both groups. Diazoxide induced vasodilation was accompanied by similar TMRE fluorescence changes in both while ‰increase in HEt fluorescence was diminished in ZO (74±19) vs. ZL (180±33, p<0.05). Thus, diazoxide induced vasodilation in cerebral arteries was partly mediated by endothelium and NO. Reduced production of NO and/or mitochondrial superoxide appears to underlie the impaired vasodilation to diazoxide in IR. Support: NIH grants HL‐030260, HL‐065380, HL‐077731.
An interventriculo‐septal ganglion mediates the vagal postganglionic control of contractility of the right ventricle of the cat heart
Authors: Dávila‐García Martha Isabel , Ardell Jeffrey L , Adetobi‐Oladele Elizabeth , Ekejiuba Sussan E. , Massari V. John
Published: June 2021

We investigated the role of the interventriculo‐septal (IVS) ganglion as a potential source of vagal postganglionic control of right ventricular contractility. Prior evidence demonstrated that this ganglion provides a major source of vagal postganglionic innervation to both right and left ventricles. Blockade of the IVS ganglion with hexamethonium significantly reduced the vagally‐induced changes in right ventricular contractility in the short (transverse) and long (longitudinal) axis, without affecting heart rate or blood pressure responses to left vagal stimulation. The central pathway controlling the IVS ganglion was investigated by neuroanatomical methods. The retrograde tracer, cholera toxin‐β subunit conjugated with horseradish peroxidase (CTB‐HRP) was injected into the region of the IVS ganglion and retrogradely labeled cells were found exclusively in the ventrolateral nucleus ambiguus (NA‐VL) with the greatest concentration at the level of the area postrema. These data indicate that the IVS exerts a predominant role in modulation of RV contractile function, and that its major pre‐ganglionic input arises from the left NA‐VL.

This research was supported in part by NIH‐NIGMCS 2S06GM008016 to MDG, NIH‐NINDS‐SNRP 2 U54 NS039407 to MDG and VJM, NIH‐NIMH 1 R24MH067627 to MDG and VJM, 2 G12 RR003048 from the RCMI Program to MDG and NIH‐HL71830 to JLA.
Cervical spinal modulation of vagal and sympathetic outflows to the ischemic heart: adverse vs cardioprotective effects
Authors: Ardell Jeffrey L. , Southerland E. Marie , Gibbons David
Published: June 2021

High cervical spinal neurons modulate the responsiveness of afferent and efferent cardiac control.Objective: To determine whether high cervical spinal neurons exert differential effects on cardiac myocyte viability and electrical stability during ischemia.Methods: The hearts of anesthetized rabbits, subjected to 30 min of LAD coronary arterial occlusion (CAO) followed by 3 hr of reperfusion (control), were compared to those with pre‐emptive SCS (starting 15 min prior to and continuing throughout the 30 min CAO). For SCS, the dorsal aspect of the C1‐C2 spinal cord was stimulated electrically (50 Hz; 0.2 ms; 90% of motor threshold). For pre‐emptive SCS, separate groups of animals were compared with 1) neuroaxis intact, 2) SCS following bilateral cervical vagotomy, and 3) SCS following muscarinic or selective adrenergic blockade. Infarct size (IS), measured by tetrazolium, was expressed as percentage of risk zone.Results: SCS reduced infarct size by 43% without increasing the incidence of sudden cardiac death (SCD). Alpha or beta adrenergic blockade returned infarct size to sham levels. Beta blockade eliminated ischemia induced ventricular fibrillation; alpha blockade doubled the incidence of sudden cardiac death. SCS mediated cardioprotection against cell death was preserved following vagotomy; it was abolished by atropine. In the presence of SCS, sudden cardiac death was eliminated by vagotomy but unaffected by atropine.Conclusions: Central neuronal influence on infarct size does not parallel that of sudden cardiac death. These data point to the underlying differential control of high cervical neurons on sympathetic and parasympathetic cardiac efferent neurons. (Supported by HL71830)
Carotid body dysfunction and altered oxygen homeostasis in models of Parkinson's disease
Authors: Gillum Matthew Paul , Azam Iram T. , Wehner Mackenzie R. , Hodges Matthew R. , Maguire‐Zeiss Kathleen A. , Federoff Howard J. , Richerson George B.
Published: June 2021

Abstract: For reasons that are not well understood, patients with Parkinson's disease (PD) are three times more likely to die from respiratory illnesses. Because a hallmark of PD is the degeneration of select dopaminergic neurons, and because PD patients do not increase ventilation normally in response to hypoxia, we investigated the hypothesis that the carotid body—a key regulator of O2 homeostasis situated at the bifurcation of the carotid artery in the neck and rich in dopamine—is susceptible to dysfunction in experimental models of Parkinsonism. Normally, mice respond to hypoxia by increasing ventilation and/or decreasing oxygen consumption (VO2). However, we found that treatment with the dopaminergic neurotoxin MPP+ completely attenuated the normal ventilatory increase in response to hypoxia. In contrast, we report that α‐synuclein double mutant mice—a transgenic PD model—fail to decrease VO2 during hypoxia and as a result must ventilate more to compensate. If carotid body dysfunction also occurs in patients with PD, then impaired sensitivity to hypoxia and further failure to reduce metabolic oxygen demand in conditions of low O2 might promote mortality, especially in cases of lung disease like pneumonia when signals from the carotid body and a global decrease in oxygen consumption become critical.
Short‐term modulation of the exercise ventilatory response is not diminished in older men
Authors: Wood Helen E. , Rowell C J , Storms C D , Klocko M N , Ranasinghe K , Mitchell G S , Babb T G
Published: June 2021

We have previously shown that the exercise ventilatory response (EVR; defined as the slope of the relationship between ventilation and CO2 production) is reversibly augmented within a single exercise trial in the presence of added dead space (DS) in healthy young men. This augmentation arises from a mechanism known as short‐term modulation (STM; FASEB J. 21, 765.18. 2007), independent of changes in chemoreceptor (CR) feedback from rest to exercise. We hypothesized that the capacity for STM to augment the EVR with added DS is diminished in healthy older men due to age‐dependent decreases in pulmonary function and increases in EVR. Eleven men (age 69 ± 3 years) were studied at rest and during cycle exercise (10, 30, 50W; not randomized) in Control conditions (no added DS) and with added DS (200, 400, 600ml; randomized). The EVR increased sufficiently with the increasing DS volume (p<0.01), e.g. at 10W, from 33 ± 6 in Control to 47 ± 10 with 600ml DS, such that CO2 was not retained with added DS vs. Control. Hence, the increase in the EVR was not due to increased CR feedback from rest to exercise. Increasing exercise intensity diminished the EVR (p<0.01), e.g. with 400ml DS, from 40 ± 7 at 10W to 34 ± 5 at 50W; and the size of this effect varied by DS level (p<0.05). A very similar response pattern was observed in younger men. We conclude that the capacity for STM to augment the EVR with added DS is not diminished with age in healthy men.

Support: The Cain Foundation, The King Foundation, and Presbyterian Hospital of Dallas
B6D2F1 mouse model to study the effects of habitual aerobic exercise on vascular endothelial dysfunction with aging
Authors: Durrant Jessica Ruth , Connell Melanie L , Folian Brian J , Russell Molly J , Seals Douglas R , Lesniewski Lisa A
Published: June 2021

Regular aerobic exercise improves vascular endothelial function in older humans by reducing oxidative stress and increasing nitric oxide (NO) bioavailability, but the potential for gaining further mechanistic insight is limited. To establish a mouse model, we studied endothelium‐dependent dilation (EDD) systemically and in isolated carotid arteries from older (29‐34 mo) cage‐control (OC) and voluntary wheel running (OVR: 1.1±0.6 km/d × 12 wk) B6D2F1 mice (n=5–6/group). OVR had lower mean arterial pressure (122±2 vs. 136±4 mmHg, P=0.02), enhanced systemic sensitivity to acetylcholine (ACh) (IC50: 0.04±0.01 vs. 0.08±0.01 mg/kg, P = 0.01) and greater maximal carotid artery dilation to ACh (96±1 vs. 76±3%, P<0.01). OVR had lower aortic nitrotyrosine (NT) abundance (1.0±0.2 vs. 2.9±0.6 NT/GAPDH, P<0.01). TEMPOL, a superoxide inhibitor, improved systemic (P=0.001) and carotid (P=0.004) EDD in OC, but not OVR. L‐NAME, a NO synthase inhibitor, reduced ACh‐mediated EDD in TEMPOL‐treated OC (−47% to 49±8%) and untreated OVR carotid arteries (−48% to 47±7%) to the same extent. Endothelium‐independent dilation to sodium nitroprusside was similar in OC and OVR. B6D2F1 OC and OVR appear to provide a useful model for future studies of the cellular and molecular mechanisms underlying the effects of habitual aerobic exercise on age‐associated endothelial dysfunction. NIH AG006537, AG013038, AG022241
Ascorbic acid increases muscle blood flow during dynamic exercise in older healthy humans
Authors: Kirby Brett S , Voyles Wyatt F , Simpson Carrie B , Carlson Rick E , Schrage William G , Dinenno Frank A
Published: June 2021

Age‐related increases in oxidative stress impair endothelium‐dependent vasodilation in humans, leading many to speculate that endothelial dysfunction contributes to impaired muscle blood flow and vascular control during exercise in older adults. We directly tested this hypothesis in 13 young (22±1 yrs) and 14 healthy older men and women (65±2 yrs). We measured forearm blood flow (FBF; Doppler ultrasound) and calculated vascular conductance (FVC) during rhythmic handgrip exercise at 10% maximum voluntary contraction. After 5 minutes of steady‐state exercise with saline, ascorbic acid (AA) was infused via brachial artery catheter for 10 minutes during continued exercise. Prior to AA, FBF (∼28%) and FVC (∼31%) were lower in older vs young adults (P<0.05). In young adults, AA administration did not significantly influence FBF and FVC, whereas FBF and FVC increased 30±4% in older adults at end exercise (P<0.05). This increase in FBF and FVC during exercise in older adults was associated with improvements in vasodilator responses to acetylcholine (ACH; endothelium‐dependent) but not sodium nitroprusside (SNP; endothelium independent). AA had no effect on ACH or SNP responses in the young. We conclude that acute AA administration increases muscle blood flow during dynamic exercise in older adults, and this is likely due to an improvement in endothelium‐dependent vasodilation.

Supported by AG‐027150
ACE2 prevention of oxidative stress in the brain is associated with a reduction in Angiotensin II‐induced sympathetic vasomodulation
Authors: Xia Huijing , Bindom Sharell M. , Feng Yumei , Raju Sammeta VM , Seth Dale , Navar Gabriel L. , Lazartigues Eric
Published: June 2021

We previously reported the presence of Angiotensin Converting Enzyme type 2 (ACE2) in brain regions involved in cardiovascular regulation. Here we test the role of central ACE2 on oxidative stress and autonomic function. ACE2 knockout (KO) mice (n=3/group) were infected with a hACE2 or control (eGFP) adenovirus (2×10e6 pfu, 200 nL) in the paraventricular nucleus (PVN) 3d before AngII infusion (osmotic pump: 600 ng/kg/min sc). After 4d infusion, mice were injected with dihydroethidium (DHE) intracardially and staining measured in brain regions (Image J, arbitrary units). Baseline blood pressure (BP) (telemetry: 112±7 vs. 111±3 mmHg) and DHE staining in the brain were not different between ACE2 KO and wildtypes. Post infusion, AngII levels in the hypothalamus were significantly higher in KO vs. wildtype mice (RIA: 2.9±0.3 vs. 2.0±0.2 fmol/mg, n=5, P<0.05) and DHE fluorescence in KO (eGFP‐infected) dramatically increased (P<0.01) vs. baseline in the PVN (194±12 vs. 101±3), NTS (52±2 vs. 20±2) and RVLM (207±22 vs. 77±12). This increase was prevented in Ad‐hACE2‐infected KO in the PVN (114±8, P<0.01) as well as in the NTS (22±2, P<0.01) and RVLM (88±8, P<0.01). Moreover, ACE2 gene therapy decreased the LF/HF ratio (BP variability) in AngII‐infused KO (5±1 vs. 9±3). These data support the protective role of ACE2 in the central regulation of autonomic function by reducing oxidative stress. (NIH NS052479 and P20RR018766)