bluuebunny/crossref_metadata_2025 huggingface dataset:
ASSOCIATION OF ARTERIAL PRESSURE OSCILLATIONS WITH MUSCLE SYMPATHETIC NERVE ACTIVITY (MSNA) DURING CENTRAL HYPOVOLEMIA
Authors: Ryan Kathy L , Rickards Caroline A. , Convertino Victor A.
Published: June 2021

We have previously demonstrated (Crit Care Med 36:1892, 2008) that spectral power at the high frequency of the R‐R interval (RRIHF) is inversely correlated with MSNA during progressive hypovolemia induced by lower body negative pressure (LBNP). We tested the hypothesis that oscillations in arterial pressure would also be associated with MSNA during LBNP. Subjects (11 M/5 F; n=16) were instrumented to record ECG, beat‐by‐beat arterial pressure (Finometer) and MSNA (peroneal nerve) during a progressive LBNP protocol taken to the point of tolerance (i.e., presyncope), followed by recovery. Systolic blood pressure (SBP) and R‐R interval oscillations were assessed in the frequency domain with Fourier transform. Application of LBNP caused reductions in R‐R interval and SBP, and progressive increases in MSNA. SBP oscillations increased during LBNP, as indicated by increases in both high frequency (SBPHF; 0.15‐0.4 Hz) and low frequency (SBPLF; 0.04‐0.15) domains, but only at the highest LBNP levels. While RRIHF was strongly and inversely correlated with MSNA (R2 = 0.97), SBPHF and SBPLF were not as highly correlated with MSNA (SBPHF R2 = 0.56, SBPHF R2 = 0.50). These results demonstrate that increases in arterial pressure oscillations during LBNP are not solely attributable to sympathetic activation.
Blood pressure regulatory system based on an automatic feedback controller
Authors: Siu Kin Lung , Ahn Jae M. , Chon Ki H.
Published: June 2021

In this work, we describe our design for a new electrohydraulic (EH) pump‐driven systemic blood pressure (SBP) regulatory system capable of implementing precise and rapid SBP regulation in experimental animals. Without this automated system, SBP is manually controlled via a blood pressure clamp, and the imprecision in this method leads to compromised SBP data. This motivated us to develop an EH pump‐driven closed‐loop blood pressure regulatory system based on flow‐mediated occlusion using the vascular occlusive cuff technique. A closed‐loop servo‐controller system based on a proportional plus integral (PI) controller was designed using the dynamic feedback SBP signal from animals. In vivo performance was evaluated via flow‐mediated SBP occlusion, maintenance, and release responses. A step change of ‐30 mmHg, referenced to normal SBP, was applied to 5 Sprague‐Dawley rats with the proposed system to assess the performance of the PI controller. The PI's performance was compared against manual control of blood pressure clamp to regulate SBP. Rapid SBP occlusion (within 3 seconds) and a release time of approximately 0.3 seconds were obtained for the PI controller, which was significantly better than manual control. We concluded that the proposed EH SBP regulatory system could fulfill in vivo needs to study various pressure‐flow relationships in diverse fields of physiology.
The medial nucleus of amygdala (MeA) mediate the cardiovascular responses evoked by noradrenaline microinjected into the lateral septal area (LSA) in rats
Authors: Scopinho América Augusto , Fortaleza Eduardo Albino Trindade , Correa Fernando Morgan Aguiar
Published: June 2021

Introduction

We demonstrated that the microinjection of noradrenaline into the LSA evoked pressor and bradycardic responses mediated by vasopressin release. Moreover, the microinjection of cobalt or selective non‐NMDA antagonist NBQX into the paraventricular nucleus (PVN) blocked the pressor response and bradycardia evoked by NA injected into the LSA, suggesting the PVN as the final structure in this pathway. Anatomical dates demonstrated that no direct connections were observed between the LSA and the PVN or the SON. The MeA is a structure involved in cardiovascular control. Projections from the LS to the MeA have also been described in the literature and MeA sends projections to PVN.

Objective

To investigate if MeA acute ablation by the injection of cobalt blocked the cardiovascular responses to NA microinjection into the LSA.

Methods and Results

Male Wistar rats were used. Cannulaes was implanted in the LSA and the ipsilaterally in the MeA using a stereotaxic apparatus A catheter was implanted into the femoral artery for blood pressure and heart rate recording. The microinjection of cobalt into the MeA blocked the pressor and bradycardiac responses evoked by NA microinjection into the LSA.

Conclusion

Our results suggest that synapses in the MeA are involved in the mediation of the pressor and bradycardiac response to the microinjection of NA into the LSA.

Financial Support

CAPES and FAEPA.
Octreotide acetate moves the volume indifferent point
Authors: Jarvis Sara Sun , Pawelczyk James
Published: June 2021

During head‐up tilt (HUT), gravity redistributes ~700 mL of blood to the legs, abdomen, and pelvis, decreasing venous return. The location of the volume indifferent point (VIP) provides a quantitative assessment of this volume redistribution and coincides with the hydrostatic indifferent point (HIP). A more superior VIP/HIP should relate to improvements in HUT tolerance as cardiac filling is enhanced. Previously we noted that administration of octreotide acetate, a selective splanchnic vasoconstrictor, improved HUT tolerance. In this study we determined the location of VIP, with and without octreotide acetate. Blood volume shifts were determined with segmental impedance and were regressed against the distance of each segment from the feet. The point where blood volume did not change was defined as the VIP. The VIP was located 64.5 ± 2.6% of an individual's height. Administration of octreotide acetate resulted in a superior shift of the VIP (1.9 ± 3.3 cm, p=0.03). Taken together, these studies suggest that selective splanchnic constriction induced by octreotide acetate alters blood volume distribution during HUT, improving tilt table tolerance by shifting the VIP to a more superior location.
Food intake, physical activity and BMI status of youth enrolled in a competitive, creative, problem solving program
Authors: Spence Marsha , Costello Carol , Moustaid‐Moussa Naima , Greer Betty , Fitzhugh Eugene , Bassett David , Maples Jill , Muenchen Bob , Gibbs Robert
Published: June 2021

The objective of this study was to determine if children who participate in a competitive, creative, problem‐solving program differ in BMI, food intake, and physical activity levels when compared to a nationally representative sample. Participants were a sample of high school‐aged youth that competed in the 2008 Destination ImagiNation® (DI) Global Finals (n=668). Height and weight measures were collected and a survey based on the Youth Risk Behavioral Survey was administered to participants. Based on the CDC age and gender BMI classifications, 1.8% of participants were underweight, 69.2% had a healthy weight, 17.7% were overweight, and 11.4% were obese. Further, 31.5% reported consuming fruits and vegetables >5 times/day, 21.9% reported drinking milk >3 times/day, 26.9% reported drinking soft drinks >1 times/day, 48.0% were physically active for at least 60 minutes/day on >5 days during the week, 14.3% reported watching >3 hours of TV on an average school day, and 15.8% reported using computers >3 hours on an average school day. Thus, compared to the 2007 Youth Risk Behavioral Surveillance data, the youth surveyed at the DI Global Finals reported overall a higher intake of fruits, vegetables and milk, a lower intake of soft drinks, less TV or computer use and a higher level of physical activity, suggesting overall healthier practices in youth participating in this creative team‐based program.
Blood‐Brain Barrier Disruption Following Hypoxic Stress Requires Activation of Myosin Light Chain Kinase
Authors: Brown Rachel C , Hicks Kali D , O'Neil Roger G
Published: June 2021

When blood‐brain barrier (BBB) endothelial cells are exposed to hypoxic stress for prolonged periods of time, they undergo a morphological change and round up, disrupting cell‐cell junctions which leads to increased paracellular permeability. In this study, we investigated the hypothesis that myosin light chain kinase (MLCK) is integral to this process, and investigated alterations in tight junction structure after hypoxic stress. iTRAQ proteomic analysis identified a number of proteins in BBB endothelial cells that are involved in modulation of the actin cytoskeleton that were significantly altered after 6 hrs of hypoxia. Western blotting confirmed the changes in actin, moesin and vasodilatory stimulated phosphoprotein (VASP). We treated cells with ML‐7, a selective MLCK inhibitor, and assessed barrier function; ML‐7 completely blocked the hypoxia‐induced barrier disruption seen in untreated monolayers. This protection of BBB integrity was correlated with a prevention of stress fiber formation and decreased ZO‐1 expression at tight junction sites, implicating contraction of the actin‐myosin cytoskeleton in BBB disruption following hypoxic stress.
Role of TRPC Channels in Mediating Calcium Signaling Response of Brain Microvessel Endothelial Cells to Mechanically‐Induced Injury
Authors: Berrout Jonathan , Jin Min , O'Neil Roger G.
Published: June 2021

Traumatic brain injury (TBI) can lead to the breakdown of the blood brain barrier (BBB). Since elevation of cytosolic calcium levels, [Ca]i, can compromise the BBB integrity, we assessed the effects of mechanical injury on [Ca]i in brain endothelial cells. Mouse brain microvessel endothelial cells (bEnd3 cells) were grown on elasticized membranes (BioFlex plates) and [Ca]i monitored using fura 2 fluorescence imaging. Application of a "moderate" injury, using a pressure/stretch pulse of 50 ms (~35% stretch; Cell Injury Controller II module), induced a rapid transient increase in [Ca]i by 110 +/‐ 17 nM (n = 6) which slowly returned to elevated basal levels over several minutes. In the absence of extracellular Ca the injury‐induced [Ca]i transient was greatly reduced, but not eliminated, implicating both Ca release from stores and Ca influx across the cell membrane. Unloading of Ca stores by treatment with thapsagargin (10 uM) did not abolish the injury transient. Addition of LOE‐908 (1 uM), a TRPC channel blocker, partially blocked the transient [Ca]i increase, while ruthenium red, a TRPV channel blocker, was without affect. It is concluded that mechanical injury of brain endothelial cells induces calcium influx, implicating [Ca]i transients in injury‐induced barrier breakdown. Further, the induced Ca influx appears to be associated with injury‐induced TRPC channel activation, but not TRPV channel activation.
To Eat or Not to Eat: AMPK and Food Intake Regulation in Hibernators
Authors: Florant Gregory L , Fenn Ashley M. , Wilkerson Gregory , Healy Jessica , Handa Robert
Published: June 2021

Many mammals that hibernate do not feed during the winter. For example, the yellow‐bellied marmot (Marmota flaviventris) does not feed for roughly seven months after nearly doubling its body mass in the autumn. AMP‐activated protein kinase (AMPK) acts as a sensor of peripheral energy balance within the hypothalamic arcuate nucleus (HARC) and is postulated to play a role in the regulation of food intake in mammals. AMPK is activated when cellular energy is low, specifically when the AMP/ATP ratio is high. 5′‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR) is a known specific agonist of AMPK as it mimics the effects of high AMP by activating AMPK. We hypothesized that infusing AICAR near the HARC of fasting marmots during their winter hibernation period would activate AMPK in the HARC leading to an increase in food intake during the fasting period. Cannulae were stereotaxically implanted into the ventricles near the HARC of five marmots. The cannulae were connected to seven‐day Alzet mini‐pumps; 4 were filled with AICAR (50µg/µl) and one filled with saline (control). The AICAR and saline were infused at a rate of 25µg/hr. During the seven days, the four marmots infused with AICAR failed to undergo torpor and significantly increased their food intake from 0.00g/day to an average of 12.2 g/day (p<.01). The control animal returned to torpor and did not increase food intake. These results suggest that AICAR activated AMPK, disrupted torpor patterns, and significantly increased food intake over the period the animals were infused. After AICAR infusion was complete, all but one animal re‐entered hibernation and animals stopped eating even though food was available. These results support our hypothesis that AMPK may be involved in the pathway(s) that regulate food intake in marmots. Supported by NIH grant (R25 DK067017) to GLF.
The threshold of central ghrelin induced anorexia is lower in chicks selected for low rather than high body weight
Authors: Calchary Wendy Alyssa , Siegel Paul , Cline Mark
Published: June 2021

Ghrelin is a gut derived 28 amino acid peptide that when centrally injected stimulates food intake in mammals, but decreases food intake in avians. The present study was designed to determine the effects of central ghrelin in lines of chickens that have undergone long term divergent selection for low (LWS) or high (HWS) body weight. In experiment one, chicks in line LWS responded to all doses of central ghrelin injected (0.083, 0.33, 1.33 nmol) with decreased food intake during the 180 min observation time , while line HWS chicks only responded to 0.33 and 1.33 nmol central ghrelin with decreased food intake. The dose of 0.33 nmol ghrelin only significantly reduced food intake in line HWS after 120 min post injection. In experiment two, central ghrelin injection was associated with a greater magnitude suppression of standing and perching time and feeding and exploratory pecks in line LWS than line HWS. Additionally, the magnitude of increased sitting and deep resting time was higher in line LWS. These results support the thesis that the magnitude of centrally induced ghrelin associated anorexia is different between lines LWS and HWS and differential behavior responses may contribute to this effect.
Litorin is associated with satiety but does not affect non‐ingestive behaviors in chicks
Authors: Cofield Stuart Alden , Cline Mark A
Published: June 2021

Litorin is a bombesin‐like peptide that causes satiety in mammals; however its mechanisms of action are poorly understood. The present study was designed to elucidate some of these mechanisms using chicks as model organisms. Chicks treated with 1 and 10 nmol intracerebroventricularly delivered litorin had reduced food intake throughout the entire 180 min observation period. However, chicks that received 0.1 nmol litorin did not experience reduced food intake. Litorin was also associated with reduced water intake, but only chicks that received 10 nmol showed this effect. The effect on water was secondary to food, since food restricted chicks did not reduce water intake when centrally injected with litorin. Additionally, whole blood glucose concentration was not affected by ICV litorin. Behaviors unrelated to injection including, exploratory pecking, escape attempts, jumping, defecations, and time spent standing, sitting, preening and in deep rest were not affected by central litorin. Thus, litorin does not appear to cause behaviors that may be competitive with appetite. The number of feeding pecks was reduced at 5 min post injection, but at no other time. This effect can be explained by decreased pecking efficiency of litorin‐treated chicks. These results provide new inside into litorin regulation of feeding.
Leptin mediates discriminate response to feed restriction in feed efficient pigs
Authors: Lkhagvadorj Sender , Qu Long , Cai Weiguo , Couture Oliver P. , Barb C. Richard , Hausman Gary J. , Nettleton Dan , Anderson Lloyd L. , Dekkers Jack C.M. , Tuggle Christopher K.
Published: June 2021

Genetic mechanisms that control feed efficiency (FE) and feed intake are not well understood. Residual feed intake (RFI) is a measure of FE, in which low RFI designates high FE. Transcriptional profiling coupled with serum metabolite analyses were used to identify genes and pathways that respond to a feed restriction (FR) in high or low RFI pigs. Low RFI (n=10) and high RFI Yorkshire pigs (n=10) of ~82 kg were fed ad lib. or fed 80% of maintenance for 7 days in a 2x2 factorial treatment design. Microarray analysis declared 6,114 genes in fat and 305 genes in liver as differentially expressed (DE) in response to FR and 311 fat genes and 147 liver genes due to RFI. Functional annotation of DE genes due to RFI in fat indicates that pathways such as K+ transport and monocarboxylic acid metabolism were overrepresented. Genes that were DE due to FR in fat overrepresented many pathways, including TCA cycle, apoptosis, and MAPK signaling. Concentrations of serum T3 and glucose were higher in the low RFI vs. high RFI pigs. Serum leptin concentration was decreased by FR in high RFI pigs but had the opposite effect in low RFI pigs. Leptin expression was 2.4‐fold down‐regulated in the low RFI pigs and in silico pathway analysis indicates that leptin had the highest number of interacting neighbors among genes that were DE due to RFI in fat. Leptin may play a key role in determining pathways that respond to FR in feed efficient pigs. USDA‐NRI‐2005‐3560415618
Is the central melanocortin system involved in mediating the actions of neuronostatin?
Authors: Yosten Gina L. C. , Samson Willis K.
Published: June 2021

Neuronostatin (NST), a recently discovered 13 amino acid peptide, is encoded in the somatostatin pro‐hormone. It is produced throughout the body, in particular in brain areas known to be important in metabolic and autonomic regulation, where it exerts direct membrane effects. NST decreases, in a dose‐related fashion, both food and water intake in ad libitum fed rats when injected into the lateral cerebroventricle (i.c.v.). Additionally, i.c.v. NST causes a biphasic increase in mean arterial pressure (MAP) in conscious rats. Because the central melanocortin system is involved in both appetite regulation and cardiovascular control, we sought to determine if the actions of NST required activation of melanocortin receptors. Rats were injected at 1650 with either the melanocortin ¾ receptor antagonist SHU9119 and NST, or NST alone. Food and water intakes were recorded. For cardiovascular studies, rats were pretreated with SHU9119 or saline, and treated with either NST or saline 15 minutes later. We found that pretreatment with SHU9119 abrogated the effect of NST on both food/water intake and MAP, indicating that the actions of NST are likely mediated by activation of central melanocortin systems. Neuronostatin can now be added to the list of neuropeptides with known orexigenic/anorexigenic potency. These data further support the concept of co‐ordinated CNS mechanisms regulating metabolic and autonomic function.
Intracerebroventricular (ICV) injections of nesfatin‐1 induces c‐fos expression in the rat forebrain.
Authors: Martin Theodore J. , Moreau Jason M. , Ajani Abdallah A. , Oiamo Tor H. , Maximos Mira , Ciriello John
Published: June 2021

The forebrain contains neuronal circuits of critical importance in the regulation of food intake. Nesfatin has been identified as a satiety peptide within central neurons. ICV injection of nesfatin‐1 decreases food intake in a dose‐dependent manner and result in a decrease in body weight. However, the central pathways activated by nesfatin‐1 are not known. In this study, the distribution of neurons expressing fos‐like immunoreactivity following ICV infusions of nesfatin‐1 (5 pmol/µl; 2 µl followed by two 1 µl injections every 30 min) was identified in the rat forebrain. Fos labeled neurons were found within the hypothalamus: paraventricular, supraoptic, arcuate, lateral, dorsomedial and posterior nuclei. In addition, the organum vasculosum of the lamina terminalis, median preoptic nucleus and subfornical organ expressed fos labeling. Finally, fos labeling was observed within the midline thalamic nuclei, lateral septum, islands of Calleja, medial preoptic nucleus and medial and central nuclei of amygdala. These data suggest that nesfatin‐1 exerts its anorexigenic effects through the activation of forebrain structures previously implicated in the regulation of food intake and energy expenditure.

Supported by Heart and Stroke Foundation of Ontario.
Increased sensitivity to AM251 in aged rats in the prevention of high‐fat diet‐induced hyperphagia
Authors: Judge Melanie Kae , Zhang Yi , Scarpace Philip J
Published: June 2021

Energy balance is regulated, in part, by the leptin and endocannabinoid (EC) systems. These systems interact, in that leptin inhibits endocannabinoid levels in the hypothalamus. Little is known about the EC system in aged, leptin resistant rats. Three‐ and 30‐month‐old male F344xBN rats were given ad libitum access to chow or 60% high‐fat (HF) diet during 6‐day daily i.p. administration of cannabinoid receptor‐1 (CB1) antagonist, AM251, and HF diet‐induced hyperphagia was examined. In young, the lower (0.83mg/kg/day) and higher (2.78mg/kg/day) doses of AM251 reduced the peak high‐fat diet‐induced hyperphagia by 24% and 100%, respectively. In aged, the lower and higher doses reduced the peak by 12% and 98%, respectively. However, when the total period of hyperphagia was considered, the low dose did not decrease cumulative caloric intake in young, but reduced it by 45% in aged rats. The lower dose of AM251 did not affect the length of hyperphagia period in young, but the period was reduced from 30 to only 6 days in aged rats. These data indicate that aged rats are more sensitive to AM251. One possible mechanism is that aged rats, which are leptin resistant, lack the leptin negative regulation of endocannabinoid levels leading to hyperactive CB1 receptors, thus increasing the susceptibility to CB1 blockade in aged rats. Supported by NIH AG‐26159, P30 AG028740, and AG00196.
New animal model for metabolic syndrome: Nocturnal binge drinking of fructose
Authors: Graham Cathy D. , Morris Mariana
Published: June 2021

A high fructose diet induces symptoms of metabolic syndrome in mice. The goal was to determine whether the timing of fructose (F) consumption alters intake patterns and metabolic function. C57Bl/6 male mice were given: 1) water, C, 2) 24 h access to 10% fructose water, F24 or 3) 12h access to %F water (dark period, DP) or water (light period, LP), F12, for 8 weeks. Regular chow was available ad libitum. Day/night fluid intake and metabolic/hormonal indices were measured. DP fructose intake was 40% higher in F12 (F12: 15.5 ± 0.3 vs F24: 9.2 ± 0.8 ml/12hr). F12 drank 90% of total F load during DP as compared to 60% for F24. Weight gain was greater in F12 (C: 6 ± 0.01, F12: 19 ± 0.01, F24: 11 ± 0.01, % weight gain over 8 weeks). Glucose tolerance was reduced in both F groups as was basal plasma glucose (C: 115.6 ± 5.7 vs. F12: 95.3 ± 5.4, F24: 94.4 ± 4.2 mg glucose/dl). There was no change in plasma insulin (C:0.8 ± 0.1, F12:0.9 ± 0.4, F24:0.9 ±0. 2 ng/ml) or leptin. Cholesterol was elevated in F groups (C:75.5 ± 6.5, F12: 87.4 ±4 .9, F24: 95.0 ± 4.4 mg/dl) as were triglycerides. Data shows that limiting fructose availability to the active period results in binge drinking behavior associated with accelerated weight gain and glucose intolerance in mice. The model provides insight into human metabolic syndrome associated with dietary excesses. Supported by R01 HL093567
Role of enkephalinergic signaling in the limbic forebrain in modulating food reward
Authors: Taha Sharif , Enquist Ida , Fields Howard
Published: June 2021

Opioid signaling promotes palatable food intake but endogenous signaling mediating this effect is poorly defined. We used behavioral, cytochemical, and electrophysiological approaches in wild‐type (WT) and preproenkephalin knockout (PPENK) mice to study the role of enkephalin in palatable food intake. Administration of the nonspecific opioid antagonist naltrexone (NTX) decreased intake of an array of palatable foods in WT mice; in PPENK mice, however, NTX administration elicited a slight trend toward increased consumption. This effect was not dependent upon caloric content, as it was observed for saccharin intake. Using cfos immunohistochemistry, we found that systemic NTX, while increasing cfos expression in the central nucleus of the amygdala (CeA) in WT mice, had no effect on cfos expression in the CeA of PPENK mice. To elucidate opioid effects on neural firing in the amygdala, we recorded from this brain region during intraoral infusion of appetitive or aversive tastants in WT and KO mice. Systemic NTX administration modulated baseline and taste‐evoked amygdalar firing in a population of neurons, including a subset that responded differentially to sucrose versus quinine. Our results demonstrate that tonic enkephalinergic signaling modulates taste‐sensitive neural responses in the amygdala.
Chronic alcohol ingestion exacerbates lung epithelial barrier dysfunction in HIV‐1 transgenic rats
Authors: Fan Xian , Joshi Pratibha C , Koval Mike , Guidot David M
Published: June 2021

We previously reported that chronic expression of HIV‐1‐related proteins in an HIV‐1 transgenic rat (HIV‐1 Tg) model altered the expression and membrane localization of the tight junction proteins, occludin and ZO‐1 in the alveolar epithelium, and decreased lung liquid clearance in vivo. Alcohol abuse and HIV‐1 infection frequently co‐exist and these individuals are at high risk for serious lung infections and respiratory failure. Therefore, in this study we evaluated the potential additive effects of alcohol ingestion on alveolar epithelial barrier dysfunction caused by chronic expression of HIV‐1‐related proteins. Chronic alcohol ingestion (6 wks) worsened lung liquid clearance in vivo and exacerbated paracellular permeability in vitro in HIV‐1 Tg rats. Further, immunoblotting and immunocytofluorescence analyses of tight junction protein expression in primary alveolar epithelial cells showed that the decrease in occludin and ZO‐1 expression was more pronounced in alcohol‐fed rats. In addition, by qPCR analysis, alcohol significantly suppressed epithelial sodium channel expression, which would be predicted to impair alveolar epithelial sodium transport. These findings suggest that alcohol ingestion augments HIV‐1‐induced defects in the alveolar epithelial barrier, which could explain the increased risk of respiratory failure in these vulnerable individuals.
P. aeruginosa ExoY Increases Lung Endothelial Permeability with a Concomitant Decrease in Lung Vascular Compliance
Authors: Prasain Nutan , Alvarez Diego F. , Frank Dara W. , Stevens Troy
Published: June 2021

The P. aeruginosa toxin, ExoY, when injected through a type III secretion system (TTSS) into endothelial cells, produces a cytosolic cAMP pool and induces endothelial cell retraction. However, it is unclear whether ExoY‐induced endothelial cell retraction increases endothelial permeability, causing alveolar flooding, alveolar hemorrhage and perivascular cuff formation in rat lungs. To test this idea, filtration coefficient (Kf), and lung vascular compliance (Cvas) were measured using two strains of P. aeruginosa that possess a: (1) functional TTSS, and only injects ExoY (ExoY+), and (2) functional TTSS, but injects a catalytically inactive ExoY (ExoYK81M) due to a single point mutation at the ATP binding site. Intra‐tracheal instillation of a bacterial concentration that causes 50% lethality to animals was performed. Lungs were isolated from animals that survived for 24, 48 and 72 hours post tracheal instillation, and Kf and Cvas were measured. The ExoY+ strain increased Kf and decreased Cvas 24 hours after instillation. Lung histology of ExoY+‐treated animals revealed extensive perivascular cuffs in larger as well as smaller vessels, with prominent alveolar fluid accumulation and hemorrhage, consistent with increased Kf and decreased Cvas at 24 hours. Thus, introduction of catalytically active ExoY by P. aeruginosa is essential to increase lung endothelial permeability.
A pro‐proliferative endothelial cell phenotype occurs late in the course of inflammation
Authors: Malleske Daniel T , Chicoine Louis G , Nelin Leif D
Published: June 2021

We hypothesized that cytokine stimulation will result in an initial impairment of endothelial cell proliferation, followed by a pro‐proliferative phenotype mediated by elevated arginase expression. Bovine pulmonary artery endothelial cells (bPAEC) were treated with lipopolysaccharide and tumor necrosis factor‐α(L/T)or vehicle and harvested at 0, 1, 2, 4, 8, 24, 48, 72 and 96 h. Levels of mRNA were measured for iNOS, arginase I, arginase II, ornithine decarboxylase (ODC) and ornithine aminotransferase (OAT). iNOS mRNA in L/T treated cells was increased at 2 h and peaked by 24 h with a return to basal levels by 48 h. Conversely, arginase II mRNA expression was increased at 4 h and remained elevated throughout the experiment. Arginase I mRNA remained low in both control and L/T cells. The mRNA levels of ODC and OAT increased at 72 and 96 h in L/T cells. We measured PCNA expression and cells exposed to L/T showed significantly greater PCNA levels than did control cells after 96 h. Endothelial cells grown for 24 and 96 h in either L/T or vehicle were used in a proliferation assay, the number of viable L/T treated cells were lower in the 24 h treated cells and greater in the 96 h treated cells versus controls. Taken together, L/T treatment results in L‐arginine metabolism to NO early, followed by a shift to an arginase‐mediated metabolism of L‐arginine to ornithine resulting in cellular proliferation.
Efficient combinatorial approach to isolating rat pulmonary endothelial cell phenotypes
Authors: Penton Buford Anna , Ayers Linn , Stevens Troy , Creighton Judy
Published: June 2021

Endothelium from discrete vascular segments within the lung displays phenotypic and functional heterogeneity. However, isolation of segment specific endothelial cells (ECs) is a time and cost intensive process. Thus the aim of this current work was to develop an efficient method for obtaining ECs from rat pulmonary artery (PAECs), pulmonary vein (PVECs) and pulmonary microvasculature (PMVECs). Following surgical removal, vessels were digested using type II collagenase and trypsin. The enzymatically obtained cells were plated in D‐valine/heparin endothelial selective growth media on standard culture dishes. Non‐ECs resistant to the D‐valine media were removed by scraping. Lectin panning and differential cell binding were utilized to remove any remaining contaminating cells. Cell cultures were characterized using an EC marker panel: platelet endothelial cell adhesion molecule 1 (PECAM1), endothelial nitric oxide synthase (eNOS), von Willebrand factor (vWF), vascular endothelial cadherin (VE‐cadherin), and acetylated LDL uptake. Specific lectin binding was used to confirm the segmental origin of the ECs. PAECs bound Helix pomatia and Sambucus nigra, while PMVECs bound Griffonia simplicifolia. This combinatorial approach to isolating rat lung endothelial cells consistently yields multiple cell lines from individual animals. Supported by 5PO1HL066299.
Role of PKCepsilon in hypoxia induced contraction of pulmonary endothelium
Authors: Bauer Eileen Maria , Cao Rong , Glorioso Joseph , Pitt Bruce R , St. Croix Claudette M
Published: June 2021

PKCepsilon (PKCε), a novel calcium independent and zinc sensitive & kinase, has been implicated in the contraction of the systemic vasculature. Furthermore, PKCε‐/‐ mice display decreased hypoxic pulmonary vasoconstriction (HPV). Our lab has previously shown that acute hypoxia leads to NO mediated zinc release that contributes to hypoxic pulmonary vasoconstriction. We therefore, hypothesized that PKCε acts as an effector molecule mediating zinc/hypoxia induced constriction. Using live cell microscopy we demonstrated that inhibition of PKCε by RO31‐8220, or infection with a herpes virus expressing dominant negative PKCε led to reduced contraction of pulmonary microvascular and pulmonary artery endothelial cells. Western blot analysis further revealed that hypoxia and zinc induced translocation of PKC&ε from the cytoplasm to the membrane fraction. Hypoxia also led to increased enzyme activity of PKCε in a zinc‐ and NO‐ dependent fashion. We used the isolated perfused rat lung to determine the effect of PKCε inhibition on HPV. While inhibition of PKCε did not completely abolish constriction, our data suggested that PKCε serves as modulator of constriction in the pulmonary vasculature. Funded by RHL081421‐01.
Lipopolysaccharide (LPS) amplifies cigarette smoke (CS)‐induced release of IL‐1 beta and TNF‐alpha from macrophages via indirectly acting on neurokinin 1 receptor (NK1R)
Authors: Xu Junyang , Xu Fadi , Zhang Cancan , Lin Yong
Published: June 2021

CS increases secretion of IL‐1 beta and TNF‐alpha from macrophages in human, which is amplified by LPS exposure. The mechanism underlying this amplification remains unclear. CS and LPS promote macrophage release of substance P (SP) that activates macrophage NK1R to secrete these cytokines, and NF‐κB participates in NK1R intracellular signaling and synthesis of these cytokines. Thus, we asked whether LPS amplified these cytokines response to CS via increasing the macrophage release of SP that binds with NK1R to activate NF‐κB in vitro. RAW264.7 macrophages were incubated with CS condensate (CSC) and DMSO alone or coupled with LPS and/or NK1R antagonist (aprepitant). We found that compared to DMSO, the responses of supernatant IL‐1 beta, TNF‐alpha, SP and nuclear NF‐κB activation to: 1) CSC were increased by 2‐, 11‐, 0.4‐ and 1‐fold; 2) LPS by 4‐, 21‐, 0.3‐, and 2‐fold; and 3) CSC+LPS by 9‐, 50‐, 2‐, and 5‐fold. The data indicate that LPS amplifies the macrophages′ responses of cytokines as well as SP and NF‐κB to CSC. Aprepitant strikingly reduced the LPS amplificatory effects on IL‐1 beta, TNF‐alpha, and NF‐κB activation by 36%, 39%, and 58%, respectively. Our study in vitro confirms that LPS amplifies the CS‐induced secretion of these cytokines from macrophages, and further demonstrates that NK1R is responsible for this amplification, presumably via the NF‐κB pathway. (Supported by NIH 74183)
Molecular mechanisms affecting LPS‐induced endothelial cells (EC) hyperpermeability
Authors: Bogatcheva Natalia V , Kolosova Irina , Kovalenkov Yevgeniy , Mirzapoiazova Tamara , Zemskova Marina , Verin Alexander D
Published: June 2021

LPS decreases transendothelial electrical resistance (TER), increases endothelium permeability for large molecules, and leads to sizeable gap formation in cells grown on glass coverslips. Forskolin suppresses LPS‐induced drop in TER and gap formation in EC monolayer, concomitant with the decrease in LPS‐induced myosin light chain (MLC) phosphorylation. Both protein kinase A activator 6‐Bnz‐cAMP and Epac activator O‐Me‐cAMP have similar effect on LPS‐induced TER changes and gap formation; however, only 6‐Bnz‐cAMP decreases LPS‐induced MLC phosphorylation and increases phosphorylation of VASP. Inhibition of MLC phosphorylation with Rho kinase inhibitor Y27632 significantly suppresses LPS‐induced gap formation. VASP depletion with siRNA exacerbates LPS‐induced changes in TER. These data suggest that MLC‐dependent actomyosin contraction contributes to LPS‐induced barrier dysfunction, whereas VASP‐dependent organization of actin near cell membrane plays barrier‐protective role. We next studied the role of p38 pathway in LPS‐induced pulmonary edema. p38 inhibitor SB203580 significantly suppresses LPS‐induced gap formation and permeability for large molecules, indicative of barrier‐disruptive role of p38 pathway. HSP27 depletion by siRNA exacerbates LPS‐induced changes in TER and gap formation, suggesting barrier‐protective role of this protein. This work was supported by HL‐067307.
Antenatal Administration of PPARγ Agonist Rosiglitazone (RGZ) Prevents Hyperoxia‐Induced Lung injury Postnatally
Authors: Rehan Virender K. , Sakurai Reiko , Shin Eugene , Corral Julia , Torday John S.
Published: June 2021

Nuclear transcription factor PPARγ plays a critical role in normal lung development and injury/repair. We have recently shown that a PPARγ agonist RGZ administered postnatally could prevent hyperoxia‐induced lung injury in a neonatal rat model. However, it is not known if PPARγ agonist administration antenatally can prevent neonatal lung injury postnatally. We hypothesized that antenatal administration of PPARγ agonist RGZ would prevent hyperoxia‐induced neonatal lung injury postnatally. Pregnant Sprague‐Dawley rat dams were administered either diluent or RGZ (3mg/kg), at embryonic day 21 (term ~ 22). After delivery at term, pups were placed in either 21% O2 or 95% O2 before sacrificing them after 24 hours. The lungs were examined for morphometry and specific markers of lung injury and repair. 24h exposure to hyperoxia alone resulted in decreased alveolar sacculation and septal wall thickness, which were blocked by RGZ administration antenatally. Similarly, hyperoxia‐induced increase in TGFβ and Wnt signaling (LEF‐1 and p‐Smad3) and decrease in PPARγ signaling were also blocked by antenatal RGZ administration. Antenatal administration of RGZ virtually blocked 24h hyperoxia‐induced lung morphometric and molecular changes postnatally, suggesting a novel antenatal intervention to protect against hyperoxia‐induced neonatal lung injury (Grants: NIH‐HL75405, HD051857).
Transactivation of Beta 2 Adrenergic Receptor by Bradykinin Receptor via Heterodimerization
Authors: Vincent Karla Kristine , Tougas Michelle , Jones Kymry , Radhakrishna Harish , McCarty Nael
Published: June 2021

G Protein‐coupled receptors (GPCRs) are the third largest family of genes in the human genome and are the target of many pharmaceuticals. Evidence has shown that many GPCRs form heteromeric complexes, although the functional consequences of such interactions are unclear. Bradykinin type 2 receptor (Bk2R) and Beta 2 adrenergic receptor ( β2AR), Gαq‐ and Gαs‐coupled receptors, respectively, are GPCRs that can heterodimerize with other members of their classes in vitro and in vivo, participate in cardiovascular regulation, and are coexpressed in many cell types, including pheochromocytoma (PC12) cells. Given these factors, we examined potential interplay between Bk2R and β2AR. We hypothesized that Bk2R and β2AR physically associate, changing native receptor signaling. Co‐immunoprecipitation studies in PC12 cells and X. laevis oocytes showed physical association of Bk2R and β2AR. In both PC12 cells and oocytes expressing these receptors, bradykinin‐induced Bk2R transactivation via both Gαs‐ and Gαq‐coupled pathways, which was significantly ablated by inverse agonists of Bk2R or β2AR; thus, conformational changes in both receptors are required for transactivation. This is the first evidence of Bk2R/β2AR physical interaction and functional cross‐talk. Given the relevance of pharmaceutics that affect Bk2R and β2AR, these exciting data may provide opportunities for drug development and refinement.
Prenatal hypoxia programs an altered β1/β2 adrenoceptor subtype ratio in the heart of adult broiler chickens
Authors: Lindgren Isa , Carreras Badosa Gemma , Altimiras Jordi
Published: June 2021

Background and purpose

β‐adrenoceptors (βARs) are essential for cardiac regulation. Adverse events in fetal life may program the adult phenotype for cardiovascular disease. Previous studies from our lab show that chronic prenatal hypoxia renders an adult animal with decreased sensitivity to cardiac βAR stimulation. To investigate whether low fetal oxygen conditions have a programming effect on the β1/β2AR ratio in the adult, causing the previously shown decrease in adult βAR sensitivity.

Method and results

We studied total β‐AR density (Bmax) and the ratio of β1/β2 subtypes in the broiler chicken heart (14 and 35 days post hatching, P14 and P35) prenatally developed in normoxia (21% O2, N) or chronic hypoxia (14% O2, H). To obtain total Bmax we incubated ventricular tissue with the tritiated β‐antagonist [3H]CGP‐12177. The β1/β2 ratio was obtained by specific inhibition of β1 by CGP‐20712A and β2 by ICI‐11855 in the presence of 2nM of [3H]CGP‐12177. Total Bmax did not differ between treatments in either P14 or P35, but βAR density decreased with age. There was significantly more β2 than β1 in P35H, while P35N showed a 50/50 ratio of the two subtypes.

Conclusion

Prenatal hypoxia programs an altered βAR subtype ratio in the adult in favor of β2 which may be the cause of the previously observed decrease in βAR sensitivity.

Supported by FORMAS (Dnr. 2004‐1331).
Plasma Soluble Cytokine Receptor Levels are Elevated during Heat Stroke Recovery in Mice
Authors: Blaha Michael D. , Helwig Bryan G. , Leon Lisa R.
Published: June 2021

Cytokines are implicated as mediators of heat stroke (HS) morbidity/mortality. As shown previously, mice show hypothermia and fever during 24h of HS recovery, which correlate with high plasma IL‐1β and IL‐6 levels. This study examined plasma cytokine soluble receptor (SR) levels (sgp130, sIL‐1RI, sIL‐1RII, sIL‐4R, sIL‐6R, sTNFRI, sTNFRII; Luminex FlowMetrix(™)) in HS mice at baseline (To), HS collapse (Tc,Max=42.7°C), hypothermia depth (HD) and fever (24h). Sgp130 was not elevated at any time. sTNFRI was the only receptor elevated at Tc,Max and HD, with recovery at 24h. sTNFRII was elevated at HD only. sIL‐1RI and II were both elevated at HD; sIL‐1RI levels increased through 24h, whereas sIL‐1RII returned to baseline at 24h. sIL‐4R was elevated at 24h only, whereas the sIL‐6R showed elevations at Tc,Max and 24h, with a robust increase at HD. These results show the dynamic nature of SR responses in HS. Despite an inability to detect increased plasma IL‐4 and TNF levels in HS mice, SRs for these cytokines changed through 24h of recovery. SRs have agonist/antagonist actions, such that the role of cytokines in the HS syndrome remains poorly understood. Author views not official US Army or DoD policy.
Sorting Nexin 1 is Required for Dopamine D5 Receptor Function in Human Proximal Tubule Cells
Authors: Villar Van Anthony Mayor , Jones John Edward , Armando Ines , Palmes‐Saloma Cynthia , Li Hewang , Pascua Annabelle Morano , Wang Xiaoyan , Lu Quansheng , Felder Robin A , Jose Pedro A
Published: June 2021

The peripheral dopaminergic system plays a crucial role in blood pressure regulation through its actions on renal hemodynamics and epithelial ion transport. The dopamine D5 receptor (D5R) interacts with sorting nexin 1 (SNX1), a protein involved in receptor retrieval from the trans‐Golgi network. In this report, we elucidated the temporal and spatial aspects of this interaction in both in vitro (human proximal tubule cells and HEK293 cells stably expressing human D5R) and in vivo (Wistar Kyoto rats) models through FRET and confocal microscopy. Inhibition of SNX1 function via a PI3 kinase inhibitor or SNX1 expression by RNAi resulted in the failure of D5R internalization, the blunting of the agonist‐induced cAMP response, and the up‐regulation of basal AT1R expression, previously shown to be negatively regulated by D5R. Basal SNX1 mRNA and protein expression were markedly decreased in renal proximal tubule cells of hypertensive subjects compared to those of normotensive controls. These data demonstrate a novel role for SNX1 in G protein‐coupled receptor trafficking and suggest that an intact SNX1 function is important in normal D5R signaling. Moreover, SNX1 down‐regulation results in D5R dysfunction and, thus, may represent a new paradigm in the pathogenesis of essential hypertension.
Rapid distal convoluted tubule and connecting tubule isolation from TRPV5 promoter‐driven EGFP expressing mice
Authors: Hofmeister Marlene Vind , Praetorius Jeppe , Fenton Robert Andrew
Published: June 2021

The renal late distal convoluted tubules (DCT2s) and connecting tubules (CNTs) are sites for the fine‐regulation of Na+‐ and Ca2+‐reabsorption. Their role in Na+‐ and K+‐homeostasis is possibly underestimated, as the tubules are technically difficult to isolate in sufficient numbers and purity for functional analysis. We have developed a transgenic mouse model expressing enhanced green fluorescent protein (EGFP) in DCT2 and CNT driven by the transient receptor potential subfamily V, member 5, promoter. EGFP expression in DCT2 and CNT was validated by double‐ and triple‐fluorescence immunolabeling with specific tubule markers. Freshly isolated DCT2s and CNTs increased their intracellular Ca2+ levels in response to the V2 receptor specific agonist, deamino‐Cys,D‐Arg8‐vasopressin (2x10−10 M), after 1 min of superfusion. In addition, 1α,25‐dihydroxyvitamin D3 concentration dependently (10−10 to 10−8 M) increased intracellular Ca2+ levels. This suggests that 1α,25‐dihydroxyvitamin D3 can act through a non‐genomic signaling pathway in these tubules. In conclusion, the mouse model is suitable for rapid isolation of viable DCT2s and CNTs, and provides an ideal tool for a more exhaustive functional characterization of these segments.
Vitamin E Modulates the Expression of the Hepatic Alpha Tocopherol Transfer Protein (TTP)
Authors: Thakur varsha , Morley Samantha , Manor Danny
Published: June 2021

Alpha tocopherol is the most biologically active form of the vitamin E, which is selectively retained in the body. The hepatic tocopherol transfer protein (TTP) regulates body‐wide levels of vitamin E by facilitating the selective secretion of ‐tocopherol from hepatocytes to circulating lipoproteins. We studied whether vitamin E affects the expression of TTP in cultured HepG2 cells engineered to inducibly express the protein. Treatment with vitamin E (100ìM RRR‐alpha‐tocopherol for 48 hours) increased the steady‐state levels of TTP by attenuating the rate at which the protein is degraded by the proteasome. Fractionation studies indicated that treatment with tocopherol induced translocation of a fraction of the TTP protein from the soluble to the insoluble fraction of the cell. In vitro,vitamin E significantly protected TTP against proteolytic degradation by trypsin.

These observations suggest that vitamin E imparts a distinct conformation on TTP that is associated with localization to a specific cellular compartment, and with decreased availability for proteasomal degradation.

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Amniotic fluid stem cells: growth, characterization and differentiation
Authors: Janz Felipe Lara , Debes Adriana , Duarte Sergio , Cavaglieri Rita , Moron Antonio Fernandes , Zugaib Marcelo , Bydlowski Sergio Paulo
Published: June 2021

Amniotic fluid (AF) present different cellular types in its composition and a specific group of these cells shows multipotent characteristics as high proliferation ratio and increased differentiation ability. The aim of our study is to better characterize these cells and evaluate their plasticity. AF cells were investigated regarding the expression of undifferentiation markers (Oct‐4, Sox‐2, Nanog) by RT‐PCR, surface protein identification by flow cytometry and differentiation assays into adipogenic, chondrogenic and osteogenic lineage, confirmed with Alizarin Red, H&E and Oil Red O stains respectively. AF cells showed high proliferation ratios and expressive doubling time. They also expressed pluripotent stem cell‐specific genes (Oct‐4, Sox‐2, Nanog) and were positive for mesenchymal membrane markers (CD29, CD44, CD133). They were also capable to differentiate into distinct lineages: adipocytes, osteocytes and chondrocytes. In conclusion, AF is a promising source of mesenchymal stem cells with high differentiation potential.

Research governmental support: by CNPq.