bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional characterization of ghrelin secreting A‐like cells isolated from rat gastric mucosa
Authors: Nakamura Eiji , Hasumura Mai , Matsumoto Takuya , Gabriel Ana San , Uneyama Hisayuki , Torii Kunio
Published: June 2021

Recent reports showed that gastric small endocrine cells, especially A‐like cells, produce orexigenic ghrelin and anorexigenic nesfatin‐1, suggesting the important roles in the regulation of food intake and satiety. However, the mechanisms in the direct regulation of ghrelin and nesfatin‐1 secretion from A‐like cells are still unknown. To clarify these, we established A‐like cell culture isolated from rat stomach using a counterflow elutriation. We successfully enriched A‐like cells from the dispersed gastric mucosal cells confirmed by real‐time RT‐PCR and immunocytochemistry. The cultured cells secreted detectable amounts of ghrelin and nesfatin‐1 in cell culture supernatants. Pharmacological analysis revealed that ghrelin secretion was strongly stimulated by adrenalin via beta receptors, but, not by leptin or endothelin‐1. In contrast, nesfatin‐1 secretion was stimulated by endothelin‐1. These results indicate that ghrelin and nesfain‐1 secretions from gastric A‐like cells were differentially regulated by endogenous substances. Thus, our A‐like cell culture is quite valuable model to clarify the direct regulation of hormone secretions from the cells. This method would help to explore the new therapeutic targets by regulating food intake and satiety via gastric hormones.
Psychological Stress and Changes in the Intestinal Microflora Impact Memory in Mice
Authors: Gareau Melanie G , Wine Eytan , Rodrigues David M , Whary Mark T , MacQueen Glenda , Sherman Philip M
Published: June 2021

The brain‐gut axis is a regulator of intestinal physiology and psychological stress is linked to altered gut barrier function, food allergies, and altered behavior.

AIM

To determine the effects of Citrobacter rodentium infection, probiotics, or the absence of a gut microflora on anxiety and memory.

METHODS

The impact of psychological stress (1h water avoidance stress; WAS) was assessed following acute bacterial infection and in germ‐free mice. Mice were infected and anxiety and memory assessed during acute infection (10d) and post‐infection (30d). Whether daily treatment with probiotics could normalize behavior was then determined. Immunohistochemistry for brain‐derived neurotropic factor (BDNF) and c‐Fos was performed.

RESULTS

Anxiety and memory were normal at 10d and 30d in C. rodentium‐infected mice. However, exposure of infected mice to WAS caused memory dysfunction at 10d and 30d post enteric infection. This defect was associated with reduced levels of BDNF and c‐Fos in the CA1 region of the hippocampus. Memory defects were prevented by probiotics, together with normalization of hippocampal c‐Fos. Memory was absent in germ‐free mice, irrespective of stress, in contrast to gut colonized controls.

CONCLUSIONS

Alterations in the intestinal microbiota exert a measurable impact on memory. These findings are relevant to post‐infectious irritable bowel syndrome in humans.

Funding provided by CIHR.
Role of Non‐muscle Myosin Light Chain Kinase in Neutrophil‐mediated Intestinal Barrier Dysfunction During Thermal Injury
Authors: Guo Mingzhang , Sun Chongxiu , Shen Qiang , Frederich Bert J. , Wu Mack H. , Yuan Sarah Y.
Published: June 2021

Neutrophils and non‐muscle myosin light chain kinase (nmMLCK) have been implicated in intestinal microvascular leakage and mucosal hyperpermeability in inflammation and trauma. The aim of the study was to characterize the role of nmMLCK in neutrophil‐dependent gut barrier dysfunction following thermal injury. Histopathological examination revealed that severe burns led to intestinal mucosa inflammation characterized by neutrophil infiltration, epithelial contraction, and narrow villi with blunt brush boarders and loss of goblet cells. These abnormalities were diminished in nmMLCK−/− mice. Intravital microscopic studies showed significant leukocyte adhesion in intestinal microvessels post‐burn, a response blunted in the absence of nmMLCK. Functionally, thermal injury significantly increased the gut lumen‐to‐blood transport of FITC‐dextran, which was attenuated by either neutrophil depletion or nmMLCK deficiency. Stimulation of Caco‐2 cells with activated neutrophils caused a decrease in transcellular electric resistance coupled with MLC phosphorylation, actomyosin ring condensation, and claudin‐1 internalization. The responses were prevented by MLCK inhibition. Taken together, the results suggest that nmMLCK plays an important role in neutrophil‐dependent intestinal barrier dysfunction during inflammatory injury. Supported by NIH RO1 HL‐96640, HL‐61507 and HL‐70752.
Tight junction protein expression by γδ intraepithelial lymphocytes (IELs) regulates interactions between lymphocytes and epithelial cells
Authors: Edelblum Karen L , Marchiando Amanda M , Shen Le , Wang Yingmin , Sperling Anne I , Turner Jerrold R
Published: June 2021

Intraepithelial lymphocytes (IEL) are found in the lateral intercellular space. IELs expressing the γδ T‐cell receptor (γδ IELs) promote to mucosal homeostasis, but their interactions with enterocytes have not been clearly defined. To define these interactions in vivo, mice expressing GFP γδ T cells and mRFP‐ZO‐1 within enterocytes were studied by video microscopy and showed that jejunal γδ IEL trafficking is highly dynamic. After crossing the basement membrane, γδ IELs migrate into villous epithelium, approach the tight junction, and exit; all within minutes. Immunofluorescence microscopy of mouse small intestinal γδ IELs, either in situ or after isolation, demonstrated ZO‐1 and occludin expression at bright cell surface punctae, suggesting a role for these proteins in IEL migration. To model this in vitro, murine IELs were applied to the underside of filter‐grown Caco‐2 monolayers. γδ IELs migrated through the filter and across an artificial basement membrane to settle, transiently, just beneath the tight junction. Interestingly, occludin accumulated at γδ IEL‐Caco‐2 contacts. Although epithelial occludin was recruited to sites of occludin knockout γδ IELs‐Caco‐2 contacts, occludin knockout γδ IELs migrated 4‐fold less efficiently than wild type γδ IELs. Thus, γδ IEL trafficking is highly dynamic, both in vitro and in vivo, and depends, in part, on occludin expressed by γδ IELs.
Functional expression in Xenopus oocytes reveals that human ferroportin is an iron exporter shared with zinc
Authors: Mitchell Colin J , Shawki Ali , Nemeth Elizabeta , Ganz Tomas , Mackenzie Bryan
Published: June 2021

Iron homeostasis is achieved by regulating the intestinal absorption of the metal and its recycling by macrophages. Export of iron from enterocytes or macrophages to the blood is thought to be mediated by ferroportin (Fpn) under the control of hepcidin. Some mutations in Fpn result in macrophage iron loading whereas others, as a result of insensitivity to hepcidin, cause systemic iron overload. Whereas Fpn was identified a decade ago, there remains a paucity of information about how it works. We expressed GFP‐tagged human Fpn in RNA‐injected Xenopus oocytes and observed using confocal microscopy exclusive plasma‐membrane localization. As a first step in its characterization, we established an assay to detect functional expression of Fpn by microinjecting oocytes with 55Fe and measuring the 55Fe efflux over 30 min. Expression of Fpn increased the first‐order rate constants describing 55Fe efflux between 4‐fold and 100‐fold over control. Fpn‐mediated 55Fe efflux was maximal when the injectate contained nitrilotriacetic acid (NTA), and when we provided apotransferrin, bathophenanthroline disulfonate (BPS) and NTA in the incubation medium, whereas inclusion of ceruloplasmin had only a modest effect. Fpn‐mediated 55Fe was accelerated in pH 8.2 medium compared with pH 7.2 or pH 6.2 (P < 0.001). Fpn expression also stimulated the efflux of 65Zn (P < 0.001) but not of 54Mn, 64Cu or 109Cd (P > 0.11). We conclude that Fpn mediates cellular iron efflux and that Zn2+ counts among its narrow substrate profile.
Autonomic control and antioxidant status during early pregnancy in formerly preeclamptic women
Authors: Janssen Ben , Courtar Dorette , Ekhart Timo , Haenen Guido , Peeters Louis
Published: June 2021

In formerly pre‐eclamptic women plasma volume is lower (LPV group) than in women who had a normal pregnancy and normal plasma volume (NPV). Blood pressure variability (BPV), heart rate variability (HRV) and baroreflex sensitivity (BRS) were compared in women with LPV (47.7 ml/kg lean body mass, n=7) and NPV (57.2 ml/kg lbm, n=7) before and during a new pregnancy at 8, 12, 16 and 20 weeks during pregnancy. The anti‐oxidant status was assessed if changes in oxidative stress were related to those in BRS. Systolic BP was slightly (about 5 mmHg) lower in LPV than in NPV before and during pregnancy. Before pregnancy low frequency LF‐BPV and LF‐HRV were slightly greater in LPV than in NPV. However, during pregnancy, both LF‐BPV and LF‐HRV were slightly lower in LPV than NPV. BRS increased during pregnancy by 20% in NPV and by 40% in LPV, but between group differences were not statistically significant. Overall these data indicate that autonomic adaptations to pregnancy and changes in BPV, HRV and BRS were comparable in women with LPV and NPV. Remarkably, plasma anti‐oxidant levels fell during pregnancy in NPV from 484 to 384 μM Trolox equivalents and in LPV and from 487 to 413 μM Trolox equivalents. These data indicate that oxidative stress increases during early pregnancy and therefore are in contrast with previous suggestions that increased oxidative stress decreases BRS.
DORSOMEDIAL HYPOTHALAMUS AND MEDULLARY RAPHE MEDIATE RESPIRATORY AROUSAL RESPONSES IN RATS
Authors: Nalivaiko Eugene , Beig Mirza I , Xavier Carlos H , Fontes Marco AP
Published: June 2021

We aimed to reveal which brain areas are involved in the generation of tachypnoeic alerting responses. In male Wistar rats, we recorded heart rate (ECG telemetry) and respiration (plethysmography). Microinjection of muscimol or vehicle was made into the dorsomedial hypothalamus (DMH, Group 1, n=6) or the medullary raphe (Group 2, n=6). Basal respiratory rate (RR, 90±5 and 88±8 cpm) and heart rate (HR, 377±10 and 354±10 bpm) did not differ between groups. Alerting stimuli (tap, side move and gbright light) did not affect HR. In Group 1 (vehicle), RR raised by 105±27, 150±27 and 183±15.03 cpm after tap, move and light stimuli respectively. Inhibition of the DMH suppressed these responses by 92, 83 and 90% (tap/move/light). In Group 2 (vehicle), RR raised by 99±14, 118±23 and 165±20 cpm after tap, move and light stimuli respectively. Inhibition of the medullary raphe suppressed these responses by 81, 75, and 71% (tap/move/light). Blockade of the DMH or raphe region had no effect on basal levels of RR or HR. We conclude that DMH and MR are not involved in the control of respiratory rate at rest, but their integrity is essential for mediating tachypnoeic arousal responses. Support: NHMRC & Fapemig/CNPq
Activated Projection from Arcuate Nucleus to Ventrolateral Periaqueductal Gray during Electroacupuncture: relation to neurons containing VGLUT3
Authors: Guo Zhiling , Liu Yu , Longhurst John C.
Published: June 2021

Our previous studies have shown that electroacupuncture (EA) at the Neiguan‐Jianshi acupoints (P5–P6, overlying the median nerve) attenuates sympathoexcitatory responses through activation of arcuate nucleus (ARC) and ventrolateral periaqueductal gray (vlPAG). Activation of the ARC leads to neuronal excitation of the vlPAG during EA. Neurons activated by EA contain vesicular glutamate transporter 3 (VGLUT3), a potential marker for glutamate. However, direct neuronal projections from the ARC to the vlPAG that could participate in central neural processing during EA have not been identified. Thus, the present study evaluated neuronal projections from the ARC to the vlPAG, especially with respect to neurons activated by EA. Seven to ten days after unilateral microinjection of a rodamine‐conjugated microsphere retrograde tracer (100 nl) into the vlPAG, rats were subjected to EA or served as a sham‐operated control. EA was performed for 30 min at P5–P6 acupoints bilaterally. Perikarya containing the microsphere tracer were found in the ARC of both groups. Compared to controls (needle placement without electrical stimulation, n=5), c‐Fos immunoreactivity and neurons double‐labeled with c‐Fos, an immediate early gene and the tracer were increased significantly in the ARC of EA‐treated rats (n=6, P<0.01). Moreover, some neurons were triple‐labeled with c‐Fos, tracer and VGLUT3 in the ARC following EA stimulation (P<0.01). These results suggest that a direct neural pathway projecting from the ARC to the vlPAG may participate in EA‐modulation of sympathetic activity and that VGLUT3‐containing neurons are an important neuronal phenotype involved in this process (supported by NIH grants HL‐72125 and HL‐63313).
The provision of the fermentable fibers soy polysaccharides and fructooligosaccharides increases short‐chain fatty acid transporter abundance in healthy piglets
Authors: Woodard Jennifer , Correa‐Matos Nancy J , Tappenden Kelly A
Published: June 2021

Glucagon‐like peptide 2 (GLP‐2) is an intestinotrophic hormone released from the distal ileum and colon shown to be stimulated by short‐chain fatty acids (SCFAs). Previous in vitro work has shown SCFA receptors and transporters play a role in mediating GLP‐2 production. Therefore, the aim of this study was to examine SLC5A8, MCT‐1, GPR43, and T2R38 abundance in vivo following SCFA production. Piglets were randomized to a control formula, formula supplemented with 7.5 g/L soy polysaccharides (SPS), or formula supplemented with 7.5 g/L fructooligosaccharides (FOS) for 14 days. Expression of GPR43 and T2R38 was not significantly different between the treatment groups. Colonic, but not ileal, MCT‐1 abundance was significantly elevated with SPS (1.35 vs. 2.67 MCT‐1/18S; p = 0.0163); expression in the FOS treated animals was intermediate, but not significantly different from either the control or SPS groups. The abundance of SLC5A8 was significantly elevated in the ileum, but not the colon, with FOS fermentation (0.388 vs. 0.880 SLC5A8/18S; p = 0.0578), while SPS was not significantly different than the control animals. Together, these results indicate that SCFAs increase transporter mRNA abundance, particularly in the intestinal segment in which the fiber was fermented, and thus may have implications for the types of fermentable substrates best suited for stimulating GLP‐2.

Grant Funding Source: NIH T32 DK59802
The influence of beat‐to‐beat changes in muscle sympathetic nerve activity on vascular conductance in humans
Authors: Fairfax Seth T. , Young Colin N. , Fadel Paul J.
Published: June 2021

Sympathetic nerve activity (SNA) is important for beat‐to‐beat regulation of arterial blood pressure (BP). Previous studies have indicated that a burst of muscle SNA results in a peak BP response of ~3 mmHg with a latency of 5.5 seconds. This delay is thought to represent the transduction time of a SNA burst into a change in vascular smooth muscle tone. However, the influence of a SNA burst on beat‐to‐beat alterations in vascular conductance has not been clearly identified. Therefore, in 5 young men, muscle SNA, diastolic BP and brachial artery blood flow were measured. Changes in diastolic BP and calculated forearm vascular conductance (FVC) were characterized for 15 cardiac cycles following each individual muscle SNA burst to describe the latency and magnitude of peak changes in diastolic BP and FVC to the burst of muscle SNA. A clear increase in DBP (Δ 2.0±0.2 mmHg) followed a burst of muscle SNA with a latency of 5.9±0.7 seconds. In contrast, decreases in FVC after a burst of muscle SNA were not readily apparent. These preliminary results suggest that a clear magnitude and latency for increases in diastolic BP following a burst of muscle SNA can be identified on a beat to beat basis; however, related decreases in FVC are not easily observed. The pattern of muscle SNA firing (single burst vs. volley of bursts) and burst size warrants additional consideration. Support: NIH RO1HL093167
Sex‐dependent molecular alterations during development of alcoholic cardiomyopathy in rat
Authors: Fogle Rachel LaDean , Vary Thomas C
Published: June 2021

Excessive, chronic alcohol consumption can result in alcoholic heart muscle disease (AHMD) and, ultimately, heart failure. Functional and structural damage to the myocardium is consistent with a shift in myocardial protein expression and activity that occurs as the disease progresses. The current study aims to link alcohol‐induced alterations in expression of specific proteins with the differences between male and female using a chronic rat model. This study uses isobaric tags for relative and absolute quantitation (iTRAQ) followed by mass spectrometry to delineate shifts in protein expression in rat myocardium as a consequence of chronic (18 week) alcohol intake. Echocardiography allowed the early detection of changes to the heart. Consuming alcohol for 16 weeks lowered stroke volume and left ventricular diastolic volume in both sexes. Males showed additional significant declines in left ventricular mass and end‐diastolic dimension. At the end of the alcohol‐feeding regimen, hearts were fractionated into myofibrillar, sarcoplasmic, mitochondrial, and nuclear fractions. Early interpretation of the proteomic data obtained from mitochondrial fractions identified variations in structural proteins as well as proteins involved in oxidative phosphorylation and electron transport.

NIAAA R01 AA012814 (TCV) and PA Dept of Health Tobacco Settlement Award (RLF)
Reduction of Na/H exchanger‐1 expression limits cardiac injury in mice with chronic intermittent hypoxia
Authors: Chen Ling , Zhang Jin , Prasad Vikram , Scharf Steven M
Published: June 2021

Obstructive sleep apnea causes chronic intermittent hypoxia (CIH) that contributes to the developments of cardiovascular complications via mechanisms yet to be defined. We hypothesized that Na/H exchanger‐1 (NHE1), the primary intracellular pH regulator, mediates cardiac injury induced by CIH. Heterozygous NHE1 knockout mice (KO) and their wild type siblings (WT) were exposed to either CIH (5% nadir O2 for 1 min, following by 21% O2 for 1 min; 10 daytime hours per day for 8 weeks) or sham control (21% O2 constantly). At 8 weeks and compared to WT/sham, WT/CIH showed a 10% increases in mean blood pressure (MBP), a 2‐fold increases in cardiac NHE1 mRNA expression, and evidence of cardiac injury as suggested by elevated left ventricular (LV) cardiomyocyte size and apoptotic cardiomyocytes, as well as increased LV posterior wall thickness and decreased LV fractional shortening measured by echocardiography. Compared to WT/CIH, KO/CIH had similar increases in MBP, 40% NHE1 expression (but a 30% higher than that in KO/sham), and reduced cardiac injury. In conclusion, CIH increases MAP and NHE1 expression, and leads to cardiac injury. Reduction of NHE1 expression limits CIH‐induced cardiac injury, but not MAP elevation.

Supported by AHA.
Effect of Digoxin on Hypoxic Pulmonary Hypertension (HPH)
Authors: Abud Edsel , Undem Clark , Sylvester J T , Semenza Gregg L , Shimoda Larissa A
Published: June 2021

Many lung diseases induce HPH, leading to increased mortality. HPH, caused by vascular contraction and remodeling, is associated with increased intracellular pH (pHi) and calcium concentration ([Ca2+]i) in pulmonary arterial smooth muscle cells (PASMCs). Hypoxia‐inducible factor 1 (HIF‐1), a transcription factor, plays a critical role in the development of HPH. Recently, digoxin (Dig) was found to inhibit HIF‐1. We hypothesized that Dig could attenuate the development of HPH. Mice were weighed and injected daily with saline or Dig (low = 0.2 mg/kg or high = 1.0 mg/kg; i.p.) and kept in hypoxia (10% O2) or room air. After 21 days, right ventricular (RV) pressures were measured via closed‐chest puncture. Hypoxia increased RV pressures in mice receiving saline. Low Dig reduced, and high Dig normalized, RV pressure in hypoxic mice. RV hypertrophy (RVH) increased in hypoxic mice treated with saline or low Dig, but was reduced in hypoxic mice receiving high Dig. Hypoxia increased hematocrit in mice treated with saline or low Dig, whereas high Dig reduced the hypoxia‐induced increase in hematocrit. Both pHi and [Ca2+]i increased in PASMCs from saline‐treated hypoxic mice, and treatment with low or high Dig prevented these changes in ion homeostasis. From these results, we conclude that digoxin attenuates pulmonary vascular responses to chronic hypoxia and may be a possible candidate for treatment of HPH.

HL67191, GM008074
Two‐compartment model of rat renal inner medullary vasculature: relevance to countercurrent exchange and medullary blood flow
Authors: Pannabecker Thomas L. , Kim Julie , Yuan Justin , Hoopes Brandi , Laufenberg Collin , Sadideen Zeyad
Published: June 2021

Functional architecture of descending and ascending vasa recta (DVR, AVR) was examined between 0.3 and 3.0 mm below the outer‐inner medullary (OM‐IM) boundary to investigate countercurrent exchange and blood flow. All UT‐B‐positive DVR (DVRUTB) lie between CD clusters, in the intercluster region (compartment one). 75% of DVRUTB have terminal fenestrated extensions that descend 16 ± 3% of the length of the contiguous DVRUTB prior to formation of the first branch. This branch point is indicative of vessel entry into the intracluster region (compartment two), the region dominated by CDs. We distinguish AVR as fenestrated vessels that lie in the intercluster region and that do not abut CDs and do abut DVR (AVR1) or that abut neither CDs nor DVR (AVR2; AVR1+2 mean length ~1.5 mm). These contrast with fenestrated vessels that lie in the intracluster region and abut CDs (AVR3; mean length ~0.6 mm). The AVR1:DVRUTB ratio is ~1.0, the AVR2:DVRUTB ratio is ~1.8, and the ratio for the full complement of vessels is ~4.0. For each vascular bundle, AVR1+2 arise from the intracluster region about 500 μm deeper than the depth to which DVRUTB descend. AVR3 link DVR with AVR1+2, and AVR3 provide a direct pathway for fluid/solute uptake from CDs as they ascend within the CD cluster. Some proportion of AVR3 pass directly into the OM, distant from vascular bundles. A two compartment model supports multiple modes of IM blood flow regulation. DK16294
Urinary excretion of angiotensinogen in male and female Sprague Dawley rats during angiotensin II infusion and high salt diet
Authors: Rands Vicky F. , Byrd Charlene , Seth Dale , Kobori Hiroyuki , Prieto Minolfa C
Published: June 2021

We have found that urinary angiotensinogen (AGT) excretion (uAGT) differs between male(♂)and female(♀) Sprague Dawley (SD) rats on a normal salt diet (♂: 60±26 vs ♀: 4.5 ±0.2ng/day, p<0.05). In this study, we examined if the effects of a high salt (HS) diet on uAGT exhibits sexual dimorphism during Angiotensin (ANG) II dependent hypertension. Four groups of age‐matched (8 weeks) male (n= 20) and female (n=17) SD rats were studied: 1) high salt diet (HS; 8%NaCl); 2) ANG II (80ng/min, sc; for 14 days); 3) ANG II + HS; 4) ANG II + HS + Candesartan. (CAND, 25 mg/L in drinking water). uAGT levels were measured by enzyme immunoassay. HS diet alone did not raise uAGT levels (♂: 107±23 vs. ♀: 5±2ng/day) from baseline; however, AGT increased significantly during ANG II infusion (♂: 1107±1061 vs ♀: 206±74ng/day, p<0.01) and was augmented even further in ANG II + HS (♂: 4363±1673 vs. ♀: 8022±1300ng/day, p=ns). Candesartan ameliorated this effect with a better response in female than in male rats.(♂: 240±28 vs. ♀: 10±4 ng/day, p< 0.01). The results demonstrate a sexual dimorphism in the uAGT excretion rate during baseline conditions and HS diet characterized by higher levels in the males. The augmentation of uAGT with HS + ANG II is ameliorated more effectively by ARB treatment in female ANG II hypertensive rats than in males.

NIH (P20 RR 017659), Tulane‐BIRCWH (K12HD043451)
Diabetic Natriuresis is Due to the Loss of Insulin Dependent Proximal Tubular Sodium Reabsorption
Authors: Manhiani Marlina , Sheppard Tuere A , Allen Ashlyn J , Cormican Michael T , Brands Michael W
Published: June 2021

This study tested the role of hypo‐insulinemia in mediating the natriuresis in Type I diabetes. A 6‐day diabetic period was induced using either decreased insulin administration to induce hyperglycemia (D group), or clamped insulin administration (DG group) at the control‐period infusion rate and hyperglycemia was induced by infusing glucose continuously i.v. During diabetes, blood glucose increased from normal to 449±40 mg/dl in the D group, and 470±56 mg/dl in the DG group. Control urinary sodium excretion averaged 71±5 mEq/day and 69±7 in the D and DG groups respectively. Urinary sodium excretion increased during diabetes in the D group averaging 125±24 by day 5 while decreased to 54±9 by day 5 in the DG group. Fractional sodium reabsorption (FNaR) decreased by day 5 in the D group (99.8±0.02 vs. 98.2±0.08%, p<0.05) but tended to increase by day 5 in the DG group. Estimated proximal fractional sodium reabsorption (PFNaR) decreased from control during diabetes in the D group while PFNaR increased during the diabetic period in the DG group. This was corroborated by fractional lithium reabsorption, an indicator of PFNaR, which increased during the diabetic period in the DG group from 73±6% to 89±4 day 2 and 92±1 day 5, p<0.05) These data suggest that the decrease in plasma insulin in Type I diabetes plays a significant role in mediating the diabetic natriuresis by decreasing proximal tubular sodium reabsorption. (HL56259)
Central mineralocorticoid receptor blockade reduces sodium appetite in rats: new evidence for an old effect
Authors: Formenti Silmara , Schoorlemmer Guus , Menani José V. , Colombari Eduardo
Published: June 2021

In a previous study we showed that chronic infusion of the mineralocorticoid hormone aldosterone into the fourth ventricle (4V) induces robust dose‐dependent daily sodium intake. In the present study we investigated the effects of the mineralocorticoid receptor (MR) antagonist, RU 28318, injected into the 4V or lateral ventricle (LV) on sodium intake induced by 24 h of sodium depletion (treatment with furosemide, 20 mg/kg b.w., s.c., followed by 24 h of sodium deficient diet), 9 days of sodium deficient diet (0.005% Na+) or chronic treatment with deoxycorticosterone acetate (DOCA, 2 mg/day, s.c. for 7 days). Male Wistar Hannover rats (280–350 g, n=4–7/group) with stainless steel guide cannulas implanted into the 4V or into the LV were used. RU 28318 (100 ng) into the 4V reduced 0.3 M NaCl intake induced by 24 h of sodium depletion (6±1 vs. vehicle: 12±2 mL/2 h) or by sodium deficient diet (1±1 vs. vehicle: 9.5±3 mL/2 h). However, injection of RU 28318 (100 ng) into the LV did not affect sodium intake induced by 24 h of sodium depletion (15±3 vs. vehicle: 15±3 mL/2h). RU 28318 (500 ng) into the 4V also produced no effect on sodium intake induced by DOCA (14±3 vs. vehicle: 11±2 mL/2 h). These data reinforce findings suggesting that hindbrain MRs are involved on aldosterone‐induced sodium appetite. However, it seems they are not involved on DOCA‐induced sodium intake.

Supported by: FAPESP, CNPq, CAPES.
Oropharyngeal regulation of urine output and cardiovascular function in rats with osmotic stimulation by sham ingestion
Authors: Gottlieb Helmut B , Knight David W , Ji Lisa , Cunningham JT
Published: June 2021

Studies indicate that oropharyngeal afferents participate in the central regulation of hydration status although the role of these afferents in the rat remains controversial. Therefore, we tested the renal effects of oropharyngeal stimulation with water, isotonic saline, and 3% hypertonic saline. We repeated these experiments in oropharyngeal nerve lesioned rats. Anesthetized rats were implanted with femoral catheters, esophageal fistulae, and bladder catheters. During the experiment, each rat was infused with isotonic saline (55 ml/min, iv) containing ketamine (1 mg/kg/min) and xylazine (20 mg/kg/min). After a 1 h baseline, rats were infused orally (1 ml/min; 2 min.) with either 0.9% isotonic saline (n=8), water (n=8), or 3% hypertonic saline (n=6) and urine was collected in 10 min. aliquots for 1 h. Intact animals infused with water produced an early natriuresis and delayed dieresis. Infusions of isotonic saline had no effect. Infusions of 3% hypertonic solution produced an antidiuresis that was concurrent with a natriuresis. Removal of the oralpharyngeal nerves abolished the responses to water and hypertonic saline. The renal effects of oropharyngeal water infusion are consistent with an osmotically‐driven regulation of AVP release. Further, the oralpharyngeal nerves may serve as an anticipatory reflex to perceived ingestion of a volume that will dilute or concentrate plasma osmolality. R01 HL062576.
The effect of hypoxia on the ventilatory and cerebral blood flow (CBF) responses to CO2
Authors: Battisti Anne , Fisher Joseph , Duffin James
Published: June 2021

Objectives

This study examines the proposed approach to a standard measurement regime for characterizing the respiratory chemoreflexes as detailed by Duffin (2007) and develops a similar approach to characterize cerebrovascular responsiveness to hypoxia and CO2. We measured the effect of hypoxia on the isoxic modified rebreathing ventilatory and CBF responses to CO2 in terms of sensitivity, ventilatory recruitment threshold and wakefulness ventilation at 2 levels of isoxia (PO2 = 50 mmHg & 150 mmHg). We also measured the effect of hypercapnia on the isocapnic steady‐state (SS) ventilatory and CBF responses to hypoxia at 3 levels of hypercapnia (PCO2 = 4, 7 & 10 mmHg above resting). Finally we compared the isoxic rebreathing measurements with the SS isocapnic measurements.

Method

Custom designed software controlling a specialized gas blending device (RespirAct™; Thornhill Research) was used to implement the steady state and modified rebreathing protocols. Ventilation (VE), PetCO2 and PetO2, middle cerebral artery velocity (MCAv), and blood pressure (BP) were recorded.

Results

9 subjects completed the tests (6 male).

This graph displays the SS response of VE, MCAv and BP to CO2 (PCO2 = 50 mmHg) first in hyperoxia (PO2 = 150 mmHg) and then in hypoxia (PO2 = 50 mmHg). We compared the SS and rebreathing ventilation and CBF responses.

Rebreathing analysis (figures above) gives us interesting results. Ventilation shows a higher slope (increase of sensitivity) as well as a lower threshold during the Hypoxic phase as expected. For the MCAv, the interindividual variability is larger than expected. This example shows same slope, as expected. Comparison with SS method is more complex. Some subjects might have limitations of the MCAv increase. Further analysis still need to be done.
Attenuated Myocardial Effects of Prenatal Stress on Female Rats
Authors: Chen Fangping , Mullenax Heather , Xie Sherry , Finkel Mitchell S.
Published: June 2021

Third trimester maternal stress (prenatal stress; PS) has been demonstrated to result in behavioral changes in male offspring consistent with mood disorders in humans. Predominant effects on male offspring are consistent findings, regardless of the PS paradigm used. We have previously reported echo and hemodynamic evidence in vivo and cardiac myocyte evidence in vitro of myocardial dysfunction and adrenergic desensitization in male PS rats followed by restraint stress (R) (PS+R) compared with matched Control offspring of non‐stressed dams (Control + R). Accordingly, we sought to determine if female PS rats were protected from myocardial effects of PS as they are from behavioral effects. Consistent with prior behavioral assessments, there were no significant differences between baseline hemodynamics (+dp/dt;−dp/dt) in awake female PS+R vs female Control+R (p=NS; n=6–8). However, more subtle effects of PS+R on contractility (+dp/dt) were demonstrable in female PS+R following beta adrenergic stimulation with isoproterenol (ISO) (p<0.05; n=6–8) or isoflurane anesthesia (p<0.05; n=6–8). In addition, cardiac myocytes isolated from female PS+R also revealed a blunted inotropic response to ISO vs female PS+R (p<0.05; n=15). However, female PS+R were more responsive to ISO than male PS+R (p<0.05; n=15). These data support a protective effect of female sex on myocardial, as well as behavioral consequences of PS.
Vitamin A value of high beta‐carotene yellow maize in humans
Authors: Muzhingi Tawanda , Tang Guangwen , Siwela Andrew , Gadaga Henry T , Gruksak Micheal
Published: June 2021

This study determined the vitaminA value of yellow maize β‐carotene in humans. Yellow maize was grown in a 23 atom% 2H2O cultural solution. Intrinsically labeled yellow maize β‐carotene showed the highest abundance of enrichment as[2H9] β‐carotene. Eight healthy men with mean age of 48.4±10, serum retinol concentration 59.2±17.l μg/dL, and BMI 22.4±3.1 volunteered and adhered to study diets. On day‐1 after a fasting blood sample draw, subjects consumed 300g of yellow maize porridge containing 1mg β‐carotene, 20g of fat and a blank oil capsule. On day‐8 a fasting blood sample was drawn, subjects consumed 1mg [13C10] retinyl acetate in oil capsule and 300g of white maize porridge with 20g of fat. Thirty six bloodsamples were collected from each subject over a span of 36‐days. Concentrations and enrichments of retinol and β‐carotenein dose and serum were determined by using HPLC, GC/MS and LC/MS.The AUC of retinol from yellow maize β‐carotene (1 mg) was 72.9nmole·d and the AUC of retinol 13C10 (1mg) was 161.1nmole·d. The conversion factor of yellow maize β‐caroteneto retinol was 2.7 ± 1.0 to 1 by weight. The conversion factor was negatively correlated with age and serum concentration of retinol (p<0.05). In eight healthy Zimbabwean men 1mg yellow maize β‐caroteneconsumed with 20 g fat has the same vitamin A activity as 0.37 mg retinol. (Funding: Nutricia Research Foundation and Pioneer Hi‐Bred International)
Transforming Growth Factor‐beta Expression in Obesity‐Linked kidney Disease
Authors: Maddox David A. , Ingebrigston Dane
Published: June 2021

Objective

To gain insight into the matrix synthesis cascade in obesity‐linked renal disease.

Methods

The study used obese and lean Zucker rats. Obese rats six weeks of age were fed either ad libitum (Ob‐ ad lib) or restricted (Ob‐Res) in food intake to that consumed by lean Zucker controls, then tested at 6, 12, and 18 weeks for each condition. Tissue mRNA was extracted from kidney cortex (~40 mg) and cDNA was synthesized. Multiplex Real‐Time PCR was performed for transforming growth factors (TGF‐β1, TGF‐ β2 and TGF‐ β3). Housekeeping genes were Rpl19 and Cyclophilin‐A.

Summary of Results

Increased protein excretion in the Ob ad lib group was evident by 12 weeks of age compared to lean animals and increased further at 18 weeks. Food restriction beginning at 6 weeks of age prevented this from occurring. TGF‐β1 gene expression was elevated at 12 weeks of age in the Ob ad‐lib animals compared to lean or Ob‐Res. TGF‐β3 gene expression was increased at 6 weeks in the Ob ad lib compared to lean and remained elevated at 12 and 18 weeks of age compared to lean or Ob‐Res groups. The TGF‐β2 (not shown) had no elevated expression levels in any group.

Conclusions

Increased gene expression levels of TGF‐β1 and TGF‐ β3 suggest a role for these growth factors early in the development of obesity‐linked kidney disease, a conclusion strengthened by the complete prevention of these changes by food restriction which also prevented proteinuria. Project funded by VA Merit Review award, Avera Research Institute, and Sanford School of Medicine of the University of South Dakota.
Cell‐Type Specific Patterns of Gene Expression Predict the Existence of VEGF and PLGF Homo‐ and Heterodimer Gradients in the Vascular Wall
Authors: Xiang Lingjin , Yin Wei , Lloyd Pamela G.
Published: June 2021

Vascular endothelial growth factor (VEGF‐A) and placenta growth factor (PLGF) are related angiogenic factors which exist as dimers in vivo. Interestingly, VEGF‐A and PLGF can form heterodimers with unique signaling properties. Thus, the PLGF:VEGF‐A ratio in vascular cells may be physiologically significant. We hypothesized that PLGF and VEGF‐A would show cell‐type specific patterns of expression in smooth muscle cells (SMC) vs endothelial cells (EC). Ten cell lines were studied (4 SMC and 6 EC), including both established and primary cell lines derived from both conduit and microvascular sources. PLGF gene expression varied over a ~4000‐fold range in the cell lines tested, with expression in EC⋙SMC (p<0.05). In contrast, VEGF‐A expression varied over a smaller range (~20‐fold), with expression in SMC>EC (p<0.05). These data suggest that gradients of PLGF and VEGF‐A homodimers and PLGF:VEGF heterodimers may exist across the vascular wall. Based on our data from cultured cells, PLGF homodimers are predicted to be the major species found in and around the EC layer, whereas VEGF homodimers and PLGF:VEGF heterodimers are expected to predominate in the media. Additional studies are needed to assess whether PLGF and VEGF‐A gradients do in fact exist in the vascular wall, and to identify the functional role of such gradients in vivo, e.g. in regulating monocyte infiltration. Support: NIH R01 HL084494 (PL).
Long lasting high fat feeding increases the capillary density in the skeletal muscle of mice
Authors: Silvennoinen Mika Matti , Rinnankoski‐Tuikka Rita , Kivelä Riikka , Lehti Maarit , Torvinen Sira , Vuento Meri , Kainulainen Heikki
Published: June 2021

The aim of our study was to find out the effects of high fat diet, voluntary running and their combination on oxidative capacity of skeletal muscle. Sixty C57BL/6J mice were randomly divided into four groups (15 in each): control diet (C), control diet + running (CR), high fat diet (HF), and high fat diet + running (HFR). All animals were housed individually and they had free access to control (10% fat) or high fat diet (60%) and water. CR and HFR groups had free access to running wheels. After 19 weeks animals were sacrificed and muscle samples were collected. The amount of subsarcolemmal mitochondria of soleus muscle was determined using electron microscopy. Capillary density was analyzed from the sections of quadriceps femoris. Capillaries were visualized by staining with Isolectin‐GS‐IB4 combined with dystrophin staining to visualize muscle fibers. Voluntary running or diet did not have significant effect on amount of mitochondria. High fat diet increased the capillary density (HF vs. C, +21%, P<0.005). Voluntary running increased the capillary density only in the control diet group (CR vs. C, +32%, P<0.01). As a novel finding, our results indicate that skeletal muscles of mice adapt to high fat diet by increasing the capillary density. The study was supported by the Finnish Ministry of Education.
The Role of IP3 Receptors in Generating Calcium Waves and Cerebral Myogenic Tone
Authors: Muftia Rania , Welsh Suzanne Brett , El‐Rahman Rasha Abd , Jones Peter , Chen Wayne , Welsh Donald
Published: June 2021

This study of the cerebral vasculature examined whether: 1) elevated intravascular pressure induces asynchronous Ca2+ waves by activating IP3 receptors; and 2) these events drive myogenic tone development by augmenting myosin light chain phosphorylation (LC20). Rat cerebral arteries pressurized between 20–100 mmHg were examined in the absence and presence of agents that deplete the sarcoplasmic reticulum (SR) of Ca2+ or block IP3 receptors. Diameter and membrane potential (VM) were assessed using conventional techniques whereas Ca2+ waves and LC20 phosphorylation were monitored using confocal microscopy and western blot analysis, respectively. Elevated intravascular pressure increased the proportion of smooth muscle cells firing Ca2+ waves as well as the frequency of these voltage‐independent events. Agents that deplete the SR of Ca2+ (ryanodine, CPA and Thapsigargin) or block IP3 receptors (2‐APB or Xestospongin‐C) eliminated Ca2+ wave generation. The loss of Ca2+ waves attenuated LC20 phosphorylation and attenuated myogenic tone development. It did not, however, alter pressure‐induced depolarization in an intact artery In summary, this study shows that pressure‐induced Ca2+ waves facilitate cerebral myogenic tone by providing a proportion of the Ca2+ required to activate myosin light chain kinase. Supported by NSERC.
Discrepancy between blood flow and tissue‐oxygen in the brain during acute cerebral hypoperfusion in anesthetized rats
Authors: Maruyama Satoshi , Sato Yoshiaki , Kemuriyama Takehito , Tandai‐Hiruma Megumi , Ohta Hiroyuki , Manabe Tomoko , Takahata Tomofumi , Nishida Yasuhiro
Published: June 2021

Acute cerebral hypoperfusion is experienced on head‐to‐foot inertial force (+Gz). However, no report has been found about redistribution of blood flow inside the brain. Then we estimate blood flow (BF) and tissue‐oxygen (PO2) level responses to +Gz stress in the cortex and hippocampus. In 13 male Sprague‐Dawley rats were anesthetized with urethane, cortical and hippocampal blood flows or these PO2 levels were measured by a laser blood flow meter with the glass fiber prove or a PO2 monitor with the polarographic oxygen electrodes, respectively. +Gz stress of 3.0 G by a centrifuge decreased arterial pressure at a level of the brain to 39.3 ± 13.5 mmHg, cortical and hippocampal BF to 77.0 ± 17.2% and 66.6 ± 15.1 %, respectively (NS), and these PO2 to 77.8 ± 14.7 % and 58.9 ± 19.7 %, respectively (p<0.05). After +Gz exposure, PO2 returned significantly slower than BF in both of the hippocampus and the cortex. These results indicate that acute cerebral hypoperfusion may induce more ischemic damages in the hippocampus than the cortex.
PAI‐1 derived peptide EEIIMD prevents impairment of hypercapnic and hypotensive cerebrovasodilation by augmenting p38 MAPK upregulation after cerebral hypoxia/ischemia
Authors: Armstead William M , Riley John , Kiessling John Willis , Cines Douglas B , Higazi Abd Al‐Roof
Published: June 2021

Babies are frequently exposed to hypoxia and ischemia (H/I) during the perinatal period as a result of stroke, problems with delivery or post delivery respiratory management. The sole approved treatment for acute stroke is tissue type plasminogen activator (tPA). H/I impairs pial artery dilation (PAD) to hypercapnia and hypotension, tPA aggravates impairment, and the PAI‐1 derived peptide EEIIMD largely prevents it. Mitogen activated protein kinase (MAPK), a family of at least 3 kinases, ERK, p38 and JNK, is upregulated after H/I and ERK contributes to impaired cerebrovasodilation. This study determined the roles of p38 and JNK MAPK in H/I dilator impairment in piglets equipped with a closed cranial window. CSF phosphorylated (activated) p38 MAPK but not JNK MAPK was elevated by H/I, an effect potentiated by EEIIMD. Hypercapnic and hypotensive PAD was blunted by H/I but dilation was maintained by EEIIMD. Hypercapnic and hypotensive PAD after H/I was further impaired by the p38 antagonist SB 203580, but unchanged by the JNK antagonist SP 600125. Isoproterenol induced PAD was unchanged by H/I, EEIIMD, SB 203580, and SP 600125. These data indicate that EEIIMD limits H/I cerebrovasodilator impairment through upregulation of p38 but not JNK. These data suggest that p38 upregulation may offer a novel approach to increase the benefit/risk ratio of thrombolytic therapy for CNS disorders associated with hypoxia/ischemia.
Androgen effects on in vitro and in vivo coronary vasomotor responses in intact and castrated swine
Authors: O'Connor Erin Kathleen , Ivey Jan R , Bowles Douglas K
Published: June 2021

Low endogenous testosterone levels have previously been shown to be a risk factor for the development of coronary artery disease; however, the effects of endogenous testosterone levels on the acute coronary vasomotor responses to androgen administration are not clear. The objective of this study was to compare the effects of acute androgen administration on in vivo coronary blood flow and in vitro coronary microvascular diameter in intact and castrated adult male swine. Pigs were anesthetized and received intracoronary infusions of physiologic levels (1–100 nM) of testosterone (T), the epimer epitestosterone (EpiT), and the metabolite 5 alpha‐dihydrotestosterone (DHT) while left anterior descending coronary blood flow and mean arterial pressure were continuously monitored. Following sacrifice, coronary arterioles were isolated, cannulated, and exposed to physiologic concentrations (1–100 nM) of T, EpiT, and DHT. In vivo, T, EpiT, and DHT produced greater increases in coronary conductance in the intact compared to the castrated males. Similarly, in vitro, percent maximal dilation of microvessels was greater in the intact compared to the castrated males for T, EpiT, and DHT. In conclusion, increases in coronary blood flow and coronary arteriolar relaxation in response to acute androgen administration are influenced by endogenous androgen status. Supported by NIH HL071574.
ENDOPLASMIC RETICULUM STRESS AND PERIPHERAL PREDIABETIC AND DIABETIC NEUROPATHY
Authors: Obrosova Irina G , Watcho Pierre , Maksimchyk Yury , Shevalye Hanna , Stavniichuk Roman , Lupachyk Sergey
Published: June 2021

Endoplasmic reticulum (ER) stress caused by accumulation of unfolded proteins in the ER lumen, contributes to beta‐cell loss, insulin resistance, and plays an important role in the pathogenesis of both Type 1 and Type 2 diabetes. We evaluated the role of this phenomenon in the pathogenesis of prediabetic and diabetic peripheral neuropathy. The experiments have been performed in 1) Zucker lean and Zucker fa/fa rats, a model of obesity and Type 2 prediabetes, and 2) control and STZ‐diabetic rats, a model of Type 1 diabetes. Both 16‐wk‐old Zucker fa/fa rats and STZ‐diabetic rats with 12‐wk duration of STZ‐diabetes displayed ER stress response manifest by overexpression of BiP/GRP78 and GRP94 in the peripheral nerve. They also had nerve conduction velocity (NCV) deficit and small sensory nerve fiber dysfunction. Treatment with the chemical chaperone trimethylamine N‐oxide (TMAO) reversed sensory NCV deficit and alleviated thermal hypoalgesia and tactile allodynia in Zucker fa/fa rats. It also prevented sensory NCV deficit and partially prevented motor NCV deficit and small sensory nerve fiber dysfunction in STZ‐diabetic rats. In conclusion, ER stress is implicated in prediabetic and diabetic peripheral neuropathy. Studies of biochemical mechanisms of ER stress‐induced peripheral nerve damage are in progress.
Ginkgo biloba extract down‐regulates LOX‐1‐mediated endothelial dysfuntion: Involvement of P38‐NF‐κB signaling pathway
Authors: Chen Kai Ling , Ou Hsiu‐Chung , Pi I , You Ya‐Ching , Lee Shin‐Da
Published: June 2021

Oxidized low‐density lipoprotein (oxLDL) is a proatherogenic molecule that accumulates in the vascular wall and contributes to the pathogenesis of vascular dysfuntion.LOX‐1, a lectin‐like oxLDL receptor, is responsible for binding and uptake of oxLDL in endothelial cells. It has been well documented that the activation of LOX‐1 can stimulate the formation of ROS and initiate a cascade of redox‐sensitive singnaling events. Ginkgo biloba extract (GbE), extracted from the leaves of the Ginkgo biloba tree, has been well known about its benefits in cardiovascular and neurological system. In this study, incubation of primary human umbilical vein endothelial cell culture (HUVECs) with oxidized low‐density lipoprotein (oxLDL) was to examine whether GbE protects against oxLDL‐induced endothelial dysfuntion and the underlying mechanisms were explore. Our results showed that GbE reduced ROS production and up‐regulation of LOX‐1 caused by oxLDL. In addition, oxLDL enhances cyclooxygenase 2 (COX‐2) expression through p38 MAPK phosphorylation and consequent NF‐κB translocation. These oxLDL‐induced endothelial dysfuntion were ameliorated dose dependently by GbE. Findings from this study suggest that GbE prevent oxidized LDL‐induced endothelial dysfuntion through down‐regulation of LOX‐1‐mediated singaling implying that GbE has a potential ability in the prevention of atherosclerotic vascular disease.