bluuebunny/crossref_metadata_2025 huggingface dataset:
MODULATION OF RENIN ANGIOTENSIN SYSTEM COMPONENTS BY HIGH GLUCOSE LEVELS IN CULTURE OF COLLECTING DUCT CELLS
Authors: Leite Ana Paula de Oliveira , Aragão Danielle , Ronchi Fernanda Aparecida , Nogueira Marie , Mendes Lys Angela , Moreira Roseli , Jara Zaira , Fiorino Patricia , Casarini Dulce , Farah Vera
Published: June 2021

The increased intrarenal renin‐angiotensin system (RAS) activity contributes to diabetic nephropathy. A high glucose (HG) extracellular concentration (EC) induces activation of RAS in mesangial cells. However, this relationship in the collecting duct was not studied yet. The aim of this study was to evaluate the modulation of RAS in collecting duct cells (mIMCD‐3) induced by HG concentration. At the subconfluence (80%) the cells were divided into three groups: Normal Glucose (5 mM D‐glucose, NG), High Glucose (30 mM D‐glucose, HG) or Mannitol (30 mM, M) and were maintained for 48h in these medium. The ACE and ACE2 activities were measured using a fluorimetric assay and the cellular localization of ACE was examined by immunofluorescence. The RAS components were analyzed by western blotting and HPLC‐UV. Our results showed that ACE activity was significantly lower at intracellular (IC) (27%) and EC (22%) content in the cells of HG group when compared with NG and M. The IC activity of ACE2 was higher in HG and M (68 and 73%, respectively). The protein expression of ACE (69kDa) was lower in groups HG and M. The renin expression was higher in HG when compared to NG and M, with no changes in in other proteins of RAS. The ANG I, ANG II and ANG 1‐7 were higher in HG compared to NG and M, both in IC and in EC content. The ACE migrated to the cell nucleus in the HG mIMCD‐3 cells, what could act like a transcription factor. We also suggest that the HG concentration activates a compensation mechanism avoiding the deleterious effects of ANG II. Financial support: CAPES, FAPESP, MACKPESQUISA.
Internalized Angiotensinogen is Secreted to the Apical Side in Renal Proximal Tubular Cells
Authors: Yeh Alice , Satou Ryousuke
Published: June 2021

Increased angiotensinogen (AGT) production and secretion in renal proximal tubular cells (PTC) contribute to spillovers of AGT in the distal nephron, enhancing intrarenal angiotensin generation and onset of renin‐angiotensin system associated diseases. Recent studies have established megalin mediates AGT endocytosis in PTC and facilitates secretion. Although this mechanism can regulate proximal tubular AGT levels, secretion of internalized AGT to the lumen has not been demonstrated. Under normal conditions, mouse S1 PTC were cultured to stimulate apical polarization for megalin expression. Cells were treated with and without 2ug/mL of histidine tagged mouse recombinant AGT (AGT‐His) up to 48 hours and were detected by an anti‐His tag antibody. Free AGT‐His in the medium was washed out and cells were incubated over a 24 hour period to determine secretion levels of internalized AGT‐His. Higher mRNA levels of megalin in the PTC compared to mouse whole kidney were detected. Immunostaining of megalin indicated localized protein levels at the plasma membrane and cytoplasmic regions in PTC. Western blot analysis showed gradual accumulation of internalized AGT‐His at 1 hour and at augmented levels at 48 hours (32.6 ± 0.11 ng). After 24‐hour secretion, AGT‐His levels were decreased to 26.3± 3.4% comparatively. His‐tagged ELISA revealed 11% of internalized AGT‐His in PTC had been secreted (3.56 ± 0.11 ng), suggesting secretion of AGT‐His and lysosomal degradation. In transwell, AGT‐His was secreted primarily to the apical compared to the basolateral side. These results establish that PTC possess a mechanism underlying secretion of internalized AGT to the apical surface, which may contribute to proximal tubular AGT regulation.
Sodium balance and resistance to Ang II‐induced hypertension, despite NCC and NKCC2 activation, in mice with global deletion of Na,K‐ATPase regulatory subunit FXYD2
Authors: Arystarkhova Elena , Liu Yi Bessie , Ralph Donna , Figtree Gemma , McDonough Alicia , Sweadner Kathleen
Published: June 2021

Na,K‐ATPase generates the driving force for sodium reabsorption in the kidney. Na,K‐ATPase functional properties are regulated by association with small proteins belonging to the FXYD family. Mice with global deletion of the inhibitory subunit, FXYD2, show 25% higher Na,K‐ATPase activity in renal cortex with no evidence of sodium retention. The hypothesis was that activation of basolateral Na,K‐ATPase in Fxyd2‐/‐ mice is compensated by adaptive reductions in apical Na+ transporters to maintain Na+ output equal to Na+ intake. Instead, there was an increase in expression and phosphorylation of sodium chloride co‐transporter, NCC, in DCT. The phosphorylation of the closely related apical NKCC2 also increased. NCC activation is commonly linked to development of renal hypertension. However, no elevation of blood pressure was evident in the knockout mice under resting conditions. Continuous infusion with Ang II (1 mg/kg, 7 days) resulted in an additional 2‐fold increase in phosphorylation of NCC at T53 and S71 in both WT and Fxyd2‐/‐ mice. Yet, an increase in the mean arterial pressure was seen only in the control group, from 88 ± 4 to 99 ± 3 mm Hg, with no changes in Fxyd2‐/‐ mice: 86 ± 5 vs 81 ± 9 mm Hg. Abundance of the total and cleaved (activated) forms of ENaC α‐subunit was not different between genotypes. Thus activation of NCC and NKCC2 may reflect a compensatory response to Na+ loss proximal to the cTAL and DCT in Fxyd2‐/‐ mice that appear to be in a desirable hypertension‐resistant state.
Sympathetic hyperactivity and AT1 receptor overexpression are associated with high‐fat diet‐induced hypertension in young rats
Authors: Americo Anna Laura , Muller Cynthia , Evangelista Fabiana , Farah Vera , Fiorino Patricia
Published: June 2021

We evaluated the autonomic nervous system (ANS) and components of the renin angiotensin system (RAS) AT1 and MAS receptors in the control of blood pressure (BP) in young rats. Male Wistar rats (n=8/group) were fed during 8 weeks with control diet (C) or high‐fat diet (HF, 30% lipids) since the weaning. The animals were catheterized to record basal BP and heart rate (HR) during 15 minutes. The ANS was evaluated by spectral analysis (SA) in the time and frequency domain. At the end of the protocol, the animals were killed by overload of anesthetic (Ketamine/Xylazine, ip.) and the heart was harvested for left ventricle (LV) isolation. The AT1 and MAS receptors in the LV were performed by Western Blot. The HF group showed increase in the basal BP (12%) and HR (6%).In the SA, systolic BP (10%) and low frequency band were higher in HF group compared with C group as revealed by the increase in the variability of pulse interval (C: 9± 1 vs. HF: 15 ± 2 Hz) and in the variability of systolic BP (C: 34 ± 2 vs. HF: 42 ± 3 Hz). No difference was observed in the high frequency band between groups.The AT1 receptor protein expression was higher in the HF group (52%) compared with C group, however the increase in MAS receptor expression (18%) in the HF group was not significantly different from C group. In conclusion, the results suggest that the sympathetic hyperactivity and the overexpression of AT1 receptor could be involved in the genesis of the hypertension observed in young rats fed a high‐fat diet. Supported by Mackpesquisa.
Stress Mis‐programming of Mesenteric Arterial Smooth Muscle is Suppressed by a‐adrenergic Receptor Blockade
Authors: Reho John , Fisher Steven
Published: June 2021

The sympathetic nervous system (SNS) programs rodent resistance artery smooth muscle (RASM) maturation from post‐natal day (PND) 14‐35. We hypothesized that stress hyper‐activation of the SNS would cause developmental mis‐programming of RASM resulting in a hyper‐contractile smooth muscle phenotype and vascular dysfunction. Rats were stressed as neonates by maternal separation (MS) for 3hrs/day (PND2‐14) and as adults by 1hr of restraint (RS). During MS, rat pups were injected IP with a1‐adrenergic blocker Terazosin (0.5mg/kg) or Vehicle. Mesenteric arteries were isolated at PND21, 35 or 3‐24hrs after RS. Smooth muscle contractile genes were measured by qPCR and correlated with functional studies by wire myography. MS accelerated the molecular maturation of RASM measured at PND21 with 2‐4 fold increases in MLCK, myosin phosphatase subunits CPI‐17 (inhibitory subunit) and smMHC as compared to un‐stressed littermates (p<0.05). This correlated with increased force to the a‐agonist phenylephrine and to calcium in permeabilized arteries (p<0.05). These effects were suppressed by treatment with Terazosin (p<0.05). Differences between MS and littermate controls normalized by PND35. RS of adult rats caused a 4‐6 fold induction of MLCK, CPI‐17, and smMHC at 3hrs and this resolved at 24hrs with no interaction between MS and RS. We conclude that stress‐induced SNS hyper‐activation in a critical developmental window or in maturity mis‐programs resistance artery smooth muscle causing vascular dysfunction, but maintained effects during or after maturation likely require additional triggers.
Anti‐Hypertrophic Effect of Estradiol in Cardiomyocytes is Mediated by GPER1
Authors: Gonçalves Gleisy , Nocchi Eduardo , Scalzo Sergio , Guatimosim Silvia , Reis Adelina
Published: June 2021

Previous data have shown that E2 prevents cardiomyocyte hypertrophy induced by endothelin‐1 (ET‐1) and Angiotensin II, acting through the increase of atrial and type‐B natriuretic peptides (ANP and BNP) levels. In recent years, a membrane‐bound E2 receptor has emerged, G protein‐coupled estrogen receptor 1 (GPER1), which mediates many estrogenic effects. In this work, we investigated whether E2 protective signaling effect on ET1‐induced hypertrophy is mediated via GPER1 receptor. Newborn rat cardiomyocytes were isolated and cultured in DMEM supplemented with 10% fetal bovine serum with antibiotic and antimycotic. Cells were treated for 48h with ET‐1 (10 nM) in the presence or not of E2 (10 nM). In some experiments GPER1 antagonist (G15, 10 nM) was added to the medium. ET‐1 induced an increase in cardiac myocyte area compared to control group. Treatment with E2 partially inhibited the hypertrophic effect of ET‐1. Treatment with G15 reversed antihypertrophic effect of E2. Moreover, ET‐1 upregulated ANP and BNP levels, an effect that was abolished by E2. G15 prevented the decrease of BNP but not ANP in cardiomyocytes treated with E2 in the presence of ET‐1. ET‐1 treated cells showed increased translocation of GRK5 to the nucleus and this effect was abolished by E2. The reduction of the contraction frequency and increase of contraction force observed in the ET‐1 group were reversed by E2. In conclusion, E2 plays a key role antagonizing the hypertrophy induced by ET1 and this effect was mediated by GPER1.

Financial Support: CNPQ, CAPES, FAPEMIG
Impact of Chronic Salt Load on Mean Arterial Pressure in Female Growth Restricted Rats at One Year of Age
Authors: Brown Andrew , Intapad Suttira , Alexander Barbara
Published: June 2021

Salt sensitivity is an risk factor for cardiovascular (CV) disease and the prevalence of salt sensitivity increases after menopause. LBW women exhibit early menopause associated with increased adiposity and enhanced CV risk including salt sensitivity. Our laboratory utilizes a rat model of intrauterine growth‐restriction (IUGR) induced via placental insufficiency. In this model female IUGR offspring develop persistent estrous by one year of age indicative of early reproductive senescence. Thus, the goal of this study was to test the hypothesis that female IUGR rats develop salt sensitivity in association with early reproductive aging. Female IUGR rats were placed on a regular rodent chow (RC) diet (0.4% salt) or a high Na+Cl2 (8%) diet for three weeks. Rats underwent weekly assessment prior to and during chronic salt load of: food and water intake and urine output via 24 hour metabolic studies, total fat and lean mass using Echo MRI, and body weight. Mean arterial pressure (MAP) was measured in conscious, chronically instrumented rats at the end of the 3 week protocol. Birth weight was significantly reduced in IUGR pups versus control (5.1±0.1 vs. 5.8±0.1 grams, P<0.05). Chronic salt load did not significantly alter MAP in female IUGR or control at one year of age (IUGR: 134±4 vs. 138±3 and Control: 130±3 vs. 128±3; RC vs. High Salt). 24 hour sodium excretion also did not differ (RC: 16±3 vs. 19±2; High Salt: 202±15 vs. 172±14 mmol, IUGR vs. Control, respectively). Thus, these data indicate that female IUGR do not significantly increase their blood pressure in response to chronic salt load indicating that they are not salt‐sensitive. NIH: HL074927, HL51971, P20GM104357, AHA GRNT19900004
Salt Overload During Postnatal Period Promotes Neurogenic‐mediated Increase of Arterial Blood Pressure in Adult Rats
Authors: Moreira Marina , Mourão Aline , Nunes Allancer , Castro Carlos , Pedrino Gustavo
Published: June 2021

The present study evaluated the effects of salt (NaCl) overload during the postnatal phases on cardiovascular parameters, cardiac function and cardiovascular responses induced by MnPO blockade in adulthood. 21 days‐old male Wistar rats were maintained on 0.3 M NaCl solution and food for 60 days (experimental group; EG); the control group was maintained on tap water and food. Later, both groups were maintained on tap water and food for 15 days (recovery). After the recovery period, baseline mean arterial pressure (MAP) and heart rate (HR) were recorded in unanaesthetized rats. The EG presented increased MAP (118.3 ± 2.7 mmHg vs. 98.6 ± 2.6 mmHg) and HR (398.2 ± 7.5 bpm vs. 365.4 ± 12.2 bpm) when compared to control group. To evaluate the cardiac function, the isolated hearts were perfused using the Langendorff technique in constant flow; after the basal recording, the hearts were submitted to ischemia/reperfusion. The salt overload did not affect the cardiac function in basal conditions. However, during the reperfusion, the hearts from EG presented increased intraventricular diastolic pressure (20.5 ± 5.16 mmHg vs. 11.5 ± 1.6 mmHg) and increased duration of reperfusion arrhythmias (208.8 ± 32.9 s vs. 75.0 ± 7.8 s), when compared to control group. Muscimol nanoinjections into MnPO promoted a decrease of MAP in both control and experimental rats, however, the decrease was greater in the EG (‐23.68 ± 1.46 mmHg vs. ‐15.58 ± 1.57 mmHg). These results demonstrate that postnatal salt overload alters cardiovascular regulation in adulthood. However, other studies are required to clarify the mechanisms by which the changes occur.

Sponsors: Fapeg, Capes, CnPq
Renal Sympathetic Nerve Activity – Controlled by Renal Afferent Sympathoexcitatory or ‐Inhibitory Nerves ?
Authors: Ditting Tilmann , Rodionova Kristina , Heinlein Sonja , Hilgers Karl , Ott Christian , Schmieder Roland , Veelken Roland
Published: June 2021

Renal sympathetic nerve activity (RSNA) is important in hypertension, volume disorders or renal disease. It is unclear if increases of RSNA in disease are due to sympathoexcitatory or impaired sympathoinhibitory renal afferent nerves. We present data from nephritic rats suggesting the latter.

Nephritis due to OX7‐antibodies. Methohexital anesthetized nephritic rats & controls instrumented to stimulate renal afferent nerve activity (ARNA) in order to influence RSNA: ipsilateral renal arterial catheter for intrarenal administration (IRA) of the TRPV1 agonist capsaicin to stimulate ARNA (CAP 6.6*10‐7M) and induce release of the tachykinin receptor agonist SP from renal afferents; contralateral stainless steel electrode for RSNA recording; before and after IRA CAP the tachykinin‐receptor blocker RP67580 was given. Baseline RSNA & ARNA were assessed. Some nephritic rats pretreated with tachykinin receptor antagonists to prove increased SP effects.

IRA CAP decreased RSNA from 67.5±12.0 µV*sec to 14.8±4.2 µV*sec (p<0.05) over 60 minutes while in nephritis RSNA suppression was abolished. Suppressed RSNA in controls was transiently reversed by the tachykinin inhibitor. Under resting conditions RSNA was higher, ARNA lower in nephritis as compared to controls. Tachykinreceptor antagonism ameliorated damage in renal nephritis suggesting increased SP release from renal afferent nerves despite lack of the tachykinin dependant sympathoinhibition seen in controls.

Our data suggest that a tachykinin dependant reno‐sympathetic reflex mechanism exerts sympathoinhibitory effects being impaired under pathophysiological circumstances.
Intracellular Renin in the Brain Contributes to Cardiovascular and Metabolic Control
Authors: Shinohara Keisuke , Weidemann Benjamin , Folchert Matthew , Liu Xuebo , Morgan Donald , Rahmouni Kamal , Grobe Justin , Sigmund Curt
Published: June 2021

Renin gene expression is regulated by two distinct promoter‐first exon combinations that target renin either for secretion (exon 1a initiates secreted renin) or cytoplasmic retention (exon 1b initiates intracellular renin, icREN). Interestingly, icREN is expressed predominantly in the brain. We selectively deleted exon 1b and developed icREN KO mice to examine the role of icREN in cardiovascular and metabolic control. Systolic blood pressure in the light phase as measured by telemetry was increased in KO mice (126±2 mmHg, n=8 vs 116±2 mmHg, n=7, P<0.01). The low‐ to high‐frequency ratio (LF/HF) derived from power spectral analysis of heart rate variability was increased in KO mice (1.24±0.21, n=7 vs 0.70±0.11, n=7, P<0.05). Body weight was normal in KO mice fed normal chow. However, high fat diet‐induced obesity was attenuated in male KO mice (body weight, 32.4±1.3 g, n=5 vs 36.6±1.4 g, n=9; relative fat mass, 26.1±2.3 %, n=5 vs 34.4±2.3 %, n=9, both P<0.05). The resting metabolic rate measured by respirometry was increased in KO mice (0.156±0.005 kcal/h, n=46, P<0.05) vs controls (0.145±0.003 kcal/h, n=53). We previously reported that the brain renin‐angiotensin system facilitates renal sympathetic nerve activity (RSNA) response to acute intracerebroventricular injection of leptin. Interestingly, the RSNA response to leptin was greater in KO mice compared to controls (214±40 % baseline, n=5 vs 114±18 % baseline, n=10, P<0.01). Further, in KO mice, AT1a receptor mRNA was upregulated in the paraventricular nucleus (P<0.05). These data suggest that this novel icREN isoform contributes to cardiovascular and metabolic control possibly through the modulation of AT1a receptor expression in the brain.
Interaction Between NCC and ENaC α, γ Subunits are Differentially Regulated‐ Role of SGK1
Authors: Wynne Brandi , Al‐Khalili Otor , Eaton Douglas , Hoover Robert
Published: June 2021

Thiazide‐sensitive NCC and ENaC are paramount in the regulation of sodium and water homeostasis. Previous data from this laboratory has extensively demonstrated a novel anatomical and biochemical interaction between these two transporters in the late distal convoluted tubule (DCT2). In this study, we sought to further our understanding of the physiological mechanisms regulating this interaction. Knowing the important role of aldosterone in their regulation, we hypothesized two downstream mediators‐ SGK1 and WNK4‐ would alter their association. We used a mammalian two‐hybrid (M2H) assay, using vectors (pBind/pAct with NCC and/or ENaC α or γ, controls and SGK1, WNK4 or NEDD4‐2) which allow for activation of the luciferase reporter vector (firefly) when interaction occurs. COS‐7 cells were transfected at 50‐70% confluency, and allowed to grow for 48 hours. Cells were lysed; M2H determined by analysis of firefly and renilla luciferase; renilla used as a transfection control. Data were normalized (firefly/renilla) and expressed as fold change (of negative, ±SEM, ANOVA, n=8‐12). Our data demonstrate a significant interaction between both ENaC α (7.7±0.8, p<0.05) or γ (4.3±0.7, p<0.05); however, with SGK1 a greater increase in NCC and ENaC α interaction was observed (14.7±1.5, p<0.05). No significant changes were observed with NCC/ENaC γ interactions (SGK1, WNK4 or NEDD4‐2). Here we confirm the direct interaction between NCC and ENaC α/γ. Furthermore, these data suggest that increases in SGK1, possibly via aldosterone, may strengthen or increase NCC and ENaC α interaction.
Regulation of the Interaction of NCC and ENaCβ by SGK1
Authors: Liu Melissa , Al‐Khalili Otor , Eaton Douglas , Wynne Brandi , Hoover Robert
Published: June 2021

Aldosterone and angiotensin II are the primary hormones mediating NCC and ENaC function, which regulate salt and water balance. Two downstream mediators include the aldosterone‐mediated activation of SGK1 and angiotensin II‐mediated activation of WNK4. Additionally, the negative regulator NEDD4‐2 is an important modulator of NCC and ENaC function. Previously, our laboratory has demonstrated that NCC and ENaC subunits (α, β or γ) are closely associated and interact in the distal convoluted tubule (DCT2); however, whether this interaction can be modulated has yet to be investigated. Herein, we investigate physiological parameters by which NCC and ENaCβ are augmented. We used a mammalian two‐hybrid (M2H) assay, using vectors (pBind/pAct with NCC and/or ENaCβ, empty pBind/pAct and SGK1, WNK4 and NEDD4‐2 expression vectors) which allow for activation of the luciferase reporter vector (firefly) when interaction occurs. COS‐7 cells were transfected and allowed to grow for 48 hours. Cells were then lysed and results of the M2H assay were determined by analysis of firefly and renilla luciferase; renilla was used as a transfection control. Data were normalized and shown as firefly/renilla luciferase values. Our data demonstrate that NCC and ENaCβ may not interact; however, with SGK1 overexpression, we observed an increase as compared to negative vectors only (70.4±20.28 vs. 6.36±1.37, p<0.0001, ANOVA, n=4). Interestingly, WNK4 and NEDD4‐2 did not seem to alter the baseline interaction of NCC and ENaCβ. These data suggest that increases in SGK1, possibly via aldosterone, may produce increased NCC and ENaCβ interaction.
Early Pulmonary and Systemic Inflammation Leads to Tissue‐Specific Recruitment of Lectin Complement Pathway Initiators
Authors: Plovsing Ronni , Berg Ronan , Munthe‐Fog Lea , Konge Lars , Iversen Martin , Møller Kirsten , Garred Peter
Published: June 2021

Objective

We wanted to clarify whether lipopolysaccharide (LPS)‐induced pulmonary and systemic inflammation induces localized and systemic recruitment of mannose‐binding lectin (MBL) and ficolins.

Methods

We performed a randomized, double‐blind, placebo‐controlled, cross‐over study in 15 healthy volunteers that received Escherichia coli endotoxin (LPS, 4 ng/kg) intravenously (IV) or in a lung subsegment on two different occasions. Groups were evaluated by blood samples and by bronchoalveolar lavage 2, 4, 6, 8, or 24 hours post‐challenge; gene expression patterns and protein levels of MBL and ficolins were determined in both blood and lung.

Results

Endobronchial challenge with LPS was associated with an increase in alveolar ficolin‐3 levels, with a peak after 24 hours (p < 0.04),

image

and an increase in alveolar MBL (p < 0.001). IV LPS challenge led to an increase in plasma ficolin‐1 protein levels (p < 0.001),

image

whereas no changes were observed in ficolin‐1 gene expression patterns or plasma protein levels of MBL, ficolin‐2, or ficolin‐3.

Conclusions

LPS induces a tissue‐specific recruitment of ficolin‐3, MBL, and ficolin‐1 in the lung and circulation, suggesting an important role of lectin complement pathway initiators in both local and systemic host defense.
Loss of Activating Transcription Factor 4 (ATF4) Alters the Homeostatic Amino Acid Response (AAR) in the Liver of Mice Treated with Asparaginase
Authors: Al‐Baghdadi Rana , Mirek Emily , Phillipson‐Weiner Lindsey , Belden William , Wek Ronald , Anthony Tracy
Published: June 2021

L‐asparaginase (ASNase) is widely used to treat leukemia in children but it causes metabolic complications and liver toxicity. ASNase activates the amino acid response (AAR), increasing gene‐specific translation of the transcription factor ATF4 to regain homeostasis via altered gene expression. We hypothesized that ATF4 deficiency would compromise the hepatic AAR to ASNase and augment hepatotoxicity. Intact (Atf4wt), heterozygous (Atf4het) and homozygous null (Atf4null) mice were administered ASNase (3 IU/g BW i.p.) or saline once daily for 8d. Blood and tissues were collected 8h after final injection. Atf4null mice treated with ASNase lost body weight and experienced premature mortality while all other strains appeared healthy. ASNase did not alter liver mass but promoted lipid accumulation in all genetic strains of mice to a similar degree. Phosphorylation of eIF2 was increased in the liver of all mice treated with ASNase and in saline‐injected Atf4null mice. Examination of the hepatic AAR showed many genes including Asns, Atf5, Chop, Fgf21, 4ebp1 and Gadd34 required ATF4 for full induction. Furthermore, inflammatory stress was evident in the liver of Atf4null but not Atf4het or Atf4wt mice treated with ASNase, similar to that previously reported in Gcn2null mice (AJP 305:E1124, 2013). These results suggest that ATF4 mediates GCN2‐driven hepatic adaptive responses to ASNase and prevents liver inflammation but not lipid accumulation. HD070487 (TGA); GM49164 (RCW)
Phosphorylation of Mineralocorticoid Receptor at Residue S839 Impairs Aldosterone‐dependent Gene Transactivation Coupling in a Dominant Negative Manner
Authors: Jimenez‐Canino Ruben , Fernandes Miguel , Martin‐Martinez Mercedes , Gonzalez‐Muñiz Rosario , Giraldez Teresa , Alvarez de la Rosa Diego
Published: June 2021

The mineralocorticoid receptor (MR), a member of the nuclear receptor family, transduces the biological effects of the corticosteroid hormone aldosterone. Aldosterone interaction with MR ligand binding domain (LBD) induces nuclear translocation, dimerization and gene transactivation. It has recently been demonstrated that phosphorylation of S843, a residue near LBD, inactivates human MR. In this work we examined the mechanisms involved in MR modulation by S843p using mouse MR phosphomimetic mutant S839D (equivalent to S843 in human). MR‐S839D is inactive and significantly decreases wild type MR activity when both forms are coexpressed, consistent with a dominant negative role of MR‐S839D in the dimer. Surprisingly, aldosterone is able to induce nuclear translocation of MR‐S839D, although at a slower rate than wild type receptors. Therefore aldosterone is still able to bind to MR‐S839D but it is inefficient for gene transactivation. In silico modeling of MR LBD with S839D mutation or S839p show the same effect, namely a small decrease in steady‐state agonist docking energy. Our results suggest that the effect of S839p cannot be fully explained by decreased aldosterone binding affinity and may imply a defect in transactivation coupling.
Transcriptional Mechanisms that Mediate Downregulation of TSP‐1 by Chromium in Vascular Smooth Muscle Cells in Response to High Glucose
Authors: Ganguly Rituparna , Sahu Soumyadip , Raman Priya
Published: June 2021

Trivalent chromium (Cr3+) is a beneficial mineral nutrient reported to lower the atherogenic index and reduce risks of vascular inflammation in diabetes. We recently reported that Cr3+ inhibits expression of a proatherogenic matricellular protein, thrombospondin‐1 (TSP‐1), in glucose‐stimulated human aortic smooth muscle cells (HASMC) in vitro. Using luciferase reporter assays, we further showed that Cr3+ significantly decreases TSP‐1 gene (THBS1) promoter activity in glucose‐stimulated cells. The goal of this study was to determine the molecular mechanisms by which Cr3+ inhibits high glucose‐induced TSP‐1 transcription in HASMC. Using protein DNA array (Panomics) that allows detection of activated transcription factors (TF) in nuclear extracts, we demonstrate that Cr3+ reduces activation of glucose‐induced TFs (IRF1, PPARγ), with predicted binding sites in the glucose‐responsive region of the THBS1 promoter. In contrast, Cr3+ did not have any effect on other glucose‐activated TFs (AhR, CEBP, Egr‐1) under similar experimental conditions. These results were further confirmed in EMSA using relevant THBS1 promoter‐specific oligonucleotide probes; specifically we show that Cr3+ attenuates THBS1 promoter‐specific IRF1 binding activity in glucose‐treated nuclear extracts. Additionally, immunoprecipitation of cell lysates with anti‐IRF1 followed by immunoblotting with RL2, an antibody that recognizes O‐linked N‐acetylglucosamine (O‐GlcNAc) moieties on proteins, shows a dramatic reduction in O‐GlcNAcylation of IRF1 by Cr3+ in glucose‐stimulated cells. Together, our data suggest that modulation of IRF1 via O‐GlcNAc modification plays a key role in downregulation of TSP‐1 transcription by Cr3+.
Naturally Occurring Anti‐oxidants As Possible Chemo‐preventives For Pancreatic Tumor Cell Proliferation
Authors: Chowdhury Parimal , Fenton Ember
Published: June 2021

Identification and limited utilization of various naturally occurring compounds in search for novel and effective cancer chemo‐preventive agents is the subject of investigation in many laboratories. This study is designed in‐vitro to evaluate the properties of some most common naturally occurring anti‐oxidants in pancreatic cancer proliferation.

Materials and methods

Cultured AR42J cells from a rat pancreatic cancer cell line were exposed to a fixed dose of nicotine (100uM) either alone or in combination with several naturally occurring and other anti‐oxidants. Cell proliferation was examined by MTT assay and the oxidative status by malondialdehyde (MDA) assay. Further characterization of the effect of nicotine and anti‐oxidants were investigated at the molecular level by Western blot and immunofluorescence analysis to identify the involvement of mitogen activated protein kinase signal transduction pathways in this process.

Results

The results show that proliferation of pancreatic cancer cell was induced by nicotine and these effects appear to have been reversed to a various degree in the presence of these compounds. Further analysis suggests that the responses by nicotine is mediated by oxidant activities since these effects were reversed in the presence of these compounds.

Conclusions

Our study suggest that proliferative activity of rat pancreatic tumor cells by nicotine is associated with its evoked oxidative response and the compounds used in the study counteracted the antioxidant activity. Our data implicate the potential role of these agents as chemo‐preventive agents to pancreatic cancer cell proliferation (supported by UAMS Foundation Fund)
The Effect of Iqmik‐derived Metals on ROS Production in Oral Epithelial Cells
Authors: Dwyer Gaelen , Knall Cindy
Published: June 2021

Smokeless tobacco (SLT) users have 4 ‐ 6 times higher risk for developing oral cancer than non‐users. The percentage of Alaska Natives (AN) who use SLT is 4 times than that of other Alaskans. Out of all adult AN who use smokeless SLT, 35% use only iqmik, a unique combination of tobacco leaves mixed with punk fungus ash. Tobacco has a large number of carcinogens including toxic metals. The ash component of iqmik contains higher levels of pro‐inflammatory toxic metals, such as cadmium (Cd), cobalt (Co) and nickel (Ni), than tobacco. These metals trigger the generation of reactive oxygen species (ROS). The purpose of this research was to determine the intracellular redox response of primary human gingival epithelial cells (HGEP) to artificial saliva extracts of iqmik, punk ash and tobacco. Levels of Cd, Co and Ni were analyzed by ICP‐MS in iqmik, tobacco and punk ash samples and in artificial saliva extracts of iqmik, tobacco and punk ash samples following microwave‐assisted acid digestion. Intracellular redox status was quantified using H2DCFDA in primary HGEP after exposure to iqmik, tobacco and punk ash artificial saliva extracts, CdCl2, CoCl2 and NiCl2. Significantly higher concentrations of Cd, Co and Ni were isolated in punk ash and iqmik samples than in tobacco samples. Artificial saliva extracts of iqmik produced a greater than additive ROS response compared to extracts of tobacco and punk ash. Treatment with Coproduced significantly greater ROS than Cd or Ni treatment, indicating that Co may account for more of the overall ROS.

The research reported was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103395.
Maximal Metabolic Rates of the Barking Frog (Craugastor augusti)
Authors: Hedrick M. , Streicher J. , Crossley D. , Fujita M.
Published: June 2021

The Barking frog (Craugastor augusti, Dugès, 1879) is a direct‐developing anuran from Mexico and the Southwestern US. Very little is known about the metabolic characteristics of this species and maximal metabolic rates of adult animals are unknown. We hypothesized that metabolic characteristics of two genetically distinct populations from TX and NM, inferred from mitochondrial and nuclear DNA (Streicher et al. 2014), might show differences in maximal metabolic rates. We used closed respirometry at 20 °C to measure resting and maximal O2 consumption (VO2max) and CO2 production (VCO2max) to compare adult animals from the two populations, and examined the effects of body mass on metabolism. VO2max was 0.40 ± 0.04 ml O2 g‐1 h‐1 and 0.46 ± 0.02 ml O2 g‐1 h‐1 for the NM and TX populations (mean mass = 46.9 g), respectively, but there was no significant difference. The respiratory exchange ratio (VCO2max/VO2max) for the combined groups was 0.85 ± 0.04 and the aerobic scope (VO2max/VO2rest) was 16.9 ‐ 18.3, indicative of a high aerobic capacity. VO2max and VCO2max were significantly related to body mass (ml O2 h‐1 = 0.47 * Mass + 3.2 & ml CO2 h‐1 = 0.35 * Mass + 3.7). Previous work with anurans (32 species) has shown that VO2max scales with body mass to the 0.97 power (Taigen et al. 1992) and C. augusti falls on the predicted line relating VO2max and body mass for anurans. These results indicate that C. augusti is a highly aerobic species, but genetically distinct populations from TX and NM do not exhibit differences in maximal metabolic rates. Supported by NSF‐IOS 0843082 (MSH) and NSF‐CAREER 0845741(DAC).
Do Late‐Instar Drosophila Larvae Experience Functional Oxygen Limitations?
Authors: Harrison Jon , Callier Viviane , Greenlee Kendra
Published: June 2021

Arthropods gain considerable mass between molts, increasing oxygen demand while parts of the respiratory system are fixed in size. We hypothesized that internal hypoxia could be a cue triggering molting in arthropods and/or be a functional reason that the animal needs to molt. In support of this hypothesis, hypoxic rearing (10 kPa) triggered an ecdysone peak and molting to adulthood at a smaller body size in Drosophila, and hyperoxia induced earlier pupariation (JEB 216:4334). We tested for evidence that late‐third instar Drosophila larvae show signs of functional oxygen‐limitation. The dorsal longitudinal tracheae increased in diameter through the instar, scaling with mass0.38, approximately as predicted to conserve oxygen delivery. Respiratory exchange ratios declined through the instar, indicating that metabolism remains aerobic and shifts away from carbohydrate catabolism. Lactate levels did not increase later in the instar and were unaffected by rearing in hyperoxia. However, hyperoxia (30 kPa) increased late‐instar metabolic rates, suggesting functional oxygen limitation. Late‐instar larvae had greater capacities to generate lactate in response to anoxia, suggesting past selection for coping with late‐instar oxygen limitation. Most late‐ but not early‐stage larvae unregulated HIF signaling. Together these data support the conclusion that Drosophila, and likely other arthropods, experience some functional oxygen limitations later in the instar that trigger HIF signaling, potentially linked to the molting cascade. This research was supported by NSF IOS 1256745.
Torpor in Tenrecs, a Basal Protoendothermic Mammal
Authors: Cahoon Brandy , Treat Michael , van Breukelen Frank
Published: June 2021

Thirty of the 33 described tenrec species are endemic to Madagascar. These basal mammals possess features such as a cloaca and variable body temperature (Tb). We measured the energetics of common tenrecs, Tenrec ecaudatus, during the hibernation season. Tenrecs are capable of variable arousal from torpor but do not perform the predictable interbout arousals characteristic of all other known small hibernators. At lower ambient temperatures, ventilation may be punctuated with periods of 30 min or more between breaths. We found discordant changes to oxygen consumption (VO2), heart rate (HR), and Tb e.g. a spontaneous 23% decrease in VO2 was accompanied by an 47% decrease in HR and no change in Tb. Marked changes in Tb could occur with little change to VO2. We hypothesize that hibernation in tenrecs may represent a plesiomorphic form that is manifested by poor coordination of key homeostatic processes.
Chronic Intermittent Hypoxia (CIH) Enhances Nucleus Tractus Solitarius (NTS) Barosensitive Neuronal Activity in Response to Arterial Pressure Increase in F344 rats
Authors: Kolpakova Jenya , Li Liang , Cheng Zixi
Published: June 2021

Previously, we showed that baroreflex control of heart rate (HR) is reduced but aortic baroreceptor afferent function is enhanced in CIH rats. The goal of this study was to determine the effect of CIH on NTS. F344 rats (4 mo) were exposed to either room air (RA; n=10) or CIH (6 minutes cycles of 21% and 10% O2, 12‐h/day, n=11) for 35‐50 days. Following CIH exposure, rats were anaesthetized with Ket/Ace. Single‐unit extracellular recording was made of NTS barosensitive neurons responding to mean arterial pressure (MAP) changes induced by descending aorta occlusion and measured from the carotid artery. The baseline MAP and HR were similar in RA and CIH. Baseline NTS neuronal firing was 5.6±0.9 spikes/s for RA (n=24) and 6.1±1.0 spikes/s for CIH (n=29). Aortic occlusion‐induced ΔMAP was similar: 26.7±1.0 mmHg (RA) and 26.7±0.8 mmHg (CIH). The time from 5% MAP increase (relative to baseline) to MAP peak was comparable (RA: 5.3±0.6 s, CIH: 5.4±0.3 s). The time from response threshold (30% increase relative to baseline) to NTS neuron peak response was shorter in CIH compared to RA (4.1±0.3 s vs 5.5±0.5s, p<0.05). The time between 5% MAP increase and NTS response threshold was not different between RA and CIH neurons (3.1±0.5s, 3.6±0.5s). Data from the neurons that had short response time (蠄3 s) were used to construct logistic sigmoidal curves (n = 16/group). Based on Gainmax, we identified two groups of neurons. CIH neurons increased NTS response Gainmax [6.4±4.4 (RA) vs 10.54±3.5 (CIH) in grp 1 and 31.9±3.7 (RA) vs 55.5±9.3 (CIH) in grp 2, p<0.05] and range [78.9±11.9 (RA) vs 131.7±21.2(CIH) in grp 1 and 211.2±34.8 (RA) vs 471.6±120.1 (CIH) in grp 2, p<0.05], but did not alter MAP50. Our data suggest that CIH increased output of NTS barosensitive neurons in rats.
Neonatal Exercise and Age‐Related Oxidative Stress of the Rat Cerebral Cortex
Authors: Lopes‐de‐Morais Andréia , Mendes‐da‐Silva Rosâgela , Ferreira Dioginis , Pedroza Anderson , Amâncio‐dos‐Santos Angela , Lagranha Claudia , Batista‐de‐Oliveira‐Hornsby Manuella
Published: June 2021

Aging is related to increased oxidative damage, since Harman proposed the “free‐radical theory of aging”. The cerebral tissue is strongly susceptible to oxidative stress (OS). Although, exercise can counteract OS by increasing anti‐oxidative defenses, it is not necessarily clear which factor of the exercise protocol is more critical. Furthermore, neonatal moderate exercise can decrease brain excitability in a long‐lasting manner (Batista‐de‐Oliveira et al 2012), however, the underlying mechanisms are still not clear. Therefore, we evaluated if the effects of neonatal moderate exercise is related to OS in cerebral cortexes of aged rats (585 days of age). At 15 days old, Wistar rats (n=20) were equally distributed in sedentary or exercised. The treadmill exercise was performed according to Batista‐de‐Oliveira et al (Exp Gerontology 47: 452–457, 2012). Malondialdehyde was measured as described by Buege & Aust (M Enzymol 52, 302‐310; 1978), Sulfhydryl content by G. Leipnitz et al. (Life Sciences 87 275–280; 2010), GSH levels by Hissin & Hilf (Analytical Biochemistry 74:214‐216; 1976), SOD activity by Misra & Fridovich (JBC 247(10): 3170‐3175), Catalase measured according to Aebi (M Enzymol 105, 121‐126; 1984), and GST by Nascimento et al. (APNM 39:1‐8). Neonatal exercise does not affect antioxidant enzymes in a long‐lasting manner (p > 0.05). Thereby, this exercise changes brain excitability later in life by other mechanism than increasing anti‐oxidative defenses.

Study conducted in conformance with the FASEB Statement and the Institutional Ethics Committee for Animal Research (23076.028310/2012‐52)
Angiotensin Converting Enzyme 1 (ACE1) Knockdown in the Median Preoptic Nucleus (MnPO) Attenuates Downstream Neural Activation following Chronic Intermittent Hypoxia
Authors: Faulk Katelynn , Cunningham J.
Published: June 2021

Sleep apnea is associated with a sustained increase in diurnal blood pressure. Chronic Intermittent Hypoxia (CIH), which simulates the arterial hypoxemia of sleep apnea, also produces a sustained increase in diurnal blood pressure. Several CNS regions contribute to CIH hypertension have been identified including the MnPO and paraventicular nucleus (PVN). We have shown that viral‐mediated shRNA knockdown of ACE1 within MnPO selectively decreases CIH hypertension during normoxia. Our hypothesis is that ACE1 knockdown in MnPO will decrease FosB, a neuronal activation marker, positive neurons in the PVN which is a downstream target of the MnPO. We tested this hypothesis using a viral vector containing shRNA to ACE1 within the MnPO. Isoflurane anesthetized adult male rats were microinjected in the MnPO with 500nl of an adeno‐associated virus containing GFP and either shRNA against ACE1 (shACE1) or scrambled shRNA (shSCM). Mean arterial blood pressure was recorded using radio telemetry. Rats were then exposed to 7 days of CIH (3 minute periods of hypoxia (10% oxygen) and 3 minute periods of normoxia (21% oxygen) for 8 hours/day) or normoxia (room air). Immunohistochemistry was used to assess FosB stained neurons within the MnPO and PVN. FosB positive neurons increased in the MnPO and PVN following CIH in both shSCM and shACE1 groups. However, shACE1 significantly decreased the FosB positive neurons in both MnPO and PVN following CIH. The shACE1 had no effects on FosB staining in normoxic controls. These results suggest that FosB activation within the MnPO and the PVN following CIH is at least partially dependent on MnPO ACE1. P01 HL88052
Losartan Injected into the Nucleus of the Solitary Tract Blunts Pressor Mechanisms Activated by High‐Fat Diet
Authors: Speretta Guilherme , Bassi Mirian , Menani José , Colombari Eduardo , Colombari Débora
Published: June 2021

High‐fat diet (HFD) increases the activity of renin‐angiotensin system, sympathetic nerve activity (SNA) and arterial pressure. In the present study, we evaluated the cardiovascular changes produced by HFD in rats treated with angiotensin type 1 receptor (AT1r) blocker injected into the nucleus of the solitary tract (NTS). Male Holtzman rats (300‐320 g, n = 5‐6/group) with stainless steel guide‐cannulas implanted into the NTS were fed with either standard rat chow diet (SD; 5.4% total fat) or HFD (26.4% total fat) for 6 weeks. Mean arterial pressure (MAP), heart rate (HR) and the low frequency (LF) of the systolic blood pressure (SBP, an index of vasomotor SNA) were evaluated in conscious rats, before and after bilateral microinjections of losartan (AT1r antagonist, 10 μg/100 nl) into the NTS. HFD rats presented an increased MAP (115 ± 2 vs. SD: 105 ± 2 mmHg, p < 0.05) and LF of the SBP (9.3 ± 0.9 vs. SD: 4.9 ± 0.6 mmHg2, p < 0.05). Losartan injected into the NTS reduced MAP and LF of SBP (respectively, 107 ± 2 mmHg and 6.6 ± 1.0 mmHg2 vs. baseline pre‐injection; p < 0.05) in HFD rats, but not in SD rats (102 ± 4 mmHg and 4.7 ± 1.0 mmHg2, p > 0.05). Losartan into the NTS in HFD or SD rats produced no significant changes in HR. These results suggest that AT1r in the NTS acts increasing MAP and sympathetic modulation of arterial pressure in HFD rats.
Glial Glutamate Transporters in PVN Suppress Sympathetic Nerve Activity (SNA) and Mean Arterial Pressure (MAP) by Buffering GluN2B‐containing NMDA Receptor Activation
Authors: Bardgett Megan , Calderon Alfredo , Toney Glenn
Published: June 2021

Recent evidence indicates that non‐selective blockade of excitatory amino acid transporters (EAAT) in the hypothalamic PVN increases SNA and MAP by activating local NMDA receptors. The glial expressed EAAT subtype EAAT2 buffers a tonic NMDA current in nearby magnocellular neurons and that EAAT2 buffering is diminished in heart failure. Here we sought to determine the role of EAAT2 in regulating PVN driven levels of renal SNA and MAP. In anesthetized male rats (n=12), bilateral PVN injection (50 nl) of the EAAT2‐selective inhibitor dihydrokainate (DHK, 1.25 pmol) increased renal SNA (DHK: 60±9% vs aCSF: 1±1%) and MAP (DHK: 19±3 mmHg vs aCSF: 0±2 mmHg). Next, ionotropic glutamate receptors (n=3), NMDA receptors (n=6) or GluN2B subunit containing NMDA receptors (n=3) were separately blocked bilaterally in PVN with kynurenic acid (3.6 pmol), AP5 (3 pmol) or ifenprodil (3 pmol), respectively. Whereas resting SNA and MAP were unaltered by KYN, Ifenprodil or AP5, KYN and ifenprodil each attenuated the DHK‐induced increase of renal SNA (DHK: 60±9% vs +KYN: 10±8% or +Ifenprodil: 7±3%) and MAP (DHK: 19±3 mmHg vs +KYN: 2±3 mmHg or +ifenprofil: 5±1 mmHg). Of interest is that AP5 had little effect (renal SNA ‐ DHK: 70±12 % vs +AP5: 55±10 %, MAP ‐ DHK: 23±4 mmHg vs +AP5: 28±3 mmHg). Results indicate that GluN2B containing NMDA receptors, which can be located extrasynaptically and mediate a tonic NMDA current, are mainly responsible for driving sympathetic outflow during PVN inhibition of EAAT2. The inability of AP5 to block responses to DHK is presently unexplained, but could relate to affinity or accessibility differences compared to KYN/Ifenprodil. HL 102310 & 088052 (GMT), HL 07446 (MEB)
Blockade of NMDA Receptors in the Dorsomedial Hypothalamic Region Attenuates the Cardiovascular Response Evoked by Cage Switch Stress
Authors: Paula Cristiane , Marins Fernanda , Guimaraes Priscila , Cordeiro Letícia , Fontes Marco Antonio
Published: June 2021

Evidence indicates that the dorsomedial hypothalamic region (DMH) plays a key role in the organization of the cardiovascular (CV) response to acute emotional stress. Inhibition of DMH and/or blockade of excitatory amino acid (EAA) receptors in this region attenuate the physiological responses evoked by stress. However, all these effects have been explored in a single stress model (air‐stress). We evaluate the contribution of NMDA receptors in the DMH on the CV response evoked by cage switch stress (CS stress). Under anesthesia (tribromoethanol, 250 mg/kg), Wistar rats received guide cannulas into DMH. Seven days after, a cannula was inserted into femoral artery for HR and BP recording. After 24 hs, bilateral nanoinjections (100nL) of vehicle (saline 0.9%, n=7), the GABAA agonist muscimol (100pmol, n=8) or the NMDA antagonist AP‐5 (100pmol, n=6) were performed into DMH; ten min later, rats were submitted to CS stress. An additional control group was also tested (no injection, intact group, n=9). In the intact group and in the vehicle group CS stress evoked a tachycardic response (Δ 119±12 bpm and 121±11 bpm) accompanied by large increases in BP (Δ 60±2 mmHg and Δ 60±3 bpm, respectively). The tachycardic and pressor responses were markedly reduced by muscimol and AP‐5 (musc: ΔHR: 62±7 bpm and Δ MAP: 37±4 mmHg; AP‐5: Δ HR: 31±7 bpm and Δ MAP: 39±3 mmHg, P<0.05 vs. vehicle). Corticosterone and glucose levels were unaffected by CS or any treatment. Data suggest that the DMH is also important for controlling CV responses in other forms of acute emotional stress. Part of this response involves activation of NMDA EAA receptors. Support: Fapemig, Capes, CNPq