bluuebunny/crossref_metadata_2025 huggingface dataset:
Inflammatory genes and antioxidant response of aortic endothelial cells to minimally modified LDL in atherosclerosis‐susceptible and ‐resistant Japanese quail
Authors: Hoekstra Kenneth A
Published: June 2021

In this study, we examined whether variations of aortic endothelial cells (AEC) of atherosclerosis‐susceptible (SUS) and –resistant (RES) Japanese quail in response to minimally modified low‐density lipoprotein (mmLDL) constitute a genetic component in atherosclerosis. AEC were isolated from the aorta of SUS and RES birds, which differ markedly in susceptibility to dietary cholesterol‐induced atherosclerosis. Responses to mmLDL were examined by measuring the expression of inflammatory genes, monocyte chemotactic protein‐1 (MCP‐1) and macrophage colony‐stimulating factor (MCS‐F), heme oxygenase‐1 (HO‐1) and its reaction products (bilirubin, catalytic iron), and endogenous antioxidants glutathione peroxidase, glutathione reductase, superoxide dismutase, and catalase. AEC from SUS strain are more susceptible than those from the RES strain to mmLDL‐induced oxidative stress, as judged by lower HO activity, HO‐1 expression, endogenous antioxidants, ferritin and bilirubin levels. AEC from SUS birds also showed higher levels of catalytic iron and dramatic induction of MCP‐1 and MCS‐F. These results indicate that genetic factors and endogenous antioxidants may be important in determining the susceptibility of SUS and RES cells to oxidative stress.
Elevation in fibrinogen content affects the tight junction proteins and increases endothelial cell permeability
Authors: Tyagi Neetu , Dean William L. , Roberts Andrew M. , Tyagi Suresh C. , Lominadze David
Published: June 2021

Mechanisms of fibrinogen (Fg)‐induced endothelial cell (EC) permeability were assessed by measuring transendothelial electrical resistance using an electrical cell‐substrate impedance system (ECIS). Rat cardiac microvascular ECs were grown either in gold plated ECIS's chambers or in regular 6‐well plates. Confluent ECs were treated with Fg (2 or 4 mg/ml), Fg (4 mg/ml) with extracellular signal‐regulated kinase (ERK) kinase inhibitor (PD98059), or medium alone for 24 hours. Resistance values from each microelectrode were collected and plotted as relative resistance vs. time. Western blot analysis was pdone to determine the changes in tight junction proteins of the ECs treated with Fg (2mg/ml or 4 mg/ml) or medium alone for 24 hours. The higher dose of Fg (4 mg/ml) induced greater EC layer permeability compared to that induced by the lower dose or by medium alone. Fg‐induced increase in EC permeability was inhibited by PD98059. Treatment of EC with 4 mg/ml of Fg significantly decreased the content of occludin in ECs. These data suggest that Fg increases EC permeability through ERK signaling, affecting occluding, which is one of the tight junction proteins that is known to be bound to actin filaments.

Supported by NIH grant# HL080394 to DL.
Mouse muscle and skin permeability coefficients (Ps) to macromolecular contrast agent using magnetic resonance imaging (MRI)
Authors: Rygh Cecilie Brekke , Salvesen Gerd , Moen Ingrid , Reed Rolf , Curry FitzRoy
Published: June 2021

We evaluated the use of MRI to measure blood to tissue clearances of Gadomer (kindly provided by Bayer Schering Pharma), a gadolinium based contrast agent with an apparent MW of 35 kDa. C57 black mice (25–30 g) were anaesthetized (Isoflurane) and Gadomer was injected i.v. during scanning using a 7T Bruker Pharmascan. A T1‐weighted dynamic sequence (TR/TE= 11.1/2.5 ms, flip angle= 25°, time resolution 0.8 s, frames = 500) was used to measure signal intensity over time in selected Regions of Interest (ROI's) in the masseter muscle and head skin. Tracer concentration in blood vessels was also measured in small ROI's placed over arteries. Controlled bolus injection of 0.1 mmol Gd/kg BW caused an initial “step” increase in tracer intensity as the vascular volume was filled. Subsequent blood to tissue tracer exchange resulted in further (initially linear) increased (slope) in tracer intensity over periods of 100–300 seconds. Ps to Gadomer in tissue ROI's was calculated from the slope and step, assuming a microvessel volume to surface ratio of 2.5 microns (Curry et al, AJP245:H496, 1983). Average Ps values (X10‐7 cm/sec) were 2.9 ±1.0 (muscle) and 15±5 (skin). Corrections for the fall in plasma concentration increased estimates by 10–20%. The method enables repeated measurements Ps in organs of individual mouse of periods of days to weeks. Our research is supported by Research Council of Norway and University of Bergen.
Vasopressin regulates phosphorylation of aquaporin‐2 (AQP2) at ser‐264 and ser‐269
Authors: Hoffert Jason , Pisitkun Trairak , McDill Brad , Chen Feng , Fenton Rob , Knepper Mark
Published: June 2021

Arginine vasopressin increases phosphorylation of the water channel AQP2 at Ser‐256 (S256). We recently discovered two novel AQP2 phosphorylation sites, S264 and S269, via shotgun phosphoproteomics. Here, we generated two distinct phospho‐specific antibodies to these sites to assess changes in AQP2 phosphorylation. Through immunoblotting we detected significant increases in pS256 (t1/2 = 41 sec), pS269 (t1/2 = 3.2 min), and pS264 (t1/2 = 4.2 min) following short‐term incubation of rat inner medullary collecting duct suspensions with 1 nM dDAVP. The magnitude of increase at steady state was: S256 3.0‐fold; S264 8.5‐fold; S269 14.9‐fold. Short‐term incubation with cpt‐cAMP (0.1 mM) produced nearly identical responses. Preincubation of tubules with the PKA inhibitor H89 (10 μM) prevented cAMP‐dependent increases in AQP2 phosphorylation at all 3 sites, suggesting coordinate regulation through the cAMP‐PKA pathway. However, incubation of synthetic AQP2 peptides with PKA in vitro resulted in phosphorylation of S256, but not S264 or S269. Both S264 and S269 phosphorylation failed to occur in mice with an S256L mutation, suggesting that S256 phosphorylation may be necessary for downstream phosphorylation events. These results demonstrate that all 3 sites are regulated by vasopressin, via cAMP‐PKA, but do not identify the kinases responsible for S264 and S269 phosphorylation. Support: ZO1‐HL001285.
Regulation of ENaC by small G proteins
Authors: Staruschenko Alexander
Published: June 2021

Small G proteins are recognized to regulate diverse cellular processes. We have shown previously that the epithelial Na+ channel (ENaC) is regulated by Ras and Rho GTPases. K‐Ras, RhoA and Rac1 markedly increase activity of ENaC reconstituted in CHO cells in a GTP‐dependent manner. However, their transductionpathways and mechanisms of activation are different; K‐Ras increases channel open probability via PI3‐kinase signaling, whereas RhoA increase channel membrane levels. Regulation of ENaC open probability by PI3‐kinaseis a direct consequence of the physical association of the product of PI3‐kinase, PI(3,4,5)P3with the channel. RhoA activates ENaC via Rho‐kinase and subsequent activation of PI(4)P5‐kinase with concomitant increases in PI(4,5)P2 levels promoting channel insertion into the plasma membrane. Recent studies reveal that multiple Rab isoforms physically interact with and/or modulate the activity of several ion channels. Rab11a is well‐established as a participant in the regulation of recycling endosomal trafficking. Rab11a is associated with vesicles in the apical portion of epithelial cells near the centrosome and beneath the apical plasma membrane. Co‐expression of Rab11a with ENaC results in a significant increase in channel activity. We conclude that Rab11a may be important in the trafficking of ENaC.

Supported by AHA.
Regulation of ENaC trafficking by Hrs
Authors: Zhou Ruifeng , Kabra Rajesh , Snyder Peter M
Published: June 2021

The epithelial Na+ channel (ENaC) is regulated in part through mechanisms that control its expression at the cell surface. Nedd4‐2, an E3 ubiquitin ligase, catalyzes ENaC ubiquitination, which increases both endocytosis and degradation of the channel. Here we tested the hypothesis that in endosomes, Hrs (Hepatocyte growth factor‐regulated tyrosine kinase substrate) functions as a receptor for ubiquitinated ENaC, targeting it for lysosomal degradation. Consistent with this hypothesis, we found that Hrs binds to ENaC; in HEK 293 cells, ENaC and Hrs coprecipitated. This binding was increased by coexpression of Nedd4‐2, but not by a mutant Nedd4‐2 lacking ubiquitin ligase activity (C821A). To test whether Hrs alters ENaC trafficking, we expressed a dominant negative Hrs construct that lacks the ubiquitin interacting motif (HrsΔUIM) and measured ENaC surface expression by biotinylation. We found that Nedd4‐2 reduced ENaC surface expression, and HrsΔUIM abolished this effect. Moreover, HrsΔUIM increased recycling of ENaC to the cell surface. We propose a model in which Nedd4‐2 induces binding of ENaC to Hrs in the endosome, which diverts ENaC from the recycling pathway, targeting it for degradation in lysosomes.
Regulation of ENaC‐mediated sodium transport by glucocorticoids in Reissner's membrane epithelium
Authors: Kim Sung Huhn , Raveendran Nithya N. , Kim Kyunghee , Harbidge Donald G. , Sanneman Joel D. , Marcus Daniel C.
Published: June 2021

Reissner's membrane (RM) epithelium forms much of the barrier that sustains the large ionic differences between cochlear endolymph and perilymph. We reported that RM contributes to normal cochlear function by absorption of Na from endolymph via the epithelial sodium channel (ENaC) in gerbil inner ear [Lee et al, Neuroscience, 2003].

In the present study, we sought in mouse RM 1) to determine the presence of genes involved in the Na transport pathway, 2) to determine whether their level of expression was regulated by corticosteroids using gene array and quantitative RT‐PCR and 3) to obtain functional evidence for these genes by electrophysiologic and pharmacologic means. Transcripts were present for the α‐, β‐, γ‐subunits of ENaC; the steroid receptors GR (glucocorticoid receptor) and MR (mineralocorticoid receptor); the GR agonist‐regulator 11β‐HSD1; Na transport control components Sgk1 and Nedd4‐2. Expression of the MR agonist‐regulator 11β‐HSD2 was near the lower limit of detection. Dexamethasone upregulated transcript expression of α‐ and β‐subunits of ENaC (∼6 and ∼3 fold). Currents directed into the endolymphatic face of RM were sensitive to amiloride and benzamil, but not EIPA. The currents were also inhibited by ouabain and K‐channel blockers (TEA and clotrimazole). These molecular and functional observations are consistent with Na absorption by RM in the mouse that is mediated by apical ENaC, basolateral Na‐pump and K channels, and that is under the control of glucocorticoids but not mineralocorticoids. These results provide an understanding and molecular definition of one possible mode of therapeutic action of glucocorticoids in the treatment of Meniere's disease.

Supported by NIH grants R01‐DC00212, P20‐PR017686.
A conserved N‐terminal serum and glucocorticoid kinase‐1 (Sgk‐1) variant with enhanced stability, preferential membrane localization and greater stimulation of epithelial Na+ transport
Authors: Raikwar Nandita S , Snyder Peter M , Thomas Christie P
Published: June 2021

Sgk1 is an aldosterone‐induced kinase that regulates epithelial sodium channel (ENaC)‐mediated Na+ transport in the collecting duct and connecting tubule of the kidney. The N‐terminus of Sgk1 contains instability motifs that are targets for ubiquitination resulting in a rapidly degraded protein. By bioinformatic analysis we identified three alternate transcripts of human Sgk1 that differed at their 5′ end. These variants are widely expressed, are conserved from rodent to man and are predicted to encode N‐terminal variants. One of these variants, Sgk1B, diverges from Sgk1 by 120 aa. When expressed in HEK293 cells, Sgk1 was less abundant than sgk1B and substantially increased with the proteasomal inhibitor ALLN but not with lysosomal inhibitor chloroquine. Protein turnover analysis demonstrated that the half life of Sgk1B was greater than Sgk1 correlating with decreased ubiquitination of Sgk1B as compared to Sgk1. Immunocytochemical studies showed that in contrast to Sgk1, Sgk1B is preferentially targeted to the plasma membrane. When expressed with ENaC subunits in FRT epithelia, Sgk1B had a significantly greater effect on amiloride‐sensitive short circuit current as compared to Sgk1. Together the data demonstrate that a conserved N‐terminal variant of Sgk1 shows improved stability, enhanced membrane association and greater stimulation of Na+ transport in a heterologous expression system. NHLBI and VA
WNK3 and WNK4 Amino Terminal Domain Defines the Effect Upon the Renal Na+:Cl− Cotransporter
Authors: San‐Cristobal Pedro , Ponce‐Coria José , Vazquez Norma , Gamba Gerardo
Published: June 2021

The activity of the renal Na‐Cl cotransporter (NCC) is regulated by WNKs. Because in Xenopus laevis oocytes the NCC activity is reduced by WNK4, but is increased by WNK3, sequences within WNKs should endow them with these properties. To gain insight into WNKs structure function relationship, we constructed chimeras between WNK3 and WNK4 by swapping the amino or carboxyl terminus domains, which flank the central kinase domain, between WNK3 and WNK4. Then, effect of chimeras upon NCC activity was analyzed by assessing thiazide‐sensitive 22Na+ uptake in oocytes injected with NCC cRNA alone or together wild type WNKs or chimeras cRNA. Our results show that NCC activity was increased by chimeras containing the amino terminal domain of WNK3 and decreased by chimeras containing the amino terminal domain of WNK4. Elimination of catalytic activity in chimeras prevented their effect upon NCC. Thus, the effect of WNK3 and WNK4 upon NCC follows the amino terminal domain. We conclude that sequences within amino terminal domain endow WNK3 and WNK4 with their activating or inhibiting properties upon NCC.
Regulation of NKCC2 by WNK3 and SPAK
Authors: Ponce‐Coria Jose , San‐Cristobal Pedro , Vazquez Norma , Pacheco‐Alvarez Diana , Muñoz Eva , Bobadilla Norma , Gimenez Igancio , Kahle Kristopher , Hebert Steve , Gamba Gerardo
Published: June 2021

Little is known about regulation of the Na‐K‐2Cl cotransporter (NKCC2) by non cAMP/PKA mechanisms. In this study, NKCC2 activity was analyzed by assessing 86Rb+ uptake in Xenopus laevis oocytes. We show that intracellular chloride depletion in oocytes, induced by co‐expression of NKCC2 with the K‐Cl cotransporter KCC2 or by low‐chloride hypotonic stress, activates NKCC2. This activation is associated with phosphorylation of three highly‐conserved threonines (96, 101, and 111) of NKCC2 amino terminus. The chloride‐sensitive activation of NKCC2 requires the interaction of two serine‐threonine kinases, WNK3 and SPAK. Elimination of WNK3's only SPAK‐binding motif prevents its binding with SPAK and activation of NKCC2. Thus, WNK3 appears to be positioned upstream of SPAK. Elimination of the threonines 96, 101, and 111 or the injection of a catalytically‐inactive WNK3 mutant also completely prevents NKCC2 activation by intracellular chloride depletion. Together, these data reveal a novel chloride‐sensing mechanism that regulates NKCC2, requiring WNK3 and SPAK. We propose that WNK3 appears to be the chloride‐sensitive kinase.
The expression of zinc transporters and metallothionein is decreased in the intestinal and alveolar epithelium of chronic alcohol‐fed rats
Authors: Joshi Pratibha C , Guidot David M
Published: June 2021

We have previously shown in animal models that chronic alcohol ingestion significantly alters the alveolar epithelial barrier, and renders the lung susceptible to acute inflammatory injury. Although alcoholics are known to be deficient in micronutrient zinc that protects cell membranes from oxidative damage, there are no reports on the effects of chronic alcohol ingestion on zinc bioavailability. In this study, we examined the expression of selected members of the zinc transporters ZIP (SLC39A), and ZNT (SLC30A), as well as the zinc storage protein metallothionein in the small intestinal and alveolar epithelium of alcohol‐fed and control‐fed rats. The expression of ZIP1 and ZIP4 was significantly decreased in the duodenum of alcohol‐fed rats, suggesting less zinc absorption from the diet. The expression of the ZNT4 was significantly decreased in the small intestine and alveolar epithelium, perhaps to conserve locally available zinc. In parallel, this downregulation of transporters resulted in decreased levels of zinc in the epithelial lining fluid from alcohol‐fed rats. Metallothionein expression, a good indicator of zinc status, was significantly decreased in the intestinal and alveolar epithelium of alcohol‐fed rats. These data suggest that chronic alcohol ingestion may affect zinc homeostasis in vivo through alterations in the zinc transporters and zinc storage protein.
Effect of ovariectomy in the renal effects of NFkB inhibition on aged DOCA hypertensive female
Authors: Fortepiani Lourdes A
Published: June 2021

The loss of ovarian function with aging is associated with increases in BP and renal dysfunction. Mineralocorticoid hypertension (DOCA) is associated with increased NF‐kB activation that regulates genes involved in BP and renal function regulation. Young female (F) DOCA rats exhibit hypertension and renal injury that is exacerbated after ovariectomy (ovx). Since NFkB inhibition lowered BP in male DOCA rat, we hypothesize that NFkB inhibition will attenuate the development of hypertension and renal damage in old ovx DOCA in a greater extent than in old F‐DOCA rats. F‐DOCA rats were ovx or sham operated at 9 mos of age and treated with NFkB inhibitor (PDTC) for 2 weeks. Ovx at 9 mos did not alter BP (F: 147.1 ± 4.2 vs Ovx: 145.4 ± 5.1mmHg) but increased glomerular filtration rate (GFR), and decreased renal vascular resistance (RVR) and proteinuria. Blockade of NFkB increased GFR (1.32 ± 0.1 vs 1.6 ± 0.1 ml/min/g) and decreased RVR and P in female (29.9 ± 0.8 vs 16.6 ± 1.5ml/min/g and 14.1 ± 0.3 vs 2.5 ± 1.0g/ml); however it decreased GFR (1.6 ± 0.1 vs 1.3 ± 0.1 ml/min/g), and increased RVR and P in ovx (23.9 ± 2.9 vs 44.8 ± 6.2 and 0.3 ± 0.01 vs 6.3 ± 1.4g/ml). These results suggest that the beneficial effects of NFkB inhibition in the kidney of aged DOCA rats are mediated by ovarian sex hormones and when ovaries are removed, the renal effects of NFkB inhibition result detrimental rather than beneficial. This work was supported by NIH AG029250‐01 award
NOP receptor stimulation by ZP120 induces anti‐natriuresis through a direct effect on epithelial sodium channel (ENaC) in the renal collecting duct
Authors: van Deurs Ulla Seehusen Kruchov , Hadrup Niels , Petersen Jørgen Søberg , Christensen Sten , Jonassen Thomas
Published: June 2021

ZP120 is a peptide analogue of the endogen peptide nociceptin, which has been shown to have marked aquaretic effects in vivo. However, in addition to this effect ZP120, at least during sodium replete conditions, seems to induce an anti‐natriuretic effect by an unknown mechanism.

The aim of this study was to identify the specific tubular segment where ZP120 facilitates its sodium retaining effects.

The acute effect of ZP120 (1 nmol/kg/min) infusion was examined in chronic catheterized, conscious rats by clearance technique. The rats received a basal infusion of 3 ml/h of isotonic saline throughout. During these conditions ZP120 induced marked diuresis (71 ± 8 vs time control: 12 ± 1 μl/min/100 g BW) whereas urinary sodium excretion decreased in the ZP120 infused compared to time controls (−42± 5 vs time control: 14± 13 umol/min/100 g BW).

90 min ZP120 infusion was followed by coadministration of bendroflumethiazide (BFTZ) or amiloride to block the thiazide sensitive cotransporter in the distal convoluted tubules or the Epithelial Na+ Channel in the collecting ducts. Rats treated with BFTZ or amiloride acted as controls. The natriuretic response to BFTZ was similar in ZP120 treated animals and controls, whereas the natriuretic response to amiloride was increased by 63 % when compared to controls.

These results strongly suggest a direct stimulatory effect of ZP120 on the Epithelial Na+ Channel in the kidney collecting ducts.
Differential Phenotype of Placodal vs Neural Crest Derived Afferent C‐fibers in Airways and Esophagus.
Authors: Undem Bradley J. , Kwong Kevin , Nassenstein Christina , Kollarik Marian
Published: June 2021

C‐fibers in the guinea pig respiratory tract comprise two distinct phenotypes, depending on whether the cell body is situated in the nodose (placodal origin) ganglion or jugular (neural crest origin) ganglion (Undem et al. J.Physiol, 2004). Both types of C‐fibers are responsive to capsaicin but only the nodose C‐fibers are responsive to purinergic P2X receptor, 5‐HT‐3 receptor, and adenosine A1 receptor agonists. The vast majority of jugular, but not nodose ganglia express neurokinins. We speculate that the C‐fiber phenotype may be determined more by their embryonic source than from molecular cues arising from the tissue they innervate. To further evaluate this, we compared lung and esophageal C‐fiber neurons arising from dorsal root ganglion (DRG), nodose, and jugular ganglia, based on the knowledge that the DRG neurons innervating these tissues are derived from the same embryonic source as the jugular neurons. Neurons labeled from the lungs and studied with single cell RT‐PCR, histology, and whole cell patch clamp recordings, revealed that DRG and Jugular C‐fiber phenotype are equivalent, but quite distinct from the nodose C‐fibers. A similar distinction was observed in esophageal‐specific afferent neurons. The results indicate that the C‐fiber phenotypes innervating the lungs and esophagus are determined more by their embryonic environment than by the environment of the tissue they ultimately innervate.
Rats bred as low intrinsic aerobic capacity runners (LCR) recover more slowly from chronic pain compared to rats bred as high intrinsic aerobic capacity runners (HCR)
Authors: Muncey Aaron R. , Saulles Adam , Baghdoyan Helen A. , Koch Lauren G. , Britton Steven L. , Lydic Ralph
Published: June 2021

Low physical fitness and obesity are associated with chronic pain (J Pain 8: 430, 2007) and the mechanisms underlying this link are difficult to explore in humans. This study is testing susceptibility to experimentally induced chronic pain in two rat strains developed by two‐way artificial selection on a treadmill to contrast for both intrinsic (untrained) fitness and obesity. Experiments were performed using generation 19 LCR (n=3) and HCR (n=3) males. Pain threshold was measured as paw withdrawal latency (PWL) to a nociceptive thermal stimulus using an IITC Hargreaves system. PWL was measured before and weekly for six weeks after chronic constriction injury (CCI) of the sciatic nerve, a validated model of neuropathic pain (Pain 33: 87, 1988). PWL did not differ between HCR and LCR rats before CCI. Two‐way ANOVA for repeated measures showed that PWL varied significantly as a function of aerobic capacity, time after CCI, and by interaction. At days 21, 28, 35, and 42 after CCI the LCR rats had a 23% shorter PWL than the HCR rats. The finding that CCI produced a shorter PWL in LCR compared to HCR rats is consistent with the hypothesis that a low aerobic capacity phenotype is associated with greater hyperalgesia. Thus, LCR and HCR rats may provide contrasting models of susceptibility to chronic pain relevant for mechanistic studies in humans.

Support: NIH grants HL6427, RR‐17718, HL57120, HL40881, MH45361 and Dept Anesthesiology
Dysregulation of Inflammatory Immune Responses: Effect of Stress Paradigms in a Rat Model of Novelty‐seeking Phenotype
Authors: Iragavarapu‐Charyulu Vijaya L. , Libreros Stephania , Garcia Ramon A. , Isgor Ceylan
Published: June 2021

Behavior and stress affect immune responses. A rat model of human sensation‐seeking/risk‐taking trait for drug and stress vulnerability, based on their exploratory behavior displaying high rates (HRs) or low rates of locomotor reactivity (LRs) to environmental stress was used to study the relationship between the neuroimmunologic systems. Exposure to chronic, variable physical stress (CVP) or chronic, variable social stress (CVS) during nicotine training resulted in the HR but not LR phenotype to develop nicotine craving with dysregulated stress axis activation following nicotine exposure. In contrast, CVP with nicotine caused emergence of an HR‐like craving in the LRs, and conversely the CVS with nicotine suppressed the craving normally observed in the HRs. Baseline immune responses differed in LR‐HR rats. These differences were further impacted by exposure to the two types of stress regimen: HR rats’ T‐cells responded to nicotine with increased tumor necrosis factor alpha (TNF‐α) and decreased interferon‐γ (IFN‐γ) production compared to LRs. Immune response impacted by stress is associated with secretion of glucocorticoids (GC) which bind to GC receptors (GRs) with inhibition of inflammatory gene transcription. A potential deficit in the GR‐mediated trans‐repression of inflammatory gene expression via the histone deacetylase 2 (HDAC2) pathways may be involved in the HR‐like vulnerability in the pro‐inflammatory cytokine response to nicotine. This project is supported by: JEK Florida Biomedical Research Grant (05NIR‐06‐5194).
Mice with targeted knockout of insulin receptor from the proximal tubule have impaired glucose tolerance
Authors: Tiwari Swasti , Gill Pritmohinder S. , Neilson Eric G. , Ecelbarger Carolyn M.
Published: June 2021

Recently, we demonstrated basolateral and brush border staining for the insulin receptor (IR) in the proximal tubule (PT) of rodents by immunofluoresence. Expression of IR was reduced in the PT of insulin resistant rats. In order to evaluate the physiological relevance of the IR in PT, we generated PT IR knockout mice using a Cre‐ loxP recombination strategy. Mice homozygous for loxP‐flanked IR gene were crossed with mice heterozygous for Cre‐recombinase driven by the type I gamma glutamyltransferase promoter, PT‐specific promoter. Young male PTKO mice exhibited a mild diabetic phenotype as indicated by their significantly higher plasma glucose levels relative to their wild‐type (WT) littermates (mmol/L): 7.8 ± 0.2 vs. 10.5 ± 1.1 for WT and PTKO respectively, p = 0.03. To determine whether these mice had slower/impaired rate of glucose clearance, mice were injected with 5 ml/kg.bw of 20% dextrose intraperitoneally followed by measurement of blood glucose levels at 15, 30, 60, 90 and 120 minutes. PTKO mice showed impaired GT as evidenced by significantly higher area under the curve for glucose over time: 15.5 ± 1.1 vs. 19.6 ± 1.4 mmol/L·hour for WT and PTKO mice respectively, p = 0.04. PTKO mice excreted significantly higher insulin levels in their urine than WT under basal state (pmol/24 hours): 0.05 ± 0.02 vs. 0.19 ± 0.05, p=0.04. Plasma insulin levels however were not significantly different 210 ± 56 vs. 361 ± 84 pmol/L, p=0.2. These results support a physiologic role for IR in the proximal tubule to affect whole body glucose tolerance; and furthermore, suggest that down regulation of IR in renal PT in obesity may have a causative influence on whole‐body glucose utilization. Supported by HL074142
Enhanced insulin sensitivity with loss of collectrin, an important mediator of renal amino acid recovery
Authors: Malakauskas Sandra , Kourany Wissam , Lu Danhong , Hohmeier Hans , Newgard Christopher , Le Thu
Published: June 2021

The metabolic syndrome is a complex constellation of disorders which places patients at increased risk of diabetes, cardiovascular disease and all‐cause mortality. The objective of this study is to examine the metabolic effect of deletion of collectrin, a novel candidate mediator of the metabolic syndrome. We previously reported that targeted deletion of collectrin in the mouse results in failed recovery of filtered amino acids at the proximal tubule. We now report that collectrin‐deficient (Tmem27−/y) mice develop enhanced insulin sensitivity with age. We observe a statistically significant decrease in blood glucose levels and a delayed hypoglycemic response during insulin tolerance testing (p 0.05). This enhanced insulin sensitivity is associated with a 25% decrease in adiposity by DEXA scanning (p=0.009), despite a 30% increase in food consumption, and persists with high‐fat feeding. Examination of the pancreas reveals no difference in morphometric variables, ‐cell proliferation by in vivo BrdU staining or glucose‐stimulated insulin secretion in isolated islets in Tmem27−/y animals compared to wild‐type. This suggests that the enhanced insulin sensitivity does not arise from a difference in pancreatic physiology. We hypothesize that the increased insulin sensitivity associated with loss of collectrin results from altered amino acid flux in metabolic pathways important in energy homeostasis.
Simvastatin and Tempol Protect Against Endothelial Dysfunction and Renal Injury in a Model of Obesity and Hypertension
Authors: Knight Sarah F , Yuan Jianghe , Roy Siddhartha , Imig John D
Published: June 2021

Obesity and hypertension are linked to the development of chronic kidney disease. We investigated fatty acid and superoxide (SO) contribution to endothelial dysfunction and renal injury in a model of obesity and hypertension. Male 8 week old WKY rats and SHR fed normal diet (NFD) (7% fat), high fat diet (HFD) (36% fat), HFD with Tempol (Tmp) (20mg/kg/day) or HFD with Simvastatin (Sim) (10mg/kg/day) up to 10 weeks (n=4). Blood pressure was unaffected by Tmp or Sim treatment. After 3 weeks, renal afferent dilatory responses to acetylcholine reached 75% of baseline diameter in NFD SHR but 47% in HFD SHR (P<0.05). In Tmp and Sim treated SHR diameters reached 79% and 78% of baseline (P<0.05). Ten weeks HFD raised albumin excretion from 199 ± 35 in NFD SHR to 870 ± 378μg/day in HFD SHR (P<0.05), Tmp and Sim treated WKY and SHR excreted just 258 ± 111 and 455 ± 76μg/day, respectively. Urinary 8‐isoprostane increased from 6 ± 2 to 27 ± 13ng/day and 12 ± 4 to 26 ± 9ng/day by HFD in WKY and SHR (P<0.05). Tmp and Sim treatment prevented this increase. Monocyte chemoattractant protein‐1 (MCP‐1) excretion was 29 ± 12 and 40 ± 5ng/day in HFD WKY and SHR compared to 11 ± 3, 15 ± 4, 16 ± 6 and 30 ± 10ng/day in NFD and Tmp WKY and SHR respectively (P<0.05). Sim WKY and SHR excreted 38 ± 7 and 43 ± 9ng/day. This indicates that Tmp and Sim prevent endothelial dysfunction and renal injury in a model of obesity and hypertension via a reduction in SO, independently from MCP‐1. (NIH)
Hepatocyte Syndecan 1 Mediates Triglyceride‐Rich Lipoprotein Clearance in the Liver
Authors: Stanford Kristin I , Bishop Joseph R , Niesman Ingrid , Esko Jeffrey D
Published: June 2021

We previously provided genetic evidence that alterations in heparan sulfate fine structure result in an accumulation of plasma triglyceride‐rich lipoproteins (MacArthur et al, J. Clin. Invest. 117:153, 2007). In this study, we sought to determine which heparan sulfate proteoglycans play a role in triglyceride‐rich lipoprotein metabolism. RT‐PCR analysis of hepatocytes demonstrated the expression of the membrane proteoglycans syndecans‐1, ‐2, and ‐4, glypicans‐1 and ‐4, and the secreted proteoglycans agrin, perlecan, and collagen XVIII. Analysis of available mice deficient in the various proteoglycans showed that only syndecan 1 and collagen XVIII mutants accumulated plasma triglycerides. Furthermore the extent of accumulation was comparable to that observed in heparan sulfate deficient strains. Immunoelectron microscopy showed expression of syndecan‐1 on plasma membrane microvilli of hepatocyte basal membranes lining the space of Disse. Mice deficient in syndecan‐1 synthesized VLDL normally, but exhibited reduced uptake of injected VLDL. Retinyl ester excursion studies showed that clearance of intestinally derived lipoproteins also depended on syndecan‐1. In contrast, collagen XVIII deficient mice did not show a defect in hepatic clearance, but rather a change in metabolism of lipoproteins in the peripheral circulation. These findings show that syndecan 1 is an hepatic proteoglycan receptor mediating the clearance of both intestinally derived and hepatic lipoprotein particles.
Proteomic analysis of rat myocardium in a model of chronic alcohol consumption
Authors: Lantry Rachel L , Vary Thomas C , Lynch Christopher J
Published: June 2021

Heavy, chronic alcohol consumption is associated with a syndrome known as alcoholic heart muscle disease (AHMD), clinically characterized by ventricular dilation, thinning of the ventricular wall, and myocardial dysfunction. These changes occur as a result of a decreased rate of protein synthesis. The clinical manifestations of AHMD are consistent with alterations in structural and myofibrillar proteins. The current study used a proteomic approach with isotope coded affinity tag (ICAT) labeling and mass spectrometry to delineate changes in protein expression in a chronic (16 wk) alcohol rat model. Chronic alcohol ingestion was associated with falls in mitochondrial proteins associated with TCA cycle (citrate synthase, isocitrate dehydrogenase, succinyl‐CoA synthetase, malate dehydrogenase) as well as enzymes involved in electron transport (ubiquinone, adenine nucleotide translocator, F1‐ATPase, H+ transporting two‐sector ATPase) and energy transfer (nucleoside‐diphosphate kinase, creatine kinase). No changes were seen in pyruvate dehydrogenase complex, NADH coenzyme Q reductase, or ubiquinol cytochrome c reductase. The results indicate myocardial mitochondrial proteins are adversely affected in chronic alcoholism. (PHS; RO1 AA012814).
Growth Factors and Oxidative Stress Act Oppositely on Chaperone‐Mediated Autophagy (CMA) in Renal Tubular Cells: Differences in FoxO1 abundance
Authors: Franch Harold A. , Brown Nikia S. , Shen Wen
Published: June 2021

In diabetic renal hypertrophy, CMA proteolysis (PD) is reduced increasing proteins important for growth (e.g., GAPDH and Pax2). Epidermal growth factor (EGF) decreases CMA activity via Akt signaling. Because oxidative stress also activates Akt, we examined how it affects CMA using EGF (10−8 M) or H2O2 (100 μM) in NRK‐52E cells. Both EGF and H2O2 increased Akt S473 phosphorylation (P)(a marker of Akt activity) and GSK III P (a substrate of Akt). EGF decreased PD by − 25%, but H2O2 increased PD by 17% (n=18). PD was unchanged by EGF+ H2O2. At 48 hrs, EGF increased pax2 protein by 2.7 fold and GAPDH protein by 74%. H2O2 only mildly reduced Pax2 or GAPDH (−10%, NS, n=3–4), but prevented EGF‐induced increases in both. However, one Akt signaling pathway showed differences: At 1 hr, EGF resulted in 5 fold phosphorylation of FoxO1 at T24 but H2O2 caused only 1 fold T24 P. FoxO1 P on this site leads to destruction by the proteasome. EGF decreased FoxO1 protein abundance by 49% at 1 hr while H2O2 increased it by 25% (n=2). EGF still decreased FoxO1 protein in the presence of H2O2. Adenoviral expression of constitutively active FoxO1 (with S/A mutations at T24 and S256 sites) reversed EGF‐induced Pax2 and GAPDH accumulation and accelerated PD by 21%. While both EGF and H2O2 lead to Akt, differences in FoxO1 P and protein abundance may account for differences in CMA.
Differences in muscle protein synthesis and anabolic signaling in the postabsorptive state and in response to food in 65–80 y old men and women
Authors: Smith Gordon Ian , Atherton Philip , Villareal Dennis T. , Rennie Michael J. , Mittendorfer Bettina
Published: June 2021

Men have more muscle than women but during aging lose muscle faster than women. To investigate potential mechanisms responsible for this we measured rates of muscle protein synthesis (MPS) with stable isotope‐labeled tracer techniques and the phosphorylation status of anabolic signaling elements during postabsorptive conditions and feeding in men (n=13) and women (n=16) who were matched on age (65–80 y) and body mass index. Postabsorptive MPS was ∼25% less in men than in women (0.045 [0.039; 0.057] vs 0.058 [0.052; 0.072]; %·h−1; medians [quartiles]; P=0.02) and feeding increased MPS in men (to 0.071 [0.059; 0.079] %·h−1; P<0.01) but not women (0.060 [0.056; 0.089] %·h−1). Postabsorptively, muscle phosphorylated AktSer473, p70s6kThr389, eIF4ESer209 and eIF4E‐BP1Thr37/46 concentrations were not different between sexes but that of eEFThr56 was less in women, consonant with more activated protein elongation. Accordingly, feeding increased eIF4ESer209 and eIF4E‐BP1Thr37/46 phosphorylation in men (both P<0.05) but not in women. Thus, there is sexual dimorphism in MPS and its control in older adults with a diminished basal rate of MPS, which operates over much of the day, potentially explaining the faster loss of muscle in older men.

This research was supported by grants from the US NIH, the University of Nottingham, the UK BBSRC, and the European Union.
FVB/N mice are hypersensitive to moderate insulin‐induced hypoglycemia while 129X1 mice are completely insensitive
Authors: Berglund Eric D. , Li Candice Y. , Lynes Sara E. , Ayala Julio E. , Wasserman David H
Published: June 2021

The phenotype of mutant mice is known to differ based on background strain. Insulin‐induced hypoglycemia was used as a metabolic stress to examine the strain‐dependence of the endocrine counter‐regulatory response. Hypoglycemic clamps were done in 5h fasted DBA/2J (DBA), C57BL/6J (B6), FVB/NJ (FVB), and 129X1/SvJ (129X1) mice (n=7–9) surgically implanted with carotid artery and jugular vein catheters 5d prior to study. During the clamp, insulin was infused (20mU/kg/min) and plasma levels increased to ~350 mU/mL in each strain. Hypoglycemia (~45 mg/dL) was maintained via variable glucose infusion rate and glucose turnover was assessed with [3‐3H]glucose. Glucagon (pg/mL) increased by 51±11, 111±21, and 304±40 in DBA, B6, and FVB, but failed to increase in 129X1. Epinephrine (pg/mL) increased by 174±38, 203±36, and 1076±149 in DBA, B6, and FVB, but did not change in 129X1. Hyperinsulinemia suppressed hepatic glucose production in DBA, B6, and 129X1, but failed to do so in FVB due to the greater counter‐regulatory drive. In summary, FVB and 129X1 provide contrasting counter‐regulatory responses during hypoglycemic clamps. These mouse models may be useful in understanding the response to insulin‐induced hypoglycemia and the impairment that frequently occurs in people with diabetes. Supported by DK50277 and DK59637.
Hypertension caused by angiotensin II infusion involves superoxide production in the RVLM resulting in enhanced sympathetic nerve activity
Authors: Braga Valdir A , Burmeister Melissa A , Sharma Ram V , Davisson Robin L
Published: June 2021

Angiotensin II (Ang‐II)‐dependent hypertension (HTN) is mediated by increased superoxide (O2−) signaling in the subfornical organ (SFO), a primary sensor for blood‐borne Ang‐II that lies outside the blood‐brain barrier. Considering the importance of the SFO‐PVN‐RVLM axis in the context of essential HTN, in the present study we hypothesized that Ang‐II infusion also leads to overproduction of O2− in the RVLM resulting in HTN. Infusion of Ang‐II (600 ng.kg. −1.min. −1) over a 2‐week period in mice caused a gradually developing HTN (Ang‐II=137±6 vs saline=103±2, n=11, P<0.01) that was correlated with marked elevations in O2− production in the RVLM (2.75±0.25‐fold in relation to saline‐infused mice, n=6, P<0.05). We also evaluated blood pressure variability using spectral analysis and we found that the chronic infusion of Ang‐II produced increase in the LF/HF ratio (+109±13%, n=9, P<0.01), suggesting enhanced sympathetic nerve activity (SNA). Adenoviral‐mediated delivery of cytoplasmically targeted superoxide dismutase (CuZnSOD) selectively to the RVLM abolished the increase in O2− production and the increase in the SNA preventing HTN. These data suggest that increased intracellular O2− production in the RVLM leads to increased SNA playing a role in the development of Ang II‐dependent HTN.
Chronic hypertension enhances the function of pre‐synaptic GABAB receptors in the NTS
Authors: Zhang Weirong , Herrera‐Rosales Myrna , Vitela Melissa , Mifflin Steve
Published: June 2021

Microinjection of the GABAB receptor agonist baclofen into the NTS increases arterial pressure, heart rate and sympathetic nerve discharge. This response is enhanced in hypertension and is associated with increased post‐synaptic GABAB receptor function. We tested whether a presynaptic mechanism also contributes to the enhanced baclofen effect in hypertension. Whole‐cell recordings of NTS second‐order neurons identified by DiA labeling of aortic nerve were obtained in a brain slice from normotensive control (NT) and renal‐wrap hypertensive (HT) rats. After 4 weeks, arterial blood pressure was 162±9 mmHg in HT rats (n=6) and 107±3 mmHg in NT rats (n=6/11) (p<0.001). Baclofen reduced amplitude of evoked EPSCs (eEPSCs) elicited by tractus stimulation and the EC50 of was significantly greater in NT rats (1.5±0.5 μM, n=6) than HT rats (0.6±0.1 μM, n=6, p<0.05). Baclofen (1 μM) elicited greater inhibition of eEPSCs in HT (58±6%, n=9) than NT cells (40±6%, n=8, p<0.05). HT cells (0.60±0.08, n=8) had greater paired‐pulse ratio (PPR) of eEPSCs than NT cells (0.48±0.06, n=5, p<0.05). After application of baclofen (1 μM) HT cells (0.92±0.07) had greater PPR than NT cells (0.67±0.05, p<0.05). The results suggest that in renal‐wrap HT rats baclofen evokes an enhanced presynaptic inhibition of glutamate release in 2nd‐order baroreceptor neurons. This enhanced inhibition could alter baroreflex function in hypertension.
Identification of Differentially‐Expressed MicroRNAs in the Paraventricular Nucleus (PVN) Following Myocardial Infarction (MI)
Authors: Guruju Mallikarjuna R. , Zhou Yi , Infanger David W. , Braga Valdir A. , Peterson Jeffrey , Sharma Ram V. , Davisson Robin L.
Published: June 2021

Alterations in gene regulation in central cardiovascular regulatory regions such as the PVN may play an important role in cardiovascular homeostasis. Given recent evidence suggesting that changes in miRNA expression may be involved in cardiovascular disease, we hypothesized that miRNA expression in the PVN is altered following MI. Microarray analyses for miRNA expression was performed on total RNA samples (n=3) isolated from PVN punches pooled from 5 brains of MI or sham mice using Sangers miRbase V8.0 array specific for mouse. Fifteen miRNAs were differentially regulated (>1.5 fold change between MI and sham mice; 11 up‐regulated, 4 down‐regulated in MI mice). MI‐induced alterations in the expression of mir‐29(a,b,c), mmu‐mir‐9 and ‐9*, and mmu‐mir‐199b were statistically significant (p<0.05; n=3). Up‐regulation of mmu‐mir29c expression was confirmed by real time PCR, and was increased 10‐fold in MI vs. sham mice (p<0.01, n=3). Sequence analysis using miRBase revealed likely interactions of the mmu‐mir‐29 family of micoRNAs with Fos, Agtr2, and MTMR7 genes, some of the highly and specifically expressed genes in neuronal cells. Further studies for confirming the binding of miRNAs to postulated mRNAs are in progress. These studies suggest that differential expression of miRNAs following MI may contribute to changes in gene expression in the PVN involved in MI‐induced heart failure.
eNOS contributes to altered nitric oxide availability and neuronal activity in the PVN during Heart Failure
Authors: Biancardi Vinicia C , Sonner Patrick M , Stern Javier E
Published: June 2021

An altered nitric oxide (NO) function within the hypothalamic paraventricular nucleus (PVN) is involved in increased neurohumoral drive during heart failure (HF). However, the precise cellular sources and NOS isoforms involved are still unknown. Here, we evaluated the expression, and functional relevance of the endothelial NOS isoform (eNOS) within the PVN of HF rats. Our studies indicate eNOS expression in astrocytes and the local microvasculature, but not in major PVN neurons. A diminished eNOS signal was apparent in the PVN of HF rats. Studies using the NO‐sensitive dye DAF2 indicated diminished NO levels in the posterior parvocellular subnucleus (13%, P<0.001), an area containing presympathetic PVN neurons. While the specific eNOS blocker L‐NIO (10 μM) diminished DAF2 levels in sham rats (14%, P<0.05) within this region, no changes were observed in HF rats. No differences in NO availability or L‐NIO effects were observed in magnocellular regions of the PVN and SON (P>0.1). L‐NIO increased the firing discharge of PVN‐RVLM neurons in sham rats (∼75%, P< 0.02), an effect that was blunted in PVN‐RVLM neurons from HF rats (P>0.1). In summary, our results support that a diminished eNOS expression/function within autonomic‐related PVN regions contributes to enhanced PVN neuronal activity, and likely neurohumoral activation, during HF. AHA 0640092N and NIH HL68725.
Enhanced Glutamatergic Inputs to Hypothalamic Presympathetic Neurons in Spontaneously Hypertensive Rats
Authors: Li De‐Pei , Yang Qing , Pan Hao‐Min , Pan Hui‐Lin
Published: June 2021

Glutamatergic inputs in the paraventricular nucleus (PVN) maintain the increased sympathetic activity in hypertension. In this study, we determined the synaptic mechanisms of increased glutamatergic inputs to presympathetic PVN neurons in spontaneously hypertensive rats (SHR). The spinally projecting PVN neurons were retrogradely labeled by injection of Fluospheres into the intermediolateral cell column of the spinal cord. The basal frequency of spontaneous and miniature glutamatergic excitatory postsynaptic currents (EPSCs) of labeled PVN neurons are significantly greater in SHR than that in WKY rats. The amplitude of EPSCs was not significantly different between WKY and SHR. Furthermore, the postsynaptic NMDA currents elicited by either electrical stimulation or puff application of NMDA are significantly higher in SHRs than in WKY rats. However, AMPA currents evoked by the electrical stimulation and puff application in were not different between WKY and SHRs. In addition, ionotropic glutamate receptors selective antagonists kynurenic acid, AP5, and CNQX significantly decreased the basal firing activity of labeled PVN neurons in SHR, but failed to alter the firing activity of the PVN neurons in WKY rats. Thus, the glutamatergic synaptic inputs to presympathetic PVN neurons are augmented and contribute to the elevated firing activity of the PVN neruons in SHR.
Altered GABAergic inhibition of the rostral ventrolateral medulla in spontaneously hypertensive rats
Authors: Schreihofer Ann M , Mobley Susan C
Published: June 2021

Spontaneously hypertensive rats (SHR) have elevated sympathetic nerve activity (SNA) and arterial pressure (AP) compared to WKY rats. Studies examining the ability of GABA to inhibit pre‐sympathetic neurons in the rostral ventrolateral medulla (RVLM) that drive SNA in SHR have yielded equivocal results, possibly due to differences in baroreflexes or endogenous GABAergic inhibition from the caudal ventrolateral medulla (CVLM). Here we examine whether the ability of GABA to decrease SNA and AP in the RVLM is altered in SHR compared to WKY rats after inhibition of the CVLM. In chloralose‐anesthetized, ventilated, paralyzed rats a glutamate antagonist (kynurenate 5.5 pmol/100 nl) was microinjected bilaterally into CVLM, followed by a microinjection of GABA (0.3, 1.0, 3.0 nmol) into an RVLM. Microinjection of kynurenate into CVLM increased splanchinc SNA less in SHR than WKY rats (326 +/− 38% vs. 713+/− 98%, P <0.05, n=13) with smaller pressor responses (79+/−3 vs. 95+/−3 mmHg, P<0.05). In contrast, microinjection of GABA into the RVLM evoked larger decreases in SNA and AP in SHR at all doses. For example, 1 nmol GABA decreased SNA 39+/− 7% in SHR vs. 8+/−3% in WKY rats. (P<0.05, n=5/group) and AP by 30+/−14 in SHR vs. 10+/−8 mmHg in WKY rats (P<0.05). These data suggest tonic inhibition of the RVLM by the CVLM is reduced in SHR, manifesting in increased sensitivity to GABA in the RVLM. NIH HL075174.