bluuebunny/crossref_metadata_2025 huggingface dataset:
Fractional Na+ excretion is a clinical description of renal tubular Na+ reabsorption
Authors: Carroll Robert Graham
Published: June 2021

All physiology textbooks indicate that the renal tubules reabsorb >99% of the filtered Na+ load. Clinical tests, however, focus on the inverse, and indicate that the fractional excretion of Na+ is <1% of the filtered load. Fractional Na+ excretion is used to distinguish between renal failure from tubular Na+ reabsorption defects vs. renal failure from pre‐renal diseases. For example, the damage to the tubular epithelia in acute tubular necrosis causes an impaired Na+ reabsorption across the tubular epithelia. This disease is characterized by a fractional Na+ excretion of > 2%, corresponding to a reduction in Na+ reabsorption from > 99% to < 98%. In contrast, renal failure from hypotension is characterized by intact epithelial transport, and since epithelial Na+ transport is not diminished, the fractional Na+ excretion remains normal at < 1%. Addition of the clinical term "fractional Na+ excretion" to the tubular transport of the medical physiology texts will allow students to more easily connect the diagnostic tests to the underlying mechanisms of diseases.
Evaluating the differentiation state of aortic vascular smooth muscle cells in the metabolic syndrome
Authors: Bradley Kiana K , Rocic Petra , Weber David S
Published: June 2021

The JCR rat, a model mimicking human metabolic syndrome exhibits altered vascular remodeling. These features make the JCR rat an excellent model for study of vascular smooth muscle cell (VSMC) phenotype switching. To date, basal contractile protein expression in VSMCs from JCR rats has not been determined. We hypothesized that the expression profile in JCR VSMCs would be indicative of a de‐differentiated, synthetic phenotype compared to VSMCs from healthy WKY rats, resulting in decreased expression of SM‐specific contractile proteins, and increased VSMC proliferation and migration. We isolated aortic VSMCs from JCR and WKY rats and compared basal contractile protein expression by Western blotting. Results indicate that SM‐myosin heavy chain and SM‐alpha‐actin expression was decreased 46% and 76%, respectively, in JCR vs. WKY VSMCs, caldesmon expression was six fold higher, and smoothelin levels remained unchanged. Proliferation rate, determined by BrdU incorporation, was increased 63% in JCR VSMCs. In response to scratch wounding, JCR VSMCs exhibited significantly greater migration than WKY VSMCs. These findings demonstrate that cultured JCR aortic VSMCs display a more de‐differentiated phenotype when compared to WKY aortic VSMC, which may underlie altered vascular remodeling in the metabolic syndrome. NIH HL 084159 and HL 093052
Nonmuscle Myosin is Regulated During Smooth Muscle Contraction
Authors: Yuen Samantha , Ogut Ozgur , Brozovich Frank
Published: June 2021

The participation of nonmuscle myosin in force maintenance is controversial. To participate in force maintenance, nonmuscle myosin activity must be regulated in smooth muscle. To investigate this question, phosphorylation of the regulatory light chains of smooth muscle myosin (SMLC) and nonmuscle myosin (NMLC) were examined by two dimensional SDS‐PAGE. Intact mouse aortic smooth muscle strips were stimulated with either KCl depolarization or angiotensin II, and 4 distinct LC spots were resolved by two dimensional SDS‐PAGE. Using mass spectroscopy, these spots were identified as four distinct proteins; phosphorylated NMLC, non‐phosphorylated NMLC, phosphorylated SMLC and non‐phosphorylated SMLC. During KCl depolarization, inhibition of Rho kinase or PKC did not affect the increases in SMLC and NMLC phosphorylation. During angiotensin II stimulation, inhibition of PKC or Rho kinase reduced SMLC phosphorylation, while only inhibition of Rho kinase reduced NMLC phosphorylation. These data suggest that in smooth muscle, nonmuscle myosin is regulated by Ca2+‐calmodulin activated MLCK during depolarization, and a Rho kinase mediated pathway during agonist stimulation.
IP3 receptors stimulate mitochondrial‐dependent TRPC1 expression through NF‐κB activation in arterial smooth muscle cells
Authors: Narayanan Damodaran , Pfeffer Lawrence M , Jaggar Jonathan H
Published: June 2021

In arterial smooth muscle cells, physiological functions of mitochondria that are distinct from their energy generating function are unclear. We show that endothelin‐1 (ET‐1), a vasoconstrictor, stimulated calcium (Ca2+) waves, increased mitochondrial Ca2+ concentration ([Ca2+]mito), depolarized mitochondria, and increased mitochondria‐derived reactive oxygen species (ROS) production in cerebral artery smooth muscle cells. Thapsigargin, a sarcoplasmic reticulum Ca2+ ATPase inhibitor and xestospongin C, an inositol 1,4,5‐trisphosphate receptor (IP3R) inhibitor, blocked ET‐1‐induced Ca2+ waves, [Ca2+]mito elevation, mitochondrial depolarization, and ROS elevation. ET‐1 increased nuclear translocation of the p50 subunit of nuclear factor‐κB (NF‐κB) and luciferase expression driven by a NF‐κB promoter, and these effects were blocked by thapsigargin. ET‐1 elevated transient receptor potential cation channel 1 (TRPC1) expression, and this effect was blocked by bortezomib, a proteasome inhibitor that inhibits NF‐κB activation. These data indicate that ET‐1‐induced IP3R activation drives TRPC1 expression by causing a [Ca2+]mito elevation, leading to mitochondrial depolarization, ROS production, and NF‐κB activation. NIH R01077678.
Maternal low protein diet: Developmental origin of adult hypertension
Authors: Goyal Ravi , Leitzke Arthur , Goyal Dipali , Longo Lawrence D.
Published: June 2021

Objective

We hypothesized that maternal isocaloric, low‐protein, diet during pregnancy programs the Renin Angiotensin System in the fetus leading to hypertension in the adult. We conducted studies on pregnant mice, on isocaloric, normal or low protein diet (50% and 33% less protein).

Methods

Proteins and mRNA levels were measured using western blot and real time PCR technique and blood pressure was recorded by the non‐invasive tail cuff system.

Results

Maternal low protein diet (MLPD) resulted in low birth weight and rapid catch up growth in both male and female offspring. MLPD induced hypertension in female mice offspring at 11 weeks of age, whereas no hypertension was observed in male offspring by 20th week of age. Our results indicate increased AGT and ACE, but decreased AT2, expression of mRNA, with no change in the protein expression of AGT but decreased protein expression of both, ACE and AT2 receptors with MLPD. Promoter methylation analysis of ACE gene showed hypo‐methylation of the CpG islands as a consequence of maternal protein deprivation.

Conclusions

Our studies establish that MLPD programs hypertension in female offspring by epigenetic programming of the renin‐angiotensin system.
Cystathionine γ‐lyase and cystathionine β‐synthase expression and activity change with age and caloric restriction
Authors: Alendy Maikel J , Predmore Benjamin Lee , Ahmed Khadija , Leeuwenburgh Christiaan , Julian David
Published: June 2021

Hydrogen sulfide (H2S), a gas signaling molecule with actions on the cardiovascular system, is endogenously produced by cystathionine γ‐lyase (CSE) and cystathionine β‐synthase (CBS). We have previously shown an increase in H2S signaling efficacy with age and caloric restriction (CR) in rat aorta and hypothesized that CSE and CBS expression would change similarly. Aorta and liver tissues were collected from rats of ages 8 to 38 months maintained on CR or ad libitum (AL) diets. Protein and RNA were extracted for western blotting and real time PCR, respectively. H2S production was determined in tissue homogenates. While age did not affect H2S production or RNA expression, diet did: aorta and liver H2S production and CSE RNA expression were higher in CR than AL animals for all ages (t‐test, p=0.041). Age and diet both affected protein expression (2‐way ANOVA, p=0.0056): in aorta, both enzymes increased with age, with expression highest in AL animals at older ages; in liver, CSE increased with age, with expression highest in CR animals at older ages, whereas CBS decreased with age with no diet affect. These data show that for aorta, enzyme expression increases with age while RNA expression does not, and CR animals with lower CSE and CBS produce more H2S than AL animals. However, this may not be true of non‐vascular tissues, such as liver. This suggests a decrease in enzyme function with age that may be attenuated by CR.
Hibernation and plasma from hibernating animals modulate the cardioprotective delta opioid system
Authors: Denning Gerene M , Ackermann Laynez W , Stoll Lynn L , Romig Sara A , Carey Hannah V , Dickson Eric W
Published: June 2021

Our goal is to harness the protective effects of mammalian hibernation to prevent and to treat cardiovascular disease (CVD). CVD is among the leading causes of morbidity and mortality in the United States. The specific objective of this study was to determine the effect of hibernation on the cardiac delta opioid system, and to determine the mechanisms by which plasma from hibernating woodchucks (HWP) is cardioprotective. Hearts were collected from summer woodchucks and squirrels, and from animals at defined times in the hibernation cycle. Tissues were analyzed by real‐time PCR for gene expression and by Western blot analysis for protein levels. Immunofluorescence microscopy was used to determine the effects of HWP on cellular localization of the delta opioid receptor (DOR). Hibernation induced dynamic changes in cardiac expression of opioid genes, and altered protein levels of the delta opioid precursor proenkephalin and of DOR. HWP increased localization of DOR to the plasma membrane of primary human vascular cells, suggesting that mobilization of DOR to cell surfaces may contribute to cardioprotection. These data support a role for delta opioids and their receptor in the hibernation phenotype, and provide insights that will further the development of therapies based on the natural protective processes of hibernation. Funded by the Department of Emergency Medicine, The University of Iowa.
The genomics of DNA replication of human chromosomes
Authors: Dutta Anindya , Karnani Neerja , Taylor Christopher M , Malhotra Ankit
Published: June 2021

The sequencing of the human genome allows us to now address questions about DNA replication in human cells at a genome‐wide level on defined sequence fragments. The time of replication of mb of sequence clearly demarcates the chromosomes into domains of hundreds of kb with different epigenetic packaging. The time of replication also identifies chromosomal segments that show inter‐allelic variation in epigenetic packaging. Origin‐centered nascent strands were isolated from human cells by two independent methods: bromodeoxyuridine (BrdU) incorporation into nascent strands followed by immunoprecipitation with anti‐BrdU antibody or resistance of RNA‐capped nascent strand DNA to digestion by lambda exonuclease. On the 1% of the genome studied, about 80 sites (of a few hundred bases each) reproducibly yielded overlapping nascent strand peaks by both methods (high confidence origins). However, there are an additional >200 origins on the same 1% of the genome that occur at variable sites separated in location by up to 10 kb between nascent strand preparations. Thus, the origins that replicate our chromosomes are either site‐specific or plastic and the biochemical basis of the differences between the two types of origins will be interesting to explore.
Effects of Metabolic Syndrome (MetS) on Male and Female Ossabaw Miniature Pig Detrusor Muscle Contractility and Compliance
Authors: Francis Samuel J , Yang David , Sharma Richa , Alloosh Mouhamad , Sturek Michael S , Packer C Subah
Published: June 2021

Diabetic bladder dysfunction is not understood. Previous work suggests that male MetS Ossabaw pigs have stiffer and more contractile detrusor muscle compared with lean male pigs. The purpose of the current study was to determine if altered detrusor muscle function in MetS pigs is gender dependent. Detrusor strips from male and female Ossabaw pigs maintained on an atherogenic or control diet were mounted at slack length in muscle baths. Following equilibration, muscle strips were stimulated with supramaximal electrical field stimulation (EFS). Following relaxation, cycles of incremental stretches (0.5‐1.5 mm) followed by EFS‐induced contractions were repeated every 5 minutes until maximum total tension was achieved or the tissue yielded. Normalized passive and active length‐tension (L‐T) curves were generated. Both mean passive and active L‐T curves for MetS bladder muscle are elevated above the respective control curves indicating increased contractility and decreased compliance of bladder muscle in both male and female MetS pigs. Stiffer bladders that are hypercontractile could explain both incomplete voiding and increased urinary frequency experienced in diabetes characteristic of metabolic syndrome.

(D Yang is an Indy Project SEED Scholar; S Francis is an IUSM Summer Research in Academic Medicine Scholar; R Sharma was a 2007 APS Undergraduate Summer Research Fellow; Support: NIH RR013223, HL062552)
Y‐27632 relaxation of carbachol‐induced smooth muscle contraction in guinea pig trachea
Authors: Hare Brendan , Kojtari Sheri , Herrera Gerald M.
Published: June 2021

Smooth muscle (SM) contractions are caused by increases in cytoplasmic Ca2+ ([Ca2+]i). SM contractility may also depend on factors that regulate the sensitivity of intracellular processes to [Ca2+]i. Increasing evidence supports a role for Rho Kinase pathway in regulating smooth muscle contractility, and modulation of Rho Kinase activity may have therapeutic use for diseases such as asthma. Pharmacological inhibition of Rho Kinase using Y‐27632 causes relaxation of airway smooth muscle preparations. However, little is known about how Rho Kinase inhibition affects the sensitivity of airway smooth muscle to a contractile agonist. In this study, the muscarinic agonist carbachol (CCh) was applied to guinea pig trachea rings in a cumulative fashion. Following an initial CCh dose‐response curve, a second CCh dose‐response curve was constructed in the presence of varying concentrations of Y‐27632 (0.01 to 100 µM). EC50 values for CCh‐induced contractions in the presence of Y‐27632 were (in µM) 0.52, 0.4, 0.15, 0.2, 0.24, 0.29, 2, 0.51, 2.2, 0.46 in presence of 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 Y‐27632, respectively. The maximum contractile response to CCh was decreased by 20‐25% in the presence of Y‐27632. Inhibition of Rho Kinase using Y‐27632 appears to decrease the maximum contractility in response to muscarinic stimulation without affecting the sensitivity of the airway SM to the muscarinic agonist.
The large conductance Ca2+‐ and voltage‐activated K+ channel is the primary K+ channel that controls cell excitability and contractility in human urinary bladder smooth muscle
Authors: Hristov Kiril L. , Chen Muyan , Kellett Whitney F. , Rovner Eric S. , Petkov Georgi V.
Published: June 2021

We examined the large conductance Ca2+‐activated K+ (BK) channel expression, electrophysiological properties, and possible physiological roles in human urinary bladder smooth muscle (UBSM). RT‐PCR with freshly isolated human UBSM cells shows mRNA expression of BK channel pore‐forming α, and regulatory β1/β4 subunits. The expression of all three subunits was confirmed by specific antibodies and immunocytochemistry on single cells. The majority of the whole‐cell outward current induced by voltage‐step depolarizations (‐40 to +80 mV) was inhibited by iberiotoxin (IBTX), a BK channel selective inhibitor. Voltage‐dependent spontaneous transient outward currents (STOCs) were recorded from ‐40 to +40 mV. STOCs were blocked by IBTX or ryanodine. In current‐clamp mode, IBTX caused ~3 mV depolarization. In whole cell configuration, in the absence of STOCs, single channels of 139.5 pS conductance with voltage‐dependent amplitude and open probability were recorded. These channels were blocked by IBTX, consistent with the BK channel. In isolated strips, IBTX increased spontaneous phasic contraction amplitude, force, and tone as well as the amplitude of electrical field induced contractions. In conclusion, the BK channel in the human bladder consists of α, β1 and β4 subunits and is the primary K+ channel that controls cell excitability and contractility of human UBSM.

Supported by NIH DK‐070909 to G.V. Petkov.
Evaluation of markers for skeletal muscle atrophy and calcineurin/NFAT signaling in acute and chronic diabetes mellitis
Authors: Roberts Tiffany Karla , Donaldson Sue K , Roberts Brian , Gooch Jennifer L , Price S Russ
Published: June 2021

Diabetes mellitus (DM) causes persistent skeletal muscle atrophy. In a streptozotocin (STZ)‐induced rat model of DM, atrophy occured both acutely (3 d) and chronically (3 wk) and was greater in the mixed MHC fiber gastrocnemius (GAST) muscle than the predominantly MHCI fiber soleus muscle. In chronic STZ‐DM atrophy, switching of muscle fibers from Type MHCI to MHCII was also observed in the soleus. In this study, we have examined several markers of muscle atrophy including atrogin‐1 (AT‐1) and PGC1α mRNAs in GAST muscle of acute and chronic STZ‐DM rats and their controls. Since calcineurin activity is necessary to maintain MHCI fiber type, we also tested the hypothesis that calcineurin (Cn)/NFAT signaling would be decreased during atrophy by measuring CnA catalytic subunit protein and the mRNA of MCIP1.4, a NFAT‐regulated gene. In acute STZ‐DM rats AT‐1 was increased and PGC1α mRNA was decreased relative to Control muscle. CnA protein content was unchanged, and MCIP1.4 mRNA was decreased. In chronic STZ‐DM rats, AT‐1 mRNA was unchanged, whereas CnA protein and both MCIP1.4 and PGC1α mRNAs were decreased. These[PC1] findings indicate that STZ‐DM rapidly initiates and sustains muscle atrophy in the GAST. The decrease in Cn/NFAT signaling is also consistent with an atrophy process that occurs preferentially in MHC II fibers. Supported by NIH DK63658.
Synchronous synaptic input to respiratory muscle motoneurons of tongue and inspiratory intercostal muscles.
Authors: Rice Amber D , Fregosi Ralph F
Published: June 2021

Short time scale, synchronous firing of two or more motor units, within or between muscles, is thought to be due to common input from the same presynaptic neuron. The extent of synchronous input can be quantified with cross‐correlation analysis of simultaneously recorded motoneurons or motor units. We examined the prevalence of synchronous discharge of motor units within genioglossus (tongue protrudor muscle), hyoglossus (tongue retractor muscle) and inspiratory intercostal muscles. 28 pairs of single motor unit potentials were recorded in urethane‐anesthetized rats, and cross correlation analysis was performed on each pair. Long‐time scale oscillations were found in all 28 pairs of simultaneously recorded motor units, reflecting shared input from the respiratory central pattern generator (CPG). 6/12 pairs of ipsilateral hyoglossus units, 2/3 pairs of ipsilateral genioglossus motor units and 6/13 pairs of ipsilateral intercostal motor units showed short time scale peaks in the cross correlation histogram. However, the strength of the correlation between pairs of intercostal motor units was higher than that for tongue muscle motor units. Tongue muscles appear to have more complex presynaptic circuitry than intercostal muscles. Supported by the NIDCD.
Skeletal muscle sensory afferents affect cardiac regulation in humans
Authors: Goswami Ruma , Jackson Patience , Frances Maria F , Fleischhauer Arlene , Shoemaker J Kevin
Published: June 2021

Elevated cardiac output (Q) during isometric contractions may be due to neural signals arising from the motor cortex or from the contracting muscle. This study tested the hypothesis that skeletal muscle sensory inputs affect cardiac function and that this effect depends on baroreceptor loading. Three‐minute segments of heart rate (HR; ECG), mean arterial pressure (MAP), and Q (Finometer) were obtained during supine rest, ‐35 mmHg lower body negative pressure (LBNP), and LBNP + electrical stimulation (ES) of the forearm (n=7 males). ES was applied at sub‐motor (group I and II afferents) and non‐fatiguing supra‐motor threshold (group III and IV fibers) levels. Parasympathetic indicators were assessed by heart rate variability (HRV) analysis. ES had no effect on HR, MAP, Q or HRV during supine rest. LBNP increased HR and decreased pulse pressure (P<0.05). Q during LBNP was reduced further with sub‐motor ES versus LBNP (‐0.142 L/min; P<0.05). Compared with LBNP alone, Q was increased during LBNP + supra‐motor ES (+0.132 L/min; P<0.05). ES during LBNP did not change HR, MAP, or HRV indicators. These results suggest that Type I and II afferents depress, and Type III and IV afferents augment Q during LBNP. As indices of HRV and parasympathetic outflow were not affected, these data suggest that muscle afferent stimulation affects Q through a sympathetic neural mechanism. Supported by the Heart and Stroke Foundation of Ontario.
Effects of aging on body temperature and several hormones (ADH, TSH, fT3, fT4 and leptin) in different seasons
Authors: Sato Maki , Kanikowska Dominika , Inukai Yoko , Shimizu Yuuki , Nishimura Naoki , Iwase Satoshi , Sugenoya Junichi
Published: June 2021

With change in the season, the patterns of body temperature regulation as well as hormonal regulation might be modified. The purpose of this study is to examine the seasonal changes in various hormones in association with temperature and metabolic regulation (ADH, TSH, fT3, fT4 and leptin) in young (21±1 yrs; mean±SD) and elder subjects (69±5 yrs; mean±SD). At each season, blood was taken in a sitting position at rest after the subject stayed for 30 min or more in the climatic chamber set at 26°C and 50%RH, and the blood osmolality and hormone concentrations were measured. The average of atmospheric temperature in experimental days were 15‐20°C in spring, 25‐30°C in summer, 15‐23°C in autumn and 5‐10°C in winter. We found that: 1) ADH concentration was significantly higher in elder than in younger in winter; 2) osmolality in elder subject was significantly higher in winter than in summer; 3) concentration of leptin in elder subjects was significantly higher in winter than in other seasons; 4) concentration of TSH, fT3 and fT4 in elder subjects was not significantly different with seasons. It was suggested that hormonal regulation was reduced in elder subjects in winter.
Subclinical Inflammation, Endothelial Progenitor Cell Depletion, and Endothelial Dysfunction in Adolescents with Obesity
Authors: Huang Hong , Groner Judith A. , Kuck Jennifer , Nicholson Lisa , Murray Robert , Bauer John A
Published: June 2021

Obesity related inflammation plays a key role in endothelial injury, which is an antecedent of adult cardiovascular disease. Few studies have attempted to define these relationships in children. We hypothesized that obesity is related to inflammation, endothelial progenitor cell (EPC) depletion, and endothelial dysfunction in adolescents. A total of 100 normal weight (n=45, BMI <85th percentile for their age and sex) and obese (n=55, BMI >95th) adolescents (9‐18 years of age) were recruited. Obese adolescents had higher glucose, total‐ and LDL‐cholesterol, and lower HDL‐cholesterol levels (p<0.05). They had higher plasma hsCRP (6.40 +/‐ 8.93 vs. 0.45 +/‐ 0.90 mg/L, p<0.001) and lower adiponectin (2410.72 +/‐ 727.69 vs. 3111.68 +/‐ 1063.18 mg/L, p<0.01) levels. Meanwhile, the obese group had fewer EPCs, measured by flow cytometry (1.76 +/‐ 0.69 vs. 2.16 +/‐ 0.81 %, p<0.01). Increased forearm vascular resistance (FVR) (mmHg/ml/100 ml/min) was also observed, both at basal condition (44.22 +/‐ 28.29 vs. 30.54 +/‐ 15.73, p<0.01) and after ischemic test (9.34 +/‐ 5.90 vs. 6.31 +/‐ 5.04, p<0.01), in obese children. Significant correlations were found between BMI and adiponectin (r= ‐0.46), EPC (r= ‐0.26), hsCRP (r=0.64), and FVR (basal: r=0.35; ischemia: r= 0.30). EPC was negatively correlated with hsCRP (r= ‐0.28), suggesting obesity‐related inflammation is involved in endothelial injury in adolescents.
NBCn1 (Slc4a7) is a major HCO3− importer in murine duodenum and essential for basal and stimulated HCO3− secretion in vivo
Authors: Chen Mingmin , Praetorius Jeppe , Wang Jian , Stieger Nicole , Zheng Wen , Riederer Brigitte , Krabbenhöft Anja , Bonhagen Janina , Rausch Brigitte , Engelhardt Regina , Dringenberg Ulrike , Aalkjaer Christian , Seidler Ursula
Published: June 2021

HCO3− secretion protects the proximal duodenum against damage by gastric acid. The molecular details of basolateral HCO3− import are unknown, but several base importers are expressed in the basolateral membrane of duodenocytes. The electroneutral sodium bicarbonate cotransporter NBCn1 (Slc4a7) is expressed in the villous region of the duodenum. To elucidate its importance for duodenal bicarbonate secretion, as well as pHi regulation, we measured basal and stimulated HCO3− secretion in the proximal duodenum of anesthetized Slc4a7‐/‐ mice and wt littermates in vivo during controlled systemic acid/base parameters, by continuous determination of the HCO3− that was secreted into the luminal perfusate, and in isolated duodenal mucosa in vitro in Ussing‐chambers by pH‐stat titration. In addition, we fluorometrically assessed base‐uptake rates after acidification of intact villi and isolated duodenocytes. Slc4a7‐/‐mice displayed significantly lower basal and forskolin‐stimulated duodenal HCO3− secretion in vivo than wt littermates. This difference was markedly larger in vivo than in isolated mucosa. The Na+‐ and HCO3−‐dependent base uptake rate was significantly decreased in Slc4a7‐/‐ (33.2%) compared to wt villus duodenocytes. The electroneutral Na+HCO3− cotransporter Slc4a7 is a major HCO3− importer in murine duodenum and is essential for basal and stimulated HCO3− secretion in vivo.
Proportion of down‐regulated in adenoma (Dra, Slc26a3) Cl−/HCO3− exchange activity coupled with Na+/H+ exchange in the lower villous epithelium of murine duodenum
Authors: Clarke Lane L , Walker Nancy M
Published: June 2021

Dra plays a dual role in the proximal small intestine. Studies of knockout mice (KO) have shown that 1) Dra couples with the Na+/H+ exchanger Nhe3 for NaCl absorption, and 2) Dra contributes importantly to basal and, in concert with the cystic fibrosis transmembrane conductance regulator (CFTR), to cAMP‐stimulated HCO3− secretion. Further, cell shrinkage of the villous epithelium induced by cAMP activation of CFTR plays a central role in the inhibition of coupled NaCl absorption. These interactions gain complexity in the lower villous epithelium where Dra is the dominant Cl−/HCO3− exchanger and both CFTR and Nhe3 are active. We sought to determine the proportion of Dra engaged in coupled NaCl absorption in the lower villous epithelium of intact murine duodenal mucosa using BCECF microfluorimetry. Cl−/HCO3− exchange was estimated by changes in intracellular pH induced by Cl− substitution in the apical bath. Lower villous epithelium exhibited robust Cl−/HCO3− exchange in wild‐type (WT) duodenum that was eliminated in Dra KO mice. In WT, cAMP stimulation or treatment with amiloride analog EIPA reduced Cl−/HCO3− exchange rates by 50%. In contrast, cAMP stimulation had no effect on Cl−/HCO3− exchange in CFTR KO duodenum. We conclude that ~50% of Dra activity in the duodenal lower villus is coupled to Nhe3 and that the two exchangers are coordinately inhibited by cAMP in a CFTR‐dependent process. Support: NIDDK and CFF.
Slow‐pressor angiotensin II (AngII)‐dependent hypertension (HTN) requires prostaglandin E2 (PGE2) type 1 receptors (EP1R) and superoxide (O2·‐) signaling
Authors: Burmeister Melissa A. , Butler Scott D. , Sharma Ram V. , Iadecola Costantino , Davisson Robin L.
Published: June 2021

Oxidant signaling in the subfornical organ (SFO), a primary CNS sensor for blood‐borne AngII, plays a causative role in slow‐pressor AngII‐dependent HTN. PGE2 and EP1R are also implicated in AngII‐induced vasopressor effects, but it is not known if they act in brain or other organs. Here we hypothesized that AngII HTN is mediated by PGE2 signaling in the brain and that blocking EP1R in the SFO would prevent AngII HTN by inhibiting O2−. production. C57BL/6 mice were implanted with telemeters for mean arterial pressure (MAP) recordings, osmominipumps for 2 wks AngII infusion (600 ng/kg/min), and ICV cannulae for infusion of the selective EP1R antagonist SC‐51089 (6 μg/h) or saline (0.25 μl/h) for 2 wks. Ang‐II infusion induced HTN in mice receiving ICV saline (baseline: MAP=101±5; day 12 AngII: MAP=129±5 mmHg, n=4, p<0.05). This HTN was abolished in mice receiving ICV SC‐51089 (baseline: MAP=100±2; day 12 AngII: MAP=101±7 mmHg, n=4). In mice treated with ICV saline, AngII infusion also produced a 1.9±0.4 fold increase in O2−. production in the SFO as measured by dihydroethidium, which was completely prevented in mice treated with ICV SC‐51089 (fold change=0.9±0.2; p>0.05). These data suggest that EP1R activation in the CNS is critical in slow pressor AngII‐induced HTN and implicate O2−. in the SFO in this effect. Supported by HL063887, HL084624, AHA 0540114N.
Sirtuin‐1‐dependent COX2 Induction is an Essential Survival Mechanism in Renal Medullary Interstitial Cells against Oxidative Stress
Authors: He Wenjuan , Zhao Min , Zhang Ming‐Zhi , Davis Linda , Harris Raymond , Breyer Matthew , Hao Chuan‐Ming
Published: June 2021

The present studies examined the role of NAD+‐dependant deacetylase sirtuin‐1 (SIRT1) in protecting the cells in renal medulla against oxidative stress. High levels of nitrosylated tyrosine were detected in renal medulla compared with renal cortex of C57BL6/j mice by immunohistochemistry, consistence with the presence of oxidative stress in renal medulla. Immunoblot and immunohistochemistry also showed abundant SIRT1 in renal medulla, mainly co‐localizing with COX2 in renal medullary interstitial cells (RMICs) by immunofluorescence. Selective knock‐down of SIRT1 in cultured mouse RMICs by shRNA significantly increased H2O2 (500 μM) induced cell death (from 48±5% to 78±4%, P<0.0001), accompanied with increased activated‐caspase‐3 levels. SIRT1 knock‐down also almost abolished H2O2‐induced COX2 expression (P<0.0001). ChIP showed physical association of SIRT1 with mouse COX2 gene promoter, supporting a role of SIRT1 in modulating COX2 transcription. Pre‐treatment with PGE2 (0.1, 1 μM) dose dependently protected cultured SIRT1‐deleted RMICs from H2O2 induced cell death (from 57±5% to 31±6% and 18±9% respectively, P<0.0001), suggesting COX2 derived PGE2 contributes to the protective effect of SIRT1. These data are consistent with an important role for SIRT1 in regulating COX2 expression and promoting cell adaptation to oxidative stress in renal medulla. Research supported by NIH.
Bradykinin stimulates renal collecting duct prorenin
Authors: Toma Ildiko , Vargas Sarah Laurin , Koo Jahoon John , Peti‐Peterdi Janos
Published: June 2021

Elevated plasma prorenin (PR) is a predictor of diabetic (DM) microvascular complications. We recently identified the renal collecting duct (CD) as the major source of PR in DM and showed that angiotensin II (ANGII) receptor blockade, effectively reduces renal and plasma PR levels. We aimed to compare the effects of the ANGII receptor 1 blocker telmisartan (ARB, T), and the angiotensin converting enzyme inhibitor (ACEI), ramipril (R), on CD PR. PR content in the intact kidney was visualized in control (C) and STZ‐DM MWF rats using in vivo multi‐photon fluorescence microscopy. DM‐rats were treated for 28 days with T (ARB, 3mg/kg/day) or R (ACEI, 1mg/kg/day). Direct effects of ANGII and bradykinin (B,10nM each) on CD PR synthesis were measured using renin immunocytochemistry. STZ‐DM caused a 3‐fold increase in CD PR content which was completely abolished by T, but not by R. Also, immunoblot and renin activity measurements found that renal tissue and plasma PR levels increased 2‐fold in STZ‐DM which were prevented by T (0.8‐fold of C), but not by R (2.2‐fold). Similarly to ANGII, B increased immunoreactive PR content in cultured CD cells 35‐fold which was prevented by the B B2 receptor blocker HOE‐140. In conclusion, elevated CD PR content in DM is most effectively inhibited by ANGII receptor blockade using T and not by the blockade of the ACE using R, which effect is at least in part due to the direct stimulatory effect of B on CD PR.
Measurement of Plasma Volume using Nanoparticles in Mice
Authors: Eisner Christoph Benedikt , Espey Mike , Ow Hooisweng , Wang Kenneth W. , Wiesner Ulrich , Schnermann Jurgen
Published: June 2021

Plasma volume (PV) is an important pathogenic factor in hypertension and cardiovascular disease, and a target of infusion and diuretic therapies. However, PV is not commonly measured because available methods (distribution V of Evans Blue, radioactively labeled albumin or indocyanine green) are not well suited for routine hospital application. To measure PV we have used precisely sized and biologically inert core‐shell silica nanoparticles comprised of a dye‐rich core (Cy5), silica shell, and a coating of polyethylene glycol ("C dots"). Size‐gradated C dots (diameter in nm: 25, 20, 18, 14, 6 and 3) injected iv were not detected in the urine (fluorescence correlation spectroscopy) down to a size of 14 nm; in contrast, 6 and 3 nm C dots were rapidly excreted. To determine PV, C57 mice received a retro‐orbital injection of 25 nm C dots under brief isoflurane anesthesia. Plasma samples were taken from conscious mice after 10, 20, 30, and 60 min. Calculated distribution volume was not significantly different between time points suggesting retention of C dots in the vascular space. At 30 min PV averaged 1056 ± 81 μl (3.7 ± 0.1 % BW) in male and 843 ± 33 μl (3.1 ± 0.1 % BW) in female mice. After 24 h only 5.4 ± 1.7 % of injected particles was found in plasma. C dots may be a useful tool for assessing PV. Their range of applicability could be extended by using different particle sizes and encapsulated dyes with different fluorescent wavelengths.
Exogenous L‐Arginine (L‐Arg) blunts Angiotensin II (AngII)‐mediated renal vasoconstriction and improves pressure‐diuresis
Authors: Das Satarupa , De Miguel Carmen , Lund Hayley , Mattson David L
Published: June 2021

Administration of exogenous L‐Arg reverses AngII‐mediated hypertension and kidney disease in conscious rats. The present study assessed renal hemodynamics and pressure‐diuresis in Inactin‐anesthetized Sprague Dawley rats (n=4‐5/group) infused with saline vehicle, AngII (20 ng/kg/min, iv) or AngII + L‐Arg (300 ?g/kg/min, iv). Increasing renal perfusion pressure (RPP) from 100±1 to 142±3 mmHg with aortic clamps resulted in an increase in urine flow in control rats from 5.3±0.6 to 56.1±12.5 ?l/min/gram kidney weight (gkwt). In comparison, the pressure‐dependent increase in urine flow was significantly depressed by 50‐65% at all levels of RPP in AngII‐infused rats. Supplementation of L‐Arg restored pressure‐dependent diuresis to levels not significantly different from control rats. In control rats, glomerular filtration rate (GFR) averaged 1.4±0.4 ml/min/gkwt and renal blood flow (RBF) averaged 6.8±0.7 ml/min/gkwt at the lowest RPP. RBF was significantly reduced by 45% in AngII‐infused rats. Co‐infusion of L‐Arg reversed the AngII‐mediated changes in RBF to values not different from control. These data indicate that AngII‐mediated vasoconstriction and blunting of pressure‐diuresis is reversed by exogenous L‐Arg, demonstrating an important role for nitric oxide to blunt the renal vasoconstrictor, anti‐diuretic, and pro‐hypertensive effects of AngII. Supported by DK‐62803.
Entanercept reduces vessel remodeling in stroke prone spontaneously hypertensive rats
Authors: Dorrance Anne M , Poulsen Kyle L , McClain Jonathon L , Pires Paulo W
Published: June 2021

Inflammation plays a key role in the pathogenesis of hypertension. Hypertension results in resistance vessel remodeling which includes an increased vessel wall thickness and wall / lumen ratio. We hypothesized that inhibition of the proinflammatory cytokine, tumor necrosis factor‐α (TNF‐α) with the etanercept (ETN) would prevent vessel remodeling in stroke prone spontaneously hypertensive rats (SHRSP). Six‐week‐old SHRSP were treated with ETN (1.25mg/kg/day I.P.) for six weeks, after which blood pressure was measured and third order mesenteric arteries were removed for assessment of vessel structure by pressure myography. The results are presented as mean ± SEM. There was no difference in the body weight (253.4±6.6 vs 246.3±4.8 grams, control vs ETN) or blood pressure (206±9 vs 198±3mmHg, control vs ETN) between the control and ETN treated rats. At an intralumenal pressure of 60mmHg, ETN treatment significantly reduced the vessel wall thickness (31.9±1.8 vs 26.5±1.3μm control vs ETN p<0.05) and wall / lumen ratio (0.13±0.01 vs 0.10±0.003 control vs ETN p<0.05). Vessel wall stiffness was also reduced in the ETN treated rats as evidenced by a reduction in the β coefficient calculated from the individual stress / strain curves (5.45±0.39 vs 4.58±0.20 control vs ETN p<0.05). These results suggest that TNF‐α mediated inflammation contributes to the vessel remodeling process in a blood pressure independent manner.
Angiotensin II induced pro‐inflammatory cytokines and oxidative stress in the brain are attenuated in mice lacking the gene for TNF‐α
Authors: Sriramula Srinivas , Francis Joseph
Published: June 2021

Angiotensin II (Ang II) has been shown to have both central and peripheral effects. Recent studies suggest a functional crosstalk between angiotensin II and the pro‐inflammatory cytokine (PIC) tumor necrosis factor‐alpha (TNF‐a) in the progression of cardiovascular diseases. Recent findings from our lab suggest that Ang II increases the expression of PICs in the PVN, and that mice lacking the gene for TNF‐a (TNF‐a−/−) have attenuated hypertensive and hypertrophic responses to Ang II infusion. However, the mechanism for this effect is not known. In this study we examined whether Ang II infusion in TNF‐a−/− mice had any effect on PICs and oxidative stress in the hypothalamus and brainstem. Male TNF‐a−/− and control B61295F2/J (WT) mice were subcutaneously implanted with osmotic minipumps containing Ang II (1µg/kg/min) or saline for 14 days. Ang II infusion resulted in increased expression of PICs such as IL‐1beta, IL‐6, and TNF‐α in the hypothalamus and brainstem of WT mice. Ang II infusion also increased the expression of the NOX‐2 subunit of NADPH oxidase, indicating increased oxidative stress. However, this increase in PICs and oxidative stress was attenuated in Ang II infused TNF‐α ‐/− mice. Findings from the present study suggest that brain inflammatory cytokines contributes to the development of Ang II induced hypertension. Grant support: NHLBI grant HL‐80544 for Dr. Joseph Francis.
Lipopolysaccharide‐induced inflammation reduces tyrosine phosphorylation of cardiac connexin 43
Authors: Ouellette Yves , Sieck Gary C.
Published: June 2021

Myocardial performance is depressed in both sepsis and endotoxin shock. Altered gap junctional coupling of ventricular myocytes may play an important role in myocardial dysfunction during sepsis. Since phosphorylation is known to regulate turnover of gap junction protein, we hypothesized that the phosphorylation status of connexin 43 (Cx43) is altered in vivo following exposure to lipopolysacccharide (LPS). Saline or LPS (25 mg/kg; E. coli 055:B5) was injected intraperitoneally into c57bl/6 mice. At 18 h, hearts were excised and snap frozen on dry ice. Tissue was homogenized in Triton X‐100 lysate buffer, centrifuged and prepared for Western blot. Cx43 was immunoprecipitated from the supernatant and tyrosine phosphorylation of Cx43, detected by immunoblot. Phosphorylated forms of Cx43 are seen as three major bands on Western blots. After LPS treatment, there is a decrease in the intensity of the fastest migrating band (Cx43‐P0) and an increase in the two slower migrating forms, (Cx43‐P1 and Cx43‐P2). This corresponds to a decrease in tyrosine phosphorylation of Cx43. LPS results in decrease in myocardial Cx43 tyrosine phosphorylation possibly mediating LPS‐induced myocardial dysfunction.
High Sodium Diet Activated NFκB Signaling Plays an Important Role in Renal Medullary COX2 Induction
Authors: He Wenjuan , Zhao Min , Yull Fiona , Davis Linda , Blackwell Timothy , Breyer Matthew , Hao Chuan‐Ming
Published: June 2021

Renal medullary COX2 plays an important role in renal response to dietary sodium. NFκB is known as an important mediator of COX2 induction. The present study examined the role of NFκB in renal medullary COX2 induction following increased dietary sodium. High sodium diet (8% NaCl) for 3 days markedly increased renal medullary COX2 expression in C57Bl/6J mice. Co‐immunofluorescence using a COX2 antibody and antibodies against AQP1, ClC‐K, AQP2 and CD31 showed that dietary sodium induced COX2 was selectively expressed in renal medullary interstitial cells. By using NFκB reporter transgenic mice, we observed a 7±2 fold (P<0.05) increase of luciferase activity in renal medulla of the NFκB‐luciferase reporter mice following high sodium diet, and a robust induction of EGFP expression mainly localized in renal medullary interstitial cells of the NFκB‐EGFP reporter mice following high sodium diet. Treating C57Bl/6J mice with selective IκB kinase inhibitor IMD‐0354 (8mg/kg bw) substantially attenuated COX2 induction in renal medulla, and also significantly reduced urinary PGE2 following high sodium diet (from 1798±578 to 809±267 μg/24h, P<0.05). These data suggest a critical role of renal medullary NFκB signaling in the renal response to increased dietary sodium, which may account for the altered sodium balance observed in patients taking NFκB‐inhibiting drugs, such as Bortezomib. Research supported by NIH.
Estradiol replacement inhibits increased expression of Cyp17a1 mRNA in residual ovarian tissue of VCD‐treated follicle‐depleted mice
Authors: Craig Zelieann Rivera , Christian Patricia J. , Marion Samuel L. , Hoyer Patricia B.
Published: June 2021

Cyp17a1 encodes the protein responsible for production of androstenedione (A) and it has been proposed to be negatively regulated at the mRNA level by estradiol (E2) via estrogen receptor alpha in a local negative feedback loop. Enriched expression of Cyp17a1 mRNA has been observed in residual ovarian tissue of an ovary‐intact mouse model of menopause (VCD‐treated mouse) when compared to cycling controls. This study was designed to test the hypothesis that increased expression of Cyp17a1 in VCD‐treated follicle‐depleted ovaries can be reversed by replacement with E2. Female B6C3F1 mice (age d28) were dosed daily (17d; i.p.) with sesame oil (n=5) or VCD (160mg/kg; n=5). On day 19 after the onset of dosing a group of VCD‐treated mice received subcutaneous E2 implants (VCD/E2 mice; 180 μg/ml; silastic) which were replaced on d47, 75, 103 and 131. Ovaries and blood were collected on d188 and processed for analysis by real‐time PCR and measurement of circulating E2, A and E1 (estrone). Relative to VCD‐treated mice, ovarian Cyp17a1 mRNA was lower (VCD/E2: 2.44±0.35/control; VCD: 8.97±0.14/control; P<0.05) in VCD/E2 mice but remained higher (P<0.05) than in controls. Circulating A and E1 were decreased (P<0.05) in VCD/E2 mice when compared to VCD‐treated and cycling controls, whereas, levels of E2 did not differ between groups (P>0.05). These results provide evidence that E2 replacement decreases expression of Cyp17a1 mRNA in residual ovarian tissue of VCD‐treated mice which results in a decrease in levels of circulating A and E1. (AG021948)
Aged‐related decrease in post‐synaptic signaling components and caveolin‐1 in membrane/lipid rafts and synaptosomal membrane fractions in CNS
Authors: Head Brian Patrick , Bonds Jacqueline A. , Roth David M. , Patel Piyush M. , Peart Jason N. , Patel Hemal H.
Published: June 2021

Spines/synaptic densities decreases in aged brains. Membrane/lipid rafts (MLRs), enriched in cholesterol, glycosphingolipids, and sphingomyelin, are essential for development/stabilization of synapses. Caveolin (Cav), a cholesterol binding protein and marker of MRLs, localize synaptic residents such as NMDARs and AMPARs to MRLs, suggesting Cav compexes with synaptosomal proteins. We assessed age‐related loss of synaptic components from MLRs and synaptosomes (Syn). Two different sucrose density fractionation methods were used to isolate MLRs and Syn from brains of wild type (WT) C57BL/6 mice [young (Yg, 3‐6 months), mature (Mt, 12 months), and aged (Ag, 24 months)] and Cav‐1 KO mice (Yg). PSD‐95 (post‐synaptic density marker), the ionotropic glutamate receptors NMDAR2A (NR2A), NR2B, AMPAR, and Cav‐1 were detected in MLRs from Yg brains, with less detection in MLRs from Mt and Ag brains. Cav‐1 and PSD‐95 immunoprecipitation (IP) of MLRs showed a decrease in NR2A, NR2B, AMPAR, PSD‐95 and Cav‐1 in both IPs from Mt and Ag MLRs compared to Yg. There was also a significant decrease in PSD‐95, NR2A, NR2B, and Cav‐1 in Syn and PSD‐95 IPs of Syn with age. Hippocampi Syn from Cav‐1 KO mice (Yg) showed reduced PSD95, NR2A, and Cav‐1 compared to WT (Yg). These data indicate that Cav‐1 expression and its ability to properly organize signaling complexes may be a key determinant of age‐related changes in synaptic signaling.
Effect of forced exercise on differential expression of caveolin isoforms in MCAo‐operated rat tissues
Authors: Park Sookyoung , Won Jinyoung , Lee Youngjeon , Hong Yonggeun
Published: June 2021

Endurance exercise has many health benefits, including improvements in muscle metabolism, cardiovascular function, and increased exercise tolerance, also induce mitochondrial biogenesis in skeletal muscle. Exercise training can improve strength and cause adaptations in the skeletal muscle and nervous systems. Caveolins are integral membrane protein, which play a role in essential cellular functions. Especially, caveolin‐1 was reported to inhibit the activation of the type I receptor for TGF‐β1, which induces growth arrest in non‐muscle cells. Cav‐3 normally suppresses the myostatin‐mediated signal, thereby preventing muscular atrophy, Thus, this study was to reveal that the changes of expression of caveolins after forced exercise using by treadmill in muscular and non‐muscular tissues. Male(n=16) and female(n=16) Wistar rat were introduced to forced exercise with 25m/min speed for 30min, 3 times/day each time points at 07:00, 15:00 and 23:00. Then, the tissues were homogenized, total RNA extracted from gastrocnemius (fast‐twitched), soleus (slow‐twitched) muscle, and non‐muscle tissue of female rat using RNeasy kit (Qiagen, Germany) for further analysis. Stable expression of caveolin‐3 was detected even in all tissues applied of exercise group compared to control. Strikingly, caveolin‐1 and ‐2 were dramatically changed at expression level in the tissues of exercise group. Also, caveolin isoforms was shown as tissue specific differential expression. Keywords: Caveolae, caveolin, forced exercise, muscle type. Supported by BioGreen 21 Program (#20070401034006) from Rural development Administration, South Korea.