bluuebunny/crossref_metadata_2025 huggingface dataset:
A combined theoretical and experimental study of dehydrogenation selectivity by hepatic P450 enzymes during tamoxifen metabolism
Authors: Shahrokh Kiumars , Cheatham Thomas E. , Yost Garold S.
Published: June 2021

The factors that govern P450 dehydrogenation reactions are not as well understood as those that govern oxygenation reactions. Thus, current in silico methods fail to accurately predict dehydrogenation pathways and the resulting drug toxicities. Here we present new findings from a combined theoretical and experimental study of the P450‐catalyzed metabolism of tamoxifen. Ab initio calculations for tamoxifen and key metabolites formed during P450‐catalyzed oxygenation and dehydrogenation reactions suggest that the quinone methide species formed via dehydrogenation reactions may be stabilized by a unique cis/trans ethylene‐quinone interaction. Additionally, molecular dynamics simulations of P450‐drug interactions provide further insight into structural constraints that contribute to reaction mechanism selectivity. Finally, LC‐MS analysis of tamoxifen metabolism by recombinant P450 enzymes demonstrates isozyme‐specific dehydrogenation reactions. These results provide encouraging insights into the electronic and conformational constraints that control the selectivity for P450‐catalyzed oxygenation and dehydrogenation reactions. Supported by NIH grants # GM074249 from the National Institute of General Medical Sciences and # NCRR 1 S10 RR17214–01 from the National Center for Research Resources and the University of Utah Center for High Performance Computing.
CYP2C9‐CYP3A4 protein‐protein interactions in a reconstituted expressed enzyme system
Authors: Subramanian Murali , Zhang Helen , Tracy Timothy S
Published: June 2021

Protein‐protein interactions between cytochrome P450 isoforms can alter catalytic activity and may confound in‐vitro in‐vivo correlations. The interaction between CYP2C9 and CYP3A4, two highly abundant P450s in human liver and responsible for 70% of the P450 oxidation reactions carried out, were studied for their ability to affect catalysis. Reconstituted, expressed human CYP2C9, CYP3A4 and P450 reductase (CPR) were co‐incubated with lipids, substrate and NADPH, and rate of metabolite formation measured. CYP3A4 inhibited the CYP2C9‐mediated S‐flurbiprofen (4’‐hydroxylation) and S‐naproxen (O‐demethylation) metabolism up to 70%, depending on enzyme ratios and amount of CPR present; these effects were only partially reversed by additional CPR. High concentrations (50 μM) of dapsone (a CYP2C9 activator) decreased CYP3A4 inhibition by 20%, whereas lower concentrations of dapsone had no effect. When evaluated in the converse, the presence of CYP3A4 appeared to enhance dapsone activation due to a lower baseline CYP2C9 activity. It is hypothesized that an active site conformational change occurs when CYP2C9 and CYP3A4 interact. In conclusion, it appears that the presence of the abundant P450 enzyme CYP3A4 may alter CYP2C9 activity and thus, potentially confound in vitro – in vivo predictions of drug metabolism and disposition. Support: NIH GM063215.
Anatomical Differences in Response to Angiogenesis
Authors: Folkman Judah , Zaslavsky Alexander , Baek Kwan Hyuck , Chen Catherine , Ryeom Sandra
Published: June 2021

Ten angiogenesis inhibitors are currently approved by the FDA in the U.S., to treat cancer and age‐related macular degeneration, and by regulatory agencies in >30 other countries.1 1,257,600 patients received prescriptions for these angiogenesis inhibitors during 2006.2 40 other antiangiogenic drugs are in late Phase III or Phase II clinical trials. Significant increases in survival have been reported for colorectal cancer, lung cancer and breast cancer. Of patients with progressive macular degeneration, 95% gain improved eyesight, and of those who are blind, 40% regain sight.3

These clinical successes have increased the need to understand basic mechanisms of angiogenesis even beyond the level of detail that was necessary to discover angiogenesis inhibitors in the first place.

One example is the differential result of angiogenic responses at different anatomic sites. In an animal model of human ovarian cancer, 5 of 5 tumor types in the lower abdominal cavity remain non‐angiogenic and do not grow beyond 1 mm.3 Tumors in the upper abdomen, are angiogenic and continue to expand to 140‐fold larger mass than lower abdominal tumors. The tumor cell proliferation rate is the same at both sites, but apoptosis is higher in non‐angiogenic tumors. In thrombospondin‐1 null mice all tumors in the lower abdomen become angiogenic and grow to an equivalent size as those in the upper abdomen. If both thrombospondin and endostatin are knocked out, the lower abdominal tumors are not only angiogenic, but are 3 times larger than the upper abdominal tumors.

Conclusion: Potent endogenous angiogenesis inhibitors are differentially distributed in the stroma according to anatomical site.
Gestation stage‐dependent changes in monoclonal antibody pharmacokinetics and function during rat pregnancy
Authors: Hubbard Jonathan J , Laurenzana Elizabeth M , Owens S. Michael
Published: June 2021

We studied monoclonal IgG1 antibody (mAb) pharmacokinetics (PCKN) and in vivo function in pregnant rats from gestation day (GD) 8 to GD21 (1–2 day antepartum). On GD8, pregnant rats (n=4) received an iv‐bolus dose of 15 mg/kg of anti‐phencyclidine mAb6B5 or anti‐methamphetamine mAb6H4 along with a [3H]‐mAb tracer. PCKN analysis of serum mAb concentration‐time data showed significant (p<0.001) gestation stage‐dependent changes in terminal elimination half‐life (t1/2λz) for both mAb's. From GD9 to ~GD15‐GD18, the t1/2λz of mAb6B5 and mAb6H4 were 3.6 ± 0.4 and 3.1 ± 0.3 days, respectively. However, between ~GD15‐GD18, the t1/2λz of these mAb's abruptly decreased to 1.0 ± 0.1 and 1.1 ± 0.2 days, respectively. When 15 mg/kg mAb6B5 was dosed on GD15, we found a similarly decreased late‐gestation t1/2λz of 1.4 ± 0.2 days. Importantly, IgG1 PCKN was dose‐independent, since a 90 mg/kg mAb6B5 dose on GD8 demonstrated the same PCKN profile from GD8‐GD21 as the 15 mg/kg mAb6B5 dose. In vivo mAb6B5 serum phencyclidine (PCP) binding in the presence of infused PCP also showed decreased mAb function starting at ~GD15. We observed that the dramatic decrease in mAb t1/2λz and function always coincided with the start of an accelerated dam weight gain. These findings represent an important step in understanding mAb PCKN and function during pregnancy. (Funded by NIDA DA07610, Kirschstien NRSA DA24522, and a GlaxoSmithKline fellowship).
Up‐Regulation of Heterologously Expressed αβγ ENaC Channel Activity by Nitric Oxide in Tissue Culture Medium
Authors: JI HONGLONG , Nie Hongguang , Su Xue‐Feng , Smith Peter R
Published: June 2021

Several groups have demonstrated that nitric oxide (NO) reduces the activity of native amiloride‐sensitive sodium channels (ENaC) and αβγ ENaC channels heterologously expressed in oocytes. However, conflicting results for the effects of NO on the biological function of cells have been observed between in vivo and in vitro studies due to the superoxide‐dependent consumption of NO in biological media (Keynes RG et al. 2003). We aimed to study the regulation of ENaC by NO under physiological conditions by applying DetaNONOate in a tissue culture medium (L‐15). The activity of αβγ ENaC channels was recorded using the two‐electrode voltage clamp techniques. Our results showed that when added to L‐15 culture medium, NO increased αβγ ENaC activity. In contrast, NO decreased αβγ ENaC activity when added to a saline medium (OR‐2), consistent with other report (DuVall MD et al. 1998). Coexpression of CFTR did not affect the opposite effects of NO exposure on αβγ ENaC activity. These results indicate that NO can regulate transepithelial ion transport in a medium‐dependent pattern. Our results suggest a novel mechanism for the regulation of apical ion transport by NO in vivo. Furthermore, our data provide an explanation why NO inhibits ENaC and alveolar fluid clearance in vitro but gaseous NO inhalation is still being used clinically to prevent and treat pulmonary edema. Supported by: NIH HL 87017 and AHA 555333B
Identification of a CFTR‐like Cl channel in A6 Distal Nephron Cells Under Hypotonic Conditions
Authors: Yu Ling , Helms My , Eaton Douglas C
Published: June 2021

The osmolarity of fluid surrounding the renal collecting duct can change dramatically by several folds, and extreme changes in hypotonicity can cause tubular cells to immediately swell. Therefore, in order to prevent dramatic changes in cell volume, tubular epithelial cells can activate various transporters, such as K and Cl channels and secret KCl. Although swelling‐activated Cl channels have been intensively studied in various cell types, in most cases, the channel characteristics recorded in vivo are not comparable to studies performed with cloned Cl channels. In the present study, we were able to characterize a hypotonic‐activated Cl channel in A6 cells using single channel patch clamp analysis. We found that putative Cl channels can be activated by hypotonic bath solution (containing mainly KCl but not NaCl). These channels conduct both inward and outward current, and has single channel conductance of about 7–10 pS. After correcting for the liquid junction potential, the channel reversal potential does not significantly change by replacing pipette KCl with NMDG‐Cl. However, the channel activity is not seen when the pipette KCl is replaced by K‐Aspartic. Interestingly, the same channel is activated by cAMP, or PMA when the cells were bathed in isotonic solution. The conductance and kinetics of the channel that we describe above is similar to the CFTR Cl channel previously described by Ling et, al. JBC 272: 594–600, 1997.

This work was supported by NIH grants HL071621‐01 and DK37963‐16 awarded to DCE.
Aquaporin 2 and Adducin Polymorphism Impact on Essential Hypertension
Authors: Procino Giuseppe , Romano Francesca , Mastrofrancesco Lisa , Tamma Grazia , Svelto Maria , Lanzani Chiara , Manunta Paolo , Bianchi Giuseppe , Valenti Giovanna
Published: June 2021

Aim: In this study we tested the hypothesis that alteration in Aquaporin 2 (AQP2) expression/trafficking may contribute to the extracellular volume expansion observed in hypertension.

Methods: To test this hypothesis we monitored the urinary AQP2 excretion in patients with essential hypertension carrying polymorphisms of the cytoskeletal protein adducin (ADD) known for increasing tubular sodium reabsorption. AQP2 urinary excretion was also measured in Milan Hypertensive Strain (MHS) rats carrying single nucleotide polymorphisms (SNPs) of ADD genes. AQP2 trafficking to the apical plasma membrane was also evaluated in MCD4 cells upon overexpression of human SNPs of ADD1 gene.

Results: AQP2 excretion was significantly higher in hypertensive patients than in controls. In addition, after two hours of acute sodium load, AQP2 excretion was significantly higher in patients carrying ADD mutation. MHS rats showed a significant increase in total AQP2 protein and an increased expression at the apical plasma membrane compared to normotensive rats. In MCD4 cells, AQP2 trafficking to the apical plasma membrane was significantly increased upon co‐expression of mutated ADD.

Conclusions: Taken together, these observations suggest that increase in AQP2 expression/trafficking possibly related to alteration of cytoskeleton structure, may contribute to the extracellular volume expansion observed in hypertension.
Brush border intestinal alkaline phosphatase inversely regulates luminal ATP signaling in rat duodenum
Authors: Mizumori Misa , Akiba Yasutada , Kaunitz Jonathan D
Published: June 2021

Duodenal bicarbonate secretion (DBS) creates a juxtamucosal alkaline microclimate, enhancing brush border intestinal alkaline phosphatase (IAP) activity. We hypothesized that IAP degrades luminal purines such as ATP, regulating DBS via P2Y receptors. We measured DBS with a pH‐stat or with pH and CO2 electrodes. We tested the effect of the perfusion of the IAP inhibitor L‐cysteine or glycerol phosphate, the ecto‐ATPase apyrase, the P2 receptor antagonist suramin or several P2 receptor agonists (2MeSATP, INS45973, UTP, UDP, ATPγS) on basal or acid‐induced DBS. Furthermore, we measured perfusate ATP content with luciferin‐luciferase. AP inhibition increased DBS, inhibited by suramin, with increased luminal ATP content. Acid augmented DBS, which was enhanced by L‐cysteine (with ATP increase) but reduced by apyrase (with ATP decrease). Increased luminal ATP content was not due to cellular injury. Immunohistochemistry localized P2Y1 receptors to the brush border membrane of duodenal villi. The P2Y1 agonist 2MeSATP, but not other agonists increased DBS. IAP inhibition increased DBS and luminal ATP content, consistent with ATP degradation by IAP. Luminal ATP stimulates DBS via P2Y1, consistent with an ecto‐purinergic regulatory system. Increased DBS augments IAP activity, which degrades ATP and decreases ATP‐mediated DBS, forming a negative feedback loop.
Apical membrane expression of NKCC2 is directed by a domain within its cytoplasmic C‐terminus
Authors: Carmosino Monica , Gimenez Ignacio , Caplan Michael , Forbush Biff
Published: June 2021

The renal Na‐K‐Cl cotransporter (NKCC2) is selectively expressed in the apical membrane of thick ascending limb (TAL) cells of the mammalian kidney, where it is the central player in NaCl reabsorption, and the target of the clinically important loop diuretic drugs. In contrast, the “housekeeping” Na‐K‐Cl cotransporter (NKCC1) is localized in the basolateral membrane of many epithelia, including the distal collecting duct in the kidney. To identify the sorting signal(s) that direct apical or basolateral trafficking of NKCCs, we generated chimeras between the two isoforms and expressed these constructs in polarized renal epithelial cell lines. Transient expression analysis in MDCK cells demonstrated that among the three large portions of the cotransporter (the N‐terminal, C‐terminal, and transmembrane domains) the C‐terminus of NKCC2 is necessary and sufficient for apical expression. Subsequent chimeras, in which sequential regions of the NKCC1 C‐terminus were exchanged with the corresponding regions of NKCC2 C‐terminus were stably expressed in MDCK cells. This analysis resulted in the identification of 70 aa residue stretch of sequence that is sufficient to determine the apical localization of NKCC2. The identification of sorting signals in NKCC2 will provide important tools with which to gain insight into the molecular basis of mis‐sorting diseases and the molecular basis of hypertension.
Fatty acid transporter expression in human myocytes
Authors: Bell Jill A , Reed Melissa A , Muoio Deborah M , Dohm G. Lynis
Published: June 2021

Long‐chain fatty acid (LCFA) transporters in skeletal muscle mediate the uptake and subsequent storage and/or oxidation of LCFA in muscle. The purpose of this study was to examine the regulation of LCFA transporter expression in skeletal muscle. We measured the mRNA expression via RT‐PCR of FAT/CD36, FABPPM, and FATP1 in myocytes that were isolated and cultured from lean and morbidly obese subjects. Skeletal muscle FAT/CD36, FABPPM, and FATP1 mRNA expression were significantly upregulated 2‐fold in the morbidly obese myocytes compared to lean. The addition of a PPARγ antagonist at either 2μM or 4μM in the morbidly obese myocytes significantly decreased FAT/CD36 mRNA expression in a dose‐dependent manner by 42% and 66%, respectively. Treatment with fatty acids (100μM) significantly increased mRNA expression of FAT/CD36 (2‐fold), FABPPM (2.5‐fold), and FATP1 (2‐fold) in lean myocytes, whereas LCFA transporter mRNA expression remained upregulated in morbidly obese myocytes. When lean myocytes were incubated in the presence of insulin (10nM), mRNA expression of FAT/CD36 (2‐fold) and FATP1 (1.7‐fold) was significantly increased. We conclude that LCFA transporter expression is upregulated in morbidly obese skeletal muscle and following treatment with fatty acids. Our findings also indicate that FAT/CD36 expression is downregulated in the presence of a PPARγ antagonist.
Arachidonic acid modulation of KV channel kinetics in retinal arteriolar myocytes
Authors: Kur Joanna , McGahon Mary K. , Scholfield C. Norman , McGeown J. Graham , Curtis Tim M.
Published: June 2021

Recent studies have suggested that arachidonic acid (AA) may act as a vasoconstrictor in the brain and retina, but the mechanisms responsible are poorly understood. To address this issue, we examined the effects of AA on ionic currents in retinal vascular myocytes using the whole‐cell perforated patch‐clamp technique. AA (10μM) inhibited membrane currents between ‐20 and +40 mV (reduced from 19.72±4.34 to 1.64±1.88pA/pF at 0mV, n=7; P<0.005; Student's t‐test), although currents were enhanced at potentials positive to +60mV (increased from 87.14±22.02 to 203.53±29.68pA/pF at +100mV; n=7; P<0.005). As a first step in deciphering how AA modulates the membrane current, we isolated the A–type Kv current, which in these cells is carried mainly by Kv1.5+Kvβ1 containing channels (McGahon et al. 2007 Am J Physiol Heart Circ Physiol 292: H1001–8). AA modulated this current in two ways. It accelerated inactivation (control τ1=0.037±0.0033ms, τ2=0.37±0.064ms and AA τ1=0.016±0.0075ms, τ2=0.077±0.019ms; P<0.05 and P<0.001; n=8, step to +40mV), and shifted the voltage–dependence of inactivation to more negative potentials (V0.5control=−38.7±2.06mV and V0.5AA=−47.99±1.61mV; P<0.001, step to +60mV). This reduced the steady‐state “window” current at physiological membrane potentials. Effects of AA were observed within 30s of exposure and reached maximal effect at 7mins. We conclude that AA may act as a vasoconstrictor in the retinal vasculature by reducing steady–state Kv current leading to membrane potential depolarisation and an increased activation of voltage‐gated Ca2+ channels.

Funded by Fight for Sight, UK, Wellcome Trust and JDRF.
Contribution of Tweety‐3 to the Ca2+‐activated chloride current in retinal arteriolar smooth muscle
Authors: McGahon Mary Kathleen , Needham Maurice , Arora Aruna R. , Scholfield C. Norman , McGeown J. Graham , Curtis Tim M.
Published: June 2021

The aim of the present study was to investigate the molecular identity of the Ca2+‐activated chloride channels in native rat retinal arteriolar smooth muscle cells using patch‐clamp, RT‐PCR, immunohistochemistry, and neutralizing antibody studies. Application of 10mM caffeine evoked large transient Ca2+‐activated Cl− currents which reversed close to ECl and were completely abolished by 100μM tetracaine or low Cl‐ Hanks’ solution containing the Cl− channel inhibitor, 9‐AC (1 mM). Transcripts encoding the large conductance Ca2+‐activated Cl− channel, Tweety 3, but not Tweety 2 were identified in freshly isolated retinal arterioles. Immunofluorescence labeling revealed a punctuate distribution of Tweety 3 localized to the plasma membrane of retinal arteriolar smooth muscle cells. Anti‐Tweety 3 antibody applied intracellularly inhibited caffeine‐evoked Ca2+‐activated Cl− currents by ~70% (peak current densities at −40mV were −39.82±10.04 and −12.88±6.30pA/pF for antibody‐free and anti‐Tweety 3 solutions, respectively; n=10–11; p<0.005, Student's t‐test), whereas a control antibody (Anti‐Tweety 1) had no effect (−40.33±9.20pA/pF n=11; p=0.97 versus antibody‐free solution). These data suggest that Tweety‐3 may represent a major component underlying the Ca2+‐activated Cl− current in retinal arteriolar myocytes. Funded by the JDRF, US and Wellcome Trust, UK.
Inhibition of human TRPC5 channel by serum albumin
Authors: Bahnasi Yahya M , Bateson Alan N , Beech David J
Published: June 2021

Classical transient receptor potential (TRPC) channels are suggested to play pivotal roles in the regulation of endothelial barrier function (Gioffi and Stevens 2006, Microcirc 13, 709–23). TRPC5, like other TRPCs, is a calcium‐permeable channel that is widely expressed, including in endothelial cells (Tiruppathi et al 2006, Microcirc 13, 693–708). It is activated by a multiplicity of signals, one of which is lysophosphatidylcholine, an inflammatory factor (Flemming et al 2005, J Biol Chem 281, 4977–82; Qiao et al 2006, Am J Physiol 291 91–101). Here we show that serum albumin, which influences vascular tone and endothelial permeability (Fuentes et al 1997, J Phyiol 504, 315–26), inhibits TRPC5. Tetracycline‐inducible TRPC5‐expressing HEK 293 cells were studied using fura‐2 to measure calcium‐entry in real‐time 96‐well plate assays. Activation of TRPC5 by lysophospholipids or gadolinium was inhibited by fatty acid‐free serum albumin or recombinant human serum albumin. Boiled albumin had no effect. Electrophysiological studies showed reversible inhibition of TRPC5‐mediated current by serum albumin. The data suggest albumin may inhibit calcium entry via TRPC5 in cell types that come into contact with albumin, either constitutively or dynamically during inflammation.
Discontinuous expression of renal tubule AQP1 and AQP2 in neonatal mouse with and without ureteral obstruction
Authors: Pannabecker Thomas Lloyd , Thornhill Barbara A , Chevalier Robert L
Published: June 2021

We have created three‐dimensional models of proximal and distal segments in a mouse model of partial unilateral ureteral obstruction (UUO) in order to better understand obstructive nephropathy. Kidneys were subjected to UUO within 1 day of birth and were harvested after 2 days. Three‐dimensional architecture of nephron segments from obstructed and contralateral kidneys was assessed with digital reconstructions of physical sections from resin‐embedded kidneys. Proximal segments and collecting ducts (CDs) from inner core to kidney surface were identified with antibodies against segment‐specific proteins associated with water transport (AQP1 and AQP2) by indirect immunofluorescence. A number of proximal segments from obstructed and contralateral kidneys express markedly reduced AQP1 beginning at variable lengths distal to tubule origin. These likely include proximal segments of short loops of Henle whose descending limbs are known to be AQP1‐null. AQP2 expression is markedly reduced at variable depths in a number of CDs from obstructed and contralateral kidneys. Reduced AQP2 expression in obstructed kidneys is notable in subcortical CDs that exhibit laterally‐expanded axial extensions. Reduced AQP2 expression along the CD axis may reflect incomplete developmental expression of AQP2 and/or obstruction‐mediated down‐regulation. (NIH DK16294, DK45179, DK52612)
ETA‐dependent natriuresis is mediated by NOS1 in renal medulla of female ETB‐deficient rat
Authors: Nakano Daisuke , Pollock David M.
Published: June 2021

We have previously reported that the renal medullary ETB receptor inhibits sodium reabsorption via nitric oxide synthase (NOS) 1. In addition, we have also demonstrated that endothelin (ET)‐1‐induced diuresis and natriuresis in female rats are mediated not only by the ETB receptor, but surprisingly, also by the ETA receptor. Therefore, the present study was conducted to determine the involvement of NOS1 in ET‐1/ETA‐dependent diuretic and natriuretic responses in renal medulla of female ETB‐deficient rats. Infusion of ET‐1 (0.45 μg/kg/h; n = 8) directly into the renal medulla of anesthetized rats markedly increased urine flow (UV: 4.6 ± 0.2 μl/min during baseline and 9.2 ± 0.4 μl/min during ET‐1 infusion; p<0.01) and urinary sodium excretion (UNaV: 0.40 ± 0.03 μmol/min during baseline and 0.98 ± 0.09 μmol/min during ET‐1 infusion; p<0.01). Pretreatment with NG‐propyl‐L‐arginine (NPA; 10 μg/kg/h, n = 4), a selective inhibitor of NOS1, suppressed ET‐1‐induced increases in UV and UNaV (5.4 ± 0.4 μl/min and 0.60 ± 0.05 μmol/min, respectively; p<0.01). Neither NPA nor ET‐1 affected renal medullary blood flow at these doses (laser‐Doppler flowmetry). These results suggest that NOS1 is responsible for the ETA receptor‐dependent water and sodium excretion in rats, and that these responses do not result from a change in medullary hemodynamics.
Perinatal asphyxia induces hyperemotionality and elevates corticosterone (CORT) release in response to novel stimuli in later life
Authors: Ronca April E , Tulbert Christina D , Winn Glenn G , Baer Lisa A , Kleven Gale A
Published: June 2021

We tested the hypothesis that perinatal asphyxia (PA) augments adult hyperemotionality and increases corticosterone (CORT) release. Term fetuses were exposed to PA (12 min) by occluding (OCCL) the arteries feeding one uterine horn. Control subjects were derived from the opposite, non‐occluded (N‐OCCL) horn, and from Vaginal delivery groups. Newborn pups were fostered to newly parturient dams. Adult male and female rats were singly housed for 24hr, placed in an open field and videographed for the duration of a 52min novelty test comprised of a concatenation of novel environmental stimuli including exposure to the open field, sustained darkness, presentation of a novel object, startle stimuli, introduction of an unfamiliar same‐gender intruder, and punctate acoustic stimuli. As compared to controls, OCCL adult rats showed a tendency to spend time near the wall rather than the center of the arena (thigmotaxis). Approaches to a novel object were slower in OCCL offspring relative to controls. Behavioral testing was associated with a significant increase in CORT, with greater magnitudes of response in OCCL offspring. Collectively, our findings provide clear evidence for magnified emotional responses and HPAA activity in response to novelty in adult offspring exposed to PA.
Chronic stress increases the production of volatile sulphur compounds in rats
Authors: Cunha Tatiana Sousa , Calil Caroline Morini , Lima Patrícia Oliveira , Ferreira Rosemary , Costa Rafaela , Tamascia Mariana Leite , Neves Vander José , Marcondes Fernanda Klein
Published: June 2021

In the great majority of cases, halitosis is originated in the mouth itself and psychological symptoms have been pointed out as one of halitosis‐inducing factor. The aim of this study was to evaluate, in laboratory animals, the effect of chronic mild stress (CMS) on the production of volatile sulfur compounds (VSC), the main component of bad breath. VSC measurements were made using a portable industrial monitor (Halimeter) once a week, during seven weeks, in control and stressed adult males Wistar rats. CMS group was submitted to the stress protocol on weeks 3, 4 and 5 (Student t test; p<0.05). Rats submitted to CMS presented an increase in the production of oral VSC, in comparison to the control group, on week 3 (22±1 vs. 18±1 ppb), week 4 (25±1 vs. 16±1 ppb), week 5 (24±1 vs. 17±1 ppb) and week 6 (26±2 vs.19±1 ppb), with no difference on week 7 (16±1 vs. 16 ± 0.6 ppb). Our data confirm the influence of stress on the production of oral VSC and demonstrate that this effect is still observed one week after the cessation of stress stimulus. Financial Support: FAPESP, CNPq
Rescuing of lethal phenotype of calcineurin A alpha knockout mice
Authors: Reddy Ramesh Narayana , Roberts Brian R , Gooch Jennifer L
Published: June 2021

Calcineurin is a calcium/calmodulin‐ dependent serine/threonine protein phosphatase discovered as a target of the immunosuppressant drug, cyclosporin A. Mice lacking α or β isoform of calcineurin have been useful in our understanding of the functions of calcineurin in the kidneys. While loss of the β isoform does not affect development or fertility, &alpha −/− mice routinely die between 3 and 4 weeks of age. Previously, we reported numerous developmental abnormalities in α −/− mice including reduced organ size and renal dysfunction. We now report the rescuing of the early lethality of α −/− mice by feeding the mice a powdered diet in gelatin. Groups of α−/− and wildtype mice were given supplemental food cups around 15 days and were fed a modified diet for 8 weeks. Weekly body weights and blood glucose levels were measured. At the end of 8 weeks postnatal, mice were sacrificed and serum, urine and tissue samples were collected. The α −/− mice regained body mass and had normal growth throughout the study period. Prior to rescue, α−/− mice were hypoglycemic consistent with malnutrition but feeding the modified diet led to slightly higher blood glucose levels than wildtype mice, suggesting an endocrine/metabolic abnormality. Despite rescue of the nutritional defect, serum BUN levels were significantly elevated with concomitant decrease in GFR in the alpha knockout mice compared to wildtype mice indicating impaired renal function.
Tonicity‐responsive activation of NFATc by calcineurin Aβ in the inner medullae
Authors: Reddy Ramesh Narayana , Roberts Brian R , Gooch Jennifer L
Published: June 2021

Previously, we found that nuclear factor of activated T cell (NFATc) is regulated by the β isoform of calcineurin (CaN). In vivo, NFATc‐driven luciferase expression is highest in the renal inner medullae (IM) and loss of the β isoform significantly decreases NFATc‐transcriptional activity. Expression and activity of NFATc in the IM suggests a role for CaNβ/NFATc in response to tonicity. To explore this, activation of CaN in response to calcium ionophore, glucose, mannitol, urea, and NaCl was examined. CaN activity in wildtype renal fibroblasts was increased by changes in tonicity in a dose‐dependent manner. CaN activity in β−/− cells, in contrast, was no longer regulated by tonicity. To examine the tonicity response in vivo, wildtype and β−/− mice were thirsted for 32 hours. Urine output and osmolarity were measured and kidneys were harvested for analyses. Wildtype mice had decreased urine output and increased urine concentration, as expected. This was accompanied by an increase in NFATc‐luciferase activity in the IM. β−/− mice, however, did not reduce urine output and did not concentrate their urine despite the period of thirsting. In addition, despite normal basal urine concentration, β−/− mice failed to increase urine osmolality in response to the vasopressin analog DDAVP. In conclusion, CaNβ is activated in vitro in response to hypertonicity and loss of β in vivo impairs urine concentration.
Cardiovascular function and metabolism in old melanocortin‐4 receptor deficient obese mice.
Authors: do Carmo Jussara M , da Silva Alexandre A , Brandon Elizabeth , Hall John E
Published: June 2021

The present study examined cardiovascular and metabolic functions in 120‐week‐old obese melanocortin‐4 receptor deficient mice (MC4R(−/−), n=8) and heterozygous control mice (MC4R(+/−), n=4) of similar age. MAP and HR were recorded 24h/day by telemetry and spontaneous baroreflex sensitivity (BRS) was assessed by the sequence method. Oxygen consumption (VO2) was continuously monitored by indirect calorimetry. Compared to control mice, the MC4R(−/−) mice were heavier (42±4 vs 52±4 g), consumed more food (5.5±0.3 vs 7.1±0.5 g/day) and hyperleptinemic (2.3±0.8 vs 55.0±9.9 ng/ml). Despite exhibiting many features of the metabolic syndrome, MC4R(−/−) mice had similar MAP and HR to MC4R (+/−) mice (112±4 vs 108±5 mmHg and 498±28 an 478±46 bpm, respectively). MC4R (−/−) also exhibited reduced motility (125.5±16.5 vs 69.5±15 cm) and lower VO2 than MC4R (+/−) (42.5±0.5 vs 38.0±1.5 ml/kg/min). Plasma glucose levels (174±12 vs 147±7 mg/dl) and BRS (4.6±0.5 vs 4.7±1.1 bpm/mmHg) did not differ between groups. These results suggest that despite having obesity and many metabolic abnormalities, 120–week‐old MC4R(−/−) mice do not have hypertension or altered BRS (NHLBI ‐ PO1 HL51971).
High fat diet reduces NOS functional activity during vasoconstriction in aorta, but not small mesenteric arteries, from Dahl rats
Authors: Kang Kyu‐Tae , Spradley Frank , Sullivan Jennifer C. , Pollock David M. , Pollock Jennifer S.
Published: June 2021

Recently, we reported that Dahl salt‐sensitive rats (DS) become hypertensive when placed on a high fat diet (HF) diet for 4 weeks. Reduced NO bioavailability is associated with hypertension. Therefore, we tested the hypothesis that HF‐induced hypertension in salt sensitive rats enhances vasoconstriction by decreasing NO synthase (NOS) functional activity. DS and normotensive control (SS.BN13) (12 weeks old) were given normal fat (NF) or HF (36% fat) diet for 4 weeks. Dose response curve to phenylephrine (PE: 1 nM to 10 μM) were generated in isolated aorta and 3rd order mesenteric arteries (MA). In both strains, HF did not change the contractile response to PE in aorta (logEC50 of NF vs HF: DS: −7.7±0.1 vs −8.3±0.4, BN13: −7.4±0.1 vs −7.6±0.1) or MA (DS: −6.0±0.1 vs −5.8±0.1, BN13: −6.1±0.1 vs −6.1±0.1). Inhibition of NOS (100 μM of L‐NAME) in both strains on NF increased sensitivity to PE in aorta (L‐NAME logEC50 of NF: DS: −8.0±0.0, BN13: −7.9±0.1; both P<0.01) and MA (DS: −6.6±0.1, BN13: −6.6±0.1; both P<0.001). Interestingly, HF diminished L‐NAME‐induced sensitivity in aorta, but not MA in both strains (L‐NAME logEC50 of HF: aorta: DS −8.0±0.6, P=0.72; BN13: −7.8±0.1, P=0.29; MA: DS −6.2±0.1, P=0.01: BN13: −6.7±0.1, P<0.001). These results indicate that HF reduces the NOS buffering capacity during vasoconstriction in aorta, but not small mesenteric arteries, independent of hypertension.
Synthesis and breakdown of Very Low Density Lipoprotein Apo‐B100 measured with stable isotope methodology
Authors: Børsheim Elisabet , Tuvdendorj Demidmaa , Chinkes David L. , Volpi Elena , Zhang Xiao‐Jun , Aarsland Asle
Published: June 2021

Increased concentration of Very Low Density Lipoprotein (VLDL) is an important and common risk factor for cardiovascular disease. VLDL concentration depends on the balance between synthesis and degradation, which can be influenced by many drugs and nutrients. The present study was designed to establish novel methods to simultaneously measure the fractional synthesis and breakdown rates of VLDL Apo‐B100. Six elderly (2 males, 4 females; 66.7 ± 2.4 (SE) years; 72.4 ± 6.8 kg) completed a study with constant infusion of two different tracers of leucine. 2H3‐leucine was infused for nine hours, whereas 1‐13C‐leucine was infused only for the first four hours. VLDL was isolated from plasma by ultracentrifugation, and Apo‐B100 was precipitated from this fraction by isopropanol. Basal Apo‐B100 fractional synthetic rate (FSR) was calculated from the linear part of the plasma enrichment slope of 2H3‐leucine vs. the plateau part of the slope. The fractional breakdown rate (FBR) of Apo‐B100 was obtained from the decay curve of the 1‐13C‐leucine, corrected for the incorporation of label. The basal FSR of Apo‐B100 was 18.33 ± 0.66 %/hr, whereas the FBR of Apo‐B100 was 16.31 ± 0.71 %/hr. These results are in agreement with previous reported measurements of Apo‐B100 secretion rates using isotope methodologies, and indicate that both fractional Apo‐B100 synthesis and breakdown can be reliably and simultaneously measured using stable isotopes.

P30 AG024832 NIH/NIA, Shriners grant 8490, M01 RR 00073.
Role and regulation of the proprotein convertase subtilisin/kexin type 9 (PCSK9) in the small intestine
Authors: Leblond François , Précourt Louis‐Philippe , Delvin Edgard , Elchebly Mounib , Seidah Nabil , Ziv Ehud , Levy Emile
Published: June 2021

Proprotein convertase subtilisin kexin type 9 (PCSK9) is playing a central role in the pathogenesis of atherosclerosis. This enzyme reduces cellular uptake of plasma LDL‐cholesterol by promoting LDL receptor degradation in liver through an unknown posttranscriptional mechanism. The current investigation was undertaken to explore the role and regulation of PCSK9 in human enterocytes. To this end, molecular strategieswere devised in the Caco‐2 cell line to determine the importance of PCSK9 in intestinalcholesterol transport and metabolism. Knockdown of PCSK9 caused a decline in the ability of Caco‐2 cells to capture micellar [14C]‐free cholesterol, and resulted in increased activity of HMG‐CoA reductase. To examine the regulation of PCSK9, various effectors were administered to the apical side of Caco‐2 cells for a 24‐h period. Taurocholic acid produced a significant decrease (30%, p<0,01) in PCSK9 gene expression, whereas inflammation agents (LPS, TNF‐α), oxidative stress inducer (iron‐ascorbate), long‐chain fatty acids (DHA, EPA) and glucose showed no effect on mRNA levels. Similarly, insulin did not modify PCSK9 gene expression in the small intestine, in contrast with the findings obtained in rat liver. Together, our data suggest that PCSK9 contributes to intestinal cholesterol homeostasis and a different regulation characterizes the intestine and the liver.
Nicotinic acid (NA) alters lipid metabolism in a pair‐fed mouse model
Authors: White Amanda T , Menke John , Krsmanovic Mihajlo , Wilsie Larissa , Taggart Andrew K P , Strack Alison , Metzger Joseph
Published: June 2021

Pharmacological doses (2–3 grams/day) of NA favorably alter the serum lipid profile by increasing HDL while decreasing VLDL and LDL cholesterol and triglycerides (TG). In rodents, the poor pharmacokinetic properties of NA (plasma half‐life on the order of minutes) necessitates admixture in chow to deliver sustained plasma exposures. In C57BL/6 mice treated with NA diet at 1 or 3% (w/w) in chow, TG levels are lowered after two days of treatment. However, food intake (FI) is reduced during this period, which could artificially decrease TG levels in NA‐treated mice by inducing a fasted state. To separate the mechanism‐based effect of NA from aversive effects of the diet we utilized a CLAM pair‐feeding (PF) system with indirect calorimetry to yoke the FI of the PF group to the NA‐treated group in 30′ intervals. Exposure to 3% NA diet altered meal pattern and decreased FI by 57, 35, 11 and 0 % on days 1–4. Body weight decreased by 17.1 % in NA‐treated mice while the PF group lost only 8.9 % (day 4, v. ad lib controls). Serum TG levels on day 4 in ad lib and PF animals did not differ (119 and 118 mg/dL, respectively) whereas NA treatment decreased serum TG by 29% (85 mg/dL). These data show that the NA effects on TG are independent of the suppression of FI. Interestingly, mice treated with NA had decreased respiratory quotient even when their FI matched that of vehicle animals, suggesting an altered use of glucose and/or an increase in beta‐oxidation.
The novel adipokine chemerin significantly increases cholesterol uptake in human macrophages
Authors: McCarthy Tanya C. , Zuniga Luis A , Zabel Brian A , Butcher Eugene C , Sinal Christopher J
Published: June 2021

Atherosclerosis is the most common pathology underlying cardiovascular disease. One of the early events in atherosclerotic plaque formation is the excessive accumulation of cholesterol by macrophages leading to the development of macrophage foam cells. The novel adipokine chemerin is an endogenous ligand for CMKLR1, a G‐protein‐coupled receptor that is highly expressed on macrophage cells. Therefore, we hypothesized that chemerin may have an important role in the pathology of atherosclerosis through the modulation of macrophage function. Cholesterol uptake and efflux experiments show a significant increase in total cholesterol in human monocyte derived macrophages (MDM) treated with chemerin. Conversely chemerin treatment had no effect on cholesterol efflux in human MDM. Interestingly, the PPAR gamma ligand rosiglitazone, caused an increase in cholesterol efflux from human MDM with a corresponding decrease in the expression of CMKLR1. These data suggest that chemerin may play a role in macrophage cholesterol uptake and foam cell formation. Supported by the Canadian Institutes of Health Research and the National Institutes of Health
Kudzu root extract improves glycemic control in non‐glucose‐impaired mice.
Authors: Carlson Scott Hayden , Peng Ning , Prasain Jeevan K. , Dai Yanying , Wyss J. Michael
Published: June 2021

The root of kudzu (Pueraria radix) is commonly used in traditional Chinese medicine. A recent study demonstrated that puerarin, the dominant isoflavone in kudzu, improved glucose handling in ob−/ob− mice (J. Agric. Food Chem. 53:8760–8767, 2005). Interestingly, daidzin (the second main isoflavone in kudzu) significantly impaired glucose handling in that model. The antagonistic actions of the two major kudzu isoflavones raised the question of whether whole kudzu root extract would affect glucose handling. Further, the effect of kudzu in non‐glucose‐impaired rodent models was unknown. The present study investigated whether dietary kudzu root extract supplementation improves glycemic control in non‐impaired mice. Male Cd‐1 mice were lightly anesthetized with isofluorane and blood glucose measured in tail blood. Mice were then orally gavaged with kudzu extract (75 mg/kg BW) or vehicle followed by an oral glucose challenge (OGTT; 2 g/kg BW), and blood glucose measured at 15‐minute intervals for 2 hours. In mice receiving kudzu extract, the peak change in blood glucose following the OGTT was significantly lower than in mice receiving vehicle (+76 vs. 109 mg/dl, respectively). Further, the overall glucose response was significantly attenuated by kudzu extract. These results suggest that kudzu extract improves glycemic control in non‐glucose‐impaired mice, and that this effect is likely mediated by puerarin.
Threonine Requirement in Neonates Receiving Total Parenteral Nutrition (TPN)
Authors: Chapman Karen P. , Courtney‐Martin Glenda , Moore Aideen M. , Ball Ronald O. , Pencharz Paul B.
Published: June 2021

Current neonatal threonine (THR) enteral requirement is 87 mg·kg‐1·d‐1. The THR requirement of the parenterally (TPN) fed human neonate has never been experimentally determined. Concentrations of THR in commercially available TPN solutions are derived from concentrations in cord blood, plasma amino acid profile during TPN feeding, or human milk, and are 30 to 80% higher than the enteral requirement. We showed in piglets that the neonatal TPN requirement for THR was 55% lower than the enteral requirement.

Our objective was to determine the human neonatal TPN requirement for THR in post‐surgical neonates using the indicator amino acid oxidation technique with L[1–13C]phenylalanine. Neonates were randomized to receive one of twelve THR intakes ranging from 10 to 100 mg· kg‐1·d‐1. Preliminary data indicate a mean THR requirement between 30 and 40 mg·kg‐1·d‐1, only 30% of the THR concentration in the lowest THR containing TPN solution. We conclude that the THR requirement for TPN fed human neonates is significantly lower than the THR present in current TPN solutions and the recommended intake for orally fed infants. High THR intakes are a concern; THR is potentially neurotoxic and neonates are unable to oxidize excess
Expression Of Epithelial Na+ Channel (ENaC) And Serum/glucocorticoid Regulated Kinase 1 (SGK1) In Brain Nuclei of Rats Post‐MI
Authors: Wang Hong‐Wei , Alfy Ahmed , Leenen Frans HH
Published: June 2021

Blockade of brain mineralocorticoid receptors (MR) or ENaC prevents sympathetic hyperactivity and improves cardiac function in rats post MI. To assess whether this response reflects increases in ENaC activity, we studied mRNA expression of ENaC (α, β, γ) subunits and SGK1, an important regulator of ENaC, in brain nuclei of male Wistar rats at 2 & 4 weeks (wks) post MI. Micro‐punches were taken from the subfornical organ (SFO), paraventricular nucleus (PVN) and supraoptic nucleus (SON). Expression of mRNA was measured by real‐time qRT‐PCR, normalized by house keeping gene phosphoglycerate kinase 1 (PGK1). In SFO, α and β subunit mRNA levels significantly increased at 4 (1.7±0.2 vs 1.2±0.1 α/PGK1 [×10−3]; 1.0±0.1 vs 0.6±0.1 β/PGK1 [×10−4]) but not 2 wks compared to sham. In PVN, α subunit mRNA levels decreased at 2 (2.9±0.5 vs 4.5±0.6 [×10−3], p<0.05) but not 4 wks post MI. In contrast, mRNA levels of γ subunit significantly increased at 4 (1.2±0.2 vs 0.6±0.2 [×10−4]) but not 2 wks. No changes of ENaC subunit mRNA was found in SON. mRNA levels of SGK1 were similar in SFO, PVN and SON between MI and sham at 2 & 4 wks. These results indicate that in rats post MI, ENaC subunit mRNA expression is differentially regulated in brain nuclei. SGK1 transcription regulation appears not to be involved. Upregulation of ENaC subunit transcription in SFO and PVN may increase ENaC activity and thereby contribute to sympathetic hyperactivity.
Contribution of brain nuclear factor‐kappa B to the pathogenesis of heart failure
Authors: Ma Ying , Kang Yu‐Ming , Yang Zhi‐Ming , Zheng Jin‐Ping , Francis Joseph
Published: June 2021

Previous studies have shown nuclear factor kappa B (NF‐κB) is a major mediator facilitating the synthesis of proinflammatory cytokines (PIC) both in the peripheral and central nervous system. In this study we determined, whether blockade of brain NF‐κB attenuates PIC and oxidative stress in heart failure (HF) rats. Adult male Sprague‐Dawley rats were implanted with intracerebroventricular (ICV) cannulae and subjected to coronary artery ligation to induce HF or sham surgery (SHAM). Subsequently, animals were ICV treated with SN50 (2μg/hr), a membrane permeable peptide which inhibits nuclear translocation of NF‐κB, or vehicle (VEH) for 4 weeks. HF‐induced increases in the expression of NF‐κB, AT1‐R and NADPH oxidase gene in paraventricular nucleus (PVN) of hypothalamus, and TNF‐α, IL‐1β, IL‐6 and angiotensin type 1 receptor (AT1‐R) in hypothalamus compared with sham rats. The HF rats also had increased plasma norepinephrine (NE), an indirect indicator of sympathetic activity. In contrast, ICV treatment with SN50 attenuated the expression of PIC, NF‐κB, AT1‐R and oxidative stress gene in the PVN compared with VEH‐treated HF rats. Treatment with SN50 also reduced plasma levels of NE, angiotensin II (ANG II) and PIC, and lung weight to body weight ratio. These findings suggest NF‐κB may be one key mediator stimulating the synthesis of brain RAS and PIC in HF.
High salt intake enhances sympathetic modulation in the rat.
Authors: Farah Vera Azevedo , Fiorino Patricia , Roque Andre Prevides , Martins André Alves , Fonteles Manassés Claudino
Published: June 2021

There is a significant association between salt intake and high blood pressure that could be related to alterations in autonomic modulation of cardiovascular system. The purpose of this study was to evaluate in rats the cardiovascular function induced by high salt intake. Male Wistar rats (n=5, each group) were divided in: 1) Control group (CG), without treatment and 2) Salt treatment (SG), salt added to the drinking water (1%) during 10 days. Arterial pressure (AP) was recorded and processed with a data acquisition system (2kHz sampling frequency). Systolic arterial pressure (SAP) and pulse interval (PI) time series were submitted to autoregressive spectral analysis to determine SAP and PI variability in time and frequency domains in order to evaluate autonomic influences on the circulation. AP was higher in SG (119 ± 2.2 mmHg) than in CG (104 ± 1.5 mmHg), whereas heart rate was not changed. There was an increase in SAP variance and its low frequency domain (9.3 ± 0.5 vs. 16.5 ± 1.5 mmHg2, variance and 6.3 ± 9.8 ± 0.5 mmHg2, LF, CG vs. SG). High salt intake also increased PI variability analyzed by either time (12 ± 0.8 vs. 27 ± 3.2 ms2, CG vs. SG) or frequency domains (9 ± 0.6 vs. 17 ± 1.5 ms2, CG vs. SG). The key findings of the present study were that high salt intake increased blood pressure that is associated with increased sympathetic modulation described herein.

Financed by CNPq and MackPesquisa‐Brazil.