bluuebunny/crossref_metadata_2025 huggingface dataset:
Attenuation of Cocaine Self‐Administration by Dual Inhibition of the Dopamine Transporter and Sigma Receptors
Authors: Hiranita Takato , Newman Amy H. , Katz Jonathan L.
Published: June 2021

Previous studies have indicated that sigma receptor (σR) antagonists decrease reinforcing effects of cocaine in place conditioning, but not self‐administration (SA) procedures. The present study further examined σR involvement in cocaine SA. Rats trained to self administer cocaine exhibited maximal increases in responding compared to vehicle at 0.32 mg/kg/injection and lower rates at other doses. The σR agonists, DTG and PRE‐084, but not standard σR antagonists (BD 1008, BD 1047, BD 1063, AC 927, NE 100), maintained SA like cocaine, with PRE‐084 being 10‐fold more potent than DTG. The σR antagonists had no effect on cocaine SA across a range of doses. DTG and PRE‐084 as well as ligands for the dopamine transporter (DAT) shifted the cocaine dose‐effect curve leftward. In addition, σR agonist SA was dose‐dependently blocked by standard σR antagonists, and analogues of rimcazole which also bind to the DAT. In contrast to standard σR antagonists, rimcazole analogues also decreased cocaine SA. Further, combinations of standard σR antagonists with the DAT inhibitor, WIN 35,428, decreased cocaine SA. Rimcazole analogues were more potent in decreasing responding maintained by cocaine than food. These findings suggest that 1) σR agonists can have reinforcing effects in rats trained to self‐administer cocaine, and 2) cocaine SA can be attenuated by dual inhibition of the DAT and σRs.
Effects of varenicline on the reinforcing and discriminative stimulus effects of cocaine in rhesus monkeys
Authors: Gould Robert Warren , Czoty Paul W , Nader Susan H , Nader Michael A
Published: June 2021

In the CNS, stimulation of nicotinic acetylcholine receptors (nAChR) can directly affect dopaminergic tone and may, therefore, modulate the reinforcing effects of abused drugs. In rhesus monkeys, we examined the nAChR partial agonist varenicline and full agonist nicotine in i.v. drug self‐administration and cocaine discrimination procedures. When substituted for cocaine in monkeys responding under a progressive‐ratio (PR) schedule, nicotine (0.03‐0.3 mg/kg) but not varenicline (0.01‐0.17 mg/kg) functioned as a reinforcer. When administered chronically, varenicline (titrated up to 0.3 mg/kg, BID p.o. by day 30) did not affect cocaine break point under the PR schedule or cocaine intake. In monkeys discriminating 0.3 mg/kg i.v. cocaine from saline, varenicline (0.03‐0.3 mg/kg, i.v.) did not engender cocaine‐like discriminative stimulus effects, but dose‐dependently potentiated the effects of cocaine when administered 60 min prior to testing. These preliminary results indicate low abuse liability for the nAChR partial agonist varenicline but suggest that varenicline may potentiate the subjective effects of cocaine. Future studies will investigate nAChR compounds of varying intrinsic efficacy and subtype specificity to further understand the role of nAChRs in mediating the reinforcing and discriminative stimulus effects of cocaine. All doses are expressed as that of the salt. DA12460.
Impulsivity and vulnerability to cocaine self‐administration in adult rhesus monkeys exposed to cocaine in utero
Authors: Hamilton Lindsey R , Czoty Paul W , Calhoun Tonya L , Nader Michael A
Published: June 2021

Impulsivity is a trait that can be assessed as a predictor of increased likelihood to self‐administer drugs of abuse. A validated method to assess impulsivity is delay discounting, a model based on choice between different magnitudes of reinforcers under different delay conditions. In this study, male and female adult rhesus monkeys prenatally exposed to cocaine (n = 10) and controls (n = 11) chose between a small immediate reinforcer, one 1‐g banana‐flavored pellet, or a larger delayed reinforcer, three pellets. The delay varied between 0 and 300 s; conditions remained constant for at least 5 consecutive sessions. The delay value at which the monkey chose the immediate and delayed reinforcers equally often (the ED50) provided an index of impulsivity. Preliminary data indicate that monkeys exposed to cocaine in utero have a smaller mean ED50 value (70 s) than controls (110 s), indicating greater impulsivity. These differences were not correlated with cerebrospinal fluid levels of serotonin or its metabolite. Sex differences were observed as only the male prenatally cocaine exposed monkeys exhibited increased impulsivity. Whether this increased impulsiveness predicts a greater vulnerability to self‐administer cocaine is currently being examined. DA 25120, DA 13370.
Age‐Related Neural Changes After Brief Exposure to Nicotine
Authors: Dao Jasmin M , Soria Aide M , Tran An‐Ha T , Leslie Frances M
Published: June 2021

Adolescence is a critical period for the onset of tobacco smoking, with more than 90% of smokers beginning at this age. Clinical studies have shown that children who tried a single cigarette by the age of 11 continue to be susceptible to smoking throughout the rest of their lives. In animal models, it has been shown that adolescents exhibit unique responses to nicotine exposure, as compared to adults. Furthermore, subchronic treatment with a low dose of nicotine in early adolescence increases subsequent cocaine self‐administration. The purpose of this study was to examine the behavioral effects and age‐related neurochemical changes that occur after exposure to nicotine. Male Sprague‐Dawley rats aged P28 or P86, were given one or four daily i.v. injections of saline or nicotine (2 x 0.03 mg/kg/0.1 ml spaced 1 min apart). Immediately after the last injection, locomotor behavior was monitored for 30 minutes and brains were collected for anatomical analysis. Acute nicotine treatment produced increased locomotor activation in adolescents, which persists through the subchronic treatment. Adults show a decrease in activity after one injection of nicotine, whereas repeat nicotine treatment has no effect. Preliminary analysis of nicotine‐induced IEG expression has shown selective nicotine activation of limbic forebrain in adolescents. In vivo microdialysis studies are currently being conducted to further elucidate changes after nicotine exposure. These findings provide evidence that nicotine administration during adolescence causes unique neural adaptations not seen following treatment of adults.
Striatal Mu Opioid Receptor Blockade Alters Methamphetamine‐Induced Stereotypy
Authors: Horner Kristen Ashley , Gilbert Yamiece E
Published: June 2021

Methamphetamine (METH) induces stereotypy, which is characterized as inflexible, repetitive behavior. Enhanced activation of the patch compartment of striatum has been correlated with stereotypy, suggesting that stereotypy may be related to preferential activation of this region. Mu opioid receptors are densely expressed in the patch compartment and may contribute to stereotypy. The goal of this study was to determine if mu opioid receptor blockade alters METH‐induced stereotypy. Animals were bilaterally infused in the striatum with the mu antagonist CTAP (10 μg/μl) or vehicle. Animals were then given METH (7.5 mg/kg) or saline, placed in locomotor activity chambers and observed for 3h. Stereotypy was scored on a scale from 1‐10 in four behavioral dimensions: repetitiveness, frequency, duration and spatial distribution. Horizontal and rearing activity was also monitored during the 3h period. CTAP pretreatment significantly reduced the degree of stereotypy induced by METH. METH‐induced stereotypy is accompanied by spatial immobility and CTAP significantly reduced this effect. In support of this observation, CTAP pretreatment significantly increased horizontal locomotor activity in METH‐treated animals. There was also an increase in rearing activity in CTAP‐pretreated METH‐treated animals. These data indicate that striatal mu opioid receptor activation contributes to METH‐induced stereotypy.
CM156, A Sigma (σ) Receptor Antagonist, Mitigates Stimulant And Neurotoxic Effects Of Methamphetamine In Mice
Authors: Kaushal Nidhi , Seminerio Michael J. , Shaikh Jamaluddin , Medina Mark A. , McCurdy Christopher R. , Matsumoto Rae R.
Published: June 2021

Methamphetamine (METH) is a psychostimulant drug of abuse, causing hyperthermia and neurotoxicity at high doses. Currently, there is no clinically proven pharmacotherapy to treat these effects. Therefore, there is a need to identify potential therapeutic targets. Radioligand binding assays showed that METH binds to sigma (σ) receptors in the brain. Antisense oligonucleotides targeting σ1 receptors antagonized the locomotor stimulant effects of METH in mice. As a potential pharmacological antagonist, CM156 was developed with high affinity and selectivity for σreceptors. CM156 antagonized the locomotor stimulant effects of METH without altering basal activity. CM156 was further tested against METH‐induced neurotoxicity, whereby male Swiss Webster mice were treated every 2 hours, a total of 4 times, with one of the following: Saline/Saline, Saline/METH (1.25‐10 mg/kg), CM156 (5‐20 mg/kg)/Saline, and CM156 (5‐20 mg/kg)/METH (5‐10 mg/kg). Body temperature was measured 1 hour after every treatment and the brains were collected for ELISA and immunohistochemical analyses one week later. CM156 significantly and dose dependently attenuated the METH‐induced hyperthermia, depletions in striatal dopamine and serotonin levels, and reductions in DAT levels, without itself causing any changes. Together, the data suggest that σreceptors represent a viable therapeutic target for mitigating METH effects.

(Support: DA023205, DA013978)
An economic analysis of alcoholic and nonalcoholic beer drinking in rats exposed to daily restraint stress or handling
Authors: Kohut Stephen J , Christensen Chesley J , Riley Anthony L
Published: June 2021

Using a behavioral economic approach, this study attempted to determine whether the reinforcing strength of alcoholic or nonalcoholic beer changes after stress or handling. Twenty‐four, male Wistar rats were trained to bar press for either alcoholic or nonalcoholic beer and assigned to one of four treatment groups: stress‐alcohol (SA), no stress‐alcohol (NA), stress‐no alcohol (SN) and no stress‐no alcohol (NN). The stressed condition consisted of 15 min of restraint stress, while non‐stressed rats were handled and placed into a novel cage for 15 min immediately prior to the session. After training under an FR1 schedule, the schedule was increased every five sessions from 3 to 32 in logarithmic steps. Consumption was plotted as a function of ratio size and modeled by the exponential‐demand equation (Hursh & Silberberg 2008). Consumption prior to the stress/handling manipulation was greater for nonalcoholic than alcoholic beer, but there was no difference between stressed and unstressed rats for either solution. When stress was applied, alcohol consumption decreased relative to that in the non‐stressed rats. Stress did not have an effect on non‐alcoholic drinking. The economic analysis showed that NA, SN and NN groups all defended beer consumption with greater vigor than the SA group. Our results suggest that stress has a suppressive effect on alcohol drinking in "naïve" rats.

This work was supported by the Mellon Foundation.
Gclm Null Mice have Drastically Increased Angiogenic Potential
Authors: Pattillo Christopher B , Pardue Sibile , Kavanagh Terrance J , Kevil Christopher G
Published: June 2021

Previous studies have provided evidence that increased levels of glutathione (GSH) attenuate VEGF‐A induced cellular ROS production, thereby decreasing cell motility. The rate limiting step in GSH production is carried out by the holoenzyme glutamate‐cysteine ligase (GCL) which is composed of catalytic (Gclc) and modifier (Gclm) subunits. A mouse Gclm null model has been developed with tissue levels of GSH between 9 and 40% of wild type (WT) mice. This null model was employed to determine the role Gclm plays in redox regulation of angiogenesis following hind limb ischemia. The ischemic leg in the Gclm null mice had blood flows returning to pre‐ligation values by day 7, whereas WT blood flow had not recovered 21 days post‐ligation, indicating an increased cellular proliferation and migration in Gclm null mice. The angiogenic index in Gclm null mice was 2 fold higher in ischemic tissue compared with non‐ischemic tissue. The proliferative index was approximately 1 in ischemic tissue indicating that the proliferating cells were likely endothelial cells. Protein carbonyls were elevated, while GSH levels were depressed in the Gclm null mice. Together, these data provide strong evidence that Gclm null mice have a more intense angiogenic response to ischemia than WT mice. This may be due to increased VEGF‐A/NADPH oxidase signaling in these mice.
Oxygen dependence of mitochondrial respiration in high and low altitude birds
Authors: Scott Graham R , Richards Jeffrey G , Milsom William K
Published: June 2021

We examined the oxygen dependence of mitochondrial respiration in the high altitude bar‐headed goose and several low altitude waterfowl species (greylag goose, barnacle goose, and mallard duck). Mitochondria were isolated from the flight muscle and their respiration was measured by high‐resolution respirometry at avian body temperature (41°C), with malate/pyruvate or malate/pyruvate/succinate fuel combinations. Mitochondria were well coupled with high phosphorylation efficiencies in all birds. Respiratory capacities, cytochrome oxidase excess capacity, and uncoupled respiration rates were all similar between species. Oxygen kinetics depended on respiration state, as half‐maximal mitochondrial respiration occurred at O2 tensions (P50) of 0.1‐0.2 Torr in State 4 (no ADP) and 0.8‐0.9 Torr in State 3 (maximal ADP). These data suggest that intramyocellular O2 tension could regulate mitochondrial respiration during flight, particularly at high altitude where O2 supply is reduced. (Supported by NSERC of Canada)
Characterization and validation of aerial respiration and central CO2 chemosensitivity in the Alaska blackfish, Dallia pectoralis
Authors: Hoffman Megan , Harris Michael B , Taylor Barbara E
Published: June 2021

Little is known of respiratory physiology in Dallia pectoralis, an arctic air‐breathing fish. We used electromyography and electromyostimulation to identify muscles involved in air breathing. We attempted to determine the hindbrain innervation of the respiratory muscles using retrograde fluorescent labeling. We identified and validated function of respiratory muscles and motoneuron activities associated with aquatic respiration but were unable to fully characterize aerial respiration. Current attempts to investigate aerial respiration employ a partial preparation with intact respiratory structures. Identification of respiratory muscles and nerves enables us to assess central CO2 chemosensitivity (cCO2‐CS) in D. pectoralis. cCO2‐CS has been identified in other air‐breathing fish of divergent phylogeny, such as gar (Holostei) and betta (Teleostei), but not in a primitive air‐breathing fish such as amia (Holostei). Preliminary data suggest that D. pectoralis is unlike its fellow teleost; they lack cCO2‐CS. With a complete data set, we will be able to assess the phylogenetic origins of cCO2‐CS. If cCO2‐CS exists in gar and both teleost species, this characteristic likely arose in a common actinopterygian ancestor of Teleostei and Holostei. In contrast, if cCO2‐CS is absent from D. pectoralis, then it likely arose independently multiple times among the air‐breathing teleosts.
Skeletal muscle hypertrophic effect of clenbuterol is additive to the hypertrophic effect of myostatin suppression
Authors: Kim Kyung Ho , Kim Yong Soo
Published: June 2021

It is well established that myostatin (MSTN) suppression or clenbuterol (CL) administration enhances skeletal muscle mass, but the interaction of MSTN suppression and CL administration on skeletal muscle mass has not been examined. To examine the interaction of MSTN suppression and CL administration on skeletal muscle mass, 35 days old female wild type and heterozygous MSTN‐pro transgenic mice that exhibits 20‐30 % enhanced muscle mass by overexpression of MSTN prodomain were administrated 0 and 20 ppm CL in drinking water for 2 wks. Four different muscle samples from hind legs were collected at 1 wk and 2 wk after CL administration. Regardless of genotype, CL significantly increased the body and muscle mass at 1 wk or 2 wk after CL administration. No significant genotype and CL interactions on body and muscle mass were observed. DNA concentration of plantaris muscle was significantly decreased by CL administration for 1 wk or 2 wk regardless of genotype. RNA concentration of plantaris muscle was significantly increased by CL administration for 1 wk in both genotypes, but no significant change in RNA concentration was observed at 2 wk of CL administration in both genotypes. The RNA/DNA ratio in plantaris muscle was significantly elevated by CL administration for 1 wk or 2 wk in both genotypes. In summary, the result of this study demonstrates that skeletal muscle hypertrophic effect of CL is additive to the hypertrophic effect of MSTN suppression, suggest that the mechanism of action of CL leading to skeletal muscle hypertrophy might be different from that of MSTN.
The absence of UCP3 leads to tighter coupling of oxidative phosphorylation in skeletal muscle at rest
Authors: Siegel Michael , Harper Mary‐Ellen , Marcinek David
Published: June 2021

The role of uncoupling protein 3 (UCP3) in mitochondrial function in skeletal muscle remains controversial. To gain insight into the role of this mitochondrial protein, we sought to determine whether UCP3 uncouples oxidative phosphorylation in vivo, as suggested by the putative function of the homologue, UCP1. We hypothesized that, under resting conditions, the presence of UCP3 would correspond with a reduction in coupling. To test this we compared wild type mice with UCP3 knockout mice using non‐invasive methods for assessing in vivo resting metabolism, 31P nuclear magnetic resonance spectroscopy and optical spectroscopy. During a bout of ischemia, the former provides a measure of the resting rate of ATP production while the latter provides a measure of the resting rate of oxygen consumption. Dividing the rate of ATP production by the rate of oxygen consumption provides a measure of coupling (P/O ratio). We found a significant increase in the P/O ratio in mice lacking UCP3 compared to wild type mice (2.81 +/‐ 0.34 for knockout and 2.00 +/‐ 0.18 for wild type). This difference appears to be due to an increase in resting mitochondrial ATP production in the absence of UCP3. Our results indicate that UCP3 plays a role in regulating coupling of oxidative phosphorylation in skeletal muscle under resting conditions. This work was supported by NIH grants AG022385 and AG028455 and the Ellison Medical Foundation. Sample Data
Thyroid hormone affects diabetes‐induced changes on cardiac myosin heavy chain gene expression
Authors: Giger Julia , Haddad Fadia , Baldwin Kenneth M.
Published: June 2021

Ventricular muscle expresses two isoforms of myosin heavy chain (MHC) genes: beta and alpha; which are arranged in tandem on the chromosome. Endogenous mRNA of beta and alpha MHCs are expressed in an antithetical manner in control, diabetic, hypo‐ and hyperthyroid rat hearts. We have identified an intergenic region between the beta and alpha genes that modulates the transcription of alpha sense and beta antisense pre‐mRNAs, we believe the intergenic bidirectional promoter controls the antithetic expression of alpha and beta MHC genes. The alpha MHC gene is predominantly expressed in adult rat heart. However, in the diabetic state, the expression of alpha is decreased and beta is increased. Thyroid hormone treatment has been shown to have a large impact on cardiac MHC gene expression and is a positive regulator of alpha MHC and negative regulator of the beta MHC gene. The pathways that regulate MHC isoform expression are unclear. By examining the sequence of beta sense, beta antisense and alpha sense promoters, we hopefully can discern the factors that alter the MHC expression in response to diabetes and thyroid hormone. We attempt to "rescue" diabetic (STZ treated) adult and neonatal rats using low levels of thyroid hormone; and show an effective response on MHC isoform expression and antisense and sense promoter activity. Supported by Amer. Diabetes Assoc and NIHHL.
Mechanical stretch induces activation of eukaryotic elongation factor 2 in C2C12 myoblast
Authors: Nakai Naoya , Kawano Fuminori , Oke Yoshihiko , Nomura Sachiko , Terada Masahiro , Ohira Takashi , Ohira Yoshinobu
Published: June 2021

It has been proposed that mechanically‐induced tension was the critical event for initiating muscle hypertrophy. However, the molecular mechanisms involved in this process are still under investigation. In the present study, effect of mechanical stretch on the protein translation initiation is studied in C2C12 myoblast. C2C12 cells were plated on fibronectin‐coated silicone elastomer chamber in DMEM containing 10% fetal bovine serum. Cells were grown in CO2 incubator and subjected to 30 min of cyclic 15% biaxial stretch at a frequency of 1Hz. 30 min of cyclic stretch increased the phosphorylation levels of p38 mitogen activated protein kinase (MAPK) and extracellular‐regulated kinase 1 and 2 (ERK1/2). Eukaryotic elongation factor 2 (eEF2), a marker for peptide chain elongation process, was also activated (dephosphorylated) by cyclic stretch. These results suggest that p38 MAPK and/or ERK1/2 pathway may be involved in stretch‐induced activation of protein translation elongation process. This study is supported by Grant‐in‐Aid for Scientific Research C (20500577 to N.N.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
Unmasking hyperactive ENaC in P2Y2 ‐/‐ mice as a molecular mechanism for their hypertension
Authors: Pochynyuk Oleh , Bugaj Vladislav , Rieg Timo , Insel Paul A , Vallon Volker , Stockand James D
Published: June 2021

ENaC activity is limiting for Na+ reabsorption across the distal nephron, thus making this channel critical for the regulation of blood pressure. As such, ENaC is under negative‐feedback control by aldosterone. Paracrine signaling, intrinsic to the kidney, also modulates ENaC: ATP released by the distal nephron tempers ENaC activity through P2Y2 receptors. Local regulation of ENaC by ATP is physiologically important for its disruption, by knockout of P2Y2 receptors (P2Y2‐/‐ ), leads to hypertension. Here, we test the relation between intrinsic and extrinsic regulation of ENaC and ask if dysfunction of intrinsic regulation potentiates extrinsic regulation. The Po and N of ENaC in the murine distal nephron, as expected, increased in response to DOCA and decreased in response to chronic salt loading with DOCA returning activity to normal in the presence of high salt intake. In contrast, Po was high under all conditions in P2Y2‐/‐ mice but N responded normally. High Po plus increases in N in response to DOCA produced an inappropriate increase in ENaC activity in P2Y2‐/‐ mice maintained on a high salt diet plus DOCA: ENaC activity was significantly greater in ‐/‐ compared to wild‐type mice. We conclude that disruption of local purinergic (P2Y2) signaling sets ENaC activity at a high level. Salt loading plus DOCA unmasks this inappropriate ENaC activity which leads to the hypertension in P2Y2‐/‐ mice.
Modulation of vasa recta calcium activated chloride channels by myosin light chain kinase
Authors: Cao Chunhua , Lin Hai , Pallone Thomas L.
Published: June 2021

Descending vasa recta (DVR) are 12 ‐ 15 μm microvessels that supply the renal medulla with blood flow. They consist of contractile smooth muscle like pericytes and an endothelial monolayer. DVR pericytes express calcium activated chloride channels (ClCa). In this study, we tested whether myosin light chain kinase regulates those channels. Using the conventional whole cell patch‐clamp technique, ClCa currents were elicited in murine pericytes by depolarizing the cells during EGTA buffering of intracellular Ca2+ to a range between 20 to 1000 nM. Pericytes, held at ‐80 mV and depolarized from ‐90 mV to +80 mV in 10 mV increments, showed outwardly rectifying ClCa currents that were both voltage and Ca2+ dependent. The ClCa currents did not rundown after initial access via ruptured patches. ClCa currents elicited in the presence of 500 nM intracellular Ca2+ were significantly inhibited by intracellular or extracellular application of the calmodulin inhibitor W‐7 (30 μM). Selective myosin light chain kinase (MLCK) inhibitors, ML‐7 or ML‐9 also inhibited ClCa currents in a concentration dependent manner. Finally, the MLCK inhibitor ML‐7 (20 μM), reversed AngII (10 nM) induced pericyte depolarization and dilated AngII preconstricted DVR. In conclusion, DVR pericyte ClCa currents, membrane potential and contraction by AngII are regulated by calmodulin and myosin light chain kinase.
Changes in tubular fluid pH and nucleotide concentration alter P2 receptor‐mediated regulation of renal ENaC
Authors: Wildman Scott S.P.
Published: June 2021

Epithelial Na channels (ENaC) control Na reabsorption along the renal collecting duct (CD) and help determine systemic BP. We have shown that ENaC activity is inhibited by activation of apical P2Y2 and/or 4 receptors and P2X4 and/or 4/6 receptors when luminal concentrations of Na are high, and that apical P2X4 and/or 4/6 receptors switch to being potentiators of ENaC activity when concentrations of Na are lowered. Here we address the effect of altering luminal pH, and concentration of P2 receptor (P2R) agonists, on P2R‐mediated regulation of ENaC in the CD.

Rats were maintained on a low Na diet to increase ENaC expression. Kidneys were microdissected, and CD split‐open to expose the apical membrane of cells for patch clamp experiments.

When concentrations of P2R agonists were high, UTP‐evoked currents (i.e. P2Y2 and/or 4‐mediated) reduced, and 2meSATP‐evoked currents (i.e. P2X4 and/or 4/6‐mediated) increased, the amplitude of subsequent ENaC‐mediated currents. Lowering pH from 7.4 to 6.5 did not alter these effects. Further acidification to pH 5.5 increased the degree of inhibition by UTP and abolished the effects of 2meSATP‐evoked currents. At pH 7.4 and 6.5, when concentrations of P2R agonists were lowered, the inhibitory effect of UTP was abolished and the potentiating effect of 2meSATP was unaltered.

Apical P2R and ENaC interactions in CD are complex and dependent on luminal Na and nucleotide concentration, and pH.
The Kir channel immunoglobulin domain: a cytoplasmic scaffold essential for (Kir1.1) ROMK channel thermodynamic stability, trafficking and gating
Authors: Denton Jerod , Banerjee Sreedatta , Sheehan Jonathan , Lewis L. Michelle , Meiler Jens , Fallen Katherine
Published: June 2021

Heritable mutations in renal potassium channel ROMK cause a severe salt and water balance disorder called antenatal Bartter syndrome (ABS). Of more than 30 ABS mutations identified, about half are located in the intracellular domain of the channel; the mechanisms underlying channel dysfunction for most of these mutations are unknown. By mapping intracellular mutations onto an atomic model of ROMK, we found that several are localized to an ancient immunoglobulin (Ig)‐like domain (IgLD) that has not been characterized previously, prompting us to examine it in detail. The IgLD is assembled from two β‐pleated sheets packed face to face, creating a β‐sheet interface, or core, populated by highly conserved side chains. Thermodynamic calculations on computationally mutated channels indicate that IgLD core residues are among the most important residues for determining cytoplasmic domain stability. We show that two ABS mutations located within the IgLD core impair channel trafficking in mammalian cells. A fraction of core mutant channels reach the cell surface, but they are electrically silent due to closure of the helix‐bundle gate. Compensatory mutation‐induced rescue of channel function revealed that IgLD core mutants fail to rectify. Our study sheds new light on the pathogenesis of ABS and provides a novel framework for understanding Kir channel cytoplasmic domain structure‐function relations.
SPARC deficiency ameliorates renal damage resulting from angiotensin II and a high salt diet
Authors: Socha Matthew John , Manhiani Marlina , Motamed Kouros , Imig John D , Inscho Edward W
Published: June 2021

Markedly elevated levels of matricellular protein SPARC (Secreted Protein Acidic and Rich in Cysteine, SP) have been found in serum of patients with fibrotic renal injury and in glomeruli of hypertensive injury. Our previous data indicate that SPARC deficiency resulted in decreased renal fibrosis, oxidative stress, and inflammation following Angiotensin II (Ang II)‐induced hypertension, with no improvement in renal function. We hypothesized that SPARC deficiency would attenuate the detrimental renal effects and protect kidney function in Ang II‐induced hypertension combined with a high salt diet. Following a 2‐week Ang II infusion combined with a high salt diet (4% w/v), and independent of blood pressure differences (SP+/+ 163 ± 4, SP−/− 162 ± 3 mmHg), SP−/− mice showed significant attenuations in albuminuria (urinary albumin decreased 70.7%), renal matrix deposition (urinary levels of active TGF‐β1 decreased 58.9%), reactive oxygen species formation (urinary 8‐isoprostane decreased 60.9%), and renal inflammatory responses (MCP‐1 and IL‐1β levels decreased 36.8% and 59.8%, respectively), relative to SP+/+ controls. We conclude that in a severe model of hypertension, SPARC deficiency protects against renal hypertensive damage at the cellular and functional levels.

This work is supported in part by National Institute of Health grants K01‐CA089689 (to K.M.) and HL59699 (to J.D.I.).
Ryanodine receptors and store‐operated calcium entry (SOCE) mediate vasopressin‐induced intracellular Ca2+ oscillations in inner medullary collecting duct (IMCD)
Authors: Yip Kay‐Pong , Sham James S.K.
Published: June 2021

It is well documented that arginine vasopressin (AVP) stimulates water permeability in IMCD through a cAMP‐dependent mechanism. Our recent studies suggest that it is also dependent on intracellular Ca2+ mobilization. Here we sought to determine the specific Ca2+ pathways and the signaling mechanisms of AVP induced Ca2+ mobilization in isolated perfused IMCD. Application of AVP (0.1 nM) activated an initial transient increase in [Ca2+]i followed by repetitive Ca2+ oscillations at 0.02 ‐ 0.1Hz. The Ca2+ response was mimicked by photo‐release of caged cAMP, suggesting that cAMP generation induced by AVP is sufficient to elicit the Ca2+ response. AVP‐induced Ca2+ mobilization was mediated by both extracellular Ca2+ influx and ryanodine receptor (RyR). Removal of extracellular Ca2+ completely abolished Ca2+ oscillations without affecting the initial Ca2+ release, which was obliterated by further inhibition of RyR with 100 μM ryanodine, but was unaffected by the IP3‐receptor antagonist xestospongin C or 2‐APB. Moreover, direct activation of RyR by flash photolysis of caged cyclic‐ADP‐ribose also mimicked the AVP‐induced Ca2+ response. To further investigate the pathway for extracellular Ca2+ influx, 0.1 nM AVP was applied to IMCD in Ca2+‐free medium in the presence of nifedipine. Readmission of extracellular Ca2+ restored the repetitive Ca2+ oscillations, which was blocked by SKF96365. SKF96365 also blocked SOCE induced by thapsigargin in IMCD. These observations suggest that AVP triggers Ca2+ oscillations through cAMP‐dependent activation of RyR, probably leading to depletion of Ca2+ in endoplasmic reticulum and subsequent SOCE in IMCD.
Methylglyoxal (MG) Contributes to the Development of Insulin Resistance and Salt Sensitivity in Sprague Dawley (SD) Rats
Authors: Guo Qi , Mori Takefumi , Jiang Yue , Osaki Yusuke , Yoneki Yoshimi , Ogawa Susumu , Miyata Toshio , Ito Sadayoshi
Published: June 2021

The present study was designed to investigate whether MG could induce insulin resistance and salt sensitivity in SD rats. Rats were given either 1% MG in tap drinking water or tap water alone. After 4‐week treatment insulin resistance was evaluated by glucose clamp technique. In another set of rats, either a high‐salt diet (HS; 4%) alone, standard rat chow with 1% MG in tap drinking water or HS plus MG was given for 4 weeks. Immunohistochemistry was performed to measure nitrotyrosine and MG‐induced advanced glycation endproducts (AGEs), Nƒ Ã‐carboxyethyl‐lysine (CEL) in the kidney. Systolic blood pressure (SBP) was measured by an tail‐cuff method. 4‐week treatment with 1% MG in drinking water significantly increased insulin resistance without altering the SBP. Co‐administration of MG and HS significantly increased SBP, urinary albumin excretion, urinary thiobarbituric acid‐reactive substances excretion and the renal nitrotyrosine expression in the kidney compared with MG or HS alone. Renal CEL was significantly increased in MG‐treated rats compared to non‐MG‐treated rats. These results indicate that MG induced insulin resistance and salt sensitivity at least in part by increasing oxidative stress and/or AGEs formation in SD rats. The present study provides further evidence for MG as one of the causative factors in the pathogenesis of insulin resistance and salt‐sensitive hypertension.
Nocturnal fructose feeding increases renal ACE2 activity in mice
Authors: Weir Nathan , Graham Cathy , Key Mary , Koles Paul , Morris Mariana
Published: June 2021

The objective was to study renal morphology and angiotensin converting enzyme (ACE) activity in an animal model of metabolic syndrome. C57Bl/6 male mice were given: 1) tap water 2) 24 h access to 10% fructose water or 3) 12 h access to fructose water (dark period) and water (light period) for 8 weeks. Regular chow was available ad libitum. Mice on 12 h fructose consumed more total fructose and gained more weight than those with 24 h access. Upon conclusion of the dietary regimen, kidneys were collected for morphologic studies using PAS staining, immunohistochemical staining for ACE and ACE2 and ACE/ACE2 activity. ACE/ACE2 enzyme activity was measured using a mass spectrometric assay with endogenous peptide substrates. Immunohistochemical staining showed a stronger presence of ACE2 in the 12 h group over control and 24 h. ACE staining appeared unchanged. ACE2 activity in the 12 h group was 4 fold higher than in control and 24 h groups (C = 0.6±0.2, 24h = 0.6±0.08 vs. 12h = 2.5±0.4; p<0.05). ACE activity was not different between groups. Pathological changes were unremarkable except for mesangial expansion in the 12 h (C = 0.84±0.01, 24h = 0.88±0.01 vs. 12h = 0.92±0.009; p<0.05). Results document that the renal effects of fructose water are dependent on the timing and volume of consumption. Increased nocturnal fructose water intake is associated with elevated ACE2 activity without changes in ACE. Supported by NIH grant R01 HL09356
Intrarenal RAS expression during Ang II‐infusions and ACE inhibition
Authors: Gonzalez‐Villalobos Romer A. , Satou Ryousuke , Semprun‐Prieto Laura C. , Ohashi Naro , Katsurada Akemi , Upchurch G. Michael , Prieto‐Carrasquero Minolfa C. , Kobori Hiroyuki , Navar L. Gabriel
Published: June 2021

ACE inhibition (ACEi) ameliorates the development of hypertension and the intrarenal Ang II augmentation observed during chronic Ang II infusions in mice, the objective of this study was to determine if these effects are associated with changes in the mouse intrarenal RAS. 9‐12 week C57BL/6J male mice were distributed as follows: controls (n=10), Ang II‐infused (Ang II=8, 400 ng/kg/min), controls + ACEi (ACEi=10, lisinopril, 100 mg/L) and Ang II‐infused + ACEi (Ang II + ACEi=11). As previously reported, ACEi attenuated the increases in MAP (monitored by telemetry) and intrarenal Ang II caused by Ang II infusions (measured by RIA). No significant changes were observed in angiotensinogen (AGT), ACE or AT1R mRNA by qRT‐PCR. AGT protein expression [determined by Western blot (WB) and Immunohistochemistry (IHC)] was increased by Ang II infusions (Ang II, 1.41 ± 0.14 by WB, 2.5‐fold increase by IHC) vs. controls (1.00, p<0.05) and this was prevented by ACEi (ACEi + Ang II, 0.80 ± .07 by WB, NS). Tubular renin protein increased in ACEi‐treated mice (ACEi, 2.3 ± 0.36; Ang II + ACEi, 4.01 ± 1.1 by IHC) vs. controls (1.00, p<0.05), but was not altered by Ang II alone. ACE protein was increased by Ang II (Ang II, 1.3 ± 0.18 by IHC) vs. controls (1.00, p<0.05) and ACEi prevented this changes (Ang II + ACEi, 0.85 ± 0.11). AT1R protein was increased by Ang II infusions (Ang II, 1.27 ± 0.06 by WB) vs. controls (1.00 p<0.05) and this was prevented by ACEi (Ang II + ACEi, 1.14 ± 0.06, NS). In summary, Ang II infusions augmented the expression of intrarenal AGT, ACE and the AT1R and did not suppress tubular renin. The findings that these changes are prevented by ACEi suggest that they are mediated by endogenous ACE‐derived Ang II.
Central ACE2 reduces blood pressure and restores baroreflex and autonomic functions in chronically hypertensive mice
Authors: Xia Huijing , Feng Yumei , Hickman Peter J , Lazartigues Eric
Published: June 2021

We previously showed reduced central ACE2 activity in RA mice, a chronically hypertensive model with elevated AngII levels. To confirm the critical role of central ACE2 in blood pressure (BP) regulation, we generated a triple transgenic model (SARA) with brain ACE2 over‐expression on a RA background. BP (radiotelemetry), baroreflex (sequence method) and autonomic function were assessed in RA, SARA and control mice (n=6/group). Mean BP (MBP: 144 ±2 vs. 97 ±1 mmHg) and daily water intake (7.4 ±0.3 vs. 3.1 ±0.1 ml) were dramatically increased, while brain ACE2 activity (17 ±7 vs. 49 ±7 AFU/mg/min) was significantly decreased in RA mice (P<0.01). SARA mice exhibit higher ACE2 activity (60 ±3 AFU/mg/min), lower MBP (133 ±2 mmHg) and a blunted water intake (4.9 ±0.2 ml) vs. RA (P<0.05), suggesting that ACE2 lowers brain AngII levels. In addition, impaired baroreflex sensitivity (1.0 ±0.2 vs. 2.0 ±0.2 msec/mmHg), parasympathetic tone (ΔHR: 75±10 vs. 129 ±15 bpm) and increased sympathetic drive (ΔHR: ‐104 ±7 vs. ‐67 ±9 bpm), observed in RA (P<0.05 vs. controls), were normalized in SARA mice (1.7 ±0.3 msec/mmHg; 128 ±18 bpm and ‐70 ±13 bpm, respectively; P<0.05 vs. RA). These data suggest that decreased central ACE2 activity contributes to the maintenance of hypertension and that ACE2 gene therapy could be used in the treatment of diseases resulting from an overactive renin‐angiotensin system. (NIH HL093178, NS052479 and RR018766)
Neurokinin 3 receptor (NK3R) immunoreactivity (IR) in supraoptic nucleus (SON): Impact of antigen epitope on immunohistochemical (IHC) localization
Authors: Sladek Celia D. , Stevens Wanida , Lawson Regan R. , Levinson S. Rock
Published: June 2021

Activation of NK3R results in receptor internalization and nuclear localization of NK3R‐IR in SON when a C‐terminal directed antibody (Ab; aa 438‐452; Novus) is used (Howe et al, J. Neurosci. 24:10103, 04). To further characterize this IR, we generated 2 Abs against each of 3 additional epitopes in the NK3R (N‐terminal aa 6‐18; C‐terminal aa 376‐396 and aa 408‐420). Western blot (WB) and IHC with confocal microscopy were used to characterize the IR. On WB, all Abs recognized ~70kD protein in microdissected rat SON and in CHO cells transfected with rat NK3R. This band was absent in non‐transfected CHO cells and following peptide preabsorption of the Ab. The larger than predicted size (e.g. ~54kD) probably reflects post‐translational modification of the receptor. IHC with the N‐terminal Abs duplicated the membrane labeling seen with the Novus Ab, but prominent membrane IR was not evident with the other Abs and none of the new Abs replicated the hypotension induced punctate nuclear staining evident with the Novus Ab. These observations were not altered by modification of the fixation protocol nor Ab dilution. These findings demonstrate that Abs generated against multiple epitopes of the NK3R recognize a similar protein on WB. In contrast, the epitope target of NK3R Ab alters IHC detected cellular localization of the receptor perhaps due to differential Ab access to the epitope in the intact cell. Supported by NIH R21‐NS059569.
Skeletal Muscle Oxygen Uptake Dynamics during the Exercise Off‐Transient
Authors: Behnke Brad J , Ferreira Leonardo F , Musch Timothy I , Poole David C
Published: June 2021

The time course of muscle V̇O2 recovery (i.e., muscle V̇O2 kinetics) from contractions, measured at the site of O2 exchange i.e., in the microcirculation, is unknown. It is known that, in the rat spinotrapezius muscle, blood flow (Qm) kinetics are slower in recovery (time constant, t ~41 s) than at the onset of contractions (t ~17 s, Ferreira et al. J. Physiol. 2006).

Purpose

We tested the hypothesis that this Qm kinetics profile was associated with a slowed m V̇O2 recovery compared with that seen at the onset of contractions (t ~23 s, Behnke et al. Resp. Physiol. 2002).

Methods

Measurements of capillary red blood cell flux and microvascular PO2 (PO2) were combined to resolve the time course of muscle V̇O2across the moderate intensity contractions‐to‐rest transition.

Results

Contrary to our hypothesis, in recovery muscle V̇O2 began to decrease immediately (i.e., time delay 0 s in all but one animal) and demonstrated rapid first order‐kinetics (t 22.4 ± 2.1 s).

Conclusions

The slowed Qm kinetics in recovery serves to elevate the Qm/ V̇O2 ratio and thus microvascular PO2. Whether this Qm response is obligatory to the rapid muscle V̇O2 kinetics and hence speeds the repletion of high‐energy phosphates by maximizing conductive and diffusive O2 flux is an important question that awaits resolution. Support NIH AG031327.
Effect of exercise training on coronary endothelin‐1‐mediated constriction and receptor expression in hypercholesterolemic swine
Authors: Alajajian Sarah K , Bender Shawn B , Laughlin M. Harold
Published: June 2021

Exercise training (EX) has beneficial effects on coronary endothelial cell function in dyslipidemic states, often characterized by enhanced endothelium‐dependent dilation. Less is known, however, concerning the effect of EX on the pathways mediating constriction to endothelin‐1 (ET‐1), a potent endothelium‐derived vasoconstrictor. A better understanding of these effects is important since vascular responses to ET‐1 are often increased by cardiovascular risk factors. Therefore, we investigated the effect of EX on left circumflex (LCX) coronary artery responsiveness to ET‐1 and expression of the endothelin type A (ETA) and type B (ETB) receptor expression in a swine model of familial hypercholesterolemia. Swine were treadmill trained (EX; n=8) or cage‐confined (control; n=8) for 16 wk. In LCX artery rings, ET‐1 responsiveness tended to be decreased following EX. This decreased ET‐1‐induced constriction was not altered by endothelial denudation. In addition, Western blot analysis revealed an EX‐induced reduction in ETB (~40%), but not ETA, receptor expression in these vessels. These changes are consistent with an EX‐induced reduction in ET‐1‐mediated vasoconstriction potentially via reduced smooth muscle ETB receptor expression. Supported by NIH grant HL52490.
Contribution of nerve growth factor to augmented TRPV1 responses of muscle sensory neurons by hindlimb ischemia
Authors: Xing Jihong , Lu Jian , Sinoway Lawrence , Li Jianhua
Published: June 2021

When metabolite‐sensitive transient receptor potential vanilloid type 1 (TRPV1) receptors are stimulated a reflex is engaged that increases sympathetic nerve activity. In rats, hindlimb ischemia enhances this response. The underlying mechanism by which TRPV1 receptor responses are increased after ischemic insult is unclear. In this experiment we tested the hypothesis that muscle ischemia/vascular insufficiency elevates nerve growth factor (NGF) levels in primary afferent neurons and thereby increasing TRPV1 responsiveness. Hindlimb muscle ischemia/vascular insufficiency induced by the femoral artery ligation significantly increased NGF in the dorsal root ganglion (DRG) as compared with sham‐controls. Furthermore, when NGF was infused into hindlimb muscles of healthy rats (72 hours using an osmotic minipump), the magnitude of the DRG neuron TRPV1 response increased (5.4±0.54 nA with NGF infusion vs. 2.5±0.55 nA in control, P<0.05). After adding NGF, the magnitude of the DRG neuron TRPV1 response induced by capsaicin was greater (5.5±0.33 nA, P<0.05 vs. control) than that seen in controls (2.2±0.37 nA). This NGF effect was seen in isolectin B4‐negative DRG neurons, a group of thin fiber nerves that contain neuropeptides, and depend on NGF for survival. Thus NGF plays an important role in augmented TRPV1 responses after femoral artery occlusion. (All procedures outlined in this study were performed in compliance with the rules and regulations described in the NIH Guide for the Care and Use of Laboratory Animals).
Normobaric hypoxia exposure during sleep does not improve time‐trial performance in hypobaric hypoxia
Authors: Fulco Charles S. , Muza Stephen R. , Beidleman Beth A. , Jones Juli , Demes Rob , Staab Janet , Lammi Eric , Elliott Leonard D. , Viskochil Richard , Rock Paul B. , Cymerman Allen
Published: June 2021

This study determined if sleeping in normobaric hypoxia (NH) induces sufficient acclimatization to improve time‐trial (TT) performance in hypobaric hypoxia (HH). Two groups of sea‐level (SL) residents having similar peak oxygen uptakes (~47 ml/kg/min), ages (~24 yr), and weights (~75 kg) slept for ~6.5 hrs/night for 7 consecutive nights in portable rooms under blinded NH (n=14) or SL (n=8) conditions. Ambient % O2 for the NH group was progressively reduced by ~0.30% O2 each night from ~16.2% O2 (2200 m equivalent) on the 1st night to ~14.4% O2 (3050 m) on the 7th night. The SL group remained at =20.5% O2 (150 m). Within 25 hrs of treatment end, all were exposed to HH (Pikes Peak, CO; 4300 m). At SL before treatment and in the first 5 hrs of HH, an all‐out, 7‐mile treadmill TT was performed. Volunteers freely adjusted speed but grade was fixed at 3%. Heart rate (HR) and oxygen saturation (SaO2) were recorded every 5 min. For both groups, TT duration increased (P<0.01) while HR and SaO2 decreased (P<0.02) from SL to HH. There were no between‐group differences at SL or HH for TT duration (SL: 75±4 vs 73±4 min; HH: 103±6 vs 106±6 min), HR (SL: 165±2 vs 168±5 b/min; HH: 154±1 vs 150±3 b/min) or SaO2 (SL: 95±1 vs 96±1; HH: 74±1 vs 73±2). These results indicate that sleeping 7 nights in NH did not induce sufficient acclimatization to improve TT performance in HH. Funding: US Army MRMC ATO IV.MD.2006.01. Authors' views; not official U.S. Army or DoD policy.
PICOT, a novel monothiol glutaredoxin, plays a critical role in postembryonic growth and cardiac function in mice under nutritional perturbation
Authors: Cheng Ning‐Hui
Published: June 2021

PICOT (Protein Kinase C‐Interacting Cousin of Thioredoxin) is a newly identified monothiol glutaredoxin. In the present study, we demonstrated that MmPICOT had conserved functions in cell proliferation and response to oxidative stresses. Furthermore, disruption of MmPICOT in mice caused embryo lethal. In Picot+/− mice, early postembryonic growth was indistinguishable to wild type littermates, but Picot+/− mice did show significantly less in body weight and lean mass than that of wild type littermates when mice were ageing. Picot+/− mice on chow diet displayed distinct difference in intraventricular septal wall thickness and LV posterior wall thickness at end‐systole in comparison to wild type littermates, but cardiac output in those Picot+/− mice remained similar to wild type littermates. However, under chronic dietary fat intake, Picot+/− mice had significant decrease in ejection fraction and fractional shortening, suggesting impaired cardiac function in those Picot+/− mice. Together, these results suggest that PICOT is essential in embryo development and may also play a critical role in postembryonic growth and heart function, particularly under oxidative stress and nutritional perturbation.