bluuebunny/crossref_metadata_2025 huggingface dataset:
Protective effects of auricularia auriculajudae polysaccharide on vascular of streptozotocin induced diabetic rats
Authors: Wang Linlin , Situ Wangmin , Fei Jiaqian , Zhang Xiaoming , Ye Tingmei , Shen Yueliang , Chen Yingying
Published: June 2021

Aim

To investigate the direct vascular effects of auricularia auriculajudae polysaccharide (AAP) and the underlying mechanisms on the thoracic aorta of streptozotocin(STZ) induced diabetic SD rats.

Methods

The thoracic aortic rings with endothelium from STZ induced diabetic rats were mounted in an organ bath. Isometric contraction of aortic rings was measured. On the other hand, isolated aortic rings from normal control SD rats were conducted with immuno‐histochemical analysis for induced nitric oxide synthase (iNOS) expression after they were pre‐incubated with APP (10 μg/ml) for 0~50 minutes.

Results

(1) Cumulative administration of AAP (2‐20 μg/ml) did not affect the vasomotion of aortic rings of diabetic rats in basal tension. (2) After incubation with APP (10 μg/ml) for 30 minutes, aorta rings with endothelium were pre‐contracted with PE (10−6 M) and then acetylcholine (Ach) were added. The vascular relaxation response to Ach was significantly impaired in diabetic group, which was completely reversed by APP treatment. (2) The expression levelof iNOS in aortic rings was markedly increased after incubated with APP. iNOS expression had a peak level at time point of 10 minutes.

Conclusion

APP could restore the vasorelexation dysfunction in diabetic rat aortic rings, in which the activation of NO pathway may probably be involved.
Effects of Radix Astragali Injection on Urinary Microalbuminuria and Insulin Signal Transduction in Kidney of Early Diabetic Nephropathy
Authors: Zhang Jingfang , Wang Guanghao , Yang Xueqin
Published: June 2021

To investigate the effects of radix astragali injection on urinary microalbuminuria and insulin signal transduction in renal tissue of early diabetic nephropathy(DN).The Wistas rats were randomly divided into 3 groups: normal control group, DN group, DA group(DN +astragali).The DN Rat model was established by intraperitoneal injection of streptozotocin(STZ).After injecting radix astragali injection 0.2ml/100g into caudal vein for 3 weeks, the urinary microalbuminuria excretion (UAE) was measured by radioimmunoassay, the fasting blood glucose(FBG) was measured by enzyme assay, and the expression of insulin receptor (ins‐R), insulin receptor substrate‐1(IR‐1), phosphatidylinositol‐3‐kinase (PI3‐K) were detected by immunohistochemistry in rat's kidney. The results showed that the UAE and the serum glucose of rats in DN group were obviously higher than normal group, there was a significant decrease in UAE and serum glucose in DA group, however, there was no effect on serum glucose of rats in normal group; the expression of ins‐R, IR‐1, PI3‐K in rat's kidney of DN group was significant lower than normal control group, and there was a significant increase in the expression of ins‐R, IR‐1, PI3‐K in DA group. The results of our research showed that radix astragali injection can significantly decrease the urinary microalbuminuria and serum glucose in early DN. The mechanism may be related to that radix astragali injection can increase the expression of ins‐R, IR‐1, PI3‐K in kidney effectively, and reduce the dysfunction of insulin‐signaling transmission and improve the glucose and lipid metabolism.
Bkca potassium channels and sympathetic transmission in the pulmonary artery from pulmonary hypertensive rats
Authors: Oriowo Mabayoje , El‐Hajj Hanadi
Published: June 2021

Pulmonary hypertension (PH) is a chronic disabling disease that affects the pulmonary vasculature. In this study, we have examined the effect of blocking potassium channels on sympathetic neurotransmission in the pulmonary artery and how this is influenced by PH.

The right ventricular pressure was 30.4±3.5 mmHg and 52.8±5.4 mmHg in control and hypertensive rats respectively. These values were significantly different from each other indicating pulmonary hypertension. Electrical stimulation of the pulmonary artery produced frequency‐dependent contractions with LogES50 values of 1.13±0.18 and 0.44±0.26 in control hypertensive rats, respectively. There was no significant difference in the maximum response between the groups. TEA (1 mM), a BKca channel blocker, produced a nonsignificant increase in maximum response in control rat but produced a significant shift of the frequency‐response curve to the left reducing the LogES50 value from 1.13±0.18 to 0.37±0.32 (n=5). In hypertensive rats TEA (1 mM) shifted the frequency‐response curve to the left and significantly increased the maximum response. It was concluded that BKCA channels modulated adrenergic transmission in the rat pulmonary artery and this was not affected by pulmonary hypertension.
Inhibition of phosphodiesterase‐4 reverses memory deficits produced by beta‐amyloid peptide in rats
Authors: Cheng Yu‐Fang , Wang Chuang , Huang Ying , Xu Jiang‐Ping , Zhang Han‐Ting
Published: June 2021

PDE4, an enzyme that catalyzes hydrolysis of cAMP, is important in mediating memory. However, little is known about its role in memory loss associated with Alzhemer's disease (AD). In the Morris water maze test, microinfusions of beta‐amyloid peptide 25‐35 (Abeta; 10 µg/side) into bilateral CA1 subregions of the hippocampus increased the escape latency during the acquisition trials and decreased the swimming time and distance in the target quadrant in the probe trial test performed 24 h after the last acquisition trial; these were reversed by chronic treatment with rolipram (0.5 mg/kg/d, i.p., 14 d). Consistent with these, in the step‐through passive avoidance test, Abeta infused into CA1 decreased the latency and increased errors entering the dark compartment, which were examined 24 h after initial training; rolipram also reversed the effects of Abeta. Immunoblot analysis revealed a decrease in hippocampal phospho‐CREB induced by Abeta, which was blocked by rolipram. Interestingly, Abeta‐induced memory deficit was reversed by CA1 infusions of lentiviral vectors containing microRNAs that targeted PDE4D4 and PDE4D5, two important PDE4 variants in the hippocampus. Taken together, these results suggest that inhibition of PDE4 reverses memory deficits induced by Abeta; PDE4, in particular its PDE4D4/5 variants, mediates AD‐related memory loss and could be a novel target for treatment of this disorder.
The interaction of delta‐9‐tetrahydrocannabinol (THC) and estradiol during adolescence has long‐term effects on the repeated acquisition and performance of response sequences in female rats
Authors: Sutton Jessie L , Hulst Jerielle L , Winsauer Peter J
Published: June 2021

THC abuse by individuals during adolescence may produce long‐term deficits in complex behavioral processes such as learning. Therefore, the present experiment tested the hypothesis that 40 daily THC injections of 5.6 mg/kg i.p. during adolescence in ovariectomized (+OVX) or sham‐operated (‐OVX) female rats would alter THC's disruptive effects on response rate and accuracy as adults. Following THC administration and training to respond under a multiple schedule with acquisition and performance components, 4 groups received 0.56‐10 mg/kg of THC acutely. With respect to response rate, THC produced similar dose‐dependent effects in both the acquisition and performance components, although +OVX had higher overall baseline rates than ‐OVX. With respect to accuracy, THC's effects differed across components and adolescent treatments. In the acquisition components, +OVX subjects had higher baseline error levels than ‐OVX subjects. The rank order for disruption by THC was ‐OVX/ +THC > +OVX/‐THC > ‐OVX/‐THC = +OVX/+THC. In the performance components, there were no baseline differences between groups and THC had little or no effect on accuracy at any dose. These results indicate that adolescent THC administration in female rats can have long‐term effects on their capacity to learn, and that these effects cannot be readily predicted from the effects of THC on response rate or on performance tasks. Supported by DA 019625.
Acute nicotine and phencyclidine increase locomotor activity of the guinea pig
Authors: Simmons Mark A. , Hudzik Thomas J.
Published: June 2021

While the behavioral effects of nicotine and phencyclidine have been well characterized in rats and mice, substantially less is known about their effects in guinea pigs. Understanding the pharmacology of these compounds has relevance to both addictive behaviors and psychoses. In some circumstances, human pharmacology may be better represented by guinea pigs than by rats or mice. The goal of this study was to determine the utility of the guinea pig as a model for assessing the behavioral actions of nicotine and phencyclidine, drugs that have locomotor stimulant activity in rat and mouse.

Nicotine (1‐3 mg/kg) dose‐dependently increased locomotor activity. The time courses of the responses were also dose‐dependent. Phencyclidine (3‐10 mg/kg) increased locomotor activity with effects that were longer‐lasting than those of nicotine.

Both the magnitude and duration of the increased locomotor activity are less than that seen with mouse or rat models under otherwise similar conditions. Additionally, higher doses of nicotine and phencyclidine are required to produce locomotor stimulation in the guinea pig. These data suggest that, in terms of responsiveness to psychomotor stimulants, the guinea pig is a less sensitive model species than rats or mice.
Characterizing the Acute Cardiovascular Toxicities of Cocaine in Freely Moving Rhesus Monkeys
Authors: Collins Gregory Thomas , Woods James Henry , Ko Mei‐Chuan
Published: June 2021

Cocaine (COC) abuse remains a significant public health problem with an estimated 2.4 million users in the United States alone (SAMHSA, 2007). COC is not only the most commonly reported illicit drug involved in emergency room (ER) visits, but COC‐related visits have increased ~160% from 1995 to 2005, with admissions most commonly attributed to chest pain resulting from increases in blood pressure (BP) and heart rate (HR). These studies were aimed at characterizing the acute cardiovascular (CV) effects of COC in freely moving rhesus monkeys (n=4). Implantable radio‐telemetric probes (DSI; D70‐PCT) were used to collect continuous measures of BP, HR, ECG parameters, body temperature, and locomotor activity for a period of 2 hrs after COC administration (0, 0.1, 0.32, 1.0, and 3.2 mg/kg; iv). BP and HR were the most reliable indicators of COC intoxication, with dose‐dependent increases in BP and HR (magnitude and duration) observed following doses of 0.32‐3.2 mg/kg COC. These changes in CV activity are not only similar to those observed in the ER, but also provide a model to compare the effectiveness of currently used, and novel therapeutics for the treatment of acute COC‐induced CV toxicity.

Supported by NIDA grant DA 023213
Mutant Bacterial Cocaine Esterase with a Three‐day Half‐life Possess Characteristics of a Cocaine Abuse Therapy
Authors: Brim Remy Leigh , Nance Mark , Tesmer John , Sunahara Roger , Woods James
Published: June 2021

Cocaine abuse is a widespread problem in the United States. A pharmacologic treatment for cocaine abuse needs to eliminate the reinforcing properties of cocaine. We have recently shown that a mutant form of bacterial cocaine esterase (CocE) with a t1/2~4.5 hrs @ 37°, is capable of blocking cocaine‐reinforced responding in a rat cocaine self‐administration model. Here we describe a novel mutant form of CocE (L169K/G173Q) with t1/2~75 hrs @ 37°, representing a more realistic abuse treatment candidate. CocE‐L169K/G173Q is more catalytically efficient at cocaine hydrolysis in vitro and more potent than wild type CocE (wt‐CocE) at protecting mice against cocaine‐induced lethality. Importantly, CocE‐L169K/G173Q given to mice 7.5 hours before a lethal dose of cocaine protects 50% of animals from lethality, representing a dramatic improvement over wt‐CocE (~10 min). The duration of CocE‐L169K/G173Q's protective effect does not correlate with the in vitro stability (t1/2), but is more consistent with the plasma half‐life of the enzyme. Overall, we have found that CocE‐L169K/G173Q displays a robust enhancement in enzyme stability at 37°C, and a significant but smaller improvement in vivo. Reducing the discrepancy between the observed in vitro and in vivo effects of CocE‐L169K/G173Q is an area of ongoing research. Research supported by USPHS grants DA021416, GM007767, DA025100, DA013930, and Reckitt Benckiser
Vibration‐induced changes in oxidative stress and expression of inflammatory factors are frequency dependent
Authors: Krajnak Kristine , Waugh Stacey , Miller Roger , Johnson Claud , Kashon Michael
Published: June 2021

Repetitive exposure to vibration through the use of powered hand tools is associated with the development of vibration white finger (VWF), a disorder that is characterized by cold‐induced vasospasms that result in blanching of the skin. The risk of developing VWF appears to be dependent upon the frequency of the exposure. The goal of this study was to assess frequency dependent changes in gene expression and oxidative stress in a rat‐tail model of VWF. Rats (n = 5/group) were exposed to vibration (63, 125, or 250 Hz) or control conditions for 4 h/day for 10 days. After the last exposure, gene expression and oxidative stress were assessed in ventral tail arteries. Transcript levels of pro‐inflammatory factors were increased in arteries of rats exposed to 63 Hz vibration compared to controls. In contrast, oxidative stress (e.g., peroxynitrite) was higher in rats exposed to 125 and 250 Hz vibration. These results demonstrate that the biological responses of peripheral arteries to vibration are frequency dependent. The frequency dependent differences in these responses may affect the risk of developing vibration‐induced vascular dysfunction.
Vascular adaptation in dura and pia mater of ovariectomized pigs employs different molecular mechanisms
Authors: Glinskii Olga , Glinsky Vladislav , Abraha Tsghe , Huxley Virginia
Published: June 2021

Cessation of the ovarian hormone production leads to a reduction in plasma estradiol (E2), and aromatization at extragonadal sites (brain, bones, adipose) becomes the primary sources of E2 production after menopause. Previously, we demonstrated that dura mater (DM) microvascular networks of ovariectomized (OVX) pigs undergo dramatic remodeling, characterized by capillary loss and increased vessel permeability. The analysis of growth factors and receptors as well as downstream signaling pathways revealed that in DM, which depends on plasma E2 supply, the initial E2‐dependent microvessels loss triggers hypoxic responses manifested by an increase in HIF‐1α expression and PDGF/VEGF system activation. Concomitant attenuation of hypoxia‐induced AMPK phosphorylation caused by reduced E2 levels contributes to activation of Akt and mTOR/p70S6K pathways promoting enhanced protein synthesis and new vessel growth. In contrast, in pia mater (PM) microvessels that benefit from local E2 production by the brain, there was no change in HIF‐1α expression or Akt activation following OVX, while estrogen receptor β was upregulated suggesting active signaling through this receptor by the locally produced E2. Our results suggest that post OVX angioadaptation in DM and PM may involve different molecular pathways depending on local E2 availability. Supported by AHA National SDG 0830287N, NIH HL078816, HL075186, RR017353.
Pro‐atherogenic stimuli initiate increased cell‐cell contacts in carotids
Authors: Straub Adam C , Rizzo Michael J , Isakson Brant E
Published: June 2021

Oxidized phospholipids (oxPL) are a significant risk factor for the development of atherosclerosis. Two oxPLs isolated from atherosclerotic plaques are PGPC and POVPC, each of which contain unique biological properties. We performed a transmission electron microscopy study to better understand ultrastructural changes of carotid arteries after oxPL application. For comparison, we also examined carotids from ApoE(‐/‐) mice. C57BL/6 mice were anesthetized and carotid arteries were exposed. Forty micrograms of POVPC or PGPC were applied to the carotid artery for 1, 3, or 6 days. In control carotids, we saw limited myoendothelial junctions (i.e., locations where endothelial and smooth muscle cells make contact), a result consistent with previous observations. However, application of either PGPC or POVPC demonstrated an increased trend in the number of myoendothelial junctions. In addition, POVPC application had an increased number of junctions between smooth muscle cell layers and after PGPC application, formation of smooth muscle cell and peri‐advential cell junctions were observed. These results confirm that carotid arteries undergo ultrastructural changes in both in intimal and advential part of the vessel that appear to indicate increased cell‐cell contact during the initiation stages of atherosclerosis. Supported by NIH 088554 and an AHA SDG.
Cell physiology of fish gill mitochondrion rich cells
Authors: Parks Scott Kenneth , Ryder Jeremy D , Tresguerres Martin , Goss Greg G
Published: June 2021

Regulation of both acid‐base and ion levels are essential for fish survival. Specialized gill mitochondrion‐rich (MR) cells are responsible for the active transport required to maintain homeostasis. We have utilized an intracellular pH (pHi) imaging system to uncover the specific cellular mechanisms responsible for ion‐transport in gill MR cells. We provide important new information on the use of apical Na+ channels coupled with basolateral electrogenic Na+/HCO3‐ co‐transporters acting in export mode to accomplish transepithelial Na+ uptake. In addition, we present evidence for Cl‐/HCO3‐ exchangers (CBE) acting apically for transepithelial Cl‐ uptake on one MR cell population and a second CBE located basolaterally for pHi regulation. We also present evidence for basolateral Cl‐ dependent Na+/H+ exchangers playing a role in pHi regulation. Our data on dopamine and PMA stimulated Na+‐dependent transport processes suggest are the first evidence for direct stimulation of ion transport in MR cells. Our results have culminated in new models for both acid and base secreting cells and their connection to overall Na+ and Cl‐ uptake respectively.

Funded by a NSERC Discovery Grant to GGG.
Proximal and distal Egr‐1 sites mediate GnRH‐responsiveness of the squirrel monkey chorionic gonadotropin β‐subunit promoter in LβT2 cells
Authors: Vasauskas Audrey Anne , Hubler Tina R. , Scammell Jonathan G.
Published: June 2021

In Old World primates, ovulation is under the control of luteinizing hormone (LH) synthesized in the pituitary gland and acting downstream through LH receptors. Chorionic gonadotropin (CG) also acts through LH receptors, but is released from the placenta and is responsible for maintaining pregnancy. Recent studies have shown that the pituitary glands of New World primates, such as squirrel monkey, do not express LHβ, but rather CGβ is the relevant pituitary hormone for ovulation. The role of CGβ released from the pituitary and placenta is different as would be expected of its regulation. Here, we investigated the cis‐acting elements that confer GnRH‐responsiveness to the squirrel monkey CGβ promoter. Several transcription factor binding sites are conserved between the human LHβ and squirrel monkey CGβ promoters: proximal and distal Egr‐1 and SF‐1 sites and a Pitx‐1 site. The response of squirrel monkey CGβ promoter mutation constructs of either the proximal or distal SF‐1 or Egr‐1 sites to stimulation with 100 nM GnRH was tested in LβT2 pituitary gonadotrope cells. Either the proximal or distal Egr‐1 mutations of the squirrel monkey CGβ promoter diminished its GnRH‐responsiveness, whereas the SF‐1 mutations showed no effect. Therefore, it appears that both the proximal and distal Egr‐1 sites contribute to GnRH‐responsiveness of the squirrel monkey CGβ promoter.
Fast and slow Ca2+ transients in intact fast‐twitch skeletal muscle: tissue photometry and 2D confocal detection.
Authors: Payne Anthony M. , Oca Jose , Oliver Scott R. , Clanton Thomas L.
Published: June 2021

We previously reported estimates of changes in intracellular [Ca2+] during contractions of mouse diaphragm (dia) bundles. In this study, we sought a fast‐twitch muscle with flat morphology for similar detection. Whole mouse epitrochlearis (epi), primarily MHC IIb, were mounted horizontally, and loaded with 5 μM Rhod‐2 AM or Fluo‐4 AM. A multi‐fiberoptic epifluorescence probe at 90° to the muscle or a 2D array scanning confocal microscope (∼33 Hz) were used for detection. We observed, 1) the signal to noise was greatly enhanced in epi, 2) like dia, epi demonstrated slow Ca+2 transients following tetany, 3) Mn+2, a quenching agent caused marked reduction in only the fast Ca+2 transients, 4) Ru360, a mito Ca+2 ‐uptake inhibitor, had no effect on the slow Ca+2 transients, 5) Slow Ca+2 transients were reduced in Fluo‐4 compared to Rhod‐2 loaded muscles. 6) Confocal microscopy demonstrated that the slow Ca+2 transients were consistent with mitochondrial Ca+2 . These results demonstrate the feasibility of measuring changes in [Ca+2 ] in intact mammalian fast skeletal muscle and that significant signals arise from mitochondrial sources that, unlike cardiac muscle, are insensitive to Ru360. Supported by NIH 53333
Postnatal PGC‐1α over‐expression improves muscle function in a mouse model of Duchenne muscular dystrophy
Authors: Selsby Joshua T. , Morine Kevin , Pendrak Klara , Tian Z. , Blanco Erica , Barton Elisabeth R , Sweeney H Lee
Published: June 2021

PGC‐1α has received a great deal of attention due to its potential to induce oxidative and slow proteins. The purpose of this investigation was to: 1) extend observations made in an initial study demonstrating transgenic over‐expression of PGC‐1α reduced dystrophic pathology 2) eliminate effects that could have taken place during development. Neonatal mdx mice were injected in the right hind limb with 1x1011 gc of AAV causing over‐expression of PGC‐1α and sacrificed at 4 (n=6) or 6 wks of age (n=5). Muscle mass was reduced in limbs over‐expressing PGC‐1α at 4 and 6 wks, however, tetantic force and specific force in the soleus and EDL were either maintained or improved when compared to control limbs. PGC‐1α over‐expression caused EDLs to be more resistant to damage at 6 wks and the soleus to be more fatigue resistant at 4 and 6 wks compared to control limbs. PGC‐1α increased expression of slow proteins as utrophin was increased nearly 2‐fold and type I myosin heavy chain nearly 3‐fold as well as expression of oxidative proteins as cytochrome C and uncoupling protein‐1 were increased approximately 2‐fold, complex IV subunit IV (cytochrome C oxidase) was increased 1.5‐fold and myoglobin was increased 3‐fold in limbs over‐expressing PGC‐1α compared to control limbs. These data demonstrate the potential therapeutic role of the PGC‐1α pathway for dystrophic skeletal muscle. U54‐AR052646, F32AR055005‐01, AR053461, and PPMD.
Discovery of small‐molecule inhibitors of ROMK: one step closer to a potassium‐sparing loop diuretic?
Authors: Denton Jerod , Lewis L. Michelle , Banerjee Sreedatta , Fallen Katherine , Redha Rey , Lornsen Katharina A. , Weaver C. David
Published: June 2021

The renal inward rectifying potassium (Kir) channel ROMK plays key roles in regulating systemic electrolyte balance and blood pressure. Because ROMK supports both NaCl reabsorption in the thick ascending limb and potassium secretion in the collecting duct of the nephron, a selective inhibitor of the channel could in principle function as the first potassium‐sparing loop diuretic. At present, however, it is not possible to test this hypothesis due to the lack of suitable channel inhibitors. We therefore developed and implemented a novel fluorescence‐based assay to enable high‐throughput small‐molecule library screening. The assay reports flux of the potassium congener thallium (Tl+) through ROMK channels using the Tl+‐sensitive fluorescent dye FluoZin‐2. We show that the assay is robust, DMSO tolerant and enables screening of more than 20,000 compounds per day for ROMK modulators. From a screen of approximately 200,000 small molecules, several new ROMK inhibitors were discovered. We are currently assessing structure‐activity relationships, potency, selectivity and molecular mechanism of action of these compounds.
Differential desensitization of Na coupled transporters with chronic parathyroid hormone (PTH) stimulation
Authors: Lederer Eleanor DeLand , Holthouser Kristine , Lesousky Nina , Yared Nicholas , Khundmiri Syed Jalal
Published: June 2021

PTH regulates renal proximal tubule apical (BBM) and basolateral membrane (BLM) transporters including Npt2a, Na+, K+ ATPase (Na‐K), sodium hydrogen exchangers 1 and 3 (NHE1, NHE3). We have shown that the acute inhibitory effect of PTH on proximal tubule Na‐K activity disappears with chronic PTH treatment, but the inhibitory effect on Npt2a activity persists. We hypothesize that chronic PTH results in desensitization of PTH actions on Na but not phosphate transport. To address this hypothesis, we compared the effect of acute and chronic PTH stimulation on expression and function of transport proteins and signaling pathways in Sprague Dawley rat kidney and in OK cells. Treatment with PTH (5 µg/kg) for 6 h decreased Na‐K activity and Na‐K, NHE1, and NHE3 expression in rat BLM, while chronic treatment with PTH (4 days) increased Na‐K‐activity and expression above control levels. BBM Npt2a expression significantly decreased with acute and chronic PTH treatment. PTH did not affect BLM or BBM PTH receptor expression, while BLM expression of NHERF1 significantly increased. Similar findings were seen in OK cells. After chronic PTH, selective stimulation of OK cell BBM with PTH did not stimulate additional PKA and PKC activity, while stimulation of BLM significantly increased PKC activity. BBM or BLM stimulation with 8‐bromo‐cAMP resulted in further PKA activation; stimulation with phorbol myristate acetate resulted in further PKC activation. We conclude that the effects of PTH on Na transport undergo desensitization at a post‐receptor level while the effects of PTH on phosphate transport do not desensitize. The role of differential NHERF1 regulation in modulating PTH receptor‐transporter signaling is unknown. Support from VAMR.
Estimation of Acid‐Base and NH+4 Transport Parameters in a TAL Cell Model Using Inverse Methods
Authors: Nieves‐Gonzalez Aniel , Marcano Mariano , Clausen Chris , Layton Harold E. , Moore Leon C.
Published: June 2021

A mathematical model of a medullary thick ascending limb (mTAL) cell was used to study cytosolic pH (pHi) responses to luminal NH4+ perturbations. TAL cells load NH4+ via apical NH4+‐permeable channels and the NKCC2 cotransporter, resulting in a rapid fall in pHi. Our goal was to use our model and published NH4+ perturbation data to estimate parameters associated with pHi regulation using an inverse method. The TAL cell model, which includes NH+4 binding by NKCC2 and NHE, also includes models for Na+,K+‐ATPase, the Cl−‐HCO3− exchanger, KCC1, and trans‐ and paracellular electrodiffusion. A least‐squares problem was formulated and solved to fit the model to the data. First, steady‐state short‐circuit current data was used to estimate parameters important in Na+ transport; this yielded reasonable cytosolic concentrations, membrane PD's and resistances. The model was then fit to time records of NH4+ perturbations, with and without inhibition of NKCC2 or K+ channels, to estimate parameters associated with NH4+ transport. The results show that our TAL cell model, when used with parameter sets obtained from optimization methods, exhibits behaviors consistent with a wide variety of experimental results, including NH4+ perturbations, in isolated perfused TAL segments. This provides validation of the TAL model, which we intend to use in a multi‐cell model of the TAL segment.
The inhibitory effect of WNK4 on ENaC is mediated by Nedd4‐2
Authors: Yu Ling , Cai Hui , Wang Dexuan , Otor Al‐Khalili , Eaton Douglas C.
Published: June 2021

The epithelial sodium channel (ENaC) plays an important role in maintaining salt homeostasis. A serine‐threonine protein kinase, WNK4, is expressed in the distal nephron, where it participates in the regulation of Na+, Cl‐, and K+ homeostasis. The effect of WNK4 on ENaC activity has been tested in Xenopus oocytes, but its effect on native ENaC is not clear. We investigated the effect of WNK4 on ENaC activity in A6 cells by patch‐clamp single channel recording. Our results revealed that the ENaC channel activity (NPo) in WNK4 over‐expressing A6 cells is about 60% of that in the control empty vector transfected cells. By analyzing the channel number (N) and open probability (Po), we found that the reduction in channel NPo is due to decrease in channel N rather than in channel Po. We also examined the co‐regulation of WNK4 and Nedd4‐2 on ENaC activity. Channel N in Nedd4‐2 over expressing cells is significantly reduced as expected; however, WNK4 expression shows no additive effect. In addition, the inhibitory effect of WNK4 on ENaC is reversed when it is co‐expressed with Nedd4‐2 dominant negative mutant. Although both WNK4 and Nedd4‐2 decrease the density of apical channels, neither regulator has an effect on ENaC Po. In conclusion, WNK4 reduces ENaC activity by decreasing its surface expression and this inhibitory effect is apparently due to a Nedd4‐2 mediated mechanism.
Disruption of Guanylyl Cyclase‐A/Natriuretic Peptide Receptor‐A Gene Leads to an Increased Synthesis of Pro‐inflammatory Cytokines and Fibrosis in the Kidney.
Authors: Das Subhankar , Krazit Stephen T. , Pandey Kailash N.
Published: June 2021

The objective of the present study was to evaluate the effect of Npr1 (coding for Guanylyl Cyclase‐A/Natriuretic Peptide Receptor‐A; GC‐A/NPRA) gene‐disruption in the development of renal hypertrophy and fibrosis. Blood pressure measurement and plasma cytokine assays were performed in 16‐weeks old mice having different copies of Npr1 gene (0‐copy, 1‐copy and 2‐copy). The 0‐copy mice showed 35‐45 mmHg higher systolic blood pressure (SBP) as compared with 2‐copy mice. A significant increase in plasma levels of tumor necrosis factor (TNF‐α; 5‐fold) and interleukin‐6 (IL‐6; 2.5‐fold) were observed in 0‐copy mice as compared with 2‐copy mice. Moreover, protein analysis also showed a significant increased expression levels of transforming growth factor (TGF‐β) and reduced expression of matrix metalloproteinases (MMP's) by Western blot analysis in 0‐copy mice as compared with 2‐copy wild‐type mice. Renal collagen content was significantly increased by 2‐fold in 0‐copy mice and was confirmed by histological study with masson's trichrome staining of kidney tissues. The increased levels of TNF‐α, IL‐6, and TGF‐β as well as a reduced levels of MMP's in 0‐copy mice indicated the ensuing process of renal fibrosis in these animals. Thus, the present results suggest that the absence of ANP/NPRA signaling exerts a stimulatory effect to cause significant increase in pro‐inflammatory cytokines to produce hypertrophy and fibrosis in the kidney.
Methyl gallate attenuate cadmium‐induced cytotoxicity by heme oxygenase‐1 in cultured mouse mesangial cells
Authors: Cha Seok Ho , Lee Woon Kyu , Suh Chang Kook
Published: June 2021

The purpose of this study is to investigate the protective effect of methyl gallate (MG), a phytochemical, on cadmium (Cd)‐induced renal toxicity related by heme oxygenase‐1 (HO‐1) expression using cultured mouse mesangial cell line (MMC).

By employing RT‐PCR and Western blotting techniques, we have examined the HO‐1 induction in MMCs that were treated with Cd or Methyl gallate. By employing a cell viability assay, we have also examined the cytoprotective effect of HO‐1 induction against the cytotoxicity caused by toxic dose of Cd. In MMCs exposed to Cd and methyl gallate, expression of HO‐1 (mRNA and protein) was increased in a dose‐ and time‐dependent manner. The up‐regulation of HO‐1 mRNA and protein expressions by Cd and methyl gallate was inhibited by a pre‐incubation of the cells with actinomycin D, an inhibitor of mRNA transcription. Upon the inhibition of elevated HO‐1 mRNA and protein expressions by the use of zinc protoporphyrin IX (ZnPP), an inhibitor of HO‐1 activity, the change of HO‐1 mRNA and protein expressions by ZnPP was not observed. The decreased viability of the cells by Cd was partially recovered by the treatment with methyl gallate. These cytotoxic and cytoprotective effect was dramatically decreased by the treatment with ZnPP.

From these results, methyl gallate have cytoprotective effect that related with heme oxygenase‐1 activity.
Amiloride normalizes arterial pressure in Cyp1a1ren‐2 transgenic rats without significant effects on small artery function
Authors: Grisk Olaf , Rohsius Robert , Schmid Catherina , Schlüter Torsten , Rettig Rainer
Published: June 2021

Activation of the vascular and renal epithelial sodium channel (ENaC) may contribute to arterial hypertension. Cyp1a1ren‐2 transgenic rats carry a renin transgene the expression of which can be induced by feeding an inductor. During transgene induction, these rats show high plasma aldosterone and tissue angiotensin II levels. We tested if hypertension is sensitive to amiloride in these rats and if transgen activation causes amiloride‐sensitive alterations in small arteries that may contribute to hypertension. Cyp1a1ren‐2 transgenic rats (n = 6‐8 per group) were randomized to control (ctr), three weeks transgene induction (ind) and three‐weeks transgene induction + amiloride (amil) during the last week. At the end of the experiment, mean arterial pressure (MAP) was 118 ± 2 (ctr); 153 ± 8 (ind) and 118 ± 2 mmHg (amil). With MAP as effective transmural pressure, third order mesenteric and renal arteries did not show differences in lumen diameter. There was no difference in vascular compliance, α1‐adrenoceptor‐ or depolarisation‐induced vasoconstriction and endothelium‐dependent vasodilation. Amiloride‐induced arterial pressure reduction in transgen‐induced rats was accompanied by increased renal sodium excretion. We conclude that amiloride normalizes arterial pressure in Cyp1a1ren2 transgenic rats likely by stimulating renal sodium excretion but without appreciable effects on small artery function.
Contrasting roles of ETA and ETB receptors in angiotensin II‐high salt diet‐induced hypertension
Authors: Boesen Erika , Pollock Jennifer , Pollock David
Published: June 2021

Endothelin (ET) receptor blockade reduces blood pressure and renal injury in angiotensin II(AngII)‐induced hypertension when treatment is initiated at the commencement of AngII infusion. This study tested if ET receptor blockade is also beneficial if treatment is initiated after the onset of hypertension. Male rats received either normal salt diet throughout the experiment (Control), 14‐day AngII infusion (65ng/min s.c.) plus 8% NaCl diet (AngII‐HS) or AngII‐HS plus the ETA receptor antagonist ABT‐627 commencing on day 8 of AngII‐HS treatment (AngII‐HS+ABT). Robust hypertension was observed in AngII‐HS and AngII‐HS+ABT groups by day 7 (mean arterial pressure, MAP 170±5 and 165±5mmHg). Further increase in hypertension was significantly attenuated by treatment with ABT‐627 from day 8 (MAP 168±5 and 191±3mmHg at day 13 in AngII‐HS+ABT and AngII‐HS, P<0.001). Urine was collected for 24 h at day 14. Treatment with ABT‐627 did not blunt the significant increases in urinary protein (19‐fold vs control), albumin (25‐fold) or MCP‐1 excretion (6 to 8‐fold) induced by AngII‐HS treatment. To determine the effect of ETB receptor blockade during AngII‐HS treatment, separate groups of rats were treated with AngII‐HS alone or with the ETB receptor antagonist A‐192621 commencing on day 8 of AngII‐HS treatment. A‐192621 significantly (P<0.05) enhanced AngII‐HS‐induced hypertension by ~14 mmHg over the final 4 days of AngII‐HS treatment but did not affect urinary protein or albumin excretion at day 14. Thus, our data indicate a role for the ETA receptor in the progression of hypertension in this model. However, ETA receptor blockade after hypertension had developed was insufficient to attenuate renal injury, at least over the timeframe studied. ETB receptor blockade exacerbates AngII‐HS hypertension, suggesting that ETB receptor blockade should be avoided in treating hypertension.
Simultaneous administration of Ang(1‐7) or A‐779 does not affect the chronic hypertensive effects of AngII in normal rats
Authors: Collister John P , Nahey David B
Published: June 2021

Despite the well described actions of Ang(1‐7) and its potential role in opposing the actions of angiotensin II (AngII), there have been relatively few studies examining the role of Ang(1‐7) in a chronic setting. It is well established that Ang(1‐7) plays a protective role in preventing deleterious chronic effects of AngII in the heart, but little is known of its role in modulating the chronic hypertensive effects of AngII. The current study was designed to test the hypothesis that simultaneous administration of either Ang(1‐7) or its antagonist A779 would attenuate or exacerbate the chronic hypertensive effects of AngII, respectively. To test this hypothesis, Sprague‐Dawley rats were instrumented with venous catheters and radio telemetric pressure transducers and begun on a normal (0.4%) NaCl diet. Arterial pressure responses were measured in rats treated with AngII (10 ng/kg/min, n=9) and compared with those treated simultaneously with AngII and Ang(1‐7) (24 µg/kg/hr, n=8), or the Ang(1‐7) antagonist A‐779 (24 µg/kg/hr, n=7) for 8 days. Mean arterial pressure rose significantly and similarly in all 3 groups of rats, such that by day 8 of AngII infusion, pressures had risen approximately 25‐30 mmHg in all rats. These results do not support the hypothesis that the chronic hypertensive effects of AngII in normal rats are altered by co‐administration of either Ang(1‐7) or its antagonist, A‐779.
Age dependent expression of angiotensin II receptors in the ovine kidney
Authors: Vinturache Angela E , Qi Wei , Smith Francine G
Published: June 2021

Previously we showed in conscious lambs that angiotensin II (ANG II) modulates renal haemoydnamics in an age‐dependent manner through activation of type 1 (AT1), but not type 2 (AT2) receptors. The present study was designed to assess the transcriptional expression (mRNA) of AT1 and AT2 receptors during post natal maturation in the ovine kidney. Kidney tissue was obtained from lambs aged 24 h, 1, 3, 6 and 12 weeks (n=3 for each age group) for measurement of AT1 and AT2 receptor mRNA expression. Corresponding cortical and medullary samples were obtained from the same location of kidney and frozen in liquid N2. Following RNA isolation from tissues, mRNA levels were assessed using RT‐PCR and validated sheep specific primers; data were normalized to β‐actin. At 24 h to 6 weeks, the expression of AT1 was higher in the medulla; at 12 weeks it was higher in the cortex. In contrast, AT2 expression was higher in the cortex than medulla for all age groups. At 6 to 12 weeks, AT2 expression appeared enhanced in the cortex and was decreased at 12 weeks in the medulla. This study provides evidence that the expression of ANG II receptors is altered with post natal age in the ovine kidney. These changes in intrarenal AT1 and AT2 receptor expression may help to explain age dependent physiological effects of ANG II on renal haemodynamics and function.

Dr. Vinturache and Dr. Qi are equal first authors.

Supported by the Canadian Institutes for Health Research.
Spinal 5‐HT7 Receptor Agonist Induced Phrenic Motor Facilitation Requires TrkB Signaling
Authors: Hoffman Michael S. , Mitchell Gordon S.
Published: June 2021

Spinal activation of Gs‐protein coupled 5‐HT7 receptors with AS‐19 (selective agonist) elicits long‐lasting phrenic motor facilitation (PMF). Other Gs‐coupled receptors (eg. adenosine A2A) induce PMF via trans‐activation of TrkB, the high affinity BDNF receptor, activating AKT but not ERK MAP kinases (Golder et al., 2008). Since AKT is a "downstream" signaling molecule from TrkB, AKT activation may underlie PMF. We hypothesized that AS‐19 elicits PMF by similar mechanisms to A2A‐induced PMF, and requires TrkB and PI3 Kinase/AKT (not ERK) activation. In anesthetized, vagotomized and ventilated Sprague Dawley rats, intrathecal (C4) injections of a tyrosine kinase (k252a, 100 μM, 15 μl) or PI3 Kinase inhibitor (PI‐828, 180 μM, 15 μl) were made prior to episodic AS‐19 injections (10 μM, 5 μl; 3x5min). AS‐19‐induced PMF (80±11% baseline, 120 min post‐injection) was inhibited by k252a (30±13%; n=5) and PI‐828 (7±5%, n=4)(both p<0.001), which were not different from vehicle controls (14±10%, n=5). Although ERK inhibition (UO126, 100 μM; 15 μl, n=4) blocked early PMF (60min: 13±13%, p<0.05), late phase PMF was unaffected (120 min: 53±7%, nsd). Thus, 5‐HT7 receptor‐induced PMF requires TrkB and AKT activity, whereas ERK activity may be necessary only in early stages of the response. An understanding of PMF may guide development of novel strategies to treat ventilatory control disorders. (NIH HL80209 and F31 HL092785)
Differential carotid baroreflex control of arterial blood pressure in young women and men at rest and during dynamic exercise
Authors: Kim Areum , Young Colin N. , Deo Shekhar H. , Fisher James P. , Fadel Paul J.
Published: June 2021

Arterial baroreceptors play a pivotal role in the rapid reflex adjustments that accompany acute cardiovascular stressors. Previous studies have demonstrated that the arterial blood pressure (BP) response of young women to cardiovascular stressors, such as exercise, was reduced compared to men. However, whether alterations in arterial baroreflex function contribute to sex differences in BP control remains unclear. To address this, 5s pulses of neck suction (NS, ‐60 Torr) and neck pressure (NP, +40 Torr) were applied to load and unload the carotid baroreflex (CBR), respectively at rest and during steady‐state cycling at 50% heart rate reserve in nine young women (22±1 yr) and nine men (22±1 yr). At rest, mean BP responses to NS were significantly greater in women (Δ‐16±1 women vs. Δ‐11±2 men mmHg; P=0.03), whereas responses to NP were similar between groups. Interestingly, exercise eliminated the group differences in mean BP responses to NS (Δ‐11±2 women vs. Δ‐11±2 men mmHg; P=0.98) however; responses to NP were significantly greater in women during exercise. These preliminary findings suggest that sex differences in CBR control of BP exist at rest and during exercise. In comparison to men, young women appear to be better able to defend against hypertensive challenges at rest, whereas during exercise women exhibit augmented CBR‐mediated BP responses to hypotension.

Supported by R01HL093167.
SB220025 a p38 inhibitor mediates neuroprotection by inhibiting transcriptional activation and apoptosis in focal cerebral ischemia
Authors: Sharma Shyam S , Thiyagarajan Meenakshisundaram
Published: June 2021

In the present study [5‐(2‐amino‐4‐pyrimidinyl)‐4‐(fluorophenyl)‐1‐(4piperidinyl)imidazole)] (SB220025) a p38 inhibitor was evaluated for its neuroprotective efficacy in transient focal cerebral ischemia (FCI) model. FCI was induced in rat by occluding middle cerebral artery for 2 h followed by 70 h of reperfusion. IR insult resulted in a prominent infarction (188.5±25.7 mm3) and edema (115.1±28.1 mm3) in the ipsilateral hemisphere of brain. Severe motor dysfunction was also observed in ischemic rats (median cumulative deficit score 9.0±1.8). The temporal pattern of p38 activation was found to be biphasic in nature. IR insult also resulted in an intense NFκB binding and apoptosis mediated neuronal loss. Intravenous administration of SB220025 (0.5 and 1 mg/kg) reduced the extent of infarct volume. SB220025 (1 mg/kg) treatment resulted in a statistically significant improvement in neurological score. Treatment with SB220025 (1 mg/kg) also resulted in a statistically significant decrease in TNFα abundance, number of TUNEL positive cells. These results demonstrate that SB220025 is an efficacious neuroprotective agent which mediates its effect at least in part by inhibiting TNFα abundance and apoptosis mediated neuronal loss.
DEGRADATION OF CYSTATIN C BY MATRIX METALLOPROTEINASES‐2 AND ‐9 AND ITS INHIBITION BY DOXYCYCLINE OR SELENIUM
Authors: KOKSOY ASLIHAN
Published: June 2021

Matrix metalloproteinases (MMPs) are proteinases with a wide range of intra and extracellular targets. MMP‐2 and ‐9 are the major MMPs in the circulation which can get activated by oxidants. Cystatin C (CysC), is a very potent inhibitor cysteine proteinases. Decreased CysC levels and increased activity of MMPs are commonly observed in diseases involving chronic oxidative and inflammatory stress such as arteriosclerosis, abdominal aortic aneurisms and multiple sclerosis. In our study we hypothesized that CysC could be a substrate for these enzymes as an explanation for the observed reduction in CysC levels in these diseases. CysC has a common cleavage motif that can be recognized by both MMP‐2 and 9. Cleavage of CysC at this site have been shown to result in amyloid formation in neural tissue. We found that CysC is degraded by both MMPs ‐2 and ‐9, by 71±6% and 78±1% respectively, after 4 hours of incubation. Pre‐treatment of the cells with selenium (an essential biological trace element) or doxycyclin (an MMP inhibitor) prevented MMP‐induced degradations, significantly. Our preliminary data suggest interaction between CysC and MMPs can be modulated with reactive oxygen species and provide grounds for a new hypothesis for explaining amyloid formation associated with oxidant stress related pathologies.
Oxidative stress induced translocation of Prdx6 to the plasma membrane in lung epithelial cells A549
Authors: KAZI ALTAF S , CHOWDHURY IBRUL , DODIA CHANDRA , FEINSTEIN SHELDON , FISHER ARON
Published: June 2021

Peroxiredoxin 6 (Prdx6), a bifunctional 25‐kDa protein with both peroxidase and phospholipase A2 (PLA2) activities, is expressed in all major organs with particularly high levels in the lung. Prdx6 can reduce phosphatidylcholine hydroperoxides with a high rate constant similar to H2O2 (~106M−1s−1). In vitro, recombinant Prdx6 binds only to oxidized phospholipid substrate at pH 7.0 but not to reduced substrate. Consistent with its antioxidant functions and preferential binding to oxidized phospholipids we hypothesized that Prdx6 can repair oxidized membrane without hydrolyzing reduced phospholipids. Plasma membranes (PM) preparations from A549 cells treated with either H2O2 (400μM) or t‐butyl hydroperoxide (800μM) for 6 hr showed translocation of Prdx6 as compared to PMs from control untreated cells. H2O2 treated A549 cells showed a 3.8 and 3.6 fold increase in DPPP fluorescence and TBARS, respectively, indicating lipid peroxidation. H2O2 induced enrichment of Prdx6 in PM, DPPP fluorescence, and TBARS values returned to basal levels when cells were either allowed to recover for 6 hr post H2O2 treatment or pretreated with catalase (100U/ml). In conclusion, using intact lung epithelial cells, we establish a mechanism by which Prdx6 mediates reduction of oxidized phospholipids such as those present in the plasma membrane of cells exposed to oxidative stress.