bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective attenuation of carotid‐cardiac responses to hypertension at the onset of static handgrip in humans
Authors: Deo Shekhar H , Young Colin N. , Fairfax Seth T. , Fadel Paul J.
Published: June 2021

Previous studies have indicated that at the onset of exercise cardiac baroreflex function is reduced in an intensity‐dependent manner, which appears to be mediated by a blunted ability to buffer hypertensive challenges. However, whether cardiac baroreflex responses to a hypotensive stimulus are altered at exercise onset is unclear. To examine this, ten subjects (25±1 years) performed multiple 1‐min bouts of static handgrip (HG) at 15 and 60% of maximal voluntary contraction (MVC), while breathing to a metronome set at eupneic frequency. Neck pressure (NP +40 Torr) or neck suction (NS −60 Torr) was applied for 5s at end expiration, to simulate carotid baroreflex (CBR) hypotension and hypertension, respectively at rest, at the onset of HG (<1 s), and at ~40 s of HG. Cardiac responses to NP at the onset of exercise were maintained at both HG intensities, whereas heart rate responses to NS were blunted at exercise onset during 60% MVC HG (−16±2 rest vs. −9±3 bpm 60% MVC; P<0.05) but preserved at 15% MVC. The response range of the carotid‐cardiac baroreflex at the onset of 60% MVC HG was reduced (25±1 rest vs. 18±3 bpm 60% MVC; P<0.05) compared to rest and 15% MVC. Heart rate responses to NP and NS applied at ~40s of 15 and 60% MVC HG were similar to rest. These results suggest that carotid‐cardiac responses to hypertension are selectively contributing to an impaired CBR control of heart rate at the onset of static HG in humans.

Support: NIH R01HL093167
Neural circuits underlying the inhibition of sympathetically‐mediated thermogenesis in brown adipose tissue (BAT) induced by atypical antipsychotic drugs
Authors: Madden Christopher J.
Published: June 2021

Administration of atypical antipsychotic drugs suppresses sympathetic activation of metabolism in BAT, which may contribute to the weight gain observed with maintenance on these medications. This study investigated the neural circuitry responsible for the antipsychotic drug‐evoked inhibition of the sympathetic outflow to BAT. In urethane/chloralose anesthetized rats, the increase in BAT sympathetic nerve activity (SNA) evoked by cooling the core temperature to ~35.5°C was completely inhibited by the atypical antipsychotic drug, Clozapine (Cloz; 3mg/kg, iv). Bilateral microinjection of muscimol (2mM, 120nl/side) into the ventrolateral medulla (VLM) reversed or prevented the Cloz‐evoked inhibition of BAT SNA. In contrast to the effect of Cloz on the cooling response, the increase in BAT SNA evoked by blockade of GABAA receptors within the rostral raphe pallidus (RPa) was not inhibited by Cloz (3–6mg/kg, iv). These results suggest that neurons within the VLM play a role in the inhibition of BAT SNA evoked by atypical antipsychotic drugs, possibly by activating a GABAergic input to the RPa. Supported by NIH grant DK082558 and an American Heart Association SDG.
Spinally‐projecting C1 neurons are not responsible for increased arterial pressure after nasal stimulation
Authors: McCulloch Paul , DiNovo Karyn
Published: June 2021

Nasal stimulation (NS) can produce a sympathetically‐mediated increase in arterial blood pressure (BPa). Our objective was to determine if the increased BPa seen after NS occurs through activation of spinally‐projecting C1 RVLM neurons. Using Fos (a marker for activated neurons) and PNMT (a marker for C1 neurons), we found that NS activates both C1 and non‐C1 RVLM neurons. Only 7–24% of activated RVLM neurons were C1 neurons. Next we used Fos and PNMT immunohistochemistry with Fluorogold injections into the T3 IML to identify spinally‐projecting neurons activated by NS. Of all spinally‐projecting RVLM neurons, 10% were activated non‐C1 neurons, while 4% were activated C1 neurons. Of all activated RVLM neurons, 12% were non‐C1 spinally‐projecting neurons, while 4% were C1 spinally‐projecting neurons. Of all C1 neurons, 9% were activated and spinally‐projecting. Only 2% of all RVLM neurons were spinally‐projecting C1 neurons activated by NS. Finally, anti‐DβH‐saporin was injected into the T3 IML to selectively kill spinally‐projecting catecholaminergic neurons. In 3 of 12 rats, 77% of the spinally‐projecting C1 neurons within the RVLM were selectively killed. We found that successful ablation of spinally‐projecting C1 neurons did not eliminate the cardiovascular responses to NS. We therefore conclude that spinally‐projecting C1 neurons are not responsible for the increased BPa seen after NS.
Angiotensin‐(1–7) increases neuronal potassium current via a nitric oxide‐dependent mechanism
Authors: Yang Rui‐Fang , Yin Jing‐Xiang , Li Yu‐Long , Zimmerman Matthew C. , Schultz Harold D.
Published: June 2021

Angiotensin 1–7 [Ang‐(1–7)] signaling in the brain may play an important protective role in cardiovascular diseases by decreasing sympathetic output. It is generally well‐accepted that nitric oxide (NO•) in the brain functions as a sympatho‐inhibitory molecule. We hypothesized that Ang‐(1–7) increases NO• in neurons and modulates neuronal activity. We tested this hypothesis in catecholaminergic CATH.a neurons. Protein analysis revealed these neurons express the Ang‐(1–7) receptor (MasR) and three isoforms of NO• synthase (nNOS, eNOS, iNOS). Expression of MasR, nNOS, and eNOS protein markedly increased in CATH.a neurons following six days of differentiation with N6,2′‐O‐Dibutyryladenosine 3′,5′‐cyclic monophosphate sodium salt (dbcAMP, 1 mM). As measured by DAF‐FM fluorescence and confocal microscopy, Ang‐(1–7) (100 nM) increased NO• levels in differentiated CATH.a neurons (31 ± 3%; P<0.05 vs. baseline) and this response was attenuated (P<0.05) by the MasR antagonist A779 (5 ± 3%), non‐specific NOS inhibitor (L‐NAME; −5 ± 1%), and nNOS inhibitor (SMTC; −5 ± 7%). Using the whole‐cell patch‐clamp technique, we observed that Ang‐(1–7) increased outward K+ current (IKv) in differentiated CATH.a neurons (53 ± 6% increase; P<0.05 vs. vehicle). These findings indicate that Ang‐(1–7) increases nNOS‐dependent NO• levels via MasR activation and elevates IKv in differentiated CATH.a neurons. NIH P01HL062222
Diet‐Induced Obesity Causes Selective Insulin Resistance
Authors: Bardgett Megan , Stocker Sean D
Published: June 2021

Peripheral insulin resistance is a hallmark characteristic of the metabolic syndrome and obesity. However, whether obesity causes insulin resistance in brain is not known. To test this hypothesis, male Sprague‐Dawley rats (200–250g) were fed a moderately high‐fat diet (32% kcal as fat) or a low fat (LF) diet (10.6% kcal as fat). After 13 weeks, the body weights of rats fed the moderately high‐fat diet segregated into obesity‐prone (OP, 793±13 g) and obesity‐resistant (OR, 596±8 g) groups. The control group consisted of young adult rats (292±22 g, n=4) or rats fed LF (612±15 g, n=4). Rats were anesthetized with α‐chloralose and infused with insulin (3.75 mU/kg/min) and 50% dextrose (.25–.75 ml/hr) for 120 min. Baseline plasma insulin levels were significantly elevated in OP compared to OR and control rats (OP: 16±2 vs OR: 8±1 and control: 11±1 ng/ml, p<0.05). As expected, glucose utilization was significantly reduced in OP rats (OP: 3.3±0.7 vs OR: 5.5±0.9 and control: 7.5±0.8 mg/kg/min, p<0.05). Interestingly, hyperinsulinemia produced similar increases in lumbar sympathetic nerve activity across groups (OP: 127±6%; OR: 128±9%, control: 133±10% P>0.05). Arterial blood pressure decreased significantly by 120 min in all groups (OP: 94±4 vs 84±3 mmHg; OR: 94±7 vs 87±6 mmHg, control: 100±7 vs 85±6, P<0.05). These findings indicate that obesity causes peripheral but not central insulin resistance.

Supported by NIH HL090826, AHA 0630202N & 0815372D
Topical application of Ibunex suppresses hyperalgesia and allodynia and inhibits sensory neuron hyperexcitability and upregulation of Nav1.7 and Nav1.8 in rats with intervertebral foramen inflammation
Authors: Song Xue‐Jun , Huang Zhi‐Jiang , Natividad Robert , Li Hao‐Chuan , Rupert Ronald L
Published: June 2021

There is lack of evidence that topical application of an anti‐inflammatory reagent on the surface of local skin can alleviate intervertebral foramen (IVF) inflammation‐induced pain. We investigated effects and neural mechanisms of topical application of an anti‐inflammatory cream Ibunex on behavioral pain, dorsal root ganglion (DRG) neuron hyperexcitability, alteration of the sodium channels and the associated pathological changes following IVF inflammation. The results showed that repetitive, topical application of Ibunex significantly reduced the severity and shortened duration of IVF inflammation‐induced thermal hyperalgesia and mechanical allodynia, and suppressed DRG neuron hyperexcitability. Western blotting analysis showed that the inflammation‐induced significant upregulation of Nav1.7 and Nav1.8 was reduced by Ibunex treatment. Further, pathological manifestations of the inflamed DRG such as the increased vascularization and satellitosis were markedly alleviated 2–3 weeks after treatment. These results provide evidence that an anti‐inflammatory reagent can be used topically to alleviate IVF inflammation‐induced pain. Such analgesic effect may be produced by inhibition of sensory neuron hypersensitivity and the pain‐related sodium channels. This study suggests a convenient and safe intervention for treating pain due to IVF inflammation and probably similar syndromes in clinic. Support by Parker Research Foundation and Core Products Laboratories, Inc.
Effect of hindlimb suspension on hippocampal neurogenesis in weaned rats
Authors: Nomura Sachiko , Kami Katsuya , Kawano Fuminori , Oke Yoshihiko , Ohira Takashi , Fujita Ryo , Nakai Naoya , Ohira Yoshinobu
Published: June 2021

Effects of inhibited antigravity activity of hindlimb muscles on the hippocampal neurogenesis were studied in newly weaned rats, using the hindlimb suspension (HS) model. Effects of ambulation recovery were also studied. Brain was fixed by infusion of phosphate buffer with 4% paraformaldehyde in the anesthetized rat and cross‐sectional samples, around the dentate gyrus (DG) of the hippocampus, were analyzed immunohistochemically. Proliferating cell nuclear antigen (PCNA) and doublecortin (DCX) were used as markers for mitotic cell and immature neuron, respectively. Both PCNA+ and DCX+ cells in DG decreased gradually following growth in unsuspended control rats. Decrease of DCX+ cells was further stimulated significantly by 14 days of HS, although the number of PCNA+ cells in the suspended rats was the same as controls. After 14 days of recovery, however, the number of DCX+ cells of the suspended rats returned to the control levels. These results suggest that HS transiently impaired hippocampal neurogenesis, especially in the differentiation of new‐born neurons. This study was supported by Grant‐in‐Aid for Scientific Research (19100009, 21700654) from Japan Society for the Promotion of Science.
DNA methyltransferase expression in sensitized and non‐sensitized mice to stimulant effect of ethanol
Authors: Goeldner Francine O , D'Almeida Vania , Formigoni Maria Lucia OS
Published: June 2021

Recent studies indicated that DNA methylation in patients with alcoholism is increased. Since the development of behavioral sensitization to ethanol does not occur uniformly, we investigated the expression of DNA methyltransferases (DNMTs) in the medial pre‐frontal cortex (mPFC), nucleus accumbens, amygdala, and ventral tegmental area (VTA) of the mice with different profiles of locomotor activity after chronic ethanol administration. Male Swiss mice received 2.2 g/kg ethanol or saline intraperitoneally daily for 21 days, were tested weekly for locomotor activity. Ethanol‐treated mice were classified into sensitized and non‐sensitized groups (n=10) according to their locomotor activity during treatment. Real time PCR assays were used to measure levels of mRNAs encoding the gene of enzymes in the tissues. In the mPFC, non‐sensitized mice presented lower levels of DNMT3a gene expression than sensitized and control groups. Moreover, in the VTA sensitized mice presented lower levels of DNMT3a gene expression than non‐sensitized and control groups. Since methylation of DNA is an important epigenetic factor in regulation of gene expression these findings may have important implications for development of behavioral sensitization to ethanol in mice. Financial support: FAPESP, CNPq and AFIP.
Environmental Enrichment Does Not Impact the Emergence of Repetitive Behavior in the C58/J Inbred Strain, A Novel Mouse Model of Autistic‐Like Behavior
Authors: Hess Christina , Johnson Ashley , Wahby Hady , Yano Hiroshi , Ryan Bryce
Published: June 2021

Autism has three core symptoms: restricted, repetitive behaviors, deficits in communication, and abnormal social interactions. The causes of autism are unknown, and no established animal model currently exists. Past research indicates that the inbred C58/J mouse strain has elevated repetitive behaviors and deficits in social behavior, making this an animal of interest in the research of autism and its underlying causes. This study expands on the earlier work and investigates the effects of environmental enrichment during development on the emergence of abnormal behaviors in the C58/J mouse. Breeding pairs of mice were placed in enriched or non‐enriched caging and the behavior of their pups was measured throughout the lactational period. Home cage activity levels of adult mice reared in different environments were also measured. C58/J pups showed elevated activity at a young age when compared to control mice and demonstrated emergence of repetitive behaviors in the second week of life. Adult C58/J mice showed no difference in home cage activity compared to control mice, but do retain elevated repetitive behaviors throughout life regardless of the level of enrichment in which they were raised.

This research was supported by NIMH 1R21MH084132‐01A1 and the University of Redlands Science Center.
Enhanced Urinary Angiotensinogen Excretion in Cyp1a1‐Ren2 Transgenic Rats with Inducible ANG II‐Dependent Malignant Hypertension
Authors: Milani Carlo J , Davis Porcha D , Mucci Jessica L , Kobori Hiroyuki , Mitchell Kenneth D
Published: June 2021

The present study was performed to determine if urinary angiotensinogen excretion is increased in Cyp1a1‐Ren2 transgenic rats [strain name: TGR(Cyp1aRen2)] with inducible ANG II‐dependent malignant hypertension. Adult male Cyp1a1‐Ren2 rats (n=6) were fed a normal diet containing 0.3% indole‐3‐carbinol (I3C) for 10 days to induce ANG II‐dependent malignant hypertension. Rats induced with I3C exhibited pronounced increases in systolic blood pressure (SBP) (208±7 vs. 127±3 mmHg, P<0.001), marked proteinuria (29.4±3.6 vs. 5.0±0.4 mg/day, P<0.01), and augmented urinary angiotensinogen excretion (996±186 vs. 199±19 mg/day, P<0.05). Chronic administration of the AT1 receptor antagonist, candesartan (25 mg/L in drinking water, n=6), prevented the I3C‐induced increases in SBP (125±5, P<0.001), proteinuria (7.3±1.5 mg/day, p<0.001) and urinary angiotensinogen excretion (488±51 mg/day, P<0.05). The present findings confirm that AT1 receptor activation contributes to the increase in SBP and proteinuria in Cyp1a1‐Ren2 transgenic rats with ANG II‐dependent malignant hypertension. These data also demonstrate that the urinary excretion of angiotensinogen is markedly augmented in ANG II‐dependent malignant hypertension. Such increased urinary angiotensinogen excretion likely reflects an AT1 receptor‐mediated stimulation of intrarenal angiotensinogen production which may contribute to augmented intrarenal ANG II levels and, thereby, to the increased blood pressure in Cyp1a1‐Ren2 transgenic rats with inducible ANG II‐dependent malignant hypertension.
Klotho Gene Delivery Suppresses Endothelin‐1 Production but Upregulates ETB Receptors in Kidneys in SHR
Authors: Wang Yuhong , Sun Zhongjie
Published: June 2021

Klotho is a newly‐discovered anti‐aging gene. We recently reported that klotho gene delivery prevents progression of spontaneous hypertension and renal damage in spontaneously hypertensive rats (SHRs). The purpose of this experiment was to further determine the effect of klotho gene delivery on the production of ET‐1 and regulation of SOD in kidneys of SHRs. AAV carrying mouse klotho full‐length cDNA (AAV.mKL) was constructed for in vivo expression of klotho. Four groups of male SHRs (5 rats/group) and 1 group of sex‐ and age‐matched WKY rats were used. Briefly, 4 groups of SHRs received IV injection of AAV.mKL, AAV.LacZ, AAV.GFP, and PBS, respectively. The WKY group received PBS and served as a control. AAV delivery of klotho gene significantly attenuated spontaneous hypertension. All animals were sacrificed at the end of week 12 after gene delivery. AAV.mKL decreased the ET‐1 levels in plasma and kidneys. Interestingly, klotho gene delivery increased ETB receptor expression in renal distal tubule and decreased the ETA/ETB receptor ratio, suggesting, for the first time, that klotho may up‐regulate renal ETB receptors. Klotho gene delivery also enhanced superoxide dismutase (SOD) expression in kidneys.

Conclusions

Klotho gene delivery suppressed the ET‐1 production and up‐regulated ETB and SOD in kidneys of SHRs. These findings may offer new insights into the relationship between renal klotho and the ET system in hypertension.
Cilia movement represses mitogen‐activated protein kinase signaling by enhancing expression of the raf kinase inhibitor protein
Authors: Sas Kelli Margot , Janech Michael G. , Amria May Y. , Stenbit Antine E. , Bell P. Darwin
Published: June 2021

Polycystic kidney disease (PKD) is characterized by the growth of fluid‐filled cysts in the kidneys and other organs. These cysts arise from uncontrolled cellular proliferation, potentially resulting from dysfunctional cilia. We hypothesized that the mechanosensory action of cilia regulates cellular proliferation by limiting signaling through the mitogen‐activated protein kinase (MAPK) pathway. Immortalized collecting duct cell lines from the Oak Ridge mouse that is a hypomorph for the Tg737 gene [cilia (−) cells] or cells in which the Tg737 gene was reinserted [cilia (+) cells] were incubated in a stationary state or subjected to 12 hr of shear stress on a rotator. Analysis of proteins from cilia (+) cells subjected to rotation revealed cilia movement increased expression of the raf kinase inhibitor protein (RKIP) and decreased expression of downstream phosphorylated proteins in the MAPK pathway when compared to all other cells. In addition, renal cortex tissue obtained from adult Tg737 conditional floxed allele mice in which cilia had been knocked out for 3 weeks [cre (+)] or in which cilia were unaffected [cre (−)] was analyzed by western blot. We found that RKIP was significantly higher in renal tissue expressing cilia. These results suggest that flow‐induced bending of cilia suppresses cellular proliferation by controlling the MAPK signaling pathway. This work was supported by NIH grants and a VA Merit Award.
Thyroid Hormone Ameliorates Diabetic Nephropathy in Type II Diabetes
Authors: Lin Yi , Sun Zhongjie
Published: June 2021

Our recent study showed that the T3 level was decreased in db/db mice, a model of type II diabetes. The purpose of this study was to test our hypothesis that treatments with T3 would improve diabetic nephropathy. We treated 16 week‐old male db/db mice with T3 for 4 weeks. T3 decreased albuminuria and plasma creatinine and increased creatinine clearance, suggesting improved renal function. T3 significantly decreased accumulation of collagenous components in cortical interstitium and expansion of mesangial matrix in glomeruli and prevented the loss of glomeruli in db/db mice. Therefore, T3 improved the renal structural damage in type II diabetes. Notably, diabetic nephropathy was accompanied by a decrease in phosphatidylinosital 3‐kinase (PI3‐kinase) activity and an increase of transforming growth factor‐β (TGF‐β) expression in kidneys. Interestingly, T3 restored PI3 kinase activity and decreased TGF‐β expression in db/db mice. Simultaneous treatments with PI3 kinase inhibitor (LY294002) abolished the beneficial effects of T3 on diabetic nephropathy and the attenuating effect of T3 on increased TGF‐β expression. These data revealed, for the first time, that T3 can prevent progressive renal insufficiency and overt diabetic nephropathy in db/db mice and that this protective effect may be mediated by up‐regulation of PI3‐kinase activity.
L‐carnitine ameliorate podocyte injury in diabetic nephropathy by increasing the glucose uptake
Authors: Han Gi Dong , Fan Jiang Ping , Kim Dongyeop , Jeong Boyoung , Kim Gyoung Woo , Kawachi Hiroshi , Ha Tae‐Sun
Published: June 2021

High glucose could change podocyte gene expression and subsequently induce podocyte damage through altered glucose metabolism. L‐carnitine is known to play a beneficial role in diabetes, however, there are no studies on the effect of L‐carnitine on podocyte alteration in high glucose condition. This study was to investigate whether L‐carnitine can attenuate diabetic podocyte injury through the prevention of slit diaphragm proteins loss. L‐carnitine treatment group showed an increased glucose uptake compared to control group, suggesting that utilization of glucose in the podocyte increased by L‐carnitine. L‐carnitine treatment also prevented the decreased mRNA expressions of nephrin and podocin in high glucose‐stimulated podocytes. However, the mRNA expression of CD2AP and ¥á‐actinin‐4 were not significantly changed by high glucose condition. Taken together, L‐carnitine could increase glucose uptake of podocyte in high glucose condition and its mechanism may be at least partly related to the upregulation of nephrin and podocin. Our results give information on the understanding the beneficial effects of L‐carnitine in diabetic nephropathy.
Urotensin II in the killifish gill: regulation of gill chloride transport
Authors: Hyndman Kelly Anne , Cornwell Emily , Buchanan Patrick , Evans David H
Published: June 2021

The endocrine, urotensin II, was first described in fishes forty years ago, and since its discovery evidence is emerging to suggest that it is a regulator of ion transport in the fish gill. The purpose of this study was to first identify if the urotensin II receptor is expressed in the euryhaline killifish gill and to elucidate its putative role in ion transport. The urotensin II receptor was partially cloned from the killifish gill, and found to be widely expressed in the tissues of the killifish. In addition, urotensin II receptor mRNA was found to significantly increase in the gill in fishes that were transferred from freshwater (FW) to sea water (SW) after 24 hrs (0.22 ± 0.05 RU vs 1.02 ± 0.4, n= 5, p= 0.05). As well, chronic acclimation of killifish to FW results in an 80% reduction of urotensin II mRNA expression (1.0 ± 0.2 RU in SW vs 0.23 ±0.04 in FW, n= 6, p = 0.014). Using the SW killifish operculum (a model for the SW teleost gill salt extrusion), urotensin II was found to significantly inhibit the short circuit current in a dose‐dependent manner. Both the nitric oxide synthase inhibitor L‐NAME and the cyclooxygenase inhibitor indomethacin reduced this inhibition, suggesting that urotensin II regulates salt transport in the SW teleost gill via intracellular production of both NO and prostaglandins. In addition, we immunolocalized the urotensin II receptor to the mitochondrion‐rich cell of the gill. Take together, we conclude that urotensin II is a regulator of transport in the gill, and the urotensin II receptor is regulated by environmental salinity with very low levels in FW acclimated fish. Funding the National Science Foundation IOB‐0519579 to DHE.
Identification of Sodium/Glucose Transport for Protein in Avian Renal Tubules
Authors: Picone Amy C , Braun Eldon J
Published: June 2021

Compared to mammals of similar body mass, birds have as a normal aspect of their metabolic physiology higher plasma glucose concentrations (Pglu). The normal Pglu of mammals ranges from 4.5 to 8.3 mM/L and for birds the range is 16.5 to 38.8 mM/L. Given that birds and mammals have similar glomerular filtration rates (GFR) based on body mass (Yokota, et al.); the filtered loads (GFR × Pglu) of glucose are much higher in birds than mammals. In spite of the high filtered loads, little or no glucose appears in the ureteral urine of birds. In mammals, two glucose transport proteins have been identified in the kidney. These are the sodium/glucose co‐transporters (SGLT 1 and 2) in the apical cell membrane of the proximal renal tubule. As the filtered load of glucose is high for the avian kidney and no glucose appears in the ureteral urine, the logical extension is that the SGLTs in the avian kidney must be up‐regulated or there is greater density of these proteins present. To our knowledge, SGLT proteins or SGLT‐like proteins have not been identified and characterized for the avian kidney. To study the nature of glucose transport of the avian nephron at the level of the proximal tubule cell, SDS‐PAGE and Western blots have been run on homogenates of the Mourning Dove kidney.
The in vitro Generation of Avian Urate‐Containing Spherules
Authors: Burnham Emily E. , Braun Eldon J.
Published: June 2021

Uricotelic animals such as birds excrete nitrogen in the form of urate‐containing spherules to prevent uric acid crystallization in the nephron. This could be a concern for uricotelic animals due to the low aqueous solubility of uric acid and its salts. Our objective was to generate in vitro the urate‐containing spherules found in avian urine. A dialysis membrane with a molecular weight cut off of 100 was used in chambers to separate two solutions. On one side of the membrane was a solution mimicking avian glomerular filtrate and on the other was a solution mimicking avian plasma. The artificial plasma had an osmotic potential higher than that of the filtrate to draw fluid from the filtrate side and thus concentrate the uric acid. Three permutations and a control were run to examine the effect of calcium and cholesterol on sphere formation. The increase in uric acid concentration lead to the formation of spheres about 1 micron in diameter. The results suggest that calcium is necessary for sphere formation and that cholesterol is not. It appears that divalent cations are necessary for the formation of the urate and protein‐containing spheres.
Three‐dimensional architecture of the renal inner medulla of the desert rodent Dipodomys merriami: potential impact on the urinary concentrating mechanism
Authors: Urity Vinoo , Nichols Austin J. , Issaian Tadeh , Braun Eldon J. , Dantzler William H. , Pannabecker Thomas L.
Published: June 2021

We hypothesize that the inner medulla (IM) of Dipodomys merriami, a desert rodent that concentrates its urine to over 6000 mOsm/Kg water, provides extreme examples of architectural features most necessary for production of highly concentrated urine. Three‐dimensional architecture of vasculature and nephron segments in the IM was assessed with digital reconstructions from physical tissue sections. Descending and ascending vasa recta (DVR, AVR), descending thin limbs (DTLs), ascending thin limbs (ATLs), and collecting ducts (CDs) were identified by indirect immunofluorescence using antibodies that label segment‐specific proteins associated with solute and water transport and lectins (UT‐B, wheat germ lectin, AQP1, ClC‐K1, and AQP2, respectively). CD clusters form the central organizing motif in the IM. Transverse sections along the corticopapillary axis show nonuniform distribution of AQP‐1‐positive DTLs and uniform distribution of ATLs. Countercurrent exchange occurs only at the perimeter of CD clusters as DVR lie distant from CDs. Electron microscopy shows fenestrated AVR adhering to CDs. This architectural arrangement raises the possibility that fluid and solute reabsorbed from CDs diffuses preferentially into AVR. An additional factor increasing and defining axial compartmentation, and thereby restricting diffusive exchange, would be interstitial cell architecture. DK16294
Electrophysiology of the isolated and perfused midgut of adult female yellow fever mosquitoes (Aedes aegypti)
Authors: Onken Horst , Fuks Yolana , Valencia Melanie , Moffett Stacia B , Moffett David F
Published: June 2021

The midgut of adult, female mosquitoes receives the blood meal. Salt and water are rapidly absorbed and secreted by the Malpighian tubules. Later, nutrients are digested and absorbed. Transport in the midgut of adult mosquitoes has never been studied with isolated midguts. Isolated midguts were transferred into a bath with mosquito saline and mounted on perfusion pipettes connected to a syringe pump. The transepithelial voltage (Vte) was measured. Mounting midguts with the posterior end on the pipette allows continuous perfusion, and a small, lumen negative Vte was observed. Addition of theophylline (10 mmol/l, increasing intracellular cAMP concentrations) to the bathing solution resulted in a small, lumen positive Vte. Because a valve is located between midgut and hindgut, the posterior midgut is inflated when the anterior part connects to the perfusion pipette. When inflated with mosquito saline the initial, lumen negative Vte and the lumen positive Vte after addition of theophylline were markedly higher, suggesting that stretch is a stimulator for the ion transport characteristics reflected in Vte. Vte is inhibited with dinitrophenol, indicating that it reflects active ion transport. Financial support by the NIH (1R01AI063463‐01A2) and by a Wagner College Faculty Research Grant is gratefully acknowledged.
CFTR and ClC‐5 Chloride Channels Modulate Plasma Membrane H+‐ATPase in Proximal Tubule Cells
Authors: Carraro‐Lacroix Luciene Regina , Lessa Lucília M. , Pessoa Thaíssa D. , Nogueira Camila , Souza‐Menezes Jackson , Morales Marcelo M. , Girardi Adriana C. , Malnic Gerhard
Published: June 2021

H+ transport across membranes by H‐ATPase creates a lumen‐positive voltage, generating an electrochemical potential gradient that limits pump activity. This DP is normally dissipated by a Cl‐ movement, which provides an electric shunt compensating for the transferred positive charge. This work was performed to identify the associated Cl−‐conducting proteins involved in the regulation of plasma membrane (PM) H‐ATPase from rat kidney proximal tubule (PT). Through in vivo stationary microperfusion, it was observed that ATPase‐mediated HCO3− reabsorption was significantly reduced in the presence of the Cl− channels inhibitor NPPB. This effect was confirmed in vitro by measuring the cell pH recovery rates after NH4Cl pulse in a cultured cell line derived from PT, IRPTC. In these cells, even after abolishing the membrane potential with valinomycin, ATPase activity was seen to be still dependent on Cl−. siRNA‐mediated CFTR and ClC‐5 channels knockdown significantly reduced H‐ATPase activity. These results indicate that PM H‐ATPase activity from PT depends on both CFTR and ClC‐5 chloride channels.

Supported by FAPESP
NBCe1‐A residues mutated in patients with pRTA: A Topological Analysis
Authors: Zhu Quansheng , Kao Liyo , Newman Debra , Liu Weixin , Azimov Rustam , Kurtz Ira
Published: June 2021

Eight missense mutations in Na+ coupled HCO3− cotransporter 1 (NBCe1‐A) (R298S, S427L, T485S, G486R, R510H, L522P, A799V and R881C) are reported to cause proximal renal tubular acidosis (pRTA). In this study, we explored location of the pRTA causing mutations and their structural importance in NBCe1‐A by substituted cysteine accessibility method (SCAM). The 8 residues were individually substituted with cysteine (cys) in a modified NBCe1‐A backbone with no free reactive cys (NBCe1‐A‐5C−). Locations of the introduced cys were determined by whole cell labeling with two cysteine specific reagents: membrane permeant biotin maleimide and a membrane impermeant MTS‐TAMRA. Results showed none of the 8 residues is on the surface of NBCe1‐A. Selective mutation of T485 revealed it resides in a unique position that is important for normal function. Further topologic analysis of NBCe1‐A by SCAM on 56 individually introduced cys in the NBCe1‐A‐5C− revealed that the first half of the transmembrane region of NBCe1‐A contains 8 transmembrane segments (TM) highly homologous to anion exchanger 1 (AE1), whereas the remainder of the cotransporter folds tightly unlike AE1. On the basis of these results, we propose a new topologic model of NBCe1‐A which allows the assignment of pRTA causing mutations to the following regions: S427L, TM1; T485S and G486R, TM2; R510H and L522P, TM3; A799V, TM10; R881C, TM12.

Source of Research Support: NIH
A new delta current method for estimating the transport stoichiometry of electrogenic transporters
Authors: Shao Xuesi Max , Kao Liyo , Kurtz Ira
Published: June 2021

The stoichiometry of electrogenic transporters is an important determinant of their function. If an electrogenic transporter transports ions in a fixed stoichiometric ratio q, q can be determined by the reversal potential Erev with known intra‐ and extra‐cellular concentrations of transporting ions given that a specific blocker is available. An alternative approach widely used is to calculate delta Erev (ΔErev) by altering concentrations of a transported ion. This approach is based on the hypothesis that the effects of other channels and transporters in cell membrane on Erev are additive. However, the Goldman‐Hodgkin‐Katz (GHK) equation suggests otherwise: Erev is a logarithmic function of the sum of different conductances rather than being additive. In the present study, we propose a delta current (ΔI) method that does not require a blocker. ΔI instead of ΔErev is determined by altering the external concentration of a transported ion thereby eliminating other currents. The ratio of ΔI at two different membrane voltages (E1 and E2) is: ΔI2/ΔI1= (E2/E1) ((exp ((q−1) FE1/RT)−1)/(exp ((q−1) FE2/RT)−1)). ΔI2/ΔI1 only depends on q, E1, and E2. We tested this new ΔI methodology in patch‐clamp experiments of HEK‐293 cells expressing NBC1e1‐A or NBC4c. Our data show that the ΔI equation accurately calculates a stoichiometry ratio of HCO3− :Na+of 2 demonstrating the utility of this new methodology.
Inhibition of seizure‐induced cell death by sodium/bicarbonate cotransporter NBCn1 (SLC4A7)
Authors: Park Hae Jeong , Choi Inyeong
Published: June 2021

Recent studies show that ablating some genes encoding bicarbonate transporters results in alteration of seizure threshold and progression, indicating a close association of neuronal pH regulation with seizure. In this study, we investigated the effect of the sodium/bicarbonate cotransporter SLC4A7 (NBCn1) on seizure and seizure‐induced neuronal injury in the hippocampus. NBCn1 knockout mice were injected with N‐methyl‐D‐aspartate (NMDA, 75 mg/kg, intraperitoneal) and the severity of seizure activity was scored (0 for normal behavior and 7 for death). Knockout mice had the mean seizure score of 1.1 ± 0.2 (n = 6), whereas wild type mice had 5.1 ± 1.8 (n = 8). The seizure behavior of knockout mice faded after 20 min and the animals behaved normally at 30 min. Determined by caspase‐3 activity after NMDA administration, knockout mice had significantly low neuronal death (40% low compared to wild type; p < 0.01). Caspase‐3 expression and TUNEL staining were also low in the hippocampus of knockout mice. Immunoblot of the hippocampus showed a 30% reduction of neuronal nitric oxide synthase in knockout mice. PSD‐95 and NR2A expression were unaffected. In primary cultures of postnatal neurons, cytotoxicity by 300 μM NMDA treatment for wild type mice was 83%, whereas cytotoxicity for knockout mice was 25%. These results demonstrate that NBCn1 contributes to seizure and seizure‐induced neuronal injury in the hippocampus.
Modulation of Cell Specific Signaling by Cross‐Linking Receptors with Multivalent Ligands
Authors: Weber Craig S , Vagner Josef , Josan Jatinder S , Gillies Robert , Lynch Ronald M
Published: June 2021

Connecting two ligands with a flexible linker produces a molecule with potential to cross‐link multiple receptors on a cells surface. We previously showed that a hetero‐multivalent ligand (htMVL) composed of melanocyte stimulating hormone (MSH) and cholecystokinin (CCK) parmacophores connected by peptidomimetic linkers exhibits a >50 fold increase in binding affinity (relative to monomers) only to cells expressing both receptors. Here we evaluate the signaling properties of this htMVL by measuring intracellular Ca2+ ([Ca2+]i) in HEK cells engineered to express both receptors. CCK by itself elevates [Ca2+]i, while MSH elevates cAMP at sub‐μM concentrations. The CCK induced Ca2+ response was abolished when both monovalent ligands were added together, suggesting synergy between the two receptors. Conversely, the MSK/CCK ligand elicited an elevation in [Ca2+]i though it was significantly attenuated compared to monovalent CCK. Since the binding affinity of the htMVL is greater than the CCK monomer, this htMVL acts as a weak agonist by itself, but a super‐antagonist of the normal CCK response. Further, the cell signaling properties of the htMVL is further evidence that this agent is binding to and activating both receptors simultaneously. Our findings indicate that htMVLs may provide new pharmacological tools for altering cell‐type specific signaling. Supported by NIH, RO1 CA097360 and CA123547.
Paracrine responses to hyperthermia: do cells warn their neighbors about environmental stress?
Authors: Linowski Angelika Kamila , Oca‐Cossio Jose A , Clanton Thomas L
Published: June 2021

We hypothesize that when cells are exposed to stress in the form of heat, paracrine signals are produced and released into the extracellular fluid. These chemicals act as warning signals to the surrounding cells. As a result, the “warned cells” promote their survival by inducing a reduction in metabolism, a mild “hibernation.” To test this hypothesis, we used oxygen polarography to measure metabolic responses in HeLa cells and in mouse C2C12 cells following hyperthermic treatment. Control cells were maintained at 37° C (37T) and hyperthermic cells exposed to 42°C for 45 min (42T). Cells (37T and 42T) and media (37M and 42M) were separated after treatment by centrifugation. Oxygen consumption was done in combinations of cells/media using the media as “respiratory buffer” in the electrode (37T‐37M, 37T‐42M, 42C‐37M and 42C‐42M). The results indicate a reduction in the cell respiration of Hela cells at 42C (42T‐42M) when compared to the ones at 37C (37T‐37M). There was also a decrease in metabolic rate of cells treated at 37T when exposed to the media from those treated at 42C (37T‐42M vs. 37C‐37M) (P<0.02) and an increase in cells treated at 42T and exposed to the media at 37 (42T‐37M vs. 42C‐42M). For C2C12 cells, similar but less consistent responses were observed (ANOVA P < 0.01). The metabolic changes by paracrine signaling may result in the improvement of survival of cells in response to local environmental stress. NHLBI 53333.
Deletion of caveolin‐1 enhances hepatic heme oxygenase‐1 (HO‐1) induction following endotoxin, especially in female mice.
Authors: Crooks Amanda , Yavelak Bianca , Culberson Cathy , Clemens Mark G
Published: June 2021

We have previously shown that caveolin‐1 knockout (Cav −/−) mice are protected against endotoxemia and this is associated with preservation of the endothelial nitric oxide synthase activity. However, protection against inflammation can also involve induction of heme oxygenase‐1 (HO‐1). Therefore we tested whether HO‐1 is differentially regulated in Cav −/− mice. Wildtype (WT) and cav −/− mice received i.p. lipopolysaccharide (LPS) or saline and liver tissue samples were taken at either 3 or 6 hours. HO‐1 mRNA levels were significantly induced at 6 but not 3 hours following LPS in male WT mice. In contrast, HO‐1 mRNA was significantly (P< .05) induced at both three and six hours in Cav −/− mice suggesting enhanced HO‐1 induction in Cav −/−. Since previous studies have shown that females are more resistant to LPS than are males, we compared induction of HO‐1 protein in male and female Cav −/− mice at 6 hours after LPS. Both genders showed significant upregulation of HO‐1 protein after LPS but the levels in females were nearly twice that observed in male cav −/− mice. These results suggest that enhanced induction of HO‐1 may contribute to the protective effect of cav‐1 deletion; moreover, the significantly enhanced induction of HO‐1 in females may contribute to their relative tolerance to endotoxemia. Supported by DK 38201‐21
TP53 polymorphisms and association with endometriosis in Brazilian population
Authors: Carvalho Cristina Valletta , Camargo‐Kosugi Cintia Meirelles , Kosugi Eduardo Macoto , D'Amora Paulo , Rojas Jéssica Vidal , Schor Eduardo , da Silva Ismael Dale Cotrim Guerreiro
Published: June 2021

Introduction

Endometriosis is a benign gynaecologycal disease, defined as the presence of functional endometrial implants out of uterus, mainly in pelvis. Cell cycle and apoptosis disturbs could be associated with this disease as well as alterations in the genes that control.

Aim

To investigate the possible association between the presence of the polymorphisms in the genes TP53: codons 11; 72; 248 with endometriosis.

Methods

Cross sectional case–control study was performed. Genomic DNA was extracted from cells collected from buccal swabs. The TP53: codons 11; 72; 248 polymorphisms were investigated using the polymerase chain reaction restriction fragment length polymorphism (PCR‐RFLP) method in a hospital‐based Brazilian population.

Results

The P53 gene, codon 11 (P53*11) polymorphism study showed that mostly of women did not present the allelic variance (p=0.255). The P53 gene, codon 72 (P53*72) polymorphism study presented significant differences between endometriosis and control groups (P=0.01; OR=2.19; 95% IC=1.196–4.039). The P53 gene, codon 248 polymorphism study showed that only one woman from endometriosis group presented heterozygosis to variate allele.

Conclusions

P53*72 polymorphisms studies could be associated with endometriosis.
Soybean phytoestrogens modulate ion transport in porcine endometrial epithelial cells
Authors: Deachapunya Chatsri , Poonyachoti Sutthasinee
Published: June 2021

Effects of the phytoestrogens genistein and daidzein on ion transport functions in primary cultured porcine endometrial epithelial cells were investigated using the Ussing chamber technique. Acute exposure of 17β‐estradiol failed to change short circuit current (Isc) whereas genistein produced a concentration‐dependent increase in Isc (EC50 37 μM). The genistein‐stimulated Isc was diminished in Cl−‐free solution or in the presence of bumetanide, but not amiloride. In amphotericin B‐permeabilized monolayers, genistein activated apical NPPB‐ and glibenclamide‐senstive Cl− current with no effect on apical K+ current. Genistein also activated basolateral barium‐sensitive K+ current which created a driving force for Cl− secretion. Treatment of monolayers with Ca2+ chelating BAPTA‐AM did not affect the genistein‐activated Isc, suggesting a Ca2+‐independent regulation of Cl− and K+ channels. In contrast, daidzein decreased Isc by inhibiting apical Cl− current with no change in basolateral K+ current. These data suggest that the Cl− secretory pathway in endometrial epithelial cells is subject to be regulated by soybean phytoestrogens genistein and daidzein. This work was supported by The Thailand Research Fund (RMU 4980026).
Role of caveolin ‐ 1 in the entry of HCMV into human extravillous trophoblast cells
Authors: Wang Lin , Zhou Li
Published: June 2021

Objective

To establish the cell model of HCMV infect human extravillous trophoblast cells (EVTs)and use this valuable cell model to study the role of caveolin‐1 in the entry of HCMV into EVTs.

Methods

EVTs separated from early gestation villous tissue (5–6 weeks gestation) was used to establish the cell model of HCMV infection. The dynamic change of caveolin‐1 in EVTs after infection of HCMV was characterized by using flow cytometry and immunocytochemistry and RT‐PCR.

Results

The positive products of HCMV immediate early protein can be observed in the nucleuses of infected cells using immunocytochemistry method, mRNA from HCMV IE gene was detected in the EVT cells 24 and 36 hours after infection. The result of flow cytometry shows that the percentage of EVTs apoptosis does not increase at early period, but improve 48 hours later after infection; The expression of caveolin‐1 in uninfected EVT cells is higher, began to reduce at 12 hours after infection, the lowest expression of caveolin‐1 occurs at 24 hours after infection. The expression of caveolin‐1 began to increase at 48 hours, with peak expression occurring at 96 hours after infection.

Conclusion

Established a cell model of HCMV infect EVT cells successfully. The dynamic change of caveolin‐1 in EVTs imply that HCMV enters EVT cells via unique endocytic pathway that involves caveolae/caveolin‐1.
Nexilin is a component of Enteropathogenic Escherichia coli (EPEC) pedestals
Authors: Law H T , Jackson Jeremy S , Guttman Julian A
Published: June 2021

Enteropathogenic Escherichia coli (EPEC) are extracellular bacteria that colonize intestinal epithelial cells, leading to severe diarrheal disease and possibly death. Following bacterial attachment to the cell surface these microbes utilize a syringe‐like apparatus, called a Type III Secretion System (T3SS), to inject various pathogenic bacterial proteins into the host cell to cause disease. One of these proteins, Tir (Transmembrane intimin receptor), inserts itself into the host plasma membrane where it functions to recruit actin beneath the attached bacteria, generating structures called “pedestals”. These pedestals are common a characteristic of EPEC pathogenesis and allow the bacteria to ‘surf’ atop infected cells. Here we report the discovery of nexilin, an actin binding protein known to localize to focal adhesions, at EPEC pedestals. We demonstrate that nexilin recruitment beneath the attached bacteria requires a functional T3SS and specifically the bacterial protein Tir. Our discovery sets the foundation for investigating its contribution during these infections and is a method of exploiting EPEC to tease out the cellular function of novel host proteins.

Grant Funding Source: NSERC
Ectopic Telomerase Expression in Dupuytren's Disease Myofibroblasts
Authors: Vaughan Melville B. , Tomasek James J. , Noon Miriam C. , Isiaho Armstrong M.
Published: June 2021

In vitro studies of disease states often require large quantities of cells; however, replicative senescence limits the availability of such resources. Ectopic telomerase expression is known to immortalize cells with little effect on their normal activites. In this study we used telomerase to immortalize fibroblasts derived from Dupuytren's disease, then studied their ability to differentiate into myofibroblasts. Cells were cultured onto coverslips or within stress‐relaxed collagen lattices in the presence or absence of transforming growth factor‐beta1 (TGF‐beta). Samples of both assays were immunostained with an anti‐smooth muscle alpha‐actin antibody to determine the percentage of myofibroblasts; cell contractility was determined using lattices. In all tests, TGF‐beta increased myofibroblast percentage and contractility. This suggests that telomerase has little effect on myofibroblast differentiation. Similar studies elsewhere determined that telomerase inhibits the myofibroblast phenotype, and so our future goals will include comparing parental to immortalized fibroblasts in their ability to differentiate into myofibroblasts. Supported by UCO Office of Research and Grants, and NIH NCRR P2PRR016478.