bluuebunny/crossref_metadata_2025 huggingface dataset:
EAST/SeSAME syndrome mutations partially disrupt trafficking of homomeric Kir4.1 channels and produce non‐functional heteromeric Kir2.3/Kir4.1 channels
Authors: Ortega Bernardo , Li Dimin , Kim Bo-Young , Wade James B , Welling Paul A
Published: June 2021

Mutations in Kir4.1 cause renal salt and Mg wasting (EAST/ SeSAME syndrome, E/S) within the DCT. We have recently found that WT Kir4.1 co‐assembles and co‐localizes with Kir2.3 to produce heteromeric channels with properties similar to native channels in distal nephron. We previously reported that E/S Kir4.1 mutants disrupt Kir2.3 function, raising the possibility that E/S Kir4.1 mutants broadly disrupt the basolateral K+ conductance by dominant negative (DN) effects. Here we further investigate the mechanisms underlying the inhibitory effects of E/S mutants on WT Kir4.1 and Kir2.3 channels. By comparing current density to cell surface expression of external epitope tagged Kir2.3 or Kir4.1 channels, we found that inhibitory effects arise from disturbances in either trafficking or gating. Co‐expression of E/S mutant channels (R65P, C140R, R199X, R297C) with WT Kir4.1 resulted in a decreased current density (20–40%). The inhibitory effect of mutants R65P, C140R and R199X resulted from impaired surface expression while others affected channel gating. By contrast, five of the six mutants (not C140R) exerted a DN effect on Kir2.3 largely by altering channel gating. In summary, Kir4.1 E/S mutant channels affect gating and trafficking of heteromeric channels at the basolateral membrane of the distal nephron. Funded by NIH.
Aldosterone modulates NCC involving MAPK ERK1/2 signaling pathway
Authors: Feng Xiuyan , Wang Yanhui , Wu Ping , Delpire Eric , Gu Dingying , Cai Hui
Published: June 2021

The thiazide‐sensitive sodium chloride cotransporter (NCC) is one of key regulators of sodium balance. Aldosterone was shown to increase NCC function via SPAK signaling pathway. We previously showed that knock‐down ERK 1/2 expression increased NCC expression. To further discrete the role of aldosterone‐mediated MAPK ERK1/2 signal pathway in regulation of NCC protein expression, we treated the mDCT cells with aldosterone at different time point and found that the ERK1/2 phosphorylation increased in 15 min but deceased in 1hr and lasted 24hr. We further determined NCC protein expression and ERK1/2 phosphorylation levels in both SPAK knockout (KO) and wild type (WT) mice. We found that NCC protein expression is decreased with increased ERK 1/2 phosphorylation in SPAK KO mice. We also found that aldosterone level is lower and ERK1/2 phosphorylation is increased with decreased total NCC protein expression in high sodium diet group compared to normal salt diet group. These results suggest that aldosterone‐mediated MAPK ERK1/2 signal pathway is involved in modulating NCC expression independent on SPAK signal pathway.
Involvement of TNF‐α receptor type 2, but not the type 1, in angiotensin II induced renal tissue injury in mice
Authors: Singh Purnima , Bahrami Laleh , Castillo Alex , Majid Dewan S.A.
Published: June 2021

Although TNF‐α has been implicated in the hypertensive and renal injury (RI) responses to angiotensin II (AngII), the role for its receptors (type 1 and 2) in these responses are not yet clearly defined. In this study, their roles were assessed by evaluating the responses to chronic administration of AngII (25 ng·min−1 by implanted minipump) for 2 weeks in knockout mice for either type 1 (TNFR1KO; n=6) or type 2 (TNFR2KO; n=6) receptors and compared these results with those in wild type (WT; C57BL6; n=6) mice. RI responses were assessed by measuring glomerulosclerosis index (GSI; PAS staining), interstitial collagen deposition index (CDI; Gomori's trichrome staining) and CD68+ macrophage cell infiltration (MCI; immunohistochemistry). Hypertensive responses (monitored by radiotelemetry) to AngII were not different in these three groups of mice (TNFR1KO, 88 ± 5 to 155 ± 6 mmHg; TNFR2KO, 90 ± 5 to 141 ± 16 mmHg; WT, 89 ± 6 to 147 ± 14 mmHg). Compared to the values in WT, RI parameters were similar in TNFR1KO (GSI, 22.6 ± 4.2% vs 26.3 ± 2.6%; CDI, 8.1 ± 1.1% vs 7.1 ± 2.0% and MCI, 2.2 ± 0.6 vs 1.1 ± 0.2 cells/mm2tissue) but were significantly lower in TNFR2KO (GSI, 17.3 ± 3.1%; CDI 5.2 ± 0.6% and MCI, 0.02 ± 0.001 cells/mm2tissue). These data demonstrate that hypertensive response to AngII is independent of activation of TNFR1 or TNFR2 but inflammatory process leading to RI is modulated by TNFR2, but not by TNFR1 receptor. (NHLBI grant #66432)
Orchidopexy protects the apoptosis of spermatogenic cells in experimentally cryptorchid rats
Authors: Jiang Yuqing , Li Ruili , Guo Yuexian
Published: June 2021

Orchidopexy is an effective method for patients with cryptorchidism to restitute fertility. To investigate the effects of orchidopexy on the pathogenesis of cryptorchidism, we compared the apoptosis of spermatogenic cells with TUNEL labeling and the expression of Bcl‐2/Bax proteins with immunohistochemistry between orchidopexy and sham groups of cryptorchid rats. We found that the weight of testis was decreased, the apoptotic spermatogenic cells increased, the peak of monokaryotic cells (1C) declined, and the percentage of 1C and 4C reduced, but the percentage of 2C increased after cryptorchidism when compared with sham group. The expression of Bcl‐2 was down‐regulated while Bax was up‐regulated after cryptorchidism. Two weeks after orchidopexy, we found that the number of apoptotic spermatogenic cells was decreased significantly, the expression of Bcl‐2 upregulated, Bax downregulated, and the peak of 1C raised. Six weeks after orchidopexy, there was no significant difference between experimental and sham groups in the weight of testis. The apoptotic spermatogenesis cells were increased but less than those without orchidopexy. The number of 1C increased to the level in sham group. These data suggest that orchidopexy inhibits apoptosis of spermatogenic cells and recovers the spermatogenesis function.

Supported by Medical Scientific Research Key Program of Hebei province, China (20100095)
Activation of malonyl‐CoA/fatty acid synthase axis is an early event in the effects of insulin in human skeletal muscle myotubes: Implication for obesity linked insulin resistance
Authors: Ritov Vladimir , Chacon Alexander , Menchikova Elizaveta , DeLany James , Dube John
Published: June 2021

Insulin simultaneously increases both glucose transport and synthesis of malonyl‐CoA by acetyl‐CoA carboxylase. Increased malonyl‐CoA concentration inhibits transport of fatty acyls into mitochondria and that shifts mitochondria from fatty acid oxidation to glucose oxidation. In the current study human myotubes were differentiated from primary myoblasts obtained from obese or athlete subjects to test effects of insulin on content of palmitoyl‐CoA, NADPH and NADH (marker for glucose consumption). Insulin initially induces the consumption of NADPH while sharply increasing the content of NADH. Analysis of long‐chain acyl‐CoAs showed that an insulin‐induced decrease in NADPH concentration is accompanied by accumulation of palmitoyl‐CoA. Accelerated synthesis of palmitate that needs two NADPH for each elongation step can explain initial insulin induced consumption of NADPH. We found increased degree of NADPH oxidation and increased synthesis of palmitoyl‐CoA in athletes. That suggests an accelerated rate of generation of malonyl‐CoA in myotubes from athletes. We hypothesize that in obesity insulin has reduced ability to increase the concentration of malonyl‐CoA in skeletal muscle and to attenuate the oxidation of fatty acids by mitochondria. As a result, the sustained oxidation of fatty acids by mitochondria in this condition generates a metabolic signal that suppresses glucose transport.
Role of L‐type voltage dependent calcium and large conductance potassium channels in adenosine A1 receptor mediated vasoconstriction through Cyp4a
Authors: Kunduri Swati S , Nayeem Mohammed A , Ponnoth Dovenia S , Tilley Stephen , Mustafa S. Jamal
Published: June 2021

Adenosine A1 receptor (A1AR) induces vasoconstriction possibly via arachidonic acid‐derived metabolite, 20‐HETE by Cyp4a. 20‐ HETE is important in the regulation of vascular tone. We hypothesize that A1AR‐mediated vasoconstriction depends on Cyp4a through activation of L‐type voltage dependent calcium channels (L‐VDCC) and inhibition of large conductance potassium (BK) channels. Using WT and A1KO mouse aortae, responses for AR agonists were obtained with inhibitors of Cyp4a (HET0016, 10−5M), L‐VDCC (nifedipine, 10−6M) and BK activator (NS1619, 10−5M). Immunoblots suggest higher Cyp4a levels in WT than A1KO (p<0.05) aortae. CCPA (A1AR agonist, 10−7M) elicited contraction was significantly blunted with HET0016 (−7.9 ± 1.9% vs. −32.5 ± 1.7%, p<0.05) and nifedipine (−11.5 ± 5.9% vs. −32.5 ± 1.7%, p<0.05) in WT and with no effect in A1KO. NECA (AR agonist, 10−7M) induced relaxation was unaltered in control and NS1619 treated WT. NS1619 enhanced NECA elicited relaxation (32.4 ± 4.2% vs. −1.7 ± 4.2%, p<0.05) in A1KO. 20‐HETE (10−7M) induced contraction was higher in WT than A1KO (−33.4 ± 2.9% vs. −23.9 ± 2.4%, p<0.05) that was significantly abated by nifedipine and NS1619 in WT and A1KO. This is the first evidence suggesting that A1AR mediates vasoconstriction via 20‐ HETE by activating L‐VDCC and inhibiting BK channels which may have important implications in cardiovascular disorders. (HL027339, HL094447, HL071802)
Endothelium modulates the contractile effect of RhoA activation in rat aorta
Authors: Stallmann-Jorgensen Inger Susanne , Goulopoulou Styliani , Webb R. Clinton
Published: June 2021

Increased signaling in the Rho/Rho kinase (ROCK) pathway is involved in the development of hypertension. Activation of ROCK was shown to increase vascular tone. For RhoA, a member of the Rho family of GTPases, translocation to the plasma membrane is required for activation and for subsequent downstream activation of ROCK. In contrast, vascular nitric oxide (NO) exerts vasodilatory effects on vascular tone in the aorta. We hypothesized that NO inhibits RhoA activation. Aortic rings were isolated from Sprague‐Dawley rats, endothelium was left intact or removed; rings were mounted on myographs and contractile force measured in response to a new RhoA activating drug (CNO3) in 10−6 M concentration. Aortic rings were pre‐treated with phenylephrine at 10−8 M concentration to stimulate the contraction. The CNO3 compound significantly increased the contractile force over baseline in rings with the endothelium removed (136%) compared to intact rings (44.1%). Our results suggest that the presence of vascular endothelium reduces the contractile response to CNO3 in the aorta. Since NO has well‐known vasodilatory effects, it is a strong candidate as an inhibitor of RhoA activation.
TGF‐β/Smad3 signaling inhibition protects from obesity and diabetes through modulation of adipocyte biology
Authors: Yadav Hariom , Quijano Celia , Kamaraju Anil K , Gavrilova Oksana , Lonning Scott , Skarulis Monica , Sumner Anne E. , Finkel Toren , Rane Sushil G
Published: June 2021

Epidemic increase of obesity and diabetes drives to develop better understanding of pathways that promote obesity and diabetes and that might unravel novel therapeutic targets against obesity and diabetes. Transforming Growth Factor beta (TGFβ) signals regulate diverse cellular processes including proliferation, differentiation and apoptosis. Surprisingly, to‐date the role of TGFβ signaling in diabetes and obesity is obscure. We found that the TGFβ/Smad3 signaling pathway plays an important role in regulating glucose and energy homeostasis. Smad3 deletion in mice protects from diet‐induced obesity and diabetes. Interestingly, these effects were associated with increased brown like phenotype in Smad3−/− white adipose tissue (WAT) that is characterized by UCP1 positive cells and acquiring brown fat/skeletal muscle gene expression profile. In addition, Smad3−/− adipocytes demonstrated increased energy metabolic function and mitochondrial biogenesis. TGFβ has shown Smad3 dependent effects on PGC1α promoter activity and Smad3 acts as a repressor of PGC‐1α expression. Circulating TGFβ1 levels significantly correlated with adiposity in rodents and humans. Further, injecting anti‐TGFβ antibody protected mice from obesity, diabetes and hepatic steatosis. These results strongly suggest that blockade of TGFβ/Smad3 signaling strategy can be an effective therapy against obesity and diabetes.
Hemeoxygenase ‐1: A potential target for management of hypertension caused by Recurrent Insulin Induced Hypoglycemia
Authors: Quadri Syed Siraj Ahmed , Prathipati Priyanka , Jackson Debra , Jackson Keith
Published: June 2021

Recurring insulin‐induced hypoglycemia (RIIH) often occurs during therapeutic management of insulin dependent diabetes mellitus. There is evidence that both cardiac and adrenergic sensitivity are altered in type 1 diabetics. Controversy currently exists in literature as to the ability of insulin to promote hypertension. Could insulin promote adverse pressor effects, if so under what conditions and through what mechanism? Thus, the current study was performed to evaluate the hypothesis that hypoglycemia promotes hypertension via an increase in Heme oxygenase‐1 (HO‐1). Male Sprague Dawley rats (200 – 225g) were treated for 2 weeks with varying doses of subcutaneous insulin injections (1, 3, 5, 7, and 9 U/kg body weight) and fed on normal chow or zinc diet (1mM) for 2 weeks. Tail‐cuff blood pressure, food/water intake and blood glucose states were monitored daily. A dose dependent decrease in blood glucose was observed. Blood pressure was significantly elevated in rats treated with 7U/Kg dose as compared to other units. However, there was no change in urine output among all the groups. Animals fed a high zinc diet and animals treated with the HO‐1 inhibitor zinc deuteroporphyrin 2,4‐ bis glycol (ZnDPBG) (20mg/kg) were shown to have reduced blood pressure as compared to animals fed on normal diet. Also, HO‐1 protein levels in heart and kidney were reduced in the animals fed on zinc diet vs control. These results demonstrate that RIIH promotes hypertension and inhibition of HO‐1 with high zinc diet and ZnDPBG reduces the blood pressure.
Antihypertensive Actions of Moderate Hyperbilirubinemia: Role of Superoxide Inhibition
Authors: Stec David , Storm Megan , Gousette Monette
Published: June 2021

Our lab has previously demonstrated that moderate (~ 2 fold) increases in plasma unconjugated bilirubin levels are able to attenuate the development of Angiotensin II (Ang II) dependent hypertension. In order to determine the specific role of decreases in superoxide production to the blood pressure lowering affects of moderate hyperbilirubinemia (MHyB), we performed the current study in which the NADPH oxidase inhibitor, apocynin, was given to Ang II infused mice in the presence and absence of moderate hyperbilirubinemia. Apocynin (14 mM) was administered in the drinking water prior to treatment with UGT1A1 antisense morpholino (ASM; 16 μg/kg) which was administered via intravenous injection every third day. Treatments were started before the implantation of Ang II containing minipumps (1μg/kg/min) and continued throughout the 7 day protocol. Ang II infusion increased blood pressure to 145 ± 2 mmHg. Apocynin treatment alone reduced blood pressure to 135 ± 5 while MHyB alone decreased blood pressure to 118 ± 5 in Ang II infused mice. Prior inhibition of NADPH oxidase with apocynin did not result in a further decrease in blood pressure in MHyB mice which averaged 117 ± 3 (n=6/group). In aortic preparations, apocynin treatment decreased Ang II mediated superoxide production from 2433 ± 120 to 1851 ± 126 RLU/min/mg (n=4/group) which was similar to levels observed in MHyB mice alone 1473 ± 132 or in combination with apocynin 1503 ± 115. Our results indicate that MHyB lowers blood pressure via a mechanism that is independent of the inhibition of superoxide production; however, we cannot exclude the possibility that other antioxidant properties of bilirubin may be important to the antihypertensive actions in Ang II dependent hypertension. This work was supported by grants from the National Heart, Lung and Blood Institute, HL088421 and PO1HL‐51971.
The apical NKCC1 cotransporter debate
Authors: Crum Jeannine Marie , Alvarez Francisco J. , Alvarez-Leefmans Francisco Javier
Published: June 2021

Choroid plexus epithelial cells (CPECs) secrete cerebrospinal fluid (CSF) and regulate its electrolyte composition. CPECs express Na+/K+ ATPase and Na+‐K+‐2Cl− cotransporter 1 (NKCC1) on their apical membrane (CSF‐facing), deviating from typical basolateral membrane location in secretory epithelia. Given this non‐canonical location of NKCC1 for a secretory epithelial cell, the direction of net ion fluxes mediated by this cotransporter and associated water fluxes, under physiological conditions, is controversial in CPECs. NKCC1‐mediated ion and water fluxes are tightly linked, thus their direction can be inferred by measuring cell volume changes following NKCC1 inactivation. Hypotheses: under physiological conditions NKCC1 is working in the uptake mode and maintains normal cell water volume in CPECs, hence knocking out NKCC1 will produce cell shrinkage due to unbalanced net efflux of solutes and water. Electron microscopy revealed NKCC1−/− CPECs are severely shrunken, forming large dilations of their basolateral extracellular spaces, yet remain attached by apical tight junctions. Nomarski microscopy confirmed that freshly dissociated CPECs from NKCC1−/− mice are half the size of CPECs from WT (p< 0.01). These results demonstrate that under physiological conditions NKCC1 is constitutively active, works in the uptake mode, and is necessary to maintain normal cell water volume.
Evidence for a Na/K pump in nematocytes isolated from sea anemones
Authors: Morabito Rossana , Marino Angela , La Spada Giuseppina , Adragna Norma C. , Lauf Peter K
Published: June 2021

Nematocytes, the stinging cells of the primitive marine animals jellyfish and sea anemones respond with regulatory volume decrease (RVD) to osmotic swelling in 35% diluted artificial sea water (65% ASW, Marino et al., Cell Physiol Biochem 2010). Since RVD is attained through K+ and Cl‐efflux via channels and cotransporters, an active pump mechanism must be required to maintain the cellular K+ gradient and expel Na, which was here explored. Cells were isolated from acontia of the anemone Aiptasia mutabilis (Strait of Messina, Italy) by applying 605mM NaSCN plus 0.01mM Ca2+ and then exposed to isosmotic or 65% hyposmotic ASW in the presence and absence of ouabain, the specific Na/K pump inhibitor. Ouabain present at 10‐4 M, but not at 10‐6 and 10‐8M, induced significant cell swelling in isosmotic ASW suggesting Na/K pump inhibition and failure to extrude Na causing an osmotic influx of water. This effect was attenuated by replacing Na with choline in the ASW. Ouabain applied at 10‐4M prior to 65% ASW did not affect the initial osmotic swelling phase but inhibited RVD consistent with loss of K required for this process. Results suggest that nematocytes possess Na/K pumps with low ouabain affinity that actively extrude Na and maintain cellular K steady state levels, necessary to perform RVD.
Rab14 GTPase downregulates urea transporter UT‐A1 activity through enhanced clathrin‐dependent endocytosis and protein degradation
Authors: Su Hua , Xu Gang , Carter Conner B , Fröhlich Otto , Sands Jeff M , Chen Guangping
Published: June 2021

Rab small GTPase proteins are involved in multiple steps in the intracellular movement of various molecules. By yeast two hybrid assay, we identified Rab14 as a binding protein of urea transporter UT‐A1. Their physical interaction was confirmed by both in vitro and in vivo co‐immunoprecipitation. GST pull‐down assay shows that the GDP‐bound inactive Rab14 (S25N) binds to UT‐A1 much more strongly than GTP‐bound active Rab14 (Q70L). Functionally, co‐injection of Rab14 in Xenopus oocytes results in decreased UT‐A1 protein expression and decreased urea transport activity. To explore the possible mechanism, we used HEK293 cells and cotransfected UT‐A1 with Rab14. The total UT‐A1 protein abundance is significantly decreased when the cells overexpress Rab14 in the presence of the translation inhibitor cycloheximide. Cell surface biotinylation experiments revealed a reduction of plasma membrane UT‐A1 expression when co‐transfected with Rab14. Concanavalin A, an inhibitor of clathrin‐dependent endocytic pathway, can ameliorate the UT‐A loss from the cell membrane caused by Rab14. Consistently, sucrose gradient fractionation by ultracentrifugation revealed Rab14 protein present in non‐lipid raft clathrin fractions. Our study shows that Rab14 directly binding to UT‐A1 promotes UT‐A1 clathrin‐dependent endocytosis and degradation, and therefore downregulates UT‐A1 activity.
Role of PKCα in Hypertonicity‐stimulated Urea Permeability
Authors: Wang Yanhua , Klein Janet D. , Froehlich Otto , Sands Jeff M.
Published: June 2021

The ability of the kidney to concentrate urine is vitally important to our quality of life. In the hypertonic environment of the kidney, water and urea transporters must be regulated in a coordinated fashion to optimize function. We previously showed that hypertonicity increases urea permeability and the PKC blockers, chelerythrine and rottlerin, decreased hypertonicity‐stimulated urea permeability in rat inner medullary collecting ducts (IMCDs). Because PKCα knockout mice have a urine concentrating defect, we tested the effect of hypertonicity on urea permeability in mouse isolated perfused IMCDs. Increasing the osmolality of perfusate and bath from 290 to 690 mOsM did not change urea permeability (39±8 vs 42±8 ×10−5 cm/s) in PKCα knockout mice, but significantly increased urea permeability (from 25±6 to 53±3 ×10−5 cm/s) in wild‐type mice. To determine if the response to PKA was similarly missing in IMCDs of PKCα knockout mice, tubules were treated with 200 pm vasopressin (AVP) and subsequently with 200nM PKC stimulator PDBu. AVP stimulated urea permeability from 24±4 to 31±5 ×10−5 cm/s in PKCα knockout mice. Unlike in rats, subsequent treatment of PDBu did not further increase urea permeability (from 31±5 to 32±5 ×10−5 cm/s) in the PKCα knockout mice. Western blot analysis showed that expression of urea transporter, UT‐A1 in IMCDs was reduced in PKCα knockout mice compared to wild‐type mice. We conclude that PKCα regulates the hypertonicity‐related stimulation of urea transport but is not necessary for vasopressin stimulation of urea permeability in IMCDs.
Reactive oxygen species production in mitochondria of pigs selected for residual feed intake
Authors: Grubbs Judson Kyle , Fritchen Aaron , Harris Amanda , Huff-Lonergan Elisabeth , Gabler Nicholas K , Lonergan Steven M
Published: June 2021

Electron transport in the mitochondria is a primary source of reactive oxygen species (ROS). Excess production of ROS may lead to increased oxidative stress, decreasing nutrient utilization and efficiency. The purpose of this study was to compare mitochondrial ROS production in pigs (n = 7 per line) divergently selected for residual feed intake (RFI). Mitochondria were freshly isolated from liver by differential centrifugation. The production of H2O2 was determined by using 2, 7 ‐Dichlorofluorescein diacetate. Glutamate or succinate was provided as an energy source for complex I and II respectively. Rotenone inhibited electron backflow when using succinate. No differences in H2O2 production between the lines were observed when using glutamate as an energy substrate. When succinate was added, H2O2 production was decreased by 8% in low RFI pigs compared to their high RFI counterparts (46.7 vs. 34.3 nmol H2O2/mg protein/min ± 8.87). Other inhibitors of electron transport, 4,4,4‐trifluoro‐1‐ [2‐thienyl]‐1,3‐butanedione, malonate, or the combination of these, yielded similar results (P<0.05). Altogether, these data indicate that less efficient, high RFI, pigs are more prone to hepatic intracellular oxidative stress. This may explain why low RFI pigs are more efficient at utilizing and partitioning nutrients for growth and metabolism. This research was supported by USDA‐AFRI grant# 2010‐65206‐20670.
Antioxidant enhanced spare capacity in cardiomyocytes as a protective response to oxidative stress induced by hypoxia and redox cycling agent
Authors: Legmann Rachel , melito Julie , Ferrick David
Published: June 2021

Increased reactive oxygen species (ROS) generation underlies many pathologies of the cardiovascular system. Strategies that enhance the cell's ability to cope with oxidative stress are being explored by many labs. The main objective of this work was to analyze the effect of antioxidant on cellular respiration in embryonic rat cardiac cells and their capability to cope with oxidative stress. The cells were adapted to hypoxia in the presence of the antioxidant, ascorbic acid, followed by exposure to the endogenous H2O2 generator redox cycling agent, 2, 3‐dimethyloxy‐ 1, 4‐naphtoquinone (DMNQ). Mitochondrial function was measured in intact cells using an Extracellular Flux (XF) analyzer. The spare respiratory capacity of the cells decreased in a dose dependent manner following DMNQ treatment. Growing the cells in the presence of an antioxidant enhanced spare respiratory capacity and therefore protected from exposure to DMNQ both in hypoxic (Fig 1) and normoxic conditions. In conclusion, the admission of antioxidant to the cardiomyocytes, either in hypoxia or normoxia, enhanced significantly both their maximal respiration and spare respiratory capacity, enabling them to protect the mitochondria within the cell against oxidative damage. Boosting spare capacity should provide a means to counteract its decrease caused by environmental toxins, aging or the acute effects of disease.
Nicotinic acetylcholine receptors (nAchRs) in supraoptic nucleus (SON) neurons: Potential role in regulation of food intake
Authors: Song Zhilin
Published: June 2021

Nicotine induces anorexia. nAchRs are expressed in SON of the hypothalamus, where oxytocin (OT) and vasopressin (VP) are synthesized. Nicotine stimulates release of VP as well as OT, an anorexic agent. Deficits in OT are associated with human obesity. Thus, nicotine may reduce food intake, in part, via modulation of OT neurons in SON. In our current studies, live‐cell calcium imaging was used to determine if acetylcholine (Ach) or nicotine altered intracellular calcium ([Ca++]i)signaling in SON neurons. Hypothalamic explants that include SON were loaded with the calcium sensitive dye, Fura‐2AM. Changes in [Ca++]i were monitored by changes in the 340/380 nm emission. A short application of Ach (5 mM) to the bath caused an increase in [Ca++]i in a subset of SON neurons, in the presence or absence of PNU‐120596 (10 μM), a positive allosteric modulator of α7 nAchRs. Ca++ response to Ach was reduced but still existed in the presence of atropine (10 μM) to block muscarinic AchRs. Nicotine (20–100 μM) also induced [Ca++]i increase in a portion of SON neurons. These findings are consistent with activation of nAchRs in SON neurons. Future studies will further characterize the specific subtypes of nAchRs in SON and identify the peptidergic phenotype of responsive neurons. Supported by AHA 0735329N.
Nesfatin‐1 Alters Synaptic Activity in Neurons in the Nucleus of the Solitary Tract
Authors: Mimee Andrea , Ferguson Alastair V
Published: June 2021

Nesfatin‐1 (nes‐1) is a recently discovered neuropeptide that decreases food intake and is expressed in the medullary nucleus of the solitary tract (NTS). In order to begin elucidating the mechanisms of nes‐1 actions, we studied the effect of nes‐1 on synaptic activity in NTS neurons using whole‐cell voltage clamp recordings. Bath applied nes‐1 (10nM) did not influence the frequency (freq, 157±49 vs 163±48 events/100s, p=0.85) nor amplitude (amp, 29±4 vs 51±13 pA, p=0.11) of EPSCs recorded from rat medial NTS neurons (n=8) in slice. Nes‐1 did, however, induce an increase in freq (144±45 vs 224±53 events/100s, p=0.03) and amp (57±9 vs 74±12 pA, p=0.02) of IPSCs in 8/9 cells. We next used tetrodotoxin to study spontaneous miniature EPSCs and IPSCs. 10nM nes‐1 did not affect freq (305±68 vs 275±45 events/100s, p=0.37) nor amp (30±2 vs 34±4 pA, p=0.25) of mEPSCs (n=7). Neither the freq (335±102 vs 385±90 events/100s, p=0.41) nor amp (85±12 vs 92±12 pA, p=0.31) of mIPSCs was affected (n=9) by nes‐1. Our findings identify NTS as a site where nes‐1 acts to selectively enhance GABAergic tone and increase inhibitory synaptic events to potentially modify food intake.

Research support: NSERC, FQRNT, CIHR.
Roux‐en‐Y Gastric Bypass Surgery Alters the Membrane Properties of Brainstem Neurons
Authors: Browning Kirsteen N , Hajnal Andras
Published: June 2021

Roux‐en‐Y gastric bypass surgery (GBS) is the most effective treatment for morbid obesity although the effects on brainstem neurons responsible for vagal control of the viscera are unknown. The aim of the present study was to investigate the effects of obesity and GBS on the membrane properties of dorsal motor nucleus of the vagus (DMV) neurons in rats.

4‐week old Sprague‐Dawley rats were fed a high fat diet (60% kcal from fat) for 44–59 weeks prior to GBS surgery. Sham rats received the same surgical protocol except intestinal manipulation was performed instead of GBS. Three weeks post‐surgery, whole‐cell patch clamp recordings were made from DMV neurons in thin (300μm) brainstem slices.

While the input resistance and action potential (AP) duration of DMV neurons was not different between Sham and GBS rats (298±32MΩ and 5.3±0.4ms (n=20) vs 284±65MΩ and 5.4±0.4ms (n=7), respectively, P>0.05), the amplitude of the after hyperpolarization was higher (16.1±1.0mV vs 11.8±2.4mV, P<0.05). By consequence, the firing frequency of Sham neurons was slower (2.6±0.4 Hz and 5.0±0.5Hz at 90 and 270pA current injection vs 4.7±1.1Hz and 8.7±1.7Hz, respectively, P<0.05).

These results suggest that the effects of GBS to reset obesity‐related alterations in ingestive behavior also involve modulation of central neurocircuits responsible for satiety and sensory afferent processing.

Supported by NIH DK080899
Roux‐en‐Y Gastric Bypass Surgery Increases The Response Of Central Vagal Neurons To Satiety Neuropeptides
Authors: Browning Kirsteen N , Hajnal Andras
Published: June 2021

Roux‐en‐Y gastric bypass surgery (GBS) is the most effective treatment for morbid obesity although the effects on brainstem neurons responsible for vagal control of the viscera are unknown. The aim of the present study was to investigate the effects of obesity and GBS on the pharmacological responses of rat dorsal motor nucleus of the vagus (DMV) neurons.

4‐week old Sprague‐Dawley rats were fed a high fat diet (60% kcal from fat) for 44–59 weeks prior to GBS surgery. Sham rats received the same surgical protocol except intestinal manipulation was performed instead. Three weeks post‐surgery, whole‐cell patch clamp recordings were made from DMV neurons in brainstem slices (300μm) and the effects of perfusion with either GLP‐1 or CCK8s (both 100nM) on neuronal excitability were measured.

In GBS rats, 5 of 5 neurons tested responded to GLP‐1 with an increase in action potential firing rate (219±35%) whereas only 2 of 9 Sham neurons were similarly responsive (P<0.05). In contrast, while 3 of 6 GBS neurons responded to CCK8s with an increase in firing rate (190±21%) this was not significantly different from the response of Sham neurons (2 of 12 responsive neurons, 230±70%, P>0.05).

These results suggest that the effects of GBS to reset obesity‐related alterations in ingestive behavior also involves modulation of the responsiveness of central vagal neurons to gastrointestinal satiety neuropeptides.

Supported by NIH DK080899
Mechanisms underlying the cardiovascular responses to cooling
Authors: Tung Che-Se , Yang Chien-Fan , Liu Yia-Ping , Chang Shang-Tang , Huang Chuen-Lin
Published: June 2021

We investigated the mechanisms of hunting response, i.e. cold‐evoked vasoconstriction (CEC) and latter vasodilatation (CED) at the lowest temperatures. Rats were immersed soles in a cage containing 2 cm iced water (4±2°C) for 10 min and then the responses through cooling (C) were monitored by changes of blood pressure and heart rate with variability in frequency domain: very low (VLF), low (LF) and high (HF). Firstly, we tested the brain dopamine‐β hydroxylase (DBH) mechanisms. Rats were given lateral ventricularly (icv) DBH‐shRNA to knock‐down DBH. Secondly, we tested the origins of CED. Rats with or without given tail‐venously (iv) a ganglion blockade, Hexamethonium (H, 10 mg/kg), were then given centrally (1 mg/h, icv) or peripherally (30 mg/kg, iv) an antagonist of nitric oxide (NO) production (L‐NAME). The results indicate (1) C causes significant CEC followed by CED; CED correlates highly with the falling of VLF, (2) icv DBH‐shRNA is dominant in attenuating CEC and CED, (3) both iv and icv L‐NAME cause reversal of the CED‐VLF changes, (4) H alone attenuates CEC but not CED; H plus iv L‐NAME attenuate the effects of iv L‐NAME on CED‐VLF changes. We conclude that brain DBH may participate in mechanisms of autonomic cardiovascular responses to C, and CED is due to both central and peripheral increased NO production. Grant in aid: National Defense Medical Center (DOD‐100‐I‐17) and Cardinal Tien Hospital (CTH‐100‐1‐2A04)
Cytokines in the Hypothalamic Paraventricular Nucleus (PVN) are Not Required for Maintenance of Angiotensin II‐Salt Hypertension
Authors: Bardgett Megan , Herrera-Rosales Myrna C. , Toney Glenn M.
Published: June 2021

Rats consuming high salt and treated with systemic angiotensin II (Ang II) develop neurogenic hypertension mediated by elevation of sympathetic nerve activity (SNA) derived from a forebrain‐hypothalamic circuit. Cytokines are proposed to increase SNA and arterial blood pressure (ABP) and we therefore sought to determine their role within PVN in maintenance of AngII+salt hypertension. We first focused on tumor necrosis factor α (TNFα), which was microinjected into the PVN (0.3 pmol in 50 nl/side) of anesthetized normotensive (NT, 0.4% NaCl diet, n=10) rats. Although this increased lumbar SNA (18±7%), splanchnic SNA (49±9%) and mean ABP (4±2 mmHg) compared to vehicle (P<0.05), interfering with TNFα binding by PVN injection of etanercept (20 μg in 100 nl/side) did not affect recorded variables either in NT rats or in Ang II (150 ng/kg/min, sc) + salt (2% NaCl diet) hypertensive (HT, n=12) rats. Given this apparent lack of TNFα “tone” in PVN, we next non‐selectively inhibited local cytokine release by microinjection of minocycline (1 μg in 100 nl/side). This too failed to alter lumbar SNA (HT: 13±9% vs NT: 0±3%), splanchnic SNA (HT: 12±4% vs NT: 6±2%) or mean ABP (HT: 3±2 mmHg vs NT: −1±1 mmHg) in NT or HT rats. These findings indicate that whereas TNFα can act in PVN to increase SNA; maintenance of Ang II‐salt hypertension does not depend on either PVN actions of TNFα or microglia derived cytokines. Support: HL102310 & HL 088052 (GMT)
Social defeat stress‐induced hyperthermia involves non‐shivering thermogenesis in brown adipose tissue
Authors: Kataoka Naoya , Nakamura Kazuhiro
Published: June 2021

Psychological stress‐induced hyperthermia is a fundamental autonomic response in mammals. However, it is controversial whether this stress response involves sympathetic thermogenesis in brown adipose tissue (BAT). We have previously reported that systemic blockade of β3‐adrenoreceptors, a major adrenoreceptor subtype in BAT, reduces hyperthermic response evoked by social defeat stress, a psychological stress model and that this stress activates medullary raphe sympathetic premotor neurons that are known to control BAT thermogenesis for cold defense and fever (Eur. J. Neurosci. 34:1442–1452, 2011). To further examine the involvement of BAT thermogenesis in psychological stress‐induced hyperthermia, here, we directly measured changes in both interscapular BAT temperature and abdominal temperature of rats using a telemetry system. Rats that received social defeat stress exhibited an immediate increase in BAT temperature and also a delayed elevation of abdominal temperature. This result provides direct evidence that non‐shivering BAT thermogenesis contributes to hyperthermic response to social defeat stress, a model close to stress in human society.
Carotid Body Artery Removal (CBX) Prevents Training‐ Induced Plasticity in Preautonomic Oxytocinergic Neurons of the Paraventricular Nucleus SHR
Authors: Cruz Josiane Campos , Cavalleri Marina Tuppy , Michelini Lisete Compagno
Published: June 2021

We observed that sinoaortic denervation prevents T‐induced plasticity in the OTergic preautonomic PVN neurons of SHR (Cavalleri et al., AJP, Regulatory, 2011). In the present study, we sought to investigate whether peripheral chemoreceptors participate in T‐induced OTergic plasticity in PVN subnuclei of SHR. CBX or SHAM were trained or kept sedentary (S) for 3 month. After cardiovascular records, rats were anesthetized and perfused (saline or PFA 4%). Tissue samples of PVN were processed for oxytocin (OT) mRNA expression (RT‐PCR) or for immunofluorescence (ir) staining with OT antibody (1:200.000). CBX abolished pressor and bradicardic responses to chemoreflex activation (KCN 40 μg, iv) in S and T rats. T induced increases in both OT mRNA (1.3±0.2 vs 2.6±0.4, ShamS vs ShamT) and OTir in PVN preautonomic subnuclei (dorsal cap, dc = 15±2 vs 23±1%; ventromedial, vm = 36±1 vs 44±1%; posterior, post = 9±1 vs 17±5 %, respectively). CBX did not change basal expression, but blocked T‐induced OT gene and protein expression (ShamT vs CBXT: PVN OT mRNA= 2.6±0.4 vs 1.4±0.6; OTir dc = 23±1 vs 15±2 %); OTir vm = 36±1 vs 22±3; OTir post = 17±2 vs 12±2%). Chemoreceptors are not tonic to drive preautonomic oxytocinergic neurons, but participate in the peripheral signaling to modulate T‐induced OTergic plasticity Financial Support: FAPESP, CNPq
The influence of transcranial direct current stimulation on cardiac autonomic function in healthy human subjects
Authors: Clancy Jennifer Ann , Johnson Robyn , Wilkie Richard , Mon-Williams Mark , Deuchars Susan , Deuchars Jim
Published: June 2021

Transcranial direct current stimulation (tDCS) of the primary motor cortex is a possible therapy to enhance motor rehabilitation after stroke. The aim of this study is to determine any potential autonomic effects of the tDCS electrode montage conventionally used in stroke therapy. The study was approved by the University of Leeds Ethics Committee and conducted in accordance with the Declaration of Helsinki. tDCS was applied over the primary motor cortex at 1mA with a reference electrode placed on the contralateral supraorbital region in 16 healthy subjects (20–48 years; 7 female, 9 male). Subjects visited the department twice and received active or sham tDCS for 10 minutes. Active tDCS was anodal (n=8) or cathodal (n=8). Heart rate (HR), blood pressure (BP) and respiration were recorded at baseline, during tDCS and recovery. ECG data underwent power spectral analysis to calculate heart rate variability (HRV). HRV did not change significantly (p>0.05) however there was a 32% increase in LF/HF ratio (a measure of sympathovagal balance) during anodal tDCS that continued into recovery reaching 42% above baseline. This indicates a trend towards increased sympathetic activity that was supported by a significant increase in LF power (p=0.048) in the anodal tDCS group. These results suggest further investigation is required before tDCS should be used for stroke therapy. This research is supported by the University of Leeds.
Vesicular release of ATP by brainstem astrocytes evoked by changes in pH
Authors: Kasymov Vitaliy , Marina N , Kasparov S , Gourine A
Published: June 2021

The mechanisms of central chemosensory function are not completely understood. Here we explored the mechanisms underlying release of ATP by brainstem astrocytes in response to changes in pH.

RESULTS

Loading of astrocytes with fluorescent ATP analogue MANT‐ATP resulted in abundant punctate fluorescent staining in the cytosol and this was prevented by bafilomycin A1, indicating vesicular accumulation of the marker. In brainstem astrocytes, TIRF imaging revealed progressive decreases in MANT‐ATP fluorescence intensity in response to a decrease in external pH indicating fusion of the labelled compartments. The number of fusion events triggered by acidification in brainstem astrocytes loaded with BAPTA‐AM was significantly reduced indicating that vesicular fusion requires intracellular Ca2+ signalling. The fusion rate in cortical astrocytes was not affected by acidification. Acidification‐evoked vesicular fusion responses in brainstem astrocytes were unaffected by blockade of ATP‐mediated signalling with MRS2179 or apyrase. Quantitative RT‐PCR analysis of genes encoding proteins associated with vesicular fusion revealed that expression levels of VAMP3, Slc1a2 and Slc17a9 are significantly higher in brainstem astrocytes as compared to cortical astrocytes.

CONCLUSIONS

Chemosensory stimulation of brainstem astrocytes triggers vesicular fusion underlying release of ATP.
JSim models of two‐dimensional concentrations in capillary‐tissue systems relating center‐of‐mass of retained tracer to washout kinetics
Authors: Raymond Gary M. , Bassingthwaighte James B.
Published: June 2021

The transport of a metabolite from a capillary into tissue surrounding it and subsequent back flux and washout is modeled. The system is radially symmetric. It is solved using JSim's Mathematical Modeling Language (MML) by subdividing the tissue into equally spaced concentric rings surrounding a cylindrical capillary. The capillary and concentric rings are modeled as partial differential equations in the axial direction. In the radial direction they are mathematically connected by second order accurate finite difference equations where the permeability‐surface area parameters are calculated from the radial diffusion coefficient. Solutions from the capillary and tissue rings are assembled into an array and contoured. The center of mass of material as a function of axial distance and absolute radial distance is calculated. Animated visualizations are constructing by linking the solutions to MatlabTM through a JSim procedure written in Java. The model was generated using the Modular Program Constructor allowing us to prototype a system where the governing equations repeat patterns of information.(Supported by NIH grants T15‐HL088516 and R01‐EB08407)
Conditional deletion of β1‐integrin from urothelium results in bladder dysfunction and abnormal voiding
Authors: Hill Warren G , von Bodungen Maximilian , Yu Weiqun , MacIver Bryce , Kalluri Raghu , Kanasaki Keizo
Published: June 2021

Integrins link cells to each other and to the extracellular matrix and function to transduce physical and sensory information from the environment into biochemical signals. We used Cre‐LoxP conditional targeting to delete β1‐integrin specifically from the urothelium in mice (β1‐cKOs). Urothelium lacking integrins was morphologically normal and maintained barrier function, however these animals were incontinent with excess urine leakage (P<0.05 for non‐corner urine spot number and spot volume). Bladder filling cystometry confirmed that β1‐cKOs exhibited excess contractility and longer voiding intervals with males having more severe symptoms than females. ATP secretion into the lumen of the bladder during filling was two‐fold greater in male β1‐cKOs than controls (P<0.05) suggesting a purinergic signaling defect. Preliminary qPCR performed on laser‐captured urothelium suggested that female controls may have more β1‐integrin than males and that knock‐down in females may be less than complete; providing an explanation for gender differences. β1‐integrin immunolocalized to the basal/intermediate cells of the urothelium and we speculate it may be part of the mechanosensory apparatus that communicates information on bladder fullness to sensory afferents. These data demonstrate that urothelium plays an essential role in regulating voiding function. Supported by NIH grant DK083299 to WGH.
Expression and Purification of the Human Xeroderma Pigmentosum F (XPF/ERCC1) Complex in Bacterial Cells for Protein Binding Assays and Crystallographic Studies
Authors: Anduaga Javier , Duprez Kevin , Hilario Eduardo , Fan Li
Published: June 2021

Mutations in dna repair enzymes of the XP complementation group, XPA‐XPG, leads to a number of genetic disorders such as Xeroderma Pigmentosum, Cockayne's Syndrome, and Trichothiodystrophy. The structure specific dna repair endonuclease responsible for the 5′ incision during dna repair, XPF, is involved in homologous recombination that assists in removing interstrand cross‐link. XP‐F is an autosomal recessive disease characterized by hypersensitivity of the skin to sunlight followed by high incidence of skin cancer and frequent neurologic abnormalities. I expressed and purified human recombinant XPF and ERCC1 proteins in E.Coli Rosetta and BL21 cells, these results will be scaled up for use in interaction and crystallographic studies with proteins of the XP family.
Herpes Simplex Virus: Manipulating DNA Damage Response Pathways
Authors: Weller Sandra K , Schumacher April J. , Mohni Kareem N.
Published: June 2021

During infection, cellular DNA damage response (DDR) factors that are beneficial to HSV are recruited while others are degraded or inactivated. For instance, the non‐homologous end joining pathway (NHEJ) is inactivated by ICP0‐mediated degradation of DNA‐PK. Furthermore, we have published that ATR phosphorylation of downstream targets is suppressed during viral infection. Using cell‐based assays we have now shown that single strand annealing (SSA) is stimulated in HSV‐1‐infected cells, while homologous recombination (HR) and classical and alternative NHEJ are not. Interestingly, HSV encodes an exonuclease (UL12) and a single strand annealing protein (ICP8) that together mediate strand exchange in vitro. UL12 and ICP8 are reminiscent of the two subunit “recombinase” of bacteriophage lambda red alpha/beta proteins. Using the cell based assays we find that UL12 can specifically stimulate SSA; whereas, a catalytically dead UL12 protein cannot. In addition, interactions between UL12, ICP8 and cellular DDR proteins including MRN, MSH2 and MSH6 suggest that the HSV “recombinase” may interact with host DDR factors to effect pathway choice and promote appropriate recombination‐dependent DNA synthesis. Concatemer formation is an essential step for the generation of progeny virus, and we are interested in whether SSA is the preferred cellular pathway utilized by HSV during infection to create concatemers. This work was supported by NIH R01‐ AI37549, NIH RO1‐AI21747 and Connecticut stem cell research grant 09‐SCA‐UCHC‐34.
Structure of Human Mad1 C‐terminal Domain Reveals Its Kinetochore‐Targeting Function
Authors: Kim Soonjoung , Sun Hongbin , Tomchick Diana , Yu Hongtao , Luo Xuelian
Published: June 2021

Accurate chromosome segregation during mitosis is critical for maintaining genomic stability. The spindle checkpoint is a cellular surveillance system that ensures the fidelity of chromosome segregation. The kinetochore‐bound checkpoint protein complex Mad1–Mad2 promotes the conformational activation of Mad2 and serves as a catalytic engine of checkpoint signaling. How Mad1 is targeted to kinetochores is not understood, however. To understand the functional importance of the C‐terminal region of Mad1, we solved the crystal structure of human Mad1 C‐terminal domain (CTD) and found that it formed a homodimer and had a fold similar to that of the known kinetochore‐targeting domain of Spc25 (a subunit of the Ndc80 complex). Indeed, Mad1‐ ∆CTD was defective in kinetochore targeting in human cells. The mutagenesis studies validate a role of Mad1 CTD in kinetochore targeting and implicate Bub1 as its receptor. Our results thus reveal novel functions of Mad1‐CTD in kinetochore targeting. Deletion of CTD does not abolish Mad1 kinetochore localization, however. Non‐overlapping Mad1 fragments retain detectable kinetochore targeting. Therefore, our results indicate that CTD is a part of an extensive kinetochore‐binding interface of Mad1, and rationalize graded kinetochore targeting of Mad1 during checkpoint signaling.