bluuebunny/crossref_metadata_2025 huggingface dataset:
Dysregulation of ryanodine receptors/Ca2+ release channels by FKBP12.6 in diabetic vascular myocytes
Authors: Zheng Yun-Min , Yang Yan , Yadav Vishal , Wang Yong-Xiao
Published: June 2021

Diabetes significantly increases mortality and morbidity from accompanying cerebral and other vascular diseases, which may result from an abnormally enhanced increase in [Ca2+]i in vascular smooth muscle cells (SMCs). However, the underlying molecular processes remain unclear. Here, we for the first time show that the activity of ryanodine receptors/Ca2+ release channels (RyRs) is greatly enhanced in diabetic cerebral artery SMCs (CASMCs). RyR‐mediated local Ca2+ release (Ca2+ spark) is significantly increased. Targeted gene knockout of FK506 binding protein 12.6 (FKBP12.6−/−), known to associate with and inhibit (stabilize) RyR2, increases Ca2+ spark as well. However, the increased Ca2+ spark is not further augmented in FKBP12.6−/−/diabetic cells. Caffeine, a classic RyR agonist, evokes an equivalent enhanced increase in [Ca2+]i (global Ca2+ release) in diabetic, FKBP12.6−/−, and FKBP12.6−/−/diabetic CASMCs. The amount of FKBP12.6 is largely decreased in SR fractions from diabetic cerebral arteries, and increased in cytosolic fractions, suggesting FKBP12.6/RyR dissociation. Supportively, FKBP12.6/RyR2 FRET signal is significantly decreased in diabetic CASMCs. Collectively, we conclude that FKBP12.6/RyR2 dissociation represents an essential mechanism in the diabetic increase in [Ca2+]i in vascular SMCs and associated vascular diseases.
Liver X receptors down‐regulate CFTR‐mediated chloride secretion in renal collecting duct cells
Authors: Soodvilai Sunhapas , Raksaseri Promporn , Chatsudthipong Varanuj
Published: June 2021

Liver X receptors (LXRs) are nuclear transcription factors that regulate several transporters. We investigated the role of LXRs on cystic fibrosis transmembrane conductance regulator (CFTR)‐mediated chloride secretion in MDCK‐C7 cells and collecting duct (CD) cells. Treatment of the MDCK‐C7 cells with TO901317, GW3965, and oxysterol significantly inhibited chloride secretion to 45+5 %, 63+12%, and 55+7% of vehicle‐control, respectively. These results were reproducible in primary CD cells. The effects of LXR agonists were abolished by cycloheximide indicated that the inhibitory effect of LXR agonists on chloride secretion was mediated by protein synthesis. Results from basolateral‐permeablized MDCK‐C7 cell showed the apical membrane was a target of inhibitory effect of LXR agonists on chloride secretion. These results correlated well with evidence that LXR agonists reduced CFTR protein expression, as evaluated by western blot analysis. CFTR is responsible for cysts enlargement in polycystic kidney disease, we future investigated the effect of LXR agonist on cyst growth using MDCK‐C7 cells. The data from cyst growth experiment revealed that LXR agonists retarded cysts enlargement. Taken together, this study provides for the first time evidence showing LXR agonists regulate CFTR function in renal CD cells. This mechanism could be new targets for for treatment of polycystic kidney disease.
Paracellular intestinal absorption increases in mice exposed to cold
Authors: Karasov William Henry , Price Edwin R. , Ruff Lisa , Guerra Alberto
Published: June 2021

Recent studies of birds and bats demonstrate high passive (paracellular) absorption of nutrients. We hypothesized that a species with low paracellular absorption (mice) could increase absorption in response to increased energy demand. We predicted that mice exposed to cold would increase intestinal size to meet the increased energy demand, but in the short term these mice would increase paracellular absorption as well. We transferred mice from 21 to 5 °C and assessed absorption of radiolabeled L‐arabinose, a paracellular absorption probe, at 3 timepoints: prior to cold exposure; after 1 day of cold exposure; and after 2 weeks in the cold. We also assessed control mice kept at 21 °C. Mean gut size increased over the 2 week cold exposure, and this was accompanied by an increase in food consumption. Absorption of an L‐arabinose dose increased from 15.8% (prior to exposure) to 19.4% (P=0.006) after 1 day in the cold, and 21.5% (P<0.001) after 2 weeks exposure. We also assessed expression (qPCR) of tight junction proteins ZO‐1, Cldn2, Cldn4, and Cldn15, relative to housekeeping gene Eef1a1 in the medial intestine. Cldn2 was differentially expressed among treatment groups (P=0.02); however, there were no treatment‐induced differences in the expression of the other tight junction proteins. Our results suggest that mice may adjust tight junction “leakiness” to meet short‐term need for increased absorptive capacity.
Mapping the claudin‐2 pore: comprehensive cysteine‐scanning mutagenesis and thiol modification of claudin‐2 first extracellular loop
Authors: Li Jiahua , Nakamura Tamako , Zhuo Min , Pei Lei , Yu Alan S.L.
Published: June 2021

Claudins are tight junction proteins that determine paracellular permeability in epithelia. The first extracellular loop of claudin‐2 forms a paracellular cation pore. To understand the structure and function of this pore, we aim to conduct a comprehensive cysteine‐scanning mutagenesis of the first extracellular loop and map amino acids that line the cation pore or are located at the intermolecular interface. Up to now, we have generated 20 cysteine mutants and made stably transfected, polyclonal MDCK I Tet‐Off cells. Protein expression was first confirmed by Western blot. Mutants S39C, T42C, T56C, G60C, T62C and L70C showed a high molecular weight band consistent with dimer formation, suggesting that these mutated residues are located near the intermolecular interface. Next, the accessibility of the introduced cysteines was assessed by adding the thiol‐reactive reagent, 2‐(trimethylammonium) ethyl methanethiosulfonate (MTSET), to the cells. Block of the pore by MTSET was monitored by recording any change of transepithelial resistance (TER). Mutants S33C, T56C, S68C and T69C showed 14%, 26%, 35% and 16% increase of TER, respectively, suggesting that these residues are located near the narrow part of the pore. We conclude that we have identified 6 new residues located near the intermolecular interface and 4 new residues located near the cation pore. Supported by NIH grants R01DK062283, R01DK062283‐07S1 and U01GM094627.
Metformin restricts the basolateral glucose dependent growth of apical Staphylococcus aureus in airway epithelia‐bacteria co‐cultures
Authors: Garnett James Peter , Naik Sonam , Baker Emma , Baines Deborah L
Published: June 2021

Glucose concentration in the airway surface liquid (ASL) is normally ~12.5 times lower than blood. In patients, raised ASL glucose increases the risk of respiratory infection, particularly with methicillin‐resistant Staphylococcus aureus. We investigated the relationship between basolateral glucose concentration and apical S. aureus growth using an in vitro model of human airway epithelium to test whether metformin (drug used to lower blood glucose) affected this relationship. H441 epithelial cells grown at air‐liquid‐interface were inoculated with 5×105CFU/cm2 S. aureus (8325‐4) on the apical surface for 7 hours. The transepithelial electrical resistance (TEER) was significantly reduced (P<0.05, n=4) and paracellular glucose flux across H441 monolayers (assessed using 14C‐L‐glucose) was greatly enhanced (P<0.001, n=4), following S. aureus addition. In addition, apical S. aureus growth (determined by Miles‐Misra) increased with basolateral glucose concentration (10, 20, 40 mM). A 12h pre‐treatment of H441 cells with metformin (1mM) significantly prevented both the reduction in TEER (P<0.001, n=4) and the increase in glucose flux (P<0.0001, n=4), as well as impairing the growth of S. aureus (P<0.05, n=4). These results indicate that metformin restricted apical S. aureus growth in airway co‐cultures by reducing the paracellular diffusion of glucose into the ASL.
Effect of alcohol on liver mitochondrial folate uptake
Authors: Biswas Arundhati , Rajan Senthilkumar Sundar , Said Hamid M
Published: June 2021

Mammalian cells obtain folate, a water‐soluble vitamin, from their surroundings via transport across the plasma membrane. Internalized folate is compartmentalized between the cytoplasm and the mitochondria. Transport of folate into mitochondria is via the mitochondrial folate transporter (MFT). Chronic alcohol use disturbs folate physiology and metabolism, but little is known about its effect on transport into mitochondria. We addressed this issue using freshly isolated mitochondria from human‐derived liver HepG2 cells and from liver of rats chronically exposed to alcohol. Chronic exposure of HepG2 cells to alcohol and chronic alcohol feeding of rats led to a significant inhibition in mitochondrial folate uptake. This was associated with a significant reduction in level of expression of MFT protein, mRNA and heterogenous nuclear RNA. Chronic alcohol exposure also inhibited promoter activity of the human SLC25A32 gene (which encodes the hMFT). These findings show for the first time that chronic alcohol exposure/feeding significantly inhibit carrier‐mediated folate transport into mitochondria, and that the inhibition is exerted at the level of transcription of the SLC25A32 gene. [Supported by NIH and DVA grants].
Molecular characterization of nonshivering thermogenesis in a ‘protoendothermic’ mammal, the Lesser‐hedgehog tenrec (Echinops telfairi)
Authors: Ölkrug Rebecca , Götze Nadja , Heldmaier Gerhard , Jastroch Martin , Meyer Carola Waltraud
Published: June 2021

The molecular events and mechanisms that facilitated the evolutionary transition from ecto‐to endothermia in vertebrates are still unknown but must have involved the development of endogenous heat production. The Lesser‐hedgehog tenrec, Echinops telfairi, is considered ‘protoendothermic’ as a result of ectothermic‐like body temperature patterns. We studied metabolic responses of E. telfairi in vivo and observed daily cycles of hypothermia ranging from 21° to 33°C body temperature (Ta: 20°C). Rewarming rates from torpor were independent of β3‐adrenergic stimulation (judged with inhibitor SR59230A), emphasizing that classical adaptation of thermogenesis as found in brown adipose tissue (BAT) of rodents was absent. However, we found brown adipose‐like tissue predominantly located in the abdominal cavity. Molecular analysis confirmed the expression of UCP1 mRNA and protein. The proton leak of isolated mitochondria could be inhibited more than 50% by GDP, suggesting UCP1‐dependent proton conductance. Taken together, BAT in E. telfairi appears to be consistent with the thermogenic function as found in rodents, but does not display the typical thermogenic adaptation. We are currently comparing UCP1 specific activity of E. telfairi with mouse (Mus musculus) in HEK293 cells to explore whether differences in physiological heat production are reflected on the molecular evolution of UCP1.
Examination of Avian Renal Tissue for the Presence of Sodium‐glucose Co‐transporters
Authors: Isaacs Jim M , Braun Eldon J
Published: June 2021

Given animals of similar body mass, birds have been shown to have much higher plasma glucose concentrations than that of mammals. However, birds appear not to show the pathological symptoms associated with high plasma glucose levels as seen in mammals. As the glomerular filtration rates (on a mass specific basis) of birds and mammals are not significantly different and with higher plasma glucose levels, birds must have a larger filtered load of glucose. Despite this there is little or no detectable glucose in the ureteral urine of birds. This suggests that the mechanisms for glucose reabsorption by the bird kidney must be significantly up‐regulated. In mammals glucose reabsorption is achieved through the sodium‐glucose co‐transport proteins (SGLTs) located in the apical cell membrane of the proximal renal tubule. The putative SGLTs within the avian kidney have not been isolated or characterized. Antibodies against mammal (rabbit) SGLT2 were used to isolate SGLTs from renal tissue harvested from Mourning doves. We have been able to demonstrate the presence of an immuno‐reactive protein in the kidneys of doves albeit not at the same intensity as that the rat kidney used as control. Using primers developed from known sequences of bovine SGLT2 DNA, RT‐PCR was used to further identify avian SGLTs.
Maturation and functional plasticity of cellular respiratory apparatus in flies’ eyes
Authors: Zupancic Gregor , Rudolf Jerneja , Meglic Andrej
Published: June 2021

Phototransduction and oxidative phosphorylation are closely related in insect photoreceptor cells. We assessed the status and plasticity of this relationship during the course of blowflies’ adult lives. Absorption spectra of the eyes exposed to anoxia and illumination were measured optically in living white‐eyed blowflies of different ages, using dynamic differential absorption spectroscopy. The changes of respiratory pigments’ redox states were then extracted using PCA‐based spectral deconvolution. Responses to anoxia gave us pigments’ dynamic ranges while 10 second illuminations showed responses of mitochondria to sudden changes in metabolic loads. In animals kept on a 12h/12h day/night cycle there was a 3.4 (haem b) to 5.6 (haem c) fold increase in the dynamic range in three weeks post‐eclosion. In dark‐reared animals the increase was from 2.0 (haem a3) to 3.1 (haem c) fold. By reversing culturing regimes we showed this dynamic range plasticity is retained troughout adult life, yet its changes never reached the amplitudes of control experiments. These results demonstrate flies’ eyes present a good model for studying the bioenergetics of aging animals.
Metabolic Oversupply and Mitochondrial Dysfunction as a Cause of Diaphragmatic Failure after Mechanical Ventilation
Authors: Picard Martin , Liang Feng , Hussain Sabah , Goldberg Peter , Godin Richard , Danialou Gawiyou , Chaturvedi Rakesh , Rygiel Karolina , Matecki Stefan , Jaber Samir , DesRosiers Christine , Karpati George , Ferri Lorenzo , Turnbull Douglass M , Taivassalo Tanja , Petrof Basil J
Published: June 2021

Mechanical ventilation (MV) unloads the diaphragm and leads to ventilator‐induced diaphragmatic dysfunction (VIDD). The cellular basis of VIDD is poorly understood, but oxidative stress is involved. A known cause of oxidative stress and skeletal muscle impairment is mitochondrial dysfunction, which can result from altered cellular energy metabolism. Therefore, we investigated indices of mitochondrial function and energy metabolism in human diaphragm after MV. Compared to controls, MV diaphragms had abnormal intramyocellular lipid accumulation (+219%, p<0.01), de‐phosphorylation of the cellular metabolic sensor AMPK (−64%, p<0.01), and downregulation of SIRT1 (−50%, p<0.01). Mitochondrial biogenesis and antioxidant gene systems were globally downregulated, as indicated by gene expression profiling. Consequently, whole muscle mitochondrial enzyme activities were decreased by 35–70%. MV diaphragms contained abnormally high levels of mitochondrial cytochrome c oxidase (COX) deficient myofibers that co‐localized with intracellular lipid accumulation, and which contained high levels of clonally‐expanded mtDNA deletions. MV‐induced contractile inactivity results in an overabundance of energy substrate supply relative to demand, a potent metabolic imbalance that triggers mitochondrial ROS production, and may lead to compromised diaphragmatic function.
Effects of epicatechin rich cocoa on REDUX status in human skeletal muscle
Authors: Ramirez-Sanchez Israel , Taub Pam , Ciaraldi Theodore , Hogan Michael , Murphy Anne , Ceballos Guillermo , Henry Robert , Maisel Alan , Villarreal Francisco
Published: June 2021

Increases in tissue oxidative stress levels are associated with alterations in mitochondrial structure/function. We performed a study in 5 heart failure, type 2 diabetes patients where treatment (Tx) with (−)‐epicatechin (Epi) rich cocoa led to the recovery of skeletal muscle (SkM) mitochondrial cristae and increases in markers of biogenesis. In this study we evaluated SkM levels of indicators of tissue oxidative stress before and after 3 months of treatment with Epi‐rich cocoa containing ~100 mg of Epi. Biopsy SkM samples were assayed for changes in total thiols, nytrotyrosine and carbonylation levels and compared to values obtained from SkM of normal subjects. Prior to Tx, there were significant decreases in levels of SkM thiols and increases in nitrotyrosine and carbonylation levels which were restored by the use of Epi‐rich cocoa to levels similar to controls. To understand the mechanisms underlying these changes, a mouse model of diabetes was used (high fat diet; HFD). Similar changes were observed in SkM thiols, nitrotyrosine and carbonylation levels which were restored by Epi Tx for 2 weeks (1 mg/kg BID). SkM levels of molecular regulators of reactive oxygen species metabolism (superoxide dismutase and catalase) were decreased with HFD and restored by Epi. These results indicate that Epi can apparently reverse metabolic alterations in SkM REDUX status by exerting effects on key enzymatic control systems.
Activation of Brain Peroxisome Proliferator‐Activated Receptor‐γ Inhibits Lipopolysaccharide‐Induced Central Inflammation and Sympathetic Excitation
Authors: Zhang Zhihua , Yu Yang , Wei Shun-Guang , Felder Robert B.
Published: June 2021

Inflammation in the brain cardiovascular regulatory regions has recently emerged as an important factor contributing to augmented sympathetic drive in hypertension and heart failure. Activation of peroxisome proliferator‐activated receptor (PPAR)‐γ, a nuclear transcription factor, has been shown to inhibit inflammation in brain and peripheral tissues. We hypothesized that activation of brain PPAR‐γ would inhibit hypothalamic inflammation and sympathetic excitation in response to a lipopolysaccharide (LPS) challenge. In anesthetized rats, intracerebroventricular (ICV) injection of LPS (5 μg) increased (p<0.05, vs baseline) renal sympathetic nerve activity (RSNA, 44.2 ± 7.2 %), mean blood pressure (MBP, 13.9 ± 2.5 mmHg ) and heart rate (HR, 97.2 ± 21.6 bpm). LPS decreased (p<0.05, vs vehicle) PPAR‐γ mRNA (−28.7 %) in hypothalamic paraventricular nucleus (PVN), and increased PVN mRNA for tumor necrosis factor‐α (311.7 %), interleukin‐1β (374.1 %), and cyclooxygenase‐2 (341.1 %), as well as cerebrospinal fluid levels of prostaglandin E2 (171.2 %). ICV pretreatment with the PPAR‐γ agonist pioglitazone (10 μg) significantly attenuated all of these LPS‐induced responses. These data demonstrate that brain PPAR‐γ activation counteracts central inflammatory mechanisms leading to sympathetic excitation and so may be an appropriate target for therapeutic intervention in heart failure and hypertension.
Colocalization of estrogen receptor β (ERβ) with spinally projecting and vasopressinergic neurons in the hypothalamic paraventricular nucleus (PVN) during pregnancy
Authors: Coldren Kevin Max , Brown Randall J , Kline David D , Hasser Eileen M , Heesch Cheryl M
Published: June 2021

Spinally projecting neurons in the rostral ventrolateral medulla (RVLM) and the PVN are sympathoexcitatory. The PVN contributes to increased sympathetic nerve activity in near‐term pregnant (P) compared to nonpregnant rats (NP). ERβ is highly expressed in RVLM‐projecting PVN neurons in male and female rats, but little is known about ERβ expression in PVN neurons in pregnancy. Immunohistochemistry was used to evaluate colocalization of ERβ with spinally projecting (retrograde tracer = fluorogold, FG) and vasopressin (AVP) cells in the PVN (−1.8 and −2.12 mm caudal to bregma) in 6 NP and 6 P female rats. Total number of AVP (primarily posterior magnocellular region) and FG labeled cells was similar in NP and P, while ERβ cells tended (P = 0.12) to be higher in NP. Total % co‐labeling with ERβ was greater in spinally projecting compared to AVP cells in both NP (33 ± 4 vs 21± 8%) and P rats (31 ± 4 vs.3 ± 8%). The number of both spinally projecting and ERβ immunoreactive cells was greater in the ventrolateral parvocellular (vlp) compared to the dorsal parvocellular subregion of the PVN in both P and NP rats. Although in the vlp the number of FG‐ERβ co‐labeled cells was greater in NP (27± 5) than P (16± 3) rats, the % of spinally projecting cells expressing ERβ was not different ( NP = 48 ±9% ; P =48 ±9%). We conclude that changes in ERβ expression alone cannot explain increased PVN mediated sympathoexcitation in P rats. NIH R01 HL091164 (CMH)
GLT‐1 over‐expression mitigates chronic bladder inflammation and GLuR1 trafficking
Authors: Roman Kenny , Stephens Robert L.
Published: June 2021

Strategies to attenuate glutamate induced changes in synaptic activity may lead to better options in visceral pain management. Previous data in our laboratory showed that animals over‐expressing the glutamate transporter GLT‐1, crucial for glutamate removal, had a visceromotor response (VMR) to urinary bladder distension (UBD) that was inhibited 57–64%. The current study showed that intrathecal pretreatment with dihydrokainate (DHK; 0.03mM), a selective GLT‐1 blocker, reversed this response and intracisternal administration of DHK was not effective. Also, a chronic bladder inflammation model was established via intraperitoneal treatment of cyclophosphamide (80mg/kg; day 0, 2, 4, and 6) and measured the VMR associated with urinary bladder distension in mice. The data showed that cyclophosphamide treatment significantly increased VMR (66%–120% at 0.15 and 0.2mL) and the effect was diminished by GLT‐1 over‐expression. To study trafficking of the GLuR1 subunit from the cytosol to the membrane in the lumbosacral spinal region of animals with chronic bladder inflammation, cyclophosphamide was administered as previously described, and the results showed a 33% increase in total GluR1 and a 27% increase in membrane GLuR1 compared to controls. A small increase was recorded in the cytosol (<10%). GLT‐1 over‐expression attenuated the increased total (9%) and membrane (11%) GLuR1expression. Overall, this study suggests that GLT‐1 over‐expression mitigates visceral nociception due to chronic bladder inflammation and trafficking of GLuR1 to the membrane may be involved. Supported by NIH DK 071839 (RLS) and APS Porter Fellowship (KR).
Acute inflammation alters the response to stretch in mouse extensor digitorum longus muscle afferents
Authors: Wilkinson Katherine A. , Hochman Shawn
Published: June 2021

Inflammatory pain alters muscle spindle responses to stretch (Wenngren, et al, 1998; Ro & Capra, 2001), but the exact mechanism of sensitization is unknown. We used an in vitro mouse hindlimb muscle‐nerve preparation to study plasticity in afferent responses to ramp and hold stretches following acute inflammation (0.5 mg/kg lipopolysaccharide (LPS) 18 hrs before experiment). Following LPS, units had lower baseline firing and more units were only active during stretch (6/16 LPS vs. 3/26 control). The LPS units also displayed a significantly greater decrease in firing frequency over the 4 s stretch. Some LPS units (3/16) are likely group III/IV instead of muscle spindle afferents; they had very small responses to stretch and did not show the characteristic frequency adaptation. Overall, the acute systemic inflammatory state induced by LPS clearly alters the response profile of muscle afferents to stretch, and therefore proprioceptive signaling. Further study on these changes are planned to provide insight into underlying mechanisms, their behavioral relevance, and to identify strategies that normalize function.

Supported by K12 GM000680 NIH/NIGMS More Division (KAW) and Pfizer (SH).
Greater autonomic support of blood pressure in older women
Authors: Barnes Jill Nicole , Hart Emma C , Curry Timothy B , Nicholson Wayne T , Eisenach John H , Wallin B Gunnar , Charkoudian Nisha , Joyner Michael J
Published: June 2021

In young women, tonic autonomic nervous system activity contributes less to resting blood pressure than that in men. It is unknown whether the autonomic support of blood pressure increases in postmenopausal women, therefore we examined the effect of autonomic blockade on blood pressure and how this relates to baseline muscle sympathetic nerve activity (MSNA) in women. Five young (23±4 yr) and 6 older (57±3 yr) women were examined before and during autonomic blockade using trimethaphan camsylate (TM). MSNA burst frequency and burst incidence were higher in older women (24±2 vs. 14±3 bursts/min; 41±3 vs. 23±5 bursts/100hb, respectively; p<0.05). Older women had a greater drop in mean arterial pressure (MAP; −25±2 vs. −8±3 mmHg; p<0.01) and total peripheral resistance (−0.7±0.1 vs. −0.2±0.1 AU; p<0.01) during autonomic blockade. MSNA bursts were abolished by TM within minutes. Baseline burst frequency and burst incidence were associated with the decrease in MAP during autonomic blockade (r=−0.64, r=−0.63; respectively; p<0.05). In conclusion, autonomic blockade reduces MAP to a greater extent in older women, and this decrease in blood pressure is associated with resting levels of MSNA. Our results suggest that autonomic support of blood pressure is greater in older women compared to young women. Elevated sympathetic nerve activity may explain the increased incidence of hypertension after menopause.
Preferential increase in splanchnic sympathetic activity induced by hepatic cirrhosis in rats
Authors: Estrela Heder Frank Gianotto , Castro Eduardo Ferreira , Naffah-Mazzacoratti Maria da Graça , Bergamaschi Cassia Toledo , Campos Ruy Ribeiro
Published: June 2021

Increased sympathetic activity and arterial hypotension are found in cirrhotic patients. The aim of this study was to evaluate the control of renal (rSNA) and splanchnic (sSNA) sympathetic nerve activity by the arterial baroreceptors in Wistar rats with cirrhosis induced by bile duct ligation (BDL), experiments were performed four weeks after BDL. Mean arterial pressure (MAP), heart rate (HR), rSNA and sSNA were recorded under anesthesia. The baroreflex sensitivity (BS) was assessed by the reflex changes of rSNA and sSNA induced by alterations in MAP in response to vasoactive drugs. Renal and hepatic noradrenaline was assessed by HPLC. Cirrhotic rats (CR, n=6) showed decreased MAP (control (CO, n=6):108±2 and CR:95±2mmHg) and increased HR (CO:352±10 and CR:396±13bpm). In CR group, significant increase in the basal sSNA (CO:36±8 and CR:115±13pps) accompanied by increased hepatic noradrenaline level (CO:0.7±0.1 and CR:1.4±0.1ng/mL), besides increase in the BS for PHE (CO: −0.47±0.1 and CR: −1.75±0.3) and for SNP (CO: −0.34±0.1 and CR: −1.01±0.2). However, in CR group a significant decrease in rSNA (CO:92±7 and CR:57±5pps) accompanied by no changes in rSNA BS or renal noradrenaline level (0.14±0.1ng/mL). The present study shows that in the initial phase of cirrhosis a preferential increase in sSNA appears independently of arterial baroreceptor dysfunction.

Financial support: CAPES and CNPq.
Molecular inhibition of endoplasmic reticulum (ER) stress targeted to the subfornical organ (SFO) prevents slow‐pressor angiotensin‐II (Ang‐II) mediated hypertension
Authors: Young Colin N. , Guruju Mallikarjuna , Butler Scott D. , Davisson Robin L.
Published: June 2021

We have shown that ICV administration of the chemical ER stress inhibitor TUDCA ameliorates slow‐pressor Ang‐II hypertension (HTN); however the precise brain nuclei involved are unknown. Since the SFO is critical in this model of HTN, we tested the hypothesis that ER stress in SFO is causally linked to Ang‐II HTN. A new adenovirus encoding the ER chaperone GRP78 was constructed (AdGRP78) in order to inhibit ER stress site‐selectively in the SFO. In vitro studies in Neuro2A cells confirmed AdGRP78 was targeted to ER and inhibited Ang‐II‐induced ER stress biomarker induction. In vivo targeting of AdGRP78 to SFO of C57 mice caused robust SFO‐selective expression of the GRP78 transgene and inhibition of the ER stress biomarker CHOP in this region following ICV injection of the chemical ER stress inducer thapsigargin (1ug; AdLacZ 1.6 ± 0.1 vs AdGRP78 1.1 ± 0.1, p<0.05). To test if AdGRP78‐mediated inhibition of ER stress in SFO prevents slow‐pressor Ang‐II HTN, mice underwent SFO‐targeted injection of AdGRP78 or AdLacZ and were instrumented with radiotelemeters (DSI) for mean arterial blood pressure (MAP) recordings. After recovery, osmominipumps were implanted for 2 wk slow‐pressor Ang‐II infusion. AdGRP78 prevented the development of slow‐pressor Ang‐II HTN (day 14 MAP: AdLacZ 138 ± 3 vs AdGRP78 106 ± 3 mmHg, p<0.05, n=4–6). These data implicate ER stress in the SFO as a key mediator of slow‐pressor Ang‐II HTN.

HL063887
Immunohistochemical expression and nitration of MnSOD in the adrenal gland and the carotid body of rats exposed to chronic intermittent hypoxia
Authors: Moya Esteban , Arias Paulina , Iturriaga Rodrigo
Published: June 2021

Oxidative stress is associated with the hypertension induced by chronic intermittent hypoxia (CIH), but the mechanisms involved in the increased ROS levels are not entirely known. Since, ROS levels are regulated by antioxidant enzymes, such as MnSOD, it is likely that a reduction in MnSOD levels or on its enzymatic activity produced by nitration of tyrosine residues may contribute to the oxidative stress. Thus, we studied the effects of CIH on the immunoreactive levels of MnSOD and 3‐nitrotyrosine (3‐NT), and the nitration of MnSOD in the carotid body and adrenal glands. Sprague‐Dawley male rats were exposed to CIH (5% O2, 12 times/hr for 8 hr/day) for 7 days. Tissues extracted from anesthetized rats, were fixed, included in paraffin, cut and processed for immunohistochemical detection of MnSOD and 3‐NT. In addition, tissues were processed for immunoblot measurements of MnSOD levels and nitration. CIH increased the MnSOD levels in the carotid body and adrenal glands, but also increased the nitration of MnSOD. Thus, our results suggest that despite of the increased MnSOD levels, CIH may also reduce the activity of the enzyme due to the formation of 3‐NT. Therefore, this novel post‐transductional modification of MnSOD induced by CIH may contribute to the oxidative stress.

Supported by FONDECYT 1100405 & AT 24110172
Carotid body afferent activity stimulation by PACAP is mediated through PKC pathway
Authors: Roy Arijit , Wilson Richard James Alfred
Published: June 2021

Pituitary adenylate cyclase‐activating polypeptide (PACAP) but not vasoactive intestinal peptide (VIP) injected into the common carotid artery stimulates ventilation, suggesting activation of PAC1 receptors within the primary peripheral respiratory chemoreceptor, the carotid body (CB). Possible biochemical pathways downstream of the PAC1 receptor include G‐proteins activation of (a) PLC which stimulates protein kinase C (PKC) and (b) cAMP which stimulates protein kinase A (PKA). Here we elucidate the relative importance of PKC and PKA in mediating PACAP's effects on carotid sinus nerve activity. We used an ex‐vivo artificially – perfused rat CB preparation and measured integrated CSN activity during normoxia (PO2 ≃ 100 Torr/PCO2 ≃ 35 Torr) and tested effects of PACAP 1–38 (100 nM) alone and against a background of : (a) PKC inhibitors – GF 109203X (GF;10 μM) and Chelerythrine chloride (CC;20 μM) and (b) PKA inhibitor – H89 (10 μM). We demonstrate PACAP stimulates CSN activity in a biphasic manner, with an initial transient stimulatory phase, followed by a steady‐state phase. PKC inhibitors, GF and CC (n=6) reduced both peak and steady state PACAP responses by over 50%. The PKA inhibitor H89 had no effect on the transient phase but reduced the steady state by 17%. In conclusion, PACAP induced stimulation of CSN activity is mediated primarily through a PLC‐IP3 ‐ PKC, not a cAMP‐PKA pathway.
Exposure to Acute Intermittent Hypoxia (IH) Prolongs Sympathoexcitatory and Pressor Responses Evoked by NMDA in the Hypothalamic Paraventricular Nucleus (PVN)
Authors: Kandlikar Sachin S. , Andrade Mary Ann , Calderon Alfredo S. , Toney Glenn M.
Published: June 2021

Exposure of rats to acute IH induces long term facilitation (LTF) of sympathetic nerve activity (SNA). Neurons of PVN subserve cardio‐respiratory function, project to brainstem and spinal cord regions controlling SNA, and are activated by short‐term (7 days) exposure to chronic IH. Here we tested the hypothesis that acute IH not only induces LTF of SNA, but facilitates sympathoexcitatory responses to acute PVN neuronal activation. Sprague Dawley rats (300–350g) were anesthetized (urethane/chloralose), ventilated (100% O2) and prepared for recording of splanchnic (n=3) or renal (n=3) SNA and mean arterial pressure (MAP, n=6). Responses to unilateral PVN injection of N‐Methyl‐D‐aspartic acid (NMDA, 200 pmol in 50 nl) were determined before and after exposure to acute IH (n=6), which was achieved by alternately changing FiO2 from 100% to 15% every 3 minutes. Compared to pre‐acute IH values, ongoing SNA, MAP and ETCO2 were unchanged after an average of 10 hypoxic cycles. Thus, LTF of SNA was not observed. Likewise, the amplitude of SNA and MAP responses to PVN NMDA was unaltered by acute IH. By contrast, whereas NMDA evoked responses of SNA and MAP prior to AIH were transient as expected, responses following acute IH were persistent (lasting >1 h). These data suggest that exposure to acute IH prolongs sympathetic and pressor responses to PVN NMDA even in the absence of sympathetic LTF. Support: HL102310 & HL 088052 (GMT)
Pattern of breathing in a mouse model of Down Syndrome (DS)
Authors: DeRuisseau Lara Roberts , Murphy Stacy L , Zabycz Sylvia A , DeRuisseau Keith C
Published: June 2021

DS results from trisomy 21 and is the most common cause of mental retardation in the US. Additionally, people with DS display muscle weakness and cardiorespiratory complications that may contribute to their altered breathing. We hypothesized that Ts65Dn mice, a model of DS, would exhibit an altered pattern of breathing compared to control mice. Unrestrained barometric plethysmography was used to quantify frequency (F; breaths/min), tidal volume (TV; mL/breath), and minute ventilation (MV; mL/min) in 12 month colony control (n=6) and Ts65Dn (n=6) mice during exposure to room air and hypercapnia (7% CO2, balanced air). During quiet breathing, Ts65Dn mice displayed reductions (control vs. Ts65Dn; mean ± sem; p<0.05) in TV (0.36 ± 0.02 vs. 0.26 ± 0.02) and MV (60.0 ± 6.1 vs. 40.0 ± 2.9), with no differences in F (168 ± 9 vs. 152 ± 7). When MV with room air was normalized to expired CO2 the differences between groups remained. The breathing response to hypercapnia was elevated compared to baseline in both groups, with differences between groups in F (371 ± 19 vs. 315 ± 15 bpm), TV (0.62 ± 0.11 vs. 0.42 ± 0.04 mL/breath), and MV (188.2 ± 9.3 vs. 135.8 ± 18 mL/min). These preliminary findings indicate the Ts65Dn mouse has an altered pattern of breathing compared to control mice. These data also suggest that Ts65Dn mice are an appropriate model to study ventilation in DS, although further investigation is warranted. LMC R&D Committee.
The role of self‐regulation in modulating the heart‐brain connection
Authors: Moravec Christine Schomisch
Published: June 2021

Cardiovascular disease is characterized by autonomic imbalance. The sympathetic nervous system is activated in order to compensate for declining cardiac function, but the increased activation actually has deleterious effects over time. Mortality in end‐stage heart failure is predicted by elevated plasma norepinephrine levels and a decrease in overall heart rate variability. Beta adrenergic blocking drugs are increasingly used to prevent the cardiac remodeling which follows myocardial infarction or other stresses to the myocardium. Together with sympathetic overdrive, the parasympathetic branch of the autonomic nervous system is correspondingly down‐regulated, and it is now clear that decreased parasympathetic input to the cardiovascular system may be equally harmful, promoting a pro‐inflammatory state which further exacerbates disease. Biofeedback‐mediated stress management is a method for training individuals to control the balance of sympathetic and parasympathetic nervous system activation. Such training can be used to increase parasympathetic and decrease sympathetic activation, with corresponding effects on remodeling and disease progression. Work in our research program focuses on testing the hypothesis that patients with cardiovascular disease can be taught to improve autonomic balance, and that this improvement will result in decreased symptoms, improved quality of life, and possibly even disease reversal. Patient populations which are currently being studied and which will be discussed include those with end‐stage heart failure awaiting transplantation and those with documented coronary artery disease.
Estradiol enhances conditioned place preference to cocaine in male rats
Authors: Silva Richard D , Amadeo Waldo , Rivera Jose G. , Ramos Tania , Torres Yvonne , Segarra Annabell C
Published: June 2021

Gender differences are reported in the subjective effects of cocaine, as well as in the response to treatment. Animal studies show that in ovariectomized rats, estradiol enhances sensitization, as well as conditioned place preference to cocaine. Furthermore, blocking estrogen receptors with ICI 182,780, in gonadally intact females blocks cocaine‐induced behavioral sensitization and conditioned place preference. We observed a similar situation in males. Gonadectomized males that receive estradiol, but not DHT, become sensitized to cocaine after repeated administration. Administration of ICI 182,780 curtails the development of conditioned place preference to repeated cocaine injections in gonadally intact males. These results indicate that estrogen receptors are essential for conditioning to the rewarding effects of cocaine. We are currently exploring the mechanism involved. This work was supported by the NINDS Specialized Neuroscience Research Program of the U54NS39405 and the NIGMS RISE Program R25Gm061838.
Anion selectivity and counter‐ion cation permeation in glycine receptor‐channels
Authors: Moorhouse Andrew , Sugiharto Silas , Carland Jane , Lewis Trevor , Barry Peter
Published: June 2021

The polarity of the permeant ion (ion charge selectivity) is a major determinant of the physiological function of cys‐loop receptor‐channels at central synapses. Our studies aim to determine the molecular and biophysical basis of ion permeation in α1 glycine receptors (GlyRs) expressed in HEK‐293 cells, using patch clamp techniques. We previously reported a mutation to a critical M2 transmembrane domain residue (A251E) converts the channel from anion to cation selective, with the mean Cl− to Na+ permeability ratio (PCl/PNa) changing from 25.1 to 0.31. Mutations to neutralize or invert the charge of basic residues within an intracellular (“portals”) or extracellular (K104) component of the extended permeation pathway did not affect PCl/PNa, supporting the notion of a discrete ion charge selectivity filter at the internal end of the M2 delineated pore. With more precise consideration of liquid junction potentials (calculated using activities), the PCl/PNa in WT GlyRs is 11.8±0.4 (n=32) in the absence of divalent cations, and 16.2±0 (n=6) in 4 mM external Ca2+. PCl/PCs is even lower (5.0±0.5, n=6). A mutant anionic GlyR with a larger pore diameter (P250Δ) shows greater relative Na+ permeation. Hence counterion cations permeate through GlyRs, the relative extent of which depends on the pore size, the hydrated cation size and the presence of divalent cations. Funded by UNSW Faculty Research Grants.
Neuronal and non‐neuronal steady‐state pHi and recovery from NH4+‐induced acid loads
Authors: Ruffin Vernon Alexander , Boron Walter F.
Published: June 2021

Regulation of neuronal function requires a tightly controlled intracellular pH (pHi) in both neurons and non‐neuronal cells by the major pHi‐regulating transport proteins, the sodium hydrogen exchangers (NHE's) and sodium‐coupled bicarbonate transporters (NCBT's). The regulation of steady‐state pHi was examined in neurons and non‐neuronal cells (presumably astrocytes) in the same primary culture of rat hippocampus. Fluorescent recordings using BCECF, a pH‐sensitive dye were taken while perfusing cultured cells on gridded coverslips, for later identified using immunocytochemistry. Neurons have a relatively wide range of steady‐state pHi values in a non‐CO2/HCO3− buffer, with a mean of 7.40, whereas non‐neuronal cells have a more narrow steady‐state range, with a mean of 7.25. When examining the recovery rate (dpHi/dt) from an NH4+‐induced acid load in the absence of CO2/HCO3−, we did not observe much difference between cell populations within the pHi range 6.9–7.1. At more acidic values, non‐neuronal cells have a greater dpHi/dt. In the presence of CO2/HCO3−, the dpHi/dt in both cell populations increased, although the increase in non‐neuronal cells was more pronounced. In summary, the recovery of pHi from NH4+‐induced acid loads in both neurons and non‐neuronal cells requires both HCO3− independent transporters (presumably NHEs) and HCO3−‐dependent transporters (presumably NCBTs).
Modulation of GABAA‐ergic synaptic currents by GABAB‐ergic presynaptic inputs in mouse locus coeruleus neurons
Authors: Jin Xin , Cui Ningren , Zhong Weiwei , Oginsky Max , Zhang Shuang , Jiang Chun
Published: June 2021

Norepinephrinergic neurons in the locus coeruleus (LC) play an important role in neuromodulation of a variety of neurons. These cells are modulated by both GABAA‐ergic and GABAB‐ergic synaptic inputs. The GABAA‐ergic input produces inhibition of LC neurons. However, the GABAB effect remains controversial. Through GIRK channels, activation of the GABAB‐ergic receptors in post‐synaptic neurons leads to hyperpolarization and an inhibition of spontaneous firing activity, whereas the presynaptic GABAB‐ergic modulation can be inhibitory or excitatory depending on the primary neurotransmitter targeted. To reveal the GABAB‐ergic modulation, we studied LC neurons in mouse brain slices. In whole‐cell current clamp, an exposure to 5 μM baclofen produced hyperpolarization, accompanied with ~20% decrease in input resistance and ~30% inhibition of firing activity. The same baclofen treatment, in contrast, produced significant depolarization with increased input resistance when 1 mM GDP‐β‐S was added to the pipette solution, suggesting a disinhibiton of the LC neurons by presynaptic GABAB‐ergic input. Therefore, the modulation of the GABAA‐ergic post‐synaptic currents by presynaptic GABAB receptors was studied in voltage clamp. The frequency, but not the amplitude, of GABAA‐ergic miniature post‐synaptic currents was strongly inhibited by 5 μM baclofen and augmented by 20 μM phaclofen, respectively. These results thus suggest that activation of GABAB receptors produces postsynaptic inhibition, whereas the presynaptic GABAB modulation seems to serve for a feedback control of the GABA release from presynaptic terminals.
Ketamine Enhances Morphine‐induced Analgesia and Suppresses Morphine Tolerance in Diabetic Neuropathic Pain
Authors: Wang Xiu-Li , Zhang Ying-Ze , Liu Fei-Fei , Cao Qian-Qian , Guo Yue-Xian
Published: June 2021

NMDA receptors play important role in neuropathic pain and morphine tolerance. Morphine analgesia is significantly reduced in painful diabetic neuropathy. We determined the effect of spinal NMDA receptor inhibition on morphine analgesia and tolerance in diabetic neuropathy. Paw withdrawal threshold (PWT) was measured by Von Frey monofilaments in rats with streptozotocin (STZ)‐induced diabetic neuropathy. The possible maximum analgesia percentage (PMAP) was calculated based on PWT. Ketamine (2, 4, 8, 16 mg/kg, i.m.) and morphine (1, 2, 3, 5 mg/kg, s.c.) dose‐dependently increased PWT. The PMAP values of ketamine and morphine were less than 40% at 30 min after injection. Pretreatment with ketamine (2, 4, 8 mg/kg) significantly increased morphine analgesic. Furthermore, ketamine (4 mg/kg) pretreatment increased the PMAP of morphine to 82% compared with that (32%) of morphine alone (P<0.01). Morphine tolerance was induced in rats with diabetic neuropathy by 5‐day administration. The PMAP values significantly decreased from 78% (1st day) to 18% (5th day) during morphine administration. However, ketamine (0.5, 1, 2 mg/kg, i.m.) dose‐dependently increased PMAP values of morphine (1 mg/kg) in morphine tolerance rats with diabetic neuropathy. Collectively, ketamine significantly increased morphine analgesia and effectively suppressed morphine tolerance in diabetic neuropathy.
Effect of neonatally induced brain serotonin depletion on anxiety‐like behavior of adult offspring wistar rats
Authors: Silveira Anderson Luiz Bezerra , Laureano-Melo Roberto , Fonseca Fabricia Viana , Lustrino Danilo , Mencalha Rodrigo , Reis Luis Carlos , Côrtes Wellington Silva
Published: June 2021

Serotonin (5‐HT) plays an important role in the central nervous system embryogenesis of mammals. Indeed, the serotonergic neurons are the earliest to be generated and can modulate neuronal plasticity. Some studies showed that brain 5‐HT depletion in the neonatal period leads to a plastic cytoarchitectonic change of the serotonergic system, which influences several brain systems ontogeny. Therefore, we investigated the effect of systemic pCPA (p‐chloro‐phenylalanine) injection (100mg/kg, sc), from the 8th at 16th post‐natal day, on the behavioral response of adults female and male rats. For this purpose, we use the forced swimming, open field and elevated plus maze tests. Male rats programmed with pCPA had longer time spent in open arms in plus maze test and lower immobility time in forced swimming test than the controls (148.8±8.8 vs. 99.7±21.4s and 109.0±7.8 vs. 158.1±18.2s, respectively). Programmed female rats had higher number of quadrants traversed in open field test, longer time spent in open arms in plus maze and lower immobility time in forced swimming tests than the control females (137.7±8.5 vs. 101.8±3.8; 140.7±3.1 vs.78.0±7.9s and 85.3±13.1 vs. 168.0±3.4s, respectively). These findings suggest that programming with pCPA alters neural circuits involved with anxiety‐like behavior in adult rats. In addition, we conjecture that the decrease in brain levels of 5‐HT during development may have led to a higher expression of 5HT1A receptors, especially in female rats.

Support: FAPERJ & CNPq
Biomechanical analysis of primary upper limb muscle contributors to a functional pulling movement
Authors: Saul Katherine R , Daly Melissa , Vidt Meghan E , Marsh Anthony P
Published: June 2021

Better understanding of the muscles that are dominant contributors to upper limb functional tasks may inform rehabilitation in older individuals at risk for disability. Our aim was to determine the muscles primarily responsible for hand movement during pulling. We used an upper limb musculoskeletal computer model that incorporated upper limb joints, physiologic force‐generating properties for all muscles crossing the shoulder, elbow, and forearm, and delays associated with neural activation. A horizontal pulling task was performed by a 50th percentile adult male (5′11″, 180lbs) against 6lb resistance; joint kinematics and EMG were recorded. In the computer simulation, contributions of 32 individual muscles in the measured movement were determined using induced acceleration analysis, which calculates the effect of each muscle's force, applied in isolation, on hand movement. The simulation successfully replicated experimental kinematics (mean error 0.0003°; max 0.06°) and muscle activations. Of the 12 muscles identified as substantial contributors to pulling, 8 cross the shoulder. Our novel simulations suggest that shoulder muscles play an important role in successful functional pulling and are more influential to this upper limb task than elbow and forearm muscles; they may warrant more attention in strengthening programs in at‐risk groups. NIH #5R24HD050821‐02, NSF CBET‐0828115, WFU SRF and CCCRF.