bluuebunny/crossref_metadata_2025 huggingface dataset:
Leptin Controls Arterial Pressure and Sympathetic Activity Through PI3K Signaling in POMC Neurons
Authors: Harlan Shannon M , Morgan Donald A , Rahmouni Kamal
Published: June 2021

Leptin is an adipocyte‐derived hormone that acts in the brain to promote weight loss and arterial pressure (AP) elevation by increasing the sympathetic nerve activity (SNA). We previously demonstrated the importance of PI3K in mediating the AP and renal SNA effects of leptin. However, the specific PI3K isoform and neuronal population that mediates these effects is unknown. We hypothesized that the catalytic subunits of PI3K in proopiomelanocortin (POMC) neurons are critical for leptin‐induced increases in SNA and AP. To test this, we generated mice lacking the catalytic p110α or p110β subunits of PI3K specifically in POMC neurons (p110αflox/flox/POMCCre and p110βflox/flox/POMCCre). In wild type mice, intracerebroventricular (ICV) injection of leptin (2 μg) increased renal SNA by 297±60% and mean AP by 18±6 mmHg. In contrast, the renal SNA and mean AP responses to ICV leptin were substantially (P<0.05) reduced in p110αflox/flox/POMCCremice (46±35% and 9±5 mmHg, respectively). Strikingly, deletion of p110β in POMC neurons (p110βflox/flox/POMCCremice) did not alter ICV leptin‐induced increase in renal SNA (297±44%) and mean AP (27±4 mmHg) indicating that loss of leptin effects is specific to the disruption of p110α subunit of PI3K. Our data identifies the p110α subunit of PI3K in POMC neurons as a critical mediator of the renal sympathetic and arterial pressure effects of leptin.
Hypothalamic Paraventricular Nucleus (PVN) contributes to diet‐induced obesity hypertension
Authors: Stocker Sean D
Published: June 2021

Diet‐induced obesity elevates sympathetic nerve activity (SNA) and arterial blood pressure (ABP). PVN neurons influence SNA and ABP through mono‐ and poly‐synaptic projections to the brainstem and spinal cord. The purpose of the present study was to determine whether PVN neurons contribute to elevated SNA and ABP in diet‐induced obesity. Male Sprague‐Dawley rats (250–300g) were placed on a low‐fat (LF) or moderate high‐fat diet for 15 weeks and segregated into obesity‐resistant (OR) and obesity‐prone (OP) groups. OP versus OR or low‐fat rats had a significantly higher body weight (OP: 774±7g, OR: 630±7g, LF: 607±8g, n=6 per group) and adiposity (OP: 7.8±0.4%, OR: 5.6±0.5%, LF: 4.4±0.6%). Inhibition of PVN neurons by bilateral microinjection of glycine (0.5M/60nL/side) produced a significantly greater fall in lumbar SNA (OP: −15±3%, OR: −7±1%, LF: −5±2%, P<0.05) and mean ABP OP: −12±3mmHg, OR: − 6±2mmHg, LF: −6±1mmHg, P<0.05) but not splanchnic SNA (OP: −2±2%, OR: −2±2%, LF: 1±1%). Inhibition of PVN neurons with the GABAA agonist muscimol (2mM/60nL/side) produced similar responses. A linear regression analysis revealed a significant correlation between adiposity and the glycine‐induced fall in lumbar SNA (r=0.462) and mean ABP (r=0.525) but not splanchnic SNA (0.046). These findings suggest PVN neurons contribute to obesity‐induced sympathoexcitation and hypertension.
Regional blood flow of the mesencephalic ventral tegmental area increases during spontaneous fictive motor activity in decerebrate cats and rats
Authors: Liang Nan , Matsukawa Kanji , Ishii Kei , Endo Kana
Published: June 2021

The mesencephalic ventral tegmental area (VTA) has been supposed to play an important role in the central generation of the cardiovascular response to exercise (Nakamoto et al. J Appl Physiol 2011), but whether the neural activity in the VTA increases accompanying the cardiovascular response during exercise remained unclear. The present study aimed to investigate whether the regional blood flow (RBF) in the VTA, which is ascribed to the neural activity, changes in association with the cardiovascular responses during spontaneous fictive motor activity. Cats and rats were decerebrated at the premammillary and precollicular level and then paralyzed. A probe of laser Doppler flowmetry was inserted into the VTA or substantia nigra, and the RBF was measured during 1) fictive motor activity, 2) transient occlusion of the abdominal aorta, and 3) stretching of the triceps surae muscle. RBF of the VTA, but not substantia nigra, increased accompanying a pressor response in all of the three conditions. Importantly, the increment of vascular conductance in the VTA was larger during fictive motor activity as compared to that during muscle stretching, while the vascular conductance decreased during aortic occlusion. The present results indicate that the neural activity in the VTA contributes to the cardiovascular response during fictive motor activity in decerebrate cats and rats.
Catecholaminergic neurons projecting to the paraventricular nucleus (PVN) of the hypothalamus are essential for adjustments to respiratory challenges
Authors: King T Luise , Heesch Cheryl M , Ruyle Brian C , Kline David D , Hasser Eileen M
Published: June 2021

Brainstem catecholamine neurons modulate sensory information and participate in control of breathing. These neurons have wide projections, including to the PVN. To test the hypothesis that responses to respiratory challenges require PVN‐projecting catecholamine neurons, we injected anti‐dopamine beta‐hydroxylase saporin (DSAP, n=6) or IgG control (n=6) into the PVN to retrogradely lesion catecholamine neurons. After 2 weeks, respiratory (plethysmography) measurements were made in awake rats during normoxia (N), increasing intensities of hypoxia (12%, 10%, 8% O2), and hypercapnia (5% CO2/95% O2). DSAP efficacy was validated by decreases in tyrosine hydroxylase immunoreactive neurons in A1/C1 (−67%) and A2/C2 neurons (− 56%). DSAP significantly decreased breathing rate during N and all intensities of hypoxia. Tidal volume and minute ventilation (VE) were impaired at higher hypoxic intensities (10, 8% O2; e.g. VE at 10% O2: IgG = 4±0.5, DSAP = 2.6±0.6 arbitrary units). Depressed ventilation in DSAP rats was associated with significantly lower arterial O2 saturation at all hypoxic intensities. DSAP also reduced ventilatory responses to 5% CO2 (VE: IgG = 4.9±0.6, DSAP = 2.5±0.1 arbitrary units). Data indicate that catecholamine neurons projecting to the PVN are critical for peripheral and central chemoreflex respiratory responses and maintain arterial blood oxygen levels during hypoxic stimuli. HL98602
TRPA1 mediates amplified sympathetic responsiveness in rats with femoral artery occlusion
Authors: Xing Jihong , Lu Jian , Li Jianhua
Published: June 2021

Muscle metabolic by‐products stimulate thin fiber muscle afferent nerves and evoke reflex increases in sympathetic nerve activity (SNA) and blood pressure (BP). It has been reported that metabolically sensitive transient receptor potential cation channel subfamily A, member 1 (TRPA1) appears on sensory afferent neurons and plays a role in regulating SNA and BP responses evoked by muscle contraction. The responses of sensory nerves (neurons) to muscle metabolites are altered under the conditions of insufficient limb blood supply in peripheral artery disease (PAD), induced by femoral artery ligation in rats. In turn, SNA and BP responses are exaggerated by an ischemic insult to the muscle. Our data show that 1) TRPA1 is expressed in C‐fiber of DRG neurons; 2) femoral occlusion increases the protein levels of TRPA1 in the dorsal root ganglion (DRG) (optical density: 0.97 ± 0.02 in control; and 1.68 ± 0.03 after ischemia, P < 0.05 vs. control; n = 6 in each group); and 3) renal SNA and mean BP responses to AITC (40μg/kg, a TRPA1 agonist) are also enhanced after chronic ischemia (61±5%, 31±5 mmHg in controls; and 89±8%, 44±3 mmHg after ischemia, P<0.05 vs. control, n=14 in each group). Thus, alternations in sensory nerves'TRPA1 likely contribute to enhanced sympathetically mediated cardiovascular responses under circumstances of chronic muscle ischemia observed in PAD patients. (Supported by NIH P01 HL096570 & AHA EIA 0840130N)
Tyrosine hydroxylase expression is elevated in the intermediolateral cell column of upper, but not lower thoracic spinal segments of rats with ischemic heart failure
Authors: Scrogin Karie Elizabeth , Samarel Allen , Koshman Yevgeniya
Published: June 2021

Sympathetic activity is often elevated in ischemic heart failure (IHF), but the target organs that receive elevated input are difficult to determine. A5 and C1 adrenergic cell groups of the brainstem project to the spinal cord and regulate sympathetic activity. Therefore, we predicted that expression of tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine synthesis, would increase in rats with IHF, primarily in spinal segments that send sympathetic projections to the heart. TH immunofluoresence was measured in the intermediolateral cell column (IML) of spinal segments T4‐T12 of male Sprague Dawley rats after sham‐ or coronary artery ligation. 8 weeks after ligation, TH intensity increased from 395–186 ± 87–36% of controls (P<0.05, MI n=4, control n=6) in spinal segments T4‐T7, but not in lower spinal segments. TH protein expression in adrenal glands was not different between groups. Sympathetic projections to the heart emanate primarily from T1‐T5, while those to the adrenal gland arise primarily from T7‐T11. Our data suggest that TH immunoreactivity in the IML may serve as a marker of patterned sympathetic output, and that sympathetic activity is not ubiquitously elevated in IHF.
Valproic acid does not affect decreased insulin secretion in WFS1‐deficient pancreatic islets
Authors: Koks Sulev , Ivask Marilin , Hugill Alison , Terasmaa Anton , Plaas Mario , Vasar Eero
Published: June 2021

Wolfram syndrome, an autosomal recessive disorder characterized by juvenile‐onset diabetes mellitus and optic atrophy, is caused by mutations in the wolframin (WFS1) gene. WFS1 negatively regulates endoplasmic reticulum (ER) stress signalling and therefore have a role in the pathogenesis of diabetes. Valproic acid (VPA), a widely used anticonvulsant and mood‐stabilizing drug, normalized the glucose intolerance in Wfs1 mutant mice, but had no effect on the course of glucose tolerance in wild‐type (WT) mice. The aim of this study was to investigate insulin secretion in isolated pancreatic islets of WFS1‐deficient (Wfs1KO) mice and also evaluate insulin secretion in the presence of VPA. In addition, the content of proinsulin in the isolated islets was measured. In general Wfs1KO pancreatic islets secreted less insulin compared to WT and Wfs1HZ islets. Differences in proinsulin amount were not statistically significant although there was a trend that Wfs1KO had an increased level of proinsulin. We found significantly reduced insulin secretion in Wfs1 mutants and no effect of VPA treatment.

This study was supported by the Frontiers of Functional Genomics and by The European Regional Development Fund.
EXPRESSION AND ACTIVITY OF NEUTRAL SPHINGOMYELINASE IN THE KIDNEY OF DIABETIC RATS.
Authors: del Rocio Bautista María , del Valle‐Mondragón Leonardo , Cano‐Mártinez Agustina , Mercado Adriana , Franco Martha , Zarate‐Calleja Efren
Published: June 2021

Background

Sphingomyelinases (SMase) are enzymes that specifically catalyze the hydrolysis of sphingomyelin into ceramide. An increase in tissue ceramide could result from either increased ceramide production (such as, SMase mediated sphingomyelin hydrolysis), or decreased ceramidase activity. However, the role of sphingolipids and key enzymes of sphingolipids metabolism in diabetes are not well understood. The purpose of this study was to determine the ceramide concentration, expression and activity of SMase in the kifney of diabetic rats.

Methods

The rats were divided into two groups: control and diabetic rats (n=10 each). Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) at 60 mg/kg body. After a followup of 4 weeks, rats were sacrificed and kidney tissue samples were processed and stored as needed. The ceramide generation and the expression of neutral‐SMase were measured by immunohistochemistry. N‐SMase activity was monitored indirectly using 10‐acetyl‐3,7‐dihydroxyphenoxazine, a sensitive fluorogenic probe for H2O2.

Results

Four weeks after treatment with STZ, in the kidney of diabetic rats increase the ceramide concentration. Conversely, the expression and activity of N‐SMase were lower en the kidney of diabetic rats than those of the control groups.

Conclusions

These results suggest that nSMase did not increase the ceramide content in the kidney of diabetic rats.
Prolonged high glucose treatment increased STIM1/Orai1 protein expression and enhanced store‐operated Ca2+ entry in human glomerular mesangial cells
Authors: Chaudhari Sarika , Wang Yanxia , Ding Min , Ding Yanfeng , Yuan Joseph , Ma Rong
Published: June 2021

Stromal interaction molecule (STIM1) and Orai1 regulate intracellular Ca2+ signaling via store operated Ca2+ entry (SOCE). Our previous study has demonstrated that STIM1 is required for SOCE in human mesangial cells (HMC) of kidneys. However, its pathological relevance in HMCs is unknown. The aim of the present study was to determine the effect of high glucose (HG) on STIM1/Orai1 protein expression and SOCE in cultured HMCs. Western blot was used to detect the expression levels of STIM1/Orai1 proteins. Fura‐2 fluorescence ratiometry was utilized to assess SOCE. Whole‐cell patch clamp was employed to measure store‐operated currents. With 25 mM HG, STIM1/Orai1 protein expressions did not significantly increase until 7 days after treatment. With 7‐day incubation, HG significantly elevated STIM1/Orai1 expressions in the range of 10–30 mM concentrations. Ca2+ imaging showed that the cyclopiazoic acid (30 μM)stimulated Ca2+ entry was significantly enhanced in HMCs with 25 mM HG treatment for 7 days, but not 3 days. The HG‐enhanced Ca2+ entry was nearly abolished by GSK‐7975 (10 μM). Similarly, the whole cell currents induced by intracellular Ca2+ store depletion were significantly augmented in the HMCs with 7 day HG, but not in HMCs with 1 day HG treatment. Taken together, our results indicate that a prolonged HG treatment enhances SOCE in HMCs by upregulating STIM1/Orai1 protein expression.(Support by NIH)
Dose‐dependent effects of losartan and salt‐sensitivity of diabetic nephropathy in ZSF1 rats
Authors: Plato Craig F. , Silva Stacia L , Joly Kristin M , Horn Todd R
Published: June 2021

Diabetic nephropathy (DN) is the leading cause of end stage renal disease. Renal complications of diabetes are influenced by hypertensive comorbidity while angiotensin receptor blockade (ARB) represents current standard of care. We hypothesized that high salt (HS) intake would exacerbate, and ARB by losartan (LOS) would attenuate progression of DN. Male obese ZSF1 (OB‐ZSF1) rats (8 wk) underwent unilateral nephrectomy, and received (po, qd for 15 wks) vehicle (VEH), LOS (3, 10, or 30 mg/kg), or 0.9% NaCl water. Male lean ZSF1 (CON) served as controls. Blood pressure data, urine and blood samples were collected every 4 weeks. Metabolic data support progression of diabetes in OB‐ZSF1. LOS attenuated while HS exacerbated arterial hypertension indicating salt‐sensitivity in OB‐ZSF1. Albuminuria was time‐dependently increased, dose‐dependently inhibited by LOS and exacerbated by HS by 18 wks of age. Endpoint renal injury urinary biomarkers Kim‐1 and osteopontin were increased (6‐, 40‐fold, respectively) in VEH relative to CON, while LOS inhibited their excretion. HS dramatically further increased urinary Kim‐1 and osteopontin excretion (~3‐fold each) relative to VEH. LOS improved, while HS worsened glomerular filtration rate and renal plasma flow relative to VEH as directly evaluated at endpoint. We conclude LOS effectively attenuates progression and severity of DN, while HS increases renal injury in OB‐ZSF1.
Nrf2 mediates the antioxidant effect of DJ‐1 in the kidney
Authors: Cuevas Santiago , Yang Yu , Asico Laureano , Escano Crisanto S , Armando Ines , Jose Pedro A
Published: June 2021

Dopamine D2 receptor (D2R) dysfunction is associated with increased oxidative stress in the kidney & high blood pressure. We have reported that DJ‐1 is regulated by D2R in the kidney. Nrf2 is a transcription factor that regulates the expression of several antioxidant genes. We hypothesize that Nrf2 is involved in the DJ‐1‐mediated regulation of ROS production in the kidney. Nrf2 activity was assessed by the expression of its prototypic target genes NQO1, and GST Yc‐2. Silencing DJ‐1 expression in mouse renal proximal tubule cells (mRPTCs) via DJ‐1 siRNA decrease the expression of DJ‐1 (75±15%, n=4, P<0.01), NQO1 (75±15%, n=4, P<0.01), GST Yc‐2 (61±8%, n=4, P<0.05), and Nrf2 (63±11%, n=3, P<0.01). Selective renal silencing of DJ‐1 in mice by the renal subcapsular infusion of DJ‐1 siRNA decreases expression of DJ‐1 (31±5.9%, n=3, P<0.01), NQO1 (46±11%, n=3, P<0.01), GST Yc2 (28±7%, n=3, P<0.05), and Nrf2 (57±6%). the promoter activity of NRF2 in mRPTCs was decreased when DJ‐1 expression was silenced(58%±1, n=5, P<0.01). Silencing of D2R expression also decreased NRF2 promoter activity (53%±2, n=5, P<0.01). Quinpirole a D2R agonist, decreases ROS in mRPTCs but does not modify the mRNA expression of Nrf2 or DJ‐1, or Nrf2 promoter activity. However in the presence of H2O2, silencing DJ‐1 increased ROS production (190.3%±33 vs control, n=6) and the effect is blocked by pretreated with quinpirole (111,1%%±10 vs control, n=6). Our results suggest that DJ‐1 can inhibit renal ROS production via the activation of antioxidant genes mediated by Nrf2, the antioxidant effect of D2R could be independent of DJ‐1 Nrf2, however, D2R is necessary for normal NRF2 and DJ‐1 activity.
INHIBITION OF NITRIC OXIDE GENERATION ENHANCES SUPEROXIDE PRODUCTION IN CULTURED HK2 and M1 CELLS
Authors: Islam Mohammed Toriqul , Maiti Arpan Kumar , Sato Ryosuke , Majid Dewan S.A.
Published: June 2021

An enhancement in superoxide (O2−) activity due to nitric oxide (NO) deficiency facilitates renal tubular sodium reabsorption leading to the condition of salt‐sensitivity. To examine the interaction between NO and O2− activity in the renal epithelial cells, cultured HK2 (proximal tubular) and M1 (distal tubular) cells were incubated with L‐NAME (1 mM) or vehicle for 2, 4 and 6 hrs in different wells (n=6 in each case) separately. O2− activity in incubated cells was determined by Lucigenin‐chemiluminescence assay. Compared to vehicle treated cells, L‐NAME treated cells showed increment in O2− activity within 2 hr of incubation in HK2 cells (973 ± 99 to 1691 ± 165 RLU/μg protein) and in M‐1 cells (1768 ± 244 to 3548 ± 622 RLU/μg protein). Such increases in O2− activity was prevented in cells co‐incubated with NO donor compound, DETA NONOate (1 mM). In separate sets, L‐NAME treated cells were co‐incubated with O2− scavenger, tempol (5 mM) or with NADPH oxidase inhibitor, apocynin (5 mM). O2− activity was minimal in cells co‐incubated with L‐NAME + tempol (189 ± 13 and 345 ± 24 RLU/μg protein) and L‐NAME + apocynin (242 ± 25 and 333 ± 27 RLU/μg protein) in HK2 and M1 cells respectively indicating that NADPH oxidase is the major source of O2− production in these cells. These data demonstrate that a reduction in NO generation leads to increases in O2− production induced by NADPH oxidase activity in renal epithelial cells.
Renal Collecting Duct Cell‐specific mVps34 Deletion Decreases Nephron Number and Increases Nephron Size
Authors: Chen Jian‐Kang , Hu Bo , Chen Jianchun
Published: June 2021

The class III PI3‐kinase, mVps34, has been implicated in autophagy and endocytosis, but its physiological role in the renal collecting duct system is unknown. We generated an mVps34 gene‐floxed mouse and crossed it with Hoxb7‐Cre mice to produce collecting duct‐specific mVps34 knockout mice (mVps34cdKO, KO) and used mVps34flox/flox;Hoxb7‐Cre(−)littermates as controls (mVps34Ctrl, Ctrl). By embryonic day 17.5, KO mice began to show dilated collecting ducts and proximal tubules, the earliest morphological alterations observed. At birth, KO mice exhibited markedly decreased kidney‐to‐body weight ratios (0.21±0.02% in KO vs. 0.42±0.18% in Ctrl; N=10, P<0.0001), with indistinguishable body weight. By 4 weeks of age, KO mice had a 48% reduction in glomerular number (N=6, P<0.0008) and exhibited unproportionally thinner renal medulla, marked glomerular and tubular hypertrophy, glomerulosclerosis, interstitial fibrosis, proteinuria, and elevated BUN. By 12 weeks of age, KO mice developed hypertension (129.12±0.5 mmHg vs. Ctrl mice: 110.54±0.01; N=7, P<0.0001). TUNEL‐ and cleaved caspase 3‐positive cells were markedly increased, with decreased Akt phosphorylation. These data demonstrate an essential role for mVps34 in the renal collecting duct dictating nephron number and maintaining normal renal structure and function through a mechanism of antagonizing programmed cell death.
Impaired renal function in Belgrade rats
Authors: Veuthey Tania V , Hoffmann Dana , Vaidya Vishal S , Wessling‐Resnick Marianne
Published: June 2021

Belgrade rats carry a disabling mutation in the iron transporter DMT1. Although DMT1 plays a major role in intestinal iron absorption, the transporter is also highly expressed in kidney, where its function remains unknown. Our goal was to characterize renal physiology of Belgrade rats. Animal protocols were approved by the Harvard Medical Area Animal Care and Use Committee. Male Belgrade rats died prematurely with ~50% survival at 20 weeks of age. Necropsy results indicate that renal failure was the major cause of death. By 15 weeks of age, Belgrade rats displayed thickening of glomerular membrane and collagen deposits in tubules and glomeruli. Creatinine clearance was significantly lower compared with heterozygote littermates. Urinary biomarkers of kidney injury, including albumin, fibrinogen and Kim‐1, were significantly elevated. Belgrade rat kidney non‐heme iron levels were not different from controls but urinary iron excretion was higher. Our results show impaired renal function and altered renal morphology in Belgrade rats, with sclerosis and fibrosis. Loss of DMT1 function results in renal injury, and studies are underway to elucidate the molecular basis. Work supported by NIH grant ES014638
Construction of a tagged truncated Selenoprotein K to determine function
Authors: Fukuyama Allyson , Norton Robert , Sonido Charles , Sanchez Gabriella , Hoffmann Peter
Published: June 2021

Selenium (Se) is an essential trace element that biologically functions as proteins, known as selenoproteins through selenocystine. Selenoproteins are categorized as antioxidant enzymes; however, the functions of several selenoproteins are still unknown. Selenoprotein K (SelK) is an endoplasmic reticulum (ER) transmembrane protein that has been shown to be important in calcium flux during immune cell activation. To understand the function of SelK, it is important to identify proteins that interact with SelK. Plasmids encoding full‐length mouse SelK were previously designed and constructed in our laboratory. This served as a template in an Inverse PCR (iPCR) reaction to create a truncated version of SelK missing the C‐terminal portion when SelK is cleaved by calpain. Two PCR products were obtained and one was a truncated product. Gel purification technique used to separate desired PCR product, that was then ligated, digested, transformed, and then purified. The final plasmid generated from the inverse PCR approach was found to contain the desired altered C‐terminal portion of SelK. iPCR is an effective approach to generate mutations or deletions for desired encoding for protein products of research interest. Construction of the plasmid encoding the truncated SelK product will facilitate structure‐function studies and allow determination of the role the C‐terminal portion of SelK in binding other proteins.
Lower limb movement related increases in simulated arterial pressure during walking
Authors: Goreham Joshua A , Peterson Clayton , Sheriff Don D , Ladouceur Michel
Published: June 2021

A study has shown the potential of rotational movement in increasing arterial blood pressure during shoulder motion (Sheriff et al,2009). The aim is to investigate the effect of motion on simulated arterial pressure during walking. Ten participants walked while wearing mid‐thigh and mid‐shank mounted strain gauges connected to water filled tubes and bony landmark markers. Pressure measured during walking by the strain gauges ranged from 0 to 130mmHg. Pressure measurements fluctuated during the gait cycle with minimums during mid‐stance and swing and peaks during early stance and swing. A kinematic model was used to evaluate the influence of different movement components on the pressure measurements. The measured and calculated pressures were found to have similar profiles with slightly different averages (measured:55mmHg; calculated:60mmHg, p=0.08). The orthostatic pressure component has a large contribution with movement related pressures having both positive and negative effects. This study shows the interaction between the different components in modulating arterial pressure and the need to investigate these mechanisms in order to optimize exercise in conditions like peripheral artery disease.
Molecular adaptation of skeletal muscle to high‐intensity resistance exercise in aged males
Authors: Rivas Donato A , Rice Nicholas P , Fielding Roger A
Published: June 2021

Impaired contractile‐induced muscle growth may contribute to sarcopenia: the age‐associated loss of muscle mass and function that is manifested by loss of strength, contractile capacity and endurance. MicroRNAs (miRNAs) are novel posttranscriptional regulators that may play an important role in contractile‐induced adaptations in human skeletal muscle. We performed a study comparing global changes in gene expression using DNA microarrays and PCR arrays targeting 60 specific microRNAs (miRNA) that regulate skeletal muscle metabolism (myo‐miRs), in young (YNG, 22±0.06 yrs) and older (OLD, 74±1.5 yrs) males 6 hours (6H) after a single bout of high‐intensity resistance exercise (RE). We observed in OLD only 48 genes were altered 6H after RE while in YNG there were 201 genes altered by RE. There was no change in the expression of myo‐miRs in OLD after RE and in contrast the expression of myo‐miRs were generally decreased in YNG. These results reveal a significant decrease in the contractile capacity of aging skeletal muscle and may highlight a significant role of myo‐miRs in this decreased response.
Nitrate supplementation augments cutaneous reactive hyperemia in healthy humans
Authors: Eder Jena K , Keen Jeremy T , Levitt Erica L , Wong Brett J
Published: June 2021

Beetroot juice (BRJ) has been shown to increase levels of nitrates in the vascular system, which can subsequently be converted to NO through either NOS‐independent or‐dependent pathways. We tested the hypothesis that nitrate supplementation would augment cutaneous reactive hyperemia (RH) via NOS‐independent mechanisms. Subjects were equipped with 2 microdialysis fibers (control and 20 mM L‐NAME to inhibit NOS) on the left forearm as well as a blood pressure cuff for arterial occlusion. Arterial occlusion was performed for 5 minutes and the cutaneous RH was continuously monitored using laser‐Doppler flowmetry (LDF). A total of 3 occlusions were performed with at least 10 min rest between occlusions. Mean arterial pressure was continuously measured and cutaneous vascular conductance (CVC) was calcluated as LDF/MAP and normalized to %CVCmax. Subjects reported to the lab for pre‐and post‐BRJ RH protocols. Subjects consumed concentrated BRJ (.45g nitrates; 5mM) for 3 days. BRJ significantly increased total RH (area under curve) at both control (pre: 2518±337 vs. post: 4562±481 %CVCmax*sec) and L‐NAME (pre: 2495±117 vs. post: 4408±901 %CVCmax*sec) sites. There was no difference in total RH between control and L‐NAME pre‐BRJ or post‐BRJ. These data suggest BRJ improves RH in the cutaneous microvasculature through NOS‐independent mechanisms.
Role of Transient receptor potential vanilloid 4 in neutrophil activation and acute lung injury
Authors: Tang Christine , Yin Jun , Kuebler Wolfgang M
Published: June 2021

Introduction

Transient receptor potential vanilloid 4 (TRPV4) is a cation channel expressed in lung parenchyma and inflammatory cells. TRP channels have been implicated in the activation of inflammatory cells and the pathogenesis of acute lung injury (ALI), thus we hypothesized that TRPV4 deficiency or inhibition may attenuate inflammatory responses and ALI.

Method

To test this, in vivo and ex vivo studies were performed using TRPV4−/− mice.

Results

Genetic deficiency or pharmacologic inhibition of TRPV4 attenuated the functional, histological and inflammatory hallmarks of ALI following intratracheal instillation of hydrochloric acid or lipopolysaccharide (LPS). TRPV4−/− compared to TRPV4+/+mice had an increased survival rate following i.p. LPS. Perfused lung studies showed decreased lung weight increase in TRPV4−/− mice in response to platelet activating factor (PAF). Transfusion of TRPV4+/+ vs. TRPV4−/− blood into perfused lungs led to greater Evans blue extravasation following PAF administration. Neutrophils from TRPV4−/− mice had attenuated increases in intracellular calcium and reactive oxygen species and showed reduced endothelial transmigration in response to PAF.

Conclusion

Our findings identify a role for TRPV4 in neutrophil activation, and suggest TRPV4 as a promising pharmacological target for the prevention and potentially treatment of ALI.

Supported by CREMS, University of Toronto and NSFC # 81000028
TrkB kinase activity is necessary for spontaneous recovery of ipsilateral rhythmic phrenic activity following cervical spinal cord hemisection
Authors: Zhan Wen‐Zhi , Mantilla Carlos , Sieck Gary
Published: June 2021

Following unilateral C2 spinal hemisection (SH), descending excitatory drive to phrenic motoneurons is disrupted causing ipsilateral diaphragm muscle paralysis. After SH, there is gradual recovery of phrenic nerve activity reflecting neuroplasticity. We hypothesized that this neuroplasticity is dependent on BDNF signaling via its TrkB receptor and kinase activity. In this study we used TrkB(F616A) knockin mice, that permit rapid and selective inhibition of TrkB kinase activity by 1NMPP1. Adult TrkB(F616A) mice were treated with 1NMPP1 (25 μM) or vehicle in the drinking water starting 3 days post‐SH. At the terminal experiment, bilateral diaphragm EMG was monitored to determine the proportion of animals displaying recovery of ipsilateral phrenic activity. At 3 days post‐SH, no animals displayed ipsilateral diaphragm EMG activity (0/10). At 14 days post‐SH, 40% (6/15) of TrkB(F616A) mice displayed ipsilateral phrenic activity, although EMG amplitude remained substantially below contralateral levels. Following 1NMPP1 treatment, no animals displayed ipsilateral diaphragm EMG activity (0/10; p<0.05). In control, wild‐type mice, 1NMPP1 treatment (for 11 days) had no evident effect on bilateral diaphragm EMG activity (n=7). These results support an essential role for TrkB kinase activity in the spontaneous recovery of rhythmic phrenic activity post spinal cord injury.

Supported by NIH grant HL096750.
Implication of nuclear progesterone receptor on respiratory control in newborn mice.
Authors: Rossignol Orlane , Potvin Catherine , Uppari NagaPraveena , Marcouiller François , Bairam Aida , Joseph Vincent
Published: June 2021

We tested the hypothesis that nuclear progesterone receptors (PR) contribute to respiratory control development by studying newborn mice lacking PR expression (PRKO) or wild type controls (WT) at 1 and 4 postnatal days (P1 – P4). Minute ventilation was assessed under normoxia and in response to hypoxia (FiO2=14%, 12%, 10%, 20 min each) using whole body plethysmography. Hypoxic ventilatory response (HVR) was expressed as percentage changes from normoxia, apnea frequency was evaluated under normoxia. In P1 and P4 PRKO mice HVR was 2–3 times lower than in WT for the whole range of FiO2, during the early (peak response) and late (steady state) phase of HVR. In P1, the frequency of spontaneous apnea was lower in PRKO than in WT, while the frequency of post‐sigh apnea frequency was similar. In P4, the frequency of spontaneous and post‐sigh apnea was higher in PRKO than in WT. We conclude that the nuclear progesterone receptor plays a prominent role in respiratory regulation in newborn mice. Both phases of HVR (early and late) are disrupted in PRKO mice, likely indicating effects on peripheral chemoreceptors and central integration. Supported by CIHR.
Acute Lung Injury Alters Synaptic Drive to Nucleus Tractus Solitarius Neurons
Authors: Getsy Paulina M , Hsieh Yee‐Hsee , Nethery David , Wilson Christopher G , Jacono Frank J
Published: June 2021

Acute lung injury (ALI) is a severe clinical problem where the progression of lung damage contributes to altered ventilatory patterns. Changes in central control of breathing may influence the pathophysiology of ALI. We have previously shown ALI induced upregulation of inflammatory cytokine production in the nucleus tractus solitarius (nTS). The effects of this immune‐modulation on central neural circuits are unclear. We hypothesized that ALI alters synaptic inputs to the nTS neurons. To test this hypothesis, we cut transverse slices (300 micrometers) of brainstem from juvenile (P15–P18) male Sprague‐Dawley rats 7d after intratracheal instillation of bleomycin (0.2 units) or saline. Whole‐cell perforated patch‐clamp recordings were done to characterize the spontaneous excitatory synaptic events in nTS neurons from bleomycin (n=4) and saline (n=2) animals. At least 200 synaptic events were analyzed from each cell and a student's t‐test was used for comparison. In bleomycin animals, the amplitude of spontaneous excitatory post‐synaptic currents (EPSCs) was significantly reduced (18.1 vs. 19.7 pA, p = 0.02) and the decay time of EPSCs was significantly longer (6.8 vs 5.7 ms, p < 0.01). Our data suggest that bleomycin lung‐injured animals have altered central processing of vagal afferent input that may contribute to breathing dysregulation in ALI.
Ibuprofen does not reverse time‐dependent increase in hypoxic ventilation in chronically hypoxic rats
Authors: De La Zerda David J , Go Ariel , Ries Andrew , Soler Xavier , Powell Frank L
Published: June 2021

Chronic sustained hypoxia (CSH) increases inflammatory cytokine gene expression in carotid bodies and medullary respiratory centers, and increases carotid body chemoreceptor and ventilatory responses to hypoxia in rats. Non‐steroidal anti‐inflammatory drugs (ibuprofen IBU) block these responses (Respir.Physiol.Neurobiol. (2011) 178: 362–369 & 381–386). We tested the hypothesis that IBU can reverse these responses after they are already established in CSH. We measured cytokine expression in medullary respiratory centers and ventilatory responses in adult male rats acclimatized to CSH (PiO2 = 70 Torr, 1 wk) before and after IBU (4 mg/kg intraperitoneal for 3 days) or sham (0.6 ml saline IP for, 3 days) treatments. We measured the effects of IBU vs. sham in normoxic animals too. Expression of mRNA for IL‐1β, IL‐6, and TNFα measured with RT‐PCR in dorsal brainstem biopsies tended to be lower with IBU compared to sham: IBU = 68, 67 and 85% of sham levels for IL‐1β, IL‐6, and TNFα, respectively. However, ventilatory and metabolic responses to hypoxia measured with barometric pressure plethysmography and stop‐flow respirometry were not significantly different between IBU and sham in CSH or normoxic rats (p>;0.1). Hence, inflammatory cytokines in the CNS and carotid bodies appear necessary for the induction but not the maintenance of an increased hypoxic ventilatory response with CSH. Supported by NIH RO1 HL‐081823.
α1G (CaV3.1) T‐type calcium channel controls NOS3 activation in pulmonary microvascular endothelial cells
Authors: Sellak Hassan , Yap Fui Chi , Alexeyev Mikhail F. , Chen Hairu , Lincoln Thomas M. , Wu Songwei
Published: June 2021

In pulmonary microvascular endothelial cells (PMVECs), Ca2+ entry through the site‐specific, voltage‐gated α1G T‐type Ca2+ channel promotes vWF release and P‐selectin surface expression. The present study seeks to determine whether α1G directly interacts with endothelial nitric oxide synthase (NOS3) and controls its activation in PMVECs. Rat NOS3 was initially cloned and confirmed to be functioning in a heterologous expression system via activation of cGMP‐dependent protein kinase that ultimately phosphorylates vasodilator‐stimulated phosphoprotein on serine239. In PMVECs over‐expressing rat NOS3 loaded with a nitric oxide (NO)‐sensitive fluorophore, DAF‐2, thrombin (2 units/mL) stimulated NO generation assessed by epifluorescence microscopy. This effect was abolished by pharmacologic T‐type Ca2+ channel blockade using mibefradil and potentiated in cells co‐expressing NOS3 and α1G. Three NOS3 deletion mutants (a.a. sequence D1: 1–450, D2: 440–860, and D3: 850–1,200) were generated and their transient expression in PMVECs revealed that D2 domain attenuated thrombin‐stimulated NO generation. Similarly, five α1G cytosolic fragments (a.a. sequence F1: 1–80, F2: 396–743, F3: 965–1272, F4: 1,538–1,610, and F5: 1,855–2,377) were cloned and expressed in PMVECs via retroviral infection. Thrombin‐stimulated NO generation was remarkably reduced in PMVECs individually expressing each of these fragments. Taken together, these data suggest a physical and functional interaction between α1G and NOS3, i.e., through binding of α1G cytosolic domains to NOS3 D2 domain, α1G controls NOS3 activation in PMVECs. Supported by HL66299.
AMP‐Kinase (AMPK) stimulation improves lung function and survival in a rat model of LPS‐induced lung injury
Authors: Jian Ming‐Yuan , Creighton Judy
Published: June 2021

Using isolated lungs, we demonstrated previously that activation of AMPK attenuated injury in a pharmacologically‐induced model of microvascular damage. LPS is known to damage the lung's microcirculation resulting in increased edema. We reasoned AMPK stimulation would improve lung function in LPS treated animals. Rats were given LPS by intraperitoneal injection (IP). One group also received AMPK activator by IP 6 hr later. Permeability was assessed via Kf measurements at 24 hr post LPS treatment. Following experiments, lungs were fixed and stained for morphometric analysis. LPS caused a 2.2 fold increase in Kf at 6hr and a 3.1 fold increase at 24 hr. Histology revealed diffuse capillary injury with thickened alveolar walls and intra‐alveolar exudates in LPS treated lungs. AMPK stimulation using metformin or AICAR following LPS‐induced injury significantly attenuated alveolar damage and decreased Kf. Furthermore, AMPK activation increased survival in LPS treated animals. 30 % of LPS treated animals died, whereas no animals in the control or metformin/AICAR treated groups died.

Conclusion

AMPK stimulation using clinically approved drugs reverses lung injury. Supported by HL102296 and HL 110803.
Ouabain activation of MAPK not dependent of caveolae: involvement on adherens junctions redistribution
Authors: Barbosa Leandro A , Souza Waldemir F , Liu Lijun , Fontes Carlos FL , Morgado‐Díaz José A
Published: June 2021

Previous studies have reported that the Na,K‐ATPase interacts with E‐cadherin to stabilize adherens junctions (AJ) and maintain the cell‐cell adhesion. In the other hands, several papers have demonstrated that the activation of signaling pathway through the cardiotonic steroids is dependent of caveolin‐1 and caveolae. Here, we have used ouabain and Caco‐2 cells, a human colorectal cancer cell that not express caveolin‐1 and not develop caveolae. We have showed that ouabain treatment (6 and 8h) resulted in significant redistribution of AJ proteins, E‐cadherin and β‐catenin from the cell‐cell contacts to the cytoplasm, concomitantly with decreased protein levels of the β1Na,K‐ATPase. Besides, ouabain treatment induced an increase of the phosphorylation levels of ERK1/2 protein, and the treatment with PD98059, a specific MAPK inhibitor abrogated this effect. Additionally, the MAPK inhibitor blocked the effect of ouabain treatment on AJ disassembly, indicating that activation of this kinase was the final event in this signaling cascade. Our findings strongly suggest that downregulation of the β1 Na,K‐ATPase and MAPK activation induced by ouabain cooperate together to mediate cell‐cell adhesion loss in Caco‐2. Also, we have demonstrated that the modulation of Na,K‐ATPase signaling pathway can be independent of caveolin‐1 and caveolae content. Supported: CNPq, FAPERJ, FAPEMIG, CAPES
Protease‐activated receptor 2 (PAR2) inactivates the pro‐apoptotic protein, BAD, via ERK1/2 and PI3K activity to decrease apoptosis in colonic epithelial cells
Authors: Iablokov Vadim , Hirota Christina Lynn , MacNaughton Wallace Keith
Published: June 2021

Colonic biopsies from inflamed colon have higher levels of epithelial apoptosis than biopsies from healthy individuals. Therapies that decrease intestinal epithelial apoptosis may decrease the inflammation observed in inflammatory bowel disease. We previously showed that signaling by PAR2, a GPCR activated by serine proteases, delayed IFN‐γ & TNF‐α‐induced apoptosis in colonic epithelial (HT‐29) cells. We aimed to determine the mechanism responsible for PAR2‐induced survival. The PAR2 agonists 2‐furoyl‐LIGRLO (2fLI), SLIGKV and trypsin all significantly reduced cleavage of caspase‐8, 9, 3 as well as PARP in response to IFNγ/TNFα. 2fLI increased phosphorylation of ERK1/2, p90RSK and Akt. 2fLI treatment stimulated the phosphorylation of pro‐apoptotic BAD at Ser112 and Ser136. Selective MAPK pathway inhibitors (U0126 & SL0101) blocked BAD Ser112 phosphorylation, whereas the PI3K‐selective inhibitor (LY294002) blocked BAD Ser136 phosphorylation. Pre‐treatment with 2fLI prior to the addition of IFNγ/TNFα increased BAD phosphorylation at both residues. PAR2 signaling reduces epithelial apoptosis in response to inflammatory cytokines via activation of pathways that inactivate the pro‐apoptotic BAD protein. Our findings may represent a mechanism whereby proteases facilitate epithelial cell survival and colonic healing after inflammation.

Funding: Crohn's and Colitis Foundation of Canada
HPLC fraction of ethylacetate extract from Orostachys japonicus induces apoptosis in human gastric cancer cells
Authors: KIM Seon‐Hee , Kwon Ji‐Hye , Ryu Deok‐Seon , Lee Dong‐Seok
Published: June 2021

Orostachys japonicus is traditionally used as anti‐inflammatory, anti‐febrile, hemostatic, anti‐dote, and anti‐cancer agent in Korea. The soluble faction of ethyl acetate (EtOAc) showed the highest anti‐cancer activity on AGS human gastric cancer cells. Further factions of EtOAc extracts were performed by HPLC. The aim of the present study was to investigate the apoptotic activity of further fraction of EtOAc fraction of O. japonicus in AGS cells. ASG cells were cultured in several concentration of further fraction of EtOAc fraction. The apoptotic effect of further fractions of EtOAc fraction was measured using MTS assay, annexin V‐FITC, PI‐labeling, and western blotting. In MTS assay, the survival rates were decreased in dose‐dependent manner. O. japonicus‐induced apoptosis was confirmed by assays with annexin V‐FITC, PI‐labeling. The increase in apoptosis level induced was associated with down‐regulation of Bcl‐2 and activation of Bax, caspase ‐3, ‐8 and ‐9.
Volume regulation in in situ human airway epithelial (calu‐3) cells: Focal Adhesion Kinase (FAK) and NKCC1
Authors: MacLellan Amber L , Marshall William S
Published: June 2021

Ion transport by FAK‐activated Na+‐K+‐2Cl− co‐transporter (NKCC1) and cystic fibrosis transmembrane conductance regulators (CFTR) is essential in cell regulatory volume responses in teleost epithelia (Marshall et al. 2005,2009). FAK‐related protein tyrosine kinase 2 (PYK2) activates CFTR in Calu‐3 cells (Liang et al. 2011), however, FAK regulation of NKCC or CFTR has not been investigated in a mammalian system. We aim to examine the possible role of FAK in regulating NKCC or CFTR ion‐transport during cell volume regulation in human airway (Calu‐3) cells. To model in vivo airway conditions, Calu‐3 cells were cultured into confluent monolayers on glass coverslips in minimal essential medium (MEM) at 37°C with 5% CO2/95% air. Cell volume regulation was examined using, 4–7 day‐old monolayers stained with BODIPY‐ceramide vital dye (1:1000 in isotonic MEM solution for 1hr) and mounted in a perfusion chamber. Monolayers were perfused with combinations of isotonic MEM 285 mOsm/kg (control), and NaCl‐equilibrated 360 mOsm/kg hypertonic MEM (hypersaline lung lavages use 3–7% NaClaq). Cell height (volume) changes were recorded over 1hr using XZt confocal micrographs. Hypertonic shock caused Calu‐3 cells shrinkage over 1–7 mins followed by regulatory volume increase (RVI) (contrasting previous reports) over 45‐mins (n=18 cells; 6 monolayers). To determine if NKCC1 or FAK‐facilitated ion transport facilitated RVI, monolayers were treated with NKCC1 inhibitor bumetanide and a FAK‐inhibitor TAE226. Inhibition of NKCC1 and FAK altered volume regulation patterns suggesting a critical mediation of airway cell volume regulation by NKCC1 and FAK. Funded by NSERC and NSHRF.
Role of phosphatidylinositol 3‐kinase in ouabain‐induced cardiac cell signaling and hypertrophy
Authors: WU JIAN , Li Daxiang , Bai Yan , Gable Marjorie , Liu Lijun
Published: June 2021

Cardiac steroid ouabain binding with Na+‐K+‐ATPase (NKA) not only induces positive inotropy in the heart, but also triggers the Phosphatidylinositol 3‐kinase (PI3K)/Akt/mTOR signaling pathway leading to cardiac hypertrophy. This study is to examine the mechanism of ouabain regulation on PI3K and NAK interaction by using cardiac‐specific PI3Kα knockout mice (KO). Adult cardiomyocytes from Cre mice (WT) and KO mice were isolated and cultured. In KO adult cardiomyocytes, PI3Kα protein and kinase activity significantly decreased, but PI3Kγ remained the same. Ouabain increased PI3Kα activity in WT myocytes; however, ouabain did not alter PI3Kγ activity in either WT or KO myocytes. Increase of Akt/mTOR phosphorylation by ouabain in WT was blunted in KO myocytes. Co‐IP study showed that ouabain increased the interaction of PI3Kα p85 and NKA α1 subunit; ouabain augmented p85‐associated NKA α1 tyrosine phosphorylation in WT myocytes. Ouabain also induced PTEN “tail” serine380 phosphorylation in WT myocytes. Moreover, ouabain increased protein synthesis rate in WT as a measurement of hypertrophy, but not in KO myocytes by [3H] leucine incorporation assay. Ouabain did not up‐regulate hypertrophic markers ANP and BNP mRNA expression. By regulating the interaction of PI3Kα and NKA, ouabain induced adult cardiomyocyte signaling and hypertrophy. (This work is supported by NIH PO1 HL036573)
Effect of Endothelial cells on RPE differentiation and Matrix Deposition
Authors: Spencer Carrie , Abend Stephanie , McHugh Kevin , Saint‐Geniez Magali
Published: June 2021

Previous studies have suggested a critical interdependence of choroidal endothelial cells (EC) and retinal pigment epithelial (RPE) cells, which are separated by the Bruch's membrane (BrM), for the maintenance of retinal function. We developed a long‐term coculture system to evaluate the effect of EC‐RPE interactions on cell behavior and matrix deposition. Human RPE cells and primary human EC were seeded on opposite sides of polyester transwells and cultured for up to 4 weeks in low serum medium. Cells and extracellular matrix deposition were analyzed using viability assay, transepithelial resistance, H&E staining, immunohistochemistry, and qPCR. Coculture with RPE sustained EC survival and proliferation in low serum for up to 2 weeks. Presence of EC induced RPE expression of matrix‐associated and visual cycle proteins. Interestingly, expression of PEDF and Thrombospondin‐1 by RPE were also significantly upregulated in coculture condition, suggesting that RPE acquired an anti‐angiogenic status. TEM and immunohistochemistry analysis revealed the accumulation of BrM‐like material on the basal side of cocultured RPE. RPE differentiation and induced deposition of certain matrix‐associated proteins in coculture further emphasizes the significance of RPE‐EC interactions in retinal homeostasis. This work was supported by the NIH Director's New Innovator Award 1DP2OD006649