bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of environment and chronic methylphenidate on anxiety‐like behavior and dopamine receptors in adolescent rodents
Authors: Gill Kathryn Estelle , Beveridge Thomas , Porrino Linda J
Published: June 2021

Environmental rearing conditions are reported to alter behavior and dopamine (DA) transmission in rodents. Administration of DA‐ergic drugs, such as methylphenidate (MPH), may have varying effects depending on environmental exposure. This study explored the interactive effects of environment and chronic MPH on DA receptors and anxiety‐like behavior in adolescent rats. Sprague‐ Dawley rats (PND 21; n=8/group) were housed in isolated/impoverished (II), pair‐housed (PH), or socially/environmentally enriched (EE) conditions for 4 weeks. At 1 week, rats were implanted with ALZET minipumps that delivered saline or MPH (2 or 8 mg/kg/day) for 3 weeks. Post treatment, rats were tested for locomotor behavior and anxiety‐like behavior in an elevated plus maze (EPM). Rats were sacrificed at PND 50; brains processed for in vitro autoradiography to quantify DA D1 and D2 receptors in striatal regions. II rats had greater locomotor activity and spent less time in the open arms of the EPM than EE and PH rats. Environment did not affect the density of D1 or D2 receptors in striatal regions. MPH did not impact any measure of behavior. However, there was a dose‐dependent decrease in DA D2 receptor density in caudate putamen and a similar trend in accumbens across housing groups. While D1 receptor density was not affected by MPH across housing groups, preliminary results suggest that D1 receptors were increased by 8 mg/kg/day MPH in II rats, but decreased in PH and EE animals. Differences in locomotor and anxiety‐like behavior between II, PH, and EE animals highlight the importance of considering housing conditions in rodent studies. MPH did not impact behavior, but did decrease D2 receptor density across housing conditions. Despite no main effect of MPH on D1 receptors, there was a trend towards an interaction between MPH and housing. Thus, chronic MPH causes alterations in DA systems, which may appear more pronounced in animals with a history of isolation.
Exploring The Molecular Mechanisms Underlying The Efficacy of Mindfulness Based Stress Reduction In Alleviating Psychological Stress in Alzheimer's Disease Caregivers
Authors: Ho Lap , Bloom Patricia A. , Vega Joan Griffiths , Pasinetti Giulio Maria
Published: June 2021

Caregiving for a dementia patient is associated with an increased risk for emotional distress, depression, dementia, physical health problems and death. Targeting caregivers as the recipients of interventions may greatly improve patient care. Mindfulness Based Stress Reduction (MBSR) is a clinical intervention based on simple meditation practices which have been shown to reduce stress and improve chronic disease outcomes for many different psychological and medical problems. We explored the effectiveness of MBSR at reducing caregiver stress, as measured by psychological and biological markers. We found that MBSR is an effective means to improve the psychological health status of caregivers for dementia patients. Interestingly, we found some of our caregiver participants benefited from MBSR while others did not. Moreover, we identified multiple biomarkers for which contents in the circulating blood were correlated with psychological indices of stress reduction following MBSR and other biomarkers for which baseline blood contents prior to MBSR were predictive of the likelihood of positive response to MBSR. Our observations provide a window into the mechanisms underlying the health benefits of MBSR intervention and provide a logical basis for developing a personalized medicine approach for applying MBSR intervention to a diverse population of caregivers. Supported by discretionary funding to GMP
Modulation of behavior by methylphenidate
Authors: Carmack Stephanie A , Howell Kristin K , Anagnostaras Stephan G
Published: June 2021

Psychostimulants containing methylphenidate (e.g. Ritalin) are the mainstay of treatment for attention deficit hyperactivity disorder. In affected individuals, low doses reduce activity and impulsivity, and improve attention. Low doses also enhance learning in control populations, which has led to off‐label use of methylphenidate as a cognitive enhancer. A reliable animal model of methylphenidate's cognitive effects has been lacking, with much research instead focusing on its addictive potential. We have developed a model using Pavlovian fear conditioning where we consistently detect methylphenidate‐induced memory enhancements at a clinically relevant dose (1 mg/kg) and memory deficits at a high dose (10 mg/kg). We also investigated the ability of these doses to induce addiction‐related behaviors. While a high dose of methylphenidate dramatically stimulated locomotor activity, produced locomotor sensitization and conditioned place preference, the therapeutic dose did not induce any of these behaviors. These results suggest that methylphenidate's cognitive effects are dose‐dependent and dissociable from its other behavioral effects. These studies were supported by NIH NIDA DA020041.
Cytochrome P450 2J2 metabolizes the endocannabinoid, anandamide
Authors: Walker Vyvyca , Hollenberg Paul F.
Published: June 2021

Cytochrome P450 2J2 (CYP2J2) is a heme containing monooxygenase that is highly expressed in hematologic malignant diseases and in several human tumor tissues and cell lines. CYP2J2 metabolizes arachidonic acid (AA) to four epoxyeicosatrienoic acids (EETs) which promote tumor cell growth by increasing cell proliferation and inhibiting apoptosis. Studies show that the concentration of anandamide (AEA), an endogenous cannabinoid related to AA, can be increased, decreased, or unchanged in cancer cells. Conflicting reports exist regarding AEA effects on cancer cell proliferation. Moreover, AEA has been shown to induce apoptosis in certain cancer cells and it is involved in the metastatic processes of migration, invasion, and angiogenesis. In addition, anandamide is metabolized by several P450s, including CYP3A4, CYP2D6, and CYP2B6. The objective of this study is to determine if CYP2J2 expressed in leukemia cells can metabolize AEA and to investigate the effects of any metabolites formed by CYP2J2 on cell proliferation and survival. LC/MS was used to identify the metabolites formed during the metabolism of AEA by CYP2J2. Studies using purified protein in the reconstituted system showed that CYP2J2 formed the 20‐hydroxyeicosatetraenoic acid ethanolamide (HETE‐EA) and several epoxygenated products, including 8,9‐, 11,12‐, and 14,15‐epoxyeicosatrienoic acid ethanolamides (EET‐EAs). The leukemia cell line, K562, metabolizes AEA to 5,6‐, 8,9‐, 11,12‐, and 14,15‐EET‐EA, in addition to 19‐HETE‐EA. Future studies utilizing CYP2J2 specific siRNA will be used to determine if the metabolites formed by the K562 cells are dependent on CYP2J2 activity. In addition, experiments will be performed to observe the effects of anandamide and its metabolites on the proliferation and apoptosis of the K562 cells. This research was supported in part by NIH grant CA16954.
Hemodynamic parameters during acute and chronic metabolic acidosis in rabbits
Authors: Celotto Andrea Carla , Ferreira Luciana Garros , Capellini Verena Kise , Albuquerque Agnes Afrodite Sumarelli , Evora Paulo Roberto Barbosa
Published: June 2021

Metabolic acidosis has profound effects on vascular tone, with impact on systemic and local flow and blood pressure (BP) control. This study investigated the effect of in vivo acute and chronic metabolic acidosis on hemodynamic parameters. Chronic metabolic acidosis (CMA) was induced by intake of NH4Cl 1% during 7 days (ad libidum), while acute metabolic acidosis (AMA) was induced by infusion of NH4Cl 6M (1ml/kg, diluted 1:10, during 3h). Dose‐response curves to Phe and ACh were performed in acidotic animals during venous and arterial BP monitoring to an indirect evaluation of vascular response. In the CMA (pH 7.15 ± 0.03) the bicarbonate reduced (13.8 ± 0.98 mmol/L) without changes on pCO2. In the AMA (pH 7.20 ± 0.01) bicarbonate decreased (10.8 ± 0.54 mmol/L) and pCO2 decreased (47.8 ± 2.54 to 23.2 ± 0.74mmHg) with simultaneous hyperventilation. No difference on arterial or venous BP was observed during Phe or ACh infusion for CMA, while for AMA the ACh infusion promoted greater arterial BP decreased (ΔBP: −28.0 ± 2.35 AMA to −14.5 ± 2.89 control mmHg). No changes on cardiac frequency or cardiac output were observed. The NOx plasma levels were normal in CMA, but were increased after AMA (25.3 ± 0.88 to 31.3 ± 0.54μM). These results point that AMA, but not CMA, was able to potentiate the ACh effect on BP accompanied by NOx increases, reinforcing the effect of pH imbalance on vascular tone and BP control. Support: FAPESP; FAEPA‐HC/FMRP.
NO‐dependent vasodilation is coupled to coordinated activation of connexin‐ and pannexin‐based hemichannels in endothelial cells
Authors: Lillo Mauricio , Gaete Pablo , Saez Juan Carlos , Figueroa Xavier
Published: June 2021

Nitric oxide(NO)activates connexin(Cx)‐based channels by S‐nitrosylation, but its effect on pannexin(Panx)channels has not been determined. We evaluated the involment of Cx‐and Panx‐based hemichannels in the endothelium‐mediated NO‐dependent vasodilation induced by acetylcholine (ACh) in rat arterial mesenteric bed of rat. Hemichannel activity was assessed by ethidium (Etd) or Lucifer Yellow(LY)uptake. Vessels did not show Etd uptake in control conditions. ACh stimulation induced a strong increase in Etd and LY uptake that was blocked by the Panx blockers probenecid and Panx10, and also carbenoxolone, but only attenuated by La3+ (Cx hemichannels blocker) and 18‐beta‐glycyrrhetinic acid. In addition, Etd uptake was also blocked by the inhibition of NO synthase with NG‐nitro‐L‐arginine (L‐NA), guanylate cyclase soluble with ODQ and PKG with KT5823. The same results were observed with the NO donor S‐nitroso‐N‐acetyl‐ D‐penicillamine (SNAP). However, the increase in Etd uptake activated by the analogue of cGMP, 8‐Br‐cGMP, was only sensitive to probenecid and KT5823, but not to La3+. Interestingly, probenecid potentiated the NO‐dependent, ACh‐induced vasodilation. These results indicate that Cx‐based hemichannels are activated directly by NO, whereas the cGMP/PKG pathway induced the opening of Panx‐formed hemichannels.

FONDECYT 1100850, 1090757, 1111033, Anillos ACT‐71
Sphingosine‐1‐phosphate receptor 1 agonist SEW2871 decreases sepsis‐induced renal microvascular permeability but not peritubular capillary hypoperfusion
Authors: Wang Zhen , Mayeux Philip R
Published: June 2021

Renal microcirculatory failure and vascular leakage are hallmarks of sepsis‐induced acute kidney injury (AKI). To investigate the mechanism of renal capillary hypoperfusion, the cecal ligation and puncture (CLP) model of sepsis was used in male 40 wk C57BL/6 mice. Since activation of sphingosine‐1‐phosphate receptor 1 (S1P1) has been reported to enhance endothelial barrier function and decrease vascular permeability, mice were administered the S1P1 agonist SEW2871 (1, 3, 10 or 30mg/kg ip) at the time of CLP. Evans blue dye leakage assay was used to measure renal microvascular permeability and intravital video microscopy was used to quantitate changes in renal peritubular capillary perfusion. CLP caused a rapid increase in capillary permeability at 2h, prior to decreased renal capillary perfusion at 4h. SEW2871 dose‐dependently decreased renal vascular permeability at 6h post CLP. However, peritubular capillary perfusion was not improved compared to CLP mice at 6h. Results suggest that renal microvascular leakage is an early event during the development of sepsis and that activation of S1P1 may decrease vascular permeability but cannot prevent the deterioration of renal capillary perfusion. Overall, our study suggested that the early renal capillary hypoperfusion is independent of the increased vascular permeability in CLP septic mice. Supported by AHA 10PRE4140065 (ZW) and NIH DK075991 (PRM).
Numerical simulation of blood flow with different red blood cell deformability
Authors: Ju Meongkeun , Low Hong Tong , Kim Sangho
Published: June 2021

The present study investigated the effect of changes of red blood cell deformability on micro‐hemodynamics by using the Lattice Boltzmann Method (LBM) with Immersed Boundary Method (IBM). The blood flow of 40% stiff red blood cells and 60% normal cells was simulated and the result was compared with that of 100% normal red blood cells. The deformability of stiff cells was 50% less than normal cells. Thus, with the stiff cells, the averaged red blood cell deformability became 20% less than normal condition, which is similar to the condition of malaria‐infected blood. The simulation was conducted at 40% hematocrit and 100 s−1 pseudo shear rate. In this study, the viscosity difference between inside and outside fluids of red blood cell membrane was considered by using a flood fill method. The results showed that the effective viscosity of blood flow with stiff red blood cells significantly increased compared with that without the stiff cells. The axial migration of the stiff cells moving away from the centerline was observed during the simulation.

This work was supported by NUS URC Grant R‐397‐000‐091‐112.
Apocynin improves exercise performance and functional vasodilation by improving KATP function in obese Zucker rats
Authors: Lu Silu , Xiang Lusha , Clemmer John , Lee Lynn , Sebai Mohamad , Hester Robert L.
Published: June 2021

An impaired exercise performance and functional vasodilation has been observed in obesity. There is an increase in reactive oxygen species (ROS), which impairs functional vasodilation in obese Zucker rats (OZ), but the mechanism by which ROS impairs vascular function remains unclear. Since our laboratory has demonstrated that KATP channel activation is required for functional vasodilation, we hypothesize that increased ROS would impair vascular KATP channels in obesity, resulting in a blunted exercise capability. Ten‐week old lean Zucker rats (LZ) and OZ were treated with apocynin (drinking water, 2 mM) for five weeks. After the treatment, maximal oxygen consumption (VO2max) was measured during treadmill running with or without glibenclamide treatment (10 mg/kg, i.p.), a KATP channel blocker. On the following day the spinotrapezius muscles were prepared for in vivo microscopy, with arcade arteriolar diameters being measured following muscle stimulation in the absence and presence of the glibenclamide (1 μM). OZ exhibited a significantly decreased VO2max (42 ± 1 ml/kg/min) and functional vasodilation (34 ± 6%) as compared with LZ (VO2max: 54 ± 1 ml/kg/min; vasodilation: 77 ± 2%). Apocynin treatment significantly increased VO2max (45 ± 1 ml/kg/min) and functional vasodilation (64 ± 1%) in OZ with no effect in LZ. Glibenclamide significantly inhibited the VO2max (46 ± 1 ml/kg/min) and functional vasodilation (39 ± 2%) in LZ and apocynin‐treated OZ (VO2max: 41 ± 1 ml/kg/min; vasodilation: 35 ± 1%) but had no effect in control OZ. These results suggest that inhibition of ROS production by apocynin improves VO2max and vascular function via improving impaired KATP channel function in OZ. (Supported by NIH HL‐89581 and HL‐51971)
Effects of photoperiod, light intensity, and their interaction on blood physiological variables of modern broilers grown to heavy weights
Authors: Olanrewaju Hammed A , Purswell Joseph L , Collier Stephanie D , Branton Scott L
Published: June 2021

This study examined the effects of photoperiod, light intensity, and their interaction on blood acid‐base balance, metabolites, and electrolytes in broilers under environmentally controlled rooms in 2 trials. The study consisted of a factorial arranged in a randomized complete block design with 9 treatments that consisted of 3 photoperiod (continuous [23L:1D] from d 8 ‐ d 56; intermittent [2L:2D], and short [8L:16D] from d 8 ‐ d 48 and 23L:1D from d 49 ‐ d 56, respectively) and exposure to 3 light intensities (10, 5.0 and 0.5 lx) from day 8 through d 56 at 50% RH. A total of 540 1‐d‐old (30 male/30 female) chicks were randomly distributed into 9 chambers. Feed and water were provided ad libitum. Venous blood samples were collected on d 14, 28, 42, and 56. Short photoperiod significantly (P ≤ 0.05) reduced BW, pH level, pO2, sO2, and Ca2+, while significantly (P ≤ 0.05) elevated pCO2, Hct, Hb, and McHc concentrations. There was only an effect of light intensity on K+. Acid‐base regulation during short photoperiod and light intensity exposure had not deteriorated despite higher pCO2 that consequently decreased blood pH, due to a respiratory acidosis. Plasma corticosterone was not affected by photoperiod, light intensity or their interaction, indicating that exposure of broiler chickens to short photoperiod markedly affects some physiological blood variables without inducing stress in modern broilers grown to heavy weights.
Dysfunction of coronary smooth muscle Ca2+ regulation in the progression of metabolic syndrome and coronary artery disease in Ossabaw miniature swine
Authors: McKenney Mikaela Lee , Kohr Meredith C , Alloosh Mouhamad , Schultz Kyle A , Bell Lauren N , Tune Johnathan D , Sturek Michael
Published: June 2021

Coronary smooth muscle (CSM) cell Ca2+ regulation was studied in the Ossabaw swine model of metabolic syndrome (MetS) and coronary artery disease (CAD). MetS was induced by excess calorie atherogenic diet for 9 or 12 months and compared to lean controls on normal diet. Isometric tension studies on isolated coronary arteries revealed that 9 months of MetS increased tension development to KCl depolarization vs. lean controls, whereas 12 months reduced tension vs. control. MetS increases CAD and CSM Ca2+ dysregulation. CSM were isolated enzymatically and digitally imaged with the fluorescent Ca2+ indicator fura‐2. There was no difference in basal Ca2+ levels between groups. We released sarcoplasmic reticulum (SR) Ca2+ stores with caffeine. The peak Ca2+ release was increased in 9 month and decreased in 12 month MetS vs. lean. This Ca2+ response largely represents the caffeine‐sensitive SR Ca2+ store capacity. After the SR store depletion, a sustained Ca2+ signal above basal levels remained in the 9 month MetS group, reflecting store‐operated Ca2+ entry and decreased Ca2+ extrusion ability. Intravascular ultrasound and micro‐computed tomography imaging showed greater atherosclerosis and extracellular calcification in 12 vs. 9 month MetS. Collectively, these data suggest that CSM undergo dedifferentiation from a contractile to an osteogenic phenotype with progressively increasing CAD. (NIH HL062552, HL092245)
Mechanism for sodium retention by insulin+glucose in diabetes may involve renal epithelial sodium channels (ENaC)
Authors: Manhiani Marlina M , Sheppard Tuere A , Brands Michael W
Published: June 2021

This study tested the role of epithelial sodium channels (ENaC) in the sodium‐retaining effect of insulin we have reported in diabetic dogs. Alloxan‐treated dogs (n=3) were maintained normoglycemic using i.v. insulin replacement. Following control, insulin replacement was stopped to begin an 8‐day diabetic period. In 2 dogs, insulin was infused 24 hr/day into the renal artery (ir.) during diabetes. Consistent with our previous results, diabetes caused sustained natriuresis in the control diabetic dog, with UNaV equaling 93 mEq/day on day 6 of diabetes vs. an average of only 52 mEq/day in the 2 diabetic dogs with ir. insulin. Amiloride (iv.) was administered in all dogs on diabetes days 7 and 8 and raised UNaV in both groups to similar levels (165 and 132 mEq/day in control and i.r. insulin dogs, respectively). In a second experiment, diabetes was induced again in all dogs, but amiloride was given during the first 2 days of diabetes. In the control diabetic dog, UNaV increased from 67 mEq/day to 199 and 111 mEq/day on days 1 and 2 of diabetes, respectively. In the 2 dogs with ir. insulin infusion, UNaV increased similarly, from an average of 70 mEq/day to 216 and 86 mEq/day on days 1 and 2 of diabetes. The elimination of the difference in diabetes‐induced natriuresis in control vs. ir. insulin‐infused dogs by amiloride suggests that ENaC contributes significantly to the sodium retaining action of insulin+glucose in diabetes.
Calmodulin and CaM kinase II govern MARCKS‐mediated PIP2‐dependent regulation of ENaC
Authors: Alli Abdel A , Bao Hui-Fang , Alli Alia A , Aldrugh Summer , Zhou Yiqun , Yu Ling , Eaton Douglas C
Published: June 2021

We have previously shown that MARCKS mediates the PIP2 dependent regulation of ENaC. Here we show that both calmodulin (CaM) and CaM kinase II may regulate the function of MARCKS within the actin cytoskeleton at the cytoplasmic face of the plasma membrane and, thereby, regulate ENaC. We mutated the calmodulin binding site within the basic effector domain of MARCKS by site‐directed mutagenesis and cotransfected the mutant or wildtype MARCKS CFP‐tagged constructs and an ENaC gamma YFP‐tagged construct into 2F3 cells. Without increasing intracellular calcium to stimulate calmodulin binding to MARCKS, we observed no difference between the two constructs in the amount of MARCKS associated with the membrane or with ENaC. We show by single channel patch clamp studies that application of the calmodulin inhibitor, calmidazolium, to 2F3 cells causes an increase in the open probability of ENaC. We also show by transepithelial current measurements that application of the CaM kinase inhibitor, KN93, to 2F3 cells causes an increase in transepithelial current. We show by sucrose density gradient ultracentrifugation, western blot, and densitometric analysis that CaM kinase II mediates actin cytoskeleton reorganization. Various signaling pathways downstream of CaM kinase II may be involved it the reorganization of the actin cytoskeleton, but further investigation will be necessary to identify the exact mechanism.
Enriched Cholesterol Diet Increases Expression of Renal Epithelial Na+ Channel Gamma Subunit in Rats
Authors: Sood Jessica , Balayan Shaina , Minas Jacqueline N. , Rodriguez Ruben , Aguil Jennifer , Nakano Daisuke , Nishiyama Akira , Awayda Mouhamed S. , Ortiz Rudy
Published: June 2021

The epithelial Na+ channel (ENaC) is a trimeric transmembrane channel expressed in multiple secretory organs. ENaC is responsible for regulation of Na+ transport and balance in the kidney with the gamma subunit (γENaC) primarily responsible for regulation. However, the impact of enriched cholesterol diet on the regulation of ENaC during insulin resistance is not well described. To examine the effects of enriched cholesterol diet and activation of the angiotensin receptor on renal ENaC protein during insulin resistance, Otsuka Long Evans Tokushima Fatty (OLETF) rats and their lean, non‐insulin resistant controls, Long Evans Tokushima Otsuka (LETO) rats were supplemented with an enriched cholesterol diet (ECD; 2% + 0.5% cholic acid) and/ or treated with an angiotensin receptor blocker (ARB; 10 mg olmesartan/kg/d) for 16 weeks. The renal ENaC alpha and gamma subunits were measured by Western blot. The results suggest that elevated dietary cholesterol is associated with a quicker increase in blood pressure and expression of the ENaC γ loop. The ENaC γ loop was suppressed in the 24‐week‐old OLETFs, further implicating that the cleavage of ENaC γ loop is involved in renal uptake of Na+ resulting in volume‐dependent hypertension.
Potassium Excretion During Antinatriuresis: Perspective from a Mathematical Model of Distal Nephron (DN)
Authors: Weinstein Alan M.
Published: June 2021

Renal excretion of Na+ and K+ is complicated by the fact that changing excretion of one solute requires adjusting the transport of both, “aldosterone (Aldo) paradox”. In this work, a DN model (Am. J. Physiol. 295:F1353, 2008), including distal convoluted tubule (DCT), connecting segment (CNT), and collecting duct (CD), is used to examine renal K+ excretion during antinatriuresis, with emphasis on hypovolemia and familial hyperkalemic hypertension (FHH). Na+ avidity is represented by modulation of DCT NaCl reabsorption, and the K+ secretion signal is an Aldo‐like effect on principal cells of CNT and CD. The first model prediction is that changes in DCT NaCl reabsorption are mediated by NaCl cotransporter density in conjunction with both peritubular Na,K‐ATPase and KCl cotransport. A second observation is that the CNT response to increased DCT NaCl reabsorption, should not only stabilize CD K+ delivery, but also compensate for increased CD K+ reabsorption, due to low CD Na+ delivery. Aldo provides this response in hypovolemia, while in FHH there is enhanced DCT NaCl transport but blunted Aldo effect. Conversely, when a K+ load increases Aldo, the model requires a concomitant decrease in DCT NaCl transport to stabilize Na+ excretion. The model emphasizes the need for two distinct DN signals, regulating Na+ excretion and K+ excretion, in contrast to a single switch apportioning NaCl reabsorption and Na+‐for‐K+ exchange.
Na‐K ATPase Regulation by Cardioglycosides: Role of AT1R
Authors: Khundmiri Syed J , Merchant Michael L , Lederer Eleanor D
Published: June 2021

Cardioglycosides are specific inhibitors of Na‐K ATPase; however, at low doses increase BP and Na‐K ATPase activity in kidney basolateral membranes. TIRF and FRET microscopy suggested that 10 pM ouabain associates with Na‐K only at cell‐cell junctions. We hypothesized that low and high molar ouabain interacts with divergent sets of surface proteins in kidney. To address this hypothesis, we treated HK11 cells with 100nM or 10pM ouabain. Cell lysates were immunoprecipitated using anti‐ouabain antibodies. Unique proteins identified by proteomics in 10pM ouabain treated samples include Ang II type 1 receptor (AT1R), ankyrin 2 and 3, Akt1, and RTK erbB‐4. Unique proteins identified from 100nM ouabain cells include RTK erbB‐2, HK ATPase, and SLC4A4 isoform 2. Na‐K ATPase, NHE1, Src kinase, and EGFR were identified at both 10pM and 100nM ouabain. Based on these data we hypothesized that 10pM ouabain stimulates Na‐K ATPase through an AT1R dependent mechanism. To address this hypothesis cells were treated with 10pM ouabain in the presence or absence of candesartan, an AT1R antagonist. Ouabain‐induced stimulation of Na‐K ATPase activity, expression, phosphorylation, and Src signaling were prevented by candesartan. These data suggest that low concentrations of ouabain may increase BP through novel interactions with proteins of the renin angiotensin system. Supported by AHA and VA
Meprins cleave the catalytic subunit of protein kinase A in a meprin isoform‐specific manner
Authors: Ongeri Elimelda Moige , Ranasinghe Ruvina , Bond Judith S
Published: June 2021

Meprins are metalloproteases that are abundantly expressed in the brush border membrane of proximal kidney tubules and small intestines. Meprins are also expressed in podocytes, skin, and leukocytes. Meprins are redistributed to the cytosol after kidney ischemia/reperfusion, and have been implicated in the pathology of diabetic nephropathy (DN). There is increasing knowledge of in vivo kidney meprin substrates, and recently villin and actin were found to be cleaved by meprins. The objective of this study was to determine whether meprins cleave the catalytic subunit of protein kinase A (PKAcat), and whether the levels of PKAcat are changed in DN. Kidney proteins from meprin αβ double knockout (KO) mice, and purified recombinant human PKAcat were incubated with activated forms of either homomeric meprin A (α‐α) or meprin B (β‐β). The proteins were resolved by SDS‐PAGE and probed by Western blot analysis using PKAcat specific antibodies. Only the meprin B isoform cleaved PKAcat. Cytosolic levels of PKAcat were elevated in kidneys from both WT and meprin KO mice with streptozotocin‐induced type 1 diabetes. Cytosolic PKAcat leveles were higher in WT diabetic kidneys than in meprin double KO diabetic kidneys. The PKA signaling pathway plays a role in glomerular extracellular matrix (ECM) metabolism. Accumulation of ECM proteins is a hallmark of DN, and meprins could modulate the pathology of DN via cleavage of PKAcat.
Insulin Signaling and Caveolae: Role of TNF alpha in the development of insulin resistance in C2C12 myocytes
Authors: Vallejo Johana , Gunn Amanda , Miraglia Gregory , Gordon Christopher , Boyack Dane
Published: June 2021

Recent research has shown that TNF‐á affects insulin signaling by up‐regulating ganglioside‐GM3 production in adipocytes; however little is known about these effects in skeletal muscle. Caveolae are specialized plasma membrane microdomains thought to serve as signaling platforms for several receptors including the insulin receptor (IR) and glucose transporters (Glut‐1, ‐4), via interaction with caveolin proteins (Cav‐1, ‐3). We hypothesized that exposure of C2C12 cells to TNFá will result in deregulation of the insulin signaling cascade and alteration of the pattern of IR and Glut‐4 localization to the plasma membrane by promoting a shift from the caveolae to other membrane domains enriched in ganglioside‐GM3 expression. Here, C2C12 myocytes were differentiated into skeletal myotubes and treated with TNF‐á, insulin, and a TNF‐á antagonist. Treated cells were subjected to Western blot analysis with antibodies against IR, phosphorylated IR, Akt, GM3‐synthase, Glut‐4, Cav‐1, Cav‐3, and actin (loading control). Western blot demonstrated higher levels of AKT, and GM3‐S after treatment with TNF‐á. Immunofuorescence analysis demonstrated a disruption of the colocalization of IR and Glut‐4 to caveolae domains in cells treated with TNF‐á, which was reversed by treatment with the antagonist. These results identify TNF‐á and gangliosides as potential therapeutic targets during insulin resistance.
Anti‐obesity effects of resveratrol: from in vivo to in vitro study
Authors: Lin Keng-Yang , Peng Kang-Yu , Hung Li-Man
Published: June 2021

Obesity is related to many chronic diseases, including type 2 diabetes, fatty liver, cardiac vascular disease, and cancers. Resveratrol (RSV, trans‐3, 4’, 5‐trihydroxystilbene), a phenolic compound, has been reported to have anti‐oxidation, anti‐inflammation, anti‐platelet aggregation, anti‐cancer, protecting against cardiac vascular disease, and promotion longevity. Recently, several researches pointed out that RSV can attenuate high‐fat‐diet (HFD) induced obesity; however, its underlying mechanisms are not fully elucidated. In the present study, the HFD‐fed mice and 3T3‐L1 preadipocytes were employed to further explore the anti‐obese mechanisms of RSV. The 10‐week‐old male C57BL/6C mice were fed with HFD and treated HFD mice with or without RSV (1, 3, 10, or 30 mg/kg) for 10 weeks simultaneously. In RSV‐treated groups, the body weight gain was reduced significantly compared to compared to chow diet fed mice, which demonstrated that RSV has anti‐obesity effect. In respect of in vitro study, RSV (10–100 μM) treatment significantly decreased cell viability and increased apoptosis in both 3T3‐L1 preadipocytes and differentiated adipocytes. RSV (3–10 μM) suppressed preadipocytes differentiation by decrease PPARγ and Perilipin proteins expressions. RSV (3 μM) also inhibited lipolysis in mature adipocyte. Taking together, these results indicate that RSV exerted anti‐obese effects may through inhibition of preadipocyte proliferation and differentiation. Moreover, the anti‐lipolysis of RSV may contribute to maintain the lipids storage function in matured adipocyte which might have potential to prevent ectopic lipid accumulation.
Diabetes mellitus modifies endothelial SERCA activity producing a prostaglandin dependent vascular hypercontractility
Authors: Morales-Loredo Juan Humberto , Espinosa-Tanguma Ricardo
Published: June 2021

To assess the underlying mechanism of diabetes mellitus (DM)‐induced vascular hypercontractility, we evaluated the participation of the sarcoendoplasmic reticulum calcium ATPase (SERCA), the protein kinase C (PKC) and prostaglandins during the contraction induced by phenylephrine (PE) of aorta rings from a 10 weeks streptozotocin (STZ)‐induced DM rat model.

PE produced a stronger contraction of DM aorta rings with endothelium vs control (CTRL), (Emax: DM .69 ± .06 vs CTRL .38 ± .07 g. p<0.02). This vascular hypercontractility was eliminated by: a) indomethacin (10μM) pretreatment (Emax: DM .64 ± .07 vs CTRL .55 ± .11 g. ns), and b) endothelium removal (Emax: DM 1 ± .07 vs CTRL .93 ± .04 g., ns). On the other hand, Gö6976 (1μM), a PKC inhibitor, decreased the contractile response in CTRL more than in DM (Emax: DM .55 ± .06 vs CTRL .28 ± .04 g., p<0.01). Also, 1μM thapsigargin (T), a SERCA blocker, increased the CTRL concentration dependent contraction (Emax: CTRL .40 ± .08 vs CTRL+T .76 ± .09, p<0.001), but was not different in DM rings (Emax: DM .66 ± .10 vs DM+T .69 ± .06 g., ns), nor between groups (Emax DM+T .69 ± .07 vs CTRL+T .76 ± .08 g., ns).

Conclusions

During STZ‐induced DM, endothelial cells release a contractile prostaglandin, perhaps TXA2, that induces vascular hypercontractility.

Supported by: P/PIFI‐2010‐24MSU0011E‐13.
Transient cyclooxygenase inhibition blocks development of chronic angiotensin II‐salt hypertension
Authors: Asirvatham-Jeyaraj Ninitha , Fink Gregory D
Published: June 2021

Hypertension (HT) is the major risk factor for cardiovascular diseases. Previously, cyclooxygenase inhibition with ketoprofen was found to prevent Angiotensin II (AngII)‐salt HT development and sympathetic over‐activity. But ketoprofen did not reverse established HT. Recently, Johnson and colleagues reported that transient, short‐term exposure to circulating AngII can “prime” (i.e. accentuate) subsequent AngII HT development. So we hypothesized that an early increase in cyclooxygenase activity accentuates AngII‐salt HT; additionally, we attempted to determine the time‐course of the effect. Radio‐telemetric recording of arterial pressure (AP) in a 22‐day protocol was made. After 3 days of control recordings, rats on 2% NaCl diet were pretreated with vehicle or ketoprofen (2mg/kg, sc) for 5 days and 11 days. From day 8, all rats received AngII (150 ng/kg/min, sc). In vehicle‐treated rats MAP rose to 147±2 mmHg. In rats pretreated with ketoprofen for 5 days and 11 days, MAP rose to 129±8 mmHg (p>0.05) and 109±7 mmHg (p<0.05) compared to vehicle treated rats. The fall in MAP using ganglionic blocker hexamethonium in 11‐day ketoprofen treated rats (35±7mmHg) was less than in vehicle treated rats (61±5mmHg) and 5‐day ketoprofen treated rats (70±2mmHg). We conclude that early AngII‐mediated cyclooxygenase pathway activation is critical for the later full development of AngII‐salt sympathoexcitation and HT.
The role of plasma IL‐6 and IL‐10 during DOCA‐salt hypertension
Authors: Ongele Michael , Duan Rong , Lee Dexter L
Published: June 2021

Interleukin‐10 (IL‐10) is an important immunoregulatory cytokine that inhibits the expression of pro‐inflammatory cytokines including interleukin‐6 (IL‐6), reduces vascular dysfunction and blood pressure in various animal models. Previous reports demonstrate an increase in plasma IL‐6 during different time periods of deoxycorticosterone acetate (DOCA) ‐salt hypertension and that increases in mean arterial pressure (MAP) are independent of plasma IL‐6. Reports also suggest that plasma IL‐10 is decreased during DOCA‐salt hypertension. We tested whether the reduction of plasma IL‐10 is dependent on increases in plasma IL‐6 and if plasma IL‐10 plays a role in attenuating DOCA‐salt hypertension. Ten to twelve week old IL‐6 knockout (KO) mice and their wild‐type (WT) controls were subjected to uninephrectomy and DOCA‐salt (1.5 mg/g) treatment for 3 weeks. Baseline MAP determined by radiotelemetry was not different between IL‐6 KO and WT mice (122 ± 2 and 129 ± 5 mmHg, respectively). The average MAP during days 16 – 20 of DOCA‐salt hypertension was similar between IL‐6 KO and WT mice (157 ± 7 and 162 ± 6 mmHg, respectively). Baseline plasma IL‐6 in WT mice was 43 ± 5 pg/mL and 96 ± 8 pg/mL on day 20 of DOCA‐salt hypertension. Baseline plasma IL‐10 was 80 ± 9 and 75 ± 10 pg/mL for WT and IL‐6 KO mice, respectively. DOCA‐salt hypertension significantly reduced plasma IL‐10 on day 20 to 2 ± 1 and 9 ± 3 pg/mL for WT and IL‐6 KO mice, respectively. Plasma levels of IL‐10 were significantly higher in IL‐6 KO mice when compared to WT controls. Our results suggest that reduction of plasma IL‐10 is independent of increases in plasma IL‐6 and plasma IL‐10 does not play a role in attenuating DOCA‐salt hypertension.
Na‐K‐ATPase isoform profile in acquired and genetic forms of hypertension favors the hypertensive phenotype
Authors: Pulgar Victor M , Rashad Hanadi M , Jeffers Anne B , Dickenson Samantha-Rae , Diz Debra I , Aileru Azeez A
Published: June 2021

The Na‐K ATPase (NK) isoform alpha 2 (α2) contributes to stress‐induced hypertension. We hypothesize that alterations in NK expression and activity contribute to the ouabain‐induced and transgenic (mRen2)27 hypertension. Sprague‐Dawley rats were implanted subcutaneously with a 60‐day 1.5 mg ouabain (OUA) pellet (n = 6 control, n = 6 treated). Blood pressure (BP) was recorded daily via tail cuff for 8 weeks. OUA increased systolic BP by 30% (p<0.05) whereas age‐matched (mRen‐2)27 rats had a 55% higher BP vs control (p<0.05). Expression of the cardiac α2 NK isoform was higher in OUA and (mRen‐2)27 rats (n = 6) vs control (p<0.05), whereas renal NK α1 expression was lower in hypertensive animals (p<0.05). Functional NK activity was analyzed as the relaxation to low KCl concentrations in pre‐constricted renal and mesenteric arteries in K+‐free media and expressed as the difference of area under the curve (dAUC%). NK activity was lower in hypertensive animals in renal [OUA 30 ± 9% vs control 56 ± 6%, p<0.05] and mesenteric vasculature [(mRen‐ 2)27 59 ± 2%; OUA 63 ± 2% vs control 73 ± 3%, p<0.05]. Greater cardiac contractility due to increased NK α2 and increased peripheral vascular tone due to lower functional NK activity would favor hypertension and may be consistent with a contribution of the NK to these two forms of hypertension with known activation of the sympathetic nervous system.

NIMHD MD 002303 and NIGMS 5T34GM070416
Use of protein chaperones to correct BMP receptor processing errors in Hereditary Pulmonary Arterial Hypertension
Authors: Frump Andrea , de Caestecker Mark
Published: June 2021

Patients with HPAH inherit heterozygous mutations in the Bone Morphogenetic Type 2 receptor (BMPR2). BMPR2 mutations subject to non‐sense mediated mRNA decay (NMD+) promote less severe pulmonary hypertension (PH) than BMPR2 mutations that escape NMD (NMD‐). Moreover, we have shown that mice carrying the NMD+ Bmpr2 mutation, Bmpr2+/−, are less susceptible to PH than mice carrying the NMD‐ Bmpr2Δex2/+mutation. To explore the mechanisms underlying these differences we evaluated expression of mutant receptor products in isolated pulmonary endothelial cells (PECs). PECs from Bmpr2Δex2/+ mice express the 150kDa wild type Bmpr2 protein product, but also express high levels of a 125kDa product that is also seen in skin fibroblasts from HPAH patients with the same BMPR2 ΔEXON2 mutation. Biochemical studies indicate that this endogenous Bmpr2Δex2 product is retained in the ER, and that BMP‐dependent Smad 1/5/8 signaling is reduced in Bmpr2Δex2/+ PECs. Moreover, treatment with chemical chaperones restores cell surface expression of Bmpr2Δex2 and rescues the BMP signaling defects in these cells. These rescue studies suggest that protein chaperones can be used to correct BMP signaling defects in HPAH patients and mice with NMD‐ BMPR2 mutations. We are currently testing this hypothesis by determining whether the chemical chaperone 4PBA also reduces susceptibility of Bmpr2Δex2/+ mice to PH in vivo.
Autonomic function decline observed in hypoglycemia is attenuated during hyperoxia: potential role of carotid bodies in mediating cardio‐vagal activity
Authors: Taylor Jennifer Lynne , Wehrwein Erica A , Curry Timothy B , Basu Rita , Basu Ananda , Joyner Michael J
Published: June 2021

Cardio‐vagal tone (CVT) and heart rate variability (HRV) are reduced during hypoglycemia. Carotid body (CB) peripheral chemoreceptors are multi‐modal; therefore we hypothesized a correlation between CB activity and CVT during hypoglycemia. Seven healthy adults (4M/3F) underwent two 180 min hyperinsulemic (2mU/kg FFM/min), hypoglycemic (3.33 mmol/L) clamps one week apart, randomized to normoxia (arterial PO2 (PaO2) 111 ± 6.3 mmHg) or hyperoxia (PaO2 345 ± 80.6 mmHg) to desensitize the CB. CVT was assessed via the high frequency (HF) component of HRV from 5 min periods of 5‐lead ECG recording under baseline (euglycemia) and hypoglycemia. HF values decreased significantly from baseline during hypoglycemia, however this reduction was attenuated under hyperoxia (Normoxia (N): −69% vs. Hyperoxia (H): −41%, p=0.04). Heart rate increased significantly during hypoglycemia (N: 59.7 bpm ± 2.4 vs. 71.9 ± 4.0; H: 60.6 bpm ± 1.9 vs. 68.5 ± 3, p<0.001), however, this increase was not different between normoxia and hyperoxia, despite differences in CVT and HRV. These data indicate that the CB chemoreceptors may be partially responsible for the reduction in CVT associated with hypoglycemia, and they provide evidence for a role of the CB chemoreceptors in parasympathetic tone to the heart. Future studies are required to determine whether inhibition of the CB may serve to improve CVT function during hypoglycemia.

T32 DK07352, F32 DK84624‐01 A1, CTSA 1 UL 1 RR024150, R01DK 29953
Post‐exercise Appetite Was Affected By Fructose Content But Not Glycemic Index of Pre‐exercise Meals
Authors: Sun Fenghua , Wong Stephen H. , Huang Wendy Y. , Chen Yajun
Published: June 2021

The purpose of the present study was to investigate whether both glycemic index (GI) and fructose content in breakfast would affect appetite during the postprandial period and recovery period after 1‐hr brisk walking. Ten healthy young males (age: 21.7 ± 1.5 y, BMI: 20.9 ± 1.1 kg·m−2, V̇ O2max: 53.7 ± 3.7 mL kg−1 min−1) completed 1‐hr brisk walking at 46% V̇ O2max 2 hours after they ate one of three isocaloric breakfasts: a low‐GI breakfast not including fructose content (LGI), a low‐GI breakfast including fructose beverage (LGIF) and a high‐GI breakfast (HGI). All breakfasts provided 1.0 g kg−1 body weight carbohydrate, and the calculated GI values for the three breakfasts were 41, 39, and 72, respectively. In the LGIF and HGI trials, about 25% energy came from either fructose or glucose beverage. Appetite scores were measured every 30 min during the 2‐hr postprandial period and 1‐hr recovery period. During the postprandial period, the incremental area under the blood response curve values of glucose and insulin were higher in the HGI trial, compared with those in the LGI and LGIF trials. At 30 and 60 min during the recovery period, the appetite scores were lower in the LGIF trial than those in the LGI and HGI trials, whereas no differences were found between the latter two trials. It appeared that fructose content, rather than GI, in a breakfast affected appetite during the recovery period after 1‐hr brisk walking.
Enhancer mediated regulation of developmental gene expression
Authors: Wysocka Joanna K
Published: June 2021

Cell‐fate transitions involve the integration of genomic information encoded by regulatory elements, such as enhancers, with cellular and signaling environments. Identification of genomic sequences that control human embryonic development represents a formidable challenge, as they are embedded within a vast non‐coding genomic space. However, enhancers share certain chromatin features, including presence of specific histone modifications and general coactivators. We have recently shown that unique chromatin signatures can be used to annotate two functionally distinct classes of enhancer elements in human embryonic stem cells (hESC): active and poised. We now extended enhancer profiling to other embryonic cell types. I will discuss our new findings on the dynamics of enhancer regulation during development and on transcription factors and chromatin modifiers involved in that process.
Estrogen protects against hypertension during autoimmune mediated hypertension
Authors: Gilbert Emily L , Venegas-Pont Marcia , Mathis Keisa W , Wasson Katie L , Ryan Michael J
Published: June 2021

Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease. Because SLE predominantly affects young women, estrogen is commonly implicated in disease development. Hypertension, a major cardiovascular risk factor, is highly prevalent in women with SLE for reasons that are unclear. Based on the presumed role of estrogen to promote SLE and the prevalent hypertension, we hypothesized that estrogen promotes hypertension during SLE. To test this, 30‐week old female mice with SLE (NZBWF1) and controls (NZW/LacJ) were subjected to an ovariectomy (OVX) or sham operation. After 4 weeks of recovery, mean arterial pressure (MAP in mmHg) was measured by carotid catheters in conscious mice. MAP was higher in SLE sham mice compared to control sham mice (133±4 vs. 120±2, n=11, p<0.05). MAP was elevated further in SLE OVX mice (153±4, n=6, p<0.05 vs. SLE sham) but not in control OVX mice (118, n=2). Albuminuria (>100 mg/dL) developed in 27% of the SLE sham mice compared to 0% of sham or OVX control mice. 100% of SLE OVX mice developed albuminuria during this time. Renal expression of RORγ, a transcription factor specific for interleukin 17 producing T cells, is increased in OVX SLE mice compared to sham SLE mice. These data suggest that estrogens protect against, rather than promote, SLE hypertension and renal injury by suppressing renal inflammation. HL085907, HL092284 and HL085907S1 (MJR), and HL051971 (UMMC‐Physiology).
OSMOLALITY, METALLOTHIONEIN AND NA/KATPASE ACTIVITY IN A MANGROVE CRAB, UCIDES CORDATUS, SUBMITTED TO ACUTE AND CHRONIC COPPER EXPOSURE IN THE WATER
Authors: Sá Marina Granado e , Zanotto Flavia Pinheiro
Published: June 2021

Ucides cordatus were submitted to acute (24h and 96h) and chronic copper (Cu) exposure (15 days), containing 5mg/L CuSO4 in the water. Anterior and posterior gills, hepatopancreas (HP), and aliquots of haemolymph and urine were collected to measure osmolality, metallothionein (MT) concentration and Na/K‐ATPase activity (NKA). Haemolymph showed an increase in osmolality for crabs submitted to 24h Cu exposure, while animals submitted for 96h and 15 days to Cu demonstrated a reduction in osmolality compared to control. There was no change in urine osmolality for animals exposed to Cu for 24h and 15 days, while in the 96h group, there was a small reduction in the osmolality. MT synthesis increased in the anterior gills in crabs exposed to 24h, 96h and 15 days of Cu exposure, while posterior gills showed increased MT concentration in the 24h and 15 days groups, but not in the 96h group, suggesting that there is a regulation between MT concentration and time of exposure to water Cu. There was an increase of NKA in the anterior gills, while in the posterior gills there was reduction in the 24h group and an increase in the 96h and 15 days group compared to controls. For HP, there was an increase of activity for 96h group, while for the 15 days group the values were similar to control. Animals exposed to Cu show a relationship between time of exposure and higher MT concentration and increased NKA in both organs as well as alterations in osmolality.