bluuebunny/crossref_metadata_2025 huggingface dataset:
Intermedin/adrenomedullin‐2 (IMD/AM2) relaxes rat pulmonary arterial rings via protein kinase G (PKG) and BKCa channel dependent pathway: The effect of chronic hypoxia induced pulmonary hypertension
Authors: Kandilci Hilmi Burak , Gumusel Bulent , Lippton Howard , Witriol Norman
Published: June 2021

Our previous results showed that when rat pulmonary arterial (PA) rings were precontracted with U‐46619 IMD relaxes PA rings in a nitric oxide and endothelial dependent manner. In this study the pulmonary vasorelaxant response (PVR) to IMD in PA was inhibited by cholera toxin, Rp‐8‐Br‐PET‐cGMPs and iberitoksin whereas rolipram, KT5720 and apamin were without effect. PVR to IMD was potentialized in the presence of sildenafile. In PA rings isolated from rats with pulmonary hypertension induced by chronic hypoxia (PHCH), PVR to both IMD and ACh response were diminished. In contrast, in the isolated, buffer‐perfused rat lungs with PHCH, the pulmonary vasodilator responses to IMD and ACh were not altered. In PA rings from rats with PHCH, the PVR to NS1619 decreased when compared to control rats. The present data suggest that cGMP‐induced PKG activation and promotion of hyperpolarization through BKCa is the major pathway for rIMD induced vasorelaxation in PA rings. CH may induce downregulation of BKCa channels in pulmonary arterial conductance segments whereas pulmonary resistance arterial segments may not be susceptible to similar changes by PHCH, it is possible other mechanisms such as selective upregulation of eNOS or downregulation in phosphodiesterase activity in response to CH in resistance arteries may explain the difference in vascular responsiveness.
Role of large conductance Ca2+‐activated K+ (BKCa) channels in local metabolic coronary vasodilation in Ossabaw swine with metabolic syndrome
Authors: Borbouse Lena , Payne Gregory A. , Dick Gregory M. , Aloosh Mouhamad , Sturek Michael , Tune Johnathan D.
Published: June 2021

This study examined the contribution of BKCa channels to local metabolic coronary vasodilation in Ossabaw swine fed a normal maintenance diet (lean) or excess atherogenic diet that induces metabolic syndrome (MetS). Coronary blood flow responses during graded exercise were measured in conscious, chronically‐instrumented swine before and after specific BKCa antagonism with penitrem A (10 μg/kg, iv) that significantly decreased coronary vasodilation to the BKCa agonist NS1619. MetS developed over 20 weeks as evidenced by elevated body weight 51%, arterial pressure 13% and plasma insulin 80% without affecting fasting glucose. The relationship between coronary venous PO2 (cvPO2) and myocardial O2 consumption (MVO2) was significantly decreased in MetS relative to lean. This impaired balance between coronary blood flow and myocardial metabolism was accompanied by a negative lactate uptake during exercise in MetS (−1.8 ± 1.3 μmol/min/g), but not in lean (1.7 ± 0.6 μmol/min/g) swine (P < 0.01). BKCa inhibition did not significantly affect arterial pressure, heart rate or diminish the cvPO2‐MVO2 relationship in either lean or MetS swine. These findings indicate that BKCa channels are not required for coronary vasodilation to exercise and that the imbalance between myocardial O2 delivery and metabolism in MetS is not related to altered functional expression of BKCa channels. (Support: HL67804, RR013223, HL062552)
Responses of nitric oxide‐cGMP releases over acupuncture points to heat stimulation in humans
Authors: Hu Jay , Li Xiyan , Saberon Lara , Ma Shengxing
Published: June 2021

The purpose of this study was to quantify nitric oxide (NO)‐cGMP concentrations over acupuncture points (acupoints) and to examine the influences of heat stimulation on NO‐cGMP releases over acupoints, meridian lines between acupoints, and non‐meridian controls in healthy volunteers. A semi‐circular plastic tube (0.5 X 7 cm) was taped to the skin surface along the Lung meridian (LU) and filled with a 2‐Phenyl‐4,4,5,5‐tetramethylimidazoline‐3‐oxide‐1‐oxyl solution. The solution was in contact with the skin surface for 20 minutes in order to absorb NO. A small warming pad (45 °C) with a probe sensor was wrapped around a finger for 20 min to measure the effects of heat. The concentrations of total nitrate and nitrite (NOx−) and cGMP in the samples were quantified using chemiluminescence and ELISA in a blinded fashion, respectively. NOx− concentrations were significantly higher during and 20 min after heat stimulation compared to the non‐stimulated side (n=15). cGMP levels over acupoints of the LU meridian were also increased by heating, but not altered in non‐meridian control (n=10). The results show that heat stimuli predominantly release/generate NO‐cGMP on the skin surface at acupoints. We conclude that heat stimulus‐evoked NO‐cGMP release enhances local circulation at the acupoints, which may mediate signaling functions to attain therapeutic effects. Supported by NIH RO1 AT 002478 and ADA 7‐07‐RA‐100 to S. Ma.
Losartan, an angiotensin II receptor blocker, fails to attenuate pulmonary dysfunction in ovine burn and smoke model
Authors: Foresman Lauren , Enkhbaatar Perenlei , Connelly Rhykka L. , Hamahata Atsumori , Traber Lillian D. , Schmalstieg Frank C. , Traber Daniel L.
Published: June 2021

Elevated levels of angiotensin II (AngII) in the lung have been shown in response to burn and smoke inhalation injury. AngII has been associated with increased nitric oxide levels, leading to nuclear oxidative damage in Type II lung epithelial cells. Losartan is an AngII receptor blocker, specific to AT1A receptors. Using an ovine model of acute respiratory distress, we hypothesized that blocking AngII receptors with losartan would improve the clinical conditions seen in response to the injury. Chronically prepared adult ewes received a 40% total body surface area third degree flame burn followed by 48 breaths of cotton smoke. The sheep were randomly divided into two groups: control (injury, no treatment, n=6) and treatment (injury + losartan, n= 6). Losartan was given intravenously as a bolus (50 mg) at 1hour post‐injury. All animals received 4ml/kg/%burn of Ringer's lactate and were mechanically ventilated. Control animals showed severe signs of acute lung injury evidenced by deterioration of pulmonary gas exchange (PaO2/FiO2, pulmonary shunt fraction), increased lung water content, and increased pulmonary transvascular fluid flux, and vascular permeability index. Although there was a trend for reduction of lung lymph flow and permeability index, these alterations could not be shown as statistically affected by this dosage of losartan in this 24‐hour ovine model of acute lung injury. SBI 8450, PPG 5P01GM066312
Different Role of Calcium‐activated Potassium Channels in Human Internal Mammary (IMA) and Radial Artery (RA)
Authors: Fan Li , Xiao Xiao‐Qiu , Grove Kevin , Yang Qin , Furnary Anthony , He Guo‐Wei
Published: June 2021

We investigated the contribution of small (SKCa) and intermediate conductance calcium‐activated potassium channel (IKCa) in human IMA and RA. Cumulative concentration (−10 ~ −5.5 log M)‐relaxation curves for acetylcholine (ACh) were established in IMA and RA rings (n=6) pretreated with/without inhibitor of SKCa (apamin, 100 nM) or IKCa (charybdotoxin, ChTX, 100 nM). Expression levels of SKCa and IKCa in the whole tissue and smooth muscle cells (SMC) of IMA and RA were quantitated with real‐time PCR. The relaxation curve to ACh was depressed significantly (p < 0.001) by apamin (29.5 ± 2.9% vs. control 55.5 ± 4.2%) and less by ChTX (44.0 ± 5.2%, p < 0.001) in IMA. In RA, the inhibition of apamin was less significant (57.1 ± 5.4%, p < 0.05) than ChTX (42.5 ± 8.2 % vs control 67.8 ± 8.9 %, p < 0.001). RT‐PCR showed that expression of SKCa was similar to IKCa in integrated IMA tissues (2.32 ± 0.20 vs. 2.24 ± 0.24, p=0.6), but higher than IKCa in IMA SMC (4.06 ± 0.60 vs 1.30 ± 0.26, p < 0.01). For RA, expression of SKCa was higher than IKCa (3.34 ± 0.44 vs 2.49 ± 0.57, p < 0.05) in both integrated tissue and SMC (30.27 ± 7.86 vs. 1.92 ± 0.58, p < 0.001). Thus, both SKCa and IKCa paly a role in IMA and RA. However, the mRNA expression of these ion channels is different in the endothelium and SMC between IMA and RA.

Supported by St. Vincent Foundation, Hong Kong RGC (CUHK4383/03M) & CUHK (4450171, 4450231, 2041305).
The calsequestrin mutation CASQ2‐K206N affects sarcoplasmic reticulum Ca handling and causes catecholaminergic polymorphic ventricular tachycardia
Authors: Kirchhefer Uwe , Wehrmeister Diana , Müller Frank U. , Postma Alex V. , Schmitz Wilhelm , Neumann Joachim
Published: June 2021

Catecholamine‐induced polymorphic ventricular tachycardia (CPVT) is a familial disorder caused by calsequestrin 2 (CASQ2) gene mutations. Here, we report the identification of a new CASQ2 mutation, K206N, which transmitted autosomally and is associated with CPVT and sudden cardiac death in a young patient. To find a causal link between K206N and the CPVT phenotype, we studied biochemical and physiological effects of K206N overexpression, in comparison to the native form (NT) of the calsequestrin protein (CSQ), in both insect cells and isolated neonatal mouse myocytes by use of recombinant baculoviruses and adenoviruses, respectively. The CSQ mutation was associated with the insertion of a second glycosylation site resulting in a higher mobility form compared to NT. Consistently, the mutated CSQ colocalized with mannosidase II, whereas NT did not. Thus, hyperglycosylation of K206N may trigger the lower Ca binding (by 54%) observed in 45Ca overlay blots. Moreover, we found a higher binding (by 40%) of the mutated CSQ to a GST‐triadin fusion protein in pull‐down assays. [3H]ryanodine binding was increased by 115% in homogenates of myocytes infected with K206N compared to NT, suggesting a higher open probability of the SR Ca release channel. In line with these findings, myocytes with K206N overexpression had a higher rate of spontaneous Ca transients, when exposed to isoproterenol.

The data of the present study demonstrate that overexpression of K206N in myocytes causes an abnormal Ca handling and therefore, may predispose to the CPVT phenotype observed in the human patient.

This study was supported by the Deutsche Forschungsgemeinschaft (Ki 653/13‐1/2).
Differential expression of Gs and Gi in mice with homocysteinemic heart failure
Authors: Vacek Thomas Paul , Moshal Karni Singh , Sen Utpal , Tyagi Neetu , Steed Mesia , Kumar Munish , Passmore John , Tyagi Suresh
Published: June 2021

High plasma homocysteine levels are a known risk factor in heart failure and sudden cardiac death. β1‐adrenergic receptor, a G protein coupled receptor (GPCR) containing only Gα s G protein subunit, undergoes down‐regulation in heart failure. The aims of this study were to examine the differential expression of Gα s G protein and Gα i (inhibitory) in the hearts of hyperhomocysteinemic (Hhcy) mice, and to determine if homocysteine acts via a paracrine mechanism in vivo or in isolation as an agonist in cell culture to mediate the change in G protein isoforms. To create Hhcy, mice were treated with Hcy (0.67mg/mL) in water for 12 weeks. Echocardiogram and EKG were performed on each mouse. Mice were sacrificed, hearts were excised, cardiac tissue homogenates were prepared, and western blots were performed to determine content of Gα s and Gαi. The results suggest that Gα s G protein was down regulated in cardiac tissue of Hcy mice to 54% that of control hearts. However, the intracellular Gα i G protein content remained the same in Hhcy mice. Mouse aortic endothelial cells (MAECs) and transformed cardiomyocyte HL‐1 cells were treated with varying concentrations of homocysteine for 24 hours. The results suggest no detectable differential Gα s and Gα i expression. This suggests that homocysteine acts via paracrine mechanism in vivo, mediating changes in intracellular Gα s G protein content related to cardiac dysfunction in Hhcy.
GENDER DIFFERENCES IN THE EFFECTS OF ANTENATAL BETAMETHASOME EXPOSURE ON RENAL FUNCTION IN ADULT SHEEP
Authors: Tang Lijun , Carey Luke , Valego Nancy , Deibel Philip , Perrott James , Figueroa Jorge , Chappell Mark , Rose James C
Published: June 2021

Exposure to clinically relevant doses of glucocorticoids during fetal life increases blood pressure in adult male and female sheep. The purpose of this study was to evaluate the effect of prenatal exposure to betamethasone at 80‐81 days of gestation on renal function in 17 ewes and 19 rams at 1.5 years of age. Basal glomerular filtration rate (GFR) and renal plasma flow (RPF), determined as clearance of inulin and p‐aminohppuric acid respectively were performed. An acute sodium load was administrated by a continuous infusion of hypertonic NaCl (0.0275 mEq/Kg/min) for 60 minutes. In males, basal GFR was decreased in betamethasone exposed group (B) (1.935 ± 0.08 ml/min/kg) compared with the vehicle (V) (2.267 ± 0.10 ml/min/kg) (P=0.028). In females, basal GFR was similar in both groups (1.832 ± 0.11 vs 2.133 ± 0.18 ml/min/kg). RPF was similar in B and V males (13.561 ± 0.88 vs 15.483 ± 0.40 ml/min/kg) and females (14.585 ± 0.92 in B vs 14.378 ± 1.20 ml/min/kg in V). Although basal urinary sodium excretion was similar, male B group excreted less of an acute sodium load (37.07 ± 4.4% of the dose of Na) compared with V group (53.65 ± 9.7%) (P=0.03). In contrast, prenatal betamethasone exposure in females had no effect on basal Na excretion or the percent of the Na dose excreted (98.08 ± 15.0% in B vs 92.22 ± 8.9% in V). GFR and RPF did not significantly change during the experiments. These data suggest that prenatal exposure to glucocorticoids alters renal function in adult male sheep but not in females by decreasing GFR and reducing excretion of an acute sodium load. The gender difference in the renal effects of prenatal steroid exposure suggests different mechanisms are involved in the antenatal steroid‐induced elevation in blood pressure in males and females.
Proteomic analysis of lithium induced changes in rat inner medulla collecting duct
Authors: Nielsen Jakob , Hoffert Jason , Knepper Mark A , Nielsen Søren , Fenton Robert A
Published: June 2021

Chronic lithium treatment induces nephrogenic diabetes insipidus (NDI) with altered expression of renal water, sodium and acid‐base transporting proteins, proliferation and apoptosis of principal cells and structural changes in the collecting duct. To identify novel and differentially expressed proteins in IMCD after 1 or 2 week lithium treatment we used differential gel eletrophoresis (DIGE) combined with mass spectrometry (MALDI‐TOF‐TOF). Urine production increased 3‐ and 6‐ fold, while AQP2 protein expression decreased to 60% and 30% of controls at 1 and 2 weeks of lithium‐treatment, respectively. Stringent analysis of the identified proteins revealed that 8 and 74 proteins were significantly changed at 1 and 2 weeks of lithium‐treatment, respectively. Bioinformatics analysis identified cell viability and morphology as the primary cellular functions likely affected by lithium‐treatment in addition to cell structure, cell death and apoptosis. Several of these lithium targets were confirmed by immunoblotting. Follow‐up studies in rats and in vitro showed marked changes in the expression of proteins involved in Wnt‐signaling, tight junction formation, the activation state of several MAPKs, as well as GSK3ß. These results identify novel pathways and proteins likely involved in lithium‐induced renal pathology and provide the basis for numerous hypothesis‐driven studies.
Blunted Renal Medullary Blood Flow Responses in Dahl S Rats Following a Hypertonic Sodium Load
Authors: Jin Chunhua , Cowley Allen W.
Published: June 2021

We have shown that increased luminal delivery of NaCl to the medullary thick ascending limb of Henle (mTAL) results in increased production of superoxide (O2−) (Am J Physiol 291: F350, 2006) and that mTAL of Dahl salt‐sensitive (SS) rats produce greater amounts of O2− in response to elevations of extracellular [NaCl] vs. salt‐resistant consomic rats (SS‐13BN). We hypothesized that an acute increase of filtered NaCl by infusion of 5% NaCl would stimulate mTAL O2− production more in SS rats and buffer the expected increases of medullary blood flow (MBF) related to the volume expansion. The left kidney was instrumented with an optical fiber in the outer medulla, a ureteral catheter, and an adjustable occluder was placed around the aorta proximal to the left renal artery to maintain renal perfusion pressure unchanged during the period of expansion. After equilibration and two 30 min. control periods, the MBF responses to 60 min infusion of 5% NaCl (4 ml/hr) were determined throughout the infusion period. MBF rose significantly in the control SS‐13BN rats (20.7%; N=6) during the 5% NaCl infusion. In contrast, MBF in SS rats (N=5) did not change. A greater amount of sodium and volume were excreted (P<0.05) in SS‐13BN than SS rats throughout the 5% NaCl infusion period. We conclude that SS rats have a reduced ability to increase MBF and sodium excretion in response to a salt load. (Support: HL‐29587 and 82798).
Tumor necrosis factor‐alpha response to shock wave lithotripsy‐induced renal injury
Authors: Clark Daniel Lynn , Handa R. K. , Johnson C. D. , Connors B. A. , Evan A. P.
Published: June 2021

Shock wave lithotripsy (SWL) induces a rapid inflammatory response in renal tissue. This study investigated the site of the inflammatory response in a porcine model of acute SWL‐induced renal injury, using tumor necrosis factor alpha (TNF) as a marker of inflammation. The lower pole left renal calyx of anesthetized female pigs (30–35 lbs) received 2000 shock waves (SW) from a Dornier HM3 lithotripter (24 kV, 120 SW/min). Blood and urine samples were taken at timed intervals. At 4 hours post‐treatment, kidneys were perfused with cold saline, excised, and tissue samples were flash‐frozen in liquid N2. TNF was measured by ELISA (BioSource). Left urinary TNF excretion peaked by 1 hr post‐SWL (23.8±20.9 pg/min vs. 0.0±0.0 pg/min for control, p<0.001) and although declining remained significantly elevated up to 4 hrs post‐SWL. Right urinary TNF excretion, renal tissue TNF, and plasma TNF levels were unchanged after SWL. These data correlate with our previously reported IL‐6 and currently observed HO‐1 data suggesting that inflammation and oxidative stress are primarily occurring in the shocked kidney. Supported by NIH grant DK67133.
P2X2 purinergic receptors (P2X2R) are responsible for ATP stimulated calcium influx in supraoptic nucleus neurons (SON)
Authors: Song Zhilin , Sladek Celia D
Published: June 2021

ATP and phenylephrine (PE, α1 adrenergic receptor agonist) co‐applied to explants of the hypothalamo‐neurohypophyseal system (HNS) act synergistically to stimulate vasopressin and oxytocin release and generate an extended elevation in intracellular calcium ([Ca++]i) in SON (AJP:Regul 291:R31,'06). The latter may be the driving force for synergism in hormone release. ATP activated both P2X and P2YRs to increase [Ca++]i in SON (AJP:Regul 292:R423, ‘07). Our current studies evaluated the relative contribution of different P2XR subtypes to ATP stimulated Ca++ influx. HNS explants were loaded with the Ca++‐sensitive dye, fura 2AM (100 uM for 10 min., then 20 uM for 50 min in 0.02% pluronic acid), and [Ca++]i in SON was monitored by ratiometric analysis of fluorescence emitted by excitation at 340 and 380 nm. Neither A317491 (300 nM, P2X3 and P2X2/3R antag.) nor BBG (1 uM, P2X7R antag.) attenuated the increase in [Ca++]i induced by ATP or ATP+PE. α,β‐MethyleneATP (100 uM, a P2X3 and P2X2/3R agonist) did not increase [Ca++]i. BzATP increased [Ca++]i at 100 and 200 uM (activates P2X2Rs), but not at 20 uM (activates only P2X7Rs). Moreover, ATP γ S (100 uM, P2X2 and P2X4R agonist) also induced an increase in [Ca++]i. Since P2X4Rs are not sensitive to PPADS, and PPADS (10uM) blocked ATP stimulated Ca++ influx, together these findings support a primary role for P2X2Rs in stimulating Ca++ influx in SON. Since the non‐desensitizing P2X2/3 and P2X7Rs are important for sustained VP release induced by ATP+PE (Gomes et al, EB'08), but do not participate in Ca++ signal generation, ATP+PE synergism in hormone release may require a shift in P2XR subtypes. Supported by NIH R01‐NS27975.
Hypertensive Dahl salt‐sensitive rats produce a blunted diuresis to central N/OFQ which is restored by CNS G‐alpha q protein knockdown
Authors: Wainford Richard David , Kapusta Daniel R
Published: June 2021

Hypertensive Dahl salt‐sensitive (DSS) rats on high NaCl intake have enhanced plasma vasopressin (AVP). We examined whether DSS rats can effectively excrete water following intracerebroventricular (i.c.v.) injection of nociceptin/orphanin FQ (N/OFQ), a peptide that evokes profound diuresis via inhibition of AVP release.

Methods Changes in CV and renal excretory function produced by i.c.v. N/OFQ (5.5 nmol) were examined in conscious chronically instrumented DSS rats maintained (3 weeks) on 8% NaCl (N=6) or normal chow (N=6). Separate animals fed 8% NaCl diet (N=6), were pretreated with a Gαq oligodeoxynucleotide (ODN) (25 μg), which we have shown participates in the recovery (AVP stimulatory) phase following the peak diuresis to N/OFQ. MAP and HR were recorded and urine collected during control and for 80‐min (10‐min periods) following i.c.v. N/OFQ.

Results Dahl SS rats fed an 8% NaCl diet became hypertensive (normal chow, MAP 128 ± 4 mmHg; 8% NaCl chow, 169 ± 7 mmHg, P< 0.05). The diuresis to i.c.v. N/OFQ was blunted in duration and magnitude in hypertensive DSS rats (peak diuresis; normotensive DSS 50‐min, 174 ± 18 μl/min; hypertensive DSS 30‐min, 110 ± 17 μl/min, P< 0.05). In hypertensive DSS rats, i.c.v. Gαq pre‐treatment fully restored the diuresis to i.c.v. N/OFQ (peak; DSS + Gαq ODN 50‐min, 170 ± 14 μl/min P< 0.05).

Conclusion Hypertensive DSS rats produced a blunted diuresis to i.c.v. N/OFQ, which was restored to a normal diuretic pattern by prior central Gαq protein knockdown. Thus, in DSS rats, high NaCl may enhance central Gαq protein expression and attenuate the inhibitory effects of this, and possibly other Gαq‐protein coupled receptor ligands on AVP secretion. DK43337, HL 071212, P20 RR018766
A proteomics approach to the mammalian kidney as a model for hyperosmotic stress
Authors: Gabert Beverly Joy , Kültz Dietmar
Published: June 2021

The papilla of the mammalian kidney must tolerate varying degrees of hyperosmotic stress during urine concentration. Though functionally distinct the papilla is in close physical proximity to the renal cortex, an isoosmotic region. Proteomic differences between the papilla and cortex of C57BL6 mice were investigated using two‐dimensional gel electrophoresis to generate a whole proteome map for each tissue region. Delta2D software was used to quantify (n = 6) two‐fold or greater expression changes between the regions leading to detection of 211 cortex spots and 70 papilla spots. MALDI‐TOF/TOF mass spectrometry was used to sequence and identify tryptic peptides from the spots identifying 34 different cortex proteins and 19 proteins overexpressed in the papilla. Region specificity was substantiated by Western Blot confirmation. Proteins overrepresented in the cortex include enoyl CoA hydratase and malate dehydrogenase. Protein identifications of the renal papilla include alpha B crystallin, HSP 90, 14‐3‐3 protein and aldose reductase, which indicates that an array of stress response proteins contributes to offset the hostile environment in the papilla. PANTHER analysis of protein identifications was used to determine cellular functions, biological processes and molecular pathways characteristic of renal cells exposed to the stressful environment found in the papilla. Supported by NIH grant DK 59470.
Caloric restriction attenuates apoptosis in larger type II fibers with aging
Authors: Carter Erin , Villarin Jason , Hepple Russell T
Published: June 2021

Apoptosis has been implicated in the age‐related decline in skeletal muscle mass known as sarcopenia. Similarly, an attenuation of apoptosis is thought to contribute to the attenuation of sarcopenia by caloric restriction (CR). On this basis, we examined markers of apoptotic activation in cross‐sections of gastrocnemius muscle (Gas) from young adult (YA) and senescent (SEN) ad libitum fed (AL) and CR rats. Although CR markedly attenuated Gas atrophy (27% vs 51% for CR vs AL) and fiber atrophy (26% vs 40% in CR vs AL) with aging, CR did not affect the proportion of nuclei that were TUNEL positive (1.9±0.2% vs 3.0±0.2% for AL vs CR, respectively). Similarly, the number of TUNEL positive nuclei per 100 fibers was not different in SEN CR (11.0±1.1) vs SEN AL (8.3±0.8). On a fiber type basis, type IIa (58%) and IIb/x (51%) fibers exhibited more atrophy with aging in AL than type I fibers (31%), whereas fiber atrophy was less severe and more uniform among fiber types in CR (21%, 11% and 25% for type I, IIa and IIb/x, respectively). Interestingly, whereas fibers with TUNEL positive nuclei were larger than the mean for both type IIa and IIb/x in SEN AL, this was not the case for CR. Collectively, these results suggest that the apoptosis of nuclei in larger type II fibers of AL may contribute to their greater atrophy with aging and that this process may be attenuated by CR.
Mitochondrial function in physically active older adults with sarcopenia
Authors: Waters Debra L , Qualls Clifford , Gasparovich Charles , Mullins Paul , Raj Dominic
Published: June 2021

Sarcopenia is prevalent during aging and has been linked to mitochondrial dysfunction. The role of physical activity in protecting against sarcopenia and preserving mitochondrial function is debated due to its dual role of increasing muscle function while increasing free radical production. To investigate these relationships, healthy normal lean (NL: n=15) and sarcopenic (SS: n=9) subjects were recruited based on body composition (DXA, Lunar DPXTM), age (78.5 ± 5 yr), sex, and self‐reported long‐term physical activity. Calf mitochondrial function was assessed using 4 T 31PMRS (Bruker Biospin). Resistance was 50% of 1MVC and total work measured by force transducer. Total work was lower in SS (p<0.001) and data were scaled to total work output. At rest [Pi] was modestly elevated in SS (p=0.02). End of exercise [Pi] was lower (p=0.007) and [ADP] and pH higher (p=0.0002, p=0.05 respectively) in the SS. The [PCr] recovery rates were not different between the groups, but [ADP] during recovery remained elevated in SS (p=0.02). Elevated [ADP] during exercise can imply impaired mitochondrial function. However, the similar [PCr] recovery rates suggest an increased proton efflux in SS, that would increase pH and raise [ADP] via increased mitochondrial respiration. Our data indicate that a relationship exists between sarcopenia, long‐term physical activity, and mitochondrial function that remains to be fully elucidated.

Supported by NIH/NIA R21 AG21560‐01, NCRR MO1‐RR‐00997
Increased mitochondrial content and evidence of dysfunction in aging extraocular muscles
Authors: McMullen Colleen A , Gamboa Jorge L , Andrade Francisco H
Published: June 2021

The high mitochondrial content of extraocular muscles (EOMs) reflects the metabolic demand imposed by their fast and constant activity. In young rats, EOM mitochondrial volume density (MVD) is 3‐fold greater than in limb muscle (IOVS 2005; 46:4541). However, we do not know how EOMs are affected by age. Their constant activity may pose life‐long metabolic stress leading to more severe age‐related mitochondrial dysfunction than in limb muscles. Therefore, we tested the hypothesis that aging EOMs have a greater decrease in mitochondrial content than limb muscles. EOM and limb muscles from 6‐, 18‐, and 30‐mo old male Fisher 344‐Brown Norway rats were used for light and electron microscopy, and mRNA and protein content of respiratory complex subunits. Histochemically, SDH and cytochrome c oxidase activity increased with age in EOM and decreased in limb muscle. Likewise, aging increased MVD in EOM. mRNAs for subunits of the respiratory complexes increased with age in EOMs and decreased in limb muscle. Content of the corresponding proteins followed a similar trend. Based on these data, we reject our hypothesis and conclude that MVD changes in the opposite direction in aging EOM compared to limb muscle. The unexpected increase in EOM mitochondrial content suggests age‐related mitochondrial dysfunction and may explain why some mitochondrial myopathies target EOMs.
The role of Heat Shock Proteins in cytoprotection of skeletal muscle
Authors: Bayer Monika Lucia , Paulsen Gøran , Ugelstad Ingrid , Hallen Jostein , Raastad Truls
Published: June 2021

The aim of this study was to examine the Heat Shock Protein (HSP) response to a repeated bout of unaccustomed eccentric exercise. 9 volunteers (mean age 25 yrs.) performed 2 bouts of 70 maximal eccentric actions of the biceps, with 3 weeks between the bouts. Muscle biopsy tissue was homogenized and fractionated subcellulary for HSP analysis. Immediately after both bouts, muscle force was ~50% lower than pre‐exercise, and a translocation of small HSPs (sHSPs) from the cytosolic to the cytoskeletal fraction was observed. Compared to Bout‐1, however, muscle function recovered faster (p<0.05), sHSP accumulation in the cytoskeletal fraction was higher (p= 0.1), and cytosolic sHSP content was greater after Bout‐2 (p<0.05). Furthermore, an increase in HSP70 in the cytoskeletal (p<0.05) and cytosolic fractions (n.s.) was observed only after Bout‐2. In conclusion, total HSP content was elevated and the accumulation in the cytoskeletal fraction was more pronounced after Bout‐2, indicating a role for HSPs in the repeated bout effect, most likely by protecting muscle cells from stress‐induced alterations in protein structure and function. This may result in improved recovery of cellular homeostasis following repeated exercise. The study was supported by the Norwegian School of Sports Sciences.
Neuropeptide‐Y and prepro‐orexin gene expression increases after continuous sleep deprivation but not sleep restriction in rats
Authors: D′Almeida Vânia , Forcina Martins Paulo J. , Tufik Sérgio
Published: June 2021

The sleep loss is characterized by increased food intake in human and rats. However, the statement that in rats the sleep deprivation‐induced hyperphagia is followed by weight loss, suggests that the physiological factors involved are different from those proposed to human. Our objective was to investigate the sleep deprivation‐induced changes on feeding behavior and body weight by the platform technique in rats and to access the repercussions on hypothalamic expression of neuropeptides related to sleep and food intake control. Rats receiving a liquid diet were sleep deprived by the platform technique for 24, 48, 72, 96 hours and for 21 days by sleep restriction. We investigated feed behavior, body weight, and hypothalamic prepro‐orexin, neuropeptide Y, insulin receptor, agouti related peptide and POMC expression. Increased gene expression of the prepro‐orexin was closer related to sleep debit than food intake changes. Despite of unchanged body weight, rats continuously sleep deprived, but not the restricted ones, showed increases in prepro‐orexin and neuropeptide Y gene expression which can interacts to food intake augmentation during sleep deprivation.

Financial support: FAPESP, CNPq and AFIP.
Hypoxia‐induced pulmonary endothelial microparticle (EMP) generation regulates pulmonary microvascular endothelial cell (PMVEC) permeability
Authors: Bauer Natalie Norwood , Moore Timothy M , Kapadia Pavan D , Townsley Mary I , McMurtry Ivan F
Published: June 2021

EMPs are 0.2–1.5 μm vesicles derived from plasma membranes of activated or apoptotic cells. Endothelial cells release different numbers and phenotypes of EMPs dependent on the stimulus. Therefore, we hypothesized that hypoxia, a known stimulus for PMVECs, would stimulate release of EMPs which may then play a role in permeability of PMVEC monolayers. PMVEC monolayers were grown to confluence, media changed and exposed to normoxia for 1 hour (20% O2). A second media change was followed by a 1 hour exposure to hypoxia (3% O2). EMPs in media harvested after normoxia and hypoxia were enumerated by flow cytometry. Hypoxia increased EMP release by approximately 30% compared to normoxia. We then used a modified Ussing‐chamber system to determine the hydraulic permeability (Lp) of normoxic PMVEC monolayers treated with 105/mL EMPs derived from either normoxic or hypoxic PMVECs. We observed that treatment with hypoxia‐generated EMPs increased Lp when compared to normoxic EMP treated or untreated monolayers. Thus, we conclude that hypoxia stimulates the generation of EMPs by PMVECs and that these EMPs can increase permeability of quiescent PMVECs. This raises the interesting possibility that the release of EMPs into the pulmonary circulation may increase the permeability of downstream vessels. Support: NHLBI HL‐066299 (M.I. Townsley), HL‐15985 (I.F. McMurtry), AHA #0765413B (N. Bauer) and 0765421B (T. Moore).
Lung microvascular endothelial cells utilize aerobic glycolysis (e.g. Warburg effect) to sustain rapid proliferation
Authors: Parra‐Bonilla Glenda , Alvarez Diego F. , Cioffi Donna L. , Stevens Troy
Published: June 2021

Oxidative phosphorylation is the main source of ATP in eukaryotic cells. However, cancer cells and rapidly growing untransformed cells preferentially utilize glycolysis to generate ATP, a so‐called Warburg effect, which may be necessary to sustain growth. Since pulmonary microvascular endothelial cells (PMVECs) display rapid proliferative behavior, we sought to determine whether PMVECs exhibit aerobic glycolysis during proliferation. Growth curves for pulmonary artery endothelial cells (PAECs) and PMVECs were performed using standard tissue culture conditions. The pH and glucose concentrations of the media were measured daily. As the PMVECs proliferated rapidly, the pH, and the glucose decreased dramatically, an effect that was not seen in the slower growing PAECs. The difference in glucose utilization and media acidification between these two cell types could be explained by differences in expression of genes involved in glycolysis. Indeed, QRT‐PCR analysis revealed an upregulation of glucose transporter‐1, hexokinase‐2, phosphofructokinase, and lactate dehydrogenase in PMVECs compared to PAECs. In conclusion, upregulation of key glycolytic regulatory enzymes, increased glucose consumption, and media acidification, suggest that under normoxic conditions, PMVECs actively utilize the glycolytic pathway to sustain their growth. HL66299.
Altered IP3 receptors, IP3‐dependent Ca2+ transients and Ca2+‐dependent gene expression in a Duchenne muscular dystrophy cell line
Authors: Jaimovich Enrique , Juretic Nevenka , García Isaac , Hartley Ricardo , Liberona Jose Luis , Figueroa Reinaldo , Riveros Nora , Molgó Jordi , Cárdenas Julio Cesar
Published: June 2021

Although deregulation of calcium handling has been recognized to play a role in Duchenne muscular dystrophy (DMD), little is known about the role of calcium release in this phenomenon. Inositol 1,4,5 trisphosphate receptors (IP3Rs) are located in the sarcoplasmic reticulum and nucleus of skeletal muscle cells. Upon electrical stimulation (400pulses, 1ms, 45Hz), these receptors mediate slow calcium transients, involved in gene regulation. Using normal (RCMH) and dystrophic (RCDMD) human skeletal muscle cell lines, we explored the distribution of IP3Rs as well as IP3‐dependent calcium dynamics. Mouse oligonucleotide microarrays alowed analysis of total RNA from RCMH and RCDM myotubes obtained 4h post‐stimulus.

Dystrophic cells show a five‐fold over expression of type‐2 IP3R; type‐2 IP3R locates in the nucleus in RCDMD cells, while type‐1 and type‐3 IP3Rs appear also to locate differently. Both cell lines displayed a slow calcium transient upon electrical stimulation, the signal being much faster and larger in RCDMD cells. IP3‐mediated calcium signals induced phosphorylation of extra cellular signal‐regulated kinase (ERK1/2) in normal but not in dystrophic cells. Statistical analysis of microarrays resulted in 132 and 150 differentially expressed genes in RCMH and RCDMD respectively. As IP3‐dependent calcium signals appear to be the first step in regulation of gene expression, impairment of this pathway could play an important role in DMD patho‐physiology.

FONDAP 15010006
Ecto‐phosphorylation of CD98 regulates lymphocytes‐epithelia interactions
Authors: Nguyen Hang Thi Thu , Yan Yutao , Dalmasso Guillaume , Charrier‐Hisamuddin Laetitia , Sitaraman Shanthi V
Published: June 2021

Ecto‐phosphorylation has been shown to play an important role in several cellular functions. Here we investigate the ecto‐phosphorylation of membrane glycoprotein CD98 by ecto‐protein kinases (ePK) and its functional consequences. Our results show that recombinant CD98 is phosphorylated by purified casein kinase 2 (CK2) and by ePK from Jurkat cells. Km and Vmax for the phosphorylation of CD98 by CK2 are 0.84 μM and 8.7e+6 μmol/min, respectively. In intestinal epithelial Caco2‐BBE cells, CD98 is also ecto‐phosphorylated by ePK from Jurkat cells. Interestingly, activation of CD98 in CHO cells expressing CD98 enhances the ecto‐phosphorylation of CD98 and increases the attachment of Jurkat cells to CHO cells, suggesting that the ecto‐phosphorylation of CD98 is important for cell‐cell interactions. To examine the role of CD98 ectodomain phosphorylation in cell‐cell interactions, we generated CD98‐CD69 and CD69‐CD98 constructs, as well as CD98 lacking PDZ‐binding domain or mutated at predicted serine phosphorylation sites and transfected these constructs into CHO cells. Cell adhesion assays show that mutation at S305‐S307‐S309 or deletion of extracellular C‐terminal or PDZ‐binding domain of CD98 significantly decreases the binding of Jurkat cells to CHO cells. In conclusion, we demonstrate, for the first time, that CD98 is ecto‐phosphorylated by ePK and its phosphorylation affects cell‐cell interactions.
Gastrointestinal (GI) infusion of bitter tastants supports conditioned flavor avoidance (CFA) and activates central neural Fos expression
Authors: Hao Shuzhen , Dulake Michelle , Espero Elvis , Sternini Catia , Raybould Helen E , Rinaman Linda
Published: June 2021

Noxious substances are detected by oral bitter taste receptors. G‐protein coupled bitter taste receptors (T2Rs) and the associated G protein, Gαgustducin also are expressed in the rodent GI tract, where Gαgustducin co‐localizes with PYY, GLP‐1 and 5‐HT in entero‐endocrine cells. The present study sought to determine whether ligand stimulation of GI T2Rs can support CFA and activate associated brain areas in rats. In a two‐bottle choice paradigm, GI infusion of T2R agonists induced a robust CFA, whereas GI water infusion did not. Immunohistochemical localization of Fos, a marker of neuronal activation, showed that GI infusion of T2R agonists recruited noradrenergic and other neurons within the nucleus of the solitary tract (NST) and ventrolateral medulla (VLM), and also activated the lateral parabrachial nucleus (LatPBN) and central amygdala (CeA). These results support the view that GI T2Rs are sufficient to activate central neural circuits that promote conditioned avoidance behavior.
Apoptosis and Surivivin gene expression during Holothuria glaberrima intestinal regeneration
Authors: Donato‐Santana Christian , Rojas‐Cartagena Carmencita , Garcia‐Arraras Jose Enrique
Published: June 2021

The sea cucumber (Holothuria glaberrima) is an excellent model to study regeneration processes in deuterostomes. H.glaberrima has the ability to regenerate most of its viscera after the process of evisceration, where most of the viscera are expelled from the body. We have showed that apoptosis takes place during intestinal regeneration. Apoptosis, is the result of DNA internucleosomal cleavage, the morphological characteristics are: cell contraction, nuclear pyknosis, chromatin condensation and cell fragmentation. By using the TUNEL assay, where the TdT enzyme catalyzes the addition of Br‐dUTP, labelling the 3′‐OH terminal groups exposed during the internucleosomal cleavage; we identified apoptotic cells by fluorescence microscopy. We showed a peak in the number of apoptotic cells throughout the first week of regeneration and the distribution of apoptotic cells in the different layers of the large intestine (serosa, sub‐mucosa, mucosa and mesentery). Moreover we have now identified the holothurian homologue of the Survivin gene that in vertebrates functions as an inhibitor of apoptosis. Preliminary results have shown a differential expression of this gene during regeneration. Molecular experiments, PCR and In‐situ hybridization are under way to asses the temporal and spatial expression of apoptosis and survivin expression. Our results prove that similar to embryonic development, apoptosis plays an important role during intestinal regeneration. These studies provide a deeper understanding of the process of intestinal regeneration in the sea cucumber and the applications to studies in other animals, including humans.
Camptothecin induced expression and phosphorylation of p53 regulates apoptosis in epithelial cells
Authors: Bhattacharya Sujoy , Ray Ramesh M. , Johnson Dianna A. , Johnson Leonard R.
Published: June 2021

p53 is an established tumor suppressor protein. In addition to preventing tumor growth, p53 plays a key role in cell cycle regulation and in the cellular response to cytotoxic stress and DNA damage. Upon induction of p53, a host of target genes are transcriptionally activated, including p21waf1, GADD45, bax, bcl‐2. At this time, the specific signals required for p53‐mediated cell cycle arrest and/or apoptosis remain complex. Polyamine depletion of intestinal epithelial cells (IEC‐6) increased p53 and p21 protein levels and these cells were resistant to TNF‐α‐, γ‐ irradiation‐, and camptothecin (CPT)‐induced apoptosis. CPT induced p53 protein levels in a time dependent manner, however, its phosphorylation decreased significantly. Increased p53 levels correlated with p21 levels indicating transcriptional activity of p53. The inhibitors of transcription (Actinomycin D) and translation (Cycloheximide) prevented CPT‐induced apoptosis. Although, cycloheximide (CHX) completely prevented p21 synthesis, p53 levels were unaltered. RITA (p53 activator III) which blocks p53‐MDM2 interaction and thereby p53 ubiquitination significantly increased apoptosis. Actinomycin D significantly increased p53 protein levels and its phosphorylation. However, p21 expression was completely abolished. These results suggest a predominant role of p53 protein in CPT‐induced apoptosis. In addition, we used retinal pigment epithelial cells (RPE), which are highly resistant to a variety of apoptosis inducers, and compared the p53 status and phosphorylation in response to CPT. Supported by NIDDK grant DK‐16505.
Inositol hexaphosphate alters mucin expression in the intestinal mucosa of mice
Authors: Onyango Edward , Madsen Cathy , Gendler Sandra
Published: June 2021

Inositol hexaphosphate (IP6 or phytic acid) is thought to be the active ingredient in legumes and cereals that provides anti‐cancer activity. Our goal is to understand the influence of IP6 on mucin expression (Muc1 and Muc2, specifically) in the mouse intestine. C57BL/6 mice were randomized into 3 groups of 6 mice. Mice were orally dosed with IP6 (0, 1, 2 g/kg BW) for 5 days and sacrificed on the sixth day. Sections of the small intestine were removed, fixed in methacarn, sectioned and stained with Alcian blue‐PAS to visualize mucins. Mucosa from the jejunum and colon were removed and total RNA prepared using Trizol®. cDNA was prepared using random hexamers and TaqMan minor groove binding probes were used for real time PCR amplification of message for Muc1 and Muc2. Expression of Muc1 in the jejunum was minimal whereas that of Muc2 increased 1.4‐fold at the higher IP6 dose rate. In the colon, expression of both Muc1 and Muc2 increased in mice dosed with IP6. Expression of Muc1 increased 1.6‐fold and 3.8‐fold in mice dosed with 1 and 2 g/kg IP6, respectively. Expression of Muc2 increased 2.0‐fold and 1.7‐fold in mice dosed with 1 and 2 g/kg IP6, respectively. Alcian blue‐PAS stained sections of the colon showed an increase in mucus. Thus, IP6 seems to increase the expression of Muc1 and Muc2 in the mucosa lining of the small intestine, particularly in the colon.

Research was supported by East Tennessee State University and Mayo Clinic.
Effects of Anandamide on Proliferation and Activation of Hepatic Stellate Cells Through Cannabinoid‐2 Receptors
Authors: Tang Wang‐Xian , Liu Hong‐Yan , Duan Ruixian , Yang Qiao
Published: June 2021

Recent studies have indicated that endogenous endocannabinoids (ECB) are importantly involved in the development of liver cirrhosis. However, the mechanisms mediating the actions of these ECB are still poorly understood. The present study tested the hypothesis that anandamide (AEA) inhibits activation and proliferation of hepatic stellate cells (HSCs) to prevent liver fibrosis via cannabinoid‐2 (CB2) receptors. Using immunocytochemistry and Western blot analysis, we found that both cannabinoid‐1 (CB1) and CB2 receptors were detectable in cultured HSCs with a higher level of CB2 than CB1. When HSCs were stimulated by PDGF, the expression of CB2 receptors was significantly enhanced. By flow cytometry and MTT (methyl thiazolyl tetrazolium) detection, PDGF was found to markedly stimulate proliferation of HSCs, which was inhibited by AEA dose‐dependently. However, this action of AEA was not associated with increased apoptosis or necrosis, and CB2 receptors were not involved. We also found that PDGF‐induced increase in various factors related to activation of HSCs such as alpha‐SMA, TGF‐β, α1 (I) collagen, and TIMP‐1 was substantially suppressed by AEA and that CB2 antagonist AM630 reversed this suppressor action of AEA. These results suggest that AEA may inhibit proliferation and activation of HSCs and CB2 receptors importantly mediate AEA‐induced inhibitory action on activation of these HSCs. This CB2 receptor‐mediated action on HSCs could be a therapeutic target against liver fibrosis. (Supported by National Nature Science Foundation of China 30571627)
Hypoxia‐inducible Factor‐dependent Production of Profibrotic Mediators by Hypoxic Hepatocytes
Authors: Welch Timothy Paul , Bustamante Juan , Moon Jeon‐Ok , Copple Bryan L.
Published: June 2021

Liver fibrosis is characterized by increased deposition of collagen in the liver during chronic injury. Previous studies have shown that hypoxia‐inducible factor‐1α (HIF‐1α) is a key regulator of profibrotic mediator production during fibrosis. The study goal was to determine whether HIFs are activated in hypoxic hepatocytes and whether they regulate production of profibrotic mediators. Exposure of primary mouse hepatocytes in vitro to either 3% or 1% oxygen (i.e., hypoxia) stimulated nuclear accumulation of HIF‐1α and HIF‐2α proteins. In addition, hypoxia increased expression of proteins implicated in liver fibrosis, including monocyte chemotactic protein‐1 (MCP‐1), vascular endothelial cell growth factor (VEGF), and plasminogen activator inhibitor‐1 (PAI‐1). Hypoxia also increased expression of adrenomedullin‐1 (ADM) and ADM2, proteins implicated in development of hemodynamic abnormalities. Next, to determine whether HIFs are required for upregulation of these mediators by hypoxia, Cre/lox technology was used to delete either HIF‐1α or HIF‐1α from mouse hepatocytes. Results from this study showed that HIFs were required for MCP‐1, VEGF, PAI‐1, ADM, and ADM2 upregulation in hypoxic hepatocytes.