bluuebunny/crossref_metadata_2025 huggingface dataset:
The intrarenal angiotensin system and inflammation in Dahl salt‐sensitive hypertension
Authors: Lin Lin , Phillips William E. , Manning R. Davis
Published: June 2021

Dahl salt‐sensitive (S) hypertension is characterized by renal damage, renal immune cell infiltration and low plasma renin activity. Recent studies have shown that the intrarenal angiotensin (ANGII) system may be activated in these rats and along with the immune system may play important roles in the development of renal injury and hypertension. However, the roles of intrarenal ANGII/immune systems in the Dahl salt‐sensitive hypertension are not clear. Dahl S rats were equipped arterial catheters and subjected to a 5‐week diet of high Na (8% NaCl) or high Na+ARB (ANGII AT1 receptor blocker, candesartan cilexetil, 15 mg/kg/day). By the end of week 5 in the high Na+ARB group: mean arterial pressure (MAP) was 145 ± 1 mmHg compared to the high Na group value of 168 ± 7 mmHg*; kidney tissue ANGII concentration, macrophages, TNFα, and MCP‐1 and urinary protein excretion were 3.2 ± 1.4 pg/mg, 7.1 ± 0.6 cells/mm2, 0.38 ± 0.01 pg/mg, 21.7 ± 1.4 pg/mg, and 168 ± 20 mg/day in ARB rats compared to the high Na rat values of 5.5 ± 0.7 pg/mg*, 22 ± 1.6 cells/mm2*, 0.52 ± 0.02 pg/mg*, 26.7 ± 1.5 pg/mg*, and 318 ± 43 mg/day*, respectively. In summary, long term ARB treatment in high Na Dahl S rats decreased renal tissue ANGII, renal inflammation, renal damage and MAP. The intrarenal angiotensin system plays an important proinflammatory role in the kidneys of Dahl S rats leading to renal damage and increased blood pressure. Support: NIH grant HL51971.
Renal vascular L‐arginine metabolism, NO release and contraction in Angiotensin II hypertensive rats
Authors: Hultström Michael , Helle Frank , Iversen Bjarne M.
Published: June 2021

The effect of Angiotensin II (AngII) hypertension on L‐arginine metabolising enzymes and AngII induced vascular contraction and nitric oxide release was investigated because we found increased expressions in vessels from both kidneys of 2K1C rats and increased nitric oxide release from the non‐clipped kidney. Four groups of rats were infused with 80 ng/min AngII or saline for 14 days, and/or given 60 mg/kg/day Losartan. Gene expression was studied in isolated preglomerular vessels by RTPCR. Arteriolar contraction was studied using 10−10, 10−9, 10−8 and 10−7 mol/L of AngII and 10−4 mol/L L‐NAME in vessels isolated using the agar infusion/enzyme digestion method. Expressions of eNOS , caveolin‐1 and Arginase‐2 were not changed by AngII infusion. CAT‐1 (0.38±0.07 to 0.73±0.12, P<0.05), CAT‐2 (1.14±0.29 to 2.74±0.48), DDAH‐2 (1.09±0.27 to 2.3±0.46) and Arginase‐1 (1.08±0.17 to 1.82±0.22) were increased in AngII infused rats and normalised by Losartan. Losartan reduced the expression of eNOS (0.97±0.26 to 0.37±0.11 in controls; 0.8±0.16 to 0.36±0.1 in AngII infused rats) and caveolin‐1 (2.49±0.59 to 0.82±0.24 in controls and 2.59±0.61 to 1.1±0.25 in AngII infused rats). AngII induced contraction was enhaced in the AngII infused animals (47±15 % vs. 7±5 % at 10−10 mol/L AngII). Contraction at 10−10 mol/L AngII was enhaced by L‐NAME in AngII infused rats (96±4 %). In conclusion, Losartan affects eNOS and caveolin‐1 expression through an AT1 receptor independent pathway. CAT‐1, CAT‐2, DDAH‐2 and Arginase‐1 expressions in renal resistance vessels are regulated through the AT1 receptor, which may be of direct importance for nitric oxide synthesis and the regulation of renal blood flow as can be seen by the buffering effect of NO on the contraction of isolated arterioles in the vessels from AngII infused animals. The study was funded by Helsevest, Norway.
Vascular Endothelial Growth Factor (VEGFA) induced phrenic motor facilitation (PMF) requires ERK activation
Authors: Dale‐Nagle Erica Arden , Mitchell Gordon S
Published: June 2021

VEGFA is a hypoxia‐induced growth factor known to regulate angiogenesis. We recently demonstrated that VEGFA is expressed in presumptive phrenic motor neurons (Dale et al., FASEB J., 2007) and that cervical spinal VEGFA receptor activation elicits long‐lasting PMF (Dale‐Nagle and Mitchell, FASEB J., 2008). Since VEGF receptor 2 signals via ERK 1/2 MAP kinase, we tested the hypothesis that ERK 1/2 activation is necessary for VEGFA‐induced PMF. Anesthetized, pump‐ventilated, paralyzed and vagotomized male, Sprague‐Dawley rats were prepared with two intrathecal catheters at C4. Recordings of integrated phrenic nerve activity were made with three groups after intrathecal injections of: 1) vehicle (10μL of 0.1% BSA in aCSF; n=6); 2) VEGFA (100ng; n=6); or 3) U0126 (100μM, 12μL; n=3), a MEK1/2 inhibitor, 30 min prior to VEGFA. VEGFA induced PMF for at least 90 min post‐injection (59% ± 7% baseline; p<0.01), an effect significantly greater (p<0.01) than in rats pretreated with U0126 (16% ± 8%; n.s.) or time controls (9% ± 8%; n.s.). Our results provide the first evidence that VEGFA elicits spinal synaptic plasticity by a mechanism requiring ERK 1/2 MAP kinase activation. Supported by NIH NS05777 & HL80209.
Exaggerations in skeletal muscle metaboreflex activity are attenuated by increasing nitric oxide production within the brainstem of spontaneously hypertensive rats
Authors: Leal Anna K , Mitchell Jere H , Smith Scott A
Published: June 2021

In hypertension, evidence suggests the skeletal muscle metaboreflex (SMM) is exaggerated and contributes significantly to the heightened blood pressure (BP) response to exercise in this disease. The mechanisms underlying SMM overactivity remain unknown. Sensory information elicited by the SMM is processed within the nucleus tractus solitarius (NTS) of the brainstem. Normally, endogenously produced nitric oxide (NO) within the NTS attenuates the BP response elicited by the SMM. Therefore, we hypothesized that a decrease in the availability of NO within the NTS mediates the exaggerated SMM activity manifest in hypertension. The SMM was preferentially activated by injecting capsaicin (0.3 μg/ 100 μL) into the hindlimb arterial supply of male normotensive Wistar Kyoto (WKY; n=4) and spontaneously hypertensive (SHR; n=5) rats before and after the dialysis of the NO precursor L‐arginine (1.0 μM) into the NTS. Capsaicin injections resulted in larger increases in BP in SHR (41±9 mmHg) than WKY (21±4 mmHg). In both groups, L‐arginine dialysis attenuated the BP response. Interestingly, the magnitude of this reduction was greater in SHR (‐12±5 mmHg) than WKY (‐3±1 mmHg). Augmenting NO availability in the NTS partially corrected the exaggerated BP response to SMM activation in SHR. This finding suggests that decreases in NO within the NTS may contribute to SMM overactivity in hypertension. Supported by NIH HL‐088422
Evidence for the possible association between calcium transient of isolated ventricular cardiomyocytes and exercise capacity in rats
Authors: Prímola‐Gomes Thales Nicolau , Lauton‐Santos Sandra , Balthazar Claudio Heitor , Guatimosim Silvia , Coimbra Cândido Celso , Cruz Jader Santos , Soares Danusa Dias , Natali Antonio José
Published: June 2021

The association between the whole cell intracellular Ca2+ transients ([Ca2+]i) of isolated cardiomyocytes and intrinsic exercise capacity in rats was evaluated. Standard performance (SP; n= 120) and high performance (HP; n= 36) rats were previous selected in a treadmill progressive exercise test accordingly to total exercise time until fatigue (TTF). The SP group encloses rats with TTF between 16.63 and 46.57 min and the HP encloses rats with TTF higher than 46.57 min. Oxygen consumption at the point of fatigue (VO2max) was measured by an open‐flow indirect calorimeter. The [Ca2+]i transients were measured by confocal microscopy in cardiomyocytes loaded with fluo‐4 AM. Statistical significance was assessed by t test, Mann‐Whitney test and linear regression (means ± SE). The study was approved by the Ethical Committee at the Federal University of Minas Gerais. The HP rats presented high VO2max (56.72 ± 0.54 mL.Kg−1.min−1, n= 6) when compared to SP rats (40.01 ± 0.42 mL.Kg−1.min−1, n= 6; P≤0.05). The [Ca2+]i transient peak was 4.18 ± 0.12 and 5.79 ± 0.34 for SP (n= 5 rats and 83 cells) and HP (n = 4 rats and 61 cells) groups, respectively (P≤0.05). The [Ca2+]i transient peak was positively correlated with the maximal treadmill speed in the progressive test (r = 0.81; P≤0.05). The data indicated that the high intrinsic exercise capacity is closely related to higher [Ca2+]i transients in isolated ventricular cardiomyocytes.
Urocortin 3 Microinjected Into The Intermediolateral Cell Column Elicits Tachycardia In The Rat
Authors: Chitravanshi Vineet C , Sapru Hreday N
Published: June 2021

Urocortin 3 (Ucn 3) is a new member of corticotropin releasing factor (CRF) family of peptides. There is limited information about the cardiovascular actions of this peptide. Urethane‐anesthetized, artificially ventilated, adult male Wistar rats, weighing between 325‐350 gm, were used in this study. L‐Glutamate (L‐Glu; 5 mM), microinjected (30 nl) into the right intermediolateral cell column (IML) at T1 or T2 level, elicited an increase (35 ± 4 bpm) in heart rate (HR). The onset and duration of these responses was 5 and 50‐60 sec, respectively. Microinjections (30 nl) of Ucn 3 (0.31, 0.62, 0.12 and 0.25 mM) into the same sites in the IML elicited tachycardia; the increases in HR were 13.6 ± 1.5, 31.8 ± 3.9, 37.9 ± 2.1 bpm and 33.6 ± 3.4 bpm, respectively. Maximum tachycardic responses were elicited by 0.12 mM concentration of Ucn 3. The tachycardic responses elicited by Ucn 3 (0.12 mM) were significantly attenuated by prior microinjections (30 nl) of a selective antagonist (K41498, 1 mM) for CRF type 2 receptors. It was concluded that Ucn 3 exerts an excitatory effect on IML neurons controlling cardiac function.

Support: NIH grants HL024347 and HL076248.
Sympathetic nerve activity during development of DOCA‐salt hypertension
Authors: Yemane Henok T , Burris Sarah K. , Knuepfer Mark M
Published: June 2021

Prior studies have provided indirect evidence suggesting sympathetic nerve activity (SNA) contributes to the development of hypertension. We hypothesized that evoked and resting SNA would be elevated 2‐3 weeks after inducing DOCA‐NaCl hypertension. We instrumented male Sprague‐Dawley rats to measure arterial pressure, ECG and renal SNA before inducing hypertension. We recorded resting SNA before and after implanting DOCA and administering 1% NaCl drinking water. Responses to acute stress were also recorded prior to inducing and during development of hypertension (2‐3 weeks). Background activity was determined with ganglionic blockade (trimethaphan). We observed that DOCA‐salt treatment (n=8) elicited an increase in arterial pressure that started within 2‐3 days. During the early phase, renal SNA did not seem to be elevated as the decrease in SNA in response to trimethaphan was unchanged (‐41± 6% control vs. ‐38 ± 6% on day 12). We examined the acute response to cold water stress (1 cm deep for 1 min) and observed that initial evoked responses during cold stress were progressively smaller in DOCA‐salt hypertension over 12 days. Our data suggest that SNA does not play a role in the development of DOCA‐salt hypertension, although it will be necessary to verify this in other sympathetic nerves. Supported by USPHS NIH R01 DA0017371, HL 091440 and 5T32 GM008306.
Vestibular effects on relative arterial blood flow to and venous return from the limbs during postural changes of conscious felines
Authors: Yavorcik Kevin J , Erwin Monica , Misra Sunil P , Cotter Lucy A , Reighard Derek A , Wilson Timothy D , Cass Stephen P , Yates Bill J
Published: June 2021

Prior studies showed that vestibular signals influence cardiovascular regulation by eliciting vasoconstriction in the dependent limbs during postural changes. These findings led to the hypothesis that loss of vestibular inputs would exacerbate blood pooling in the body regions below the heart during alterations of body position. We tested this hypothesis by comparing blood flow measured using transit‐time ultrasound technology from the femoral vein and artery in conscious cats subjected to head‐up tilt (HUT) at amplitudes up to 60°. Responses were recorded before and after bilateral ablation of vestibular afferents. Before vestibular lesions, blood flow decreased 24‐35% in the femoral artery by ≈9 sec following 60° HUT, presumably due to vasoconstriction, while venous blood flow decreased 55‐65%. After lesions, arterial blood flow dropped only 0‐14% at the time maximal vasoconstriction previously occurred, while venous blood flow decreased 57‐87%. The alterations were shown to be significant by ANOVA (P<0.05). Because vestibular lesions resulted in more blood flow to the leg in the femoral artery and less venous return in the femoral vein, we concluded that the lesions resulted in hindlimb blood pooling during HUT.
PPARγ activation reverses hypertensive remodeling of cerebral arteries
Authors: Cipolla Marilyn J. , Godfrey Julie A. , Smith Jeremiah
Published: June 2021

Peroxisome proliferator‐activated receptor gamma (PPARγ) is expressed in vascular smooth muscle and is thought to have beneficial effects on the vasculature. We tested the hypothesis that PPARγ activation could reverse pre‐existing remodeling of cerebral arteries induced by hypertension. Female SD rats were hypertensive for 5 weeks by NOS inhibition with L‐NAME (0.5 g/ml in drinking water; HTN, n=11) and compared to untreated controls (CTL, n=7). A separate group of HTN rats were given the PPARγ activator rosiglitazone (20 mg/kg in food) after 2 weeks on L‐NAME for the remaining 3 weeks (HTN+Rosi, n=8). L‐NAME increased MAP for the entire 5 weeks in both HTN and HTN+Rosi groups vs. CTL (mmHg): 134 ± 5 and 132 ± 2 vs. 111 ± 2 (p<0.01). Elevated pressure in the HTN group was associated with inward hypertrophic remodeling of posterior cerebral arteries, demonstrated by reduced inner diameters and increased wall thickness. PPARγ activation reversed remodeling and had similar diameters and wall thickness as controls. At 75 mmHg, inner diameters of isolated arteries from CTL, HTN and HTN+Rosi were (μm): 224 ± 9, 199 ± 5 (p<0.01 vs. CTL) and 217 ± 8 (ns vs. CTL) and wall:lumen ratios were 0.04 ± 0.002, 0.06 ± 0.002 (p<0.01 vs. CTL and HTN‐Rosi) and 0.04 ± 0.002 (ns vs. CTL). These data demonstrate that PPARγ activation reverses hypertensive remodeling of cerebral arteries despite significantly elevated blood pressure.
The expression pattern of pannexin1 after global cerebral ischemia/reperfusion in rats
Authors: Zhu Yujin , Sun Lina , Wang Qian , Su Fang , Wang Jinye , Xia Qiang
Published: June 2021

Pannexin1 (Panx1) is a protein found recently in vertebrate and expresses highly in central nervous system. Recent studies showed that Panx1 maybe form gap junction hemichannels in neurons and participate in hypoxia induced hippocampal pyramidal neurons injury in vitro, but its function toward cerebral ischemia/reperfusion (I/R) injury in vivo is unclear. In this study we tested the expression pattern of Panx1 after global cerebral ischemia and reperfusion in rat hippocampus. Global cerebral ischemia was induced by four‐vessel occlusion on male Sprague‐Dawley rats. Proteins for Western Blotting were isolated from rat hippocampus after ischemia for 2, 3, 5, 10, 15, 20, 30 min, or after reperfusion for 1/6, 1/2, 1, 2, 4, 6, 8, 12, 24, 48, 72 h following 20 min ischemia, respectively. We found that the content of Panx1 showed biphasic alterations in different I/R periods. The protein content curve in I/R temporal coordinate dropped suddenly upon the onset of ischemia with the first valley (32.1% of pre‐ischemia) detected at 10 min of ischemia. Then Panx1 content increased gradually and reached peak of 138.5% at about 1/2 h reperfusion. The second valley of Panx1 appeared at 8 h of reperfusion. After then the content of Panx1 increased slightly but kept in lower level till 72 h of reperfusion. The time‐dependent alterations of Panx1 content during global cerebral ischemia and reperfusion suggests that it may play an important role in the development of cerebral I/R injury.
Protective effect of hypothermia and mannitol postconditioning on mouse brain suffered from ischemic/reperfusion injury
Authors: SHEN Jia , LI Menghan , BAI Xiaoyin , XIA Qiang
Published: June 2021

OBJECTIVE

The aim of the study was to determine the synergistic protective effect of mild hypothermia combined mannitol therapy against global cerebral ischemia/reperfusion (I/R) injury and the underlying mechanisms.

METHODS

Male C57BL/6 mice (22‐28g) were randomly divided into sham group (Gourp S), control (ischemia/reperfusion) group (Group C), mild hypothermia (31‐33°C, 2h after reperfusion) group (Group Ht), mannitol (20% mannitol, caudal intravenous infusion) group (Group Ma), mild hypothermy combined mannitol group (Group Ht+Ma). Learning and memory abilities were detected by Morris water maze task, the CA1 region neuron survival rates were detected by Nissl staining, the activities of superoxide dismutase (SOD) of hippocampus tissue were measured.

RESULTS

The average escape latency and swimming distance of Group Ht+Ma were significantly shorter than Group C, also shorter than those of Group Ht and Group Ma. The average neuron survival rates were markedly increased in Group Ht+Ma, Group Ht and Group Ma, more than that in Group C. The SOD activity of Group Ht+Ma was remarkably higher than control group, and higher than group Ht and group Ma.

CONCLUSION

The therapies of mild hypothermia and mannitol have protective effect against global ischemia/reperfusion injury. The combination of mild hypothermia and mannitol therapy enhances the protection effect by increasing the SOD activity.
Protective effect of luteolin on neurons against cerebral ischemia/reperfusion injury via regulating the activity of ATPase
Authors: Fang Lumei , Xia Qiang , Zhang Xiong
Published: June 2021

The aim of this study was to determine whether luteolin protects neurons against cerebral ischemia/reperfusion injury and its underlying mechanism. We cultivated hippocampal neurons from new‐born Sprague‐Dawley rats, and established the model of oxygen‐glucose deprivation/reperfusion (2 h/24 h). Neuronal viability was assessed by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay, and cell injury was evaluated by lactate dehydrogenase (LDH) leakage rate. The percentage of apoptotic cells was measured by using Hoechst 33258 staining. Adult male Sprague‐Dawley rats were subjected to four‐vessel‐occlusion for 10 min followed by reperfusion for 24 h, and the activities of Na+, K+‐ATPase and Ca2+, Mg2+‐ATPase were measured by spectrophotometry. Oxygen‐glucose deprivation/reperfusion decreased the cell viability and increased LDH leakage rate and percentage of apoptotic cells. Compared with oxygen‐glucose deprivation/reperfusion group, luteolin (1‐100 μmol/L) treatment significantly increased the cell viability, decreased LDH leakage rate and the percentage of apoptotic cells in a dose‐dependent manner. Luteolin (200 mg·kg−1, i.p.) markedly inhibited the decrease of ATPase activities induced by global cerebral ischemia/reperfusion. These results demonstrate that luteolin protects neurons against ischemia/reperfusion‐induced cell injury via, at least partly, regulating the activity of ATPase.
Increased cerebral ischemic damage during chronic alcohol consumption: role of NAD(P)H oxidase
Authors: Zhao Honggang , Mayhan William G. , Sun Hong
Published: June 2021

We hypothesized that NAD(P)H oxidase may contribute to increased cerebral ischemic damage during chronic alcohol consumption. Sprague‐Dawley rats were randomly divided into four groups: nonalcohol‐fed, alcohol‐fed, nonalcohol‐fed plus apocynin‐treated, and alcohol‐fed plus apocynin‐treated. Alcohol (8 g/kg/day) was mixed into a liquid diet and given to rats for 8 weeks. Apocynin (7.5 mg/kg/day) was orally administrated for 4 weeks prior to the experiment. Rats were subjected to 2 hours of right middle cerebral artery occlusion (MCAO). Infarct volume and neurological score were assessed 24 hours after reperfusion. Superoxide production from ischemic border area was measured by lucigenin‐enhanced chemiluminescence. In addition, expression of NAD(P)H oxidase membrane subunit gp91phox in ischemic border area was analyzed by Western blot. Compared with nonalcohol‐fed rats, alcohol‐fed rats had a larger infarct volume, worse neurological score and higher superoxide production. Apocynin did not alter infarct size, neurological deficit or superoxide production in nonalcohol‐fed rats, but significantly reduced infarct size, improved neurological outcome and attenuated superoxide production in alcohol‐fed rats. Chronic alcohol consumption significantly increased gp91 phox expression compared with nonalcohol‐fed rats, and cerebral ischemia also significantly enhanced gp91 phox expression compared with nonischemic side. Apocynin treatment did not alter gp91 phox expression in either nonalcohol‐fed rats or alcohol‐fed rats. Our findings suggest that NAD(P)H oxidase may play an important role in increased cerebral ischemic damage during chronic alcohol consumption.
Cerebroprotective role of Tetrahydro Curcumin in hyperhomocysteinemic ischemic mice by regulating NF‐kappa B
Authors: Kumar Munish , Givvimani Srikanth , Puspakumar SB , Mishra Paras K , Kundu Soumi , Rodriguez‐Alvarez Walter E. , Tyagi Neetu , Sen Utpal , Tyagi Suresh
Published: June 2021

Vasospasm is a major cause of cerebrovascular diseases. Hyperhomocysteinemia exacerbates stroke in part by increasing oxidative stress and vascular remodeling. Cystathionine βsynthase heterozygous (CBS ‐/+) mice develop high level of Hcy. Tetra hydro curcumin (THC) polyphenolic compound from plant Curcuma longa, is a commonly used remedies. Present study determines the effect of THC in hyperhomocysteinemic mice after an ischemia episode. There were six groups of mice: WT, WT/Ischemia, WT/Ischemia treated THC, CBS (‐/+), CBS (‐/+)/Ischemic and CBS (‐/+)/Ischemic treated THC. Middle cerebral Artery Occlusion (MCAO) was performed for 1 hour. After 24 hours, mice were injected with THC (300mg/kg) I.P. The Infarct area (IA) was assessed using TTC stain. Real Time PCR, Immunohistchemistry and Western blot were used to determine the expression of SOD‐I, SOD‐II, Phospho 22, Phospho 47. Neurological assessments were done in above group. The Electrophoretic Mobility Shift Assay (EMSA) was performed using NF‐kappa B probe. The result suggested THC significantly decreases IA and oxidative damage in Ischemic mice. The EMSA result suggest that there was significant decrease in the NF‐kappa B binding interaction to protein. In addition, there was significant improvement in neurological behavior in THC treated ischemic mice compared to Ischemic groups.
Effects of hypoxic exercise training on exercise‐induced thrombin generation
Authors: Chen Yu‐Wen , Wang Jong‐Shyan , Chen Jan‐Kan
Published: June 2021

Platelet‐derived microparticles (PDMP) are important players in thrombogenesis. This study investigates whether hypoxic exercise training affects PDMP‐mediated thrombin generation (TG) at performing strenuous exercise. Twelve sedentary males were trained on a bicycle ergometer at about 60%VO2max under 15% O2 air condition for 20 minutes per day, 5 days per week for 4 weeks. TG was determined by a novel calibrated, automated thrombinography assay, whereas PDMP count, procoagulant factor binding, and phosphatidylserine (PS) exposure were evaluated by a two‐color flow cytometry. Our results showed that strenuous exercise increased TG via enhancing circulating PDMP concentration. Pathological shear stress (108 dynes/cm2) increased the PS exposure of the PDMP, the binding of the PDMP to procoagulant factor V/Va and VIII, and PDMP‐induced TG peak and rate. However, 4 weeks of hypoxic exercise training remained unchanged in exercise‐induced PDMP secretion and TG. Therefore, we conclude that strenuous exercise magnifies shear stress‐induced TG by eliciting PDMP secretion and enriching procoagulant factors binding to PDMP, which remain unchanged after hypoxic exercise training.
High altitude acclimatization responses in cerebral artery PKC‐dependent signaling pathway proteins: Changes with development
Authors: Goyal Ravi , Mittal Ashwani , Shi Lijun , Wedhas Nia , Longo Lawrence D.
Published: June 2021

Objective

High altitude, long‐term hypoxia (LTH) is associated with a number of changes in pre‐ and post‐junctional signaling in both fetal and adult cerebrovasculature. Because PKC and downstream pathways play a key role in Ca2+ sensitization of vascular contraction, and are less abundant in fetus, we tested the hypothesis that LTH alters PKC activation pathways differently in the fetus, as compared to the adult.

Methods

In cerebral arteries we quantified both total and activated (phosphorylated) PKC, ERK1/2, caldesmon, Rho A, CPI‐17, and myosin light chain20 (MLC20), and PDBu‐ (PKC agonist) induced contractions and their responses to blockade of ERKs (U‐0126) or Rho kinase (Y‐27632).

Results

In adult cerebral arteries, LTH acclimatization was associated with significant decreases in total and activated PKC, with increases in activated ERK1/2, CPI‐17, and MLC20 and its kinase. In contrast, in fetal arteries LTH increased PKC expression and increased ERK1/2 activation, with no change in caldesmon, Rho A, CPI‐17, or MLC20.

Conclusions

In adult cerebral arteries, acclimatization to LTH is associated with significant up‐ or down‐regulation of a number of elements of the PKC‐dependent Ca2+ sensitization pathway. In contrast, acclimatization to LTH in the fetus is associated with relatively minor changes in the Ca2+ sensitization pathway. (Supported by USPHS HD 31226)
Exercise performance in moderate hypoxia in rats with impaired carotid bodies
Authors: Meyers Kristen A , Young Kristen M , Bavis Ryan W
Published: June 2021

Previous studies have shown a positive correlation between the magnitude of the resting hypoxic ventilatory response (HVR) and exercise performance at high altitude in humans. We employed a rodent model to experimentally test this relationship. Rats exposed to chronic hyperoxia during postnatal development exhibit smaller carotid bodies and reduced HVR as adults. Therefore, rats were exposed to 60% O2 for the first 2 postnatal weeks. At 2‐3 months of age, these rats were run on a treadmill at various work rates in both room air and moderate hypoxia (12% O2) to assess endurance (time to exhaustion at 20 m/min, 5° incline) and maximal rate of O2 consumption (VO2,max). Running times and VO2,max were reduced by hypoxia in both control and hyperoxia‐treated rats (P<0.001). In hypoxia, hyperoxia‐treated rats exhibited somewhat shorter times to exhaustion than controls (21±2 (SEM) vs. 29±2 min; P<0.01). In contrast, VO2,max during moderate hypoxia did not differ between treatment groups. Although HVR was not measured in this study, histology confirmed a significant reduction in carotid body volume in hyperoxia‐treated rats (P<0.01). We conclude that HVR and/or carotid body size may have modest effects on hypoxic exercise performance in rats. Supported by NIH grant P20 RR‐016463 (Maine INBRE).
Critical Evaluation of DHE Fluorescence and Cytochrome c Absorbance Measurement of Superoxide in the Presence of Nitric Oxide and Superoxide Dismutase
Authors: Rogers Steven Clay , Kavdia Mahendra
Published: June 2021

Excessive superoxide levels can disrupt vascular homeostasis and lead to increased formation of hydrogen peroxide, peroxynitrite and decreased vasodilation. An accurate detection of superoxide in vivo is necessary to better understand a number of vascular pathologies. In this study, we measure the effect of nitric oxide and superoxide dismutase (SOD), which are two of the most effective scavengers of superoxide in vivo, presence on the fluorescence and absorbance measurement of superoxide. We used xanthine oxidase/hypoxanthine and sodium nitroprusside to produce superoxide and NO, respectively. A microplate reader was used for two common methods to measure superoxide: the fluorescence of dihydroethidium (DHE) and absorbance of ferricytochrome‐c. SOD significantly decreased superoxide concentrations in both the absorbance and fluorescence assays. NO showed only an effect on superoxide production at higher concentrations in the fluorescence measurements. Kinetic analysis of DHE‐superoxide interaction resulted in a reaction rate of 1.1 × 103 M−1s−1 that is ~250× lower than the previously reported reaction rate of 2.6 × 105 M−1s−1. The results suggest that accurate in vivo measurements of superoxide production may be extremely difficult due to competitive interference from varying NO and SOD levels, however the superoxide concentration may be quantified. Supported by: AHA 0530050N & NIH R01 HL084337.
Radioprotection by inactivation of deoxyribonuclease I
Authors: Apostolov Yevgeniy O. , Soultanova Izoumroud , Savenka Alena V. , Bagandov Osman O. , Yin Xiaoyan , Stewart Anna G. , Walker Richard B. , Basnakian Alexei G.
Published: June 2021

Gamma radiation‐induced cell death is associated with and mediated by enzymatic DNA fragmentation. A nuclease that produces this fragmentation of DNA is unknown. To determine whether deoxyribonuclease I (DNase I), a major cytotoxic/apoptotic nuclease expressed in all cells and tissues, can mediate gamma radiation‐induced tissue injury, we used DNase I knockout (KO) mice and a zinc chelate of 3,5‐diisopropylsalicylic acid (Zn‐DIPS) that, as we show, has DNase I inhibiting activity in vitro. Our study demonstrated that inactivation or inhibition of DNase I strongly suppressed radiation injury (8 Gy, LD50/30) to white pulp of spleen, intestine villi, and bone marrow as measured using quantitative TUNEL assay. Spleen and intestine of DNase I KO mice were additionally protected from radiation by Zn‐DIPS due to the broad radioprotective efficacy of zinc. It is interesting that main DNase I‐producing tissues such as salivary gland, pancreas, and kidney showed no effect of DNase I inactivation. These results suggest that DNase I‐mediated mechanism of DNA damage and tissue injury has important regulatory role in radiation‐mediated cell death in radiosensitive organs. The study was supported by NIH/NIDDK and VA Merit Review grants to A.G.B.
Mdm2 antagonist Nutlin‐3 induces apoptosis in p53 mutant human colon carcinoma cells
Authors: Bhattacharya Sujoy , Ray Ramesh M. , Johnson Leonard R.
Published: June 2021

Mdm2 antagonist Nutlin‐3 disrupts p53‐Mdm2 interaction and activates p53 signaling. Most human cancers have p53 mutations and are resistant to apoptosis induced by chemotherapeutic drugs. This study investigated the role of Nutlin‐3 in apoptosis of human colon carcinoma cells (Caco‐2) with a mutant p53. Caco‐2 cells were insensitive to TNF‐α/CHX and camptothecin (CPT)‐mediated apoptosis. However, Nutlin‐3 induced DNA fragmentation and apoptosis in Caco‐2 cells by itself. Induction of apoptosis by Nutlin‐3 correlated with increased levels of E2F1 protein, which led to increased expression of its target p73, a homologue of p53. High basal levels of the anti‐apoptotic Bcl‐xL protein normally present in Caco‐2 cells, decreased in response to Nutlin‐3. E2F1 and p73 activation by Nutlin‐3 increased SIVA protein levels, which sequestered Bcl‐xL, caused mitochondrial permeability changes and activated caspases‐9, and ‐3. Pre‐treatment of cells with cycloheximide (CHX) prevented Nutlin‐3‐induced increase of SIVA and protected cells from apoptosis. Our results indicate that Nutlin‐3 can induce apoptosis in the absence of functional p53 by modulating levels of E2F1 and p73 and may have therapeutic implications in various forms of human cancer. Supported by NIDDK grant DK‐16505.
The effect of life‐long caloric restriction on alpha motoneuron numbers in aged rats
Authors: Chopek Jeremy William , Gardiner Phillip
Published: June 2021

The objective of this study was to determine if caloric restriction (CR) attenuates the age‐related loss of alpha‐motoneurons (α‐mns). Rats were either fed ad libitum (oldAL) or a fortified CR (40% reduction) diet from 14 weeks of age (oldCR). A control group of young free eating rats (youngAL) was also studied. Rat lumbar enlargements were sectioned at 15 μm and every sixth section for 3mm was used for immunohistochemistry. Choline acetyltransferase (ChAT) ‐ positive neurons in the ventrolateral horn larger than 15 micrometers ( μm) in diameter and each having a clear soma and nucleus were sized and counted. Results demonstrated a significant age effect (p < 0.05) on the total number of α‐mns (diameter = 25 μm). A decrease of 28% in α‐mn numbers was found in the old (31 months) rats compared to the young (8.5 months) rats. No age effect was evident on the total number of gamma‐mns (diameter < 25 μm). While CR attenuated the loss of muscle mass with age, there was no effect on age‐related mn loss since there was no difference between the total number of alpha or gamma mns between the oldCR and oldAL rats. Caloric restriction, while attenuating age‐associated changes in muscle mass and brain apoptosis markers, does not attenuate the decline in mn numbers in the rat lumbar spinal cord.

Supported by funds from CIHR and MHRC
Ceramide impairs apoptotic cells phagocytosis (efferocytosis) by alveolar macrophages
Authors: Petrusca Daniela Nicoleta , Fisher Amanda , Rush Natalia I. , Hubbard Walter C. , Smith Patricia , Presson Robert G. , Petrache Irina
Published: June 2021

Pulmonary emphysema is characterized by a loss of alveolar cells via apoptosis along with an inflammatory response, which may be explained by decreased efferocytosis of apoptotic cells. We studied the role of ceramides, pro‐apoptotic sphingolipids markedly increased in emphysema lungs, in the regulation of alveolar macrophage (AM) efferocytosis. Engulfment experiments were performed with rat AM obtained via bronchoalveolar lavage. Labeled apoptotic Jurkat cells or carboxylated beads were used as targets. AM were treated with various ceramide species and phagocytosis was quantified by flow cytometry. Ceramide treatment (10µM Cer 6:0, 2‐18h) significantly impaired AM efferocytosis (33% and 55% inhibition at 2h and 18h, respectively) in a dose‐dependent manner, without inducing AM apoptosis. Interestingly, ceramide treatment had no effect on monocyte‐derived macrophage efferocytosis or on AM Fc‐mediated phagocytosis. The mechanism of ceramide‐induced efferocytosis impairment was independent of the RhoA ‐ Rho kinase pathway, but required endogenous new ceramide synthesis, primarily via the neutral sphingomyelinase pathway. Since there were increased levels of ceramide in AM after exposure to cigarette smoke extract, we conclude that ceramides may be the molecular mediators of the inhibitory effect of cigarette smoke on AM efferocytosis in emphysema. AHA 0826103G (DP) and RO1HL077328 (IP).
DOES H2S TRANSDUCE THE EFFECTS OF HYPOXIA ON METABOLIC AND RESPIRATORY CONTROL IN MAMMALS?
Authors: Haouzi Philippe A , Bell Harold , Ferguson Carrie , Notet Veronique
Published: June 2021

Endogenous H2S appears to mediate some of the cardio‐vascular effects of hypoxia (H) in non mammalian species, while exposure to non toxic concentration of H2S reduces the metabolism in small mammals just like with H. Could H2S be an O2 sensor in mammals? We compared the ventilatory and metabolic effects of exposure to 60 ppm H2S and to 10 % O2 in small and large freely behaving rodents which responses to H differ according to body mass, i.e. 20 g mice and 500g rats. H2S and H produced a similar depression in metabolism in the mice (‐58 %), but not in the large rats. The relative hyperventilation produced by H in the mice (+23 %) was replaced by a ventilatory depression with H2S (‐53%) which paralleled the drop in metabolism. In the rats, ventilation was stimulated in H (+64%), but not with H2S. Adding H2S on top of H in the mice abolished the relative hyperventilation of H and provoked a much larger respiratory (and metabolic) depression. H2S does not mediate the effects of H on the arterial chemoreflex. The depressive effects of H2S and H on breathing, which is linked to the metabolic depression, is only expressed in small sized mammals and is compatible with the hypothesis.
Glutamatergic neurotransmission in the Locus coeruleus modulates CO2‐drive to breathing
Authors: Gargaglioni Luciane H , Taxini Camila L , Cintia Puga I , Bicego Kenia C
Published: June 2021

The Locus coeruleus (LC) has been suggested to be a CO2 chemoreceptor site in mammals and contains L‐glutamate receptors. In the present study, we assessed the role of glutamatergic receptors in the LC on cardiorespiratory and thermal responses to hypercapnia. Pulmonary ventilation (Ve, plethismography), mean arterial blood pressure (MAP), heart rate (HR) and body temperature (Tb, data loggers) were measured before and after microinjection of kynurenic acid (KYN, an ionotropic glutamatergic receptors antagonist; 5 nmol/100 nL,) and alpha‐methyl‐4‐carboxyphenylglycine (MCPG, a metabotropic glutamatergic receptors antagonist; 10 nmol/100 nL,) into the LC, followed by a 60‐min period of hypercanic exposure (7%CO2 in air). KYN or MCPG into the LC did not change Ve, MAP, HR and Tb under normocapnia. Hypercapnia caused an increase in Ve, but did not change MAP, HR and Tb. KYN increased the hypercapnic ventilatory response [2269±405 (KYN) vs 1374.2±337 (vehicle) mL kg−1min−1] due to a higher tidal volume, but did not change MAP, HR and Tb. MCPG did not affect cardiorespiratory and thermal responses to hypercapnia. These results suggest that ionotropic glutamatergic receptors in the LC are involved in the ventilatory response to hypercapnia. Financial Support: FAPESP, CNPq.
Angiotensin 1‐7 treatment induces apoptosis in Human Umbilical Vein Endothelial Cells.
Authors: Anene‐Maidoh Ogugua T. , Greene Andrew S.
Published: June 2021

Angiotensin 1‐7 (ANG1‐7), a physiologically active product of the Renin‐Angiotensin system, plays an important role in the regulation of the cardiovascular system. Acting through the Mas receptor, ANG1‐7 counter‐regulates the actions of Angiotensin II (ANGII) mediated by AT1. ANGII is protective in certain cell types but induces apoptosis in human umbilical vein endothelial cells (HUVECs). We therefore hypothesized that ANG1‐7 would have proliferative effects on HUVECs.

HUVECs were incubated for 24 hours in M199 media, treated with ANG1‐7 (0.1nM ‐ 10uM) in serum free media for 24 hours, and then allowed to recover for 3 hours in M199 media. A cell viability assay was performed. Compared to control cells left in complete media for the duration of the experiment, all serum starved cells showed a significant decrease in cell number. ANG1‐7 caused a further decrease in cell number compared to serum starved non‐treated controls. These data suggest that ANG1‐7 treatment does not rescue HUVECs during serum starvation but instead stimulates the increased induction of apoptotic pathways. These experiments demonstrate a dose dependent and cell type specific action of ANG1‐7 that can either augment or counter the effects of ANGII. Support NIH HL 29587
Renin‐Angiotensin System Blockade Improves Skeletal Muscle Glucose Uptake In Burn Injury
Authors: Kasper Sherry Oden , West Lori Gail , Desai Jayashree , Phillips Erin , Benavides Jeshurun , Lepard Jacob , Wayne Michelle , Enderson Blaine L , Daley Brian J , Karlstad Michael D
Published: June 2021

Insulin resistance after burn is associated with alterations in postreceptor insulin signaling and abnormal glucose homeostasis. We have previously shown that renin‐angiotensin system blockade using losartan, an AT1 receptor blocker, improves insulin signaling and glucose tolerance in burn injured rats. In this study, we examined the effects of losartan on 2‐deoxy‐glucose uptake into the soleus muscle of burned rats. A 30% body surface area burn was induced by immersion of the dorsum into 90ºC water for 15s. Sham burned rats were immersed in 23ºC water. Losartan (30 mg/kg/day) or placebo (water) was given by gavage for three days post‐burn resulting in sham burn (n= 8), burn placebo (n= 8) burn losartan (n= 8). Insulin (100 nM) stimulated glucose uptake was impaired in the soleus muscle of burn placebo animals as determined by 2‐deoxy‐glucose uptake, in vitro (77% decrease compared with sham). However, losartan treatment completely reversed this effect returning the uptake to the level of the sham burn animals. This suggests that the renin‐angiotensin system is involved in the insulin resistance of skeletal muscle that occurs in burn injury.
Effects of growth hormone‐releasing peptide on [Ca2+]i and NO in rat cardiomyocytes
Authors: Tian Guozhong , Li Meixiu , Zhong Zhenya , Bai Shuling , Kang Yu‐Ming
Published: June 2021

Growth hormone‐releasing peptide (GHRP) is an oligopeptide which can promote the release of growth hormone. GHRP has protective effects on the heart. We observed the effects of GHRP on subcellular Ca2+ and NO dynamics of the cardiomyocytes in Sprage‐Dawley rats. Cardiomyocytes were isolated by the constant‐flow Langendorff system and enzymic methods. Effects of GHRP on [Ca2+]i and NO in crdiomyocytes of rats were observed by LSCM after [Ca2+]i and NO of cardiomyocytes were double‐labeled by molecular probes. GHRP induced a transient increase of [Ca2+]i then followed by a plateau phase in the cardiomyocytes of normal rats, the similar response was found in the heart failure (HF) rats. Acute ejection of adriamycin (ADR) into perfusion solution induced an abnormal elevation of the fluorescence intensity of [Ca2+]i in normal cardiomyocytes, GHRP moderately reduced the elevation and decreased the Ca2+ oscillation rate. There was no change in NO signal after ADR stimulation. Rhod‐2/AM and DAF‐FM/DA could act as [Ca2+]i and NO molecular probes respectively. GHRP increased [Ca2+]i of crdiomyocytes in both HF rats and normal rats. GHRP has an inhibitory effect on calcium overload caused by ADR. GHRP has bidirectional modulatory effects on cardiac contractility. Both GHRP and ADR did not affect NO signal in cardiomyocytes of rats.
Analysis of Signalling Pathways Triggering Transcriptional Changes in Adrenal Medulla with Single and Repeated Stress
Authors: Tillinger Andrej , Liu Xiaoping , Papanikolaou Nickolaos A , Serova Lidia I , Sabban Esther L
Published: June 2021

The "fight or flight" response of the adrenal medulla (AM) is crucial to restoring homeostasis with acute stress. However, when prolonged or repeated, stress is a major contributor to disease. Here, we examined the changes in gene expression in rat AM with immobilization stress for 2 hr once (IxIMO) or 6 consecutive days (6x IMO). Microarray data from RAE 230 2.0 (Affymetrix), were analyzed by GeneSifter (VizXLabs), and GeneSpringGX (Agilent), and selective genes confirmed by RT‐PCR and western blots. We concentrated on changes in signalling pathways leading to transcriptional responses. Gene Ontology of transcripts significantly (p<0.01) changed showed 248 related to transcription and 315 to signal transduction. The analysis suggested central roles for Fos and Egr1 with single IMO. As Egr family members were induced by both 1x and 6x IMO, Gene Set Enrichment (GSEA) was used to identify pathways and the genes within them enriched with respect to Egr1 expression status and indicated the importance of JAK/STAT and MAP kinase pathways. Panorama Antibody Microarray found different activation of MAPKs by single and repeated IMO, with increased protein levels of ERK 1/2, ERK 5 and p38 with 1xIMO, but not 6xIMO. Several phosphatases were down regulated, especially with single IMO. The results reveal a transition in cell signalling in the AM with repeated exposure to stress. Supported by NIH NS28869.
Elevated interstitial ATP concentrations stimulate adenosine production in resting rat skeletal muscle
Authors: Zhao Yi , Smith Ashley A. , Fabris Sergio , Tai T. C. , MacLean David
Published: June 2021

It has been suggested that muscle contraction is needed for ATP release into the interstitial space and subsequently adenosine production. However, whether elevated interstitial ATP alone in the absence of muscle contraction can increase interstitial adenosine production has yet to be examined. Anesthetized rats had microdialysis fibers inserted into the gastrocnemius muscle of each leg and perfused with saline (control), saline plus 60 uM of ATP (treatment) and then saline (recovery) for 20 min at a rate of 5 uL/min. In vitro probe loss of ATP was determined to be 10.7±0.4%, resulting in a loss of >6 uM of ATP into the interstitium. Dialysate adenosine concentrations increased (P<0.05) from 0.144±0.026 to 0.401±0.049 uM during ATP perfusion, but returned to baseline (0.168±0.22 uM) during recovery. The magnitude of increase in interstitial adenosine concentrations are comparable to those seen during muscle contraction as well as during hypoxia. These results clearly demonstrate that elevated interstitial ATP concentrations are sufficient to stimulate adenosine production without muscle activation. This is most likely due to breakdown of adenine nucleotides by ecto‐5′‐nucleotidase. In summary, adenosine production can be stimulated in resting skeletal muscle independent of muscle contraction. Supported by NSERC.
Interstitial nitric oxide levels do not increase following L‐Arginine or ATP perfusion into the interstitium of rat hind‐limb skeletal muscle
Authors: Smith Ashley A. , Zhao Yi , Fabris Sergio , MacLean David A.
Published: June 2021

It has been proposed that increased extracellular L‐Arginine (L‐Arg) or ATP stimulates nitric oxide synthase activity producing nitric oxide (NO). In the present study we used the microdialysis technique to alter the interstitial concentration of L‐Arg and ATP to examine whether elevations in these compounds results in nitric oxide formation in rat skeletal muscle. Male Sprague‐Dawley rats were anaesthetized and had microdialysis fibers (1 cm diffusion length) inserted into the gastrocnemius muscle of each leg. Following 20 min. of baseline collection (saline), the microdialysis probes were perfused at a rate of 5 uL/min with either 20 mM L‐Arg (20 min., n = 10) or 60 uM ATP (20 min., n = 15). Interstitial NO concentrations were 7.2±1.1 and 5.2±0.7 uM during saline perfusion and did not change (P>0.05) following L‐Arg (6.5± 0.8 uM) or ATP (5.6±0.9 uM) perfusion. These data clearly demonstrate that elevated interstitial L‐Arg as well as ATP concentrations did not alter the basal interstitial concentrations of NO. Furthermore, these data suggest that the independent manipulation of L‐Arg and ATP in the interstitial space alone does not promote NO production and release from surrounding tissues.

Supported by NSERC.
Antioxydant treatment prevents endothelial dysfunction and alteration of the eNOS pathway associated with severe dyslipidemia in mouse cerebral arteries
Authors: Drouin Annick , Thorin Eric
Published: June 2021

Endothelial dysfunction observed in cardiovascular diseases has been linked with an excess in free radical formation. We hypothesized that a chronic treatment with the antioxydant catechin would preserve the endothelium by maintaining eNOS function in dyslipidemic mice (DL). DL (C57Bl/6‐LDLR−/−;ApoB+/+) 3 months‐old (m/o) male mice were treated with catechin (30 mg/kg/day) for 3 months. C57Bl/6 (WT) were used as controls. Endothelium‐dependent dilations to acetylcholine (EDD; % max diameter) were recorded in pressurized cerebral arteries. Results are mean±SEM of n = 6 mice. Myogenic tone (MT) increased in DL at 3 and 6 m/o (from 14±5% and 22±4% in WT, to 44±4% and 48±5% in DL; p<.05) but was restored by catechin (29±4%). eNOS inhibition with N‐nitro‐L‐arginine (L‐NNA) increased MT at both ages in WT (p<.05) but not in DL (43±6%, 34±4%, 58±6% and 52±5%, respectively). EDD was reduced at 3 and further at 6 months in DL compared to WT (26±2%, 17±2%, 45±6% and 38±4%, respectively; p<.05). L‐NNA reduced EDD in 3 and 6 m/o WT and in 3 m/o DL (24±2, 20±4% and 13±4%, respectively; p<0.5) but not in 6 m/o DL (18±2%). Catechin improved EDD in DL (31±3%; p<0.5) and restored eNOS function: L‐NNA reduced EDD (9±1%; p<.05) and increased MT (44±4%; p<.05). In conclusion, chronic treatment with catechin limits endothelial dysfunction due to its protective effect on the eNOS pathway in DL mouse cerebral arteries. This work is supported by CIHR.