bluuebunny/crossref_metadata_2025 huggingface dataset:
In‐vivo genetic inactivation of CuZn‐superoxide dismutase (SOD1) prevents vascular protective effects of erythropoietin
Authors: d'Uscio Livius V. , Smith Leslie , Katusic Zvonimir S.
Published: June 2021

Previous studies demonstrated that treatment with erythropoietin (EPO) prevents endothelial dysfunction and medial thickening after wire‐induced injury of mouse carotid artery. In the present study, we tested the hypothesis that stimulatory effect of EPO on expression and activity of SOD1 is an important mechanism underlying the vascular protective effect of EPO. This hypothesis was based on observation that treatment with recombinant human EPO (1000 U/kg, s.c., biweekly for 2 weeks) significantly enhanced SOD1 protein expression by 31% in arteries of wild‐type mice (P<0.05; n=3). Analysis of the vascular effects of EPO was performed in SOD1‐deficient (SOD1−/−) mice. Two weeks after wire‐induced injury vascular reactivity of carotid arteries was studied in‐vitro under pressurized condition, and the arteries were histologically assessed. Endothelium‐dependent relaxations to acetylcholine were impaired in carotid arteries of SOD1−/− mice (P<0.05 vs. control SOD1−/− mice; n=7‐8) while endothelium‐independent relaxations to nitric oxide donor, DEA‐NONOate, were unaffected. This was associated with increased medial cross sectional area (33±2 x10−3 mm2; P<0.05 vs. controls: 28±1 x10−3 mm2; n=9‐10). Treatment with EPO did not affect endothelial dysfunction or medial thickening of injured carotid arteries in SOD1−/− mice (n=7‐10). Furthermore, EPO did not prevent increased superoxide anion production in aortas of SOD1−/− mice. These findings suggest that the vascular protective effects of EPO are dependent on activation of SOD1.
Cardiac anti‐fibrotic effect of Ang (1‐7) in L‐NAME‐treated mice does not involve AT2 receptor stimulation
Authors: Phua Damie Sher‐Maine , Jones Emma Susan , Widdop Robert Edward
Published: June 2021

The aim of this study was to determine the chronic effects of Ang (1‐7) on L‐NAME‐induced cardiac remodelling in both wildtype (WT), and AT2 knockout mice (AT2KO). Cardiac remodelling was induced by treatment with L‐NAME (100mg/kg/day) for 4 weeks. Animals (n=10 in both WT and AT2KO, 16 wks old) were treated with L‐NAME in combination with Ang (1‐7) (24µg/kg/hr), the Mas receptor antagonist, A779 (48µg/kg/hr) and/or the COX inhibitor, indomethacin (2mg/kg/day). At the conclusion of treatment, hearts and aorta were removed to examine cardiovascular structure. Indices of cardiac (left ventricular to body weight ratio) hypertrophy were not influenced by any drug treatments. As expected, L‐NAME significantly increased blood pressure (BP) and cardiac interstitial fibrosis in both WT and AT2KO mice. However, Ang (1‐7) attenuated the increase in BP and interstitial fibrosis due to L‐NAME by a similar magnitude in both WT and AT2KO. Indomethacin, but not A779 inhibited the ability of Ang (1‐7) to prevent the L‐NAME‐induced hypertension implicating vasodilator prostaglandins in this effect. Thus, Ang (1‐7) exhibits an anti‐fibrotic effect that does not involve AT2R stimulation in this model.

NHMRC (Australia)
Hyperhomocysteinemia Potentiates Endothelial Dysfunction in Diabetes by Calpain and NADPH Oxidase Activation
Authors: Cheng Zhongjian , Jiang Xiaohua , Sidorov Stanislav , Fang Pu , Durante William , Yang Xiaofeng , Wang Hong
Published: June 2021

We studied the effect of hyperhomocysteinemia (HHcy) on endothelial function in type 1 diabetes. Severe and moderate HHcy were developed in male cystathionine beta‐synthase (CBS−/+ and CBS+/+) mice fed a high methionine (HM) diet at 8‐weeks of age for 8 weeks. Diabetes was induced by streptozotocin injection 4 weeks after dietary intervention. Severe HHcy markedly increased 24 h food and water intake, urine excretion, kidney and liver weight, and reduced blood glucose tolerance in diabetic mice. Endothelium‐dependent vascular relaxation to acetylcholine (ACh) was impaired by moderate HHcy and further potentiated by severe HHcy in thoracic aorta from HHcy diabetic mice. Endothelium‐independent vascular relaxation to sodium nitroprusside was impaired. HHcy impaired vascular relaxation to ACh was reversed by a superoxide scavenger tempol, a combination of PEG‐SOD and catalase, a NADPH oxidase inhibitor apocynin, and calpain inhibitors (MDL28170, ALLM and calpeptin) in diabetic mice. Severe HHcy also increased the protein levels of calpain and caspase 12, a marker of calpain activity, in the aortas of diabetic mice or the aortas of control mice incubated with L‐homocysteine (500 µM) and D‐glucose (44 mM) for 48h. Our data suggest that HHcy potentiates organ damage, metabolic disorders and endothelial dysfunction in diabetes, at least in part, by calpain and NADPH oxidase activation.
Inhibition of NADPH oxidase restores NO availability and migratory function in diabetic CD34 cells
Authors: Jarajapu Yagna , Verma Amrisha , Kent David , Li Qiuhong , Grant Maria
Published: June 2021

Migration of circulating endothelial progenitor cells (CD34) to sites of vascular injury is critical for the reparative function and is mediated by NO. CD34 cell migration is defective in diabetes. Production of reactive oxygen species (ROS) by NADPH oxidase (NOX) is known to decrease NO availability involving endothelial dysfunction. We hypothesized that migratory defect in diabetic CD34 cells could be corrected by the inhibition of NOX. Peripheral blood was obtained from healthy and Type 1‐diabetic individuals and CD34 cells were isolated by immunomagnetic selection. Real time PCR detected higher expression of NOX2 mRNA in diabetic CD34 cells. Diabetic CD34 cells showed increased ROS production and decreased NO release in response to SDF1 (100 nM). Apocynin (300 µM) treatment decreased ROS production and increased NO release. Similar effects were observed with pretreatment of cells with gp91ds‐tat that selectively inactivates gp91phox. Pretreatment with apocynin or gp91ds‐tat increased migration of diabetic CD34 cells in response to SDF1 and VEGF (25 ng/ml). These results suggest that pretreatment of diabetic CD34 cells with NOX inhibitors may enhance their ability of in vivo engraftment.
PKCα REGULATION OF TRPM2 CHANNEL ACTIVATION OF Ca2+ ENTRY IN ENDOTHELIAL CELLS
Authors: Hecquet Claudie M , Vogel Stephen M , Malik Asrar B
Published: June 2021

Oxidant signaling, through the production of oxygen metabolites such as hydrogen peroxide (H2O2), regulates multiple endothelial functions. We have previously shown that the transient receptor potential melastatin (TRPM) channel 2 (TRPM2) mediates H2O2‐induced Ca2+ entry and endothelial hyper‐permeability (Hecquet et al., Circ Res 2008; 102: 347‐355). Here, we provide evidence that PKCα regulates oxidant‐induced Ca2+ entry by modulation of TRPM2 channel activity. H2O2‐elicted Ca2+ entry via TRPM2 channels was significantly reduced by PKCα‐silencing or inhibition, suggesting that PKCα positively regulates Ca2+ entry through TRPM2. The H2O2‐elicited decrease in transendothelial electrical resistance (TER) was also reduced by TRPM2‐silencing (by 39 ± 9 %), PKCα‐silencing (by 36 ± 6 %) and to a similar degree by PKCα‐ or TRPM2‐silencing (by 40 ± 8 %), signifying that PKCα plays a permissive role in the opening of TRPM2 channels. In addition, H2O2 induced rapid PKCα co‐localization with TRPM2‐S (the dominant‐negative splice variant of TRPM2 that associates with TRPM2) and the phosphorylation of TRPM2‐S. Thus, PKCα phosphorylation of TRPM2‐S regulates the opening of TRPM2 channels enabling H2O2‐induced Ca2+ entry in endothelial cells.
Effect of Uridine (U), Melatonin (MLT) and Ramelteon (RMT) on edema formation following traumatic brain injury (TBI) in rats
Authors: Kabadi Shruti Vasudeo , Maher Timothy J.
Published: June 2021

TBI is a significant cause of disability and death. We investigated post‐treatment with U (a pyrimidine) and MLT (a pineal indoleamine hormone and free radical scavenger), individually and in combination, on regional brain edema following TBI via fluid percussion. Male rats underwent craniotomy and were divided into groups: Sham+Vehicle (V), Sham+Treatment (TX), TBI+V [moderate TBI on right (impacted) side], and TBI+TX [received i.p. (mg/Kg): U (16 & 32), MLT (100 & 200) or U (16/32) + MLT (200)]. After 48 hrs, edema (%) was determined on both sides. Impacted side edema was higher in TBI+V vs Sham+V and treatments did not affect edema levels in Sham+TX vs Sham. Our previous studies showed that U (16 and 32) and MLT (200), individually reduced impacted side striatal edema vs TBI+V. Combination U (32) + MLT (200) reduced edema in impacted and non‐impacted hippocampus and striatum vs TBI+V. U (16) + MLT (200) also reduced edema in impacted hippocampus, and impacted and non‐impacted striatum. To determine whether the neuroprotection of MLT in this model involved MT1 and MT2 receptors in addition to its anti‐oxidant properties, we tested RMT (10 and 20 mg/Kg, i.p.), a selective MT1/MT2 receptor agonist. RMT failed to reduce edema levels in cortex, hippocampus and striatum on impacted as well as non‐impacted side vs TBI+V. Thus, neuroprotection by MLT in this TBI model is not likely MT1/MT2 receptor‐mediated.
AAV9 mediates more specific cardiac gene transfer in the rat than AAV2, AAV5, AAV7, and AAV8
Authors: Qi Yanfei , Liu Xuan , Li Hongwei , Shenoy Vinayak , Li Qiuhong , Hauswirth William W. , Sumners Colin , Katovich Michael J.
Published: June 2021

Cardiac gene transfer may serve as a novel therapeutic approach for heart disease in the USA. Numerous serotypes of AAV have been identified with variable tropisms to cardiac tissue. In vitro and in vivo experiments were undertaken to compare cardiac tropisms of AAV‐2, 5, 7, 8, and 9. For the in vitro study, 107 genome copies (gc) of AAV‐2, 5, 7, 8, or 9, expressing GFP under the CBA promoter, transfected both rat neonatal cardiac myocytes (RNCM) and fibroblasts (RNCF). 3 days post viral transduction, GFP expression was visualized under a microscope. In vitro results indicated that AAV9 and AAV2 showed the highest transducing efficiency in RNCM. Only AAV2 produced any transduction in the RNCF, which was minimal. In the in vivo studies, 5‐day‐old rats were intra‐cardiacly injected with 4x1010 gc of either AAV‐2, 5, 7, 8, or 9. 1 and 2 months post gene delivery, rats were killed and tissues (heart, liver, lung, and kidney) were collected. GFP distribution was visualization using the microscope. The level of GFP gene expression was also measured by Real‐Time PCR. In vivo results indicated that transduction efficiency order in the heart was: AAV9>AAV8>AAV7>>>>AAV2=AAV5. The transduction efficiency order in the liver was: AAV2>AAV5>AAV7>AAV8>AAV9. No to minimal expression was found in the lung and kidney for all serotypes. Collectively these results suggest that AAV9 provides the best transduction efficiency in cardiac tissue.
Effect of ethanol extract of Cynanchum wilfordii on vascular dysfunction in rat fed high fat diet
Authors: Choi Deok Ho , Kim Eun Ju , Lee An Sook , Kang Dae Gill , Lee Ho Sub
Published: June 2021

Hyperlipidemia and hypercholesterolemia are critical pathogenic factors for the development and maintenance of atherosclerosis. This study was designed to investigate whether the ethanol extract of Cynanchum wilfordii (ECW) suppresses atherogenesis in rat fed with high fat diet (HFD). Treatment of HFD with low (100 mg/day/kg) or high (200 mg/day/kg) doses of ECW for 8 weeks markedly attenuated plasma level of triglyceride (TG) and augmented plasma level of high density lipoprotein (HDL)‐cholesterol. Treatment of ECW also lowered the systolic blood pressure (SBP) in atherogenic diet rats. Furthermore, the impairment of acetylcholine (ACh)‐ and sodium nitroprusside (SNP)‐induced relaxation of aortic rings in the HFD‐rat was improved by chronic treatment of ECW. Treatment of ECW also suppressed the HFD‐induced increase in expression levels of intracellular cell adhesion molecule (ICAM)‐1, vascular cellular adhesion molecule (VCAM)‐1, and E‐selectin as well as matrix metalloproteinase (MMP)‐2 in aorta. The decrease in production of cGMP induced by HFD was recovered by administration with ECW in aortic tissue. Taken together, the present study suggests that ECW prevents vascular inflammatory process and vascular dysfunction in rat fed high fat diet.
Effects of Oenothera odorata on vasorelaxation in thoracic and carotid artery
Authors: Kim Hye Yoom , Choi Deok Ho , Kang Dae Gill , Lee Ho Sub
Published: June 2021

An ethanol extract of seed of Oenothera odorata (EOE) induced dose‐dependent relaxation of phenylephrine‐precontracted aorta, which was abolished by removal of functional endothelium. Therefore, the aim of the present study was to define the mechanism of the ethanol extract‐induced vascular relaxation on rat thoracic artery and carotid artery. Among the four solvent partitions of methanol extract, ethylacetate, n‐buthanol and water, water and ethanol partitions induced vascular relaxation, respectively. Pretreatment of the endothelium‐intact aortic tissues with NG‐nitro‐L‐arginine methyl ester (L‐NAME), 1H‐[1,2,4] ‐oxadiazole‐[4,3‐α]‐quinoxalin‐1‐one (ODQ) inhibited the relaxation induced by EOE, respectively. However, the relaxation effect of EOE was not blocked by indomethacine, glibenclamide, verapamil. diltiazem, tetraethylammonium (TEA), atropine, and propranolol, respectively. Moreover, EOE‐induced cGMP production was blocked by pretreatment with L‐NAME or ODQ. These results suggest that EOE dilates vascular smooth muscle via endothelium‐dependent NO/cGMP signaling.
Nuclear receptor coactivators: Regulation of turnover and function
Authors: O'Malley Bert W.
Published: June 2021

Steroid receptor coactivators, such as the SRC‐family of molecules, amplify nuclear receptor induction of gene expression by modifying promoter chromatin structure and implementing all of the downstream subreactions required for efficient transcription. Induced post‐translational modifications such as phosphorylations 'acivate' the SRC‐3 coactivator by allowing formation of active coactivator multimeric complexes with the receptors. Two regulable sets of co‐coactivator molecules control the degradation of SRC‐3 coactivator. E3 ubiquitin ligases control the lifetime of 'transcriptionally activated SRC‐3' by virtue of a 'ubiquitin transcriptional clock', which begins by a monoubiquitin‐dependent activation and ends via a polyubiquitylatin‐dependent degradation. In contrast, the REGγ proteasomal Cap controls the cellular levels of 'inactive SRC‐3 coactivator' via an ubiquitin‐independent and ATP‐independent mechanism. Turnover of coactivators is additionally regulated by specific kinases, such as atypical PKC, which modify the C‐terminal region of SRC‐3 and prevent the entrance of the coactivator into the 20S proteasome core. Since many coactivators are oncogenes (or tumor suppressors), the mechanisms for controlling their levels are crucial for the regulation of all hormone‐mediated physiological and pathological consequences.
Mechanisms for Differential Effects of Estrogen on Tyrosine Hydroxylase (TH) Gene Transcription
Authors: Maharjan Shreekrishna , Serova Lidia I , Sabban Esther L
Published: June 2021

There are conflicting findings regarding the ability of estrogen to regulate TH gene expression in vivo, with some reports of elevation, reduction or no change. The basis for these discrepancies is unclear and may result from differences in dose, duration, mode of administration, or estradiol receptor (ER) subtype expressed. We found that in the nucleus of the solitary tract of OVX female rats, the response of the TH gene depends on the mode of estrogen administration. Daily injections of estradiol benzoate (EB) at 25 μg/kg for 16 days led to a greater than 2‐fold rise in TH mRNA, while administration of 17‐β estradiol (E2) (0.1 mg, 21 day release) for a similar time by pellets, triggered a 90% reduction in TH mRNA compared to vehicle treated controls. Another important parameter is the ER subtype. Many catecholaminergic cells express both ERα and ERβ. To study the mechanism and effect of ER subtypes, we co‐transfected PC12 cells with expression vectors for ERα or ERβ and TH reporter constructs. E2 elevated TH promoter driven reporter activity with ERα, while it triggered a reduction with ERβ. Further studies found that in the presence of ERα, the membrane impermeable E2‐BSA triggered a similar elevation of TH promoter activity requiring the CRE/CaRE motif. CREB and its phosphorylation at Ser133 were required for the response to E2 or E2‐BSA. The findings reveal several ways whereby estrogen can regulate TH transcription.
DOCKING SIMULATIONS ON THE HUMAN beta‐2 ADRENOCEPTOR HELPS TO EXPLAIN THE ENANTIOSELECTIVITY IN BINDING PROCESS.
Authors: Correa‐Basurto Jose , Soriano‐Ursúa Marvin A , Trujillo‐Ferrara Jose G
Published: June 2021

Stereoselectivity has been observed in the interaction between some ligands on the adrenoceptors suggested by observations about in vitro studies. This is an attractive phenomenon in biochemistry, physiology and drug development. In this work, the role of enantioisomery of the ligands on the human β2 adrenoceptor (β2AR) binding was analyzed. For this objective, a set of ligands including well‐known inverse, partial and full agonists and antagonists were tested on an in silico model. These ligands, previously built and geometrically optimized by Gaussian 98 software at the B3LYP/6‐31G* level, were tested on a β2AR model recently obtained by X‐ray crystallography (PDB ID:2rh1). We carried out docking simulation to identify the best complexes ligand‐β2AR under a 60X60X60 Åbox GRID was built centered at Cα of Asp113 (constant residue in putative binding site) for determining the best binding energy. The binding sites on β2AR for the ligands in their minimal interaction energy were identified and explored using Autodock tools v1.4 software. Obtained results were compared with that described in previous in vitro tests. Results showed a greater affinity for the R‐enantiomers compared with the S‐enantiomers in the case of the agonists, but not appears to be a good model for antagonists binding study. Interactions with specific residues involved in binding and receptor activation were found to be in agreement with in vitro and by mutagenesis reported.
Mechanisms regulating the dopamine D1‐D2 receptor heteromeric signaling complex
Authors: Verma Vaneeta , O'dowd Brian , George Susan
Published: June 2021

We previously identified that co‐activation of a Gq‐linked heteromeric D1‐D2 dopamine receptor signaling complex results in phospholipase C (PLC)‐dependent intracellular Ca2+ release. We have demonstrated agonist specificity for the D1‐D2 triggered Ca2+ signal and the D1 stimulated cyclic AMP production with three D1 agonists, SKF83959, SKF81297, and SKF83822 that differentially activate either the adenylate cyclase (AC) or PLC pathway. Because the three agonists can activate different signaling pathways, we propose that they are effective pharmacological tools for assessing the mechanisms regulating the D1‐D2 signaling complex. Pretreatment of D1‐D2 expressing cells with each agonist lead to 60% desensitization of the Ca2+ signal within 5 minutes. Intriguingly, occupancy of the D1 binding pocket by SKF 83822 that could not activate the D1‐D2 complex still permitted desensitization of the Ca2+ response. Results from co‐application of the agonists with a D1 or D2 specific antagonist, suggest the desensitization elicited by each agonist is through selective occupancy of the D1 receptor. Furthermore, over‐expression of wild type G‐protein receptor kinase 2 (GRK2) and its catalytically inert construct, but not its regulator of G‐protein signaling (RGS) domain impaired construct, decreased the Ca2+ signal, suggesting the RGS domain of GRK2 is involved in regulating the D1‐D2 receptor signaling cascade.

This work is supported by the NIH National Institute on Drug Abuse
Sodium channel activation modulates synchronized calcium oscillations and neuronal morphology in immature cerebrocortical neurons.
Authors: George Joju , Murray Thomas F
Published: June 2021

Neuronal activity regulates the morphology and connectivity of neurons during development. In the present study we used brevetoxin‐2 (PbTx‐2), an activator of voltage‐gated sodium channels, to mimic the influence of neuronal activity. PbTx‐2 exposure was used to explore the relationship between intracellular sodium ([Na+]i) and NMDA receptor‐dependent structural plasticity. PbTx‐2 (30 nM) elevates [Na+]i, augments NMDA‐induced Ca2+ influx and enhances NMDA receptor‐mediated neurite outgrowth in cerebrocortical neurons. Here we assessed the influence of PbTx‐2 on spontaneous synchronized Ca2+ oscillations: a manifestation of neuronal connectivity in cerebrocortical neuron cultures. These oscillations become prominent from day in vitro (DIV)‐6 onwards. PbTx‐2 accelerated the emergence of synchronized Ca2+ oscillations by 24 h (DIV‐5). Inasmuch as the synchronized Ca2+ oscillations reflect formation of functional synapses and a neuronal network, we assessed the effect of PbTx‐2 on neuronal morphology. PbTx‐2 enhanced spinogenesis at DIV‐5 by stimulating both dendritic and axonal filopodia formation followed by increased thin/stubby spines at DIV‐6. The influence of PbTx‐2 on filopodia and spine density correlated temporally with synchronized Ca2+ oscillations. These findings provide evidence for a role of [Na+]i in activity‐dependent regulation of neuronal morphology and development.
MODELING COUPLED DIAMETER AND HEMODYNAMIC OSCILLATIONS IN ARTERIOLAR NETWORKS
Authors: Goldman Daniel , MacKie SJ Meaghan , Dias Gemma M , Milkovich Stephanie , Ellis Christopher G
Published: June 2021

Regulation of convective O2 supply to capillary beds in organs is accomplished by coordinated changes in upstream arteriolar diameter. To study this regulation system, in vivo experiments have been performed using a gas exchange chamber to sinusoidally oscillate the O2 environment at the surface of a rat skeletal muscle. Capillary hematocrit (H) changes were delayed by several seconds relative to changes in red cell velocity (V) and the delay varied with the animal suggesting geometry dependence. To assess how alterations in diameters led to observed hemodynamics, a computational model was developed that couples variations in network blood flow to conducted diameter changes. The model uses an arteriolar tree that was constructed based on measured data in rat skeletal muscle and a two‐phase (red cells and plasma) time‐dependent model of microvascular blood flow. The initial response to imposed surface O2 oscillations was simulated by applying diameter oscillations at the distal ends of selected terminal arterioles. The model was used to calculate hemodynamic oscillations in the network and, in particular, examine how the transit time for red cells in the network could lead to the observed delay in changes in H relative to those in V. Initial results show similarities to experimental observations, including decreases in hemodynamic response amplitude with increases in forcing frequency and a delay in H relative to V.
Contribution of carotid body chemoreceptors to the eupneic drive to breathe in the intact awake canine
Authors: Blain Gregory M , Smith Curtis A , Henderson Kathleen S , Demspey Jerome A
Published: June 2021

We used extracorporeal perfusion of the reversibly isolated carotid sinus to determine the effects of specific carotid body (CB) chemoreceptor inhibition on eupneic ventilation (VI) in the resting, awake, unanesthetized, intact dog. 4 female dogs were studied when CB was perfused with 1) normoxic and normocapnic blood, and 2) hyperoxic (> 500 mmHg) and hypocapnic (20 mmHg) blood to maximally inhibit the CB tonic activity. We found that perfusion per se (normoxic ‐ normocapnic) had no effect on VI. In contrast, CB inhibition caused an immediate and substantial reduction in VI which reached a nadir (‐56%) within 29 sec and a steady state (‐24%) within 100 sec. At steady state, this VT‐induced (‐17%) hypoventilation persisted throughout continued CB perfusion (up to 25 min) despite a marked CO2 retention (+9 mmHg) and respiratory acidosis (pHa = 7.27). Consistent with hypoventilation, mean electrical activity of diaphragm EMG was also reduced (‐24%) during CB inhibition. Ventilation returned toward control values within ∼27 sec when exogenous CB perfusion was terminated. We interpret these data to mean that CB chemoreceptor contributes as much as one‐half or more to the total drive to eupnea in the normoxic, intact, awake animal, and that this contribution consists of both a tonic sensory input to respiratory controller as well as a strong modulatory effect on the central chemoreceptor responsiveness to CO2. NHLBI/NIH ‐ AHA
Distensibility of the common carotid and carotid sinus during lower body negative pressure
Authors: Hodges Gary J , Norton Katelyn N , Chia Justin , Zuj Kathryn A , Shoemaker J Kevin
Published: June 2021

Changes in common carotid distensibility (CCD) do not correlate well with reductions in baroreflex sensitivity (BRS). This might be due to assessment of distensibility in the common carotid (CC) rather than the carotid sinus (CS), the location of the baroreceptors. To test this, two studies were performed. In Part 1 (n=10), pulsatile pressure (tonometry) and diameter (Ultrasound) of the CC and CS were assessed on both sides of the neck. This study characterized the bilateral similarities of these vascular segments and confirmed that simultaneous diameter and pressure measures from contralateral CC and CS could be used for distensibility calculations. In Part 2 (n=3), changes in the CCD and CS distensibility (CSD) measured during supine rest and with an orthostatic stress of ‐40 mmHg lower body negative pressure (LBNP). Bilateral comparisons of the CCD (R 6.81±1.39, L 6.17±0.59 units; P = 0.62) or the CSD (R 9.56±2.25, L 9.74±2.88 units; P = 0.97) were not different. Pulse pressures in the CC were 43±5 mmHg supine and 46±1 in LBNP and in the CS were 50±13 mmHg while supine and 28±7 mmHg during LBNP. Nonetheless, CSD was reduced from supine (9.3±0.75 units) during LBNP (3.79±1.51 units; P = 0.04) whereas CCD was unaffected (7.49±3.37 supine vs. 7.79±1.65 units in LBNP; P = 0.92). LBNP has a larger effect on distensibility of the CS than the CC. Supported by Canadian Diabetes Association and Canadian Institutes of Health Research.
PHYSICAL (IN)ACTIVITY AND GLUTAMATE RECEPTOR ACTIVATION IN THE ROSTRAL VENTROLATERAL MEDULLA OF SPONTANEOUSLY HYPERTENSIVE RATS
Authors: Mancini Eric J. , Flack‐Valdez Amanda B. , Mueller Patrick J.
Published: June 2021

A sedentary lifestyle is a major risk factor for hypertension. Treatment of hypertension with regular exercise can be beneficial if prescribed appropriately. Hypertension is associated with sympathetic overactivity and excessive glutamatergic excitation of the rostral ventrolateral medulla (RVLM). We have reported that sedentary versus physically active conditions enhance sympathoexcitation elicited from the RVLM in otherwise "normal" rats. We hypothesized that sedentary conditions may also enhance sympathoexcitation elicited from the RVLM of spontaneously hypertensive rats (SHRs). To test this hypothesis, RVLM microinjections were performed in Inactin anesthetized SHRs that remained sedentary or were physically active (running wheels; 8‐10 wks; 7.9±0.5 km/day). Preliminary data suggest that mean arterial pressure and lumbar sympathetic nerve responses to glutamate (1‐100mM) varied over time and responses to AMPA (1‐10μM) and NMDA (1‐100μM) also differed over time in both sedentary and physically active SHRs. These preliminary data support the possibility of glutamate receptor mediated alterations in the RVLM of hypertensive rats under both physically active and sedentary conditions. These potential mechanisms may be involved in the development of hypertension in sedentary individuals, and in the treatment of hypertension with exercise prescription. (AHA‐0650161Z)
Cellular substrates for angiotensin1‐7 (Ang1‐7) action in the rostral ventro‐lateral medulla (RVLM) of the normotentsive and spontaneously hypertensive rat (SHR)
Authors: Guo Fang , Souslova Ekaterina , Chudakov Dmitry , Paton Julian F.R. , Kasparov Sergey
Published: June 2021

Ang1‐7 is an important component of the central renin‐angiotensin system and affects haemodynamics when injected into the RVLM. The cellular substrate(s) that Ang1‐7 targets in RVLM is unknown., We studied the effects of Ang1‐7 in organotypic brainstem slice cultures of Wistar rat (WR) and SHR. Using cell‐selective promoters incorporated into adeno viral vectors, we expressed green fluorescent protein in catecholaminergic (CA‐) neurones and non‐CA neurones and a Ca2+ indicator Case12(1) in astroglia. . Ang1‐7 (200 nM) depolarised non‐CA neurones in both WR and SHR (by 5 and 4 mV, correspondingly; p<0.05), which in SHR, but not WR, was accompanied by an increase in [Ca2+]i of 61 ± 28% (P<0.05). In contrast, Ang 1‐7 (200 nM) had no measurable effect on either membrane potential or [Ca2+ ]i in CA neurones of either SHR or WR. In astrocytes Ang1‐7 also triggered increases in [Ca2+ ]i: 200nM Ang1‐7 evoked increases of 62±23% in [Ca2+ ]i in 26% of WR astrocytes whereas 600 nM Ang1‐7 activated more astrocytes (54%) but the average amplitude of response was similar. In SHR, Ang1‐7 effects were also similar. Effect of Ang1‐7 could be blocked by co‐application of its antagonist, A779 (200 nM) but not losartan (1 μM). We conclude that non‐CA neurones and glia in RVLM are the primary targets for Ang1‐7 and its effect on [Ca2+]i in non‐CA neurones is enhanced in SHR.

F.G. was supported by The Royal Society. SK and JFRP are supported by BHF.

1. Souslova EA et al. BMC Biotechnol 7:37, 2007
Electroacupuncture Enhances Preproenkephalin mRNA Expression in Rostral Ventrolateral Medulla of Rats
Authors: Li Min , Tjen‐A‐Looi Stephanie C. , Longhurst John C.
Published: June 2021

Electroacupuncture (EA) causes prolonged suppression of reflex elevations in blood pressure for at least 80 min in anesthetized preparations. Since the opioid system may play a role in prolonged inhibition of neuronal activity in rVLM, we postulated that EA increases preproenkephalin (PPE) mRNA for prolonged periods of time. Under a‐chloralose anesthesia, rats received EA (1‐2 mA, 2 Hz, 0.5 ms) at P5‐P6 acupoints (overlying median nerve) or sham (absence of needle stimulation) for 30 min. PPE mRNA also was evaluated in control rats that received surgery but no EA or sham treatment. Then, 1.5 hr or 4 hr following EA or sham, brains were removed quickly, stored in RNA Later solution and punch biopsies of the rVLM were obtained for assay. PPE mRNA copies were analyzed by reverse transcription and absolute quantitative real‐time PCR. Relative ratios of PPE mRNA levels (normalized with house keeping gene 18s) were increased 1.5 hr after treatment in EA (9.13 ± 1.54, n=4) vs sham (2.83 ± 0.36, n=5). By 4 hr PPE mRNA levels in EA (3.25 ± 0.56, n=2) and sham (3.65 ± 0.07, n=2) groups were similar. The baselines of PPE at 1.5 hr and 4 hr after 30 min in the absence of treatment were 2.43 ± 0.2, n=2; 3.65 ± 0.01, n=2 respectively. Thus, PPE mRNA increased 1.5hr after EA, relative to baseline, while PPE of sham groups was not altered. In conclusion, EA increased PPE mRNA expression at 1.5hr after EA, suggesting a prolonged inhibitory effect of EA to increase opioid mRNA expression in the rVLM (Supported by NIH HL‐63313 and HL‐072125).
Sinoaortic denervation differentially reverses iliac, renal and mesenteric vasoconstriction evoked by stimulation of A1 adenosine receptors in the Nucleus of the Solitary Tract (NTS)
Authors: McClure Joseph Martin , O'Leary Donal S. , Scislo Tadeusz J.
Published: June 2021

Activation of NTS A1 adenosine receptors evokes transient vasodilation followed by differential neural and vasopressinergic vasoconstriction in the iliac, mesenteric and renal vascular beds (FASEB, 21: A466, 2007). These responses may be partially mediated via inhibition of NTS baroreflex mechanisms (Am J Physiol 294: H172‐82, 2008). Therefore, in the present study we assessed the contribution of baroreflex mechanism to the regional vascular responses evoked by stimulation of NTS A1 adenosine receptors (CPA 330pmol/50nl) in urethane/chloralose anesthetized male Sprague Dawley rats. We compared the responses in iliac, mesenteric and renal vascular conductance evoked in intact vs. sinoaortic denervated (SAD) animals. SAD abolished vasoconstriction and accentuated vasodilation (iliac=mesenteric>renal). Inasmuch as NTS A1 adenosine receptor mediated activation of the adrenal medulla partially persisted after SAD plus vagotomy or blockade of glutamatergic transmission in the NTS (Am J Physiol 283: H1588‐99, 2002) we conclude that the differential regional vasodilation observed in SAD animals is mediated mostly via non‐baroreflex activation of the adrenal medulla.
Rap1 mediates protective effects of iloprost against lung endothelial dysfunction and vascular leak
Authors: Birukov Konstantin G , Fu Panfeng , Xing Junjie , Birukova Anna A
Published: June 2021

Prostaglandin I2 has been implicated in the regulation of vascular function, inflammation, and acute lung injury. However molecular mechanisms of prostaglandin I2 protective effects on pulmonary endothelial cells (EC) are not well understood. We tested a role of novel cAMP‐activated Epac‐Rap1 pathway in barrier protective effects of synthetic analogue of prostaglandin I2 iloprost using in vivo and in vitro models of lung vascular leak. Iloprost dramatically reduced bronchoalveolar lavage (BAL) protein content, neutrophil accumulation in BAL and lung tissue, capillary filtration coefficient, and Evans blue albumin extravasation caused by high tidal volume ventilation (HTV, 30 ml/kg, 4 hrs). SiRNA‐based Rap1 knockdown inhibited protective effects of iloprost against HTV‐induced lung injury. In vitro, iloprost induced sustained TER increase in lung microvascular endothelium, enhanced intercellular junctions and formation of actin cortical rim, and attenuated thrombin‐induced EC barrier disruption. Consistent with in vivo experiments, Rap1 depletion attenuated protective effects of iloprost in the thrombin model of EC permeability. These data demonstrate that Rap1‐dependent signaling is a major contributor to protective effects of iloprost against lung endothelial barrier dysfunction and vascular leak.

HL87823, HL58064
Interleukin 1β (IL‐1β) expression is required for lysophosphatidic acid (LPA)‐induced lymphangiogenesis in endothelial cells
Authors: Lin Chih hsin , Lee Hsinyu
Published: June 2021

Lysophosphatidic acid (LPA) is a lipid mediator which binds to G‐protein‐coupled receptors and regulates various cellular functions. Interleukin‐1β (IL‐1β), a pro‐inflammatory cytokine, is elevated upon LPA treatment in human umbilical vein endothelial cells (HUVECs). Previous studies indicated that LPA upregulated vascular endothelial growth factor C (VEGF‐C) and lymphatic markers expression in HUVECs. However, the role of IL‐1β in the lymphangiogenesis process has not been studied. In this study, we demonstrated that in the presence of AF12198, an interleukin‐1 receptor (IL‐1R) inhibitor, LPA‐induced VEGF‐C and lymphatic markers expression were inhibited in HUVECs. Furthermore, LPA‐induced in vitro tube formation of HUVECs were also suppressed by pretreatment of AF12198. Our results suggested that IL‐1 plays a role in LPA‐mediated lymphangiogenesis in HUVECs.
Altered renal hemodynamic and excretory function in rats treated with a COX2 inhibitor during the nephrogenic period
Authors: Reverte Virginia , Saez Fara , Salazar Francisco , Llinas Maria T , Salazar F. Javier
Published: June 2021

The administration of a COX‐2 inhibitor during the perinatal period (COX2 Inh‐pp) reduces nephron number by 17%, and leads to the development of hypertension. However, there are important gender differences in the aging‐dependent renal structural changes in COX2 Inh‐pp‐treated rats. Our objective was to examine whether the renal response to an amino acid (aas) infusion, and the renal excretory response to an acute volume expansion (AVE) are altered in COX2 Inh‐pp‐treated rats. Newborn SD rats were treated with vehicle or a COX2 Inh‐pp (Rofecoxib, 2.4 mg/kg/day). The responses to aas infusion or to AVE (6%, bw) were examined at 3 months of age in male and female rats treated with vehicle or the COX2 Inh‐pp. No changes in arterial pressure were found in any group during the experiments. An increase (P<0.05) in GFR (47%), RPF (28%) and sodium excretion (152%) was found in vehicle‐treated males in response to aas. However, this renal hemodynamic and excretory response to aas was completely abolished in COX2 Inh‐pp‐treated males. The renal response to aas was also prevented in COX2 Inh‐pp‐treated females. The renal excretory ability to eliminate an AVE was also reduced in male (28%, P<0.05) and female (38%, P<0.05) COX2 Inh‐pp‐treated rats.

Our results demonstrate that the COX2 inhibition during the perinatal period leads to an important impairment of the renal hemodynamic and excretory function during the adult age.
Absorption and biosynthesis of D‐chiro‐inositol in mice
Authors: Gopalan Chaya , Lin Xiaobo , Ma Lina , Ostlund Richard
Published: June 2021

D‐chiro‐inositol (DCI) is known to possess insulin‐like properties. Mice were fed with either regular chow (control group) or DCI‐free diet over a five‐week period. The levels of DCI declined significantly in plasma (1.91 ± 0.17 to 0.056 ± 0.0 μM), stool (20.2 ± 6 to 0.147 ± 0.05 nmoles) and urine (27.39 ± 8.2 to 0.88 ± 0.09 μM) in mice fed with DCI‐free diet at the five‐week period compared to control (plasma 2.47 ± 0.3 to 2.41 ± 0.29 μM; stool 25.7 ± 8.67 to 31.6 ± 9.7 nmoles and urine 39.9 ± 10.3 to 51.5 ± 6.8 µM).

Whether DCI could be synthesized endogenously was tested next in the mice maintained on DCI‐free diet for 10 weeks where deuterated water was administered (20 % w/w deuterium oxide) for one week followed by a 24‐hour urine collection. The concentration of deuterium oxide was 8.7 + 0.52 % but there was no natural or deuterated DCI (<0.0067 nmoles/mL) in urine. Instead, there was deuterated myo‐inositol (dMI; 34.4 + 2.8 %) synthesis. To test whether MI could be converted to DCI in mice treated with DCI‐free diet, dMI (1mg MI‐d6) was injected intraperitoneally and urine was collected. Although MI‐d6 constituted 38.7+11.5 % of urinary MI, there was no natural or deuterated DCI present (<0.0067 nmoles/mL). Thus, DCI appears to be derived solely from the diet and may represent additional nutritional factor related to insulin action.
Oral NF‐κB blockade attenuates pro‐inflammatory cytokines in the hypothalamic paraventricular nucleus of streptozotocin‐induced diabetes
Authors: Kang Yu‐Ming , Lei Jing‐Hui , Ma Ying , Guo Zheng , Sun Yao‐Jun , Qin Da‐Nian , Zheng Jin‐Ping , Francis Joseph
Published: June 2021

A growing body of evidence suggests that inflammatory molecules contribute to the pathogenesis of diabetes. In this study, we explored the role played by inflammatory molecules in the central nervous system in the progression of streptozotocin‐induced diabetes. Adult male Sprague‐Dawley rats were injected with streptozotocin (STZ, 65 mg/kg, ip) to induce diabetes, and treated for 4 weeks with the NF‐κB inhibitor pyrrolidine dithiocarbamate (PDTC, 150mg/kg/day) orally or vehicle. Streptozotocin‐induced diabetic rats had higher levels of PICs (TNF‐α, interleukin (IL)‐1β and IL‐6) in plasma and in the hypothalamic paraventricular nucleus (PVN), and higher levels of Fra‐LI (an indicator of chronic neuronal activation), superoxide, p47phox (a subunit of NAD(P)H oxidase) and NF‐κB activation (determined by phosphorylated IKKβ) in the PVN when compared with control rats. Treatments with PDTC for 4 weeks reduced the plasma levels of PICs, decreased Fra‐LI, superoxide, PICs, p47phox and phosphorylated IKKβ in the PVN of the STZ‐induced diabetic rats. These findings indicate that PTDC, as a NF‐κB inhibitor, may have a beneficial effect on the diabetes by attenuating the pro‐inflammatory cytokines and inhibiting oxidative stress. Supported by NIH HL‐080544.
Rapamycin prevents the post‐exercise increase in protein synthesis and alters cell signaling in human skeletal muscle
Authors: Drummond Micah John , Fry Christopher S. , Glynn Erin L. , Dreyer Hans C. , Dhanani Shaheen , Timmerman Kyle L. , Volpi Elena , Rasmussen Blake B.
Published: June 2021

Resistance exercise stimulates mTOR signaling and muscle protein synthesis (MPS) during post‐exercise recovery in humans. Rapamycin is a specific inhibitor of mTOR and is often used to inhibit protein synthesis in cell models and rodent skeletal muscle in vivo. We hypothesized that the administration of rapamycin to human subjects prior to a bout of resistance exercise would prevent the increase in MPS during early post‐exercise recovery. We studied young men that ingested rapamycin (12mg) 2 hr prior to exercise (Rapamycin; N=7) and a Control group that did not receive rapamycin (N=8). Rapamycin and Control subjects performed a bout of heavy resistance exercise and muscle biopsies were obtained at 1 and 2 hours post‐exercise. Stable isotope and immunoblotting techniques were used to determine MPS and phosphorylation of proteins involved in the regulation of MPS. We found that MPS increased during post‐exercise recovery in the Control group (P<0.05) but was unchanged in the Rapamycin group (P>0.05). In addition, phosphorylation of mTOR (Ser2448), S6K1 (Thr389) and S6 (Ser240/244 and 235/236) were partially inhibited and S6K1 (Thr421/Ser424), eEF2 (Thr56) and ERK1/2 (Thr202/Tyr204) and MNK1 (Thr197/202) were completely blocked during post‐exercise recovery in the Rapamycin group. Raptor association with mTORC1 also tended to be higher in the Control group (as compared to the Rapamycin group) at 2 hours post‐exercise (P=0.06). We conclude that the increase in MPS following an acute bout of resistance exercise appears to be rapamycin dependent indicating that mTOR signaling is playing an important role in regulating the post‐exercise protein anabolic response.

Supported by NIH/NIAMS grant # RO1 AR049877 and P30AG024832.
Anti‐inflammatory activities of new steroidal antedrugs isoxazoline derivatives
Authors: Errahali Younes Jean , Gassett Sandi , Lee Henry Joung
Published: June 2021

Antedrug design represents a new approach projected to create safer drugs by inactivating the metabolites after biotransformation. Our primary objective is to synthesis a new group of corticosteroids that have an anti‐asthmatic and anti‐inflammatory purpose without systemic adverse effects.

A new antedrugs isoxazoline derivative is synthesized from prednisolone. Various concentrations of isoxazoline derivatives synthesis was tested by ELISA in vitro: human 549 alveolar epithelium and leukocyte macrophage cells after stimulation with cytomix‐induced proinflammatory molecular release and in vivo: receptor‐binding assay using rat lung and liver cytosol. The metabolism of biotransformation was studied in rat plasma using HPLC technique.

Most of isoxazoline derivatives showed high binding affinities to the glucocorticoid receptors; five times more potent than prednisolone and significant inhibitory effects of the antedrugs on the nitric oxide (NO) and other proinflammatory cytokines such IL8 cell release. The metabolism study in rat plasma showed that a new isoxazoline with 21‐acetyl groups were hydrolyzed rapidly to inactive metabolites, with half lives 5 minutes.

These results suggest that isoxazolines derivatives compared to conventional steroids improve topical anti‐inflammatory activity without concomitant increase in adverse systemic activity. Support by NIH grants S06 GM08111
The role of HSP70 and HSP90 in the trafficking of hIK1
Authors: Pittner Amie Lynn , White Colleen Annette , Jones Heather Marie
Published: June 2021

The ability of proteins to traffic properly to the cell surface requires that they be properly folded in the ER. The cell contains chaperone proteins which assist in this process. Chaperones will bind to both properly assembled proteins and mis‐folded proteins to determine whether they can traffic out of the ER to the plasma membrane or be retained and eventually degraded. Chaperones may also regulate ion channel cell surface expression by retaining improperly folded channels intracellularly. Co‐immunoprecipitation of hIK1 with HSP70 and HSP90 antibodies resulted in co‐assembly of the proteins in an immune‐complex. Inhibitors of the chaperones will decrease association of these proteins with hIK1 and alter protein expression. Recent studies have shown that chaperone related interactions with ion channels is dictated via charged amino acid residues. Similar residues near the hIK1 selective pore possibly regulate hIK1 association with HSP70 and HSP90. These experiments demonstrate the role of HSP's in hIK1 trafficking and cell surface expression.
The Axial Musculature of the Red Panda (Ailurus fulgens)
Authors: Julik Emily Jean , Smith Heather F. , Adrian Brent , Barton Michael , Fisher Rebecca E.
Published: June 2021

The red panda (Ailurus fulgens) is an endangered, scansorial carnivore found in the forests of the Himalayas and southern China. The phylogeny of the red panda has been the subject of debate, with molecular and morphological studies indicating a number of possible relationships, including affinities to ursids, procyonids, mustelids, and mephitids. The myology of the red panda, however, remains largely undocumented. The axial muscles of the red panda were evaluated to determine whether any evolutionary affinities with suggested relatives existed, and whether any adaptations to the red panda's scansorial lifestyle were present in these muscles. Two adult red pandas were dissected and photodocumented. The axial musculature of Ailurus was then compared to other carnivores. The dissections revealed several interesting findings. For example, m. biventer cervicis was found to have six tendinous intersections, similar to those found in the mm. rectus abdominis and likely allowing precise movement of the neck just as the rectus muscles allow for precise movements of the trunk. The comparison of the axial musculature of the red panda to that of other carnivores also revealed interesting findings; for example, the red panda had several differences from the giant panda (Ailuropoda melanoleuca), including a more superficial m. splenius with a significantly more cranial origin.

Support from the Howard Hughes Medical Institute through the Undergraduate Science Education Program and from the ASU School of Life Sciences.