bluuebunny/crossref_metadata_2025 huggingface dataset:
Circulating Bone Marrow‐Derived Mesenchymal Precursors Robustly Contribute to Lung Vascular Remodeling in Pathogenesis of Pulmonary Hypertension
Authors: Frid Maria G , Gnanaskharan Meena , Burke Danielle L , Iivanainen Antti , Niku Mikael , Stenmark Kurt R
Published: June 2021

Pulmonary hypertension (PH) is characterized by marked fibroproliferative changes in the pulmonary artery (PA). We studied if circulating bone‐marrow (BM)‐derived mesenchymal precursors contribute to vascular remodeling in PH. In rat and calf models of PH, chronic hypoxia induced a robust influx of circulating mesenchymal precursors (fibrocytes) into the PA wall. Their in vivo depletion in circulation resulted in attenuation of PA remodeling. BM origin and differentiation fate of these cells were studied in female freemartin calves, natural XX‐XY BM chimeras. In hypoxic freemartins, numerous Y‐chr+ (BM‐derived) cells were observed in the PA, some differentiated into mesenchymal cells. Using laser capture microdissection and RT‐PCR, we observed an early, persistent, and pulmonary‐specific upregulation of factors potentially involved in recruitment, retention and differentiation of fibrocytes (C5, FIZZ‐1, MCP‐1, IL‐18, ‐6, VEGF, SDF‐1, MMP‐9, ET1, TGFb1). In culture, fibrocyte‐like cells from PA of hypoxic, but not control, calves exhibited augmented growth, migratory capabilities, and secreted potent mitogens for resident PA SMC. These data demonstrate that circulating BM‐derived fibrocytes robustly contribute to pulmonary vascular remodeling in PH, and could serve as a selective pharmacological target in the treatment of PH.

Funding: (1) NIH HL084923‐1, HL014985‐35; (2) (210978/10.11.2004)
Effects of sibutramine and rimonabant in rats trained to discriminate 22 from 2 hours food deprivation
Authors: Jewett David C. , Hahn Thomas W. , Smith Travis R. , Levine Allen S.
Published: June 2021

We tested the effects of sibutramine, a norephinephrine and serotonin reuptake inhibitor, and rimonabant, a CB1 antagonist, in rats trained to discriminate 22 hr food deprivation from 2 hr food deprivation in a two‐lever, operant choice task. After rats acquired the discrimination, subjects were food restricted for 22 hr and administered sibutramine (0.3–10 mg/kg, p.o.) or rimonabant (0.3–10 mg/kg, s.c.). One hour later, the discriminative stimulus effects were assessed. Sibutramine significantly decreased the discriminative stimulus effects of 22 hf deprivation; 3.2 mg/kg reduced the effects of deprivation by 50%. A larger dose eliminated responding and decreased food intake. Rimonabant did not affect the discriminative effects of deprivation, but significantly reduced response rates and decreased food intake. While rimonabant decreases intake and rate of responding it does not alter “hunger” discrimination. Such data support the idea sibutramine affects “hunger‐driven” feeding and that the CB1 receptor is involved in feeding associated with affect/reward, rather than feeding stimulated by energy needs.
Mice lacking fatty acid amide hydrolase have facilitated acquisition of anandamide discriminative stimulus,
Authors: Vann Robert Edward , Warner Jonathan A. , Nguyen Thanh K. , Grainger Darren B. , Lichtman Aron H. , Martin Billy R. , Wiley Jenny L.
Published: June 2021

Anandamide (AEA), an endocannabinoid (eCB), shares some, but not all, pharmacological properties with THC. This pharmacological dissociation is in part due to the rapid catabolism of AEA by fatty acid amide hydrolase (FAAH). With inhibitors of FAAH, studies have demonstrated that AEA shares THC's discriminative stimulus (DS) properties providing the leading evidence for the involvement of the eCB system in the subjective effects of marijuana. However, this remains to be fully elucidated without demonstrating that these DS effects are cross symmetrical. Thus, the current study investigated whether FAAH (−/−) mice, those lacking the ability to breakdown AEA, could be trained to discriminate AEA (6 mg/kg) versus VEH in a 2‐lever discrimination. AEA was successfully trained and dose dependently generalized. THC fully and dose dependently substituted whereas oleamide, another FAA, failed to substitute. THC's and AEA's DS effects were blocked by SR141716. This study demonstrates that: AEA's DS can be trained in FAAH (−/−) mice, THC and AEA cross generalize, and these effects are CB1 receptor mediated. Oleamide's lack of AEA‐like DS effects provides further support for the specificity of this model. Taken together, this research demonstrates that THC or AEA activation of the eCB system results in similar DS effects. Research supported by NIH grant DA‐09789.
Comparison of antagonist potency to evoke an acute withdrawal stimulus 4 h after pretreatment with morphine in rats
Authors: Hazel Kweku Dodoo , Fluck Jonathan , Jones Nicholas T. , Evola Marianne , Young Alice M.
Published: June 2021

In vivo behavioral experiments evaluated potency of opioid antagonists to evoke discriminative stimulus effects associated with acute morphine withdrawal. Rats were trained, on a food‐reinforced task, to discriminate naltrexone (Ntx; 0.32 mg/kg) 4 h after pretreatment with either 5.6 mg/kg morphine (MS) or saline. Dose‐effect tests assessed the ability of other opioid antagonists to mimic the MS/Ntx stimulus; all injections were given by subcutaneous injection. Ntx itself (0.001 – 1.0 mg/kg) and naloxone (0.001 – 0.32 mg/kg) both dose‐dependently mimicked the MS/Ntx stimulus at doses that did not suppress response rates. Surprisingly, naloxone produced full MS/Ntx stimulus effects at lower doses than did Ntx. In contrast, 6‐β naltrexol (0.32 – 10 mg/kg) evoked a maximum of 60% generalization to the MS/Ntx stimulus and did not suppress response rates markedly. The partial agonist nalbuphine (0.32 – 32 mg/kg) produced full MS/Ntx stimulus effects at a dose of 32 mg/kg but suppressed responding markedly. These data suggest that opioid antagonists differ in potency to evoke acute withdrawal and may differ in their ability to evoke an acute maximal withdrawal stimulus effect. [Supported by NIH DA03796, GM 08167, and the Howard Hughes Medical Institute.]
Effects of a novel kappa opioid receptor agonist, TRK‐820, on intrathecal morphine‐induced itch and analgesia in monkeys
Authors: Ko Mei‐Chuan , Husbands Stephen
Published: June 2021

Itch (Pruritus) is the most common side effect associated with spinal administration of morphine given to humans for analgesia. Recent studies have suggested that kappa opioid receptor (KOR) agonists can attenuate intrathecal morphine‐induced itch/scratching responses. TRK‐820 has been shown to be a potent high efficacy KOR agonist with a low efficacy mu opioid agonist action. The aim of the study was to investigate the effectiveness of TRK‐820 as an antipruritic and to elucidate the receptor mechanisms underlying TRK‐820's antipruritic effect. Adult rhesus monkeys were used in various behavioral assays for measuring itch/scratching, analgesia, and respiratory depression. Systemic administration of TRK‐820 (0.1–1 μg/kg, i.m.) dose‐dependently attenuated intrathecal morphine (30 μg)‐induced scratching responses without affecting morphine‐induced antinociception. Pretreatment with 1 μg/kg of TRK‐820 did not significantly change the dose‐response curves of systemic morphine‐induced antinociception and respiratory depression. In addition, a selective KOR antagonist, GNTI (1 mg/kg), significantly reversed the antipruritic effect of TRK‐820. Taken together, these results indicated that TRK‐820's antipruritic action was mediated by a KOR agonist action without a mu opioid antagonist action. This receptor mechanism‐based finding supports the clinical potential of KOR agonists as antipruritics to treat mu opioid receptor‐mediated pruritus (Supported by USPHS Grant DA‐013685).
Pharmacological characterization of 3H‐HYDIA a new tritiated ligand selective for mGlu2/3 receptors
Authors: Lundström Linda , Beck Jennifer , Wettstein Joseph G , Huguenin Philipp , Woltering Thomas J , Gatti Silvia
Published: June 2021

Metabotropic glutamate receptor 2 and 3 have been involved in dementia, depression and schizophrenia and selective ligands are under development. This study contains the pharmacological characterization of a new radioligand 3H‐HYDIA: the selective and competitive mGlu2/3 antagonist (1S,2R,3R,5R,6S)‐2‐amino‐3‐hydroxy‐3‐tritio‐bicyclo[3.1.0]hexane‐2,6‐dicarboxylic acid RO0653479‐002‐002. This radioligand was generated after the observation that the introduction of the R‐configurated 3‐hydroxy‐group was sufficient to turn the potent mGlu2/3 agonist LY354740 into a selective mGlu2/3 antagonist (see Woltering T. J. et al. ChemMedChem 2007). The in vitro binding of 3H‐HYDIA was characterized in recombinant rat and human mGlu2 and mGlu3, in rat cortical and hippocampal membranes as well as in rat brain sections. Ionotropic glutamate receptor ligands, group I and III selective mGlu receptor ligands and negative allosteric modulators for group II mGlus did not inhibit 3H‐HYDIA binding. Several known mGluII ligands e.g. LY341495, LY354740, MGS0039, (2S,2′R,3′R)‐2‐(2′,3′‐dicarboxycyclopropyl)glycine, glutamate, completely inhibited the specific binding. This radioligand, a unique molecule interacting at the ortosteric binding site of the receptors, may serve as useful tools for the study of the molecular determinants of activity in the glutamate binding pocket of mGlu2/3.
Relationship between methamphetamine exposure and matrix metalloproteinase 9 expression
Authors: Matsumoto Rae R. , Liu Yun , Shaikh Jamaluddin , Brown Sheketta
Published: June 2021

Matrix metalloproteinase (MMP) 9 is up regulated following various types of insults to the brain. Recent studies have also implicated MMP9 in neural remodeling after stimulant doses of methamphetamine (METH). The involvement of MMP9 in METH‐induced neurotoxicity, however, remains unclear and was thus evaluated in the present study. Male, Swiss Webster mice were injected with stimulant or toxic doses of METH. MMP9 gene expression was up regulated in the brain within 5 min. By 24 h, MMP9 gene expression returned to control levels in the stimulant treated mice, but remained elevated in animals exposed to toxic doses of METH. The development of neurotoxicity was evidenced between 1­7 days following METH exposure as depletions in striatal dopamine levels. However, this marker of METH neurotoxicity was not accompanied by concomitant changes in striatal MMP9 gene expression. In MMP9 knock out mice, neurotoxic dosing with METH produced depletions in striatal dopamine levels that were comparable to wild type mice. Although the absence of MMP9 did not affect METH neurotoxicity, the knock out mice exhibited the expected decrease in behavioral sensitization following repeated administration of stimulant doses of METH. Together, the data suggest that changes in MMP9 expression do not affect METH‐induced neurotoxicity, and may instead be contributing to remodeling of the nervous system.
Sex differences in the corticotropin‐releasing factor receptor and its regulation by stress
Authors: Bangasser Debra A , Bethea Thelma T. , Parastatidis Ioannis , Valentino Rita J
Published: June 2021

Depression, a common stress‐related mental illness, affects twice as many women as men. Dysfunctions in corticotropin‐releasing factor (CRF) have been linked to depression, but sex differences in the CRF system are not well characterized. Previous studies from our laboratory demonstrated that the postsynaptic sensitivity of locus coeruleus (LC) neurons to CRF was greater in female vs. male rats. Moreover, prior stress sensitized LC neurons of male but not female rats. These findings suggested the existence of sex differences in CRF receptor (CRFr) coupling and regulation. To test this, the CRFr was immunoprecipitated from cortical samples of stressed (15min swim) or handled male and female rats. Under unstressed conditions, the CRFr is more highly coupled to Gs in females vs. males. Stress increases coupling of CRFr to Gs in male but not female rats. Additionally, we found that following stress, CRFr association with βarrestin2— which targets the CRFr for desensitization and internalization—was increased in male but not female rats. Thus, sex differences in CRFr coupling and/or desensitization may contribute to differential neuronal sensitivity of females to CRF and stress. This may translate to increased vulnerability of females to depression. Finally, sex differences in CRFr should be considered in the development of CRF antagonists as therapeutic agents. Supported by PHS Grants MH 40008 and MH 63267 to RJV.
Perinatal iron deficiency results in increased visceral adiposity in male Wistar rats
Authors: Bourque Stephane , Komolova Marina , Nakatsu Kanji , Adams Michael
Published: June 2021

Perinatal iron deficiency (ID) has adverse programming effects in adult offspring manifested as alterations in cardiovascular function as well as glucose and lipid metabolism. Given the importance of increased visceral adipose tissue (VAT) as a likely mediator of these sequalae, in the present study we determined the effect of perinatal ID on VAT in male wistar rats, and its association with other obesity‐related disturbances, such as salt sensitive arterial pressure (AP). Dams were fed a low iron diet (<10ppm Fe) prior to and throughout gestation. At delivery, dams were fed a normal iron diet (270ppm Fe). ID offspring had lower body weights (−11%) and markedly reduced hematocrits (−40%) at birth compared to controls. At 16 weeks of age, these animals had 20% more VAT (normalized to body weight) than controls. Furthermore, the elevation of AP (assessed by radiotelemetry) in response to increased sodium intake was 79% greater in the ID offspring compared to controls. In summary, perinatal ID resulted in a persistent increase in visceral adiposity associated with an enhanced hypertensive response to dietary salt. Supported by the Canadian Institutes of Health Research and the Bickell Foundation of Canada.
Cerebral infarct size is gender‐dependent following transient but not permanent middle cerebral artery occlusion in mice
Authors: Brait Vanessa H , Jackman Katherine A , Mast Anja E , Drummond Grant R , Sobey Christopher G
Published: June 2021

Estrogen mediates protection of females in cerebral ischemia‐reperfusion (I‐R), and inhibits atherosclerosis via TGFβ signalling. We tested whether females are also protected during cerebral ischemia with no reperfusion (I‐NR), and whether this may be related to TGFβ activity. Stroke was induced by 0.5 h middle cerebral artery occlusion in 6–8 week old female (n=22) and male (n=30) C57Bl6J mice. After 24 h, brain infarct volume was measured in thionin‐stained coronal sections. After I‐R, females had a smaller infarct volume than males (24±6 vs 55±11 mm3, n=7–8; P<0.05), whereas there was no gender difference following I‐NR (88±9 vs 88±11 mm3, n=8–9). Preliminary data (n=1–3) indicate that expression of p‐SMAD, a marker of TGFβ signalling, is 47% higher in brains of sham‐operated females vs males. Furthermore, in females p‐SMAD expression was reduced by 37% after I‐R and by 67% after I‐NR. By contrast, neither model of stroke had any effect on p‐SMAD expression in males. Thus, cerebral infarct size is gender‐dependent following I‐R but not I‐NR in mice. The lack of protection in females in I‐NR may be related to reduced TGFβ signalling.
Fenoterol derivatives show multitarget interactions with beta2 adrenergic and 5‐HT receptors
Authors: Jozwiak Krzysztof , Wainer Irving W
Published: June 2021

Stereoisomers of fenoterol and eight fenoterol derivatives modified at the N‐alkyl moiety (Fig. 1) have been synthesized to determine their binding affinities for the β2 adrenergic receptor (β2–AR) and the subtype selectivity relative to the β1–AR. Sub‐micromolar affinities were observed for (R,R)‐fenoterol, the (R,R) isomer of the p‐methoxy, (R,R) and (R,S) isomers of 1‐naphthyl and (R,R) and (R,S) isomers 4‐methoxy‐1‐napthyl derivatives. Most of these compounds show also high β2/β1 subtype selectivity. Off‐target screening panel revealed that some naphtyl derivatives show additional high binding affinities towards 5‐HT2 and 5‐HT3 subtypes of serotonin receptor determined in radioligand displacement assays.

The binding data were analyzed using 3‐D QSAR and the resulting pseudo‐receptor models identified binding fields responsible for asymmetric recognition of diastereoisomers. The steric restriction on the second chiral center is responsible for observed β2/β1 subtype selectivity. Molecular models of β2‐AR and β1‐AR macromolecules were used to analyze ligand‐receptor interactions. Docking and molecular dynamics simulations on the β2‐AR model revealed that S204, S207 and T118 residues form the network of hydrogen bonds interacting with the catechol moiety; residues N293 and D113 mainly affect the asymmetric recognition of diastereoisomers and T308 and H296 residues are responsible for β2/β1 subtype selectivity profile of studied compounds. Molecular modeling investigations of ligand interactions with serotonin receptors were also performed.

The data from the binding and modeling studies indicate that this set of compounds can be used to explore a wide variety of ligand‐GPCR binding mechanisms.
Investigation of the site of action of McN‐A‐343 at the M1 muscarinic receptor based on the binding profile of its nitrogen mustard analogue.
Authors: Figueroa Katherine Wendy , Ehlert Frederick J
Published: June 2021

To date, the precise location of the binding site for McN‐A‐343 on the M1 muscarinic receptor has yet to be determined, although prior studies on the M2 receptor suggest binding to both the orthosteric and allosteric sites. To address this question, we investigated the mode of interaction of a nitrogen mustard derivative of McN‐A343 (BR384; 4‐[(2‐bromoethyl)methyl‐amino]‐2‐butynyl N‐(3‐chlorophenyl)carbamate) with the M1 receptor using radioligand binding methods. The modulatory effects of known competitive and allosteric muscarinic ligands on the irreversible alkylation of the receptor were investigated in stably transfected CHO‐K1 cells and homogenized tissue. In both brain tissue homogenates and CHO‐K1 M1 cells gallamine exhibited allosteric inhibition of M1 receptor alkylation by BR384, whereas N‐methylscopolamine, atropine and Mc‐N‐A‐343 competitively inhibited alkylation. WIN 62,777 had no significant effect on the rate of M1 receptor alkylation by BR384. Our data suggest that both BR384 and McN‐A‐343 bind to the orthosteric site of the M1 receptor.

Supported by NIH grant 69829 (Frederick J. Ehlert) and Pre‐Doctoral Fellowship from The PhRMA Foundation (Katherine W. Figueroa).
Modified Silicon‐Coated Nylon Suture in A Rat Middle Cerebral Artery Occlusion Model
Authors: Zhao Honggang , Mayhan William G , Sun Hong
Published: June 2021

Due to anatomical variations of the arterial circle the infarct size is not uniform in traditional nylon suture‐produced a rat middle cerebral artery occlusion (MCAO) model. We modified nylon suture by coating silicon to 4–0 nylon suture 3 mm from the tip for 17 mm, and tested the modified suture in a rat MCAO model. Sprague‐Dawley rats were divided into four groups (Group A, traditional suture under katemine/xylazine anesthesia; Group B, modified suture under katemine/xylazine anesthesia; Group C, traditional suture under isoflurane anesthesia; Group D, modified suture under isoflurane anesthesia) and subjected to 2 hours MCAO. Regional cerebral blood flow (rCBF) was monitored through a Laser‐Doppler flow probe attached to the lateral aspect of the skull. Neurological evaluation and ischemic region were assessed 24 hours after reperfusion. The total ischemic volume (infarct and penumbra) was similar among all four Groups (Group A, 56.0 ± 5.2%; Group B, 66.2 ± 3.4%; Group C, 59.4 ± 2.8%; Group D, 59.2 ± 5.2%). However, the infarct size was significantly larger in modified suture‐produced MCAO rats under either ketamine/xylazine anesthesia (Group A, 20.3 ± 2.3% vs. Group B, 35.4 ± 5.1%) or isoflurane anesthesia (Group C, 6.4 ± 0.9% vs. Group D, 12.9± 1.6%). In addition, neurological evaluation demonstrated that modified suture‐produced MCAO groups showed greater neurological deficits than traditional suture‐produced MCAO groups. We conclude that modified silicon‐coated nylon suture provides more uniform occlusion of the middle cerebral artery than the traditional nylon suture.
Polymorphonuclear (PMN) leukocytes in type 2 diabetes are hyper‐responsive to activation induced by myocardial ischemia‐reperfusion
Authors: Davis‐Gorman Grace F. , La Bonte Laura R. , McDonagh Paul
Published: June 2021

Reperfusion of ischemic myocardium induces an acute inflammatory response. This response may cause leukocyte activation, increasing leukocyte adhesion, sequestration and a PMN‐mediated reperfusion injury. This study was performed to determine if the expression of leukocyte adhesion protein expression was increased during reperfusion following ischemia. We also were interested in determining if this response is enhanced in the setting of type 2 diabetes. Neutrophil (PMN) adhesion protein CD11b expression was measured by flow cytometry before and after ischemia in Zucker Lean Control (ZLC) and Zucker Diabetic Fatty (ZDF) rats subjected to 30 minutes of regional (LAD) ischemia followed by two hours of reperfusion (I/R). We found that PMN CD11b expression was significantly increased in ZDF blood prior to ischemia, indicative of a chronic, low‐level activation. During reperfusion, PMN CD11b increased significantly with time for both groups, but the increase was greater for the ZDF group (P<0.05, RMANOVA). These results indicate that ischemia‐reperfusion itself causes an acute activation of circulating PMNs and that the PMNs from diabetic animals were hyper‐responsive. The difference in the activation response may enhance PMN adhesion and the PMN‐contribution to I/R injury in the type 2 diabetic heart. Supported by NIH HL 58859 and AHA 0610018Z.
Ischemic skeletal muscle protection by direct delivery of ATP – the role of purinergic and adenosine receptors
Authors: Pushpakumar Sathnur Basappa , Perez‐Abadia Gustavo , Arumugam Sengodagounder , Todnem Nathan , Barker John , Maldonado Claudio
Published: June 2021

Fusogenic Lipid vesicles loaded with ATP (FLVs‐ATP) have been used to conserve critical levels of intracellular ATP to minimize tissue damage in ischemia/reperfusion injury. Although beneficial its precise mechanism was unclear. The purpose of this study was to determine the role of purinergic and adenosine receptors during ATP delivery to ischemic skeletal muscles. We hypothesized that the ATP from FLVs‐ATP activates purinergic and adenosine receptors on cells, and has an ATP sparing effect. We used phosphocreatine (PCr) half life as an index of internal depletion of ATP in order to distinguish between intracellular and extracellular ATP (FLVs‐ATP). Extensor Digitorum Longus muscles (n=10/group) were perfused: Heparinized Lactated Ringer's (HLR); Free ATP; ATPgamma; Adenosine; FLVs; FLVs‐ATP; FLVs‐ATP + purinergic blocker; FLVs‐ATP + adenosine blockers. 31 Phosphate Nuclear Magnetic Resonance was used to measure PCr levels and calculate its half life. We found that FLVs‐ATP with purinergic receptor blockers was beneficial illustrating that purinergic receptors may have played a minor/no role in prolonging the PCr half life. In contrast FLVs‐ATP + adenosine receptors blockers showed significant difference compared to purinergic blockers in the PCr half life (p<0.05) but not significant to HLR indicating that the beneficial effects of exogenous ATP are being mediated by adenosine receptors.
NADPH oxidase mediates the mesenteric inflammation initiated by alveolar macrophages in alveolar hypoxia
Authors: Chao Jie , Allen Julie , Blanco Victor , Wood John , Gonzalez Norberto
Published: June 2021

Alveolar hypoxia (AH), induced by reducing inspired O2 to 10%, rapidly produces inflammation in skeletal muscle, brain, and mesentery of rats. Data from our laboratory suggest that the inflammation is initiated by an agent (s) released into the circulation by alveolar macrophages (AM). This agent stimulates mast cells (MC) and the renin‐angiotensin system (RAS) to produce inflammation. Plasma from AH rats produces inflammation in normoxic muscle; the inflammation is attenuated by MC stabilization and by blockade of the RAS. Depletion of AM in vivo attenuates the inflammation in AH rats; furthermore, supernatant of AM cultured in hypoxia activates MC and produces inflammation in normoxic muscle. The present experiments investigated the interaction: AM‐MC‐inflammation using intravital microscopy; specifically, we asked whether NADPH oxidase is involved in the RAS‐mediated inflammation. Supernatant from AM cultured in hypoxia induced leukocyte endothelial adherence (LEA) and MC degranulation (MCD) in mesentery of normoxic rats. Angiotensin II receptor blocker and NADPH oxidase inhibitor (apocynin) blocked LEA but not MCD, indicating that activation of RAS and of NADPH oxidase are downstream of MCD. The data support the hypothesis that an agent(s) released from AM in AH initiate MCD; this, in turn, stimulates the RAS which leads to inflammation via activation of NADPH oxidase. Supported by NIH HL39443‐17
High Glucose Concentrations via Activating Rho‐kinase Leads to Augmented and Sustained Angiotensin II‐induced Arteriolar Constrictions
Authors: Bagi Zsolt , Kaley Gabor , Koller Akos
Published: June 2021

We tested the hypothesis that functional availability of type 1 angiotensin II (Ang II) receptors (AT1) is enhanced and more sustained in arterioles exposed to high glucose concentration. In isolated, pressurized rat skeletal muscle arterioles (~ 150 μm) Ang II‐induced (10−8 M) constrictions were measured upon repeated applications in normal glucose (NG, 5.5 mM) or high glucose conditions (HG, 25 mM for 1 hour). Ang II‐induced constrictions to first applications were significantly augmented in HG arterioles (max: 74±6%), compared to NG vessels (max: 56±7%). Upon second applications Ang II‐induced constrictions decreased in NG arterioles (30±6%), but remained substantial in HG vessels (65±9%). In similar protocols constrictions to norepinephrine (10−7 M) were unaffected by HG exposure. In arterioles, HG caused Rho‐kinase activation, as detected by the increased membrane‐bound RhoA with Western blots. In the presence of Rho‐kinase inhibitor, Y27632 (1 μM) constrictions to first application of Ang II were not significantly different in NG and HG vessels. Moreover, Y27632 reduced constrictions to second applications of Ang II in HG arterioles to the level of NG vessels. These findings indicate that high glucose via activation of Rho‐kinase leads to augmented and maintained Ang II‐induced arteriolar constrictions, which is likely due to a sustained availability of AT1 receptors. (Grants: Hungarian Sci. Res. Funds/OTKA T48376; Am Heart Assoc., NE Aff. T‐0555897, T‐0735540, NIH HL43023).
Soluble Epoxide Hydrolase Inhibition Modulates Gene Expression of Anti‐Apoptotic and Apoptotic Factors
Authors: Simpkins Alexis N , Elmarakby Ahmed A. , Quigley Jeffery E. , Schreihofer Derek A. , Hammock Bruce D. , Imig John D.
Published: June 2021

Epoxyeicosatrienoic acids cerebral protective properties are attenuated by degradation to less active diols by the soluble epoxide hydrolase enzyme. We previously demonstrated that chronic SEH inhibition via adamantanyl dodecanoic acid (AUDA) protects against cerebral ischemia. Here we test the hypothesis that AUDA provides neural protection via altering gene expression of apoptotic and anti‐apoptotic factors. We compared expression of 30 anti‐apoptotic and 54 apoptotic genes in brain samples collected from controls and AUDA treated (2mg/day, 6weeks) SHRSPs and WKYs using an apoptosis PCR microarray (n=3 per group). Gene expression of 18 apoptotic and 8 anti‐apoptotic factors increased ≥2 fold in SHRSP control versus (vs) WKY control. While AUDA treated SHRSP had only 1 upregulated apoptotic gene ≥2 fold vs WKY control. Also, AUDA reduced gene expression of 29 apoptotic and 8 anti‐apoptotic factors ≥1.48 fold in SHRSP vs SHRSP control. In contrast, gene expression of 8 anti‐apoptotic and 14 of apoptotic factors increased ≥1.48 fold in AUDA treated WKY vs WKY control. Interestingly, AUDA increased gene expression of anti‐apoptotic Mapk8ip in SHRSPs and WKYs, which is reportedly neural protective in ischemia. These results suggest that SEH inhibition is neural protective due to dampening of apoptotic gene expression in SHRSPs and upregulation of anti‐apoptotic gene expression in WKYs and SHRSPs. (AHA and NIH)
Responsiveness to acute angiotensin II is increased in intrauterine growth restricted rats
Authors: Ojeda Norma Beatriz , Royals Thomas P , Alexander Barbara T
Published: June 2021

Numerous epidemiological studies indicate birth weight is inversely correlated with blood pressure. Our laboratory has developed a unique rat model of placental insufficiency that results in intrauterine growth restriction (IUGR) and hypertension in adult IUGR. Although renal and systemic components of the renin angiotensin system (RAS) do not differ between IUGR and control rats, RAS blockade abolishes IUGR hypertension. Therefore, we hypothesized that an increased responsiveness to angiotensin II (ANG II) may contribute to hypertension in this model of IUGR. Physiological parameters were determined before and after acute infusion of ANG II (100 ng/kg/min for 30 minutes) in rats pretreated with RAS blockade (250 mg/L enalapril for 1 week). Mean arterial pressure (MAP) and renal vascular resistance (RVR) were increased; glomerular filtration rate (GFR) and the pressure natriuresis function (PNF) slope were decreased following acute infusion of ANG II in IUGR compared to control. Thus, these data demonstrate that an increased responsiveness to ANG II is observed in IUGR offspring and that impairment in sodium excretion may serve as an underlying mechanism in IUGR hypertension.
High Salt Diet During Pregnancy Alters Kidney Renin Granules In The Adult Rats Offspring.
Authors: Costa Nauilo Lima , Ramos Débora Rothstein , Lopes Karen Lucasechi , Furukawa Luzia Naoko Shinohara , Heimann Joel Claudio
Published: June 2021

Objective: To study the influence of dietary salt diet during pregnancy on body weight (BW), blood pressure (BP) and the kidney renin granules (RG) of the adult offspring Wistar rats.

Methods: Dam groups were fed with normal salt – NSD (1.3%), high salt 1‐ HSD1 (4%) or high salt 2 HSD2 (8% NaCl, ) since 8‐week‐old and pregnancy until delivery day. Female and male offsprings were fed standard salt diet since birthday to 12 week‐old. The BW was measured weekly and BP every 2 weeks. Low salt diet (LSD – 0.15% NaCl) + captopril (100mg/kg) in drink water [LSD+C]; LSD + furosemide (1mg/kg, one dose) [LSD+F] and HSD2 tests for 7 days carried out at 12 week‐old. The kidney was removed and RG were quantified.

Results (mean±SEM, p<0.05, n=7): The BP and BW did not differ among offsprings from dams fed different salt diet. Positive arterioles RG in HSD2 (70±3.4%‐LSD+C; 59.3±4.9%‐LSD+F; 59.8±5.0%‐HSD2); and HSD1 (63.8±3.4%‐LSD + C; 50.7±5.2%‐HSD2) female offsprings were higher in three and two tests respectively compared to NSD (44.3±3.0%‐LSD + C; 37.2±1.9%; LSD +F, 34.2±3.3%‐HSD2). However, the same did not occur on the male offspring.

Conclusion: Modifications of salt dietary content for pregnancy on dams can program differently RG secretion in both genders of offspring through adult late life.

Support by FAPESP
The role of a gel filled tip in maintaining the frequency response of telemetry based blood pressure measurements
Authors: McCormick Daniel , Lim Matthew , Kirton Robert , Van Vliet Bruce , Nielsen Poul , Budget David , Malpas Simon
Published: June 2021

In recent years, measurement of blood pressure via telemetry in a variety of laboratory animals has become an indispensable part of cardiovascular physiology, drug development and safety pharmacology. The solution provided by Data Sciences Inc almost 20 years ago is in the form of a catheter attached to a pressure sensor and wireless link to a remote receiver. This design features a catheter made of compliant tubing and filled at the tip with a gel material. This gel was suggested to contribute to the frequency response of blood pressure telemetry system. However, the actual influence of the specialized tip on the dynamic response of the pressure sensor has not been reported. In the present experiment, we investigated the role of the specialized gel‐filled catheter tip in the frequency response of fluid filled catheters. Experiments were carried out using a custom made sinusoidal pressure generator and the data analyzed using Labview software. The experiments reveal that the bandwidth of the DSI system is ultimately limited to less than 100 Hz by the low power transmission system and not the gel tip catheter. When a high bandwidth transmission system is available, −3 dB bandwidths of 400 Hz are attainable for rat sized catheters. The results of the study suggest that the gel tip reduces the frequency response slightly when compared to a tip‐less catheters but not to the extent that the blood pressure measurements are compromised.
BAROREFLEX IMPAIRMENT IS CORRELATED WITH HIGH SUPEROXIDE DISMUTASE ACTIVITY IN METHIONINE‐TREATED RATS
Authors: Mendes Roberta Hack , Candido Georgia Orsi , Mostarda Cristiano , De la Fuente Raquel Nitrosi , Jorge Luciana , Silva Ivana C.M. , Belló‐Klein Adriane , Rigatto Katya , Irigoyen Maria Claúdia Costa
Published: June 2021

Introduction:Homocysteine (Hcy) is a suphur amino acid in methionine‐cysteine metabolism. Hyperhomocysteinemia is an independent risk factor for atherosclerosis.

Purpose:To evaluate the effect of methionine treatment (ME) on systolic arterial pressure (SAP), heart rate (HR), baroreflex sensitivity (BRS) and superoxide dismutase (SOD) activity.

Methods:Male wistar rats (n=5/group) were treated by gavage for 9 weeks (water=CO or ME=0,1g/kg in water). After treatment, rats were cannulated for cardiovascular measurements. BRS was evaluated by bradycardic (BR) and tachycardic responses (TR) to vasoactive drugs. Rats were killed and blood samples were collected, prepared and stored at −80ºC for SOD activity measurements.

Results: Our results showed that ME increased SAP (117±8 vs 136±9 mmHg) and there was no difference in HR between groups (CO=324±13; M=337±11bpm). TR and BR in ME were decreased (2.4±0.4 vs 1.4±0.6; 1.9±0.5 vs 1.3±0.2 bpm/mmHg, respectively). SOD activity was higher in ME (14.8±1.8 vs 13±1.9 USOD/mg protein) and it was negatively correlated with BRS for both, TR (r=−0.8) and BR (r=−0.7).

Conclusion: These data suggest that ME treatment increased SOD activity and induced a decrease on BRS, leading to an impairement on blood pressure control. Therefore, superoxide anion may participate in the baroreflex impairment.
The effect of AT1 receptor downregulation in the paraventricular nucleus (PVN) on chronic angiotensin II (AngII)‐salt hypertension in the rat
Authors: King Andrew J , Haywood JR , Fink Gregory D , Northcott Carrie A
Published: June 2021

Previous studies have shown silencing AT1a receptor expression with small hairpin RNA delivered by adenovirus (AT1x) in the PVN attenuated acute systemic AngII‐mediated increases in arterial pressure (AP). In the present study, it was hypothesized that chronic AngII‐salt hypertension would be attenuated after AT1x in PVN. LacZ (a marker gene) or AT1x were injected into the PVN [1 × 109 pfu/ml, bilateral (200 nl/site)] of male Sprague Dawley rats (on 2% NaCl diet) instrumented with radiotelemetry transmitters to measure AP and heart rate (HR). Ten days post virus, AngII (150 ng/kg/min) or vehicle was given subcutaneously by minipump for 14 days. Final mean AP was the same in AngII rats treated with AT1x and LacZ (AngII AP: AT1x: 146±11 mmHg, Lac Z: 147±7 mmHg). No differences between groups in HR, weight gain, sodium or water intake were observed. Sympathetic nervous system (SNS) pressor effects were tested using the fall in AP to hexamethonium (30 mg/kg) on infusion day 14. In vehicle rats depressor responses were similar in AT1x (−42±5 mmHg) and Lac Z (−33±4 mmHg) groups. PVN AT1x treatment did not interfere with SNS activation by AngII (AT1x: −93±19 mmHg, LacZ: −80±13 mmHg). Western analyses showed a 34% reduction in AT1a receptors in the PVN (p<0.05). In conclusion, partial downregulation of PVN AT1a receptors did not affect the development of AngII‐salt hypertension or SNS activation in chronic Ang II‐salt hypertension.
Ovariectomy impairs estrogen‐induced relaxation of mesenteric microvessels
Authors: Royal Crista Ruth , White Richard E
Published: June 2021

The effect of estrogen on the cardiovascular health of postmenopausal women has become highly controversial, particularly in light of recent clinical trials such as the Women's Health Initiative. Prior to such trials estrogen was believed to be primarily cardio‐protective, an effect at least partially due to its ability to relax blood vessels. We had demonstrated that 17β‐estradiol (E2) relaxed porcine coronary arteries by an endothelium‐independent mechanism involving stimulation of neuronal nitric oxide synthase (nNOS) in smooth muscle cells. We now report that artificially inducing menopause by ovariectomy (OVX) almost completely abolishes E2–induced, endothelium‐independent relaxation of rat mesenteric arteries. In contrast, estrogen relaxed mesenteric microvessels from OVX rats in the presence of an intact endothelium. Inhibition of NOS activity with either 0.1mM L‐NAME or 1mM L‐NMMA abolished this relaxant effect of E2 in intact vessels from an OVX rat. Our results indicate endothelium‐independent, estrogen‐induced relaxation of resistance arteries is attenuated in postmenopausal rats compared to age‐matched control animals. (supported by HL073890)
Inhibiting vascular ceramide synthesis prevents arterial dysfunction and hypertension in mice with diet‐induced obesity
Authors: Symons J. David , Holland William , Tanner Jason , Kearns Devin , Cahoon Judd , Palionyte Milda , Duncan Bradlee , Losee Jason , Narra Krishna , Hillas Elaine , Rohrwasser Andreas , Abel E. Dale , Summers Scott
Published: June 2021

Ceramide accumulation in vascular smooth muscle might contribute to obesity‐related arterial dysfunction and hypertension. To test this hypothesis, mice were treated with myriocin (M), an inhibitor of an enzyme required for ceramide biosynthesis (i.e., serine palmitoyltransferase). 10‐week old mice consumed chow containing 45% (HF, n=20) or 10% (Con, n=20) fat ± M (0.3 mg/kg i.p., n=10) or vehicle (V; saline i.p., n=10) for 14 weeks. Mean (MAP), systolic (SBP), and diastolic (DBP) blood pressures then were recorded via telemetry for 6 × 24‐h periods followed by excision of the aorta and femoral arteries. Vascular ceramide (pmol ceramide/mg aorta) increased (p<0.05) from 17±4 in Con‐V mice to 30±3 in HF‐V animals, and was suppressed in HF‐M animals (10±1). Elevated (*p<0.05) MAP, SBP, and DBP in HF‐V vs. Con‐V mice was prevented in HF‐M animals. Values in the Table represent the mean±SE of 6 dark/active cycles. Impaired (*p<0.05) acetylcholine (ACh)‐evoked vasorelaxation (wire‐myography) in femoral arteries from HF‐V vs. Con‐V mice was prevented in HF‐M animals. Sodium‐nitroprusside‐evoked vasorelaxation was similar among groups. ACh values represent % vasorelaxation.

These results suggest that vascular ceramide accumulation may contribute to arterial dysfunction and hypertension in mice with diet‐induced obesity. AHA GIA 06‐55222Y
Perivascular adipose tissue impairs coronary endothelial function via protein kinase C‐beta dependent phosphorylation of nitric oxide synthase
Authors: Payne Gregory Allen , Borbouse Lena , Dincer U. Deniz , Bohlen H. Glenn , Dick Gregory M. , Tune Johnathan D.
Published: June 2021

Our laboratory recently demonstrated that perivascular adipose tissue significantly diminishes coronary nitric oxide production and endothelial‐dependent dilation. However, the exact mechanisms by which adipose‐derived factors impair coronary endothelial function have not been fully elucidated. Accordingly, this study was designed to delineate the cellular/molecular pathways involved in adipose‐induced coronary endothelial dysfunction. Experiments were conducted in isolated canine circumflex coronary arteries with and without perivascular adipose tissue. Adipose tissue significantly decreased coronary endothelial‐dependent vasodilation to bradykinin (1 – 30 nM) ∼ 30% and increased EC50 2.4‐fold (n = 4). This effect of adipose tissue was not observed in arteries pretreated with the general protein kinase C (PKC) inhibitor Ro 31‐8220 (1 μM, n = 7) or the PKC‐β specific inhibitor ruboxistaurin (1 μM, n = 6). Western blot analysis revealed phosphorylation of endothelial nitric oxide synthase (eNOS) at threonine‐495 in arteries with adipose tissue that was reversed by inhibition of PKC‐β with ruboxistaurin and was not present in arteries without adipose tissue (n = 4). Taken together these data indicate that perivascular adipose tissue impairs coronary endothelial nitric oxide production via a PKC‐β dependent, site‐specific phosphorylation of eNOS at threonine‐495. (Support: HL67804)
Density of the oxidized low‐density lipoprotein (OX‐LDL) receptor, LOX1, correlates with OX‐LDL reactivity of various arterial muscles
Authors: Odya Erin C. , Dominguez Maria H. , Dominguez Jesus H. , Rice Ami E. , Temm Constance J. , Packer C. Subah
Published: June 2021

The causal relationship of the hypertension associated with metabolic syndrome and diabetes mellitus (NIDDM) is unknown. Elevated OX‐LDL, characteristic of NIDDM causes dose‐dependent arterial muscle contraction but the signaling pathway is not understood. The OX‐LDL receptor (LOX1) has been identified on arterial smooth muscle but its role is unknown. Interestingly, the caudal artery, a muscular systemic vessel, is more reactive to OX‐LDL than is either the conduit aorta or the low pressure pulmonary artery in the rat. The purpose of the current study was to quantify the relative LOX1 density on the three arterial muscles using Metamorph software (Universal Imaging) analyses of immunofluorescence and confocal microscopy images of sections treated with a polyclonal antibody raised in rabbits against recombinant LOX1. Although quantitative analysis did not yield statistically significant differences, qualitative data suggests that LOX1 density on the three arterial muscles correlates with OX‐LDL reactivity. Larger sample sizes are needed for quantitative confirmation that vascular smooth muscle LOX1 density correlates with OX‐LDL reactivity supporting a role for LOX1 activation in mediating OX‐LDL induced vasoconstriction.

(Supported, in part, by a VA Merit Award, PI: JH Dominguez. EC Odya is a 2007‐08 APS Frontiers in Physiology Professional Development Fellow)
Differential expression of BK‐channel isoforms and beta‐subunits in rat neuro‐vascular tissues
Authors: Poulsen Asser Nyander , Wulf Helle , Hay‐Schmidt Anders , Jansen‐Olesen Inger , Olesen Jes , Klaerke Dan A.
Published: June 2021

The Ca2+‐activated K+ channel (BK, MaxiK, Slo, KCa1.1) is involved in regulation of smooth muscle tone and shaping of action‐potentials. It appears in several functional forms due to alternative splicing and the presence of modulatory β‐subunits. The expression of these has been suggested to be tissue‐specific, yet the relative abundance in tissues including trigeminal nerve and cerebral blood‐vessels has not been thoroughly determined. By RT‐PCR we investigated the expression of the BK channel splice‐variants and β‐subunits in rat cerebral blood‐vessels, larger arteries, heart, the trigeminal ganglion, brain, inner organs and skeletal muscle. A 58 or 29 aa insert (STREX and e22) was found at splice‐sites two in most vascular tissues and in skeletal muscle respectively, while a 27 aa insert (Slo27) at splice‐site four was found predominantly in brain and trigeminal ganglion. A surprising 92 bp insert was found in Sagital Sinus at a splice‐site (X2), inducing a shift in reading‐frame and truncation of the protein (stop codon). β1 and β4 were found in all tissues except skeletal muscle, and β2 mostly in brain, trigeminal ganglion and cerebral vessels. In conclusion, the Slo27 insert, β2 and β4 were more abundant in nerve than in vascular tissues (cerebral vessels), while the STREX insert and β1 were less abundant in nerve tissue (trigeminal ganglion), suggesting differential expression of functional BK channel forms.
Role of VDAC in vascular responses to isoflurane.
Authors: Weihrauch Dorothee , Sepac Ana , Warltier David C. , Bosnjak Zeljko J. , Brzezinska Anna K.
Published: June 2021

Permeability transition pore (mPTP) is a protein complex located within the mitochondrial membranes; the voltage dependent anion channel (VDAC) is a component of mPTP. Volatile anesthetics such as isoflurane have been shown to play the critical role in survival of cardiac cells. We hypothesized that in human coronary artery endothelial cells (HCAECs) isoflurane increases cell survival via direct interaction with VDAC protein. The presence of VDAC protein was determined by western blot analysis and immunocytochemistry. The exposure of HCAECs to isoflurane (1MAC) initiated a translocation of VDAC from mitochondria to the plasma membrane (n=2). The translocation of VDAC was NO‐independent. The structure of VDAC protein comprises a chloride channel. Using MAQE a chloride flux indicator, we determined that the isoflurane‐induced translocation of VDAC caused increase in chloride flux across the plasma membrane. In isoflurane‐treated cells chloride influx was increased by 21% (n=3, P<0.05, 25 000 cells per well). In control and isoflurane‐treated cells, a chloride channel inhibitor, DIDS (100 μM) caused the complete inhibition of Cl− influx (n=2). Next, isoflurane treated cells were exposed to 1 hr of 0% hypoxia, 24 hr of reperfusion and 2nd cycle of hypoxia (1hr). Isoflurane treated cells survived significantly better the second hypoxic insult. The rate of ATP production was 36% higher (n=3, P<0.05, 25 000 cells per well) in isoflurane treated cells as compared to controls. Taken together, the ability of volatile anesthetic to initiate a translocation of mitochondrial protein (VDAC) combined with improved survival of vascular cells could be an important mechanism likely to abolish vascular damage.
Sirtuin, an oxidant sensitive deacetylase, is post‐translationally modified and degraded by the proteasome in response to cigarette smoke in lung epithelial cells
Authors: Caito Samuel William , Cook Suzanne , Yang Se‐Ran , Rajendrasozhan Saravanan , Rahman Irfan
Published: June 2021

Sirtuin1 (Sirt1) is a class III histone/protein deacetylase that has anti‐aging and anti‐inflammatory properties. We have previously shown that cigarette smoke (CS) decreased Sirt1 protein level in rat lung and human monocyte/macrophages leading to increased NF‐κB dependent pro‐inflammatory cytokine release. As CS contains greater than 1015–17 free radicals, we hypothesized that the cell's redox status influences Sirt1 protein level in response to CS, leading to post‐translational modifications and degradation. Human bronchial epithelial cells (BEAS.2B) were exposed for 24 hrs to different concentrations of cigarette smoke extract (CSE 0.5–1%) in the presence or absence of buthionine sulfoximine (BSO) or N‐acetyl‐L‐cysteine (NAC), an inhibitor and precursor for glutathione biosynthesis respectively. CSE decreased nuclear Sirt1 levels; co‐treatment with BSO further decreased Sirt1, whereas treatment with NAC rescued CSE depletion. Furthermore, CSE caused 4‐hydroxy‐2‐nonenol adduct formation on Sirt1, which was increased by BSO treatment and decreased by NAC. Decreased Sirt1 level was also found to be due to proteasomal degradation as proteasomal inhibitors (ALLN and MG132) attenuated CSE‐mediated degradation of Sirt1. These data suggest that oxidants in CSE modify Sirt1 post‐translationally, marking the protein for proteasomal degradation in lung epithelial cells, which may contribute to the pro‐inflammatory effect of cigarette smoke.

Supported by the NIEHS Center Grant ES01247 and Toxicology Training Program Grant ES07026.