bluuebunny/crossref_metadata_2025 huggingface dataset:
Antiobesity and lipid profile improvement with Inonotus Obliquus extract supplementation in C57BL/6J mice with high fat diet induced obesity
Authors: Soh Ju‐Ryoun , Moon Yeon‐Jeong , Jeong Yong‐Seob , Cha Youn‐Soo
Published: June 2021

Inonotus Obliquus extracts(fruiting body, mycelium) were evaluated for protective effects against the adverse effects of a chronic high fat diet. High fat diet‐induced obese C57BL/6J mice were divided into six groups; normal diet control group (ND), high fat diet control group (HD), HD+fruiting body 80°C hot water extract supplementation group(F80), HD+fruiting body 150°C hot water extract supplementation group(F150), HD+mycelium 80°C hot water extract supplementation group(M80) and HD+mycelium 150°C hot water extract supplementation group(M150) and fed for 12 weeks. Final body weight, epididymal fat % and serum total cholesterol concentrations were significantly higher in the HD than ND, but were lower when the high fat diet was combined with fruiting body extract or mycelium extract. Blood glucose levels after fasting for 4hr in F80, F150 and M150 were significantly lower than those of the HD. Inonotus Obliquus extract supplements altered levels of ACS, CPT‐1, UCP‐1 and ACC mRNA expression in liver and fat tissue. These results suggest that Inonotus Obliquus extract supplementation has beneficial effects in preventing diet‐induced obesity and related metabolic abnormalities. (This study was supported by Technology Development Program for Agriculture and Forestry, Ministry of Agriculture and Forestry, Republic of Korea)
Intraparenchymal supratentorial anaplastic ependymoma with massive intratumoral hemorrhage: a case report
Authors: Rishi Kirtee , Cherqui Alice , Zhang Yi , Takei Hidehiro , Powell Suzanne Z
Published: June 2021

Supratentorial intraparenchymal ependymomas with no attachment to the ventricular system are rare and generally do not present with hemorrhage which may be the presenting finding in oligodendrogliomas. We report an unusual case of a 60 year old female with a large frontoparietal supratentorial anaplastic ependymoma with massive hemorrhage. The patient presented to our institution with progressive right sided weakness after having been treated elsewhere for an intraparenchymal hemorrhage several weeks prior. Computed tomography, magnetic resonance imaging and cerebral angiography on presentation revealed a 7 cm partly calcified right frontoparietal lesion with acute hemorrhage and significant mass effect. The patient underwent a craniotomy for hematoma evacuation and resection of possible neoplasm. The frozen and permanent sections revealed tumor forming pseudorosettes with mitoses and focal areas of necrosis. The MIB‐1 labeling index was 14% and a diagnosis of anaplastic ependymoma was rendered. The tumor cells stained positively with GFAP and showed dot like immunoreactivity with epithelial membrane antigen (EMA). Ependymomas usually arise from the cells lining the ventricles or central canal of the spinal cord. Anaplastic ependymoma should be included in the differential diagnosis of parenchymal hemorrhagic masses and may confer a higher risk for intratumoral hemorrhage.
Primary central nervous system lymphoma: a review of 21 cases
Authors: Silva Aline Helen da , Pullarkat Sheeja T. , Yong Willian H. , Vinters Harry V.
Published: June 2021

We retrospectively analyzed 21 cases of primary central nervous system lymphoma (PCNSL) diagnosed during 2002–2007. Immunocompromised and HIV positive patients were excluded from the study. The study included 10 men and 11 women with a median age of 55 years. Of the total 21 cases included 20 biopsy cases and 1 autopsy case. Most of the patients (43%) showed an ECOG (Eastern Cooperative Oncology Group) Performance Status 2 at diagnosis. Radiologically, a total of 34 contrast‐enhancing lesions were observed among affected patients. Tumor sites most frequently included frontal lobe, cerebellum, basal ganglia and thalamus. In the pathologic analysis, 18/21 (85%) were diffuse large B‐cell lymphoma (DLBCL) and 3/21 (15%) were T‐cell lymphoma. The median Ki67 labeling index in DLBCL cases was 75% and in T‐cell Lymphoma cases was 20%. Treatment included surgical resection (3 patients), chemotherapy (11 patients) and/or radiotherapy (6 patients). 11 patients received at least one course of high‐dose methotrexate. Two patients received combination chemotherapy and radiotherapy. At the time of last contact 5/12 patients for whom follow‐up was available (42%) had died, all from PCNSL or from complications due to treatment. The median survival was 20 months. Our data are similar to those previously described except for the equal representation of males and females, most reports have shown a male preponderance.
Quantitative analyses of drug combinations using a benzodiazepine discrimination procedure in rhesus monkeys
Authors: Gerak Lisa R , France Charles P
Published: June 2021

Adverse effects of benzodiazepines limit their clinical use; these effects might be reduced without altering therapeutic effects by administering other positive GABAA modulators (i.e., neuroactive steroids) concurrently with benzodiazepines. To begin to understand combined effects of positive modulators, the ability of flumazenil to attenuate effects of 2 benzodiazepines, midazolam and flunitrazepam, administered alone or in combination, was studied in 4 monkeys discriminating midazolam. Although receptor theory predicts that concurrent administration of benzodiazepines will not alter interactions with their receptors, this notion has not been rigorously tested. Midazolam, flunitrazepam or a combination of the 2 drugs produced >80% midazolam‐lever responding, and flumazenil dose‐dependently antagonized each effect. Schild analyses yielded slopes that did not deviate from unity, regardless of whether benzodiazepines were administered alone or concurrently, indicating simple, competitive interactions; the pA2 value for flumazenil was 7.58. Thus, these data conform to theory in a quantifiable manner, allowing other notions regarding drug/drug interactions to be tested. Combinations of drugs acting at different sites, such as benzodiazepines and neuroactive steroids, could be studied to develop strategies for reducing adverse effects. Supported by USPHS grant DA09157 and DA17918 (CPF).
Inhibition of Angiotensin Converting Enzyme (ACE) by Captopril Protects in Animal Models of Parkinson's Disease
Authors: Sonsalla Patricia K. , Coleman Christal G.
Published: June 2021

The brain possesses a complete renin angiotensin system which, like that in the periphery, contributes to the regulation of blood pressure. Additionally, angiotensin has been implicated in contributing to neurodegeneration as blockade of ACE or of angiotensin receptors provides protection in ischemia models. Many of the factors contributing to neurodegeneration in ischemia or stroke may also contribute to degeneration of dopamine (DA) neurons in Parkinson's disease (PD). Thus, we hypothesized that blockade of the angiotensin system would protect against DA neurodegeneration. The ability of captopril to protect against DA neurodegeneration was tested in the acute MPTP mouse model as well as the chronic MPP+ rat model. Concurrent treatment of mice with captopril and MPTP attenuated the neurotoxicant‐induced decreases in DA and tyrosine hydroxylase content in the neostriatum. Likewise, concurrent daily treatment of rats with captopril attenuated the decrease in striatal DA caused by the infusion of MPP+ into the left cerebral ventricle for 28 days. These findings demonstrate protection by captopril in two different PD models and indicate that ACE and angiotensin exert a significant role in neurodegeneration in these models. Thus, blockade of brain angiotensin actions may be a viable pharmacological strategy to reduce neuronal degeneration in PD.
Death and decoy receptors in neuronal preconditioning
Authors: Panneerselvam Mathivadhani , Patel Hemal H , Head Brian P , Chin‐Lee Blake , Patel Piyush M , Davis Daniel P
Published: June 2021

Ischemic preconditioning (IPC) is a phenomenon where brief periods of ischemia confer protection against subsequent severe ischemic injury. The tumor necrosis factor (TNF) family of cytokines and receptors are involved in diverse signaling pathways regulating cell death and survival. Decoy receptors are a unique subset of these TNF receptors that can bind death ligands but do not contain cytoplasmic domains necessary to transduce signals. Therefore, decoy receptors serve to effectively compete for death ligands to promote cell survival. We hypothesized that decoy receptors may be upregulated after IPC to confer neuronal protection. Bilateral carotid occlusion in rats in vivo or oxygen‐glucose deprivation (OGD) in mixed cortical neurons from neonatal rats in vitro was used to induce neuronal death. IPC was induced by sublethal ischemia/OGD exposure (3 min in vivo or 20 min in vitro) one day prior to lethal ischemia/OGD (6 min in vivo or 1 hr in vitro). In the intact rat an increase in decoy molecule expression was observed in IPC versus controls in both CA1/3 and cortex. IPC induced increase in decoy expression was reduced by prior treatment with N‐methyl‐D‐aspartate receptor or nuclear factor‐kappa B inhibitors. Cell death and receptor expression was assessed 24–48 hrs after lethal ischemia in vitro. IPC resulted in reduced neuronal death when compared to controls. IPC followed by lethal ischemia resulted in altered regulation of decoy receptors and death receptors when compared to controls not undergoing IPC. Our results suggest that expression of decoy molecules and suppressed expression of death receptors may mediate neuronal survival induced by IPC.
The determination of the medical and annual needs of ephedrine, pseudoephedrine and phenylpropanolamine as required by the enactment of the Combat Methamphetamine Epidemic Act of 2005 (CMEA)
Authors: Walker Michelle D , Strait Matthew J. , Carr Susan M , Sannerud Christine A
Published: June 2021

The CMEA was enacted on March 9, 2006 and it amended the Controlled Substances Act (CSA) by adding provisions to the regulatory control of ephedrine (EPH), pseudoephedrine (PSE), and phenylpropanolamine (PPA). EPH, PSE, and PPA are List I chemicals which can be used as precursor chemicals in the illicit manufacture of certain controlled substances, e.g. methamphetamine and amphetamine. CMEA required DEA to establish quotas for these List I chemicals and to prevent the use of these chemicals in the clandestine manufacture of methamphetamine. DEA obtained assistance from a private independent contractor, IMS Health Government Solutions (IMS), to develop the proposed initial estimate of the medical needs of EPH and PSE for the U.S. For this analysis, IMS utilized the following types of data: (1) sales to retail establishments, (2) sales to patients, and (3) medical insurance claims. A weighted average of the three individual estimates was used to derive the final estimates of medical need. The weighted average was determined based on IMS’ confidence in each individual estimate. The medical needs of EPH and PSE were determined to be 5,998 kg and 350,700 kg, respectively. After DEA considered the medical needs of the EPH and PSE, and PPA (veterinary products) the assessment of annual needs was determined using other required factors. The methodology used to determine these estimates will be discussed.
Botanical Drug Development: Waiver of Preclinical Pharm/Tox Studies and Importance of Initial Cinical Trial Design
Authors: Wu Kuei‐Meng , Ghantous Hanan , Birnkrant Debra B.
Published: June 2021

Development of botanical drug products in the U.S. may be entitled for a waiver of preclinical Pharm/Tox (P/T) studies prior to an initial clinical trial, contingent upon existing previous human experience. The regulatory perspective for this measure is to allow the sponsor to conduct a well‐designed Phase I/II trial to detect initial signals of efficacy without prior animal studies. Based on botanical INDs submitted to the FDA, the “initial” trials proposed have some of the following design characteristics: (1) the treatment duration for certain indications often lasted longer than a typical Phase I/II trial (e.g., ≥6 months for hepatitis and HIV); (2) the dose proposed sometimes far exceeded that recommended historically or that used under dietary supplement, without supporting P/T studies; (3) the sample size was limited and dosage often arbitrarily selected without a supporting dose ranging trial. These issues on the initial trial design, particularly in regard to the sample size, duration and dosage, may compromise the study, resulting in outcomes that may inadequately address the original question as to whether the botanical is efficacious, and mislead conclusions on the botanical's pharmacological effects in patients. These variables and factors could be part of the reasons why most of the botanical INDs submitted to the FDA become inactive, and do not progress to the advanced phase of drug development.
Functional domains of the mouse β3‐adrenoceptor associated with differential G‐protein coupling
Authors: Sato Masaaki , Hutchinson Dana S , Evans Bronwyn A , Summers Roger J
Published: June 2021

Alternative splicing of the mouse β3‐adrenoceptor (AR) produces two isoforms of the receptor: the β3a‐AR couples to Gs and the β3b‐AR couples to both Gs and Gi. A truncated receptor that lacks the C‐terminus of the β3a‐ or the β3b‐AR showed pertussis toxin (PTX) sensitivity in a cyclic AMP (cAMP) accumulation assay, suggesting that the β3a‐AR is restrained from coupling to Gi by residues in the C‐terminus. This view was supported by studies using a cell‐penetrating peptide transportan‐10 (Tp10) coupled to the C‐terminal tail of the β3a‐ARτhat caused the β3a‐AR to become PTX sensitive.

Site‐directed mutagenesis was used to identify residues in the C‐terminus of the β3a‐AR responsible for inhibition of coupling to Gi. Mutation of a putative caveolin binding site caused β3a‐AR mediated cAMP accumulation to become PTX sensitive (control, 1.00±0.16; PTX, 1.75±0.14, n=4, P<0.0001). Furthermore, treatment with filipin III, an agent that disrupts lipid rafts, also caused the wild type β3a‐AR to become PTX sensitive (control, 1.00±0.19; PTX, 1.56±0.04, n=4, P<0.0001), but in addition concentration‐response curves to CL316243 were markedly shifted to the right (pEC50 control: 9.76±0.03, +filipin III: 8.73±0.21). These observations suggest that β3a‐ARs are restricted to caveolae and that localization of the receptor may play a specific role in G‐protein mediated signalling.

This research was supported by National Health and Medical Research Council Project Grant 236884.
Investigation of the inverse agonism effect of nadolol on the Gs signaling pathway
Authors: Peng Hui , Bond Richard A. , Knoll Brian
Published: June 2021

Nadolol is a nonselective beta‐adrenergic receptor (¦ÂAR) blocker with inverse agonist activity at ¦Â2‐ARs. Previous studies in human embryonic kidney 293 cells over‐expressing human ¦Â2AR showed an initial increase in the number of inactive state of receptors (R) after acute nadolol treatment, and an increase in the number of active state of receptors (R*) with decreased receptor degradation after chronic treatment. In this study, further experiments indicated that chronic treatment with isoproterenol significantly decreased cellular level of Gs while chronic nadolol treatment had no effect. In organ bath experiments using the tracheal rings from an asthmatic mouse model we investigated the effects of nadolol on other Gs‐coupled receptors. Nadolol treatment produced an enhanced relaxation response mediated by the prostacyclin receptor (IP) agonist cicaprost and prostaglandin E2 receptor (EP2) agonist CAY 10399, in addition to the ¦ÂAR agonist isoproterenol. In isolated airway smooth muscle cells, western blot data suggested chronic treatment with nadolol increased the levels of IP and EP2 receptors. These data suggest that nadolol enhances Gs signaling through the ¦Â2‐AR, IP and EP2 receptors and this enhancement of bronchorelaxant responses may contribute to the beneficial effects of chronic nadolol treatment in murine asthma model.
Use of 4‐[(2‐bromoethyl)methyl‐amino]‐2‐butynyl N‐(3‐chlorophenyl)carbamate (BR 384), to study allosteric interactions at M2 muscarinic receptor
Authors: Suga Hinako , Ehlert Frederick James
Published: June 2021

We explored the interaction of a nitrogen mustard derivative of McN‐A‐343 (BR 384) with the human M2 muscarinic receptor expressed in CHO cells using the muscarinic radioligand, [3H]N‐methylscopolamine ([3H]NMS). BR 384 caused a concentration‐dependent, first order loss of [3H]NMS binding at 37°C, with the half maximal rate constant occurring at 8.6 μM BR 384. We examined the effects of various ligands on the rate of alkylation of M2 receptors by BR 384. NMS competitively slowed the rate of alkylation, whereas the inhibition by gallamine and McN‐A‐343 reached a plateau at high concentrations, indicating an allosteric inhibition. In contrast, WIN51708 had no effect. We also measured the inhibition of [3H]NMS binding by BR 384 at 0°C, conditions under which covalent binding is reduced. In these experiments, the dissociation constants of NMS, gallamine, McN‐A‐343 and the aziridinium ion of BR 384 for their binding sites were 0.0001, 0.64, 18 and 3.7 μM, respectively. In contrast, the parent mustard and alcoholic hydrolysis product of BR 384 were without effect. Our results show that the aziridinium ion of BR 384 binds covalently to the orthosteric site of the M2 receptor, whereas McN‐A‐343 binds to an allosteric site. Measurement of the effects of ligands on the rate of inactivation of muscarinic receptors by a covalent orthosteric ligand is a powerful method for detecting allosterism.

Supported by a NIH Grant GM 69829.
A single conserved leucine residue in the first intracellular loops is required for endoplasmic reticulum export of G protein‐coupled receptors
Authors: Duvernay Matthew T , Wu Guangyu
Published: June 2021

The intrinsic structural determinants for export trafficking of G protein‐coupled receptors (GPCRs) have been mainly identified in the termini of the receptors. In this report, we determined the role of the first intracellular loop (ICL1) in the transport from the endoplasmic reticulum (ER) to the cell surface of GPCRs. The α2B‐AR mutant lacking the ICL1 is unable to traffic to the cell surface. Mutagenesis studies identify a single Leu48 residue in the ICL1 absolutely essential for the export of α2B‐AR from the ER. The export function of Leu48 can be substituted by Phe, but not Ile, Val, Tyr and Trp, and is unlikely involved in correct folding or dimerization of α2B‐AR in the ER. Importantly, the isolated Leu residue is remarkably conserved in the center of the ICL1 amongst the family A GPCRs and is also required for ER export of β2‐AR, α1B‐AR and angiotensin II type 1 receptor. These data indicate an essential role of a single Leu residue within the ICL1 in ER export of GPCRs and provide strong evidences for the functional role of the intracellular loops in GPCR export trafficking (R01GM076167).
Characterization of receptor internalization and inhibition of cAMP in cell lines expressing amphibian or human mu opioid receptors
Authors: Stevens Craig W , Brasel Christopher M , Sawyer Gregory W
Published: June 2021

A fundamental goal in pharmacology is to correlate the primary amino acid sequence of receptors with observed ligand binding and function. After cloning the mu opioid receptor expressed in the frog, Rana pipiens, (rpMOR), we found that selective and non‐selective opioid receptor antagonists and morphine had a greater affinity for rpMOR compared to hMOR in transiently‐transfected CHO cells. In contrast, the MOR‐selective agonist DAMGO had an 11‐fold greater affinity for hMOR. Collectively, these data supported the hypothesis that opioid receptors are less divergent, and therefore less selective, in non‐mammalian vertebrates. Here we present a comparison of internalization and signaling in intact CHO cells stably expressing either rpMOR or hMOR. Using saturating concentrations of morphine and DAMGO, we found that the half‐life for MOR internalization was faster for morphine in rpMOR‐CHO compared to hMOR‐CHO cells. However, the half‐life of internalization after DAMGO incubation was faster in hMOR‐CHO compared to rpMOR‐CHO cells. Morphine produced less maximum internalization than DAMGO in both cell lines. Functional studies using inhibition of forskolin‐mediated cAMP increase showed that morphine and DAMGO were less efficacious in rpMOR‐CHO compared to hMOR‐CHO cells as shown both by maximum inhibition and IC50 values. These data suggest that the minor sequence differences in particular domains of MOR proteins expressed in amphibians and humans mediate the differences observed in ligand binding and function. Supported by a National Institutes of Health grant, DA0124482, awarded to CWS.
Inhibition of cyclic AMP production is not reduced in HEK293 cells expressing the alpha‐2C adrenergic receptor Del322–325 polymorphism
Authors: Montgomery Megan D. , Bylund David B.
Published: June 2021

The human alpha‐2C adrenergic receptor inhibits norepinephrine release in the sympathetic nervous system. A four residue deletion in the third intracellular loop of this receptor, Del322–325, has been reported to exhibit reduced signaling in vitro (Small, et al., 2000) and be a potential risk factor for heart failure, presumably due to excess norepinephrine release and action. We attempted to replicate the reduced signaling, with the hypothesis that decreased signaling is due to decreased coupling of Del322–325 to Gi proteins. Our objective was to compare cyclic AMP inhibition of alpha‐2C wild type and Del322–325 receptors stably expressed in HEK293 cells. Cyclic AMP production was stimulated by 30 μM forskolin, and alpha‐2 agonists were used to inhibit this production in clones with high or low receptor expression levels. WT and Del322–325 clones with high expression levels (Bmax = 1500 and 2300 fmol/mg) did not differ in their percent inhibition (94 and 98%) or in their potencies (0.5 and 0.4 nM) for UK‐14,304. Clonidine also showed no difference between WT and Del322–325 clones with high Bmax (93 and 97%, 2.2 and 1.7 nM EC50s). Similar results were obtained in clones with low Bmax (20 and 20 fmol/mg). Therefore, in HEK293 cells, Del322–325 does not have a reduced ability to inhibit cyclic AMP production in comparison to WT.

Supported by a donation from Procter and Gamble.
Molecular changes in the cytochrome P450 4A isoforms in the Sprague Dawley rat kidney following ischemia/reperfusion
Authors: Akbulut Talha , Steinman Averia , Roman Richard J. , Park Frank
Published: June 2021

20‐HETE is a major eicosanoid in the kidney, but little is known about the molecular regulation of the enzymes that produce this molecule in the kidney following ischemia‐reperfusion (I/R) injury. We designed the following study to determine the changes in the cytochrome P450 4A isoforms, specifically CYP4A1, 4A2, 4A3, and 4A8, following I/R in uninephrectomized Sprague Dawley (SD) rats. SD rats were sham‐operated or exposed to ischemia by clamping of renal artery and vein for 45 minutes, and then reperfused for 1, 3 or 7 days. Medullary CYP4A protein levels determined by Western blot analysis started to decrease 1 day after reperfusion and reduction levels reached to ~10% (P<0.01; n=5) of the control levels at day 3 post‐I/R. No changes in the cortical protein levels were detected. To determine the mRNA changes in the medulla, quantitative RT‐PCR was performed. CYP4A2, CYP4A3 and CYP4A8 mRNA levels decreased significantly following I/R, and reached their nadir at day 3 post‐I/R between 74–93% (P<0.05). These findings would suggest that CYP4A isoforms are differentially regulated in the kidney following I/R and may have a possible role in mediating biological effects in response to this type of injury.
Induction of heme oxygenase‐1 attenuates cerebral ischemia/reperfusion injury: role of heme
Authors: Volti Giovanni Li , La Delia Francesco , Zappalà Agata , Serapide Maria Francesca , Puglisi Giovanni , Cicirata Valentina , Cicirata Federico
Published: June 2021

We evaluated heme metabolism in different brain regions of transiently ischemized rats (TI). Rats underwent TI for 20 min and following 72h they were sacrificed, brains were dissected and analyzed for heme oxygenase‐1 (HO‐1) distribution. Heme content was determined by HPLC. We firstly assessed the effects of SnCl2, an inducer of HO‐1. ELISA showed an increase of HO‐1 protein expression which was followed by increased activity in selected brain regions. We also showed that HO‐1 was induced in neuronal cells of the hippocampus (HP), cerebellum, and brainstem. Increased expression of HO‐1 was also associated to a reduction of heme levels. We evaluated the expression of iNOS, a heme dependent protein. TI strongly increased iNOS expression overall the brain. Pre‐treatment with SnCl2 decreased both iNOS expression and neuronal cell loss. The effects of SnCl2 were abolished by SnMP, an inhibitor of HO activity. The results showed that SnCl2 induces HO‐1 in neuronal cells of selected brain areas, modulates heme metabolism and iNOS expression in TI rats.
Delayed preconditioning with NS1619 protects cultured cortical neurons against both apoptotic and necrotic cell deaths
Authors: Gaspar Tamas , Katakam Prasad VG , Domoki Ferenc , Snipes James A , Kis Bela , Bari Ferenc , Busija David W
Published: June 2021

NS1619, a potent activator of the large conductance Ca2+ activated potassium (BKCa) channel, has been demonstrated to induce preconditioning in the heart. The aim of our study was to test the delayed preconditioning effect of NS1619 in rat cortical neuronal cultures against oxygen‐glucose deprivation, H2O2, or glutamate excitotoxicity. We also investigated its actions on reactive oxygen species (ROS) generation, and on mitochondrial and plasma membrane potentials. In addition, we tested the activation of the phosphoinositide 3‐kinase (PI3K) signaling pathway, and the effect of NS1619 on caspase‐3/7. NS1619 dose‐dependently protected the cells against the toxic insults, and the protection was blocked by a superoxide dismutase mimetic and a PI3K antagonist, but not by BKCa channel inhibitors. Furthermore, NS1619 increased ROS generation, depolarized isolated mitochondria, hyperpolarized the neuronal cell membrane, and activated the PI3K signaling cascade. Finally, NS1619 inhibited the activation of capase‐3/7. In summary, NS1619 is a potent inducer of delayed neuronal preconditioning. However, the neuroprotective effect seems to be independent of cell membrane and mitochondrial BKCa channels. Rather it is the consequence of ROS generation, activation of the PI3K pathway, and inhibition of caspase activation. Supported by the NIH Grants HL‐030260, HL‐065380, and HL‐077731.
Lack of glutamate excitotoxicity in primary rat and piglet cerebromicrovascular endothelial cell cultures
Authors: Domoki Ferenc , Kis Béla , Gáspár Tamás , Bari Ferenc , Busija David W
Published: June 2021

Cerebromicrovascular endothelial cells (CMVECs) have recently been claimed to be vulnerable to excitotoxic levels of L‐glutamate (L‐glut) normally present in the blood plasma, and be involved in L‐glut‐induced vasodilation. Our purpose was to clarify the effects of L‐glut and N‐methyl‐D‐aspartate (NMDA) on CMVECs of two species. Primary CMVEC cultures were obtained from 2 week old Wistar rat pups and 1 day old piglets as described (Am J Physiol 288:R897–902, 2005). Experiments were done on confluent CMVECs within 4–5 days after isolation. Both in rat and piglet CMVCs, L‐glut and NMDA (1‐1mM) 1) did not elevate intracellular Ca2+ levels unlike calcimycin using Fluo‐4 fluorescence; 2) caused no changes in the characteristic cobblestone appearance of cultures; 3) did not affect transendothelial electrical resistance; 4) did not decrease cell viability; and 5) did not augment cell death induced by 8h medium withdrawal or 12h oxygen‐glucose deprivation. In conclusion, CMVECs in vitro are resistant to glutamatergic stimuli and maintain prominent blood brain barrier features even at doses exceeding the physiological range of L‐glut. Our findings are consistent with previous studies in piglets showing little or no effect of NMDA or L‐glut on pial arterioles in vitro (Microvasc Res 70:76–83, 2005) or damage in vivo (Am J Physiol 257:H1200‐3, 1989). Supported by OTKA K68976, K63401 and by NIH HL30260, HL65380, HL77731.
Mechanism of homocysteine‐induced dementia/spasm
Authors: Kumar Munish , Tyagi Neetu , Pushpakumar SB , Moshal Karni S , Sen Utpal , Lominadze David , Tyagi Suresh C
Published: June 2021

Although individuals with homozygous deficiency in cystathionine beta synthase (CBS), known as hyperhomocysteinemia, develop mental retardation and die in teens, the heterozygous live a normal life but develop vascular dementia (VA/spasm) and Alzheimer's disease (AD). The treatment with muscimol, a gamma amino butyric acid receptor‐A (GABA‐A) agonist, mitigates the AD syndrome. We tested the hypothesis that homocysteine (Hcy) antagonizes the GABA‐A receptor and behaves as an exitotoxic neurotransmitter and causes blood brain barrier (BBB) permeability and VA. The BBB permeability was measured by infusing evan's blue dye through tail vein. After three hours, excess dye was removed from blood circulation. The brain homogenates were prepared from GABA‐A (−/ −), CBS (−/+), CBS/GABA‐A double knockouts, and wild type (WT) mice in 50% trichloroacetic acid and the amount of evan's blue was measured on spectophotometer. To determine vascular dementia/spasm, carotid artery blood flow was measured by an ultrasonic flow meter. The level of Evans blue significantly increases in CBS/GABA‐A double knockouts > CBS(−/+) > GABA‐A (−/ −) > compared to WT. The carotid artery flows were 0.42 and 0.55 ml/min in GABA‐A (−/ −) and CBS/GABA‐A double knockouts, where as in WT it was 0.73 (ml/min). These results suggest that Hcy masks the GABA‐A receptor and decrease vascular flow causing (VA/spasm). In conclusion results are in coordination with increase in BBB permeability in cerebroventricle interstitium.
Vasomotor sympathetic neural responses during upright tilt in early human pregnancy
Authors: Fu Qi , Shibata Shigeki , VanGundy Tiffany B , Creson Daniel L , Levine Benjamin D
Published: June 2021

Pregnancy‐induced hypertension (PIH) and preeclampsia (PE) is proposed to be associated with a hyperadrenergic state. However, the state of sympathetic neural control in normal pregnancy, especially in early pregnancy, is unknown. We tested the hypothesis that sympathetic neural responses during orthostasis are enhanced in early pregnancy in humans. Six healthy young early pregnant (4–7 wk of gestation) women and 12 age, race and BMI‐matched non‐pregnant women (controls) were studied. Muscle sympathetic nerve activity (MSNA), blood pressure (BP), heart rate (HR), and respiration (nasal cannula) were recorded continuously supine and during a graded upright tilt (30° and 60° for 8 min each). We found that MSNA was much greater in early‐pregnant women than in controls supine (33±7 vs 14±9 bursts/min, P<0.001) and during the graded tilt (44±6 and 56±5 vs 25±13 and 35±10 bursts/min, P<0.001 for both group and protocol). However, changes of MSNA (ΔMSNA) from supine to 30° (11±8 vs 12±5 bursts/min, P=0.746) and 60° tilt (23±10 vs 22±6 bursts/min, P=0.640) were similar between early pregnant women and controls (P=0.690 for interaction). BP responses did not differ between the groups, while increases in HR at 60° tilt tended to be greater in early‐pregnant women than in controls (ΔHR, 23±13 vs 16±8 beats/min, P=0.087). These results suggest that vasomotor sympathetic activity increases markedly, but sympathetic neural responses during acute orthostasis remain unchanged in early human pregnancy. It seems likely that sympathetic hyperactivity is a universal characteristic in normal pregnancy and marked sympathetic activation alone may not be the cause for PIH or PE.
Role of the Median Preoptic Nucleus in Arterial Pressure Regulation and Sodium and Water Homeostasis during Chronic Changes in Dietary Salt.
Authors: Ployngam Trasida , Collister John P
Published: June 2021

Proper adjustment of renin‐angiotensin system activity is believed to be important to maintain normal blood pressure during high salt intake. The median preoptic nucleus (MnPO) receives afferent inputs from the subfornical organ and organum vasculosum of lamina terminalis, the circumventricular organs that have been shown to be necessary in central effects of angiotensin II involving sodium and water homeostasis. In this study, we hypothesized that an intact MnPO is necessary for the long‐term control of arterial pressure during chronic changes in dietary salt. To test this hypothesis, male Sprague Dawley rats were subjected to either sham (SHAM) or electrolytic lesion of the entire MnPO (MnPOx). After a 1‐wk recovery period, rats were instrumented with radiotelemetric transducers, and aortic flow probes, for the 24 h‐measurement of mean arterial pressure (MAP) and heart rate, and cardiac output, respectively. After another 10 days of recovery period, the protocol was started with a 7‐day control period (1% NaCl diet) followed by a 14‐day high salt period (4% NaCl diet), and 7 day recovery period (1% NaCl diet). Daily water and sodium intake, output, and balance were recorded throughout the protocol. By day 10 of the high salt period, MAP in SHAM (n=3) and MnPOx (n=3) lesion rats had increased from baseline control level (8.5 ± 2.0 and 7.4 ± 1.2, respectively). The level of MAP during high salt intake was not significantly different between the two groups. These results do not support the hypothesis that the MnPO is necessary for normal arterial pressure regulation during chronic changes in dietary salt.
Selective blockade of 5‐HT2A receptors suppresses hyperthermic but not cardiovascular responses elicited by social defeat in rats
Authors: Beig Mirza Irfan , Nalivaiko Eugene
Published: June 2021

Our aim was to determine whether 5‐HT2A receptors are involved in mediating autonomic responses to psychological stress. For this purpose, using telemetry, we assessed changes in the heart rate, arterial pressure and core body temperature elicited in Wistar rats during social defeat. Fifteen min prior to entering the resident's cage, intruders received s.c. injection of either selective 5‐HT2A receptor antagonist SR‐46379B (0.3, 1.0 or 3.0 mg/kg) or vehicle. After vehicle, social defeat induced substantial hyperthermic (+1.1±0.1°C), tachycardic (+142±11 bpm) and pressor (+22±3 mmHg) responses. SR46349B at doses 0.3 and 1.0 mg/kg abolished hyperthermia, and at the dose of 3.0 mg kg reverted it to the hyporthermia (−0.41±0.1°C; n=7, p<0.01 for all doses). The drug reduced tachycardia (to +115±13 bpm, n=15, p<0.05) only when applied at the highest dose, and did not affect pressor response at any dose (n=8). Anti‐hyperthermic effect was still well expressed 1.5 h after injection, whereas anti‐tachycardic effect waned at the end of defeat (30 min after injection). We conclude that activation of 5‐HT2A receptors is essential for the expression of stress‐induced hyperthermia, that contribution of these receptors to the stress‐induced tachycardia is minor, and that they do not mediate rise in arterial pressure during stress.
Tempol, a SOD‐mimetic, improves rat pharyngeal dilator muscle performance during hyperoxia and hypoxia
Authors: Skelly James Richard , Mitchell Gordon S , Bradford Aidan , O'Halloran Ken D.
Published: June 2021

Agents that improve respiratory muscle performance may be useful as an adjunct treatment for obstructive sleep apnoea. The aim of this study was to examine the effects of antioxidants on rat pharyngeal dilator muscle contractile and endurance properties.

Isometric contractile and endurance properties of isolated strips of sternohyoid muscle were examined in tissue baths under hyperoxic (95%O2/5%CO2) or hypoxic (95%N2/5%CO2) conditions in the absence (control) or presence of the antioxidants: N‐acetyl cysteine (NAC, 10mM), Tiron (10mM) or Tempol (10mM). Specific force (Sf) was measured at stimulus frequencies from 10–100Hz and throughout a standard fatigue trial (40Hz for 300msec every 2 seconds for 2 minutes).

Under hyperoxic conditions, Tempol had an inotropic effect over initial 90s of fatigue (Sf at 90s was 3.7 ± 0.5 vs. 7.7 ± 0.4 N/cm2, control (n=9) vs. Tempol‐incubated (n=9) muscles, P<0.05 ANOVA). In hypoxia, Tempol significantly increased force over the initial 50s of fatigue (Sf at 50s was 1.3 ± 0.2 vs. 2.9 ± 0.5 N/cm2, control (n=9) vs. Tempol‐incubated (n=9) muscles, P<0.05 ANOVA). NAC had no significant effect and Tiron had weak inotropic effects on sternohyoid muscle function.

This study illustrates that the SOD mimetic, Tempol, has inotropic effects on sternohyoid muscle during fatiguing and non‐fatiguing contractions. We conclude that antioxidants may be beneficial in the treatment of OSA.
Nitric oxide modulates rat pharyngeal dilator muscle force and endurance during hypoxia
Authors: McMorrow Clodagh , Mitchell Gordon S , Bradford Aidan , O'Halloran Ken D
Published: June 2021

Nitric Oxide (NO), a highly reactive second messenger, plays an important role in skeletal muscle physiology. The aim of this study was to investigate the effects of the NO synthase inhibitor, N‐nitro‐L‐arginine (L‐NNA) on the contractile and endurance properties of the sternohyoid (upper airway dilator) muscle under hyperoxic and hypoxic conditions in vitro.

Adult male Wistar rats were killed humanely and sternohyoid muscle strips were mounted isometrically in an organ bath containing aerated physiological salt solution maintained at 30degC. Four groups were assessed: control muscle strips exposed to hyperoxic (95%O2, 5% CO2; n=9) or hypoxic (95%N2, 5%CO2; n=8) conditions and L‐NNA‐treated strips (1mM) studied under hyperoxic (n=9) or hypoxic conditions (n=8). Force‐frequency relationship was assessed.

L‐NNA had no effect on sub‐maximal forces but significantly decreased peak tetanic force at 100Hz under both hyperoxic (17+/−1.5 vs. 13.8+/−1.2 N/cm2; mean+/−SEM, control vs. L‐NNA, P<0.05 ANOVA) and hypoxic conditions (9.3+/−0.9 vs. 6.6+/−0.7, control vs. L‐NNA, P<0.05).

We conclude that NO facilitates rat sternohyoid muscle contractile function especially under hypoxic conditions. Although NO is reported to inhibit diaphragm contractile function, a positive inotropic effect of NO donors has been observed in limb muscle.
Vestibulosympathetic reflex during the early follicular and mid‐luteal phases of the menstrual cycle
Authors: Lawrence Johnathan E , Lucas Angela K , Ray Chester A , Carter Jason R
Published: June 2021

The influence of the menstrual cycle on the vestibulosympathetic reflex is unknown. We examined muscle sympathetic nerve activity (MSNA), mean arterial pressure (MAP), and heart rate responses to head‐down rotation (HDR) in 10 healthy females during the early follicular (EF) and mid‐luteal (ML) phases of the menstrual cycle. The menstrual cycle had no effect on resting MSNA, MAP, and heart rate (EF: 13±3 bursts/min, 81±3 mmHg, 65±2 beats/min versus ML: 14+3 bursts/min, 83±3 mmHg, 64±3 beats/min). During the EF phase, HDR increased MSNA (Δ3±1 bursts/min; P<0.05), but did not change MAP or heart rate (Δ0±1 mmHg and Δ1±1 beats/min). During the ML phase, HDR increased both MSNA and MAP (Δ4±1 bursts/min and Δ3±1 mmHg; P<0.05) with no change in heart rate (Δ0±1 beats/min). MSNA and heart rate responses to HDR were not different between the EF and ML phases, but MAP responses to HDR were augmented during the ML phase (Interaction, P=0.029). Our results demonstrate that the menstrual cycle does not influence the vestibulosympathetic reflex, but appears to alter MAP responses to HDR during the ML phase. The augmented arterial blood pressure during the ML phase of the menstrual cycle may be due to increased sympathetic transduction to the vascular smooth muscle. Supported by REF‐060605.
3‐Iodothyronamine is a novel suppressor of fetal sheep cardiomyocyte proliferation in vitro
Authors: Chattergoon Natasha N , Louey Samantha , Grandy David K , Scanlan Thomas S , Hohimer Roger , Giraud George D , Thornburg Kent L
Published: June 2021

OBJECTIVES:

Fetal cardiomyocyte (CMC) maturation results in cells losing ability to divide. In late‐gestation sheep CMCs thyroid hormone (T3) decreased proliferation and changed signaling protein levels in favor of cell cycle arrest (Chattergoon et al, 2007). 3‐Iodothyronamine (T1AM) is a naturally occurring relative of T3 that does not bind classical thyroid receptors (TRs). We hypothesized that T1AM may depress CMC proliferation similar to T3.

METHODS:

Isolated right and left ventricular fetal sheep CMCs were cultured with T1AM (1nM, 10nM) and Bromodeoxyuridine (BrdU; 10μM) for 48 hours. Two ages were studied—100 days gestational age (dGA) and 135dGA—to determine developmental variations. BrdU uptake (index of proliferation) in fixed cells was analyzed.

RESULTS:

Basal BrdU uptake in serum free medium (SFM) was 10–12% in 100dGA CMCs versus 1–2% in 135dGA CMCs (p<0.01). BrdU uptake in response to 10% serum medium (SM) increased to 20% in 100dGA cells versus 8–10% in 135dGA CMCs (p<0.01). Similar to T3, T1AM does not affect cells exposed to SFM but decreases BrdU uptake by CMCs exposed to SM by 50% (p<0.05).

CONCLUSIONS:

These data suggest T1AM is as potent an inhibitor of fetal CMC proliferation as T3. Because T1AM does not activate TRs, non‐classical signaling of T1AM and T3 may be important in CMC growth and maturation.

Grant support: NICHD (P01HD34430) and Minority Supplement (3PO1 HD034430‐09S1).
Expression of delta opioids in heart and other tissues from summer‐active and hibernating woodchucks
Authors: Denning Gerene M. , Carraway Robert E. , Dickson Eric W.
Published: June 2021

In response to the severe environmental challenges of reduced temperature and limited food, mammalian hibernators enter into an extraordinarily protective state of decreased activity, temperature, and metabolism. Cardioprotection is transferred to hearts from non‐hibernators by treatment with plasma and plasma fractions from hibernating animals, and this protection is blocked by delta opioid receptor antagonists. To further elucidate the role of opioids in hibernation, we measured proenkephalin (PENK), the precursor protein for endogenous delta opioids, the delta opioid receptor (DOR), and plasma enkephalin levels in woodchuck tissues and blood. PENK and DOR were widely distributed in woodchuck tissues, including heart and skeletal muscle, and circulating levels of enkephalins were relatively high (> 300 pM). Additional studies are underway to compare levels of these proteins in samples from summer‐active and hibernating animals. The overall goal of these studies is to gain additional insight into the mechanism(s) by which hibernation and enkephalins increase cardiac ischemic tolerance. This insight in turn may contribute to future therapeutic approaches that mimic these naturally, and sometimes profound, protective processes to improve prevention and treatment of ischemic disease. Funding for these studies was provided by the University of Iowa Department of Emergency Medicine.
High‐Fat Feeding Increases Pro‐Inflammatory Cytokines in Type 2 Diabetes
Authors: Sachidanandam Kamakshi , Eto Chidimma , Hutchinson Jim , Ergul Adviye
Published: June 2021

Obesity leads to increased circulating levels of pro‐inflammatory cytokines that are secreted by adipocytes, thus increasing cardiovascular risk. Type 2 diabetes often presents along with obesity, insulin resistance and hypertension, leading to a condition called the metabolic syndrome. We hypothesized that diet‐induced hyperlipidemia would worsen inflammation in the Goto‐Kakizaki (GK) rat, a lean model of Type 2 diabetes. Plasma adiponectin, leptin, IL‐6, TNF‐α and MCP‐1 levels were measured in Control and GK rats on regular or high‐fat diet. Adiponectin was decreased in diabetes, although high‐fat feeding normalized it. Leptin, IL‐6 and TNF‐α levels were increased in GK rats, and diet‐induced hyperlipidemia caused further elevation. This correlated tightly with the weight gain and adiposity (fat weight/body weight) in these subsets. Circulating inflammatory markers are good predictors of cardiovascular disease. It is thus important to target obesity/hyperlipidemia and diabetes early on in order to prevent further worsening.
Induction of NOS and PDE5 isozymes in resistance arteries under chronic high and low flow
Authors: Lee Hyon Jae , Zhang Haiying , Fisher Steven
Published: June 2021

Myosin phosphatase (MP), the primary effector of smooth muscle relaxation, is a key target of signaling pathways that regulate vascular tone. Using the mesenteric artery ligation high flow/low flow model (HF/LF), we previously showed changes in MP isoform expression associated with increased sensitivity of VSM to NO/cGMP signaling. We hypothesized that altered expression of genes proximal in this pathway also influence NO/cGMP signaling under sustained HF/LF.

Methods: Every other second order mesenteric artery (MA2) was ligated to induce LF in upstream MA1s and HF in adjacent MA1s. Rat MA1s were harvested 1‐28 days after ligation and assayed for gene expression by real time PCR.

Results: Fold change vs. Control, Day4

Changes in NOS and PDE5 expression were confirmed at the protein level by Western blotting.

Conclusion: This and the functional studies presented in the accompanying abstract suggest that induction of PDE5 de‐sensitizes vascular smooth muscle to NO signaling under sustained HF/LF conditions. These results provide a novel mechanism for the dynamic changes in NO signaling in HF and LF disease states.

Supported by NIH R01 HL‐66171 and Case School of Medicine Crile Fellowship.
Carbon monoxide inhibits myocardial oxygen free radical generation following the onset of ischemia
Authors: Zuo Li , Talukder M.A. Hassan , Palmer Brian , Clanton Thomas L.
Published: June 2021

Reperfusion of the ischemic heart causes a burst of oxygen free radical (OFR) formation and secondary myocardial damage. However, questions remain on the mechanisms of OFR generation during ischemia. Recently, it has been reported that superoxide can derive from heme‐protein oxidation immediately following the onset of ischemia. Therefore, we tested the hypothesis that if OFR generation following ischemia involves heme‐protein oxidation then it should be inhibited by carbon monoxide (CO). Surface fluorometry was used to measure OFR generation during the onset of global ischemia in isolated rat heart. The fiber optic probe facing the left ventricle (LV) obtained the fluorescence signal from the heart. Hydroethidine (HE) was used as a sensitive probe to measure superoxide. When exposed to superoxide, HE is converted to ethidium (ET). Hearts were perfused with 44μM HE for 1.5 −5 min. We observed a rapid increase in ET signal with the onset of ischemia and that reached to a peak within 5 min. ET signal was completed blocked by a SOD mimic, MnPyP (250μM, P<0.01). Importantly, CO (100μM) treatment significantly reduced ET signal to 30% of control (P<0.01). Thus, there is a burst of OFR generation during the onset of myocardial ischemia in isolated rat heart. We report for the first time that CO inhibits OFR generation during myocardial ischemia, indicating that heme‐protein oxidation plays a crucial role in OFR generation.