bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of hemodynamic responses and the proliferative effect of the novel angiotensin peptide Angiotensin A
Authors: Badejo Adeleke , Greco Anthony J , Casey David B , Cook Julia L , Murthy Subramanyam N , Kadowitz Philip J
Published: June 2021

A novel angiotensin peptide known as angiotensin A (Ang A) has been recently identified in the human plasma. Ang A has been reported to have a higher affinity for AT2 receptors and therefore may modulate the effects of Ang II. The in vivo responses to Ang A as well as the effects of this peptide on proliferation of vascular smooth muscle cells (VSMCs) have not been studied and therefore were investigated. The iv injections of Ang A 0.1, 0.3, and 1.0 μg/kg increased systemic arterial pressure of 30.0 ± 2.0, 46 ± 2, and 60 ± 4 mm Hg, respectively. On the other hand, the iv injections of Ang II at doses of 0.1, 0.3, and 1.0 μg/kg produced increases in systemic arterial pressure of 38.7 ± 3.3, 50 ± 2, and 63 ± 4 mm Hg, respectively. At the doses tested the responses to both Ang A and Ang II were significantly attenuated by the AT1 receptor blocker losartan, but were not altered by the AT2 receptor antagonist PD123,319. The proliferation effect of Ang A when tested in A10 VSMCs cultured in media containing 100 nM or 1 μM showed increases by 75 ± 10% and 99 ± 5% respectively, as determined by BrdU incorporation. In contrast, the proliferation of cells in media containing 100 nM Ang II the increase was only 31 ± 4% over cells grown in control medium. These data demonstrate that both Ang A and Ang II have similar hypertensive activity and Ang A has a greater proliferative effect than Ang II on rat A10 VSMC.
Gender influences cerebral vascular responses to angiotensin II through Nox2‐derived reactive oxygen species
Authors: De Silva Travice Michael , Broughton Brad , Drummond Grant , Sobey Christopher , Miller Alyson
Published: June 2021

This study tested whether gender influences cerebrovascular responses to angiotensin II (AngII) and the role(s) of Nox2‐containing NADPH oxidase. AngII (0.1 μmol/L)‐stimulated superoxide (O2−) production by cerebral arteries from male and female C57Bl6/J (WT) and Nox2−/− mice was measured using lucigenin (5 μM)‐ and L‐012 (100 μM)‐ enhanced chemiluminescence. Hydrogen peroxide (H2O2) production was measured by Amplex Red fluorescence. Contractile responses of middle cerebral arteries (MCA) to AngII (0.1‐1 μM) were measured in a perfusion myograph. Protein expression of Nox2, O2− dismutases (SOD1‐3), angiotensin receptors (AT1, AT2) and endothelial nitric oxide synthase (eNOS) was measured by Western Blotting. Immunofluorescence was used to localize Nox2. AngII‐stimulated O2− and H2O2 production by cerebral arteries from female WT mice was ~75% lower vs. males (n=7; P<0.05). AngII elicited smaller contractions of MCA from female WT mice vs. males (n=6; P<0.05). ROS production and contractions were markedly reduced in male, but not female, Nox2−/− mice (n=6; P<0.05). Expression of Nox2, SOD, AT1, AT2 and eNOS were similar between genders (n=5‐14). In both genders, Nox2 was localized to adventitial and endothelial cells of MCA. Thus, the female gender is associated with lower cerebrovascular ROS production and smaller contractions to AngII. These gender differences are dependent on Nox2. NHMRC (491133).
Nitroxyl (HNO) exerts antioxidant actions in carotid arteries of mice
Authors: Ravi Ravina Maru , Miller Alyson A , Bullen Michelle , Ritchie Rebecca H , Sobey Christopher G , Kemp‐Harper Barbara K
Published: June 2021

HNO, the reduced and protonated congener of nitric oxide (NO), displays distinct pharmacology and therapeutic advantages over NO•. Unlike NO•, HNO has positive cardiac inotropic effects, is resistant to scavenging by superoxide (.O2−) and displays antioxidant properties in yeast and cardiomyocytes. Here we explored the potential antioxidant effects of HNO in the vasculature. Vascular .O2− levels were measured in freshly isolated and angiotensinII(AngII;1nM,24hrs)‐treated common carotid arteries from C57BL/6J mice using lucigenin(5μM)‐enhanced chemiluminescence. Acutely, the HNO donor Angeli's salt (AS;1 μmol/L) decreased NADPH(100μM)‐stimulated .O2− production in carotid arteries (control: 2536±304 vs AS: 1191±422counts/mg dry tissue weight, P<0.05;n=6‐8). Overnight treatment of arteries with AngII caused a significant increase in .O2− levels (control 110±30 vs AngII 430±70counts/mg, P<0.05;n=8‐12). Treatment of arteries with AS(1 μmol/L) during the 24hr period prevented the large increase in .O2− (150±20counts/mg, P<0.01;n=7). In conclusion, HNO appears to suppress the activity of vascular NADPH‐oxidase both acutely and chronically. The mechanisms via which this occurs remain to be elucidated, however such antioxidant actions of HNO may confer therapeutic advantages in the treatment of oxidative stress associated with cardiovascular disease.
Enzymatic sources of basal hydrogen peroxide (H2O2) levels in rat arterial and venous tissues
Authors: Szasz Theodora , Sattler‐Leja Rebecca L , Watts Stephanie W
Published: June 2021

Reactive oxygen species (ROS) production in vascular tissues is linked with the initiation and progression of diseases such as hypertension and atherosclerosis. An important but less studied sector of the vasculature is the venous system. We have shown that ROS production is greater in rat vena cava (VC) compared to aorta (Ao). Here we investigated the relative contribution of ROS enzymes to basal vascular H2O2 levels, hypothesizing that xanthine oxidase (XO) would be the most important H2O2 source in veins. Using two rat artery‐vein pairs: Ao‐VC and carotid artery (CA)‐jugular vein (JV), we examined H2O2 production in the presence of inhibitors for NADPH oxidase (100μM apocynin), XO (100μM allopurinol/oxypurinol), eNOS (100μM LNNA), mitochondrial complex I (10μM rotenone), and cyclooxygenase (COX, 10μM indomethacin/10mM ketorolac). Aortic H2O2 production was abolished by apocynin, allopurinol and indomethacin. Allopurinol also abolished H2O2 production in the CA and decreased it by 88% in the JV, but did not significantly lower VC H2O2 production. Surprisingly, COX inhibition with indomethacin decreased H2O2 levels of the JV by 82% and of the CA by 41%, while ketorolac decreased H2O2 production in the VC by 38%. LNNA and rotenone had no effect in any tissue. These findings highlight the importance of investigating non‐classical sources of ROS in vascular tissues.

Funding: PO1 HL70687 (SWW), AHA 0715679Z (TS).
Insulin Analogs Reduce Carotid Artery Intimal Hyperplasia Potentially via Insulin‐like Growth Factor‐1 Mediated Reduction in Oxidant Stress
Authors: Murthy Subramanyam N , Sukhanov Sergiy , McGee Jennifer , Casey David , Chandra Surabhi , Delafontaine Patrice , Kadowitz Philip J , McNamara Dennis B , Fonseca Vivian A
Published: June 2021

Glargine and glulisine (long and short acting insulin analogs respectively), used in the treatment of diabetes can activate insulin‐like growth factor‐1 (IGF‐1) dependent signaling in vitro. IGF‐1 is known to decrease atherosclerotic lesion in ApoE‐null mice. Glargine or glulisine (0.45 mg/kg/d) or vehicle were sc injected in Zucker fatty rats for 7 days before and 21 days after injuring the carotid artery (CA) to study the effects on intimal hyperplasia. Intima/media ratio (I/M), in CA sections was significantly reduced in glargine group (glargine, 0.4±0.1, glulisine, 0.6±0.1, control, 0.9±0.1, P<0.05). This correlated with increase in serum IGF‐1 (glargine, 1059±150 ng/ml, glulisine, 763±161 ng/ml, control, 567 ±121 ng/ml, P<0.05). As IGF‐1 suppresses oxidative stress in vivo, aortic 8‐isoprostane (8IP, ELISA) and N‐tyrosine (NT, immunostaining of CA sections) were measured. Glargine and glulisine significantly reduced 8IP and NT compared to control [8IP (ng/mg protein): glargine, 0.92±0.42, glulisine, 1.12±0.29, control, 2.57±0.4, P<0.05; NT immunopositivity, glargine, 55% decrease; glulisine, 34% decrease]. Glargine and to a lesser extent glulisine reduced IH in rat CA and suppressed vascular oxidative stress with an increase in IGF‐1. These effects are possibly mediated by IGF‐1, and its action as an atheroprotective agent.
Alterations in β‐adrenergic receptors in frontal cortex and amygdala in rats with heart failure: Effects of chronic antidepressant treatment
Authors: Lundberg Peter , Henze Marcus , Glasgow Jaimee , Battaglia George , Scrogin Karie
Published: June 2021

Chronic treatment with the serotonin reuptake inhibitor, fluoxetine (FLX), decreases cardiac sensitivity to β‐adrenergic receptor stimulation in rats with congestive heart failure (CHF). To determine if β‐adrenergic receptors are altered in brain regions important in anxiety, frontal cortex and amygdale were dissected and β‐adrenergic receptors were determined at a single concentration of 0.2 nM 125Iodocyanopindolol in 4 groups of rats (CHF‐vehicle, n=8‐9; CHF‐FLX, n=6‐8; Sham‐Vehicle, n=5; Sham‐FLX, n=6) in the absence and presence of propranolol (100 µM) to determine specific binding. 10 µM serotonin was included to preclude binding of 5‐HT receptors. β‐adrenergic receptors in the frontal cortex were significantly decreased in rats with CHF (19.3%, P<0.05) while FLX had no effect. In contrast, β‐adrenergic receptors were increased in the amygdala of rats with CHF (27.3%, P<0.05), while FLX caused an overall reduction in binding (13.2%, P<0.05). Thus, alterations in β‐adrenergic receptor binding develop in brain regions involved in behavioral and autonomic responses to stress during CHF and chronic antidepressant treatment. Supported by Loyola University.
Effect of a methanol extract of Arctium lappa on penile erection in penile smooth muscle
Authors: Li Xiang , Kim Ho Tae , Kang Dae Gill , Lee Ho Sub
Published: June 2021

The aim of the present study was to investigate whether a methanol extract of Arctium lappa (EAL) augments penile erection. In isolated endothelium‐intact rabbit corpus cavernosum preconstricted with phenylephrine, EAL relaxed penile smooth muscle in a dose‐dependent manner, which was inhibited by pretreatment with a nitric oxide synthase inhibitor, NG‐nitro‐L‐arginine methyl ester (L‐NAME), and a soluble guanylyl cyclase inhibitor, 1H‐[1,2,4]‐oxadiazole‐[4,3‐α]‐quinoxalin‐1‐one (ODQ), L‐type Ca2+ channel blockers, verapamil, and diltiazem, respectively. On the other hand, penile smooth muscle relaxation induced by EAL was not blocked by several inhibitors including indomethacin, atropine, propranolol, tetraethylammonium (TEA), and glibenclamide, respectively. In addition, incubation of endothelium intact rabbit corpus cavernosum tissues with EAL increased cGMP production in a dose‐dependent manner. Taken together, these results suggest that EAL may be induce penile erection through the activation of penile tissue NO/cGMP system and/or Ca2+channels in rat corpus cavernosum.
An ethanol extract of Lycopus lucidus Turcz has the cardiotonic effect in beating rabbit atria
Authors: Cui Hao Zhen , Choi Hye Ran , Kang Dae Gill , Cho Kyung Woo , Lee Ho Sub
Published: June 2021

Although Lycopus lucidus Turcz has long been used in the Oriental traditional medicine, effects of Lycopus lucidus on the cardiac function have not well been documented. The purpose of the present study was to test the effects of ethanol extract of Lycopus lucidus Turcz (ELT) on atrial dynamics in beating rabbit atria. ELT increased atrial dynamics including stroke volume and pulse pressure in a concentration‐dependent manner without changes in cAMP efflux. ELT‐induced positive inotropic effect was blocked by diltiazem, an inhibitor of L‐type Ca2+ channel. Blockade of the Ca2+ release from the sarcoplasmic reticulum by combined treatment with ryanodine, a SR Ca2+ release‐selective inhibitor, and thapsigargin, a SR Ca2+ ATPase‐selective inhibitor, significantly attenuated ELT‐induced inotropic effect. In addition, betulinic aicd (BA), a well known pentacyclic triterpenes constituent of ELT, also increased atrial stroke volume and pulse pressure without alteration of the cAMP efflux level.Increases in atrial dynamics by BA were blocked by pre‐treatment with diltiazem, but was not altered by pre‐treatment with staurosporine. These results suggest that the ELT‐induced positive inotropic effect in beating rabbit atria may, at least in part, be due to the activity of L‐type Ca2+ channels and be related with BA.
Pharmacologic response of regenerated bladders in a preclinical model
Authors: Bertram Tim , Christ George J , Andersson Karl‐Erik , Aboushwareb Tamer , Fuellhase Claudius , Soler Roberto , Wagner Belinda J , Jain Deepak , Ludlow John W , Payne Richard , Jayo Manuel J
Published: June 2021

Purpose

Neo‐bladder pharmacology was evaluated in animals after total cystectomy and implant of Autologous Neo‐Bladder Replacement constructs (NBR) made from bladder smooth muscle cells (SMC).

Methods

NBR were seeded with SMC to form three test groups in which 25, 12, or 4x106 SMC were seeded per construct (n= 8/grp). A fourth group (n=8) in which radically cystectomized bladders were immediately reimplanted (R) served as a control. Ex‐vivo pharmacological and histological studies were conducted on neo‐bladder tissues at study termination.

Results

At 9 mo post‐implantation, neo‐bladders of all groups had tissue histology (including mucosal and serosal linings, detrusor muscle, vasculature, and nerve components) consistent with native bladder. Contractile responses to parasympathomimetic (carbachol) and sympathomimetic (phenylephrine) agonists were similar among all groups. Logistic analysis of bladder tissue strips subjected to electrical field stimulation (EFS) revealed similar EC50 and slope factor values for all groups.

Conclusions

An Autologous Neo‐bladder Replacement construct is capable of regenerating a complete urinary bladder as a total organ with structural, urodynamic, and pharmacological features similar to native bladder. There was no evidence of abnormal tissue development, immune response, or systemic response to the neo‐bladder regeneration in any group.
The protective effect of tetrahydrobiopterin on rat hearts during long‐term hypothermic preservation
Authors: Zheng Ming‐zhi , ZHU Li , CHEN Ying‐ying , SHEN Yue‐liang
Published: June 2021

Objective

To assess the protective effect of tetrahydrobiopterin (BH4) on rat hearts suffered from long‐term hypothermic preservation.

Methods

The Langendorff model of isolated rat heart was used. Forty SD rat hearts were divided randomly into five groups (n=8): control group, hypothermic preservation groups (preserved in celsior solution for 6 or 12 h), BH4 groups (preserved in celsior solution containing 20 μmol/L of BH4 for 6 or 12 h). The myocardial contents of formazan, nitric monoxide (NO), and malonaldehyde (MDA) were measured. The myocardial ultrastructure was observed by transmission electron microscope.

Results

Compared with control group, hypothermic preservation of isolated hearts for 6 or 12 h could reduce the formazan and NO contents, and increase the MDA contents of myocardium. BH4 partly inhibited the hypothermic preservation‐induced decrease in formazan and NO contents, and enhancement of MDA content (P<0.05). After hypothermic preserved rat hearts for 6 or 12 h, the myocardial ultrastructure was impaired. Treatment with BH4 resulted in improvement in myocardial ultrastructure in hypothermic preserved hearts.

Conclusion

BH4 could reduce rat myocardial injury that induced by long‐term hypothermic preservation. The mechanism might be through the enhancement of myocardial NO production and inhibition of the generation of reactive oxygen species.
Relationship between expression of PARs and infection with H. pylori in the stomach of gastric cancer patients
Authors: Fukami Kazuki , Sekiguchi Fumiko , Takaoka Kaori , Miyake Kensaku , Yamamoto Takatsugu , Tanaka Shogo , Ohno Kohichi , Ishikura Hiroyasu , Naruse Mitsuhide , Kawabata Atsufumi
Published: June 2021

Helicobacter pylori (H. pylori) is involved in the pathogenesis of gastric ulcer and cancer, and might secret proteinases. Given emerging roles of proteinase‐activated receptors (PARs) in the gastric mucosa, we investigated the correlation between expression of PARs and H. pylori infection in the stomach isolated from gastric cancer patients. The extent of H. pylori infection was evaluated as regional protein levels of ureases A and B, and analyzed in relation to expression levels of PAR1 and PAR2 in the corresponding regions. In the cancer area, expression of PAR1, but not PAR2, was positively correlated with protein levels of either urease, whereas no such a correlation was observed in the non‐cancer area. There was no significant difference in levels of any proteins between the non‐cancer and cancer areas. Together, expression of PAR1, but not PAR2, appears to be associated with H. pylori infection in gastric cancer tissues. It is likely that the increased expression of PAR1 might be involved in the development of H. pylori‐related gastric cancer.
3,3′‐Diindolylmethane Enhances the Efficacy of Butyrate in Colon Cancer Prevention through Down‐regulation of Survivin
Authors: Bhatnagar Namrata , Li Xia , Chen Yue , Zhou Xudong , Garrett Scott H , Guo Bin
Published: June 2021

Butyrate is an inhibitor of histone deacetylase (HDAC) and has been extensively evaluated as a chemoprevention agent for colon cancer. We recently demonstrated that mutations in the adenomatous polyposis coli (APC) gene confer resistance to HDAC inhibitor‐induced apoptosis in colon cancers (Huang and Guo, Cancer Research, 66(18), 9245‐9251, 2006). Here we show that APC mutation rendered colon cancer cells resistant to butyrate‐induced apoptosis due to the failure of butyrate to down‐regulate survivin in these cells. Another cancer preventive agent, 3,3′‐Diindolylmethane (DIM), was identified to be able to down‐regulate survivin in colon cancers expressing mutant APC. Pre‐treatment with DIM enhanced butyrate‐induced apoptosis. Down‐regulation of survivin by DIM occurred at both transcription and post‐translation level through increased protein degradation. We further demonstrated that DIM was able to down‐regulate survivin and enhance the effects of butyrate in apoptosis induction and prevention of familial adenomatous polyposis in APCmin/+ mice. Thus, the combination of DIM and butyrate is potentially an effective strategy for the prevention of colon cancer.

This research was supported by Grants 1R03CA130062, 5R21CA111765 and 2P20RR015566 from the National Institutes of Health (NIH).
Pleiotrophin and Transgelin, Two Proteins Important in Cell Migration, Are Targeted by Non‐Steroidal Anti‐Inflammatory Drugs
Authors: Silver Kristopher , Lillich James D. , Freeman Lisa C.
Published: June 2021

Non‐steroidal anti‐inflammatory drugs (NSAIDs) are among the most widely used drugs today. Chronic treatment with NSAIDs can result in gastrointestinal (GI) toxicity. Microarray analysis of intestinal epithelial cell (IEC‐6) monolayers revealed that mRNA expression of transgelin (TAGLN), an actin binding protein of the calponin family, and pleiotrophin (PTN), a heparin binding protein involved in angiogenesis, are suppressed by NSAID‐treatment. qRT‐PCR and sequence analysis confirmed that PTN and TAGLN messages are down significantly in the presence of indomethacin and NS‐398, NSAIDs that inhibit IEC‐6 migration, but not SC‐560, an NSAID that does not inhibit migration. NSAIDs also suppress protein expression of TAGLN and PTN in IEC‐6 cells. Additionally, TAGLN mRNA expression is up‐regulated in migrating cells versus non‐migrating cells. Furthermore, there was a significant reduction in protein expression of PTN from mucosa of NSAID‐treated equine stomach, small intestine, cecum, and large intestine. Our results indicate that expression of PTN and TAGLN are down‐regulated by NSAIDs. Thus, PTN and TAGLN may have an important role in cell migration, and NSAID‐induced suppression of PTN and TAGLN expression may account for at least a portion of the inhibition of cell migration caused by NSAIDs in IEC‐6 cells. Finally, reduced PTN may play a role in the clinical development of NSAID induce GI ulceration.
Antenatal Betamethasone Causes Alterations in Angiotensin Receptors in Sheep Brain in a Gender Specific Manner
Authors: Shaltout Hossam A. , Figueroa Jorge P , Rose James C , Chappell Mark C , Averill David B , Diz Debra I.
Published: June 2021

We showed recently that antenatal betamethasone (Beta) exposure at mid gestation (80th day) elevates mean arterial pressure and attenuates baroreflex sensitivity in adult sheep of both sexes (1.8 yrs old). We now examined the effects of fetal Beta exposure on AT1, AT2, and Ang‐(1‐7) receptors in autonomic nuclei of the brain medulla in 1.8 yr old sheep using in vitro receptor autoradiography for binding with the non‐selective Ang receptor antagonist [125I‐Sar1‐Thr8]‐Ang II. Receptor subtypes were defined by competition with losartan for AT1, PD 123319 for AT2, [D‐Ala7]‐Ang‐(1‐7) for Ang‐(1‐7) in nucleus tractus solitarii (nTS), dorsal motor nucleus of vagus (dmnX) and area postrema (AP). There were no significant differences in overall binding density between Beta‐treated male or female sheep and their controls. There was a significant increase in the ratio of AT1 to A1‐7 receptors in Beta‐treated females, but not treated males, towards AT1 in nTS (0.7 ± 0.2 vs 3 ± 1), dmnX (0.6 ± 0.4 vs 4 ± 2) and AP (0.6 ± 0.2 vs 3 ± 1) versus the control group. In contrast, there was a 50‐60% reduction (p<0.03) in AT2 binding in dmnX and AP of Beta‐treated male sheep compared with controls. These sex‐specific changes in receptor subtypes are consistent with different mechanisms underlying the fetal programmed hypertension in male and female animals overall favoring a greater role for Ang II via the AT1 subtype. HD47584, HL51952
Estradiol (βE) Promotes Adhesion and Mitogenesis in CD34+ Human Progenitor Endothelial Cells (PECs)
Authors: Baruscotti Isabella , Barchiesi Fedreica , Jackson Edwin K. , Gillespie Delbert G. , Imthurn Bruno , Dubey Raghvendra K.
Published: June 2021

Here we investigated whether βE promotes endothelium recovery by promoting PECs adhesion and proliferation. PECs were isolated from blood using CD34+‐antibody‐based magnetic separation and placed in culture or used for adhesion studies. For adhesion studies, early PECs, late differentiated PECs or mononuclear cells (MNC) labelled with fluorescent‐dye calcein were layered over monolayers of endothelial cells (ECs) pre‐treated with βE, and the number of adherent cells analyzed by ELISA. βE promoted adhesion of early PECs, but not late differentiated PECs or MNC, to ECs. Increased PECs colony formation and proliferation were observed in cultures of MNC isolated from IVF subjects where βE synthesis was induced. PECs expressed functional ERα and ERβ, and βE induced PECs growth (DNA/cell number) The mitogenic effects of âE were mimicked by PPT (ERα agonist), but not DPN (ERβ agonist), moreover these effects were blocked by ICI (ER antagonist) and MPP (ERα antagonist), βE treatment induced ERK1/2 phosphorylation and upregulated positive growth regulators including cyclin D1, A, B and E and downregulated the negative growth regulator p21. The effects of βE were mimicked by PPT but not DPN, moreover, ICI and MPP blocked the effects, suggesting the effects are ERα mediated.

Conclusion

βE selectively promotes the adhesion of early CD34+ PECs to ECs and promotes their proliferation via ERα mediated ERK1/2 activation.
Cannabinoid Receptor Interacting Protein (CRIP1a) attenuates CB1 signalling
Authors: Hardt Tricia Ann , Elphick Maurice , Lewis Deborah L , Selley Dana E.
Published: June 2021

A recently discovered protein, Cannabinoid Receptor Interacting Protein (CRIP1a) interacts with the C‐terminal tail of the CB1 receptor (aa 418‐473). Initial studies found that CRIP1a decreased the effects of the inverse agonist SR141716A (SR1) on Ca2+ currents in superior cervical ganglion neuronal expression system, while leaving the effects of the full agonist WIN 55,212‐2 unchanged, suggesting a decrease in constitutive activity (Niehaus et. al. Molecular Pharmacology (2007) 72:1557‐1566). To examine CRIP1a, previous studies in this laboratory have characterized HEK cells transfected with CB1, with and without CRIP1a co‐transfection. Stable co‐transfection of CRIP1a had no effect on CB1 expression levels in HEK cells stably expressing CB1 receptor (hCB1‐HEK) or CB1 receptor and CRIP1a (hCB1‐HEK CRIP1a). CRIP1a protein expression was verified using immunoblot analysis. The effect of CRIP1a on G‐protein activity was examined in the presence of Na+ concentrations, a negative allosteric modulator of G‐protein‐coupled receptor activity. [35S]GTPγS binding in hCB1‐HEK cells ± CRIP1a showed that CRIP1a significantly reduced WIN‐stimulated G‐protein activity at all NaCl concentrations. CRIP1a significantly decreased basal, THC‐stimulated and SR1‐inhbited G‐protein activity at low NaCl concentrations (0‐25 mM NaCl). In conclusion, CRIP1a attenuates G‐protein activation by agonists and inhibition by inverse agonists when spontaneous receptor activity is high, suggesting a decrease in constitutive activity. Funded by F31‐DA023747 from NIDA (TAH) and a Jeffress Memorial Trust Research Award (DES).
Pharmacological profile of constitutively active NOP opioid receptors heterologously expressed in rat stellate ganglion neurons
Authors: Ruiz‐Velasco Victor , Trapella Claudio , Calo Girolamo , Margas Wojciech
Published: June 2021

We have previously shown that opioid receptor‐like 1 (NOP) receptors are expressed in rat stellate ganglion (SG) neurons and that activation of the receptors with nociceptin/orphanin FQ (N/OFQ) results in voltage‐dependent (VD) inhibition of N‐type Ca2+ channel currents. In the present study, we examined the effect of NOP receptor overexpression in SG neurons on the pharmacological response to five high affinity NOP receptor blockers on Ca2+ channel currents employing the whole‐cell patch‐clamp technique. In control neurons, 0.1 μM N/OFQ inhibited Ca2+ currents 41.0 ± 3.6% (n=29). On the other hand, all five NOP receptor antagonists (1 μM) significantly (P < 0.01) blocked the N/OFQ‐mediated Ca2+ current inhibition, being per se inactive. In neurons overexpressing NOP receptors, application of UFP‐101 resulted in Ca2+ channel inhibition of 39.9 ± 2.4% (n=5). On the other hand, exposure of cells to Compound 24, JTC‐801 and TRAP‐101 enhanced the Ca2+ current amplitude by 60.8 ± 8.7 (n=6), 51.0 ± 13.9 (n=5) and 59.9 ± 6.8% (n=5), respectively. Finally, application of (±)J‐113397 resulted in a small increase in Ca2+ current amplitude of 11 ± 5.1% (n=6). The data suggest that the effect of NOP receptor blockers is modulated when the receptors are constitutively active such that partial, neutral or inverse agonistic effects can be observed.
Identification of binding determinants for arrestin on D2 dopamine receptor intracellular loops
Authors: Neve Kim A , Lan Hongxiang , Liu Yong , Teeter Martha M , Gurevich Vsevolod V
Published: June 2021

We explored the ability of arrestins to bind to GST fusion proteins containing D2 receptor intracellular loops (IC2 and IC3). We identified an important determinant of the binding of arrestin in IC3. Alanine mutations of this determinant (IYIV212‐215) in the full‐length D2 receptor generated a signaling‐biased receptor with intact ligand binding and G‐protein coupling and activation, but deficient in receptor‐mediated arrestin3 translocation to the membrane and receptor internalization. Arrestin3 bound to GST fusion proteins of the D2 receptor IC2 more avidly than to the D3 receptor IC2, consistent with the lower affinity of the D3 receptor for arrestins. We identified a residue at the C‐terminus of IC2, K149, that was important for the preferential binding of arrestin3 to D2‐IC2. Mutating this lysine in the full‐length D2 receptor to cysteine decreased the ability of the D2 receptor to mediate agonist‐induced arrestin3 translocation to the membrane and decreased agonist‐induced receptor internalization in human embryonic kidney 293 cells. The reciprocal mutation in the D3 receptor increased receptor‐mediated translocation of arrestin3. (MH045372 and VA Merit Review)
α1 Adrenergic receptor regulation of interneuron function
Authors: Jurgens Chris WD , Knudson Chris A , Carr Pat A , Perez Dianne M , Doze Van A
Published: June 2021

Adrenergic receptors (ARs) have been shown to regulate neuronal function in diverse ways, from altering membrane potential and transmitter release to effects on gene expression. Activation of α1ARs produces a membrane depolarization in a specific subset of hippocampal interneurons. Evidence from transgenic mice either lacking (KO) or overexpressing (CAM) α1A or α1BARs suggests that the majority of this response is due to activation of α1AARs. Electrophysiological recordings during application of α1AR agonists produced a concentration‐dependent increase in action potential frequency in hippocampal interneurons from normal and α1BAR KO mice, but not α1AAR KO mice. α1AR agonists also increased the frequency and amplitude of IPSCs in CA1 pryamidal neurons. Mice overexpressing α1AARs showed increased hippocampal interneuron density. BrdU immunohistochemistry revealed enhanced neurogenesis in CAM α1AAR animals. This result was also observed in normal mice treated with the α1AAR agonist, cirazoline. Furthermore, when compared to controls, CAM α1AAR mice showed an increase in latency periods preceding seizures when exposed to the epileptogenic agent, flurothyl. These findings suggest that activation of α1AARs increases inhibitory tone not only via increased interneuron firing but possibly by increased numbers of interneurons. These findings are potentially very significant as they link α1AAR‐induced proliferation of interneurons to the antiepileptic actions of the adrenergic system. Supported by NIH R01HL61438, NIH COBRE, NSF ND EPSCoR, NSF CAREER, Epilepsy Foundation.
Cerebroprotective action of angiotensin 1‐7 in a rat model of ischemic stroke
Authors: Mecca Adam Peter , O'Connor Timmothy E , Dooies Kimberly A , Katovich Michael J , Sumners Colin
Published: June 2021

Recent progress in cardiovascular therapy suggests that stimulation of Angiotensin Converting Enzyme 2 (ACE2), production of Angiotensin 1‐7 (Ang 1‐7), and activation of the Ang 1‐7 receptor, Mas, are viable targets for disease prevention and treatment. We proposed that central administration of Ang 1‐7 via lateral ventricular cannula will provide cerebroprotection in a rat model of Endothelin‐1 (ET‐1)‐induced middle cerebral artery occlusion (MCAO). Prior to ET‐1 induced MCAO, rats were treated with intracerebroventricular Ang 1‐7 (1µg/h) or artificial Cerebrospinal Fluid (aCSF) for 5 days. Ang 1‐7 treatment reduced neurological deficits and infarct size measured 72 h after MCAO induction. Infarct size was reduced to 15.23 ± 5.77% (n = 9) of ipsilateral gray matter in the Ang 1‐7 treated rats compared with 36.94 ± 7.56% (n = 12) in artificial Cerebrospinal Fluid (aCSF) controls. Neurological deficits were also reduced in Ang 1‐7 treated rats as indicated by a lower Benderson Exam Score of 0.22 ± 0.15 (n = 9) compared to 1.08 ± 0.31 (n = 12) in control rats, a higher Garcia Exam Score of 17.56 ± 0.18 (n = 9) compared to 14.00 ± 1.10 (n = 12) in control rats, and improved sunflower seed eating from 146 ± 23 s and 14 ± 1 (n = 9) shell pieces compared to 293 ± 7 s and 29 ± 1 shell pieces (n = 12) in control rats. This provides the first demonstration of the cerebroprotective properties of Ang 1‐7 during ischemic stroke.
Antiepileptogenic and Antiepileptic Activity of Progesterone in the Rapid Kindling Model in PRKO Mice
Authors: Briyal Seema , Reddy Doodipala S.
Published: June 2021

Presently progesterone is undergoing NIH‐sponsored, multicenter clinical evaluation as treatment for epilepsy in women. Although the anticonvulsant effect of progesterone has long been known, the molecular mechanism underlying its protection in the brain is obscure. Progesterone‐derived GABA‐A receptor‐modulating neurosteroids could be involved in its anticonvulsant actions. However, the role of progesterone receptor (PR) is not clearly defined in the protective effects of progesterone. Here we utilized PR knockout (PRKO) mice to evaluate the antiepileptogenic and antiepileptic activity of progesterone. Progesterone was examined in female PRKO and WT controls in the kindling model under conditions of rapid kindling. Mice were subjected to stimulations via implanted electrode in the hippocampus. Progesterone treatment (100 mg/kg) prior to rapid kindling significantly suppressed subsequent kindling progression indicating antiepileptogenic effect of the steroid. In fully kindled mice, progesterone reduced the occurrence of generalized seizures. In all seizure protocols, the antiepileptogenic and antiepileptic potency of progesterone was undiminished in PRKO mice. These studies provide strong evidence that progesterone exerts both antiepileptogenic and antiepileptic effects in the kindling model, and the PR is not required for these effects of progesterone.
Transfer of Nitric Oxide by Blood From Upstream to Downstream Resistance Vessels Causes Microvascular Dilation
Authors: Bohlen Harold Glenn , Zhou Harry , Miller Steve , Unthank Joe
Published: June 2021

Oxygenated hemoglobin does not destroy nitric oxide (NO), but carries and releases NO in blood from upstream to downstream microvessels. Given that the circulation time from small arteries to capillaries is ~2‐4 seconds, NO in plasma or bound to proteins likely survives to downstream locations. To test this hypothesis, the vessel wall versus intravascular [NO] was measured in the in vivo sequential mesenteric and intestinal vessels of rats. NO microelectrodes demonstrated vessel wall [NO] was 25‐40% higher than blood [NO] in all vessels. Flow in mesenteric arteries was elevated by occlusion of collateral arteries to induce a flow mediated increase in arterial NO production. Both arterial wall and downstream arteriolar [NO] increased and the arterioles dilated as the blood [NO] was increased. To study receptor mediated NO generation, bradykinin was locally applied to upstream large arterioles and NO measured there and in downstream arterioles. At both sites, the [NO] and vessel diameters increased. When isoproterenol was applied to upstream vessels, they dilated but their [NO] and that of downstream arterioles did not increase and minor downstream dilation occurred. These observations indicate that NO can move in blood from upstream to downstream resistance vessels. This phenomenon may allow larger vessels to influence vascular tone in downstream vessels when local NO production is altered. (Support NIH HL‐20605)
Acute Antioxidant (AOX) Treatment Increases Muscle Microvascular O2 Extraction in Young Rats
Authors: Copp Steven W , Hirai Daniel M , Schwagerl Peter J , Herspring Kyle F , Musch Timothy I , Poole David C
Published: June 2021

AOX treatment (76 mg/kg vitamin C, 52 mg/kg tempol) elevates microvascular O2 pressures (PO2mv) at rest and following the onset of muscle contractions in aged (A) rats reducing fractional O2 extraction (Herspring et al. J. Appl. Physiol. 2008).

PURPOSE

To test the hypothesis that this specific AOX treatment would elevate PO2mv in the spinotrapezius muscle of young (Y) rats at rest and during contractions.

METHODS

PO2mv, which reflects the dynamic balance between O2 delivery/utilization (fractional O2 extraction), was measured via phosphorescence quenching in the spinotrapezius of 12 Y (6‐8 mo) and 15 A (26‐30 mo) Fisher 344 x BN rats. Electrically‐induced contraction bouts (1 Hz twitch) were performed with and without (CON) intra‐arterial AOX administration. The PO2mv response was fitted with an exponential model and compared using two‐way ANOVA (age x condition).

RESULTS

The CON baseline PO2mv was not different (P>0.05) between Y (31±4 mmHg) and A (30±3 mmHg). However, AOX decreased baseline PO2mv in Y (25±5 mmHg) whilst it was increased in A (36±3 mmHg) (both P<0.05). Moreover, throughout contractions in Y, AOX decreased PO2mv below CON values by 21+6% on average (P<0.05).

CONCLUSIONS

The impact of AOX supplementation on PO2mv is age‐dependent. In contrast to A rats, in Y rats, AOX reduced PO2mv (increased fractional O2 extraction) at rest and during contractions.

Support: AHA, Heartland Affiliate 0750090Z
Myostatin gene knockout increases vascular function in skeletal and cardiac muscle tissues
Authors: Ali Mohammed Irfan , Mintz James D , Elms Shawn , Hamrick Mark W , Stepp David W
Published: June 2021

Exercise improves vascular health through mechanisms yet to be defined. Exercise enhances both muscle mass and metabolism, in part by decreasing myostatin (MS). Genetic MS deletion increases muscle mass and metabolism but vascular effects of these changes are unknown. This study tested the hypothesis that MS deletion would alter vascular function in the microcirculation. To test this hypothesis, we compared Ms−/− mice on an ICR background with Ms+/+ ICR as controls. Gracilis (SA), coronary (CA), gonadal (GA) and mesenteric (MA) resistance arteries were analyzed in vitro (±Ca2+, i.d. < 150μm). Acetylcholine (ACh) reactivity was used to assess endothelial relaxation. Similarity was seen between Ms−/− and Ms+/+ in mean arterial pressure (90±9 vs. 90±10 mmHg), heart rate (600±136 vs. 545±93 bpm) but in Ms−/− visceral adiposity and plasma leptin (2.4±0.3 vs. 0.6±0.1 ng/ml, p<0.01) were reduced. Maximum passive i.d. was similar in both strains in all arteries. In Ms−/−, dilation to ACh was increased in SA (88±1 vs. 71±4%, p<0.01) and CA (63±2 vs. 51±4%, p<0.01), unchanged in GA and reduced in MA (45±2 vs. 63±4%, p<0.01). Myogenic tone was reduced in CA and SA but unchanged in MA or GA. Taken together, these data indicate that increased muscle mass has pro‐dilatory effects on the microcirculation of muscular organs. These findings may explain, in part, improvements in vascular health seen with exercise training.

HL76533
Differentiation of mechanical forces in perfused artery
Authors: Takahashi Maki , Mell Blair M , AbouAlaiwi Wissam A , Jones Thomas J , Ratnam Shobha , Kolb Robert J , Nauli Surya M
Published: June 2021

Background

Mechanical forces in the blood vessel can be biophysically observed in the forms of stretch due to distention of surrounding muscle and shear stress due to drag force generated by the blood flow. We report here a novel approach to distinguish these two forces.

Methods and Results

Aorta of an adult mouse is isolated and cleaned from connective tissues. The aorta, which is to be perfused, is then submerged freely in phosphate buffer saline solution or placed inside a capillary tubing. The aorta inside the capillary tube has very limited room for stretching or expanding by the perfusate pressure. Our data shows that the changes in cytosolic calcium are much more prolonged in freely‐placed than in capillary‐enclosed aortas. The perfuse‐induced calcium signal can be inhibited by apyrase more substantially in freely‐placed than in capillary‐enclosed aortas.

Conclusion

Different forms of mechanical forces may be physiologically impossible to differentiate in vivo because of the complex nature of the vascular system and its surrounding tissue. Here, we are introducing a new setup that allows us to distinguish the mechanical force of stretching and shear stress in an ex vivo study.
Proteinuria is associated with increased systemic and glomerular water permeability
Authors: Ferguson Joanne K , Neal Chris R , Bates David O , Harper Steven J , Salmon Andrew H J
Published: June 2021

Proteinuria is associated with an increased risk of cardiovascular death. Evidence suggests that proteinuria reflects systemic endothelial dysfunction as well as renal pathology. The Munich Wistar Fromter (MWF) rat strain develops spontaneous proteinuria, permitting study of altered endothelial function under conditions of increased proteinuria. Male MWF rats (15‐24 wks) were significantly proteinuric (MWF:15.0±1.5 n=14 Wistar: 1.8±0.20 n=10, p<0.0001). Hydraulic conductivity (Lp) and effective oncotic pressure (σδπ) were measured in individually perfused microvessels of the mesentery. Male MWF rats had significantly increased Lp (x10−7 cm s−1 cmH20−1) but unchanged σδπ (cmH20) compared with age matched wistar controls (Lp MWF:6.4±1.8 n=7; Wistar: 2.8±0.4 n=15, p<0.05 σδπ MWF:24.6±2.0 n=7; Wistar:23.7±0.9 n=7). Normalised glomerular ultrafiltration coefficient (LpA/Vi) was measured using an oncometric assay. The increase in systemic Lp was mirrored by a significant increase in glomerular LpA/Vi (MWF: 1.5±0.1 min−1mmHg−1 n=58; Wistar: 1.0±0.1 min−1mmHg−1 n=24, p<0.01). These results suggest proteinuria is associated with increased glomerular and systemic water permeability. The mechanistic links require further investigation and may provide insight into the predictive link between proteinuria and increased cardiovascular mortality.

Supported by BHF
Platelet‐activating factor (PAF) and bradykinin (BK) induce venular hyperpermeability in part by decreasing cAMP levels and Epac activity
Authors: Gonzalez Francisco G , Eller Gisela , Duran Walter N , Velarde Victoria , Boric Mauricio P
Published: June 2021

We have established earlier that PAF causes hyperpermeability via eNOS‐derived NO. We investigate here the alternative hypothesis that PAF and BK diminish cAMP levels and Epac activity and thus reduce the barrier properties of the endothelium. We studied the hamster cheek pouch microcirculation by intravital microscopy and measured FITC‐Dx clearance (Cl‐FITC‐Dx) as an index of macromolecular transport. We homogenized the pouches and determined cAMP content (ELISA kit) at defined intervals related to changes in Cl‐FITC‐Dx. We applied 8‐CPT‐2′‐O‐Me‐cAMP (8‐CPT) topically 2 min after PAF application to stimulate Epac activity. PAF (10−7M) and BK (10−6M) elevated Cl‐FITC‐Dx from 2.66 + 0.4 and 2.21 + 0.76 to 112.28 + 43.02 and 67.76 + 10.03 nl/min, respectively. Application of 8‐CPT reduced the PAF and BK‐induced elevation in Cl‐FITC‐Dx by 50%. cAMP levels in tissues collected at 5 min, 15 min and 30 min after PAF application were both reduced by 30% compared to baseline (Basal; 29.5 ± 4.5, PAF 30 min; 18.7 ± 1.3 pmol cAMP/g). We speculate that PAF and BK stimulate hyperpermeability via two complementary mechanisms: a) eNOS‐derived NO and b) reduction of cAMP levels and Epac activity. We demonstrate that the PAF‐ and BK‐induced hyperpermeability can be deactivated by increasing Epac activity. (Supported by Fondecyt 1040816, Proyecto puente VRAID y NIH 5RO1 HL070634‐06).
Extracellular acidosis promote endothelium‐independent and nitric oxide‐dependent vasodilation
Authors: Celotto Andrea Carla , Capellini Verena Kise , Baldo Caroline Floreoto , Restini Carolina Baraldi , Evora Paulo Roberto
Published: June 2021

Extracellular acidosis (EA) has profound effects on vascular homeostasis since EA‐induced tone alterations are specific to vascular bed. In the present study, we examined the in vitro ("organ chambers") effects of acidosis on dilatory responses of isolated rat thoracic aorta. pH‐response curves (7.4 to 6.5) were generated using HCl (1M) in phenylephrine pre‐contracted endothelium intact and denuded rings. The acidosis pH‐response curves were obtained by gradual addition of HCl to the preparation. The curves were performed with and without tetraethylamonium (TEA), NG‐nitro‐L‐arginina metil ester (L‐NAME) and/or indomethacin (Indo). Relaxation mediated by HCl was increased in endothelium intact rings (73.12±3.66) compared to denuded rings (39.56±3.78) during mild (pH 7.0), but not severe acidosis (pH 6.5) (94.33±1.96 to 91.42±0.95). Mild acidosis‐dilatory effect in endothelium intact rings was markedly reduced by L‐NAME (32.54±5.38) and L‐NAME + Indo (22.80±7.36). In denuded rings, relaxation was reduced only by L‐NAME in mild (11.52±4.18) and in severe acidosis (53.32±6.44). TEA and Indo failed to alter the relaxing effect of acidosis. These results point that acidosis promoted nitric oxide (NO)‐mediated relaxation in rat aorta. Moreover, other factors, besides NO, can be involved in relaxation mediated by EA. Support: FAPESP; FAEPA‐HC/FMRP.
Superoxide from uncoupled nitric oxide synthase reduces endothelium‐dependent arteriolar dilation in mice fed high salt
Authors: Boegehold Matthew A. , Nurkiewicz Timothy R.
Published: June 2021

We have previously reported that the impaired endothelium‐dependent dilation of arterioles in rodents on a high salt diet is due to oxidation of nitric oxide (NO) by superoxide anion (O2−) in the arteriolar wall. The aim of this study was to test the hypothesis that "uncoupled" nitric oxide synthase (NOS) is the predominant source of this O2−. Arterial pressure in C57BL/6J mice fed a high salt (4%, HS) diet for 4 weeks was not significantly different from that in mice fed a normal salt (0.45%, NS) diet. However, compared to NS mice, spinotrapezius muscle arterioles in HS mice had elevated arteriolar wall oxidant activity (assessed by hydroethidine oxidation) and a large (75‐80%) reduction in dilator responses to acetylcholine. In HS mice, arteriolar oxidant stress was abolished by acute application of the NOS inhibitor NG monomethyl L‐arginine and by long‐term supplementation of L‐arginine intake (1.51% in drinking water for 4 weeks), which has been shown to increase vascular tetrahydrobiopterin (BH4) levels. L‐arginine supplementation also prevented the reduction in arteriolar ACh responses in HS mice but had no effect on these responses in NS mice. These findings suggest that NOS uncoupling due to reduced BH4 availability is primarily responsible for O2− accumulation and the resultant impairment of NO‐dependent arteriolar dilation in mice fed a high salt diet. (Support AHA Grant‐in‐Aid 0755264B and NIH ES‐015022)
BKCa and KV channels modify remote Ca2+ responses to local depolarization in rat mesenteric arterioles
Authors: Jensen Lars Jørn , Braunstein Thomas Hartig , Inoue Ryuji , Ito Yushi , Holstein‐Rathlou Niels‐Henrik
Published: June 2021

Conducted vasoconstriction plays an important role in determining microvascular resistance. The spread of depolarization along an arteriole occurs through cell‐cell coupling via gap junctions. The decay of conducted depolarization is characterized by the length constant λ, and depends on the membrane (rm) and junctional resistances (rj) as follows: λ=v(rm/rj). We hypothesized that this decay can be diminished by increasing rm through inhibition of K+ channels. We investigated this hypothesis in rat mesenteric terminal arterioles (<40 µm) by monitoring local and remote Ca2+ responses to localized delivery of 155 mM KCl. Ca2+ responses were recorded simultaneously at 6 positions along a short arteriolar segment and λ was estimated by non‐linear curve‐fitting. The KV channel blocker 4‐AP (1 mM) augmented the remote Ca2+ responses (λ increased from 296 ± 30 to 417 ± 64 µm; P=0.05, n=5). The BKCa channel blocker IbTx (100 nM) increased λ from 254 ± 37 to 343 ± 54 µm (P<0.01, n=6). Preconstriction of arterioles (norepinephrine + neuropeptide Y) increased λ from 289 ± 37 to 336 ± 40 µm (P<0.05; n=6), and subsequent addition of IbTx further increased λ to 425 ± 59 µm (P<0.01; n=6). BKCa, KV1.5, and KV2.1 channels were immuno‐localized to VSMC. These data are consistent with the idea that BKCa and KV channel expression modulates conduction of depolarization through an effect on smooth muscle rm in mesenteric arterioles.