bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleotide coronary vasodilation in guinea pig hearts
Authors: Gorman Mark W. , Ogimoto Kayoko , Savage Margaret V. , Jacobson Kenneth A. , Feigl Eric O.
Published: March 2015

The role of P1 receptors and P2Y1 receptors in coronary vasodilator responses to adenine nucleotides was examined in the isolated guinea pig heart. Bolus arterial injections of nucleotides were made in hearts perfused at constant pressure. Peak increase in flow was measured before and after addition of purinoceptor antagonists. Both the P1 receptor antagonist 8-( p-sulfophenyl)theophylline and adenosine deaminase inhibited adenosine vasodilation. AMP-induced vasodilation was inhibited by P1 receptor blockade but not by adenosine deaminase or by the selective P2Y1 antagonist N6-methyl-2′-deoxyadenosine 3′,5′-bisphosphate (MRS 2179). ADP-induced vasodilation was moderately inhibited by P1 receptor blockade and greatly inhibited by combined P1 and P2Y1 blockade. ATP-induced vasodilation was antagonized by P1 blockade but not by adenosine deaminase. Addition of P2Y1 blockade to P1 blockade shifted the ATP dose-response curve further rightward. It is concluded that in this preparation ATP-induced vasodilation results primarily from AMP stimulation of P1 receptors, with a smaller component from ATP or ADP acting on P2Y1 receptors. ADP-induced vasodilation is largely due to P2Y1 receptors, with a smaller contribution by AMP or adenosine acting via P1 receptors. AMP responses are mediated solely by P1 receptors. Adenosine contributes very little to vasodilation resulting from bolus intracoronary injections of ATP, ADP, or AMP.
Adenosine and opioid receptor-mediated cardioprotection in the rat: evidence for cross-talk between receptors
Authors: Peart Jason N. , Gross Garrett J.
Published: March 2015

The relative roles of free-radical production, mitochondrial ATP-sensitive K+ (mitoKATP) channels and possible receptor cross-talk in both opioid and adenosine A1 receptor (A1AR) mediated protection were assessed in a rat model of myocardial infarction. Sprague-Dawley rats were subjected to 30 min of occlusion and 90 min of reperfusion. The untreated rats exhibited an infarct of 58.8 ± 2.9% [infarct size (IS)/area at risk (AAR), %] at the end of reperfusion. Pretreatment with either the nonselective opioid receptor agonist morphine or the selective A1AR agonist 2-chloro-cyclopentyladenosine (CCPA) dramatically reduced IS/AAR to 41.1 ± 2.2% and 37.9 ± 5.5%, respectively ( P < 0.05). Protection afforded by either morphine or CCPA was abolished by the reactive oxygen species scavenger N-(2-mercaptopropionyl)glycine or the mitoKATP channel blocker 5-hydroxydecanoate. Both morphine- and CCPA-mediated protection were attenuated by the selective A1AR antagonist 1,3-dipropyl-8-cyclopentylxanthine and the selective δ1-opioid receptor (DOR) antagonist 7-benzylidenealtrexone. Simultaneous administration of morphine and CCPA failed to enhance the infarct-sparing effect of either agonist alone. These data suggest that both DOR and A1AR-mediated cardioprotection are mitoKATP and reactive oxygen species dependent. Furthermore, these data suggest that there are converging pathways and/or receptor cross-talk between A1AR- and DOR-mediated cardioprotection.
A3adenosine receptor agonist IB-MECA reduces myocardial ischemia-reperfusion injury in dogs
Authors: Auchampach John A. , Ge Zhe-Dong , Wan Tina C. , Moore Jeannine , Gross Garrett J.
Published: March 2015

We examined the effect of the A3adenosine receptor (AR) agonist IB-MECA on infarct size in an open-chest anesthetized dog model of myocardial ischemia-reperfusion injury. Dogs were subjected to 60 min of left anterior descending (LAD) coronary artery occlusion and 3 h of reperfusion. Infarct size and regional myocardial blood flow were assessed by macrohistochemical staining with triphenyltetrazolium chloride and radioactive microspheres, respectively. Four experimental groups were studied: vehicle control (50% DMSO in normal saline), IB-MECA (100 μg/kg iv bolus) given 10 min before the coronary occlusion, IB-MECA (100 μg/kg iv bolus) given 5 min before initiation of reperfusion, and IB-MECA (100 μg/kg iv bolus) given 10 min before coronary occlusion in dogs pretreated 15 min earlier with the ATP-dependent potassium channel antagonist glibenclamide (0.3 mg/kg iv bolus). Administration of IB-MECA had no effect on any hemodynamic parameter measured including heart rate, first derivative of left ventricular pressure, aortic pressure, LAD coronary blood flow, or coronary collateral blood flow. Nevertheless, pretreatment with IB-MECA before coronary occlusion produced a marked reduction in infarct size (∼40% reduction) compared with the control group (13.0 ± 3.2% vs. 25.2 ± 3.7% of the area at risk, respectively). This effect of IB-MECA was blocked completely in dogs pretreated with glibenclamide. An equivalent reduction in infarct size was observed when IB-MECA was administered immediately before reperfusion (13.1 ± 3.9%). These results are the first to demonstrate efficacy of an A3AR agonist in a large animal model of myocardial infarction by mechanisms that are unrelated to changes in hemodynamic parameters and coronary blood flow. These data also demonstrate in an in vivo model that IB-MECA is effective as a cardioprotective agent when administered at the time of reperfusion.
Hypoxia-induced cleavage of caspase-3 and DFF45/ICAD in human failed cardiomyocytes
Authors: Todor Anastassia , Sharov Victor G. , Tanhehco Elaine J. , Silverman Norman , Bernabei Alvise , Sabbah Hani N.
Published: March 2015

It has been proposed that the hemodynamic deterioration associated with heart failure (HF) may be due in part to ongoing loss of viable cardiac myocytes through apoptosis. Hypoxia has been shown to promote apoptosis in normal cardiomyocytes. Adaptation and maladaptations inherent to heart failure can modify the susceptibility of cells to different stress factors. We hypothesized that HF modifies the threshold of cardiomyocytes to hypoxia-induced apoptosis. Cardiomyocytes were isolated from 18 human hearts explanted at the time of cardiac transplantation due to either ischemic cardiomyopathy (ICM) ( n = 9) or idiopathic dilated cardiomyopathy (IDC) ( n = 9). Tissue from five normal donor hearts (NL) for whom no suitable recipient was available was used as control. Cardiomyocytes were incubated for 3 h under normoxic (95% air-5% CO2) or hypoxic (95% N2-5% CO2) conditions. Expression of caspase-3 and DNA fragmentation factor-45 (DFF45)/inhibitor of caspase-3-activated DNase (ICAD) was detected by Western blot analysis. Three hours of hypoxia did not affect the expression of these proteins in NL cardiomyocytes. In contrast, hypoxia led to cleavage of caspase-3 and DFF45/ICAD both in ICM and IDC. In conclusion, failing cardiomyocytes exhibit increased susceptibility to hypoxia-induced apoptosis.
Activation of δ- and κ-opioid receptors by opioid peptides protects cardiomyocytes via KATPchannels
Authors: Cao Zhiping , Liu Lijuan , Van Winkle Donna M.
Published: March 2015

To examine the receptor specificity and the mechanism of opioid peptide-induced protection, we examined freshly isolated adult rabbit cardiomyocytes subjected to simulated ischemia. Cell death as a function of time was assessed by trypan blue permeability. Dynorphin B (DynB) and Met5-enkephalin (ME) limitation of cell death (expressed as area under the curve) was sensitive to blockade by naltrindole (NTI, a δ-selective antagonist) and 5′-guanidinyl-17-(cyclopropylmethyl)-6,7-dehydro-4,5α-epoxy-3,14-dihydroxy-6,7-2′,3′-indolomorphinan (GNTI dihydrochloride, a κ-selective antagonist): 85.7 ± 2.7 and 142.9 ± 2.7 with DynB and DynB + NTI, respectively ( P < 0.001), 94.1 ± 4.2 and 164.5 ± 7.3 with DynB and DynB + GNTI, respectively ( P < 0.001), 111.9 ± 7.0 and 192.1 ± 6.4 with ME and ME + NTI, respectively ( P < 0.001), and 120.2 ± 4.3 and 170.0 ± 3.3 with ME and ME + GNTI, respectively ( P < 0.001). Blockade of ATP-sensitive K+channels eliminated DynB- and ME-induced protection: 189.6 ± 5.4 and 139.0 ± 5.4 for control and ME, respectively ( P < 0.001), and 210 ± 5.9 and 195 ± 6.1 for 5-HD and ME + 5-HD, respectively ( P < 0.001); 136.0 ± 5.7 and 63.4 ± 5.4 for control and ME, respectively ( P < 0.001), and 144.6 ± 4.5 and 114.6 ± 7.7 for HMR-1098 and ME + HMR-1098, respectively ( P < 0.01); 189.6 ± 5.4 and 139.0 ± 5.4 for control and ME, respectively ( P < 0.001), and 210 ± 5.9 and 195 ± 6.1 for 5-HD and ME + 5-HD, respectively ( P < 0.001); and 136.0 ± 5.7 and 63.4 ± 5.4 for control and ME, respectively ( P < 0.001), and 144.6 ± 4.5 and 114.6 ± 7.7 for HMR-1098 and ME + HMR-1098, respectively ( P < 0.01). We conclude that opioid peptide-induced cardioprotection is mediated by δ- and κ-receptors and involves sarcolemmal and mitochondrial ATP-sensitive K+channels.
Pattern formation of vascular smooth muscle cells subject to nonuniform fluid shear stress: mediation by gradient of cell density
Authors: Liu Shu Q. , Tang Dalin , Tieche Christopher , Alkema Paul K.
Published: March 2015

Smooth muscle cells (SMCs) are organized in various patterns in blood vessels. Whereas straight blood vessels mainly contain circumferentially aligned SMCs, curved blood vessels are composed of axially aligned SMCs in regions with vortex blood flow. The vortex flow-dependent feature of SMC alignment suggests a role for nonuniform fluid shear stress in regulating the pattern formation of SMCs. Here, we demonstrate that, in experimental models with vascular polymer implants designed for the observation of neointima formation and SMC migration under defined fluid shear stress, nonuniform shear stress possibly plays a role in regulating the direction of SMC migration and alignment in the neointima of the vascular implant. It was found that fluid shear stress inhibited cell growth, and the presence of nonuniform shear stress influenced the distribution of total cell density and induced the formation of cell density gradients, which in turn directed SMC migration and alignment. In contrast, uniform fluid shear stress in a control model influenced neither the distribution of total cell density nor the direction of SMC migration and alignment. In both the uniform and nonuniform shear models, the gradient of total cell density was consistent with the alignment of SMCs. These observations suggest that nonuniform shear stress may regulate the pattern formation of SMCs, possibly via mediating the gradient of cell density in the neointima of vascular polymer implants.
Pharmacological preconditioning with resveratrol: role of nitric oxide
Authors: Hattori Reiji , Otani Hajime , Maulik Nilanjana , Das Dipak K.
Published: March 2015

Resveratrol ( trans-3,4′,5-trihydroxystilbene), a recently described grape-derived polyphenolic antioxidant, has been found to protect the heart from ischemic-reperfusion injury. The present study sought to determine the mechanism of cardioprotection by investigating the ability of resveratrol to precondition the heart. Isolated perfused rat hearts were randomly divided into six groups: group Iwas perfused for 15 min with Kreb-Henseleit buffer (KHB) only; group II was perfused with 10 μM resveratrol; group III was perfused with 10 μM resveratrol plus 100 μM N G-nitro-l-arginine methyl ester (l-NAME), a nonselective nitric oxide (NO) synthase (NOS) inhibitor; group IV was perfused with 10 μM resveratrol plus 100 μM aminoguanidine (AG), an inducible NOS (iNOS) blocker; and groups V and VI consisted of hearts perfused withl-NAME and AG, respectively. The perfusion was then switched to working mode, and all hearts were made globally ischemic for 30 min followed by 2 h of reperfusion. Preconditioning of the hearts with resveratrol provided cardioprotection as evidenced by improved postischemic ventricular functional recovery (developed pressure and aortic flow) and reduced myocardial infarct size and cardiomyocyte apoptosis. Resveratrol-mediated cardioprotection was completely abolished by both l-NAME and AG. In a separate study, hearts were examined for iNOS mRNA induction. Resveratrol caused an induction of the expression of iNOS mRNA beginning at 30 min after reperfusion, increasing steadily up to 60 min of reperfusion, and then decreasing progressively up to 2 h after reperfusion. Preperfusion of the hearts with AG almost completely blocked the induction of iNOS. The results of our study demonstrate that resveratrol can pharmacologically precondition the heart in a NO-dependent manner.
Reactive oxygen species modulate coronary wall shear stress and endothelial function during hyperglycemia
Authors: Gross Eric R. , LaDisa John F. , Weihrauch Dorothee , Olson Lars E. , Kress Tobias T. , Hettrick Douglas A. , Pagel Paul S. , Warltier David C. , Kersten Judy R.
Published: March 2015

Hyperglycemia is associated with generation of reactive oxygen species (ROS), and this action may contribute to accelerated atherogenesis. We tested the hypothesis that hyperglycemia produces alterations in left anterior descending coronary artery (LAD) wall shear stress concomitant with endothelial dysfunction and ROS production in dogs ( n = 12) instrumented for measurement of LAD blood flow, velocity, and diameter. Dogs were randomly assigned to receive vehicle (0.9% saline) or the superoxide dismutase mimetic 4- hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (tempol) and were administered intravenous infusions of d-glucose to achieve target blood glucose concentrations of 350 and 600 mg/dl (moderate and severe hyperglycemia, respectively). Endothelial function and ROS generation were assessed by coronary blood flow responses to acetylcholine (10, 30, and 100 ng/kg) and dihydroethidium fluorescence of myocardial biopsies, respectively. Indexes of wall shear stress were calculated with conventional fluid dynamics theory. Hyperglycemia produced dose-related endothelial dysfunction, increases in ROS production, and reductions in oscillatory shear stress that were normalized by tempol. The results suggest a direct association between hyperglycemia-induced ROS production, endothelial dysfunction, and decreases in oscillatory shear stress in vivo.
Novel complexes of guanylate cyclase with heat shock protein 90 and nitric oxide synthase
Authors: Venema Richard C. , Venema Virginia J. , Ju Hong , Harris M. Brennan , Snead Connie , Jilling Tamas , Dimitropoulou Christiana , Maragoudakis Michael E. , Catravas John D.
Published: March 2015

Soluble guanylate cyclase (sGC) is an important downstream intracellular target of nitric oxide (NO) that is produced by endothelial NO synthase (eNOS) and inducible NO synthase (iNOS). In this study, we demonstrate that sGC exists in a complex with eNOS and heat shock protein 90 (HSP90) in aortic endothelial cells. In addition, we show that in aortic smooth muscle cells, sGC forms a complex with HSP90. Formation of the sGC/eNOS/HSP90 complex is increased in response to eNOS-activating agonists in a manner that depends on HSP90 activity. In vitro binding assays with glutathione S-transferase fusion proteins that contain the α- or β-subunit of sGC show that the sGC β-subunit interacts directly with HSP90 and indirectly with eNOS. Confocal immunofluorescent studies confirm the subcellular colocalization of sGC and HSP90 in both endothelial and smooth muscle cells. Complex formation of sGC with HSP90 facilitates responses to NO donors in cultured cells (cGMP accumulation) as well as in anesthetized rats (hypotension). These complexes likely function to stabilize sGC as well as to provide directed intracellular transfer of NO from NOS to sGC, thus preventing inactivation of NO by superoxide anion and formation of peroxynitrite, which is a toxic molecule that has been implicated in the pathology of several vascular diseases.
Novel 3D culture system for study of cardiac myocyte development
Authors: Evans Heather J. , Sweet Janea K. , Price Robert L. , Yost Michael , Goodwin Richard L.
Published: March 2015

Insufficient myocardial repair after pathological processes contributes to cardiovascular disease, which is a major health concern. Understanding the molecular mechanisms that regulate the proliferation and differentiation of cardiac myocytes will aid in designing therapies for myocardial repair. Models are needed to delineate these molecular mechanisms. Here we report the development of a model system that recapitulates many aspects of cardiac myocyte differentiation that occur during early cardiac development. A key component of this model is a novel three-dimensional tubular scaf-fold engineered from aligned type I collagen strands. In this model embryonic ventricular myocytes undergo a transition from a hyperplastic to a quiescent phenotype, display significant myofibrillogenesis, and form critical cell-cell connections. In addition, embryonic cardiac myocytes grown on the tubular substrate have an aligned phenotype that closely resembles in vivo neonatal ventricular myocytes. We propose that embryonic cardiac myocytes grown on the tube substrate develop into neonatal cardiac myocytes via normal in vivo mechanisms. This model will aid in the elucidation of the molecular mechanisms that regulate cardiac myocyte proliferation and differentiation, which will provide important insights into myocardial development.
Paradoxical overexpression and translocation of connexin43 in homocysteine-treated endothelial cells
Authors: Li Hong , Brodsky Sergey , Kumari Sindu , Valiunas Virginijus , Brink Peter , Kaide Jun-Ichi , Nasjletti Alberto , Goligorsky Michael S.
Published: March 2015

Hyperhomocysteinemia is an established cause of defective vasorelaxation. Gene expression screening of human umbilical vein endothelial cells (HUVEC) treated with homocysteine (Hcy) revealed that connexin43 (Cx43) was upregulated. Expression of Cx43 was increased more than twofold in Hcy-treated HUVEC. Gap junctional communication (Lucifer yellow and whole cell patch clamp) was not enhanced in Hcy-treated HUVEC. HUVEC expressing chimeric Cx43-green fluorescent protein exhibited it at cell-cell contacts in control but showed redistribution to the intracellular compartment(s) in Hcy-treated cells. Confocal microscopy of HUVEC stained with anti-Cx43, mitochondrial, and endoplasmic reticulum fluorescent markers showed the localization of Cx43 to the plasma membrane of control cells and its colocalization with the mitochondrial marker in Hcy-treated HUVEC. Studies of isolated mitochondria confirmed overexpression of Cx43 in the mitochondria of Hcy-treated HUVEC. Microdissected renal interlobar arteries, which normally exhibit endothelium-derived hyperpolarizing factor-induced vasorelaxation, showed reduced nitric oxide synthase- and cyclooxygenase-independent vasorelaxation to acetylcholine after pretreatment with Hcy. In summary, Hcy-induced upregulation of Cx43 transcript and protein expression are associated with unaltered intercellular communication, redistribution of Cx43 in HUVEC, and reduced nitric oxide- and prostanoid-independent vascular responses to acetylcholine in Hcy-treated arteries.
Tyrosine kinases regulate intracellular calcium during α2-adrenergic contraction in rat aorta
Authors: Carter Rebecca W. , Kanagy Nancy L.
Published: March 2015

We have demonstrated enhanced contractile sensitivity to the α2-adrenoreceptor (α2-AR) agonist UK-14304 in arteries from rats made hypertensive with chronic nitric oxide synthase (NOS) inhibition (LHR) compared with arteries from normotensive rats (NR); additionally, this contraction requires Ca2+entry. We hypothesized that tyrosine kinases augment α2-AR contraction in LHR arteries by increasing Ca2+. The tyrosine kinase inhibitor tyrphostin 23 significantly attenuated UK-14304 contraction of denuded thoracic aortic rings from NR and LHR. However, tyrphostin 23 did not alter UK-14304 contraction in ionomycin-permeabilized aorta, which indicates that tyrosine kinases regulate intracellular Ca2+concentration. The Src family inhibitor PP1 and the epidermal growth factor receptor kinase inhibitor AG-1478 did not alter α2-AR contraction, whereas the mitogen-activated protein kinase extracellular signal-regulated kinase kinase inhibitor PD-98059 attenuated the contraction. Contraction to CaCl2in ionomycin-permeabilized LHR rings was greater than in NR rings. UK-14304 augmented CaCl2contraction in ionomycin-permeabilized rings from both groups but to a greater extent in LHR aorta. Together, these data suggest that α2-AR stimulates contraction via two pathways. One, which is enhanced with NOS inhibition hypertension, activates Ca2+sensitivity and is independent of tyrosine kinases. The other is tyrosine kinase dependent and regulates intracellular Ca2+concentration.
Ca2+oscillations, gradients, and homeostasis in vascular smooth muscle
Authors: Lee Cheng-Han , Poburko Damon , Kuo Kuo-Hsing , Seow Chun Yong , van Breemen Cornelis
Published: March 2015

Vascular smooth muscle shows both plasticity and heterogeneity with respect to Ca2+signaling. Physiological perturbations in cytoplasmic Ca2+concentration ([Ca2+]i) may take the form of a uniform maintained rise, a transient uniform [Ca2+]ielevation, a transient localized rise in [Ca2+]i(also known as spark and puff), a transient propagated wave of localized [Ca2+]ielevation (Ca2+wave), recurring asynchronous Ca2+waves, or recurring synchronized Ca2+waves dependent on the type of blood vessel and the nature of stimulation. In this overview, evidence is presented which demonstrates that interactions of ion transporters located in the membranes of the cell, sarcoplasmic reticulum, and mitochondria form the basis of this plasticity of Ca2+signaling. We focus in particular on how the junctional complexes of plasmalemma and superficial sarcoplasmic reticulum, through the generation of local cytoplasmic Ca2+gradients, maintain [Ca2+]ioscillations, couple these to either contraction or relaxation, and promote Ca2+cycling during homeostasis.
15-Deoxy-Δ12,14-prostaglandin J2 and laminar fluid shear stress stabilize c-IAP1 in vascular endothelial cells
Authors: Taba Yoji , Miyagi Megumi , Miwa Yoshikazu , Inoue Hiroyasu , Takahashi-Yanaga Fumi , Morimoto Sachio , Sasaguri Toshiyuki
Published: March 2015

Laminar shear stress strongly inhibits vascular endothelial cell apoptosis by unknown mechanisms. We reported that shear stress stimulates endothelial cells to produce 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) by elevating the expression level of lipocalin-type prostaglandin D synthase. To investigate the role of 15d-PGJ2 produced in the vascular wall, we examined the effect of 15d-PGJ2 on endothelial cell apoptosis. We induced apoptosis in human umbilical vein endothelial cells (HUVECs) by growth factor deprivation. 15d-PGJ2 strongly inhibited DNA ladder formation, nuclear fragmentation, and caspase-3-like activity in HUVECs. To elucidate the mechanism by which 15d-PGJ2 inhibits endothelial cell apoptosis, we examined expression of the inhibitor of apoptosis proteins (IAP) cellular-IAP1 (c-IAP1), c-IAP2, x-linked IAP, and survivin in HUVECs. In parallel with the inhibition of apoptosis, 15d-PGJ2 elevated the expression level of c-IAP1 protein in a dose- and time-dependent manner without changing the mRNA level. Laminar shear stress also induced c-IAP1 expression. Chase experiments with the use of cycloheximide revealed that 15d-PGJ2 and shear stress both inhibited the proteolytic degradation of c-IAP1 protein. These results suggested that 15d-PGJ2 inhibits endothelial cell apoptosis through, at least in part, c-IAP1 protein stabilization. This mechanism might be involved in the antiapoptotic effect of laminar shear stress.
Regulation of CO production in cerebral microvessels of newborn pigs
Authors: Leffler Charles W. , Balabanova Liliya , Sullivan C. Dale , Wang Xiaohu , Fedinec Alexander L. , Parfenova Helena
Published: March 2015

Carbon monoxide (CO) is produced from heme by heme oxygenase-2 (HO-2) in cerebral blood vessels. Gas chromatography-mass spectrometry was used on piglet cerebral microvessels to address the hypothesis that CO production is regulated by heme delivery and HO-2 catalytic activity. CO production appears to be substrate limited because heme and its precursor aminolevulinate increase CO production. Ionomycin also increases CO production. However, CO production from exogenous heme was the same in Ca-replete medium, Ca-free medium with ionomycin, and Ca-replete medium with ionomycin. Phorbol myristate acetate increases CO production but does not change the catalytic activity of HO-2. Also, the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine has no effect on the HO-2 catalytic activity. Protein tyrosine kinase inhibition reduces HO-2 catalytic activity. Inhibition of protein tyrosine phosphatases increased HO-2 catalytic activity. Therefore, regulation of CO production by cerebral microvessels can include changing heme availability and HO-2 catalytic activity. HO-2 catalytic activity is stimulated by tyrosine phosphorylation.
Hypoxia and acidosis impair cGMP synthesis in microvascular coronary endothelial cells
Authors: Agulló Luis , Garcia-Dorado David , Escalona Noelia , Inserte Javier , Ruiz-Meana Marisol , Barrabés José A. , Mirabet Maribel , Pina Pilar , Soler-Soler Jordi
Published: March 2015

To characterize the effects of ischemia on cGMP synthesis in microvascular endothelium, cultured endothelial cells from adult rat hearts were exposed to hypoxia or normoxia at pH 6.4 or 7.4. Cellular cGMP and soluble (sGC) and membrane guanylyl cyclase (mGC) activities were measured after stimulation of sGC ( S-nitroso- N-acetyl-penicillamine) or mGC (urodilatin) or after no stimulation. Cell death (lactate dehydrogenase release) was negligible in all experiments. Hypoxia at pH 6.4 induced a rapid ∼90% decrease in cellular cGMP after sGC and mGC stimulation. This effect was reproduced by acidosis. Hypoxia at pH 7.4 elicited a less pronounced (∼50%) and slower reduction in cGMP synthesis. Reoxygenation after 2 h of hypoxia at either pH 6.4 or 7.4 normalized the response to mGC stimulation but further deteriorated the sGC response; normalization of pH rapidly reversed the effects of acidosis. At pH 7.4, the response to GC stimulation correlated well with cellular ATP. We conclude that simulated ischemia severely depresses cGMP synthesis in microvascular coronary endothelial cells through ATP depletion and acidosis without intrinsic protein alteration.
EDHF, but not NO or prostaglandins, is critical to evoke a conducted dilation upon ACh in hamster arterioles
Authors: Hoepfl Bernd , Rodenwaldt Barbara , Pohl Ulrich , de Wit Cor
Published: March 2015

Vasomotor reactions upon focal stimulation of arterioles have been shown to be conducted along the vascular wall. Such a conduction, which is assumed to reflect the spread of electrical signals, may contribute to coordination of responses within a vascular segment. We aimed to identify which endothelial autacoid(s) act as mediators of the local and conducted dilator responses, respectively. To this end, arterioles in the hamster cremaster microcirculation were locally stimulated with endothelium-dependent [acetylcholine (ACh)] or endothelium-independent dilators [sodium nitroprusside (SNP)], and the resulting changes in diameter were measured using a videomicroscopy technique at the site of application and up to 1.4 mm upstream at distant sites. Experiments were also performed after blockade of nitric oxide (NO) synthase, cyclooxygenase, P-450 monooxygenase, or K+ channels. Dilations upon ACh (71 ± 3%) were conducted rapidly (<1 s) to upstream sites (at 1.4 mm: 37 ± 5%). Although the NO donor SNP induced a similar local dilation (71 ± 7%), this response was not conducted. Maximal amplitudes of ACh-induced dilations were not attenuated after inhibition of NO synthase and cyclooxygenase at the local and remote sites. However, additional treatment with a P-450 monooxygenase blocker (sulfaphenazole) strongly attenuated the local response (from 62 ± 9 to 17 ± 5%) and abrogated dilations at distant sites (at 0.67 mm: from 23 ± 4% to 4 ± 3%). Likewise, 17-octadecynoic acid strongly attenuated local and remote responses. Blockers of Ca2+-dependent K+ channels (charybdotoxin or iberiotoxin) attenuated dilations at the local and remote sites after focal application at the ACh stimulation site. In marked contrast, treatment of the upstream site with these blockers was without any effect. We conclude that upon local stimulation with ACh, a cytochrome P-450 monooxygenase product is generated that induces local dilation via the activation of Ca2+-dependent K+ channels and initiates conduction of the dilation. In contrast to the local site, neither activation of these K+channels nor the synthesis of NO or prostaglandins is necessary to dilate the arterioles at remote, distant sites. This suggests that endothelium-derived hyperpolarizing factor serves as an important mediator to initiate conducted dilations and, by doing so, may act as a key player in the coordination of arteriolar behavior in the microcirculatory network.
Role of USF1 phosphorylation on cardiac α-myosin heavy chain promoter activity
Authors: Xiao Qianxun , Kenessey Agnes , Ojamaa Kaie
Published: March 2015

Contractile activity of the cardiac myocyte is required for maintaining cell mass and phenotype, including expression of the cardiac-specific α-myosin heavy chain (α-MHC) gene. An E-box hemodynamic response element (HME) located at position −47 within the α-MHC promoter is both necessary and sufficient to confer contractile responsiveness to the gene and has been shown to bind upstream stimulatory factor-1 (USF1). When studied in spontaneously contracting cardiac myocytes, there is enhanced binding of USF1 to the HME compared with quiescent cells, which correlates with a threefold increase in α-MHC promoter activity. A molecular mechanism by which contractile function modulates α-MHC transcriptional activity may involve signaling via phosphorylation of USF1. The present studies showed that purified rat USF1 was phosphorylated in vitro by protein kinase C (PKC) and cAMP-dependent protein kinase (PKA) but not casein kinase II. Phosphorylated USF1 by either PKC or PKA had increased DNA binding activity to the HME. PKC-mediated phosphorylation also leads to the formation of USF1 multimers as assessed by gel shift assay. Analysis of in vivo phosphorylated nuclear proteins from cultured ventricular myocytes showed that USF1 was phosphorylated, and resolution by two-dimensional gel electrophoresis identified at least two distinct phosphorylated USF1 molecules. These results suggest that endogenous kinases can covalently modify USF1 and provide a potential molecular mechanism by which the contractile stimulus mediates changes in myocyte gene transcription.
Adenosine A1 receptor mediates late preconditioning via activation of PKC-δ signaling pathway
Authors: Kudo Mitsuhiro , Wang Yigang , Xu Meifeng , Ayub Ahmar , Ashraf Muhammad
Published: March 2015

Protein kinase C (PKC) plays a central role in both early and late preconditioning (PC) but its association with inducible nitric oxide synthase (iNOS) is not clear in late PC. This study investigates the PKC signaling pathway in the late PC induced by activation of adenosine A1 receptor (A1R) with adenosine agonist 2-chloro- N 6-cyclopentyladenosine (CCPA) and the effect on iNOS upregulation. Adult male mice were pretreated with saline or CCPA (100 μg/kg iv) or CCPA (100 μg/kg iv) with PKC-δ inhibitor rottlerin (50 μg/kg ip). Twenty-four hours later, the hearts were isolated and perfused in the Langendorff mode. Hearts were subjected to 40 min of ischemia, followed by 30 min reperfusion. After ischemia, the left ventricular end-diastolic pressure (LVEDP) was significantly improved and the rate-pressure product (RPP) was significantly higher in the CCPA group compared with the ischemia-reperfusion (I/R) control group. Creatine kinase release and infarct size were significantly lower in the CCPA group compared with the I/R control group. These salutary effects of CCPA were abolished in hearts pretreated with rottlerin. Immunoblotting of PKC showed that PKC-δ was upregulated (150.0 ± 11.4% of control group) whereas other PKC isoforms remained unchanged, and iNOS was also significantly increased (146.2 ± 9.0%, P < 0.05 vs. control group) after 24 h of treatment with CCPA. The data show that PKC is an important component of PC with adenosine agonist. It is concluded that activation of A1R induces late PC via PKC-δ and iNOS signaling pathways.
Urocortin protects the heart from reperfusion injury via upregulation of p42/p44 MAPK signaling pathway
Authors: Schulman Daniel , Latchman David S. , Yellon Derek M.
Published: March 2015

Reperfusion of ischemic myocardium is essential for tissue salvage but paradoxically contributes to cell death. We hypothesized that activation of potential survival pathways such as p42/p44 MAPK may prevent lethal reperfusion injury. Urocortin is a peptide factor that affects the p42/p44 MAPK signaling pathway. Both isolated and in vivo rat heart models were used to examine the potential for urocortin to prevent reperfusion injury. Isolated rat hearts underwent 35-min regional ischemia and 2-h reperfusion, with urocortin perfused for 20 min from the onset of reperfusion. In the in vivo study, urocortin was administered as an intravenous bolus 3 min before reperfusion with a protocol of 25-min regional ischemia and 2-h reperfusion. Blockade of the p42/p44 MAPK pathway with the inhibitor PD-98059 was used in both models. Urocortin attenuated lethal reperfusion-induced injury both in vitro and in vivo via a p42/p44 MAPK-dependent mechanism. Furthermore, Western blot analysis demonstrated the ability of urocortin to directly upregulate this signaling pathway. In conclusion, we believe that the p42/p44 MAPK-dependent signaling pathway represents an important survival mechanism against reperfusion injury.
Presence of cellular renin-angiotensin system in chromaffin cells of bovine adrenal medulla
Authors: Wang Jun Ming , Slembrouck Dirk , Tan Junhui , Arckens Lut , Leenen Frans H. H. , Courtoy Pierre J. , De Potter Werner P.
Published: March 2015

The presence of a local renin-angiotensin system has been established in organs that serve as angiotensin targets. In this study, the expression of angiotensinogen mRNA and subcellular localization of renin, angiotensin-converting enzyme, and angiotensin II were investigated in bovine adrenal medullary cells in primary culture. By light microscopy, expression of angiotensinogen mRNA, immunoreactive renin, angiotensin-converting enzyme, and angiotensin II were readily detectable only in the chromaffin cells. The density distribution of renin and angiotensin II in sucrose gradients suggested a concentration in chromaffin granules, a localization directly confirmed by immunoelectron microscopy. Reverse transcriptase-polymerase chain reaction and sequencing confirmed the expression of angiotensinogen in bovine chromaffin cells and the adrenal medulla. In addition, in vitro autoradiography indicated that both angiotensin-converting enzyme and angiotensin type 1 receptors were present in the adrenal medulla. These results provide the first direct evidence that chromaffin cells in the adrenal medulla are not only the target for angiotensin but should also be considered as potential local angiotensin-generating and -storing cells.
pH-induced changes in calcium: functional consequences and mechanisms of action in guinea pig portal vein
Authors: Smith R. D. , Eisner D. A. , Wray Susan
Published: March 2015

The effects of changing extracellular (pHo) and intracellular pH (pHi) on force and the mechanisms involved in the guinea pig portal vein were investigated to better understand the control of tone in this vessel. When pHo was altered, the effects on force and calcium were the same irrespective of whether force had been produced spontaneously by high-K depolarization or by norepinephrine; alkalinization increased tone, and acidification reduced it. Because pHo changes also lead to changes in pHi, we determined whether the effects on force could be explained by these induced pHi changes. It was found, however, that only with spontaneous activity did intracellular alkalinization increase force. In depolarized preparations, force was decreased, and, with norepinephrine, force was initially decreased and then increased. Thus the effects of pHo cannot be explained solely by changes in pHi. The role of the sarcoplasmic reticulum (SR) and surface membrane Ca2+-ATPase on the mechanism were investigated and shown not to be involved. Therefore, it is concluded that both pHo and pHi can have powerful modulatory effects on portal vein tone, that these effects are not identical, and that they are likely to be due to effects of pH on ion channels rather than the SR or plasma membrane Ca2+-ATPase.
Development of electrical activity in cardiac myocyte aggregates derived from mouse embryonic stem cells
Authors: Banach K. , Halbach M. D. , Hu P. , Hescheler J. , Egert U.
Published: March 2015

Embryonic stem cells differentiate into cardiac myocytes, repeating in vitro the structural and molecular changes associated with cardiac development. Currently, it is not clear whether the electrophysiological properties of the multicellular cardiac structure follow cardiac maturation as well. In long-term recordings of extracellular field potentials with microelectrode arrays consisting of 60 substrate-integrated electrodes, we examined the electrophysiological properties during the ongoing differentiation process. The beating frequency of the growing preparations increased from 1 to 5 Hz concomitant to a decrease of the action potential duration and action potential rise time. A developmental increase of the conduction velocity could be attributed to an increased expression of connexin43 gap junction channels. Whereas isoprenalin elicited a positive chronotropic response from the first day of spontaneous beating onward, a concentration-dependent negative chronotropic effect of carbachol only developed after ∼4 days. The in vitro development of the three-dimensional cardiac preparation thus closely follows the development described for the mouse embryonic heart, making it an ideal model to monitor the differentiation of electrical activity in embryonic cardiomyocytes.
Nitric oxide-induced inhibition of aortic smooth muscle cell motility: role of PTP-PEST and adaptor proteins p130cas and Crk
Authors: Lin Yi , Ceacareanu Alice Corina , Hassid Aviv
Published: March 2015

Vascular injury increases nitric oxide (NO) levels, and this effect may play a counterregulatory role in neointima formation, by decreasing vascular smooth muscle cell motility. However, the mechanisms underlying this effect are not well established. We tested the hypothesis that NO decreases cell motility by increasing the activity of a protein tyrosine phosphatase (PTP), PTP-PEST, in cultured rat aortic smooth muscle cells. Two NO donors increased the activity of PTP-PEST. A cGMP analog mimicked the effect of NO, whereas a guanyl cyclase inhibitor blocked it, indicating that elevated cGMP is both necessary and sufficient to induce PTP-PEST activity. Overexpression of wild-type PTP-PEST induced antimotogenesis, whereas expression of dominant negative PTP-PEST blocked the antimotogenic effect of NO, indicating that increased PTP-PEST activity is both sufficient and necessary to explain the effect of NO. Overexpression of PTP-PEST mimicked NO-induced dephosphorylation of adapter protein p130cas, whereas dominant negative PTP-PEST blocked the effect of NO, indicating that upregulation of PTP-PEST is both necessary and sufficient to explain NO-induced p130cas dephosphorylation. Expression of a substrate domain-deleted p130cas decreased motogenesis, whereas overexpression of wild-type p130cas blocked the antimotogenic effect of NO, indicating the functional importance of p130cas dephosphorylation. NO induced dissociation of the Cas-Crk complex, an effect that was mimicked by overexpression of PTP-PEST and opposed by expression of dominant negative PTP-PEST. Our results indicate that NO decreases aortic smooth muscle cell motility via a cGMP-mediated mechanism, involving upregulation of PTP-PEST, in turn inducing dephosphorylation of p130cas, and likely involving Cas-Crk dissociation as a downstream event.
Angiotensin II modulates nitric oxide-induced cardiac fibroblast apoptosis by activation of AKT/PKB
Authors: Tian Bin , Liu Jian , Bitterman Peter , Bache Robert J.
Published: March 2015

Previously we found that interleukin-1β (IL-1β)-activated inducible nitric oxide (NO) synthase (iNOS) expression and that NO production can trigger cardiac fibroblast (CFb) apoptosis. Here, we provide evidence that angiotensin II (ANG II) significantly attenuated IL-1β-induced iNOS expression and NO production in CFbs while simultaneously decreasing apoptotic frequency. The anti-apoptotic effect of ANG II was abolished when cells were pretreated with the specific ANG II type 1 receptor (AT1) antagonist losartan, but not by the AT2 antagonist DP-123319. Furthermore, ANG II also protected CFbs from apoptosis induced by the NO donor diethylenetriamine NONOate and this effect was associated with phosphorylation of Akt/protein kinase B at Ser473. The effects of ANG II on Akt phosphorylation and NO donor-induced CFb apoptosis were abrogated when cells were preincubated with the specific phosphatidylinositol 3-kinase inhibitors wortmannin or LY-294002. These data demonstrate that ANG II protection of CFbs from IL-1β-induced apoptosis is associated with downregulation of iNOS expression and requires an intact phosphatidylinositol 3-kinase-Akt survival signal pathway. The findings suggest that ANG II and NO may play a role in regulating the cell population size by their countervailing influences on cardiac fibroblast viability.
PECAM-directed delivery of catalase to endothelium protects against pulmonary vascular oxidative stress
Authors: Christofidou-Solomidou Melpo , Scherpereel Arnaud , Wiewrodt Rainer , Ng Kimmie , Sweitzer Thomas , Arguiri Evguenia , Shuvaev Vladimir , Solomides Charalambos C. , Albelda Steven M. , Muzykantov Vladimir R.
Published: March 2015

Targeted delivery of drugs to vascular endothelium promises more effective and specific therapies in many disease conditions, including acute lung injury (ALI). This study evaluates the therapeutic effect of drug targeting to PECAM (platelet/endothelial cell adhesion molecule-1) in vivo in the context of pulmonary oxidative stress. Endothelial injury by reactive oxygen species (e.g., H2O2) is involved in many disease conditions, including ALI/acute respiratory distress syndrome and ischemia-reperfusion. To optimize delivery of antioxidant therapeutics, we conjugated catalase with PECAM antibodies and tested properties of anti-PECAM/catalase conjugates in cell culture and mice. Anti-PECAM/catalase, but not an IgG/catalase counterpart, bound specifically to PECAM-expressing cells, augmented their H2O2-degrading capacity, and protected them against H2O2toxicity. Anti-PECAM/catalase, but not IgG/catalase, rapidly accumulated in the lungs after intravenous injection in mice, where it was confined to the pulmonary endothelium. To test its protective effect, we employed a murine model of oxidative lung injury induced by glucose oxidase coupled with thrombomodulin antibody (anti-TM/GOX). After intravenous injection in mice, anti-TM/GOX binds to pulmonary endothelium and produces H2O2, which causes lung injury and 100% lethality within 7 h. Coinjection of anti-PECAM/catalase protected against anti-TM/GOX-induced pulmonary oxidative stress, injury, and lethality, whereas polyethylene glycol catalase or IgG/catalase conjugates afforded only marginal protective effects. This result validates vascular immunotargeting as a prospective strategy for therapeutic interventions aimed at immediate protective effects, e.g., for augmentation of antioxidant defense in the pulmonary endothelium and treatment of ALI.
Lung endothelial heparan sulfates mediate cationic peptide-induced barrier dysfunction: a new role for the glycocalyx
Authors: Dull Randal O. , Dinavahi Ramani , Schwartz Lawrence , Humphries Donald E. , Berry David , Sasisekharan Ram , Garcia Joe G. N.
Published: March 2015

The endothelial glycocalyx is believed to play a major role in microvascular permeability. We tested the hypothesis that specific components of the glycocalyx, via cytoskeletal-mediated signaling, actively participate in barrier regulation. With the use of polymers of arginine and lysine as a model of neutrophil-derived inflammatory cationic proteins, we determined size- and dose-dependent responses of cultured bovine lung microvascular endothelial cell permeability as assessed by transendothelial electrical resistance (TER). Polymers of arginine and lysine >11 kDa produced maximal barrier dysfunction as demonstrated by a 70% decrease in TER. Monomers of l-arginine and l-lysine did not alter barrier function, suggesting a cross-linking requirement of cell surface “receptors”. To test the hypothesis that glycosaminoglycans (GAGs) are candidate receptors for this response, we used highly selective enzymes to remove specific GAGs before polyarginine (PA) treatment and examined the effect on TER. Heparinase III attenuated PA-induced barrier dysfunction by 50%, whereas heparinase I had no effect. To link changes in barrier function with structural alterations, we examined actin organization and syndecan localization after PA. PA induced actin stress fiber formation and clustering of syndecan-1 and syndecan-4, which were significantly attenuated by heparinase III. PA-induced cytoskeletal rearrangement and barrier function did not involve myosin light chain kinase (MLCK) or p38 MAPK, as ML-7, a specific MLCK inhibitor, or SB-20358, a p38 MAPK inhibitor, did not alter PA-induced barrier dysfunction. In summary, lung endothelial cell heparan sulfate proteoglycans are key participants in inflammatory cationic peptide-induced signaling that links cytoskeletal reorganization with subsequent barrier dysfunction.
Enhanced calcium signaling to bradykinin in airway smooth muscle from hyperresponsive inbred rats
Authors: Tao F. C. , Shah S. , Pradhan A. A. , Tolloczko B. , Martin J. G.
Published: March 2015

Inbred Fischer 344 rats display airway hyperresponsiveness (AHR) in vivo compared with the normoresponsive Lewis strain. Fischer AHR has been linked with increased airway smooth muscle (ASM) contraction ex vivo and enhanced ASM cell intracellular Ca2+mobilization in response to serotonin compared with Lewis. To determine the generality of this association, we tested whether bradykinin (BK) also stimulates greater contraction of Fischer airways and greater Ca2+mobilization in Fischer ASM cells. Explants of Fischer intraparenchymal airways constricted faster and to a greater degree in response to BK than Lewis airways. BK also evoked higher Ca2+transients in Fischer than in Lewis ASM cells. ASM cell B2receptor expression was similar between the two strains. BK activated both phosphatidylinositide-specific phospholipase C (PI-PLC) and phosphatidylcholine-specific PLC to mobilize Ca2+in Fischer and Lewis ASM cells. PI-PLC activity, as measured by inositol polyphosphate accumulation, was similar in the two strains. PKC inhibition with GF109203X, Go6973, or Go6983 attenuated BK-mediated Ca2+transients in Fischer cells, whereas GF109203X potentiated while Go6976 and Go6983 did not affect Ca2+transients in Lewis cells. Enhanced Ca2+mobilization in ASM cells can arise from variations in PKC and may be an important component of nonspecific, innate AHR.