bluuebunny/crossref_metadata_2025 huggingface dataset:
In vivo expression and function of recombinant GTPCH I in the rabbit carotid artery
Authors: Hynes Sean O. , Smith Leslie A. , Richardson Darcy M. , Kovesdi Imre , O'Brien Timothy , Katusic Zvonimir S.
Published: January 2004

Tetrahydrobiopterin (BH4) is an essential co-factor for endothelial nitric oxide synthase enzymatic activity. GTP cyclohydrolase I (GTPCH I) is the rate-limiting enzyme in BH4 synthesis. This study set out to test the hypothesis that in vivo gene transfer of GTPCH I to endothelial cells could increase bioavailability of BH4, enhance biosynthesis of nitric oxide and thereby enhance endothelium-dependent relaxations mediated by nitric oxide. In vivo gene transfer was carried out by adenovirus (Ad)-mediated delivery into rabbit carotid arteries. Each artery was transduced by 20-min intraluminal incubation of 109plaque-forming units of Ad-encoding GTPCH I (AdGTPCH) or β-galactosidase as a control. The rabbits were euthanized 72 h later, and vasomotor function of isolated arteries was assessed by isometric force recording. GTPCH I enzymatic activity, BH4, and oxidized biopterin levels were detected with the use of HPLC, and cGMP was measured with the use of radioimmunoassay. Expression of recombinant proteins was detected predominantly in endothelial cells. Both GTPCH I activity and BH4 levels were increased in arteries transduced with AdGTPCH. However, contraction to phenylephrine (10–5to 10–9M), endothelium-dependent relaxation to acetylcholine (10–5to 10–9M) and cGMP levels were not significantly affected by increased expression of GTPCH I. Our results suggest that expression of GTPCH I in vascular endothelium in vivo increases intracellular concentration of BH4. However, under physiological conditions, it appears that this increase does not affect nitric oxide production in endothelial cells of the carotid artery.
Phenotypic differences in transient outward K+ current of human and canine ventricular myocytes: insights into molecular composition of ventricular Ito
Authors: Akar Fadi G. , Wu Richard C. , Deschenes Isabelle , Armoundas Antonis A. , Piacentino Valentino , Houser Steven R. , Tomaselli Gordon F.
Published: January 2004

The Ca2+-independent transient outward K+ current ( Ito) plays an important electrophysiological role in normal and diseased hearts. However, its contribution to ventricular repolarization remains controversial because of differences in its phenotypic expression and function across species. The dog, a frequently used model of human cardiac disease, exhibits altered functional expression of Ito. To better understand the relevance of electrical remodeling in dogs to humans, we studied the phenotypic differences in ventricular Ito of both species with electrophysiological, pharmacological, and protein-chemical techniques. Several notable distinctions were elucidated, including slower current decay, more rapid recovery from inactivation, and a depolarizing shift of steady-state inactivation in human vs. canine Ito. Whereas recovery from inactivation of human Ito followed a monoexponential time course, canine Ito recovered with biexponential kinetics. Pharmacological sensitivity to flecainide was markedly greater in human than canine Ito, and exposure to oxidative stress did not alter the inactivation kinetics of Ito in either species. Western blot analysis revealed immunoreactive bands specific for Kv4.3, Kv1.4, and Kv channel-interacting protein (KChIP)2 in dog and human, but with notable differences in band sizes across species. We report for the first time major variations in phenotypic properties of human and canine ventricular Ito despite the presence of the same subunit proteins in both species. These data suggest that differences in electrophysiological and pharmacological properties of Ito between humans and dogs are not caused by differential expression of the K channel subunit genes thought to encode Ito, but rather may arise from differences in molecular structure and/or posttranslational modification of these subunits.
CO modulates pulmonary vascular response to acute hypoxia: relation to endothelin
Authors: Zhang Fan , Kaide Jun Ichi , Yang LiMing , Jiang Houli , Quan Shuo , Kemp Rowena , Gong Weiying , Balazy Michael , Abraham Nader G. , Nasjletti Alberto
Published: January 2004

Pulmonary intralobar arteries express heme oxygenase (HO)-1 and -2 and release carbon monoxide (CO) during incubation in Krebs buffer. Acute hypoxia elicits isometric tension development (0.77 ± 0.06 mN/mm) in pulmonary vascular rings treated with 15 μmol/l chromium mesoporphyrin (CrMP), an inhibitor of HO-dependent CO synthesis, but has no effect in untreated vessels. Acute hypoxia also induces contraction of pulmonary vessels taken from rats injected with HO-2 antisense oligodeoxynucleotides (ODN), which decrease pulmonary HO-2 vascular expression and CO release. Hypoxia-induced contraction of vessels treated with CrMP is attenuated ( P < 0.05) by endothelium removal, by CO (1–100 μmol/l) in the bathing buffer, and by endothelin-1 (ET-1) receptor blockade with L-754142 (10 μmol/l). CrMP increases ET-1 levels in pulmonary intralobar arteries, particularly during incubation in hypooxygenated media. CrMP also causes a leftward shift in the concentration-response curve to ET-1, which is offset by exogenous CO. In anesthetized rats, pretreatment with CrMP (40 μmol/kg iv) intensifies the elevation of pulmonary artery pressure elicited by breathing a hypoxic gas mixture. However, acute hypoxia does not elicit augmentation of pulmonary arterial pressure in rats pretreated concurrently with CrMP and the ET-1 receptor antagonist L-745142 (15 mg/kg iv). These data suggest that a product of HO activity, most likely CO, inhibits hypoxia-induced pulmonary vasoconstriction by reducing ET-1 vascular levels and sensitivity.
Xanthine oxidase and activated neutrophils cause oxidative damage to skeletal muscle after contractile claudication
Authors: Judge A. R. , Dodd S. L.
Published: January 2004

We previously showed oxidative damage and edema within skeletal muscle after contractile claudication. To investigate the sources of this oxidative damage in the gastrocnemius muscle, we administered allopurinol (Allo, to inhibit xanthine oxidase) and cyclophosphamide (Cyclo, to deplete neutrophils) before inducing contractile claudication in male Sprague Dawley rats. Contractile claudication (ligated stimulated, LS) caused a significant increase in xanthine oxidase activity [sham ligated stimulated (SS) = 2.57 ± 0.07; LS = 3.22 ± 0.07] and neutrophil infiltration (SS = 0.47 ± 0.03; LS = 0.91 ± 0.10) compared with controls (SS), and this was associated with increased lipid peroxidation, protein oxidation, muscle damage, and edema. Pretreatment with Allo attenuated the increase in xanthine oxidase activity and attenuated lipid hydroperoxides (control LS = 12.85 ± 0.50; Allo LS = 9.96 ± 0.71), muscle damage, and neutrophil infiltration (control LS = 0.91 ± 0.10; Allo LS = 0.61 ± 0.07). This latter finding suggests that xanthine oxidase-derived oxidants are chemotactic to neutrophils. Pretreatment with Cyclo reduced neutrophil infiltration (control LS = 0.91 ± 0.10; Cyclo LS = 0.55 ± 0.02) and attenuated lipid peroxidation (control LS = 12.85 ± 0.50; Cyclo LS = 6.462 ± 0.62), protein oxidation (control LS = 2.59 ± 0.47; Cyclo LS = 1.77 ± 0.60), muscle damage, and edema. Together, these data indicate that contractile claudication causes an increase in xanthine oxidase activity and neutrophils in muscle and that inhibition of these oxidant sources protects against oxidative stress, muscle damage, and edema.
Vasoactive effects of methylamine in isolated human blood vessels: role of semicarbazide-sensitive amine oxidase, formaldehyde, and hydrogen peroxide
Authors: Conklin D. J. , Cowley H. R. , Wiechmann R. J. , Johnson G. H. , Trent M. B. , Boor P. J.
Published: January 2004

It is hypothesized that methylamine (MA) and semicarbazide-sensitive amine oxidase (SSAO) activity are involved in the cardiovascular complications in human diabetics. To test this, we 1) determined the acute vasoactive effects of MA (1–1,000 μmol/l) in uncontracted and norepinephrine (NE; 1 μmol/l)-precontracted human blood vessels used for coronary artery bypass grafts [left internal mammary artery (LIMA), radial artery (RA), and right saphenous vein (RSV)]; 2) tested whether MA effects in LIMA and RSV were dependent on SSAO activity using the SSAO inhibitor semicarbazide (1 mmol/l, 15 min); 3) determined the effects of MA metabolites formaldehyde and hydrogen peroxide in LIMA and RSV; 4) tested whether the MA response was nitric oxide, prostaglandin, or hyperpolarization dependent; 5) measured the LIMA and RSV cGMP levels after MA exposure; and 6) quantified SSAO activity in LIMA, RA, and RSV. In NE-precontracted vessels, MA stimulated a biphasic response in RA and RSV (rapid contraction followed by prolonged relaxation) and dominant relaxation in LIMA (mean ± SE, %relaxation: 55.4 ± 3.9, n = 30). The MA-induced relaxation in LIMA was repeatable, nontoxic, and age independent. Semicarbazide significantly blocked MA-induced relaxation (%inhibition: 82.5 ± 4.8, n = 7) and SSAO activity (%inhibition: 98.1 ± 1.3, n = 26) in LIMA. Formaldehyde (%relaxation: 37.3 ± 18.6, n = 3) and H2O2(%relaxation: 55.6 ± 9.0, n = 9) at 1 mmol/l relaxed NE-precontracted LIMA comparable with MA. MA-induced relaxation in LIMA was nitric oxide, prostaglandin, and possibly cGMP independent and blocked by hyperpolarization. We conclude that vascular SSAO activity may convert endogenous amines, like MA, to vasoactive metabolites.
Differential modulation of citrate synthesis and release by fatty acids in perfused working rat hearts
Authors: Vincent Geneviève , Bouchard Bertrand , Khairallah Maya , Des Rosiers Christine
Published: January 2004

The objective of this study was to test the effect of increasing fatty acid concentrations on substrate fluxes through pathways leading to citrate synthesis and release in the heart. This was accomplished using semirecirculating work-performing rat hearts perfused with substrate mixtures mimicking the in situ milieu (5.5 mM glucose, 8 nM insulin, 1 mM lactate, 0.2 mM pyruvate, and 0.4 mM oleate-albumin) and13C methods. Raising the fatty acid concentration from 0.4 to 1 mM with long-chain oleate or medium-chain octanoate resulted in a lowering (∼20%) of cardiac output and efficiency with unaltered O2consumption. At the metabolic level, beyond the expected effects of high fatty acid levels on the contribution of pyruvate decarboxylation (reduced >3-fold) and β-oxidation (enhanced ∼3-fold) to citrate synthesis, there was also a 2.4-fold lowering of anaplerotic pyruvate carboxylation. Despite the dual inhibitory effect of high fatty acids on pyruvate decarboxylation and carboxylation, tissue citrate levels were twofold higher, but citrate release rates remained unchanged at 11–14 nmol/min, representing <0.5% of citric acid cycle flux. A similar trend was observed for most metabolic parameters after oleate or octanoate addition. Together, these results emphasize a differential modulation of anaplerotic pyruvate carboxylation and citrate release in the heart by fatty acids. We interpret the lack of effects of high fatty acid concentrations on citrate release rates as suggesting that, under physiological conditions, this process is maximal, probably limited by the activity of its mitochondrial or plasma membrane transporter. Limited citrate release at high fatty acid concentrations may have important consequences for the heart's fuel metabolism and function.
Changes in NO bioavailabilty regulate cardiac O2consumption: control by intramitochondrial SOD2 and intracellular myoglobin
Authors: Li Wei , Jue Thomas , Edwards John , Wang Xipping , Hintze Thomas H.
Published: January 2004

The aim of this study was to investigate the significance of two intracellular scavengers of nitric oxide (NO): 1) superoxide dismutase (SOD) (SOD2) to scavenge intramitochondrial superoxide anion, and 2) cytosolic myoglobin (Mb) in the regulation of tissue O2consumption. O2consumption was measured in vitro using a Clark-type O2electrode. SOD heterozygous mice (SODHZ) ( n = 13) and SOD wild-type (SODWT) ( n = 5) mice were used. Bradykinin (BK, 10–4mol/l) reduced O2consumption by 15% ± 1 in hearts of SODHZ mice, which was significantly different from SODWT (reduced by 24 ± 0.4%). Tiron significantly increased the inhibition of O2consumption by BK in male mice from 15 ± 1% ( n = 13) to 29 ± 1.2% ( n = 4) at 10–4mol/l concentration ( P < 0.05). The effect of carbachol was similar to BK. S-nitroso- N-acetyl penicillamine (SNAP, 10–4mol/l) reduced O2consumption by 39 ± 1.3% in hearts of SODHZ mice, which was not significantly different from SODWT. But at 10–7mol/l, SNAP caused significantly less inhibition of O2consumption in SODHZ mice. Mb knockout (MbKO; Mb wild-type n = 6) and (MbWT) mice ( n = 6) were also used. Kidney cortex was studied as the negative control because it does not contain Mb. BK (10–4mol/l) reduced O2consumption by 32 ± 2, 29 ± 1, and 26 ± 1% in the heart, skeletal muscle, and kidney of MbKO mice, which was also not significantly different from MbWT. SNAP (10–4mol/l) reduced O2consumption by 39 ± 3, 42 ± 4, and 46 ± 2% in the heart, skeletal muscle, and kidney of MbKO mice, which was also not significantly different from MbWT. NG-nitro-l-arginine methyl ester ( P < 0.05) inhibited the reduction in O2consumption induced by BK in the MbKO mouse heart (15 ± 1%), skeletal muscle (17 ± 1%), and kidney (17 ± 1%) as in the MbWT mice. These results suggest that the role of Mb as an intracellular NO scavenger is small, and the increase in mitochondrial superoxide in SODHZ mice may cause a decrease NO bioavailability and alter the control of myocardial O2consumption by NO.
Heat shock protein 90 and tyrosine kinase regulate eNOS NO· generation but not NO· bioactivity
Authors: Ou Jingsong , Fontana Jason T. , Ou Zhijun , Jones Deron W. , Ackerman Allan W. , Oldham Keith T. , Yu Jun , Sessa William C. , Pritchard Kirkwood A.
Published: January 2004

An increase in the association of heat shock protein 90 (HSP90) with endothelial nitric oxide (NO) synthase (eNOS) is well recognized for increasing NO (NO·) production. Despite the progress in this field, the mechanisms by which HSP90 modulates eNOS remain unclear due, in part, to the fact that geldanamycin (GA) redox cycles to generate superoxide anion ([Formula: see text]) and the fact that inhibiting HSP90 with GA or radicicol (RAD) destabilizes tyrosine kinases that rely on the chaperone for maturation. In this report, we determine the extent to which these side effects alter vascular and endothelial cell function in physiologically relevant systems and in cultured endothelial cells. Vascular endothelial growth factor (VEGF)-stimulated vascular permeability, as measured by Evans blue leakage in the ears of male Swiss mice in vivo, and acetylcholine-induced vasodilation of isolated, pressurized mandibular arterioles from male C57BL6 mice ex vivo were attenuated by Nω-nitro-l-arginine methyl ester (l-NAME), GA, and RAD. Z-1[ N-(2-aminoethyl)- N-(2-ammonoethyl)amino]diazen-1-ium-1,2-dioate (DETA-NONOate), a slow releasing NO· donor, increased vasodilation of arterioles pretreated with GA, RAD, and l-NAME equally well except at 10–5 M, the highest concentration used, where vasodilation was greater in pressurized arterioles treated with l-NAME than in arterioles pretreated with GA or RAD alone. Both GA and RAD reduced NO· release from stimulated endothelial cell cultures and increased [Formula: see text] production in the endothelium of isolated aortas by an l-NAME-inhibitable mechanism. Pretreatment with RAD increased stimulated [Formula: see text] production from eNOS, whereas pretreatment with genistein (GE), a broad-spectrum tyrosine kinase inhibitor, did not; however, pretreatment with GE + RAD resulted in a super-induced state of uncoupled eNOS activity upon stimulation. These data suggest that the tyrosine kinases, either directly or indirectly, and HSP90-dependent signaling pathways act in concert to suppress uncoupled eNOS activity.
Differing roles of mitochondrial nitric oxide synthase in cardiomyocytes and urothelial cells
Authors: Kanai Anthony , Epperly Michael , Pearce Linda , Birder Lori , Zeidel Mark , Meyers Susan , Greenberger Joel , de Groat William , Apodaca Gerard , Peterson James
Published: January 2004

The existence of mitochondrial nitric oxide (NO) synthase (mtNOS) has been controversial since it was first reported in 1995. We have addressed this issue by making direct microsensor measurements of NO production in the mitochondria isolated from mouse hearts. Mitochondrial NO production was stimulated by Ca2+and inhibited by blocking electrogenic Ca2+uptake or by using NOS antagonists. Cardiac mtNOS was identified as the neuronal isoform by the absence of NO production in the mitochondria of mice lacking the neuronal but not the endothelial or inducible isoforms. In cardiomyocytes from dystrophin-deficient ( mdx) mice, elevated intracellular Ca2+, increased mitochondrial NO production, slower oxidative phosphorylation, and decreased ATP production were detected. Inhibition of mtNOS increased contractility in mdx but not in wild-type cardiomyocytes, indicating that mtNOS may protect the cells from overcontracting. mtNOS was also implicated in radiation-induced cell damage. In irradiated rat/mouse urinary bladders, we have evidence that mitochondrially produced NO damages the urothelial “umbrella” cells that line the bladder lumen. This damage disrupts the permeability barrier thereby creating the potential to develop radiation cystitis. RT-PCR and Southern blot analyses indicate that mtNOS is restricted to the umbrella cells, which scanning electron micrographs show are selectively damaged by radiation. Simultaneous microsensor measurements demonstrate that radiation increases NO and peroxynitrite (ONOO–) production in these cells, which can be prevented by transfection with manganese superoxide dismutase (MnSOD) or instillation of NOS antagonists during irradiation or irradiation of bladders devoid of mtNOS. These studies demonstrate that mtNOS is in the cardiomyocytes and urothelial cells, that it is derived from the neuronal isoform, and that it can be either protective or detrimental.
Role of calcium and superoxide dismutase in sensitizing mitochondria to peroxynitrite-induced permeability transition
Authors: Brookes Paul S. , Darley-Usmar Victor M.
Published: January 2004

The mitochondrial permeability transition pore (PTP) is a membrane protein complex assembled and opened in response to Ca2+and oxidants such as peroxynitrite (ONOO–). Opening the PTP is mechanistically linked to the release of cytochrome c, which participates in downstream apoptotic signaling. However, the molecular basis of the synergistic interactions between oxidants and Ca2+in promoting the PTP are poorly understood and are addressed in the present study. In isolated rat liver mitochondria, it was found that the timing of the exposure of the isolated rat liver mitochondria to Ca2+was a critical factor in determining the impact of ONOO–on PTP. Specifically, addition of Ca2+alone, or ONOO–and then Ca2+, elicited similar low levels of PTP opening, whereas ONOO–alone was ineffective. In contrast, addition of Ca2+and then ONOO–induced extensive PTP opening and cytochrome c release. Interestingly, Cu/Zn-superoxide dismutase enhanced pore opening through a mechanism independent of its catalytic activity. These data are consistent with a model in which Ca2+reveals a molecular target that is now reactive with ONOO–. As a test of this hypothesis, tyrosine nitration was determined in mitochondria exposed to ONOO–alone or to Ca2+and then ONOO–, and mitochondrial membrane proteins were analyzed using proteomics. These studies suggest protein targets revealed by Ca2+include dehydrogenases and CoA-containing enzymes. These data are discussed in the context of the role of mitochondria, Ca2+, and ONOO–in apoptotic signaling.
Dynamics of protein nitration in cells and mitochondria
Authors: Aulak Kulwant S. , Koeck Thomas , Crabb John W. , Stuehr Dennis J.
Published: January 2004

Nitric oxide is a precursor of reactive nitrating species such as peroxynitrite and nitrogen dioxide that modify proteins to generate 3-nitrotyrosine. Many diseases are associated with increased levels of protein-bound nitrotyrosine, and this is used as a marker for oxidative damage. However, the regulation of protein nitration and its role in cell function are unclear. We demonstrate that biological protein nitration can be a specific and dynamic process. Proteins were nitrated in distinct temporal patterns in cells undergoing inflammatory activation, and protein denitration and renitration occurred rapidly in respiring mitochondria. The targets of protein nitration varied over time, which may reflect their sensitivity to nitration, expression pattern, or turnover. The dynamic nature of the nitration process was revealed by denitration and renitration of proteins occurring within minutes in mitochondria that were subject to hypoxiaanoxia and reoxygenation. Our results have implications that are particularly important for ischemia-reperfusion injury.
A mitochondrial role for catabolism of nitric oxide in cardiomyocytes not involving oxymyoglobin
Authors: Peterson Jim , Kanai Anthony J. , Pearce Linda L.
Published: January 2004

The maximal concentration of nitric oxide (NO) developing in cultured cells following stimulation of endogenous NO synthases was shown to be submicromolar by NO-selective microelectrode measurements. In electron paramagnetic resonance experiments with isolated and finely divided pericardium, NO was found to react with oxymyoglobin to form metmyoglobin provided that NO was supplied at concentrations in excess of a few micromolar. However, at NO concentrations achievable by endogenous sources, this reaction did not take place to any measurable extent. Oxidative conversion of NO to nitrite ion by cytochrome c oxidase appears to be the most plausible route for cellular catabolism of NO.
A novel vein graft model: adaptation to differential flow environments
Authors: Jiang Zhihua , Wu Lizhen , Miller Brett L. , Goldman Darin R. , Fernandez Chessy M. , Abouhamze Zaher S. , Ozaki C. Keith , Berceli Scott A.
Published: January 2004

Accelerated intimal hyperplasia in response to altered flow environment is critical to the process of vein bypass graft failure. Lack of a reproducible animal model for dissecting the mechanisms of vein graft (VG) remodeling has limited progress toward solving this clinically significant problem. Combining a cuffed anastomotic technique with other surgical manipulations, we developed a well-defined, more robust method for studying hemodynamic factors in VG arterialization. VG with fistula placement, complete occlusion, or partial distal branch ligation (DBL) was performed in the carotid artery of 56 rabbits. Extensive hemodynamic and physiological analyses were performed to define the hemodynamic forces and histological adaptations of the wall at 1–28 days. Anastomotic time averaged 12 min, with 100% patency of bilateral grafts and unilateral grafts plus no adjunct or delayed fistula. Bilateral VG-DBL resulted in an immediate disparity in wall shear (0.8 ± 0.1 vs. 12.4 ± 1.1 dyn/cm2, ligated vs. contralateral graft). Grafts exposed to low shear stress responded primarily through enhanced intimal thickening (231 ± 35 vs. 36 ± 18 μm, low vs. high shear). High-shear-stress grafts adapted through enhanced outward remodeling, with a 24% increase in lumen diameter at 28 days (3.0 ± 0.1 vs. 3.7 ± 0.2 mm, low vs. high shear). We have taken advantage of the cuffed anastomotic technique and combined it with a bilateral VG-DBL model to dissect the impact of hemodynamic forces on VG arterialization. This novel model offers a robust, clinically relevant, statistically powerful small animal model for evaluation of high- and low-shear-regulated VG remodeling.
Aspects, mechanism, and biological relevance of mitochondrial protein nitration sustained by mitochondrial nitric oxide synthase
Authors: Elfering S. L. , Haynes V. L. , Traaseth N. J. , Ettl A. , Giulivi Cecilia
Published: January 2004

The goal of this study was to explore the occurrence of nitrated proteins in mitochondria given that these organelles are endowed with a mitochondrial nitric oxide (NO·) synthase and considering the important role that mitochondria have in energy metabolism. Our hypothesis is that nitration of proteins constitutes a posttranslational modification by which NO· exhibits long-term effects above and beyond those bioregulatory ones mediated through the interaction with cytochrome c oxidase. Our studies are aimed at understanding the mechanisms underlying the nitration of proteins in mitochondria and the biological significance of such a process in the cellular milieu. On promoting a sustained NO· production by mitochondria, we investigated various aspects of protein nitration. Among them, the localization of nitrated proteins in mitochondrial subfractions, the identification of nitrated proteins through proteomic approaches, the characterization of affected pathways, and depiction of a target sequence. The biological relevance was analyzed by considering the turnover of native and nitrated proteins. In this regard, mitochondrial dysfunction, ensuing nitrative stress, may be envisioned as the result of accumulation of nitrated proteins, resulting from an overproduction of endogenous NO· (this study), a failure in the proteolytic system to catabolize modified proteins, or a combination of both. Finally, this study allows one to gain understanding on the mechanism and nitrating species underlying mitochondrial protein nitration.
Absence of OX-43 antigen expression in invasive capillary sprouts: identification of a capillary sprout-specific endothelial phenotype
Authors: Anderson Christopher R. , Ponce Ana M. , Price Richard J.
Published: January 2004

Endothelial cells exhibit a number of unique phenotypes, some of which are angiogenesis dependent. To identify a capillary sprout-specific endothelial phenotype, we labeled angiogenic rat mesentery tissue using a microvessel and capillary sprout marker (laminin), selected endothelial cell markers (CD31, tie-2, and BS-I lectin), and the OX-43 monoclonal antibody, which recognizes a 90-kDa membrane glycoprotein of unknown function. In tissues that were stimulated through wound healing and compound 48/80 application, double-immunolabeling experiments with an anti-laminin antibody revealed that the OX-43 antigen was expressed strongly in all microvessels. However, the OX-43 antigen was completely absent from a large percentage (>85%) of the capillary sprouts that were invading the avascular tissue space. In contrast, sprouts that were introverting back into the previously vascularized tissue retained high levels of OX-43 antigen expression. Double-labeling experiments with endothelial markers indicated that the OX-43 antigen was expressed by microvessel endothelium but was absent from virtually all invasive capillary sprout endothelial cells. We conclude that the absence of OX-43 antigen expression marks a novel, capillary sprout-specific, endothelial cell phenotype. Endothelial cells of this phenotype are particularly abundant in capillary sprouts that invade avascular tissue during angiogenesis.
The intermediary metabolite pyruvate attenuates stunning and reduces infarct size in in vivo porcine myocardium
Authors: Kristo Gentian , Yoshimura Yukihiro , Niu Jianli , Keith Byron J. , Mentzer Robert M. , Bünger Rolf , Lasley Robert D.
Published: January 2004

The intermediary metabolite pyruvate has been shown to exert significant beneficial effects in in vitro models of myocardial oxidative stress and ischemia-reperfusion injury. However, there have been few reports of the ability of pyruvate to attenuate myocardial stunning or reduce infarct size in vivo. This study tested whether supraphysiological levels of pyruvate protect against reversible and irreversible in vivo myocardial ischemia-reperfusion injury. Anesthetized, open-chest pigs ( n = 7/group) underwent 15 min of left anterior descending coronary artery (LAD) occlusion and 3 h of reperfusion to induce stunning. Load-insensitive contractility measurements of regional preload recruitable stroke work (PRSW) and PRSW area (PRSWA) were generated. Vehicle or pyruvate (100 mg/kg iv bolus + 10 mg·kg–1·min–1 intra-atrial infusion) was administered during ischemia and for the first hour of reperfusion. In infarct studies, pigs ( n = 6/group) underwent 1 h of LAD ischemia and 3 h of reperfusion. Group I pigs received vehicle or pyruvate for 30 min before and throughout ischemia. In group II, the infusion was extended through 1 h of reperfusion. In the stunning protocol, pyruvate significantly improved the recovery of PRSWA at 1 h (50 ± 4% vs. 23 ± 3% in controls) and 3 h (69 ± 5% vs. 39 ± 3% in controls) reperfusion. Control pigs exhibited infarct sizes of 66 ± 1% of the area at risk. The pyruvate I protocol was associated with an infarct size of 49 ± 3% ( P < 0.05), whereas the pyruvate II protocol was associated with an infarct size of 30 ± 2% ( P < 0.05 vs. control and pyruvate I). These findings suggest that pyruvate attenuates stunning and decreases myocardial infarction in vivo in part by reduction of reperfusion injury. Metabolic interventions such as pyruvate should be considered when designing the optimal therapeutic strategies for limiting myocardial ischemia-reperfusion injury.
Carbon monoxide activates KCachannels in newborn arteriole smooth muscle cells by increasing apparent Ca2+sensitivity of α-subunits
Authors: Xi Qi , Tcheranova Dilyara , Parfenova Helena , Horowitz Burton , Leffler Charles W. , Jaggar Jonathan H.
Published: January 2004

Carbon monoxide (CO) is a gaseous vasodilator produced by many cell types, including endothelial and smooth muscle cells. The goal of the present study was to investigate signaling mechanisms responsible for CO activation of large-conductance Ca2+-activated K+(KCa) channels in newborn porcine cerebral arteriole smooth muscle cells. In intact cells at 0 mV, CO (3 μM) or CO released from dimanganese decacarbonyl (10 μM), a novel light-activated CO donor, increased KCachannel activity 4.9- or 3.5-fold, respectively. KCachannel activation by CO was not blocked by 1-H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (25 μM), a soluble guanylyl cyclase inhibitor. In inside-out patches at 0 mV, CO shifted the Ca2+concentration-response curve for KCachannels leftward and decreased the apparent dissociation constant for Ca2+from 31 to 24 μM. Western blotting data suggested that the low Ca2+sensitivity of newborn KCachannels may be due to a reduced β-subunit-to-α-subunit ratio. CO activation of KCachannels was Ca2+dependent. CO increased open probability 3.7-fold with 10 μM free Ca2+at the cytosolic membrane surface but only 1.1-fold with 300 nM Ca2+. CO left shifted the current-voltage relationship of cslo-α currents expressed in HEK-293 cells, increasing currents 2.2-fold at +50 mV. In summary, data suggest that in newborn arteriole smooth muscle cells, CO activates low-affinity KCachannels via a direct effect on the α-subunit that increases apparent Ca2+sensitivity. The optimal tuning by CO of the micromolar Ca2+sensitivity of KCachannels will lead to preferential activation by signaling modalities, such as Ca2+sparks, which elevate the subsarcolemmal Ca2+concentration within this range.
Cortisol-mediated regulation of uterine artery contractility: effect of chronic hypoxia
Authors: Xiao DaLiao , Huang XiaoHui , Bae Soochan , Ducsay Charles A. , Longo Lawrence D. , Zhang Lubo
Published: January 2004

We previously demonstrated that cortisol regulated α1-adrenoceptor-mediated contractions differentially in nonpregnant and pregnant uterine arteries. Given that chronic hypoxia during pregnancy has profound effects on maternal uterine artery reactivity, the present study investigated the effects of chronic hypoxia on cortisol-mediated regulation of uterine artery contractions. Pregnant ( day 30) and nonpregnant ewes were divided between normoxic control and chronically hypoxic [maintained at high altitude (3,820 m), arterial Po2: 60 mmHg for 110 days] groups. Uterine arteries were isolated and contractions measured. In hypoxic animals, cortisol (10 ng/ml for 24 h) increased norepinephrine-induced contractions in pregnant, but not in nonpregnant, uterine arteries. The 11β-hydroxysteroid dehydrogenase inhibitor carbenoxolone did not change cortisol effects in nonpregnant uterine arteries, but abolished it in pregnant uterine arteries by increasing norepinephrine pD2(–log EC50) in control tissues. The dissociation constant of norepinephrine-α1-adrenoceptors was not changed by cortisol in nonpregnant, but decreased in pregnant uterine arteries. There were no differences in the density of glucocorticoid receptors between normoxic and hypoxic tissues. Cortisol inhibited the norepinephrine-induced increase in Ca2+concentrations in nonpregnant arteries, but potentiated it in pregnant arteries. In addition, cortisol attenuated phorbol 12,13-dibutyrate-induced contractions in normoxic nonpregnant and pregnant uterine arteries, but had no effect on the contractions in hypoxic arteries. The results suggest that cortisol differentially regulates α1-adrenoceptor- and PKC-mediated contractions in uterine arteries. Chronic hypoxia suppresses uterine artery sensitivity to cortisol, which may play an important role in the adaptation of uterine vascular tone and blood flow in response to chronic stress of hypoxia during pregnancy.
Role of cGMP in carbon monoxide-induced cerebral vasodilation in piglets
Authors: Koneru Padmaja , Leffler Charles W.
Published: January 2004

The hypothesis was addressed that CO-induced cerebral vasodilation requires a permissive cGMP signal that can be produced by nitric oxide (NO). Anesthetized piglets were implanted with cranial windows for measurement of pial arteriolar responses to stimuli. Pial arterioles dilated in response to isoproterenol (Iso), sodium nitroprusside (SNP), and CO or the CO-releasing molecule Mn2(CO)10 [dimanganese decacarbonyl (DMDC)]. 1 H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (ODQ), a soluble guanylyl cyclase inhibitor, decreased cerebrospinal fluid (CSF) cGMP and selectively inhibited dilations to SNP and DMDC without affecting the dilation to Iso. However, DMDC did not cause an increase in cortical periarachnoid CSF cGMP concentration. cGMP clamp with a threshold dilator level of 8-bromo-cGMP (10–4 M) and ODQ restored the dilation to DMDC that had been blocked by ODQ alone. Under these conditions, cGMP was present but could not increase. Inhibition of the pial arteriolar dilation to glutamate by N-nitro-l-arginine, which blocks NO synthase, was similar to that by heme oxygenase inhibitors, which block endogenous CO production. The dilation to glutamate, similar to dilation to DMDC, was partially restored by 8-bromo-cGMP and completely restored by SNP (5 × 10–7 M). These data suggest that the permissive role of NO in CO- and glutamate-induced vasodilation involves maintaining the minimum necessary cellular level of cGMP to allow CO to cause dilation independently of increasing cGMP.
Glucocorticoids act in the dorsal hindbrain to increase arterial pressure
Authors: Scheuer Deborah A. , Bechtold Andrea G. , Shank Sylvan S. , Akana Susan F.
Published: January 2004

Glucocorticoid receptors (GRs) are present at a high density in the nucleus of the solitary tract (NTS), an area of the dorsal hindbrain (DHB) that is critical for blood pressure regulation. However, whether these receptors play any role in the regulation of blood pressure is unknown. We tested the hypothesis that glucocorticoids act in the DHB to increase arterial pressure using two experimental strategies. In one approach, we implanted pellets of corticosterone (Cort) or sham pellets onto the DHB over the NTS. Compared with rats with sham pellets, rats with DHB Cort pellets had an increased ( P < 0.05) mean arterial pressure (111 ± 2 vs. 104 ± 1 mmHg) and heart rate (355 ± 9 vs. 326 ± 5 beats/min) after 4 days. In the second approach, we implanted subcutaneous Cort pellets to increase the systemic Cort concentration and then subsequently implanted pellets of the GR antagonist mifepristone (Mif; previously RU-38486) or sham pellets onto the DHB. Two days of DHB Mif treatment reduced ( P < 0.05) mean arterial pressure in those rats with elevated plasma Cort levels (118 ± 2 vs. 108 ± 1 mmHg for sham vs. Mif DHB pellets). Cort and Mif pellets placed on the dura had no effects on arterial pressure or heart rate, ruling out systemic cardiovascular effects of the steroids. DHB Cort treatment had no effects on plasma Cort concentration or adrenal weight, indicating that the contents of the DHB Cort pellet did not diffuse into the systemic circulation or into the forebrain areas that regulate plasma Cort concentration in concentrations sufficient to produce physiological effects. Immunohistochemistry for the occupied GRs demonstrated that the Cort and Mif from the DHB pellets were delivered to the DHB with minimal diffusion to the ventral hindbrain or forebrain. We conclude that glucocorticoids act in the DHB to increase arterial pressure.
Effect of bronchial artery blood flow on cardiopulmonary bypass-induced lung injury
Authors: Dodd-o Jeffrey M. , Welsh Laura E. , Salazar Jorge D. , Walinsky Peter L. , Peck Eric A. , Shake Jay G. , Caparrelli David J. , Bethea Brian T. , Cattaneo Stephen M. , Baumgartner William A. , Pearse David B.
Published: January 2004

Cardiovascular surgery requiring cardiopulmonary bypass (CPB) is frequently complicated by postoperative lung injury. Bronchial artery (BA) blood flow has been hypothesized to attenuate this injury. The purpose of the present study was to determine the effect of BA blood flow on CPB-induced lung injury in anesthetized pigs. In eight pigs (BA ligated) the BA was ligated, whereas in six pigs (BA patent) the BA was identified but left intact. Warm (37°C) CPB was then performed in all pigs with complete occlusion of the pulmonary artery and deflated lungs to maximize lung injury. BA ligation significantly exacerbated nearly all aspects of pulmonary function beginning at 5 min post-CPB. At 25 min, BA-ligated pigs had a lower arterial Po2at a fraction of inspired oxygen of 1.0 (52 ± 5 vs. 312 ± 58 mmHg) and greater peak tracheal pressure (39 ± 6 vs. 15 ± 4 mmHg), pulmonary vascular resistance (11 ± 1 vs. 6 ± 1 mmHg·l–1·min), plasma TNF-α (1.2 ± 0.60 vs. 0.59 ± 0.092 ng/ml), extravascular lung water (11.7 ± 1.2 vs. 7.7 ± 0.5 ml/g blood-free dry weight), and pulmonary vascular protein permeability, as assessed by a decreased reflection coefficient for albumin (σalb; 0.53 ± 0.1 vs. 0.82 ± 0.05). There was a negative correlation ( R = 0.95, P < 0.001) between σalband the 25-min plasma TNF-α concentration. These results suggest that a severe decrease in BA blood flow during and after warm CPB causes increased pulmonary vascular permeability, edema formation, cytokine production, and severe arterial hypoxemia secondary to intrapulmonary shunt.
MRI and echocardiographic assessment of the diastolic dysfunction of normal aging: altered LV pressure decline or load?
Authors: Hees Paul S. , Fleg Jerome L. , Dong Sheng-Jing , Shapiro Edward P.
Published: January 2004

Changes in diastolic indexes during normal aging, including reduced early filling velocity ( E), lengthened E deceleration time (DT), augmented late filling ( A), and prolonged isovolumic relaxation time (IVRT), have been attributed to slower left ventricular (LV) pressure (LVP) decay. Indeed, this constellation of findings is often referred to as the “abnormal relaxation” pattern. However, LV filling is determined by the atrioventricular pressure gradient, which depends on both LVP decline and left atrial (LA) pressure (LAP). To assess the relative influence of LVP decline and LAP, we studied 122 normal subjects aged 21–92 yr by Doppler echocardiography and MRI. LVP decline was assessed by color M-mode ( Vp) and the LV untwisting rate. Early diastolic LAP was evaluated using pulmonary vein flow systolic fraction, pulmonary vein flow diastolic DT, color M-mode ( E/ Vp), and tissue Doppler ( E/ Em). Linear regression showed the expected reduction of E, increase in A, and prolongation of IVRT and DT with advancing age. There was no relation of age to parameters reflecting the rate of LVP decline. However, older age was associated with reduced E/ Vp ( P = 0.008) and increased pulmonary vein systolic fraction ( P < 0.001), pulmonary vein DT ( P = 0.0026), and E/ Em ( P < 0.0001), all suggesting reduced early LAP. Therefore, reduced early filling in older adults may be more closely related to a reduced early diastolic LAP than to slower LVP decline. This effect also explains the prolonged IVRT. We postulate that changes in LA active or passive properties may contribute to development of the abnormal relaxation pattern during the aging process.
Effects of Hct and norepinephrine on segmental vascular resistance distribution in isolated perfused rat livers
Authors: Kamikado Chiaki , Shibamoto Toshishige , Hongo Minoru , Koyama Shozo
Published: January 2004

We studied the effects of blood hematocrit (Hct), blood flow, or norepinephrine on segmental vascular resistances in isolated portally perfused rat livers. Total portal hepatic venous resistance ( Rt) was assigned to the portal ( Rpv), sinusoidal ( Rsinus), and hepatic venous ( Rhv) resistances using the portal occlusion (Ppo) and the hepatic venous occlusion (Phvo) pressures that were obtained during occlusion of the respective line. Four levels of Hct (30%, 20%, 10%, and 0%) were studied. Rpv comprises 44% of Rt, 37% of Rsinus, and 19% of Rhv in livers perfused at 30% Hct and portal venous pressure of 9.1 cmH2O. As Hct increased at a given blood flow, all three segmental vascular resistances of Rpv, Rsinus, and Rhv increased at flow >15 ml/min. As blood flow increased at a given Hct, only Rsinus increased without changes in Rpv or Rhv. Norepinephrine increased predominantly Rpv, and, to a smaller extent, Rsinus, but it did not affect Rhv. Finally, we estimated Ppo and Phvo from the double occlusion maneuver, which occluded simultaneously both the portal and hepatic venous lines. The regression line analysis revealed that Ppo and Phvo were identical with those measured by double occlusion. In conclusion, changes in blood Hct affect all three segmental vascular resistances, whereas changes in blood flow affect Rsinus, but not Rpv or Rhv. Norepinephrine increases mainly presinusoidal resistance. Ppo and Phvo can be obtained by the double occlusion method in isolated perfused rat livers.
Depletion of pulmonary intravascular macrophages inhibits acute lung inflammation
Authors: Singh Baljit , Pearce Jacqueline W. , Gamage Lakshman N. , Janardhan Kyathanahalli , Caldwell Sarah
Published: January 2004

Pulmonary intravascular macrophages (PIMs) are present in ruminants and horses. These species are highly sensitive to acute lung inflammation compared with non-PIM-containing species such as rats and humans. There is evidence that rats and humans may also recruit PIMs under certain conditions. We investigated precise contributions of PIMs to acute lung inflammation in a calf model. First, PIMs were recognized with a combination of in vivo phagocytic tracer Monastral blue and postembedding immunohistology with anti-CD68 monoclonal antibody. Second, gadolinium chloride depleted PIMs within 48 h of treatment ( P < 0.05). Finally, PIMs contain TNF-α, and their depletion reduces cells positive for IL-8 ( P < 0.05) and TNF-α ( P < 0.05) and histopathological signs of acute lung inflammation in calves infected with Mannheimia hemolytica. The majority of IL-8-positive inflammatory cells in lung septa of infected calves were platelets. Platelets from normal cattle contained preformed IL-8 that was released upon in vitro exposure to thrombin ( P < 0.05). These novel data show that PIMs, as the source of TNF-α, promote recruitment of inflammatory cells including IL-8-containing platelets to stimulate acute inflammation and pathology in lungs. These data may also be relevant to humans due to our ability to recruit PIMs.
Activation of K+channels: an essential pathway in programmed cell death
Authors: Remillard Carmelle V. , Yuan Jason X.-J.
Published: January 2004

Cell apoptosis and proliferation are two counterparts in sharing the responsibility for maintaining normal tissue homeostasis. In recent years, the process of the programmed cell death has gained much interest because of its influence on malignant cell growth and other pathological states. Apoptosis is characterized by a distinct series of morphological and biochemical changes that result in cell shrinkage, DNA breakdown, and, ultimately, phagocytic death. Diverse external and internal stimuli trigger apoptosis, and enhanced K+efflux has been shown to be an essential mediator of not only early apoptotic cell shrinkage, but also of downstream caspase activation and DNA fragmentation. The goal of this review is to discuss the role(s) played by K+transport or flux across the plasma membrane in the regulation of the apoptotic volume decrease and apoptosis. Attention has also been paid to the role of inner mitochondrial membrane ion transport in the regulation of mitochondrial permeability and apoptosis. We provide specific examples of how deregulation of the apoptotic process contributes to pulmonary arterial medial hypertrophy, a major pathological feature in patients with pulmonary arterial hypertension. Finally, we discuss the targeting of K+channels as a potential therapeutic tool in modulating apoptosis to maintain the balance between cell proliferation and cell death that is essential to the normal development and function of an organism.
Evaluation of lung injury in rats and mice
Authors: Parker James C. , Townsley Mary I.
Published: January 2004

Lung injury is a broad descriptor that can be applied to conditions ranging from mild interstitial edema without cellular injury to massive and fatal destruction of the lung. This review addresses those methods that can be readily applied to rats and mice whose small size limits the techniques that can be practically used to assess injury. The methodologies employed range from nonspecific measurement of edema formation to techniques for calculating values of specific permeability coefficient for the microvascular membrane in lung. Accumulation of pulmonary edema can be easily and quantitatively measured using gravimetric methods and indicates an imbalance in filtration forces or restrictive properties of the microvascular barrier. Lung compliance can be continuously measured, and light and electron microscopy can be used regardless of lung size to detect edema and structural damage. Increases in fluid and/or protein flux due to increased permeability must also be separated from those due to increased filtration pressure for mechanistic interpretation. Although an increase in the initial lung albumin clearance compared with controls matched for size and filtration pressure is a reliable indicator of endothelial dysfunction, calculated alterations in capillary filtration coefficient Kf,c, reflection coefficient σ, and permeability-surface area product PS are the most accurate indicators of increased permeability. Generally, PS and Kf,cwill increase and σ will decrease with vascular injury, but derecruitment of microvascular surface area may attenuate the affect on PS and Kf,cwithout altering measurements of σ.