Myoglobin may serve a variety of functions in muscular oxygen supply, such as O 2 storage, facilitated O 2 diffusion, and myoglobin-mediated oxidative phosphorylation. We studied the functional consequences of a myoglobin deficiency on cardiac function by producing myoglobinknockout (myo ؊͞؊ ) mice. )]. These data demonstrate that disruption of myoglobin results in the activation of multiple compensatory mechanisms that steepen the pO 2 gradient and reduce the diffusion path length for O 2 between capillary and the mitochondria; this suggests that myoglobin normally is important for the delivery of oxygen.

Abstract-To investigate the role of adenosine formed extracellularly in vascular homeostasis, mice with a targeted deletion of the cd73/ecto-5Ј-nucleotidase were generated. Southern blot, RT-PCR, and Western blot analysis confirmed the constitutive knockout. In vivo analysis of hemodynamic parameters revealed no significant differences in systolic blood pressure, ejection fraction, or cardiac output between strains. However, basal coronary flow measured in the isolated perfused heart was significantly lower (Ϫ14%; PϽ0.05) in the mutant. Immunohistochemistry revealed strong CD73 expression on the endothelium of conduit vessels in wild-type (WT) mice. Time to carotid artery occlusion after ferric chloride (FeCl 3 ) was significantly reduced by 20% in cd73 Ϫ/Ϫ mice (PϽ0.05). Bleeding time after tail tip resection tended to be shorter in cd73mice (Ϫ35%). In vivo platelet cAMP levels were 0.96Ϯ0.46 in WT versus 0.68Ϯ0.27 pmol/10 6 cells in cd73 Ϫ/Ϫ mice (PϽ0.05). Under in vitro conditions, platelet aggregation in response to ADP (0.05 to 10 mol/L) was undistinguishable between the two strains. In the cremaster model of ischemia-reperfusion, the increase in leukocyte attachment to endothelium was significantly higher in cd73 Ϫ/Ϫ compared with WT littermates (WT 98% versus cd73 Ϫ/Ϫ 245%; PϽ0.005). The constitutive adhesion of monocytes in ex vivo-perfused carotid arteries of WT mice was negligible but significantly increased in arteries of cd73 Ϫ/Ϫ mice (PϽ0.05). Thus, our data provide the first evidence that adenosine, extracellularly formed by CD73, can modulate coronary vascular tone, inhibit platelet activation, and play an important role in leukocyte adhesion to the vascular endothelium in vivo. Key Words: transgenic mice Ⅲ adenosine Ⅲ ecto-5Ј-nucleotidase Ⅲ vascular inflammation Ⅲ thrombosis C D73/ecto-5Ј-nucleotidase, a 70-kDa glycosylphosphatidylinositol (GPI)-anchored cell surface molecule, is expressed on the vascular endothelium and catalyzes the extracellular conversion of 5Ј-AMP to adenosine. 1,2 CD73 is the final step of the extracellular nucleotide breakdown cascade that also involves membrane-associated CD39/ATPdiphosphohydrolase. 3 The product of CD73 is adenosine, a purine nucleoside that has been implicated in many physiological and pathophysiological events. 4 There are four known G-coupled adenosine receptors: A 1 , A 2A , A 2B , and A 3 , each of which operates via different intracellular signaling mechanisms and exhibits distinct patterns of tissue distribution. 5 In human neutrophils, adenosine A 1 and A 2 receptor occupancy mediate opposing roles for adenosine in inflammation: A 1 activation is proinflammatory, whereas the A 2 receptor plays an anti-inflammatory role. 6 A 2 receptor activation inhibits the neutrophil oxidative burst, whereas the A 3 receptor inhibits neutrophil degranulation 7 and may play an important role in inflammation by inhibiting eosinophil migration. 8 Recently, deletion of the A 2A receptor in transgenic mice revealed that this receptor is critical for the limitation a...

The present study explored the role of myoglobin (Mb) in cardiac NO homeostasis and its functional relevance by employing isolated hearts of wild-type (WT) and myoglobin knockout mice. 1 H NMR spectroscopy was used to measure directly the conversion of oxygenated Mb (MbO2) to metmyoglobin (metMb) by reaction with NO. NO was applied intracoronarily (5 nM to 25 M), or its endogenous production was stimulated with bradykinin (Bk; 10 nM to 2 M). We found that infusion of authentic NO solutions dose-dependently (> 2.5 M NO) increased metMb formation in WT hearts that was rapidly reversible on cessation of NO infusion. Likewise, Bk-induced release of NO was associated with significant metMb formation in the WT (>1 M Bk and IIa skeletal and cardiac muscle tissue (1). As a major breakthrough in understanding globular protein structure, its tertiary structure was derived from x-ray diffraction studies by John Kendrew and his colleagues as early as the 1950s (2). Mb is a relatively small (M r 16,700) and densely packed protein consisting of a single polypeptide chain of 153 amino acid residues. It contains an iron-porphyrin heme group identical to that of hemoglobin (Hb), and like Hb is capable of reversible oxygenation and deoxygenation. In mammals, half O 2 saturation of Mb is achieved at an intracellular O 2 partial pressure as low as 2.4 mmHg (1 mmHg ϭ 133 Pa; ref.3), suggesting a predominance of oxygenated Mb (MbO 2 ) under basal conditions. Mb's function as an oxygen store is well accepted. Mb serves as a short-term O 2 reservoir in exercising skeletal muscle and in the beating heart, tiding the muscle over from one contraction to the next (4). In diving mammals, the concentrations of Mb exceed those of terrestrial mammals up to 10-fold, and Mb most likely serves for the extension of diving time when pulmonary ventilation ceases (5). Similarly, in mammals and humans adapted to high altitudes, Mb is expressed in high concentrations in skeletal muscle (6).It has been proposed that Mb facilitates intracellular delivery of O 2 , in that Mb adjacent to the cell membrane picks up oxygen, traverses the cytosol by translational diffusion to unload O 2 in the vicinity of mitochondria, and finally diffuses back to the cell membrane in the deoxygenated state (7). This circuit, termed ''facilitated O 2 diffusion,'' may be a critical link between capillary O 2 supply and O 2 -consuming cytochromes within mitochondria in the steady state. Facilitated O 2 diffusion has been unambiguously demonstrated in concentrated Mb solutions (8), but experiments carried out in isolated cells, papillary muscle, and at the whole organ level have yielded conflicting results (9-11). Likewise, model calculations have both refuted and supported the contribution of Mb-bound O 2 to total O 2 flux (11, 12).The recent generation of transgenic mice lacking Mb has shed new light on the role of Mb in the intracellular delivery of O 2 (13,14). Loss of Mb led to a surprisingly benign phenotype, with exercise and reproductive capacity, as well as cardiac and ...

Abstract-For the specific analysis of endothelial NO synthase (eNOS) function in the coronary vasculature, we generated a mouse homozygous for a defective eNOS gene (eNOSϪ/Ϫ). Western blot as well as immunohistochemical staining revealed the absence of eNOS protein in eNOSϪ/Ϫ mice. Aortic endothelial cells derived from eNOSϪ/Ϫ mice displayed only background levels of NO x formation compared with wild-type (WT) cells (88 versus 1990 pmol). eNOSϪ/Ϫ mice were hypertensive (mean arterial pressure, 135Ϯ15 versus 107Ϯ8 mm Hg in WT) without the development of cardiac hypertrophy. Coronary hemodynamics, analyzed in Langendorff-perfused hearts, showed no differences either in basal coronary flow or in maximal and repayment flow of reactive hyperemia. Acute NOS inhibition with N -nitro-L-arginine methyl ester (L-NAME) in WT hearts substantially reduced basal flow and reactive hyperemia. The coronary response to acetylcholine (ACh) (500 nmol/L) was biphasic: An initial vasoconstriction (flow, Ϫ35%) in WT hearts was followed by sustained vasodilation (ϩ190%). L-NAME significantly reduced vasodilation in WT hearts (ϩ125%) but did not alter the initial vasoconstriction. In eNOSϪ/Ϫ hearts, the initial vasoconstriction was augmented (Ϫ70%), whereas the ACh-induced vasodilation was not affected. Inhibition of cyclooxygenase with diclofenac converted the ACh-induced vasodilation into vasoconstriction (Ϫ49% decrease of basal flow). This effect was even more pronounced in eNOSϪ/Ϫ hearts (Ϫ71%). Our results demonstrate that (1) acute inhibition of eNOS reveals a role for NO in setting the basal coronary vascular tone as well as participation in reactive hyperemia and the response to ACh; (2) chronic inhibition of NO formation in eNOSϪ/Ϫ mutant mice induces no changes in basal coronary flow and reactive hyperemia, suggesting the activation of important compensatory mechanisms; and (3) Key Words: heart Ⅲ gene targeting Ⅲ reactive hyperemia Ⅲ coronary flow Ⅲ blood pressure E ndothelial NO synthase, also called type III NO synthase, is the major NOS isoenzyme that is widely expressed in endothelial cells throughout the vascular bed. It is generally accepted that endothelium-derived NO is an important factor in the control of basal vascular tone.1 NO is also involved in receptor-mediated vasodilation in response to various agonists such as ACh, ATP, thrombin, bradykinin, and others. Through experiments using NOS inhibitors 2,3 but also by use of genetically modified animals, 4,5 it has been demonstrated that functional inactivation of eNOS activity results in hypertension. In addition to the control of vascular tone, NO inhibits platelet aggregation and leukocyte adhesion to the vessel wall as well as proliferation and migration of smooth muscle cells. Thus, eNOS is considered to play an important role in maintaining the antiatherogenic surface of the vessel wall. 6In the heart, eNOS is expressed primarily in the coronary and endocardial endothelia. In addition, eNOS has been localized to cardiac myocytes and the specialized cells of s...

To elucidate the physiological role of the AMP-adenosine metabolic cycle and to investigate the relation between AMP and adenosine formation, the O2 supply of isolated guinea pig hearts was varied (95% to 10% O2). The net adenosine formation rate (AMP-->adenosine) and coronary venous effluent adenosine release rate were measured; free cytosolic AMP was determined by 31P-nuclear magnetic resonance. Switching from 95% to 40% O2 increased free AMP and adenosine formation 4-fold, whereas free cytosolic adenosine and venous adenosine release rose 15- to 20-fold. In the AMP range from 200 to 3000 nmol/L, there was a linear correlation between free AMP and adenosine formation (R2 = .71); however, adenosine release increased several-fold more than formation. At 95% O2, only 6% of the adenosine formed was released; however, this fraction increased to 22% at 40% O2, demonstrating reduced adenosine salvage. Selective blockade of adenosine deaminase and adenosine kinase indicated that flux through adenosine kinase decreased from 85% to 35% of adenosine formation in hypoxia. Mathematical model analysis indicated that this apparent decrease in enzyme activity was not due to saturation but to the inhibition of adenosine kinase activity to 6% of the basal levels. The data show (1) that adenosine formation is proportional to the AMP substrate concentration and (2) that hypoxia decreases adenosine kinase activity, thereby shunting myocardial adenosine from the salvage pathway to venous release. In conclusion, because of the normal high turnover of the AMP-adenosine metabolic cycle, hypoxia-induced inhibition of adenosine kinase causes the amplification of small changes in free AMP into a major rise in adenosine. This mechanism plays an important role in the high sensitivity of the cardiac adenosine system to impaired oxygenation.

The intravoxel incoherent motion (IVIM) method was implemented in closed-chest dogs to obtain measurements on microcirculation in the left ventricular wall in vivo. Specifically, it enabled us to measure the mean microflow velocity (400 ؎ 40 m/s) and the vascular volume fraction (VVF) (11.1% ؎ 2.2%), and observe the directional preference of capillary orientation. The apparent diffusion coefficients (ADCs) of water along and perpendicular to myofibers were also measured. With vasodilatation by adenosine infusion, a 25% increase in the VVF and a 7% increase in the mean microflow velocity were observed, while no change in the ADC was detected. Microcirculation in tissue has generated great interest in recent years (1-3) for clinical purposes and in studies of oxygen delivery. Magnetic resonance imaging (MRI) is one of the few noninvasive methods that are being used to investigate this topic. MRI techniques employ either exogenous contrast agents or endogenous contrast mechanisms to delineate blood flow at the capillary level. Gd-DTPAbased studies give estimates of perfusion rates (4), while strictly intravascular agents provide estimates of perfusion rates, vascular volume, and water exchange rates (4,5). Two endogenous contrast methods-remote arterial spinlabeling and local spin-labeling-are used to measure perfusion rates, water distribution between vascular and extravascular compartments, and flow transit times (6 -8).We used an endogenous contrast technique based on the intravoxel incoherent motion (IVIM) phenomenon (9,10) to measure the vascular volume fraction (VVF) and microflow velocity, and to detect directional anisotropy in the capillary morphology in the myocardium. The IVIM technique is based on the assumption that in diffusionweighted images, blood flow in the capillaries and small arterioles/venules also appears as incoherent movement when observed at the scale of an image pixel, and causes a decrease in the image intensity in addition to the effect of Brownian diffusion of the water molecules. Because both flow and diffusion measurements can be obtained with this technique, we also investigated the correlation between the two by increasing blood flow with adenosine infusion.We conducted for the first time IVIM-based microcirculation measurements in the canine heart in vivo. This technique has been used successfully in vascular brain perfusion studies (11-13) and renal perfusion imaging (14,15), but IVIM-based flow detection has not been attempted in the heart-mainly because of the technical difficulties caused by heartbeat motion. Diffusionweighted sequences are critically affected by bulk deformation and rotation, and therefore they require careful registration of the heart position and strain status. Nevertheless, the IVIM method is a useful tool for examining the relationship between vascular anatomy and flow distribution in the myocardium (16), and between microcirculation and diffusion (17).The directional preference of capillary orientation in the myocardium has been observed in many optical ...

Aims: The teachers' perspectives of the educational environment have as yet only been sparsely considered. This study aimed at validating the first German version of the Dundee Ready Education Environment Measure (DREEM) from the points of view of both students and teachers. Methods: Data from 1119 students and 258 teachers were available for analysis. Psychometric validation included the analysis of homogeneity and discrimination at item level as well as reliability (Cronbach's ), criterion and construct validity at test level. Effect sizes were calculated and the independent samples t-test was used for statistical inference testing of mean differences between two groups. Results: Item characteristics were satisfactory in both samples. Reliability was high with ¼ 0.92 (students) and 0.94 (teachers), respectively. Factor analyses revealed five dimensions which slightly diverged from the five subscales postulated by the DREEM authors though. The environment was evaluated significantly ( p 5 0.001) more positively by teachers (M ¼ 117.63) than by students (M ¼ 109.75). Further significant differences were observed with regard to gender, mother language, stage of studies and previous professional training among others. Conclusions: With convincing psychometric properties at item and test levels, the suitability of DREEM not only for students but also for teachers to assess the educational environment has been demonstrated.

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