过氧化氢对血管平滑肌细胞增殖和明胶酶A及其抑制因子基因表达的影响

目的:探讨过氧化氢(H₂O₂)对体外培养大鼠血管平滑肌细胞(VSMC)增殖和明胶酶A(MMP-2)、及其抑制因子(TIMP-2)基因表达的影响。方法:先用四甲基偶氮唑(MTT)法筛选出H₂O₂对VSMC的毒性作用浓度和增殖作用浓度,在此基础上用逆转录聚合酶链反应(RT-PCR)法测定H₂O₂对MMP-2、TIMP-2mRNA表达的影响。结果:H₂O₂浓度大于300μmol/L后显示出对VSMC的致死毒性作用;H₂O₂浓度在0.01-5.0μmol/L内可以刺激VSMC的增殖,且呈时间依赖性;1μmol/LH₂O₂、10μmol/LH₂O₂对MMP-2基因转录有明显的促进作用;不同浓度的H₂O₂对TIMP-2基因转录无明显促进作用。结论:适当浓度的H₂O₂可以促进VSMC的增殖和MMP-2mRNA表达,但对TIMP-2mRNA表达无明显作用,提示H₂O₂参与了血管重塑的不同病理环节。     

Role of endothelial nitric oxide synthetase in arteriogenesis after stroke in mice

Arteriogenesis supports restored perfusion in the ischemic brain and improves long-term functional outcome after stroke. We investigate the role of endothelial nitric oxide synthetase (eNOS) and a nitric oxide (NO) donor, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1, 2-diolate (DETA-NONOate), in promoting arteriogenesis after stroke. Adult wild-type (WT, n=18) and eNOS-knockout (eNOS^−/−, n=36) mice were subjected to transient (2.5 h) right middle cerebral artery occlusion (MCAo) and were treated with or without DETA-NONOate (0.4 mg/kg) 24 h after MCAo. Functional evaluation was performed. Animals were sacrificed 3 days after MCAo for arterial cell culture studies, or 14 days for immunohistochemical analysis. Consistent with previous studies, eNOS^−/− mice exhibited a higher mortality rate (P<0.05, n=18/group) and more severe neurological functional deficit after MCAo than WT mice (P<0.05, n=12/group). Decreased arteriogenesis, was evident in eNOS^−/− mice compared with WT mice, as demonstrated by reduced vascular smooth muscle cell (VSMC) proliferation, arterial density and diameter in the ischemic brain. eNOS^−/− mice treated with DETA-NONOate had a significantly decreased mortality rate and improved functional recovery, and exhibited enhanced arteriogenesis identified by increased VSMC proliferation, and upregulated arterial density and diameter compared to eNOS^−/− mice after stroke (P<0.05, n=12/group). To elucidate the mechanisms underlying eNOS/NO mediated arteriogenesis, VSMC migration was measured in vitro. Arterial cell migration significantly decreased in the cultured common carotid artery (CCA) derived from eNOS^−/− mice 3 days after MCAo compared to WT arterial cells. DETA-NONOate-treatment significantly attenuated eNOS^−/−-induced decrease of arterial cell migration compared to eNOS^−/− control artery (P<0.05; n=6/group). Using VSMC culture, DETA-NONOate significantly increased VSMC migration, while inhibition of NOS significantly decreased VSMC migration (P<0.05; n=6/group). Our data indicated that eNOS not only promotes vascular dilation but also increases VSMC proliferation and migration, and thereby enhances arteriogenesis after stroke. Therefore, increase eNOS may play an important role in regulating of arteriogenesis after stroke.     

Effect of sodium azide on hydrogen peroxide production by zymosan-activated human neutrophils

Stimulated neutrophils (PMNs) produce large quantities of superoxide anion, which is the precursor for hydrogen peroxide (H₂O₂). We developed a new fluorimetric assay to measure the H₂O₂ released by zymosan A-activated PMNs utilizing the oxidation ofp-hydroxyphenylacetic acid by H₂O₂ to its fluorescent dimer in the presence of horseradish peroxidase. Zymosan-activated PMNs isolated from nine healthy volunteers and 20 patients with acute hypoxemic respiratory failure (AHRF) released after 90 min 2.3±0.3 and 2.4±1.3 nmol H₂O₂/10⁶ PMNs, respectively. Inhibition of the heme enzymes by 1.0 mM sodium azide (NaN₃) increased the H₂O₂ production to 21.6±4.4 nmol H₂O₂/10⁶ PMNs in the control group (P<0.001), and to 22.5±14.7 nmol H₂O₂/10⁶ PMNs in patients with AHRF (P<0.001). Incubation temperature, room temperature or 37C, did not change the total amount of H₂O₂ produced after 90 min by zymosan-activated PMN. Addition of NaN₃ improved both the sensitivity and reproducibility of the measurement of H₂O₂ and allowed detection of H₂O₂ released by PMNs with coefficients of variation of less than 5% at PMN concentrations as low as 0.1×10⁶ cells/ml. The amount of H₂O₂ released by activated PMNs did not distinguish healthy controls from patients with AHRF.     

Neutrophils may directly synthesize both H2O2 and O2 − since surface stimuli induce their release in stimulus-specific ratios

Many stimuli induce neutrophils to undergo an oxidative burst and generate toxic oxygen metabolites. The major products are O₂ ^– and H₂O₂, the latter being presumed to arise by spontaneous dismutation of the former. If H₂O₂ were indeed derived exclusively from released O₂ ^– according to the equation 2O₂ ^– + 2H⁺ H₂O₂ + O₂, one would expect that relationship to be reflected in the ratio of the two metabolites detectable in the extracellular mileu of stimulated neutrophils. A second corollary is that H₂O₂ should not form when cytochromec is present to scavenge O₂ ^– before it can dismutate. Although H₂O₂ cannot be measured directly in the presence of cytochromec because it is consumed in reoxidizing reduced cytochromec, its presence can be detected indirectly by the ability of catalase to improve the apparent yield of reduced cytochromec. We found that the relative amounts of extracellular H₂O₂ and O₂ ^– that could be measured in the environment of stimulated neutrophils varied with the stimulus and that catalase protected reduced cytochromec from H₂O₂ oxidation when some stimuli were used but not with others. For example, the ratio of O₂ ^– to H₂O₂ produced by neutrophils exposed to PMA was about 2:1, the expected result if H₂O₂ were derived from O₂ ^–. However when cytochalasin B was added to the cells before the stimulus, the yield of H₂O₂ was reduced but not the yield of O₂ ^–. When cells were allowed to settle and spread on tissue culture plastic they produced equimolar amounts of O₂ ^– and H₂O₂. Coating the plastic with IgG doubled cytochromec reduction without effecting H₂O₂. In contrast, coating with albumin reduced H₂O₂ without effecting cytochromec reduction. Soluble IgG aggregates induced production of mostly O₂ ^– whereas immune complexes resulted in release of both metabolites.FMLP and A23187 were similar to the soluble IgG aggregates in their effects and induced release of proportionately more O₂ ^– than H₂O₂. The addition of catalase to the cytochromec solution improved the yield of reduced cytochromec when PMA or IgG was used to stimulate the cells but not when FMLP was used. These and other data suggest that H₂O₂ release is not a linear function of the amount of O₂ ^– generated and that either a variable fraction of O₂ ^– spontaneously dismutates to H₂O₂ or the neutrophil NADPH oxidase, in a manner analogous to xanthine oxidase, is capable, under some circumstances, of producing H₂O₂ as well as O₂ ^–. If the latter were true, the pathologic consequences of neutrophil activation would vary depending on whether O₂ ^– was the primary product (chemotactic activation) or whether H₂O₂ was released as well (immune complex stimulation).     

Effects of low and high levels of moderate hypoxia on anaerobic energy release during supramaximal cycle exercise

The purpose of this study was to investigate whether hypoxia can alter anaerobic energy release during supramaximal exercise. Seven male subjects performed 12 submaximal cycling tests to establish the relationship between workload and O₂ demand. The subjects also performed 40 s Wingate tests (WT) under normoxia (room air), two levels of moderate hypoxia of 16.4% O₂ and 12.7% O₂. We measured the power output and oxygen uptake (VO₂) during each test and estimated the O₂ demand, O₂ deficit and percentage of anaerobic energy release (%AnAER). These data were analyzed for each 20 s interval. At all intervals, there were no differences in Pmean·body mass (BM)⁻¹, O₂ demand·BM⁻¹ or O₂ deficit·BM⁻¹ among the three O₂ conditions. However, under hypoxia of 12.7%, VO₂·BM⁻¹ was significantly decreased and %AnAER was significantly increased in the late phase (20–40 s) of the WT, compared to normoxia (P<0.05). There were no such significant differences between normoxia and hypoxia of 16.4%. Thus, the present results show that the degree of hypoxia affects the magnitude of the hypoxia-induced increase in anaerobic energy release in the late phase of the WT and suggest that certain degrees of hypoxia induce significant increases in the amount of anaerobic energy released, compared to normoxia.     

Baculovirus mediated expression of human phagocytic cell oxidase cytochrome b558 in sf9 insect cells

Chronic granulomatous disease (CGD) results from deficient production of components of the phagocyte NADPH oxidase. Most commonly affected is cytochrome b₅₅₈, a heterodimer composed of a 22-kDa protein (p22^phox noncovalently bound to a 91-kDa transmembrane glycoprotein (gp91^phox). CGD phagocytes lack both p22^phox and gp91^phox peptides when either gene is affected, suggesting that both peptides must be produced for individual subunit stability. Both genes have been cloned, but eukaryotic expression of recombinant gp91^phox has not been reported. To investigate the stability and interaction of cytochrome b₅₅₈ subunits, we introduced p22^phox and gp91^phox cDNA into recombinant baculoviruses. Recombinant gp91^phox (rgp91^phox) and p22^phox (rp22^phox) were detected individually and together in the same cells by in situ immunofluorescence and by SDS-PAGE immunoblotting of membranes from sf9 cells infected with baculovirus constructs. Formation of rp22^phox/ rgp91^phox complexes was demonstrated by coprecipitation using subunit-specific antibodies. This study demonstrates for the first time that cDNA encoding either subunit is capable of initiating production of stable recombinant cytochrome b₅₅₈ subunits in eukaryotic cells.     

Gp91<Superscript>phox</Superscript> (NOX2) in classically activated microglia exacerbates traumatic brain injury

Background  

We hypothesized that gp91^phox (NOX2), a subunit of NADPH oxidase, generates superoxide anion (O₂ ^-) and has a major causative role in traumatic brain injury (TBI). To evaluate the functional role of gp91^phox and reactive oxygen species (ROS) on TBI, we carried out controlled cortical impact in gp91^phox knockout mice (gp91^phox-/-). We also used a microglial cell line to determine the activated cell phenotype that contributes to gp91^phox generation.     

The Saccharomyces cerevisiae gene PSO5/RAD16 is involved in the regulation of DNA damage-inducible genes RNR2 and RNR3

The expression of β-galactosidase from DNA damage-inducible RNR2-lacZ and RNR3-lacZ fusion constructs was compared in wild-type (WT) and pso5/rad16 mutant strains after treatment with five mutagens/oxidative stressors. While exposure to the mutagens UVC, 4NQO and H₂O₂ induced expression of the RNR2-lacZ and RNR3-lacZ fusion constructs in two WT strains, treatment with the two oxidative stressors tBOOH and paraquat did not. In the pso5-1 mutant induction of RNR2-lacZ was largely reduced after UVC and H₂O₂ while there was no significant induction of β-galactosidase expression after 4NQO treatment for this construct. For RNR3-lacZ there was strongly reduced expression of pso5-1 after UVC and 4NQO while H₂O₂ failed to induce expression of β-galactosidase. In the WT strains the ranking of the inducing power of the mutagens at 90% survival (as measured in the pso5-1 mutant) was 4NQO>UVC>H₂O₂. Though the WT strains were clearly more resistant that the pso5-1 mutant to the two oxidative stressors paraquat and tBOOH, these substances failed to significantly enhance expression of the RNR2-lacZ and RNR3-lacZ fusion constructs in both the WT and the pso5-1 mutant. Our data suggest that Pso5p/Rad16p has a function in the signal transducing pathway controlling DNA damage-inducible components of nucleotide excision repair. Received: 16 April / 3 June 1998     

NADPH oxidases and MMP-9/TIMP-1 balance in human glomeruli: putative links to diabetic nephropathy

Study aim Glomerular basement membrane thickening, the hallmark of diabetic nephropathy, is thought to be related to an enhanced oxidative stress and reduced matrix proteolysis. Our study concerned the mRNA and protein expression of NADPH oxidase (NOX) components, MMP‐2, MMP‐9 and TIMP‐1 in freshly isolated human glomeruli as well as enzymatic activities and their modulation by glucose, H₂O₂ and angiotensin‐2. Material and methods NOX, cytosolic and membrane‐bound associated proteins and mRNA were analysed by RT‐PCR and Western blotting after glomerular extraction. Oxidase activity was identified by cytochrome c reduction and chemiluminescence. Gelatinases and inhibitors were semiquantitatively assessed by RT‐PCR, gelatin zymography and ELISA in a model of glomerular conditioned survival. Results NOX‐2, NOX‐4 and membrane‐bound and cytosolic factors could be observed in freshly extracted glomeruli (RNA + protein). p40^phox, p67^phox and p47^phox molecular weights were increased compared to their phagocytic counterparts advocating for specific glomerular analogues, and a slight specific oxidase activity was retrieved in isolated glomeruli. Also, mRNA coding for MMP‐2, ‐9 and TIMP‐1, ‐2 were detected. High glucose concentrations (25 mm) reduced TIMP‐1 release in glomerular survival media and MMP‐2 activity in glomerular extracts. On the opposite, angiotensin‐2 significantly induced MMP‐2 and ‐9 activities in the survival media as well as H₂O₂ in glomerular extracts, while addition of 25 mm glucose blunted these findings. Conclusion Glomerular matrix remodelling, the backbone of renal fibrosis in diabetic patients, could be induced by H₂O₂ from specific glomerular NADPH oxidases under the influence of extra‐cellular glucose and angiotensin‐2 and could participate in the control of MMP activities.     

Up-regulation of thromboxane A<Subscript>2</Subscript> impairs cerebrovascular eNOS function in aging atherosclerotic mice

We previously reported that in healthy mouse cerebral arteries, endothelial nitric oxide synthase (eNOS) produces H₂O₂, leading to endothelium-dependent dilation. In contrast, thromboxane A₂ (TXA₂), a potent pro-oxidant and pro-inflammatory endogenous vasoconstrictor, is associated with eNOS dysfunction. Our objectives were to elucidate whether (1) the cerebrovascular eNOS–H₂O₂ pathway was sensitive to oxidative stress associated with aging and dyslipidemia and (2) TXA₂ contributed to cerebral eNOS dysfunction. Atherosclerotic (ATX = LDLR^−/−; hApoB^+/+) and wild-type (WT) control mice were used at 3 and 12 months old (m/o). Three-m/o ATX mice were treated with the cardio-protective polyphenol catechin for 9 months. Dilations to ACh and the simultaneous eNOS-derived H₂O₂ production were recorded in isolated pressurized cerebral arteries. The age-associated decrease in cerebral eNOS–H₂O₂ pathway observed in WT was premature in ATX mice, decreasing at 3 m/o and abolished at 12 m/o. Thromboxane synthase inhibition by furegrelate increased dilations at 12 months in WT and at 3 and 12 months in ATX mice, suggesting an anti-dilatory role of TXA₂ with age hastened by dyslipidemia. In addition, the non-selective NADP(H) oxidase inhibitor apocynin improved the eNOS–H₂O₂ pathway only in 12-m/o ATX mice. Catechin normalized the function of this pathway, which became sensitive to L-NNA and insensitive to furegrelate or apocynin; catechin also prevented the rise in TXA₂ synthase expression. In conclusion, the age-dependent cerebral endothelial dysfunction is precocious in dyslipidemia and involves TXA₂ production that limits eNOS activity. Preventive catechin treatment reduced the impact of endogenous TXA₂ on the control of cerebral tone and maintained eNOS function.     

The effect of normocapnic hypoxia and the duration of exposure to hypoxia on supramaximal exercise performance

Summary Two investigations were designed that (a) evaluated the effect of the respiratory alkalosis that accompanies breathing an hypoxic (H) gas mixture and (b) examined the influence of the duration of breathing this H mixture on the subsequent performance of 45 s supramaximal dynamic exercise. In experiment 1, 12 men performed a 45-s Wingate Test (WT) on three occasions breathing a normoxic (N; 20.9% 02), H (11.3% 02), or normocapnic hypoxic (H + CO₂; 11.5070 O₂, 2.25% CO₂) gas mixture for 20 min prior to performing the WT. For experiment 2, nine men performed a 20-min normoxic (N20) and three hypoxic WT trials which consisted of breathing an 11070 O₂ balance N₂ gas mixture for 10 min (H10), 20 min (H20) or 30 min (H30) prior to the WT. For experiment 1,VO₂ was significantly reduced during the 45-s H [mean (SD); 1.22 (0.23) 1] and H + CO₂ [1.12 (0.18)1] trials compared with the N trial [1.78 (0.18) 1]. Peak power output (W _peak) during WT was similar across trials. However, a small (less than 3070) but significant reduction in the mean power output (W) was observed in both the H and H + CO₂ trials [6.8 (0.6) W · kg^–1] compared with the N trial [7.0 (0.6) W · kg^–1]. Prior to performing the WT, blood pH andPCO₂ were similar [7.40 (0.02) and 5.3 (0.3) kPa, respectively] for the N and H + CO₂ trials. A respiratory alkalosis accompanied the H condition [7.46 (0.02) for pH and 4.6 (0.3) kPa forPCO₂. For experiment 2,VO₂ also was significantly lower during the 45-s WT for H10 [1.16 (0.16) 1], H2O [1.17 (0.16)1], and H30 [1.18 (0.26) 1] compared with N20 [1.84 (0.41) 1].W _peak was similar across trials but a significant reduction in meanW was observed again for the H trials [7.1 (0.4) W · kg^–1] compared with N20 [7.4 (0.4) W · kg^–1]. These data conflict with our previous findings and suggest that breathing an 11% H gas mixture will reduce the mean power produced during 45 s of supramaximal exercise.     

Assessment of ferrocytochrome C oxidation by hydrogen peroxide

The reduction of ferricytochrome C is commonly employed for the quantitation of O ₂ ^– . H₂O₂ arising from the dismutation of O ₂ ^– is capable of oxidizing ferrocytochrome C. In order to assess whether this may interfere with O ₂ ^– quantitation, the amount of H₂O₂ required for the oxidation of ferrocytochrome C was determined. While H₂O₂ concentrations below 10^–5 M were ineffective, one half of the reduced cytochrome was oxidized by 5×10^–5 M H₂O₂ within 15 min. H₂O₂ in the concentration range at which ferrocytochrome C is oxidized is generated upon interaction of hypoxanthine with xanthine oxidase and upon stimulation of human polymorphonuclear neutrophilic granulocytes by phorbol myristate acetate or the phagocytosis of opsonized zymosan. It is suggested that O ₂ ^– quantitation by cytochrome C reduction is routinely performed in the presence of catalase.     

Histamine release with intravenous narcotics: Protective effects of H1 and H2-receptor antagonists

Summary High dose narcotic anesthesia with fentanyl or morphine is not associated with significant direct myocardial depression. Morphine is reported to produce arteriolar dilatation and a decrease in SVR (probably due to histamine release) while fentanyl is not. Studies were undertaken to determine if morphine or fentanyl caused histamine release; if such a release correlated with hemodynamic changes, and if H₁ and H₂ antagonists could provide protection. In a randomized double blind study of 40 patients in 4 groups, patients who received morphine (1 mg/kg) demonstrated significant increases in plasma histamine (880±163 to 7,437±2,684 pg/ml–p<0.01) accompanied by an increase in CI (2.4±0.2 to 3.0±0.2 l/min/m²–p<0.01) and decreases in (88±4 to 61±4 torr–p<0.01) and SVR (15.5±1 to 9.0±1 torr-l-min^–1 p<0.01). The prior administration of H₁ (dyphenhydramine 1 mg/kg) and H₂ (cimetidine 4 mg/kg) antagonists provided significant protection (SVR 17.4±1 to 14.6±1 torr-l-min^–1–p<0.05) although histamine increased comparably (1,059±22 to 7,653±4,242 pg/ml–p<0.05). In a separate study, seven patients receiving fentanyl 50 µg/kg showed no histamine changes (935±51 to 685±51 pg/ml) and no significant hemodynamic response. Eight patients receiving morphine 1 mg/kg again showed significant increases in plasma histamine (880±163 to 7,480±2,230 pg/ml–p<0.05) which collelated with the decrease in SVR (r=0.81). These data demonstrate that morphine releases histamine in amounts which correlate with the hemodynamic changes seen. Prior administration of H₁ and H₂ histamine antagonists provide significant protection — more so than either alone. Fentanyl produced no histamine release which may account for much of the cardiovascular stability reported with this drug.     

Maximal lactate steady state,respiratory compensation threshold and critical power

Critical power (CP) and the second ventilatory threshold (VT₂) are presumed to indicate the power corresponding to maximal lactate steady state (MLSS). The aim of this study was to investigate the use of CP and VT₂ as indicators of MLSS. Eleven male trained subjects [mean (SD) age 23 (2.9) years] performed an incremental test (25 W·min⁻¹) to determine maximal oxygen uptake (V˙O_2max), maximal aerobic power (MAP) and the first and second ventilatory thresholds (VT₁ and VT₂) associated with break points in minute ventilation (V˙_E), carbon dioxide production (V˙CO₂), V˙_E/V˙CO₂ and V˙_E/V˙O₂ relationships. Exhaustion tests at 90%, 95%, 100% and 110% of V˙O_2max and several 30-min constant work rates were performed in order to determine CP and MLSS, respectively. MAP and V˙O_2max values were 344 (29) W and 53.4 (3.7) ml·min⁻¹·kg⁻¹, respectively. CP [278 (22) W; 85.4 (4.8)% V˙O_2max] and VT₂ power output [286 (28) W; 85.3 (5.6)% V˙O_2max] were not significantly different (p=0.96) but were higher (p<0.05) than the MLSS work rate [239 (21) W; 74.3 (4.0)% V˙O_2max] and VT₁ power output [159 (23) W; 52.9 (6.9)% V˙O_2max]. MLSS work rate was significantly correlated (p<0.05) with those noted at VT₁ and VT₂ (r=0.74 and r=0.93, respectively). VT₂ overestimated MLSS by 10.9 (6.3)% V˙O_2max which was significantly higher than VT₁ [+21.4 (5.6)% V˙O_2max; p<0.01]. CP calculated from a given range of exhaustion times does not correspond to MLSS. Electronic Publication     

Lung oxidative stress as related to exercise and altitude. Lipid peroxidation evidence in exhaled breath condensate: a possible predictor of acute mountain sickness

Lung oxidative stress (OS) was explored in resting and in exercising subjects exposed to moderate and high altitude. Exhaled breath condensate (EBC) was collected under field conditions in male high-competition mountain bikers performing a maximal cycloergometric exercise at 670 m and at 2,160 m, as well as, in male soldiers climbing up to 6,125 m in Northern Chile. Malondialdehyde concentration [MDA] was measured by high-performance liquid chromatography in EBC and in serum samples. Hydrogen peroxide concentration [H₂O₂] was analysed in EBC according to the spectrophotometric FOX₂ assay. [MDA] in EBC of bikers did not change while exercising at 670 m, but increased from 30.0±8.0 to 50.0±11.0 nmol l⁻¹ (P<0.05) at 2,160 m. Concomitantly, [MDA] in serum and [H₂O₂] in EBC remained constant. On the other hand, in mountaineering soldiers, [H₂O₂] in EBC under resting conditions increased from 0.30±0.12 μmol l⁻¹ at 670 m to 1.14±0.29 μmol l⁻¹ immediately on return from the mountain. Three days later, [H₂O₂] in EBC (0.93 ±0.23 μmol l⁻¹) continued to be elevated (P<0.05). [MDA] in EBC increased from 71±16 nmol l⁻¹ at 670 m to 128±26 nmol l⁻¹ at 3,000 m (P<0.05). Changes of [H₂O₂] in EBC while ascending from 670 m up to 3,000 m inversely correlated with concomitant variations in HbO2 saturation (r=−0.48, P<0.05). AMS score evaluated at 5,000 m directly correlated with changes of [MDA] in EBC occurring while the subjects moved from 670 to 3,000 m (r=0.51, P<0.05). Lung OS may constitute a pathogenic factor in AMS.     

Noninvasive quantitative magnetization transfer MRI reveals tubulointerstitial fibrosis in murine kidney

Excessive tissue scarring, or fibrosis, is a critical contributor to end stage renal disease, but current clinical tests are not sufficient for assessing renal fibrosis. Quantitative magnetization transfer (qMT) MRI provides indirect information about the macromolecular composition of tissues. We evaluated measurements of the pool size ratio (PSR, the ratio of immobilized macromolecular to free water protons) obtained by qMT as a biomarker of tubulointerstitial fibrosis in a well‐established murine model with progressive renal disease. MR images were acquired from 16‐week‐old fibrotic hHB‐EGF^Tg/Tg mice and normal wild‐type (WT) mice (N = 12) at 7 T. QMT parameters were derived using a two‐pool five‐parameter fitting model. A normal range of PSR values in the cortex and outer stripe of outer medulla (CR + OSOM) was determined by averaging across voxels within WT kidneys (mean ± 2SD). Regions in diseased mice whose PSR values exceeded the normal range above a threshold value (tPSR) were identified and measured. The spatial distribution of fibrosis was confirmed using picrosirius red stains. Compared with normal WT mice, scattered clusters of high PSR regions were observed in the OSOM of hHB‐EGF^Tg/Tg mouse kidneys. Moderate increases in mean PSR (mPSR) of CR + OSOM regions were observed across fibrotic kidneys. The abnormally high PSR regions (% area) detected by the tPSR were significantly increased in hHB‐EGF^Tg/Tg mice, and were highly correlated with regions of fibrosis detected by histological fibrosis indices measured from picrosirius red staining. Renal tubulointerstitial fibrosis in OSOM can thus be assessed by qMT MRI using an appropriate analysis of PSR. This technique may be used as an imaging biomarker for chronic kidney diseases.     

Oxygen uptake kinetics during severe intensity running and cycling

The purpose of this study was to investigate the effect of exercise mode on the characteristics of the oxygen uptake ( V̇O₂) response to exercise within the severe intensity domain. Twelve participants each performed a treadmill running test and a cycle ergometer test to fatigue at intensities selected to elicit a mode-specific V̇O₂max and to cause fatigue in ~5 min. The tests were at 234 (30) m·min⁻¹ and 251 (59) W, and times to fatigue were 297 (15) s and 298 (14) s, respectively. The overall rapidity of the V̇O₂response was influenced by exercise mode [ V̇O₂max was achieved after 115 (20) s in running versus 207 (36) s in cycling; p<0.01]. V̇O₂ responses were fit to a three-phase exponential model. The time constant of the primary phase was faster in treadmill tests than in cycle ergometer tests [14 (6) s versus 25 (4) s; p<0.01], and the amplitude of the primary phase was greater in running than in cycling when it was expressed in absolute terms [2327 (393) ml·min⁻¹ versus 2036 (301) ml·min⁻¹; p=0.02] but not when it was expressed as a percentage of the total increase in V̇O₂ [86 (6)% versus 82 (6)%; p=0.09]. When quantified as the difference between the end-exercise V̇O₂ and the V̇O₂ at 2 min, the amplitude of the slow component was ~40% smaller in running [177 (92) ml·min⁻¹ versus 299 (153) ml min⁻¹; p=0.03]. It is concluded that exercise modality affects the characteristics of the V̇O₂ response at equivalent intensities in the severe domain.     

Stimulation of human neutrophilic granulocytes by two monocytederived cytokines

Human monocytes upon stimulation with bacterial lipopolysaccharide release two cytokines which modulate the functions of neutrophilic granulocytes (PMN), a monocyte-derived chemotaxic (MOC) and tumor necrosis factor (TNF). Both cytokines stimulated the adherence of PMN on protein-coated nylon-fibers. Whereas MOC is one of the four most potent chemoattractants known, TNF was a most powerful inhibitor of PMN chemotactic migration towards several chemotactic factors including MOC. Neither cytokine stimulated the release of superoxide anion (O ₂ ^– ) or hydrogen peroxide (H₂O₂) from PMN in suspension. However, TNF, but not MOC, caused the release of considerable amounts of H₂O₂ and O ₂ ^– from PMN attached to nylon fibers. The two cytokines have similar effects on the adherence, opposing effects on chemotactic migration and different effects on the oxidative burst of PMN.     

Die Rolle der Chemoreceptoren des Carotisgebiets der narkotisierten Katze für die Antwort der Atmung auf isolierte Änderung der Wasserstoffionen-Konzentration und des CO2-Drucks des Blutes

1. In 16 vagotomized cats anesthetized with chloralose-urethane CO₂ response curves [V=f (pCO_2A)] were determined at elevated inspiratory O₂ (35%) during normal acid base balance and in metabolic acidosis induced by i.v. infusion of HCl or in metabolic alcalosis induced by i.v. infusion of NaHCO₃. The experiments were repeated after severing the sinus nerves. By a graphical method using the data in acidosis and normal state or alcalosis and normal state, the isolated drives of CO₂ and H⁺ were determined.2. Cutting the vagosympathetic and depressor nerves caused the total CO₂ sensitivity (slope of CO₂ response curve) to increase. At the same time the intercept of the CO₂ response curve with the abszissa (pCO_2A) was shifted to the right. This was interpreted as suggesting the disappearance of a (possibly nonspecific) respiratory drive.3. Severing sinus nerves caused only minor changes of the isolated CO₂ (0–13%) and H⁺ effects (< 33%). It must be stated, however, that an accurate determination is much hampered by scattering of data. According to the small effect on isolated H⁺ and CO₂ drives the slope of the CO₂ response curve was only slightly diminished (from 49 to 44 ml min^–1 Torr^–1).4. Additionally to the (small) decrease of slope, severing of the sinus nerves caused a shift of the CO₂ response curve to the right (to higher pCO₂) of 5,6 Torr in the average of the animals in normal acid base balance. The shift is possibly smaller during metabolic alcalosis and bigger during metabolic acidosis.5. It is concluded that in the presence of sufficiently high arterial pO₂ the role in respiratory regulation of isolated CO₂ and isolated H⁺ effects on the chemoreceptors of the carotid region is small compared to the more central effects. The role of a possibly nonspecific respiratory drive conducted in the sinus nerves, however, is considerable. The nature of this drive is not yet clear.

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Mit 5 Textabbildungen

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.

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Diese Veröffentlichung ist ein Teil der der Medizinischen Fakultät Göttingen vorgelegten Habilitationsschrift.