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Biomedical subjects

U Flögel

Publications and source records attributed to U Flögel.

At least 19 recordsLinked to original sources
PubMed2001

Myoglobin: A scavenger of bioactive NO.

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 (MbO(2)) to metmyoglobin (metMb) by reaction with NO. NO was applied intracoronarily (5 nM to 25 microM), or its endogenous production was stimulated with bradykinin (Bk; 10 nM to 2 microM). We found that infusion of authentic NO solutions dose-dependently (>/= 2.5 microM 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 microM Bk). Hearts lacking Mb reacted more sensitively to infused NO in that vasodilatation and the cardiodepressant actions of NO were more pronounced. Similar results were obtained with Bk. The lower sensitivity of WT hearts to changes in NO concentration fits well with the hypothesis that in the presence of Mb, a continuous degradation of NO takes place by reaction of MbO(2) + NO to metMb + NO(3)(-), thereby effectively reducing cytosolic NO concentration. This breakdown protects myocytic cytochromes against transient rises in cytosolic NO. Regeneration of metMb by metMb reductase to Mb and subsequent association with O(2) leads to reformation of MbO(2) available for another NO degradation cycle. Our data indicate that this cycle is crucial in the breakdown of NO and substantially determines the dose-response curve of the NO effects on coronary blood flow and cardiac contractility.

Animals↗
PubMed1999

Disruption of myoglobin in mice induces multiple compensatory mechanisms.

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 myoglobin-knockout (myo(-/-)) mice. To genetically inactivate the myoglobin gene, exon 2 encoding the heme binding site was deleted in embryonic stem cells via homologous recombination. Myo(-/-) mice are viable, fertile, and without any obvious signs of functional limitations. Hemoglobin concentrations were significantly elevated in myo(-/-) mice. Cardiac function and energetics were analyzed in isolated perfused hearts under resting conditions and during beta-adrenergic stimulation with dobutamine. Myo(-/-) hearts showed no alteration in contractile parameters either under basal conditions or after maximal beta-adrenergic stimulation (200 nM dobutamine). Tissue levels of ATP, phosphocreatine ((31)P-NMR), and myocardial O(2) consumption were not altered. However, coronary flow [6.4 +/- 1.3 ml.min(-1).g(-1) [wild-type (WT)] vs. 8.5 +/- 2.4 ml.min(-1).g(-1) [myo(-/-)] [and coronary reserve [17.1 +/- 2.1 (WT) vs. 20.8 +/- 1.1 (myo(-/-) ml. min(-1).g(-1) were significantly elevated in myo(-/-) hearts. Histological examination revealed that capillary density also was increased in myo(-/-) hearts [3,111 +/- 400 mm(-2) (WT) vs. 4,140 +/- 140 mm(-2) (Myo(-/-)]. 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.

Adenosine↗
PubMed1999

Contribution of NO to ischemia-reperfusion injury in the saline-perfused heart: a study in endothelial NO synthase knockout mice.

The contribution of endogenous NO to ischemia-reperfusion injury was studied in isolated perfused hearts of wild-type (WT) and endothelial NO synthase knockout (eNOS-) mice. The hearts were subjected to a 16-min period of global no-flow ischemia and were subsequently reperfused for 1 h. Cardiac contractile function was evaluated and 31P-NMR spectroscopy was used to monitor myocardial energy status and the intracellular pH. During both baseline and ischemia, there were neither significant differences in mechanical function nor in energetic parameters between the two groups, for example at baseline left ventricular developed pressure (LVDP) was 56.5+/-5.4 mmHg in WT and 58.7+/-5.2 mmHg in eNOS-and phosphocreatine (PCr) level was 12.9+/-1.3 m m in WT and 12.7+/-1.7 m m in eNOS-. In reperfusion, however, a significant improvement of the post-ischemic functional and metabolic recovery became apparent in the eNOS-hearts. While in the WT group, LVDP recovered only to 38. 4+/-5.3 mmHg, LVDP in the eNOS-group attained 49.4+/-5.5 mmHg at the end of 60 min reperfusion (P<0.05, n=8). Similarly, the recovery of PCr was significantly enhanced in the transgenic hearts as compared to WT (10.4+/-1.6 vs 8.1+/-1.3 m m, P<0.05). eNOS-hearts also showed a better restoration of dP/d t and a significant lower left ventricular enddiastolic pressure. In an additional series of wild-type hearts, the NO synthase inhibitor NG-monomethyl-L-arginine methyl ester (100 microm) also tended to improve the recovery of both LVDP (43.8+/-6.8 mmHg) and PCr (9.5+/-1.6 m m) in reperfusion (1 h), but the restoration of functional and metabolic parameters was less pronounced when compared with eNOS-. The results provide clear evidence that endogenously formed NO significantly contributes to ischemia-reperfusion injury in the saline-perfused mouse heart, most likely by peroxynitrite formation from NO.

Animals↗
PubMed1998

Expression of aquaporins in Xenopus laevis oocytes and glial cells as detected by diffusion-weighted 1H NMR spectroscopy and photometric swelling assay.

Expression of aquaporins (AQP) and water permeability were studied in Xenopus laevis oocytes and immobilized glial cells by a pulsed-field gradient spin echo NMR technique and a photometric swelling assay. Oocytes injected with poly(A) RNA from C6-BU-1 cells showed increased swelling behavior under hypoosmotic stress due to expressed water channels as compared to control oocytes. The swelling could be reversibly inhibited by HgCl2. Furthermore, the intracellular relaxation time and the apparent intracellular diffusion coefficient of water in oocytes were determined by diffusion-weighted 1H NMR experiments to be T2=36 ms and Dapp, intra=0.18x10-3 mm2/s. In immobilized C6 and F98 cells the mean exchange time of intracellular water was found to be 51 ms which increased to 75 ms upon chronic treatment (4 days) in hypertonic medium. Additional hybrid depletion experiments with antisense oligonucleotides directed against AQP1 were performed on oocytes and C6 cells. Moreover, different water channel subtypes of glial cells were assessed by a reverse transcriptase polymerase chain reaction assay. With this, the mRNA encoding AQP1 could be detected in primary cultures and glial cell lines, whereas AQP4 mRNA was found in astroglia-rich primary cultures, but not in F98 and C6 cells. Our results show that water permeability in glial cells is mainly mediated by water channels which play an important role in the regulation of water flow in brain under normal and pathological conditions.

Animals↗
PubMed1998

Monitoring of cell volume and water exchange time in perfused cells by diffusion-weighted 1H NMR spectroscopy.

Diffusion of intracellular water was measured in perfused cells embedded in basement membrane gel threads. F98 glioma cells, primary astrocytes, and epithelial KB cells were used and were exposed to osmotic stress, immunosuppressiva, the water channel blocker p-chloromercuriobenzenesulfonate (pCMBS), and apoptotic conditions. With diffusion-weighted 1H NMR spectroscopy changes in the intracellular signal could be monitored and quantified with single signal (ss), constant diffusion time (ct), and constant gradient strength (cg) experiments. The temporal resolution of the ss monitoring was 3.5 s with a standard deviation of 0.5% of the signal intensity and 32 s (3%) with ct monitoring, respectively. A mean intracellular residence time of water was determined with the cg experiment to about 50 ms. Changes of this exchange time from (51.9 +/- 1.0) to (59.0 +/- 1.1) ms were observed during treatment with pCMBS. The changes in the diffusion attenuated signal could be simulated analytically varying the intracellular volume fraction and exchange time by combination of restricted diffusion (Tanner model) and water exchange (Kärger model). This sensitive and noninvasive NMR method on perfused cells allows to determine changes in the intracellular volume and residence time in a simple and accurate manner. Many further applications as anoxia, volume regulation, ischemia and treatment with various pharmaceuticals are conceiveable to follow up their effect on the cell volume and the exchange time of intracellular water.

Animals↗
PubMed1998

Restricted diffusion and exchange of intracellular water: theoretical modelling and diffusion time dependence of 1H NMR measurements on perfused glial cells.

Intracellular diffusion properties of water in F98 glioma cells immobilized in basement membrane gel threads, are investigated with a pulsed-field-gradient spin-echo NMR technique at diffusion times from 6 to 2000 ms and at different temperatures. In extended model calculations the concept of 'restricted intracellular diffusion at permeable boundaries' is described by a combined Tanner-Kärger formula. Signal components in a series of ct experiments (constant diffusion time) are separated due to different diffusion properties (Gaussian and restricted diffusion), and physiological as well as morphological cell parameters are extracted from the experimental data. The intracellular apparent diffusion coefficients strongly depend on the diffusion time and are up to two orders of magnitude smaller than the self diffusion constant of water. Propagation lengths are found to be in the range of 4-7 microns. Hereby intracellular signals of compartments with a characteristic diameter could be selected by an appropriate gradient strength. With cg experiments (constant gradient) a mean intracellular residence time for water is determined to be about 50 ms, and the intrinsic intracellular diffusion constant is estimated to 1 x 10(-3)mm2/s. Studying the water diffusion in glial cells provides basic understanding of the intracellular situation in brain tissue and may elucidate possible influences on the changes in the diffusion contrast during ischemic conditions.

Animals↗
PubMed1998

A 1H/13C inverse 2D method for the analysis of the polyamines putrescine, spermidine and spermine in cell extracts and biofluids.

The polyamines putrescine, spermidine and spermine are involved in the regulation of various metabolic processes. It is therefore desirable to detect and quantify the polyamines with NMR. We present the proton and carbon assignments for all polyamine signals obtained from PCA extracts of F98 glioma cells with high resolution using a semi-selective HSQC 2D-experiment. The biosynthesis of the polyamines in cell culture was examined using the labeled substrates [U-13C]glucose and [U-13C]glutamate. In such studies the high resolution of the semi-selective HSQC experiment at very high magnetic fields (14-19 T) allows the analysis of carbon-carbon couplings, and isotopomer patterns. The different effects of osmotic stress on the concentrations of polyamines and amino acids are also reported.

Animals↗
PubMed1998

Rat brain primary neurons immobilized in basement membrane gel threads: an improved method for on-line 13C NMR spectroscopy of live cells.

In vivo MRS studies on intact brain reflect the metabolism of all cells present, but do not distinguish between different cell types. NMR studies of immobilized cultured primary cells, such as neurons and astrocytes, are a useful model to monitor the specific differences in metabolism of the various cell types in the brain. The present study shows that primary rat neuronal cells can be cultured in basement membrane gel threads. After 4 days of incubation the threads are filled with viable cells, and represent a population of morphologically differentiated neuronal cells with less than 5% of non-neuronal cells, i.e., astrocytes. These threads were placed into a NMR tube and used for on-line monitoring of neuronal metabolism. Under these conditions cells remained viable and metabolically active for several days. The energy status was monitored by using 31P NMR spectroscopy. To study neuronal glucose metabolism [1-13C]glucose was added to the perfusion medium and 30 min later 13C-labeled metabolites were detectable by 13C NMR spectroscopy. Immobilized neurons synthesized glycolytic products such as [3-13C]lactate and [3-13C]alanine, as well as several tricarboxylic acid cycle products, i.e., [2-13C]glutamate, [3-13C]glutamate, [4-13C]glutamate, [2-13C]aspartate, and [3-13C]aspartate. In summary, 31P and 13C NMR spectra can be recorded from live neuronal cells for up to 24 h using the newly designed procedure described in the present communication.

Animals↗
PubMed1998

Alterations in glial cell metabolism during recovery from chronic osmotic stress.

NMR spectroscopy of F98 glioma cell extracts showed that chronic hypertonic conditions largely increased the intracellular content of small, osmotically active molecules. Moreover, hypertonic stress decreased the incorporation of 13C-labeled amino acids into the cellular proteins albeit their cytosolic concentrations were increased, which reflects an inhibition of protein synthesis under these conditions. Reincubation with isotonic medium restored almost completely the control values for the cytosolic metabolites but not for amino acid incorporation into the protein. An increased amount of 13C label was found in the phospholipids, which indicates stimulation of membrane synthesis processes due to the recovery-induced cell swelling. On the other hand, chronic hypotonic conditions largely decreased the steady state concentration and synthesis of small, cytosolic molecules, whereas the effect on the incorporation of 13C-labeled amino acids into the cellular proteins was variable. Reincubation with isotonic medium partially restored the depressed cytosolic metabolite content and also the incorporation of labeled amino acids into cellular protein, but induced an inhibition of phospholipid synthesis. The results verify that 'readaptation' of glial cell metabolism during recovery from chronic osmotic stress is impaired or at least seriously retarded.

Adaptation, Physiological↗
PubMed1997

Study of the interactions of cadmium and zinc ions with cellular calcium homoeostasis using 19F-NMR spectroscopy.

The effects of the heavy-metal ions Cd2+ and Zn2+ on the homoeostasis of intracellular free Ca2+ in E367 neuroblastoma cells were examined using 19F-NMR spectroscopy with the fluorinated chelator probe 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N', N'-tetra-acetic acid (5F-BAPTA). First, the technique was used to quantify the uptake and intracellular free concentrations of the heavy metals after treatment of the cells with 20 microM CdCl2 or 100 microM ZnCl2. Secondly, metal-induced transients in intracellular free Ca2+ were recorded. Addition of 20 microM CdCl2, but not 100 microM ZnCl2, evoked a transient increase in Ca2+ from a resting level of 84 nM to approx. 190 nM within 15 min after addition of the metal. Zn2+ at 20 microM completely prevented the induction of a Ca2+ transient by Cd2+. Ca2+ was mobilized by Cd2+ from intracellular organelles, since depletion of these stores by thapsigargin abolished the effect of the toxic metal. Furthermore, 20 microM Cd2+ evoked a transient rise in cellular Ins(1,4,5)P3, reaching a maximum level within 5 min after addition of the metal. These results demonstrate that perturbation of the Ins(1,4,5)P3/Ca2+ messenger system is an early and discrete cellular effect of Cd2+.

Animals↗
PubMed1997

Determination of de novo synthesized amino acids in cellular proteins revisited by 13C NMR spectroscopy.

13C nuclear magnetic resonance spectroscopy was used to determine the absolute amounts to de novo synthesized amino acids in both the perchloric acid extracts and the hydrolyzed protein fractions of F98 glioma cells incubated for 2 h with 5 mmol/l [U-13C]glucose. 13C NMR spectra of the hydrolyzed protein fraction revealed a marked incorporation of 13C-labelled alanine, aspartate and glutamate into the proteins of F98 cells within the incubation period. Additionally, small amounts of 13C-labelled glycine, proline and serine could unambiguously be identified in the protein fraction. Astonishingly, approximately equal amounts of 13C-labelled glutamate and aspartate were incorporated into the cellular proteins, although the cytosolic steady-state concentration of aspartate was below 13C NMR detectability. Hypertonic stress decreased the incorporation of 13C-labelled amino acids into the total protein, albeit their cytosolic concentrations were increased, which reflects an inhibition of protein synthesis under these conditions. On the other hand, hypotonic stress increased the amount of 13C-labelled proline incorporated into the cellular proteins even though the cytosolic concentration of 13C-labelled proline was largely decreased. Apparently, hypoosmotic conditions stimulate the synthesis of proteins or peptides with a high proline content. The results show that already after 2 h of incubation with [U-13C]glucose there is a pronounced flux of 13C label into the cellular proteins, which is usually disregarded if cytosolic fluids are examined only. This means that calculations of metabolic fluxes based on 13C NMR spectroscopic data obtained from perchloric acid extracts of cells or tissues and also from in vivo measurements consider only the labelled 'NMR visible' cytosolic metabolites, which may have to be corrected for fast label flowing off into other compartments.

Amino Acids↗
PubMed1997

Assessment of the mechanism of astrocyte swelling induced by the macrolide immunosuppressant sirolimus using multinuclear nuclear magnetic resonance spectroscopy.

The toxic effect of the macrolide immunosuppressant sirolimus on cell metabolism of primary astrocytes was studied by multinuclear NMR spectroscopy of viable cells and perchloric acid (PCA) extracts and compared to the effects of the immunosuppressant cyclosporine. The addition of 5 mg/L sirolimus (5.5 mumol/L) induced swelling of primary astrocytes to 110% of the original volume. Alteration in astrocyte volume in the presence of sirolimus was accompanied by reduction of the following important cell osmolytes and amino acid metabolites: myo-inositol, -58 +/- 12% (mean +/- standard deviation, n = 5); taurine, -44 +/- 5%; glutamine, -13 +/- 2%; compared with control. Sirolimus altered glucose metabolism and partially inhibited the tricarboxylic acid (TCA) cycle: sigma TCA/sigma glycolyse = 1.36 +/- 0.09 (control, n = 3), 0.96 +/- 0.08 (with sirolimus). The increased concentration of phosphodiesters by sirolimus addition (glycerophosphoethanolamine, 52 +/- 18%; glycerophosphocholine, 61 +/- 14%; compared with control, n = 5) indicated disorders in phospholipid metabolism of cellular membranes. Addition of sirolimus led to a decline of the energy state in astrocytes: the concentration of phosphocreatine (PCr) decreased to 75% of control value within 60 min of perfusion with sirolimus and the nucleotide triphosphate (NTP) concentration to 85% within 90 min (n = 3). The effect of sirolimus on the cell metabolism of astrocytes equals that of the immunosuppressants cyclosporine and tacrolimus, the neurotoxicity of which is well-established in clinical studies. The results of this in vitro study indicate that sirolimus possesses neurotoxic potential as well.

Animals↗
PubMed1996

Adaptation of cellular metabolism to anisosmotic conditions in a glial cell line, as assessed by 13C-NMR spectroscopy.

13C-NMR spectroscopy of perchloric acid and lipid extracts of F98 glioma cells showed that volume-regulatory processes under anisosmotic conditions were accompanied by marked alterations in cellular metabolism. Production of alanine, glutamate, and glycine from [U-13C]-glucose is decreased under hypotonic stress and is oppositely increased under hypertonic stress. In contrast, degradation of these molecules is raised under hypotonic conditions and reduced under hypertonic conditions. Furthermore, phospholipid synthesis is decreased under hypertonic stress and increased under hypotonic stress. Obviously, glial metabolism is directed under hypertonic conditions to maintain a high level of small, osmotically active molecules, whereas under hypotonic conditions molecular fragments are increasingly incorporated into the phospholipids and so do not contribute to the osmotic pressure. The latter is evoked by the activation of membrane synthesis process to compensate for stretching and/or damaging of the membranes due to cell swelling.

Adaptation, Physiological↗
PubMed1996

Immobilization of primary astrocytes and neurons for online monitoring of biochemical processes by NMR.

Immobilization of primary astrocytes and cerebellar granule neurons in basement membrane gel threads and on porous microcarriers, respectively, is described. Five different porous microcarriers were examined for their capabilities to support maturation of neurons, as evaluated by determinations of protein content and glucose uptake. These systems proved suitable for perfusion in the NMR tube for long periods of time. The energetic status of the cells was monitored by 31P-NMR, and a very good temporal resolution (less than 30 min) was achieved with superfused astrocytes; thus, this provides a valuable method for real time NMR investigation of biochemical processes in primary astrocytes. Significantly longer acquisition times (approximately 8 h) were required in order to obtain a spectrum of neurons with a reasonably good signal to noise ratio, even so, the results presented here and obtained with this difficult system are very encouraging.

Animals↗
PubMed1995

Changes in organic solutes, volume, energy state, and metabolism associated with osmotic stress in a glial cell line: a multinuclear NMR study.

Diffusion-weighted in vivo 1H-NMR spectroscopy of F98 glioma cells embedded in basement membrane gel threads showed that the initial cell swelling to about 180% of the original volume induced under hypotonic stress was followed by a regulatory volume decrease to nearly 100% of the control volume in Dulbecco's modified Eagle's medium (DMEM) but only to 130% in Krebs-Henseleit buffer (KHB, containing only glucose as a substrate) after 7 h. The initial cell shrinkage to approx. 70% induced by the hypertonic stress was compensated by a regulatory volume increase which after 7 h reached almost 100% of the control value in KHB and 75% in DMEM. 1H-, 13C- and 31P-NMR spectroscopy of perchloric acid extracts showed that these volume regulatory processes were accompanied by pronounced changes in the content of organic osmolytes. Adaptation of intra- to extracellular osmolarity was preferentially mediated by a decrease in the cytosolic taurine level under hypotonic stress and by an intracellular accumulation of amino acids under hypertonic stress. If these solutes were not available in sufficient quantities (as in KHB), the osmolarity of the cytosol was increasingly modified by biosynthesis of products and intermediates of essential metabolic pathways, such as alanine, glutamate and glycerophosphocholine in addition to ethanolamine. The cellular nucleoside triphosphate level measured by in vivo 31P-NMR spectroscopy indicated that the energy state of the cells was more easily sustained under hypotonic than hypertonic conditions.

Animals↗