Biomedical subjects

G Navon

Publications and source records attributed to G Navon.

At least 55 records · Page 3Linked to original sources

PubMed1996

A 23Na multiple-quantum-filtered NMR study of the effect of the cytoskeleton conformation on the anisotropic motion of sodium ions in red blood cells.

Recently, it has been shown that 23Na double-quantum-filtered NMR spectroscopy can be used to detect anisotropic motion of bound sodium ions in biological systems. The technique is based on the formation of the second-rank tensor when the quadrupolar interaction is not averaged to zero. Using this method, anisotropic motion of bound sodium in human and dog red blood cells was detected, and the effect was shown to depend on the integrity of the membrane cytoskeleton. In the present study, multiple-quantum-filtered techniques were applied in combination with a quadrupolar echo to measure the transverse-relaxation times, T2f and T2s. Line fitting was performed to obtain the values of the residual quadrupolar interaction, which was measured for sodium in a variety of mammalian erythrocytes of different size, shape, rheological properties, and sodium concentrations. Human unsealed white ghosts were used to study sodium bound at the anisotropic sites on the inner side of the RBC membrane. Modulations of the conformation of the cytoskeleton by the variation of either the ionic strength or pH of the suspending medium caused drastic changes in both the residual quadrupolar interaction and T2f due to changes in the fraction of bound sodium ions as well as changes in the structure of the binding sites. By combining the two spectroscopic parameters, structural change can be followed. The changes in the structure of the sodium anisotropic binding sites deduced by this method were found to correlate with known conformational changes of the membrane cytoskeleton. Variations of the medium pH affected both the fraction of bound sodium ions and the structure of the anisotropic binding sites. Sodium and potassium were shown to bind to the anisotropic binding sites with the same affinity.

Algorithms↗

PubMed1996

31P NMR methods for the direct determination of ADP in the presence of ATP.

A new method is presented for the direct measurement of the amount of ADP in the presence of ATP by selectively eliminating the alpha- and gamma-ATP signals. The method is compared with other methods, both experimentally and theoretically, using the product-operator formalism. An analysis of the effect of beta 1 inhomogeneity on the efficiency of the methods is also given. Experimental results obtained using adiabatic pulses to compensate for such effects are shown. The accuracy of this new method is demonstrated by measuring various ADP concentrations in a series of solutions containing ADP and ATP.

Adenosine Diphosphate↗

PubMed1996

Volume-related activities of sodium ion transporters: multinuclear NMR studies of isolated rat hearts.

The present study aims to determine the volume-related activities of sodium ion transporters in the rat heart. Intracellular volumes were measured in isolated hearts by 1H of water and 59Co nuclear magnetic resonance (NMR) of the extracellular marker cobalticyanide. Inhibition of the Na-K-adenosinetriphosphatase pumps with 50 microM ouabain did not affect the extent of cellular swelling during 30 min of ischemia: cells swelled by 0.37 ml/g dry wt compared with the controls (0.38 ml/g dry wt). After perfusion with 400 microM ouabain or 200 microM iodoacetate, the cells shrank during ischemia (from 2.50 +/- 0.06 to 2.20 +/- 0.09 and 2.28 +/- 0.07 ml/g dry wt, respectively). Inhibition of passive sodium ion transporters reduced cellular swelling during ischemia: pretreatment (10 min) with 100 microM furosemide (Na-K-2Cl cotransport), 1.5 microM ethylisopropylamiloride (Na/H antiport), and 50 microM lidocaine (sodium channels) led to swelling of 0.27, 0.21, and 0.13 ml/g dry wt, respectively. The extent of cellular water accumulation was apparently correlated with the onset and maximal force of the ischemic contracture, unlike the data of hearts treated with ouabain and iodoacetate. The blockage of each of the passive sodium transporters improved the recovery of intracellular volumes at reperfusion, indicating that in the heart these pathways are responsible for the sustained reperfusion cellular edema. It is concluded that acute cellular swelling during myocardial ischemia is not caused by insufficiency of the Na-K pumps but is partially mediated by systems that transport sodium into the cells.

Animals↗

PubMed1996

Nuclear magnetic resonance (NMR) analysis of ligand receptor interactions: the cholinergic system--a model.

Elucidation of the molecular mechanisms that govern ligand-receptor recognition is essential to the rational design of specific pharmacological reagents. Whereas often the receptor and its binding site are the target of investigation, study of the ligand in its free and bound state can also reveal important information regarding this recognition process. Nuclear magnetic resonance (NMR) spectroscopy can be extremely useful for such studies. In this review, we discuss the attributes of NMR in the study of ligand receptor interactions. The cholinergic receptor and its binding to the neurotransmitter, acetylcholine, and cholinergic antagonists serve as a model system, illustrating the power of ligand analysis by NMR. The results discussed prove that the region of residues alpha 180-205 of the nicotinic acetylcholine receptor are an essential component of the cholinergic binding site and that ligand binding involves a positively charged hydrophobic motif.

Acetylcholine↗

PubMed1995

The nature of the thermal equilibrium affecting the iron coordination of ferric cytochrome c.

In cytochrome c, ligation of the heme iron by the methionine-80 sulfur plays a major role in determining the structure and the thermodynamic stability of the protein. In the ferric state, this bond is reversibly broken by moderately acid or alkaline pH's (pK's 2.5 and 9.4, respectively) and by exogenous ligands. NMR studies of horse ferricytochrome c in which the Met-65 and Met-80 methyl groups were chemically enriched with 13C demonstrate that, at 59 degrees C, a temperature at which the protein is still folded, the sulfur-iron bond is already partially broken. This structural change corresponds to the reversible disappearance upon moderate heating of the 695 nm band, characteristic of the sulfur-iron coordination of this protein. The thermal effect results from a shift in the alkaline pK from 9.4 at 25 degrees C to 8.2 at 59 degrees C. The exchange rate from iron-bound to free methionine-80 at 59 degrees C is 1.8 s-1, as measured by saturation transfer experiments. The free and bound methionine-80 epsilon-methyl groups in the 1H spectrum are assigned as (1.87, 2.25) and -21.43, respectively; in the 13C spectrum they are assigned as 15.6 and 12.8, respectively (all these values are in ppm from 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, sodium salt).

Animals↗

PubMed1995

Mechanism of action of the antineoplastic drug lonidamine: 31P and 13C nuclear magnetic resonance studies.

The mechanism of action of the antineoplastic drug lonidamine (LND) on MCF-7 human breast cancer cells was studied with the use of 31P and 13C nuclear magnetic resonance (NMR) spectroscopy. The cells were embedded in alginate microcapsules, perfused with growth media and LND at physiological conditions in the NMR tube, and continuously monitored in vivo for the effects of LND. 31P NMR demonstrated intracellular acidification after LND perfusion concomitant with ATP depletion and changes in phospholipid metabolites. 13C NMR showed marked LND-induced accumulation of lactate, and spectra of the perfusate disclosed that LND inhibited lactate transport. Kinetic 13C NMR also furnished information on LND effects on glucose metabolism; LND decreased initial glucose uptake and lactate formation, although the final intracellular glucose levels were higher compared with those in controls. Combined administration of LND and the metabolic inhibitor 2-deoxyglucose yielded additive but not synergistic cytotoxicity and enabled assessment of hexokinase activity. Overall, the results indicate that the major metabolic changes induced by LND are inhibition of lactate transport and its accumulation, which lead to intracellular acidification.

Antineoplastic Agents↗

PubMed1995

Intracellular volume measurement and detection of edema: multinuclear NMR studies of intact rat hearts during normothermic ischemia.

The present study describes the cell volume dynamics in intact rat hearts, during ischemia and after reperfusion. Cell volumes were measured in isolated hearts by either 13C or 59Co NMR of mannitol or cobalticyanide, respectively, as extracellular markers and 1H NMR of water as the aqueous space marker. A constant volume chamber was built inside a 15-mm NMR tube; the contents of the chamber were measured with and without a heart. The intracellular volume of isolated rat hearts was estimated to be 2.45 +/- 0.13 ml/g dry weight. In the perfused heart, adenosine triphosphate (ATP) and phosphocreatine (PCr) concentrations were calculated to be 12.2 +/- 0.7 and 16.1 +/- 1.0 mM, respectively. Consecutive volume measurements showed cell swelling of 16% during 30 min of ischemia, which was reduced at reperfusion to 7%. After 30 min of reperfusion, ATP and PCr concentrations were 4.5 +/- 0.8 and 8.1 +/- 0.9 mM. It is concluded that: (1) cell swelling is an ischemic event, which is partially reversed by reperfusion; and (2) continuous measurement of cell volumes provides intracellular molar concentrations of metabolites, which are the physiologically significant parameters.

Animals↗

PubMed1995

The effect of furosemide on calcium ion concentration in myocardial cells.

The effect of furosemide and ouabain on the intracellular concentration of Ca2+ was studies in myocardial cell cultures using Fura-2, a fluorescent agent, as an intracellular Ca2+ indicator. Introduction of 200 microM ouabain to the cultured cells increased the intracellular calcium concentration from an average of 236 nM up to an average of 833 nM. Introduction of 100 microM furosemide, prior to the administration of ouabain, decreased the ouabain induced Ca2+ elevation to an average of only 473 nM. Introduction of 2.5 mM EGTA prior to the administration of ouabain abolished the ouabain induced Ca2+ increase.

Animals↗

PubMed1995

Improved hypothermic preservation of rat hearts by furosemide.

The effect of furosemide, a blocker of the Na+/K+/Cl- cotransporter, on hypothermic preservation of rat hearts was studied with use of the Langendorff perfusion system and electron microscopy. Furosemide significantly improved the mechanical recovery and the coronary flow of the hearts preserved for 8 hours in St. Thomas' Hospital cardioplegic solution at a temperature of 4 degrees C. Furosemide at the concentration of 100 mumol/L was found to have an optimal effect, whereas at high concentrations (1000 mumol/L) it was found to have toxic effects. In addition, furosemide reduces the time elapsed between the end of the preservation time and the resumption of myocardial contractions. Ultrastructural evaluations were done in which the presence of swollen mitochondria was chosen as a criterion of hypothermic ischemic damage to the myocardium. Morphometric analysis indicated that the mitochondrial volume of hearts stored for 8 hours in St. Thomas' Hospital cardioplegic solution increased by 72% as compared with the mitochondrial volume of hearts that were not exposed to the hypothermic ischemic conditions (control group). The addition of 100 mumol/L furosemide to the cardioplegic solution resulted in a significant reduction of mitochondrial swelling during the period of 8 hours' storage, which amounted only to 28% as compared with the figure for the control group. The reduction of mitochondrial swelling by furosemide and the improved mechanical and coronary flow recoveries are thought to be related to the blocking of the sarcolemmal Na+/K+/Cl- cotransporter and consequently the reduction of the Na+ influx during hypothermic ischemic storage.

Animals↗

PubMed1995

Cardiac energetics, cell volumes, sodium fluxes, and membrane permeability: NMR studies of cold ischemia.

Intracellular sodium accumulation, cellular swelling, and energy deficiency are ischemia-associated processes that participate in the transition to irreversible ischemic injury. This study aims to determine the relationship among these parameters in intact rat hearts during global ischemia at 4 degrees C. High-energy phosphates were determined by 31P nuclear magnetic resonance, intracellular sodium accumulation was measured by 23Na spectroscopy with the shift reagent dysprosium triethyl tetraaminohexaacetic acid [Dy(TTHA)3(-)], and cell volumes were measured by 59Co and 1H spectroscopy with use of the extracellular marker Co(CN)3-(6). Intracellular sodium flux rates were 1.53 +/- 0.17, 0.17 +/- 0.05, and 0.30 +/- 0.06 mumol.g dry wt-1.min-1 at 0-1.5, 2-7, and 9-12 h, respectively. Sodium influx resulted in accumulation of the ion: 10% after 4 h, 16% after 10 h, and 29% after 12 h. Water followed sodium into the cells at two constant molar ratios (Na+/H2O): 3.80 +/- 0.15 x 10(-3) during the first 8 h of ischemia and 7.8 x 10(-3) at 8-12 h. Relative to initial intracellular volume, cells swelled by 38% after 8 h and 46% after 12 h; reperfusion reduced cellular swelling to 25 and 36%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

NMR analysis reveals a positively charged hydrophobic domain as a common motif to bound acetylcholine and d-tubocurarine.

A complete 1H assignment of d-tubocurarine was carried out using 1D and 2D NMR techniques. Geometries of free acetylcholine (ACh) and d-tubocurarine were compared with those of the ligands bound to a recombinant cholinergic binding site (T alpha 184-200 expressed as a fusion protein in Escherichia coli). The conformations of the free ligands were determined by NOESY experiments while those of the bound molecules were obtained by transferred NOESY. The complete relaxation matrix was solved yielding distance constraints which were further refined by a sigma back-calculation. ACh bound to recombinant T alpha 184-200 closely resembled the conformation previously reported for ACh bound to the intact receptor. d-Tubocurarine in the bound state undergoes extensive induced conformational rearrangements generating a "cup"-shaped structure. A unique positively charged hydrophobic domain is identified as characteristic of both bound cholinergic ligands.

Acetylcholine↗

PubMed1994

31P NMR and triple quantum filtered 23Na NMR studies of the effects of inhibition of Na+/H+ exchange on intracellular sodium and pH in working and ischemic hearts.

The triple quantum filtered 23Na NMR method is applied here to measure the effects of EIPA, a specific inhibitor of the Na+/H+ antiporter, on relative intracellular sodium concentrations in isolated working hearts at baseline, during ischemia, and at subsequent reperfusion. In analogy to the spectrophotometric isosbestic point, an approach is developed that defines a value of tau at which the effect of the relaxation times on the TQF signal intensities is minimized, and the signals are proportional to the sodium concentration for both ischemic and working hearts. EIPA at 1.5 microM significantly inhibited (P < 0.01) the influx of intracellular Na+ during 20 min of ischemia at 36.2 degrees C in this rat heart model. In parallel 31P NMR studies, EIPA had no effect on either the development of acidosis during ischemia or on the recovery of pHi during reperfusion despite its profound effect on intracellular Na+ influx. Thus, under our conditions the Na+/H+ antiporter did not play a critical role in the maintenance of intracellular pH. EIPA treatment resulted in improved recovery (P < 0.005) of mechanical function after 20 min of ischemia. [ATP] was higher in treated hearts during ischemia and reperfusion.

Acidosis↗

PubMed1994

A new method for proton detection of H2(17)O with potential applications for functional MRI.

A method is presented for the sensitive detection of minute amounts of H2(17)O. The method is based on the increase of the T2 of the water protons following an irradiation of the 17O resonance frequency, due to the partial or full decoupling of the 1H-17O spin-spin interaction. It is demonstrated that when 17O concentrations are low, full decoupling is achieved, and at short echo times the increase in the amplitude of the proton echo signal is proportional to the 17O content of the sample. The potential of the method for indirect 17O imaging is substantiated by a simple one dimensional projection of cylindrical phantoms containing various 17O concentrations. Using interleaved sequences with and without 17O decoupling, errors due to time dependent effects are minimized.

Deuterium Oxide↗

PubMed1994

Analysis of double-quantum-filtered NMR spectra of 23Na in biological tissues.

Double-quantum-filtered NMR spectra of 23Na in bovine nasal cartilage tissue and its constituents were measured. The presence of even-rank tensors was detected in the cartilage tissue and in a suspension of collagen fibers, indicating anisotropic motion of the sodium ions. Quantitative analysis of the spectra was performed by calculating the time evolution of the second- and third-rank tensors by solving a modified Redfield equation. Analytical expressions for the spectra were obtained. It is shown that the anisotropy stems from local rather than macroscopic order. A good fit to the observed spectra was obtained for several models that assume isotropic distribution of the directors of the locally ordered sites. The local residual quadrupolar interaction was found to be 550 Hz, demonstrating the sensitivity of double-quantum filtration in the detection of anisotropic motion.

Animals↗

PubMed1993

Complete elimination of the extracellular 23Na NMR signal in triple quantum filtered spectra of rat hearts in the presence of shift reagents.

A method is suggested whereby the shifted extracellular triple quantum filtered 23Na signal of an isolated organ is completely eliminated. The method is based on the long relaxation time of the triple quantum coherence and on its fast evolution rate. When the carrier frequency is set on top of the intracellular sodium signal and the time interval between the last two pulses to (12 delta nu)-1 (delta nu is the frequency difference between the intracellular and the extracellular signals), a complete elimination of the extracellular 23Na signal is achieved. The method is demonstrated for isolated rat hearts and the quantification of intracellular sodium using triple quantum filtered spectroscopy is discussed.

Animals↗

PubMed1993

The effect of vitamin D3 deficiency on the isolated chick heart: hemodynamic, P-31 NMR and membrane studies.

The purpose of this study was to investigate whether vitamin D3 deficiency affects the cardiac function of chick hearts directly or whether the influence is secondary through the hormone's effect on serum calcium levels. To this end, three experimental groups were studied: (a) the control group of vitamin D3 supplemented chicks, Ds, (b) vitamin D3 deficient chicks, Dd, and (c) vitamin D3 supplemented hypocalcemic chicks raised on decreased calcium levels, Dh. The three groups were compared by checking hemodynamic, metabolic and membrane parameters. Total and ionized serum calcium concentrations in the Dh and Dd groups were found to be lower than in the Ds group. Perfusion of the isolated hearts with solutions containing various calcium concentrations (1, 1.5 and 2.5 mM) induced enhanced contractility levels, the magnitude of which was dependent on the difference between the in vivo and perfusate calcium levels. Thus, the inotropic effect was similar and more enhanced for the two hypocalcemic groups. The differences in hemodynamic behaviour could not be explained by variations in the levels of the high energy compounds and acidity, since similar ATP, creatine phosphate and intracellular pH levels were detected for both Ds and Dd groups. However, membrane studies revealed an increase in the number of slow calcium channels for the two hypocalcemic groups and this may be the possible mechanism for the differences in the contractile activity. In conclusion, our study strongly suggests that the effects of vitamin D3 on the heart is mediated only indirectly through its effect on serum calcium levels.

Animals↗

PubMed1993

Inhibition of sodium influx and improved preservation of rat hearts during hypothermic ischemia by furosemide and bumetanide: a 23Na- and 31P-NMR study.

The importance of the Na+/K+/Cl- co-transport system of the rat myocardial sarcolemma was studied under hypothermic ischemia by investigating the effect of the co-transport blockers furosemide and bumetanide on the sodium influx into the myocardium. The intracellular Na+ accumulation during hypothermic ischemia was followed by 23Na-NMR. For this purpose the shift reagent [Dy(TTHA)3-] (SR) was added to the Krebs-Henseleit (KH) perfusion solution. The same solution was also present during the hypothermic preservation. A significant reduction in the intracellular Na+ accumulation after 12 h was found when 100 microM furosemide was present during the perfusion and preservation periods. The intracellular Na+ levels returned to the pre-ischemic values after 1 h of reperfusion with KH in both the treated and control groups. Dose-response studies have indicated that 1-100 microM furosemide or 0.1 microM bumetanide added to the KH-SR solution reduced the Na+ influx significantly over 4 h of hypothermic ischemia. No statistically significant effect was found with furosemide concentration of 0.1 microM or with bumetanide concentrations higher or lower than 0.1 microM. 31P-NMR measurements showed no effect of the 100 microM furosemide on the intracellular ATP, the sum of inorganic phosphate and phosphomonoester, or pH levels over 4 h or after 12 h of hypothermic ischemia. Hearts treated with KH containing 100 microM furosemide showed, significantly higher functional recovery after 12 h of hypothermic ischemia than hearts treated only with KH. This study strongly indicates the existence of the Na+/K+/Cl- co-transport system in the intact rat heart sarcolemma, and its major role in sodium influx during hypothermic ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Sodium interaction with ordered structures in mammalian red blood cells detected by Na-23 double quantum NMR.

Na-23 double and triple quantum filtered NMR spectra of intact dog and human red blood cells were measured with the pulse sequence 90 degrees-tau/2-180 degrees-tau/2-theta degrees-t1-theta degrees-t2(Acq). For theta = 90 degrees the triple quantum filtered spectra exhibited the typical multiple quantum filtered lineshape, characteristic of isotropic media, while the double quantum filtered ones presented a superposition of two signals, whose proportion depended on the creation time tau. This effect is due to the formation of both second and third rank tensors. The formation of the second rank tensor, T21 results from non-zero residual quadrupolar interaction and is related to the anisotropic motion of sodium ions. Measurements of the double quantum filtered spectra with theta = 54.7 degrees enabled the detection of the contribution of T21 exclusively. No residual quadrupolar interaction was detected for sodium in the cytoplasm, while unsealed ghosts displayed the double quantum filtered spectral pattern, similar to that of intact cells. The anisotropy of motion of the sodium at the plasma membrane of mammalian erythrocytes depended on the integrity of the cytoskeleton network. Theoretical analysis of the double quantum filtered spectra gave a value of residual quadrupolar splitting of approximately 20 Hz for intact unsealed ghosts. The data presented prove that double quantum filtering is a sensitive technique for detection of motional anisotropies in biological systems.

Animals↗