Ching-San Lai

SPIN TRAPPING OF NITRIC OXIDE PRODUCED IN VIVO IN SEPTIC-SHOCK MICE

A nitric oxide (•NO) spin‐trapping technique combined with electron paramagnetic resonance (EPR) spectroscopy has been employed to measure the in vivo production of •NO in lipopolysaccharide (LPS)‐treated mice. The in vivo spin‐trapping of •NO was performed by injecting into mice a metal—chelator complex, consisting of N‐methyl‐d‐glucamine dithiocarbamate (MGD) and reduced iron (Fe2+), that binds to •NO and forms a stable, water‐soluble [(MGD)2‐Fe2+‐NO] complex, and by monitoring continuously the in vivo formation of the latter complex using an S‐band EPR spectrometer. At 6 h after intravenous injection of LPS, a three‐line EPR spectrum of the [(MGD)2‐Fe2+‐NO] complex, was observed in the blood circulation of the mouse tail; the [(MGD)2‐Fe2+] complex was injected subcutaneously 2 h before EPR measurement. No signal was detected in control groups. Administration of N G‐monomethyl‐l‐arginine, an •NO synthase inhibitor, caused a marked reduction in the in vivo EPR signal of the [(MGD)2‐Fe2+‐NO] complex, suggesting that the •NO detected is synthesized via the arginine‐nitric oxide synthase pathway. The results presented here demonstrated, for the first time, the in vivo real time measurement of •NO in the blood circulation of conscious, LPS‐treated animals.

DETECTION OF NITRIC OXIDE PRODUCTION IN MICE BY SPIN-TRAPPING ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPY

We describe here a spin-trapping method combined with X-band electron paramagnetic resonance (EPR) spectroscopy for ex vivo measurement of nitric oxide (.NO) levels in the urine of both normal and lipopolysaccharide (LPS)-induced shock mice. Normal or LPS-treated mice were injected subcutaneously with a metal-chelator complex, N-methyl-D-glucamine dithiocarbamate-ferrous iron, [(MGD)2/Fe], which binds to .NO and forms a water-soluble [(MGD)2/Fe-NO] complex. At 2 h after injection of the [(MGD)2/Fe] complex, a three-line EPR signal characteristic of the [(MGD)2/Fe-NO] complex was detected in the urine of either normal or LPS-treated mice. It is estimated that the concentrations of the [(MGD)2/Fe-NO] complex in normal and LPS-treated mouse urine were 1.3 and 35 microM, respectively. This 25-fold increase in .NO levels in the LPS-treated mouse urine provides the direct evidence that LPS challenge induces the overproduction of .NO in mice. Administration of N-monomethyl-L-arginine (NMMA; 50 mg/kg) inhibited the ex vivo signal intensities of the [(MGD)2/Fe-NO] complex in the urine of either normal or LPS-treated mouse urine. Furthermore, after injection of 15N-arginine (10 mg per mouse), a composite EPR spectrum, consisting of a three-line spectrum of the [(MGD)2/Fe-14NO] complex and a two-line spectrum of the [(MGD)2/Fe-15NO] complex, was detected in the urine. These isotopic tracer experiments further confirm that the detected .NO levels in the mouse urine are produced via the arginine-nitric oxide pathway. This ex vivo spin-trapping method should readily be adapted to experiments on larger animals and provide a noninvasive way of measuring both constitutive and inducible .NO synthase activities in living animals under physiological as well as pathophysiological conditions where .NO is overproduced.

Continuous monitoring of cellular nitric oxide generation by spin trapping with an iron-dithiocarbamate complex

Nitric oxide (NO) generation in murine macrophages was determined in real time using the electron paramagnetic resonance (EPR) spin trapping method. An iron complex of N-methyl D-glucamine dithiocarbamate was utilized as the spin trap. This spin trapping compound reacts with NO in solution to form a specific room-temperature stable, mononitrosyl complex which is readily detected and identified by EPR spectroscopy. Mouse peritoneal macrophages were placed in an EPR sample-cell and activated by lipopolysaccharide and gamma-interferon at 37 degrees C, followed by an additional incubation in oxygenated medium without these activation agents. After various incubation periods, spin trap solution was infused to replace the medium in the sample-cell, and the time-evolution of the EPR signal of the spin adduct (NO-complex) was recorded. Rates of NO generation were calculated based upon the initial slopes of the increase in the EPR intensity with time. In comparison to the NO (or NO2-) generation rate obtained under similar experimental conditions using the Griess reaction assay, the spin trapping method was found to be more sensitive, with a lowest limit of the detection of 3 pmol/min. In addition, by using the spin trapping method, NO generation from the same cells could be measured consecutively during various stages of activation, because infusion of the spin trap solution did not affect the viability of macrophages.

Dolichol induces membrane leakage of liposomes composed of phosphatidylethanolamine and phosphatidylcholine

Dolichol promotes the leakage of membranes in liposomes composed ofphosphatidylethanolamine and phosphatidylcholine but not liposomes composed only of phosphatidylcholine. The membrane leakage was assayed by measuring the entrapment of TEMPOcholine, a cationic spin probe, in liposomes using ESR methods. The percent of membrane leakage induced by dolichol was found to be linearly proportional to the concentrations of dolichol. It is proposed that dolichol enhances the formation of non‐bilayer configurations in liposomes containing phosphatidylethanolamine, thereby inducing membrane leakage.

Cytotoxicity of commonly used nitroxide radical spin probes

Since nitroxide radical spin probes are used frequently to test biophysical properties of cells, their use should be restricted to conditions that do not perturb normal cell growth and viability. Eight commonly used nitroxide radical spin probes have been tested for their effects on the survival of CHO cells. These include water-soluble spin probes Tempol, Tempamine, CTPO, CTPC and 4-maleimido-Tempo, and lipid soluble spin probes 5-Doxyl-, 12-Doxyl-, and 16-Doxylstearates. With the exception of 4-maleimido-Tempo, none of the water soluble spin labels inhibited cell survival at concentrations as high as 1 mM. At concentrations of 75 microM and higher, 4-maleimido-Tempo inhibited cell survival in a dose dependent manner. At concentrations commonly used for spin labeling of cells (30-50 microM) none of the lipid soluble spin probes tested was cytotoxic. At 100 microM only 5-Doxylstearate inhibited cell survival, whereas 12-Doxylstearate and 16-Doxylstearate had no effect.

Fluorescence energy transfer detects changes in fibronectin structure upon surface binding

We report here the changes in intramolecular distances in human plasma fibronectin (Fn) detected, upon adsorption of the protein to the surface of the Cytodex dextran microcarrier, using a fluorescence energy transfer technique. The glutamine-3 residue, near the amino terminus of each chain, was labeled enzymatically with either monodansylcadaverine (dansyl) or monofluoresceinyl-cadaverine (fluorescein) by use of coagulation factor XIIIa. Using this donor (dansyl)-acceptor (fluorescein) pair, and steady-state measurements, we demonstrated previously that the two amino termini of plasma fibronectin in solution were juxtaposed and separated by 23 A (C. Wolff and C.-S. Lai (1988) Biochemistry 27, 3483-3487). Upon adsorption to the microcarrier, the energy transfer was found to be completely abolished, suggesting that the surface binding induces a conformational change by which the distance between the two amino termini is increased to more than 70 A. Moreover, we have labeled the amino terminus of each chain with fluorescein and the two free sulfhydryl groups of each chain with coumarinyl-phenylmaleimide which serves as an energy donor. The emission spectra of the double-labeled protein in solution showed the occurrence of energy transfer, indicating that the relative distances between the amino termini and the free sulfhydryl group(s) are within 70 A. Upon surface binding, a decrease in the energy transfer between this donor-acceptor pair was also noted. The results presented here are consistent with the notion that plasma Fn undergoes a drastic conformational change upon surface binding, perhaps changing from a compact form to an extended form. This process may be important for the surface activation of the fibronectin molecule.

Effect of oxygen and the lipid spin label TEMPO-laurate on fluorine-19 and proton relaxation rates of the perlluoroehemical blood substitute, FC-43 emulsion

The 19F and 1H spin-lattice relaxation (T1−1) rates of a liquid perfluorochemical, perfluorotributylamine, and its emulsified form, FC-43 emulsion, were studied using the inversion-recovery method. Both 19F and 1H relaxation rates were found to be linearly proportional to the partial pressure of oxygen, indicating that oxygen solubility in FC-43 emulsion follows Henrys law and that no specific binding between molecular oxygen and perfluorochemicals occurs. TEMPO-laurate, a water-insoluble lipid spin probe, was found to partition preferentially into perfluorotributylamine. At a concentration of 4.5 × 10−4 M, it enhanced the relaxation rate of 19F nuclei in FC-43 emulsion by 50% (in the absence of oxygen) and by 32% (in the presence of t atm oxygen) at 37°C. TEMPO-laurate also enhanced the proton relaxation rate in FC-43 emulsion by 18% (in the absence of oxygen) and by 10% (in the presence of 1 atm oxygen) at 37°C. The sensitive response of the 19F spin-lattice relaxation rate to oxygen leads to the hypothesis that this rate may be a sensitive parameter for in situ determinations of oxygen tension in blood vessels perfused with perfluorochemical blood substitutes.

An evaluation of paramagnetic broadening agents for spin probe studies of intact mammalian cells.

Six transition metal ion complexes have been examined for their effects on the cell survival as well as their effectiveness in inducing the broadening of the electron spin resonance (ESR) spectra of nitroxide spin probes. These paramagnetic species are Ni(EDTA), Ni(DTPA), potassium tris(oxalato) chromate (chromium oxalate), K3Fe(CN)6, Cu(DTPA), and NiCl2. At 100 mM concentration, the typical concentration used in cell studies to broaden the extracellular nitroxide ESR signal, only Ni(EDTA) and Ni(DTPA) are found to be non-toxic to Chinese hamster ovary cells. The relative cytotoxicities of the six metal ion complexes are Cu(DTPA) greater than K3Fe(CN)6 greater than NiCl2 greater than chromium oxalate greater than Ni(DTPA) greater than Ni(EDTA). Thus, potassium ferricyanide and NiCl2, two most commonly used paramagnetic broadening agents, are relatively toxic to the cell. In contrast, among the six paramagnetic species tested here, chromium oxalate appears to be the most effective agent at non-toxic concentrations in inducing the broadening of the ESR spectra of both cationic and neutral nitroxide spin probes. By considering both their cytotoxicity and their effectiveness in causing line broadening of the nitroxide ESR spectra, chromium oxalate is a good paramagnetic broadening agent for spin probe studies of intact mammalian cells.

Heparin modulates conformational states of plasma fibronectin: An electron spin resonance spin label approach

We have examined the interaction between heparin and human plasma fibronectin using electron spin resonance (ESR) spin label methods. The titratable sulfhydryl groups of plasma fibronectin were modified with a maleimide spin label [Lai and Tooney (1984) Arch. Biochem. Biophys. 228, 465-473]. Addition of heparin resulted in a decrease in the maximum splitting value of the ESR spectrum of spin-labeled fibronectin from 66.8 to 64.3 G, suggesting that heparin induces a conformational alteration of plasma fibronectin. This heparin effect was noticeable at a heparin-to-fibronectin ratio of 20 to 1 and reached a plateau at about 100 to 1. Other sulfated carbohydrates were tested; dextran sulfate was found to be as effective as heparin but chondroitin sulfates were ineffective. The results presented suggest that the binding of heparin changes the molecular conformation of plasma fibronectin to a more relaxed or flexible state.

Conformational change in thrombospondin induced by removal of bound Ca2+ A spin label approach

The effect of removal of Ca2+ bound to thrombospondin (TSP) on the protein structure in solution has been investigated using ESR spin‐label techniques. A maleimide spin label was selectively attached to the free thiol group presumably near the carboxyl‐terminal domain in which Ca2+‐binding sites are situated. The ESR spectra of spin‐labeled TSP showed that the bound label undergoes a relatively fast rotational motion with an effective rotational correlation time in the nanosecond time regimes. Removal of bound Ca2+ in TSP by dialyzing spin‐labeled TSP from a Ca2+‐containing buffer into an EDTA‐containing buffer resulted in an increase in the mobility of the bound label by a factor of 2.3. The data suggest that EDTA chelation of bound Ca2+ in TSP induces a conformational change of TSP at least near the site of spin labeling.