In search of the astrocytic factor(s) modulating blood-brain barrier functions in brain capillary endothelial cells in vitro.

(1) The blood-brain barrier (BBB) is formed by brain capillary endothelial cells (ECs). There are various cell types, in particular astrocytes, but also pericytes and neurons, located in close vicinity to the capillary ECs which may influence formation and function of the BBB. Based on this consideration, this paper discusses various aspects of the influence of the surrounding cells on brain capillary ECs with special focus on the role of astrocytes. (2) Based on the morphology of the BBB, important aspects of brain EC functions are summarized, such as transport functions and maintenance of low paracellular permeability. Moreover, various facets are discussed with respect to the influence of astrocytes, pericytes, microglia, and neurons on the BBB. Data on the role of glial cells in the ontogenesis of the BBB are presented subsequently. The knowledge on this subject is far from being complete, however, these data imply that the neural/neuronal environment rather than glial cells may be of importance in the maturation of the barrier. (3) The role of glial cells in the induction and maintenance of the BBB is discussed under physiological as well as pathological conditions. Although the literature presents manifold evidence for a great variety of effects induced by astroglia, there are also many controversies, which may result from different cellular models and experimental conditions used in the respective studies. Numerous factors secreted by astrocytes have been shown to induce a BBB phenotype. On the molecular level, increased expression of barrier-relevant proteins (e.g., tight junction proteins) is documented in the presence of astrocyte-derived factors, and many studies demonstrate the improvement of physiological parameters, such as increased transendothelial resistance and decreased paracellular permeability, in different in vitro models of the BBB. Moreover, one has to take into account that the interaction of brain ECs and astrocytes is bi-directional, and that the other cell types surrounding the brain microvasculature also contribute to BBB function or dysfunction, respectively. (4) In conclusion, it is expected that the present and future research focused on molecular mechanisms and signaling pathways will produce new and exciting insights into the complex network of BBB regulation: the cornerstone is laid.

Nitronyl nitroxides, a novel group of protective agents against oxidative stress in endothelial cells forming the blood-brain barrier.

Nitronyl nitroxides (NN) effectively decompose free radicals (. As brain endothelium, forming the blood-brain barrier (BBB), is both the main source and the target of reactive species during cerebral oxidative stress, we studied the effect of NN on brain endothelial cells injured by the mediator of oxidative stress H(2)O(2) (. H(2)O(2) caused hydroxyl radical generation, lipid peroxidation, membrane dysfunction, membrane leak and cell death, concentration dependently. Due to 0.5 mM H(2)O(2), oxy-radical-induced membrane phospholipid peroxidation (malondialdehyde) increased to 0.61+/-0.04 nmol/mg protein vs control (0.32+/-0.03, p<0.05), cells lost cytosolic proteins into the medium and viability decreased to 28+/-2% of control (p<0.05). Permeability through the endothelial monolayer (measure for the tightness of the BBB) rose to 250+/-40% after 0.15 mM H(2)O(2) (p<0.001). Addition of 10 microM of the NN 5,5-dimethyl-2,4-diphenyl-4-methoxy-2-imidazoline-3-oxide-1-oxyl (NN-2), 1 mM phenylbutyl nitrone (PBN), or 10 microM of the lazaroid U83836E improved cell viability during incubation with 0.5 mM H(2)O(2) to 57+/-1%, 49+/-2%, and 42+/-3% (p<0.05, vs drug-free H(2)O(2) group). The permeability enhancement by 0.15 mM H(2)O(2) was reduced to 171+/-21%, 170+/-25%, and 118+/-32% (p<0.05 vs drug-free H(2)O(2) group). Generally, the assumption is supported that during cerebral oxidative stress the protection should also be directed to the cells of the BBB, which can be provided by antioxidative approaches. NN represent a new group of antioxdatively acting cytoprotectiva improving the survival and function of the endothelium against oxidative stress.

*NO and oxyradical metabolism in new cell lines of rat brain capillary endothelial cells forming the blood-brain barrier.

To investigate the relevance of *NO and oxyradicals in the blood-brain barrier (BBB), differentiated and well-proliferating brain capillary endothelial cells (BCEC) are required. Therefore, rat BCEC (rBCEC) were transfected with immortalizing genes. The resulting lines exhibited endothelial characteristics (factor VIII, angiotensin-converting enzyme, high prostacyclin/thromboxane release rates) and BBB markers (gamma-glutamyl transpeptidase, alkaline phosphatase). The control line rBCEC2 (mock transfected) revealed fibroblastoid morphology, less factor VIII, reduced gamma-glutamyl transpeptidase, weak radical defence, low prostanoid metabolism, and limited proliferation. Lines transfected with immortalizing genes (especially rBCEC4, polyoma virus large T antigen) conserved primary properties: epitheloid morphology, subcultivation with high proliferation rate under pure culture conditions, and powerful defence against reactive oxygen species (Mn-, Cu/Zn-superoxide dismutase, catalase, glutathione peroxidase, glutathione) effectively controlling radical metabolism. Only 100 microM H2O2 overcame this defence and stimulated the formation of eicosanoids similarly as in primary cells. Some BBB markers were expressed to a lower degree; however, cocultivation with astrocytes intensified these markers (e.g., alkaline phosphatase) and paraendothelial tightness, indicating induction of BBB properties. Inducible NO synthase was induced by a cytokine plus lipopolysaccharide mixture in all lines and primary cells, resulting in *NO release. Comparing the cell lines obtained, rBCEC4 are stable immortalized and reveal the best conservation of properties from primary cells, including enzymes producing or decomposing reactive species. These cells can be subcultivated in large amounts and, hence, they are suitable to study the role of radical metabolism in the BBB and in the cerebral microvasculature.

An optical method for the detection of oxidative stress using protein-RNA interaction.

The cytosolic 4Fe-4S protein aconitase can be converted under the influence of reactive oxygen species into an iron-regulatory protein (IRP1). Therefore, the IRP1 level is considered as an indirect marker of oxidative stress. An experimental approach is presented here to detect the concentration of this marker protein by surface plasmon resonance. The optical method exploits the natural binding affinity of IRP1 to an iron-responsive element (IRE) which was in vitro transcribed with a linker sequence and subsequently immobilized on a BIACORE sensor chip. The detection was found to be reproducible and sensitive in the range 20-200 nM IRP. Conditions of the binding process, such as pH and thiol concentration, were characterized. Feasibility of the method to detect and quantify IRP1 in physiological media was demonstrated.

Phosphorylation of vasodilator-stimulated phosphoprotein: a consequence of nitric oxide- and cGMP-mediated signal transduction in brain capillary endothelial cells and astrocytes.

There is contradictory information on the relevance of nitric oxide (NO) and cGMP for the function of brain capillary endothelial cells (BCEC) forming the blood-brain barrier (BBB). Therefore, NO/cGMP-mediated signal transduction was investigated in cell cultures of BCEC and of astrocytes (AC) inducing BBB properties in BCEC. Constitutive, Ca2+-activated isoforms of NO synthase (NOS) were found in BCEC (endothelial NOS: eNOS) and in AC (neuronal NOS: nNOS), leading to increased NO release after incubation with the Ca2+-ionophore A23187. Both cell types expressed inducible NOS (iNOS) after incubation with cytokines. Soluble guanylate cyclase (sGC) was detected in both cell types. NO-dependent cGMP formation were observed in BCEC and, less pronounced, in AC. Furthermore, both cell types formed cGMP independently of NO via stimulation of particulate guanylate cyclase (pGC). cGMP-dependent protein kinase (PKG) type Ibeta, but not type II, was expressed in BCEC and AC. In BCEC, vasodilator-stimulated phosphoprotein (VASP) was detected, an established substrate of PKG and associated with microfilaments and cell-cell contacts. Phosphorylation of VASP was intensified by increased intracellular cGMP concentrations. The results indicate that BCEC and, to a smaller degree, AC can form NO and cGMP in response to different stimuli. In BCEC, NO/cGMP-dependent phosphorylation of VASP is demonstrated, thus providing a possibility of influencing cell-cell contacts.

PBN spin trapping of free radicals in the reperfusion-injured heart. Limitations for pharmacological investigations.

Post-ischemic reperfusion causes cardiac dysfunction and radical-induced lipid peroxidation (LPO) detectable by ESR spin trapping. This study deals with the applicability of the spin trapping technique to pharmacological investigations during myocardial reperfusion injury. The use of the spin trap phenylbutylnitrone (PBN, 3 mM) in isolated rat hearts demonstrated the release of alkoxyl radicals (aN = 1.39 mT, aHbeta = 0.19 mT) formed particularly within the first 15 min of reperfusion following 30 min of ischemia. The decline of radicals, after 10 min of reperfusion, was accompanied by recovery of function in 80% of the hearts. The radical concentration in the coronary effluent (maximum after 7.5 min) was reduced by the infusion of 1 mM mercaptopropionylglycine (MPG, 2.7+/-0.5 U/ml, p < 0.001) or 5 microM vitamin E (11.7+/-0.8 U/ml, p < 0.001), compared to the (PBN-containing) control (29.7+/-4.3 U/ml). Moreover, functional recovery (left ventricular developed pressure, LVDP 91.6 +/-20% of pre-ischemic level, p < 0.05) was improved by the hydrophilic radical scavenger MPG, compared to the (PBN-containing) control (LVDP 50.5+/-15.7% of baseline). PBN alone led to higher functional recovery (p < 0.05) and reduced VF (duration of ventricular fibrillation; 7.10+/-0.36 min/30 min, p < 0.05), compared to the untreated (PBN-free) control (LVDP 26.6+/-11.8%; VF 19.42+/-3.64 min/30 min). The Ca antagonist verapamil (0.1 microM), MPG, and the lipophilic vitamin E showed cardioprotection in the absence of PBN: post-ischemic recovery of LVDP was 25.4+/-6.8% (p < 0.05), 39.6+/-12.7% (p < 0.05) and 52.4+/-2.6% (p < 0.01), respectively, compared to the corresponding untreated control (13.3+/-6.6%). Whereas verapamil and vitamin E were able to protect the heart when present alone, they offered no additive effect in the presence of PBN. Therefore, PBN can be used to estimate the radical scavenger properties of an agent in the heart. However, because of the protective properties of PBN itself, the results of simultaneous investigations of the effects of other compounds, such as Ca antagonists or lipophilic radical scavengers, on heart function may be limited.

Protective effects of the thiophosphate amifostine (WR 2721) and a lazaroid (U83836E) on lipid peroxidation in endothelial cells during hypoxia/reoxygenation.

Little is known about pharmacological interventions with thiophosphates or lazaroids in endothelial cells injured by hypoxia/reoxygenation with respect to membrane lipid peroxidation (LPO) caused by reactive oxygen species. Therefore, a cell line of bovine aortic endothelial cells was studied after 120-min hypoxia followed by 30-min reoxygenation, resulting in moderate and predominantly reversible injury (energy depression/cytosolic Ca2+-accumulation during hypoxia, which almost normalized during reoxygenation; membrane blebs, an increasing amount of lysosomes, vacuolization, lipofuscin formation, alterations in mitochondria size, some lyzed cells). 18.9 +/- 4.3% of the cells died. Radical-induced LPO measured as malondialdehyde continuously increased to 2.18 +/- 0.17 nmol/mg of protein after reoxygenation vs control (0.41 +/- 0.13, P < 0.05). Simultaneously, the content of 4-hydroxynonenal, a novel indicator of LPO, increased from 0.02 +/- 0.01 to 0.11 +/- 0.02 nmol/mg of protein (P < 0.01). The results support the assumption that reoxygenation injury is accompanied by an increase in membrane LPO, causing structural and functional disturbances in the monolayer. The thiophosphate WR 2721 [S-2-(3-aminopropylamino) ethylphosphorothioic acid] and the lazaroid U83836E [(-)-2-[[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl] methyl]-3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran-6-ol (dihydrochloride)] were effective scavengers of .OH, being more efficient than trolox C (6-hydroxy-2,5,7,8-tetramethylchroman-2-carbon acid) used as standard (EC50: 12, 5 and 15 microM, respectively, measured by electron spin resonance spectroscopy). One mM WR 2721, 10 microM U83836E, and 5 microM trolox C reduced formation of malondialdehyde during hypoxia/reoxygenation to 53 +/- 7, 51 +/- 10 and 48 +/- 6%, respectively (P < 0.05 each, versus control). In general, WR 2721 and U83836E prevent radical-induced membrane LPO in a model of endothelial cells injured by hypoxia/reoxygenation. The use of these two agents is a new approach to protect the endothelium against oxidative stress.

Nitric oxide protects blood-brain barrier in vitro from hypoxia/reoxygenation-mediated injury.

A cell culture model of blood-brain barrier (BBB, coculture of rat brain endothelial cells with rat astrocytes) was used to investigate the effect of nitric oxide (.NO) on the damage of the BBB induced by hypoxia/reoxygenation (H/R). Permeability coefficient of fluorescein across the endothelium was used as a marker of BBB tightness. The permeability coefficient increased 5.2 times after H/R indicating strong disruption of the BBB. The presence of the .NO donor S-nitroso-N-acetylpenicillamine (SNAP, 30 microM), authentic .NO (6 microM) or superoxide dismutase (50 units/ml) during H/R attenuated H/R-induced increase in permeability. 30 microM SNAP or 6 microM .NO did not influence the function of BBB during normoxia, however, severe disruption was observed using 150 microM of SNAP and more than 24 microM of .NO. After H/R of endothelial cells, the content of malondialdehyde (MDA) increased 2.3 times indicating radical-induced peroxidation of membrane lipids. 30 microM SNAP or 6 microM authentic .NO completely prevented MDA formation. The results show that .NO may effectively scavenge reactive oxygen species formed during H/R of brain capillary endothelial cells, affording protection of BBB at the molecular and functional level.

Thiol-induced nitric oxide release from 3-halogeno-3,4-dihydrodiazete 1,2-dioxides.

In this work we studied the mechanism of nitric oxide (NO) release underlying the vasorelaxant and antiaggregant effect of 3,4-dihydrodiazete 1,2-dioxides (DD). Six derivatives were included in the investigations, namely, 3-bromo- and 3-chloro-3,4,4-trimethyl-DD (1a,b), 3-bromo- and 3-chloro-4-methyl-3,4-hexamethylene-DD (2a,b), 3,3,4,4-tetramethyl-DD (3), and 3-methyl-3,4-hexamethylene-DD (4), and their reactivity toward thiols was analyzed. The 3-bromo- and 3-chloro-DD derivatives were found to react with thiols; this reaction can lead to NO formation, DD 2a being the most reactive compound. 2-(Hydroxyamino)-2-methylbutan-3-one oxime (5a) and 2-hydroxy-2-methylbutan-3-one oxime (6) were the main products isolated from the reaction of 1a with cysteine. Reaction rates of DD with thiols were dependent upon pH and concentration of the reagents. Maximum rates of NO release corresponded to thiol concentrations in the range of 1 mM. Consistent with reaction kinetics data and products isolated, a reaction mechanism was proposed. Addition of 2a to bovine aortic endothelial cells led to strong NO release indicating a reaction with endogenous thiols. In rat mesenterial arteries, the vasorelaxant action of 2a was only slightly influenced by addition of thiol to the incubation medium. For the most reactive DD derivatives, cytotoxic effects were observed at concentrations roughly 2 orders of magnitude higher than those inducing vasorelaxation.

Cytotoxicity of spin trapping compounds.

Spin trapping compounds are used frequently to detect free radicals released by cells. Their cytotoxicity has to be considered in order to prevent perturbations of normal cell growth and viability. Eleven spin traps (eight nitrones and three nitroso traps) have been tested for their effects on bovine aortic endothelial cells (toxicity range, 50% survival rate). The lowest cytotoxicity was found for 5,5-dimethylpyrroline-1-oxide and 2,2,4-trimethyl-2H-imidazole-1-oxide whereas nitrosobenzene and 2-methyl-2-nitrosopropane exerted the strongest cytotoxic effects. In addition, three nitronyl nitroxides were tested. Their cytotoxicity was found to be dependent on substitution, and the toxic concentration of a lipophilic derivative was found to be more than two orders lower as compared to a hydrophilic derivative. The results of this study indicate that most spin traps can be used in cell cultures at customary (i.e. millimolar) concentrations; caution is recommended when nitroso spin traps are applied to cells.

Nitroxides increase the detectable amount of nitric oxide released from endothelial cells.

Nitroxides are known to exert superoxide dismutase-mimetic properties and to decrease O-2- and H2O2-mediated cytotoxicity. However, the effect of nitroxides on .NO homeostasis has not been studied yet. The present study investigates the effect of nitroxides on the detectable amount of .NO released by 3-morpholinosydnonimine (SIN-1) and cultured endothelial cells. Cultured bovine aortic and atrial endothelial cells stimulated with 10 microM A23187 released a stable flux of .NO, as detected by .NO chemiluminescence. Addition of 100 units/ml SOD or 10 microM of the nitroxides 4-hydroxy-2,2,6, 6-tetramethylpiperidine-N-oxyl (TEMPOL), 3-carboxy-proxyl, and 3-ethoxycarbonyl-proxyl, increased the chemiluminescence signal. The effect of these nitroxides on the amount of .NO released from cell monolayers was dose-dependent, with the highest efficacy between 30 and 100 microM. EPR spin trapping in SIN-1 solutions revealed the formation of .OH adducts from spontaneous dismutation of O-2 and concomitant reaction with H2O2. Both SOD and TEMPOL increased the signal intensity of the .OH adduct by accelerating the dismutation of O-2. The results of this study demonstrate that the SOD-mimetic activity of nitroxides increases the amount of bioavailable .NO in vitro.

Synthesis and spin trapping applications of 2,2-dimethyl-d6-4-methyl-2H-imidazole-1-oxide-1-15N.

A new spin trap, 2,2-dimethyl-d6-4-methyl-2H-imidazole-1-oxide-1-15N (lTMIO), was synthesized and characterized. Hyperfine splitting (HFS) constants of spin adduct ESR spectra of this compound with oxygen-centered, carbon-centered, thiyl and sulfite-derived radicals were determined and compared with the data of the unsubstituted compound. The increase in ESR spectral intensity and the accompanying decrease of the spectral linewidth result in resolution of the HFS due to interaction with alpha-protons of alkyl radicals trapped by lTMIO. Trapping of the formate radical in deoxygenated aqueous solution revealed a very low spectral linewidth (delta Bpp = 0.028 mT) of the corresponding adduct. A strong dependence of the ESR spectra on pH was observed when the autoxidation product of sulfite, SO3-, was trapped. The pKa was found to be 5.8 +/- 0.3. In comparison to other nitrones, application of this spin trap provides more detailed information on the structure of the species trapped, especially for carbon-centered radicals.

Superoxide-mediated reduction of the nitroxide group can prevent detection of nitric oxide by nitronyl nitroxides.

Nitronyl nitroxides (NN), a class of compounds which react with nitric oxide forming imino nitroxides, were applied in different systems for the detection of nitric oxide. Addition of a NN to planar monolayers of bovine aortic endothelial cells (BAEC) activated by Ca2+ ionophore A23187 immediately resulted in a strong decrease of the ozone-mediated .NO chemiluminescence. Simultaneously, a rapid diminution of the electron spin resonance (ESR) signal intensity of the NN (without detectable formation of the corresponding imino nitroxide) was observed; superoxide dismutase partially inhibited this decrease in the NN concentration. Model experiments using hypoxanthine/xanthine oxidase in aqueous solution and KO2 in dimethylsulfoxide as sources of O2.- revealed that there is a rapid reduction of nitronyl nitroxides by superoxide. The second order rate constant for the reaction of the water soluble NN with O2.- was determined to be 8.8 x 10(5) M-1s-1, which is more than two orders of magnitude higher than the value reported previously for reaction with .NO (Woldman et al., BBRC 202, 195-203, 1994). Reduction of the nitronyl nitroxide was also observed in the presence of glutathione, ascorbic acid or rabbit liver microsomes. Incorporation of both nitronyl and imino nitroxides into liposomes strongly decreased reduction by superoxide and other reductants, however, in the presence of microsomes, there was no protective effect by liposomal encapsulation of NN. The results indicate that in biological systems (in addition to other reducing agents) the presence of superoxide can prevent the detection of nitric oxide using nitronyl nitroxides.

4-Hydroxynonenal, a novel indicator of lipid peroxidation for reperfusion injury of the myocardium.

4-Hydroxynonenal (HNE) has been proposed as an important marker of radical-induced lipid peroxidation (LPO) during postischemic reperfusion injury of the myocardium. Therefore, the liberation of HNE into the effluent of isolated perfused rat hearts was investigated. For the first time, the formation of the aldehyde is demonstrated in myocardium. During control perfusion, 1.28 +/- 0.33 pmol HNE.min-1.mg protein-1 were formed by the hearts of 18-mo-old Wistar-Kyoto (WKY) rats and 2.74 +/- 1.12 pmol.min-1.mg protein-1 by those of 18-mo-old spontaneously hypertensive (SHR) rats, respectively. In the WKY group, HNE release increased to 3.35 +/- 1.13 pmol.min-1.mg protein-1 2 min after the onset of reperfusion following 30 min of total and global ischemia compared with the preischemic control period (P < 0.05). In the SHR group, HNE liberation was higher during reperfusion (8.66 +/- 1.33 pmol.min-1.mg protein-1, maximum at 2 min reperfusion) compared with both the respective preischemic control and the respective reperfusion interval of the WKY group (P < 0.05 each). The SHR rats showed signs of congestive cardiac failure of a decompensated hypertrophy in comparison to the normotensive WKY rats. Moreover, the SHR rat hearts exhibited a lower release of adenine nucleotide degradation products (adenine, inosine, hypoxanthine plus uric acid: 48.1 +/- 10.2 nmol.30 min-1.mg protein-1; P < 0.05) and a diminished functional recovery (left ventricular developed pressure, 32 +/- 16 mmHg; P < 0.05) during 30 min of reperfusion compared with the WKY group (77.9 +/- 14.4 nmol.30 min-1.mg protein-1; 90 +/- 21 mmHg).(ABSTRACT TRUNCATED AT 250 WORDS)

Oxygen radicals attenuate the contractility of skinned muscle fibres from the pig myocardium.

This study deals with the influence of oxygen radicals on the contraction of skinned muscle fibres from pig myocardium. The radicals were generated by xanthine-xanthine oxidase (X/XO) or by Fe2+/H2O2 (Fenton system). Addition of the X/XO to the incubation medium (KCl/imidazole) induced a depression of the contractility which was dependent from the incubation time and the X/XO concentration. The maximum contraction in the presence of high concentrations of free calcium ions (pCa 4.32) decreased to 52.0 +/- 15.5% (p < 0.01). The EC50 of calcium ions inducing fibre contraction increased from 2.82 +/- 0.66 mumol/l to 5.47 +/- 2.06 mumol/l (p < 0.05). The Hill plot of contraction versus concentration of calcium ions was shifted to the right and the maximum of contractility was attenuated. Replacement of X/XO by the Fenton system was without significant effects on the fibre contractility. Addition of 5.10(-4) mol/l APP 210-533 (3-amino-6-methyl-5-phenyl-1,2- dihydropyrid-2-on), a known "calcium sensitizer", increased the fibre contractility in radical impaired fibres, too. This may indicate that the radicals did not impair the troponine complex. Oxygen radicals were detected by electron spin resonance spectroscopy spin trapping using 5,5-dimethylpyrroline-1-oxide. Superoxide radicals were found in the presence of X/XO whereas addition of Fe2/H2O2 to the incubation medium resulted in the formation of hydroxyl radical adducts. The appearance of additional adducts observed in both system is discussed. The experiments indicate that free radicals can interact with components of the skinned fibre (probably with contractile proteins of the myocardial muscle cells) resulting in an impairment of the contractility.(ABSTRACT TRUNCATED AT 250 WORDS)

Spin trapping using 2,2-dimethyl-2H-imidazole-1-oxides.

The ability of novel cyclic nitrones, 4-substituted 2,2-dimethyl-2H-imidazole-1-oxides (IMO's) to trap a variety of short-lived free radicals has been investigated using ESR spectroscopy. IMO's scavenge oxygen-, carbon- and sulfur-derived free radicals to give persistent nitroxides. Compared to the spin trap 5,5-dimethyl-pyrroline-1-oxide, a higher lifetime of hydroxyl radical adducts and a higher selectivity related to the trapping of carbon-centered radicals was found. A reaction between IMO's and superoxide was not observed. ESR parameters of 4-carboxyl-2,2-dimethyl-2H-imidazole-1-oxide (CIMO) spin adducts are highly sensitive to the structure of the trapped radical, e.g., different spectra were detected with radicals derived from Na2SO3 and NaHSO3. From the data obtained, a successful application of these new spin traps in biological systems can be expected.

Does the antiarrhythmic effect of DMPO originate from its oxygen radical trapping property or the structure of the molecule itself?

Using the isolated perfused rat heart with transient (30 min) normothermic global ischemia, it was shown that DMPO (5,5-dimethyl-pyrroline-N-oxide), an organic spin trap agent designed specifically to trap free radicals, dramatically reduced the vulnerability of the myocardium to reperfusion-induced ventricular fibrillation (VF) and ventricular tachycardia (VT). DMPO (concentration range 30-500 mumol/l) infused in the heart at the moment and during the first 10 min of reperfusion exerted a dose-dependent antiarrhythmic effect. Thus, the doses of 30, 100, and 500 mumol/l of DMPO reduced the incidence of reperfusion-induced VF and VT from their control values of 100% and 100% to 83% and 91%, 50% (p < 0.05) and 67%, 25% (p < 0.01) and 50% (p < 0.05), respectively. Furthermore, the recovery of myocardial function was improved during postischemic reperfusion. A modification in the molecular structure of DMPO leading to HMIO (1,2,2,4,5,5-hexamethyl-3-imidazoline-oxide), so-called inactive DMPO which does not trap free radicals in the presence of a radical generating system or in the effluent of reperfused hearts, failed to reduce the incidence of reperfusion-induced arrhythmias or improve the recovery of postischemic reperfused myocardium. These findings suggest that the free radical trapping properties of DMPO or the effects of the formed DMPO-OH, a stable nitroxyl radical adduct, are responsible for the reduction of reperfusion-induced arrhythmias, and not the molecular structure of DMPO itself. Finally, it is of interest to note that the detection of free radicals was observed in fibrillating hearts, but not in nonfibrillating hearts. This consideration should be taken into account when making therapeutic interventions and risk assessments of a radical scavenger in this setting.