Biomedical subjects

A Mülsch

Publications and source records attributed to A Mülsch.

At least 37 records · Page 2Linked to original sources

PubMed1996

In vivo nitrate tolerance is not associated with reduced bioconversion of nitroglycerin to nitric oxide.

BACKGROUND: In vitro data suggest that reduced bioconversion of nitroglycerin (NTG) to nitric oxide (NO) contributes to the development of vascular and hemodynamic tolerance to NTG. We examined the in vivo validity of this hypothesis by measuring NTG-derived NO formation by in vivo spin-trapping of NO in vascular tissues from nitrate-tolerant and -nontolerant rats. METHODS AND RESULTS: Five groups (n = 6 to 8 each) of conscious chronically catheterized rats received NTG (0.2 or 1 mg/h IV) for 72 hours (nitrate-tolerant groups). Four other groups received either NTG vehicle (placebo, for 72 hours) or were left untreated (control). Nitrate tolerance was substantiated by a reduced (55% to 85%) hypotensive response to NTG in vivo and a reduced relaxation to NTG in isolated aortic rings. NTG-derived NO formation in aorta, vena cava, heart, and liver was measured as NOFe(DETC)2 and NO-heme complexes formed in vivo during 35 minutes combined with ex vivo cryogenic electron spin resonance spectroscopy. NO formation was significantly (P < .05) increased in all tissues in nitrate-tolerant rats in an NTG dose-dependent manner. Furthermore, the amount of NO formed from a bolus dose of NTG (6.5 mg/kg over 20 minutes) was similar in nitrate-tolerant and -nontolerant rats. CONCLUSIONS: The results suggest that vascular and hemodynamic NTG tolerance occurs despite high and similar rates of NO formation by NTG in tolerant and nontolerant target tissues. This finding is compatible with the assumption that reduced biological activity of NO, rather than reduced bioconversion of NTG to NO, contributes to in vivo development of nitrate tolerance.

Animals↗

PubMed1996

Effect of cyclic GMP-dependent vasodilators on the expression of inducible nitric oxide synthase in vascular smooth muscle cells: role of cyclic AMP.

1. In the present study we examined whether interleukin-1 beta (IL-1 beta) increases the activity of adenylyl cyclase in vascular smooth muscle cells and determined its role in the cytokine-induced expression of the inducible nitric oxide synthase (iNOS) and activation of nuclear transcription factor-kappa B (NF-kappa B). In addition the interaction between cyclic AMP- and cyclic GMP-elevating agonists on the IL-1 beta-stimulated expression of iNOS was examined. 2. Exposure of vascular smooth muscle cells to IL-1 beta stimulated the formation of cyclic AMP but not of cyclic GMP. The intracellular level of cyclic AMP reached a maximum within 1 h and then gradually declined over the next 5 h. This IL-1 beta (60 u ml-1)-stimulated formation of cyclic AMP was modest (about 3 fold at 60 u ml-1 for 1 h) compared to that evoked by isoprenaline (about 9 fold at 3 x 10(-6) M for 2 min). 3. The IL-1 beta (60 u ml-1 for 24 h)-stimulated accumulation of nitrite, which was taken as an index of NO production, was concentration-dependently increased by preferential inhibitors of cyclic AMP-dependent phosphodiesterases (rolipram and trequinsin). This effect was reproduced by a specific activator of the cyclic AMP-dependent protein kinase(s) A, Sp-8-CPT-cAMPS (10(-4) M) but was prevented by a specific inhibitor of cyclic AMP-dependent protein kinase(s) A, Rp-8-CPT-cAMPS (10(-4) M). These compounds alone [rolipram (10(-6) M), trequinsin (3 x 10(-6) M) and Sp-8-CPT-cAMPS (10(-4) M)] slightly but significantly increased the release of nitric oxide while Rp-8-CPT-cAMPS elicited no such effect. 4. Inducible NOS protein was expressed in IL-1 beta (30 u ml-1, 24 h)-stimulated smooth muscle cells as assessed by Western blot analysis. The level of iNOS protein was markedly increased in smooth muscle cells which had been exposed to IL-1 beta in combination with either rolipram (3 x 10(-6) M) or Sp-8-CPT-cAMPS (10(-4) M) but was reduced in those exposed to IL-1 beta and Rp-8-CPT-cAMPS (10(-4) M). A weak expression of iNOS protein was found in smooth muscle cells which had been exposed to either Sp-8-CPT-cAMPS or rolipram alone for 24 h while Rp-8-CPT-cAMPS elicited no such effect. 5. Exposure of smooth muscle cells to IL-1 beta (30 u ml-1) for 30 min increased the level of NF-kappa B-DNA complexes in nuclear extracts as detected by electrophoretic mobility shift assay. Similar levels of NF-kappa B-DNA complexes were found in cells which had been exposed to IL-1 beta in combination with either Sp-8-CPT-cAMPS (10(-4) M), trequinsin (10(-6) M) or rolipram (10(-6) M). None of the modulators alone affected the basal level of NF-kappa B binding activity. 6. NO-donors [sodium nitroprusside (SNP) 10(-4) M; dinitrosyl-iron-di-L-cysteine-complex (DNIC), 10(-4) M; 3-morpholino-sydnonimine (SIN-1), 10(-4) M] and atrial natriuretic factor (10(-6) M) significantly increased the IL-1 beta (30 or 60 u ml-1, 24 h)-stimulated expression of iNOS protein and activity as assessed indirectly by the conversion of oxyhaemoglobin to methaemoglobin. In the absence of IL-1 beta, SNP (10(-4) M, 24 h) but not the other cyclic GMP-dependent vasodilators caused a modest expression of iNOS protein. No such effect was found in smooth muscle cells exposed to SNP in combination with Rp-8-CPT-cAMPS (10(-4) M) while an increased level of iNOS protein was found in those exposed to SNP in combination with either Sp-8-CPT-cAMPS (10(-4) M) or rolipram (3 x 10(-6) M). 7. Exposure of vascular smooth muscle cells to either S-nitroso-L-cysteine (Cys-SNO, 10(-4) M), SNP (10(-4) M) or SIN-1 (10(-4) M) for 35 min affected minimally the basal activation of NF-kappa B but abolished that evoked by IL-1 beta (30 u ml-1 added during the last 30 min). However, addition of Cys-SNO following the stimulation with IL-1 beta (during the last 5 min of the 30 min exposure period) reduced the level of NF-kappa B-DNA complexes only slightly. 8. These data indicate that the cyclic AMP-dependent pathway plays a decisi

Animals↗

PubMed1995

S-nitrosation of serum albumin by dinitrosyl-iron complex.

The objective of this study was to identify a potential mechanism for S-nitrosation of proteins. Therefore, we assessed S-nitrosation of bovine serum albumin by dinitrosyl-iron-di-L-cysteine complex [(NO)2Fe(L-cysteine)2], a compound similar to naturally occurring iron-nitrosyls. Within 5-10 min, (NO)2Fe(L-cysteine)2 generated paramagnetic albumin-bound dinitrosyl-iron complex and S-nitrosoalbumin in a ratio of 4:1. Although S-nitroso-L-cysteine was concomitantly formed in low amounts, its concentration was not sufficient to account for formation of S-nitrosoalbumin via a trans-S-nitrosation reaction. Low oxygen tension did not affect S-nitrosation by the dinitrosyl-iron complex thus excluding the involvement of oxygenated NOx-species in the nitrosation reaction. Blockade of albumin histidine residues by pyrocarbonate, which prevented formation of dinitrosyl-iron-albumin complex, did not inhibit S-nitrosation of albumin. Thus, S-nitrosation of albumin by (NO)2Fe(L-cysteine)2 can proceed by direct attack of a nitrosyl moiety on the protein thiolate, without previous binding of the iron. We conclude that protein-bound dinitrosyl-iron complexes detected in high concentrations in certain tissues provide a reservoir of S-nitrosating species, e.g. low molecular dinitrosyl iron complexes.

Animals↗

PubMed1995

In vivo spin trapping of glyceryl trinitrate-derived nitric oxide in rabbit blood vessels and organs.

BACKGROUND: The objectives of this study were (1) to assess glyceryl trinitrate (GTN)-derived nitric oxide (NO) formation in vascular tissues and organs of anesthetized rabbits in vivo, (2) to establish a correlation between tissue NO levels and a biological response, and (3) to verify biotransformation of GTN to NO by cytochrome P-450. METHODS AND RESULTS: NO was trapped in tissues in vivo as a stable paramagnetic mononitrosyl-iron-diethyldithiocarbamate complex [NOFe(DETC)2]. After removal of the tissues, NO was determined by cryogenic electron spin resonance spectroscopy. NO formation in vitro was assessed by spin trapping and by activation of soluble guanylyl cyclase. The GTN-elicited decrease in coronary perfusion pressure was monitored in isolated, constant-flow perfused rabbit hearts. NO was not detected in control tissues. In GTN-treated rabbits, NO formation was higher in organs than in vascular tissues and higher in venous than in arterial vessels. In isolated hearts, ventricular NO levels and decreases in coronary perfusion pressure achieved by GTN were closely correlated. Purified cytochrome P-450 catalyzed NO formation from GTN in a P-450-NADPH reductase- and NADPH-dependent fashion. CONCLUSIONS: Since GTN-derived NO formation in myocardial tissue correlates to the GTN-elicited vasodilator response, we conclude that GTN-derived NO detected in vivo correlates with the systemic effects of GTN. Therefore, the higher rate of NO formation detected in veins compared with arteries explains the preferential venodilator activity of GTN. High NO formation in cytochrome P-450-rich organs in vivo and efficient NO formation from GTN by cytochrome P-450 in vitro highlights the importance of this pathway for NO formation from GTN in the intact organism.

Animals↗

PubMed1995

Coordinate up- and down-modulation of inducible nitric oxide synthase, nitric oxide production, and tumoricidal activity in rat bone-marrow-derived mononuclear phagocytes by lipopolysaccharide and gram-negative bacteria.

Simultaneous incubation of primary rat bone-marrow-derived mononuclear phagocytes (BMMo) and tumor cells with gram-negative agents triggers within 24 h interferon gamma (IFN gamma)- and tumor necrosis factor (TNF alpha)-independent tumoricidal activity. On the other hand, BMMo that had been incubated for 24 h with gram-negative agents prior to re-exposure to the same agent had largely lost their ability to generate tumoricidal activity, although their ability to bind lipopolysaccharide (LPS) was not diminished. Parallel measurements of the kinetics of inducible nitric oxide synthase (iNOS), nitrite secretion, and tumoricidal activity triggered in primary BMMo by LPS revealed that these parameters take a coordinate course, reaching a peak within 24 h and then rapidly decaying. Down-regulation of expression of NOS protein and iNOS activity could be attributed neither to down-regulation of LPS receptors nor to L-arginine depletion.

Amino Acid Oxidoreductases↗

PubMed1995

Characterization of the stable L-arginine-derived relaxing factor released from cytokine-stimulated vascular smooth muscle cells as an NG-hydroxyl-L-arginine-nitric oxide adduct.

The nature of an L-arginine-derived relaxing factor released from vascular smooth muscle cells cultured on microcarrier beads and stimulated for 20 h with interleukin 1 beta was investigated. Unlike the unstable relaxation elicited by authentic nitric oxide (NO) in a cascade superfusion bioassay system, the effluate from vascular smooth muscle cells induced a stable relaxation that was susceptible to inhibition by oxyhemoglobin. Three putative endogenous NO carriers mimicked this stable relaxing effect: S-nitroso-L-cysteine, low molecular weight dinitrosyl-iron complexes (DNICs), and the adduct of NG-hydroxy-L-arginine (HOArg) with NO. Inactivation of S-nitroso-L-cysteine by Hg2+ ions or trapping of DNICs with agarose-bound bovine serum albumin abolished their relaxing effects, whereas that of the vascular smooth muscle cell effluate remained unaffected. In addition, neither S-nitrosothiols nor DNICs were detectable in the effluate from these cells, as judged by UV and electron spin resonance (ESR) spectroscopy. The HOArg-NO adduct was instantaneously generated upon reaction of HOArg with authentic NO under bioassay conditions. Its pharmacological profile was indistinguishable from that of the vascular smooth muscle cell effluate, as judged by comparative bioassay with different vascular and nonvascular smooth muscle preparations. Moreover, up to 100 nM HOArg was detected in the effluate from interleukin 1 beta-stimulated vascular smooth muscle cells, suggesting that sufficient amounts of HOArg are released from these cells to spontaneously generate the HOArg-NO adduct. This intercellular NO carrier probably accounts for the stable L-arginine-derived relaxing factor released from cytokine-stimulated vascular smooth muscle cells and also from other NO-producing cells, such as macrophages and neutrophils.

Acetylcholine↗

PubMed1995

Specificity of different organic nitrates to elicit NO formation in rabbit vascular tissues and organs in vivo.

1. In the present study we assessed the formation of nitric oxide (NO) from classical and thiol-containing organic nitrates in vascular tissues and organs of anaesthetized rabbits, and established a relationship between the relaxant response elicited by nitroglycerin (NTG) and NO formation in the rabbit isolated aorta. Furthermore, the effect of isolated cytochrome P450 on NO formation from organic nitrates was investigated. 2. Rabbits received diethyldithiocarbamate (DETC; 200 mg kg-1 initial bolus i.p. and 200 mg kg-1 during 20 min, i.v.) and either saline, or one of the following organic nitrates: nitroglycerin (NTG, 0.5 mg kg-1), isosorbide dinitrate (ISDN), N-(3-nitratopivaloyl)-L-cysteine ethylester (SPM 3672), S-carboxyethyl-N-(3-nitratopivaloyl)-L-cysteine ethylester (SPM 5185), at 10 mg kg-1 each. After 20 min the animals were killed, blood vessels and organs were removed, and subsequently analyzed for spin-trapped NO by cryogenic electron spin resonance (e.s.r.) spectroscopy. 3. In the saline-treated control group, NO remained below the detection limit in all vessels and organs. In contrast, all of the nitrates tested elicited measurable NO formation, which was higher in organs (liver, kidney, heart, lung, spleen) (up to 4.8 nmol g-1 20 min-1) than in blood vessels (vena cava, mesenteric bed, femoral artery, aorta) (up to 0.7 nmol g-1 20 min-1). Classical organic nitrates (NTG, ISDN) formed NO preferentially in the mesenteric bed and the vena cava, while the SPM compounds elicited comparable NO formation in veins and arteries. 4. Using a similar spin trapping technique, NO formation was assessed in vitro in phenylephrine-precontracted rabbit aortic rings. The maximal relaxation elicited by a first exposure (10 min) to NTG (0.3 to 10 microM) was positively correlated (r = 0.8) with the net increase (NTG minus basal) of NO spin-trapped during a second exposure to the same concentration of NTG in the presence of DETC. 5. Cytochrome P450 purified from rabbit liver enhanced NO formation in a NADPH-dependent fashion from NTG, but not from the other nitrates, as assessed by activation of purified soluble guanylyl cyclase. 6. We conclude that the vessel selective action of different organic nitrates in vivo reflects differences in vascular NO formation. Thus, efficient preload reduction by classical organic nitrates can be accounted for by higher NO formation in venous capacitance as compared to arterial conductance and resistance vessels. In contrast, NO is released from cysteine-containing nitrates (SPMs) to a similar extent in arteries and veins, presumably independently of an organic nitrate-specific biotransformation. Limited tissue bioavailability of NTG and ISDN might account for low NO formation in the aorta, while true differences in biotransformation seem to account for differences in NO formation in the other vascular tissues.

Animals↗

PubMed1994

Nitric oxide promotes seizure activity in kainate-treated rats.

L-Arginine-derived nitrogen monoxide (NO) formation was determined in different regions of the rat brain during kainate-induced seizures. NO was trapped in vivo as a paramagnetic mononitrosyl-iron diethyldithiocarbamate complex, the concentration of which was determined ex vivo by cryogenic electron spin resonance spectroscopy. Basal NO formation (0.3-0.8 nmol g-1 tissue 30 min-1) was detected in the brain of control rats. In kainate-injected rats NO formation was increased six-fold within 30-60 min in the amygdala/temporal cortex region, and up to 12-fold, though more slowly, in the remaining cortex. The kainate-elicited convulsions and NO formation were attenuated in animals pretreated with either 7-nitroindazole, a specific inhibitor of neuronal NO synthase, or diazepam. These findings identify NO as a proconvulsant mediator in kainate-evoked seizures.

Amino Acid Oxidoreductases↗

PubMed1994

Pyrrolidine dithiocarbamate selectively prevents the expression of the inducible nitric oxide synthase in the rat aorta.

Exposure of rat aortic rings without endothelium to interleukin-1 beta for 5 h significantly attenuated the contractions due to phenylephrine and increased the tissue content of guanosine 3',5'-cyclic monophosphate (cyclic GMP) due to the induction of nitric oxide synthase. The presence of pyrrolidine dithiocarbamate, a specific inhibitor of nuclear transcription factor kappa B activation, during the exposure of the rings to interleukin-1 beta prevented these responses to interleukin-1 beta. Rat aortic rings which had been incubated for 5 h with interleukin-1 beta in the absence and presence of pyrrolidine dithiocarbamate prior to the organ chamber experiment had a similar concentration-dependent relaxation curve for acetylcholine in rings with endothelium, and for 3-morpholino-sydnonimine (SIN-1) in rings without. Pyrrolidine dithiocarbamate applied acutely did not alter the tone elicited by phenylephrine in rings with or without endothelium and had no effect on the subsequent relaxation induced by acetylcholine in rings with endothelium or by SIN-1 in rings without endothelium. These observations suggest that pyrrolidine dithiocarbamate prevents the interleukin-1 beta-mediated expression of the inducible nitric oxide synthase without affecting the activity of the constitutive enzyme in the rat aorta.

Acetylcholine↗

PubMed1994

Epr evidence of nitric oxide production by the regenerating rat liver.

Nitric oxide (NO) production in the regenerating liver was estimated from the intensity of the electron paramagnetic resonance (e.p.r.) signal of the mononitrosyl complexes of iron and diethylthiocarbamate (DETC). Preformed complexes of intracellular non-heme Fe2+ and added DETC served as a trap for endogenously produced NO. The time-dependent changes of NO production were connected with the periodicity of liver regeneration. The first increase in NO production occurred ca. 1 h after partial hepatectomy (PHE). The second and more pronounced peak of NO production was observed about 6 h after PHE, when the hepatocytes entered the first cell cycle; it originated mainly from these cells. The following minimum of NO synthesis coincided with the maximal rate of DNA synthesis. The third gradual rise of NO production was seen at the end of the investigated period that covered the G2 + M phases, the transit from the first to the second cell cycle of the hepatocytes and the entrance of the nonparenchymal cells into proliferation.

Animals↗

PubMed1994

Subcellular localization and characterization of nitric oxide synthase(s) in endothelial cells: physiological implications.

Endothelial cells (EC) contain a constitutive Ca2+/calmodulin-dependent nitric oxide (NO) synthase (cNOS) which plays an important role in the local control of vascular tone. We compared the subcellular distribution of this enzyme in cultured and freshly isolated pig EC by determination of specific cNOS activity and immunoblot analysis. Similar studies were also performed with cultured and freshly isolated bovine and cultured human EC. Enzyme activity was predominantly (> 70%) associated with the particulate fraction of all EC types tested and was highest in freshly isolated porcine EC. Both specific cNOS activity and immunoreactivity were substantially higher (> 3-fold) in the microsomal as compared with the soluble fraction of all EC types tested. In freshly isolated pig EC, these two fractions also differed in terms of their Ca(2+)-dependency, pH optimum and inhibitor specificity. EC may thus contain either two different cNOS isoenzymes or a single enzyme, the conformation of which differs between the soluble and membrane-bound state. Moreover, detailed subcellular fractionation of freshly isolated pig EC revealed that the distribution of cNOS activity closely resembled that of the plasma membrane marker 5'-nucleotidase, suggesting that most, if not all, of the cNOS activity in these cells is associated with the plasma membrane. This localization might render the enzyme more susceptible to activation by physical stimuli, such as a shear stress-induced change in the fluidity of the plasma membrane. Moreover, the continuous exposure to shear stress in vivo may also upregulate cNOS expression in EC, since specific enzyme activity, immunoreactivity and basal NO release were significantly higher in freshly isolated EC as compared with cultured EC.

Amino Acid Oxidoreductases↗

PubMed1994

Synthesis of 15N omega-hydroxy-L-arginine and ESR and 15N-NMR studies for the elucidation of the molecular mechanism of enzymic nitric oxide formation from L-arginine.

N omega-Hydroxy-L-arginine (2) was prepared by a multi-stage synthesis; the key step was the addition of hydroxylamine to the protected cyanamide 8. The presence of N-hydroxyguanidines was confirmed, above all, by 15N-NMR investigations. 15N omega-Hydroxy-L-arginine (2) was converted quantitatively to 15NO by NO synthases from macrophages. 15NO was identified by ESR-spectroscopy. These experiments confirm that 15N omega-hydroxy-L-arginine (2) is an intermediate in the biosynthesis of NO from arginine (1) and that the N-hydroxylated N-atom is present in the NO formed.

Amino Acid Oxidoreductases↗

PubMed1994

Subcellular localization and characterization of neuronal nitric oxide synthase.

In contrast to the predominantly particulate, Ca2+/calmodulin-dependent nitric oxide (NO) synthase in endothelial cells, the corresponding neuronal isoenzyme is considered to be mainly soluble, presumably owing to the lack of a posttranslational myristoylation. However, preliminary findings from this and other laboratories suggest that a substantial portion of the neuronal NO synthase activity may in fact be membrane bound. We have therefore investigated the distribution of this enzyme among subcellular fractions of the rat and rabbit cerebellum in more detail. Up to 60% of the total NO synthase activity was found in the particulate fraction and, according to density gradient ultracentrifugation, associated mainly with the endoplasmic reticulum fraction. There was no apparent difference between the soluble and particulate enzymes with respect to their specific activity, Ca2+ and pH dependency, inhibitor sensitivity, or immunoreactivity, suggesting that both rat and rabbit cerebella contain a single Ca2+/calmodulin-dependent NO synthase. The inhibition by the cytochrome P450 inhibitor SKF-525A of the NO synthase activity in these subcellular fractions (IC50 = 90 microM) and the fact that mammalian cytochrome P450 enzymes are endoplasmic reticulum-bound proteins support the notion that the cerebellar NO synthase is a cytochrome P450-type hemoprotein. Moreover, the aforementioned findings suggest that posttranslational myristoylation may not be the only factor determining the intracellular localization of NO synthase.

Amino Acid Oxidoreductases↗

PubMed1994

Nitrogen monoxide transport mechanisms.

The present article focuses on the possible existence of transport forms of nitrogen monoxide (NO) in biological tissues. Stimulated by the present controversy on the identity of L-arginine-derived endothelium-derived relaxing factor (EDRF) experiments were designed to clarify whether or not EDRF is a nitrosyl-iron complex. Synthetic dinitrosyl-iron-di-L-cysteine--(NO)2Fe(L-cysteine)2, a low molecular mass dinitrosyl-iron-dihiolate complex (DNIC)--exhibits similar pharmacological properties as EDRF. It is a potent (EC50 10 nmol/l) endothelium-independent, labile (< 2 min), superoxide radical-sensitive vasodilator and a direct activator of soluble guanylyl cyclase. In stimulated endothelial cells a paramagnetic DNIC associated with intracellular proteins was detected by electron spin resonance (ESR) spectroscopy. In the presence of N-acetyl-L-cysteine a low molecular mass DNIC released from endothelial cells was specifically trapped and detected in the extracellular medium as a paramagnetic albumin-DNIC. Due to release of DNIC the content of endothelial non-heme iron decreased after prolonged (1 h) agonist-induced stimulation in iron-free medium. Therefore, it is conceivable that endothelial NO forms high molecular (protein-bound) and low molecular DNIC. The protein-bound DNIC may serve as a reservoir for low molecular DNIC. The low molecular DNIC permeates the cell membrane and may thus act as an EDRF. The biological significance of DNIC may reside less in stabilization of free NO than in modulation of its reactivity. Thus, it is conceivable that DNIC reacts more selectively with proteins essential for NO-mediated signal transduction. By this mechanism signal transduction by NO will become more efficient and directed to certain pathways.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Formation and release of dinitrosyl iron complexes by endothelial cells.

The release of dinitrosyl non-heme iron complexes from cytotoxic macrophages accounts for NO-mediated iron loss. We have now investigated whether or not a similar mechanism operates in endothelial cells. Following stimulation with bradykinin or calcium ionophore A23187 NO and intracellular dinitrosyl iron complexes were detected by ESR spectroscopic analysis of frozen cells. In addition, endothelial cells released dinitrosyl iron complexes which bound to extracellular albumin. In transferrin and iron-free medium stimulation of endothelial cells by bradykinin or thimerosal resulted in a loss of non-heme iron. These effects were prevented by inhibition of NO synthase. Thus NO generated by the constitutive NO synthase appears to be incorporated into dinitrosyl iron complexes, which potentially account for endothelium-dependent relaxation.

Animals↗

PubMed1993

The relationship between L-arginine-dependent nitric oxide synthesis, nitrite release and dinitrosyl-iron complex formation by activated macrophages.

We identified the source of the nitrogen included into nitric oxide (NO) and studied the relationship between formation of NO, intracellular dinitrosyl ferrous iron complex (DNIC) and release of nitrite by murine bone-marrow-derived macrophages stimulated with E. coli lipopolysaccharide (LPS). NO was trapped in the cell membrane by iron-diethyldithiocarbamate complex (FeDETC) and was detected as a paramagnetic NOFe(DETC)2 complex by electron paramagnetic resonance (EPR) spectroscopy. Macrophages stimulated for 7 h up to 48 h with LPS and then incubated for 2 h with DETC exhibited an anisotropic EPR signal of axial symmetry with g-factor values g perpendicular = 2.035, g parallel = 2.02 and a triplet hyperfine structure (hfs) at g perpendicular characteristic for NOFe(DETC)2. In cells incubated with [15NG]L-arginine instead of [14NG]L-arginine the EPR signal of [15N]OFe(DETC)2 was detected with a doublet hfs at g perpendicular, indicating that NO was generated exclusively from the terminal guanidino-nitrogen of extracellular L-arginine. The ratio of NO formation and of nitrite release changed with time of exposure to LPS, nitrite exceeding NO at early stages of macrophage activation, and NO exceeding nitrite at later stages. DNIC with thiolate ligands (0.5 nmol/10(7) cells) was observed in stimulated macrophages not loaded with DETC. Furthermore, DNIC released from macrophages was trapped in the extracellular medium by bovine serum albumin (BSA) (1 nmol/10(7) cells per 2 h) by formation of a paramagnetic DNIC with BSA. DNIC release not only provides a route for iron loss from activated macrophages, but may also play a role in the cytotoxic and microbiostatic activity of macrophages.

Animals↗

PubMed1993

Diethyldithiocarbamate inhibits induction of macrophage NO synthase.

We investigated whether sodium diethyldithiocarbamate (DETC), an inhibitor of the nuclear transcription factor kappa B (NFkappa B), modulates induction of NO synthase (NOS) in murine bone marrow-derived macrophages. A short exposure (between 1 and 16 h) of L929-cell medium-preconditioned macrophages to E. coli lipopolysaccharide (LPS) significantly increased the level of NOS mRNA, and elicited NO formation as detected by electron spin resonance spectroscopy and by the release of nitrite. DETC (0.1-1 mM) present during stimulation with LPS prevented the increase in NOS mRNA and the expression of NOS activity. These findings suggest that NFkappa B is involved in the signal transduction pathway linking stimulation of macrophages by LPS with transcription of the gene encoding inducible NOS.

Amino Acid Oxidoreductases↗

PubMed1993

Enzymic and nonenzymic release of NO accounts for the vasodilator activity of the metabolites of CAS 936, a novel long-acting sydnonimine derivative.

The molecular mechanism(s) underlying the vasodilator activity of CAS 936 (3-(cis-2,6-dimethylpiperidino)-N-(4-methoxybenzoyl)-sydn oni mine) and its metabolites 3-(cis-2,6-dimethylpiperidino)-sydnonimine (C87 3754) and N-(cis-2,6-dimethylpiperidino)-N-nitroso-2-aminoacetonitrile (C873786) was investigated. These compounds were tested for their relaxant activity in isolated rabbit arterial segments, activation of purified soluble guanylyl cyclase and release of nitric oxide (NO) in vitro and in vivo. C873754 and C873786 inhibited the noradrenaline-induced contraction and increased the cyclic GMP content of endothelium-denuded rabbit aortic and femoral segments, whereas CAS 936 was without effect. Similarly, both metabolites, but not CAS 936, activated purified soluble guanylyl cyclase (EC50 about 30 microM) and released NO in buffered aqueous solutions, as detected by electron spin resonance (esr) spectrometry. Both in vitro and in vivo an accumulation of NO was detected by esr spectrometry in vascular tissues exposed to the metabolites of CAS 936, whereas a significant release of NO from CAS 936 was only detected in the isolated rabbit liver, but not in vascular tissue. It is conceivable, therefore, that the metabolites of CAS 936 appearing in the systemic circulation after hepatic biotransformation induce vasodilatation by release of NO and activation of soluble guanylyl cyclase in vascular smooth muscle. Moreover, the activation of soluble guanylyl cyclase in vitro by the metabolites of CAS 936 was significantly enhanced by co-incubation with certain particulate fractions from bovine aortic endothelial and smooth muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗