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C Thiemermann

Publications and source records attributed to C Thiemermann.

At least 109 records · Page 6Linked to original sources
PubMed1995

Amidines are potent inhibitors of nitric oxide synthases: preferential inhibition of the inducible isoform.

We evaluated the ability of simple alkyl amidines to inhibit the activity of the inducible isoform of nitric oxide (NO) synthase in vitro. In immunostimulated J774 macrophages, 2-iminopiperidine (EC50 = 10 microM) and butyramidine (EC50 = 60 microM) were more potent than NG-methyl-L-arginine (EC50 = 70 microM) in inhibiting nitrite formation. The five amidines tested for their ability to inhibit the conversion of L-arginine to L-citrulline by bovine endothelial cell homogenates (a source of the constitutive, endothelial NO synthase isoform) were less effective than NG-nitro-L-arginine or NG-methyl-L-arginine. The rank-order of the potencies of the amidines against the endothelial NO synthase was, in general, similar to the rank-order of the pressor effects of these agents in anesthetized rats. Thus, certain amidines are potent inhibitors of NO synthase, and are more selective towards the inducible NO synthase than the commonly used L-arginine based NO synthase inhibitors.

Amidines↗
PubMed1995

The cell wall components peptidoglycan and lipoteichoic acid from Staphylococcus aureus act in synergy to cause shock and multiple organ failure.

Although the incidence of Gram-positive sepsis has risen strongly, it is unclear how Gram-positive organisms (without endotoxin) initiate septic shock. We investigated whether two cell wall components from Staphylococcus aureus, peptidoglycan (PepG) and lipoteichoic acid (LTA), can induce the inflammatory response and multiple organ dysfunction syndrome (MODS) associated with septic shock caused by Gram-positive organisms. In cultured macrophages, LTA (10 micrograms/ml), but not PepG (100 micrograms/ml), induces the release of nitric oxide measured as nitrite. PepG, however, caused a 4-fold increase in the production of nitrite elicited by LTA. Furthermore, PepG antibodies inhibited the release of nitrite elicited by killed S. aureus. Administration of both PepG (10 mg/kg; i.v.) and LTA (3 mg/kg; i.v.) in anesthetized rats resulted in the release of tumor necrosis factor alpha and interferon gamma and MODS, as indicated by a decrease in arterial oxygen pressure (lung) and an increase in plasma concentrations of bilirubin and alanine aminotransferase (liver), creatinine and urea (kidney), lipase (pancreas), and creatine kinase (heart or skeletal muscle). There was also the expression of inducible nitric oxide synthase in these organs, circulatory failure, and 50% mortality. These effects were not observed after administration of PepG or LTA alone. Even a high dose of LTA (10 mg/kg) causes only circulatory failure but no MODS. Thus, our results demonstrate that the two bacterial wall components, PepG and LTA, work together to cause systemic inflammation and multiple systems failure associated with Gram-positive organisms.

Animals↗
PubMed1995

Lipid A and the lipid A analogue anti-tumour compound ONO-4007 induce nitric oxide synthase in vitro and in vivo.

The ability of lipid A and the antitumour compound, ONO-4007 (sodium2-deoxy-2-[3S-(9-phenylnonanoyloxy)tetradecanoyl] amino-3-O-(9phenylnonanoyl)-D-glucopyranose 4-sulphate) to induce nitric oxide (NO) synthase was investigated in vitro and in vivo, in comparison to the effects of lipopolysaccharide and di- and monophosphoryl lipid A. In J744.2 macrophages, lipopolysaccharide, di-and monophosphoryl lipid A and ONO-4007 (10(-9) - 10(-5) g/ml) alone, or in combination with interferon-gamma, induced NO synthase (order of potency: lipopolysaccharide > diphosphoryl lipid A > monophosphoryl lipid A > ONO-4007). ONO-4007 increased the activity of the inducible NO synthase in the lung of anesthetised rats (20% of the increased caused by bacterial lipopolysaccharide). Thus, ONO-4007 is a weak inducer of the inducible isoform of NO synthase in vitro and in vivo. The finding that di- and monophosphoryl lipid A also induce NO synthase indicates that the lipid A moiety of lipopolysaccharide contributes to the induction of NO synthase by lipopolysaccharide. The induction of NO synthase by ONO-4007, resulting in the formation of cytotoxic NO may contribute to the antitumour activity of the compound.

Adult↗
PubMed1995

Lipocortin 1 mediates the inhibition by dexamethasone of the induction by endotoxin of nitric oxide synthase in the rat.

Administration of Escherichia coli lipopolysaccharide (LPS; 10 mg/kg i.v.) to male Wistar rats caused within 240 min (i) a sustained fall (approximately 30 mmHg) in mean arterial blood pressure, (ii) a reduction (> 75%) in the pressor responses to norepinephrine (1 microgram/kg i.v.), and (iii) an induction of nitric oxide synthase (iNOS) as measured in the lung. Dexamethasone (1 mg/kg i.p. at 2 h prior to LPS) attenuated the hypotension and the vascular hyporeactivity to norepinephrine and reduced (by approximately 77%) the expression of iNOS in the lung. These effects of dexamethasone were prevented by pretreatment of LPS-treated rats with a neutralizing antiserum to lipocortin 1 (anti-LC1; 60 mg/kg s.c. at 24 h prior to LPS) but not by a control nonimmune sheep serum. Stimulation of J774.2 macrophages with LPS (1 microgram/ml for 24 h) caused the expression of iNOS and cyclooxygenase 2 (COX-2) protein and significantly increased nitrite generation; this was prevented by dexamethasone (0.1 microM at 1 h prior to LPS), which also increased cell surface lipocortin 1. Pretreatment of J774.2 cells with anti-LC1 (1:60 dilution at 4 h prior to LPS) also abolished the inhibitory effect of dexamethasone on iNOS expression and nitrite accumulation but not that on COX-2 expression. A lipocortin 1 fragment (residues 1-188 of human lipocortin 1; 20 micrograms/ml at 1 h prior to LPS) also blocked iNOS in J774.2 macrophages activated by LPS (approximately 78% inhibition), and this too was prevented by anti-LC1. We conclude that the extracellular release of endogenous lipocortin 1 (i) mediates the inhibition by dexamethasone of the expression of iNOS, but not of COX-2, and (ii) contributes substantially to the beneficial actions of dexamethasone in rats with endotoxic shock.

Amino Acid Oxidoreductases↗
PubMed1995

Comparison of the induction of cyclooxygenase and nitric oxide synthase by endotoxin in endothelial cells and macrophages.

Endotoxin causes the expression of inducible nitric oxide (NO) synthase and cyclooxygenase-2. We have compared the ability of endotoxin to increase the activities of these enzymes in bovine aortic endothelial cells and the macrophage cell line (J774.2). Endotoxin (1 microgram ml-1; for 24 h) caused a time-dependent increase in the accumulation of cyclooxygenase metabolites from endogenous arachidonic acid, in both cell types. Cyclooxygenase activity towards exogenous arachidonic acid (30 microM; for 15 min) was also increased in both cell types. Endothelial cells and macrophages also contained comparable amounts of cyclooxygenase-2 protein after incubation with endotoxin for 24 h which was prevented by pretreatment with cycloheximide (10 micrograms ml-1; 30 min prior to endotoxin). Endotoxin for 24 h caused a time-dependent increase in nitrite accumulation in macrophages, but not in endothelial cells. Thus, endotoxin increased cyclooxygenase activity and induced cyclooxygenase-2 protein in endothelial cells and macrophages. Endotoxin also increased NO synthase activity in macrophages, but not in endothelial cells.

Amino Acid Oxidoreductases↗
PubMed1995

The induction of cyclooxygenase-2 elicited by endotoxin in endothelial cells and macrophages is inhibited by prostaglandin E1 and 13,14-dihydro prostaglandin E1.

The effects of PGE1 or 13,14-dihydro PGE1 (PGE0) on the expression of COX-2 protein and COX activity elicited by LPS (1 microgram/ml for 24 h) in bovine aortic endothelial cells (BAEC) and J774.2 macrophages were investigated. PGE1 or PGE0 (0.001 to 10 micrograms/ml) caused a dose-dependent decrease of COX activity elicited by LPS in both cell types. Western blot analysis showed that PGE1 or PGE0 (1 microgram/ml) inhibited the expression of COX-2 protein in LPS-activated BAEC and J774.2 macrophages. Thus, PGE1 or (its metabolite) PGE0 decrease the formation of COX metabolites by inhibiting the induction of COX-2 protein by LPS.

Alprostadil↗
PubMed1995

Effects of nitric oxide synthase inhibition combined with nitric oxide inhalation in a porcine model of endotoxin shock.

1. The present investigation compares the effects of intravenous infusion of the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) with that of an inhalation with NO gas in a porcine model of endotoxin (lipopolysaccharide, LPS) shock. In addition, the effects of the combination of these two treatments were also investigated. 2. Male pigs were anaesthetized and instrumented for the measurement of haemodynamic parameters. Blood samples were withdrawn at different time intervals for determination of blood gases, pH, and plasma levels of nitrite/nitrate and tumour necrosis factor. 3. Endotoxin infusion (15 micrograms kg-1 h-1 for 3 h) caused a progressive fall in mean arterial blood pressure (MABP) and cardiac output (CO) and a biphasic increase in mean pulmonary arterial pressure (MPAP) and pulmonary vascular resistance (PVR). A continuous infusion of L-NMMA (0.1 mg kg-1 min-1) significantly attenuated the fall in MABP, but did not affect MPAP, CO and PVR. NO-inhalation (50 p.p.m.) did not affect MABP, but significantly blunted the biphasic increase in MPAP and PVR and significantly delayed the fall in CO. The combination of L-NMMA infusion (0.1 mg kg-1 min-1) with NO-inhalation (50 p.p.m.) completely prevented the fall in MABP, significantly improved CO, and attenuated the biphasic increase in MPAP and PVR. 4. Endotoxin also caused a decline in PaO2 and a rise of PaCO2. Infusion of L-NMMA neither affected the fall in PaO2 nor the increase in PaCO2. In contrast, inhalation with NO gas alone as well as the combined administration of L-NMMA infusion and NO-inhalation completely prevented the fall in Pao2 and significantly protected against the increase in Paco2.5. Infusion of endotoxin for 180 min resulted in a mortality of 58%, which was not affected by L-NMMA (63%). In contrast, treatment of LPS-animals with either NO-inhalation alone or NO inhalation plus L-NMMA completely prevented mortality.6. This investigation demonstrates that treatment with NO-inhalation, in order to prevent the dramatic increase in MPAP, PVR and the alterations in peripheral blood gases combined with systemic L-NMMAto improve systemic MABP and thus organ perfusion, may be a new therapeutic regimen in the treatment of septic shock.

Administration, Inhalation↗
PubMed1995

Isothioureas: potent inhibitors of nitric oxide synthases with variable isoform selectivity.

1. The induction of a calcium-independent isoform of nitric oxide (NO) synthase (iNOS) and a subsequent enhanced formation of NO has been implicated in the pathophysiology of a variety of diseases including inflammation and circulatory shock. Here we demonstrate that the S-substituted isothioureas, S-methylisothiourea (SMT), S-(2-aminoethyl)isothiourea (aminoethyl-TU), S-ethylisothiourea (ethyl-TU) and S-isopropylisothiourea (isopropyl-TU) potently inhibit iNOS activity in J774.2 macrophages activated with bacterial endotoxin with EC50 values 8-24 times lower than that of NG-methyl-L-arginine (MeArg) and 200-times lower than that of NG-nitro-L-arginine (L-NO2Arg). 2. The inhibition of iNOS activity by these S-substituted isothioureas is dose-dependently prevented by excess of L-arginine suggesting that these isothioureas are competitive inhibitors of iNOS at the L-arginine binding site. 3. Ethyl-TU and isopropyl-TU are 4-6 times more potent than MeArg in inhibiting the constitutive NOS activity in homogenates of bovine aortic endothelial cells (eNOS) and are more potent pressor agents than MeArg in the anaesthetized rat. SMT is equipotent with MeArg, whereas aminoethyl-TU is 6-times less potent in inhibiting eNOS activity in vitro. Both SMT and aminoethyl-TU, however, elicit only weak pressor responses (approximately 15 mmHg at 10 mg kg-1, i.v.) in vivo. 4. A comparison of the potencies of ethyl-, iso-propyl-, n-propyl-, t-butyl- and n-butyl-isothioureas on iNOS activity shows that the inhibitory activity of S-substituted isothioureas declines sharply if the side chain exceeds 2 carbon atoms in length. Similarly, substitution of the ethylene side chain of ethyl-TU also results in a diminished potency. Substitution of either one or both nitrogens of SMT with either amino or alkyl groups also substantially reduces its NOS inhibitory potency.5. In conclusion, isothioureas represent a new class of NOS inhibitors which includes the most potent inhibitors of iNOS activity reported to date. Some members of this class (ethyl-TU and isopropyl-TU)are potent inhibitors of eNOS and iNOS with little selectivity towards either isoform, while others (SMT and aminoethyl-TU) are relatively selective inhibitors of iNOS activity. These latter agents may become useful tools for studying the role of iNOS in various disease models and may be useful in the therapy of diseases that are associated with an enhanced formation of NO due to iNOS induction, such as inflammation, circulatory shock or cancer.

Amino Acid Oxidoreductases↗
PubMed1995

Glibenclamide-induced inhibition of the expression of inducible nitric oxide synthase in cultured macrophages and in the anaesthetized rat.

1. We have investigated whether glibenclamide, an inhibitor of ATP-sensitive potassium channels, influences the induction of the calcium-independent isoform of nitric oxide synthase (iNOS) in cultured J774.2 macrophages activated by bacterial endotoxin (E.coli lipopolysaccharide; LPS), as well as in the lung and aorta of rats with endotoxic shock. 2. Pretreatment of J774.2 macrophages with glibenclamide (10(-7) to 10(-5) M for 30 min) dose-dependently inhibited the accumulation of nitrite caused by LPS (1 microgram ml-1). In contrast, pretreatment of macrophages with tetraethylammonium (10(-4) to 10(-2) M for 30 min), a non-selective inhibitor of potassium channels, did not affect the rise in nitrite caused by LPS. At the highest concentration (10(-5) M) used, cromakalim, an opener of ATP-sensitive potassium channels, caused a small, but significant inhibition of nitrite formation in macrophages activated with LPS, while lower concentrations (10(-7) to 3 x 10(-6) M) were without effect. 3. The inhibition by glibenclamide (3 microM) of the increase in nitrite induced by LPS in J774.2 macrophages was weaker when glibenclamide was given several hours after LPS, indicating that glibenclamide inhibits the induction, but not the activity, of iNOS. In contrast, the degree of inhibition of nitrite formation caused by the nitric oxide synthase (NOS) inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) was similar when this agent was given up to 10 h after LPS. 4.In anaesthetized rats, LPS caused a fall in mean arterial blood pressure (MAP) from 120 +/-(time 0)to 98 +/- mmHg at 180 min (P<0.05, n = 6). Treatment of LPS-rats with glibenclamide (1 mg kg-1, i.v.at 60 min after LPS) caused a rapid and sustained rise in MAP (e.g. MAP at 180 min after LPS:122 +/-4 mmHg; n =6, P <0.05 when compared to LPS-rats). The maximum of the rise in MAP produced by glibenclamide (1 mg kg-1 , i.v.) was similar when the drug was given either at 60 or 180 min after LPS. However, the duration of the pressor response was significantly longer when glibenclamide was given at 60 min, rather than at 180 min after LPS.5. LPS-treatment caused a significant reduction of the pressor responses elicited by noradrenaline (NA,1 microg kg-1, i.v.) from 35 +/- 2 to 19 +/- 1 mmHg at 60 min and 20 +/- 2 mmHg at 180 min (P<0.05).Treatment of LPS-rats with glibenclamide (1 mg kg-1, i.v. at 60 min) caused a significant restoration of the pressor responses elicited by NA from 19 +/- 1 mmHg at 60 min (prior to glibenclamide injection) to 29 +/- 3 mmHg at 180 min (P<0.05).6. Endotoxaemia for 180 min resulted in a significant increase in a calcium-independent NOS activity(which was taken to represent iNOS activity) in the lung from 0.17 +/- 0.1 (control, n =4) to 6.21 +/- 0.48 pmol mg-1 min-1 (n =6, P<0.05). Injection of glibenclamide (1 mg kg-1, i.v.) at 60 min after LPS attenuated the increase in iNOS activity caused by endotoxaemia in the lung by 43 +/- 7%(n = 6, P <0.05). In contrast, injection of glibenclamide at 180 min after LPS did not result in a significant inhibition of iNOS activity (n = 6, P <0.05. 7. Thoracic aortae obtained from rats at 180 min after LPS showed a significant reduction in the contractions elicited by noradrenaline (NA, 10-9 to 10-6 M). Treatment of LPS-rats with glibenclamide(1 mg kg-1, i.v. at 60 min after LPS) significantly alleviated this LPS-induced hyporeactivity to NA ex vivo. In contrast, when aortic rings from LPS-rats were incubated in vitro with glibenclamide (10 microM for 20 min), glibenclamide did not reverse the vascular hyporeactivity to NA. However, L-NAME (300 microM for 20 min) significantly enhanced the contractile response to NA in aortic rings obtained from LPS-rats(P<0.05, n=6).8. No significant amounts of tumour necrosis factor-alpha (TNF alpha) were detectable in the plasma before the injection of LPS. Endotoxaemia for 90 min resulted in a significant rise in plasma TNFalpha levels(0.05 +/- 0.05 ng ml-1 at time 0, 3.78 +/- 0.24 ng ml-1 at 90 min, n = 6, P < 0.05). Treatment of LPS-rats with glibenclamide (1 mg kg-1, i.v. at 15 min prior to LPS, n = 5) did not significantly reduce the rise in plasma TNF alpha levels caused by endotoxin.9. Thus, glibenclamide inhibits the induction, but not the activity, of iNOS in vitro and in vivo. This inhibition of iNOS induction may contribute to the beneficial haemodynamic effects of glibenclamide in endotoxic shock.

Amino Acid Oxidoreductases↗
PubMed1995

Delayed circulatory failure due to the induction of nitric oxide synthase by lipoteichoic acid from Staphylococcus aureus in anaesthetized rats.

1. This study investigates the effect of lipoteichoic acid (LTA) from the cell wall of Staphylococcus aureus, a micro-organism without endotoxin, on haemodynamics and induction of nitric oxide synthase (iNOS) in the anaesthetized rat. 2. Intravenous injection of LTA (10 mg kg-1) resulted in a decrease in blood pressure from 123 +/- 1 mmHg to 83 +/- 7 mmHg after 270 min (P < 0.001) and a reduction of the pressor response to noradrenaline (1 microgram kg-1) from 33 +/- 1 mmHg.min to 23 +/- 3 mmHg.min after 270 min (P < 0.05). 3. The delayed circulatory failure (hypotension and vascular hyporeactivity) caused by LTA was prevented by pretreatment of rats with dexamethasone (10 mg kg-1, 60 min prior to LTA) or the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 10 mg kg-1 h-1, i.v. infusion starting 30 min prior to LTA). 4. In contrast, treatment of rats with polymyxin B (0.05 mg kg-1), an agent which binds endotoxin (lipopolysaccharides, LPS), did not affect the delayed circulatory failure caused by LTA. Polymyxin B, however, attenuated the hypotension and vascular hyporeactivity to noradrenaline afforded by endotoxaemia (2 mg kg-1 LPS, i.v.) for 270 min. 5. The delayed circulatory failure caused by LTA was associated with a time-dependent increase in (i) the expression of iNOS protein in the lung (Western blot analysis), and (ii) iNOS activity. This increase in iNOS protein and activity was prevented by pretreatment of LTA-rats with dexamethasone (10 mg kg-1). 6. Intravenous injection of LTA resulted in an increase in serum tumour necrosis factor (TNF)-alpha(maximum at 90 min after LTA), which was attenuated by pretreatment of rats with dexamethasone(10 mg kg-1, 60 min prior to LTA). The magnitude of the rise in TNF-alpha caused by LTA was similar to the one elicited by LPS (10mgkg-', i.v.).7. Thus, an enhanced formation of nitric oxide following the induction of iNOS contributes importantly to the delayed vascular failure (hypotension and vascular hyporeactivity) caused by LTA in the anaesthetized rat. We suggest that the endogenous release of TNF-alpha contributes to the induction ofiNOS caused by LTA in vivo.

Amino Acid Oxidoreductases↗
PubMed1995

Aminoguanidine attenuates the delayed circulatory failure and improves survival in rodent models of endotoxic shock.

1. We have investigated the effects of aminoguanidine, a relatively selective inhibitor of the cytokine-inducible isoform of nitric oxide synthase (iNOS), on the delayed circulatory failure, vascular hyporeactivity to vasoconstrictor agents, and iNOS activity in a rat model of circulatory shock induced by bacterial endotoxin (E. coli lipopolysaccharide; LPS). In addition, we have evaluated the effect of aminoguanidine on the 24 h survival rate in a murine model of endotoxaemia. 2. Male Wistar rats were anaesthetized and instrumented for the measurement of mean arterial blood pressure (MAP) and heart rate (HR). Injection of LPS (10 mg kg-1, i.v.) resulted in a fall in MAP from 115 +/- 4 mmHg (time 0, control) to 79 +/- 9 mmHg at 180 min (P < 0.05, n = 10). The pressor effect of noradrenaline (NA, 1 microgram kg-1, i.v.) was also significantly reduced at 60, 120 and 180 min after LPS injection. In contrast, animals pretreated with aminoguanidine (15 mg kg-1, i.v., 20 min prior to LPS injection) maintained a significantly higher MAP (at 180 min, 102 +/- 3 mmHg, n = 10, P < 0.05) when compared to rats given only LPS (LPS-rats). Cumulative administration of aminoguanidine (15 mg kg-1 and 45 mg kg-1) given 180 min after LPS caused a dose-related increase in MAP and reversed the hypotension. Aminoguanidine also significantly alleviated the reduction of the pressor response to NA: indeed, at 180 min, the pressor response returned to normal in aminoguanidine pretreated LPS-rats. 3. Thoracic aortae obtained from rats at 180 min after LPS showed a significant reduction in the contractile responses elicited by NA (10-9- 10-6 M). Pretreatment with aminoguanidine (15 mg kg- 1, i.v.,at 20 min prior to LPS) significantly prevented this LPS-induced hyporeactivity to NA ex vivo.4. Endotoxaemia for 180 min resulted in a significant increase in iNOS activity in the lung from 0.6 +/- 0.2 pmol mg-1 min-1 (control, n = 4) to 4.8 +/- 0.3 pmol mg-1 min-1 (P<0.05, n = 6). In LPS-rats treated with aminoguanidine, iNOS activity in the lung was attenuated by 44+/- 5% (n = 6, P <0.05).Moreover, when added in vitro to lung homogenates obtained from LPS-rats, aminoguanidine and N omega-nitro-L-arginine methyl ester (L-NAME; 10-8 to 10-3 M) caused a concentration-dependent inhibition of iNOS activity (n = 3-6, IC50: 30 +/- 12 and 11 +/- 6pEM, respectively P>0.05). In contrast,aminoguanidine was a less potent inhibitor than L-NAME of the constitutive nitric oxide synthase in rat brain homogenates (n = 3-6, IC50 is 140 +/- 10 and 0.6 +/- 0.1 I1M, respectively, P<0.05). In addition, the inhibitory effect of aminoguanidine on iNOS activity showed a slower onset than that of L-NAME(maximal inhibition at 90 min and 30 min, respectively).5. Treatment of conscious Swiss albino (T/O) mice with a high dose of endotoxin (60 mg kg-1, i.p.)resulted in a survival rate of only 8% at 24 h (n = 12). However, therapeutic application of aminoguanidine (15 mg kg-1, i.p. at 2 h and 6 h after LPS) increased the 24 h survival rate to 75%(n = 8), whereas L-NAME (3 mg kg-1, i.p. at 2 h and 6 h after LPS) did not affect the survival rate(11%, n=9).6 Thus, aminoguanidine inhibits iNOS activity and attenuates the delayed circulatory failure caused by endotoxic shock in the rat and improves survival in a murine model of endotoxaemia. Aminoguanidine,or novel, more potent selective inhibitors of iNOS may be useful in the therapy of septic shock.

Amino Acid Oxidoreductases↗
PubMed1995

Inhibition by N-acetyl-5-hydroxytryptamine of nitric oxide synthase expression in cultured cells and in the anaesthetized rat.

1. Induction of the calcium-independent isoform of nitric oxide (NO) synthase (iNOS) in various cell types has been implicated in the circulatory failure in experimental models of septic shock. Tetrahydrobiopterin (BH4) appears to be an essential co-factor for NO formation and therefore an inhibition of its biosynthesis represents a feasible therapeutic target. We have investigated the effects of an inhibitor of BH4 synthesis, N-acetyl-5-hydroxytryptamine (N-acetylserotonin, NAS), on the expression of iNOS in cultured macrophages and smooth muscle cells in vitro, and on the hypotensive response to bacterial lipopolysaccharide (LPS) in the anaesthetized rat in vivo. 2. NAS (0.01-5 mM) caused a concentration-dependent inhibition of the accumulation of nitrite in the conditioned medium of LPS/interferon-gamma (IFN gamma)-stimulated RAW 264.7 macrophages and interleukin-1 beta (IL-1 beta)-activated vascular smooth muscle cells (VSMC). This effect was paralleled by a similar decrease in the iNOS protein content of these cells, as determined by immunoblot analysis. 3. Pretreatment of RAW 264.7 macrophages with the BH4 precursor, dihydrobiopterin (BH2, 0.1 mM) did not restore nitrite formation in the presence of NAS (1 mM). 4. Intravenous administration of NAS (1 mg kg-1 min-1 for 30 min) in anaesthetized rats significantly reduced the fall in mean arterial blood pressure, restored the pressor response to noradrenaline (1 micrograms kg-1), and ameliorated the increase in plasma nitrite following exposure to LPS (10 mg kg-1). 5. NAS pretreatment also attenuated iNOS activity in lung homogenates, as determined by the conversion of radiolabelled L-arginine to L-citrulline, and partially restored the constrictor effect of noradrenaline in aortic rings isolated from LPS-treated rats. Moreover, NAS significantly reduced the rise in the plasma concentration of tumour necrosis factor alpha (TNFalpha) in response to LPS.6. These findings suggest that NAS inhibits the expression rather than the activity of iNOS in cultured macrophages and smooth muscle cells. This effect of NAS appears to be independent of the availability of BH4, but may be related to an attenuation of the release of TNFalpha following LPS administration, as shown in the anaesthetized rat. This mechanism may also account for the beneficial haemodynamic effect of NAS in our experimental model of endotoxaemia.

Anesthesia↗
PubMed1995

Effect of interleukin-4 and interleukin-10 on leucocyte migration and nitric oxide production in the mouse.

1. The effect of systemic treatment of mice with murine recombinant interleukin-4 (IL-4) or interleukin-10 (IL-10) on neutrophil infiltration into a specific tissue site and nitric oxide (NO) production from peritoneal macrophages was investigated. 2. Intravenously (i.v.) administered IL-4 (0.01-10 micrograms per mouse, approximately 0.3-300 micrograms kg-1, i.v.) and IL-10 (0.01-1 micrograms per mouse, approximately 0.3-30 micrograms kg-1, i.v.) dose-dependently inhibited neutrophil accumulation into a 6-day-old murine air-pouch induced by local application of interleukin-1 beta (IL-1 beta, 5 ng), with approximate ED50s of 0.35 and 0.90 micrograms, respectively. Neither IL-4 (1 micrograms, 30 micrograms kg-1, i.v.) nor IL-10 (1 micrograms, 30 micrograms kg-1, i.v.) prevented leucocyte accumulation in the mouse air-pouches when interleukin-8 (IL-8, 1 micrograms) was used as chemoattractant. Similarly, neither cytokine had any effect on the in vitro up-regulation of CD11b antigen on the surface of murine circulating neutrophils. 3. Treatment of mice with lipopolysaccharide (LPS, 0.3 mg kg-1, i.p.) caused an increase in the formation of NO (measured as nitrite accumulation) in the supernatant of peritoneal macrophages ex vivo. Pretreatment of mice with IL-4 (0.01-1 micrograms i.v., 20 min before LPS), but not with IL-10 (1 micrograms i.v., 20 min before LPS), caused a dose-dependent reduction in this LPS-stimulated formation of nitrite by peritoneal macrophages ex vivo. 4. Activation of murine macrophages with LPS (1 microgram ml-1 for 24 h) in vitro caused a significant increase in nitrite release in the supernatant of these cells. Pretreatment of either J774.2 or peritoneal macrophages with IL-4 (0.1-1 microg ml-1, 20 min before LPS), but not with IL-1O (1 microg ml', 20 min before LPS) caused a concentration-related attenuation of this LPS-stimulated nitrite formation.5 Thus, both IL-4 and IL-10 inhibit the migration of leucocytes (stimulated by IL-1beta>) in vivo; IL-4 (but not IL-10) inhibits the induction of NO synthase caused by LPS in murine macrophages in vitro and ex vivo.

Animals↗
PubMed1995

Reduction by prostaglandin E1 or prostaglandin E0 of myocardial infarct size in the rabbit by activation of ATP-sensitive potassium channels.

1. This study examined whether pretreatment of rabbits with infusions of prostaglandin E1 (PGE1) or prostaglandin E0 (PGE0) (which were terminated prior to the onset of ischaemia) reduce myocardial infarct size arising from coronary artery occlusion (60 min) and reperfusion (120 min). In addition, we investigated whether the observed cardioprotective effects of these two prostaglandins were due to the activation of ATP-sensitive potassium (KATP) channels. 2. In the anaesthetized rabbit, infarct size (expressed as a percentage of the area at risk) after 60 min of coronary artery occlusion followed by 2 h of reperfusion was 59 +/- 4% (n = 10). PGE1 or PGE0 treatment (1.0 micrograms kg-1 min-1), administered as 1 h pretreatments (0.05 ml min-1, i.v.), significantly reduced infarct size to 44 +/- 6% (n = 6) or 42 +/- 1% (n = 6), respectively. PGE1 or PGE0 pretreatment resulted in a significant reduction in mean arterial blood pressure, which returned to baseline within 15 min of discontinuation of the infusion (i.e. prior to LAL ligation). 3. The reduction in infarct size afforded by PGE1 was abolished by pretreatment of rabbits with the KATP channel blockers, glibenclamide (60 +/- 4%; n = 8) or 5-hydroxydecanoate (58 +/- 6%; n = 6). Similarly, glibenclamide also largely attenuated the reduction in infarct size afforded by PGE0 (52 +/- 3%; n = 8). 4. We propose that a 1 h pretreatment of PGE1 or PGE0 reduces infarct size by activating protein kinase C resulting in the opening of KATP channels.

Adenosine Triphosphate↗
PubMed1995

Role for intracellular platelet-activating factor in the circulatory failure in a model of gram-positive shock.

1. This study investigates the effects of two structurally different antagonists of platelet-activating factor (PAF), BN52021 and WEB2086, on the circulatory and renal failure elicited by lipoteichoic acid (LTA) from Staphylococcus aureus (an organism without endotoxin) in anaesthetized rats. 2. Administration of LTA (10 mg kg-1, i.v.) caused hypotension and vascular hyporeactivity to noradrenaline (1 microgram kg-1, i.v.) WEB2086 (5 mg kg-1, i.v., 20 min before and 150 min after LTA) inhibited the delayed fall in mean arterial blood pressure (at 300 min: 99 +/- 6 mmHg vs. 75 +/- 6 mmHg, P < 0.01) and prevented the decrease in pressor response to noradrenaline (at 300 min: 36 +/- 5 mmHg min vs. 17 +/- 5 mmHg min, P < 0.01). Surprisingly, BN52021 (20 mg kg-1, i.v., 20 min before and 150 min after LTA) neither prevented the hypotension (74 +/- 6 mmHg) nor the vascular hyporeactivity (21 +/- 5 mmHg min). However, BN52021 inhibited the hypotension to injections of PAF as well as the circulatory failure elicited by lipopolysaccharides (10 mg kg-1, i.v.). 3. LTA caused an increase in plasma concentration of creatinine from 39 +/- 5 microM (sham-operated) to 70 +/- 8 microM and urea from 4.7 +/- 0.1 to 13.1 +/- 1.6 mM. The renal failure elicited by LTA was significantly inhibited by WEB2086 (creatinine: 45 +/- 4 microM and urea: 5.7 +/- 0.7 mM), but not by BN52021. 4. The induction of nitric oxide synthase activity in lungs by LTA was attenuated by WEB2086 from 98 +/- 17 to 40 +/- 15 pmol L-citrulline 30 min-1 mg-1 protein (P < 0.01), but not by BN52021 (148 +/- 21 pmol L-citrulline 30 min-1 mg-1 protein). Similarly, WEB2086, but not BN52021, inhibited the increase in plasma nitrite concentration associated with the delayed circulatory failure caused by LTA. The release of tumour necrosis factor-alpha (TNF-alpha) after injection of LTA was not attenuated by WEB2086. 5. The induction of nitrite release by cultured macrophages activated with LTA (10 micrograms ml-1 for 24 h) was inhibited by 74 +/- 4% by WEB2086 (3 x 10(-4) M), but not by BN52021, indicating that only WEB2086 acts on intracellular PAF receptors. 6. Thus, the intracellular release of PAF contributes to the circulatory and renal failure and induction of nitric oxide synthase elicited by LTA in anaesthetized rats. The difference between the two structurally different PAF antagonists in our septic shock models using either LTA or lipopolysaccharide (LPS), shows the importance of models for Gram-positive sepsis in the elucidation of the pathophysiology of septic shock and for the evaluation of potential drugs.

Acute Kidney Injury↗
PubMed1995

The contribution of tumour necrosis factor-alpha and endothelin-1 to the increase of coronary resistance in hearts from rats treated with endotoxin.

1. Inflammatory disease states predispose to myocardial infarction. Here we have investigated the effects of a systemic inflammatory response syndrome, i.e. lipopolysaccharide (LPS)-induced circulatory shock in rats, on coronary vascular tone. 2. Anaesthetized rats were given LPS (10 mg kg-1, i.v.) and the hearts excised 180 min later for isolated perfusion at constant flow by the Langendorff technique. Once the ex vivo perfusion was started, the perfusion pressure strongly increased in these hearts compared to hearts from control rats (130 +/- 3 vs. 49 +/- 3 mmHg after 10 min). This increase in coronary resistance was not associated with a reduction in endothelial cell function, for the vasodilator responses to bradykinin were unchanged. 3. When hearts were removed 30 min after the injection of LPS, the LPS-induced rise in perfusion pressure was delayed. No changes in perfusion pressure were seen when the hearts were removed 15 min after the injection of LPS. Pre-treatment with cycloheximide or an anti-tumour necrosis factor-alpha (TNF-alpha) antibody or continuous infusion in vivo and in vitro of the endothelin ETA receptor selective antagonist FR 139317, greatly decreased the increase in coronary vascular resistance induced by LPS. 4. These data suggest that TNF-alpha may induce the release of endothelin-1 (ET-1) and that this mediates at least part of the coronary vasoconstriction. This hypothesis is supported by the demonstration that LPS administration increased the circulating levels of both TNF-alpha and ET-1. 5. We conclude, therefore, that in inflammatory disease states, such as LPS-induced septic shock, there is the sequential release of TNF-alpha and endothelin-1 which leads to an increase in coronary vascular tone and so a predisposition to myocardial ischaemia. Inactivation of TNF-alpha by an antibody as well as ETA receptor blockade by a selective antagonist may effectively interfere with this pathway.

Animals↗
PubMed1995

The multiple organ dysfunction syndrome caused by endotoxin in the rat: attenuation of liver dysfunction by inhibitors of nitric oxide synthase.

1. We have investigated whether (i) endotoxaemia caused by E. coli lipopolysaccharide in the anaesthetized rat causes a multiple organ dysfunction syndrome (MODS; e.g. circulatory failure, renal failure, liver failure), and (ii) an enhanced formation of nitric oxide (NO) due to induction of inducible NO synthase (iNOS) contributes to the MODS. In addition, this study elucidates the beneficial and adverse effects of aminoethyl-isothiourea (AE-ITU), a relatively selective inhibitor of iNOS activity, and NG-methyl-L-arginine (L-NMMA), a non-selective inhibitor of NOS activity on the MODS caused by endotoxaemia. 2. In the anaesthetized rat, LPS caused a fall in mean arterial blood pressure (MAP) from 117 +/- 3 mmHg (time 0) to 97 +/- 4 mmHg at 2 h (P < 0.05, n = 15) and 84 +/- 4 mmHg at 6 h (P < 0.05, n = 15). The pressor effect of noradrenaline (NA, 1 micrograms kg-1, i.v.) was also significantly reduced at 1 to 6 h after LPS (vascular hyporeactivity). Treatment of LPS-rats with AE-ITU (1 mg kg-1, i.v. plus 1 mg kg-1 h-1 starting at 2 h after LPS) caused only a transient rise in MAP, but significantly attenuated the delayed vascular hyporeactivity seen in LPS-rats. Infusion of L-NMMA (3 mg kg-1, i.v. plus 3 mg kg-1 h-1) caused a rapid and sustained rise in MAP and attenuated the delayed vascular hyporeactivity to NA. Neither AE-ITU nor L-NMMA had any effect on either MAP or the pressor effect elicited by NA in rats infused with saline rather than LPS. 3. Endotoxaemia for 6 h was associated with a significant rise in the serum levels of aspartate or alanine aminotransferase (i.e. GOT or GPT), gamma-glutamyl-transferase (gamma GT), and bilirubin, and hence, liver dysfunction. Treatment of LPS-rats with AE-ITU significantly attenuated this liver dysfunction (rise in GOT, GPT, gamma GT and bilirubin) (P < 0.05, n = 10). In contrast, L-NMMA reduced the increase in the serum levels of gamma GT and bilirubin, but not in GOT and GPT (n = 5). Injection of LPS also caused a time-dependent, but rapid (almost maximal at 2 h), increase in the serum levels of urea and creatinine, and hence, renal dysfunction. This renal dysfunction was not affected by either AE-ITU (n = 10) or L-NMMA (n = 5). In rats infused with saline rather than LPS, neither AE-ITU (n = 4) nor L-NMMA (n = 4) had any significant effect on the serum levels of GOT, GPT, gamma GT, bilirubin, creatinine or urea. 4. Endotoxaemia for 6 h resulted in a 4.5 fold rise in the serum levels of nitrite (9.13 +/- 0.77 microM, P < 0.01, n = 15), which was significantly reduced by treatment with AE-ITU (6.32 +/- 0.48 microM, P < 0.05, n = 10) or L-NMMA (5.10 +/- 0.40 microM, P < 0.05, n = 5). In addition, endotoxaemia for 6 h was also associated with a significant increase in iNOS activity in lung and liver homogenates, which was significantly reduced in lung or liver homogenates obtained from LPS-rats treated with either AE-ITU or L-NMMA. 5. Thus, AE-ITU or L-NMMA (i) inhibits iNOS activity in LPS-rats without causing a significant increase in MAP in rats infused with saline and, hence inhibition of endothelial NOS activity, and (ii) attenuates the delayed circulatory failure as well as the liver dysfunction caused by endotoxaemia in the rat. Thus, an enhanced formation of NO may contribute to the development of liver failure in endotoxic shock.

Animals↗