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J R Vane

Publications and source records attributed to J R Vane.

At least 91 records · Page 5Linked to original sources
PubMed1994

The mechanism of the inhibitory effect of polyamines on the induction of nitric oxide synthase: role of aldehyde metabolites.

1. We have recently found that in the presence, but not in the absence, of foetal calf serum, spermine inhibits the production of nitric oxide (NO) in cultured J774.2 macrophages stimulated with bacterial endotoxin (lipopolysaccharide; LPS) or with gamma-interferon (IFN), showing that polyamines may act as suppressants of NO-mediated immune functions. Here, we have studied the mechanisms and the specificity of this inhibitory action. 2. Other polyamines, as well as spermine, inhibit the formation of NO in cultured J774.2 macrophages, with the order of potency being spermine > spermidine >> putrescine = cadaverine. This inhibition of NO formation is not due to any cytotoxic effect of these agents for they neither reduced mitochondrial respiration nor increased the release of lactate dehydrogenase into the supernatant. 3. Spermine is not a direct inhibitor of the activity of iNOS in induced J774.2 cells as measured by its lack of effect on the conversion of L-arginine to L-citrulline in homogenates. Neither spermine, nor its metabolites, interfere with the production of nitrite from NO or act as scavengers of NO. Thus, spermine is an inhibitor of the induction of iNOS. 4. Spermine inhibits nitrite formation in the presence of foetal, newborn or adult bovine serum, but not rat or human serum. 5. The effect of sper mine on nitrite production can be prevented by isoniazid, hydrazine or hydroxylamine, inhibitors of spermine oxidase, as well as by phenylhydrazine, an aldehyde inhibitor. We have, therefore, tested the effects of spermine dialdehyde or malon dialdehyde on the induction of iNOS. Spermine dialdehyde (SDA, 10(-5) M) inhibits nitrite formation by IFN-activated J774.2 cells in the absence of serum when given as a pretreatment but not when given 6 h after stimulation. In contrast, malon dialdehyde was ineffective. Thus, aldehyde metabolites of spermine, such as SDA, account for the inhibitory effect of polyamines on the induction of NOS in vitro. 6. The inhibitory effect of polyamines on iNOS induction appears to be fairly specific to iNOS, for spermine does not inhibit LPS-induced production of prostaglandin F2 alpha or tumour necrosis factor.

Aldehydes↗
PubMed1994

Induction of cyclo-oxygenase-2 by cytokines in human pulmonary epithelial cells: regulation by dexamethasone.

1. Cyclo-oxygenase metabolizes arachidonic acid to prostaglandin H2 (PGH2) and exists in at least two isoforms. Cyclo-oxygenase-1 (COX-1) is expressed constitutively whereas COX-2 is induced by lipopolysaccharide (LPS) and some cytokines in vitro and at the site of inflammation in vivo. Epithelial cells may be an important source of prostaglandins in the airways and we have, therefore, investigated the expression of COX-1 or COX-2 isoforms in primary cultures of human airway epithelial cells or in a human pulmonary epithelial cell line (A549). 2. COX-1 or COX-2 protein was measured by western blot analysis using specific antibodies to COX-2 and selective antibodies to COX-1. The activity of COX was assessed by the conversion of either endogenous or exogenous arachidonic acid to four metabolites, PGE2, PGF2 alpha, thromboxane B2 or 6-oxo PGF1 alpha measured by radioimmunoassay. Thus, COX-1 or COX-2 activity was measured under two conditions; initially the accumulation of the COX metabolites formed from endogenous arachidonic acid was measured after 24 h. In other experiments designed to measure COX activity directly, cells were treated with cytokines for 12h before fresh culture medium was added containing exogenous arachidonic acid (30 microM) for 15 min after which COX metabolites were measured. 3. Untreated primary cells or A549 cells contained low amounts of COX-1 or COX-2 protein. Bacterial LPS (1 micro g ml-1 for 24 h) induced COX-2 protein in the primary cells, a process which was enhanced by interferon-gamma, with no further increase in the presence of a mixture of cytokines (interleukin-1 beta, tumour necrosis factor-alpha and interferon-gamma, 10 ng ml-1 for all). In contrast, A549 cells contained only low levels of COX-2 protein after exposure to LPS or LPS plus interferon-y, but contained large amounts of COX-2 protein after exposure to the mixture of cytokines.4. Untreated human pulmonary primary cells or A549 cells released low levels of all COX metabolites measured over a 24 h incubation period. This release was enhanced by treatment of either cell type with the mixture of cytokines (interleukin-1 beta , tumour necrosis factors- and interferon-gamma, 10 ng ml-1 for all).PGE2 was the principal COX metabolite released by cytokine-activated epithelial cells. The release of PGE2 induced by cytokines occurred after a lag period of more than 6 h.5. The glucocorticosteroid, dexamethasone (1 micro M; 30 min prior to cytokines) completely suppressed the cytokine-induced expression of COX-2 protein and activity in both primary cells and A549 cells.6. In experiments where COX-2 activity was supported by endogenous stores of arachidonic acid,treatment of A549 cells with interleukin-l beta but not tumour necrosis factor a or interferon-gamma alone caused a similar release of PGE 2 to that seen when the cytokines were given in combination. However, both interleukin-l beta and necrosis factor- alone produced similar increases in COX-2 activity (measured in the presence of exogenous arachidonic acid) as seen when the mixture of interleukin-l beta, tumour necrosis factor- alpha and interferon-gamma were used to stimulate the cells.7. These findings show that COX-2 expression correlates with the exaggerated release of prostaglandins from cytokine-activated human pulmonary epithelial cells and that the induction of the enzyme is suppressed by a glucocorticosteroid. These findings may be relevant to inflammatory diseases of the lung, such as asthma.

Arachidonic Acid↗
PubMed1994

Nitric oxide, and not vasoactive intestinal peptide, as the main neurotransmitter of vagally induced relaxation of the guinea pig stomach.

1. Nitric oxide synthase (NOS) was localized in the guinea pig stomach by immunocytochemistry. In vitro experiments were carried out on the isolated stomach of the guinea pig to study any possible links between nitric oxide (NO) and vasoactive intestinal peptide (VIP) in mediating relaxations induced by vagal stimulation. 2. NOS was localized to nerve cell bodies and nerve fibre varicosities of the myenteric plexus in wholemounts of the longitudinal muscle-myenteric plexus of the stomach fundus. The NOS-positive cells had a Dogiel type I morphology characteristic of motor neurones. 3. The cross-sections of the stomach wall showed NOS-positive neurones mainly in the myenteric plexus ganglia and NOS-positive nerve fibre varicosities in the circular muscle layer. 4. Relaxations induced by vagal stimulation were almost completely prevented by L-NAME with an IC50 value of 5.5 x 10(-6) M. This inhibition was reversed by L-arginine (2 mM). 5. VIP (100 nM) induced reproducible relaxations of the stomach. These were unaffected by tetrodotoxin (2 microM) or N omega-nitro-L-arginine methyl ester (L-NAME, 100 microM). 6. Desensitization to the relaxant effect of VIP partially reduced relaxations induced by vagal stimulation, glyceryl trinitrate or sodium nitroprusside but not noradrenaline. 7. These results show that NO has a neuronal origin in the guinea pig stomach, and support NO, and not VIP, as the major neurotransmitter of vagally induced gastric relaxation in the guinea pig.

Amino Acid Oxidoreductases↗
PubMed1994

Involvement of tyrosine kinase in the induction of cyclo-oxygenase and nitric oxide synthase by endotoxin in cultured cells.

1. Cyclo-oxygenase (COX) and nitric oxide synthase (NOS) are two enzymes which have distinct cytokine-inducible isoforms (COX-2 and iNOS). Many cytokine receptors have an intracellular tyrosine kinase domain. Here we have used the tyrosine kinase inhibitors, erbstatin and genistein, to investigate the potential role of tyrosine kinase activation in the induction on COX-2 and iNOS caused by endotoxin (lipopolysaccharide; LPS) in bovine aortic endothelial cells (BAEC) and J774.2 macrophages. 2. The main COX metabolites, 6-oxo-prostaglandin F1 alpha (6-oxo-PGF1 alpha) (for BAEC) and PGF2 alpha (for 774.2 macrophages) were measured by radioimmunossay: (i) accumulation of COX metabolites from endogenous arachidonic acid was measured at 24 h after addition of LPS (1 microgram ml-1); (ii) in experiments designed to measure 'COX activity', COX metabolites generated by BAEC or J774.2 macrophages activated with LPS were assayed (at 12 h after LPS administration) after incubation of the washed cells with exogenous arachidonic acid (30 microM for 15 min). Western blot analysis with a specific antibody to COX-2 was used to determine the expression of COX-2 protein caused by LPS in cell extracts. Accumulation of nitrite (measured by the Griess reaction) was used as an indicator of NO formation and, hence, iNOS activity. 3. Erbstatin (0.05 to 5 micrograms ml-1) or genistein (0.5 to 50 micrograms ml-1) caused a dose-dependent inhibition of the accumulation of COX metabolites in the supernatant of BAEC or J774.2 macrophages activated with LPS. Erbstatin or genistein also caused a dose-dependent inhibition of 'COX activity' in both cell types. Western blot analysis showed that erbstatin (5 ig ml1') or genistein (50gg ml-') inhibited the expression of COX-2 protein in BAEC and J774.2 macrophages activated with LPS (lLgml-' for 24 h).4. Erbstatin or genistein also caused a dose-dependent inhibition of nitrite accumulation in J774.2 macrophages activated with LPS (1 sg ml-' for 24 h). In contrast to J774.2 macrophages, BAECstimulated with LPS (1 pg ml-' for 24 h) did not produce detectable amounts (<1PiM) of nitrite.5. These results suggest that tyrosine phosphorylation is part of the signal transduction mechanism that mediates (i) the induction of COX-2 and iNOS elicited by LPS in J774.2 macrophages, and (ii) the induction of COX-2 by LPS in BAEC.

Amino Acid Oxidoreductases↗
PubMed1993

Endothelin ETA and ETB receptors mediate vasoconstriction and prostanoid release in the isolated kidney of the rat.

Endothelin-1 or sarafotoxin 6c (10(-12) to 10(-9) M) induced concentration-dependent increases in perfusion pressure in the isolated perfused kidney of the rat, and increased the release of 6-keto-prostaglandin F1 alpha, prostaglandin E2 and prostaglandin F2 alpha. The pressor effects of endothelin-1 were partially antagonised by BQ-123 (10(-6) M) and more strongly so by the endothelin ETA/B receptor non-selective antagonist PD 145065 (10(-6) M). PD 145065, but not BQ-123, also completely blocked the pressor effects of sarafotoxin 6c. The releases of prostanoids induced by endothelin-1 were greatly reduced by infusion of either BQ-123 or PD 145065, whereas PD 145065, but not BQ-123, strongly antagonised the releases induced by sarafotoxin 6c. These results indicate that the vasoconstrictions and the prostanoid releases induced by the endothelin/sarafotoxin peptides are mediated by the activation of both endothelin ETA and endothelin ETB receptors in the isolated perfused kidney of the rat.

6-Ketoprostaglandin F1 alpha↗
PubMed1993

Selectivity of nonsteroidal antiinflammatory drugs as inhibitors of constitutive and inducible cyclooxygenase.

Constitutive cyclooxygenase (COX-1; prostaglandin-endoperoxide synthase, EC 1.14.99.1) is present in cells under physiological conditions, whereas COX-2 is induced by some cytokines, mitogens, and endotoxin presumably in pathological conditions, such as inflammation. Therefore, we have assessed the relative inhibitory effects of some nonsteroidal antiinflammatory drugs on the activities of COX-1 (in bovine aortic endothelial cells) and COX-2 (in endotoxin-activated J774.2 macrophages) in intact cells, broken cells, and purified enzyme preparations (COX-1 in sheep seminal vesicles; COX-2 in sheep placenta). Similar potencies of aspirin, indomethacin, and ibuprofen against the broken cell and purified enzyme preparations indicated no influence of species. Aspirin, indomethacin, and ibuprofen were more potent inhibitors of COX-1 than COX-2 in all models used. The relative potencies of aspirin and indomethacin varied only slightly between models, although the IC50 values were different. Ibuprofen was more potent as an inhibitor of COX-2 in intact cells than in either broken cells or purified enzymes. Sodium salicylate was a weak inhibitor of both COX isoforms in intact cells and was inactive against COX in either broken cells or purified enzyme preparations. Diclofenac, BW 755C, acetaminophen, and naproxen were approximately equipotent inhibitors of COX-1 and COX-2 in intact cells. BF 389, an experimental drug currently being tested in humans, was the most potent and most selective inhibitor of COX-2 in intact cells. Thus, there are clear pharmacological differences between the two enzymes. The use of such models of COX-1 and COX-2 activity will lead to the identification of selective inhibitors of COX-2 with presumably less side effects than present therapies. Some inhibitors had higher activity in intact cells than against purified enzymes, suggesting that pure enzyme preparations may not be predictive of therapeutic action.

Animals↗
PubMed1993

Nitric oxide is a mediator of the decrease in cytochrome P450-dependent metabolism caused by immunostimulants.

Bacterial lipopolysaccharide (LPS) and a diverse array of other immunostimulants and cytokines suppress the metabolism of endogenous and exogenous substances by reducing activity of the hepatic cytochrome P450 mixed-function oxidase system. Although this effect of immunostimulants was first described almost 40 yr ago, the mechanism is obscure. Immunostimulants are now known to cause NO overproduction by cells via induction of nitric oxide synthase. We have investigated whether NO overproduction is involved in suppressing hepatic metabolism by LPS. In vitro treatment of hepatic microsomes with NO, produced by chemical decomposition of 3-morpholinosydnonimine or by nitric oxide synthase, substantially suppressed cytochrome P450-dependent oxygenation reactions. This effect of NO was seen with hepatic microsomes prepared from two species (rat and chicken) and after exposure to chemicals that induce distinct molecular isoforms of cytochromes P450 (beta-naphthoflavone, 3-methylcholanthrene, and phenobarbital). Spectral studies indicate that NO reacts in vitro with both Fe(2+)- and Fe(3+)-hemes in microsomal cytochromes P450. In vivo, LPS diminished the phenobarbital-induced dealkylation of 7-pentoxyresorufin by rat liver microsomes and reduced the apparent P450 content as measured by CO binding. These LPS effects were associated with induction of NO synthesis; LPS-induced NO synthesis showed a strong positive correlation with the severity of cytochrome P450 inhibition. The decrease in both hepatic microsomal P450 activity and CO binding caused by LPS was largely prevented by the selective NO synthase inhibitor N omega-nitro-L-arginine methyl ester. Our findings implicate NO over-production as a major factor mediating the suppression of hepatic metabolism by immunostimulants such as LPS.

Adjuvants, Immunologic↗
PubMed1993

Interleukin-1 contributes to the induction of nitric oxide synthase by endotoxin in vivo.

We investigated the role of interleukin-1 in the induction of a Ca(2+)-independent nitric oxide (NO) synthase by bacterial endotoxin in vivo. In anaesthetized rats, pretreatment with interleukin-1 receptor antagonist (interleukin-1ra; 16 mg kg-1 i.v., followed by an infusion of 2.4 mg kg-1 h-1) ameliorated the delayed hypotension and tachycardia in response to endotoxin (2 mg kg-1 i.v.). Endotoxaemia for 3 h induced a Ca(2+)-independent NO synthase activity in the lung and reduced the contractions to noradrenaline in the thoracic aorta ex vivo. Treatment with interleukin-1ra attenuated both the induction of NO synthase in the lung (by 46 +/- 5%) and the endotoxin-induced hyporeactivity to noradrenaline in the aorta. Thus, endogenous interleukin-1 contributes to the induction of NO synthase in response to endotoxin in vivo.

Amino Acid Oxidoreductases↗
PubMed1993

Dihydropyridine antagonists and agonists of calcium channels inhibit the induction of nitric oxide synthase by endotoxin in cultured macrophages.

Here we investigate the effects of the dihydropyridine-type antagonists of calcium channels nitrendipine, nimodipine, nisoldipine and the calcium channel agonist BAY K 8644 on the induction of nitric oxide synthase (NOS) by bacterial endotoxin (lipopolysaccharide; LPS) in J774.2 macrophages cultured in vitro. Pretreatment of J774.2 cells with these dihydropyridines (10(-8) -3 x 10(-6) M for 30 min) dose-dependently inhibited the LPS-stimulated (1 microgram/ml, 24 h) nitrite formation. For instance, at 10(-6) M, the inhibition was 59 +/- 3% for nitrendipine; 47 +/- 5% for nimodipine and 42 +/- 3% for nisoldipine (n = 9; p < 0.05). BAY K 8644 caused a moderate, but significant inhibition of nitrite accumulation (by 16 +/- 3% at 10(-7) M, n = 9; p < 0.05). The inhibition of LPS-stimulated nitrite accumulation produced by nitrendipine, nimodipine, and BAY K 8644 was significantly smaller when they were applied 2 or 4 h after LPS, indicating that these agents inhibit the induction, but not the activity of the induced NOS. At concentrations which caused a significant inhibition of the LPS-stimulated nitrite accumulation, the dihydropyridine calcium channel modulators did not inhibit mitochondrial respiration. Thus, dihydropyridine calcium channel modulators (antagonists and an agonist) inhibit the induction of the calcium-independent isoform of NOS produced by LPS in J774.2 macrophages. This effect is not related to the modulation of intracellular calcium levels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1993

Inhibition of the production of nitric oxide and vasodilator prostaglandins attenuates the cardiovascular response to bacterial endotoxin in adrenalectomized rats.

Bacterial lipopolysaccharide (LPS) is the toxic moiety of the gram-negative bacterial outer membrane which is responsible for many of the pathophysiological events that occur during endotoxic shock. Here we investigate the hypothesis that endogenous glucocorticoids modulate the formation of nitric oxide (NO) and of vasodilator cyclooxygenase metabolites in response to LPS. Intravenous administration of a small dose of Escherichia coli LPS (0.1 mg kg-1) to normal Wistar rats caused a moderate fall in blood pressure, and 120 min of endotoxaemia was not associated with an attenuation of the rise in blood pressure elicited by intravenous injection of noradrenaline (NA; vascular hyporeactivity). When adrenalectomized (ADX) rats, which lack endogenous glucocorticoids, were subjected to the same dose of LPS, they developed a much more severe form of circulatory shock, which was characterized by a profound fall in blood pressure and a vascular hyporeactivity to NA. Both hypotension and vascular hyporeactivity were prevented by pre-treatment with dexamethasone. Inhibition of NO biosynthesis with NG-methyl-L-arginine significantly attenuated the hypotension and the vascular hyporeactivity to NA caused by LPS in ADX rats. Similarly, the cyclooxygenase inhibitor indomethacin significantly attenuated the circulatory failure elicited by LPS in the ADX rats. Interestingly, 120 min of endotoxaemia resulted in a de novo biosynthesis of an induced isoform of NO synthase in the lungs of ADX, but not normal Wistar, rats. This induction of NO synthase was prevented by dexamethasone pre-treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenalectomy↗
PubMed1993

Modification of energy metabolism and radiation response of a murine tumour by changes in nitric oxide availability.

The nitric oxide donor, SIN-1 and nitric oxide synthase inhibitor, nitro-L-arginine were examined for their effects on energy metabolism using 31P magnetic resonance spectroscopy, and on radiation sensitivity in the transplantable murine tumour, SCCVII/Ha. SIN-1 at 2 mg/kg i.v. reduced Pi/total by 40-50% within 10 min and increased X-ray sensitivity 3 fold, consistent with increased tumour oxygenation. Nitro-L-arginine at 10 mg/kg i.v. increased Pi/total by 250% at 45 min and was maintained for at least 2 hr. Nitro-L-arginine also increased radiation resistance 3-5 fold, consistent with the induction of tumour hypoxia. The results indicate that tumour energy metabolism may be altered through drug induced modification of nitric oxide availability, and that these changes are sufficient to modify tumour sensitivity to X-rays.

Animals↗
PubMed1993

Vascular hyporeactivity to vasoconstrictor agents and hemodynamic decompensation in hemorrhagic shock is mediated by nitric oxide.

This study investigates the role of nitric oxide (NO) and the induction of a calcium-independent NO synthase (NOS) in development of vascular hyporeactivity to norepinephrine (NE) and vascular decompensation associated with hemorrhagic shock (HS) in the anesthetized rat. HS for 120 min caused a time-dependent reduction of the pressor responses to NE. This hyporeactivity is mediated by an enhanced release of NO by the constitutive NOS, for it was reversed by NG-nitro-L-arginine methyl ester (NO2Arg), an inhibitor of both constitutive and inducible NOS, but it was not prevented by dexamethasone, an inhibitor of NOS induction. Vascular decompensation following prolonged periods of HS was characterized by a failure of control animals to maintain arterial blood pressures despite reinfusion of blood. This progressive decrease in blood pressure is mediated by enhanced formation of NO by the inducible NOS, for it was prevented by NO2Arg or dexamethasone. A strong increase in calcium-independent (inducible) NOS activity was observed in several organs after 150 and 330 min of HS, being most pronounced in lung, liver, and spleen. HS for 330, but not 150, min also caused hyporeactivity of rat aortic rings to vasoconstrictors, which was associated with induction of calcium-independent NOS activity in this tissue. Aortic hyporeactivity was prevented by dexamethasone pretreatment in vivo and reversed by NO2Arg in vitro. HS was not associated with an increase in plasma endotoxin levels, showing that endotoxin does not account for induction of NOS in this model. Thus, excessive NO formation induces vascular hyporeactivity and decompensation in HS, indicating that NOS inhibitors, particularly of the inducible NOS, may improve the therapeutic outcome of patients suffering from HS.

Amino Acid Oxidoreductases↗
PubMed1993

Calcium ionophores inhibit the release of endothelin-1 from endothelial cells.

We have determined the effect on endothelin-1 (ET-1) release of stimulating cultured endothelial cells for 24 h with Ca2+ ionophores. The results obtained using cultured bovine aortic endothelial cells (BAECs) were compared with those using the human endothelial cell line (EA.hy 926). ET-1 was measured using a radioimmunoassay specific for the C-terminal ET[16-21] sequence. Ionomycin (1 microM) lessened ET-1 accumulation over 24 h in the conditioned medium from BAECs (-37%) and EA.hy 926 (-25%). A23187 was a more potent inhibitor of ET-1 release. With BAECs, ET-1 release was inhibited by 28% and 77% at 0.1 microM and 1 microM A23187, respectively. The corresponding decreases for EA.hy 926 were 20% and 98%. A23187 at 0.2 microM lowered ET-1 concentrations in the medium after 24 h by 44% and 24% for BAECs and EA.hy 926, respectively. Neither NG-monomethyl-L-arginine (0.2 mM) nor indomethacin (10 microM), either alone or in combination, affected the inhibition by A23187. Thus, this inhibitory effect is not dependent on the stimulation of nitric oxide or prostacyclin synthesis. Additional factors in endothelial cells, activated by elevated Ca2+ levels, may be important in exerting an inhibitory influence on ET-1 formation.

Animals↗
PubMed1993

Evidence for vesicles that transport endothelin-1 in bovine aortic endothelial cells.

To facilitate studies of the intracellular processing of endothelin-1 (ET-1) in endothelial cells we developed an enrichment procedure for the isolation of transport or secretory vesicles containing ET-1. Cultured bovine aortic endothelial cells (BAECs) were disrupted using a tight-fitting Dounce homogenizer, and subcellular fractions were isolated by sucrose-density-gradient ultracentrifugation. ET-1 immunoreactivity (ET-IR) in the fractions was measured with a specific ET[16-21] radio-immunoassay (RIA). The major peak of ET-IR was consistently localized at the 1.0/1.2 M sucrose interface (10.96 fmol/10(6) cells) in each preparation of subcellular fractions. The mean level of ET-IR at this interface, expressed as a percentage of the total, was 47.9 +/- 3.5% (n = 6). This finding provides strong evidence for the existence of a vesicle that transports ET-1 within BAECs, which may be an important site for endogenous ET-1 processing. A number of studies have reported that the final processing step in ET-1 biosynthesis by the hypothetical endothelin-converting enzyme (ECE) is inhibited by phosphoramidon (PHOS). Therefore, levels of PHOS-sensitive ECE activity in each fraction were assessed in parallel with ET-IR levels. The ECE activity associated with the peak of ET-IR (2.67 pmol ET-1/h/10(6) cells) was insensitive to 100 microM PHOS. However, the peak of ECE-like activity (10.4 pmol ET-1/h/10(6) cells) localized in the 0.8 M sucrose band was inhibited by 79% in the presence of 100 microM PHOS.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗