Jane A. Mitchell

Nitric oxide-mediated hyporeactivity to noradrenaline precedes the induction of nitric oxide synthase in endotoxin shock

1 The role of an enhanced formation of nitric oxide (NO) and the relative importance of the constitutive and inducible NO synthase (NOS) for the development of immediate (within 60 min) and delayed (at 180 min) vascular hyporeactivity to noradrenaline was investigated in a model of circulatory shock induced by endotoxin (lipopolysaccharide; LPS) in the rat. 2 Male Wistar rats were anaesthetized and instrumented for the measurement of mean arterial blood pressure (MAP) and heart rate. In addition, the calcium‐dependent and calcium‐independent NOS activity was measured ex vivo by the conversion of [3H]‐arginine to [3H]‐citrulline in homogenates from several organs obtained from vehicle‐ and LPS‐treated rats. 3 E. coli LPS (10 mg kg−1, i.v. bolus) caused a rapid (within 5 min) and sustained fall in MAP. At 30 and 60 min after LPS, pressor responses to noradrenaline (0.3, 1 or 3 μg kg−1, i.v.) were significantly reduced. The pressor responses were restored by NG‐nitro‐l‐arginine methyl ester (l‐NAME, 1 mg kg−1, i.v. at 60 min), a potent inhibitor of NO synthesis. In contrast, l‐NAME did not potentiate the noradrenaline‐induced pressor responses in control animals. 4 Dexamethasone (3 mg kg−1, i.v., 60 min prior to LPS), a potent inhibitor of the induction of NOS, did not alter initial MAP or pressor responses to noradrenaline in control rats, but significantly attenuated the LPS‐induced fall in MAP at 15 to 60 min after LPS. Dexamethasone did not influence the development of the LPS‐induced immediate (within 60 min) hyporeactivity to noradrenaline. However, dexamethasone pretreatment prevented the hypotension and vascular hyporeactivity at 180 min. 5 At 60 min after LPS a moderate increase in the activity of a calcium‐independent (inducible) NOS activity was detected in the aorta, but not in any of the other tissues studied. However, at 180 min after LPS, a significant NOS induction was observed in the lung, liver, spleen, mesentery, heart and aorta. This NOS induction was substantially prevented by pretreatment with dexamethasone. 6 These results suggest that the immediate hypotension and vascular hyporeactivity to noradrenaline in endotoxin shock is caused by an enhanced formation of NO due to activation of the constitutive enzyme. The delayed hypotension and vascular hyporeactivity, however, is due to enhanced NO formation by the LPS‐induced enzyme.

Co-induction of nitric oxide synthase and cyclo-oxygenase: interactions between nitric oxide and prostanoids

1 Lipopolysaccharide (LPS) co‐induces nitric oxide synthase (iNOS) and cyclo‐oxygenase (COX‐2) in J774.2 macrophages. Here we have used LPS‐activated J774.2 macrophages to investigate the effects of exogenous or endogenous nitric oxide (NO) on COX‐2 in both intact and broken cell preparations. NOS activity was assessed by measuring the accumulation of nitrite using the Griess reaction. COX‐2 activity was assessed by measuring the formation of 6‐keto‐prostaglandin F1α (6‐keto‐PGF1α) by radioimmunoassay. Western blot analysis was used to determine the expression of COX‐2 protein. We have also investigated whether endogenous NO regulates the activity and/or expression of COX in vivo by measuring NOS and COX activity in the lung and kidney, as well as release of prostanoids from the perfused lung of normal and LPS‐treated rats.

Phosphorylation by calcium calmodulin-dependent protein kinase II and protein kinase C modulates the activity of nitric oxide synthase

Nitric oxide synthase purified from rat brain, which is Ca2+ and calmodulin dependent, was phosphorylated by calcium calmodulin-dependent protein kinase II as well as protein kinase C. Phosphorylation by calcium calmodulin-dependent protein kinase II resulted in a marked decrease in enzyme activity (33% of control) without changing the co-factor requirements, whereas a moderate increase in enzyme activity (140% of control) was observed after phosphorylation by protein kinase C. These findings indicate that brain nitric oxide synthase activity may be regulated not only by Ca2+/calmodulin and several co-factors, but also by phosphorylation.

Cyclo-oxygenase and nitric oxide synthase isoforms in rat carrageenin-induced pleurisy.

1 The profiles of cyclo‐oxygenase (COX) and nitric oxide synthase (NOS) isoforms were determined in the rat carrageenin‐induced pleurisy model of acute inflammation. 2 The enzymes were assessed in peripheral blood leucocyte (PBL) cell pellets taken from untreated animals and at 2, 6 and 24 h after injection of the irritant in pleural exudate cell pellets and lung homogenates. 3 COX activity was assessed by the generation of prostacyclin (PGI2, measured as the stable metabolite, 6‐keto prostaglandin F1α) and prostaglandin E2 (PGE2). Western blot analysis and immunohistochemistry were also carried out. 4 NOS activity was based on the conversion of [3H]‐L‐arginine to [3H]‐L‐citrulline in the presence (total NOS activity) or absence of Ca2+ (inducible NOS; iNOS). 5 Peripheral blood leucocyte samples contained low levels of COX activity. In pleural exudate cell pellets, COX activity peaked at 2 to 6 h after injection of the carrageenin. At 24 h, COX activity was significantly reduced. 6 Western blot analysis demonstrated that the inducible isoform of COX (COX‐2), was the predominant enzyme at all time points. Low levels of COX‐2 were seen in PBLs. In pleural exudate cell pellets maximal COX‐2 protein levels were seen at 2h. 7 Immunohistochemistry confirmed the findings of Western blot studies. Approximately 10% of polymorphonuclear neutrophils (PMNs) in PBLs from untreated animals were immunopositive for COX‐2. In cell pellet smears from carrageenin‐induced pleurisy taken 2 h after injection of the irritant, PMNs were also the major source of COX‐2 immunoreactivity. A small proportion of macrophages and mesothelial cells were also immunolabelled for COX‐2. 8 Low levels of NOS activity were seen in PBLs. In pleural exudates NOS activity was maximum at 6h and greatly reduced by 24 h. This activity was solely attributable to iNOS. 9 The present results illustrated a similar profile of COX and NOS activity in the carrageenin‐induced pleurisy model of acute inflammation. It was demonstrated that COX‐2 and iNOS were the predominant isoforms of their respective enzymes.

Endothelial cells metabolize NG-monomethyl-L-arginine to L-citrulline and subsequently to L-arginine

NG-monomethyl-L-arginine (MeArg) inhibits the release of endothelium-derived relaxing factor (EDRF) from endothelial cells (EC) and the formation of nitric oxide (NO) from L-arginine (Arg) in EC and activated macrophages. We have compared the inhibitory potency of MeArg to that of N omega-nitro-L-arginine (NO2Arg), a more potent inhibitor of EDRF synthesis in vitro. NO2Arg (100 microM) was significantly more potent than MeArg in inhibiting the endothelium-dependent relaxation of rabbit aorta induced by acetylcholine. MeArg and NO2Arg (10 and 30 microM) also inhibited the release of EDRF from bovine aortic cultured EC. In the anaesthetized rat in vivo, the pressor effect of NO2Arg (3 and 10 mg kg-1) was significantly larger and longer lasting than that of MeArg. These differences in potency could be due to the extensive metabolism of MeArg but not NO2Arg to L-citrulline (Cit) and subsequently to Arg by EC. The enzyme responsible for the conversion of MeArg to Cit had the characteristics of a novel deiminase, NG,NG-dimethylarginine dimethylaminohydrolase, recently isolated from rat kidney.

Platelet-activating factor contributes to the induction of nitric oxide synthase by bacterial lipopolysaccharide.

This study investigates the role of endogenous platelet-activating factor (PAF) in the production of nitric oxide (NO) by constitutive and inducible isoforms of NO synthase (NOS) in endotoxin shock. In anesthetized rats, 3 hours of endotoxemia resulted in a fall in mean arterial blood pressure (MAP) from 127 +/- 5 (control) to 61 +/- 7 mm Hg and a reduction of the pressor responses to norepinephrine (NE, 1 microgram.kg-1) from 33 +/- 3 (control) to 17 +/- 2 mm Hg. Endotoxemia for 3 hours also resulted in a significant reduction in the contractile effects of NE (10(-8) to 10(-6) mol/L) in thoracic aortas ex vivo. This hyporeactivity to NE was due to an enhanced formation of NO, for it was restored by the NOS inhibitor NG-nitro-L-arginine methyl ester. Animals pretreated with the PAF receptor antagonist WEB 2086 maintained higher MAP (MAP at 180 minutes, 98 +/- 6 mm Hg) and exhibited more pronounced pressor responses to NE at 180 minutes after LPS injection. Moreover, WEB 2086 attenuated by 58% the lipopolysaccharide (LPS)-induced hyporeactivity of the rat aortic rings ex vivo. At 3 hours after LPS injection, calcium-independent NOS activity was induced in the lung. The activity of inducible NOS was significantly lower (by 31%) in lungs of rats pretreated with WEB 2086. The hypothesis that WEB 2086 attenuates the induction of NOS in vivo was substantiated in vitro by the finding that pretreatment with WEB 2086 for 30 minutes inhibited the LPS-stimulated NO production in cultured murine macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Characterization of nitric oxide synthases in non-adrenergic non-cholinergic nerve containing tissue from the rat anococcygeus muscle.

Tissue homogenates prepared from rat anococcygeus muscle converted l‐arginine to l‐citrulline indicating the presence of nitric oxide (NO) synthase. NO synthase activity was also found in crude and partially‐purified soluble and particulate fractions prepared from the homogenates. Both soluble and particulate NO synthase were dependent on NADPH, 5,6,7,8‐tetrahydrobiopterin and calcium, and inhibited by NG‐nitro‐l‐arginine. Tissue homogenates or crude cytosolic and membrane fractions from rat vas deferens, which does not contain NO releasing non‐adrenergic non‐cholinergic neurones, had no NO synthase activity.

Effects of Cyclic GMP on Smooth Muscle Relaxation

Cyclic GMP levels within smooth muscle are affected then by a number of different pathways. Physiologically NO and ANF are probably the two most important regulators for smooth muscle function, but a variety of other mediators and pharmacological agents may also influence this system. Because of the important role that cyclic GMP plays in the control of smooth muscle tone, which clearly includes vascular smooth muscle, it is now and will continue to be in the future an important physiological and biochemical target for research and a pharmacological target for therapeutic agents.

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 F1α (6‐oxo‐PGF1α) (for BAEC) and PGF2α (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 μg 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 μg for 15min). 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 μg ml−1) or genistein (0.5 to 50 μg 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 μg ml−1) or genistein (50μg ml−1) inhibited the expression of COX‐2 protein in BAEC and J774.2 macrophages activated with LPS (1 μg ml−1 for 24 h). 4 Erbstatin or genistein also caused a dose‐dependent inhibition of nitrite accumulation in J774.2 macrophages activated with LPS (1 μg ml−1 for 24 h). In contrast to J774.2 macrophages, BAEC stimulated with LPS (1 μg ml−1 for 24 h) did not produce detectable amounts (> 1μm) 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.