Annette Tomlinson

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.

Differential effects of inhibitors of cyclooxygenase (cyclooxygenase 1 and cyclooxygenase 2) in acute inflammation

The anti-inflammatory activity of drugs more selective for cyclooxgenase isoform inhibition (cyclooxygenase 1, cyclooxygenase 2), were compared in rat carrageenin-induced pleurisy. Suppression of inflammation by cyclooxygenase 2-selective inhibitors, NS-398 (N-[-2-cyclohexyloxy]-4-nitrophenyl methanesulphonamide) and nimesulide (4-nitro-2-phenoxy-methanesulfonanilide), and by piroxicam and aspirin, more selective for cyclooxygenase 1, was measured. Piroxicam and aspirin significantly inhibited inflammatory cell influx, exudate and prostaglandin E2 formation, 6 h after carrageenin injection. Cyclooxygenase 2 inhibitors had little effect on these parameters with NS-398 alone reducing prostaglandin E2 levels, but increasing levels of leukotriene B4. In contrast, at 3 h after carrageenin injection, cyclooxygenase 2 inhibitors significantly inhibited all inflammatory parameters however suppression with piroxicam and aspirin was greater, and more pronounced than at 6 h. NS-398 and nimesulide dosing did not reduce thromboxane B2 production from platelets isolated from rats with carrageenin-induced pleurisy, demonstrating that at the doses used, cyclooxygenase 2 inhibitors did not inhibit cyclooxygenase 1, as platelets contain only this isoform. Therefore, in the rat carrageenin-induced pleurisy, drugs more selective for the inhibition of cyclooxygenase 1 attenuate inflammation over a wider time frame than cyclooxygenase 2-selective drugs, suggesting a significant role for cyclooxygenase 1 in this model. Inhibition of cyclooxygenase 2 by NS-398 however, resulted in an increase in the potent chemoattractant leukotriene B4.

Induction of Cyclo-Oxygenase and Nitric Oxide Synthase in Inflammation

Publisher Summary This chapter focuses on the role of prostanoids and NO at each stage of the inflammatory response with particular emphasis given to the cellular source and the factors that may modulate the activity of their respective enzymes. Inhibition of prostanoid formation by use of nonsteroidal anti-inflammatory drugs (NSAIDs) ameliorates the classical signs of inflammation, indicating their pivotal role in the inflammatory response. Recently, a second isoform of cyclooxygenase (COX), the enzyme that liberates the prostanoids, has been identified. This discovery has given new impetus as to the role of COX isoforms in inflammation and has fueled the search for selective inhibitors free from side effects. Once liberated, arachidonic acid is converted to the biologically active PGs and thromboxanes (TXs), collectively termed prostanoids, by the enzyme COX, also known as prostaglandin H synthase or prostaglandin endoperoxidase synthase. COX has two functions: first, cyclo-oxygenase activity that catalyzes PGG 2 formation, and second, peroxidase activity that reduces the 15-hydroperoxyl group of PGG, to PGH 2 . The chapter keeps space for discussion on both COX-1 and COX-2. The biological activity of the elusive molecule, termed endothelium-derived relaxing factor (EDRF), involved in vascular relaxation, could be accounted for by nitric oxide (NO). It was found that NO, synthesized by endothelial cells (ECs), acted as an intercellular effector molecule, causing vasodilatation by activation of guanylate cyclase and the elevation of cGMP levels in vascular smooth muscle cells. Although NO is indubitably involved in inflammation its role cannot be defined as pro- or anti-inflammatory but rather depends on the prevailing circumstances. The products of the NOS and COX pathways as well as having potent effects on various cellular systems can also modulate the activity of their respective enzymes. Although the mutual effects between COX activity and action of NO effects are not clear cut as both stimulatory and inhibitory actions have been ascribed.

Colocalization of nitric oxide synthase and NADPH-diaphorase in rat adrenal gland

The distribution and colocalization of nitric oxide synthase (NOS) and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase was studied in the neuronal elements of the adrenal gland of the rat. Ganglion cells and many nerve fibres in the gland showed both NOS-immunoreactivity and NADPH-diaphorase staining. The adrenal cortical cells showed NADPH-diaphorase staining but were not immunoreactive for NOS. Positive labelling for both NADPH-diaphorase and NOS was found in bundles and in single fibres with varicosities, preferentially located around the noradrenaline (NA)-storing cells. Adrenaline (A)-storing cells and ganglion cells in the medulla, along with the cortical cells and blood vessels in the zona glomerulosa, received relatively fewer positive fibres.

Nitric Oxide Synthase Inhibitors Have Opposite Effects on Acute Inflammation Depending on Their Route of Administration

The bulk of published data has shown that NO is proinflammatory. However, there also exists the conflicting notion that NO may be protective during an inflammatory insult. In an attempt to resolve this issue, we have compared the effects on inflammation of a range of NO synthase (NOS) inhibitors given either directly to the site of the inflammatory lesion or systemically. It was found that in the carrageenin-induced pleurisy, a single intrapleural injection of the selective inducible NO inhibitors S-(2-aminoethyl) isothiourea (AE-ITU; 3 and 10 mg/kg) and N-(3-(aminomethyl)-benzyl) acetamidine (1400W; 10 mg/kg) or the selective endothelial cell NOS inhibitor l-N5(1-iminoethyl)-ornithine (10 mg/kg) not only exacerbated inflammation at the very early stages of the lesion (1–6 h), but also prevented inflammatory resolution. By contrast, administering NOS inhibitors systemically ameliorated the severity of inflammation throughout the reaction. To elucidate the mechanisms by which inhibition of NO synthesis locally worsened inflammation, we found an increase in histamine, cytokine-induced neutrophil chemoattractant, superoxide, and leukotriene B4 levels at the inflammatory site. In conclusion, this work shows that the local production of NO is protective by virtue of its ability to regulate the release of typical proinflammatory mediators and, importantly, that NOS inhibitors have differential anti-inflammatory effects depending on their route of administration.

Distribution and colocalization of nitric oxide synthase and NADPH-diaphorase in adrenal gland of developing, adult and aging Sprague-Dawley rats

The distribution and colocalization of nitric oxide synthase and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-diaphorase) was investigated in the adrenal gland of developing, adult and aging rats with the use of immunohistochemical and histochemical techniques. Nitric oxide synthase-immunoreactive neurons within the adrenal gland were found from the 20th day of gestation onwards. During early development the neurons were found as small clusters of smaller-size cells compared to those observed in the adult gland. Their number reached that of adult level by the 4th day after birth, and in the glands from aging rats a 28.6% increase was observed. Whilst no immunofluorescence was seen in chromaffin cells during early development, some cells from glands of aging rats showed nitric oxide synthase-immunoreactivity with varying intensity. The immunoreactive neurons from postnatal rat adrenals were also positive for NADPH-diaphorase, whilst those in prenatal rats were negative or lightly stained. Nitric oxide synthase-immunoreactive nerve fibres were present in all adrenal glands examined from the 16th day of gestation onwards. A considerable degree of variation in the distribution of immunoreactive fibres both in medulla and outer region of cortex at the different age groups was observed and described. Most, but not all, nitric oxide synthase-immunoreactive nerve fibres also showed NADPH-diaphorase staining.

Synergy between cyclo-oxygenase-2 induction and arachidonic acid supply in vivo: consequences for nonsteroidal antiinflammatory drug efficacy

Prostanoids produced via the action of cyclo‐oxygenase‐2 (COX‐2) appear central to many inflammatory conditions. Here we show in LPS‐treated rats, however, that COX‐2 induction alone does not greatly increase prostanoid production in vivo. For this, a second, arachidonic acid liberating stimulus is also required. Thus, only after intravenous injection of bradykinin or exogenous arachidonic acid was a marked increase in prostanoid formation seen. There is, therefore, synergy between proinflammatory mediators: both induction of COX‐2 protein and an increase in the supply of arachidonic acid are required to greatly enhance prostanoid production. Second, we show that supplying arachidonic acid to increase prostanoid production reduces the effectiveness of both currently used nonsteroidal antiinflammatory drugs (NSAIDs) (diclofenac) and novel COX‐2‐selective inhibitors (NS‐398, celecoxib) as inhibitors of COX‐2 activity. Our data lead to two important conclusions. First, increased prostanoid production in inflammation is a two‐component response: increased COX‐2 expression and increased arachidonic acid supply. Second, the supply of arachidonic acid to COX‐2 determines the effectiveness of NSAIDs. NSAIDs and selective COX‐2 inhibitors, therefore, will generally be less effective at more inflamed sites, providing a rationale for the very high doses of NSAIDs required in human conditions such as rheumatoid arthritis.—Hamilton, L. C., Tomlinson, A. M., Mitchell, J. A., Warner, T. D. Synergy between cyclo‐oxygenase‐2 induction and arachidonic acid supply in vivo: consequences for nonsteroidal antiinflammatory drug efficacy. FASEB J. 13, 245–251 (1999)

An immunohistochemical study of endothelial cell heterogeneity in the rat: observations in “en face” Häutchen preparations

Summary“En face” sheets of endothelium, in which cellular spatial relationships were maintained, were prepared from proximal pulmonary and femoral arteries and aortae of the Wistar rat, in order to visualise patterns of heterogeneity in populations of endothelial cells. These preparations, termed Häutchen, were immunolabelled with antibodies to angiotensin II, endothelin and Factor-VIII-related antigen, and visualised by an avidin/biotin peroxidase complex. Clusters of cells, which accounted for approximately 30% of the total endothelial cell population, and which were positively immunostained for angiotensin II, were found perpendicular to the longitudinal axis of the aorta and femoral artery. Cells in the pulmonary artery were immunonegative for angiotensin II. The majority of cells in all three vessels were immunopositive for endothelin; groups of intensely stained cells were present in both the femoral artery and aorta, but not in the pulmonary artery. Immunoreactivity to Factor-VIII-related antigen was heterogeneous, with intensely stained amorphous patches of endothelial cells present in the femoral artery and aorta. Häutchen preparations present an opportunity for the investigation of endothelial cell heterogeneity, both within and between vessels; this may provide a basis for the interpretation of the heterogeneity of endothelium-dependent responses in vessels of differing origin.

Localization of arginine-vasopressin in endothelial cells of rat pulmonary artery.

SummaryThe localization of arginine-vasopressin in the endothelial cells of rat pulmonary artery was investigated by immunocytochemistry at the light and electron microscopic levels. The immunogold silver staining method was used for light microscopy of sheets of endothelium, removed from the artery, and the pre-embedding peroxidase-antiperoxidase technique was used for electron microscopy of cross sections of the artery. With both of the methods used, numerous vasopressin-positive endothelial cells were observed. None of the subendothelial elements showed labelling for vasopressin. The results are discussed in terms of the involvement of the endothelium in local control of the pulmonary circulation.

Inducible enzymes in the inflammatory response

Overview of COX-2 in inflammation - from the biology to the clinic, Michel Pairet et al overview of HO-1 inflammatory pathologies, Dean Willis inducible enzymes in the pathogenesis of rheumatoid arthritis, Vivienne R. Winrow and David R. Blake INOS and COS-2 in atherosclerosis, Lee D.K. Buttery and Julia M. Polak the role of the inducible enzymes cyclooxygenase-2, nitric oxide synthase and hemeoxygenase in angiogenesis of inflammation, Michael P. Seed et al role of the inducible forms of cyclooxygenase and nitric oxide synthase in inflammatory pain, Sergio H. Ferreira et al neuroinflammation, Bernd C. Kieseier and Hans-Peter Hartung inducible enzymes in inflammation - advances, interactions and conflicts, Annette Tomlinson and Derek A. Willoughby.