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Biomedical subjects

T D Warner

Publications and source records attributed to T D Warner.

At least 55 records · Page 3Linked to original sources
PubMed1999

A771726, the active metabolite of leflunomide, directly inhibits the activity of cyclo-oxygenase-2 in vitro and in vivo in a substrate-sensitive manner.

1. The immunosuppressive and anti-inflammatory drug leflunomide has several sites of action, although its precise mode of action is unknown. 2. Here we show in vitro and in vivo that leflunomide and/or its active metabolite A771726, inhibit the activity of cyclo-oxygenase (COX) at doses below those that affect protein expression. 3. In J774.2 macrophages treated with endotoxin for 24 h to induce COX-2 and iNOS, leflunomide and A771726 inhibited more potently the accumulation of PGE2 (A771726, IC50 3.5 microg ml-1) than of NO2 (A771726, IC50 380 microg ml-1). At high concentrations (>300 microg ml-1) A771726 also exhibited the expression of COX-2 and iNOS proteins. 4. In A549 cells treated for 24 h with interleukin-1beta, to induce COX-2, A771726 potently inhibited PGE2 synthesis (IC50 0.13 microg ml-1). In the same cells, A771726 was notably less active (IC50, 52 microg ml-1) at inhibiting the formation of PGE2 stimulated by exposure to 30 microM arachidonic acid. 5. In a human whole blood assay, measuring the accumulation of TxB2 in response to calcium ionophore as a measure of COX-1 activity and in response to incubation with bacterial endotoxin as a measure of COX-2 activity, leflunomide inhibited COX-1 and COX-2 with IC50 values of 31 and 185 microg ml-1; for A771726 the corresponding values were 40 and 69 microg ml-1. 6. Pre-treatment of rats with leflunomide or A771726 (10 mg kg-1, i.p.) inhibited the plasma accumulation of 6-keto-PGF1alpha but not NO2/NO3 following infusion of endotoxin. Injection of a bolus of arachidonic acid following 6 h infusion of endotoxin caused a marked acute rise in plasma 6-keto-PGF1alpha which was inhibited only by higher doses of A771726 (50 mg kg-1, i.p.). 7. In conclusion, leflunomide via A771726 can directly inhibit the activity of COX, an effect that appears blunted both by increases in substrate supply and possibly by plasma binding. Only at much higher drug levels does leflunomide and/or A771726 inhibit the induction of COX-2 or iNOS proteins.

6-Ketoprostaglandin F1 alpha↗
PubMed1999

Cyclo-oxygenase-2: pharmacology, physiology, biochemistry and relevance to NSAID therapy.

Cyclo-oxygenase is expressed in cells in two distinct isoforms. Cyclo-oxygenase-1 is present constitutively whilst cyclo-oxygenase-2 is expressed primarily after inflammatory insult. The activity of cyclo-oxygenase-1 and -2 results in the production of a variety of potent biological mediators (the prostaglandins) that regulate homeostatic and disease processes. Inhibitors of cyclo-oxygenase include the nonsteroidal anti-inflammatory drugs (NSAIDs) aspirin, ibuprofen and diclofenac. NSAIDs inhibit cyclo-oxygenase-2 at the site of inflammation, to produce their therapeutic benefits, as well as cyclo-oxygenase-1 in the gastric mucosa, which produces gastric damage. Most recently selective inhibitors of cyclo-oxygenase-2 have been developed and introduced to man for the treatment of arthritis. Moreover, recent epidemiological evidence suggests that cyclo-oxygenase inhibitors may have important therapeutic relevance in the prevention of some cancers or even Alzheimer's disease. This review will discuss how the new advancements in NSAIDs research has led to the development of a new class of NSAIDs that has far reaching implications for the treatment of disease.

Alzheimer Disease↗
PubMed1999

Selective COX-2 inhibitors and human inflammatory bowel disease.

BACKGROUND: Much recent effort has been made to produce selective inhibitors of cyclo-oxygenase-2 (COX-2) in the belief that these will lack the gastrointestinal damaging effects of traditional non-steroidal anti-inflammatory drugs (NSAIDs). Inflammatory bowel disease is associated with increased local production of prostanoids. These prostanoids, particularly PGE2 and PGI2, may well be protective as inflammatory bowel disease is aggravated by NSAID use. AIM: To examine the effects of a traditional NSAID and a highly selective COX-2 inhibitor on the production of these prostanoids in human inflammatory bowel disease. METHODS: Colonic mucosal biopsies were obtained from patients undergoing routine colonoscopy and biopsy for diagnostic or surveillance purposes. Biopsies were incubated in culture medium containing 10% foetal calf serum and antibiotics, plus test drugs or vehicle for 24 h, after which time the medium was removed and the content of PGE2, PGI2 (measured as 6 keto-PGF1alpha) and thromboxane (Tx) A2 (measured as TxB2) determined. RESULTS: Biopsies obtained from diseased colonic mucosa produced significantly more PGE2, PGI2 and thromboxane A2 than did controls (for example, PGE2: ulcerative colitis, 4.17+/-1.06; Crohn's disease, 3.97+/-1.66; control, 0.12 +/-0.13 ng/mL, n = 8-12). These increases were inhibited to a similar extent by either a highly selective COX-2 inhibitor (L-745,337) or a traditional non-selective NSAID (indomethacin). CONCLUSIONS: Until selective COX-2 inhibitors have been assessed adequately in human inflammatory bowel disease, these compounds should not be assumed to be safe for the gastrointestinal tract in inflammatory bowel disease.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1999

Relationships between the endothelin and nitric oxide pathways.

1. In the normal blood vessel, the vascular endothelium regulates the tone of the underlying smooth muscle and the reactivity of blood elements, such as platelets and neutrophils, by the release of mediators, in particular nitric oxide (NO) and endothelin-1 (ET-1). 2. Nitric oxide is a potent vasodilator that also inhibits platelet and neutrophil aggregation and adhesion; ET-1 is the most potent mammalian vasoconstrictor peptide yet found. Recently, much research effort has focused on examining the interactions between these two important mediators. At a simple level, ET-1 acts on specific receptors on the endothelium to increase the release of NO, while NO depresses the production and/or release of ET-1 from endothelial cells. 3. While ET-1 appears to have a relatively small influence on the basal regulation of blood pressure, NO appears central. For example, inhibition of NO production in normotensive animals produces a marked elevation in blood pressure. 4. Conversely, numerous vascular disease states have been associated with elevations in the production and/or release of ET-1 and it has been implicated in the deleterious changes associated with ischaemia-reperfusion injury, subarachnoid haemorrhage and hypertension. In these conditions, NO production may also be increased by the induction of NO synthetic pathways within the vascular smooth muscle. Endothelin-1 may also be produced by the vascular smooth muscle under similar circumstances. 5. Therefore, in pathological states, a new balance between NO and ET-1 production may be central to changes in blood vessel reactivity, smooth muscle proliferation and blood coagulability.

Animals↗
PubMed1999

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.

Animals↗
PubMed1999

Mechanisms of prostaglandin E2 release by intact cells expressing cyclooxygenase-2: evidence for a 'two-component' model.

Prostaglandin (PG) release in cells expressing constitutive cyclooxygenase-1 is known to be regulated by liberation of arachidonic acid by phospholipase A2 followed by metabolism by cyclooxygenase. However, the relative contribution of phospholipase A2 to the release of PGs in cells expressing cyclooxygenase-2 is not clear. We addressed this question by using radioimmunoassay to measure PGE2 release by human cells (A549) induced to express cyclooxygenase-2 (measured by Western blot analysis) by interleukin-1beta. Cells were either unstimulated or stimulated with agents known to activate phospholipase A2 (bradykinin, Des-Arg10-kallidin, or the calcium ionophore A23187) or treated with exogenous arachidonic acid. When cells were treated to express cyclooxygenase-2, the levels of PGE2 released over 15 min were undetectable; however, in the same cells stimulated with bradykinin, A23187, or arachidonic acid, large amounts of prostanoid were produced. Using selective inhibitors/antagonists, we found that the effects of bradykinin were mediated by B2 receptor activation and that prostanoid release was due to cyclooxygenase-2, and not cyclooxygenase-1, activity. In addition, we show that the release of PGE2 stimulated by either bradykinin, A23187, or arachidonic acid was inhibited by the phospholipase A2 inhibitor arachidonate trifluoromethyl ketone. Hence, we have demonstrated that PGE2 is released by two components: induction of cyclooxygenase-2 and supply of substrate, probably via activation of phospholipase A2. This is illustrated in A549 cells by a clear synergy between the cytokine interleukin-1beta and the kinin bradykinin.

Adrenergic beta-Agonists↗
PubMed1999

Endothelin-1 is induced by cytokines in human vascular smooth muscle cells: evidence for intracellular endothelin-converting enzyme.

Endothelin-1 (ET-1) is the predominant endothelin isopeptide generated by the vascular wall and therefore appears to be the most important peptide involved in regulation of cardiovascular events. Many pathologic conditions are associated with elevations of ET-1 in the blood vessel wall. Because these conditions are often cytokine driven, we examined the effects of a mixture of cytokines on ET-1 production in human vascular smooth muscle cells (VSMCs) derived from internal mammary artery and saphenous vein (SV). Incubation of IMA and SV VSMCs with tumor necrosis factor-alpha (10 ng/ml) and interferon-gamma (1000 U/ml) in combination for up to 48 h markedly elevated the expression of mRNA for prepro-ET-1 and the release of ET-1 into the culture medium. This cytokine-stimulated release of ET-1 was inhibited by a series of dual endothelin-converting enzyme (ECE)/neutral endopeptidase inhibitors, phosphoramidon, CGS 26303, and CGS 26393, with an accompanying increase in big ET-1 release but with no effect on expression of mRNA for prepro-ET-1. These same compounds were 10 times more potent at inhibiting the conversion of exogenously applied big ET-1 to ET-1. ECE-1b/c mRNA is present in SV VSMCs, however no ECE-1a is present in these cells. Thus VSMCs most probably contain, like endothelial cells, an intracellular ECE responsible for the endogenous synthesis of ET-1. Under the influence of pro-inflammatory mediators the vascular smooth muscle can therefore become an important site of ET-1 production, as has already been established for the dilator mediators nitric oxide, prostaglandin I2, and prostaglandin E2.

Aspartic Acid Endopeptidases↗
PubMed1998

Regulation of cyclo-oxygenase gene expression in rat smooth muscle cells by catalase.

We have studied, in detail, the effect of catalase, one of the naturally occurring antioxidant enzymes, on the expression of cyclo-oxygenase (COX) mRNA and protein in rat aortic smooth muscle cells (RASMC). The activity of COX enzyme within the cells was also determined. Catalase either alone or in combination with interleukin-1beta (IL-1beta) enhanced mRNA and protein expression for cyclo-oxygenase 2 (COX-2) in a concentration-dependent manner. However, it did not affect the expression of mRNA or protein for cyclo-oxygenase 1 (COX-1). The expression of mRNA for COX-2 induced by catalase was blocked completely by actinomycin D (ACT) or cycloheximide (CHX). In comparison, expression of mRNA for COX-2 stimulated by IL-1beta was inhibited by actinomycin D, but not by cycloheximide. This suggests that induction of the synthesis of mRNA for COX-2 by catalase and IL-1beta involves different mechanisms. In particular, the induction of mRNA for COX-2 by catalase requires on-going protein and RNA synthesis, but the induction following exposure to IL-1beta does not. The increase in expression of mRNA for COX-2 induced by catalase may be related to the ability of catalase to stimulate cyclic AMP response element (CRE) and NF-IL6 transcription factors, but not nuclear factor kappa B (NF-kappaB), for electrophoretic mobility shift assays (EMSA) showed that catalase enhanced nuclear factor binding to cyclic AMP response element and NF-IL6 but not to NF-kappaB. Catalase exerted a biphasic effect on prostaglandin synthesis. At low concentrations it enhanced prostaglandin production, but at high concentrations it tended to inhibit it. These findings suggest that catalase has differential and multiple effects on COX expression and activity in rat aortic smooth muscle cells.

Animals↗
PubMed1998

Role of AT2 receptors in the cardiovascular events following microinjection of angiotensin II into the superior colliculus of anaesthetised rats.

Central injection of angiotensin II (ANGII) induces pressor and regional haemodynamic effects. Here we have investigated the systemic and regional cardiovascular changes induced by injection of ANGII into the superficial layer of the superior colliculus (SC) of male rats anaesthetised with urethane. In addition, we have used the AT1 receptor-selective antagonist, losartan, and the AT2 receptor-selective antagonist, PD123319 to characterise the receptor(s) mediating these effects. Injection of ANGII (0.1, 1 and 100 nmol/rat) into the superficial layer of the SC significantly (P < 0.05) increased, in a dose-dependent manner, the mean arterial blood pressure (MAP) while decreasing the heart rate, (e.g. by 37+/-4 beats min(-1), at 1 nmol, P < 0.05). The increases in blood pressure induced by ANGII (1 nmol; 43+/-6 mmHg, n = 5) were greatly reduced (> 85%) by pre-administration to the SC of PD123319 (50 nmol/rat), but were unaffected by losartan (50 nmol/rat). Similarly, PD123319 prevented the decrease in heart rate induced by ANGII while losartan did not affect it. Injection of ANGII (1 nmol) also increased (P < 0.01) total peripheral resistance (TPR; control, 2.36+/-0.1 mmHg ml(-1) min 100 g body weight) by 130+/-10% (n = 5) and reduced the cardiac output (CO; control, 99.8+/-1.3 ml min[-1]) by 51+/-3% (n = 5), as determined by radioactive microspheres. The increase in TPR was associated with increases in the vascular resistances of organs, such as the left and right kidney (390+/-15%, P < 0.01 and 352+/-12%, P < 0.01 respectively), the skeletal muscle (91+/-7%, P < 0.05, n = 5), the stomach (43+/-2%, P < 0.01, n = 5), the colon (50+/-3%, P < 0.05, n = 5) and the caecum (65+/-5%, P < 0.05, n = 5). Pre-treatment of the SC with PD123319 reduced (P < 0.01) the increases in TPR and vascular resistance, and the reduction in CO caused by ANGII. Losartan did not affect the responses to ANGII. Thus, injection of ANGII into the SC causes complex haemodynamic changes which are sensitive to AT2 receptor antagonism. AT2 receptors are, therefore, the predominant mediators of the actions of ANGII into the superior colliculus of the rat.

Anesthesia, General↗
PubMed1998

Interactions between inducible isoforms of nitric oxide synthase and cyclo-oxygenase in vivo: investigations using the selective inhibitors, 1400W and celecoxib.

1. Exposure of tissues to endotoxin (LPS) and/or cytokines leads to the induction of both inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2). It has previously been reported that there is 'cross-talk' between these two systems. However, such previous studies have been limited by the availability of highly selective inhibitors. Here we have investigated the interactions between iNOS and COX-2 in vivo using 1400W, an iNOS-selective inhibitor, and celecoxib, a COX-2-selective inhibitor. 2. Infusion of LPS to rats for 6 h caused a time-dependent increase in the plasma concentrations of 6 keto-prostaglandin F1alpha (6 keto-PGF1alpha) and nitrite/nitrate (NO2/NO3), consistent with the induction of iNOS and COX-2. Bolus injection of arachidonic acid (AA) at t=6 h resulted in a further increase of circulating levels of 6 keto-PGF1alpha in LPS-treated animals. 3. Treatment of rats with 1400W or the non-selective NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA) inhibited the increase in plasma NO2/NO3 but were both without effect on the plasma concentration of 6 keto-PGF1alpha before or after AA. 4. Treatment with the non-steroidal anti-inflammatory drugs (NSAIDs), A771726 or diclofenac, or with celecoxib significantly reduced the increase in circulating 6 keto-PGF1alpha caused by LPS, and the large increase in 6 keto-PGF1alpha following injection of AA. None of the COX inhibitors affected the increase in plasma NO2/NO3. Dexamethasone, however, significantly inhibited both the increase in 6 keto-PGF1alpha and the increase in NO2/NO3. 5. In conclusion, the use of selective inhibitors does not support the concept of cross talk in vivo between iNOS and COX-2.

6-Ketoprostaglandin F1 alpha↗
PubMed1998

Cytokine and lipopolysaccharide stimulation of endothelin-1 release from human internal mammary artery and saphenous vein smooth-muscle cells.

Many pathologic conditions are associated with elevations in the production of endothelin-1 (ET-1) in the blood vessel wall. Because many of these conditions are cytokine-driven we examined the effects of a mixture of cytokines and lipopolysaccharide on ET-1 production in human vascular smooth-muscle (VSMC) cells derived from the internal mammary artery (IMA) and saphenous vein (SV). Incubation of VSMCs from IMA and SV with a combination of tumor necrosis factor-alpha (10 ng/ml), interferon-gamma (1,000 U/ml), interleukin-1 beta (500 U/ml) and lipopolysaccharide (10 micrograms/ml) for up to 48 h markedly elevated the expression of mRNA for ET-1 and the release of ET-1 into the culture medium. We conclude that low levels of ET-1 mRNA and peptide production in human VSMCs are markedly increased by exposure to cytokines and LPS. This suggests that during inflammatory states the VSMC, as well as the endothelium, may be a site of significant ET-1 production in the blood vessel wall.

Cells, Cultured↗
PubMed1997

Internal medicine, psychiatry, and emergency medicine residents' views of assisted death practices.

BACKGROUND: Although studies have revealed conflicting attitudes within the medical community regarding assisted death practices in the United States, the views of current resident physicians have not been described. OBJECTIVE: To investigate the perspectives of residents from 3 medical specialty fields regarding the acceptability of assisted suicide and euthanasia practices as performed by 4 possible agents (the resident personally, a referral physician, physicians in general, or nonphysicians in general) in 6 patient scenarios. METHODS: An anonymous survey exploring responses to 6 patient vignettes was conducted with a convenience sample of all residents in the internal medicine, psychiatry, and emergency medicine training programs. RESULTS: A total of 96 residents, 72% of those asked, participated in this study. Overall, residents expressed opposition or uncertainty regarding assisted suicide and euthanasia. The residents were disinclined to directly perform such practices themselves and did not support the conduct of assisted suicide practices by nonphysicians. Respondents were somewhat more accepting of other physicians' involvement in assisted death activities. Conflicting views were expressed by residents, with emergency medicine residents more likely to support assisted suicide practices in 4 of 6 patient vignettes than either internal medicine or psychiatry residents. Residents who reported being influenced by religious beliefs (21 respondents [22%]) did not support assisted death practices, whereas those influenced by personal philosophy (74 respondents [77%]) expressed less opposition. CONCLUSIONS: This study explores the uncertainty and differing views of residents from 3 fields about physician-assisted suicide practices. Study findings are considered within the larger literature on clinician attitudes toward assisted suicide and euthanasia.

Adult↗
PubMed1997

Regulation of blood pressure by L-arginine-nitric oxide pathway within the superior colliculus of rats.

Injection into the superior colliculus of anaesthetised rats of the nitric oxide (NO) synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 1 micromol), but not its inactive enantiomer N(omega)-nitro-D-arginine methyl ester (D-NAME; 1 micromol), significantly (P < 0.01) increased the mean arterial blood pressure. Injection to the superior colliculus of L-arginine (L-Arg; 1 micromol), the substrate for NO synthase, significantly (P < 0.01) lessened the pressor effect of L-NAME, while D-arginine (D-Arg; 1 micromol) did not affect it. L-Arg (7.5 micromol), but not D-Arg (7.5 micromol) administered at the peak of the pressor response to L-NAME (1 micromol) also partially reversed this pressor response (P < 0.05). These data would suggest that endogenously produced NO acts within the superior colliculus to modulate the arterial blood pressure.

Analysis of Variance↗
PubMed1997

Contraction of human airway smooth muscle by endothelin-1 and IRL 1620: effect of bosentan.

We have examined whether 4-tert-butyl-N-[6-(2-hydroxy-ethoxy)-2,2'-bipyrimidin-4-yl]-benzen esulfonamide (bosentan; endothelin ET(A/B) receptor antagonist) and (R)2-[(R)-2-[(S)-2-[[1-(hexahydro-1H-azepinyl)]carbonyl] amino-4-methylpentanoil]amino-3-[3-(1-methyl-1H-indoyl)]prop ionyl]amino-3-(2-pyridyl) propionic acid (FR 139317; endothelin ET(A) receptor antagonist) inhibit contractions of human airway smooth muscle induced by endothelin-1 or Suc-[Glu9,Ala(11,15)]enthothelin-1-(8-21) (IRL 1620; endothelin ET(B) receptor agonist). Endothelin-1 and IRL 1620 were equipotent. Bosentan and FR 139317 (each 10 microM) produced a small shift in response curves to endothelin-1 (1.6- and 1.5-fold, respectively). However, bosentan was more potent against contractions elicited by IRL 1620 (10 microM, 11.2-fold shift) suggesting that these agonists exhibit different kinetic interactions with endothelin receptors or implying an interaction with a novel endothelin ET(B) receptor subtype in human airways.

Adolescent↗
PubMed1997

Activation of ET(A) receptors is partially responsible for the rapid increase in haematocrit induced by bacterial lipopolysaccharide in the rat.

It is believed that changes in the production and release of endothelin-1 (ET-1) are mediated over prolonged periods, and, therefore, that it is unlikely that ET-1 mediates rapid responses within the circulation. Here we show that ET-1 is involved in the rapid changes produced by injection of LPS in vivo. In anaesthetised rats, a bolus of LPS induced an increase in haematocrit and a fall in blood pressure within 10 min. The increase in haematocrit was reduced by administration of antagonists selective for endothelin ET(A) receptors, while the accompanying decrease in MAP was potentiated. Thus, activation of ET(A) receptors is partially responsible for the rapid increase in haematocrit seen in this model. This clearly demonstrates that ET-1 can act as a rapid responder to acute cardiovascular challenges.

Animals↗