A unique role for p53 in the regulation of M2 macrophage polarization.

P53 is critically important in preventing oncogenesis but its role in inflammation in general and in the function of inflammatory macrophages in particular is not clear. Here, we show that bone marrow-derived macrophages exhibit endogenous p53 activity, which is increased when macrophages are polarized to the M2 (alternatively activated macrophage) subtype. This leads to reduced expression of M2 genes. Nutlin-3a, which destabilizes the p53/MDM2 (mouse double minute 2 homolog) complex, promotes p53 activation and further downregulates M2 gene expression. In contrast, increased expression of M2 genes was apparent in M2-polarized macrophages from p53-deficient and p53 mutant mice. Furthermore, we show, in mice, that p53 also regulates M2 polarization in peritoneal macrophages from interleukin-4-challenged animals and that nutlin-3a retards the development of tolerance to Escherichia coli lipopolysaccharide. P53 acts via transcriptional repression of expression of c-Myc (v-myc avian myelocytomatosis viral oncogene homolog) gene by directly associating with its promoter. These data establish a role for the p53/MDM2/c-MYC axis as a physiological 'brake' to the M2 polarization process. This work reveals a hitherto unknown role for p53 in macrophages, provides further insight into the complexities of macrophage plasticity and raises the possibility that p53-activating drugs, many of which are currently being trialled clinically, may have unforeseen effects on macrophage function.

Utilizing hydrogen sulfide as a novel anti-cancer agent by targeting cancer glycolysis and pH imbalance.

BACKGROUND AND PURPOSE: Many disparate studies have reported the ambiguous role of hydrogen sulfide (H2 S) in cell survival. The present study investigated the effect of H2 S on the viability of cancer and non-cancer cells. EXPERIMENTAL APPROACH: Cancer and non-cancer cells were exposed to H2 S [using sodium hydrosulfide (NaHS) and GYY4137] and cell viability was examined by crystal violet assay. We then examined cancer cellular glycolysis by in vitro enzymatic assays and pH regulator activity. Lastly, intracellular pH (pHi ) was determined by ratiometric pHi measurement using BCECF staining. KEY RESULTS: Continuous, but not a single, exposure to H2 S decreased cell survival more effectively in cancer cells, as compared to non-cancer cells. Slow H2 S-releasing donor, GYY4137, significantly increased glycolysis, leading to overproduction of lactate. H2 S also decreased anion exchanger and sodium/proton exchanger activity. The combination of increased metabolic acid production and defective pH regulation resulted in an uncontrolled intracellular acidification, leading to cancer cell death. In contrast, no significant intracellular acidification or cell death was observed in non-cancer cells. CONCLUSIONS AND IMPLICATIONS: Low and continuous exposure to H2 S targets metabolic processes and pH homeostasis in cancer cells, potentially serving as a novel and selective anti-cancer strategy.

Hydrogen sulphide is pro-inflammatory in haemorrhagic shock.

OBJECTIVE: H(2)S is pro-inflammatory in inflammatory models, thus we investigated whether H(2)S plays a role in haemorrhagic shock (HS)-associated inflammation. METHODS: Male, Sprague-Dawley rats were given an inhibitor of H(2)S biosynthesis, DL-propargylglycine (PAG, 50 mg/kg, i. v.) or saline (1 ml/kg) 30 min before blood withdrawal and subjected to HS (mean arterial pressure (MAP) of 40 mM Hg for 90 min) followed by reinfusion of shed blood. Animals were killed at 5, 90, 270 and 630 min after reinfusion. RESULTS: Pre-treatment of animals with PAG 1) increased the HR recovery rate (n = 6 - 12, P < 0.05); 2) attenuated the increase in plasma levels of TNF-alpha and IL-6 and reduced lung iNOS expression levels (n =5 P, < 0.05); and 3) attenuated the increase in plasma levels of ALT and reduced HS-induced increase in liver and lung myeloperoxidase (MPO) activity (n = 5, P < 0.05). CONCLUSIONS: H(2)S is pro-inflammatory in HS and inhibition of H(2)S biosynthesis may reduce some HS-induced inflammatory responses and organ injury.

A nociceptive-intensity-dependent role for hydrogen sulphide in the formalin model of persistent inflammatory pain.

The present study investigated the hypothesis that hydrogen sulfide (H2S) is pro-nociceptive in the formalin model of persistent inflammatory pain in the adult rat. Hind paw injection of formalin evoked a concentration-dependent increase in the hind paw concentration of H2S. Increased concentration of H2S was found in homogenates prepared from hind paws injected with 5% (but not 1.25%) formalin. Correspondingly, animal nociceptive flinching and hind paw edema were maximal with 5% formalin. Both nociceptive flinching and hind paw edema induced by injection of 5% formalin were attenuated by pretreatment with DL-propargylglycine (PPG; 50 mg/kg, i.p.) which is an inhibitor of the H2S synthesizing enzyme cystathionine-gamma-lyase (CSE). The effect of pretreatment with PPG was selective and the drug did not influence animal behavior or hind-paw edema with injection of 1.25% formalin. Furthermore, PPG pretreatment attenuated the induction of c-Fos in spinal laminae I-II following injection of 5% formalin. In contrast, co-injection of 1.25% formalin with sodium hydrogen sulfide (NaHS; 1 nmol/0.1 ml), a H2S donor, into the hind paw increased animal nociceptive behavior. Collectively, these findings show that the effect of peripheral H2S in the pathogenesis of inflammatory pain depends, at least in part, on the nociceptive intensity level.

Putative biological roles of hydrogen sulfide in health and disease: a breath of not so fresh air?

The past two decades have seen an upsurge in interest in the biology of naturally occurring gases, starting with nitric oxide and extending through to carbon monoxide. The latest addition to the list of biologically relevant gases is hydrogen sulfide. In the past few years, hydrogen sulfide has transited rapidly from environmental pollutant to biologically relevant mediator with potential roles in several physiological processes and disease states. Further, interest is now being shown in developing drugs which either mimic its effects or block its biosynthesis. Similarly to its gaseous cousins, the biology of hydrogen sulfide is proving to be complex and difficult to unravel.

Hydrogen sulphide in the hypothalamus causes an ATP-sensitive K+ channel-dependent decrease in blood pressure in freely moving rats.

Hydrogen sulfide (H2S) is a naturally occurring gas that may act as an endogenous signaling molecule. In the brain, H2S is mainly produced by cystathionine beta-synthase (CBS) and its cellular effects have been attributed to interactions with N-methyl-D-aspartate (NMDA) receptors and cyclic adenosine 3',5'-monophosphate (cAMP). In contrast, direct vasodilator actions of H2S are most probably mediated by opening smooth muscle ATP-sensitive K+ (K(ATP)) channels. In the hypothalamus, K(ATP) channel-dependent mechanisms are involved in CNS-mediated regulation of blood pressure. In this report, we investigated the hypothesis that H2S may act via K(ATP) channels in the hypothalamus to regulate blood pressure. Mean arterial blood pressure (MAP) and heart rate were monitored in freely moving rats via a pressure transducer placed in the femoral artery. Drugs were infused via a cannula placed in the posterior hypothalamus. Infusion of 200 microM sodium hydrogen sulfide (NaHS), an H2S donor, into the hypothalamus of freely moving rats reduced MAP and heart rate. Infusion of 300 nM to 3 microM gliclazide dose-dependently blocked the effect of 200 microM NaHS. Infusion of the CBS activator, s-adenosyl-L-methionine (0.1 mM and 1 mM), likewise decreased MAP. Infusion of the CBS inhibitors aminooxyacetic acid (10 mM) and hydroxylamine (20 mM) increased MAP but did not block the effects of infusion of 200 microM NaHS. These data indicate that actions of H2S in the hypothalamus decrease blood pressure and heart rate in freely moving rats. This effect appears to be mediated by a K(ATP) channel-dependent mechanism and mimicked by endogenous H2S.

An overview of the biological significance of endogenous gases: new roles for old molecules.

Biologically active gases that occur naturally in the body include nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H(2)S). Each of these molecules is synthesized by enzymes which have been characterized biochemically and pharmacologically, and each acts, via well-established molecular targets, to effect physiological and/or pathophysiological functions within the body. Major biological roles that appear to be common to all three gases include the regulation of vascular homoeostasis and central nervous system function. It is becoming increasingly clear that both the synthesis and the biological activity of each gas are, to some extent, regulated by the presence of the others, and as such it is necessary to consider these molecules not in isolation but acting together to control cell function. Additional, more speculative candidates for gaseous cell signalling molecules include ammonia, acetaldehyde, sulfur dioxide and nitrous oxide. Whether such molecules also play a role in regulating body function remains to be determined.

Synergism between hydrogen sulfide (H(2)S) and nitric oxide (NO) in vasorelaxation induced by stonustoxin (SNTX), a lethal and hypotensive protein factor isolated from stonefish Synanceja horrida venom.

Stonustoxin (SNTX) is a 148 kDa, dimeric, hypotensive and lethal protein factor isolated from the venom of the stonefish Synanceja horrida. SNTX (10-320 ng/ml) progressively causes relaxation of endothelium-intact, phenylephrine (PE)-precontracted rat thoracic aortic rings. The SNTX-induced vasorelaxation was inhibited by L-N(G)-nitro arginine methyl ester (L-NAME), suggesting that nitric oxide (NO) contributes to the SNTX-induced response. Interestingly, D, L-proparglyglycine (PAG) and beta-cyano-L-alanine (BCA), irreversible and competitive inhibitors of cystathionine-gamma-lyase (CSE) respectively, also inhibited SNTX-induced vasorelaxation, indicating that H(2)S may also play a part in the effect of SNTX. The combined use of L-NAME with PAG or BCA showed that H(2)S and NO act synergistically in effecting SNTX-induced vasorelaxation.

Flurbiprofen and its nitric oxide-releasing derivative protect against septic shock in rats.

OBJECTIVE: Flurbiprofen and nitroflurbiprofen were evaluated in a caecal ligation puncture (CLP) model of septic shock in the rat. METHODS AND RESULTS: CLP (12 h) reduced blood pressure (72.5 +/- 1.0 mm Hg c. f. 101.0 +/- 3.6 mm Hg, P < 0.05), and increased plasma NOx (153.0 +/- 11.5 muM c. f. 36.2 +/- 3.2 microM, P < 0.05), IL-1beta (534.0 +/- 93.1 pg/mL c. f.; 9.6 +/- 9.6 pg/mL, P < 0.05), TNF-alpha (88.0 +/- 13.6 pg/mL, P < 0.05), inflammatory damage in lung and liver, and mortality. Both flurbiprofen (21 mg/kg, p. o.) and nitroflurbiprofen (30 mg/kg, p. o.) prevented the fall in blood pressure (e. g. 80.4 +/- 2.1 mm Hg and 79.8 +/- 1.2 mm Hg respectively, 12 h, P < 0.05), reduced organ damage and prolonged survival. Nitroflurbiprofen (but not flurbiprofen) increased plasma NOx and reduced plasma TNF-alpha concentration at all time points (except 1 h). Neither drug affected plasma IL-1beta-levels. CONCLUSIONS: These results suggest a protective effect of flurbiprofen and nitroflurbiprofen in septic shock.

Regulation of vascular nitric oxide in vitro and in vivo; a new role for endogenous hydrogen sulphide?

BACKGROUND AND PURPOSE: The aim of these experiments was to evaluate the significance of the chemical reaction between hydrogen sulphide (H2S) and nitric oxide (NO) for the control of vascular tone. EXPERIMENTAL APPROACH: The effect of sodium hydrosulphide (NaHS; H2S donor) and a range of NO donors, such as sodium nitroprusside (SNP), either alone or together, was determined using phenylephrine (PE)-precontracted rat aortic rings and on the blood pressure of anaesthetised rats. KEY RESULTS: Mixing NaHS with NO donors inhibited the vasorelaxant effect of NO both in vitro and in vivo. Low concentrations of NaHS or H2S gas in solution reversed the relaxant effect of acetylcholine (ACh, 400 nM) and histamine (100 microM) but not isoprenaline (400 nM). The effect of NaHS on the ACh response was antagonized by CuSO(4) (200 nM) but was unaffected by glibenclamide (10 microM). In contrast, high concentrations of NaHS (200-1600 microM) relaxed aortic rings directly, an effect reduced by glibenclamide but unaffected by CuSO4. Intravenous infusion of a low concentration of NaHS (10 micromol kg(-1) min(-1)) into the anaesthetized rat significantly increased mean arterial blood pressure. L-NAME (25 mg kg(-1), i.v.) pretreatment reduced this effect. CONCLUSIONS AND IMPLICATIONS: These results suggest that H2S and NO react together to form a molecule (possibly a nitrosothiol) which exhibits little or no vasorelaxant activity either in vitro or in vivo. We propose that a crucial, and hitherto unappreciated, role of H2S in the vascular system is the regulation of the availability of NO.

Effect of a nitric oxide releasing derivative of paracetamol in a rat model of endotoxaemia.

BACKGROUND AND PURPOSE: Nitroparacetamol is a nitric oxide-releasing paracetamol with novel anti-inflammatory properties compared to the parent compound. This study has investigated the anti-inflammatory activity of nitroparacetamol in a model of endotoxaemia in rats to probe the mechanisms underlying this effect. EXPERIMENTAL APPROACH: Nitroparacetamol (92 mg kg(-1)), paracetamol (50 mg kg(-1)) or vehicle were administered to male, Wistar rats 15 min prior to or 3 h after lipopolysaccharide (0.5 mg kg(-1), serotype 0127:B8). Mean arterial pressure and heart rate were measured for 5 h and plasma and organs were then obtained to determine organ dysfunction, inducible nitric oxide synthase and cyclooxygenase-2 expression (lung, liver and kidney tissue) and plasma nitrate/nitrite. In separate experiments, nitroparacetamol, paracetamol or vehicle was administered 1 h before acetylcholine (0.1 microg kg(-1)) or sodium nitroprusside (0.25 microg kg(-1)) to determine if nitroparacetamol desensitizes responses to exogenous/endogenous nitric oxide. KEY RESULTS: Nitroparacetamol prevented but did not reverse the lipopolysaccharide-induced hypotension. There was no effect on heart rate or plasma markers of organ dysfunction. Nitroparacetamol prevented the increased plasma nitrate/nitrite and expression of COX-2 and iNOS, whereas paracetamol exerted partial inhibition of COX-2 in lung alone. Nitroparacetamol also reduced responses to acetylcholine and sodium nitroprusside. CONCLUSIONS AND IMPLICATIONS: NO is the active component of nitroparacetamol in this model of endotoxaemia. Pro-inflammatory processes targeted by nitroparacetamol have been shown to include iNOS/COX-2 induction and possibly vascular soluble guanylyl cyclase. Precise mechanisms underlying the NO effect are unclear but inhibition of cytokine formation may be important.

Nitric oxide releasing acetaminophen (nitroacetaminophen).

The nitric oxide releasing derivative of acetaminophen (nitroacetaminophen) exhibits potent anti-inflammatory and anti-nociceptive activity in a variety of animal models. On a mol for mol basis nitroacetaminophen is some 3-20 times more potent than acetaminophen. Nitroacetaminophen exhibits little or no hepatotoxicity following administration in rat or mouse and indeed protects against the hepatotoxic activity of acetaminophen. Nitroacetaminophen does not affect blood pressure or heart rate of anaesthetised rats but has similar potency to acetaminophen as an anti-pyretic agent. The enhanced anti-inflammatory and anti-nociceptive activity of nitroacetaminophen and the reduced hepatotoxicity in these animal models is likely to be secondary to the slow release of nitric oxide from the molecule. As yet the precise molecular mechanism(s) underlying these actions of nitroacetaminophen are not clear. Evidence for inhibition of cytokine-directed formation of pro-inflammatory molecule production (e.g. COX-2, iNOS) by an effect on the NF-kappaB transduction system and/or nitrosylation (and thence inhibition) of caspase enzyme activity has been reported. Data described in this review indicate that the profile of pharmacological activity of nitroacetaminophen and acetaminophen are markedly different. The possibility that nitroacetaminophen could be an attractive alternative to acetaminophen in the clinic is discussed.

Pharmacology and potential therapeutic applications of nitric oxide-releasing non-steroidal anti-inflammatory and related nitric oxide-donating drugs.

This review examines the biological significance, therapeutic potential and mechanism(s) of action of a range of nitric oxide-releasing non-steroidal anti-inflammatory drugs (NO-NSAID) and related nitric oxide-releasing donating drugs (NODD). The slow release of nitric oxide (NO) from these compounds leads to subtle changes in the profile of pharmacological activity of the parent, non-steroidal anti-inflammatory drugs (NSAID). For example, compared with NSAID, NO-NSAID cause markedly diminished gastrointestinal toxicity and improved anti-inflammatory and anti-nociceptive efficacy. In addition, nitroparacetamol exhibits hepatoprotection as opposed to the hepatotoxic activity of paracetamol. The possibility that NO-NSAID or NODD may be of therapeutic benefit in a wide variety of disease states including pain and inflammation, thrombosis and restenosis, neurodegenerative diseases of the central nervous system, colitis, cancer, urinary incontinence, liver disease, impotence, bronchial asthma and osteoporosis is discussed.

The smooth muscle relaxant effect of hydrogen sulphide in vitro: evidence for a physiological role to control intestinal contractility.

1. Sodium hydrogen sulphide (NaHS), a donor of hydrogen sulphide (H(2)S), produced dose-related relaxation of the rabbit isolated ileum (EC(50), 76.4+/-7.9 microM) and rat vas deferens (EC(50), 64.8+/-5.4 microM) and reduced ACh-mediated contraction of the guinea-pig isolated ileum. 2. NaHS also reduced the response of the guinea-pig (EC(50), 80.0+/-5.7 microM) and rat (EC(50), 108.2+/-11.2 microM) ileum preparations to electrical stimulation of the intramural nerves. In guinea-pig ileum this effect was spontaneously reversible and mimicked by sodium nitroprusside (SNP, EC(50), 2.1 microM). Combination of NaHS (20 microM) with SNP (0.5 microM) produced a greater than additive inhibition of the twitch response of the ileum to electrical stimulation. 3. The inhibitory effect of NaHS on the field-stimulated guinea-pig ileum was unaffected by pretreatment with L-NAME (100 microM), indomethacin (10 microM), naloxone (1 microM) or glibenclamide (100 microM). Furthermore, NaHS (200 microM) did not affect the contractile response of the ileum to KCl (10 to 60 mM). 4. Propargylglycine (PAG, 1 mM) and beta-cyanoalanine (BCA, 1 mM) (inhibitors of cystathionine-gamma-lyase) but not aminooxyacetic acid (AOAA, 1 mM) (inhibitor of cystathionine-beta-synthetase) caused a slowly developing increase in the contraction of the guinea-pig ileum to field stimulation. This effect was reversed by cysteine (1 mM). 5. These results show that NaHS relaxes gastrointestinal and urogenital smooth muscle and suggest that H(2)S is responsible for these effects. The possibility that endogenous H(2)S, formed as a consequence of activation of intramural nerves, plays a part in controlling the contractility of the guinea-pig ileum is discussed.

Vasorelaxant effect of nitric oxide releasing steroidal and nonsteroidal anti-inflammatory drugs.

The effect of several nitric oxide releasing-non-steroidal anti-inflammatory drugs (NO-NSAID) and nitroprednisolone on blood vessel relaxation in vitro and in vivo was studied. Nitroflurbiprofen (NOF; EC(50), 688.8+/-93.8 microM), nitroaspirin (NOA; EC(50), 57.9+/-6.5 microM), nitroparacetamol (NOPARA; EC(50), 71.5+/-14.6 microM) and nitroprednisolone (EC(50), 15.1+/-1.4 microM) caused concentration-related relaxation of noradrenaline (NA)-contracted rat aortic rings. All NO releasing compounds tested were approximately three orders of magnitude less potent than sodium nitroprusside (SNP, EC(50), 35.7+/-3.5 nM). The vasorelaxant effect of NOF and NOPARA in the rat aorta was potentiated by zaprinast (5 microM) and reduced by ODQ (5 microM). Flurbiprofen and paracetamol (100 microM) caused minimal (<10%) relaxation of the rat aorta and did not affect the response to SNP. The effect of NOF was unchanged in the presence of L-NAME (100 microM; EC(30), 181.8+/-35.1 microM cf. EC(30), 125.1+/-17.0 microM, P>0.05) but increased by removal of the endothelium (EC(30), 164.3+/-26.3 microM cf. EC(50), 688.8+/-93.8 microM, P<0.05). NOF (0.1 - 50 microM) produced a small but not concentration-related vasodilation of the NA-preconstricted (i.e. "high tone") perfused rat mesentery preparation (cf. SNP, EC(30), 4.4+/-0.7 microM). In contrast, NOF (1 - 100 microM) produced concentration-related vasodilation of the "high tone" perfused rat kidney with an EC(50) of 33.1+/-4.4 microM. Neither NOF (74 mg kg(-1), i.p.) nor NOA (91.9 mg kg(-1), i.p.) nor equimolar doses of flurbiprofen (50 mg kg(-1), i.p.) or aspirin (50 mg kg(-1), i.p.) affected mean arterial blood pressure (MAP) or heart rate (HR) of pentobarbitone-anaesthetized rats over a 1 h period. NO-NSAID relax blood vessels in vitro by an NO-dependent mechanism. The absolute vasorelaxant effect of NO releasing drug varies greatly with the choice of compound and between blood vessel preparations.

A comparison of the effect of nitroparacetamol and paracetamol on liver injury.

Paracetamol (5 mmol kg(-1), i.p.) caused liver damage in rats as indicated by increased plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT) and glutamate dehydrogenase (GDH) activities. No change in plasma bilirubin or creatinine was noted. An equimolar dose of nitroparacetamol (a nitric oxide (NO)-releasing derivative of paracetamol) did not alter plasma levels of any of the markers of liver/kidney damage. No difference in plasma or liver paracetamol was apparent in animals injected with paracetamol or nitroparacetamol. These results indicate that NO released from nitroparacetamol exhibits hepatoprotective activity in these animals and suggest that nitroparacetamol may therefore be considered as a safer alternative to paracetamol in the clinic.

Nitroparacetamol exhibits anti-inflammatory and anti-nociceptive activity.

Nitroparacetamol (NCX-701) is a newly synthesized nitric oxide-releasing derivative of paracetamol. Following i.p. administration, nitroparacetamol inhibits carrageenan-induced hindpaw oedema formation (ED(50), 169.4 micromol kg(-1)) and mechanical hyperalgesia (ED(50), 156 micromol kg(-1)) in the rat. In contrast, the parent compound, paracetamol, exhibits no significant anti-oedema activity in this model (ED(50)>1986 micromol kg(-1), i.p. ) and is markedly less potent than nitroparacetamol as an inhibitor of carrageenan-mediated hyperalgesia (ED(50), 411.6 micromol kg(-1), i.p.). In a second model of nociception (inhibition of acetic acid induced abdominal constrictions in the mouse), nitroparacetamol administered orally (ED(50), 24.8 micromol kg(-1)), was again considerably more potent than paracetamol (ED(50), 506 micromol kg(-1), p.o.). Thus, compared with paracetamol, nitroparacetamol not only exhibits augmented antinociceptive activity in both rat and mouse but, intriguingly, is also anti-inflammatory over a similar dose range.

A comparison of the anti-inflammatory and anti-nociceptive activity of nitroaspirin and aspirin.

1. Nitroaspirin (2.5 - 50 mg kg(-1), i.p. or 2.5 - 100 mg kg(-1), p.o.) and aspirin (2.5 - 100 mg kg(-1), i.p. or p.o.) exhibit anti-inflammatory activity in the carrageenan-induced hindpaw oedema model in the rat. When administered i.p., nitroaspirin was a more effective anti-oedema agent than aspirin particularly in the 'early' phase (i.e. up to 60 min) of the response. The ED(50) values for nitroaspirin and aspirin as inhibitors of the 'late' phase response (measured at 180 min) were 64.3 micromol kg(-1) and >555 micromol kg(-1), respectively. When administered p.o., neither nitroaspirin nor aspirin exhibited significant anti-inflammatory activity in the 'early' phase and were of similar potency in the 'late' phase. Thus, at the highest dose used (100 mg kg(-1), 360 min) orally administered nitroaspirin (aspirin in parenthesis) inhibited oedema formation by 46.9+/-1.6% (47.2+/-3.8%, both n=6, P<0.05). 2. Nitroaspirin and aspirin (25 - 200 mg kg(-1), p.o.) caused dose-related inhibition of the hyperalgesia to mechanical stimulation following intraplantar injection of carrageenan in the rat. ED(50) values were 365 micromol kg(-1) and 784 micromol kg(-1), respectively. Neither drug influenced the threshold for mechanical stimulation in the contralateral (i.e. untreated) hindpaw. 3. Nitroaspirin and aspirin (2.5 - 100 mg kg(-1), p.o.) caused dose-related inhibition of acetic acid induced abdominal constrictions in the mouse (ED(50) values of 154.7 micromol kg(-1) and 242.8 micromol kg(-1), respectively). 4. Nitroaspirin and aspirin (>200 mg kg(-1), p.o.) reduced the 'late' phase (but not the 'early' phase) of the formalin-induced hindpaw licking assay in the mouse. Similarly, nitroaspirin and aspirin (>50 mg kg(-1), p.o.) prolonged tail withdrawal latency following application of a noxious heat stimulus in the mouse.