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.

Gastric protection against cold restraint stress-induced lesions by amoxycillin in rats.

AIMS: Recent investigations have shown that amoxycillin possesses gastric protection properties in addition to its known antimicrobial effects. Therefore, this study was undertaken to investigate the potential gastric protection effects of amoxycillin and to determine its possible mechanism(s) of action in rats. METHODS: The cold restraint stress model was used to produce gastric mucosal lesions. The gastric secretion studies were undertaken by using Shay's pylorus ligation technique. The antioxidant effect was studied by luminol dependent chemiluminescence technique in vitro. RESULTS: Amoxycillin dose-dependently prevented cold restraint stress-induced mucus depletion and afforded protection. It inhibited indomethacin-stimulated gastric acid secretion with a high dose without affecting basal secretion. Furthermore, amoxycillin dose-dependently inhibited the phorbol myristate acetate-stimulated luminol-dependent chemiluminescence responses of isolated human poylmorphonuclear leukocytes in vitro. CONCLUSIONS: These results suggest that mechanisms of gastric protection effects of amoxycillin may include inhibition of stimulated acid secretion, prevention of depletion of mucus and antioxidant properties.

Effect of ablation of capsaicin-sensitive neurons on gastric protection by honey and sucralfate.

BACKGROUND/AIMS: Accumulating evidence indicates that capsaicin-sensitive afferent neurons play a pivotal role in acute gastroprotection by liberating vasodilator substances. The mechanism of gastric protection by honey and sucralfate has been shown to be mediated through the vasodilator nitric oxide and cytoprotective sulphydryl-sensitive pathways in the stomach. The aim of the present study was to investigate the role of capsaicin-sensitive afferent neurons in the protective mechanism of honey and sucralfate against ethanol-induced gastric lesions. METHODOLOGY: Ablation of capsaicin-sensitive afferent neurons was carried out by treating rats with neurotoxic doses of capsaicin (50 + 50 mg/kg subcutaneously over 2 consecutive days). The control groups received equal volumes of the vehicle (10% ethanol + 10% Tween 80 + 80% normal saline). The non-protein sulphydryl level was determined spectrophotometrically. RESULTS: Afferent sensory nerve ablation significantly aggravated ethanol-induced gastric lesions and caused a greater depletion of non-protein sulphydryl levels. Pretreatment with honey (0.078-0.625 g/kg, orally) or sucralfate (0.062-0.250 g/kg, orally) 30 min before administration of the ethanol prevented ethanol-induced gastric lesions and the depletion of non-protein sulphydryls in vehicle-treated rats. However, honey failed to afford protection or reverse non-protein sulphydryl depletion, while sucralfate was effective in the capsaicin-treated animals. The protective effect of sucralfate was lost in both groups following pretreatment with indomethacin (10 mg/kg, subcutaneously), while honey-induced protection was unaffected. CONCLUSIONS: These results suggest that the gastric protection by honey is solely dependent on the presence of intact afferent sensory neurons, whereas sucralfate-induced gastroprotection is mediated through the afferent sensory neuron and prostaglandin systems. It seems that the non-protein sulphydryl level is affected by the ablation of sensory neurons and plays an important regulatory role in gastric protection.

Protective effect of amoxycillin on ethanol-induced gastric lesions in rats.

Recent studies have suggested that amoxycillin may possess gastroprotective effects in addition to its known antimicrobial properties. Therefore, the present study was undertaken to investigate the potential gastric protective effects of amoxycillin and to determine its possible mechanism(s) of action in rats. Ethanol-induced gastric mucosal lesions in rats were used as an animal model. Nonprotein sulphydryl levels were measured spectrophotometrically. The antioxidant effect of amoxycillin was studied by luminol-dependent chemiluminescence technique in-vitro. Amoxycillin produced marked protection against ethanol-induced gastric lesions. The protective effect of amoxycillin was lost by prior treatment with a dose of indomethacin that is known to inhibit prostaglandin biosynthesis without induction of gastric ulcers. Furthermore, the concentration of nonprotein sulphydryls decreased significantly in gastric mucosa after administration of ethanol. Treatment with amoxycillin prevented this depletion. Additionally, amoxycillin dose-dependently inhibited the phorbol myristate acetate stimulated luminol dependent chemiluminescence responses of isolated human polymorphonuclear leukocytes in-vitro. These data indicate that the gastric protection effects of amoxycillin against ethanol-induced lesions may include generation of mucosal prostaglandins, prevention of nonprotein of sulphydryl depletion and antioxidant properties.

Sucralfate attenuates gastric mucosal lesions and increased vascular permeability induced by ischaemia and reperfusion in rats.

Recent evidence suggests that oxygen-derived free radicals are involved in mediating gastric microvascular and parenchymal cell injuries induced by ischaemia and reperfusion. Therefore, the effect of the locally acting anti-ulcer drug, sucralfate, was studied on ischaemia and reperfusion (e.g. induced gastric lesions, intraluminal bleeding, changes in vascular permeability and non-protein sulfhydryl levels in the rat stomach). Allopurinol was used as a known standard antioxidant drug. Rats were subjected to 30 min of gastric ischaemia in the presence of 100 mmol/L hydrochloric acid and reperfusion periods of 15, 30 or 60 min duration. The gastric lesions were assessed microscopically under an inverted microscope. The vascular permeability was quantified by measuring the extravasated Evans blue in the stomach. There were significantly greater numbers of gastric lesions, intraluminal bleeding and leakage of Evans blue during all reperfusion periods as compared with those of ischaemia, with maximum effects occurring at 60 min following reperfusion. Pretreatment with sucralfate (31.25-250 mg/kg, p.o.) or allopurinol (12.5-50 mg/kg, i.p.) 30 min before the procedure, dose-dependently reduced the gastric lesions, intraluminal bleeding, and decreased the vascular permeability induced by ischaemia and reperfusion. Furthermore, sucralfate dose-dependently reverses the ischaemia and reperfusion-induced depletion of mucosal non-protein sulfhydryl levels and inhibited the superoxide radical production in both cell-free xanthine-xanthine oxidase and in the stimulated polymorphonuclear cellular systems. These results suggest that the protection produced by sucralfate against gastric injury may be due to its antioxidant effects.

Natural honey prevents ethanol-induced increased vascular permeability changes in the rat stomach.

The effect of honey on ethanol-induced increased vascular permeability changes was studied in the rat stomach. Sucralfate and allopurinol were used as standard gastroprotective and antioxidant drugs, respectively. Extravasation of intravenously administered Evans blue dye into the stomach following 30 min exposure to ethanol was used as an indicator of vascular permeability. The amounts of the extravasated dye were quantified spectrophotometrically. Ethanol produced concentration and time-dependent increase in the extravasation of Evans blue. Oral administration of honey (0.078-0.625 g/kg) 30 min before ethanol dose-dependently attenuated ethanol-induced increased vascular permeability. Pretreatment with a sulfhydryl blocker, N-ethylmaleimide (0.050 g/kg, subcutaneously), caused enhancement of ethanol-induced vascular permeability changes. Treatment with N-ethylmaleimide before honey reduced the protective effects of honey. Similarly, sucralfate (0.031-0.250 g/kg) orally and allopurinol (0.025-0.050 g/kg) intravenously inhibited vascular permeability caused by ethanol and treatment with N-ethylmaleimide before sucralfate or allopurinol reduced their inhibitory effects. These results suggest that the protective effect of honey may be mediated through sulfhydryl-sensitive processes and it may also possess antioxidant properties. It is also suggested that endogenous sulfhydryl may facilate and mediate beneficial effects of gastroprotective and antioxidant drugs.

Enhancement of anti-inflammatory effects of calcium channel blockers by allopurinol and dimethylsulphoxide.

The effects of the calcium channel blockers, nifedipine, verapamil and flunarizine, and the antioxidants, allopurinol and dimethylsulphoxide, were investigated on carrageenan-induced rat paw oedema and changes in vascular permeability. Paw volume was measured by using a plethysmometer and vascular permeability was quantified by measuring the extravasated Evans blue dye 3 h after injecting the phlogistic agent. Intraperitoneal administration of nifedipine (1,2 and 4 mg/kg), verapamil (5, 10 and 20 mg/kg), flunarizine (2.5, 5 and 10 mg/kg), allopurinol (6.25, 12.5 and 25 mg/kg) and dimethylsulphoxide (20, 40 and 80 mg/kg) 30 min before carrageenan, dose dependently inhibited oedema formation and increased vascular permeability. Co-administration of the lowest doses of calcium channel blockers with the lowest doses of antioxidants produced synergistic inhibitory effects. These results indicate that both calcium influx and oxygen-derived free radicals are involved in carrageenan-induced inflammatory responses. Thus, the synergistic effects of their combination may be due to the blockade of calcium entry and reduction in the generation of oxygen-derived free radicals.

Selenium protects against ischemia-reperfusion-induced gastric lesions in rats.

Recent studies have shown that selenium afforded protection against ethanol and stress-induced gastric lesions in rats. The present study was undertaken to investigate the effect of selenium on ischemia-reperfusion-induced gastric injuries in which rats were subjected to 30 minutes of ischemia in the presence of 100 mM HCI and a reperfusion for 60 minutes duration. Intraluminal bleeding was assessed macroscopically and gastric lesions were graded microscopically under an inverted microscope. Nonprotein sulphydryl levels were measured spectrophotometrically. The severity of gastric lesions, intraluminal bleeding as well as the depletion of nonprotein sulphydryls during the reperfusion periods was significantly different from that of control. Pretreatment with selenium (0.125-2.0 mg/kg, intraperitoneally) 30 minutes before the ischemia-reperfusion, dose-dependently attenuated the gastric lesions, reduced the severity of intraluminal bleeding and prevented the depletion of nonprotein sulphydryls in the stomach. These results suggest that the gastric protection effect of selenium may be due to its antioxidant properties. Furthermore, endogenous nonprotein sulphydryls may play a significant role in the protective mechanisms of selenium.

Natural honey prevents ischaemia-reperfusion-induced gastric mucosal lesions and increased vascular permeability in rats.

OBJECTIVE: It has been proposed that natural honey may contain a 'sucralfate-like' substance. Recent studies have shown that sucralfate affords protection against ischaemia-reperfusion-induced injuries in the rat stomach. Therefore, the effect of honey was studied on ischaemia-reperfusion-induced gastric lesions, intraluminal bleeding, vascular permeability and non-protein sulphhydryls (NP-SH) in the rat stomach. METHODS: Rats were subjected to 30 min of gastric ischaemia in the presence of 100 mM HCl and reperfusion period of 60 min. Intraluminal bleeding was assessed macroscopically and the gastric lesions were graded microscopically under an inverted microscope. Vascular permeability was quantified by measuring spectrophotometrically the extravasated Evans blue dye in the stomach. NP-SH levels were measured spectrophotometrically. A luminol-dependent chemiluminescence method was used to assess antioxidant effects of honey in vitro. RESULTS: There were significantly more gastric lesions, more severe intraluminal bleeding, more leakage of Evans blue and depletion of NP-SH during the reperfusion period as compared to controls. Pre-treatment with honey (0.078-0.625 g/kg, orally) or dimethyl sulphoxide (0.02-0.08 g/kg, intraperitoneally) 30 min before the ischaemia-reperfusion dose-dependently reduced the gastric lesions and intraluminal bleeding and decreased the vascular permeability. Furthermore, honey reversed the ischaemia-reperfusion-induced depletion of NP-SH levels and inhibited the luminol-dependent chemiluminescence induced in a cell-free xanthine-xanthine oxidase system. CONCLUSION: These results suggest that gastric protection by honey may be a result of its antioxidant effect. It is suggested that this property of honey may be due to the presence of a 'sucralfate-like' substance.

Role of oxygen-derived free radicals on gastric mucosal injury induced by ischemia-reperfusion.

A free radical is an unstable and highly-reactive chemical species capable of independent existence that contained one or more unpaired electrons in its outer orbital. A number of oxygen-derived free radicals (ODFRs) have been identified. However, superoxide (O(-)(2) and hydroxyl (OH*) radicals are extensively studied. The univalent reduction of oxygen to water produces a number of highly-reactive chemical intermediates such as O(-)2 and OH*, which are commonly-known as oxygen-derived free radicals. ODFRS may be formed from several sources as follows: a) mitochondrial cytochrome oxidase, b) xanthine oxidase, c) neutrophils and d) transitional metals. There are several important defense mechanisms to limit or to prevent the damage caused by excessive ODFRs activity. These antioxidant defenses can be divided into a) enzymatic defense mechanisms such as: superoxide dismutase (SOD): catalase: selenium-containing glutathione peroxidase and b) non-enzymatic defense mechanisms including: alpha-tocopherol; ascorbic acid; glutathione and any sulfhydryl-containing compounds.

Rabbit brain contains an endogenous inhibitor of endothelium-dependent relaxation.

1. Supernatants prepared from the rabbit brain, lung and liver caused an endothelium-dependent and volume-related contraction of the phenylephrine-pretreated rabbit aorta and inhibited relaxation due to acetylcholine (ACh). 2. Perfusion in situ of the rabbit lung or liver with Krebs solution substantially reduced or removed the endothelium-dependent inhibitor. Spectrophotometric analysis revealed the presence of substantial amounts of haemoglobin (1.8-2.1 microM) in these organ supernatants. 3. Supernatants prepared from the Krebs-perfused rabbit brain retained the ability to contract the phenylephrine-pretreated rabbit aorta and to inhibit relaxation due to ACh and substance P (SP). Rabbit brain supernatant did not reduce the vasodilator effect of sodium nitroprusside (NP) or nitric oxide (NO). 4. Rabbit brain supernatant contained low (less than 0.35 microM) concentrations of haemoglobin. 5. The inhibitory effect of rabbit brain supernatant was reversed by L-arginine (500 microM) but not D-arginine (500 microM). 6. The inhibitor of endothelium-dependent vasodilatation present in rabbit brain was not removed by dialysis (24 h, 4 degrees C) but was partially precipitated by ammonium sulphate (30% w/v). 7. Rabbit brain contains an endogenous inhibitor of vascular NO biosynthesis. The identity of this inhibitor is not known although it seems likely to be a large peptide or protein.

L-NG-nitro arginine (L-NOARG), a novel, L-arginine-reversible inhibitor of endothelium-dependent vasodilatation in vitro.

1. The effect of L-NG-nitro arginine (L-NOARG) was compared with that of L-NG-monomethyl arginine (L-NMMA) on vasodilatation of the isolated aorta of the rabbit and perfused mesentery of the rat in response to acetylcholine (ACh) and sodium nitroprusside (NP). 2. L-NOARG (1.5-100 microM) and L-NMMA (3-100 microM) produced concentration-related contraction of the rabbit aorta precontracted with phenylephrine (700-900 nM). Similarly, L-NOARG (10-200 microM) and L-NMMA (30-100 microM) elevated perfusion pressure of the noradrenaline (NA, 0.6-2.5 mM)-preconstricted rat mesentery preparation. 3. L-NOARG (1.5-100 microM) and L-NMMA (3-100 microM) caused concentration-related inhibition of the vasodilator effect of ACh (0.01-1.0 microM) on the rabbit aorta without influencing responses to NP (0.03-0.5 microM). L-NOARG methyl ester (30 microM) also inhibited ACh-induced vasorelaxation with similar potency to NOARG. L-arginine (30-150 microM) but not D-arginine (100 microM) caused graded reversal of the inhibitory effect of both L-NOARG (15 microM) and L-NMMA (30 microM). Complete reversal of the effect of both inhibitors was achieved with 150 microM L-arginine. L-Alanine (50 microM), L-arginosuccinic acid (5 microM), L-citrulline (50 microM), L-methionine (50 microM) and L-ornithine (50 microM) failed to reverse the inhibitory effect of L-NOARG (15 microM). 4. L-NOARG (10-200 microM) and L-NMMA (30-100 microM) inhibited the vasodilator effect of ACh (0.006-18.0 nmol) in the rat mesentery without affecting vasodilatation due to NP (1.1-11.1 nmol). L-Arginine (100 microM) but not D-arginine (100 microM) produced partial reversal of the effect of L-NOARG (30 microM) and L-NMMA (30 microM). 5. L- and D-N'-butyloxycarbonyl No-nitro arginine (100 microM) produced modest (approximately 20%) inhibition of the effect of ACh on the rabbit aorta; this effect was not reversible with L-arginine (100 microM). L-Namonocarbobenzoxy arginine (L-NMCA, 5O microM), L-N-NG-dicarbobenzoxy arginine (L-NDCA, 5 microM) and L-NG-tosyl arginine (50 microM) were inactive. 6. These results identify L-NOARG as a potent, L-arginine reversible inhibitor of endothelium-dependent vasodilatation. The available data suggests that L-NOARG, like L-NMMA, inhibits endothelial nitric oxide (NO) biosynthesis.

Amino acids dilate resistance blood vessels of the perfused rat mesentery.

The vasodilator effect of several L-amino acids in the perfused, noradrenaline-preconstricted rat mesentery preparation has been investigated. N-alpha-Benzoyl-L-arginine ethyl ester (BAEE) (ED50, 1.4 +/- 0.09 mumol) and L-alanine methylester (ED50, 0.9 +/- 0.007 mumol) were the most potent although L-arginine methylester, hydroxamate and hydrochloride, N-alpha-benzoyl-L-arginine methyl ester (BAME), L-methionine methylester, L-lysine hydroxamate and L-glutamic acid methylester exhibited similar potency with ED50 values in the range 2.4-3.7 mumol. L-Homoarginine chloride was inactive at doses up to 20 mumols. D-Arginine hydrochloride and D-lysine hydroxamate were inactive at doses up to 50 mumols whilst D-methionine methylester (50 mumols) produced small falls in perfusion pressure in only 3 out of 7 preparations studied. Responses to BAEE, BAME, L-arginine hydrochloride, L-alanine methylester, L-methionine methylester, L-lysine hydroxamate and acetylcholine (but not nitroprusside) were significantly inhibited by CHAPS (4.7 mg mL-1, 30 s) de-endothelialization as well as pretreatment of mesentery preparations with gossypol (3 microM). Responses to BAEE, BAME, L-arginine hydrochloride, L-alanine methylester and acetylcholine were similarly selectively reduced by NDGA (10 microM) pretreatment. We propose that these L-amino acids exhibit vasodilator activity in the perfused rat mesentery by virtue of releasing endothelium-derived nitric oxide (EDNO).