Br. J. Pharmacol. (1991), 104, 811-816

,"-.

Macmillan Press Ltd, 1991

Effect of carbenoxolone on the biological activity of nitric oxide: relation to gastroprotection 'A. Dembinska-Kiec, D. Pallapies, Th. Simmet, *B.M. Peskar & 2B.A. Peskar Department of Pharmacology and Toxicology and *Department of Experimental Clinical Medicine, Ruhr-University of Bochum, Universitatsstr. 150, D-4630 Bochum 1, Germany 1 The interactions between carbenoxolone and nitric oxide (NO) were examined by investigating their effects on human platelet aggregation, on rat aortic strips precontracted by phenylephrine and on protection of rat gastric mucosa against ethanol-induced injury. 2 Carbenoxolone (100-300,UM) caused a significant and concentration-dependent potentiation of rat peritoneal neutrophil (RPN)-, 3-morpholino-sydnonimine (SIN-1)- or iloprost-induced inhibition of platelet aggregation. Higher concentrations (5001uM) of carbenoxolone alone markedly inhibited platelet aggregation. Pretreatment with carbenoxolone (100-300pM) antagonized the reversal of the RPN- or SIN-1induced antiaggregatory effect by oxyhaemoglobin (10Mm). 3 Rat aortic strips with intact endothelium precontracted by phenylephrine (0.1-0.31uM) were relaxed by carbenoxolone (100-300,uM) in a concentration-dependent manner. Relaxations were abolished by mechanical removal of the endothelium or by incubation with methylene blue (10pM) or NG-nitro-L-arginine (LNNA, 100pgM). Sodium nitroprusside (10 nM)-induced relaxations of endothelium-denuded rat aortic strips were potentiated by carbenoxolone (100 UM). 4 The carbenoxolone (200mg kg-1, p.o.)-induced gastroprotection against ethanol was antagonized by L-NNA (5-4Omgkg- 1) in a dose-dependent manner. Pretreatment of rats with indomethacin (lOmgkg 1, s.c.) increased the effect of L-NNA. 5 The results suggest that the activity of carbenoxolone in the experimental systems tested is due to phosphodiesterase inhibition, although radical scavenging properties of the drug could contribute to some of the effects observed. In the rat gastric mucosa both increased prostaglandin levels and effects on the NO system could contribute to the protective action of carbenoxolone. Keywords: Carbenoxolone; nitric oxide; gastroprotection; platelet aggregation; rat aortic strips; platelet leukocyte interaction; NG-nitro-L-arginine (L-NNA); 3-morpholino-sydnonimine (SIN-1)

Introduction Carbenoxolone is the synthetic hemisuccinate of the triterpenoid 18fl-glycyrrhetinic acid, which in turn is derived from glycyrrhizic acid, a biologically active ingredient of the liquorice root. Carbenoxolone protects the gastric mucosa against a variety of noxious agents in experimental animals (Derelanko & Long, 1981; Martin et al., 1984) and accelerates peptic ulcer-healing in man (Doll et al., 1962) without inhibiting acid secretion (Baron, 1977). The gastroprotective effect is diminished, but not abolished, by the cyclo-oxygenase inhibitor, indomethacin (Wan & Gottfried, 1985) suggesting that endogenous prostanoids mediate part of the gastroprotection. Other mediators could contribu~te to the carbenoxolone effect. We have, therefore, investigated the effect of carbenoxolone on the biological activity of endothelium-derived relaxing factor/nitric oxide (NO), another gastroprotective mediator system (Peskar et al., 1991). Carbenoxolone was tested in two NO-producing experimental models, rat peritoneal neutrophils (RPN) and rat aorta. In addition, we have studied whether the protective effect of carbenoxolone against ethanol-induced gastric mucosal damage in rats is affected by pretreatment with an inhibitor of endogenous NO biosynthesis.

Methods Preparation of rat peritoneal neutrophils RPN were harvested from male Wistar rats (200-250g) by

peritoneal lavage 6 h after i.p. injection of 15 ml of a 0.6% sol1 Present address: Department of Pharmacology, N. Copernicus Academy of Medicine, Cracow, Poland. 2 Author for correspondence.

ution of oyster glycogen similar to the procedure described by Palmer et al. (1980). After hypoosmotic lysis of contaminating erythrocytes in 0.2% saline, RPN were resuspended in oxygenated calcium-free Tyrode solution at a final concentration of 2 x 107 cells ml- l. The RPN preparation contained at least 95% polymorphonuclear neutrophils and the viability was more than 97% as assessed by the trypan blue exclusion test.

Preparation of washed human platelets and aggregation bioassay Blood was obtained from healthy volunteers who had not taken any medication for at least 2 weeks. Washed platelets were prepared as described by Radomski & Moncada (1983). The cells were suspended in oxygenated calcium-free Tyrode solution. After addition of calcium and magnesium ions (1 mm each), 1 ml aliquots of the platelet suspensions were incubated at 370C for 3 min in an Eppendorf aggregometer. Aggregation was induced by addition of a submaximal (30-60mumlP ) concentration of bovine thrombin. The decrease in optical density was monitored for 4min after thrombin addition. In experiments with oxyhaemoglobin the aggregation was monitored for up to 15min after addition of thrombin. Inhibition of platelet aggregation was calculated as the percentage decrease of optical density determined after 4min induced by thrombin alone.

Inhibition of aggregation by rat peritoneal neutrophils, 3-morpholino-sydnonimine (SIN-I) or iloprost RPN (0.5-8.0 x 105 cells in a volume of 10-401u) or SIN-1 (final concentration 0.35-7,pM) or iloprost (final concentration 0.05-1 nM) were added to the platelet suspensions 1 min before thrombin. In experiments with carbenoxolone the drug was added to the platelets 30s before RPN, SIN-1 or iloprost,

812

A. DEMBINSKA-KIEC et al.

respectively. Oxyhaemoglobin (1OpM) was added either 30s or 10min after thrombin. SIN- I was dissolved immediately before use in distilled water acidified to pH 5.2 with 1 N HCL. Carbenoxolone disodium salt and oxyhaemoglobin were dissolved in saline. A stock solution of iloprost (lOOgml-') was diluted in saline to the concentrations desired. In control experiments the solvents were added to the platelet suspensions instead of the drugs.

F.R.G. Methylene blue, phenylephrine, carbachol, sodium nitroprusside, N0-nitro-L-arginine (L-NNA), L-arginine HCl, D-arginine HCI, indomethacin and oyster glycogen (type II) were purchased from Sigma Chemicals, St. Louis, MO, U.S.A. Haemoglobin was purified from bovine haemoglobin type I (Sigma) as described by Martin et al. (1985). Bovine thrombin was obtained from Behringwerke, Marburg, F.R.G.

Drug effects on endothelium-dependent relaxations ofrat aortic strips

Results are expressed as means + s.e.mean. Statistical analysis of parametric data was performed by use of Student's t test for unpaired data. In the case of multigroup comparisons tstatistics were corrected by the Bonferroni method. The Wilcoxon rank test was used for statistical comparison of non-parametric data. A probability of P < 0.05 was considered as statistically significant.

Spirally cut strips of rat thoracic aorta with intact endothelium were mounted in 5 ml organ baths containing KrebsHenseleit solution of the following composition (mM): NaCl 118, KCl 4.7, KH2PO4 1.2, NaHCO3 25, MgSO4 1.2, CaCl2 2.5 and glucose 11. The solution was continuously gassed with 95% 02/5% CO2 at 37°C. The preload applied to the strips was 0.7 g. Contractions and relaxations were recorded with an isotonic lever transducer (Hugo Sachs, Hugstetten, F.R.G.) and displayed on a Watanabe multipen recorder. The tissues were allowed to equilibrate for 60 min with buffer changes every 10-15min. Then the strips were precontracted by phenylephrine (0.10.3pM) and the functional preservation of endothelium was tested by the induction of relaxations with carbachol (0.11 pM). Carbenoxolone was used in concentrations between 100 and 300pM. In all experiments sodium nitroprusside (NaNP, 1-20 nM), which elicits endothelium-independent relaxations (Rapoport & Murad, 1983), was used as a reference compound. Results are expressed as NaNP equivalents. In further experiments, the effects of a 1 h preincubation with methylene blue (10pM) or NG-nitro-L-arginine (L-NNA, 100pM) on drug-induced relaxations of the rat aortic strips were investigated. In an additional series of experiments, the relaxing effects of carbachol and carbenoxolone were tested before and after mechanical removal of the endothelium from the aortic strips.

Studies on gastric mucosal protection Male Wistar rats (180-220g) were fasted overnight with free access to water. Carbenoxolone disodium salt (200mgkg-', suspended in 0.25% carboxymethylcellulose) was administered by oral intubation. Controls received the vehicle (2 ml kg '). Groups of 5-7 rats received i.v. injections of graded doses of L-NNA (5-40mgkg-', dissolved in saline) or solvent 20min before carbenoxolone. Thirty min after carbenoxolone administration, 1.5ml of absolute ethanol was instilled into the stomach. Rats were killed 5 min later, the stomachs were removed, opened and gross mucosal damage was assessed by calculating a lesion index based on the number and length of haemorrhagic mucosal necroses as described previously (Peskar et al., 1986). In additional experiments, rats were

Statistics

Results Effects of carbenoxolone and oxyhaemoglobin on the inhibition ofplatelet aggregation by rat peritoneal neutrophils, SIN-I and iloprost RPN (0.5-6.0 x 105 cells) added to 1 ml of platelet suspension, SIN-1 (0.5-3#M) and iloprost (0.1-1.0nM) inhibited thrombin (30-60 mu)-induced platelet aggregation. The sensitivity of platelets varied between donors and a concentration of the inhibitors was selected that moderately inhibited aggregation (Figure la-c). The effects of RPN, SIN-1 and iloprost were significantly and concentration-dependently enhanced by carbenoxolone (Figure la-c). Carbenoxolone itself inhibited thrombin-induced platelet aggregation to a variable extent. Thus, at concentrations of 100 to 300pM, inhibition by 10-100% was observed (data not shown). Sensitivity of platelets to carbenoxolone was constant within one experiment, but varied between donors. For the experiments shown in Figure 1, only platelets were used that exhibited maximally 15% inhibition of aggregation on exposure to 300pM carbenoxolone. Complete inhibition of aggregation by RPN (1.58 x 105 cellsmlP ) or SIN-1 (3-10pM) was abolished by oxyhaemoglobin (10pM). This compound was effective, when added 30s, but not 10min, after thrombin. Pretreatment with a concentration of carbenoxolone (100-300pM) that potentiated the antiaggregatory effects of RPN, but inhibited platelet aggregation maximally by 15% when given alone, antagonized the action of oxyhaemoglobin (Figure 2). Similarly, carbenoxolone antagonized the oxyhaemoglobin effect on the inhibition of platelet aggregation by SIN-1 (data not shown). On the other hand, ox~rhaemoglobin did not influence the antiaggregatory effect of high concentrations of carbenoxolone alone (300-500 pM).

treated with L-arginine HCI or D-arginine HCI (100mgkg-t each) immediately prior to injection of 40mgkg-' L-NNA. L-Arginine HCI and D-arginine HCl were freshly dissolved in

Drug effects on the phenylephrine-induced tone of rat aortic strips

isotonic saline and administered i.v. in a volume of 2 ml kg-1. Further groups of rats received indomethacin (10mgkg-', s.c.) 60min before i.v. injection of L-NNA (30mgkg-1) or saline. Ten min after administration of L-NNA, rats were treated with carbenoxolone (200mgkg-1, p.o.) followed by intragastric instillation of 1.5ml of absolute ethanol after a further 30 min period.

Rat aortic strips with intact endothelium precontracted with phenylephrine (0.1-0.3 pM) were relaxed by carbachol (0.11 pM, data not shown) and carbenoxolone (Figure 3) in a concentration-dependent manner. On a molar basis carbenoxolone was about 300-500 times less potent than carbachol. Mechanical removal of the endothelium or incubation of the aortic strips with methylene blue (10pM) or L-NNA (100pM) inhibited the carbachol- (data not shown) and carbenoxolone (300 pM)-induced (Figure 4) relaxations. Carbenoxolone (100puM) potentiated the NaNP-induced relaxations of endothelium-denuded rat aortic strips. When NaNP (10nM)induced relaxations were taken as 100%, relaxations in the presence of carbenoxolone (100pM) were 153 + 17% (P < 0.02, n = 6).

Reagents 3-Morpholino-sydnonimine (SIN-1) was a gift from CassellaRiedel-Pharma, Frankfurt, F.R.G. Carbenoxolone disodium salt was a gift from Biorex Laboratories, Enfield, Middlesex, and iloprost was kindly provided by Schering AG, Berlin,

CARBENOXOLONE, NITRIC OXIDE AND GASTROPROTECTION a

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RPN + Oxy-Hb CX(100 AM) + Cx(300pM) Figure 2 Effect of carbenoxolone (Cx, 100 and 300,pM) on the reversal of the antiaggregatory effect of rat peritoneal neutrophils (RPN, 1.5-8.0 x 105 cellsmlP ) by oxyhaemoglobin (Oxy-Hb, 10pM) added 30s after thrombin. In these experiments a concentration of RPN was selected that induced 100% inhibition of platelet aggregation, while exposure to 300pM carbenoxolone alone caused maximally 15% inhibition of aggregation. Results show % reversion of inhibition of the antiaggregatory effect of RPN and are expressed as mean of 4-5 experiments per group; vertical bars show s.e.mean. *P < 0.02, **P < 0.002 as compared to the effect of oxyhaemoglobin alone.

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Drug effects on ethanol-induced gastric mucosal damage Gastric mucosal damage caused by ethanol was dosedependently aggravated by i.v. administration of L-NNA (540mgkg-1) (Figure 5). Treatment with 200mgkg-1

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Figure 1 Effect of carbenoxolone (Cx, 100 and 300pM) on the inhibition of thrombin (30-60 mu ml - )-induced aggregation of human platelets by rat peritoneal neutrophils (RPN, 0.5-6.0 x 101 cells, a), 3-morpholino-sydnonimine (SIN-1, 0.5-3 pM, b) and iloprost (0.11.OnM, c). In these experiments 300puM carbenoxolone alone caused maximally 15% inhibition of aggregation. Results, shown as % inhibition of thrombin-induced platelet aggregation, are expressed as mean of 4-9 experiments per group; s.e.mean shown by vertical bars. Significant difference from RPN, SIN-i and iloprost, alone is shown as *P < 0.02, **P < 0.01 and ***P < 0.002, respectively.

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Cx(100 FM) Cx(150 FM) Cx(300 FM) Figure 3 Relaxation of rat aortic strips with endothelium by carbenoxolone (100-300AM). Results are given in sodium nitroprusside (NaNP)-equivalents (nM) and represent the mean of 8 experiments per group; vertical bars show s.e.mean.

Table 1 Effects of administration of N0-nitro-L-arginine (L-NNA), L-arginine, D-arginine and indomethacin on the gastroprotective action of carbenoxolone Dose Treatment Controls + saline + L-NNA + indomethacin Carbenoxolone + saline + L-NNA + L-NNA + L-NNA + L-NNA + indomethacin + indomethacin

(mgkg -)

(mgkg 1)

40 10 200

40 40 40 30 10 10

+L-arginine +D-arginine

+L-NNA

100 100

30

Lesion index

(n)

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(6)

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(7) (6) (6) (8)

(10)

(7)

Results, shown as lesion index of gross mucosal damage, are mean _ s.e.mean of n animals. Statistical difference is shown as aP < 0.01 and bP < 0.001 vs. saline controls; CP < 0.001 vs. carbenoxolone + L-NNA (40mgkg-1); *P

Effect of carbenoxolone on the biological activity of nitric oxide: relation to gastroprotection.

1. The interactions between carbenoxolone and nitric oxide (NO) were examined by investigating their effects on human platelet aggregation, on rat aor...
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