The lazaroid, U-74389G, inhibits inducible nitric oxide synthase activity, reverses vascular failure and protects against endotoxin shock

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Abstract

The aim of our study was to investigate the effect of the 21-aminosteroid U-74389G [21-<4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl-pregna-1,4,9,(11) triene-3,20-dione(z)-2-butenedionate] on the l-arginine-nitric oxide (NO) pathway in a rat model of endotoxin shock. Endotoxin shock was produced in male rats by a single intravenous (i.v.) injection of 20 mg/kg of Salmonella Enteritidis lipopolysaccharide (LPS). Rats were treated with U-74389G (7.5, 15 and 30 mg/kg i.v.) or vehicle (1 ml/kg i.v.) 5 min after endotoxin challenge. Lipopolysaccharide administration reduced survival rate (0%, 72 h after endotoxin administration) decreased mean arterial blood pressure, enhanced plasma concentration of bilirubin and alanine aminotransferase and increased plasma nitrite concentrations. Lipopolysaccharide injection also increased the activity of inducible NO synthase in the liver and in the aorta. Furthermore aortic rings from shocked rats showed a marked hyporeactivity to phenylephrine (1 nM–10 μM). In addition lipopolysaccharide (50 μg/ml for 4 h) in vitro stimulation significantly increased nitrite production in peritoneal macrophages harvested from normal rats. Treatment with U-74389G (15 and 30 mg/kg i.v., 5 min after endotoxin challenge) significantly protected against lipopolysaccharide-induced lethality (90% survival rate 24 h and 80% 72 h after lipopolysaccharide injection, respectively, following the highest dose of the drug), reduced hypotension, ameliorated liver function, decreased plasma nitrite levels, restored the hyporeactivity of aortic rings to their control values and inhibited the activity of inducible NO synthase in the liver and in the aorta. Finally, U-74389G in vitro (12.5, 25 and 50 μM) significantly inhibited nitrite production in endotoxin stimulated peritoneal macrophages. The data suggest that U-74389G may exert beneficial effects in an experimental model of septic shock by inhibiting the activity of the inducible NO synthase.

Introduction

There is evidence to suggest that, among the various mediators implicated in endotoxemia, nitric oxide (NO), together with tumor necrosis factor and oxygen-derived free radicals, is involved in the pathogenesis of endotoxin shock (Morgan et al., 1987; Thiemermann and Vane, 1990). The l-arginine/NO pathway also plays an important role in other forms of circulatory shock such as haemorrhagic and splanchnic artery occlusion shock (Wright et al., 1992; Squadrito et al., 1994; Szabò and Thiemermann, 1994).

As far as endotoxin shock is concerned, the administration of bacterial lipopolysaccharide to experimental animals induces severe metabolic and physiological disturbances which lead to shock, vascular failure and death by multi-organ system failure. These pathological sequelae are induced by endotoxin which triggers the release of a large number of mediators, including cytokines, arachidonic acid metabolites, reactive oxygen metabolites and more specifically NO from several cell types.

In the last few years, there has been increasing interest in the physiological and pathophysiological roles of NO in sepsis and experimental endotoxemia.

NO is produced by three different synthases: the inducible NO synthase, the constitutive brain NO synthase, and the constitutive NO synthase in endothelial cells.

Controversial results have been reported regarding the positive effects of increasing or decreasing NO production during endotoxic shock (Wang et al., 1994; Gundersen et al., 1997).

It has been suggested that NO derived from endothelial NO synthase plays a protective role in endotoxin-induced gastrointestinal damage; on the other hand a reduced formation of NO in the endothelium can induce vasoconstriction, microvascular thrombosis and death (Hutcheson et al., 1990). Nitric oxide produced in macrophages after the induction of inducible NO synthase by lipopolysaccharide (or other agents) may have an important role in the antimicrobial activity of activated macrophages (Nathan, 1992), but the increased production of NO by inducible NO synthase is responsible for some pathological features (hypotension, myocardial dysfunction and a decreased response to vasoconstrictors) of endotoxic shock (Nava et al., 1992). Thus, it has been suggested that modulation of NO produced by inducible NO synthase could be beneficial in experimental models of sepsis.

The 21-aminosteroid, U74389G [21-<4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl-pregna-1,4, 9,(11)-triene-3,20-dione(z)-2-butenedionate] has been shown to inhibit lipid peroxidation (McCall et al., 1987). Moreover, it has been demonstrated that the inhibition of lipid peroxidation improves the survival rate of endotoxemic rats and sheeps (Kunimoto et al., 1987; Semrad et al., 1993; Remmers et al., 1996).

In a rat model of splanchnic artery occlusion shock (Squadrito et al., 1995) and during ischemia and reperfusion of rat myocardium (Campo et al., 1996), U-74389G has been reported to have anti-shock, endothelial and cardiac protective actions.

Thus, our study was carried out to further evaluate the effects of this compound on the l-arginine-nitric oxide (NO) pathway in a rat model of endotoxin shock. U-74389G was found to reduce the pathological sequelae associated with endotoxemia by inhibiting inducible nitric oxide activity.

Section snippets

Endotoxin shock procedure

Male Sprague–Dawley rats (200–250 g) fed on a standard diet and with water ad libitum, were used. Environmental conditions were standardized, including a room temperature of 22±2°C and 12 h artificial lighting. The experiments were approved by the Ethical Committee of the University of Messina. Salmonella enteritidis lipopolysaccharide was dissolved in sterile 0.9% NaCl at a concentration of 20 mg/ml. Endotoxin shock was induced, under light anaesthesia with ether, by administering a single

Survival rate

Table 1 shows the ratio of animals surviving in each group to the total number of animals throughout the experimental period. All control rats survived the entire period of the study. The endotoxin treated group had 1 and 0 survivors out of 10 within 24 and 72 h, respectively. U-74389G, administered 5 min after LPS injection, significantly protected against endotoxin induced lethality. The endotoxin shocked group treated with 30 mg/kg of U-74389G had 9 survivors and 8 survivors out of 10 after

Discussion

Our data confirmed that U-74389G has beneficial effects in experimental shock, as previously shown (Squadrito et al., 1995; Remmers et al., 1996) and furthermore add new information regarding the underlying mechanism of action.

We found that U-74389G administration was able to increase the resistance of rats to the pathophysiological consequences of endotoxin shock. The effect was marked in terms of survival rate, protection of liver function and improvement in vascular dysfunction.

The latter

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