Beneficial effects of 5-aminoisoquinolinone, a novel, potent, water-soluble, inhibitor of poly (ADP-ribose) polymerase, in a rat model of splanchnic artery occlusion and reperfusion

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Abstract

Poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated by strand breaks in DNA, plays an important role in the tissue injury associated with ischemia–reperfusion and inflammation. Splanchnic artery occlusion and reperfusion causes an enhanced formation of reactive oxygen species which contribute to the pathophysiology of shock. The aim of the present study was to investigate the effects of 5-aminoisoquinolinone (5-AIQ), a potent water-soluble inhibitor of poly(ADP-ribose) polymerase (PARP), in the pathogenesis of splanchnic artery occlusion shock. Splanchnic artery occlusion shock was induced in rats by clamping both the superior mesenteric artery and the celiac artery for 45 min, followed thereafter by release of the clamp (reperfusion). At 60 min after reperfusion, all animals were sacrificed for histological examination and biochemical studies. Treatment of rats with 5-AIQ (3 mg/kg i.v.), attenuated the fall of mean arterial blood pressure caused by splanchnic artery occlusion shock. 5-AIQ also attenuated the ileum injury as well as the increase in the tissue levels of myeloperoxidase and malondialdehyde caused by splanchnic artery occlusion shock in the ileum. The immunohistochemical examination also demonstrated a marked increase in the immunoreactivity to PAR, nitrotyrosine, and intercellular adhesion molecule (ICAM-1) in the necrotic ileum from splanchnic artery occlusion-shocked rats. 5-AIQ treatment significantly reduced the increase of positive staining for PAR, nitrotyrosine and ICAM-I. In conclusion, these results show that 5-AIQ, a new water-soluble potent inhibitor of poly(ADP-ribose) polymerase, exerts multiple protective effects in splanchnic artery occlusion/reperfusion shock.

Introduction

Poly(ADP-ribose) polymerase-1 (PARP-1) is a member of the PARP enzyme family consisting of PARP-1 and several recently identified novel poly(ADP-ribosylating) enzymes. PARP-1 is an abundant nuclear protein functioning as a DNA nick sensor enzyme. Upon binding of DNA breaks, activated PARP cleaves NAD+ into nicotinamide and ADP-ribose and polymerizes the latter onto nuclear acceptor proteins including histones, transcription factors, and PARP itself. Oxidative stress-induced overactivation of PARP consumes NAD+ and consequently ATP, culminating in cell dysfunction or necrosis (Du et al., 2003). PARP inhibitors, such as nicotinamide and 3-aminobenzamide have been used previously in both in vivo and in vitro studies investigating their beneficial role in various pathophysiological conditions ranging from ischemia–reperfusion injury to inflammation (Cuzzocrea et al., 1999).

There is good evidence that various chemically distinct inhibitors of PARP activity including 3-aminobenzamide, nicotinamide and 1,5-dihydroxyisoquinolinone 5-hydroxyisoquinolin-1-2H-one reduce the degree of tissue injury associated with regional ischemia and reperfusion of the heart Thiemermann et al., 1997, Zingarelli et al., 1997a, Zingarelli et al., 1997b, Bowes et al., 1998b, Docherty et al., 1999, the brain (Eliasson et al., 1997), the gut (Cuzzocrea et al., 1997) and the kidney (Chatterjee et al., 2000). Most notably, the degree of tissue injury caused by ischemia and reperfusion of the heart Grupp et al., 1999, Pieper et al., 2000, brain (Eliasson et al., 1997) and ileum (Liaudiet et al., 2000b) is attenuated in mice in which the gene for PARP has been disrupted by gene targeting PARP knock mice.

However, 3-aminobenzamide is a weak inhibitor of PARP activity that does not readily cross cell membranes Bowes et al., 1998a, Bowes et al., 1999. Although 1,5-dihydroxyisoquinoline and 3,4-dihydro-5-[4-(piperidin-1-yl)butoxy]isoquinolin-1(2H)-one (DPQ) are more potent inhibitors of PARP activity, these agents have to be dissolved in dimethylsulfoxide (DMSO). DMSO itself is a potent scavenger of hydroxyl radicals and inhibits PARP activity. Thus, there is still a great need for the development of potent, water-soluble inhibitors of PARP activity. In 1991, Suto et al. (1991) reported that 5-aminoiso-quinolin-1(2H)-one (5-AIQ) is a water-soluble inhibitor of PARP activity in a cell-free preparation (enzyme purified 900-fold from calf thymus). Recently, we have reported a novel route for the synthesis of 5-aminoisoquinoline (5-AIQ) and demonstrated that this compound is a water-soluble, potent inhibitor of PARP activity in human cells (Mc Donald et al., 2000). Most notably, recent study have clearly demonstrated that 5-AIQ reduced tissue injury in various experimental models including hemorrhagic shock, liver (Mota-Filipe et al., 2002) and heart (Mc Donald et al., 2000) ischemia and reperfusion and in acute inflammation. Based on these previous studies, we have investigated the effect of 5-AIQ in a model of gut ischemia and reperfusion injury (splanchnic artery occlusion shock). The results of the current study confirm the important role of PARP activation in the pathophysiology of splanchnic artery occlusion shock and support the previous suggestion that pharmacological inhibition of PARP may be a novel approach or therapeutic potential in ischemia–reperfusion injury.

Section snippets

Animals

Male Sprague Dawley rats (250–300 g; Charles River, Milan, Italy) were housed in a controlled environment and provided with standard rodent chow and water. Animal care was in compliance with the Italian Law relating to the protection of animals used for experimental and other scientific purposes (D.M. 116192) as well as with the EEC regulations (O.J. of E.C. L 358/1 12/18/1986). The experiments were performed in adherence to the National Institutes of Health Guidelines on the Use of Laboratory

Protective effects of 5-AIQ in splanchnic artery occlusion shock

Occlusion of the splanchnic arteries produced a continuous decline in mean arterial blood pressure (Fig. 1A). At histological examination of the small intestine at 60 min of reperfusion (see representative section at Fig. 2), we found the following pathologic changes. The ileum showed infiltration with neutrophils, lymphocytes and plasma cells, extending through the entire wall, with a number of cells being concentrated below the epithelial layer. We found some evidence of focal ulceration,

Discussion

Occlusion of the splanchnic arteries followed by reperfusion in anaesthetized rats results in an irreversible circulatory failure and shock. This model of shock is characterized by a marked decrease in systemic blood pressure and leucopenia as well as by disturbances in reticuloendothelial system activity, increased macrophage and plasma levels of thromboxane B2 and elevated plasma levels of platelet-activating factor Lefer and Lefer, 1993, Zimmermann et al., 1993, Squadrito et al., 1994.

There

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