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Anesth Analg 2005;101:1799-1804 © 2005 International Anesthesia Research Society doi: 10.1213/01.ANE.0000184188.33998.AE
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Abstract |
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The role of poly (adenosine diphosphate-ribose) synthetase (PARS) in the contractile and relaxant responses of pulmonary arteries injured by ischemia and reperfusion (IR) of splanchnic artery has not been evaluated. We examined these responses by using 3-aminobenzamide, a pharmacological inhibitor of PARS. IR models in rats were induced by clamping the superior mesenteric artery for 60 min, followed by release of the clamp for 60 min. In the 2 treated groups, 5 or 10 mg/kg of 3-aminobenzamide was administered as an IV bolus at 10 min before reperfusion, followed by infusion rates of 5 and 10 mg ·kg–1 · h–1, respectively, during the period of reperfusion (IR + PARS inhibitor 5 and 10 groups). In the vehicle-treated group, 3-aminobenzamide was not given, but IV saline was administered (IR group). In the control group, surgery was performed, but the superior mesenteric artery was not occluded (sham group). The pulmonary arteries were isolated, and effects of drugs were evaluated in vitro. The IR group showed no attenuation of the contractile responses of the pulmonary artery to phenylephrine. The relaxant responses to endothelium-dependent vasodilators, acetylcholine, and A23187 in the IR group were significantly inhibited when compared with the sham group. The reduction in the relaxant response to endothelium-dependent vasodilators was improved in the IR + PARS inhibitor 5 and 10 groups when compared with the IR group. We concluded that IR attenuated the relaxant responses of the pulmonary artery to endothelium-dependent vasodilators and that PARS inhibitors ameliorate the reduction in the relaxant response.
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Introduction |
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Poly (adenosine diphosphate [ADP]-ribose) synthetase (PARS) is a nuclear enzyme and activated by DNA strand breakage. It has been demonstrated that cell damage, such as ischemic-reperfusion (IR) injury, produces reactive oxygen species, nitric oxide (NO), and peroxynitrite, resulting in activation of PARS. Activation of PARS is thought to deplete cellular levels of nicotinamide adenine dinucleotide and adenosine triphosphate, thus resulting in irreversible cell death (1).
PARS plays an important role in cell death occurring after various types of cell injury, including IR injury, and PARS inhibitors may decrease the degree of this cell damage by inhibiting PARS (1,2). In an investigation of splanchnic artery occlusion and reperfusion in animal models, PARS inhibitors significantly improved arterial blood pressure after reperfusion, improved the contractile responsiveness to noradrenaline, and enhanced endothelium-dependent relaxation in the aortic rings (1). However, septic shock often accompanies increases in pulmonary vascular resistance. However, the role of PARS inhibitors in pulmonary arterial responses after IR has not yet been evaluated. We thus examined these responses using 3-aminobenzamide, a pharmacological inhibitor of PARS.
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Methods |
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The present experimental protocol was approved by the Laboratory Animal Care Committee. Male Sprague-Dawley rats weighing 350–450 g were quarantined in quiet, humidified rooms for 2–3 wk before use. Rats were allowed proper access to food and water up to the time of experimentation.
Tracheotomy was performed under general anesthesia with intraperitoneal pentobarbital sodium 60 mg/kg. Subsequently, the rats were mechanically ventilated with a fraction of inspired oxygen of 0.3, and mechanical ventilation was performed to maintain Paco2 at approximately 35 mm Hg (Ugo Basile Muromachi Kikai CO, Ltd., Japan). The right femoral artery was cannulated with a polyethylene tube for continuous arterial blood pressure monitoring. The right jugular vein was cannulated with a polyethylene tube for continuous IV fluid infusion (0.9% normal saline at 15 mL · kg–1 · h–1) and drug administration. Anesthesia was maintained throughout the experimental protocol (pentobarbital sodium, 10 mg · kg–1 · h–1 IV continuously). Rectal temperature was continuously monitored and maintained at 37°C through the use of a heating blanket. Laparotomy was performed, and the superior mesenteric artery (SMA) was isolated at its origin from the abdominal aorta. The SMA was occluded with a microvascular clamp. The abdominal contents were then covered with a sterile plastic wrap. After 60 min of SMA occlusion, the microvascular clamp was removed. After 60 min of mesenteric reperfusion, median sternotomy was performed. The heart/lung block was rapidly excised. In the two groups of rats treated with the PARS inhibitor, 3-aminobenzamide was given as a IV bolus 10 min before reperfusion (5 or 10 mg/kg) followed by an infusion of 5 and 10 mg · kg–1 · h–1, respectively, during the period of reperfusion (IR + PARS inhibitor 5 and 10 groups; n = 10 in each group). In the vehicle-treated group, 3-aminobenzamide was not given, but IV saline (IR group; n = 10) was administered. In the control group, surgery was performed, but the SMA was not occluded (sham group; n = 10).
Median sternotomy was performed, and heparin sulfate (200 U) was injected to the right ventricle. After removing the heart/lung block, the right and left pulmonary arteries were dissected out and placed in Earle’s balanced salts solution (EBSS) at 4°C. Under microscopic magnification, the surrounding tissue was carefully dissected from the pulmonary arteries. The right and left main branch of pulmonary arteries were cut into rings of 3–4 mm width. Two pulmonary arterial rings were obtained from each rat. EBSS contains (in mM) 1.80 CaCl2, 0.83 MgSO4 (anhydrous), 5.36 KCl, 116.34 NaCl, 0.40 NaPO4 (dibasic), 5.50 D-glucose, and 19.04 NaHCO3. The pulmonary arterial rings were then mounted in 20-mL organ chambers filled with 37°C warmed EBSS. Each organ chamber had a continuous bubbling gas flow of 21% O2, 5% CO2, and 74% N2. This flow produced a pH value of 7.35–7.45, which had been confirmed in preliminary investigation. Isometric force was measured with isometric transducers (TB-652T, Nihon Kohden, Tokyo, Japan), digitalized by a MacLab A/D converter (AD Instruments, Milford, MA), and was displayed and stored on a Macintosh personal computer (PowerBook 1400C). A tension of 500 mg was applied, and the rings were equilibrated for 60 min. Fresh EBSS was provided at 20 min intervals.
Study 1
To determine whether IR was associated with alterations in the contractility of pulmonary arteries, the rings from the IR group (n = 10) and the sham group (n = 10) were constricted with phenylephrine at 10–6 M, and the isometric tension of the pulmonary arterial rings was observed for 30 min. Response curves were obtained.
Study 2
To investigate whether IR was associated with alterations in the relaxation of pulmonary arteries, rings from the IR group (n = 10) and sham group (n = 10) were preconstricted with 10–6 M of phenylephrine, and cumulative concentration-response curves were generated over a concentration range of 10–9 to 10–6 M of acetylcholine (Ach; receptor-dependent vasodilator), A23187 (receptor-independent vasodilator), and sodium nitroprusside (SNP; endothelium-independent vasodilator). Rings were allowed to reach a steady-state before advancing to the next concentration. Concentration-response curves were expressed as the ratio of tension remaining in the rings in response to each dose of Ach, A23187, and SNP to phenylephrine-induced maximal tension. In a separate study, pulmonary arterial rings from the IR group (n = 10) and the IR + PARS inhibitor 5 and 10 groups (n = 10 in each group) were preconstricted with 10–6 M of phenylephrine. Endothelium-dependent relaxation was measured by concentration-response curves for Ach and A23187 (10–9 to 10–6 M, respectively).
To examine the effect of 3-aminobenzamide in the control group, sham group, and sham + PARS inhibitor 10-mg group (n = 10) were preconstricted with 10–6 M of phenylephrine, and cumulative concentration-response curves were generated over a concentration range of 10–9 to 10–6 M of Ach and A23187. Relaxations were calculated as percent of precontractile vascular tone.
Values are expressed as mean ± sd. Analysis of variance test was used to compare the mean values of the various experimental groups, followed by Bonferroni test for multiple comparison. Differences were considered significant when P value was <0.05.
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Results |
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There were no significant differences between the IR group and the sham group in the time course of pulmonary arterial ring contractility in response to phenylephrine (Fig. 1).
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The relaxations of the rings induced with the endothelial-dependent vasodilators, Ach, and A23187 were apparent at the concentrations of 10–7 M and 10–6 M, both in the IR group and the sham group. However, relaxant responses in the IR group were significantly inhibited in comparison with relaxant responses in the sham group (Fig. 2, A and B). Relaxation caused by SNP occurred both in the IR group and the sham group and depended upon the concentration of SNP. There were no significant differences between the IR group and the sham group at each concentration (Fig. 2C). Rings from the IR + PARS inhibitor 5- and 10-mg groups were significantly dilated in Ach (10–7 to 10–6 M) and A23187 (10–7 to 10–6 M) when compared with the rings from the IR group at each concentration (Fig. 3, A and B). There were no significant differences between the IR + PARS inhibitor 5 and the IR + PARS inhibitor 10 groups in relaxant response to Ach and A23187 at any concentration. There were no significant differences between the sham group and the sham + PARS inhibitor 10 group in relaxant response to Ach and A23187 at any concentration (Fig. 4, A and B).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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IR did not reduce the isometric contraction of pulmonary arterial rings in response to phenylephrine. Previous studies reported that IR leads to a reduction in the contractile response to norepinephrine in thoracic aortic rings (1,3). The reason for the differences between previous reports and our results is unclear, but minor differences between the protocols of the previous experiments and ours, or a vascular difference (pulmonary or aortic artery), may be responsible for the difference in results.
IR attenuated the isometric relaxant response to endothelial-dependent vasodilators, both receptor-dependent (Ach) and receptor-independent (A23187), in pulmonary arterial rings. The results indicate that the Ach receptors were not selectively damaged by IR. However, they did not indicate that Ach receptors are not damaged. However, IR did not attenuate isometric relaxant responses to endothelial-independent vasodilators (SNP) in the pulmonary arterial rings.
PARS inhibitors improved the attenuation of endothelial-dependent relaxant responses in pulmonary arterial rings. The maintenance dose of 3-aminobenzamide at 5 mg · kg–1 · h–1 has the same effect as 10 mg · kg–1 · h–1 in both Ach and A23187. In two reports (1,2), PARS inhibitors at 10 mg · kg–1 · h–1 were used, but the present study showed that PARS inhibitors at 5 mg · kg–1 · h–1 seemed to be effective as well as the rate of 10 mg · kg–1 · h–1 in reducing the activation of PARS. Also, the present study showed that 3-aminobenzamide itself had no effect on the relaxation of pulmonary arterial rings in the sham group. These results demonstrated that 3-aminobenzamide improved pulmonary arterial endothelium-dependent relaxation after IR injury of SMA. In addition, PARS inhibitors had no effect on the relaxation caused by endothelium-derived NO (2) and are not inhibitors of NO synthase (1). PARS inhibitors do not scavenge peroxynitrite or superoxide (1). In our study, the relaxant responses to vasodilators were investigated in isolated vascular rings placed in an artificial environment. However, the pulmonary endothelial cell damage caused by the IR of SMA would remain in rings placed in such an environment. We believe that the present study can provide useful information about what occurs in vivo.
Intestinal IR is associated with both local and systemic changes. Local functional alterations include polymorphonuclear neutrophil adhesion and activation, intestinal hyperpermeability, and changes in the vascular reactivity of the splanchnic vessels (4). Systemic alterations include the release of proinflammatory mediators from the reperfused intestinal tissue into the systemic circulation, alterations in the function of remote organs, such as the heart and lungs, and alterations in the reactivity of both splanchnic and nonsplanchnic blood vessels (1,4). The proinflammatory mediators from reperfused intestinal tissue and activated adherent polymorphonuclear neutrophils, such as oxygen-derived free radicals, injure the endothelial cells of pulmonary arteries and other systemic arteries. PARS is present in the nuclei of various cells and is activated by IR injury. PARS inhibitors improve both local and systemic changes induced by IR of the splanchnic artery (1,4). Therefore, it is considered that the PARS inhibitor had effects both in the pulmonary artery and intestinal tract.
Our results suggest that activation of PARS may be a potent cause of the reduction in the endothelial-dependent relaxant response after IR injury. The DNA strand breakage and activation of PARS play an important role in the endothelial dysfunction associated with IR. PARS inhibitors may be effective in treating disorders such as septic shock, which is accompanied by an increase in pulmonary vascular resistance. Studies have shown that PARS inhibitors may be useful for improving morbid alterations in relation to activation of PARS. PARS enhanced endothelial-dependent relaxation in aortic rings and reduced the reperfusion-induced increase in epithelial permeability (1). PARS inhibitors prevented the infiltration of neutrophils into reperfused intestine, improved the histological status of reperfused tissues, reduced the production of peroxynitrite in the reperfusion phase, and improved survival (1,4,5). PARS inhibitors exert protective effects in myocardial reperfusion injury. This is probably because PARS inhibitors preserve myocardial adenosine triphosphate and nicotinamide adenine dinucleotide levels (6–8) and prevent the infiltration of neutrophils into reperfused myocardial cells (6). PARS inhibitors significantly ameliorated the decrease in arterial blood pressure and cardiac output in the pathophysiology of hemorrhagic shock (9–11). Hemorrhagic shock may lead to activation of PARS, and at least in part, activation of PARS may be the cause of hypotension.
In conclusion, inhibition of PARS improves pulmonary arterial endothelium-dependent relaxation after IR injury of the splanchnic artery. It is thus believed that PARS inhibitors may be useful for improving pulmonary circulation accompanied by pulmonary hypertension, which is seen in diseases such as septic shock.
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Footnotes |
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Supported, in part, by Grants in Aid for Scientific Research from Japan Society for the Promotion of Science (No. 09771134).
Accepted for publication June 9, 2005.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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- Cuzzocrea S, Zingarelli B, Costantino G, Szabo A. Beneficial effects of 3-aminobenzamide, an inhibitor of poly (ADP-ribose) synthetase in a rat model of splanchnic artery occlusion and reperfusion. Br J Pharmacol 1997;121:1065–74.[Web of Science][Medline]
- Chabot F, Mitchell JA, Quinlan GJ, Evans TW. Characterization of the vasodilator properties of peroxynitrite on rat pulmonary artery: role of poly (ADP-ribose) synthase. Br J Pharmacol 1997;121:485–90.[Web of Science][Medline]
- Carey C, Siegfried MR. Antishock and endothelial protective actions of a NO donor in mesenteric and reperfusion. Circ Shock 1992;38:209–16.[Web of Science][Medline]
- Liaudet L, Szabo A, Soriano FG, Zingarelli B. Poly (ADP-ribose) synthetase mediates intestinal mucosal barrier dysfunction after mesenteric ischemia. Shock 2000;14:134–41.[Web of Science][Medline]
- Taner AS, Cinel I, Ozer I, Onde U. Poly (ADP-ribose) synthetase inhibition reduces bacterial translocation in rats after endotoxin challenge. Shock 2001;16:159–62.[Web of Science][Medline]
- Bowes J, Ruetten H, Martorana PA, Stockhausen H. Reduction of myocardial reperfusion injury by an inhibitor of poly (ADP-ribose) synthetase in the pig. Eur J Pharmacol 1998;359:143–50.[Web of Science][Medline]
- Zingarelli B, Cuzzocrea S, Zsengeller Z, Salzman AL. Protection against myocardial ischemia and reperfusion injury by 3-aminobenzamide, an inhibitor of poly (ADP-ribose) synthetase. Cardiovasc Res 1997;36:205–15.
[Abstract/Free Full Text] - Docherty JC, Kuzio B, Silvester JA, Bowes J. An inhibitor of poly (ADP-ribose) synthetase activity reduces contractile dysfunction and preserves high energy phosphate levels during reperfusion of the ischemic rat heart. Br J Pharmacol 1999;127:1518–24.[Web of Science][Medline]
- John JS, Barbee RW, Sonin N, Clemens MG. Inhibition of poly (ADP-ribose) synthetase improves vascular contractile responses following trauma-hemorrhage and resuscitation. Shock 1999;12:188–95.[Web of Science][Medline]
- Szabo C. Potential role of the peroxynitrite-poly (ADP-ribose) synthetase pathway in a rat model of severe hemorrhagic shock. Shock 1998;9:341–4.[Medline]
- Szabo A, Hake P, Salzman AL, Szabo C. 3-aminobenzamide, an inhibitor of poly (ADP-ribose) synthetase, improves hemodynamics and prolongs survival in a porcine model of hemorrhagic shock. Shock 1998;10:347–53.[Web of Science][Medline]
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