Introduction

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Early reperfusion after coronary obstruction is the most effective means of limiting ischemic myocardial injury. However, abundant evidence suggests that reperfusion may cause additional cell death, and the cause of this "reperfusion injury" is apparently multifactorial (Lucchesi, 1994; Maynard et al., 1993). Among the many proposed mechanisms, PMN accumulation, platelet aggregation and an imbalance between the vasodilators and vasoconstrictors have been demonstrated to play central roles in causing or aggravating postischemic myocardial injury (Kilgore and Lucchesi, 1993; Hansen, 1995; Maxwell and Lip, 1997).

·NO, a gas molecule that was originally identified as EDRF, plays a critical role in cardiovascular homeostasis. In addition to its vasodilator properties, NO inhibits neutrophil adhesion, attenuates platelet aggregation and quenches superoxide anion (Moncada and Higgs, 1995). Replacement of endogenous ·NO, which has been found to be decreased significantly after reperfusion (Lefer et al., 1991), by an exogenous source of ·NO at reperfusion could theoretically attenuate reperfusion injury. In this regard, organic nitrates, such as NTG, have been used in myocardial ischemia for >100 years. However, most of these classic nitrates are poor ·NO donors and quickly induce tolerance, presumably because they must be metabolized intracellularly to release ·NO. Part of the metabolic degradation of NTG and other nitrates may require reducing equivalents (e.g., thiols), and a lack of reduced thiols has been implicated in tolerance (Needleman et al., 1973; Flaherty, 1989).

SP/W-5186 [N-(3-nitratopivaloyl)-S-(N'-acetylglycyl)-L-cysteine ethylester] is a newly developed ·NO donor that contains both an NO group and a source of thiols (i.e., cysteine) (fig. 1). Preliminary pharmacological experiments have demonstrated that, in contrast to the classic nitrate NTG, no tolerance developed after a 3-day treatment with high doses of SP/W-5186 administration (Schwarz Pharma AG, nonpublished observations). Moreover, as a prodrug, SP/W-5186 has a relatively long half-life, and its vasodilator effect lasts ~3 hr after a single bolus injection at a dose of 1.68 µmol/kg in dog (Schwarz Pharma AG, nonpublished observations). The purposes of the present study were (1) to determine the effects of different doses of SP/W-5186 on cardiac functional and myocardial cellular injury associated with ischemia and reperfusion when given at different doses and (2) to investigate the mechanisms by which SP/W-5186 may exert its cardioprotective effects.

View larger version (6K): [in this window] [in a new window]   Fig. 1.   Structures of SP/W-5186 and SP/W-6373.

	Experimental Procedures

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Materials. SP/W-5186 and SP/W-6373 were kindly provided by Schwarz Pharma AG (Monheim, Germany). All other chemicals were purchased from Sigma Chemical (St. Louis, MO) unless otherwise indicated. A total of 61 adult male New Zealand White rabbits (2.8-3.5 kg) were included in this study. The experiments were performed in adherence to National Institutes of Health Guidelines on the Use of Laboratory Animals and were approved by the Thomas Jefferson University Committee on Animal Care.

Experimental preparation. Rabbits were anaesthetized with sodium pentobarbital (30 mg/kg body weight) intravenously. An intratracheal tube was inserted through a midline incision, and all rabbits were given intermittent positive-pressure ventilation with O₂-enriched room air using a Harvard small animal respirator (Harvard Apparatus, South Natick, MA). Arterial blood gases were measured using a blood gas analyzer (CIBA-CORNINE 288 Blood Gas Analyzer, Ciba-Cornine Corp., Norwood, MA) and arterial PO₂ and PCO₂ were maintained at 100 to 120 mm Hg and 35 to 45 mm Hg, respectively, by adjusting the oxygen flow and ventilation rates. The pH was adjusted to 7.35 to 7.45 with sodium bicarbonate as necessary. A polyethylene catheter was inserted into the right external jugular vein for supplemental pentobarbital injection to maintain a surgical plane of anesthesia and for administration of drugs. An additional polyethylene catheter was inserted through the left femoral artery and positioned in the abdominal aorta for measurement of ABP via a Statham P23AC pressure transducer (Spectromed, Critical Care Division, Oxnard, CA). After a midline thoracotomy was performed, the pericardium was opened and the heart was exposed. A 4-0 silk ligature was carefully placed around the major marginal branch of the left circumflex coronary artery located on the dorsal surface of the heart, 10 to 12 mm from its origin. A Millar-tip catheter transducer (5F) was inserted into the LV cavity through the apex, and the LVP was obtained. ABP, LVP and standard lead II of the scalar ECG were digitized at 250 Hz using a 12-bit analog-to-digital converter (Data Translation Devices, Marlboro, MA) and a Dell 486-66 computer. MABP, HR, LVSP, LVEDP and the instantaneous first derivative of LVP (dP/dt) were continuously monitored during the entire experimental period. The PRI, calculated as the product of MABP and HR divided by 1000, was used as an approximation of myocardial oxygen demand.

Analysis of myocardial injury. Ischemia-reperfusion-induced cardiac contractile dysfunction was monitored during the ischemia and reperfusion period. LVP, MABP and ECG were acquired over 10 sec at the following time points: immediately before coronary occlusion (0 min); 20 and 45 min after coronary occlusion; and 5, 30, 60, 120 and 180 min after reperfusion. Data were stored on computer hard disk for later retrieval and analysis. All acquisitions were obtained in duplicate. The LVSP, LVEDP, dP/dt and HR were obtained using computer algorithms and an interactive videographics program (Bowman Gray School of Medicine, Winston-Salem, NC) (Sato et al., 1995).

Studies on coronary endothelial dysfunction. After injection of Evans blue, hearts were excised and placed in ice-cold Krebs-Henseleit (K-H) buffer. The marginal coronary artery was carefully isolated and 10- to 12-mm-long segments were removed both above and below the ligature. The segment above the ligature (i.e., the proximal segment) was used as a nonischemic, nonreperfused control, and the portion of the vessel that had undergone ischemia and reperfusion (i.e., the distal segment) was used as an ischemic vessel. These segments were placed in warmed K-H solution consisting of (in mM): NaCl 118; KCl 4.75; CaCl₂, 2.54; KH₂PO₄ 1.19; MgSO₄, 1.19; NaHCO₃ 25, and glucose 10.0. Within 5 min, isolated vessels were cleaned of adhering fat and connective tissue and cut into rings 2 to 3 mm in length. The rings were then quickly mounted on stainless steel hooks, suspended in water-jacked tissue baths and connected to FORT-10 force transducers (WPI, Sarasota, FL) to record changes in force on WindoGraf Recorders (Gould, Valley View, OH). The baths were filled with 7 ml of K-H buffer and aerated with a gas mixture of 95% O₂ and 5% CO₂. They were then stretched to a preload of 0.5 g, which was determined to be optimal in previous experiments in this laboratory, and allowed to equilibrate for 60 min. During this period, the KH buffer in the tissue bath was replaced every 15 min, and the tension of vascular rings was adjusted until 0.5 g of preload was maintained.

Measurement of PMN accumulation in cardiac tissue. MPO, an enzyme occurring virtually exclusively in neutrophils, was determined in cardiac tissue by the method of Bradley et al. (1982), as modified by Mullane et al. (1985), and was used as an index of PMN accumulation in the heart. Cardiac tissue samples were homogenized in 0.5% HTAB and dissolved in 50 mmol/L potassium phosphate buffer at pH 6 using a PRO 200 homogenizer (PRO Scientific, Monroe, CT) for 60 seconds at 7000 rpm. The homogenates were centrifuged for 30 min at 12,500 × g and 4°C. The supernatants were then collected and reacted with 0.167 mg/ml o-dianisodine dihydrochloride and 0.0005% H₂O2 in 50 mmol/L phosphate buffer at pH 6. The measurement was performed at 460 nm at 25°C using a spectrophotometer (Beckman DU 640, Fullerton, CA). One unit of MPO activity was defined as that quantity of enzyme hydrolyzing 1 mmol of peroxide/min at 25°C.

Myocardial malondialdehyde assay. Myocardial tissue was hemogenized in distilled water (2% wt/v) using a PRO 200 homogenizer (PRO Scientific Inc.) for 60 sec at 7000 rpm. The homogenates were centrifuged for 30 min at 5000 × g and 4°C. The supernatants were then collected, and MDA content was measured using a modification of the method of Ohkawa et al. (1979).

Ex vivo platelet aggregation. Immediately before and 30 min after each drug or vehicle administration, 5 ml of blood samples were drawn into a polyethylene tube containing 4% sodium citrate (1:9 v/v). PRP was prepared by centrifugation of blood at 200 × g for 15 min, and PPP was obtained by centrifugation of the remaining blood at 1600 × g for 20 min at room temperature. Autologous PPP was used to dilute the PRP to a final concentration of 3 × 10⁸ platelets/ml. All tests were performed within 90 min after blood sampling.

PMN adhesion to cultured ECs. Rabbit peripheral blood (20 ml) was collected from the central ear artery and mixed with 3.0 ml of anticoagulation agents, which included 1.6% citric acid and 2.5% sodium citrate at pH 5.4, and 17 ml of Hespan solution (6% hetastarch in 0.9% saline; DuPont Pharmaceutical, Wilmington, DE). PMNs were isolated by a procedure originally described by Doerschuk et al. (1989) and modified by Todd et al. (1996). PMN preparations obtained by this method are typically >95% pure (hematoxylin/eosin staining) and >95% viable (trypan blue exclusion).

Statistical analysis. All values in the text, tables and figures were presented as mean ± S.E.M. of n independent experiments. All data were subjected to ANOVA followed by the Bonferroni correction for post-hoc t tests. Probabilities of P < 0.05 were considered to be statistically significant.

	Results

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HR, MABP and PRI. There were no significant differences among the groups in HR and MABP before coronary occlusion. Immediately after occlusion of the coronary artery, the portion of the ventricle that had been perfused by the occluded artery became cyanotic and dyskinetic. Simultaneous with these visual alterations, MABP decreased significantly. When the coronary arterial ligature was loosened after 45 min of ischemia, ventricular reperfusion was confirmed visually by resumption of normal ventricular color and wall motion, as well as a decline in S-T segment elevation on the ECG. MABP slightly declined again during the first 20 min of reperfusion and gradually returned toward the prereperfusion level thereafter. There were no significant differences in MABP after reperfusion among the five groups, with the exception that high-dose SP/W-5186-treated rabbits had a significantly lower MABP after drug injection.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Time course of PRI, LVEDP and dP/dtmax in rabbits subjected to 45-min regional ischemia (I) and 180 min of reperfusion (R). Vehicle (0.9% NaCl) or drugs (SP/W-5186 or SP/W-6373) were given as a bolus intravenously 5 min before reperfusion (D). All values are mean ± S.E. of 10 to 12 rabbits. *P < .05 vs. the vehicle-treated group.

Cardiac functional injury. The maximal rate of dP/dt_max is a commonly used index of myocardial contractility. Because dP/dt_max is critically related to LV preload (i.e., LVEDP) and afterload (i.e., MABP), we measured LVEDP, MABP and dP/dt_max simultaneously in this experiment. As described above, there were no significant differences among vehicle, low-dose SP/W-5186 and either dose of SP/W-6373-treated groups at any time point observed. Although administration of high-dose SP/W-5186 significantly decreased the MABP, this decrease lasted for a short period only and MABP returned to a level comparable to that in the other groups 30 min after drug administration. Therefore, the difference in dP/dt_max, if any, among the groups, could not be attributed to a difference in afterload (i.e., MABP).

Myocardial cellular injury. Plasma CK activity was measured before coronary occlusion, at the end of coronary occlusion and hourly thereafter. At the end of the 45-min ischemic period, plasma CK activity only slightly increased and there was no significant difference among groups. However, plasma CK activity increased markedly after reperfusion and reached 173 ± 7.1 U/g protein at the end of reperfusion in the vehicle-treated group (5.2 ± 0.8 U/g protein before ischemia, P < .0001). Treatment with inactive control compound, SP/W-6373 at either dose did not change plasma CK activity at any time point observed. In contrast, MI rabbits treated with SP/W-5186 developed significantly lower plasma CK activities compared with MI rabbits treated with either the vehicle or SP/W-6373 at every time point after reperfusion (fig. 3). Moreover, at 180 min after reperfusion, plasma CK activity in the high-dose SP/W-5186-treated group was significantly lower than the low-dose SP/W-5186-treated group, indicating that the higher dose of SP/W-5186 exerted a more pronounced cardioprotective effect. The decrease in plasma CK activity in the SP/W-5186-treated groups was not due to a direct effect of this compound on the CK assay, as addition of SP/W-5186 directly to rabbit plasma did not affect the CK assay results (i.e., duplicate values with and without exogenously added SP/W-5185 were within 5% of each other). CK values in the four plasma samples drawn from untreated MI rabbits at 3 hr after reperfusion were 166 ± 8.7 vs. 172 ± 7.9 IU/g protein in these same samples reassayed in the presence of SP/W-5186 up to a concentration of 100 nM.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Plasma CK activity expressed as IU/g of protein measured before ischemia (0), at the end of ischemia (I45) and hourly thereafter for all five groups. All values are mean ± S.E. of 10 to 12 rabbits. **P < .01 vs. the vehicle-treated group. P < .05 vs. low-dose SP/W-5186-treated group.

View larger version (51K): [in this window] [in a new window]   Fig. 4.   Tissue wet weight of AAR as a percentage of the total LV wet weight (left five bars) and of necrotic tissue as a percentage of AAR (middle five bars) and of the total LV (right five bars) for the five groups. Height of bars are mean values; brackets represent ±S.E.M. **P < .01 vs. the vehicle-treated group. P < .05 vs. low-dose SP/W-5186-treated group.

Neutrophil accumulation in the myocardial tissue. Accumulation of neutrophils in the ischemic region during reperfusion has been thought to be one of the major mechanisms responsible for reperfusion injury. We therefore measured MPO activity in the three different regions of the myocardium as a marker for neutrophil accumulation in ischemic tissue. In the nonischemic myocardium (i.e., AANR), MPO activity was very low in all groups and there was no significant difference among them, indicating that few neutrophils were present in the nonischemic myocardium. However, MPO activity in the ischemic region was markedly increased. Treatment with inactive control compound, SP/W-6373, had no effect on MPO activity either in the ischemic non-necrotic area or necrotic area. In contrast, SP/W-5186-treated ischemic rabbits exhibited a significantly lower MPO activity in ischemic non-necrotic myocardial tissue as well as in necrotic tissue (fig. 5). There was no significant difference in MPO activity between the two SP/W-5186-treated groups. These results indicate that adherence and accumulation of neutrophils in ischemia-reperfused myocardium was markedly inhibited by treatment with SP/W-5186.

View larger version (36K): [in this window] [in a new window]   Fig. 5.   Tissue MPO activity in ANAR, AAR, and NEC in U/100 mg tissue wet weight for the four groups. Height of bars are mean values; brackets represent ±S.E.M. **P < .01 vs. the vehicle-treated group.

Effect of SP/W-5186 on myocardial lipid peroxidation after ischemia and reperfusion. In a separate series experiment, the effect of ·NO on lipid peroxidation was studied and the results were summarized in table 1. Myocardial ischemia followed by reperfusion caused significant lipid peroxidation, as evidenced by marked increase in MDA content in ischemic-reperfused myocardial tissue (AAR). Administration of SP/W-5186 (0.3 µmol/kg), but not SP/W-6373, before reperfusion significantly attenuated MDA elevation, suggesting that supplementation of exogenous ·NO prevented myocardial lipid peroxidation induced by coronary occlusion and reperfusion.

Isolated neutrophil adhesion to cultured microvessel endothelial cells. To further determine the effect of SP/W-5186 on PMN-EC interaction, we investigated the effects of SP/W-5186 on isolated PMN adhesion to cultured microvascular ECs. Exposing ECs to hypoxia followed by reoxygenation resulted in a 2.5- to 3-fold increase in PMN adhesion (not shown). Addition of SP/W-5186, but not SP/W-6373, at the time of reoxygenation inhibited PMN adhesion in a concentration-dependent manner with an EC₅₀ of 53 nM. Maximal inhibition (75.4 ± 1.6% inhibition) was observed at a SP/W-5186 concentration of 300 nM (fig. 6). These results further demonstrate that SP/W-5186 is a potent and effective inhibitor of PMN-EC interaction.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Concentration-inhibition effect of SP/W-5186 or SP/W-6373 on isolated rabbit PMN adhesion to cultured microvascular endothelial cells. Confluent endothelial cells were exposed to hypoxia (45 min) and reoxygenation (180 min). At the time of reoxygenation, PMNs (5 × 105/well) and drugs were added. At the end of the 3-hr reoxygenation period, nonadherent PMNs were removed by three consecutive washings. The number of adhered PMNs to ECs was quantified by measuring MPO activity using a Thermomax microplate reader. **P < .01 vs. the vehicle-treated group.

Coronary endothelial function. Because endothelial dysfunction is an early and critical event in reperfusion and plays an important role in reperfusion injury, we tested endothelial function by comparing the vasoactivity of isolated coronary artery rings in response to the endothelium-dependent vasodilator, ACh, with the response of an endothelium-independent vasodilator, acidified NaNO₂. Figure 7 summarizes the vasorelaxant responses of isolated coronary artery rings from the five groups of rabbits obtained with ACh and NaNO₂. In control coronary artery rings, ACh induced a concentration-dependent vascular relaxation with >90% relaxation occurring at a concentration of 10 µmol/l. In contrast, the concentration-response curve to ACh showed a significant shift to the right in the coronary artery rings from vehicle-treated MI rabbits. At the highest concentration ACh tested, relaxation was markedly impaired (46 ± 4.2%, P < .001 vs. 94 ± 2.9% in sham). Treatment with SP/W-6373 did not improve vasorelaxation responses of coronary artery rings to ACh. However, coronary artery rings from MI rabbits treated with SP/W-5186 demonstrated a significant improvement in endothelium-dependent vasorelaxation. When exposed to 10 µM ACh, the coronary artery rings showed a relaxation of 78.6 ± 2.4 and 80.2 ± 2.9 in low-dose and high-dose SP/W-5186 groups, respectively (P < .001 compared with vehicle-treated MI rabbits).

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Concentration-relaxation response of coronary artery rings to the endothelium-dependent vasodilator ACh (left) and to the endothelium-independent vasodilator acidified NaNO2 (right). The rings were constricted by U-46619 (50 nM). Once a stable vasoconstriction was obtained, cumulative concentrations of ACh (1 nM to 10 µM) or acidified NANO2 (10 nM to 100 µM) were added, and the vasorelaxation to each concentration of vasodilator was calculated as a percent of the maximal vasoconstriction induced by U-46619. Each point represents the mean value of 13 to 15 rings isolated from 4 or 5 rabbits from each experimental group. **P < .01 vs. the vehicle group.

Effects of SP/W-5186 on platelet aggregation. To determine whether administration of SP/W-5186 may inhibit the platelet aggregation that may contribute to the attenuation of ischemia/reperfusion injury, two blood samples were taken from each animal (i.e., immediately before and 30 min after each drug or vehicle administration). The effects of SP/W-5186 on platelet aggregation were then tested in PRP ex vivo. As illustrated in figure 8 and summarized in figure 9, collagen-stimulated platelet aggregation was comparable in PRP obtained before or after vehicle administration, indicating that platelet function did not significantly change during the observation period. Moreover, because the first blood was sampled ~6 min before reperfusion and the second 25 min after reperfusion, this result also suggested that reperfusion per se did not significantly alter platelet aggregability. However, when 0.3 µmol/kg SP/W-5186 was administered 5 min before reperfusion, platelet aggregation was significantly inhibited compared with blood withdrawn before drug administration. The inhibitory effect of SP/W-5186 on platelet aggregation was slightly increased when 1 µmol/kg SP/W-5186 was given. This difference, however, was not statistically significant compared with low-dose SP/W-5186. Administration of SP/W-6373, the inactive control compound of SP/W-5186, had no effect on platelet aggregation.

View larger version (11K): [in this window] [in a new window]   Fig. 8.   Superimposed tracings of collagen-induced platelet aggregation and its alteration by SP/W-5186. Arterial blood was drawn immediately before (A) or 30 min after (B) each drug or vehicle administration. In the vehicle group, the amplitude of platelet aggregation was comparable before or 30 min after vehicle injection (left). Intravenous administration of SP/W-5186 at a dose of 0.3 µmol/kg significantly decreased platelet aggregation (middle). In contrast, administration of SP/W-6373, a nitrate-free analog of SP/W-5186, had no effect on platelet aggregation (right).

View larger version (40K): [in this window] [in a new window]   Fig. 9.   Summary of the effects of inhibition by SP/W-5186 and SP/W-6373 on platelet aggregation in PRP ex vivo. Height of bars are mean values; brackets represent ±S.E.M. **P < .01 vs. the vehicle-treated group.

	Discussion

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SP/W-5186 was designed as a prodrug and does not release ·NO directly. The compound is stable in solution at pH 5 but decomposes at pH 7.4 and 37°C with a half-life of ~10 hr. The major hydrolysis product of SP/W-5186, SP/W-3672, a molecule with a free SH group, releases ·NO spontaneously (Koijda et al., 1995). In contrast to the classic organic nitrates, addition of cysteine does not further increase ·NO release. After a bolus i.v. injection of SP/W-5186, SP/W-3672 is detectable in plasma within 1 min and peaks at 5 min, indicating that SP/W-5186 is quickly converted to SP/W-3672 in plasma (Schwarz Pharma, unpublished data).

The data presented in this study demonstrate that SP/W-5186 exerts significant protective effects against reperfusion injury as evidenced by improved cardiac functional recovery, decreased plasma creatine kinase concentration and reduced infarct size. Although improvement of cardiac function was not significantly different between the low- and high-dose treatment groups, myocardial cellular injury as measured by plasma CK activity and necrotic size was more potently attenuated in the group treated with the high dose of SP/W-5186. Administration of SP/W-6373, a nitrate-free analog of SP/W-5186, at either dose failed to exert significant protective effects in this ischemia-reperfusion model. Since the only chemical difference between SP/W-5186 and SP/W-6373 is that the latter lacks the nitrate moiety, the present results clearly demonstrate that ·NO released from this type of NO donor is responsible for the cardioprotective effects observed in this study.

There are several possible explanations for the marked improvement of cardiac function and the attenuation of myocardial injury seen with SP/W-5186 treatment. It is well documented that reperfusion involves a typical inflammatory response and PMNs are known to play a pivotal role in tissue injury. Many studies have documented a correlation between infarct size secondary to reperfusion and extent of PMN infiltration (Lucchesi, 1994). PMNs can induce endothelial and myocardial reperfusion injury by various mechanisms. Activated PMNs release a variety of cytotoxic substances, including proteases, collagenases, cytokines, leukotrienes and cationic proteins, capable of causing tissue damage (Lucchesi, 1994). Adhering and aggregating PMNs can physically obstruct capillary flow and induce a "no-reflow phenomenon." This mechanical obstruction causes a regional permanent ischemia and ultimately leads to an increase in necrosis (Duran et al., 1994). A large body of evidence exists that indicates that PMNs are the major source of reactive oxygen species (ROS) and thus are primarily responsible for free radical-induced endothelial and myocardial injury after myocardial ischemia and reperfusion (Lucchesi, 1994; Duran et al., 1994). ROS not only cause direct myocardial injury through the formation of hydroxyl radical (OH·) (Duncan, 1994), but more importantly, activate ECs and PMNs, thus promoting further PMN adhesion and initiating a vicious cycle.

Numerous studies have demonstrated that ·NO possesses potent antineutrophil activity (Provost et al., 1994), and thus may exert its cardioprotective effects through attenuation of PMN-mediated reperfusion injury (Egdell et al., 1994). In a previous study, we have shown that diminished basal ·NO release from coronary endothelium after myocardial ischemia and reperfusion promotes adherence of PMNs in vitro through a CD11/CD18-dependent mechanism (Ma et al., 1993). Increasing endogenous ·NO production by supplying exogenous L-arginine (Weyrich et al., 1992) has been shown to decrease PMN accumulation and attenuate myocardial injury. The cellular mechanism for the antiadhesive effects of ·NO is not clearly understood at present. However, recent studies indicate that ·NO may attenuate PMN-EC adhesion by decreasing endothelial expression of adhesion molecules. Gauthier et al. (1994) reported that ·NO decreases P-selectin expression after splanchnic ischemia and reperfusion. De Caterina et al. (1995) demonstrated that ·NO significantly inhibited cytokine-induced up-regulation of the vascular cell adhesion molecule-1 (VCAM-1) as well as ICAM-1 in cultured human saphenous vein ECs. In the present study, we have clearly shown that SP/W-5186 markedly decreases PMN accumulation in ischemia/reperfused myocardial tissue when administered in vivo and significantly inhibits PMN adhesion when exposed to hypoxia/reoxygenated ECs in vitro. The novel ·NO donor SP/W-5186 may thus protect myocardial tissue from reperfusion injury via its antineutrophil effects. Interestingly, it appears that the anti-PMN effects of SP/W-5186 are more potent when administered in vivo than in vitro. Although the administration of SP/W-5186 at a dose of as low as 0.3 µmol/kg markedly decreased MPO activity in ischemic-reperfused myocardial tissue, the minimal concentration that exerted significant inhibition of PMN adhesion to endothelial cells in vitro was 30 nM, a concentration ~6 to 8 times higher than the estimated minimal plasma concentration after 0.3 µmol/kg administration. Because SP/W-5186 needs to be converted to the activate metabolite SP/W-3672 to release ·NO, it is possible that the differences in potency of SP/W-5186 needed to inhibit the PMN-EC interaction in vitro and in vivo reflect the faster metabolism of SP/W-5186 in vivo.

Substantial evidence exists indicating that endothelial dysfunction manifested as decreased vasorelaxant response to endothelium-dependent vasodilators and increased microvascular leakiness is one of the earliest pathological responses after reperfusion (Lefer et al., 1991). It contributes significantly to the subsequent myocardial functional and cellular injury in a variety of pathological pathways. Endothelial dysfunction disturbs the balance between vasorelaxation and vasoconstriction and thus may promote vasoconstriction and contribute to the "no-reflow phenomena" seen after myocardial ischemia and reperfusion. Endothelial dysfunction may also exacerbate myocardial injury indirectly by increasing platelet aggregation and promoting PMN accumulation in ischemia/reperfused myocardial tissue. Although we cannot determine precisely the mechanisms by which SP/W-5186 preserves endothelial function, it is very likely that this effect of SP/W-5186 plays an important part in ultimately protecting against the myocardial dysfunction and cellular injury associated with ischemia and reperfusion.

It is well known that platelet and platelet-derived mediators play a significant role in acute myocardial ischemic injury in the absence of reperfusion (Stamler and Loscalzo, 1991). Recent experiments, however, have revealed that platelets may also contribute significantly to reperfusion injury via a platelet-PMN interaction (Nash, 1994). It has been reported that platelet activation significantly facilitates PMN adhesion to endothelial cells and thus increases PMN accumulation in inflammatory tissue (Diacovo et al., 1996). Moreover, the platelet-PMN interaction markedly increases superoxide anion generation by PMNs (Colli et al., 1996). Our present results clearly demonstrated that the administration of SP/W-5186 significantly inhibits platelet aggregation. Therefore, SP/W-5186 might protect myocardial tissue from PMN-induced tissue injury by decreasing the platelet-PMN interaction.

Alternatively, SP/W-5186 may have exerted its cardioprotective effects through systemic or coronary vasodilatation. However, it is unlikely that this vasodilator effect contributes significantly to the protective effects observed with the low dose of SP/W-5186 because no significant hemodynamic effects were observed at this dose regimen. In contrast, when high-dose SP/W-5186 was administered, a significant vasodilation effect occurred, and the PRI, an index of cardiac oxygen demand, was significantly decreased. This may explain the more significant decrease in plasma CK activity and necrotic size observed in the high-dose SP/W-5186-treated group.

Free radical-induced lipid peroxidation is a major component of reperfusion injury. Recent studies have suggested that ·NO may simultaneously have both pro- and anti-oxidant effects depending on the relative concentration of individual reactive species present (Lipton et al., 1993; Crow and Beckman, 1996). At lower relative concentrations, ·NO may act as a pro-oxidant by reacting with O₂^·, resulting in the formation of a more reactive oxidant, peroxynitrite (ONOO), and enhancing lipid peroxidation (Yang and Mehta, 1997). Inhibiting NO synthase thus may prevent ONOO formation and reduce lipid peroxidation. In contrast, at high relative concentrations, ·NO can act as an antioxidant by reacting with lipid-derived radicals and thus resulting in termination of the lipid peroxidation chain reaction and stop further initiation. By this mechanism, ·NO has been shown to inhibit O₂^·, ONOO, H₂O2, ·OH and lipoxygenase-dependent lipid peroxidation in vitro (Gutierrez et al., 1996; Laskey and Mathews, 1996). Moreover, Engelman et al. (1995) has recently reported that L-arginine infusion significantly reduces lipid peroxidation and attenuates reperfusion injury in vivo. Our present study has demonstrated that administration of SP/W-5186 significantly reduced myocardial lipid peroxidation associated with ischemia and reperfusion. This result provides direct evidence that in addition to its well documented vasodilatory and antineutrophil effects, ·NO may inhibit free radical-induced lipid peroxidation and thus reduces myocardial reperfusion injury.

In summary, our present study demonstrates that the novel ·NO donor SP/W-5186 exerts marked cardioprotective effects in rabbit ischemia-reperfusion injury. Several lines of experimental evidence indicate that this protective effect may be mediated by a combination of decreased PMN adherence to the coronary endothelium, decreased platelet aggregation, preserved endothelial function and direct antioxidant effects. At higher doses, a vasodilator component may also contribute to the observed cardioprotective effects of this ·NO donor.