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CARDIOVASCULAR

The Aminotetraline Derivative (±)-(R,S)-5,6-Dihydroxy-2-methylamino-1,2,3,4-tetrahydro-naphthalene Hydrochloride (CHF-1024) Displays Cardioprotection in Postischemic Ventricular Dysfunction of the Rat Heart

Department of Pharmacological Sciences, University of Milan, Milan, Italy (G.R.); Department of Pharmacology, Chemotherapy and Medical Toxicology, University of Milan, Milan, Italy (G.R., B.M., F.B.); Department of Cardiology, Istituto di Ricovero e Cura a Carattere Scientifico, Centro Cardiologico "I. Monzino" Foundation, University of Milan, Milan, Italy (V.C., G.L.P.); and Department of Pharmacology, Chiesi Farmaceutici, Parma, Italy (R.R., S.B.)

Received May 19, 2003; accepted August 6, 2003.

	Abstract

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To analyze the protective effects of the aminotetraline derivative (±)-(R,S)-5,6-dihydroxy-2-methylamino-1,2,3,4-tetrahydro-naphthalene hydrochloride (CHF-1024), a compound endowed with DA₂-dopaminergic/₂-adrenergic receptor agonistic activity, in myocardial ischemia/reperfusion damage. A model of isolated and perfused (15 ml/min) electrically driven (300 beats/min) rat heart subjected to global ischemia (1 ml/min for 20 min) and reperfusion (15 ml/min for 30 min) was followed. Cardiac mechanics changes were evaluated together with biochemical markers of cardiac ischemia in perfusate and tissue tumor necrosis factor- (TNF-). CHF-1024, perfused through the heart for 15 min before ischemia at different molar concentrations (1-100 nM), significantly improved left ventricle developed pressure during reperfusion, and normalized left ventricular end-diastolic pressure and coronary perfusion pressure. This anti-ischemic effect of CHF-1024 was associated to a decrease in creatine kinase and lactate dehydrogenase, both released during heart reperfusion. These events were concomitant with maintenance of a higher production of 6-keto-prostaglandin F₁ The ability of CHF-1024 to improve postischemic ventricular dysfunction was correlated with a dose-dependent inhibition of the release of both norepinephrine (NE), from sympathetic nerve endings, and TNF- from cardiac tissue. The effect of CHF-1024 on NE release was almost completely antagonized by specific antagonists of presynaptic inhibitory receptors domperidone and rauwolscine. The finding that this new aminotetraline derivative possesses anti-ischemic properties and limits NE release from cardiac nerve endings may bear some therapeutic potential in cardiovascular diseases.

The aminotetraline derivative CHF-1024 is the active metabolite of Nolomirole, a new oral compound in phase III development for heart failure (McMurray and Pfeffer, 2002). It is endowed with selective agonist activity on DA₂-dopaminergic and ₂-adrenergic receptors (Pastore et al., 2000). The stimulation of these prejunctional receptors leads to diminished norepinephrine (NE) release from sympathetic nerve endings. In a rat model of left ventricular dysfunction after permanent coronary artery ligation, as well as in a model of pressure-overload hypertrophy it has been shown that CHF-1024, when given alone or in combination with an ACE inhibitor, reduces adrenergic cardiac drive and catecholamines excretion and limits myocardial fibrosis (Latini et al., 1998; Masson et al., 1999, 2001). These beneficial activities of CHF-1024 seemed of potential significance in the treatment of heart failure (Remme, 2001).

On the basis of this information and considering that the most common cause of heart failure with depressed systolic function is ischemic heart disease (Massie and Shah, 1997; Packer and Cohn, 1999), experiments have been carried out with the aim to evaluate the pharmacological activity of CHF-1024 in postischemic ventricular dysfunction of the rat heart and massive catecholamines release.

	Materials and Methods

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Animals. Male Wistar rats (Charles River Italica, Calco, Italy), weighing 260 to 280 g, were used. The animals were housed in a conditioned environment (22 ± 1°C, 55 ± 5% relative humidity, 12-h light and 12-h dark cycle) and were given free access to food and tap water. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication 85-23, revised 1996).

Perfusion of Hearts. Perfusion of rat heart was performed as described previously (Rossoni et al., 1998). In brief, the rats were anesthetized with sodium pentobarbital (60 mg/kg) given by intraperitoneal injection. The chest was opened and the heart was rapidly excised and placed in cold (4°C) Krebs-Henseleit solution (KHS) of the following composition: 118 mM NaCl, 4.8 mM KCl, 1.2 mM KH₂PO₄, 1.6 mM CaCl₂, 1.2 mM MgSO₄, 25 mM NaHCO₃, and 11.5 mM glucose. The heart was mounted within 2 min after thoracotomy on the experimental setup and perfused retrogradely at 15 ml/min (Minipuls-3 peristaltic pump; Gilson, Villiers-Le Bel, France) via the aorta with KHS, which was maintained at 37°C and aerated with 95% O₂ + 5% CO₂ to maintain normal pH, pO₂, and pCO₂ parameters. Coronary perfusion pressure (CPP) and left ventricular pressure (LVP) were measured with two HP-1280C pressure transducers (Hewlett Packard, Waltham, MA) connected to a Hewlett Packard dynograph (HP-7754A). Left ventricular pressure was recorded with a polyethylene catheter, with a small latex balloon on the tip (size no. 4; Hugo Sachs Elektronik, March-Hugstetten, Germany), inserted in the left ventricular cavity through the mitral valve opening. The volume of the balloon was adjusted to give a peak left ventricular systolic pressure of 90 ± 5 mm Hg with a left ventricular enddiastolic pressure (LVEDP) of 5 to 7 mm Hg. Hearts that could not achieve this level of contractile performance (8-10% of the hearts) were excluded. Left ventricular developed pressure (LVDevP; peak left ventricular systolic pressure minus LVEDP) was also evaluated.

Ischemia and Reperfusion. After equilibration of 15 min, hearts were paced at 300 beats/min with an electrical stimulator (S-88; Grass Instruments., Quincy, MA) via two silver electrodes attached to the right atrium and an additional 30 min of perfusion was carried out (preischemic period). Ischemia was induced by reducing the flow rate from 15 ml/min to 1 ml/min for 20 min (ischemic period). Normal flow rate (15 ml/min) was then restored and the perfusion was continued for another 30 min (reperfusion period). Throughout the experiment, a thermoregulated chamber maintained the heart temperature at 37°C to avoid hypothermia-induced cardioprotection. The total duration for each experiment did not exceed 2 h, during which time the experimental preparation was quite stable.

Experimental Protocols. In groups of eight hearts each, CHF-1024 (1, 10, and 100 nM) was perfused through the hearts for the final 15 min of preischemic period. Experiments were also carried out perfusing the hearts with 100 nM CHF-1024 during the first 15 min of reperfusion. Furthermore, in separate experiments (six hearts per group), 100 nM CHF-1024 was perfused during the preischemic period in combination with the DA₂-dopaminergic or ₂-adrenergic receptor antagonists domperidone (1 µM) or rauwolscine (1 µM), respectively (Kohli et al., 1983; Du et al., 1991). The concentration selected for both antagonists was highly selective in different in vitro preparations and was in line with other studies (Tanaka et al., 1978; Hilditch and Drew, 1985). In these hearts, the antagonists were administered upstream of the coronary bed (for 10 min before CHF-1024) by a SP-101i microdialysis pump (2Biological Instruments, Besozzo, Varese, Italy) with an infusion rate adjusted to 1/75th of the coronary flow rate.

Creatine Kinase (CK) and Lactate Dehydrogenase (LDH) Activities in Heart Perfusates. The perfusate, eluted from the heart during preischemic and reperfusion periods, was collected in an ice-cooled beaker as 2.5-min samples. Each sample was used for the determination of CK and LDH activities according to the method of Bergmeyer et al. (1970) and Hohorst (1963), respectively. The total amount of these enzymes activity was measured spectrophotometrically (-16; PerkinElmer Italia, Monza, Milan, Italy) at 37°C by using kits from the Sigma-Aldrich (St. Louis, MO). (CK procedure no. DG147-UV; LDH procedure 228-UV). Data are expressed as milliunits per minute.

Prostacyclin (PGI₂) Determination in Heart Perfusates. PGI₂ was measured directly in the coronary effluent collected in an ice-cooled beaker for 10 min immediately before ischemia and during the first 10 min of reperfusion. PGI₂ was determined as its stable metabolite 6-keto-prostaglandin F₁ (6-keto-PGF₁) according to the enzyme-linked immunosorbent assay (ELISA; detection limit, 3 pg/ml) described by Pradelles et al. (1985). The rate of formation of this arachidonic acid metabolite was expressed as picograms per milliliter.

NE and TNF- Determinations in Heart Perfusates. NE and TNF- were measured in the coronary effluent collected during the last 2 min of the preischemic period and over the first 2 min of the reperfusion period in an ice-cooled beaker as 30-s samples. After isolation by the alumina adsorption method, NE concentration was determined by a high-pressure liquid chromatography technique (Wu and McComb, 1983; Chahine et al., 1994). The chromatographic system consisted of a pump (model 542; ESA, Chelmsford, MA), a C18 reverse phase column (MD-150 x 3.2 mm; ESA) and an electro-chemical detector (CoulArray model 5011; ESA). Diydroxybenzylamine was used as internal standard and the detection limit of the method was about 3 pg/ml NE. The concentrations of NE were expressed as picograms per milliliter. TNF- was assessed with an ELISA kit. Briefly, the coronary effluent collected as reported above was immediately frozen on liquid nitrogen and stored at -80°C until assay (<4 weeks). This was performed according to the method described by Gurevitch et al. (1996). Each assay included a standard curve of recombinant rat TNF- (detection limit, 10 pg/ml). The concentrations of TNF- were expressed as picograms per milliliter.

Drugs. The following drugs were used: (±)-(R,S)-5,6-dihydroxy-2-methylamino-1,2,3,4-tetrahydro-naphthalene hydrochloride (CHF-1024) (Chiesi Farmaceutici, Parma, Italy), domperidone and rauwolscine (Sigma/RBI, Natick, MA), kits for CK and LDH determinations (Sigma-Aldrich), ELISA kit for 6-keto-PGF₁ (RPN-221; Amersham Italia, Milan, Italy), ELISA kit for TNF- (rat TNF- OptEIA; BD PharMingen, San Diego, CA). Drugs were dissolved in KHS and prepared daily.

Statistical Analysis. Each value represents the mean ± S.E.M. Statistical significance was evaluated by analysis of variance followed by Bonferroni's multiple comparisons. Differences with a probability of 5% or less were considered to be statistically significant. The area under the curve (AUC) was estimated according to the trapezoid method (Yeh and Kwan, 1978; Purves, 1992) and was assessed using a computerized program MicroCal Origin 3.5 (OriginLab Corp., Northampton, MA).

	Results

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Influence of CHF-1024 on the Perfused Isolated Heart. The time course of changes in LVEDP, LVDevP, and CPP of ischemic-reperfused rat hearts treated with 1, 10, or 100 nM CHF-1024 is depicted in Figs. 1 and 2 and summarized as AUC values in Table 1. Addition of CHF-1024 to the perfusion medium for the final 15 min of preischemic period had no effect on LVEDP, LVDevP, or CPP (Fig. 2).

View larger version (23K): [in this window] [in a new window]   Fig. 1. LVEDP in isovolumic left rat heart preparations submitted to 20-min low flow ischemia (1 ml/min) and 30-min reperfusion (15 ml/min). Points are means ± S.E.M. of eight different hearts per group. Vehicle or CHF-1024 was perfused for 15 min during the preischemic period. For statistical comparisons see the area under the curve values reported in Table 1.

View larger version (29K): [in this window] [in a new window]   Fig. 2. LVDevP and CPP in isovolumic left rat heart preparations submitted to 20-min low flow ischemia (1 ml/min) and 30-min reperfusion (15 ml/min). Points are means ± S.E.M. of eight different hearts per group. Vehicle or CHF-1024 was perfused for 15 min during the preischemic period. For statistical comparisons, see the area under the curve values reported in Table 1.

During the ischemic period, the LVEDP values of the vehicle-treated hearts after standstill began progressively to rise reaching the peak approximately at 15 min (from 5.5 ± 0.4 to 22.7 ± 1.3 mm Hg; P < 0.001). However, LVEDP declined slightly during reperfusion, and at the end of this period was still significantly elevated (14.3 ± 0.8 mm Hg; P < 0.01 versus preischemic value) (Fig. 1). In this regard, it is known that in isovolumic left heart preparation, LVEDP is considered as an index of both contractility and ventricular stiffness (Henry et al., 1977).

Treatment with 1, 10, or 100 nM CHF-1024 before ischemia attenuated the rise in LVEDP during ischemic and reperfusion periods in a concentration-dependent manner (Fig. 1; Table 1).

In vehicle-treated hearts, LVDevP during reperfusion was significantly depressed and at the end of this period the heart contractility recovered only 40% (36 ± 4 mm Hg) of the preischemic values (90 ± 6.5 mm Hg) (Fig. 2). In hearts treated with various concentrations of CHF-1024 (1, 10, or 100 nM), LVDevP was partially recovered in a concentration-dependent manner at the end of reperfusion. In this instance, CHF-1024 at 10 and 100 nM caused a recovery of LVDevP of 64 ± 5 and 82 ± 7% (P < 0.01 versus vehicle-treated group) of the preischemic value, respectively (Fig. 2; Table 1).

The CPP of the vehicle-treated heart increased upon reperfusion and reached the maximum level at 3 min, indicating both stiffness of the ventricular myocardium, and coronary vasoconstriction. These effects observed during reperfusion were reduced in a concentration-dependent manner in hearts treated with 1, 10, or 100 nM CHF-1024 (Fig. 2; Table 1).

When the hearts were treated for 15 min with 100 nM CHF-1024 only during reperfusion, the changes in LVEDP, LVDevP, and CPP caused by ischemia and reperfusion injury were not significantly different from those obtained in vehicle-treated preparations (Table 1).

Because treatment with 100 nM CHF-1024 resulted in the maximum recovery of heart mechanics and CPP, the same dosage was used in separate experiments to characterize the cardioprotective activity of this compound. Pretreatment of the hearts in the preischemic period (for 10 min before 100 nM CHF-1024) with domperidone (1 µM) or rauwolscine (1 µM) significantly reduced the cardioprotective activity of CHF-1024 on ischemia and reperfusion injury. As shown in Table 1, the AUC values related to LVEDP, LVDevP, and CPP obtained with the combination of domperidone or rauwolscine plus CHF-1024 were significantly different (P < 0.001) from those shown in the hearts treated with CHF-1024 alone. Neither rauwolscine nor domperidone perfused alone for 10 min at a concentration of 1 µM did not worsen postischemic ventricular dysfunction (Table 1).

CK and LDH Activities in Heart Perfusates. CK and LDH, two indicators of myocardial damage, were determined in the coronary effluent collected from each heart in a 2.5-min sample during preischemic and reperfusion periods. As shown in Fig. 3, there were no differences among the various groups of hearts in CK and LDH release during the preischemic period. However, during 30-min reperfusion CK and LDH activities measured in vehicle-treated groups were 8.7-fold and 9.6-fold higher (P < 0.001) than those found in the preischemic period, respectively (Fig. 3). Treatment with 1, 10, or 100 nM CHF-1024, only when perfused before ischemia but not during reperfusion, significantly reduced in a concentration-dependent manner the CK and LDH release at reperfusion compared with vehicle-treated hearts (Fig. 3; Table 2). Pretreatment of the hearts with domperidone or rauwolscine attenuated the effect of CHF-1024 on CK and LDH release from reperfused heart (Table 2).

View larger version (26K): [in this window] [in a new window]   Fig. 3. CK and LDH release profile in isovolumic left rat heart preparations submitted to 20-min low flow ischemia (1 ml/min) and 30-min reperfusion (15 ml/min). Points are means ± S.E.M. of eight different hearts per group. Vehicle or CHF-1024 was perfused for 15 min during the preischemic period. For statistical comparisons, see the area under the curve values reported in Table 1.

PGI₂ Release in Heart Perfusates. It is well known that PGI₂ is the major eicosanoid produced by jeopardized myocardium (Van Bilsen et al., 1989) and the rate of formation of this lipidic material increases particularly during the first 5 to 10 min of reperfusion declining thereafter rapidly (Engels et al., 1990; Rossoni et al., 1997). In the present study, in vehicle-treated hearts the generation of 6-keto-PGF₁ (the stable metabolite of PGI₂) during reperfusion was enhanced 4.1-fold (587 ± 27 pg/ml; P < 0.001) compared with the preischemic period (142 ± 20 pg/ml) (Fig. 4). When the hearts were perfused with CHF-1024 at the highest concentration (100 nM) for 15 min before ischemia, a further slight increase (1.3-fold; P < 0.05 versus vehicle-treated hearts) in 6-keto-PGF_1a rate of formation was observed during reperfusion (Fig. 4). Compared with vehicle-treated hearts, no changes in 6-keto-PGF₁ generation were observed when CHF-1024 was given during reperfusion (data not shown).

View larger version (29K): [in this window] [in a new window]   Fig. 4. Rate of 6-Keto-PGF1 formation in isovolumic left rat heart preparations submitted to 20-min low flow ischemia (1 ml/min) and 30-min reperfusion (15 ml/min). Values are means ± S.E.M. of eight different hearts per group. Perfusates were collected during before ischemia (preischemia) and during the first 10 min of reperfusion. *, P < 0.05 versus vehicle-treated preparations in the reperfusion period.

NE and TNF- Release in Heart Perfusates. In preliminary experiments, and in accordance with Wollenberger and Shahab (1965), it was found that the bulk of NE outflow occurs within the first 2 min of the reperfusion period, and thereafter it declines to baseline within 10 to 15 min (data not shown). NE release was almost undetectable in 2-min samples of coronary effluent taken immediately before ischemia (4.1 ± 0.5 pg/ml), but it significantly increased during the first 2 min of reperfusion (258 ± 16 pg/ml). CHF-1024 perfused for 15 min through the heart in the preischemic period at the concentrations of 1, 10, or 100 nM significantly prevented the increase in NE outflow by 66% (P < 0.01), 94% (P < 0.001), and 98% (P < 0.001), respectively, compared with vehicle-treated hearts (Fig. 5). No effect was instead observed when 100 nM CHF-1024 was perfused during the reperfusion period (data not shown).

View larger version (30K): [in this window] [in a new window]   Fig. 5. Release of both NE (top) and TNF- (bottom) in the perfusates collected in the first 2 min of reperfusion period. Values are means ± S.E.M. of eight different hearts per group. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 versus vehicle-treated hearts; , P < 0.001 versus 100 nM CHF-1024-treated preparations.

Significant amounts of TNF- (107.1 ± 5.8 pg/ml) were detected in the coronary effluent only during the first 2 min of reperfusion, because 10 min later its concentration was below the sensitivity of the assay (<10 pg/ml; data not shown). CHF-1024 given at different concentrations before ischemia prevented TNF- release from the heart in a concentration-dependent manner. In particular, at 10 and 100 nM, CHF-1024 reduced the rate of TNF- release by 19% (P < 0.05) and 41% (P < 0.01), respectively, compared with the corresponding values of vehicle-treated hearts (Fig. 5).

In the experiments when perfusion with 100 nM CHF-1024 was preceded by 10-min infusion of domperidone (1 µM) or rauwolscine (1 µM), NE outflow during the first 2 min of heart-reperfusion was reduced only by 19% (P < 0.05) and 23% (P < 0.05), respectively, compared with the corresponding values of vehicle-treated hearts (Fig. 5). Similar results were obtained for TNF- release; in this case, the combined treatment of domperidone or rauwolscine plus 100 nM CHF-1024 resulted in a slight but nonsignificant reduction (P > 0.05) of TNF- release compared with vehicle-treated group (Fig. 5). Neither rauwolscine nor domperidone infused alone for 10 min at a concentration of 1 µM altered per se the basal release of NE and TNF- (data not shown).

	Discussion

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Neuroendocrine activation is one of the hallmarks of decompensated coronary heart failure; elevated plasma levels of pressor hormones, such as NE, the renin-angiotensin-aldosterone axis, and vasopressin have been repeatedly documented in various studies (Pouleur et al., 1994). Although this activation may be theoretically effective in restoring circulatory pressure and heart mechanics in the short term, in the long term it leads to increased afterload and preload and further decompensation (Rector et al., 1987).

In the present study, despite the different experimental conditions used compared with decompensated coronary heart failure, the selective DA₂-dopaminergic/₂-adrenergic receptor agonist CHF-1024 prevented in a dose-related manner NE release from myocardial sympathetic nerve endings, an event that is particularly marked during reperfusion of rat hearts subjected in vitro to global reduction of coronary flow. The activity of this compound was very marked because in preparations perfused for 15 min with 100 nM CHF-1024, the rate of NE release during reperfusion was inhibited by 94% (P < 0.01 versus vehicle-treated hearts). This effect of CHF-1024 may be explained by the ability of the compound to stimulate sympathetic prejunctional inhibitory receptors (Pastore et al., 2000; Razzetti et al., 2000). In fact, both the DA₂-dopaminergic antagonist domperidone (Kohli et al., 1983; Hilditch A and Drew, 1985) and the ₂-adrenoceptor antagonist rauwolscine (Tanaka et al., 1978; Du et al., 1991) almost completely blocked the CHF-1024-induced inhibition of NE release from reperfused hearts. These results are in line with those of Razzetti et al. (2000) who reported that domperidone and rauwolscine significantly antagonize the hypotensive response of CHF-1024 in anesthetized rats.

Another interesting point emerging from these experiments is the ability of 100 nM CHF-1024 to partially but significantly reduce the increased production of TNF- during heart reperfusion. Increasing evidence indicates that this polypeptide hormone is implicated in the pathogenesis of several cardiovascular diseases, including chronic heart failure, atherosclerosis, and sepsis associated to cardiac dysfunction (Meldrum, 1998). It is produced by the host in response to inflammation, tissue injury, and shock and has recently been recognized in perfusates of rat hearts undergoing ischemia and reperfusion in direct connection with the postischemic deterioration in myocardial mechanical performance and amount of cellular necrosis (Gurevitch et al., 1997). Furthermore, the ability of both domperidone and rauwolscine to lessen the CHF-1024-induced inhibition of TNF- generation during heart reperfusion, suggests a possible link between activation of sympathetic drive and augmentation of this polypeptide release from reperfused myocardium. This finding may be clinically relevant, because circulating levels of TNF- in congestive heart failure patients have been shown to correlate with the severity of heart failure and to predict long-term prognosis (Bolger and Anker, 2000).

These findings may also explain the cardioprotection afforded by CHF-1024 against postischemic ventricular dysfunction of the rat myocardium. The anti-ischemic activity, which is particularly marked at 100 nM CHF-1024, is also supported by concomitant and noticeable reduction of CK and LDH enzymes released in the perfusate during heart reperfusion with consequent decrease in both heart stiffness and CPP. In this respect, the DA₂-dopaminergic receptor agonist Z1046 has been shown to reduce ischemia severity, including ST-segment elevation and ventricular ectopic activity in a canine model of coronary artery occlusion (Vegh et al., 1998). As for CHF-1024, the anti-ischemic effects of this compound were attributed to presynaptic inhibitory receptor activation, because they were partly attenuated by domperidone (Vegh et al., 1998). Indeed, CHF-1024 dose dependently inhibited ventricular contracture of the hearts during ischemia and significantly improved LVDevP during reperfusion compared with vehicle-treated preparations.

Another point that speaks in favor of the cardioprotective affect of CHF-1024 is the maintenance of higher rate of 6-keto-PGF₁ release during reperfusion. In the heart, PGI₂ production during reperfusion is a critical cytoprotective mechanism for resisting the damage caused by ischemia (Ogletree et al., 1979; Berti et al., 1987). Stabilization of cardiac lysosomes provided by normal generation of PGI₂ is of paramount importance in ischemic myocardium since leakage of lysosomal enzymes (proteases and phospholipases) may contribute to irreversible damage of cardiomyocytes (Wildenthal et al., 1978).

In conclusion, the results obtained with CHF-1024 in the present study clearly indicate that this compound displays beneficial effects against myocardial ischemia and reperfusion damage. This can be referred to the reduction of release of NE from sympathetic nerve endings and of TNF- from cardiac tissues during reperfusion. Considering that CHF-1024, alone or in combination with an ACE inhibitor, has been shown to reduce adrenergic overactivity and collagen deposition in an experimental model of myocardial infarction (Latini et al., 1998; Masson et al., 1999), the finding that this compound is also endowed with anti-ischemic properties for the heart, is worth further studies and may widen its therapeutic implications in cardiovascular diseases.

	Footnotes

 
DOI: 10.1124/jpet.103.054700.

ABBREVIATIONS: ACE, angiotensin-converting enzyme; CHF-1024, (±)-(R,S)-5,6-dihydroxy-2-methylamino-1,2,3,4-tetrahydro-naphthalene hydrochloride; DA, dopamine; NE, norepinephrine; CPP, coronary perfusion pressure; KHS, Krebs-Henseleit solution; LVEDP, left ventricular end-diastolic pressure; LVDevP, left ventricular developed pressure; CK, creatine kinase; LDH, lactate dehydrogenase; ELISA, enzyme-linked immunosorbent assay; TNF-, tumor necrosis factor-; AUC, area under the curve; Z1046, (S)-6[[6-[[2-(2-methoxyphenoxy)ethyl]amino]propyl]amino]-5,6,7,8-tetra-hydro-1,2-naphthalenediol dihydrochloride.

Address correspondence to: Dr. Giuseppe Rossoni, Department of Pharmacology, Chemotherapy and Medical Toxicology, University of Milan, Via Vanvitelli 32, 20129 Milan, Italy. E-mail: giuseppe.rossoni{at}unimi.it

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