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The effect of carvedilol on enhanced ADP-ribosylation and red blood cell membrane damage caused by free radicals

Tamas Habon, Eszter Szabados, Gabor Kesmarky, Robert Halmosi, Tibor Past, Balazs Sumegi, Kalman Toth
DOI: http://dx.doi.org/10.1016/S0008-6363(01)00359-5 153-160 First published online: 1 October 2001

Abstract

Objective: Previous studies have reported that the beta and alpha adrenoceptor blocker carvedilol has unique protective effects on free radical-induced myocardial injury. The aim of this study was to examine how carvedilol regulates reactive-oxygen-species-mediated signaling and decreases red blood cell membrane damage in heart perfusion and in a rheological model. Methods: The ischemia–reperfusion-induced oxidative cell damage, and changes in the intracellular signaling mediated by reactive oxygen species and peroxynitrite were studied on rat hearts in a Langendorff perfusion system (n=15). The effect of carvedilol on red blood cell suspension viscosity (hematocrit: 60%) incubated with free radical generator (phenazine methosulphate) was also investigated (n=10). The measurements were performed on a capillary viscosimeter. Results: In both studies a protective effect of carvedilol was found, as the decrease of red blood cell suspension viscosity and K+ concentration in the supernatant indicated. Carvedilol significantly decreased the ischemia–reperfusion-induced free radical production and the NAD+ catabolism and reversed the poly- and mono(ADP-ribosyl)ation. Carvedilol also decreased the lipid peroxidation and membrane damages as determined by free malondialdehyde production and the release of intracellular enzymes. The self ADP-ribosylation of isolated poly(ADP-ribose) polymerase was also significantly inhibited by carvedilol. Conclusion: Our results show that carvedilol can modulate the reactive-oxygen-species-induced signaling through poly- and mono(ADP-ribosyl)ation reactions, the NAD+ catabolism in postischemic perfused hearts and has a marked scavenger effect on free radical generator-induced red blood cell membrane damage. All these findings may play an important role in the beneficial effects of carvedilol treatment in different cardiovascular diseases.

Keywords
  • Free radicals
  • Ischemia
  • Microcirculation
  • Reperfusion
  • Signal transduction
Abbreviations
  • CK, creatine kinase
  • DHR, dihydrorhodamine123
  • ECL, enhanced chemiluminescence
  • GOT, glutamate oxaloacetate transaminase
  • LDH, lactate dehydrogenase
  • MDA, malondialdehyde
  • NAD+, nicotinamide adenine dinucleotide (oxidized form)
  • NADH, nicotinamide adenine dinucleotide (reduced form)
  • PARP, poly(ADP-ribose) polymerase
  • PMS, phenazine methosulphate
  • RBC, red blood cell
  • ROS, reactive oxygen species

Time for primary review 23 days.

1 Introduction

Reperfusion of ischemic myocardium results in the generation of reactive oxygen species (ROS), such as H2O2, superoxide radicals and hydroxyl radicals formed in the Fenton and Haber–Weiss reaction [1]. ROS and peroxynitrite (formed from superoxide and NO) initiate lipid peroxidation [2], oxidation of proteins [3] and DNA damage [4–6], and these ROS are believed to be responsible, at least in part, for myocardial damage associated with infarction and reperfusion [7].

If ROS and peroxynitrite concentrations are permanently high, these, in turn, may cause a significant amount of single-strand DNA breaks which activate the nuclear poly(ADP-ribose) polymerase (PARP), a chromatin bound enzyme involved in the repair of DNA breaks [8,9]. Although, the PARP has a role in genome stability and defense against genomic mutation [10–12], mice having an inactivated PARP gene are still viable and fertile [11] indicating that the presence of PARP activity in different cells is not vital. On the other hand there is evidence showing that the activation of the nuclear PARP accelerates NAD+ catabolism, and the depletion of NAD+ compromises the mitochondrial energy metabolism [13] which can be one of the major causes of cell death in postischemic cardiomyocytes.

The assumption that PARP activation is a major cause of ischemia–reperfusion-induced cell death is based on the observation that PARP inhibitors and the disruption of the PARP gene partially prevents ROS and NO toxicity [14–16]. The known PARP inhibitors display several other pharmacological activities [17–19], that makes most of them unacceptable for clinical practice.

If free radical production is extensive enough to overwhelm endogenous anti-radical defenses (e.g. glutathione, superoxide dismutase, catalase, vitamin E) tissue injury can occur. The activation of these unfavorable reactions could be protected by antioxidants that reduce the intracellular level of ROS and peroxynitrite.

Additionally, many other tissues and cells can be damaged by oxygen free radicals, especially red blood cells (RBC), which are very susceptible to oxidative damage [20]. Oxidative stress changes RBC mechanical properties, decreases deformability and increases aggregability and thus increases apparent blood viscosity. In the microcirculation the ability of RBCs to deform when passing through narrow capillaries is absolutely essential for tissue perfusion and viability [21]. Antioxidant therapy can protect these detrimental rheological changes [22].

Carvedilol is a multiple-action neurohormonal antagonist combining non-selective beta-blockade and vasodilatation through alpha-blockade. Apart from the beta-blocking and vasodilating activities, carvedilol has also antioxidant properties, which may inhibit both the direct cytotoxic actions of ROS as well as oxygen radical-induced activation of transcription factors and genes [23–26]. These may primarily account for the marked cardioprotective effects observed in animal models.

In this work we studied the effect of ischemia–reperfusion on mono- and poly(ADP-ribosyl)ation of cardiac proteins, the ROS production, lipid peroxidation, DNA breaks, NAD+ catabolism and membrane integrity in a Langendorff heart perfusion system. We also investigated the effect of superoxide radicals generated within the RBCs by phenazine methosulphate (PMS) [27–30] on the viscosity of RBC suspension, measured by capillary viscosimetry. The questions of how carvedilol regulates ROS-mediated signaling in a heart perfusion model and decreases RBC membrane damage in a rheological model were addressed.

2 Methods

2.1 Chemicals

NAD+, dihydrorhodamine123 (DHR), IgG peroxidase and PMS were purchased from Sigma Chemical Co. Carvedilol was obtained from Boehringer Mannheim (Mannheim, Germany) and malondialdehyde-bis-(diethylacetal) from Merck (Darmstadt, Germany). Anti(ADP-ribose) antibody was a kind gift from Alexander Buerkle (Heidelberg, Germany) and Masanao Miwa (Tsukuba, Japan). An LMW Electrophoresis Kit was purchased from Pharmacia. All other reagents were of the highest purity commercially available.

2.2 Animals

The hearts of adult male Wistar rats weighing 300–350 g were used for the Langendorff heart perfusion experiments. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996), and approved by the Animal Research Review Committee of the University of Pecs Medical School.

2.3 Heart perfusion

Rats were anesthetized with ketamine (200 mg/kg) intraperitoneally and heparinized with sodium heparin (100 IU/rat i.p.). Hearts were perfused via the aorta according to the Langendorff method [31] at a constant pressure of 70 mmHg, at 37°C. The perfusion medium was a modified phosphate-free Krebs–Henseleit buffer consisting of 118 mM NaCl, 5 mM KCl, 1.25 mM CaCl2, 1.2 mM MgSO4, 25 mM NaHCO3, 11 mM glucose and 0.6 mM octanoic acid and in the treated group 1 μM carvedilol was added. The perfusate was bubbled with 95% O2/5% CO2 through a glass oxygenator and adjusted to pH 7.40. After a stabilization period of 15 min, hearts were either perfused under normoxic conditions for 15 or 30 min, or were subjected to a global ischemia of 1 h by closing the aortic influx and reperfused for 15 or 30 min. During ischemia hearts were submerged into perfusion buffer at 37°C. At the end of perfusion hearts were freeze clamped.

2.4 Assay of NAD+

The concentration of NAD+ in the neutralized perchloric acid extract of the cardiac muscle was measured by using alcohol dehydrogenase reaction [32]. The freshly prepared reaction buffer contained 0.1 M Tris, pH 8.40, 1 mM EDTA, 4 mM l-cysteine chloride, and 2% ethanol. Each cuvette contained 300 μl of the tissue extract, 650 μl of the reaction buffer and 4 units of enzyme. The reaction was initiated by the addition of enzyme, and the exact tissue NAD+ concentrations were determined from a calibration curve.

2.5 Detection of ROS and peroxynitrite

Hydrogen peroxide in the presence of peroxidase, iron or cytochrome C, and hydroxyl radical as well as peroxynitrite oxidizes the nonfluorescent DHR to form fluorescent rhodamine123 [33]. In normoxic hearts after 15 min washout, DHR (1 μM) was added to the perfusate and perfusion was continued for an additional 15 min. In the case of hearts subjected to 60 min ischemia and 15 min reperfusion, DHR (1 μM) was added to the perfusate just before reperfusion. In all cases, hearts were perfused with DHR for 15 min, and freeze clamped at the end of perfusion. For the extraction of rhodamine123, 90 mg of heart pieces were homogenized in 2 ml 20 mM Tris buffer pH 7.40, and an equal amount of ice-cold 70% ethanol containing 0.1 M HCl was then added. The precipitated proteins were removed by centrifugation at 3000×g for 15 min. The precipitate was extracted once again, and the unified supernatants’ aliquots were neutralized with NaHCO3 and centrifuged at 6000×g. The rhodamine123 content in the clear supernatant was determined using a Perkin Elmer fluorescence spectroscope at an excitation wavelength of 500 nm and an emission wavelength of 536 nm.

DHR can be oxidized to rhodamine123 not only by ROS but also by peroxynitrite, which can be formed, from superoxide and NO [33]. To determine the contribution of peroxynitrite to rhodamine123 formation, we perfused hearts with N(ω)-methyl-l-arginine, a well-known NO-synthase inhibitor [34], that caused only very small, statistically non-significant decrease in the rhodamine123 production indicating that NO-related compounds like peroxynitrite have only a very small contribution to the oxidation of DHR to rhodamine123. Since DHR is cell permeable and the oxidized rhodamine123 is preferentially retained by the mitochondria, the above system seems to be appropriate to detect ROS in situ in a perfused heart.

2.6 Lipid peroxidation

Lipid peroxidation was estimated from the direct determination of free malondialdehyde (MDA) by high performance liquid chromatography in the neutralized acid extract of the heart tissue and in the perfusate after acetonitrile addition as described by Lazzarino [35], using a Tosoh, Tsk-6011 HPLC system and a C-18, reverse-phase, 25 cm×4.6 mm column. The absorption was measured at 266 nm.

2.7 Assessment of cell membrane integrity

The release of lactate dehydrogenase EC 1.1.1.27 (LDH), creatine kinase EC 2.7.3.2 (CK) and glutamate oxaloacetate transaminase EC 2.6.1.1 (GOT) enzymes were measured in the perfusate of Langendorff perfused hearts under normoxic and ischemic conditions. Enzyme activities were measured by standard methods as described by Lazzarino [36] for LDH, and by Sherry [31] for GOT and CK.

2.8 ADP-ribosylation assay

Fifty mg cardiac muscle were homogenized with Ultra-Turrax in 500 μl 50 mM Tris pH 7.80 and 500 μl 2× Laemmli sample buffer were added, homogenized with Potter and cleared by centrifugation for 5 min at 10,000 rev./min. In some experiments the extraction buffer contained 8 M urea, 20 mM Tris, 4 mM EDTA. Samples were subjected to SDS–polyacrylamide gel electrophoresis using a 10% gel and blotted to nitrocellulose membrane for Western blot analysis. ADP-ribosylated proteins were detected by anti(ADP-ribose) monoclonal antibody and anti-mouse IgG peroxidase complex and visualized by enhanced chemiluminescence (ECL) method.

2.9 In vitro PARP inhibition on isolated enzyme

PARP was isolated from rat liver based on the method described before [37]. The PARP activity was determined in 130 μl reaction mixture that contained 100 mM Tris–HCl buffer, pH 8.0, 10 mM MgCl2, 10% glycerol, 1.5 mM DTT, 100 μM [32P], or [3H]NAD+, 10 μg activated DNA, 10 μg histones. The incubation time was 10 min, and the reaction was stopped by the addition of trichloroacetic acid (8%). After addition of 0.5 mg albumin, precipitation was allowed for at least 20 min on ice, and the protein was precipitated with centrifugation (10 min, 10,000×g). The precipitate was washed three times with 8% trichloroacetic acid, and the protein-bound radioactivity was determined with a Beckman scintillation counter.

2.10 Preparation of blood samples for rheological study and measurement of RBC suspension viscosity

Venous blood samples were obtained from healthy volunteers and anticoagulated with heparin. RBCs were separated from blood by centrifugation at 1500×g for 10 min and washed twice with phosphate buffered saline (PBS; pH 7.4; osmolality 310 mOsm; contains 10 mmol/l glucose). After the last centrifugation the supernatant was removed, and the hematocrit was measured (it was about 90%). The washed RBCs were resuspended in PBS at a hematocrit of 20%, divided and incubated (1) control; (2) in the presence of 10 μmol/l carvedilol; (3) 1 mmol/l PMS; (4) 1 mmol/l PMS+1 μmol/l carvedilol; (5) 1 mmol/l PMS+10 μmol/l carvedilol. All suspensions (8 ml) were incubated at 37°C for 2 h in a water bath. At 0 and 120 min of the incubation period blood samples were collected for extracellular Na+ and K+ measurements. At the end of the incubation the samples were centrifugated and RBCs resuspended in PBS and the hematocrit was adjusted to 60%. The viscosity of RBC suspensions was measured on a capillary viscosimeter (Hevimet 40, Hungary — the data are given at 90 l/s shear rate). All measurements were performed within 20 min after the preparation.

3 Results

3.1 ROS determination in perfused heart

The oxidation of DHR to rhodamine123 was detectable in normoxic perfused hearts, and the oxidation of DHR to rhodamine123 by ROS was significantly increased as a consequence of ischemia–reperfusion (Table 1). Perfusion of hearts with carvedilol significantly decreased the ischemia–reperfusion-induced rhodamine123 formation (Table 1) showing that carvedilol is an excellent antioxidant, directly decreasing the steady-state level of ROS in postischemic hearts.

3.2 Activation of PARP by ischemia–reperfusion

ADP-ribosylation of the high molecular weight nuclear PARP was detectable by Western blot analysis if the extraction buffer contained 8 M urea (Fig. 1). The formation of ischemia–reperfusion-induced ROS caused DNA damage, including single-strand DNA breaks, which activated the self ADP-ribosylation of PARP (Mw∼116 kDa). The presence of carvedilol in the perfusate significantly decreased ROS level and the single-strand DNA breaks in the reperfused hearts, and decreased the self ADP-ribosylation of the nuclear PARP (Fig. 1).

View this table:
Table 1

Effect of carvedilol on free radical formation during ischemia and reperfusion cycle in the Langendorff perfused rat heart system

Rhodamine123 fluorescence
in arbitrary units in situ
Normoxic21.4±1.7
Ischemia–reperfusion28.7±1.8a
Ischemia–reperfusion +1 μmol/l carvedilol23.8±2.4
  • ROS and peroxynitrite formation was followed by the oxidation of DHR to rhodamine123. Hearts were freeze clamped after 1 h ischemia and handled as detailed in Section 2. Values are shown as mean±S.E.M. for five heart preparations.

  • a Values are different from the other respective normoxic values and ischemia–reperfusion+carvedilol values at the significance P<0.01.

Fig. 1

Ischemia–reperfusion-induced self poly(ADP-ribosyl)ation of nuclear poly(ADP-ribose) polymerase. Proteins were separated with SDS polyacrylamide (10%) gel electrophoresis, and the level of ADP-ribosylation was determined by Western blotting using antibodies developed against ADP-ribose. Extraction of heart proteins was performed in the presence of 8 M urea, see Section 2. Lane 1, control heart (10 μg protein). Lane 2, postischemic heart (1 h ischemia and 30 min reperfusion) (10 μg protein). Lane 3, postischemic heart (1 h ischemia and 30 min reperfusion) in the presence of 1 μM carvedilol (10 μg protein).

3.3 Ischemia–reperfusion-induced NAD+ catabolism in perfused rat hearts

The activation of PARP can cause a significant loss of the intracellular NAD+, therefore, we determined the NAD+ content of heart tissues in normoxic hearts, in ischemic hearts and in the hearts subjected to ischemia–reperfusion (Table 2). It was found that 1 h ischemia caused only a slight decrease in the NAD++NADH content (Table 2), but a significant decrease was seen in the NAD+/NADH ratio (data not shown). One hour ischemia followed by 5, 30 and 60 min reperfusion induced a significant loss of the intracellular NAD+ (Table 2). The perfusion of hearts with carvedilol provided a partial protection against the ischemia–reperfusion-induced NAD+ loss (Table 2).

View this table:
Table 2

Effect of carvedilol on the ischemia–reperfusion-induced NAD+ loss in Langendorff perfused rat hearts at different reperfusion times

ConditionsReperfusion
0 min5 min30 min60 min
μmol (NAD+)/g wet tissue
Control0.49±0.03
Ischemia0.38±0.04b
Ischemia–reperfusion0.29±0.030.26±0.020.24±0.02
Ischemia–reperfusion0.39±0.05a0.30±0.03a0.29±0.03a
+1 μmol/l carvedilol
  • Experimental conditions and NAD+ determination were performed as described under Section 2. Values are shown as mean±S.E.M. for five heart preparations.

  • a Values are different from values without carvedilol at significance P<0.05.

  • b Because a significant fraction of NAD+ was reduced to NADH, under ischemic condition we determined the sum of NAD+ and NADH.

3.4 Lipid peroxidation in postischemic hearts

Under our experimental conditions free malondialdehyde was not detectable by the HPLC method in the heart tissue neither in normoxic (tissue contains sufficient antioxidants) nor in ischemic hearts (no oxygen available). However, a significant quantity of free malondialdehyde was detected in the reperfusion phase during the first couple of minutes. Perfusion of the hearts with carvedilol markedly decreased the amount of free malondialdehyde in the perfusate of postischemic hearts (Table 3).

3.5 Release of cytoplasmic enzymes

The release of CK, LDH and GOT were determined in normoxic hearts, postischemic hearts and postischemic hearts in the presence of 1 μM carvedilol (Table 3). The releases of these enzymes were very low in normoxic perfusion, but ischemia–reperfusion caused a significant release of CK, LDH and GOT into the perfusate (Table 3). When the hearts were perfused with carvedilol a significantly reduced release of CK, LDH and GOT was seen in the perfusate of postischemic rat hearts (Table 3).

3.6 Direct inhibition of nuclear PARP

In our heart perfusion system under in situ conditions carvedilol significantly decreased the self ADP-ribosylation of PARP (Fig. 1), that could be either a direct or an indirect effect. It is possible that carvedilol may directly inhibit the PARP. The effect of carvedilol on the self ADP-ribosylation of PARP was determined on isolated enzyme, and the result is shown in Table 4.

View this table:
Table 3

The effect of carvedilol on lipid peroxidation and intracellular enzyme release during ischemia and reperfusion cycle in the Langendorff perfused rat heart system

GOTLDHCKMDA
mU/mlnmol/ml
Normoxia2±11±14±2<1
Ischemia–reperfusion96±8a419±36a148±17a23±2.7b
Ischemia–reperfusion32±1244±148±47.6±2.7
+1 μmol/l carvedilol
  • Lipid peroxidation was estimated by the MDA production measured in the effluent during the first 5 min by HPLC system. Membrane integrity was followed by the determination of intracellular enzyme release into the perfusate after 15 min reperfusion. The details of experimental protocols are in Section 2. Values are shown as mean±S.E.M. for five heart preparations.

  • a Values are different from respective normoxic values and ischemia–reperfusion+carvedilol values at significance P<0.001.

  • b Values are different from the respective ischemia–reperfusion+carvedilol values at significance P<0.01.

3.7 Effect of PMS and carvedilol on RBC membrane and RBC suspension viscosity

During the incubation period the RBC suspension was stable as the similar K+ concentration and viscosity of untreated samples at 0 and 120 min indicated. Carvedilol itself had no significant effect on the K+ efflux and viscosity value. Incubation of the RBC suspension at a hematocrit of 20% with PMS significantly increased K+ concentration in the supernatant. Carvedilol in a 1 and 10 μM concentration dose dependently protected the effect of PMS as the decreased efflux of K+ indicated. The viscosity of the 60% hematocrit RBC suspension was considerably increased by the effect of PMS, which was partially protected by different concentrations of carvedilol (Table 5).

View this table:
Table 4

Effect of carvedilol on the activity of nuclear poly(ADP-ribose) polymerase, isolated from rat liver

PARP activity
Control1858±34
Carvedilol
1 μM961±82a
10 μM675±24a
Nicotinamide
1 mM933±42a
5 mM138±79a
  • Detailed in Section 2. Values are shown as mean±S.E.M. of 10 samples.

  • a Values are different from control values at significance P<0.001.

View this table:
Table 5

Effect of carvedilol on the K+ concentration of the supernatant and the viscosity of 60% HTC RBC suspension

SampleK+ concentrationViscosity
(mmol/l)(mPa s at 90 l/s)
Control at 0 min3.9±0.14.1±0.1
Control at 120 min4.0±0.14.2±0.1
Carvedilol 1 μM4.0±0.24.2±0.2
PMS 1 mM6.2±0.4a5.8±0.3a
PMS 1 mM+carvedilol 1 μM5.7±0.55.5±0.4
PMS 1 mM+carvedilol 10 μM5.5±0.3b4.9±0.4b
  • The viscosity of RBC suspensions were measured on a capillary viscosimeter (Hevimet 40, Hungary, the data are given at a 90 l/s shear rate) as detailed in Section 2. Values are shown as mean±S.E.M. for 10 blood samples.

  • a Values are different from the respective control values at the significance P<0.01.

  • b Values are different from the respective PMS-treated values at the significance P<0.05.

4 Discussion

Oxygen free radicals and their metabolites play an important role in ischemia–reperfusion injury [7]. Additionally, this oxidative attack damages RBCs, resulting in important functional alterations in both membrane and cytoplasmic structures [20]. It is important to understand how ROS affect cellular metabolism and signaling through mono- and poly(ADP-ribosyl)ation reactions [8,9,13]. In a Langendorff heart perfusion model, ischemia–reperfusion-induced ROS and peroxynitrite formation was determined by the oxidation of the nonfluorescent DHR to fluorescent rhodamine123. Ischemia–reperfusion increased the formation of these ROS (Table 1). The permanently high level of predominantly mitochondrially formed ROS can also affect other cell structures (like the nucleus and the ER), and can cause damage to DNA in several forms including single-strand DNA breaks. In our previous work we found a significant increase in the amount of single-strand DNA breaks as a consequence of increased ROS level and a parallel activation of the nuclear PARP [38].

Although there are data about the physiological function of PARP, it became evident that ROS and ischemia–reperfusion-induced cell damage can be diminished by inhibition of PARP [14,39] or the disruption of PARP gene [4,11]. The dramatic protection of cells from oxidative damage was shown in cultured cells, perfused organs and on living animals making clear that inhibition, or the complete abolishment of PARP-catalyzed reaction remarkably increases the survival of oxidatively damaged cells [14,40]. Activation of PARP plays a critical role in neurotoxicity, myocardial infarction, cytokine and oxidant damage to pancreatic islet cells and hepatocytes, and peroxynitrite toxicity to pulmonary epithelium, macrophages and smooth muscle cells [9,14–16,41].

The activation of the nuclear PARP (Fig. 1) can affect the whole cellular metabolism by decreasing the intracellular NAD+ pool (Table 2). The ischemia–reperfusion-induced decrease in the NAD+ pool (Table 2) is probably mainly the consequence of the activation of PARP, because the other NAD+ catabolizing enzymes (such as the NAD+-glycohydrolase) are not activated under the conditions of ischemia–reperfusion [42]. The lack of activation of the nuclear PARP in rat heart subjected to ischemia–reperfusion in the presence of carvedilol is probably the consequence of the decrease in the steady-state concentration of ROS (hydroxyl radical and peroxynitrite) (Table 1) and presumably the decreased amount of single-strand DNA breaks. The decreased ROS level and low activity of PARP in the presence of carvedilol (1 μM) can also explain the reduced amount of damage to cardiomyocytes (Table 3) and the better recovery of the postischemic heart [8].

Under our experimental conditions ischemia–reperfusion induced a large increase in malondialdehyde production in perfused hearts which was diminished considerably by carvedilol treatment (Table 3) that protected intracellular lipids from peroxidation. Parallel with lipid peroxidation ischemia–reperfusion increased the release of cytoplasmic enzymes in perfused hearts (Table 3) which was also markedly decreased by carvedilol treatment, showing that carvedilol through its antioxidant effect protected membrane integrity of cardiomyocytes from ischemia–reperfusion-induced damage [43,44].

The in vitro kinetic study on isolated PARP showed that carvedilol inhibits the PARP-catalyzed reaction. Besides the well-known antioxidant effect of carvedilol, the direct interaction with PARP can explain the observed decrease in ADP-ribosylation and partial protection against ischemia-reoxygenation-induced cardiac injury.

There are data in the literature about the beneficial effect of carvedilol on hemorheological parameters [45]. Oxygen free radicals and their derivates are known to damage RBCs. This causes functional alteration and changes in the microrheological properties of RBCs (i.e. increased rigidity). These changes in deformability are related to membrane protein alterations [46], like spectrin-hemoglobin cross linking. Protein cross-linking in RBC membranes caused by oxygen free radicals has also been reported [20] and increased lipid peroxidation is the other possible mechanism [47]. These unfavorable changes in hemorheological parameters (increased rigidity, aggregation, and blood viscosity) can cause alterations in the microcirculation and may play a role in the no-reflow phenomenon after reperfusion.

In our in vitro RBC-rheological model the RBC suspension viscosity was determined only by the changes of deformability, since hematocrit level is standard, no plasma and therefore no aggregation is present [48]. Carvedilol itself had no effect on the K+ efflux and RBC deformability, as RBCs were collected from healthy volunteers, and no membrane damage was detected during the 120-min incubation period. Oxidative damage generated by incubation with 1 mM PMS causes RBC membrane damage as the significant increase of K+ concentration of the supernatant indicated. This membrane damage and structural alterations of RBCs caused a significant rise in the rigidity which considerably increases the RBC suspension viscosity. Both effects can be partially protected by different concentrations of carvedilol.

Besides the known effects of carvedilol [49], this new data suggests carvedilol’s PARP inhibitory and positive rheological effects, and can give a further explanation to its advantageous role in clinical practice. All this data requires further in vitro and in vivo examinations.

Acknowledgements

We wish to thank Bertalan Horvath and Laszlo Giran for their excellent technical help. This work was supported by grants from the Hungarian Science Foundation OTKA T020622, T26624, T025432, T034320 and from the Hungarian Ministry of Health and Welfare ETT 382/96, 06030/96, T35/2000 and from the Hungarian Ministry of Education FKFP 1393/1997.

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View Abstract