Biphasic protective effect of oxytocin on cardiac ischemia/reperfusion ...

Peptides 30 (2009) 2301–2308

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Biphasic protective effect of oxytocin on cardiac ischemia/reperfusion injury in anaesthetized rats Fariba Houshmand a, Mahdieh Faghihi a,*, Saleh Zahediasl b a b

Department of Physiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran Endocrine Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Islamic Republic of Iran

A R T I C L E I N F O

A B S T R A C T

Article history: Received 16 March 2009 Received in revised form 6 September 2009 Accepted 8 September 2009 Available online 15 September 2009

Oxytocin (OT) is well known for its role in reproduction. However, evidence has emerged suggesting a role in cardiovascular system. The aim of this study was to investigate the cardioprotective effect of oxytocin on ischemia/reperfusion (I/R) injury in an in vivo rat. Myocardial ischemia, was surgically induced by means of left anterior descending coronary artery occlusion for 25 min followed by reperfusion for 120 min. Infarct size was evaluated using the staining agent 2,3,5-triphenyltetrazolium chloride. Creatine kinase-MB isoenzyme (CK-MB) and lactate dehydrogenase (LDH) levels in plasma were analyzed to assess the degree of cardiac injury. Intraperitoneal administration of OT 0.001, 0.01 and 0.1 mg significantly reduced infarct size, LDH and CK-MB levels as compared to control (I/R) group and it had a biphasic effect on the reduction of ischemia/reperfusion injury. This biphasic effect was revealed as a U-shaped curve in which efficacy was optimal between very low and very high doses. Furthermore there were no significant differences in mean arterial pressure or heart rate between the OT treatment groups and control group during I/R. Blockade of specific OT receptors by atosiban (10 6 M) abolished or attenuated the effect of OT preconditioning. The result of this study shows that OT possess a dosedependent cardioprotective effect against ischemia/reperfusion injury and so study of OT preconditioning may provide a new target site for therapeutic exploitation. ß 2009 Elsevier Inc. All rights reserved.

Keywords: Oxytocin Preconditioning Ischemia/reperfusion Infarct size LDH CK-MB

1. Introduction Intensive research is underway in effort to develop pharmacological means to control the morbidity and mortality arising from the ischemic heart disease. It has been suggested that the beneficial effects of reperfusion on the myocardium might be in part reversed by the occurrence of reperfusion injury and since the reperfusion is initiated by the treatment of myocardial infarction, it is important to limit the extent of this injury [36]. Ischemic preconditioning, in which brief episodes of ischemia are used to protect the heart, can limit the myocardial damage caused by subsequent sustained ischemic reperfusion. Evidences have indicated that the cardioprotection of ischemic preconditioning is mediated by endogenous active substances, including neurotransmitters and autacoids [1]. Few studies have considered the possible relation between hormones and preconditioning [13,31,39]. More recently, oxytocin (OT) has been considered to be a cardiovascular hormone [15,20].

* Corresponding author. Tel.: +98 21 66419484; fax: +98 21 66419484. E-mail address: [email protected] (M. Faghihi). 0196-9781/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2009.09.010

Oxytocin, a nanopeptide, was the first hormone to have its biological activities established and chemical structure determined [12,35]. It was believed that OT is released from hypothalamic nerve terminals of the posterior pituitary into the circulation where it stimulates uterine contractions during parturition and milk ejection during lactation. However, equivalent concentrations of OT were found in the male pituitary, and stimuli for OT release was determined for both genders, suggesting other physiological functions for the hormone [12]. Indeed, recent studies indicate that OT is involved in cognition, tolerance, adaptation and complex sexual and maternal behavior as well as cardiovascular function [8,15]. Oxytocin is produced and released by the heart and acts on its cardiac receptors to decrease heart rate and force of contraction [15]. Systemic administration of OT has significant effects on blood pressure, vascular tone and cardiovascular regulation. Both increase and decrease in blood pressure in response to oxytocin have been reported [15,20,21,34]. Systemically administered oxytocin induces a short-lasting increase in blood pressure in rats [7,33] whereas the opposite is seen in humans [23,26,41]. Costa-e-Sousa et al. have reported that oxytocin is also able to modulate mechanical activity of atria and exerts negative inotropic and chronotropic effects both in unanesthetized rats and in

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F. Houshmand et al. / Peptides 30 (2009) 2301–2308

Male Sprague–Dawley rats (weighing 270–360 g) were maintained in the animal quarters under standardized conditions (12-h light/day cycle, 20–22 8C room-temperature and 40–50% humidity) and received standard laboratory rat chow and water ad libitum.

Apparatus, USA) with a stroke volume of approximately 1.2 ml 100 g 1 body weight at a rate of 60–70 stroke min 1. Atelectasis was prevented by the maintenance of positive endexpiratory pressure of 3–5 cm H2O. Catheter filled with heparinized saline (100 U/ml) was placed into the right carotid artery for blood sampling and blood pressure monitoring. A standard limb lead-II electrocardiogram (ECG) and arterial blood pressure were continuously monitored and recorded on a computer throughout the experiment, using a computerized data acquisition system (Power Lab data acquisition system, four channel, ADInstruments). For all groups, heart rate was extracted from the electrocardiogram recordings. The tail vein was cannulated for the administration of Evans blue to evaluate area at risk. The left thoracotomy was performed to expose the heart by sectioning of the fourth rib approximately 3 mm from the sternum. The pericardium was incised and a sling (6-0 silk Ethicon) was placed around the left anterior descending coronary artery close to its origin immediately the left atrial appendage to the right part of the left ventricle (LV). Both ends of the ligature were passed through a small plastic tube and then chest was partially closed and the animal was allowed to recover for 30 min. Heart rate and blood pressure were allowed to stabilize for 30 min before protocols were initiated. Following stabilization, baseline and intervention periods, the coronary artery was occluded by applying tension to the plastic tube-silk string arrangement to induce transient regional myocardial ischemia for 25 min. Reperfusion was initiated by releasing the ligature and removing the tension. Coronary artery occlusion was verified by epicardial cyanosis and subsequent decrease in blood pressure, and reperfusion was confirmed by epicardial hyperemia.

2.1. Animals and surgical preparation

2.2. Experimental protocol

The animals were anaesthetized with sodium pentobarbital (60 mg kg 1, i.p.) and anesthesia was maintained with supplementary injections (30 mg kg 1, i.p.) as required. Body temperature was monitored by rectal thermometer and maintained at 37 1 8C. Following tracheotomy, the rats were ventilated with air-andoxygen mixture by a rodent ventilator (model 683, Harvard

The present study consisted of three protocols. Rats were randomly divided into eight groups and underwent LAD occlusion for 25 and 120 min of reperfusion, as shown in Fig. 1. On the day of the experiment, following stabilization period, basal hemodynamic parameters were measured for 15 min before drug administration. In protocol I (control group), saline was administered intraperitoneally 30 min before hearts were subjected to LAD

isolated rat heart [7]. Studies about the role of OT in cardiovascular regulation are limited and the mechanism by which OT induces its cardiovascular effects is not yet known [15]. A major goal of cardiovascular research is currently the identification of a reliable cardioprotective intervention that can salvage ischemic myocardium. Unfortunately, the clinical value of ischemic preconditioning itself is limited and none of several identified pharmacologic agents that appear to limit reperfusion injury is available for clinical use [49]. On the other hand, OT is produced and released by the heart and large vessels. It has been suggested that intrinsic OT system may play an important physiological role in regulation of vascular tone, as well as control of cardiac function [21]. OT is well known to exert potent physiological anti-stress effects [29] and stress increases the severity of cardiovascular disease [43]. Recently, increasing attention has been given to the potential role of oxytocin in cardiovascular functions [7,15,20,27]. Therefore, on the basis of this background the present study was designed: (1) to investigate the possible protective effect of OT against regional ischemic reperfusion (I/R) injury in heart, (2) to determine the dose– response relationship of OT on its cardioprotective effect and (3) to investigate the role of OT specific receptors in protection by three indices of injuries, infarct size, hemodynamic and biochemical factors in the same in vivo anaesthetized rats. 2. Materials and methods

Fig. 1. Illustration of the experimental protocols. Animals in control group (protocol I) were subjected to 25 min of coronary artery occlusion followed by 120 min reperfusion (I/R). In protocol II, the second to sixth groups were given i.p. infusions of different doses of oxytocin (OT) 30 min before I/R. In protocol, the selective antagonist atosiban was given i.p. 10 min before the infusions of 0.001, 0.01 and 0.1 mg oxytocin. TTC, triphenyltetrazolium chloride.


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occlusion and reperfusion. In OT dose-response studies, in protocol II, oxytocin 0.0001, 0.001, 0.01,0.1 and 1 mg (Sigma Chemical Co. St. Louis, MO, USA) was given as an i.p. bolus 30 min before hearts were subjected to LAD occlusion and reperfusion. During protocol II it was found that OT had a biphasic (revealed as a U-shaped curve) shape: a low-phase, which was significantly effective in 0.001 mg and a high phase reappearing in 0.1 mg during the descending and ascending limbs of the dose–response curves respectively and optimal effects induced in 0.01 mg of OT. Therefore, in protocol III in order to explore the role of OT receptor-mediated effects, atosiban (Sigma Chemical Co. St. Louis, MO, USA), an OT-selective receptor antagonist, was used in 2 additional groups (OT 0.001 and 0.1) beside in OT 0.01 group. It was given as a bolus injection with a dose of 10 6 M intraperitoneally 10 min prior to the treatment of 0.001, 0.01 and 0.1 mg of OT [14,37].

0.1 M phosphate buffer, pH 7.4) stain for 15 min at 37 8C, to visualize the infarct area and after that fixed for 2 days in 10% formalin to enhance the contrast of the Evans blue and TTC staining. Both surfaces of each transverse section were scanned and downloaded into Adobe PhotoShop (Adobe Systems Seattle, WA). The areas of the normal ventricle non-risk region (blue), area at risk (AAR, brick red) and area of necrosis (AN, pale) were determined by calculating the number of pixels occupying each area using the Adobe PhotoShop software. Total area at risk was expressed as a percentage of the left ventricles (AAR/LV). Infarct size was expressed as a percentage of the area at risk (AN/AAR). All experimental procedures used in this study were approved by the guidelines of the animal and human ethical committee of Tehran Medical Sciences University.

2.3. Cardiac area at risk and infarct size determination

To measure the myocardial specific enzymes, including the activity of creatine kinase-MB isoenzyme (CK-MB) and lactate dehydrogenase (LDH), blood samples were collected at the end of each experiment. The heparinized samples were centrifuged at 5000 rpm, 4 8C, for 15 min, and the plasma was removed and stored at 70 8C until the time they were assayed. The activity of CK-MB and LDH were analyzed using commercial kits (Pars Azmoon, Iran) by employing an autoanalyzer (Roche Hitachi Modular DP Systems, Mannheim, Germany).

The preparation used in the present study was as described previously [17]. In brief, after the completion of the experimental protocols, blood samples were taken, then the coronary artery was reoccluded and 2 ml of Evans blue dye in deionized water (2%) was injected intravenously to the tail vein for identification of area at risk (AAR). The Evans blue solution stains the perfused myocardium, while the occluded vascular bed remains unstained. Then the heart was excised, and both atria and the roots of the great vessels were removed. The heart was frozen for 24 h at 20 8C and then cut into slices of 2-mm-thick from the apex to the base. All slices were incubated with a 1% solution of 2,3,5-triphenyltetrazolium chloride (Sigma Chemical Co. St. Louis, MO, USA) (TTC, in

2.4. Biochemical analysis

2.5. Hemodynamic functions Hemodynamic data (arterial blood pressure and heart rate) were continuously monitored and recorded on a computer

Table 1 Hemodynamic values for OT dose–response studies. Group

n

Baseline

MAP (mmHg) Control OT 0.0001 OT 0.001 OT 0.01 OT 0.1 OT 1 OT 0.1 + atosiban OT 0.01 + atosiban OT 0.001 + atosiban

10 6 5 5 8 6 9 6 7

109 4 107 5 105 9 109 6 120 3 114 9 126 10 107 2 106 2

Heat rate (beat min 1) Control OT 0.0001 OT 0.001 OT 0.01 OT 0.1 OT 1 OT 0.1 + atosiban OT 0.01 + atosiban OT 0.001 + atosiban

10 6 5 5 8 6 9 6 7

380 12 375 16 341 15 369 11 368 8 345 16 405 10 345 10 370 5

RPP (beat min 1 mmHg 103) Control OT 0.0001 OT 0.001 OT 0.01 OT 0.1 OT 1 OT 0.1 + atosiban OT 0.01 + atosiban OT 0.001 + atosiban

10 6 5 5 8 6 9 6 7

43 3 44 2 39 4 44 2 46 1 44 5 56 8 40 1 42 1

Atosiban

Oxytocin

LAD occlusion 25 min

Reperfusion 120 min

115 5 103 4 102 5

99 6 114 9 120 5 121 5 122 5 121 3 115 4 112 3*

86 11 74 6** 99 15 112 6 99 10 91 11 88 10 107 7 96 8

83 11* 97 5 85 10 105 7 88 10* 87 4 80 10* 108 4 73 11*

377 14 314 9 347 9

302 13* 338 11 371 10 375 7 352 4 380 12 320 6 353 7

365 15 276 11*,### 330 16 382 17 362 8 345 2 350 10* 320 10+ 353 6

315 16* 291 5* 277 15* 359 15 326 17* 332 3 339 10** 322 8 323 15*

47 3 35 2 37 2

33 3* 42 4 49 3 48 2 47 2 49 2 40 1 43 9

37 4 24 2** 35 3 47 4 40 4 36 4 35 4* 37 3 38 2

27 2*** 31 1** 26 2* 41 3# 32 4** 34 2 30 4** 37 2## 31 3*

Data are presented as mean SE. n: number of animals in each group; MAP: mean arterial pressure (mmHg); RPP: rate pressure product (beat min 1 mmHg 103); control: prolonged equilibration time of 40 min prior to the 25 min period of regional ischemia followed by 120 reperfusion. Oxytocin (OT) at different doses (0.0001, 0.001, 0.01, 0.1 and 1 mg) was given as bolus intraperitoneally 30 min prior to the coronary occlusion in OT groups. Atosiban: 10 6 M a specific antagonist of OT receptor was given as bolus intraperitoneally 10 min prior to the OT treatment. *P < 0.05, **P < 0.01, ***P < 0.001 vs. baseline within the same group. +P < 0.05, vs. OT 0.01 group. #P < 0.05, ###P < 0.001 vs. control.

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Table 1 shows the time course of heart rate, mean arterial pressure and rate pressure product during the experiments. There were no significant differences among groups at baseline before treatment. At the end of reperfusion, heart function was

significantly decreased in control group, as indicated by MAP, HR and RPP as compared with its baseline. The results depicted in Table 1 show that at the end of the experiment, MAP decreased slightly in most OT groups but only in OT 0.1 group mean arterial pressure after 120 min reperfusion was significantly decreased compared to its baseline. Although in some groups, administration of OT nonsignificantly increased HR, MAP and RPP at preocclusion period, only in OT 0.0001 group the heart rate and RPP were significantly decreased compared to its baseline. On the whole, oxytocin itself had no significant effect on hemodynamic values at preocclusion. No significant differences were observed in any of the hemodynamic parameters between control and OT treatment groups during ischemia and reperfusion with two exceptions: a significant drop in HR in OT 0.0001 group in LAD occlusion period, and a significant increase in RPP in OT 0.01 group at the end of 120 min. In protocol III (Table 1) atosiban significantly decreased heart rate, mean arterial pressure and rate pressure product in OT 0.001 + atosiban and OT 0.1 + atosiban as compared to the baseline. Atosiban administration in OT 0.01 group did not cause any significant changes in hemodynamic values throughout the experimental protocol (Table 1). There were no significant differences in hemodynamic between oxytocin (0.001, 0.01 and 0.1), OT + atosiban and control groups. Only compared with OT

Fig. 2. Dose–response studies of oxytocin (OT) on infarct size (A) and area at risk (B). All values are expressed as means SE. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. control.

Fig. 3. The effect of atosiban on infarct size (A) and area at risk (B). All values are expressed as means SE. OT, oxytocin. *P < 0.05 vs. control, #P < 0.05 vs. OT 0.001, P < 0.05 vs. OT 0.01, +P < 0.05 vs. OT 0.1.

throughout the experiment: baseline, interaction period, 25 min LAD occlusion, and 120 min reperfusion. At the same time-points, the rate pressure product (RPP), defined as systolic blood pressure multiplied by the heart rate also were calculated by a computerized data acquisition system (Power Lab data acquisition system, four channels, ADInstruments). 2.6. Statistical analysis Statistical analysis of hemodynamic data within and between groups was performed with analysis of variance for repeated measures followed by the Dunnett’s test. Differences in infarct size, plasma LDH and CK-MB levels were determined by one-way analysis of variance. Individual groups were compared using Dunnett’s test. All data were expressed as mean SE and statistical significance was defined as P < 0.05. 3. Results 3.1. Hemodynamic functions


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0.01 group, HR value was significantly reduced in OT 0.01 + atosiban group at LAD occlusion process. Also in OT 0.01 + atosiban group, atosiban caused significant changes in RPP after reperfusion for 120 min compared with the control group.

There were no significant differences in the areas at risk between the groups (Fig. 3B).

3.2. Area at risk and infarct size measurements

The activity of LDH and CK-MB in plasma was used to monitor the damage of myocardium. In protocol II, compared to the control group, LDH activity in plasma of treatment groups markedly decreased after the ischemia-reperfusion process. Treatment with oxytocin 0.001 (P < 0.05), 0.01 (P < 0.01) and 0.1 mg (P < 0.05) could prevent elevation of LDH activity in plasma after ischemia/ reperfusion injury (Fig. 4A). Also OT 0.01 and 0.001 groups had significantly decreased CK-MB activity (P < 0.01, P < 0.05 respectively) as compared to the control group (Fig. 4B). In protocol III, in addition the OT 0.01 + atosiban group, administration of the antagonist attenuated the cardioprotective effects of OT in both phases during descending and ascending limbs of the dose-response curve respectively (Fig. 5). The LDH activity in OT + atosiban groups significantly was increased compared to oxytocin 0.01 and 0.1 groups (P < 0.05, Fig. 5A). The LDH activity in OT 0.01 + atosiban and OT 0.1 + atosiban groups were even higher than control group (P < 0.05) after ischemia-reperfusion injury as shown in Fig. 5A.

Fig. 2B presents risk zone size data. There were no significant differences in this parameter among the groups. Fig. 2A shows the infarct size for each group. Infarct size as a percentage of area at risk was 41.9 1.8% in control group, whereas preconditioning with 0.001–0.1 mg oxytocin (OT 0.001, 0.01 and 0.1 groups) significantly and dose-dependently reduced infarct size to 25.3 6.1% (P < 0.01), 16.2 2.8% (P < 0.001), and 30.3 2.2% (P < 0.05) respectively vs. control group (Fig. 2A). In protocol III, the reduction in infarct size (AN/AAR) by OT 0.01 was completely abolished by pretreatment with atosiban (P < 0.05, Fig. 3A). Also atosiban abolished the anti-ischemic actions of oxytocin in OT 0.001 group (low-phase) and in OT 0.1 group (high phase) during descending and ascending limbs of the dose–response curves respectively (P < 0.05, Fig. 3A). Atosiban also increased infarct size when administered before 0.1 mg oxytocin (OT 0.1 group) as compared to the control group (55.7 2.2%, and 41.9 1.8%, respectively; P < 0.05, Fig. 3A).

Fig. 4. Dose–response studies of oxytocin (OT) on plasma levels of LDH (A) and CKMB (B) of rats in ischemia/reperfusion injury. All values are expressed as means SE. *P < 0.05, **P < 0.01 vs. control.

3.3. Biochemical analysis

Fig. 5. The effect of atosiban on plasma levels of LDH (A) and CK-MB (B) in ischemia/ reperfusion injury. All values are expressed as means SE. OT, oxytocin. *P < 0.05 vs. control, #P < 0.05 vs. OT 0.001, P < 0.05 vs. OT 0.01, +P < 0.05 vs. OT 0.1.

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There was a tendency towards a significant increase in the level of CK-MB in OT + atosiban groups as compared to oxytocin groups (P < 0.05) (Fig. 5B). Administration of the antagonist before OT preconditioning also significantly (P < 0.05) altered the CK-MB levels as compared to the control (Fig. 5B). 3.4. Dose–response studies Oxytocin decreased post-ischemic infarct size, LDH and CK-MB in a U-shaped dose-dependent manner. The minimum dose of OT which could reduce myocardial infarction and levels of the enzymes was 0.001 mg. The dose of OT that caused a maximum reduction in infarct size and enzymes concentrations was 0.01 mg (Figs. 2 and 4). 4. Discussion The results of the present study show that pretreatment with OT can protect hearts from ischemic injury in anesthetized rats and to our knowledge, this is the first experimental evidence showing that OT can mimic preconditioning in a dose-dependent manner. OT with two exceptions caused no significant changes in the recorded hemodynamic profile, such as blood pressure, HR and RPP compared to the control group. This observation suggests that the effects of OT preconditioning on myocardial infarct are not related to alterations in hemodynamic parameters. So the in vivo effects of OT on the ischemic myocardium are likely dependent upon direct cytoprotection at the cellular level. Furthermore, our study demonstrates that the treatment with oxytocin before regional myocardial ischemia reperfusion can provide myocardial protection against reperfusion injury by decreasing infarct size in a dose depending manner. The present in vivo observations are in agreement with and extend previous data obtained in isolated perfused hearts, which indicate that exogenous OT has cardioprotective effect against infarction [30]. Blockage of the cardioprotective effect of OT by atosiban, a selective OT receptor antagonist, suggests that this effect was mediated via activation of the OT specific receptors. In addition, administration of OT receptor antagonist (OT 0.1 + atosiban) increased infarct size compared to control group, indicating that endogenous OT contributes to protection against I/R injury. In addition, OT reduced biochemical parameters in our experiments. Elevated levels of creatine kinase-MB (CK-MB) have been regarded as biochemical markers of myocyte necrosis [50] and OT when administrated before ischemia could induce a significant reduction in the level this enzyme compared to control group. Increase in plasma LDH level, which plays an important role in systemic tissue damage [9], was also attenuated by oxytocin treatment. In support of the current data, Tugtepe et al. showed that OT treatment attenuated the renal I/R-induced LDH response [42]. Biyikli et al. have reported that elevated serum LDH levels in the pyelonephritic rats were reversed by OT treatment [3]. Similarly Iseri et al. showed that in the acetic acid-induced colitis, serum LDH level was elevated and this increase was significantly decreased by OT treatment [18]. In this study we clearly demonstrated that OT had a cardioprotective role against I/R injury in open chest anesthetized rats and induced its protective effect dose-dependently. Oxytocin treatment reveals a biphasic pattern, i.e., increasing doses correlate with the increasing efficacy of injury inhibition until an optimal dose is attained beyond which higher doses show less activity. Optimal effect in the reduction of the infarct size, biochemical changes and hemodynamic improvement was induced by i.p. bolus injection of 0.01 mg oxytocin. For all indices of protection, higher and lower doses of oxytocin showed a less protective effect. This pattern of the effect of OT has been shown in other reports, too. For

example it was reported that oxytocin induced biphasic dosedependent changes in mean arterial pressure [27], arteriolar dilation [2] and chronotropic/inotropic effects [7,8,27]. The effects of 0.01 mg oxytocin were abolished by atosiban, a selective OT-receptor antagonist. These observations provided evidence that OT receptor mediates the cardioprotective effects of OT. On the other hand, because in the present study, pretreatment with the OT antagonist, atosiban, abolished both the low-phase and high-phase beneficial effects of OT on ischemia/reperfusion injury, the mechanism of OT-cardioprotection in both phases after ischemia appears to be related to the OT specific-receptor activity. In general, the receptor systems display such biphasic dose responses when a single agonist has differential affinities for two opposing receptor subtypes, a concept that was first described in detail by Szabadi [40]. By conducting more research about this concept, it is likely that even more mechanisms will be discovered that operate at the molecular, cellular and tissue levels, or total organism [6]. In spite of OT tendency for biphasic effects in different studies, it is not yet clarified whether different types of receptors are involved in the ‘‘low-’’ and ‘‘high’’-dose effects of oxytocin [44] and also it remains to be determined whether receptor-dependent activity is the only cause related to the biphasic cardioprotection induced by OT against I/R injury. In addition, atosiban, the OT antagonist, increased the severity of injury as compared to control group, suggesting a protective role for endogenous OT in the ischemia/reperfusion injury under experimental conditions. Therefore, the action of OT may be mediated, at least partly, via the cardiac OT receptors. Oxytocin receptors are cell surface receptors coupled with G-proteins and until now only one receptor is recognized [12,22]. But Breton et al. and Petersson et al. have reported that the OT effect may be mediated by an unidentified OTR subtype that differs from the known OTR [4,32]. Thus, the results of the present study add to the previous data [30] showing that OT under certain experimental conditions exerts a cardioprotective action. There are only limited numbers of published studies regarding the anti-ischemic effects of OT [10,42,46], and these reported that OT exerts its beneficial effects in the different aspects of protection such as anti-inflammatory actions and prevention of free radical damaging cascades. Oxytocin protects sepsis-induced oxidative damage by acting as an antioxidant agent [19] and its protective effect in the colon, liver and kidney appears to be dependent on its inhibitory effect on neutrophil infiltration and associated production of reactive oxygen species [10,42,48]. In accordance with these, the anti-inflammatory and antioxidant effects of OT on cardiac tissue may be the cause of its protection. Recent reports suggest that the protective effects of OT pretreatment on ischemia-reperfusion damage may be caused by the release of nitric oxide (NO) [10,42]. The protective effects of NO on cardiac I/R have been repeatedly described [25,28]. Another possibility to consider is that OT may release atrial natriuretic peptide (ANP) [14], which is a vasodilator and has antioxidative properties [14]. In previous studies, it was shown that ANP, released by heart in response to volume increase, protects kidney [10] and liver [11] from I/R injury. Since ANP acts as a mediator of ischemic preconditioning in rat [38], there is the possibility that the cardioprotective effects of OT preconditioning may be mediated through the release of ANP. Oxytocin has also been suggested to be involved in the modulation of immune and inflammatory processes. Specifically, oxytocin decreases the release of interleukin-6 and releases prostacyclin, which inhibit platelet aggregation [18,47]. On the other hand, it is generally accepted that OT activates two types of receptors, OT and V1 vasopressin receptors [7]. Both OT receptor [15] and vasopressin receptor [16], are present in the heart.


Activation of the V1 receptor evokes severe coronary constriction whereas activation of the endothelial OT receptors leads to vasodilatation [16,45]. However OT as a partial agonist of vasopressin may also evoke vasoconstriction in different vascular beds. The action of high concentrations of OT may be caused by activation of V1 receptors [21]. Petty et al. [34] reported that the intravenous administration of 10 mg OT produced an acute increase in mean arterial pressure of unanesthetized rats. Also, Costa-e-Sousa demonstrated that the intravenous administration of 1 mg OT (1 mg/ml to 10 6 M) produced a rise in mean arterial pressure of unanesthetized rats and that this effect resulted from the coupling of oxytocin to vasopressinergic V1 receptors [7]. Supporting this, the in vitro vasoconstrictor effect of OT can be completely blocked by a V1a receptor antagonist [5,26]. In our study we showed that administration of a specific OT antagonist could not induce significant changes on hemodynamic parameters as compared to its absence. Our results are also in agreement with the results of Petersson and Uvnas-Moberg who found that the increase in blood pressure in response to peripherally administered oxytocin was only partially antagonized by an oxytocin antagonist. Thus it probably also involves vasopressin (V1a)-receptors, since oxytocin in high concentrations also binds to these receptors [33]. In addition, a possible mechanism of action may be that OT modulates the interaction between energy demand and the contractile state of the myocardium and thereby myocardial oxygen consumption [44]. Recently, it was demonstrated that OT exerted negative inotropic and chronotropic effects [7] and also exerted an energy-saving effect via its receptor, which might be of importance for the cardioprotection effect of OT under the present experimental conditions. Another possibility is the involvement of central nervous system. Since 1–2% of peripherally given dose of OT passes the blood–brain barrier [32,44], the possible action of the hormone via central nervous system cannot be ruled out. Moreover, on the basis of coronary effects of vasopressin during ischemia and reperfusion Martinez et al. suggested that vasopressin might be involved in the adverse effects of ischemia/ reperfusion on coronary vasculature [24]. Therefore, it is possible that the interaction between exogenous OT in high doses and vasoconstrictor V1 receptors during ischemia and reperfusion induces unfavorable effects in the ischemic/reperfused myocardium in OT 1 mg. Supporting this, previous studies have demonstrated that the vasoconstrictor effect of OT of more than 10 6 M could be completely blocked by V1 receptor antagonists [7,26,34]. Thus, OT may induce PC-like cardioprotection via several different mechanisms. However, the exact mechanism(s) behind the cardioprotective effects of OT are beyond the aim of the present investigation and remain to be explored in future studies. These findings illustrate the dose-dependent nature of OT protection against infarction and highlight the need to perform dose–response studies when determining the potential cardioprotective properties of novel agents. 5. Conclusion In summary, the administration of OT before the induction of regional myocardial ischemia and reperfusion reduced the extent of irreversible myocardial injury by a mechanism involving activation of the OT specific receptors. The level of protection conferred by OT was manifested at a dose that did not result any changes in mean arterial blood pressure and heart rate values. Also in our study, reduction in infarct size was observed immediately after treatment with OT, indicating that induction of new genes is not necessary for its cardioprotective effect.

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Our results suggest that OT protects the heart and may represent a novel approach for the protection of the heart during open surgeries. Acknowledgments This study was supported by the grants from Tehran University of Medical Sciences. The authors wish to thank Professor Farokh Shadan and Professor Ahmadreza Dehpour for their critical assessments of the manuscript and valuable comments. References [1] Altup S, Demiryurek AT, Ak D, Tungel M, Kanzik I. Contribution of peroxynitrite to the beneficial effects of preconditioning on ischaemia-induced arrhythmias in rat isolated hearts. Eur J Pharmacol 2001;415:239–46. [2] Bari F, Errico RA, Louis TM, Busija DW. Influence of hypoxia/ischemia on cerebrovascular responses to oxytocin in piglets. J Vasc Res 1997;34:312–20. [3] Biyikli NK, Tugtepe H, Sener G, Velioglu-Ogunc A, Cetinel S, Midillioglu S, et al. Oxytocin alleviates oxidative renal injury in pyelonephritic rats via a neutrophil-dependent mechanism. Peptides 2006;27:2249–57. [4] Breton C, Pechoux C, Morel G, Zingg HH. Oxytocin receptor messenger ribonucleic acid: characterization, regulation, and cellular localization in the rat pituitary gland. Endocrinology 1995;136:2928–36. [5] Chen Y, Shepherd C, Spinelli W, Lai F. Oxytocin and vasopressin constrict rat isolated uterine resistance arteries by activating vasopressin V1A receptors. Eur J Pharmacol 1999;376:45–51. [6] Cook R, Calabrese E. The importance of hormesis to public health. Environ Health Perspect 2006;114:1631–5. [7] Costa-e-Sousa RH, Pereira-Junior PP, Oliveira PF, Olivares EL, Werneck-deCastro JP, Mello DB, et al. Cardiac effects of oxytocin: is there a role for this peptide in cardiovascular homeostasis? Regul Pept 2005;132:107–12. [8] Coulson CC, Thorp Jr JM, Mayer DC, Cefalo RC. Central hemodynamic effects of oxytocin and interaction with magnesium and pregnancy in the isolated perfused rat heart. Am J Obstet Gynecol 1997;177:91–3. [9] Devi R, Banerjee SK, Sood S, Dinda AK, Maulik SK. Extract from Clerodendron colebrookianum Walp protects rat heart against oxidative stress induced by ischemic-reperfusion injury (IRI). Life Sci 2005;77:2999–3009. [10] Du¨su¨nceli F, Iseri S, Ercan F, Gedik N, Yegen C, Yegen B. Oxytocin alleviates hepatic ischemia-reperfusion injury in rats. Peptides 2008. [11] Gerbes A, Vollmar A, Kiemer A, Bilzer M. The guanylate cyclase-coupled natriuretic peptide receptor: a new target for prevention of cold ischemiareperfusion damage of the rat liver. Hepatology (Baltimore MD) 1998;28: 1309. [12] Gimpl G, Fahrenholz F. The oxytocin receptor system: structure, function, and regulation. Physiol Rev 2001;81:629–83. [13] Grist M, Wambolt RB, Bondy GP, English DR, Allard MF. Estrogen replacement stimulates fatty acid oxidation and impairs post-ischemic recovery of hearts from ovariectomized female rats. Can J Physiol Pharmacol 2002;80:1001–7. [14] Gutkowska J, Jankowski M, Lambert C, Mukaddam-Daher S, Zingg H, McCann S. Oxytocin releases atrial natriuretic peptide by combining with oxytocin receptors in the heart. Proc Natl Acad Sci USA 1997;94:11704–9. [15] Gutkowska J, Jankowski M, Mukaddam-Daher S, McCann SM. Oxytocin is a cardiovascular hormone. Braz J Med Biol Res 2000;33:625–33. [16] Hupf H, Grimm D, Riegger GA, Schunkert H. Evidence for a vasopressin system in the rat heart. Circ Res 1999;84:365–70. [17] Imani A, Faghihi M, Sadr SS, Keshavarz M, Niaraki SS. Noradrenaline reduces ischemia-induced arrhythmia in anesthetized rats: involvement of alpha1adrenoceptors and mitochondrial K ATP channels. J Cardiovasc Electrophysiol 2008;19:309–15. [18] Iseri S, Sener G, Saglam B, Gedik N, Ercan F, Yegen B. Oxytocin ameliorates oxidative colonic inflammation by a neutrophil-dependent mechanism. Peptides 2005;26:483–91. [19] Iseri SO, Sener G, Saglam B, Gedik N, Ercan F, Yegen BC. Oxytocin protects against sepsis-induced multiple organ damage: role of neutrophils. J Surg Res 2005;126:73–81. [20] Jankowski M, Hajjar F, Kawas SA, Mukaddam-Daher S, Hoffman G, McCann SM, et al. Rat heart: a site of oxytocin production and action. Proc Natl Acad Sci USA 1998;95:14558–63. [21] Jankowski M, Wang D, Hajjar F, Mukaddam-Daher S, McCann SM, Gutkowska J. Oxytocin and its receptors are synthesized in the rat vasculature. Proc Natl Acad Sci USA 2000;97:6207–11. [22] Kiss A, Mikkelsen JD. Oxytocin—anatomy and functional assignments: a minireview. Endocr Regul 2005;39:97–105. [23] Light K, Smith T, Johns J, Brownley K, Hofheimer J, Amico J. Oxytocin responsivity in mothers of infants: a preliminary study of relationships with blood pressure during laboratory stress and normal ambulatory activity. Health Psychol 2000;19:560–7. [24] Martinez MA, Fernandez N, Climent B, Garcia-Villalon AL, Monge L, Sanz E, et al. Coronary effects of vasopressin during partial ischemia and reperfusion in anesthetized goats. Role of nitric oxide and prostanoids. Eur J Pharmacol 2003;473:55–63.

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[25] Masini E, Salvemini D, Ndisang J, Gai P, Berni L, Moncini M, et al. Cardioprotective activity of endogenous and exogenous nitric oxide on ischaemia reperfusion injury in isolated guinea pig hearts. Inflamm Res 1999;48: 561–8. [26] Miller M, Davidge S, Mitchell B. Oxytocin does not directly affect vascular tone in vessels from nonpregnant and pregnant rats. Am J Physiol Heart Circ Physiol 2002;282:1223–8. [27] Mukaddam-Daher S, Yin YL, Roy J, Gutkowska J, Cardinal R. Negative inotropic and chronotropic effects of oxytocin. Hypertension 2001;38:292–6. [28] Nakamura K, Al-Ruzzeh S, Gray C, Yacoub M, Amrani M. Effect of myocardial reperfusion on the release of nitric oxide after regional ischemia. Tex Heart Inst J 2006;33:35–9. [29] Neumann ID, Wigger A, Torner L, Holsboer F, Landgraf R. Brain oxytocin inhibits basal and stress-induced activity of the hypothalamo–pituitary– adrenal axis in male and female rats: partial action within the paraventricular nucleus. J Neuroendocrinol 2000;12:235–43. [30] Ondrejcakova M, Ravingerova T, Bakos J, Pancza D, Jezova D. Oxytocin exerts protective effects on in vitro myocardial injury induced by ischemia and reperfusion. Can J Physiol Pharmacol 2009;87:137–42. [31] Peng WJ, Yu J, Deng S, Jiang JL, Deng HW, Li YJ. Effect of estrogen replacement treatment on ischemic preconditioning in isolated rat hearts. Can J Physiol Pharmacol 2004;82:339–44. [32] Petersson M, Lundeberg T, Uvnas-Moberg K. Short-term increase and longterm decrease of blood pressure in response to oxytocin-potentiating effect of female steroid hormones. J Cardiovasc Pharmacol 1999;33:102–8. [33] Petersson M, Uvnas-Moberg K. Effects of an acute stressor on blood pressure and heart rate in rats pretreated with intracerebroventricular oxytocin injections. Psychoneuroendocrinology 2007;32:959–65. [34] Petty MA, Lang RE, Unger T, Ganten D. The cardiovascular effects of oxytocin in conscious male rats. Eur J Pharmacol 1985;112:203–10. [35] Pournajafi-Nazarloo H, Perry A, Partoo L, Papademeteriou E, Azizi F, Carter CS, et al. Neonatal oxytocin treatment modulates oxytocin receptor, atrial natriuretic peptide, nitric oxide synthase and estrogen receptor mRNAs expression in rat heart. Peptides 2007;28:1170–7. [36] Rao PR, Kumar VK, Viswanath RK, Subbaraju GV. Cardioprotective activity of alcoholic extract of Tinospora cordifolia in ischemia-reperfusion induced myocardial infarction in rats. Biol Pharm Bull 2005;28:2319–22.

[37] Reversi A, Rimoldi V, Marrocco T, Cassoni P, Bussolati G, Parenti M, et al. The oxytocin receptor antagonist atosiban inhibits cell growth via a ‘‘biased agonist’’ mechanism. J Biol Chem 2005;280:16311–8. [38] Sangawa K, Nakanishi K, Ishino K, Inoue M, Kawada M, Sano S. Atrial natriuretic peptide protects against ischemia-reperfusion injury in the isolated rat heart. Ann Thorac Surg 2004;77:233–7. [39] Song X, Li G, Vaage J, Valen G. Effects of sex, gonadectomy, and oestrogen substitution on ischaemic preconditioning and ischaemia-reperfusion injury in mice. Acta Physiol Scand 2003;177:459–66. [40] Szabadi E. A model of two functionally antagonistic receptor populations activated by the same agonist. J Theor Biol 1977;69:101. [41] Thomas J, Koh S, Cooper G. Haemodynamic effects of oxytocin given as iv bolus or infusion on women undergoing Caesarean section. Br J Anaesth 2007;98:116. [42] Tugtepe H, Sener G, Biyikli N, Yu¨ksel M, Çetinel S, Gedik N, et al. The protective effect of oxytocin on renal ischemia/reperfusion injury in rats. Regul Pept 2007;140:101–8. [43] Ueyama T, Yoshida K, Senba E. Stress-induced elevation of the ST segment in the rat electrocardiogram is normalized by an adrenoceptor blocker. Clin Exp Pharmacol Physiol 2000;27:384–6. [44] Uvnas-Moberg K. Antistress pattern induced by oxytocin. News Physiol Sci 1998;13:22–5. [45] Vincent JL, Su F. Physiology and pathophysiology of the vasopressinergic system. Best Pract Res Clin Anesthesiol 2008;22:243–52. [46] Wenwen Z, Zhang J, Xu M, Zhang Y. Effect of oxytocin on gastric ischemiareperfusion injury in rats. Front Med China 2007;1433–7. [47] Williams K, El Tahir K. Effects of uterine stimulant drugs on prostacyclin production by the pregnant rat myometrium. I. Oxytocin, bradykinin and PGF2 alpha. Prostaglandins 1980;19:31. [48] Xie D, Chen L, Liu C, Liu K. The inhibitory effects of oxytocin on distal colonic contractile activity in rabbits are enhanced by ovarian steroids. Acta Physiol 2006;186:141–9. [49] Yang XM, Proctor JB, Cui L, Krieg T, Downey JM, Cohen MV. Multiple, brief coronary occlusions during early reperfusion protect rabbit hearts by targeting cell signaling pathways. J Am Coll Cardioll 2004;44:1103–10. [50] Yilmaz A, Yalta K, Turgut OO, Yilmaz MB, Ozyol A, Kendirlioglu O, et al. Clinical importance of elevated CK-MB and troponin I levels in congestive heart failure. Adv Ther 2006;23:1060–7.