The Role of Nitric Oxide, Reactive Oxygen Species, and Protein Kinase C In

Home , Chelerythrine

Peptides 37 (2012) 314–319

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Peptides

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The role of nitric oxide, reactive oxygen species, and protein kinase C in

oxytocin-induced cardioprotection in ischemic rat heart

a b,∗ c d e

Mahdieh Faghihi , Ali Mohammad Alizadeh , Vahid Khori , Mostafa Latifpour , Saeed Khodayari

Department of Physiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

Cancer Research Center, Tehran University of Medical Sciences, Tehran, Iran

Department of Pharmacology, Golestan Cardiovascular Research Center, Golestan University of Medical Sciences, Gorgan, Iran

Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

Islamic Azad University, Tehran Medical Beranch, Tehran, Iran

a r t i c l e i n f o a b s t r a c t

Article history: Ischemia–reperfusion injury is a common complication of heart disease that is the leading cause of death

Received 15 June 2012

worldwide. Here, we plan to elucidate oxytocin cardioprotection effects against ischemia–reperfusion

Received in revised form 1 August 2012

via nitric oxide (NO), reactive oxygen species (ROS), and protein kinase C (PKC) in anesthetized rat pre-

Accepted 1 August 2012

conditioned myocardium. Forty-eight Sprague-Dawley rats were equally divided into eight groups. All

Available online 8 August 2012

animals were subjected to 25 min ischemia and 120 min reperfusion. Oxytocin (OT), L-NAME (LNA, a

nitric oxide synthase inhibitor), chelerythrine (CHE, a PKC enzyme inhibitor), and N-acetylcysteine (NAC,

Keywords:

a ROS scavenger) were used prior to ischemia. Results showed that mean arterial pressure signiﬁcantly

Oxytocin

Cardioprotection reduced during the ﬁrst 10 min of ischemia and reperfusion in IR, LNA, CHE, and NAC groups (p < 0.05). OT

NO prevented mean arterial pressure decline during early phase of ischemia and reperfusion. Cardioprotec-

ROS tive effects of OT in infarct size, plasma levels of creatine kinase-MB and lactate dehydrogenase, severity

PKC and incidence of ventricular arrhythmias were abolished by L-NAME, chelerythrine, and N-acetylcysteine

(p < 0.05). The present study showed that OT pretreatment reduces myocardial infarct size and ventricular

arrhythmias, and improves mean arterial pressure via NO production, PKC activation, and ROS balance.

These ﬁndings provide new insight into therapeutic strategies for ischemic heart disease.

1. Introduction The potential intracardiac role of OT during

ischemia–reperfusion is conﬁrmed by its receptor expression

In spite of advances in management and care of heart diseases, in cardiomyocytes [1–3], and direct cardioprotection or stimulated

ischemia–reperfusion (IR) injury continues to be a major cause protection via cellular mediators such as natriuretic peptides

of mortality and morbidity. There has been increased interest in (NPs) [14] and nitric oxide (NO) [9,10]. Previous studies have

the mechanisms involved in oxytocin-induced cardioprotection in indicated that NO mediates the activation of mitoKATP channels

recent years. It is not carefully understood how oxytocin (OT) pro- in cardiomyocytes [25,27]. Increased NO bioavailability may play

tects the myocardium from ischemia–reperfusion (IR) injury, but an important role in OT-dependent cardioprotective mechanisms

the overall mechanism is likely to be multifactorial. In previous through the regulation of mitoKATP channels and NO producing

studies, we showed protective effects of OT on myocardial injury enzymes [10]. In addition, we had previously showed the media-

in ischemic reperfused heart of anesthetized rats, mediatory roles tory roles of mitoKATP channels and mPTP in protective effects of

of mitochondrial ATP-dependent potassium (mitoKATP) channel OT on ischemic reperfused heart [1,2]. Indeed, pretreatment with

[2] and the mitochondrial permeability transition pore (mPTP) [1]. a mitoKATP channel opener appears to increase ROS production

We have also depicted that cardioprotective effects of ischemic and PKC activity before ischemia and reduce IR injuries in adult

preconditioning (IPC) can be induced by endogenous OT [3]. isolated cardiomyocyte [19,24]. Moreover, the protection afforded

by mitoKATP channel openers against the reperfusion-induced

apoptosis or necrosis appears to necessitate stimulation of PKC

activity by ROS [18] and NO-dependent pathways [15,25]. A

∗ further detailed understanding of the many signaling steps and

Corresponding author at: Cancer Research Center, Cancer Institute of Iran, Imam

the ﬁnal cytoprotective mechanisms underlying oxytocin-induced

Khomeini Hospital, Keshavarz Blvd., Tehran, Iran.

preconditioning may lead to improvements in the management

Tel.: +98 21 61192501/912 5790941.

E-mail address: [email protected] (A.M. Alizadeh). and care of ischemia–reperfusion injury. Therefore, in order to

M. Faghihi et al. / Peptides 37 (2012) 314–319 315

−1

clarify the mechanisms leading to OT-mediated cardioprotection, oxide synthase inhibitor (2 mg kg , IP) 10 min prior to OT admin-

we investigated the OT effects against ischemia–reperfusion via istration, (iv) LNA; L-NAME was used 35 min prior to ischemia,

NO, PKC, and ROS modulation in preconditioned myocardium. (v) CHE + OT; chelerythrine (CHE) was used as a PKC enzyme

−1

inhibitor (5 mg kg , IP) 10 min prior to OT administration, (vi) CHE;

2. Materials and methods chelerythrine was used 35 min prior to ischemia, (vii) NAC + OT; N-

−1

acetylcysteine (NAC) was used as a ROS scavenger (200 mg kg , IP)

2.1. Materials 10 min prior to OT administration, and (viii) NAC; N-acetylcysteine

was used 35 min prior to ischemia.

Oxytocin, 2,3,5-triphenyltetrazolium chloride, N -nitro-l-

arginine methyl ester (L-NAME), N-acetylcysteine, chelerythrine,

2.6. Hemodynamic functions

and Evans blue were obtained from Sigma Chemical Co. (St. Louis,

MO, USA).

Hemodynamic parameters such as mean arterial pressure (MAP)

and heart rate (HR) were continuously monitored and recorded by

2.2. Animals

Power Lab data acquisition system.

Male Sprague-Dawley rats (300–350 g) were maintained in ani-

mal quarters under standardized conditions 12 h light/dark cycle,

◦ 2.7. Cardiac area at risk and infarct size determination

20–22 C ambient temperature, and 40–50% humidity with free

access to rat chow and water. All experimental procedures were

To identify area at risk (AAR), the coronary artery was reoc-

done according to the guidelines of animal and human ethical com-

cluded. Evans blue dye (2 ml, 2%) was then injected through lateral

mittee of Tehran University of Medical Sciences.

tail vein. The heart was excised; atria and the roots of the great ves-

sels were removed. Remaining tissues were frozen for 24 h. Then

2.3. Surgical preparation

they were cut into 2 mm slices. All pieces were incubated in 1%

solution of 2,3,5-triphenyltetrazolium chloride (TTC, 0.1 M phos-

The preparation used in the present study was as previ-

phate buffer, pH 7.4, Sigma Chemical Co., St. Louis, MO, USA) at

ously described [2]. The animals were anesthetized with Sodium ◦

−1 37 C for 15 min to visualize the infarct area. Then they were ﬁxed

Thiopental (60 mg kg , IP) and maintained with supplementary

−1 for 2 days in 10% formalin to enhance the contrast. Non ischemic

doses (∼30 mg kg , IP) if required. Body temperature was mea-

◦ area, area at risk and infarcted area were colored blue, brick red,

sured by rectal thermometer and maintained at 37 C.

and pale respectively. Sections were scanned to determine normal

The rats were ventilated through a tracheotomy tube with

area, AAR, and infarct size (IS) by calculating pixels occupying each

air-and-oxygen mixture by a rodent ventilator (model 683, Har-

−1 area using Adobe PhotoShop software (Adobe Systems Seattle, WA).

vard Apparatus) (stroke volume approximately 1.2 ml 100 g ,

−1 Total AAR and IS are expressed as the percentage of total ventricle

60–70 stroke min ). A positive end-expiratory pressure was

and AAR, respectively.

applied to prevent alveolar atelectasis (3–5 cm H2O).

Heparinized catheter (100 U/ml) was ﬁxed into the right carotid

artery for blood sampling and pressure monitoring. The lateral tail 2.8. Assessment of ventricular arrhythmias

vein was cannulated to inject Evans blue dye and other drugs. Elec-

trocardiogram standard limb lead-II and arterial blood pressure Ischemia-induced ventricular arrhythmias were counted dur-

were continuously monitored using a computerized data acquisi- ing the occlusion period and determined in accordance with the

tion system (Power Lab data acquisition system, four channels, AD Lambeth Conventions [30]. Ventricular ectopic beats (VEBs) were

Instruments). diagnosed as a distinctive and identiﬁable premature QRS com-

The fourth rib was cut 3 mm below left lateral sternal border. The plexes. Ventricular tachycardia (VT) was deﬁned as a run of four

pericardium was incised and a sling (6-0 silk Ethicon) was placed or more consecutive VEBs. Ventricular ﬁbrillation (VF) was deﬁned

around the left anterior descending (LAD) artery close to its origin as unidentiﬁable and low voltage QRS complexes. Other multipart

right below the left atrial appendage. Both ends of the ligature were forms of VEBs, such as bigeminy and salvos (two or three consec-

passed through a small plastic tube. Heart rate and blood pressure utive VEBs), were not separately determined and were included

were allowed to stabilize for 20 min before the intervention proto- as single VEB. VF lasting for more than 5 min was measured as

cols. To induce transient myocardial ischemia, LAD was occluded irreversible [2]. The incidence, time of occurrence, and duration

by applying tension to the plastic tube-silk string. Reperfusion was of arrhythmias were used to identify arrhythmias severity accord-

then initiated by releasing the ligature and removing the tension. ing to the following scoring system [23]; 0: 0–49 VEBs, 1: 50–499

Regional ischemia and reperfusion were conﬁrmed by myocardial VEBs, 2: >499 VEBs and/or 1 episode of spontaneously reverting

cyanosis and hyperemia, respectively. VT or VF, 3: >1 episode of VT or VF or both with a total duration

<60 s, 4: VT or VF or both 60–120 s total duration, 5: VT or VF or

2.4. Exclusion criteria both >120 s duration, 6: fatal VF starting at >15 min after occlu-

sion, 7: fatal VF starting between 4 and 14 min 59 s, 8: fatal VF

Data were excluded from the ﬁnal analysis if arterial blood pres- starting between 1 and 3 min 59 s, 9: fatal VF starting <1 min after

sure was less than 80 mmHg before drug administration or when occlusion.

the O2 saturation dropped below 90% in the presence of O2 admin-

istration.

2.9. Biochemical analysis

2.5. Experimental protocols

Blood samples were collected at the end of each experiment.

◦

Plasma samples were extracted and stored at −70 C till ﬁnal assay.

Forty eight rats were divided into eight groups (n = 6): (i) IR;

Lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB)

hearts were subjected to 25 min ischemia and 120 min reperfu-

isoenzyme level were analyzed using ELECSYS System (ELECSYS

sion, (ii) OT; oxytocin was administered (0.03 ␮g/kg, IP) 25 min

2010, Roche, Germany) and commercial kit (Pars Azmoon, Iran).

prior to ischemia, (iii) LNA + OT; L-NAME (LNA) was used as a nitric

316 M. Faghihi et al. / Peptides 37 (2012) 314–319

2.10. Statistical analysis

Statistical analysis of hemodynamic data within and between

groups was performed with two-way analysis of variance. Dif-

ferences in infarct size, LDH and CK-MB plasma levels were

determined by one-way analysis of variance. Groups were indi-

vidually compared using Dunnet’s test. Arrhythmia scores were

analyzed with Kruskal–Wallis test, and the incidences of VT or VF

were compared by Fisher exact test. All data were expressed as

mean ± SEM. Statistical signiﬁcance was deﬁned as p < 0.05.

3. Results

3.1. Hemodynamic data

Table 1 shows heart rate and mean arterial pressure during

20 min baseline, immediately before OT administration, 15 min

post OT administration, the ﬁrst 10 min of ischemia and reperfu-

sion, and at the end of reperfusion. No signiﬁcant differences were Fig. 1. Myocardial area at risk (AAR/V%) and infarct size (IS/AAR%) in IR, OT, LNA + OT,

LNA, CHE + OT, CHE, NAC + OT, and NAC groups. Data are presented as mean ± SEM.

seen between groups at baseline. Mean arterial pressure signiﬁ-

*p < 0.05 vs. IR group. p < 0.05 vs. OT group. OT, oxytocin; LNA, L-NAME; CHE, chel-

cantly reduced during the ﬁrst 10 min of ischemia and reperfusion

erythrine; NAC, N-acetylcysteine.

in IR, L-NAME, CHE, and NAC groups (p < 0.05). Oxytocin adminis-

tration prevented mean arterial pressure decline during early phase

of ischemia and reperfusion (Table 1). Hemodynamic parameters

slightly but not signiﬁcantly decreased in all groups in comparison Administration of L-NAME, chelerythrine, and N-acetylcysteine in

with baseline at the end of reperfusion. LNA + OT, CHE + OT, and NAC + OT groups was intensiﬁed severity of

arrhythmias compared to OT group (3.4 ± 0.8, 3.7 ± 0.6 and 3.5 ± 0.6

3.2. Area at risk and infarct size measurement vs. 0.9 0.2 respectively, p < 0.05). L-NAME, N-acetylcysteine, and

chelerythrine alone had no signiﬁcant effect on arrhythmias sever-

AAR was not different among groups. Infarct size was sig- ity compared to IR group (Fig. 2).

niﬁcantly decreased in OT group compared to IR (p < 0.05).

Administration of L-NAME, chelerythrine, and N-acetylcysteine in

LNA + OT, CHE + OT, and NAC + OT groups increased infarct size to 3.3.2. Incidences of VT and VF

44 ± 4, 41 ± 1.9%, and 42 ± 2.5% respectively in comparison with In IR group, all animals were experienced VT, while VF occurred

OT group (p < 0.05). L-NAME, N-acetylcysteine, and chelerythrine in 67% of cases. OT use attenuated VT incidence to 22% and abol-

alone had no signiﬁcant effect on infarct size compared to IR group ished VF incidence completely compared to IR group (p < 0.05).

(Fig. 1). In comparison with OT group, administration of L-NAME, chel-

erythrine, and N-acetylcysteine restored the incidence of VT to

100%, 100%, and 100%, and VF to 57%, 57%, and 67% in LNA + OT,

3.3. Ventricular arrhythmias during ischemia

CHE + OT, and NAC + OT groups respectively (p < 0.05). L-NAME,

N-acetylcysteine, and chelerythrine alone did not signiﬁcantly

3.3.1. Severity of arrhythmias

change incidences of VT and VF in comparison with IR group

OT use prior to ischemia signiﬁcantly declined ventric-

(Fig. 3).

ular arrhythmias severity compared to IR group (p < 0.05).

Table 1

Hemodynamic parameters.

Groups 20 min baseline Before administer OT 15 min after administer OT First 10 min ischemia First 10 min reperfusion End of reperfusion

IR 347 ± 11 345 ± 16 331 ± 14 325 ± 19 325 ± 19

OT 350 ± 13 347 ± 14 331 ± 13 325 ± 21 335 ± 15 335 ± 15

LNA + OT 342 ± 11 345 ± 11 337 ± 16 328 ± 19 336 ± 21 333 ± 18

LNA 345 ± 15 340 ± 13 338 ± 22 335 ± 25 327 ± 19

CHE + OT 347 ± 10 347 ± 11 332 ± 16 322 ± 24 333 ± 16 333 ± 16

CHE 340 ± 15 340 ± 13 333 ± 15 327 ± 20 327 ± 20

NAC + OT 342 ± 12 345 ± 12 335 ± 12 325 ± 17 335 ± 18 324 ± 18

NAC 339 ± 15 343 ± 17 335 ± 25 330 ± 22 321 ± 22 MAP

* *

IR 126 ± 6 124 ± 8 84 ± 12 95 ± 11 105 ± 9

OT 124 ± 4 120 ± 6 118 ± 9 104 ± 11 107 ± 8 110 ± 10

LNA + OT 125 ± 9 122 ± 8 118 ± 7 109 ± 13 105 ± 10 102 ± 14

* *

LNA 118 ± 10 121 ± 7 83 ± 11 88 ± 12 97 ± 10

CHE + OT 121 8 124 9 120 ± 8 105 ± 12 104 ± 9 107 ± 9

* *

CHE 116 ± 6 120 ± 5 80 ± 8 85 ± 11 98 ± 8

NAC + OT 118 ± 9 110 ± 14 114 ± 12 100 ± 11 101 ± 13 111 ± 13

* *

NAC 125 ± 5 118 ± 11 82 ± 10 85 ± 10 99 ± 10

Data are presented as mean SEM; Number of six animals in each group; MAP, mean arterial pressure (mmHg); HR, heart rate (beats/min); IR, ischemia–reperfusion; OT,

oxytocin; LNA, L-NAME; CHE, chelerythrine; NAC, N-acetylcysteine.

p < 0.05 vs. baseline.

M. Faghihi et al. / Peptides 37 (2012) 314–319 317

Fig. 4. The effect of oxytocin, L-NAME, chelerythrine, and N-acetylcysteine on cre-

atine kinase-MB (CK-MB) isoenzyme plasma level at the end of reperfusion in IR,

OT, LNA + OT, LNA, CHE + OT, CHE, NAC + OT, and NAC groups. Data are presented

Fig. 2. Distribution of the arrhythmia score during 25 min ischemia in IR, OT, #

as mean ± SEM. *p < 0.05 vs. IR group. p < 0.05 vs. OT group. OT, oxytocin; LNA,

LNA + OT, LNA, CHE + OT, CHE, NAC + OT, and NAC groups. Data are presented as

L-NAME; CHE, chelerythrine; NAC, N-acetylcysteine.

mean SEM. *p < 0.05 vs. IR group. p < 0.05 vs. OT group. OT, oxytocin; LNA, L-

NAME; CHE, chelerythrine; NAC, N-acetylcysteine.

Fig. 5. The effect of oxytocin, L-NAME, chelerythrine, and N-acetylcysteine on lac-

tate dehydrogenase (LDH) isoenzyme plasma level at the end of reperfusion in IR,

OT, LNA + OT, LNA, CHE + OT, CHE, NAC + OT, and NAC groups. Data are presented

± #

as mean SEM. *p < 0.05 vs. IR group. p < 0.05 vs. OT group. OT, oxytocin; LNA,

L-NAME; CHE, chelerythrine; NAC, N-acetylcysteine.

Fig. 3. The incidence of ventricular ﬁbrillation (VF) and ventricular tachycardia (VT)

during 25 min ischemia in IR, OT, LNA + OT, LNA, CHE + OT, CHE, NAC + OT, and NAC

groups. Data are presented as mean ± SEM. *p < 0.05 vs. IR group. p < 0.05 vs. OT

In previous studies, we showed the role of mitoKATP channels

group. OT, oxytocin; LNA, L-NAME; CHE, chelerythrine; NAC, N-acetylcysteine.

and mPTP in infarct-sparing effects of OT [1,2]. The role of mitoKATP

channels activation in cardioprotective signaling mechanisms has

been supported by the ﬁndings that PKC and NO-mediated mech-

3.4. Biochemical analysis

anisms facilitate the opening of mitoKATP channels [17,25,26]. In

the present study, pretreatment with L-NAME abrogated cardio-

Oxytocin signiﬁcantly decreased plasma LDH and CK-MB lev-

protective effects of OT in anesthetized rat; indicating NO may

els at the end of reperfusion period compared with IR group

involve in OT cardioprotective effects. Furthermore, OT is known

(p < 0.05). L-NAME, N-acetylcysteine, and chelerythrine adminis-

to stimulate NO release during IR [12,22]. Three recent reports

tration in LNA + OT, NAC + OT, and CHE + OT groups signiﬁcantly

suggest that the protective effects of OT pretreatment during IR

increased LDH and CK-MB plasma level compared to OT group

injury may be caused by NO release [10,12,29]. Consistent with

(p < 0.05). L-NAME, N-acetylcysteine, and chelerythrine alone had

these, our results conﬁrm mediatory role of NO in OT-induced

no signiﬁcant effect on LDH and CK-MB plasma level compared to

cardioprotection.

IR group (Figs. 4 and 5).

On the other hand, a board of experimental evidence suggests

that the opening of mitoKATP channel generates ROS, and that

4. Discussion these radicals represent a part of the cascade leading to cardio-

protection [11,16,20,21]. Here, we revealed that N-acetylcysteine,

The results showed that the protective effects of OT on a ROS scavenger and chelerythrine, a PKC inhibitor eliminated the

infarct size, CK-MB, and LDH plasma level, severity and inci- protective effects of OT in rat heart. Chen et al. [7] showed that

dence of ventricular arrhythmia were abolished by L-NAME, a N-acetylcysteine blocks the effects of IPC and other cardioprotec-

nitric oxide synthase inhibitor, chelerythrine, a PKC inhibitor, and tion agents. In addition, PKC is known to be activated by ROS [4]

N-acetylcysteine, a ROS scavenger. These ﬁndings suggest that acti- hence; it may work downstream to ROS and to close mPTP as

vation of NO, ROS, and PKC is a requisite for the infarct-sparing end effectors in cardioprotection phenomena. Furthermore, cardio-

effects of OT in IR injuries. protective effects of PKC have been shown to omit by mitoKATP

318 M. Faghihi et al. / Peptides 37 (2012) 314–319

channel inhibitors [13] and mPTP openers [8]. Consistent with Acknowledgment

these, our results conﬁrm mediatory role of ROS and PKC in OT-

induced cardioprotection. This study was supported by grant of Tehran University of Med-

In the present study, OT administration before ischemia led ical Sciences.

to suppression of life-threatening arrhythmias. In previous study,

we reported that 5-HD, a speciﬁc mitoKATP channel blocker, pre- References

vented OT beneﬁts effects on life-threatening arrhythmias. Das and

Sarkar [10] showed the activation of mitoKATP due to OT in pre- [1] Alizadeh AM, Faghihi M, Khori V, Sohanaki H, Pourkhalili K, Moham-

madghasemi F, et al. Oxytocin protects cardiomyocytes from apoptosis induced

vention of ventricular arrhythmia during reperfusion. The authors,

by ischemia–reperfusion in rat heart: role of mitochondrial ATP-dependent

however, did not report arrhythmia score during ischemic phase

potassium channel and permeability transition pore. Peptides 2012;36:71–7.

preceding reperfusion. In this study, a new manifestation of OT [2] Alizadeh AM, Faghihi M, Sadeghipour H, Mohammadghasemi F, Imani A,

anti-arrhythmic effects has demonstrated during ischemic phase. Houshmand F, et al. Oxytocin protects rat heart against ischemia–reperfusion

injury via pathway involving mitochondrial ATP-dependent potassium chan-

Previous studies proved different mechanisms of arrhythmogenic-

nel. Peptides 2010;31:1341–5.

ity in ischemia and reperfusion phases [5]. In fact, occurrence of

[3] Alizadeh AM, Faghihi M, Sadeghipour HR, Mohammadghasemi F, Khori V.

arrhythmia in ischemic phase is dependent to release of endoge- Role of endogenous oxytocin in cardiac ischemic preconditioning. Regul Pept

2011;167(1):86–90.

nous triggering factors originated from biochemical mediatory

[4] Baines CP, Goto M, Downey JM. Oxygen radicals released during ischemic pre-

compounds such as NO and ROS [5]. Furthermore, pharmacolog-

conditioning contributes to cardioprotection in the rabbit myocardium. J Mol

ical agents such as diazoxide [28] and pravastatine [6] would Cell Cardiol 1997;29:207–16.

[5] Brown DA, O’Rourke B. Cardiac mitochondria and arrhythmias. Cardiovasc Res

prevent arrhythmia in ischemic phase by a mechanism mediated

2010;88(2):241–9.

through ROS and PKC pathways [5]. Consistent with these, in the

[6] Chen J, Shen H, Nagasawa Y, Mitsui K, Tsurugi K, Hashimoto K. Pravastatin

present study, OT administration induced similar anti-arrhythmic inhibits arrhythmias induced by coronary artery ischemia in anesthetized rats.

J Pharmacol Sci 2007;103(3):317–22.

proﬁle as IPC and pharmacologic preconditioning during ischemia.

[7] Chen W, Gabel S, Steenbergen C, Murphy E. A redox-based mechanism for car-

In addition, pretreatment with L-NAME, N-acetylcysteine, and chel-

dioprotection induced by ischemic preconditioning in perfused rat heart. Circ

erythrine abrogate beneﬁcial anti-arrhythmic effects of OT which Res 1995;77(2):424–9.

[8] Costa AD, Jakob R, Costa CL, Andrukhiv K, West IC, Garlid KD. The mechanism by

brings more focus on critical role of NO and ROS in antiarrhythmic

which the mitochondrial ATP-sensitive K channel opening and H2O2 inhibit

effects of OT. On the other hand, ROS and infarct size are two fun-

the mitochondrial permeability transition. J Biol Chem 2006;281:20801–8.

damental determinants of arrhythmogenicity during reperfusion [9] Danalache BA, Paquin J, Donghao W, Grygorczyk R, Moore JC, Mummery CL,

phase [5]. In the present study, we did not measure superoxide and et al. Nitric oxide signaling in oxytocin-mediated cardiomyogenesis. Stem Cells

2007;25(3):679–88.

ROS levels in cardiomyocytes, but in preliminary experiment, we

[10] Das B, Sarkar C. Is preconditioning by oxytocin administration mediated by

showed OT-induced decrease in plasma malondialdehyde activity

iNOS and/or mitochondrial K(ATP) channel activation in the in vivo anes-

(2.1 ± 0.3 vs. 3.7 ± 0.4 nM/ml). In addition, hemodynamic instabil- thetized rabbit heart? Life Sci 2012;90(19–20):763–9.

[11] Diazoxide-induced cardioprotection requires signaling through a redox-

ity shown as MAP drop in the ﬁrst phase of reperfusion is the

sensitive mechanism. Circ Res 2001;88(8):802–9.

sign of ROS release from mitochondria. Here, decreasing infarct

[12] Dusunceli F, Iseri SO, Ercan F, Gedik N, Yegen C, Yegen BC. Oxytocin alleviates

size as well as compensation of hypotension during ﬁrst 10 min hepatic ischemia–reperfusion injury in rats. Peptides 2008;29:1216–22.

[13] Garg V, Hu K. Protein kinase C isoform-dependent modulation of ATP-sensitive

of reperfusion possibly indicates ROS suppression during reperfu-

K channels in mitochondrial inner membrane. Am J Physiol Heart Circ Physiol

sion phase by OT, and indirectly emphasizes to anti-arrhythmic role 2007;293:322–32.

of OT in reperfusion phase. Therefore, a decrease in ROS release [14] Gutkowska J, Jankowski M. Oxytocin: old hormone, new drug. Pharmaceuticals

2009;2:168–83.

may be partly involved in OT inhibitory effect on life-threatening

[15] Lebuffe G, Schumacker PT, Shao ZH, Anderson T, Iwase H, Vanden Hoek TL.

arrhythmias.

ROS and NO trigger early preconditioning: relationship to mitochondrial KATP

In summary, we may infer that OT as a triggering agent can ini- channel. Am J Physiol Heart Circ Physiol 2003;284:H299–308.

[16] Matejikova J, Kucharska J, Pinterova M, Pancza D, Ravingerova T. Protection

tiate mitochondrial signaling cascade including NO, ROS, and PKC.

against ischemia-induced ventricular arrhythmias and myocardial dysfunction

Nevertheless, without analyzing the inﬂuences of OT in the pres-

conferred by preconditioning in the rat heart: involvement of mitochondrial

ence of several inhibitors or direct kinase activity assay, we cannot KATP channels and reactive oxygen species. Physiol Res 2009;58(1):9–19.

determine whether the interactions among multiple mediators are [17] Murphy E. Primary and secondary signaling pathways in early precondition-

ing that converge on the mitochondria to produce cardioprotection. Circ Res

in parallel or in sequence. Future studies are required to provide

2004;94(1):7–16.

sufﬁcient evidence related to downstream signaling pathways of

[18] Murriel CL, Mochly-Rosen D. Opposing roles of delta and epsilon PKC in cardiac

OT and their possible links. ischemia and reperfusion: targeting the apoptotic machinery. Arch Biochem

Biophys 2003;420:246–54.

[19] Oldenburg O, Yang XM, Krieg T, Garlid KD, Cohen MV, Grover GJ, et al.

P1075 opens mitochondrial KATP channels and generates reactive oxygen

species resulting in cardioprotection of rabbit hearts. J Mol Cell Cardiol

5. Conclusion 2003;35:1035–42.

[20] Pain T, Yang XM, Critz SD, Yue Y, Nakano A, Liu GS, et al. Opening of mito-

chondrial KATP channels triggers the preconditioned state by generating free

Our results indicate that pharmacological preconditioning of

radicals. Circ Res 2000;87(6):460–6.

rat heart by pretreatment with L-NAME, a non-speciﬁc NOS

[21] Patel HH, Gross GJ. Diazoxide induced cardioprotection: what comes ﬁrst, KATP

inhibitor, chelerythrine, a PKC inhibitor and N-acetylcysteine, a channels or reactive oxygen species? Cardiovasc Res 2001;51(4):633–6.

[22] Pournajaﬁ-Nazarloo H, Perry A, Partoo L, Papademetriou E, Azizi F, Carter CS,

ROS scavenger abolished the beneﬁcial effects of oxytocin in

et al. Neonatal oxytocin treatment modulates oxytocin receptor, atrial natri-

ischemia–reperfusion heart model. This may suggest that the bene-

uretic peptide, nitric oxide synthase and estrogen receptor mRNAs expression

ﬁts were achieved via NO release, PKC activation, and ROS balance. in rat heart. Peptides 2007;28:1170–7.

These observations have considerable relevance for future thera- [23] Sarraf G, Barrett TD, Walker MJ. Tedisamil and lidocaine enhance each other’s

antiarrhythmic activity against ischemia-induced arrhythmias in rats. Br J Phar-

peutic approaches for oxytocin in myocardial ischemic injuries.

macol 2003;139:1389–98.

[24] Sarre A, Lange N, Kucera P, Raddatz E. mitoKATP channel activation in the

postanoxic developing heart protects E–C coupling via NO-, ROS-, and PKC-

dependent pathways. Am J Physiol Heart Circ Physiol 2005;288(4):H1611–9.

Conﬂict of interest [25] Sasaki N, Sato T, Ohler A, O’Rourke B, Marban E. Activation of mito-

chondrial ATP-dependent potassium channels by nitric oxide. Circulation

2000;1:439–45.

The author(s) declare(s) that they have no conﬂict of interest to +

[26] Sato T, O’Rourke B, Marban E. Modulation of mitochondrial ATP-dependent K

disclose. channels by protein kinase C. Circ Res 1998;83(1):110–4.

M. Faghihi et al. / Peptides 37 (2012) 314–319 319

[27] Shinbo A, Iijima T. Potentiation by nitric oxide of the ATP-sensitive K current [29] Tugtepe H, Sener G, Biyikli NK, Yuksel M, Cetinel S, Gedik N, et al. The protec-

induced by K channel openers in guinea-pig ventricular cells. Br J Pharmacol tive effect of oxytocin on renal ischemia/reperfusion injury in rats. Regul Pept

1997;120(8):1568–74. 2007;140:101–8.

[28] Thuc LC, Teshima Y, Takahashi N, Nagano-Torigoe Y, Ezaki K, Yufu K, [30] Walker MJ, Curtis MJ, Hearse DJ, Campbell RW, Janse MJ, Yellon

et al. Mitochondrial KATP channels-derived reactive oxygen species acti- DM. The Lambeth conventions: guidelines for the study of arrhyth-

vate pro-survival pathway in pravastatin-induced cardioprotection. Apoptosis mias in ischemia, infarction, and reperfusion. Cardiovasc Res 1988;22: 2010;15(6):669–78. 447–55.