Central oxytocin modulation of acute stress-induced cardiovascularresponses after myocardial infarction in the rat

AGNIESZKA WSOŁ, AGNIESZKA CUDNOCH-JEDRZEJEWSKA, EWA SZCZEPANSKA-

SADOWSKA, STANISŁAW KOWALEWSKI, & JAKUB DOBRUCH

Department of Experimental and Clinical Physiology, Medical University of Warsaw, Warsaw, Poland

(Received 2 September 2008; revised 28 November 2008; accepted 12 December 2008)

AbstractThe present study was aimed at determining the role of centrally released oxytocin in regulation of blood pressure and heartrate (HR) under resting conditions and during an acute air-jet stress in rats with a myocardial infarction and controls infarcted.Four weeks after ligation of a coronary artery or sham surgery, conscious Sprague Dawley rats were subjected to one of thefollowing intracerebroventricular (ICV) infusions: (1) 0.9% NaCl (control), (2) oxytocin, (3) oxytocin receptor antagonist{desGly-NH2-d(CH2)5[D-Tyr2Thr4]OVT}(OXYANT). Resting arterial blood pressure and HR were not affected by any ofthe ICV infusions either in the infarcted or sham-operated rats. In the control experiments, the pressor and tachycardicresponses to the air jet of infarcted rats were significantly greater than in the sham-operated rats. OXYANT significantlyenhanced the cardiovascular responses to stress only in the sham-operated rats whereas oxytocin significantly attenuated bothresponses in the infarcted but not in the sham-operated rats. The results suggest that centrally released endogenous oxytocinsignificantly reduces the cardiovascular responses to the acute stressor in control rats. This buffering function of the brain-oxytocin system is not efficient during the post-myocardial infarction state, however it may be restored by centraladministration of exogenous oxytocin.

Keywords: Heart failure, myocardial infarct, neuropeptides, oxytocin-antagonist, stress, vasopressin

Introduction

Until recently, oxytocin, the neurohypophysial neuro-

peptide, has been known for its pivotal role in the

progress of parturition and initiation of milk ejection

(Russell et al. 2003; Leng et al. 2005). The regulatory

spectrum of oxytocin is now known to be substantially

wider. An increasing amount of data in the literature

strongly suggests that oxytocin may exert an analgesic

effect (Robinson et al. 2002) and participate in the

control of cognitive functions, affective behaviour and

stress-related reactions (Engelmann et al. 1999; Windle

et al. 1997; Neumann et al. 2000; Nakashima et al.

2002; Choleris et al. 2007; Blume et al. 2008; Carter

et al. 2008; Neumann 2008). In addition, some

investigators have provided evidence that oxytocin

may be involved in regulation of the cardiovascular

system by means of direct peripheral and indirect

central actions (Petersson et al. 1996; Braga et al. 2000;

McCann et al. 2002; Costa e Sousa et al. 2005).

Anatomical studies of oxytocin pathways in the brain

have revealed extensive innervation of the brain stem

structures regulating the cardiovascular, behavioural

and neuroendocrine responses to stress by oxytocin

fibres projecting from the paraventricular nucleus

(PVN) (Sawchenko and Swanson 1982; Bergquist

and Ludwig 2008). Expression of oxytocin receptors in

the same regions of the brain stem has been also well

documented (Sofroniew and Schrell 1981; Barberis

and Tribollet 1996), and there is evidence that oxytocin

acting on the neurones of the solitary vagal complex

modulates reflex control of the heart rate (HR) (Higa

et al. 2002). The studies of Braga et al. (2000) and

Michelini (2001) suggest that during physical exercise

central oxytocin decreases modulation of tachycardia

Correspondence: E. Szczepanska-Sadowska, Department of Experimental and Clinical Physiology, Medical University of Warsaw,Krakowskie Przedmiescie 26/28, 00-927 Warsaw, Poland. Tel: 4822 826 0778. Fax: 4822 826 8092. E-mail: eszs@amwaw.edu.pl

Stress, November 2009; 12(6): 517–525q Informa Healthcare USA, Inc.ISSN 1025-3890 print/ISSN 1607-8888 onlineDOI: 10.3109/10253890802687688

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by the neurones located in the nucleus of the solitary

tract. Moreover, it has been found that content of

oxytocin in the brain stem and the PVN is significantly

altered in the spontaneously hypertensive rats (Mohr-

ing et al. 1983; Morris et al. 1985). Engagement of

oxytocin in the control of neuroendocrine responses to

stress and its putative contribution to the regulation of

cardiovascular parameters raises the question whether

or not the centrally released oxytocin may also be

involved in modulation of the cardiovascular responses

to stress. Sudden, alarming stress evokes rapid resetting

of the autonomic control of the HR and blood pressure

(Lampert et al. 2000; Kario et al. 2003). It is generally

known that in each individual the same stressors may

evoke markedly different cardiovascular responses

under different circumstances, however, the exact

mechanisms responsible for this variation are not yet

fully understood. Previous studies have implicated the

involvement of brain angiotensin II AT1 receptors and

vasopressin V1a receptors in regulation of the

cardiovascular responses to acute air-jet stress

(Mayorov and Head 2003; Dobruch et al. 2005;

Mayorov et al. 2004; Cudnoch-Jedrzejewska et al.

2007; Stojicic et al. 2008). Moreover, it has been shown

that engagement of these receptors in cardiovascular

responses to stress increases remarkably during the

post-infarct state. Thus far, the role of oxytocin

receptors in the regulation of cardiovascular responses

to stress has not been clarified. The only study devoted

to this problem (Petersson and Uvnas-Moberg 2007)

did not provide convincing results. The present

investigation was designed to determine whether

centrally released endogenous oxytocin is involved in

regulation of the arterial blood pressure and HR under

resting conditions and during an alarming stress

(air-jet), and whether its function is altered during the

post-myocardial infarction state. The results provide

evidence that centrally released endogenous oxytocin

reduces the pressor and tachycardic responses to the

acute air jet stress in the control non-infarcted rats and

that this action is abolished after a myocardial

infarction. Preliminary data from this study have been

presented at a scientific meeting (Szczepanska-

Sadowska et al. 2008).

Methods

Animals and surgical procedures

Adult, male Sprague Dawley rats (SPRD/Mol/Lod)

weighing 250–300 g were used in the study. The rats

were raised in the Department of Animal Breeding at

the Medical University of Warsaw. They were housed

in a room with regulated temperature (range 22–

258C) and had free access to tap water and a

commercial rat diet containing 0.45% NaCl. The

rats were maintained on a 12 h/12 h light/dark cycle

(light on at 7.00 am). During the experimental

sessions, water and food were withdrawn to avoid

incidental changes in cardiovascular parameters due to

feeding and drinking activity. All surgical procedures

and experimental protocols were in accordance with

the international/European Union guidelines and

regulations on the use and care of laboratory animals.

The experimental protocol was approved by the

Ethical Committee on the Animal Research of the

Warsaw Medical University. Before the experimental

sessions all rats were subjected to the following

surgical procedures: ligation of a coronary artery at

10–12 weeks of age, or a sham procedure, implan-

tation of a guide tube into the left cerebral ventricle at

14–16 weeks of age, and insertion of an aortic catheter

at 15–17 weeks of age. Each of these procedures was

performed under pentobarbital anaesthesia (Pento-

barbital, Biowet, Puławy; 15 mg/ml, 50 mg/kg i.p.).

Induction of myocardial infarction and sham procedure.

The rats were divided at random into two groups. One

group was subjected to the myocardial infarction

procedure and the other group to sham surgery.

The myocardial infarct was produced according to the

technique described by Selye et al. (1960) and

modified by Dobruch et al. (2005). In brief: a

surgical incision was made between the fourth and the

fifth intercostal space while the lungs were ventilated

by means of air puffs applied from a small rubber

balloon. The infarction was produced by ligation of the

left coronary artery with a 6-0 prolen stich (Ethicon).

In the sham-infarcted rats, the cardiac pericardium

was touched with the needle but the coronary artery

was not ligated. At the end of both procedures, the

wound was closed with surgical sutures (Ethicon,

Sommerville, NJ, USA 4.0) and spontaneous

ventilation was re-established. After surgery, the rats

were given analgesic (Buprenorphine sulphate, Polfa,

Rzeszow, Poland; 9 mg/ml, 30 mg/kg s.c.) and

antibiotic (Penicillin, Polfa, Pabianice, Poland;

30,000 U in 1 ml/rat i.m), and placed in individual

cages. The rate of survival of the infarcted and the

sham-operated rats was 53 and 97%, respectively.

Implantation of the intracerebroventricular guide tube.

The rat’s head was placed in a Kopf’s stereotaxic device

and the stainless steel guide tube (o.d., 0.81 mm,

MIFAM S.A. Milanowek, Poland) was implanted into

the brain using the following stereotaxic coordinates:

1.3 mm posterior to the bregma and 2 mm lateral from

the midline, 4.5 mm below the surface of the skull.

The guide tube was secured in the skull with acrylic

cement (Duracryl, SPOFA-DENTAL, Jicin, Czech

Republic) and closed with a stainless steel stylet (o.d.,

0.46 mm). After surgery, the rats received antibiotic

(Penicillin, Polfa; 30,000 U in 1 ml/rat i.m.) and were

placed in their home cages.

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Implantation of the arterial catheter. The intraarterial

catheter consisted of an intraarterial portion (3.5–4.0

cm long; i.d., 0.12 mm; o.d., 0.25 mm) and an external

portion (i.d., 0.25 mm; o.d., 0.4 mm) made from

polyvinyl tubing (Scientific Commodities, Inc., Lake

Havasu City, AZ, USA). The internal part was inserted

into the aorta through a femoral artery, so that its end

was 2 cm below the renal arteries. The external part was

pushed under the skin and exteriorised dorsally on the

neck. The catheter was filled with 0.9% NaCl

containing 500 U of heparin/ml and plugged with a

stopper. The experiments were performed 24–48 h

after the surgery when the rats had fully recovered from

the anaesthesia and could move freely.

Experimental protocol

Six groups of experiments were performed in order to

determine the cardiovascular effects of stimulation

and blockade of oxytocin receptors in the brain under

resting conditions and during stress in the infarcted

and the sham-operated rats. At the beginning of each

experimental session, the arterial catheter was

connected to the blood pressure and HR recording

system (BIOPAC, MP100, Santa Barbara, CA, USA).

The stylet was removed from the guide tube and the

stainless steel tube (o.d., 0.46 mm) that was 0.1 mm

longer than the stylet was inserted into the guide tube

and connected via the polyvinyl tubing to a micro-

syringe placed in a Harvard 22 Infusion

Pump (Harvard, Smith Natick, MA, USA). The

intracerebroventricular (ICV) infusion started after a

15–30 min rest period, allowed for stabilisation of the

cardiovascular parameters. Each ICV infusion was

delivered at the rate of 5 ml/h (0.083 ml/min) and

lasted until the end of the experiment. The rats were

assigned at random into six experimental groups. The

control sham-operated (n ¼ 7) and control infarcted

(n ¼ 8) groups were infused with vehicle (0.9%

NaCl). The sham-operated (n ¼ 6) and infarcted

(n ¼ 7) groups receiving oxytocin were infused with

0.9% NaCl for the first 10 min and subsequently with

oxytocin (Phoenix Pharmaceuticals, Strasbourg,

France) at the rate of 100 ng/5ml/h (1.66 pmol/0.083

ml/min) during the remaining part of the experiment.

A similar experimental design was applied in the

sham-operated (n ¼ 7) and infarcted (n ¼ 6) rats

receiving the OXYANT except that DesGly � NH2 �

dðCH2Þ5 ½D � Tyr2,Thr4]OVT was infused at the rate

of 4.3 mg/5 ml/h (71.6 nmol/0.083ml/min). The

antagonist was kindly provided by Prof. Maurice

Manning, Medical College of Ohio, USA). In the case

of oxytocin, the rationale for the choice of the dose of

oxytocin used in the present study was based on our

previous investigation in which we found that an

equimolar dose of vasopressin produces significant

changes in baseline blood pressure and in cardiovas-

cular responses to stress in rats after a myocardial

infarction. Because oxytocin and vasopressin are

frequently released together we aimed to compare

their central cardiovascular effects using comparable

doses. The dose of OXYANTwas calculated basing on

a published effective dose of DesGly-NH2-

d(CH2)5[D-Tyr2,Thr4]OVT (Manning et al. 1995).

In each group measurements of mean arterial blood

pressure (MABP) and HR were continued for 50 min

under resting conditions and during 10 min after

application of the air jet which served as the acute

stress. The air jet was applied using the technique

described by Zhang et al. (1999) and modified by

Dobruch et al. (2005). The procedure involves

blowing air for 1 s onto the top of the rat’s had from

a tank containing compressed air (10 atm) through a

funnel (i.d., 41.5 mm) held above the rat’s head and

connected with the tank by plastic tubing (i.d.,

3.0 mm). The cardiovascular responses to the stressor

were evaluated by measuring the maximum increases

in MABP and HR occurring during the first 10 s after

application of the stressor. MABP and HR immedi-

ately preceding application of the air jet were used as

the reference level. The experiments were concluded

with measurements of the end-diastolic ventricular

pressure (EDVP) in the left ventricle. To this end the

rats were anaesthetised (Pentobarbital, Biowet,

Puławy, Poland; 15 mg/ml, 50 mg/kg i.p.) and a

catheter (Dural Plastics and Engineering, Auburn,

Australia; i.d., 0.5 mm; o.d., 0.8 mm) was inserted

through the right carotid artery and the aortic arch

into the left ventricle.

Measurements

MABPand HR were recorded continuously by means of

a BIOPAC system (MP100) which determines MABP

as the area under the arterial pressure curve divided by

the cardiac cycle duration. The system calculates HR

(beats/min) from the number of systolic pressure peaks.

The BIOPAC system was also used to determine left

ventricle end-diastolic pressure (LVEDP).

Post mortem examination. At the end of the experiment,

the rats were killed by an overdose of 5% chloral

hydrate (50 mg/ml, 500–667 mg/kg i.p.) and the heart

and brain were harvested for evaluation of the size of

the myocardial infarct and contact of the ICV cannula

with the cerebroventricular system.

Size of the myocardial infarct. The size of the infarct

was determined planimetrically (Zhang et al. 1999)

with some modifications (Dobruch et al. 2005).

Briefly, the heart was excised from the thorax and

gently washed with saline. The wall of the left ventricle

(including septum) was separated from the right

ventricle along the longitudinal axis and placed flat

on transparent graph paper with 1 mm squares.

Brain oxytocin, stress and circulation 519

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The circumferences of the ventricle and of the infarct

scar were outlined on the internal and external

surfaces. The surface area of the infarct expressed as

the number of mm squares was estimated on both sites

of the ventricle and averaged. The size of the infarct

scar was expressed as the percentage of the total left

ventricle wall surface. Previous studies have shown

that estimation of the infarct size with this method

corresponds well to evaluation of the infarct size

during histological examination (Dobruch et al. 2005;

Cudnoch-Jedrzejewska et al. 2007, 2008).

Verification of the contact of the ICV cannula with the

cerebroventricular system. To verify the position of the

infusing cannula in the lateral cerebral ventricle 5ml of

Evans blue was injected through the guide tube via the

needle used for the ICV infusions. Sagittal sections of

the brain were made for visual inspection of the

ventricular system for presence of the dye. The

inspection showed that all rats used in the experiments

had appropriate locations of the infusing cannula.

Statistical analysis. Statistica software (Version 7)

was used for statistical analysis of the data. Five min

averages covering the first 50 min of the ICV infusion

were used in the statistical analysis to determine

changes in MABP and HR under resting conditions.

Three-way ANOVA on repeated measurements was

used to evaluate changes of cardiovascular parameters

at rest, as recommended by Ludbrook (1994) and

Curran-Everet and Benos (2004). Two-way ANOVA

was applied to determine the significance of differ-

ences between the air jet stress-induced maximum

changes of the cardiovascular parameters in the

different groups of experiments. The horizontal and

vertical multiple pairwise comparisons (Ludbrook

1994; Curran-Everet and Benos 2004) were made

using the post-hoc Tukey test. The differences were

considered significant if P was ,0.05. All values

presented in the text, figures and table are mean ^

standard error of mean.

Results

The size of the myocardial infarct in the rats assigned to

the groups receiving ICV infusions of 0.9% NaCl,

oxytocin or OXYANT did not differ significantly and

amounted to 33.79 ^ 2.40, 32.57 ^ 2.30 and

31.00 ^ 1.06% of the total left ventricle wall surface.

Left ventricle end-diastolic pressure was significantly

higher in the infarcted (23.9 ^ 1.0 mm Hg; n ¼ 18)

than in the sham-operated rats (4.2 ^ 0.4 mm Hg;

n ¼ 17; [F(1,33) ¼ 313.69; P , 0.001]. Significant

differences were also found when the individual groups

of the infarcted and sham-operated rats were included

in the analysis [F(5,29) ¼ 57.950; P , 0.001]

(Table I). MABP and HR at the beginning of the ICV

infusions were similar in all experimental groups. As

shown in Figure 1 and Table I resting MABP and HRTab

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were not appreciably affected by any of the ICV

infusions either in the infarcted or in the sham-

operated rats. The results of the three way ANOVA on

changes of MABP and HR from baseline during ICV

infusions of 0.9% NaCl, oxytocin or OXYANTwere as

follows: DMABP: [F(35,245) ¼ 0.606; P ¼ 0.962];

DHR: F(35,245) ¼ 0.659; P ¼ 0.931].

Application of the air jet elicited significant

increases of MABP and HR in each experimental

group. As illustrated in Figure 2, changes in MABP

and HR appeared immediately after application of the

stressor and their duration was similar in each

experimental group. The overall two-way ANOVA

performed on maximum changes in MABP from

baseline (immediately before application of the air jet

stressor) revealed significant interaction for data

obtained in the infarcted and the sham-operated rats

with the type of the experiment [F(5,35) ¼ 15.701;

P , 0.001].

Figure 3 shows significant differences between the

individual groups for maximum increases in MABP.

In rats given ICV vehicle, the air-jet stressor increased

the maximal change in MABP more in infarcted rats

than in sham-operated controls (P , 0.001); ICV

oxytocin reduced the maximum increase in MABP in

the infarcted, but not the sham-operated rats

(P , 0.001); in contrast, ICV OXYANT increased

the maximum increase in MABP in the sham rats, but

not in the infarcted rats (P , 0.001). The maximum

increases in MABP expressed as percentage of

baseline MABP in the individual groups of rats were

as follows: sham-operated control: 6.8 ^ 0.9%,

infarcted control: 13.1 ^ 0.9% (P , 0.001);

sham-operated þ oxytocin group: 7.4 ^ 0.8%,

infarcted þ oxytocin group: 7.9 ^ 1.0% (n.s.);

sham-operated þ OXYANT group: 11.4 ^ 0.8%,

infarcted þ OXYANT group 13.8 ^ 0.4% (n.s.).

Similarly, significant differences were found in

maximum changes of HR after application of the air

jet stressor [F(5,35) ¼ 9.676; P , 0.001]. Differences

between the individual groups are shown in Figure 3.

In rats given ICV vehicle, the air-jet stressor increased

the maximal change in MABP more in infarcted rats

than in sham-operated controls (P , 0.01); ICV

oxytocin reduced the maximum increase in MABP

in the infarcted, but not the sham-operated rats

(P , 0.001); in contrast, ICV OXYANT increased the

maximum increase in MABP in the sham rats, but not

in the infarcted rats (P , 0.05). The maximum

increases in HR expressed as percentage of baseline

HR in the individual groups of rats amounted to:

4.4 ^ 0.7% in the sham-operated controls,

7.6 ^ 0.4% in the infarcted controls (P , 0.01);

4.8 ^ 0.4% in the sham operated oxytocin-treated,

3.8 ^ 0.6% in the infarcted oxytocin-treated

group (n.s.); 6.8 ^ 0.4% in the sham-operated

OXYANT group, and 7.9 ^ 1.5% in the infarcted

OXYANT group (n.s.).

Figure 1. Non-significant changes of MABP and HR from baseline under resting conditions in the sham-operated (open symbols) and

infarcted (filled symbols) rats receiving ICV infusions of 0.9% NaCl (triangles), OXYANT (squares) or oxytocin (OXY, circles). The arrow

indicates the start of ICV infusion. Number of rats per group: sham-operated 0.9%: n ¼ 7; sham-operated OXY: n ¼ 6; sham-operated

OXYANT: n ¼ 7; infarcted 0.9% NaCl: n ¼ 8; infarcted OXY: n ¼ 6; infarcted OXYANT: n ¼ 6.

Brain oxytocin, stress and circulation 521

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Discussion

The present investigation provides strong evidence for

a significant role of centrally released oxytocin in the

control of cardiovascular responses to an acute mild

stressor and for impairment of this function after a

myocardial infarction. Specifically, it is demonstrated

that (1) blockade of oxytocin receptors in the brain of

the control rats significantly enhances blood pressure

and HR elevations evoked by an unexpected stressor,

(2) administration of the same dose of antagonist does

not modify the cardiovascular responses to the stressor

in the infarcted rats and (3) administration of

exogenous oxytocin at a dose which does not

modulate function of the cardiovascular system in

the sham-operated rats effectively reduces the exag-

gerated cardiovascular responses to stress in the

infarcted rats.

Buffering role of endogeneous oxytocin

Oxytocin acting centrally has been previously

described as an important anxiolytic peptide (Windle

et al. 2006; Blume et al. 2008). It has been also

demonstrated that it reduces the hypothalamic-

pituitary-adrenal axis responses to stress (Windle

et al. 1997; Neumann et al. 2000). Our finding

showing that oxytocin buffers the cardiovascular

responses to stress uncovers another previously

unknown function of this peptide. Namely, the results

with central blockade of oxytocin receptors indicate

that centrally released endogenous oxytocin signifi-

cantly attenuates the intensity of the cardiovascular

responses to acute stress in intact rats. It is also shown

that the role of endogenous oxytocin in buffering the

cardiovascular responses to stress is impaired in rats

after myocardial infarction.

Figure 2. Representative illustrations of changes in MABP and HR after application of the air jet stressor (arrow) in the sham-operated and

infarcted rats during ICV infusion of 0.9% NaCl, OXYor OXYANT. The Figure shows that (1) during infusion of 0.9% NaCl MABP and HR

responses to stress are markedly greater in the infarcted than in the sham-operated rat, (2) ICVadministration of OXYANT intensifies MABP

and HR responses to stress in the sham-operated but not in the infarcted rat and (3) ICVadministered OXY reduces MABP and HR responses

to stress in the infarcted but not in the sham-operated rat.

A. Wsoł et al.522

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Interestingly, our results demonstrate that although

the cardiovascular responses to stress are significantly

enhanced in the infarcted rats they are not further

elevated by blockade of oxytocin receptors. Reduction

of cardiovascular responses to stress in the infarcted

rats by centrally applied exogenous oxytocin strongly

suggests that the lack of effects observed from

oxytocin receptor blockade on the cardiovascular

responses to stress during the post-infarct state is

mainly caused by an inadequate release of oxytocin in

the brain. The apparent impairment of the buffering

role of oxytocin during the post-infarct state is

obviously an unfavourable factor. Sudden stress

causes significant acceleration of the HR, cardiac

contractility and total peripheral resistance (Zhang

et al. 1999). Consequently, cardiac work and oxygen

consumption may markedly increase and current

clinical studies in the literature frequently demon-

strate a coincidence of exposure to a sudden stress

with serious cardiovascular complications (Clarke

et al. 2000; Kario et al. 2003).

Effects of exogenous oxytocin

In the present study, ICV infusion of oxytocin at the

rate of 100 ng/h did not evoke significant changes of

arterial blood pressure and HR under resting

conditions. Previous studies assessing the role of

centrally released oxytocin in the regulation of arterial

blood pressure and HR provided controversial results.

In a study performed on anaesthetised rats, Tran et al.

(1982) demonstrated that the intracisternal injection

of oxytocin (1–10 mU/kg) elicited significant pressor

responses not associated with any changes of HR.

Alternatively, Feuerstein et al. (1984) reported that

ICV injections of 0.15, 1.0 or 10 nmol of oxytocin did

not produce significant changes of blood pressure,

however, higher doses elicited significant tachycardia.

In contrast, some investigators provided evidence that

oxytocin stimulates cardiovascular neurones respon-

sible for hypotensive responses. For instance, Versteeg

et al. (1983) reported that oxytocin blunted the

pressor component of the neurogenic hypertension

evoked by electrical stimulation of the mesencephalic

reticular formation. Oxytocin was also found to

stimulate neurones of the dorsal vagal complex

(Charpak et al. 1984), enhance baroreflex bradycardia

and reduce exercise-induced cardioacceleration

(Michelini 2001; Higa et al. 2002). Recently,

Petersson and Uvnas-Moberg (2007) reported that

repeated ICV injections of 300 ng of oxytocin for

5 days significantly reduced arterial blood pressure in

Sprague Dawley rats. Thus, it seems that the final

effect of oxytocin depends on several factors such as

the dose, mode and site of application, presence of

anaesthesia or hypertension. The rate of ICV infusion

of oxytocin used in the present study (100 ng/h) may

be considered to be relatively low in comparison to the

doses administered by other authors. Oxytocin was

infused at this rate in order to match the rate of

vasopressin infusion that has been used in our

previous investigations (Dobruch et al. 2005;

Cudnoch-Jedrzejewska et al. 2008). Therefore, it

cannot be excluded that a higher dose of oxytocin

would affect function of the cardiovascular system

under baseline conditions. It is important that in the

infarcted rats ICV infusion of oxytocin at the same rate

(100 ng/h) effectively reduced the cardiovascular

responses to the acute stressor. This finding suggests

that there is some deficit of endogenous oxytocin in the

brain during the post-infarct state and at the same

time indicates that the pool of the cardiovascular

neurones engaged in regulation of the pressor and

tachycardic responses to stress might be different from

that involved in regulation of arterial blood pressure

and HR under resting conditions.

Possible interaction of oxytocin and vasopressin

Because vasopressin V1 receptors antagonists are not

entirely specific and show some affinity to oxytocin

receptors, it is sometimes claimed that the effects of

blockade of V1 receptors result from the blockade of

oxytocin receptors. The present study does not

support such a possibility with regard to the

cardiovascular responses to stress. The OXYANT

used in the present study has much greater affinity

to oxytocin ( pA2 ¼ 7.37 ^ 0.07) than to vasopressin

V1 ( pA2 ¼ 5.39 ^ 0.04) and V2 ( pA2 , 5.5) recep-

tors (Manning et al. 1995). Besides, administration

Figure 3. Mean maximal changes of MABP and HR from baseline

after application of air jet stress in the sham-operated (open

columns) and infarcted (filled columns) rats receiving ICV infusions

of 0.9% NaCl, OXYor OXYANT. Number of rats per group: sham-

operated 0.9%: n ¼ 7; sham-operated OXY: n ¼ 6; sham-operated

OXYANT: n ¼ 7; infarcted 0.9% NaCl: n ¼ 8; infarcted OXY:

n ¼ 6; infarcted OXYANT: n ¼ 6. Asterisks indicate significant

differences between the experimental groups *P , 0.05;

**P , 0.01; ***P , 0.001.

Brain oxytocin, stress and circulation 523

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of the OXYANT {DesGly-NH2-d(CH2)5[D-Tyr2,-

Thr4]OVT} produced opposite effects to those

observed after administration of antagonist for V1

receptors in our previous studies (Dobruch et al.

2005; Cudnoch-Jedrzejewska et al. 2007). Similarly,

oxytocin produced opposite effects to vasopressin

(Dobruch et al. 2005; Cudnoch-Jedrzejewska et al.

2008; Stojicic et al. 2008). For instance, blockade of

oxytocin receptors enhanced the pressor and tachy-

cardic responses to stress in the sham-operated

rats while being ineffective in the infarcted rats,

whereas blockade of V1 receptors with d(CH2)5

Tyr(Me)2,Ala-NH29]AVP was not effective in the

sham-operated rats but it markedly strengthened the

pressor and tachycardic responses to stress during

the post-infarct state (Dobruch et al. 2005; Cudnoch-

Jedrzejewska et al. 2007). Thus, it may be concluded

that the effects observed in the present study after

administration of OXYANT or oxytocin were specifi-

cally caused by an interaction with oxytocin receptors.

However, it cannot be excluded that oxytocin and

vasopressin can mutually interact at the receptor level.

Such interaction could be especially important during

enhanced release of one or both of these peptides.

Summary and perspectives. Our results suggest that

centrally released oxytocin protects against excessive

increases of blood pressure and HR during sudden

stress. The buffering function of this endogenous

oxytocin appears to be significantly reduced during

the post-infarct state. Previous studies demonstrated

that exaggerated stimulation of the cardiovascular

system by air jet stress in rat models of coronary

disease is caused by enhanced stimulation of AT1

angiotensin and V1 vasopressin receptors (Zhang et al.

1999; Dobruch et al. 2005; Cudnoch-Jedrzejewska

et al. 2007). The present study reveals that an

inadequate action of the endogenous oxytocin system

in the brain may also contribute to this phenomenon.

In the present study, we administered ICV infusions of

only single doses of oxytocin and OXYANT.

Therefore, we cannot exclude that administration of

higher doses of these compounds could elicit some

additional central cardiovascular effects, for instance

change in the resting blood pressure or HR. Our study

opens several new questions regarding engagement of

oxytocin in adjustment of the cardiovascular system to

other types of stress, including chronic stress, as well

as possibility of use of oxytocin agonists for treatment

of exaggerated cardiovascular responses to stress.

Acknowledgements

The authors wish to express their gratitude to

Professor Maurice Manning from the Department of

Biochemistry and Molecular Biology, Medical College

of Ohio, Toledo, USA for the generous supply of

oxytocin antagonist used in the present study, to

Marzanna Tkaczyk for her skilful technical assistance,

and to Marcin Kumosa for preparation of illustrations.

This study was supported by a grant from the Medical

University of Warsaw (1MAW2/2006-2008).

Declaration of interest: The authors report no

conflicts of interest. The authors alone are responsible

for the content and writing of the paper.

References

Barberis C, Tribollet E. 1996. Vasopressin and oxytocin receptors in

the central nervous system. Crit Rev Neurobiol 10:119–154.

Bergquist F, Ludwig M. 2008. Dendritic transmitter release: A

comparison of two model systems. J Neuroendocrinol 20:

677–686.

Blume A, Bosch OJ, Miklos S, Torner L, Wales L, Waldherr M,

Neumannn ID. 2008. Oxytocin reduces anxiety via ERK1/2

activation: Local effect within the rat hypothalamic paraventric-

ular nucleus. Eur J Neurosci 27:1947–1956.

Braga DC, Mori E, Higa KT. 2000. Central oxytocin modulates

exercise-induced tachycardia. Am J Physiol Regul Integr

Comp Physiol 278:R1474–R1482.

Carter CS, Grippo AJ, Pournajafi-Nazarioo M, Ruscio MG,

Porges SW. 2008. Oxytocin, vasopressin and sociality. Progr

Brain Res 170:331–336.

Charpak S, Armstrong WE, MUhlethaler M, Dreifuss JJ. 1984.

Stimulatory action of oxytocin on neurones of the dorsal motor

nucleus of the vagus nerve. Brain Res 300:83–89.

Choleris E, Little SR, Mong JA, Puram SV, Langer R, Pfaff DW.

2007. Microparticle based delivery of oxytocin receptor

antisense DNA in the medial amygdala blocks social recognition

in female mice. Proc Natl Acad Sci USA 104:4670–4675.

Clarke SP, Frasure-Smith N, Lesperance F, Bourassa MG. 2000.

Psychosocial factors as predictors of functional status at 1 year

patients with left ventricular dysfunction. Res Nurs Health 23:

290–300.

Costa e Sousa RH, Pereira-Junior PP, Oliveira PF, Olivares EL,

Werneck de Castro JPS, Mello DB, Nascimento JHM, Campos

de Carvalho AC. 2005. Cardiac effects of oxytocin: Is there a role

for this peptide in cardiovascular homeostasis? Regul Pept 132:

107–112.

Cudnoch-Jedrzejewska A, Dobruch J, Puchalska L, Szczepanska-

Sadowska E. 2007. Interaction of AT1 receptors and V1a

receptors-mediated effects in the central cardiovascular control

during the post-infarct state. Regul Pept 142:86–94.

Cudnoch-Jedrzejewska A, Szczepanska-Sadowska E, Dobruch J,

Puchalska L, Ufnal M, Kowalewski S, Wsoł A. 2008. Differential

sensitisation to central effects of angiotensin II in rats with a

myocardial infarct: Relevance to stress and interaction with

vasopressin. Stress 11:290–301.

Curran-Everet D, Benos DJ. 2004. Guidelines for reporting

statistics in journals published by the American Physiological

Society. Physiol Genom 18:249–251.

Dobruch J, Cudnoch-Jedrzejewska A, Szczepanska-Sadowska E.

2005. Enhanced involvement of brain vasopressin V1 receptors

in cardiovascular responses to stress in rats with myocardial

infarction. Stress 8:273–284.

Engelmann M, Ebner K, Landgraf R, Hoisboer F, Wotjak CT. 1999.

Emotional stress triggers intrahypothalamic but not peripheral

release of oxytocin in male rats. Neuroendocrinol 11:867–872.

Feuerstein G, Zerbe RL, Faden AI. 1984. Central cardiovascular

effects of vasotocin, oxytocin and vasopressin in conscious rats.

J Pharmacol Exp Ther 228:348–353.

Higa KT, Mori E, Viana FF, Morris M, Michelini LC. 2002.

Baroreflex control of heart rate by oxytocin in the solitary-vagal

A. Wsoł et al.524

Stre

ss D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y O

saka

Uni

vers

ity o

n 11

/19/

14Fo

r pe

rson

al u

se o

nly.

complex. Am J Physiol Regul Integr Comp Physiol 282:

R537–R545.

Kario K, McEwen BS, Pickering TG. 2003. Disasters and the heart:

A review of the effects of earthquake induced stress on

cardiovascular disease. Hypertens Res 26:355–367.

Lampert R, Jain D, Burg MM, Batsford WP, McPherson CA. 2000.

Destabilizing effects of mental stress on ventricular arrhythmia in

patients with implantable cardioverter-defibrillators. Circulation

101:158–164.

Leng G, Caquineau C, Sabatier N. 2005. Regulation of oxytocin

secretion. Vitam Horm 71:27–58.

Ludbrook J. 1994. Repeated measurements and multiple compari-

sons in cardiovascular research. Cardiovasc Res 28:303–311.

Manning M, Miteva K, Pancheva S, Stoyev S, Wo NC, Chan WY.

1995. Design and synthesis of highly selective in vitro and in vivo

uterine receptors antagonists of oxytocin: Comparisons with

Atosiban. Int J Peptide Protein Res 46:244–252.

Mayorov DN, Head GA. 2003. AT1 receptors in the RVLM

mediate pressor responses to emotional stress in rabbits.

Hypertension 41:1168–1173.

Mayorov DN, Head GA, De Matteo R. 2004. Tempol attenuates

excitatory actions of angiotensin II in the rostral ventrolateral

medulla during emotional stress. Hypertension 44:101–106.

McCann SM, Antunes-Rodrigues J, Jankowski M, Gutkowska J.

2002. Oxytocin, vasopressin and atrial natriuretic peptide

control body fluid homeostasis by action on their receptors in

brain, cardiovascular system and kidney. Prog Brain Res 139:

309–326.

Michelini LC. 2001. Oxytocin in the NTS. A new modulator of

cardiovascular control during exercise. Ann NY Acad Sci 940:

206–219.

Morris M, Ross J, Sundberg DK. 1985. Catecholamine biosynthesis

and vasopressin and oxytocin secretion in the spontaneously

hypertensive rat: An in vitro study of localized brain regions.

Peptides 6:949–955.

Mohring J, Schoun J, Kintz J, Robinson IC, McNeill JR. 1983.

Vasopressin and oxytocin content are decreased in the brain

stems of spontaneously hypertensive rats. Neuroendocrinology

36:457–461.

Nakashima T, Nogfuchi T, Furukawa T, Yamasaki M, Makino S,

Miyata S, Kiyohara T. 2002. Brain oxytocin augments stress-

induced long-lasting plasma adrenocorticotropic hormone

elevation in rats. Neurosci Lett 22:161–164.

Neumann ID. 2008. Brain oxytocin: A key regulator of emotional and

social bevavior in both females and males. J Neuroendocrinol

20:858–865.

Neumann JD, Kromer SA, Toschi N, Ebner K. 2000. Brain

oxytocin inhibits the (re)activity of the hypothalamo-pituitary-

adrenal axix in male rats: Involvement of hypothalamic and

limbic brain regions. Regul Pept 96:31–38.

Petersson M, Alster P, Lundeburg T, Uvnas-Moberg K. 1996.

Oxytocin causes a long time decrease of blood pressure in female

and male rats. Physiol Behav 60:1311–1315.

Petersson M, Uvnas-Moberg K. 2007. Effects of an acute stressor

on blood pressure and heart rate in rats pretreated with

intracerebroventricular oxytocin injections. Psychoneuroendo-

crinology 32:959–965.

Robinson DA, Wei F, Wang GD, Li P, Kim SJ, Vogt SK, Muglia LJ,

Zhuo M. 2002. Oxytocin mediates stress-induced analgesia in

adult mice. J Physiol 540:593–606.

Russell JA, Leng G, Douglas AJ. 2003. The magnocellular oxytocin

system, the fount of maternity: Adaptations in pregnancy. Front

Neuroendocrinol 24:27–61.

Sawchenko PE, Swanson LW. 1982. Immunochistochemical

identification of neurons in the paraventricular nucleus of

hypothalamus that project to the medulla or to the spinal cord in

the rat. J Comp Neurol 205:260–272.

Selye H, Bajusz E, Grasso S, Mendell P. 1960. Simple techniques

for the surgical occlusion of coronary vessels in the rat.

Angiology 11:398–407.

Sofroniew MV, Schrell U. 1981. Evidence for direct projections

from oxytocin and vasopressin neurons in the hypothalamic

paraventricular nucleus in the medulla oblongata: Immunohis-

tochemical visualization of both the horseradish peroxidase

transported and peptide produced by the same neurons.

Neurosci Lett 22:211–217.

Stojicic S, Milutinovic-Smiljanic S, Sarenac O, Milosavljevic S,

Paton JF, Murphy D, Japundic-Zigon N. 2008. Blockade of

central vasopressin receptors reduces the cardiovascular

responses to acute stress in freely moving rats. Neuropharma-

cology 54:824–836.

Szczepanska-Sadowska EK, Wsol A, Kowalewski S, Cudnoch-

Jedrzejewska A. 2008. Oxytocin reduces pressor and tachycardic

response to the alarming stress in the infarcted rats. FASEB J 22:

952.2.

Tran LD, Montastruc JL, Montastruc P. 1982. Effects of lysine-

vasopressin and oxytocin on central cardiovascular control.

Br J Pharmacol 77:69–73.

Versteeg CA, Cransberg K, De Jong W, Bohus B. 1983. Reduction

of centrally induced pressor response by neurohypophyseal

peptides: The involvement of lower brainstem mechanisms. Eur

J Pharmacol 94:133–140.

Windle RJ, Shanks N, Lightman SL, Ingram CD. 1997. Central

oxytocin administration reduces stress-induced corticosterone

release and anxiety behavior in rats. Endocrinology 138:

2829–2834.

Windle RJ, Gamble LE, Kershaw YM, Wood SA, Lightman SL,

Ingram CD. 2006. Gonadal steroid modulation of stress-

induced hypothalamo-pituitary-adrenal activity and anxiety

behavior: Role of central oxytocin. Endocrinology 147:

2423–2431.

Zhang W, Huang BS, Leenen FH. 1999. Brain renin-angiotensin

system and sympathetic hyperactivity in rats after myocardial

infarction. Am J Physiol Heart Circ Physiol 276:H1608–H1615.

Brain oxytocin, stress and circulation 525

Stre

ss D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y O

saka

Uni

vers

ity o

n 11

/19/

14Fo

r pe

rson

al u

se o

nly.