http://informahealthcare.com/sts ISSN: 1025-3890 (print), 1607-8888 (electronic) Stress, 2014; 17(1): 117–125 ! 2014 Informa UK Ltd. DOI: 10.3109/10253890.2013.872620
ORIGINAL RESEARCH REPORT
Oxytocin differently regulates pressor responses to stress in WKY and SHR rats: the role of central oxytocin and V1a receptors A. Wsol, E. Szczepanska-Sadowska, S. Kowalewski, L. Puchalska, and A. Cudnoch-Jedrzejewska
Abstract
Keywords
The role of central oxytocin in the regulation of cardiovascular parameters under resting conditions and during acute stress was investigated in male normotensive Wistar-Kyoto (WKY; n ¼ 40) and spontaneously hypertensive rats (SHR; n ¼ 28). In Experiment 1, mean arterial blood pressure (MABP) and heart rate (HR) were recorded in WKY and SHR rats at rest and after an airjet stressor during intracerebroventricular (ICV) infusions of vehicle, oxytocin or oxytocin receptor (OTR) antagonist. In Experiment 2, the effects of vehicle, oxytocin and OTR antagonist were determined in WKY rats after prior administration of a V1a vasopressin receptor (V1aR) antagonist. Resting MABP and HR were not affected by any of the ICV infusions either in WKY or in SHR rats. In control experiments (vehicle), the pressor response to stress was significantly higher in SHR. Oxytocin enhanced the pressor response to stress in the WKY rats but reduced it in SHR. During V1aR blockade, oxytocin infusion entirely abolished the pressor response to stress in WKY rats. Combined blockade of V1aR and OTR elicited a significantly greater MABP response to stress than infusion of V1a antagonist and vehicle. This study reveals significant differences in the regulation of blood pressure in WKY and SHR rats during alarming stress. Specifically, the augmentation of the pressor response to stress by exogenous oxytocin in WKY rats is caused by its interaction with V1aR, and endogenous oxytocin regulates the magnitude of the pressor response to stress in WKY rats by simultaneous interaction with OTR and V1aR.
Air-jet stress, blood pressure, hypertension, neuropeptides, oxytocin antagonist, vasopressin antagonist
Introduction Numerous lines of evidence indicate that the hypothalamic neuropeptide oxytocin known from its role in parturition, lactation and formation of maternal behavior (Lee et al., 2009) plays multiple roles in the regulation of the cardiovascular system. Hence, administration of oxytocin may cause hypotension, natriuresis and negative chronotropic and inotropic effects (Feuerstein et al., 1984; Gutkowska et al., 1997; Gutkowska & Jankowski, 2012; Petersson et al., 1996). The anti-inflammatory and cardioprotective role of this peptide in ischemia-reperfusion-induced myocardial injury has been emphasized (Alizadeh & Mirzabeglo, 2013; Authier et al., 2010). There is also evidence that oxytocin may play a buffering role in behavioral and humoral reactions to stress. Experimental procedures employing acute and chronic stress paradigms have revealed elevated oxytocin concentration in the paraventricular nucleus (PVN) and blood (Grippo et al., 2007; Nishioka et al., 1998). In addition, oxytocin reduces behavioral responses to chronic immobilization or isolation (Viviani & Stoop, 2008; Windle et al., 2006). Furthermore, several reports strongly suggest that oxytocin reduces anxiety Correspondence: A. Wsol, Department of Experimental and Clinical Physiology, Medical University of Warsaw, Pawinskiego 3C, 02-106 Warsaw, Poland. Tel: +0048-22-572-07-08. Fax: +0048-22-572-07-82. E-mail: [email protected]
History Received 8 August 2013 Revised 27 November 2013 Accepted 3 December 2013 Published online 19 December 2013
and exerts an antidepressive effect (Blume et al., 2008; Engelmann et al., 1999; Neumann & Landgraf, 2012; Windle et al., 1997), and the expression of oxytocin receptors (OTRs) has been found in different structures of the limbic system that play an important role in stress, anxiety and depression (Bao et al., 2008; Barberis & Tribollet, 1996; Boccia et al., 2013). Thus far, the role of centrally released oxytocin in the regulation of blood pressure during stress is unclear. Neuroanatomical studies revealed the presence of oxytocin and oxytocinergic receptors (OTRs) in the regions of the lower brain stem involved in the regulation of cardiovascular responses to stress (Barberis & Tribollet, 1996, Boccia et al., 2013, Sofroniew, 1985). In the study by Petersson & Uvna¨sMoberg (2007), repeated intracerebroventricular (ICV) administration of oxytocin reduced the baseline blood pressure in rats, however the oxytocin-treated animals responded with blood pressure and heart rate (HR) increases to unexpected noise. The latter response was absent in control rats (Petersson & Uvna¨s-Moberg, 2007). In our previous study, blockade of the brain OTR was found to exaggerate significantly the pressor response to an alarming (air jet) stressor and it was concluded that endogenous oxytocin buffers the pressor responses to stress (Wsol et al., 2009). We also showed that the buffering role of oxytocin in the regulation of blood pressure during stress is impaired
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Department of Experimental and Clinical Physiology, Medical University of Warsaw, Warsaw, Poland
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in Sprague Dawley rats with a myocardial infarction. Other studies revealed that arterial hypertension is associated with some abnormalities in the central oxytocinergic system (Gaida et al., 1985; van Tol et al., 1988). Namely, Gaida et al. (1985) found significantly reduced content of oxytocin in the hypothalamus, mid-brain and brain stem of stroke prone spontaneously hypertensive rats (SHR-SP). The same group did not find significant differences in oxytocin content in the neuro-intermediate lobe of the pituitary between SHR-SP and WKY rats. van Tol et al. (1988) showed that SHR manifest lower expression of oxytocin mRNA in the hypothalamic paraventricular and supraoptic nuclei, as well as in the posterior pituitary. The above findings implied the possibility that the abnormal content of oxytocin and OTR in the SHR brain may have an effect on the central regulation of blood pressure in this strain. To date the role of oxytocin in the regulation of blood pressure in hypertension was assessed only in the experimental model of neurogenic hypertension evoked by electrical stimulation of the mesencephalic reticular formation (Versteeg et al., 1983). In this model, the administration of oxytocin into the fourth cerebral ventricle significantly reduced blood pressure elevation. The role of oxytocin in the regulation of blood pressure in the SHR model of spontaneously developing hypertension has not been investigated previously. Based on the literature cited above, we hypothesized that the OTRs in the brain play an essential role in the regulation of blood pressure under baseline conditions and during acute alarming stress in normotensive Wistar-Kyoto (WKY) and SHR rats. We expected that oxytocin may be an essential hypotensive agent, effectively buffering cardiovascular responses to this acute stressor both in WKY and in SHR rats. After an analysis of our preliminary data, we tested whether some of the cardiovascular effects of oxytocin are caused by its interaction with V1a receptors. The rationale for this was based on previous findings showing that oxytocin stimulates both OTR and vasopressin V1a receptors (V1aR) (Akerlund et al., 1999; Gimpl et al., 2008). To find out whether endogenous oxytocin plays an essential role in the regulation of pressor responses to stress we determined whether the magnitude of cardiovascular responses to stress in WKY rats can be affected by simultaneous blockade of central oxytocin and vasopressin V1a receptors.
Methods Animals Adult male SHR and their parent normotensive strain WKY rats weighing 250–300 g were bred in the Department of Animal Breeding of the Medical University of Warsaw. The rats were housed three per cage at a temperature of 22–25 C with 12 h:12 h light–dark rhythm (lights on between 07.00 h and 19:00 h). They were provided with a rodent dry pellet diet containing 0.45% of NaCl and had free access to tap water. 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 Animal Research of the Warsaw Medical University.
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Surgical procedures The rats entered the study at the age 10–12 weeks. They were implanted with a guide tube into the left cerebral ventricle for the ICV infusions and an aortic catheter for recording the hemodynamic parameters. Each of the surgical procedures was performed under general anesthesia with pentobarbital sodium (Pentobarbital, Biowet, Puławy, Poland; 15 mg/ml, 50 mg/kg body wt, 20 mmol/100 g body wt., i.p.), which was followed by administration of antibiotic (Penicillin, Polfa, Pabianice, Poland; 30,000 U in 1 ml/rat, i.m). After each surgical procedure, the rats were housed singly. After full recovery from anesthesia they were returned to the animal house. Implantation of ICV cannula The implantation of the ICV cannula was performed according to the procedure described previously (Dobruch et al., 2005). The rat’s head was placed in a stereotaxic device (Kopff) and the stainless steel cannula (o.d., 0.81 mm, MIFAM S.A., Milano´wek, Poland) was implanted into the left cerebral ventricle according to the following stereotaxic coordinates: 1.3 mm posterior to bregma, 2 mm lateral to the midline and 4.5 mm below the surface of the skull. The cannula was secured on the external surface of the skull with acrylic cement (Duracryl, SPOFA-DENTAL, Jicin, Czech Republic), and closed with a stainless steel stylet (o.d., 0.46 mm). Implantation of the arterial catheter Seven days after implantation of the ICV cannula the arterial catheter was inserted into the abdominal aorta through the femoral artery. The catheter consisted of the intra-arterial (3.5–4.0 cm long; i.d., 0.12 mm; o.d., 0.25 mm) and external (i.d., 0.25 mm; o.d., 0.4 mm) portions made from polyvinyl tubing (Scientific Commodities, Inc., Lake Havasu City, AZ). The extravascular part of the catheter was exteriorized on the rat’s neck in the interscapular region. The catheter was filled with 0.9% NaCl containing 500 IU of heparin/ml and closed with a plastic stopper. Experimental protocol The experiments were performed 24–48 h after the implantation of the arterial catheter between 10:00 h and 18:00 h. At the beginning of the experiment, the arterial catheter was connected to a BIOPAC MP100 system (BIOPAC, MP100, Santa Barbara, CA) for continuous recording of mean arterial blood pressure (MABP) and HR. The study was performed on 68 rats divided into two experiments. Experiment 1 was designed to compare the effects of ICV administration of vehicle, oxytocin and blockade of OTR on the cardiovascular parameters in the WKY and SHR strains. The protocol of this experiment is shown in Figure 1(A). In each experimental session the ICV infusion was started after a 15- to 30-min period of stabilization of resting cardiovascular parameters (MABP and HR), at a rate of 5 ml/h (0.083 ml/min) by means of a microsyringe pump (Harvard 22 Infusion Pump, Harvard Smith, Natick, MA). The measurements of MABP and HR
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Figure 1. The sequence of experimental procedures in Experiment 1 (oxytocin and OTR antagonist experiments) (A) and Experiment 2 (oxytocin and V1aR antagonist experiments) (B). ICV: intracerebroventricular, MABP: mean arterial blood pressure, HR: heart rate, OTR: oxytocin receptor, V1aR: vasopressin V1a receptor.
were continued for 50 min under resting conditions and for 10 min after application of the air jet, which was used as the alarming stressor. The air-jet stressor was applied by directing the air stream onto the top of the rat’s head for 1–1.5 s from a tank containing compressed air (10 atm) according to the modified technique of Zhang et al. (1999). In each rat the air-jet stressor was applied only once in order to avoid adaptation. The cardiovascular responses to acute stress during the first 10 s after the application of the air jet were evaluated by measuring the maximum increases in MABP and HR from the level immediately preceding the application. Rats undergoing Experiment 1 were divided into six experimental groups. Groups 1.1 (WKY-vehicle; n ¼ 8) and 1.2 (SHR-vehicle; n ¼ 8) received ICV infusion of 0.9% NaCl as the vehicle (5 ml/h). Groups 1.3 (WKY-oxytocin; n ¼ 7) and 1.4 (SHR-oxytocin; n ¼ 10) were infused with vehicle for the first 10 min and subsequently with oxytocin. Groups 1.5 (WKY-OTR antagonist n ¼ 7) and 1.6 (SHR-OTR antagonist; n ¼ 10) received infusion of vehicle for 10 min. This was followed by ICV infusion of OTR antagonist. Experiment 2 was performed to determine whether the regulation of the cardiovascular responses to stress by oxytocin is influenced by its interaction with V1a receptors. Experiment 2 was performed on 18 WKY rats divided into three groups. The sequence of the experimental procedures is shown in Figure 1(B). After stabilization of the cardiovascular parameters Group 2.1 (V1aR antagonist þ vehicle; n ¼ 6) received ICV infusion of V1aR antagonist in vehicle for 15 min. This was followed by ICV infusion of vehicle for 60 min. The air-jet stressor was applied 50 min after the start of infusion of the vehicle. The experimental design for Group 2.2 (V1aR antagonist þ oxytocin; n ¼ 6) was similar as
that of Group 2.1 except the vehicle was infused together with oxytocin. Group 2.3 (V1aR antagonist þ OTR antagonist; n ¼ 6) received ICV infusion of OTR antagonist together with the vehicle. Ligands used in the study Oxytocin (CysTyrIleGlnAsnCysProLeuGly-NH2) was purchased from Phoenix Pharmaceuticals (Strasbourg, France) and was infused at 100 ng/5 ml/h (1.66 pmole/0.083 ml/min). The OTR antagonist [DesGly-NH2-d(CH2)5[D-Tyr2,Thr4] OVT was supplied by Prof. Morris Manning, University of Toledo, Ohio (Manning et al., 1995). OTR antagonist was infused at 4.3 mg/5 ml/h (71.6 nmole/0.083 ml/min). The doses of oxytocin and OTR antagonist were established previously (Wsol et al., 2009). Vasopressin V1aR non-peptide antagonist (SSR49059) was provided by Sanofi Aventis (Montpellier, France) and was infused at 2 mg/40 ml/h (0.052 nmole/0.67 ml/ min) (Serradeil-Le Gal et al., 1993). The dose of V1aR antagonist was established in a preliminary series of experiments. Postmortem examination At the end of the experiment, the rats were euthanized by an overdose of pentobarbital (30 mg/ml, 100 mg/kg body wt., 40 mmol/100 g body wt., i.p.). The heart and the brain were excised for postmortem examination. The left cardiac ventricle (including the septum) was separated from the right ventricle and the weight of both ventricles was determined. Evans blue (5 ml) was injected through the guide tube into the ICV system and the position of the tip of the cannula in the brain was verified by visual inspection.
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Table 1. Mean arterial blood pressure (MABP) and heart rate (HR) at the beginning of experiment (0 min) and before application of the air-jet stressor (50 min) in WKY and SHR rats during ICV infusions.
Experimental groups WKY-vehicle Group 1.1 (n ¼ 8) SHR-vehicle Group 1.2 (n ¼ 8) WKY-oxytocin Group 1.3 (n ¼ 7) SHR-oxytocin Group 1.4 (n ¼ 10) WKY-OTR antagonist Group 1.5 (n ¼ 7) SHR-OTR antagonist Group 1.6 (n ¼ 10) WKY-V1aR antagonist þ vehicle Group 2.1 (n ¼ 6) WKY-V1aR antagonist þ oxytocin Group 2.2 (n ¼ 6) WKY-V1aR antagonist þ OTR antagonist Group 2.3 (n ¼ 6)
Body mass (g)
MABP (0 min) (mm Hg)
MABP (50 min) (mm Hg)*
HR (0 min) (bpm)
HR (50 min) (bpm)*
298 12.1 310 8.5 289 3.4 292 5,4 308 15.5 294 14.6 307 8.7 292 9.6 294.2 9.0
102 3.4 156 5.0# 112 3.7 165 4.6# 112 4.9 155 4.6# 124 2.0 127 1.3 118 1.6
106 3.0 155 5.3# 112 4.0 165 4.6# 113 6 153 4.6# 138 2.2 128 2.2 139 2.6
358 8.2 397 13.4 364 20.3 381 14.1 354 3.8 422 13.1 411 21 383 18 340 16
356 9.9 412 16.9 364 17.3 371 18.3 353 14.7 388 18.7 378 21 350 21 306 13
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HR: heart rate; MABP: mean arterial blood pressure; OTR: oxytocin receptor; V1aR: vasopressin V1a receptor; WKY: Wistar-Kyoto rats; SHR: spontaneously hypertensive rats; n: number of rats; bpm: beats per min. Means SEM are shown. *Fifty minutes after the beginning of infusions of oxytocin or OTR antagonist MABP and HR did not differ from values under baseline conditions (i.e. before the onset of ICV infusion) (ANOVA followed by the Tukey test). #Significant difference between SHR group and the corresponding WKY group (ANOVA followed by the Tukey test).
Statistical analysis STATISTICA software (Version 9.1, Chicago, IL) was used for statistical analysis of the data. Five-minute averages of MABP and HR were entered into the statistical analysis to assess changes in cardiovascular parameters during the resting period. Normal distribution of the data was established by means of the Shapiro–Wilk test. As no significant dispersion of the data from normality was found, ANOVA was applied. Two-way ANOVA on repeated measurements was used to evaluate changes in cardiovascular parameters at rest as recommended by Curran-Everett & Benos (2004) and Ludbrook (1994). One-way ANOVA was employed to determine the significance of differences between the maximum changes of MABP and HR evoked by the air-jet stressor in different groups of experiments. Horizontal and vertical multiple pair-wise comparisons were performed using the post-hoc Tukey test to determine the significance of differences between individual groups of experiments. The differences were considered significant if p50.05. All values presented in the text, figures and table are means standard error of the mean (SEM).
Results The resting MABP was significantly higher in SHR than in WKY rats (F(5,44) ¼ 38.61; p50.001) (means of individual groups are shown in Table 1), while resting values of HR were similar in all experimental groups. The left ventricle weight was significantly higher in SHR (Experiment 1: 0.281 0.004 g/100 g body weight) than in the normotensive rats (Experiment 1: 0.245 0.004 g/100 g body weight) (F(1,51) ¼ 38.549; p50.001). The right ventricle weight was lower in SHR (Experiment 1: 0.06 0.002 g/100 g body weight) than in WKY rats (Experiment 1: 0.068 0.002 g/100 g body weight) (F(1,51) ¼ 8.567; p50.01). Total body weights were similar in all experimental groups (Table 1). Resting MABP values during ICV infusions The resting MABP values were not significantly affected by ICV infusions for 50 min of 0.9% NaCl, oxytocin or
OTR antagonist either in WKY or in SHR rats (Table 1, time fluctuations not shown). Changes in MABP elicited by air-jet stressor in Experiment 1 In Experiment 1, an analysis of differences in air-jet stressorinduced DMABPmax revealed a significant difference between WKY and SHR rats (F(5,44) ¼ 6.91; p50.001). Significant differences between experimental groups were also found when DMABPmax was compared separately in WKY (F(2,19) ¼ 5.20; p50.05) and SHR rats (F(2,25) ¼ 11.63; p50.001). Figure 2 shows typical changes of MABP and HR in individual experiments after the application of the air jet in the different experiments. As shown in Figure 3(A) the response to the air-jet stressor in rats receiving ICV infusion of vehicle was significantly higher in SHR rats (p50.05; Tukey test). ICV infusion of oxytocin reduced the pressor response to the air-jet stressor only in SHR rats. The stressor-induced DMABPmax was significantly lower during ICV infusion of oxytocin (SHR-vehicle versus SHR-oxytocin: p50.05; Tukey test). In contrast, in WKY rats, the DMABPmax response to stress was significantly higher during ICV infusion of oxytocin than during infusion of the vehicle (WKY-vehicle versus WKY-oxytocin, p50.05; Tukey test). Changes in MABP elicited by air-jet stressor in Experiment 2 Significant differences in DMABPmax responses to stress were found between groups of Experiment 2 (F(2,15) ¼ 34.49; p50.001). As shown in Figure 4(A) a posteriori analysis revealed that combined administration of V1aR antagonist and oxytocin (Group 2.2) completely abolished the air-jet stressor-induced blood pressure elevation (V1aR antagonist þ vehicle versus V1aR antagonist þ oxytocin: p50.001; Tukey test). Significant differences were also found between the maximum increases in DMABPmax in Groups 2.1 (V1aR antagonist þ vehicle) and 2.3 (V1aR antagonist þ OTR antagonist: p50.05; Tukey test) and between the Groups 2.2 (V1aR antagonist þ oxytocin) and
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Figure 2. Typical recordings of mean arterial blood pressure (MABP) and heart rate (HR) fluctuations in (A) spontaneously hypertensive rats (SHR) and (B) WKY in response to the air-jet stressor during intracerebroventricular (ICV) infusions of 0.9% NaCl (vehicle), oxytocin, oxytocin receptor antagonist (OTR antagonist), V1a receptor antagonist (V1aR antagonist) and combined infusion of oxytocin and V1aR antagonist.
2.3 (V1aR antagonist þ OTR; (Figure 4A).
p50.001;
Tukey
test)
HR at rest and after application of air-jet stressor The resting HR was neither significantly affected by any of the ICV infusions at 50 min after the beginning of infusion (Table 1, time fluctuations not shown), nor were there significant differences between the stress-induced changes in DHRmax. In Experiment 1, the overall ANOVA result was: (F(5,44) ¼ 1.65); in Experiment 2 the corresponding ANOVA result was (F(2,15) ¼ 0.002) (Figures 2–4).
Discussion The present investigation shows that (1) oxytocin enhances the pressor response to stress in WKY rats and reduces this response in the SHR strain and that (2) the augmentation of the pressor response to the stressor by centrally applied oxytocin in WKY rats results from interaction of this peptide with central V1a receptors. Our results do not provide evidence for a significant role of oxytocin in the maintenance of resting blood pressure but the results show that this peptide significantly, though differently, regulates blood pressure responses to stress in WKY and SHR rats. We demonstrated
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Figure 3. Maximum increases in mean arterial blood pressure (DMABP max) (A) and heart rate (DHRmax) (B) from baseline after application of the air-jet stressor in WKY and spontaneously hypertensive rats (SHR) of Experiment 1 during intracerebroventricular (ICV) infusions of 0.9% NaCl (vehicle), oxytocin and oxytocin receptor (OTR) antagonist. Means standard errors of mean are shown. ANOVA followed by Tukey test *p50.05, ***p50.001. WKY-vehicle n ¼ 8; SHR-vehicle n ¼ 8; WKY-oxytocin n ¼ 7; SHR-oxytocin n ¼ 10; WKYOTR antagonist n ¼ 7; SHR-OTR antagonist n ¼ 10.
that centrally applied exogenous oxytocin buffers the pressor response to stress in the SHR strain by means of the OTR and unexpectedly, exaggerates the pressor reaction to stress in normotensive WKY rats via an interaction with vasopressin V1a receptors. Our results indicate that in WKY rats endogenously released oxytocin plays a significant role in the regulation of the pressor response to an emotional stressor by means of both OTRs and V1aRs and that the buffering effect exerted by OTRs dominates over the exaggerating effect mediated by V1aRs. In addition, the present study confirms previous reports showing that during the administration of ICV vehicle the SHR strain respond with significantly greater increases of blood pressure to stress than WKY rats. Most likely this effect results from significantly greater activity of the sympathetic system in SHR rats (Krieger et al., 1999). Oxytocin and regulation of blood pressure under resting conditions Our results do not provide evidence for an involvement of centrally released oxytocin in the regulation of the resting blood pressure in undisturbed WKY or SHR rats. This is indicated by a lack of changes in resting MABP and HR after ICV application of OTR antagonist or oxytocin. In this respect our results are similar to those obtained in Sprague Dawley rats (Wsol et al., 2009). However, it should be noted that in
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Figure 4. Maximum increases in mean arterial blood pressure (DMABPmax) (A) and heart rate (DHRmax) (B) from baseline after application of air-jet stress in WKY rats of Experiment 2 after pretreatment with vasopressin V1a receptor antagonist – V1aR antagonist and subsequently during ICV infusions of 0.9% NaCl (vehicle), oxytocin and oxytocin receptor antagonist (OTR antagonist). Means standard errors of mean are shown. ANOVA followed by Tukey test *p50.05, ***p50.001. V1aR antagonist þ vehicle n ¼ 6; V1aR antagonist þ oxytocin n ¼ 6; V1aR antagonist þ OTR antagonist n ¼ 6.
the present study, the rats were exposed to ICV infusion of oxytocin at a rate of 100 ng/h for 50 min. Other authors reported that repeated ICV injections of oxytocin at 300 ng and 1 mg/kg body weight elicit prolonged hypotension (Petersson et al., 1996; Petersson & Uvna¨s-Moberg, 2007); this hypotensive effect was found 1 d after the first injection of oxytocin (Petersson et al., 1996). Based on these studies, it may be speculated that the central hypotensive effect of oxytocin requires involvement of some indirect, long-lasting steps, such as inhibition or activation of synthesis of some other neuroactive substances (for instance reduced synthesis of vasopressin or increased synthesis of nitric oxide). Another possibility would be reorganisation of neuronal networks, such as decreased synthesis of V1a receptors or reduced synaptic contacts on pressor cardiovascular neurones. Further studies are needed to address these questions. Oxytocin and regulation of blood pressure responses to the stressor in WKY rats When the effect of ICV administration of oxytocin on the cardiovascular reaction to the air-jet stressor in normotensive
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WKY rats was tested, we obtained results that were opposite to our expectations. Namely, WKY rats responded to the sudden stressor with significantly greater increases of blood pressure during infusion of oxytocin than during infusion of vehicle. Previously, increased pressor and tachycardic responses to acute stress (alarming noise) were found in normotensive Sprague-Dawley rats after repeated ICV administrations of oxytocin (300 ng) for 5 d by Petersson & Uvna¨sMoberg (2007). However, a stressor-induced increase of blood pressure was found during the hypotension produced by the prior administration of oxytocin, and the blood pressure level during the exposure to the stressor did not exceed the resting blood pressure level, recorded before injections of oxytocin. The present results differ from those obtained in our previous investigation (Wsol et al., 2009), in which ICV application of the same dose of oxytocin in Sprague Dawley rats in a similar experimental design did not have significant influence on the magnitude of the pressor response to the stressor, whereas administration of OTR antagonist significantly potentiated this response. The previous study indicated that endogenous oxytocin buffers the pressor responses to a stressor in Sprague Dawley rats. Evidently, oxytocin regulates the pressor responses to stress differently in WKY and Sprague Dawley rats. An analysis of the preliminary data of Experiment 1 led us to test the hypothesis that the enhancement of the pressor response to the alarming stressor in WKY rats is caused by binding of oxytocin to V1a receptors. The structure of oxytocin and vasopressin molecules and the mechanism of activation of V1a and OTRs are similar (Barberis et al., 1998; Gimpl & Fahrenholz, 2001; Zingg & Laporte, 2003). It has been shown that OTRs bind oxytocin and vasopressin with similar affinity (Akerlund et al., 1999; Gimpl et al., 2008). The results of Experiment 2 supported the hypothesis. In particular, the data presented in Figure 4(A) show that administration of oxytocin after prior blockade of central V1a receptors completely abolishes the blood pressure increase that normally occurs after application of the air-jet stressor. To determine whether endogenous oxytocin plays a significant role in the regulation of the pressor response to stress in otherwise undisturbed WKY rats, we measured the magnitude of the pressor response to the stressor during simultaneous blockade of OTR and V1aR. A comparison of the results presented in Figure 4(A) shows that the pressor responses to the stressor after combined blockade of V1aR and OTR and after administration of vehicle significantly differ; specifically, the pressor response to the stressor was significantly higher after the combined blockade of these receptors. This finding indicates that endogenous oxytocin plays a significant role in the regulation of blood pressure responses to an emotional stressor and that its blood pressure elevation buffering effect via OTR receptors slightly, but significantly, dominates over the blood pressure enhancing effect exerted by V1aR receptors. Oxytocin and regulation of blood pressure responses to the stressor in SHR rats Our study shows that central administration of oxytocin significantly reduced the pressor response to the stressor in
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SHR and essentially abolished the difference between WKY and SHR rats that was evident during administration of the vehicle. The central administration of OTR antagonist did not have a significant effect on the pressor response to stress in the SHR strain. The latter finding indicates that endogenously released oxytocin does not play a significant role in the regulation of blood pressure responses to an emotional stressor in the SHR strain. In this respect, the present results are similar to those obtained previously in Sprague Dawley rats with a myocardial infarction, in which central administration of oxytocin reduced pressor responses to a stressor while blockade of OTRs did not significantly affect cardiovascular responses to the stressor (Wsol et al., 2009). At the same time, a significant reduction of the pressor response to the stressor during ICV administration of oxytocin indicates that in the SHR strain the central OTRs respond effectively to exogenous oxytocin. Thus, it can be speculated that the relative ineffectiveness of endogenous oxytocin in the regulation of the pressor responses to the alarming stressor in the SHR strain results from an inappropriate release of oxytocin in the SHR brain. This possibility is supported by previous studies of Gaida et al. (1985) and Martins et al. (2005) who found a significantly lower content of oxytocin in the hypothalamus and brain stem and reduced expression of oxytocin mRNA in the PVN and dorsal brain stem of SHR in comparison to WKY rats. Possible interaction of oxytocin with central a-2 adrenergic receptors should also be taken into consideration among the factors responsible for different pressor responses to a stressor in WKY and SHR rats. In the SHR strain, subchronic treatment with oxytocin was found to intensify the hypotensive response to centrally administered clonidine, which interacts with a-2 adrenergic receptors (Petersson et al., 1999). Oxytocin treatment also increased the density of high affinity a-2 adrenergic receptors (Petersson et al., 2005). Thus, it is possible that a-2 adrenergic receptors could play some role in the mechanism of reduction of the pressor response to stress that was observed in the present study in SHR rats during ICV administration of oxytocin. Specificity of antagonists used and possible involvement of other receptors The OTR antagonist ([DesGly-NH2-d(CH2)5[D-Tyr2,Thr4] OVT), used in our previous study (Wsol et al., 2009), was chosen on the basis of data showing its greater affinity to OTR (pA2 ¼ 7.37) than to V1R and V2R (pA2 ¼ 5.39, pA255.5, respectively) (Manning et al., 1995). In the present experiments, the highly-selective non-peptide V1aR antagonist SSR 49059 (pA2 ¼ 9.2) was used according to recommendations for rat experimental models (Manning et al., 2012). It corresponds to d(CH2)5[Tyr(Me)2]AVP (Manning compound), which was described as a molecule with high affinity, selectivity and efficacy for the rat V1aR. However, SSR 49059 displays low affinity to OTR (rat mammary gland: Ki 1080 115 nM) and to V1bR (Serradeil-Le Gal et al., 1993). Based on these results it can be assumed that oxytocin and vasopressin receptors were selectively blocked by the antagonists used in the present experiments. Furthermore, the present study and our previous studies showing importance of central V1aR for the regulation of pressor responses to
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stress (Cudnoch-Jedrzejewska et al., 2007, 2010; Dobruch et al., 2005; Wsol et al., 2009), strongly indicate that oxytocin may regulate pressor responses by means of both central OTR and V1aR and the resultant pressor response probably depends on the proportions of activated receptors. Interestingly, suckling, a well-known stimulus for the release of oxytocin (Neumann et al., 1996; Schafer & MacKenzie, 1911), appears to intensify the pressor responses to stress. For instance, exaggeration of cardiovascular responses to different stressors was reported in breastfeeding women (cold pressor, mental arithmetic) and in lactating Wistar rats (enhanced tachycardic responses during immobilization) (Mezzacappa et al., 2001, 2003). The V1aR has a similar structure to the V1bR, which is present mainly in the anterior pituitary but also in the brain (Hernando et al., 2001). The role of V1bR in the regulation of blood pressure was not addressed in the present investigation. Thus far, the role of V1bR in the regulation of blood pressure has not been recognized. However, a significantly lower expression of V1bR mRNA in the pontine brain region in renin transgenic (TGR (mRen2)27) rats in comparison to normotensive Sprague Dawley rats (Gozdz et al., 2003) indicates that V1bR may participate in this regulation. Thus, at present, the involvement of V1bR in the regulation of pressor responses to stress in WKY or SHR rats cannot be excluded.
Summary In conclusion, the present study demonstrates that SHR respond with significantly greater increases of blood pressure to an alarming stressor than Wistar Kyoto rats. Exogenous oxytocin acts in an opposite way in the regulation of blood pressure in response to this stressor in WKY and SHR rats, evoking exaggeration of the pressor response in WKY and reduction in the SHR strain. The results provide evidence that these differences may at least partly result from interaction of oxytocin with V1aR in WKY rats. The results indicate that endogenous oxytocin regulates the magnitude of the pressor response to stress in WKY rats by simultaneous interaction with OTR and V1aR.
Acknowledgements The authors would like to express their gratitude to Mrs. Marzanna Tkaczyk for her technical assistance and to Mr. Marcin Kumosa for the preparation of illustrations.
Declaration of interest The authors report no financial or other conflict of interest relevant to the subject of this publication. The study was supported by a grant from the National Science Centre (N N401 191439) and the Medical University of Warsaw (grant 1MA/ 2010-2013).
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ORIGINAL RESEARCH REPORT
Oxytocin differently regulates pressor responses to stress in WKY and SHR rats: the role of central oxytocin and V1a receptors A. Wsol, E. Szczepanska-Sadowska, S. Kowalewski, L. Puchalska, and A. Cudnoch-Jedrzejewska
Abstract
Keywords
The role of central oxytocin in the regulation of cardiovascular parameters under resting conditions and during acute stress was investigated in male normotensive Wistar-Kyoto (WKY; n ¼ 40) and spontaneously hypertensive rats (SHR; n ¼ 28). In Experiment 1, mean arterial blood pressure (MABP) and heart rate (HR) were recorded in WKY and SHR rats at rest and after an airjet stressor during intracerebroventricular (ICV) infusions of vehicle, oxytocin or oxytocin receptor (OTR) antagonist. In Experiment 2, the effects of vehicle, oxytocin and OTR antagonist were determined in WKY rats after prior administration of a V1a vasopressin receptor (V1aR) antagonist. Resting MABP and HR were not affected by any of the ICV infusions either in WKY or in SHR rats. In control experiments (vehicle), the pressor response to stress was significantly higher in SHR. Oxytocin enhanced the pressor response to stress in the WKY rats but reduced it in SHR. During V1aR blockade, oxytocin infusion entirely abolished the pressor response to stress in WKY rats. Combined blockade of V1aR and OTR elicited a significantly greater MABP response to stress than infusion of V1a antagonist and vehicle. This study reveals significant differences in the regulation of blood pressure in WKY and SHR rats during alarming stress. Specifically, the augmentation of the pressor response to stress by exogenous oxytocin in WKY rats is caused by its interaction with V1aR, and endogenous oxytocin regulates the magnitude of the pressor response to stress in WKY rats by simultaneous interaction with OTR and V1aR.
Air-jet stress, blood pressure, hypertension, neuropeptides, oxytocin antagonist, vasopressin antagonist
Introduction Numerous lines of evidence indicate that the hypothalamic neuropeptide oxytocin known from its role in parturition, lactation and formation of maternal behavior (Lee et al., 2009) plays multiple roles in the regulation of the cardiovascular system. Hence, administration of oxytocin may cause hypotension, natriuresis and negative chronotropic and inotropic effects (Feuerstein et al., 1984; Gutkowska et al., 1997; Gutkowska & Jankowski, 2012; Petersson et al., 1996). The anti-inflammatory and cardioprotective role of this peptide in ischemia-reperfusion-induced myocardial injury has been emphasized (Alizadeh & Mirzabeglo, 2013; Authier et al., 2010). There is also evidence that oxytocin may play a buffering role in behavioral and humoral reactions to stress. Experimental procedures employing acute and chronic stress paradigms have revealed elevated oxytocin concentration in the paraventricular nucleus (PVN) and blood (Grippo et al., 2007; Nishioka et al., 1998). In addition, oxytocin reduces behavioral responses to chronic immobilization or isolation (Viviani & Stoop, 2008; Windle et al., 2006). Furthermore, several reports strongly suggest that oxytocin reduces anxiety Correspondence: A. Wsol, Department of Experimental and Clinical Physiology, Medical University of Warsaw, Pawinskiego 3C, 02-106 Warsaw, Poland. Tel: +0048-22-572-07-08. Fax: +0048-22-572-07-82. E-mail: [email protected]
History Received 8 August 2013 Revised 27 November 2013 Accepted 3 December 2013 Published online 19 December 2013
and exerts an antidepressive effect (Blume et al., 2008; Engelmann et al., 1999; Neumann & Landgraf, 2012; Windle et al., 1997), and the expression of oxytocin receptors (OTRs) has been found in different structures of the limbic system that play an important role in stress, anxiety and depression (Bao et al., 2008; Barberis & Tribollet, 1996; Boccia et al., 2013). Thus far, the role of centrally released oxytocin in the regulation of blood pressure during stress is unclear. Neuroanatomical studies revealed the presence of oxytocin and oxytocinergic receptors (OTRs) in the regions of the lower brain stem involved in the regulation of cardiovascular responses to stress (Barberis & Tribollet, 1996, Boccia et al., 2013, Sofroniew, 1985). In the study by Petersson & Uvna¨sMoberg (2007), repeated intracerebroventricular (ICV) administration of oxytocin reduced the baseline blood pressure in rats, however the oxytocin-treated animals responded with blood pressure and heart rate (HR) increases to unexpected noise. The latter response was absent in control rats (Petersson & Uvna¨s-Moberg, 2007). In our previous study, blockade of the brain OTR was found to exaggerate significantly the pressor response to an alarming (air jet) stressor and it was concluded that endogenous oxytocin buffers the pressor responses to stress (Wsol et al., 2009). We also showed that the buffering role of oxytocin in the regulation of blood pressure during stress is impaired
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Department of Experimental and Clinical Physiology, Medical University of Warsaw, Warsaw, Poland
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in Sprague Dawley rats with a myocardial infarction. Other studies revealed that arterial hypertension is associated with some abnormalities in the central oxytocinergic system (Gaida et al., 1985; van Tol et al., 1988). Namely, Gaida et al. (1985) found significantly reduced content of oxytocin in the hypothalamus, mid-brain and brain stem of stroke prone spontaneously hypertensive rats (SHR-SP). The same group did not find significant differences in oxytocin content in the neuro-intermediate lobe of the pituitary between SHR-SP and WKY rats. van Tol et al. (1988) showed that SHR manifest lower expression of oxytocin mRNA in the hypothalamic paraventricular and supraoptic nuclei, as well as in the posterior pituitary. The above findings implied the possibility that the abnormal content of oxytocin and OTR in the SHR brain may have an effect on the central regulation of blood pressure in this strain. To date the role of oxytocin in the regulation of blood pressure in hypertension was assessed only in the experimental model of neurogenic hypertension evoked by electrical stimulation of the mesencephalic reticular formation (Versteeg et al., 1983). In this model, the administration of oxytocin into the fourth cerebral ventricle significantly reduced blood pressure elevation. The role of oxytocin in the regulation of blood pressure in the SHR model of spontaneously developing hypertension has not been investigated previously. Based on the literature cited above, we hypothesized that the OTRs in the brain play an essential role in the regulation of blood pressure under baseline conditions and during acute alarming stress in normotensive Wistar-Kyoto (WKY) and SHR rats. We expected that oxytocin may be an essential hypotensive agent, effectively buffering cardiovascular responses to this acute stressor both in WKY and in SHR rats. After an analysis of our preliminary data, we tested whether some of the cardiovascular effects of oxytocin are caused by its interaction with V1a receptors. The rationale for this was based on previous findings showing that oxytocin stimulates both OTR and vasopressin V1a receptors (V1aR) (Akerlund et al., 1999; Gimpl et al., 2008). To find out whether endogenous oxytocin plays an essential role in the regulation of pressor responses to stress we determined whether the magnitude of cardiovascular responses to stress in WKY rats can be affected by simultaneous blockade of central oxytocin and vasopressin V1a receptors.
Methods Animals Adult male SHR and their parent normotensive strain WKY rats weighing 250–300 g were bred in the Department of Animal Breeding of the Medical University of Warsaw. The rats were housed three per cage at a temperature of 22–25 C with 12 h:12 h light–dark rhythm (lights on between 07.00 h and 19:00 h). They were provided with a rodent dry pellet diet containing 0.45% of NaCl and had free access to tap water. 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 Animal Research of the Warsaw Medical University.
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Surgical procedures The rats entered the study at the age 10–12 weeks. They were implanted with a guide tube into the left cerebral ventricle for the ICV infusions and an aortic catheter for recording the hemodynamic parameters. Each of the surgical procedures was performed under general anesthesia with pentobarbital sodium (Pentobarbital, Biowet, Puławy, Poland; 15 mg/ml, 50 mg/kg body wt, 20 mmol/100 g body wt., i.p.), which was followed by administration of antibiotic (Penicillin, Polfa, Pabianice, Poland; 30,000 U in 1 ml/rat, i.m). After each surgical procedure, the rats were housed singly. After full recovery from anesthesia they were returned to the animal house. Implantation of ICV cannula The implantation of the ICV cannula was performed according to the procedure described previously (Dobruch et al., 2005). The rat’s head was placed in a stereotaxic device (Kopff) and the stainless steel cannula (o.d., 0.81 mm, MIFAM S.A., Milano´wek, Poland) was implanted into the left cerebral ventricle according to the following stereotaxic coordinates: 1.3 mm posterior to bregma, 2 mm lateral to the midline and 4.5 mm below the surface of the skull. The cannula was secured on the external surface of the skull with acrylic cement (Duracryl, SPOFA-DENTAL, Jicin, Czech Republic), and closed with a stainless steel stylet (o.d., 0.46 mm). Implantation of the arterial catheter Seven days after implantation of the ICV cannula the arterial catheter was inserted into the abdominal aorta through the femoral artery. The catheter consisted of the intra-arterial (3.5–4.0 cm long; i.d., 0.12 mm; o.d., 0.25 mm) and external (i.d., 0.25 mm; o.d., 0.4 mm) portions made from polyvinyl tubing (Scientific Commodities, Inc., Lake Havasu City, AZ). The extravascular part of the catheter was exteriorized on the rat’s neck in the interscapular region. The catheter was filled with 0.9% NaCl containing 500 IU of heparin/ml and closed with a plastic stopper. Experimental protocol The experiments were performed 24–48 h after the implantation of the arterial catheter between 10:00 h and 18:00 h. At the beginning of the experiment, the arterial catheter was connected to a BIOPAC MP100 system (BIOPAC, MP100, Santa Barbara, CA) for continuous recording of mean arterial blood pressure (MABP) and HR. The study was performed on 68 rats divided into two experiments. Experiment 1 was designed to compare the effects of ICV administration of vehicle, oxytocin and blockade of OTR on the cardiovascular parameters in the WKY and SHR strains. The protocol of this experiment is shown in Figure 1(A). In each experimental session the ICV infusion was started after a 15- to 30-min period of stabilization of resting cardiovascular parameters (MABP and HR), at a rate of 5 ml/h (0.083 ml/min) by means of a microsyringe pump (Harvard 22 Infusion Pump, Harvard Smith, Natick, MA). The measurements of MABP and HR
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Figure 1. The sequence of experimental procedures in Experiment 1 (oxytocin and OTR antagonist experiments) (A) and Experiment 2 (oxytocin and V1aR antagonist experiments) (B). ICV: intracerebroventricular, MABP: mean arterial blood pressure, HR: heart rate, OTR: oxytocin receptor, V1aR: vasopressin V1a receptor.
were continued for 50 min under resting conditions and for 10 min after application of the air jet, which was used as the alarming stressor. The air-jet stressor was applied by directing the air stream onto the top of the rat’s head for 1–1.5 s from a tank containing compressed air (10 atm) according to the modified technique of Zhang et al. (1999). In each rat the air-jet stressor was applied only once in order to avoid adaptation. The cardiovascular responses to acute stress during the first 10 s after the application of the air jet were evaluated by measuring the maximum increases in MABP and HR from the level immediately preceding the application. Rats undergoing Experiment 1 were divided into six experimental groups. Groups 1.1 (WKY-vehicle; n ¼ 8) and 1.2 (SHR-vehicle; n ¼ 8) received ICV infusion of 0.9% NaCl as the vehicle (5 ml/h). Groups 1.3 (WKY-oxytocin; n ¼ 7) and 1.4 (SHR-oxytocin; n ¼ 10) were infused with vehicle for the first 10 min and subsequently with oxytocin. Groups 1.5 (WKY-OTR antagonist n ¼ 7) and 1.6 (SHR-OTR antagonist; n ¼ 10) received infusion of vehicle for 10 min. This was followed by ICV infusion of OTR antagonist. Experiment 2 was performed to determine whether the regulation of the cardiovascular responses to stress by oxytocin is influenced by its interaction with V1a receptors. Experiment 2 was performed on 18 WKY rats divided into three groups. The sequence of the experimental procedures is shown in Figure 1(B). After stabilization of the cardiovascular parameters Group 2.1 (V1aR antagonist þ vehicle; n ¼ 6) received ICV infusion of V1aR antagonist in vehicle for 15 min. This was followed by ICV infusion of vehicle for 60 min. The air-jet stressor was applied 50 min after the start of infusion of the vehicle. The experimental design for Group 2.2 (V1aR antagonist þ oxytocin; n ¼ 6) was similar as
that of Group 2.1 except the vehicle was infused together with oxytocin. Group 2.3 (V1aR antagonist þ OTR antagonist; n ¼ 6) received ICV infusion of OTR antagonist together with the vehicle. Ligands used in the study Oxytocin (CysTyrIleGlnAsnCysProLeuGly-NH2) was purchased from Phoenix Pharmaceuticals (Strasbourg, France) and was infused at 100 ng/5 ml/h (1.66 pmole/0.083 ml/min). The OTR antagonist [DesGly-NH2-d(CH2)5[D-Tyr2,Thr4] OVT was supplied by Prof. Morris Manning, University of Toledo, Ohio (Manning et al., 1995). OTR antagonist was infused at 4.3 mg/5 ml/h (71.6 nmole/0.083 ml/min). The doses of oxytocin and OTR antagonist were established previously (Wsol et al., 2009). Vasopressin V1aR non-peptide antagonist (SSR49059) was provided by Sanofi Aventis (Montpellier, France) and was infused at 2 mg/40 ml/h (0.052 nmole/0.67 ml/ min) (Serradeil-Le Gal et al., 1993). The dose of V1aR antagonist was established in a preliminary series of experiments. Postmortem examination At the end of the experiment, the rats were euthanized by an overdose of pentobarbital (30 mg/ml, 100 mg/kg body wt., 40 mmol/100 g body wt., i.p.). The heart and the brain were excised for postmortem examination. The left cardiac ventricle (including the septum) was separated from the right ventricle and the weight of both ventricles was determined. Evans blue (5 ml) was injected through the guide tube into the ICV system and the position of the tip of the cannula in the brain was verified by visual inspection.
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Table 1. Mean arterial blood pressure (MABP) and heart rate (HR) at the beginning of experiment (0 min) and before application of the air-jet stressor (50 min) in WKY and SHR rats during ICV infusions.
Experimental groups WKY-vehicle Group 1.1 (n ¼ 8) SHR-vehicle Group 1.2 (n ¼ 8) WKY-oxytocin Group 1.3 (n ¼ 7) SHR-oxytocin Group 1.4 (n ¼ 10) WKY-OTR antagonist Group 1.5 (n ¼ 7) SHR-OTR antagonist Group 1.6 (n ¼ 10) WKY-V1aR antagonist þ vehicle Group 2.1 (n ¼ 6) WKY-V1aR antagonist þ oxytocin Group 2.2 (n ¼ 6) WKY-V1aR antagonist þ OTR antagonist Group 2.3 (n ¼ 6)
Body mass (g)
MABP (0 min) (mm Hg)
MABP (50 min) (mm Hg)*
HR (0 min) (bpm)
HR (50 min) (bpm)*
298 12.1 310 8.5 289 3.4 292 5,4 308 15.5 294 14.6 307 8.7 292 9.6 294.2 9.0
102 3.4 156 5.0# 112 3.7 165 4.6# 112 4.9 155 4.6# 124 2.0 127 1.3 118 1.6
106 3.0 155 5.3# 112 4.0 165 4.6# 113 6 153 4.6# 138 2.2 128 2.2 139 2.6
358 8.2 397 13.4 364 20.3 381 14.1 354 3.8 422 13.1 411 21 383 18 340 16
356 9.9 412 16.9 364 17.3 371 18.3 353 14.7 388 18.7 378 21 350 21 306 13
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HR: heart rate; MABP: mean arterial blood pressure; OTR: oxytocin receptor; V1aR: vasopressin V1a receptor; WKY: Wistar-Kyoto rats; SHR: spontaneously hypertensive rats; n: number of rats; bpm: beats per min. Means SEM are shown. *Fifty minutes after the beginning of infusions of oxytocin or OTR antagonist MABP and HR did not differ from values under baseline conditions (i.e. before the onset of ICV infusion) (ANOVA followed by the Tukey test). #Significant difference between SHR group and the corresponding WKY group (ANOVA followed by the Tukey test).
Statistical analysis STATISTICA software (Version 9.1, Chicago, IL) was used for statistical analysis of the data. Five-minute averages of MABP and HR were entered into the statistical analysis to assess changes in cardiovascular parameters during the resting period. Normal distribution of the data was established by means of the Shapiro–Wilk test. As no significant dispersion of the data from normality was found, ANOVA was applied. Two-way ANOVA on repeated measurements was used to evaluate changes in cardiovascular parameters at rest as recommended by Curran-Everett & Benos (2004) and Ludbrook (1994). One-way ANOVA was employed to determine the significance of differences between the maximum changes of MABP and HR evoked by the air-jet stressor in different groups of experiments. Horizontal and vertical multiple pair-wise comparisons were performed using the post-hoc Tukey test to determine the significance of differences between individual groups of experiments. The differences were considered significant if p50.05. All values presented in the text, figures and table are means standard error of the mean (SEM).
Results The resting MABP was significantly higher in SHR than in WKY rats (F(5,44) ¼ 38.61; p50.001) (means of individual groups are shown in Table 1), while resting values of HR were similar in all experimental groups. The left ventricle weight was significantly higher in SHR (Experiment 1: 0.281 0.004 g/100 g body weight) than in the normotensive rats (Experiment 1: 0.245 0.004 g/100 g body weight) (F(1,51) ¼ 38.549; p50.001). The right ventricle weight was lower in SHR (Experiment 1: 0.06 0.002 g/100 g body weight) than in WKY rats (Experiment 1: 0.068 0.002 g/100 g body weight) (F(1,51) ¼ 8.567; p50.01). Total body weights were similar in all experimental groups (Table 1). Resting MABP values during ICV infusions The resting MABP values were not significantly affected by ICV infusions for 50 min of 0.9% NaCl, oxytocin or
OTR antagonist either in WKY or in SHR rats (Table 1, time fluctuations not shown). Changes in MABP elicited by air-jet stressor in Experiment 1 In Experiment 1, an analysis of differences in air-jet stressorinduced DMABPmax revealed a significant difference between WKY and SHR rats (F(5,44) ¼ 6.91; p50.001). Significant differences between experimental groups were also found when DMABPmax was compared separately in WKY (F(2,19) ¼ 5.20; p50.05) and SHR rats (F(2,25) ¼ 11.63; p50.001). Figure 2 shows typical changes of MABP and HR in individual experiments after the application of the air jet in the different experiments. As shown in Figure 3(A) the response to the air-jet stressor in rats receiving ICV infusion of vehicle was significantly higher in SHR rats (p50.05; Tukey test). ICV infusion of oxytocin reduced the pressor response to the air-jet stressor only in SHR rats. The stressor-induced DMABPmax was significantly lower during ICV infusion of oxytocin (SHR-vehicle versus SHR-oxytocin: p50.05; Tukey test). In contrast, in WKY rats, the DMABPmax response to stress was significantly higher during ICV infusion of oxytocin than during infusion of the vehicle (WKY-vehicle versus WKY-oxytocin, p50.05; Tukey test). Changes in MABP elicited by air-jet stressor in Experiment 2 Significant differences in DMABPmax responses to stress were found between groups of Experiment 2 (F(2,15) ¼ 34.49; p50.001). As shown in Figure 4(A) a posteriori analysis revealed that combined administration of V1aR antagonist and oxytocin (Group 2.2) completely abolished the air-jet stressor-induced blood pressure elevation (V1aR antagonist þ vehicle versus V1aR antagonist þ oxytocin: p50.001; Tukey test). Significant differences were also found between the maximum increases in DMABPmax in Groups 2.1 (V1aR antagonist þ vehicle) and 2.3 (V1aR antagonist þ OTR antagonist: p50.05; Tukey test) and between the Groups 2.2 (V1aR antagonist þ oxytocin) and
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Figure 2. Typical recordings of mean arterial blood pressure (MABP) and heart rate (HR) fluctuations in (A) spontaneously hypertensive rats (SHR) and (B) WKY in response to the air-jet stressor during intracerebroventricular (ICV) infusions of 0.9% NaCl (vehicle), oxytocin, oxytocin receptor antagonist (OTR antagonist), V1a receptor antagonist (V1aR antagonist) and combined infusion of oxytocin and V1aR antagonist.
2.3 (V1aR antagonist þ OTR; (Figure 4A).
p50.001;
Tukey
test)
HR at rest and after application of air-jet stressor The resting HR was neither significantly affected by any of the ICV infusions at 50 min after the beginning of infusion (Table 1, time fluctuations not shown), nor were there significant differences between the stress-induced changes in DHRmax. In Experiment 1, the overall ANOVA result was: (F(5,44) ¼ 1.65); in Experiment 2 the corresponding ANOVA result was (F(2,15) ¼ 0.002) (Figures 2–4).
Discussion The present investigation shows that (1) oxytocin enhances the pressor response to stress in WKY rats and reduces this response in the SHR strain and that (2) the augmentation of the pressor response to the stressor by centrally applied oxytocin in WKY rats results from interaction of this peptide with central V1a receptors. Our results do not provide evidence for a significant role of oxytocin in the maintenance of resting blood pressure but the results show that this peptide significantly, though differently, regulates blood pressure responses to stress in WKY and SHR rats. We demonstrated
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Figure 3. Maximum increases in mean arterial blood pressure (DMABP max) (A) and heart rate (DHRmax) (B) from baseline after application of the air-jet stressor in WKY and spontaneously hypertensive rats (SHR) of Experiment 1 during intracerebroventricular (ICV) infusions of 0.9% NaCl (vehicle), oxytocin and oxytocin receptor (OTR) antagonist. Means standard errors of mean are shown. ANOVA followed by Tukey test *p50.05, ***p50.001. WKY-vehicle n ¼ 8; SHR-vehicle n ¼ 8; WKY-oxytocin n ¼ 7; SHR-oxytocin n ¼ 10; WKYOTR antagonist n ¼ 7; SHR-OTR antagonist n ¼ 10.
that centrally applied exogenous oxytocin buffers the pressor response to stress in the SHR strain by means of the OTR and unexpectedly, exaggerates the pressor reaction to stress in normotensive WKY rats via an interaction with vasopressin V1a receptors. Our results indicate that in WKY rats endogenously released oxytocin plays a significant role in the regulation of the pressor response to an emotional stressor by means of both OTRs and V1aRs and that the buffering effect exerted by OTRs dominates over the exaggerating effect mediated by V1aRs. In addition, the present study confirms previous reports showing that during the administration of ICV vehicle the SHR strain respond with significantly greater increases of blood pressure to stress than WKY rats. Most likely this effect results from significantly greater activity of the sympathetic system in SHR rats (Krieger et al., 1999). Oxytocin and regulation of blood pressure under resting conditions Our results do not provide evidence for an involvement of centrally released oxytocin in the regulation of the resting blood pressure in undisturbed WKY or SHR rats. This is indicated by a lack of changes in resting MABP and HR after ICV application of OTR antagonist or oxytocin. In this respect our results are similar to those obtained in Sprague Dawley rats (Wsol et al., 2009). However, it should be noted that in
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Figure 4. Maximum increases in mean arterial blood pressure (DMABPmax) (A) and heart rate (DHRmax) (B) from baseline after application of air-jet stress in WKY rats of Experiment 2 after pretreatment with vasopressin V1a receptor antagonist – V1aR antagonist and subsequently during ICV infusions of 0.9% NaCl (vehicle), oxytocin and oxytocin receptor antagonist (OTR antagonist). Means standard errors of mean are shown. ANOVA followed by Tukey test *p50.05, ***p50.001. V1aR antagonist þ vehicle n ¼ 6; V1aR antagonist þ oxytocin n ¼ 6; V1aR antagonist þ OTR antagonist n ¼ 6.
the present study, the rats were exposed to ICV infusion of oxytocin at a rate of 100 ng/h for 50 min. Other authors reported that repeated ICV injections of oxytocin at 300 ng and 1 mg/kg body weight elicit prolonged hypotension (Petersson et al., 1996; Petersson & Uvna¨s-Moberg, 2007); this hypotensive effect was found 1 d after the first injection of oxytocin (Petersson et al., 1996). Based on these studies, it may be speculated that the central hypotensive effect of oxytocin requires involvement of some indirect, long-lasting steps, such as inhibition or activation of synthesis of some other neuroactive substances (for instance reduced synthesis of vasopressin or increased synthesis of nitric oxide). Another possibility would be reorganisation of neuronal networks, such as decreased synthesis of V1a receptors or reduced synaptic contacts on pressor cardiovascular neurones. Further studies are needed to address these questions. Oxytocin and regulation of blood pressure responses to the stressor in WKY rats When the effect of ICV administration of oxytocin on the cardiovascular reaction to the air-jet stressor in normotensive
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WKY rats was tested, we obtained results that were opposite to our expectations. Namely, WKY rats responded to the sudden stressor with significantly greater increases of blood pressure during infusion of oxytocin than during infusion of vehicle. Previously, increased pressor and tachycardic responses to acute stress (alarming noise) were found in normotensive Sprague-Dawley rats after repeated ICV administrations of oxytocin (300 ng) for 5 d by Petersson & Uvna¨sMoberg (2007). However, a stressor-induced increase of blood pressure was found during the hypotension produced by the prior administration of oxytocin, and the blood pressure level during the exposure to the stressor did not exceed the resting blood pressure level, recorded before injections of oxytocin. The present results differ from those obtained in our previous investigation (Wsol et al., 2009), in which ICV application of the same dose of oxytocin in Sprague Dawley rats in a similar experimental design did not have significant influence on the magnitude of the pressor response to the stressor, whereas administration of OTR antagonist significantly potentiated this response. The previous study indicated that endogenous oxytocin buffers the pressor responses to a stressor in Sprague Dawley rats. Evidently, oxytocin regulates the pressor responses to stress differently in WKY and Sprague Dawley rats. An analysis of the preliminary data of Experiment 1 led us to test the hypothesis that the enhancement of the pressor response to the alarming stressor in WKY rats is caused by binding of oxytocin to V1a receptors. The structure of oxytocin and vasopressin molecules and the mechanism of activation of V1a and OTRs are similar (Barberis et al., 1998; Gimpl & Fahrenholz, 2001; Zingg & Laporte, 2003). It has been shown that OTRs bind oxytocin and vasopressin with similar affinity (Akerlund et al., 1999; Gimpl et al., 2008). The results of Experiment 2 supported the hypothesis. In particular, the data presented in Figure 4(A) show that administration of oxytocin after prior blockade of central V1a receptors completely abolishes the blood pressure increase that normally occurs after application of the air-jet stressor. To determine whether endogenous oxytocin plays a significant role in the regulation of the pressor response to stress in otherwise undisturbed WKY rats, we measured the magnitude of the pressor response to the stressor during simultaneous blockade of OTR and V1aR. A comparison of the results presented in Figure 4(A) shows that the pressor responses to the stressor after combined blockade of V1aR and OTR and after administration of vehicle significantly differ; specifically, the pressor response to the stressor was significantly higher after the combined blockade of these receptors. This finding indicates that endogenous oxytocin plays a significant role in the regulation of blood pressure responses to an emotional stressor and that its blood pressure elevation buffering effect via OTR receptors slightly, but significantly, dominates over the blood pressure enhancing effect exerted by V1aR receptors. Oxytocin and regulation of blood pressure responses to the stressor in SHR rats Our study shows that central administration of oxytocin significantly reduced the pressor response to the stressor in
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SHR and essentially abolished the difference between WKY and SHR rats that was evident during administration of the vehicle. The central administration of OTR antagonist did not have a significant effect on the pressor response to stress in the SHR strain. The latter finding indicates that endogenously released oxytocin does not play a significant role in the regulation of blood pressure responses to an emotional stressor in the SHR strain. In this respect, the present results are similar to those obtained previously in Sprague Dawley rats with a myocardial infarction, in which central administration of oxytocin reduced pressor responses to a stressor while blockade of OTRs did not significantly affect cardiovascular responses to the stressor (Wsol et al., 2009). At the same time, a significant reduction of the pressor response to the stressor during ICV administration of oxytocin indicates that in the SHR strain the central OTRs respond effectively to exogenous oxytocin. Thus, it can be speculated that the relative ineffectiveness of endogenous oxytocin in the regulation of the pressor responses to the alarming stressor in the SHR strain results from an inappropriate release of oxytocin in the SHR brain. This possibility is supported by previous studies of Gaida et al. (1985) and Martins et al. (2005) who found a significantly lower content of oxytocin in the hypothalamus and brain stem and reduced expression of oxytocin mRNA in the PVN and dorsal brain stem of SHR in comparison to WKY rats. Possible interaction of oxytocin with central a-2 adrenergic receptors should also be taken into consideration among the factors responsible for different pressor responses to a stressor in WKY and SHR rats. In the SHR strain, subchronic treatment with oxytocin was found to intensify the hypotensive response to centrally administered clonidine, which interacts with a-2 adrenergic receptors (Petersson et al., 1999). Oxytocin treatment also increased the density of high affinity a-2 adrenergic receptors (Petersson et al., 2005). Thus, it is possible that a-2 adrenergic receptors could play some role in the mechanism of reduction of the pressor response to stress that was observed in the present study in SHR rats during ICV administration of oxytocin. Specificity of antagonists used and possible involvement of other receptors The OTR antagonist ([DesGly-NH2-d(CH2)5[D-Tyr2,Thr4] OVT), used in our previous study (Wsol et al., 2009), was chosen on the basis of data showing its greater affinity to OTR (pA2 ¼ 7.37) than to V1R and V2R (pA2 ¼ 5.39, pA255.5, respectively) (Manning et al., 1995). In the present experiments, the highly-selective non-peptide V1aR antagonist SSR 49059 (pA2 ¼ 9.2) was used according to recommendations for rat experimental models (Manning et al., 2012). It corresponds to d(CH2)5[Tyr(Me)2]AVP (Manning compound), which was described as a molecule with high affinity, selectivity and efficacy for the rat V1aR. However, SSR 49059 displays low affinity to OTR (rat mammary gland: Ki 1080 115 nM) and to V1bR (Serradeil-Le Gal et al., 1993). Based on these results it can be assumed that oxytocin and vasopressin receptors were selectively blocked by the antagonists used in the present experiments. Furthermore, the present study and our previous studies showing importance of central V1aR for the regulation of pressor responses to
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stress (Cudnoch-Jedrzejewska et al., 2007, 2010; Dobruch et al., 2005; Wsol et al., 2009), strongly indicate that oxytocin may regulate pressor responses by means of both central OTR and V1aR and the resultant pressor response probably depends on the proportions of activated receptors. Interestingly, suckling, a well-known stimulus for the release of oxytocin (Neumann et al., 1996; Schafer & MacKenzie, 1911), appears to intensify the pressor responses to stress. For instance, exaggeration of cardiovascular responses to different stressors was reported in breastfeeding women (cold pressor, mental arithmetic) and in lactating Wistar rats (enhanced tachycardic responses during immobilization) (Mezzacappa et al., 2001, 2003). The V1aR has a similar structure to the V1bR, which is present mainly in the anterior pituitary but also in the brain (Hernando et al., 2001). The role of V1bR in the regulation of blood pressure was not addressed in the present investigation. Thus far, the role of V1bR in the regulation of blood pressure has not been recognized. However, a significantly lower expression of V1bR mRNA in the pontine brain region in renin transgenic (TGR (mRen2)27) rats in comparison to normotensive Sprague Dawley rats (Gozdz et al., 2003) indicates that V1bR may participate in this regulation. Thus, at present, the involvement of V1bR in the regulation of pressor responses to stress in WKY or SHR rats cannot be excluded.
Summary In conclusion, the present study demonstrates that SHR respond with significantly greater increases of blood pressure to an alarming stressor than Wistar Kyoto rats. Exogenous oxytocin acts in an opposite way in the regulation of blood pressure in response to this stressor in WKY and SHR rats, evoking exaggeration of the pressor response in WKY and reduction in the SHR strain. The results provide evidence that these differences may at least partly result from interaction of oxytocin with V1aR in WKY rats. The results indicate that endogenous oxytocin regulates the magnitude of the pressor response to stress in WKY rats by simultaneous interaction with OTR and V1aR.
Acknowledgements The authors would like to express their gratitude to Mrs. Marzanna Tkaczyk for her technical assistance and to Mr. Marcin Kumosa for the preparation of illustrations.
Declaration of interest The authors report no financial or other conflict of interest relevant to the subject of this publication. The study was supported by a grant from the National Science Centre (N N401 191439) and the Medical University of Warsaw (grant 1MA/ 2010-2013).
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