Original article

P. SOWA1,2, M. ADAMCZYK-SOWA1,3, K. ZWIRSKA-KORCZALA1,
K. PIERZCHALA3, D. ADAMCZYK2, Z. PALUCH2, M. MISIOLEK2

PROOPIOMELANOCORTIN BUT NOT VASOPRESSIN OR RENIN-ANGIOTENSIN SYSTEM INDUCES RESUSCITATIVE EFFECTS OF CENTRAL 5-HT1A ACTIVATION IN HAEMORRHAGIC SHOCK IN RATS

1Department of Physiology, Medical University of Silesia, Zabrze, Poland; 2ENT Department, Medical University of Silesia, Zabrze, Poland; 3Department of Neurology, Medical University of Silesia, Zabrze, Poland
The aim of this study was to determine the effectory mechanisms: vasopressin, renin-angiotensin system and proopiomelanocortin-derived peptides (POMC), partaking in the effects of serotonin through central serotonin 1A receptor (5-HT1A) receptors in haemorrhagic shock in rats. The study was conducted on male Wistar rats. All experimental procedures were carried out under full anaesthesia. The principal experiment included a 2 hour observation period in haemorrhagic shock. Drugs used - a selective 5-HT1A agonist 8-OH-DPAT (5 µg/5 µl); V1a receptor antagonist [β-mercapto-β, β-cyclo-pentamethylenepropionyl1,O-me-Tyr2,Arg8]AVP (10 µg/kg); angiotensin type I receptor antagonist (AT1) ZD7155 (0.5 mg/kg, i.v.); angiotensin-converting-enzyme inhibitor captopril (30 mg/kg, i.v.); melanocortin type 4 (MC4) receptor antagonist HS014 (5 µg, i.c.v.). There was no influence of ZD715, captopril or blocking of the V1a receptors on changes in the heart rate (HR), mean arterial pressure (MAP), peripheral blood flow or resistance caused by the central stimulation of 5-HT1A receptors (Pł0.05). However, selective blocking of central MC4 receptors caused a slight, but significant decrease in HR and MAP (P<0.05). POMC derivatives acting via the central MC4 receptor participate in the resuscitative effects of 8-OH-DPAT. The angiotensin and vasopressin systems do not participate in these actions.
Key words:
hypovolaemia, shock, proopiomelanocortin-derived peptides, serotonin, 5-HT1A, 8-OH-DPAT, serotonin 1A receptor, hemorrhagic shock, vasopressin

INTRODUCTION

The total blood volume in the human body is a rather constant value amounting to approximately 7% of the total body mass (1). In physiological conditions this volume is regulated primarily by hormonal mechanisms, including the effects of arginine vasopressin, naturietic peptides, aldosterone, angiotensin II and III and adrenal cortex glucocorticoids. Moreover, the balance between the supply and loss of water has a significant influence on the total body blood volume (2).

The body's response to blood loss is two-phased. In the first phase, called the sympathoexcitatory phase, the blood pressure is maintained within the proper range. With further blood loss it comes to a severe drop in the arterial blood pressure and a decrease in the heart rate, which is the beginning of the second phase of the regulation of the circulatory system in haemorrhagic shock, called the sympathoinhibitory phase (3). Both phases of the circulatory regulation in haemorrhagic shock are connected with a considerable increase in angiotensin II and vasopressin concentration in the blood (4, 5). Vasopressin is a well known vasoconstrictive drug in the treatment of septic shock, and this effect is connected with a substantial decrease in the heart rate with a maintained cardiac output. Moreover, no significant hemodynamic differences between patients treated with vasopressin and noradrenaline have been observed (6). Furthermore, in clinical studies it was shown that vasopressin used together with catecholamines has a kidney protective function compared with treatment with catecholamines only, and has no impact on the mortality rate (7, 8). In an experimental study it was noticed that resuscitative effects similar to that of vasopressin are triggered by the stimulation of the serotonergic system. Tiniakov and Scrogin (9) have noted that the administration of 8-OH-DPAT produces a stronger resuscitative effect than the one achieved by vasopressin.

Although noradrenaline, other sympathomimetics and vasopressin are broadly used in clinical treatment of shock, some authors suggest, that the renin-angiotensin-aldosterone system inhibition seems to be more attractive (10). Laboratory experiments have shown that after the use of angiotensin II, AT1 and AT2 receptor antagonists and angiotensin-converting-enzyme inhibitor (ACE) a considerable decrease in blood pressure, dependent on the type of the blocked receptor was observed in hypovolaemia. It was noticed that this effect is mainly dependent on the AT1 receptor (11).

There is evidence for a close link of the effects of serotonin with the renin-angiotensin-aldesterone axis (12, 13), and therefore their interaction in the regulation of blood circulation. The precise mechanism of action, however, appears to be insufficiently understood.

In hypovolaemic animals, especially in critical hypovolaemia, it comes to an increase in the concentration of proopiomelanocortin derivatives - adrenocorticotropic hormone and endorphines in the blood (14, 15). Also, well known is the resuscitative effect of derivatives of proopiomelanocortin (16-19) in which melanocortin type 4 receptor is the mediator (20, 21).

The aim of the following study was to determine the effectory mechanisms, such as: vasopressin, renin-angiotensin system and proopiomelanocortin-derived peptides (POMC), partaking in the effects of serotonin through central 5-HT1A receptors in haemorrhagic shock in rats.

MATERIAL AND METHODS

The study was conducted on male Wistar rats, weighing in the range of 210–280 g. The animals were kept in metal cages, five per cage, under the 12 hour day/night cycle, water and food ad libitum. All experimental procedures were carried out under full anesthesia. The lateral ventricle cannulation was performed at 5–7 days before the main experiment, using ketamine (100 mg/kg) (Gedeon Richter, Budapest, Hungary) and ksylazine (10 mg/kg) (Research Biochemicals Inc., Natick, MA, USA) given intraperitoneally (i.p.). The 8 mm diameter cannule, after skin preparation, was placed in the skull according to Hilliard et al. (22), 4–4.5 mm deep, and fixed with acrylic glue. All substances administered intracerebroventricularly (i.c.v.) were in the volume of 5 µl using a Hamilton syringe (type Microliter # 702, Hamilton-Bonaduz, Switzerland).

On the day of the main experiment the animals were anesthetized using ethylurethane (Riedel-de Haen, Seelze, Germany) (1.25 g/kg; i.p.). After superficial tissue preparation the femoral artery and femoral vein were cannuled with catheters filled with heparinized 0.9% NaCl (300 IU/ml) (Polfa, Warsaw, Poland). In animals in which the peripheral blood flow was measured, the carotid communal artery and internal jugular vein were cannuled.

For the peripheral blood flow measurement, after previous laparotomy, the renal artery, upper mesenteric artery and the distal part of abdominal aorta were preparated.

Systolic, diastolic and mean arterial blood pressure (MAP) was measured using RMN-201 (Temed, Zabrze, Poland). Heart rate (HR) was automatically calculated as QRS rate in electrocardiography Diascope 2 (Unitra Biazet, Bialystok, Poland). Peripheral blood pressure was measured using electromagnetic sensors (1RB2006, Hugo Sachs Elektronik, March-Hugstetten, Germany) connected to Time Flowmeter Type 700 (Hugo Sachs Elektronik, March-Hugstetten, Germany).

Peripheral resistance was calculated as a ratio of MAP to peripheral flow in the renal artery (RBF) - renal vascular resistance (RVR), upper mesenteric artery (MBF) - mesenteric vascular resistance (MVR) and the distal part of the abdominal aorta (hindlimb blood flow - HBF) - hindlimb vascular resistance (HVR). The principal experiment included a 2 hour observation period after the stabilisation of cardiovascular parameters in haemorrhagic shock.

Haemorrhagic shock was evoked according to Guarini et al. (18-20, 23), by controlled bleeding from the femoral or internal jugular vein to a calibrated drain at maximal speed of 1 ml/min, over the period of 15–25 min, reaching and stabilizing MAP at between 20–25 mmHg. The haemorrhage protocol used has already been employed in the previous studies by our team (24, 25). The average blood volume required for the indication of critical hypovolaemia was about 2.19±0.27 ml/100 g body mass.

To examine the influence of the central serotonin 1A receptor (5-HT1A) in cardiovascular regulation under haemorrhagic shock conditions, a selective 5-HT1A agonist - 8-hydroxy-2-(di-n-propylamino)tetralin, 1-(2,5-dimethoxy-4-iodophenyl)-aminopropane (8-OH-DPAT) (Riedel-de Haen, Seelze, Germany) (5 µg/5 µl) was used i.c.v. Similarly to other authors and according to our previous study we used 8-OH-DPAT in concentration of 5 µg (24-26).

The role of vasopressin in the effect of 8-OH-DPAT was examined through the use of V1a receptor antagonist - [β-mercapto-β, β-cyclopentamethylenepropionyl1,O-me-Tyr2,Arg8]AVP administered intravenously in the dose of 10 µg/kg.

In order to determine the role of the renin-angiotensin system in the effects of centrally acting 8-OH-DPAT the peripheral blood flow and vascular resistance after the administration of angiotensin type I receptor antagonist (AT1) - ZD7155 (0.5 mg/kg, i.v.) were examined. Furthermore, angiotensin-converting-enzyme inhibitor - captopril (30 mg/kg, i.v.) was used.

The participation of POMC derived peptides in the impact of 8-OH-DPAT on the circulatory regulation in critical hypovolaemia was observed through the use of melanocortin type 4 (MC4) receptor antagonist - HS014 (5 µg, i.c.v.).

Chemicals used: captopril, [β-mercapto-β, β-cyclopentamethylenepropionyl1,O-me-Tyr2,Arg8]AVP, HS014 (Ac-Cys-Glu-His-D-2-Nal-Arg-Trp-Gly-Cys-Pro-Pro-Lys-Asp-NH2) (Sigma Chemical Co., St. Louis, MO, USA), ZD7155 hydrochloride (5,7-Diethyl-3,4-dihydro-1-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl]-1,6-naphthyridin-2(1H)-one hydrochloride) (Tocris Cookson Inc., Ellisville, MO, USA).

Statistical analysis was performed using Statistica 7.0 (StatSoft Inc, USA). The parametric t-Student test and non-parametric U-Man-Whitney, χ2 and Wilcoxon tests were used for significance analysis. The values were presented as mean ± standard deviation (SD). Values of P<0.05 were accepted as statistically significant.

All experimental protocols were carried out with measures to minimize animal pain and discomfort in accordance with the European Communities Council Directive and were approved by the local Ethical Committee of Silesian Medical University in Katowice (No 46/05, 26th of July 2005).

RESULTS

Centrally administrated 8-OH-DPAT caused resuscitative effects already described before, also by our research team (24, 25). These effects were presented by an increase in HR and MAP, as well as an increase in peripheral blood flow. Moreover, survival time of study animals increased from 30 min to 2 hours (the total study observation time).

The role of angiotensin type II in hemodynamic changes triggered by central 8-OH-DPAT administration

In the first phase of the experiment the role of angiotensin type II in the effects of centrally acting 8-OH-DPAT was examined. The peripheral AT1 receptors were blocked through the intravenous administration of a selective antagonist of these receptors, ZD7155 (0.5 mg/kg, i.v.), 5 minutes prior to the i.c.v. administration of

8-OH-DPAT. Moreover, the study was expanded by angiotensin-converting-enzyme inhibitor - captopril (30 mg/kg, i.v.) administered in the same manner.

No influence, of either selective blocking of the AT1 receptor (ZD7155), or the blocking of angiotensin-converting enzyme (captopril), on the changes in HR (Fig. 1A) and MAP (Fig. 1B) caused by the central stimulation of 5-HT1A receptors (P≥0.05) was observed. Separate administration of ZD7155 or captopril in the control group had no impact on the examined circulatory parameters (Fig. 1, Fig. 2 and Fig. 3).

Figure 1 Fig. 1. Changes in HR (A) and MAP (B) after i.c.v. administration of 8-OH-DPAT (5 µg) (p˜) and i.v. 0.5 mg/kg of ZD7155 (k) or i.v. 30 mg/kg of captopril (t) premedication. In control groups i.v. ZD7155 with i.c.v. 0.9% saline (k2), i.v. captopril with i.c.v. 0.9% saline (t2) or i.v. and i.c.v. 0.9% saline (r) were used. The moment of i.v. premedication with ZD7155, captopril or 0.9% saline was marked with the dashed arrow, while the moment of i.c.v. 8-OH-DPAT or 0.9% saline administration was marked as the solid arrow. The values were presented as mean ± standard deviation (S.D.), n=6, * P<0.05 vs. control group, # P<0.05 vs. 8-OHDPAT only treated group.
Table 1. The influence of premedication with examined substances on the changes in the heart rate (HR), mean arterial pressure (MAP), renal blood flow (RBF), mesenteric blood flow (MBF), and the blood flow in the distal part of the abdominal aorta (HBF) as well as renal vascular resistance (RVR), mesenteric vascular resistance (MVR) and hindlimb vascular resistance (HVR) triggered by the central administration of a selective 5-HT1A receptor agonist - 8-OH-DPAT.
Table 1
↑- increase, ↓ - decrease, ↔- no change; t - average survival time; ACE - angiotensin-converting enzyme; Ant-V1a - [β-merkapto-β, β-cyklopentametylenopropionyl1,O-me-Tyr2,Arg8]AVP; P<0.05.

Similarly, no influence of the abovementioned substances on the changes in peripheral vascular flow in the RBF (Fig. 2A), MBF (Fig. 2B) and HBF (Fig. 2C), as well as peripheral resistance: renal vascular resistance (RVR) (Fig. 3A), mesenteric vascular resistance (MVR) (Fig. 3B) and hindlimb vascular resistance (HVR) (Fig. 3C) was found.

Figure 2
Fig. 2. Peripheral blood flow RBF (A), MBF (B) and HBF (C) changes after i.c.v. administration of 5 µg of 8-OH-DPAT (p˜) and i.v. 0.5 mg/kg of ZD7155 (k) or i.v. 30 mg/kg of captopril (t) premedication. In control groups i.v. ZD7155 with i.c.v. 0.9% saline (k2), i.v. captopril with i.c.v. 0.9% saline (t2) or i.v. and i.c.v. 0.9% saline (r) were used. The moment of i.v. premedication with ZD7155, captopril or 0.9% saline was marked with the dashed arrow, while the moment of i.c.v. 8-OH-DPAT or 0.9% saline administration was marked as the solid arrow. The values were calculated as percent of initial values and presented as mean ± standard deviation (S.D.), n=6, * P<0.05 vs. control group, # P<0.05 vs. 8-OH-DPAT only treated group.
Initial values in controls treated with 0.9% saline: RBF 5.76 ± 0.67 ml/min, MBF 7.48 ± 1.71 ml/min, HBF 8.91 ± 1.58 ml/min.
Figure 3
Fig. 3. Peripheral resistance RVR (A), MVR (B) and HVR (C) changes after i.c.v. administration of 5 µg of 8-OH-DPAT (p˜) and i.v. 0.5 mg/kg of ZD7155 (k) or i.v. 30 mg/kg of captopril (t) premedication. In control groups i.v. ZD7155 with i.c.v. 0.9% saline (k2), i.v. captopril with i.c.v. 0.9% saline (t2) or i.v. and i.c.v. 0.9% saline (r) were used. The moment of i.v. premedication with ZD7155, captopril or 0.9% saline was marked with the dashed arrow, while the moment of i.c.v. 8-OH-DPAT or 0.9% saline administration was marked as the solid arrow. The values were calculated as percent of initial values and presented as mean ± standard deviation (S.D.), n=6, * P<0.05 vs. control group, # P<0.05 vs. 8-OH-DPAT only treated group.
Initial values in controls treated with 0.9% saline: RVR 14.11 ± 3.98 mmHg/ml/min, MVR 10.41 ± 3.62 mmHg/ml/min, HVR 9.01 ± 1.84 mmHg/ml/min.

The role of vasopressin in hemodynamic changes triggered by central 8-OH-DPAT administration

In order to determine the effects of vasopressin in the resuscitative effect triggered by the central stimulation of 5-HT1A receptors, peripheral V1a receptors were inhibited through the intravenous administration of [β-mercapto-β, β-cyclopentamethylenepropionyl1,O-me-Tyr2,Arg8]AVP, a selective antagonist of those receptors, in the dose of 10 µg/kg.

No influence of the selective blocking of the V1a receptor on the changes in HR (Fig. 4A) and MAP (Fig. 4B) caused by the central stimulation of the 5-HT1A receptors was found.

Figure 4 Fig. 4. Changes in HR (A) and MAP (B) after icv administration of 8-OH-DPAT (5 µg) (p˜) and i.v. 10 µg/kg of [β-mercapto-β, β-cyclopentamethy-lenepropionyl1,O-me-Tyr2,Arg8]AVP premedication (k) and in control groups in which i.v. [β-mercapto-β, β-cyclopentamethylenepropionyl1,O-me-Tyr2,Arg8]AVP with i.c.v. 0.9% saline (k2) or i.v. and i.c.v. 0.9% saline were used (r). The moment of i.v. premedication with [β-mercapto-β, β-cyclopentamethylenepropionyl1, O-me-Tyr2,Arg8]AVP or 0.9% saline was marked with the dashed arrow, while the moment of i.c.v. 8-OH-DPAT or 0.9% saline administration was marked as the solid arrow. The values were presented as mean ± standard deviation (S.D.), n=6, * P<0.05 vs. control group, # P<0.05 vs. 8-OHDPAT only treated group.

The administration of [β-mercapto-β, β-cyclo-pentamethylenepropionyl1,O-me-Tyr2,Arg8]AVP in the control group did not affect the examined circulatory system parameters (Fig. 4, Fig. 5 and Fig. 6).

No influence of the premedication with the V1a vasopressin receptor antagonist on the changes in peripheral vascular flow RBF (Fig. 5A), MBF (Fig. 5B) and HBF (Fig. 5C), as well as peripheral resistance RVR (Fig. 6A), MVR (Fig. 6B) and HVR (Fig. 6C), triggered by the central administration of 8-OH-DPAT, was found.

Figure 5
Fig. 5. Peripheral blood flow RBF (A), MBF (B) and HBF (C) changes after i.c.v. administration of 5 µg of 8-OH-DPAT (p˜) and i.v. 10 µg/kg of [β-mercapto-β, β-cyclopentamethylenepropionyl1,O-me-Tyr2,Arg8]AVP premedication (k) and in control groups in which i.v. [β-mercapto-β, β-cyclopentamethylenepropionyl1,O-me-Tyr2,Arg8]AVP with i.c.v. 0.9% saline (k2) or i.v. and i.c.v 0.9% saline were used (r). The moment of i.v. premedication with [β-mercapto-β, β-cyclopentamethylenepropionyl1,O-me-Tyr2,Arg8]AVP or 0.9% saline was marked with the dashed arrow, while the moment of i.c.v. 8-OH-DPAT or 0.9% saline administration was marked as the solid arrow. The values were calculated as percent of initial values and presented as mean ± standard deviation (S.D.), n=6, * P<0.05 vs. control group, # P<0.05 vs. 8-OH-DPAT only treated group.
Initial values in controls treated with 0.9% saline: RBF 5.76 ± 0.67 ml/min, MBF 7.48 ± 1.71 ml/min, HBF 8.91 ± 1.58 ml/min.
Figure 6
Fig. 6. Peripheral resistance RVR (A), MVR (B) and HVR (C) changes after i.c.v. administration of 5 µg of 8-OH-DPAT (p˜) and i.v. 10 µg/kg of [β-mercapto-β, β-cyclopentamethylenepropionyl1,O-me-Tyr2,Arg8]AVP premedication (k) and in control groups in which i.v. [β-mercapto-β, β-cyclopentamethylenepropionyl1,O-me-Tyr2,Arg8]AVP with i.c.v. 0.9% saline (k2) or i.v. and i.c.v. 0.9% saline were used (r). The moment of i.v. premedication with [β-mercapto-β, β-cyclopentamethylenepropionyl1,O-me-Tyr2,Arg8]AVP or 0.9% saline was marked with the dashed arrow, while the moment of i.c.v. 8-OH-DPAT or 0.9% saline administration was marked as the solid arrow. The values were calculated as percent of initial values and presented as mean ± standard deviation (S.D.), n=6, * P<0.05 vs. control group, # P<0.05 vs. 8-OH-DPAT only treated group.
Initial values in controls treated with 0.9% saline: RVR 14.11 ± 3.98 mmHg/ml/min, MVR 10.41 ± 3.62 mmHg/ml/min, HVR 9.01 ± 1.84 mmHg/ml/min.

The role of central MC4 melanocortin receptors in hemodynamic changes triggered by central 8-OH-DPAT administration

The last phase of the experiment was to determine the role of central MC4 melanocortin receptors in the resuscitative effect of the central stimulation of 5-HT1A receptors. For this purpose, 5 minutes prior to the i.c.v. administration of 8-OH-DPAT, an i.c.v. injection of a selective MC4 receptor antagonist, HS014 in the dose of 5 µg was performed.

The selective blocking of central MC4 receptors caused a partial reduction of the effect of the centrally administered 8-OH-DPAT on MAP (P<0.05) (Fig. 7B) with a simultaneous lack of impact on the HR (Fig. 7A) (P≥0.05). Furthermore, HS014 considerably delayed the changes in MAP observed after the administration of 8-OH-DPAT (the increase appeared 10 minutes later, 15 min. vs. 5 min.) (Fig. 7B). No influence of the central administration of HS014 on the survival time of the animals was reported. The administration of HS014 in the control group did not affect the examined circulatory system parameters. (Fig. 7, Fig. 8 and Fig. 9).

Figure 7 Fig. 7. Changes in HR (A) and MAP (B) after i.c.v. administration of 8-OH-DPAT (5 µg) (p˜) and i.c.v. 5 µg of HS014 premedication (k) and in control groups in which i.c.v. HS014 with i.c.v. 0.9% saline (k2) or i.c.v. 0.9% saline in 0 min and 5 min time points were used (r). The moment of i.c.v. premedication with HS014 or 0.9% saline was marked with the dashed arrow, while the moment of i.c.v. 8-OH-DPAT or 0.9% saline administration was marked as the solid arrow. The values were presented as mean ± standard deviation (S.D.), n=6,
* P<0.05 vs. control group, # P<0.05 vs. 8-OHDPAT only treated group.
Figure 8
Fig. 8. Peripheral blood flow RBF (A), MBF (B) and HBF (C) changes after i.c.v. administration of 5 µg of 8-OH-DPAT (p˜) and i.c.v. 5 µg of HS014 premedication (k) and in control groups in which i.c.v. HS014 with i.c.v. 0.9% saline (k2) or i.c.v. 0.9% saline in 0 min and 5 min time points were used (r). The moment of i.c.v. premedication with HS014 or 0.9% saline was marked with the dashed arrow, while the moment of i.c.v. 8-OH-DPAT or 0.9% saline administration was marked as the solid arrow. The values were calculated as percent of initial values and presented as mean ± standard deviation (SD), n=6, * P<0.05 vs. control group, # P<0.05 vs. 8-OH-DPAT only treated group.
Initial values in controls treated with 0.9% saline: RBF 5.76 ± 0.67 ml/min, MBF 7.48 ± 1.71 ml/min, HBF 8.91 ± 1.58 ml/min.

A slight, but statistically significant decrease in the peripheral resistance RVR (Fig. 9A), MVR (Fig. 9B) and HVR (Fig. 9C) triggered by the icv administration of 8-OH-DPAT after i.c.v. premedication with HS014 (P<0.05) was observed. The changes in vascular resistance were not accompanied by changes in peripheral flow RBF (Fig. 8A), MBF (Fig. 8B) and HBF (Fig. 8C), which remained the same in both groups (P≥0.05).

Figure 9
Fig. 9. Peripheral resistance RVR (A), MVR (B) and HVR (C) changes after i.c.v. administration of 5 µg of 8-OH-DPAT (p˜) and i.c.v. 5 µg of HS014 premedication (k) and in control groups in which i.c.v. HS014 with i.c.v. 0.9% saline (k2) or i.c.v. 0.9% saline in 0 min and 5 min time points were used (r). The moment of i.c.v. premedication with HS014 or 0.9% saline was marked with the dashed arrow, while the moment of i.c.v. 8-OH-DPAT or 0.9% saline administration was marked as the solid arrow. The values were calculated as percent of initial values and presented as mean ± standard deviation (S.D.), n=6, * P<0.05 vs. control group, # P<0.05 vs. 8-OH-DPAT only treated group.
Initial values in controls treated with 0.9% saline: RVR 14.11 ± 3.98 mmHg/ml/min, MVR 10.41 ± 3.62 mmHg/ml/min, HVR 9.01 ± 1.84 mmHg/ml/min.

DISCUSSION

The role of sympathetic nervous system as a trigger of resuscitation effects of 5-HT1A stimulation was presented previously (9, 25). This is why we focused on other systems.

In earlier research it was noted that the renin-angiotensin system participates in the resuscitative effects of the central stimulation of the histaminergic system (27). Due to the fact that in the research of other authors it has been noted that the effect of angiotensin in haemorrhagic shock is dependent exclusively on the AT1 receptors (11, 28), an antagonist of those receptors - ZD7155 was used in the current research. Unlike in the histaminergic system, we found no influence of selective blocking of the AT1 receptors on the effects triggered by the stimulation of the central 5-HT1A receptors. Similarly, in all the examined parameters, no impact of the earlier administration of captopril on the effects triggered by the administration of 8-OH-DPAT into the lateral ventricle was registered. Data obtained from our study reveals that central activation of 5-HT1A receptors does not influence the renin-angiotensin system. The above results are consistent with the reports of Van de Kar et al. (29), who concluded that serotonin, through the 5-HT2 receptors, increased the plasma renin activity. However, stimulation of 5-HT1A receptors did not produce such a result.

The selection of the blocked receptor was made upon the basis of the existing reports in the available literature. In the research of Jochem (5) it was noted that V1a receptors participate in the resuscitative effect connected with the activation of the histaminergic system, while the V1b and V2 receptors take no part in this effect. In our study no influence of blocking the V1a vasopressin receptors on the effects of the stimulation of central 5-HT1A was found. Those results are similar to those reported by Jorgensen et al., that although the central administration of serotonin has increased mRNA expression for vasopressin, this effect was dependent on the stimulation of 5-HT2 receptors. Central administration of the selective 5-HT1A agonist (8-OH-DPAT) did not induce such effects (30).

Stimulation of the MC4 receptors triggers potent resuscitative effects in haemorrhagic shock (23, 31). Intravenous administration of a non-selective agonist of melanocortin MC1, MC3, MC4 and MC5 receptors NDP- MSH or selective MC4 receptor agonists RO27-3225 and PG-931 has caused an increase in the HR, MAP and the survival time of rats in critical hypovolaemia. The intraperitoneal administration of a selective MC4 receptor antagonist HS024 has inhibited the resuscitative effect of the NDP-αMSH administered in the same manner (32). The administration of a selective MC4 receptor antagonist HS014 into the lateral brain ventricle has completely blocked the anti-shock effects of the intravenously administered adrenocorticotropic hormone (ACTH) (23).

In this study, i.c.v. premedication with HS014 prior to the i.c.v. administration of 8-OH-DPAT caused a statistically significant decrease in MAP, with no effects on the HR. What is more, the peripheral resistances were considerably lower. Peripheral blood flow was without substantial changes, most probably due to the extremely low blood volume. These results suggest the role of POMC derivatives and the MC4 receptor in the resuscitative effects of the central stimulation of 5-HT1A receptors. Our findings remain consistent with the reports of other authors. In the research of Jorgensen et al. (33) it was noticed that the peripheral administration of serotonin receptor agonists increases secretion of ACTH. Moreover, the i.c.v. administration of 8-OH-DPAT caused a significant increase in the mRNA expression for corticotropin-releasing hormone and mRNA for POMC, as well as a 5-fold increase in the ACTH plasma concentration (34).

Therefore, the present results show that the central melanocortin MC4 receptor partially participates in the resuscitative effect of central 5-HT1A activation. However, the angiotensin and vasopressin systems do not participate in this activity (Table 1).

On the other hand, it would be interesting to study the influence of serotonin in other extreme conditions, like anti-diuretic phenomenon during spaceflights, for which the anti-diuretic hormone system activation seems to play an important role (35).

Of course, the measured parameters used in our study (HR, MAP, blood flow and vascular resistance) do not fully examine the resuscitative effects. Perhaps other parameters like cardiac output or renal perfusion still need to be examined for a deeper understanding of the phenomenon. Also, it has to be mentioned that the study was still an experimental one and the clinical implications may, needless to say, differ from ours. Like for example the hypotensive effect of some antidepressants (36). However, the results of this study contribute new data on effectory mechanisms through which serotonin acts in haemorrhagic shock.

In conclusion, the central stimulation of the 5-HT1A receptors triggers a resuscitative effect linked with an increase in the HR and MAP, as well as the regulation of peripheral blood flows and vascular resistance. The POMC derivatives and the central MC4 receptor are partial effectory mechanisms of this action. The renin-angiotensin system and vasopressin do not participate in the resuscitative effect evoked by the central stimulation of 5-HT1A receptors.

Acknowledgements: The authors would like to thank Professor Jerzy Jochem for his invaluable assistance during the planning of the experiment as well as his indispensable input he contributed in extensive discussion with the authors.

Conflict of interests: None declared.

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R e c e i v e d : March 18, 2014
A c c e p t e d : July 22, 2014
Author’s address: Dr. Pawel Sowa, ENT Department, 10 Curie-Sklodowskiej Street, 41-800 Zabrze, Poland e-mail: paw.sowa@gmail.com