Skip Navigation

Cardiovascular Research 2001 49(1):226-233; doi:10.1016/S0008-6363(00)00204-2
© 2001 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Nijsen, M. J.M.A
Right arrow Articles by de Wildt, D. J
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nijsen, M. J.M.A
Right arrow Articles by de Wildt, D. J
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2000, European Society of Cardiology

Effect of {gamma}-melanocyte-stimulating hormones on baroreflex sensitivity and cerebral blood flow autoregulation in rats

Marjoleen J.M.A Nijsen*, Gerrit J.W de Ruiter, Carina M Kasbergen and Dick J de Wildt

Department of Medical Pharmacology, Rudolf Magnus Institute for Neurosciences, Utrecht University, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands

* Corresponding author. Tel.: +31-30-253-3634; fax: +31-30-253-9032 nijsen{at}med.ruu.nl

Received 1 March 2000; accepted 7 August 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Materials and methods
 3 Experimental design
 4 Results
 5 Discussion
 References
 
Objective: In the present paper, we are interested in the effects of {gamma}-melanocyte-stimulating hormones ({gamma}-MSHs) on cardiovascular regulatory systems. Methods: Mean arterial pressure (MAP), cerebral blood flow (CBF) and heart rate (HR) were measured in urethane-anaesthetised rats after intravenous administration of lys{gamma}2-MSH, {gamma}2-MSH, {gamma}2-MSH(6–12) or phenylephrine. Results: The {gamma}-MSHs caused an increase in MAP, CBF and HR, whereas phenylephrine caused an increase in MAP and CBF and baroreceptor reflex-mediated bradycardia. All tested {gamma}-MSHs showed a significant impairment of the baroreceptor reflex sensitivity and CBF autoregulation as compared to the phenylephrine group. {gamma}2-MSH shows identical effects on the baroreceptor reflex and CBF as the endogenous occurring lys{gamma}2-MSH. In addition, the C-terminal fragment of {gamma}2-MSH, {gamma}2-MSH(6–12), induced similar effects as {gamma}2-MSH. The level of increase in MAP was comparable between the {gamma}-MSHs and the phenylephrine group. Conclusions: The present study suggests that {gamma}2-MSH and the shorter fragment {gamma}2-MSH(6–12) impair baroreceptor reflex sensitivity, due to a strong increase in sympathetic tone and/or change in baroreceptor reflex setpoint, and induce cerebrovasodilatation, which can counteract an autoregulation-mediated cerebrovasoconstriction due to systemic pressor effects. Furthermore, the results indicate that the C-terminal site of {gamma}2-MSH is relevant for its central-mediated inhibitory effects on the baroreceptor reflex and CBF.

KEYWORDS Blood pressure; Heart rate (variability); Hormones; Cerebrovascular disorders


   1 Introduction
Top
 Abstract
 1 Introduction
2 Materials and methods
 3 Experimental design
 4 Results
 5 Discussion
 References
 
Melanocortins ({alpha}-melanocyte-stimulating hormone ({alpha}-MSH), β-MSH, {gamma}-MSH and adrenocorticotropic hormone) belong to a family of peptides derived from the precursor pro-opiomelanocortin. In addition to their effects on the hormonal system, behaviour, temperature and feeding regulation, {gamma}-MSHs have strong effects on the cardiovascular system. Systemic administration of {gamma}2-MSH to conscious rats causes a dose-dependent increase in mean arterial pressure (MAP) and heart rate (HR) [1–5]. In our laboratory, we found that the shorter fragment of {gamma}2-MSH, {gamma}2-MSH(6–12), shows the highest potency and efficacy in inducing cardiovascular effects [4]. In the pithed rat systemically administered {gamma}2-MSH does not exert a cardiovascular action [3,6], suggesting that the central nervous system is involved in {gamma}2-MSH-induced cardiovascular effects. This hypothesis agrees with findings of Callahan et al. [1], who reported that the pressor response to systemic administration of {gamma}2-MSH in conscious rats was blocked by the ganglionic blocking agent chlorisondamine or by blockade of the catecholamine release from sympathetic nerve terminals with bretylium tosylate. They suggested that {gamma}2-MSH activates the sympathetic nervous system. In addition, we showed previously that application of {gamma}2-MSH on isolated rat heart preparations caused no cardiac effects [6]. These findings strongly suggest that cardiovascular effects of {gamma}2-MSH are mediated via the central nervous system and not via a peripheral site of action. In the present paper, we are interested in the centrally mediated effects of {gamma}2-MSH on hemodynamics.

Arterial pressure is normally maintained within physiological limits by the baroreceptor reflex. The baroreceptor reflex is a well-studied mechanism, in which the sympathetic nervous system plays an important role i.e. reflex reduction or activation of tonic sympathetic activity due to pressor or depressor effects, respectively. From our laboratory, we reported in several studies that {gamma}-MSHs cause an increase in both MAP and HR in conscious rats [4,5], suggesting that these peptides impair the baroreceptor reflex and activate the sympathetic nervous system. So far, the exact mechanisms of action for {gamma}2-MSH on the baroreceptor reflex are unclear.

Another hemodynamic regulatory defense system is the cerebral blood flow (CBF) autoregulation, which helps to maintain the constancy of CBF in spite of fluctuations in systemic arterial pressure. This mechanism is necessary to protect the brain against hypoxia at low perfusion pressures and the risks of brain oedema at high arterial pressures. However, if systemic blood pressure is very high or low the upper or lower limit of autoregulation respectively is surpassed. Then, CBF will follow the pressor changes [7–9]. Sandor et al. [10] reported that intracerebroventricular administered {gamma}2-MSH impaired blood flow autoregulation in the hypothalamus as it facilitated the haemorrhage-induced decrease in hypothalamic blood flow in urethane-anaesthetised rats. In a previous study [11] we reported that systemically injected {gamma}2-MSH impaired CBF autoregulation as it caused a simultaneous increase in MAP and CBF in urethane-anaesthetised rats. The question remains whether the impairment of CBF autoregulation is caused by direct central effects of {gamma}2-MSH or indirectly by the {gamma}2-MSH-induced high pressor level, which in turn will lead to a breakthrough of CBF autoregulation. Therefore, the present study was performed in a more quantitative approach to examine in greater detail the effects of {gamma}2-MSH on CBF autoregulation.

In the present paper, we are interested in the influence of {gamma}-MSHs on both regulatory hemodynamic systems (baroreceptor reflex and CBF autoregulation). Therefore, we studied the effects of increasing doses of intravenously administered {gamma}2-MSH, {gamma}2-MSH(6–12) and lys{gamma}2-MSH (an endogenously occurring {gamma}-MSH [12]) in comparison to the sympathomimetic pressor agent phenylephrine on MAP and HR. Thereby a possible role of {gamma}-MSHs in the baroreceptor reflex is considered. Furthermore, we compare the impact of increasing pressor levels induced by phenylephrine or {gamma}-MSHs on CBF.


   2 Materials and methods
Top
 Abstract
 1 Introduction
 2 Materials and methods
3 Experimental design
 4 Results
 5 Discussion
 References
 
2.1 Animals
Naive male albino Wistar rats (U:WU/CPB) weighing 230–320 g at the beginning of the experiments were used. Prior to surgery, two or three rats were housed per Macrolon cage (230x170x140 mm) containing a layer of woodshavings under conditions of constant ambient temperature (21±1°C), constant humidity (60±15%), and light/dark rhythm (with lights on from 7 a.m. to 7 p.m.). Food (complete laboratory chow: Hope Farms, Woerden, The Netherlands) and water were accessible ad libitum throughout the experiment.


   3 Experimental design
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Experimental design
4 Results
 5 Discussion
 References
 
Rats were surgically equipped with a femoral artery and vein cannula for measurements of blood pressure (BP) and heart rate (HR) and for intravenous (iv) administration of vasoactive drugs, respectively. Baseline BP and HR were recorded before each iv injection. One drug was injected per rat in a volume of 100 µl and an interval of 10 min was used between each dose of injection to allow stabilisation of BP and HR. In order to flush the cannula each injection of a drug was followed by 50 µl of saline. All experiments were performed under urethane anaesthesia and were terminal.

The experiments were approved by the ethical committee for animal experimentation of the Medical Faculty, Utrecht University, The Netherlands. The investigation conforms the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

3.1 Experiment 1: Effects of phenylephrine and sodium nitroprusside on the baroreceptor reflex and cerebral blood flow
Rats (n = 5) were iv injected with increasing doses of phenylephrine and sodium nitroprusside. The dose range for phenylephrine and sodium nitroprusside was 0, 0.5, 1, 5, 10, 50 and 100 µg/kg.

3.2 Experiment 2: Effects of {gamma}2-MSHS on the baroreceptor reflex and cerebral blood flow
Rats were iv injected with increasing doses of phenylephrine (n = 5), {gamma}2-MSH (n = 6), lys {gamma}2-MSH (n = 6) or {gamma}2-MSH(6–12) (n = 7). The dose range for phenylephrine was 0, 0.5, 1, 5, and 10 µg/kg; for the {gamma}2-MSHs 0, 1.5, 5, 15, 50 and 100 nmol/kg.

3.3 Surgery
Rats were anaesthetised with urethane (10% dissolved in 0.9% NaCl solution; Sigma Chemical, St. Louis, MO, USA) at a dose of 1.3 ml/100 g body weight intraperitoneally. During the operation body temperature of the rats was maintained at 37°C with a heated pad (Homeothermic Blanket System, Harvard Apparatus, Harvard, Massachusetts, USA). Tracheotomy was performed with polyethylene tubing (I.D. 1.57 mm, O.D. 2.08 mm; Portex, The Hague, The Netherlands) and the rats were artificially ventilated with room air using a small animal respirator (Model 683, Harvard Apparatus) delivering 75 strokes per min at 2.5–3.0 ml per stroke. When necessary, ventilation was adjusted to maintain arterial CO2 pressure (PaCO2) and pH within the range of 33.0–38.0 mm Hg and 7.35–7.45, respectively.

For BP and HR measurements, the femoral artery was cannulated with a polyethylene tubing (i.d. 0.28 mm, o.d. 0.61 mm; Portex), which was melted to another polyethylene tubing (i.d. 0.58 mm, o.d. 0.96 mm; Portex) with a 540° loop in the middle. The latter was melted to a third polyethylene tubing (I.D. 0.86 mm, O.D. 1.52 mm; Portex). The cannula was filled with a heparin solution (5000 IU/ml, Leo Pharmaceutical Products BV, Weesp, The Netherlands).

For iv administration of drugs the ipsilateral femoral vein was cannulated with polyethylene tubing (I.D. 0.58 mm, O.D. 0.96 mm, Portex). The cannula was filled with saline.

3.4 Measurements of blood pressure and heart rate and intravenous administration
The arterial cannula was connected to a pressure transducer (Viggo-Spectramed, disposable DTX/plus, Ohmeda, Bilthoven, The Netherlands) by a polyethylene tubing (I.D. 0.86 mm, O.D. 1.52 mm; Portex). The pressure transducer was connected to a DC-preamplifier and biotachometer (Instrument service, Utrecht University, The Netherlands) coupled to a P75-computer. Data were continuously recorded and measured with the Haemodynamic Data Acquisition System (Instrumental Department, University of Limburg, Maastricht, The Netherlands) and DATVIEW program (Instrumental Department, University of Limburg). Mean arterial pressure (MAP) was calculated according to the formula: (2xPd+Ps)/3, in which Pd is diastolic pressure and Ps systolic pressure.

The jugular vein cannula was attached to a syringe (1000 µl, Inacom Instruments, Veenendaal, The Netherlands) and microinfusion pump (Harvard Apparatus) by a polyethylene tubing (I.D. 0.58 mm, O.D. 0.96 mm, Portex). Systemic injections were performed at 500 µl/min.

3.5 Measurement of cerebral blood flow
Microcirculatory cerebral blood flow (CBF) was continuously measured by laser-Doppler flowmetry. A Perimed flowmeter (Periflux PF3, Stockholm, Sweden) was used according to the method described by Iadecola and Reis [13]. The flowmeter was equipped with a 2 mW helium–neon laser with a wavelength of 632.8 nm. Flow values are expressed in arbitrary units (perfusion units, PU). The contralateral parietal bone was exposed and a small square (3x2 mm) was drilled 1-mm laterally and 1-mm caudally to the bregma. The dura mater was left intact. The square was frequently cooled with saline during drilling to prevent excessive heating. A needle probe (PF 302, tip diameter 0.45 mm) was placed in a micromanipulator and positioned above the dural surface. The position of the probe corresponded to a collateral blood supplying area of 2–3 mm lateral of the midline, which is relatively devoid of large surface vessels [14]. The exact site of probe positioning in the square was chosen so that basal CBF was between 100 and 150 PU. The Doppler-shift frequency range was chosen from 20 Hz to 12 kHz. The flow signal was averaged with a 0.2-s time constant. The analogue output from the instrument was fed into a data acquisition/processing system (a Bio Signal Processing System; Instrumental Department, University of Limburg, Maastricht, The Netherlands). The collected data were sent to a P75-computer, sampled at 500 Hz and processed on a real-time base.

3.6 Drug treatment
The vasoconstrictor L-phenylephrine hydrochloride was purchased from Sigma Chemical; the vasodilator agent sodium nitroprusside from E.Merck, Darmstadt, Germany; {gamma}2-MSH from Bachem, Bubendorf, Switzerland; Lys{gamma}2-MSH and {gamma}2-MSH(6–12) were synthesised and kindly donated by Dr. P. Hoogerhout, National Institute for Public Health and Environmental Protection, Bilthoven, The Netherlands. All drugs were dissolved in bidistilled water prior to use.

3.7 Statistics
In experiment 1, mean baseline MAP (mmHg), HR (beats per min) and CBF (PU), recorded prior to phenylephrine and sodium nitroprusside injection and maximum cardiovascular effects at the highest given dose, were analysed by a two-tailed Student t-test. A linear regression was performed on changes in HR and CBF following phenylephrine- and sodium nitroprusside-induced changes in MAP. In experiment 2, mean baseline MAP, HR and CBF (recorded before saline injection) and maximum cardiovascular effects at the highest given dose were analysed by a one-way Analysis of Variance (ANOVA) and post-hoc Tuckey HSD test. The changes in MAP, HR and CBF levels to drug injections are presented as MAP ratio, HR ratio and CBF ratio respectively (ratio=post-injection level/pre-injection level; a ratio of 1 indicates no change of the pre-injection value). A linear regression was performed on MAP ratio–HR ratio and MAP ratio–CBF ratio per experimental group (mean linear regression; depicted in Figs. 3 and 4Go) and for each individual rat (individual linear regression). The resulting individual regression coefficients were analysed between the experimental groups by an ANOVA and post-hoc Tuckey HSD test.


Figure 3
View larger version (24K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Correlation between heart rate (HR) and mean arterial pressure (MAP) after administration of intravenous phenylephrine (PE; 0.5–10 µg/kg), lys{gamma}2-MSH (1.5–100 nmol/kg), {gamma}2-MSH (1.5–100 nmol/kg) and {gamma}2-MSH(6–12) (1.5–100 nmol/kg) in urethane-anaesthetised rats. Both HR and MAP are expressed as mean ratios (=post-injection level/pre-injection level; a ratio of 1 indicates no change of the pre-injection value)±S.E.M. (n = 5–7). The linear regression is indicated for each drug in the figure.

 

Figure 4
View larger version (25K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Correlation between cerebral blood flow (CBF) and mean arterial pressure (MAP) after administration of intravenous phenylephrine (PE; 0.5–10 µg/kg), lys{gamma}2-MSH (1.5–100 nmol/kg), {gamma}2-MSH (1.5–100 nmol/kg) and {gamma}2-MSH(6–12) (1.5–100 nmol/kg) in urethane-anaesthetised rats. Both CBF and MAP are expressed as mean ratios (=post-injection level/pre-injection level; a ratio of 1 indicates no change of the pre-injection value)±S.E.M. (n = 5–7). The linear regression is indicated for each drug in the figure.

 
The data are presented as mean±S.E.M. The data in the figures represent maximum cardiovascular effects by each administered dose of peptide or drug. A value of P<0.05 was considered significant.


   4 Results
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Experimental design
 4 Results
5 Discussion
 References
 
4.1 Experiment 1: Effects of phenylephrine and sodium nitroprusside on the baroreceptor reflex and cerebral blood flow
4.1.1 Baseline levels
Table 1 shows the basal levels of MAP, HR and CBF prior to phenylephrine (PE) and sodium nitroprusside (SNP) injection. Baseline levels were not significantly different between the phenylephrine and sodium nitroprusside group.


View this table:
[in this window]
[in a new window]

  		Table 1 Baseline levels of mean arterial pressure (MAP; mm Hg), heart rate (HR; beats per min) and cerebral blood flow (CBF; PU) prior to phenylephrine (PE) and sodium nitroprusside (SNP) injection (n = 5)a

 
4.1.2 Baroreceptor reflex
Injection of the vasoconstrictor phenylephrine increased baseline MAP to 191 mm Hg, whereas the vasodilator sodium nitroprusside decreased baseline MAP to 52 mm Hg. Fig. 1 shows the mean (±S.E.M) of phenylephrine- and sodium nitroprusside-induced changes in MAP and corresponding changes in HR. In the MAP range of 52–126 mm Hg, baseline HR remained relatively unchanged (no linear regression between MAP and HR), whereas the baroreceptor reflex was distinctly present at the higher blood pressure range of 126–191 mm Hg (HR=–1.1 MAP+545, P<0.001). Statistical analyses over the total MAP range of 52–191 mm Hg revealed a linear regression between MAP and HR (HR=–0.6 MAP+458, P<0.001).


Figure 1
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Heart rate (HR) as a function of mean arterial pressure (MAP) after administration of intravenous phenylephrine (PE; 0.5–100 µg/kg) or sodium nitroprusside (SNP; 0.5–100 µg/kg) in urethane-anaesthetised rats (n = 5). The data are expressed as mean±S.E.M; the square represents the baseline level of HR and MAP; the dotted line represents the starting point of phenylephrine and sodium nitroprusside injection; the arrow indicates the MAP value at which HR starts to decrease.

 
4.1.3 CBF autoregulation
Fig. 2 shows the mean (±S.E.M) of phenylephrine- and sodium nitroprusside-induced changes in MAP and corresponding changes in CBF. In the MAP range of 52–142 mm Hg, baseline CBF was markedly constant (CBF=0.5 MAP+73, P<0.001), whereas CBF autoregulation was absent at the higher blood pressure range of 142–191 mm Hg (CBF=1.4 MAP–45, P<0.001).


Figure 2
View larger version (13K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Cerebral blood flow (CBF) as a function of mean arterial pressure (MAP) after administration of intravenous phenylephrine (PE; 0.5–100 µg/kg) or sodium nitroprusside (SNP; 0.5–100 µg/kg) in urethane-anaesthetised rats. The data are expressed as mean±S.E.M (n = 5); the square represents the baseline level of CBF and MAP; the dotted line represents the starting point of phenylephrine and sodium nitroprusside injection; the arrow indicates the MAP value at which CBF starts to increase profoundly (=upper limit of CBF autoregulation).

 
4.2 Experiment 2: Effects of {gamma}2-MSHs on the baroreceptor reflex and cerebral blood flow
4.2.1 Baseline levels
Table 2 shows the basal levels of MAP, HR and CBF prior to phenylephrine (PE), lys{gamma}2-MSH, {gamma}2-MSH and {gamma}2-MSH(6–12) injection. Baseline levels were not significantly different between the four experimental groups.


View this table:
[in this window]
[in a new window]

  		Table 2 Baseline levels of mean arterial pressure (MAP; mm Hg), heart rate (HR; beats per min) and cerebral blood flow (CBF; PU) prior to phenylephrine (PE; n = 5), lys{gamma}2-MSH (n = 6), {gamma}2-MSH (n = 6) and {gamma}2-MSH(6–12) (n = 7) injectiona

 
4.2.2 Baroreceptor reflex
Rats were iv injected with increasing doses of phenylephrine (0.5, 1, 5, and 10 µg/kg), {gamma}2-MSH (1.5, 5, 15, 50 and 100 nmol/kg), lys{gamma}2-MSH (1.5, 5, 15, 50 and 100 nmol/kg) or {gamma}2-MSH(6–12) (1.5, 5, 15, 50 and 100 nmol/kg) to study the baroreceptor reflex. Fig. 3 shows the mean correlation between HR and MAP after drug injection. The mean MAP and HR ratio were negatively correlated during phenylephrine injection (HR ratio=–0.05 MAP ratio +1.05; P<0.001), whereas the {gamma}-MSHs showed a positive correlation between MAP and HR (HR ratio=0.17 MAP ratio+0.83 ({gamma}2-MSH); HR ratio=0.16 MAP ratio+0.84 ({gamma}2-MSH(6–12)); HR ratio=0.13 MAP ratio+0.87 (lys{gamma}2-MSH); P<0.001). The average of the individual regression coefficients in the lys{gamma}2-MSH group was 0.13±0.02, in the {gamma}2-MSH group 0.15±0.04, in the {gamma}2-MSH(6–12) group 0.14±0.04 and in the phenylephrine group –0.04±0.03. The averaged regression coefficients between the {gamma}-MSH groups were not significantly different, whereas that of the phenylephrine group was significantly different from all {gamma}-MSH groups (P<0.01).

4.2.3 CBF autoregulation
Rats were iv injected with increasing doses of phenylephrine (0.5, 1, 5, and 10 µg/kg), {gamma}2-MSH (1.5, 5, 15, 50 and 100 nmol/kg), lys{gamma}2-MSH (1.5, 5, 15, 50 and 100 nmol/kg) or {gamma}2-MSH(6–12) (1.5, 5, 15, 50 and 100 nmol/kg) to study CBF autoregulation. Fig. 4 shows the mean correlation between CBF and MAP after drug injection. The mean MAP and CBF ratio showed a linear regression during phenylephrine (CBF ratio=0.5 MAP ratio+0.49; P<0.001), lys{gamma}2-MSH (CBF ratio=1.3 MAP ratio –0.27; P<0.001), {gamma}2-MSH(6–12) (CBF ratio=1.4 MAP ratio –0.37; P<0.001) and {gamma}2-MSH (CBF ratio=1.5 MAP ratio –0.51; P<0.001) injection. The average of the individual regression coefficients in the lys{gamma}2-MSH group was 1.2±0.1, in the {gamma}2-MSH group 1.6±0.3, in the {gamma}2-MSH(6–12) group 1.4±0.2 and in the phenylephrine group 0.5±0.1. The averaged regression coefficients between the {gamma}-MSH groups were not significantly different, whereas that of the phenylephrine group was significantly smaller as compared to all {gamma}-MSH groups (P<0.01).


   5 Discussion
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Experimental design
 4 Results
 5 Discussion
References
 
5.1 Baroreceptor reflex sensitivity
This study shows that intravenous administration of 0.5–100 µg/kg phenylephrine increased resting MAP from 97 to 191 mm Hg, whereas a similar dose of sodium nitroprusside decreased resting MAP from 90 to 52 mm Hg in urethane-anaesthetised rats. In the MAP range of 126–191 mm Hg, baseline HR showed a reflex decrease (i.e. reflex bradycardia) due to elevations in MAP, whereas HR remained relatively unchanged to lower pressure levels (52–126 mm Hg). Baroreceptor reflex sensitivity was indicated by the correlation coefficient for linear regression of changes in HR following changes in MAP. The regression coefficient was –0.6, which is in agreement with that found by Fluckiger and colleagues in urethane-anaesthetised rats [12]. However, a much higher baroreceptor reflex sensitivity has been reported in conscious rats [15]. Several studies showed that general anaesthetics profoundly depress the baroreceptor reflex sensitivity [16,17]. This is not surprising as the tonic sympathetic nervous system activity is suppressed during anaesthesia and thus reflex reduction of tonic sympathetic activity due to pressor effects or activation of the sympathetic nervous system during depressor conditions is decreased. This knowledge has to be taken into account for the interpretation of the effects of the {gamma}-MSHs on the baroreceptor reflex.

In the present paper, we found that all tested {gamma}-MSHs caused an increase in both MAP and HR in urethane-anaesthetised rats, an effect which has been reported previously in conscious rats by our laboratory [4,5] and others [1,2]. The relationship between MAP and HR ratio was not significantly different between the lys{gamma}2-MSH (regression coefficient=0.13±0.02), {gamma}2-MSH (regression coefficient=0.15±0.04) and {gamma}2-MSH(6–12) (regression coefficient=0.14±0.04) group, whereas all tested {gamma}-MSHs showed a significant impairment of the baroreceptor reflex sensitivity as compared to the phenylephrine group (regression coefficient=–0.04±0.03). As mentioned above, the baroreceptor reflex sensitivity in our study is weakened due to the anaesthesia. Still, {gamma}-MSHs show a further decrease of the baroreceptor reflex sensitivity. This finding leads us to the following three conclusions: (1) {gamma}-MSH induces a strong increase in sympathetic nervous system tone [1,2], probably due to stimulation of hypothalamic efferent projections to cardiovascular centres within the brain stem [18]. A baroreceptor reflex-mediated reduction in efferent sympathetic nervous system activity, as a consequence of {gamma}-MSH-induced pressor effects, might be overruled by this hypothalamic initiated rise in sympathetic tone; (2) {gamma}-MSH might change the setpoint of the baroreceptor reflex at the level of the nucleus tractus solitarius (NTS) or induce inhibitory effects at the level of afferent barosensory systems in the carotid sinus or the heart; (3) {gamma}-MSH might act peripherally at the level of the blood vessels, inducing vasoconstriction and at the level of the heart, inducing tachycardia that can overcome a baroreceptor reflex-mediated bradycardia. The latter suggestion is not plausible as we reported previously that in the pithed rat systemically administered {gamma}2-MSH does not exert a cardiovascular action and application of {gamma}2-MSH on isolated rat heart preparations caused no cardiac effects [6]. These findings strongly suggest that cardiovascular effects of {gamma}-MSHs are mediated via the central nervous system and not via a peripheral site of action. We hypothesise that {gamma}-MSHs act in central areas located outside the blood–brain barrier (circumventricular organs: area postrema/nucleus tractus solitarius in the lower brain stem and arcuate nucleus in the hypothalamus) which are easily accessible.

Furthermore, the results indicated that {gamma}2-MSH, which is mostly used in cardiovascular studies, shows identical effects on baroreceptor reflex sensitivity as the endogenous occurring lys{gamma}2-MSH. In addition, the C-terminal fragment of {gamma}2-MSH, {gamma}2-MSH(6–12), induced similar effects on baroreceptor reflex sensitivity as {gamma}2-MSH. This suggests that the C-terminal site of {gamma}2-MSH is relevant for its central-mediated inhibitory effects on the baroreceptor reflex.

5.2 Cerebral blood flow autoregulation
Microcirculatory CBF was continuously measured by laser-Doppler flowmetry. Laser-Doppler flowmetry allows continuous and non-invasive measurements of the microvascular blood flow in superficial brain regions. Even though this technique is not a strict measurement of blood flow, but rather movements of red blood cells in the microcirculation [19], the values obtained in the rat cerebral cortex correlate well with established methods for measuring CBF [20,21].

This study shows that in the MAP range of 52–142 mm Hg, baseline CBF was markedly constant, whereas it increased to higher pressure levels (142–191 mm Hg) in urethane-anaesthetised rats. These results are in agreement with those found in conscious rats by Hoffman et al. [7]. They reported that the mean autoregulatory coefficient (mean change in blood flow/change in MAP) was 0.6 in the cortex for the MAP range of 47–146 mm Hg. In the present paper, the degree of CBF autoregulation (=regression coefficient of changes in CBF following changes in MAP) showed a comparable level of 0.5 for the MAP range of 52–142 mm Hg. This indicates that, in contrast to the baroreceptor reflex, CBF autoregulation is not profoundly impaired by urethane anaesthesia. Other anaesthetics like halothane, isoflurane and sevoflurane have been reported to attenuate CBF autoregulation in rats [7,8,22].

In the present paper, we found that intravenous administration of lys{gamma}2-MSH, {gamma}2-MSH and {gamma}2-MSH(6–12) caused an increase in both MAP and CBF in urethane-anaesthetised rats, an effect which has been reported previously by our laboratory for {gamma}2-MSH [11]. The relationship between MAP and CBF ratio was not significantly different between the lys{gamma}2-MSH (regression coefficient=1.2±0.1), {gamma}2-MSH (regression coefficient=1.6±0.3) and {gamma}2-MSH(6–12) (regression coefficient=1.4±0.2) group, whereas all tested {gamma}-MSHs showed a significant impairment of CBF autoregulation as compared to the phenylephrine group (regression coefficient=0.5±0.1). The level of increase in MAP was comparable between the {gamma}-MSHs and phenylephrine, indicating that the high pressor level itself was not responsible for the attenuation of CBF autoregulation in {gamma}-MSH-treated rats. This suggests that {gamma}-MSHs impair CBF autoregulation by direct central effects and not indirectly due to the {gamma}-MSH-induced increase in MAP. We hypothesise that {gamma}-MSHs directly induce vasodilatation in the cortex, which can overcome an autoregulation-mediated cerebrovasoconstriction induced by an increase in systemic blood pressure. Sandor et al. [10] reported that intracerebroventricular administered {gamma}2-MSH impaired blood flow autoregulation in the hypothalamus as it facilitated the haemorrhage-induced decrease in hypothalamic blood flow in urethane-anaesthetised rats. In contrast to the present findings, the latter study suggests that {gamma}2-MSH can induce vasoconstriction of cerebral hypothalamic vessels to counteract for the autoregulation-mediated vasodilatation induced by a decrease in MAP. This contradiction can be explained by heterogeneity of blood flow autoregulation in the brain. Baumbach and Heistad [23] demonstrated regional heterogeneity of CBF autoregulation: autoregulation was impaired in the brain stem as compared to the cerebrum. Furthermore, they demonstrated segmental heterogeneity: autoregulation occurred predominantly in small vessels during acute hypotension, whereas the autoregulatory response was more important in the large cerebral arteries during acute hypertension.

In agreement with the results on baroreceptor reflex sensitivity, the latter findings indicated that {gamma}2-MSH(6–12) induced similar effects on CBF autoregulation as {gamma}2-MSH. This suggests that the C-terminal site of {gamma}2-MSH is relevant for its cerebrovasodilatory effects. Furthermore, {gamma}2-MSH shows identical effects on CBF autoregulation as the endogenous occurring lys{gamma}2-MSH. With respect to a possible role of {gamma}2-MSH in pathophysiology, Ekman et al. [24] and Edvinsson et al. [25] reported that plasma levels of immunoreactive {gamma}2-MSH in humans is increased in severe forms of heart failure and suggested that {gamma}2-MSH is released in response to stress caused by cardiac failure. It has been reported that CBF is also increased during stress [26]. Therefore, a hormonal role for {gamma}2-MSH, released during cardiovascular stress, in the regulation of CBF can be hypothesised.

For a greater part, the cerebromicrovasculature contributes to the brain vascular resistance in the rat within the autoregulatory range of blood pressure [27]. In an earlier study we have measured the internal carotid flow quantitatively [11] and we could demonstrate that internal carotid flow was increased in a parallel fashion with the laser-Doppler flowmetry measurements of cerebral blood flow. Therefore we think that laser-Doppler flowmetry measurement of cortical blood flow is suitable to measure autoregulatory processes within the brain circulation. Although we cannot exclude confounding flow rates from extracranial compartments with superficial laser-Doppler flowmetry measurements, it appears not an important disturbing factor in the overall interpretation of the results.

In summary, the present study suggests that {gamma}2-MSH and the shorter fragment {gamma}2-MSH(6–12) impair baroreceptor reflex sensitivity because (1) a baroreceptor reflex-mediated reduction in efferent sympathetic nervous system activity, as a consequence of centrally mediated pressor effects of {gamma}-MSHs, might be overruled by a strong increase in sympathetic tone induced by {gamma}-MSHs and/or (2) {gamma}-MSHs change the setpoint of the baroreceptor reflex at the level of the NTS or afferent barosensory systems, accompanying its centrally mediated pressor effects. Furthermore, it can be concluded that {gamma}2-MSH and {gamma}2-MSH(6–12) induce cerebrovasodilatation, which can counteract the autoregulation-mediated cerebrovasoconstriction due to systemic pressor effects.

Time for primary review 30 days.


   Acknowledgements
 
We thank Dr. Ir. P. Hoogerhout, Laboratory for Vaccine Development and Immune Mechanisms at the National Institute for Public Health and Environmental Protection, Bilthoven, The Netherlands for the synthesis and analysis by mass spectrometry of the peptides lys{gamma}2-MSH and {gamma}2-MSH(6–12).


   References
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Experimental design
 4 Results
 5 Discussion
 References
 

  1. Callahan M.F., Kirby R.F., Wolff D.W., et al. Sympathetic nervous system mediation of acute cardiovascular actions of {gamma}2-melanocyte-stimulating hormone. Hypertension (1985) SI:145–150.
  2. Callahan M.F., Kirby R.F., Johnson A.K., Gruber K.A. Sympathetic terminal mediation of the acute cardiovascular response of {gamma}2-MSH. J Auton Nerv System (1988) 24:179–182.[CrossRef][Web of Science][Medline]
  3. Sun X.Y., Feng Q.P., Gong Q.L., Edvinsson L., Hedner T. Cardiovascular and renal effects of gamma-MSH in spontaneously hypertensive and normotensive Wistar Kyoto rats. Am J Physiol (1992) 262:R77–R84.[Web of Science][Medline]
  4. Van Bergen P., Janssen P.M., Hoogerhout P., De Wildt D.J., Versteeg D.H. Cardiovascular effects of gamma-MSH/ACTH-like peptides: structure–activity relationship. Eur J Pharmacol (1995) 294:795–803.[CrossRef][Web of Science][Medline]
  5. Van Bergen P., De Winter T.Y., De Wildt D.J., Versteeg D.H. Influence of blockade of alpha 1-adrenoceptors, beta 1-adrenoceptors and vasopressin V1A receptors on the cardiovascular effects of gamma 2-melanocyte-stimulating hormone (gamma 2-MSH). Naunyn Schmiedebergs Arch Pharmacol (1997) 355:720–726.[CrossRef][Web of Science][Medline]
  6. Van Bergen P., Vleeming W., Van Heijst B.G., Versteeg D.H., De Wildt D.J. A study on possible modulating and direct effects of gamma2-MSH and ACTH-(1–24) on the cardiovascular system of the rat. Naunyn Schmiedebergs Arch Pharmacol (1998) 358:220–229.[CrossRef][Web of Science][Medline]
  7. Hoffman W.E., Edelman G., Kochs E., et al. Cerebral autoregulation in awake versus isoflurane-anaesthetised rats. Anest Analg (1991) 73:753–757.[Abstract/Free Full Text]
  8. Lu H., Werner C., Engelhard K., Scholz M., Kochs E. The effects of sevoflurane on cerebral blood flow autoregulation in rats. Anest Analg (1998) 87:854–858.[Abstract/Free Full Text]
  9. Tsai M.L., Lee C.Y., Lin M.T. Responses of cerebral circulation produced by adrenoceptor agonists and antagonists in rats. Neuropharmacology (1989) 28:1075–1080.[CrossRef][Web of Science][Medline]
  10. Sandor P., de Jong W., Cox-van Put J., de Wied D. Influence of centrally administered {alpha}- and {gamma}2-melanocyte-stimulating hormone on hypothalamic blood flow autoregulation in the rat. Brain Res (1987) 424:189–192.[CrossRef][Web of Science][Medline]
  11. De Wildt D.J., Kasbergen C.M., Versteeg D.H. Effect of {gamma}2-melanocyte-stimulating hormone on cerebral blood flow in rats. J Cardiovasc Pharmacol (1995) 25:898–905.[Web of Science][Medline]
  12. Eberle A.N. The melanotropins. Chemistry, physiology and mechanisms of action. (1988) New York: S. Karger Publishers.
  13. Iadecola C., Reis D.J. Continuous monitoring of cerebrocortical blood flow during stimulation of the cerebellar fastigal nucleus: a study by laser-Doppler Flowmetry. J Cerebr Blood Flow Metab (1990) 10:608–617.[Web of Science][Medline]
  14. Coyle P., Jokelainen P.T. Dorsal cerebral arterial collaterals of the rat. Anat Rec (1982) 203:397–404.[CrossRef][Medline]
  15. Farah V.M., Moreira E.D., Pires M.D., Irigoyen M.C., Krieger E.M. Comparison of three methods for the determination of baroreflex sensitivity in conscious rats. Braz J Med Biol Res (1999) 32:361–369.[Web of Science][Medline]
  16. Fluckiger J.P., Sonnay M., Boillat N., Atkinson J. Attenuation of the baroreceptor reflex by general anesthetic agents in the normotensive rat. Eur J Pharmacol (1985) 109:105–109.[CrossRef][Web of Science][Medline]
  17. Stornetta R.L., Guyenet P.G., McCarty R.C. Autonomic nervous system control of heart rate during baroreceptor activation in conscious and anaesthetised rats. J Auton Nerv System (1987) 20:121–127.[CrossRef][Web of Science][Medline]
  18. Gruber K.A., Callahan M.F. ACTH-(4–10) through {gamma}-MSH: evidence for a new class of central autonomic nervous system-regulating peptides. Am J Physiol (1989) 257:R681–R694.[Web of Science][Medline]
  19. Edvinsson L., McKenzie E.T., McCulloch J. Cerebral blood flow and metabolism. (1993) New York: Raven Press.
  20. Haberl R.L., Heizer M.L., Marmarou A., Ellis E.F. Laser-Doppler assessment of brain microcirculation: effect of systemic alterations. Am J Physiol (1989) 256:H1247–H1254.[Web of Science][Medline]
  21. Skarphedinsson J.O., Hårding H., Thorén P. Repeated measurements of cerebral blood flow in rats. Comparison between the hydrogen clearance method and laser Doppler flowmetry. Acta Physiol Scand (1988) 134:133–142.[Web of Science][Medline]
  22. Lee J.G., Hudetz A.G., Smith J.J., et al. The effects of halothane and isoflurane on cerebrocortical microcirculation and autoregulation as assessed by Laser-Doppler flowmetry. Anest Analg (1994) 79:58–65.[Abstract/Free Full Text]
  23. Baumbach G.L., Heistad D.D. Regional, segmental, and temporal heterogeneity of cerebral vascular autoregulation. Ann Biomed Eng (1985) 13:303–310.[Web of Science][Medline]
  24. Ekman R., Bjartell A., Lisander B., Edvinsson B. {gamma}2-MSH immunoreactivity in the human heart. Life Sci (1989) 45:787–792.[CrossRef][Web of Science][Medline]
  25. Van Bergen P., De Winter T.Y., De Wildt D.J., Versteeg D.H. Influence of blockade of alpha 1-adrenoceptors, beta 1-adrenoceptors and vasopressin V1A receptors on the cardiovascular effects of gamma 2-melanocyte-stimulating hormone (gamma 2-MSH). Naunyn Schmiedebergs Arch Pharmacol (1997) 355:720–726.[CrossRef][Web of Science][Medline]
  26. Bryan R.R. Cerebral blood flow and energy metabolism during stress. Am J Physiol (1990) 259:H269–H280.[Web of Science][Medline]
  27. Harper A.M., Desmukh V.D., Rowan J.O., Jenett W.B. The influence of sympathetic nervous activity on cerebral blood flow. Arch Neurol (1972) 27:1–6.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Nijsen, M. J.M.A
Right arrow Articles by de Wildt, D. J
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nijsen, M. J.M.A
Right arrow Articles by de Wildt, D. J
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?