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INTEGRATIVE |
1 Department of Physiology, University College Cork, Cork, Ireland
2 Department of Environmental Health, Life Science and Human Technology, Nara Women's University, Nara, Japan
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Abstract |
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(Received 21 February 2006;
accepted after revision 6 May 2006;
first published online 11 May 2006)
Corresponding author E. J. Johns: Department of Physiology, Aras Windle, University College Cork, Cork, Republic of Ireland. Email: e.j.johns{at}ucc.i.e
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Introduction |
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Administration of angiotensin II into the brain has been found to have diverse effects on mean blood pressure and sympathetic outflow and is dependent on the site of administration. In some areas, the peptide has been reported to be vasopressor and sympatho-excitatory while in others, a hypotension and a decrease in sympathetic nerve activity has been observed which has been taken to demonstrate an action of angiotensin II on either excitatory or inhibitory pathways. It is also important to recognize that the responses obtained are dependent on the status of the animals and whether they are anaesthetized or conscious (McKinley et al. 2003). In terms of the cartotid sinus baroreflex-mediated control of renal sympathetic nerve activity, angiotensin II has been shown to exert an attenuating influence on the sensitivity of the reflex in the anaesthetized rat (Johns, 2002). It is less certain whether a similar situation pertains with regard to other sympathetic outflows or whether angiotensin II has the same action in the conscious state. By contrast, angiotensin II exerts a central nervous system facilitatory action to allow the somatosensory stimulation of renal sympathetic outflow to occur (Zhang et al. 1997; Huang & Johns, 2000). Thus, angiotensin II has important diverse influences in many of the pathways regulating sympathetic outflow.
There is evidence that in the PVN exposure to a high dietary sodium intake increases AT1 receptor expression in the brains of Dahl salt-sensitive and resistant rats, albeit with a different time course related to length of exposure (Wang et al. 2003). Furthermore, Sandberg et al. (1994) demonstrated that 3 weeks of a high salt intake in Wistar rats increased AT1a mRNA levels in decorticated brain homogenates. There are also reports that in the spontaneously hypertensive rat (SHR), AT1 receptor densities are increased compared to the levels in normotensive control rats (Veerasingham & Raizada, 2003; Gao et al. 2004). It is unclear how these increases in AT1 receptor densities induced by the high salt intake and hypertension may impact on the sensitivity or magnitude of the reflex regulation of renal sympathetic nerve activity. If receptor densities are elevated and the impact of locally generated angiotensin II is raised following a high-sodium diet or in hypertension, then it can be hypothesized that blockade of endogenous angiotensin II receptors would lead to enhanced effects under these conditions.
This situation was investigated in two ways in the present study. Firstly, the rats were fed a high-sodium diet for 4 weeks and the impact of blockade of brain angiotensin II receptors on the baroreflex gain curves for renal sympathetic nerve activity were evaluated. Secondly, studies were undertaken in a rat model of hypertension, the stroke-prone spontaneously hypertensive rat (SHRSP), which is a substrain of the SHR, where there is evidence that brain angiotensin II and angiotensin receptor levels are elevated (Veerasingham & Raizada, 2003; Gao et al. 2004).
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Methods |
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Implantation procedures
The techniques and approaches used in the present study have been described in full previously (Miki et al. 2002). The experiments were performed under asceptic conditions and all instruments were autoclaved, cannulae and electrodes were sterilized with formaldehyde gas, and all sutures and drugs were taken from commercially supplied sterile packs. All skin areas subjected to incisions were swabbed with cotton wool soaked in 75% alcohol prior to surgery. Animals were anaesthetized with sodium pentobarbital (60 mg kg–1, I.P.) and supplemental doses were given regularly thereafter if deemed necessary as a result of the pedal withdrawal reflex and frequency of respiration. Under asceptic conditions, the right carotid artery and left jugular vein were cannulated and the cannulae were taken subcutaneously to exit between the ears. The head was placed in a stereotaxic frame, a hole drilled in the skull and a guide cannula inserted into the right cerebral ventricle at a site 1.0 mm posterior to bregma, 2.5 mm lateral to the midline, and 2.55 mm ventral to the surface of the dura. The cannula was secured in place using two small jeweller's screws, inserted into the skull to form a base, and dental cement. Using a flank incision, the renal sympathetic nerves were isolated, placed on bipolar recording electrodes and sealed into place with Wacker silgel 932 (Miki et al. 2002) and, via a subcutaneous route, the recording electrodes were tunnelled to exit between the ears. The animals were then given an analgesic, buprenorphine hydrochloride (3 µg (100 g body weight)–1, S.C.), and taken to their home cage for the remainder of the study. On recovery from the anaesthetic, the animals were observed for adverse reactions at least twice per day. The cannulae and recording electrodes were attached to a tethering mechanism containing a swivel device which allowed the animal to move freely within its home cage. Each day the venous and arterial cannulae were flushed with heparin (100 and 200 i.u. ml–1, respectively) in sterile saline solution to maintain patency. Experiments were begun at least 3 days after surgery and at a time when blood pressure had decreased from the immediate postoperative high values to a lower stable level.
Blood pressure was measured using a pressure transducer (Spectromed, Oxnard, CA, USA) and an amplifier (Grayden Electronics, Birmingham, UK). Renal sympathetic nerve activity was amplified with a gain of 100 000 and high- and low-pass filters were set at 0.2 and 2 kHz. Both blood pressure and renal nerve signals were displayed on an oscilloscope and stored on videotape. The signals were digitized with a sampling frequency of 1000 Hz to generate mean blood pressure, heart rate and renal sympathetic nerve activity which were integrated, displayed and stored on the hard disk every 1 s and were used for off-line analysis using LabVIEW (National Instruments, Austin, TX, USA) software.
Experimental protocol
The experiments took place in the mornings after the rats had been connected to the transducers and nerve amplifiers and when they were in a quiet resting state. Baroreflex regulation of renal sympathetic nerve activity and heart rate was evaluated by giving doses of phenylephrine and nitroprusside (10 µg in a volume of 0.2 ml saline for each) which were infused manually (0.05 ml every 10 s) over 40 s to increase and decrease blood pressure by 50–60 mmHg, respectively. This allowed the generation of a dynamic baroreflex gain curve for both renal sympathetic nerve activity and heart rate. The minimum value for renal sympathetic nerve activity was that achieved when mean blood pressure reached its highest level and this was removed from all readings taken that day before (baseline) and during the generation of the baroreflex curves. The baseline value was a 3.5-min recording taken prior to the first vasopressor or vasodepressor injection and was taken as 100% and all values were calculated as percentages of this value (Miki et al. 2003; Nagura et al. 2004; Miki & Yoshimoto, 2005). This was done to obviate the differences apparent in the absolute levels of renal sympathetic nerve activity obtained from these multifibre recordings. The absolute values of renal sympathetic nerve activity recorded were very dependent upon the surgical skill of the operator and the anatomical presentation of the nerves while a further consideration was the gradual deterioration in the signal each day of the study. A four-parameter logistic equation (Kent et al. 1972) was used to generate a baroreflex gain curve in which average values of renal sympathetic nerve activity and heart rate were calculated for each 5-mmHg change in blood pressure using the 1-s values stored on the hard disk. This allowed calculation of the range (A1) over which the control operated, the sensitivity or curvature coefficient of the relationship (A2), the mid-point mean blood pressure of the curve (A3) and the lowest point to which the renal sympathetic nerve activity or heart rate could be driven (A4). On completion of the generation of the first baroreflex curve, the animals were given a bolus intracerebroventricular (I.C.V.) dose of 50 ng angiotensin II, which initiated a response whereby the animals immediately sought water and drank on average 8.7 ± 1.1 ml water in the subsequent 20 min. Losartan was given I.C.V., 15 µg in 2 µl saline over 2 min. A second bolus of angiotensin II was given I.C.V. approximately 60 min after the losartan infusion I.C.V., and at this stage the angiotensin II failed to elicit any drinking behaviour. This was taken to confirm the effective delivery of drugs into the central nervous system. A second baroreflex curve was then generated approximately 20 min later. At the end of the study the animals were killed humanely using an overdose of anaesthetic.
Statistical analysis
The mean values of all data were calculated from individual rats, and are presented as means ± S.E.M. On each day, the background or minimum noise level in the renal nerve signal was determined as that obtained during the peak increase in blood pressure in response to the bolus dose of phenylephrine, and this value was subtracted from all readings. Comparisons were undertaken using a two-way ANOVA (SuperANOVA, Abacus Software Concepts Inc., Berkley, CA, USA) to test for differences between groups, losartan and losartan-group interactions and one-way ANOVA within a group to evaluate the impact of losartan. Significance was taken when P < 0.05.
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Results |
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Discussion |
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Mean blood pressure was elevated on the first postoperative day but over the subsequent 2 days decreased by 15–20% to a lower stable value. Over this period, heart rate fell slightly by approximately 2%, but not significantly, and over the period of study baseline values remained at approximately 460 beats min–1. These values are close to those reported previously by Miki et al. (2002) and Nagura et al. (2004) in the conscious rat and indeed, in these studies, it was demonstrated that exercise was able to increase heart rate to much higher levels. The heart rates of the conscious rats reported here are higher than those reported by others using comparable conscious preparations (Bexis & Docherty, 2006; Milutinovic et al. 2006) which suggests that the animals may remain in a somewhat stressed state over the period of observation.
The absolute values for the multifibre renal sympathetic nerve activity recording varied greatly between animals and tended to decrease on a day by day basis and therefore in the baroreflex gain curve studies, the nerve activity was presented in the Fig. 1 as percentage changes based on basal levels recorded each day at the start of the experiment. This was the approach that had been used and validated in earlier studies (Miki et al. 2003; Nagura et al. 2004). Nonetheless, the parameters were also calculated in absolute terms and these are given in the Tables. It was also important to be sure that the I.C.V. cannula was in place and was effective. Indeed, the observation that the angiotensin II-induced drinking response was blocked by the I.C.V. administration of the angiotensin AT1 receptor antagonist losartan provided evidence that the cannula was functional and that physiological responses could be obtained.
It has been observed that in the conscious animal the behavioural state can importantly impact on the characteristics of the baroreflex curve for renal sympathetic nerve activity. In earlier studies it was shown that in rapid eye movement sleep there was a shift in the gain curve to lower levels of renal sympathetic nerve activity but at the same level of blood pressure (Nagura et al. 2004), whereas with increasing intensity of exercise the curve was not only shifted to a higher level, but was also moved to the right indicating a raised sensitivity (Miki et al. 2003; Miki & Yoshimoto, 2005). Because of these concerns, every effort was taken to ensure that the measurements were carried out while the animals were in a quiet awake state. The characteristics of the baroreflex gain curve and the values of the four parameters generated for the renal sympathetic nerve activity and heart rate obtained under basal conditions in the rats on the normal salt diet were closely comparable to those published earlier (Miki et al. 2003) in the same behavioural state.
It was apparent in the animals maintained on the normal sodium diet that, following administration of the losartan I.C.V., the sensitivity of the baroreflex gain curve for renal sympathetic nerve activity was increased. This would be consistent with angiotensin II exerting a tonic inhibitory action on the baroreflex regulation of sympathetic outflow to the kidney. These findings would be compatible with the observations of Hayashi et al. (1988) and Matsumura et al. (1998) using the splanchnic sympathetic nerves of anaesthetized rats, and of Johns (2002, 2005) using renal sympathetic nerves in the anaesthetized SHR and Wistar rat, demonstrating that the impact of the baroreceptors to activate sympathetic outflow is attenuated by angiotensin II within the brain. The sites at which angiotensin II might exert these effects are ill-defined and undoubtedly complex, as receptors have been demonstrated on cell bodies in PVN, RVLM, CVLM and NTS while the presence of angiotensin II has been detected in the nerve axons of pathways linking these areas (McKinley et al. 2003; Saigusa et al. 2003; Tan et al. 2005). At each nucleus, local application of angiotensin II can be either excitatory or inhibitory (McKinley et al. 2003; Saigusa et al. 2003; Tan et al. 2005) which is of inherent interest but does not aid in explaining the overall impact of angiotensin II on the baroreflex pathway when its levels are raised or lowered by dietary salt manipulation or pharmacological blockade.
It is interesting that in the rats fed the high-salt diet, the value of the baroreceptor gain curve parameters for renal sympathetic nerve activity were very similar to those obtained in the rats maintained on the normal salt diet. Moreover, blockade of the AT1 receptors in the cerebroventricles with losartan increased the sensitivity of the relationship by the same magnitude as that observed in the rats maintained on the normal diet. These observations were somewhat surprising given the evidence that the level of angiotensin II receptor gene expression (Strehlow et al. 1999) and the density of AT1 receptors, evaluated by quantitative autoradiography (Wang et al. 2003), have been shown to be elevated by subjecting the animals to a high dietary sodium intake. The reasons underlying this inability to demonstrate a functional enhancement of the responses to the exogenously administered angiotensin II are unclear. It may be dependent on the duration of exposure and the level of sodium content in the diet as Huang & Leenen (1992) demonstrated that there was a depression of the baroreceptor regulation of renal sympathetic nerve activity after 3 weeks of a high-salt diet. However, the nerve recordings in that study were undertaken in conscious rats, but only 4–5 h after surgery when the effects of surgical stress and residual anaesthetics could have impacted on the findings. On the other hand, recent studies by DiBona & Jones (2001a,b) examining the effect of dietary sodium manipulation on the action of angiotensin II at the PVN and RVLM in anaesthetized rats, showed that elevation in dietary sodium intake for 2 weeks had little effect on the magnitudes of the blood pressure and renal sympathetic nerve activity responses which contrasted with the enhancement observed in the rats maintained on a low-sodium diet for the same period of time. Thus from a functional point of view, it may be that although receptor densities in various nuclei and nerve tracts may be enhanced by the different dietary sodium intakes, interactions between the various pathways mean that in an integrative sense, no overall change can be detected.
The administration of losartan I.C.V. had no influence on basal levels of blood pressure or heart rate. This observation was somewhat surprising given that the brain renin–angiotensin system appears activated in the SHR, which is also likely to occur in the SHRSP substrain, and there have been reports that blockade of the brain renin–angiotensin system reduces blood pressure (Berecek et al. 1987; Yang et al. 1992; Allen, 2002). The reasons underlying this lack of effect of losartan on blood pressure are unclear but may be related to the dose of compound used, the time frame and whether the animals were anaesthetized or conscious at the time of study. It was evident from the present study that in the SHRSPs, the sensitivity of the baroreflex curves for renal sympathetic nerve activity, reflected as the curvature coefficient, was depressed compared with that of the normotensive Wistar control rats. This finding supports our earlier observations using anaesthetized and conscious SHRs (Bristow & Johns, 1996, 1997), albeit with losartan being given systemically rather than I.C.V., and similar observations by Gao et al. (2004) in the anaesthetized SHR. The blockade of brain angiotensin II receptors with losartan caused a marked increase in the baroreflex curvature coefficient for renal sympathetic nerve activity in the SHRSPs which was proportionately greater than that obtained in the Wistar rats maintained on the normal sodium diet. This would suggest that the baroreflex control of renal sympathetic nerve activity is more under the influence of angiotensin II in the SHRSPs than in normal rats. This view would be supported by the observations in rat hypertension models, that angiotensin II receptor expression is elevated within the brain (Veerasingham & Raizada, 2003; Gao et al. 2004) and that angiotensin II is able to exert a greater action in the hypertensive state. A further point of note was that the baroreflex control of heart rate was not altered by the I.C.V. losartan. Again, there have been varying reports as to the role of brain angiotensin II in regulating sympathetic and parasympathetic control of heart rate and because of the antagonistic nature of the two systems on the control of heart rate it becomes difficult to obtain a clear understanding of how they interact.
In this study we examined the impact of feeding a high-sodium diet, from weaning to maturity, and the development of hypertension on the baroreceptor regulation of renal sympathetic nerve activity and heart rate. Under these conditions, it has been reported that the brain renin–angiotensin system is activated. Using conscious, chronically instrumented rats, the contribution of angiotensin II within the brain to the sensitivity of the baroreceptor gain curves for renal sympathetic nerve activity and heart rate, determined as the curvature coefficient, was measured and the effect of dietary salt manipulation was determined. It was evident that blockade of angiotensin II receptors in the brain with I.C.V. losartan increased the sensitivity of the baroreceptor gain curves for renal sympathetic nerve activity, but to the same degree in the rats on the regular and the high-sodium diets. Moreover, the baroreceptor regulation of heart rate was unchanged by the I.C.V. losartan irrespective of the level of dietary sodium intake. Comparative studies in the SHRSP demonstrated that the baroreflex regulation of renal sympathetic nerve activity was depressed and also tonically inhibited by brain angiotensin II, and to a greater degree than that in Wistar rats. Again, angiotensin II in the brain did not influence the baroreceptor control of heart rate. Together, these data support the view that angiotensin II in the brain exerts a tonic inhibitory action on the baroreceptor control of renal sympathetic nerve activity which was not influenced by elevation in dietary sodium intake, but was exaggerated in the hypertensive state.
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Acknowledgements |
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