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Am J Physiol Heart Circ Physiol 278: H558-H566, 2000;
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Vol. 278, Issue 2, H558-H566, February 2000

Effects of central infusion of ANG II and losartan on the cardiac baroreflex in rabbits

Elisabeth Gaudet, Shirley J. Godwin, and Geoffrey A. Head

Baker Medical Research Institute, Melbourne, Victoria 8008, Australia


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The effect of chronic activation or inhibition of central ANG II receptors on cardiac baroreflex function in conscious normotensive rabbits was examined. Animals received a fourth ventricular (4V) infusion of ANG II (30 and 100 ng/h), losartan (3 and 30 µg/h), or Ringer solution (2 µl/h) for 2 wk. After 1 and 2 wk, ANG II (100 ng/h) decreased cardiac baroreflex gain by 20 and 37%, respectively (P = 0.015), whereas losartan (30 µg/h) increased baroreflex gain by 24 and 58%, respectively (P = 0.02). Within 1 wk of the end of the infusions, cardiac baroreflex gain had returned to control. Ringer solution or the lower doses of ANG II or losartan did not modify the cardiac baroreflex function. Blood pressure and heart rate were not altered by any treatment, nor was their variability affected. These data demonstrate a novel long-term modulation of cardiac baroreflexes by endogenous ANG II that is independent of blood pressure level.

blood pressure; angiotensin type 1 receptors; fourth ventricular administration; long-term infusion


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE IMPORTANCE OF SPECIFIC regions within the brain stem, such as the nucleus tractus solitarius (NTS), the dorsal motor nucleus of the vagus nerve, and the rostral and caudal ventrolateral medulla, for central cardiovascular control is well known. These regions contain high densities of ANG II binding sites (30), but because they are behind the blood-brain barrier, they are presumably not accessible to circulating ANG II (36). They are most likely activated by the peptide derived from a brain renin-angiotensin system. Indeed, there has been considerable interest in the function of a putative renin-angiotensin system in the brain stem that can influence the sympathetic nervous system through direct actions on the bulbospinal premotor neurons (1). Injection of ANG II into the NTS decreases the sensitivity of the cardiac baroreflex (11), and inhibition of endogenous ANG II in the NTS facilitates the baroreceptor reflex sensitivity (9).

Studies from our laboratory found that injections of low doses of ANG II into the fourth ventricle (4V) of conscious rabbits produced dose-dependent increases in sympathetic activity and blood pressure (18, 21) and that removal of baroreceptor afferent input by sinoaortic denervation markedly increased the sensitivity to ANG II (13). Short-term infusion of ANG II produced a marked activation of neurons within the rostral ventrolateral medulla, NTS, and A5 (23), and direct microinjection of ANG II into the caudal ventrolateral medulla induced sympathoinhibition (35). Given these findings, we have been particularly interested in the possible involvement of the brain stem renin-angiotensin system in baroreflex regulation. Dorward and Rudd (12) showed that infusion of ANG II into the 4V produced a marked facilitation of the renal sympathetic reflex, but there was no effect on cardiac baroreflexes. Our more recent findings with the angiotensin AT1 receptor antagonist losartan and the angiotensin-converting enzyme inhibitor enalaprilat revealed an important role of ANG II in the brain stem to modulate sympathetic function in a dual sympathoinhibitory and sympathoexcitatory manner involving central catecholaminergic pathways (4, 14). However, the AT1 receptor antagonist or converting enzyme inhibitors of ANG II had no effect on cardiac baroreflexes (4, 14). This lack of effect was somewhat surprising, given that the dose of losartan that we used abolished the pressor response to ANG II injected into the 4V (14), and suggests that there is little tonic activation of the AT1 receptors in the brain stem of conscious normotensive animals.

In a recent study in conscious rabbits made hypertensive by a continuous infusion of ANG II intravenously for 6 wk, we observed the usual resetting of the baroreflex that has been commonly reported in hypertension, but we also observed an initial reduction in the gain of the cardiac baroreflex after 1 wk (28). Most forms of hypertension result in an impairment of cardiac baroreflexes, whereas sympathetic reflexes appear to be much less affected (16). However, the reduction in gain was due to a reduction in the curvature of the reflex rather than the range of the reflex, which is typical of hypertension and probably due to changes in cardiac hypertrophy (16). In our chronic infusion study with intravenous ANG II, the change in the gain was independent of the development of cardiac hypertrophy and had the characteristics of an effect within the central nervous system (17). This suggests that although under normal conditions in conscious rabbits cardiac baroreflexes are not influenced by endogenous or exogenous ANG II, there may be an influence with long-term alteration to levels of ANG II such as we have observed. However, it remained to be established whether this was independent of the hypertension. Thus in the present study we wished to investigate the chronic effect of administration of ANG II and the AT1 receptor antagonist losartan on cardiac baroreflex function in conscious animals. We chose central, rather than systemic, administration, since long-term infusions of low subacute pressor doses of ANG II can result in hypertension, whereas pilot studies showed that this was not the case with chronic 4V administration. In addition to the assessment of baroreflexes, we used power spectral analysis to examine the influence of the various treatments on short-term blood pressure and heart rate (HR) variability.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal preparation. Experiments were performed in 29 rabbits (2.6-2.9 kg) of either gender bred and housed at the Baker Medical Research Institute. The colony is derived from multicolored English and Dutch belted rabbits. All procedures were performed in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (2). All rabbits were housed under controlled temperature and humidity and a 12:12-h dark-light cycle and were fed a restricted diet of pellets (0.5% sodium chloride) and vegetables but with water available ad libitum.

At least 6 wk before experiments, rabbits were implanted with a chronic vinyl 4V catheter (ID 0.28 mm, OD 0.61 mm; model SV10, Dural Plastics and Engineering, Auburn, NSW, Australia) through the atlantooccipital membrane, as described previously (19). The rabbits were intubated after induction of anesthesia with administration of propofol (Diprivan, ICI Pharmaceutical, Australia; 1 mg/kg iv) and then placed on halothane anesthesia (Fluothane, ICI Pharmaceuticals) with the use of a vaporizer (Goldman). At least 4 wk later, in anesthetized animals, an inflatable occluding balloon (Silastic, Dow Corning, Midland, MI) was implanted around the intrathoracic vena cava for later baroreflex experiments, as described previously (27).

Experimental preparation. On the day of the experiment, the rabbit was placed in a standard single-rabbit holding box. The end of the 4V catheter and the perivascular balloon catheter were retrieved from under the skin, which had been anesthetized locally (1% prilocaine; Citanest, Astra Pharmaceuticals). A 24-gauge, 19-mm Teflon catheter (Jelco, Critikon, Pomezia, Italy) was inserted into a marginal vein for drug injections. The central ear artery was cannulated transcutaneously with a 22-gauge, 25-mm Teflon catheter (Jelco) also under local anesthesia. The catheter was then connected to a Statham P23Dc pressure transducer for continuous measurement of mean arterial pressure (MAP) and HR, and the animal was allowed a 1-h recovery period before commencement of the experiment. The basal MAP and HR levels were recorded for 40 min. Arterial pressure was digitized at 200 Hz with a data acquisition card (PC plus, National Instruments, Austin, TX) and a computer program written in Labview graphical programming language (National Instruments). The program calculated MAP instantaneously from the detected systolic and diastolic pressures as well as heart period (interval between beats) and a corresponding instantaneous HR. All parameters for each heartbeat, as well as 2-s averages, were saved onto disk. Cardiovascular parameters were also recorded on a polygraph (model 7D, Grass Instruments, Quincy, MA) during the experiment, in which case the arterial pressure was dampened to derive the MAP and also used to trigger an HR meter (model 173B, Baker Institute).

Experimental protocol. Each animal underwent five experiments, each separated by 1 wk. The two first experiments were control experiments (i.e., before the start of the infusions), in which basal measurements, MAP-HR baroreflex assessment, and initial ANG II dose-response curves were performed. At the end of the second control experiment, rabbits were randomly assigned to one of five groups and infused for 2 wk with 1) a low dose of ANG II (30 ng · 2 µl-1 · h-1, n = 6), 2) a high dose of ANG II (100 ng · 2 µl-1 · h-1, n = 6), 3) a low dose of losartan (3 µg · 2 µl-1 · h-1, n = 7), 4) a high dose of losartan (30 µg · 2 µl-1 · h-1, n = 5), and 5) Ringer solution (2 µl/h, n = 5). ANG II (human Ile form) was purchased from Peninsula Laboratories (Belmont, CA); losartan was obtained from DuPont (Wilmington, DE). Infusions were made with an osmotic minipump (model 2ML, Alzet, Palo Alto, CA) connected to the 4V catheter and placed under the skin at the back of the neck under local anesthesia. The third and fourth experiments were performed 1 and 2 wk after the onset of the drug infusions, and the final fifth experiment, "recovery," was performed 1 wk after cessation of the infusions.

On each experimental day, MAP and HR were recorded for 30 min, and the HR baroreflex was assessed. Dose-response relationships for ANG II (0.00032-125 pmol in 25 µl) administered via the 4V catheter were examined on the 1st experimental day and also after the 2-wk infusion period (4th experiment). Nine cumulative increasing doses of ANG II were administered at 15-min intervals (4). An average 2-min reading of MAP was recorded during the peak increase in MAP, and the change of MAP from the resting level was calculated. To determine whether the central losartan infusion affected peripheral vascular ANG II receptors, the pressor response to a bolus of ANG II (0.6 µg/25 µl iv) was examined on one occasion before the infusion and also at the end of the 2-wk infusion in losartan- and Ringer solution-treated animals. At the completion of the experiments, animals were killed by overdose with pentobarbitone sodium (Laboratories, Hornsby, NSW, Australia), and the position of the 4V catheter was confirmed by visual inspection.

Analysis of the MAP-HR baroreflex relationship. The HR baroreflex was assessed by a single slow ramp rise in MAP by infusion of phenylephrine hydrochloride (0.5 mg/ml iv; Sigma Chemical, St. Louis, MO) or a fall in MAP produced by caval balloon inflation. Injections/inflations lasted 0.5-1 min, and the rate of change in MAP was controlled between 0.5 and 1 mmHg/s. MAP and HR were averaged over 2-s intervals and fitted to a sigmoidal logistic function to produce MAP-HR curves, as previously described (34). The equation has two curvature parameters, which allows for a nonsymmetrical sigmoidal curve.

Spectral analysis. The beat-to-beat signals of instantaneous MAP and HR corresponding to each 30-min resting period recorded on each experimental day were analyzed for spectral analysis with a computer program written in Labview (National Instruments) by Dr. E. Lukoshkova (National Cardiology Research Center, Moscow, Russia). Data were displayed and artifacts were eliminated by spline interpolation and low-pass filtering (cut-off period = 200 ms). A moving 30-point standard deviation (SD) was calculated for each variable, and by a threshold cursor, periods of continuously low SD were selected for further analysis. The data were resampled at 5.12 Hz, partitioned into 100-s lengths (512 points), detrended using a linear regression, windowed with a tapered cosine function, padded with zeros up to 1,024 points, and then subjected to a 50% overlapping fast Fourier transform (5). The average power spectrum was calculated in a midfrequency 0.2- to 0.4-Hz range and a low-frequency 0.025- to 0.2-Hz range.

Statistical analysis. Values are means ± SE. For the baroreflex and hemodynamic parameters, a split-plot (nested) ANOVA, which combines within-animal and between-group comparisons, was used (26). The total sums of squares (SS) were divided into between- and within-groups SS. The latter contained the between-experiment days SS, between-animals SS, and animal × day interaction for each of the five groups. Comparisons within each group were made by using a set of four orthogonal contrasts as follows: 1) the treatments (weeks 3 and 4) were compared with control experiments (weeks 1 and 2), 2) recovery (week 5) was compared with the two comparison control experiments, 3) comparison between control experiments (week 1 vs. week 2), and 4) comparison between treatment weeks (week 3 vs. week 4). The F ratio for each contrast was calculated as the mean square (MS) for the contrast divided by the total residual MS of the five groups. Thus the estimate of the within-group variance was made with a contribution from all the groups. Between-group comparisons were made using the F ratio of the between-group MS divided by the rows × groups interaction. A comparison of the different control periods was also made between groups. For the dose-response curve data, a similar approach was used, except only the effects of the three highest doses of ANG II were used in the ANOVA. P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Hemodynamic resting values and parameters of the cardiac baroreflex curves. Basal MAP and HR values obtained before any treatments were similar among the five experimental groups and averaged 85.9 ± 1.3 mmHg and 215 ± 8 beats/min, respectively, on the first experiment (F4,24 between groups < 1.4, P = 0.4, n = 29). Cardiac baroreflexes were sigmoidal, with an average upper plateau of 348 ± 7 beats/min, a lower plateau of 123 ± 4 beats/min, a range of 225 ± 8 beats/min, and an average gain of -7.1 ± 0.5 beats · min-1 · mmHg-1. The baroreflex curves were not symmetrical, with the curvature being 1.9 times greater near the tachycardia plateau than at the bradycardia plateau (P < 0.001). This asymmetry was consistently observed across the five groups. For all hemodynamic and baroreflex parameters, no differences were observed between the first and the second control experiment (F1,28 < 1.8, P > 0.5). Body weight of the 29 rabbits in the study was 2.67 ± 0.03 kg at the beginning of the experiments and did not vary during the 5 wk of the study in any of the treatment groups. The infusion of Ringer solution into the 4V did not affect the MAP, HR, or any of the parameters of the cardiac baroreflex curve (Fig. 1).


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  		Fig. 1.   Left: cardiac baroreflex curves before (open circle , dashed line) and after (, solid line) 2 wk of central infusion of Ringer solution (A) or ANG II [30 ng/h (B) or 100 ng/h (C)]. Circles on curves are basal values. Error bars, average SE. HR, heart rate; MAP, mean arterial pressure. Right: gain of cardiac baroreflex before, during (solid bars), and after 2 wk of infusion of Ringer solution (A) or ANG II [30 ng/h (B) or 100 ng/h (C)]. C, control value taken as average of 2 control experiments; 1 and 2, 1 and 2 wk of treatment; P, posttreatment. * P < 0.05, control vs. 1 and 2 wk of treatment.

Cardiovascular and baroreflex effects of 4V infusion of ANG II. To determine the chronic effect of exogenous central ANG II on cardiac baroreflexes, a low dose (30 ng/h) and a high dose (100 ng/h) of ANG II were constantly infused into the 4V of separate groups of rabbits for 2 wk. Neither treatment altered MAP or HR during this period. The lower dose did not elicit any changes in the cardiac baroreflex, whereas the higher dose caused a reduction in the gain of the reflex at 7 and 14 days after the onset of the infusion by 20 and 37%, respectively (F1,20 = 7, P = 0.015; Fig. 1, Table 1). Although there was a tendency for the effect to be greater at 14 days, this did not reach statistical significance (P = 0.2). The reduction in gain was solely due to a 43% decrease in the leftward curvature, which is near the tachycardia plateau (F1,20 = 15.2, P < 0.001). The other parameters of the cardiac baroreflex curve were not modified (Table 1). One week after the end of the infusion of ANG II, the gain of the baroreflex returned to a level close to that observed before the start of the infusion.

                              
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  		Table 1.   Resting values and parameters describing MAP-HR baroreflex curves before, during, and after 4V infusion of ANG II

Cardiovascular and baroreflex effects of 4V infusion of losartan. We examined the chronic effect of blocking endogenous ANG II in the brain stem on cardiac baroreflexes with a low (3 µg/h) and a high dose (30 µg/h) of losartan administered into the 4V to rabbits over a 2-wk period. The lower dose increased the lower plateau slightly (P = 0.04, Table 2) but did not elicit any other changes in the cardiac baroreflex. The higher dose caused an increase in the gain of the reflex by 24% 7 days after the onset of the infusion, which was further augmented after 14 days of infusion to 58% (P = 0.02; Fig. 2, Table 2). The change in gain was due to a 58% increase in the leftmost curvature parameter (i.e., close to the tachycardia plateau; P = 0.01) and a 55% increase in the rightward curvature parameter (close to the bradycardia plateau; P = 0.02). None of the other cardiac baroreflex parameters were affected by losartan treatment. Neither dose altered MAP or HR during this period. One week after the end of the losartan infusion, baroreflex gain returned to the values similar to those observed before the start of the infusion.

                              
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  		Table 2.   Resting values and parameters describing MAP-HR baroreflex curves before, during, and after 4V infusion of losartan



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  		Fig. 2.   Left: cardiac baroreflex curves before (open circle , dashed line) and after (, solid line) 2 wk of central infusion of losartan [3 µg/h (A) or 30 µg/h (B)]. Circles on curves are basal values. Error bars, average SE. Right: gain of cardiac baroreflex before, during (solid bars), and after 2 wk of infusion of losartan [3 µg/h (A) or 30 µg/h (B)]. See Fig. 1 legend for definition of abbreviations. * P < 0.05, control vs. 1 and 2 wk of treatment.

Effect of the 4V treatments on dose-response curves to 4V infusion of ANG II. To test whether the 4V infusions of the different drugs were able to alter the response to 4V injection of ANG II, dose-response curves to 4V administration of ANG II were performed during the initial control experiment and 2 wk after the onset of the drug infusions. ANG II produced similar dose-dependent increases in MAP in the five experimental groups of rabbits (Fig. 3). The average maximum increase produced by the dose-response curves was 12-15 mmHg. After 2 wk of central infusion of Ringer solution, the dose-response curve to 4V ANG II was similar to the control curve. After the 2-wk 4V infusion of the low dose of ANG II (30 ng/h), the maximum MAP rise to 4V ANG II (taken as the average of the 3 last doses of ANG II) decreased by 28% (P < 0.001). The 2-wk 4V infusion of the high dose of ANG II (100 ng/h) did not modify the dose-response curve to 4V ANG II.


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  		Fig. 3.   Average change in MAP (Delta MAP) in response to 4th ventricular ANG II before (open circle ) and after () 2 wk of central infusion of Ringer solution (2 µl/h, A), ANG II [30 ng/h (B) or 100 ng/h (C)], or losartan [3 µg/h (D) or 30 µg/h (E)]. * P < 0.05, ** P < 0.001, before vs. after treatment (with 3 last doses of ANG II).

Both doses of losartan infused into the 4V for 2 wk markedly attenuated the dose-response curve to 4V ANG II. The maximum pressor responses were decreased by 30% (P < 0.05) and 61% (P < 0.001) for 3 and 30 µg/h, respectively.

Effect of the 4V treatments on intravenous administration of ANG II. To determine whether the central losartan infusion affected peripheral vascular ANG II receptors, the pressor response to a bolus of ANG II (0.6 µg/25 µl) was examined in Ringer solution- and losartan-treated animals. In the group receiving the Ringer solution, the increase in MAP was 16 ± 3 mmHg before the onset of the infusion and was similar 2 wk after the start of the infusion (14 ± 4 mmHg). In the losartan-treated groups (3 and 30 µg/h), the increase in MAP in response to intravenous ANG II was 14 ± 2 and 12 ± 3 mmHg, respectively, before the infusions and was not different at the end of the 2-wk infusion (18 ± 4 and 16 ± 3 mmHg, respectively).

Effect of 4V infusions on power spectral analysis of MAP and HR. Spectral analysis was performed on the MAP and HR recordings obtained on each experimental day, before, during, and after the 4V infusion of the different drugs. We did not observe any changes in the power spectrum of the MAP and HR in the mid- (Table 3) or the low-frequency band (data not shown).

                              
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  		Table 3.   Midfrequency components of MAP and HR before, during, and after 4V infusion of Ringer solution, ANG II, and losartan


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The principal finding of the present study was that long-term infusion of ANG II into the 4V of conscious normotensive rabbits reduced the gain of the cardiac baroreflex, whereas long-term treatment with the AT1 receptor antagonist losartan produced an increase in the baroreflex gain. Both effects were relatively slow to develop: they were maximal after 2 wk of treatment and occurred without any change in blood pressure or HR. The opposite effects of ANG II and losartan suggest that the effect on the cardiac baroreflex was a specific effect mediated by AT1 receptors that inhibits the cardiac baroreflex. The effect of losartan suggests that, in the conscious normotensive rabbit, there was activation of these receptors, presumably from an endogenous ANG II or ANG II-like peptide.

The intriguing feature of this study was the significant effect of high doses of ANG II and losartan on the gain and curvature of the baroreflex, with the maximum effect seen at the end of the 2-wk treatment period. We previously observed that an acute dose of losartan or ANG II into the 4V of conscious normotensive rabbits did not modify the cardiac baroreflex function (4, 12, 14), suggesting that the time frame for the manifestation of the effect of ANG II and losartan to activate or block, respectively, AT1 receptors and influence the cardiac baroreflex was on the order of weeks. The reason for this is not clear, since activation of neuronal AT1 receptors by ANG II occurs relatively rapidly, being due to a reduction in K+ current via protein kinase C and raised intracellular Ca2+ and stimulation of Ca2+ current (37). The delay of response that we have seen is orders of magnitude greater than this process. Nevertheless, long-term changes in the kinetics of channel activation and inactivation, changes in the rate of sensitization and desensitization, or possible alteration of expression of AT1 receptors cannot be ruled out as a mechanism. Another possibility may involve alteration to the levels of biosynthetic/degradative enzymes that, in turn, influence the levels/release or actions of other peptides or regulation of other receptors. Such is the case with converting enzyme inhibitors that prevent the formation of ANG II from ANG I but also result in an increase in bradykinin levels. Indeed, an interaction has been observed between ANG-(1-7) and bradykinin influencing the HR baroreflex, mediated, at least in part, by the release of kinins (6).

The changes in the sensitivity of the cardiac baroreflex were observed with relatively low doses of drugs. Previous observations performed on Wistar-Kyoto and spontaneously hypertensive rats (32) also demonstrated the ability of losartan to improve the cardiac baroreflex function after a 3-h central infusion without modifying MAP. However, the dose that they used was more than 3 times higher than ours and 30 times higher on a per kilogram body weight basis. Furthermore, the doses we used were not on the top of the dose-response curve, since the lower doses of either drug were not effective at altering the baroreflex within the time frame of the experiment. The highest dose of losartan we used reduced the pressor response to ANG II by 60%, suggesting that this is the threshold dose, whereas the lower dose reduced the pressor response by 30%. Thus it appears that a significant proportion of the AT1 receptors must be blocked before the chronic effect on the baroreflex is seen. The nonhypotensive effect of centrally administered losartan has been previously observed (4, 14, 32) and suggests that the antihypertensive effect of intravenous or oral administration of losartan involves peripheral AT1 receptor blockade or central AT1 receptors localized in sites not accessible by 4V administration. The finding of the lack of effect of 4V administration of losartan on MAP after intravenous injection of ANG II indicates that the increase in the sensitivity of the baroreflex produced during central infusion of losartan is a result of action on neuronal elements within the central nervous system involved with baroreceptor regulation, rather than the result of leakage of these compounds into the systemic circulation. We do not believe that the changes in baroreflex function produced by losartan and ANG II are a result of a nonspecific effect of the treatments, since there were no changes in body weight of the animals, suggesting that body fluid homeostasis was well maintained during the treatment period.

The mechanism of the reduction in the gain of the baroreflex by ANG II was due to a reduction in the leftward curvature, i.e., close to the tachycardia plateau, rather than a change in the range of the reflex. All baroreflex curves showed a degree of asymmetry, with the leftward curvature being nearly double the rightward curvature. The cause of this has been shown to be due to the different methods used to obtain the pressor or depressor parts of the curve (25). Thus the effect of ANG II was to make the baroreflex curves symmetrical. By contrast, losartan increased the gain by increasing both curvature parameters. The finding that activation or blockade of AT1 receptors influences the gain by altering the curvature is consistent with a central action on the cardiac baroreflex, since many agents that are given into the cerebrospinal fluid surrounding the brain stem, such as endothelin or clonidine, affect the curvature parameter rather than the HR range (17). In our recent study, where the rabbits were made hypertensive by an intravenous infusion of ANG II, we also observed that the impairment of the gain of the baroreflex resulted from a modification in the curvature (28). Given that this observation was very similar to the present one, we can speculate that the effect observed in our hypertensive rabbits probably resulted from a central effect of ANG II and was independent of the hypertension state.

From our results, it appears that ANG II and losartan did not modify the maximum sympathetic activation and the maximal vagal activation, inasmuch as the upper and lower plateaus of the baroreflex curves were not altered (20). When baroreflex gain is modified, the variability in blood pressure and HR would be expected to be altered as well, each in the opposite direction. However, in the present study, although the gain of the baroreflex was decreased or increased with the central infusion of ANG II or losartan, surprisingly, the HR and blood pressure variability remained unchanged. The sympathetic and the vagus are involved in the baroreflex sensitivity of the cardiac baroreflex, with the vagus being predominant (20). At rest, the midfrequency oscillations of HR are small, owing to a predominant vagal tone, whereas the midfrequency oscillations of blood pressure mainly seem to result from rhythmic fluctuations in the vasomotor sympathetic tone (33). We could speculate that the changes in the sensitivity of the cardiac baroreflex may result from an effect on the cardiac sympathetic component, which may not be a major determinant of the midfrequency oscillations of blood pressure and HR. Alternatively, central pathways that influence the shape of the curve, such as the contribution from nonarterial cardiopulmonary receptors (38), may be preferentially affected by the endogenous ANG II system. The lack of effect of both drugs on MAP and HR variability indicates that the relationship between HR variability and the baroreflex gain determined by large excursions in pressure needs further clarification. Nevertheless, the use of the five-parameter fitting method, which permits the analysis of the asymmetry of the baroreflex curves, has been most useful in the present study to pinpoint the exact nature of the changes in the shape of the curves and indicate the direction of further experiments.

From our study, we did not determine the site of action of ANG II and, hence, can only speculate on the location responsible for the cardiac baroreflex impairment by ANG II. The decrease of the cardiac baroreflex gain observed after intravenous infusion of ANG II in previous studies (7, 10, 28) suggests that the mechanism of action of ANG II on the gain of the cardiac baroreflex may be different whether ANG II is given intravenously or centrally and indicates that the ANG II receptors involved in the modification of the gain of the cardiac baroreflex are present on different sites within and outside the blood-brain barrier. Using the expression of c-fos as a marker of neuronal activity, we previously identified the neurons within the medullary regions that are activated after 4V infusion of ANG II in conscious rabbits (23). We found that ANG II increased the number of c-fos cells in the NTS, the area postrema, and the caudal, intermediate, and rostral ventrolateral medulla. This was in agreement with the work of Sasaki and Dampney (35), who described a tonic excitatory action on sympathoexcitatory and sympathoinhibitory neurons within the rostral and caudal parts of the ventrolateral medulla. Although the resetting of the baroreflex by ANG II involves the area postrema (29), the NTS has been shown to be an important site for ANG II to act, by inhibiting the baroreceptor reflex sensitivity (11), and also for ANG II antagonism in improving sensitivity (9). There is evidence to suggest that ANG II modulation of the baroreflex may occur in the NTS, since this region receives direct input from the baroreceptors (24) and has a very high concentration of ANG II receptors (30). Moreover, ANG II infusions into the NTS attenuate baroreflex control of HR (11). In our study, several areas can be stimulated by ANG II; it appears that the NTS is probably the most sensitive area to the dose and the route of administration of ANG II that we used. Hegarty et al. (22) defined three populations of NTS neurons that respond to increased levels of circulating ANG II. Another potential site could involve the area postrema. This region is sensitive to 4V administration of ANG II (23), and electrical stimulation of the area postrema activates individual NTS neurons (15). Administration of ANG II receptor antagonists has been shown to improve the baroreflex function in animal models in which baroreflex function is impaired, such as spontaneously hypertensive rats (3), and in rabbits with pacing-induced heart failure (31). In the present study the finding that central administration of losartan enhances baroreflex sensitivity in normotensive rabbits has extended these observations and provided additional confirmation for a long-term involvement of endogenous ANG II in the central regulation of the HR component of the baroreceptor reflex.

In summary, the present results show that long-term central administration of ANG II at subpressor doses is able to diminish the sensitivity of the cardiac baroreflex in conscious normotensive rabbits, whereas blockade of the AT1 receptors with losartan induces the opposite effect. Our data suggest that endogenous ANG II in the brain stem elicits a tonic inhibitory effect on the cardiac baroreflex that is independent of the prevailing MAP level.

Perspectives

Our novel observations that the inhibition of the baroreflex control of the heart by endogenous ANG within the brain stem occurs over several weeks indicate that peptide modulation of the cardiovascular system can occur over a very wide time frame. Previously, actions of peptides such as angiotensin within the central nervous system have been thought to occur relatively quickly. Although the effect appears to be mediated by AT1 receptors, the mechanism for this long-term modulation is not yet clear. The implications, however, of such a time frame may be an important feature of the central nervous system's ability to modulate the cardiovascular control system in the long term. With such a mechanism, baroreflex control of the heart would be relatively unaffected via short-term activation of the brain stem area angiotensin system. However, with chronic elevation in activity or repeated bursts of activation, baroreflexes would be affected in the long term. The implications of this are considerable in terms of the development of hypertension, considering the view that salt sensitivity may be directly related to attenuated baroreceptor reflexes (8). The stimulus for setting the level of endogenous angiotensin in the brain stem that can affect cardiac baroreflexes is not clear. Whether it is a result of the projections from angiotensinergic fibers from the forebrain is also not known. However, it would appear that whatever drives the system may have the long-term effect of not only altering short-term baroreflex control but also increasing the salt sensitivity of the individual and, hence, may predispose them to the development of salt-induced hypertension.


    ACKNOWLEDGEMENTS

The authors thank DuPont Merck Pharmaceutical for the gift of losartan.


    FOOTNOTES

This study at the Baker Medical Research Institute was supported by a Block Institute Grant from the Australian National Health and Medical Research Council.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: G. A. Head, Neuropharmacology Laboratory, Baker Medical Research Institute, PO Box 6492, St. Kilda Rd. Central, Melbourne, Victoria 8008, Australia (E-mail: geoff.head{at}baker.edu.au or elisabeth.gaudet{at}baker.edu.au).

Received 24 May 1999; accepted in final form 8 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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