This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Thomas, C. J. Articles by Woods, R. L. Search for Related Content PubMed PubMed Citation Articles by Thomas, C. J. Articles by Woods, R. L.

(Hypertension. 1997;29:1126-1132.)
© 1997 American Heart Association, Inc.

Articles

ANP Enhances Bradycardic Reflexes in Normotensive but Not Spontaneously Hypertensive Rats

Colleen J. Thomas; Andrew J. Rankin; Geoffrey A. Head; ; Robyn L. Woods

From the Baker Medical Research Institute, Prahran, Victoria, Australia.

Correspondence to Dr R.L. Woods, Baker Medical Research Institute, Commercial Road, PO Box 348, Prahran, Victoria 3181, Australia. E-mail robyn.woods{at}baker.edu.au

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract Baroreflex control of heart rate in spontaneously hypertensive rats (SHR) is defective, largely because of a poor vagal contribution to the reflex. We have demonstrated previously that atrial natriuretic peptide (ANP) enhances reflex bradycardia in normotensive rats through an action on nonarterial vagal afferent pathways. In the present study, we investigated whether ANP could reverse the baroreflex abnormality in SHR. Heart rate reflexes were activated by three different methods in conscious, instrumented SHR and Wistar-Kyoto rats (WKY) in the presence of intravenous infusions of vehicle (saline) or rat ANP (150 ng/kg per minute). Heart rate responses were measured by (1) the steady-state changes in blood pressure after alternating slow infusions (over approximately 15 to 30 seconds) of a pressor (methoxamine) and depressor (nitroprusside) drug (stimulating predominantly arterial baroreceptors), (2) the ramp method of rapid infusion of methoxamine (over <10 seconds; stimulating arterial and cardiopulmonary baroreceptors), and (3) the von Bezold–Jarisch method of activating chemically sensitive cardiac receptors through serotonin injections. ANP enhanced the heart rate range of the arterial baroreflex (steady-state method) by 13±3% in WKY but had no significant effect on the sensitivity or any other parameter of the steady-state baroreflex. When a very rapid rise in blood pressure was elicited by the ramp method in WKY, ANP significantly enhanced baroreflex bradycardia (sensitivity increased by 29±9%, P<.05). ANP also enhanced the bradycardia of the von Bezold–Jarisch reflex (by 33±16%, P<.05) in WKY. By contrast, ANP did not influence baroreceptor or chemoreceptor heart rate reflex responses in SHR. We conclude that in normotensive rats, ANP facilitates cardiopulmonary bradycardic reflexes. The lack of effect of ANP in SHR may be related to an underlying structural or genetic alteration in their cardiac sensors, perhaps associated with cardiac hypertrophy, that prevents the ANP-induced activation of cardiac sensory afferents, resulting in cardioinhibition.

Key Words: reflex, von Bezold–Jarischatrial • natriuretic factor • baroreflex

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
It is well documented in humans^{1 2} and animals^{3 4 5} that ANP has an effect on the baroreflex control of HR. In general, ANP appears to reset baroreflex control of HR in a way that favors bradycardia. In a recent study in conscious, instrumented rats, we showed that ANP infusion resulted in nonuniform baroreflex responses, depending on the method used to measure reflex function.⁶ In the presence of ANP, we observed an enhanced bradycardic response to a very rapid rise in BP but normal bradycardic responses to steady-state changes in BP. We interpreted these findings as a selective effect of ANP to sensitize cardiac vagal afferent pathways, since the rapid ramp method invokes a greater proportion of nonarterial (cardiopulmonary) versus arterial afferent pathways.⁷

In hypertensive humans and animals, the baroreflex control of HR is impaired.^{8 9 10} In the SHR model of genetic hypertension, it has been demonstrated in vitro that baroreceptor sensitivity is reset¹¹ and in vivo that baroreflex sensitivity is reduced.¹² With a steady-state method for assessment of the baroreflex BP-HR relationship, SHR manifest a rightward shift of the sigmoidal curve (resetting) and a lesser bradycardic plateau (reduction in the vagal component of the HR range).¹² Minami and Head¹³ found that this vagal deficit in SHR depended on the degree of cardiac hypertrophy rather than the hypertension and suggested that these animals have a dampened input to the baroreflex from nonarterial baroreceptors compared with normotensive WKY. If ANP selectively enhances nonarterial afferent pathways, we hypothesized that ANP could restore baroreflex responses in SHR through an action on those cardiac pathways. Therefore, in the present study, we examined the effects of infused ANP on HR baroreflexes in conscious SHR and WKY using the two methods we previously employed to evoke different proportions of baroreflex afferent pathways.^{6 7 14}

In 1994, Meyrelles et al¹⁵ demonstrated that in rats with phenylephrine-induced cardiac hypertrophy, but without hypertension, the enlarged heart was associated with an insensitivity to the von Bezold–Jarisch reflex. Given that this bradycardic reflex method activates purely nonarterial (particularly ventricular) chemosensitive sensory afferents,¹⁶ we proposed that ANP may enhance this reflex, particularly in the SHR. Thus, in addition to comparing the actions of ANP on baroreflex changes in HR between SHR and WKY, we studied the effects of infused ANP on the von Bezold–Jarisch reflex in these animals.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Animals
Studies were carried out in WKY and SHR of both sexes, aged 12 to 14 weeks and weighing 297±7 g, bred at the Baker Medical Research Institute. Experiments were approved by the Alfred Hospital/Baker Medical Research Institute Animal Experimentation Committee. Twelve WKY and 9 SHR were studied. All of these rats completed both the steady-state and ramp baroreflex studies except for one WKY, which, for technical reasons, was omitted from the steady-state study. Six rats from each strain were used for the von Bezold–Jarisch reflex tests.

Surgical Preparation: Arterial and Venous Catheters
The rats were anesthetized with a mixture of short-acting methohexital sodium (Brietal Sodium, Eli Lilly; 40 mg/kg), pentobarbital (Nembutal, Boehringer Ingelheim; 30 mg/kg), and atropine (Astra; 0.5 mg/kg) injected intraperitoneally. An abdominal aortic catheter and vena caval catheters were inserted as previously described.¹⁷ Briefly, the lower abdominal aorta was exposed through a midline incision. A polytetrafluoroethylene-tipped (OD=0.45 mm, ID=0.3 mm; Small Parts) catheter was inserted 2 mm into the temporarily clamped lower abdominal aorta, 1 to 2 cm below the kidneys, through a puncture made by a 25-gauge needle. The catheter was held in place by a small droplet of tissue glue (Loctite, Prism 406), and the aortic clamps were removed. In the same operation, an incision was made in the skin lateral to the larynx, and a triple-lumen catheter (OD=1.5 mm, ID=0.5 mm; TV4, Dural Plastics) was passed down the right common jugular vein to the vena cava, with the tip at heart level. All catheters were filled with heparin (100 U/mL) in 0.9% saline, passed subcutaneously to emerge at the back of the neck, secured, and occluded with pins.

Hemodynamic Measurements and Experiments
Experiments were performed 7 and 8 days after the arterial and venous cannulations. The rats were brought to the laboratory in individual home cages in the morning and allowed to acclimate for approximately 1 hour after catheters were connected for measurements and infusions. Phasic aortic BP was measured with a disposable pressure transducer (Cobe), and phasic pressure and MAP were recorded continuously on an eight-channel recorder (Linearcorder No. WR3310, Graphtec). HR was measured with a meter triggered by the arterial pulse pressure signal. In addition, MAP and HR data for the ramp experiments were digitized continuously via an analog-to-digital computer program on an Olivetti M-280 computer with a Metrabyte data-acquisition card at a 300-Hz sampling rate, with binning of data at 0.5 Hz.

Each rat had two experimental days. On one, steady-state baroreflex curves were constructed, and on the other, the ramp baroreflex alone or together with the von Bezold–Jarisch reflex were measured, with the order of the days alternated. Each method was tested in the presence of vehicle (0.9% saline, 270 µL/h) and rat ANP(8-33) (Peninsula Laboratories; 150 ng/kg per minute IV) infusions, with the order of vehicle and ANP alternated in each rat. At least 30 minutes was allowed between the end of the first infusion (either vehicle or ANP) and the start of the second infusion (alternate solution).

Steady-State Baroreflex Curves
MAP-HR curves were obtained as previously described, with intravenous injections of vasopressor and vasodepressor drugs.¹⁷ Intravenous injections of 1 to 50 µL methoxamine hydrochloride (2 mg/kg per milliliter; Wellcome Research Laboratories), resulting in doses of 2 to 100 µg/kg, or 1 to 50 µL sodium nitroprusside (1 mg/kg per milliliter; Nipride, Roche Products), resulting in doses of 1 to 50 µg/kg, were used to produce a series of graded steady-state increases and decreases in MAP in each animal. Pressure rises and falls were in the range of 5 to 70 mm Hg. Each pressure change was maintained for approximately 30 seconds by a bolus injection followed by a slow infusion of the drug, and the mean values over the final 10 to 15 seconds were taken as the steady-state MAP and HR values. The triple-lumen venous catheter (one for each drug, and one for saline or ANP) allowed pressor and depressor responses to be alternated without the need to flush the catheter after each injection. The steady-state changes in MAP and HR were fitted to a sigmoid logistic equation: HR=P₁+P₂/[1+e^P3(MAP-P4)], where P₁ is lower HR plateau; P₂ is HR range; P₃ is a curvature coefficient and also the normalized gain, as it indicates the gain of the curve that is independent of the HR range; and P₄ is the MAP at half the HR range (BP₅₀).

The average gain (G) or slope of the curve between the two inflection points is a product of the range and normalized gain and is given by G=-P₂xP₃/4.56, and Upper Plateau=P₁+P₂. The curve of best fit was obtained with a personal computer program (SIGMOID) as described previously,⁶ using a least-squares iterative routine based on the Marquardt algorithm.¹⁷

Ramp Baroreflex Technique
With the use of a modification of the beat-to-beat analysis method described by Struyker-Boudier and colleagues,¹⁸ the rats were given quick (over 4 to 6 seconds) intravenous infusions (25 to 50 µL) of methoxamine (50 to 100 µg/kg doses), resulting in rapid increases in MAP and falls in HR.⁶ In each experiment, three control ramps were performed, followed by three ramps in the presence of ANP, or vice versa. The increases in BP of 40 to 70 mm Hg were comparable to those of the maximal responses with the steady-state method. To obtain the best correlation between the HR responses to BP changes, we performed linear regressions of HR versus MAP using MAP values at 0.5, 1.0, 1.5, 2.0, 2.5, or 3.0 seconds earlier than the corresponding HR value. The time delay with the highest regression correlation was selected. In the majority of cases, this was the 1-second delay, which is consistent with the delay described previously with the beat-to-beat analysis technique in rats.¹⁸ The slope of the regression line indicated the ramp baroreflex gain.

Von Bezold–Jarisch Reflex
On the basis of previous experiments by Widdop and colleagues,¹⁹ cardiopulmonary receptors were activated by intravenous bolus injections of 5-HT in the range 5 to 50 µg/kg (serotonin, creatinine sulfate complex, Sigma Chemical Co). The chemosensitive receptors responded rapidly (5 to 10 seconds), producing dose-dependent reductions in HR and variable changes in BP. Change in BP was always measured at the same time as the maximal bradycardia. These HR and BP responses were obtained with different doses of 5-HT at 5-minute intervals between doses in the presence of both saline and ANP infusions. The exact doses of 5-HT varied between rats because of different sensitivities. In individual rats, however, the same doses of 5-HT were given in the presence and absence of ANP. In addition, SHR generally required a greater dose of 5-HT to elicit HR responses similar to those in WKY. For greater accuracy with slowed HR elicited by 5-HT, heart period was recorded and later converted to HR for comparison with the other HR reflex methods.

Statistics
Steady-state baroreflex curve parameters were analyzed by two-way, two-factor ANOVA. Orthogonal partitioning of the between-column sums of squares was used for determination of the between-strains effect and treatment effect (ANP or saline). Significant effects were taken at a value of P<.05. Both the ramp and von Bezold–Jarisch data were analyzed by two-way ANOVA. Orthogonal partitioning of the sums of squares was used for determination of the effects of ANP on the ramp regression parameters and HR and BP responses to 5-HT injections.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Steady-State Baroreflex
The mean baroreceptor-HR reflex curves of conscious WKY and SHR derived from individual lines of best fit are shown in Fig 1, and the corresponding parameters are provided in Table 1. In both WKY and SHR, the sigmoidally shaped baroreflex curves displayed resting HR near the midpoint of the curves (Fig 1), similar to those we reported previously in Sprague-Dawley¹⁷ and Munich-Wistar rats.⁶ In addition, SHR curves were shifted to the right of the WKY curves, corresponding to the higher resting BP in these animals (Table 1). The HR range in SHR was approximately 25% lower than that of WKY, largely because of the elevated lower plateau, which was more than 100 beats per minute higher than in the WKY (Table 1). These differences in baroreflex parameters in the two strains are similar to those in a previous report.²⁰ The BP range over which the steady-state baroreflex responses were measured was approximately 100 mm Hg in both strains (Fig 1).

View larger version (11K): [in this window] [in a new window]   Figure 1. Mean steady-state baroreflex curves in 11 conscious WKY (left) and 9 SHR (right) determined on the same day during vehicle (control; dotted line) and ANP (solid line) infusion. Circles and error bars represent mean±SEM resting BP and HR during vehicle () and ANP () infusions. Error bars at each end of the curves refer to ±SEM of upper and lower plateau, respectively. *Significant effect of ANP (see Table 1 for details) (P<.05).

View this table: [in this window] [in a new window]   Table 1. Effect of Atrial Natriuretic Peptide on Steady-State Baroreflex Parameters in Conscious SHR and WKY

ANP infusion caused a small but significant enhancement of the HR range by 13±3% in WKY (P<.05, Table 1), with the increase occurring at the bradycardic (vagal) end of the curve (Fig 1). The peptide did not significantly alter any other steady-state baroreflex parameters (Table 1). In SHR, ANP infusion caused a small but significant reduction in both the resting BP and the value for BP at half the HR range (P<.05, Table 1), which was seen as a parallel leftward shift in the curve (Fig 1). Otherwise, the baroreflex curve with ANP was closely similar to that without ANP in the SHR (Fig 1).

Ramp Baroreflex
The gain of the ramp baroreflex in the WKY was approximately 30% lower than the gain measured with the steady-state baroreflex method (Tables 1 and 2). The slope or gain of the ramp relationship between BP and HR in SHR was approximately 65% lower than the gain in WKY (Table 2 and Fig 2).

View this table: [in this window] [in a new window]   Table 2. Effect of Atrial Natriuretic Peptide on Ramp Baroreflex Parameters in Conscious SHR and WKY

View larger version (18K): [in this window] [in a new window]   Figure 2. Mean HR responses to ramp increases in MAP after rapid infusions of methoxamine in 12 conscious WKY and 9 SHR. A, Averaged regression lines during vehicle (gray lines) and ANP (black lines) infusions. B, Average gain (or sensitivity) of reflex bradycardia during vehicle (gray bars) and ANP (black bars) infusions. Values are mean±SEM. *Significant effect of ANP on ramp baroreflex (P<.05).

The average number of BP and corresponding HR observations for each ramp regression in WKY was 10±1 and in SHR was 8±1. Parameters from the combined regression analyses that determined the best relationship between BP and HR in response to a rapid infusion of methoxamine (over approximately 5 seconds) are given in Table 2. The rate of change in BP and the range in BP over which the ramp baroreflex was measured in WKY and SHR were similar (Table 2 and Fig 2).

ANP infusion produced a significant (P<.05) increase in the ramp baroreflex gain of 28.8±8.5% in WKY (Table 2 and Fig 2). In these animals, there was a small but significant (P<.05) reduction in resting BP of 7±2% during ANP infusion and a concomitant rise in resting HR of 9±3% (Table 2). In SHR, ANP infusion did not alter the ramp baroreflex gain (Table 2 and Fig 2). In this strain, ANP caused a small but significant (P<.05) reduction in resting BP of 7±2% (Table 2), but resting HR did not change (Table 2). Over all the ramp experiments, the range in BP and the rate of change in BP over which the ramp responses were measured were similar regardless of strain or whether ANP was infused.

Von Bezold–Jarisch Reflex
In WKY and SHR, intravenous injections of 5-HT produced rapid, reflex-mediated, dose-dependent reductions in HR (F value between doses, for WKY, 5.51; for SHR, 12.00; Fig 3A and 3B). Bradycardia began within 5 seconds after 5-HT injection, reached a maximum in 1 to 2 seconds, and was sustained for a further 2 to 3 seconds, occasionally longer at the highest doses. In WKY, the average doses of 5-HT administered (termed "Lo," "Med," and "Hi" in Fig 3) were 4±1, 8±1, and 14±2 µg/kg, respectively. In SHR, however, the doses of 5-HT required to elicit bradycardia similar to that in WKY were 12±2, 22±3, and 34±2 µg/kg, respectively. Over all the doses, the mean falls in HR did not differ significantly in SHR and WKY (178±24 and 147±23 beats per minute, respectively; Fig 3B, gray shading). ANP infusion enhanced the magnitude of the HR response to 5-HT at each of the three doses in WKY (Fig 3A, left), with an increase in the average bradycardia over all the doses of 33±16% during ANP infusion (to -196±16 beats per minute, P<.05, Fig 3B). By contrast, ANP infusion did not significantly alter the HR response to 5-HT in SHR (Fig 3A and 3B). The average reflex bradycardia to 5-HT in SHR during ANP infusion was -152±22 beats per minute or -15±11% of response during saline infusion (Fig 3B).

View larger version (22K): [in this window] [in a new window]   Figure 3. HR responses to von Bezold–Jarisch reflex tested over three doses of 5-HT (5 to 50 µg/kg IV) in six WKY (left) and six SHR (right). Exact doses of 5-HT varied among rats because of different sensitivities, but in each animal, the same doses were administered before and after ANP infusion. A, Averaged HR changes from resting () with three doses of 5-HT during vehicle (gray lines) and ANP (black lines) infusions. B, Average bradycardia over the three doses during vehicle (gray bars) and ANP (black bars) infusions. Values are mean±SEM. *Significant effect of ANP on the von Bezold–Jarisch reflex (P<.05).

BP changes with 5-HT administration were somewhat variable between animals but were generally a complex triphasic response consisting of an initial depression and then a rise followed by a more prolonged fall. The BP responses were not dose dependent (F value between doses, for WKY, 0.25; for SHR, 1.11), but in general, the maximal bradycardia to 5-HT was associated with the initial hypotensive phase. Average BP changes evoked by 5-HT with saline infusion were -8±4 mm Hg in WKY and -10±2 mm Hg in SHR. ANP infusion did not significantly alter hypotensive responses to 5-HT in both WKY and SHR (F=1.84 and 1.23, respectively).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Two major findings arose from the present study in which we compared the effects of ANP on reflex changes in HR, particularly those leading to cardiac slowing, between conscious SHR and WKY. First, ANP significantly enhanced reflex bradycardia by around 30% in normotensive WKY when either chemosensitive cardiac afferents or cardiopulmonary baroreceptor afferents were activated but had no effect on the sensitivity of the arterial baroreceptor HR-BP reflex. These findings extend our recent observations in normotensive Munich-Wistar rats from which we hypothesized that ANP acts selectively on nonarterial afferent pathways to enhance cardiac slowing.⁶ Second, in conscious SHR, infused ANP was without effect on bradycardic reflexes stimulated through either mechanosensory or chemosensory afferent pathways, whether in cardiopulmonary or arterial regions. Thus, we suggest that SHR are resistant to the sensitizing actions of ANP on cardiac vagal afferents, although the mechanism for this insensitivity remains unknown.

Consistent with previous reports, we found SHR to have reduced baroreflex sensitivity, which was manifest as a rightward shift of the steady-state curve and a lesser bradycardic capacity (or HR range) compared with normotensive WKY.^{13 20} Impaired reflex control of the heart in hypertension is well documented and is mainly due to a reduced maximal capacity of the cardiac vagal component of the baroreflex rather than an alteration to the sympathetic component.^{10 19 21} Other studies have shown that an increase in baroreflex sensitivity is part of the development of a normotensive cardiovascular system, whereas in SHR, responsiveness of the baroreceptor-reflex system remains depressed during the development and stabilization of the hypertension.^{18 20} There is some doubt as to the integrity of nonarterial baroreceptor afferents, such as those from cardiopulmonary receptors, which have a selective input to the pathways controlling the vagus in SHR^{19 22} and in hypertensive patients.²³ Minami and Head¹³ recently showed that the deficit in the baroreceptor-HR reflex of genetically hypertensive rats was related to the level of cardiac hypertrophy because there was a strong correlation between HR range and the ratio of left ventricular weight to body weight. Therefore, increased stiffness of the cardiac chambers may be responsible for altered cardiac baroreceptor function. Given that ANP appears to act selectively on cardiac vagal pathways, our findings of total insensitivity in SHR to the effects of ANP may indicate that the site of action of ANP is structurally altered in these animals in association with their hypertension-induced cardiac hypertrophy. Alternatively, there may be an inherited structural defect in the myocardium of SHR, not directly related to the hypertension, which is yet to be elucidated. A link between ANP production and myocardial structure, independent of BP level, was suggested from studies demonstrating that overexpression of ANP mRNA and the concentration of ANP in plasma were associated with the level of cardiac hypertrophy in established hypertension and after regression from renovascular hypertension.²⁴ If the bradycardic reflex action of ANP is a cardiac compensatory mechanism for the ventricle exposed to rapid changes in afterload, the insensitivity to this action in SHR may contribute to the pathology of the hypertension.

The three methods used in the present study to stimulate bradycardic reflexes in conscious rats relied on selective activation of different afferent pathways. The steady-state technique^{6 7 12 13 14 17 20} provides a means of determining and defining the full relationship between BP and HR controlled largely through arterial baroreceptors. The ramp method^{5 6 7 14} for measuring baroreflex function is a rapid pressure change technique that invokes a different proportion of cardiopulmonary and arterial afferent inputs to the baroreflex pathway that respond to large, rapid changes in arterial pressure. This was well demonstrated by Faris and colleagues,⁷ who showed that in rabbits in which arterial baroreceptors were denervated, modest increases in BP after infusion of phenylephrine (similar to our steady-state responses) resulted in no change in HR, whereas rapid infusion of phenylephrine (similar to our ramp responses) caused profound bradycardia. In our present and previous⁶ studies with ANP, it was the responses to rapid changes in afterload, presumably the result of activation of nonarterial, cardiopulmonary afferents, that the cardiac peptide selectively influenced.

The von Bezold–Jarisch reflex is an inhibitory cardiovascular reflex originating in cardiac sensory receptors, largely in the ventricles, associated with vagal afferent neurones and producing profound bradycardia.¹⁶ These receptors are activated by a range of chemical substances, including veratrum alkaloids, phenyldiguanide, certain prostaglandins, capsaicin, and 5-HT.²⁵ The present study confirmed the previous report from Widdop and colleagues¹⁹ which showed that SHR have an impaired cardiac vagal reflex, as measured by reduced sensitivity to phenyldiguanide. An average dose of 22 µg/kg 5-HT in our SHR, compared with 8 µg/kg in WKY, was needed to evoke a comparable fall in HR. The BP responses to 5-HT were complex, resulting from the differential activation of cardiac and vascular 5-HT receptors²⁶ and changes in cardiac dynamics associated with the initial profound bradycardia. Our main interest was in the cardiac chemosensitive afferents, rather than in peripheral 5-HT receptor responses, and the von Bezold–Jarisch HR reflex was unlikely to be influenced by changes in BP because these changes were small and not affected by 5-HT dose, rat strain, or ANP. The action of ANP on 5-HT–sensitive cardiac vagal sensory afferents to enhance reflex bradycardia was very similar to the effect of the peptide on cardiopulmonary baroreflex pathways. This similarity provides a basis for the suggestion that ANP acts by a mechanism that may be common to mechanosensitive and chemosensitive ventricular receptors.

The HR range (difference between maximum and minimum HR) observed during the steady-state baroreflex assessment reflects input from both arterial and nonarterial pathways.²¹ Where there is a greater contribution from cardiopulmonary baroreceptors (due to the method used or to structural features of the myocardium, such as innervation or wall tension), there is a resultant greater HR range, which occurs mainly because of facilitation of the vagal end of the reflex curve.²¹ In the present study, normotensive WKY had an HR range of approximately 228 beats per minute, which was considerably higher than the HR range we previously found in Munich-Wistar rats (164 beats per minute).⁶ ANP was without influence on the HR range in Munich-Wistar rats⁶ but enhanced the HR range by approximately 13% in WKY. These data provide indirect evidence that responsiveness to ANP may be related to a signal from the myocardium influencing cardiac reflex activity that is reflected in the HR range. When greater drive was provided to the cardiac receptors with the ramp method, a differential effect of ANP between the two normotensive strains was no longer evident; ANP enhanced the bradycardic response to rapid rises in pressure by about 30% in both strains. In SHR, however, reduced HR reflex activity was associated with all reflex methods, and more importantly, ANP was completely without effect on any measure of reflex activity. Thus, it is possible that the insensitivity of the SHR is related specifically to some cardiac structural alteration such as that caused by hypertension-induced cardiac hypertrophy.

Although not measured in the present experiments, we would expect SHR to have normal to high endogenous plasma ANP levels because there are many reports of elevated plasma ANP levels in human and experimental hypertension.^{27 28 29 30} The role of ANP in the development and maintenance of hypertension in SHR is not fully understood, although ANP concentrations have been shown to increase with the onset of hypertension, becoming more marked as the animals grow older and the hypertension progresses.^{28 30} Indeed, mRNA for ANP production is upregulated in cardiac tissue in SHR compared with WKY.³¹ Elevated circulating or local tissue levels of ANP in the SHR may have led to receptor downregulation, and this could be a mechanism for the insensitivity in this strain. However, a recent review of the evidence for alterations in ANP receptors and signaling in hypertension³² indicated that in SHR, the total number of ANP binding sites (B_max) was enhanced in cultured aortic vascular smooth muscle cells but reduced in mesenteric vessels, kidney, platelets, and spleen as well as variably altered in neuronal tissue, whereas the K_d for ANP binding sites was increased, decreased, or unaltered. Thus, it is uncertain what the effects of elevated circulating levels of ANP in SHR may be on binding sites in the cells responsible for transducing the HR signal. An additional possibility to explain the insensitivity of SHR to the actions of exogenous ANP is that higher endogenous levels of ANP in the SHR were already producing a maximal effect. This seems unlikely because endogenous ANP levels in SHR are elevated only about twofold (eg, see References 28, 29, and 30^{28 29 30} ) compared with the 10- to 50-fold rise that occurs in pathological conditions such as heart failure. The infused dose we used in the present and previous studies⁶ would be expected to greatly overwhelm any differences in endogenous levels between strains.

It is not known which, if any, of the cloned natriuretic peptide receptors (NPRs) may be involved in the action of ANP on HR reflexes. Recently, genes for NPR-A, NPR-B, and NPR-C were identified in isolated cardiac myocytes and nonmyocytic cells of the rat heart.³² Indeed, NPR-C gene expression occurred in Purkinje fibers,³² where ANP itself can be produced.³³ This adds support to the notion that endogenous ANP may act locally on the heart, but to date there is no known function for these receptors in either myocytes or nerve fibers from the heart. It is possible that ANP may act through NPR-C to sensitize cardiac vagal afferent neurones. NPR-C has been demonstrated to have an altered signaling mechanism in the SHR (ie, coupling to cAMP), with enhanced inhibition of adenylate cyclase activity in heart tissue but a complete loss of the inhibitory action on adenylate cyclase activity in platelets.³⁴ NPR-C signaling in nerve fibers in SHR is unknown, but if it is attenuated, then such altered receptor signaling could account for the insensitivity to the cardiac reflex actions of ANP in these animals. Alternatively, if the receptor responsible for the reflex bradycardic actions of ANP is on cells such as cardiac myocytes, on which the sensory nerve endings impinge, it is unlikely that NPR-C is involved.

As an alternative to a peripheral site of action of ANP, a central action cannot be ruled out. ANP and its specific receptors have been localized in circumventricular organs outside the blood-brain barrier and to brain areas involved in cardiovascular and fluid regulation.³⁵ Emirio and colleagues³⁶ observed colocalization of ANP-responsive sites and sites where baroreceptors and chemoreceptors terminate within the caudal nucleus tractus solitarius. They showed that ANP caused hypotension and bradycardia when microinjected onto nucleus tractus solitarius neurones receiving a baroreceptor input, suggesting a role for ANP in the transmission of baroreceptor information within the nucleus tractus solitarius. Various studies of SHR brain regions report an altered ANP content and altered expression and responsiveness to ANP compared with normotensive animals.³⁷ Support for a central role for ANP in baroreflex control of HR comes from recent microinjection studies in rat brain using direct administration of the ANP hormone and a monoclonal antibody to block the actions of ANP.^{37 38 39} These workers demonstrated that ANP administered directly into the region of the nucleus tractus solitarius did not affect bradycardic responses to ramp increases in BP in WKY but blunted ramp baroreflex responses in HR in salt-sensitive SHR. Additionally, microinjection of the ANP antibody improved baroreceptor reflex control of HR in salt-sensitive SHR but not in WKY. The central effects of ANP to blunt the baroreflex in SHR were mediated via inhibition of sympathetic nerve activity.³⁹ Thus, it is highly unlikely that our present findings can be explained by a central action of the peptide because the effects of central administration of ANP are directly opposite to those we observed with peripheral administration of the peptide.

In summary, in the present experiments, we assessed the effect of peripherally administered ANP on the reflex control of HR in conscious SHR and WKY using three different methods to activate cardiac reflexes. Our findings in the normotensive WKY are consistent with a selectivity of ANP action on nonarterial vagal afferent pathways to enhance bradycardia and increase baroreflex sensitivity to a rapid rise in afterload or chemosensory activation with 5-HT. From the results in SHR, we suggest that underlying structural and/or genetic differences in these animals preclude them from responding to the influence of ANP on the reflex control of HR.

	Selected Abbreviations and Acronyms

 

ANP = atrial natriuretic peptide BP = blood pressure HR = heart rate 5-HT = serotonin MAP = mean aortic pressure SHR = spontaneously hypertensive rat(s) WKY = Wistar-Kyoto rat(s)

	Acknowledgments

 
This work was supported by a Block Grant from the National Health and Medical Research Council of Australia. We thank Maarten van den Buuse for his assistance in surgical preparation of some of the rats and Simon Fitzpatrick for his excellent technical assistance.

Received August 20, 1996; first decision September 30, 1996; accepted November 13, 1996.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Ebert TJ, Cowley AW Jr. Atrial natriuretic factor attenuates carotid baroreflex-mediated cardioacceleration in humans. Am J Physiol. 1988;254:R590-R594.[Abstract/Free Full Text]
2. Volpe M. Atrial natriuretic peptide and the baroreflex control of circulation. Am J Hypertens. 1992;5:488-493.[Medline] [Order article via Infotrieve]
3. Thorén P, Mark AL, Morgan DA, O'Neill TP, Needleman P, Brody MJ. Activation of vagal depressor reflexes by atriopeptins inhibits renal sympathetic nerve activity. Am J Physiol. 1986;251:H1252-H1259.
4. Volpe M, Cuocolo A, Vecchione F, Mele AF, Condorelli M, Trimarco B. Vagal mediation of the effects of atrial natriuretic factor on blood pressure and arterial baroreflexes in the rabbit. Circ Res. 1987;60:747-755.[Abstract/Free Full Text]
5. Ferrari AU, Daffonchio A, Sala C, Gerosa S, Mancia G. Atrial natriuretic factor and arterial baroreceptor reflexes in unanesthetized rats. Hypertension. 1990;15:162-167.[Abstract/Free Full Text]
6. Woods RL, Courneya CA, Head GA. Nonuniform enhancement of baroreflex sensitivity by atrial natriuretic peptide in conscious rats and dogs. Am J Physiol. 1994;267:R678-R686.[Abstract/Free Full Text]
7. Faris IB, Iannos J, Jamieson GG, Ludbrook J. Comparison of methods for eliciting the baroreceptor-heart rate reflex in conscious rabbits. Clin Exp Pharmacol Physiol. 1980;7:281-291.[Medline] [Order article via Infotrieve]
8. Bristow JD, Honour AJ, Pickering GW, Sleight P, Smyth HS. Diminished baroreflex sensitivity in high blood pressure. Circulation. 1969;39:48-54.[Abstract/Free Full Text]
9. Mancia G, Ferrari AU, Zanchetti A. Reflex control of the circulation in experimental and human hypertension. In: Zanchetti A, Tarazi RC, eds. Pathophysiology of Hypertension: Regulatory Mechanisms. Handbook of Hypertension. New York, NY: Elsevier Science Publishing Co; 1986;8:47-68.
10. Korner PI. Cardiac baroreflex in hypertension: role of the heart and angiotensin II. Clin Exp Hypertens. 1995;17:425-439.
11. Andresen MC, Kuraoka S, Brown AM. Baroreceptor function and changes in strain sensitivity in normotensive and spontaneously hypertensive rats. Circ Res. 1980;47:821-828.[Free Full Text]
12. Head GA, Adams MA. Characterization of the baroreceptor heart rate reflex during development in spontaneously hypertensive rats. Clin Exp Pharmacol Physiol. 1992;19:587-597.[Medline] [Order article via Infotrieve]
13. Minami N, Head GA. Relationship between cardiovascular hypertrophy and cardiac baroreflex function in spontaneously hypertensive and stroke-prone rats. J Hypertens. 1993;11:523-533.[Medline] [Order article via Infotrieve]
14. Weinstock M, Korner PI, Head GA, Dorward P. Differentiation of cardiac baroreflex properties by cuff and drug methods in two rabbit strains. Am J Physiol. 1988;255:R654-R664.[Abstract/Free Full Text]
15. Meyrelles SS, Cabral AM, Vasquez EC. Impairment of the Bezold-Jarisch reflex in conscious rats with myocardial hypertrophy. Braz J Med Biol Res. 1994;27:1065-1069.[Medline] [Order article via Infotrieve]
16. Hainsworth R. Reflexes from the heart. Physiol Rev. 1991;71:617-658.[Free Full Text]
17. Head GA, McCarty R. Vagal and sympathetic components of the heart rate range and gain of the baroreceptor-heart rate reflex in conscious rats. J Auton Nerv Syst. 1987;21:203-213.[Medline] [Order article via Infotrieve]
18. Struyker-Boudier HAJ, Evenwel RT, Smits JFM, Van Essen H. Baroreflex sensitivity during the development of spontaneous hypertension in rats. Clin Sci. 1982;62:589-594.[Medline] [Order article via Infotrieve]
19. Widdop RE, Verberne AJM, Jarrott B, Louis WJ. Impaired arterial baroreceptor reflex and cardiopulmonary vagal reflex in conscious spontaneously hypertensive rats. J Hypertens. 1990;8:269-275.[Medline] [Order article via Infotrieve]
20. Head GA, Adams MA. Time course of changes in baroreceptor reflex control of heart rate in conscious SHR and WKY: contribution of the cardiac vagus and sympathetic nerves. Clin Exp Pharmacol Physiol. 1988;15:289-292.[Medline] [Order article via Infotrieve]
21. Head GA. Baroreflexes and cardiovascular regulation in hypertension. J Cardiovasc Pharmacol. 1995;26(suppl 2):S52-S61.
22. Verberne AJM, Young NA, Louis W. Impairment of inhibitory cardiopulmonary vagal reflexes in spontaneously hypertensive rats. J Auton Nerv Syst. 1988;23:63-68.[Medline] [Order article via Infotrieve]
23. Grassi G, Giannattasio C, Cléroux J, Cuspidi C, Sampieri L, Bulla GB, Mancia G. Cardiopulmonary reflex before and after regression of left ventricular hypertrophy in essential hypertension. Hypertension. 1988;12:227-237.[Abstract/Free Full Text]
24. Matsubara H, Yamamoto J, Hirata Y, Mori Y, Oikawa S, Inada M. Changes of atrial natriuretic peptide and its messenger RNA with development and regression of cardiac hypertrophy in renovascular hypertensive rats. Clin Res. 1990;66:176-184.
25. Thorén P. Role of cardiac vagal C-fibres in cardiovascular control. Rev Physiol Biochem Pharmacol. 1979;86:1-94.[Medline] [Order article via Infotrieve]
26. Saxena PR, Villalón CM. Cardiovascular effects of serotonin agonists and antagonists. J Cardiovasc Pharmacol. 1990;15(suppl 7):s17-s34.
27. Sagnella GA, Markandu ND, Shore AC, MacGregor GA. Raised circulating levels of atrial natriuretic peptides in essential hypertension. Lancet. 1986;1:179-181.[Medline] [Order article via Infotrieve]
28. Imada T, Takayanagi R, Inagami T. Changes in the content of atrial natriuretic factor within the progression of hypertension in spontaneously hypertensive rats. Biochem Biophys Res Commun. 1985;133:759-765.[Medline] [Order article via Infotrieve]
29. Cantin M, Garcia R, Thibault G, Kutchel O, Gutkowska P, Larochelle P, Hamet P, Schiffrin EL, Genest J. Atrial natriuretic factor in experimental and human hypertension. Eur Heart J. 1988;9(suppl 2):21-27.
30. Gutkowska J, Horky K, Lachance C, Racz K, Garcia R, Thibault G, Kuchel O, Genest J, Cantin M. Atrial natriuretic factor in spontaneously hypertensive rats. Hypertension. 1986;8(suppl I):I-137-I-140.
31. Boluyt MO, O'Neill L, Meredith AL, Bing OHL, Brooks WW, Conrad CH, Crow MT, Lakatta EG. Alterations in cardiac gene expression during the transition from stable hypertrophy to heart failure. Circ Res. 1994;75:23-32.[Abstract/Free Full Text]
32. Marcil J, Anand-Srivastava MB. Defective ANF-R₂/ANP-C receptor-mediated signalling in hypertension. Mol Cell Biochem. 1995;149-150:223-231.
33. Lin X, Hanze J, Heese F, Sodmann R, Lang RE. Gene expression of natriuretic peptide receptors in myocardial cells. Circ Res. 1995;77:750-758.[Abstract/Free Full Text]
34. Anand-Srivastava MB, Thibault G, Sola C, Fon E, Ballak M, Charbonneau C, Haile-Meskel H, Garcia R, Genest J, Cantin M. Atrial natriuretic factor in Purkinje fibers of rabbit heart. Hypertension. 1989;13:789-798.[Abstract/Free Full Text]
35. Saavedra JM, Kurihara M. Autoradiography of atrial natriuretic peptide (ANP) receptors in the rat brain. Can J Physiol Pharmacol. 1991;69:1567-1575.[Medline] [Order article via Infotrieve]
36. Emirio R, Ruggeri P, Cogo CE, Molinari C, Calaresu FR. Neuronal and cardiovascular responses to ANF microinjected into the solitary tract. Am J Physiol. 1989;256:R577-R582.[Abstract/Free Full Text]
37. Oparil S, Wyss JM. Atrial natriuretic factor in central cardiovascular control. News Physiol Sci. 1993;8:223-228.[Abstract/Free Full Text]
38. Jin H, Yang RH, Calhoun DA, Wyss JM, Oparil S. Atrial natriuretic peptide modulates baroreceptor reflex in spontaneously hypertensive rat. Hypertension. 1992;20:374-379.[Abstract/Free Full Text]
39. Zhu S-T, Chen Y-F, Wyss JM, Nakao K, Imura H, Oparil S, Calhoun DA. Arterial natriuretic peptide blunts arterial baroreflex in spontaneously hypertensive rats. Hypertension. 1996;27:297-302.[Abstract/Free Full Text]

This article has been cited by other articles:

				C. J. Thomas, R. M. McAllen, L. M. Salo, and R. L. Woods Restorative Effect of Atrial Natriuretic Peptide or Chronic Neutral Endopeptidase Inhibition on Blunted Cardiopulmonary Vagal Reflexes in Aged Rats Hypertension, October 1, 2008; 52(4): 696 - 701. [Abstract] [Full Text] [PDF]

				E. Toader, R. M. McAllen, A. Cividjian, R. L. Woods, and L. Quintin Effect of systemic B-type natriuretic peptide on cardiac vagal motoneuron activity Am J Physiol Heart Circ Physiol, December 1, 2007; 293(6): H3465 - H3470. [Abstract] [Full Text] [PDF]

				C. J Thomas and R. L Woods Do natriuretic peptides modify arterial baroreflexes in sheep? Exp Physiol, November 1, 2004; 89(6): 709 - 715. [Abstract] [Full Text] [PDF]

				S. G. Hood and R. L. Woods Vagal reflex actions of atrial natriuretic peptide survive physiological but not pathological cardiac hypertrophy in rat Exp Physiol, July 1, 2004; 89(4): 445 - 454. [Abstract] [Full Text] [PDF]

				C. J. Thomas and R. L. Woods Guanylyl Cyclase Receptors Mediate Cardiopulmonary Vagal Reflex Actions of ANP Hypertension, February 1, 2003; 41(2): 279 - 285. [Abstract] [Full Text] [PDF]

				C. J. Thomas, C. N. May, A. D. Sharma, and R. L. Woods ANP, BNP, and CNP enhance bradycardic responses to cardiopulmonary chemoreceptor activation in conscious sheep Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2001; 280(1): R282 - R288. [Abstract] [Full Text] [PDF]

				C. J. Thomas, G. A. Head, and R. L. Woods ANP and Bradycardic Reflexes in Hypertensive Rats : Influence of Cardiac Hypertrophy Hypertension, September 1, 1998; 32(3): 548 - 555. [Abstract] [Full Text] [PDF]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Thomas, C. J. Articles by Woods, R. L. Search for Related Content PubMed PubMed Citation Articles by Thomas, C. J. Articles by Woods, R. L.