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1 Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Victoria 3010, Australia
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(Received 3 June 2004;
accepted after revision 24 August 2004; first published online 13 September 2004)
Corresponding author R. L. Woods: Howard Florey Institute, University of Melbourne, Victoria 3010, Australia. Email: r.woods{at}hfi.unimelb.edu.au
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Introduction |
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It was recognized from early studies that ANP may interact with reflexes from the heart to regulate sympathetic and cardiac parasympathetic function (Thoren et al. 1986; Ackermann et al. 1988). Its actions on reflexes from arterial baroreceptors, however, have been subject to differing interpretations. Some workers show augmented reflex bradycardic responses with ANP (Parkes et al. 1990; Ferrari et al. 1990; Volpe et al. 1987a,b) whereas others, including ourselves, show no effect of the peptide (Ebert & Cowley, 1988; Woods et al. 1994; Thomas et al. 1997, 1998; 2002,). On its own, ANP does not alter aortic baroreceptor discharge (Yang & Andresen, 1990) or aortic baroreceptor responses to changes in arterial blood pressure caused by phenylephrine or nitroglycerine (Hirooka et al. 1988).
For BNP, there is indirect evidence of an action on arterial baroreflexes, since in humans the expected reflex increase in whole-body noradrenaline spillover does not appear in response to BNP-induced hypotension (Brunner-La Rocca et al. 2001). There is only one study of the effects of this peptide directly on arterial baroreflexes. Morita et al. (1989), using drug-induced changes in blood pressure, demonstrated that BNP did not alter sigmoid curves describing the mean arterial pressure–heart rate (MAP–HR) or MAP–renal sympathetic nerve activity relationships in conscious rabbits. To our knowledge there are no reports of the action CNP on arterial baroreflexes. CNP is a likely candidate to influence arterial baroreceptors since other factors released from endothelial cells, such as nitric oxide (Minami et al. 1995) and endothelin (Chapleau et al. 1992), appear to alter the activity of arterial baroreceptors by acting in a paracrine manner. If the action of CNP on arterial baroreflexes differs from those of ANP and BNP, this would be consistent with other examples where the biological actions of CNP are not like those of the other natriuretic peptides (Nicholls, 1994), due most likely to the fact that CNP acts through a different receptor (Suga et al. 1992).
In the present study we have used a conscious animal model to make the first test of the ability of CNP to alter arterial baroreflex function, and to re-examine the actions on this reflex of BNP. For comparison, the effects of an equimolar dose of ANP have been measured in the same animals. All three natriuretic peptides were infused at a dose which we have previously found to potentiate the Bezold-Jarisch reflex in conscious sheep (Thomas et al. 2001).
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Methods |
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Animal instrumentation
Studies were performed in oophorectomised, adult, crossbred merino ewes (n
= 6; 38–51 kg) previously prepared with carotid arterial loops. One to three days before experimentation, under local anaesthesia (0.5 ml S.C. of 2% xylocaine; Astra Pharmaceuticals, North Ryde, Australia), three polyethylene cannulae (‘P47’, o.d. 0.96 mm, i.d. 0.58 mm; Critchley Electrical Products, Silverwater, Australia) were introduced into a jugular vein. One catheter was used for continuous infusion of natriuretic peptide or vehicle (Haemaccel). The remaining two catheters were used to inject drugs to elicit arterial baroreceptor–HR reflex responses. A tygon cannula (‘52B’, o.d. 1.7 mm, i.d. 1.18 mm; Critchley Electrical Products, Silverwater, Australia) was also inserted 
15 cm proximally into one of the exteriorized carotid arteries to measure arterial blood pressure and HR. All cannulae were secured at the back of the neck and, when not in use, were constantly infused with heparinized saline (25 IU ml–1 at 3 ml h–1).
Experimental protocol
On each experimental day the arterial catheter was connected to a pressure transducer (Cobe, Lakewood, CO, USA) to measure phasic arterial blood pressure (both phasic and mean arterial pressure were recorded). The pressure was corrected to compensate for the height of the transducer above the level of the heart. Recording leads were connected to three stainless-steel wires, previously inserted under the sheep's skin, for ECG measurement (ECG 100; Biopac Systems Inc., Goleta, CA, USA). HR was measured using a tachometer (Baker Heart Institute, Melbourne, Australia) triggered by the R-wave of the ECG signal. All haemodynamic variables were recorded continuously on an 8-channel Graphtec chart recorder (Linearcorder no. WR3310; Graphtec, Yokohama, Japan). In addition, the signals were digitized and recorded at a sampling rate of 50 Hz using the AcqKnowledge data acquisition system (Biopac Systems Inc.) connected to a Pentium computer. For each sheep, HR reflex studies were conducted on three separate days, with 1 week between experiments.
On a given experimental day, two steady-state baroreflex curves were constructed. Following a 60 min stabilization period after all cannulae and recording leads were connected, the first baroreflex curve was measured during vehicle infusion (Haemaccel, a plasma substitute; Hoechst, Auckland, New Zealand; 12 ml h–1). Infusion of one of the following natriuretic peptides (each at 10 pmol kg–1 min–1 in sterile Haemaccel; 12 ml h–1) was then begun: 
-human-ANP (1–28) (Auspep, Melbourne, Australia), porcine-BNP-32 (Bachem; Bubendorf, Switzerland), or human-, porcine-, rat-CNP (32–53) (Bachem, Bubendorf, Switzerland). Infusions of the natriuretic peptides generally lasted 1–1
h, including a 20 min run-in period for the peptides to reach steady-state levels in the circulation, prior to construction of the second baroreflex curve. These peptides have strong sequence homology with ovine forms of the natriuretic peptides (Aitken et al. 1999). The order of experimental days for each natriuretic peptide infusion was randomised between sheep.
Steady-state baroreflex technique
Sigmoid MAP–HR curves were constructed in each sheep, using methodology similar to that previously described (Woods et al. 1994). Briefly, alternate randomised intravenous injections of 1–15 µg/kg L-phenylephrine hydrochloride (Sigma, St Louis, MO, USA) and 1–15 µg kg–1 sodium nitroprusside (David Bull Laboratories, Mulgrave, Australia) produced graded steady-state increases and decreases in MAP, respectively, with changes in the range 5–45 mmHg. Each pressure change was maintained for 30–50 s by a bolus injection followed by a slow infusion of the drug, and the mean values over the final 10–12 s (when MAP had plateaued) were taken as steady-state MAP and corresponding HR values. In general, 10–15 individual MAP–HR responses were used to construct each baroreflex curve. Using a personal computer program (SIGMOID; Baker Heart Institute, Melbourne, Australia), the steady-state changes in MAP and HR were fitted to a sigmoid logistic equation:
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Plasma levels of BNP
Arterial blood was collected from each animal into chilled EDTA tubes to measure plasma BNP levels in the absence and presence of BNP infusion. Plasma was separated immediately using a refrigerated centrifuge and stored at –20°C until measurement. BNP was determined following extraction of plasma, as we have previously described (Woods & Jones, 1999). The radioimmunoassay for BNP was performed using commercial BNP-32 (porcine) antiserum (Peninsula Laboratories, San Carlos, CA, USA), 125I-BNP-32 (porcine) tracer (Peninsula Laboratories) and the same BNP-32 (porcine) standard that was used in the experiments (Bachem, Bubendorf, Switzerland).
Statistical analysis
Effects of the natriuretic peptides on resting MAP and HR, as well as each of the arterial baroreflex parameters, were determined by two-way repeated measures analysis of variance (ANOVA), with a Bonferroni adjustment of probability value for multiple comparisons where appropriate. For each arterial baroreflex parameter, mean ± S.E.D. results are reported in the Table, where S.E.D. is calculated from the residual mean square and provides the average standard error of the difference for comparisons between control and natriuretic peptides infusions from the ANOVA. In the text, pooled control values (C1, C2 and C3) are given as means ± S.E.M. (standard error of the mean), as between-animal comparison. Figure 2 error bars represent S.E.M. (between animal comparison). Differences were considered significant when P < 0.05.
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Results |
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10-fold (from 8 ± 1 to 76 ± 31 pmol l–1) during BNP infusion. Arterial baroreflex changes in HR were not significantly influenced by any of the natriuretic peptides (Fig. 2). Comparison of the sigmoid curve parameters revealed no effect of ANP, BNP or CNP on any of these directly measured or calculated values (Table 1). Of particular note for defining the limits and characteristics of arterial baroreflex function, HR range, curvature and average gain were not significantly different during infusion of any of the natriuretic peptides, when compared with their Haemaccel-infused control curves (Table 1). |
Discussion |
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Negative results invite questions of adequate dose and stimulus. Batches of the natriuretic peptides used in the present study are known to be biologically effective since we previously demonstrated that at this 10 pmol kg–1 min–1 dose, all three peptides enhanced Bezold-Jarisch reflex bradycardia in conscious sheep (Thomas et al. 2001). From previous measurements in sheep, this dose should produce an 10-fold rise in resting plasma ANP levels (Charles et al. 1996). Our direct measurements of plasma BNP by radioimmunoassay in the present study showed a 10-fold increase in BNP levels. These plasma concentrations are in accord with levels needed to mimic endogenous release of the peptides perfusing cardiac and nearby arterial baroreceptors (Sudhir et al. 1989). Finally, the approach used is a well-characterized and accepted method to describe arterial baroreceptor–HR reflexes in experimental animals (Head & McCarty, 1987). Essential requirements of the technique were complied with, including: (1) evoking a wide range of blood pressure changes (for the sheep ±5–45 mmHg from resting MAP of 80 mmHg); (2) including sufficient points (10–15) to adequately define upper (sympathetic) and lower (vagal) HR plateaux, as well as the high-gain area along the linear portion of the curve; and (3) alternating the raising and lowering of blood pressure to avoid baroreceptor resetting (Head & McCarty, 1987).
This is the first study to examine the action of CNP on reflexes driven from arterial baroreceptors. The lack of effect of CNP on MAP–HR curves in conscious sheep is somewhat unexpected, given the action of other factors made in the endothelium on arterial baroreflexes. Nitric oxide and endothelin, for example, have been suggested to act in a paracrine manner to suppress the activity of arterial baroreceptors in other species (Chapleau et al. 1992; Minami et al. 1995). The present findings with CNP were also surprising in light of recent evidence demonstrating a direct action of CNP on guinea-pig cardiac vagal nerve activity to enhance neurotransmission and bradycardia, and that particulate guanylyl-cyclase natriuretic peptide receptors (NPA and/or NPB) are responsible for these effects of CNP (Herring et al. 2001). We not only observed that CNP was without effect on arterial baroreflex bradycardia but we also found that CNP had no bradycardic effect in the resting state. The in vitro studies of Herring et al. (2001) used an ambient concentration of CNP (50–500 nM) to influence isolated atrial vagal nerve activity. This was considerably higher than the plasma levels expected in sheep with a 10 pmol kg–1 min–1 infusion (estimated at <120 pM; Charles et al. 1996). It is possible that at much higher doses of CNP, the enhancement of vagal neurotransmission might have become evident as a reduction in the lower plateau of the steady-state reflex.
Our second major new finding was that BNP did not modulate arterial baroreflex function in conscious sheep. Morita et al. (1989), in the one previous study in experimental animals, found that human BNP was without influence in conscious rabbits on the sensitivity of sigmoid curves relating arterial baroreflex changes in renal sympathetic nerve activity or HR to drug-induced changes in MAP. Those findings are now confirmed in a second species. This makes the suggestion that the relative sympathoinhibitory effects of BNP could be due to an action on arterial baroreflexes, based on indirect evidence from human studies (Brunner-La Rocca et al. 2001), most unlikely.
The present experiments in sheep confirm previous observations in other species that ANP does little to the normal arterial baroreceptor reflex control of HR (Ebert & Cowley, 1988; Woods et al. 1994; Thomas et al. 1997, 1998, 2002). By contrast, Parkes et al. (1990), using perivascular occluders on aorta and vena cava to evoke changes in blood pressure in conscious sheep, reached a different conclusion. In their study, acute restriction of aortic flow rapidly increased arterial pressure proximal to the cuff, resulting in a reflex bradycardia which was enhanced in the presence of ANP infusion at 100 µg h–1 for 2 h (15 pmol kg–1 min–1; Parkes et al. 1990). We believe that this result can be explained on the basis of the sensitizing action of ANP on high-pressure cardiac mechanoreceptors (Thomas et al. 2002), which would have been activated by the strong stimulus of aortic occlusion.
A final consideration is how the plasma concentrations achieved with natriuretic peptide infusions in the present study compare with endogenous physiological or pathophysiological levels of these peptides. In the case of ANP and BNP, physiological manipulations, such as postural change or modest exercise, increase circulating concentrations up to 2-fold (e.g. Solomon et al. 1986; Sudhir et al. 1990; Tanabe et al. 1999), although BNP is less reactive to these physiological manoeuvres than is ANP (Richards et al. 1995). In cardiovascular disease, such as hypertension, the plasma levels increase 2-fold and in mild to moderate heart failure the increases are up to 10-fold, whereas in advanced stage congestive heart failure the levels can increase 30-fold (see review by Sagnella, 1998). We cannot rule out the possibility that natriuretic peptides may influence arterial baroreflex function in severe cardiovascular disease although ANP, at least, does not improve baroreflex function in experimental hypertension (Thomas et al. 1997, 1998). As for CNP, endogenous plasma CNP levels alter little under physiological conditions (Bentzen et al. 2004), but they can increase up to 10-fold during septic shock (Hama et al. 1994). Thus, the plasma levels of natriuretic peptides during infusions into sheep in the present study, either measured directly or inferred from the measurements of others, are considerably higher than those likely to be achieved physiologically and are within the range of pathophysiology. Although the present data cannot exclude the possibility that higher doses of natriuretic peptides might alter arterial baroreflex function, such doses generally would be beyond the range achievable by endogenous release. Moreover, any reflex comparisons at higher infusions of the natriuretic peptides would have to cope with the confounding effects of substantial haemodynamic changes.
In summary, we report for the first time that CNP and BNP did not alter arterial baroreceptor–HR reflex function in conscious sheep. By contrast with the present negative results, the same doses of BNP and CNP, as well as ANP, enhance Bezold-Jarisch reflex bradycardia (Thomas et al. 2001). The present data further support the emerging theme that natriuretic peptides selectively enhance reflexes originating in the heart itself, and not those activated by arterial baroreceptors.
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References |
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|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Aitken GD, Raizis AM, Yandle TG, George PM, Espiner EA & Cameron VA (1999). The characterization of ovine genes for atrial, brain, and C-type natriuretic peptides. Domest Anim Endocrinol 16, 115–121.[CrossRef][Medline]
Bentzen H, Pedersen RS, Nyvad O & Pedersen EB (2004). Effect of exercise on natriuretic peptides in plasma and urine in chronic heart failure. Int J Cardiol 93, 121–130.[CrossRef][Medline]
Brunner-La Rocca
HP, Kaye
DM, Woods
RL, Hastings
J
&
Esler
MD (2001). Effects of intravenous brain natriuretic peptide on regional sympathetic activity in patients with chronic heart failure as compared with healthy control subjects. J Am Coll Cardiol
37, 1221–1227.
Chapleau MW, Hajduczok G & Abboud FM (1992). Suppression of baroreceptor discharge by endothelin at high carotid sinus pressure. Am J Physiol 263, R103–R108.
Charles CJ, Espiner EA, Richards AM, Nicholls MG & Yandle TG (1996). Comparative bioactivity of atrial, brain, and C-type natriuretic peptides in conscious sheep. Am J Physiol 270, R1324–R1331.
Dodic M, Peers A, Coghlan JP, May CN, Lumbers E, Yu Z & Wintour EM (1999). Altered cardiovascular haemodynamics and baroreceptor-heart rate reflex in adult sheep after prenatal exposure to dexamethasone. Clin Sci 97, 103–109.[Medline]
Ebert TJ & Cowley AW Jr (1988). Atrial natriuretic factor attenuates carotid baroreflex-mediated cardioacceleration in humans. Am J Physiol 254, R590–R594.
Ferrari
AU, Daffonchio
A, Sala
C, Gerosa
S
&
Mancia
G (1990). Atrial natriuretic factor and arterial baroreceptor reflexes in unanesthetized rats. Hypertension
15, 162–167.
Hama N, Itoh H, Shirakami G, Suga S, Komatsu Y, Yoshimasa T, Tanaka I, Mori K & Nakao K (1994). Detection of C-type natriuretic peptide in human circulation and marked increase of plasma CNP level in septic shock patients. Biochem Biophys Res Commun 198, 1177–1182.[CrossRef][Medline]
Head GA & McCarty R (1987). Vagal and sympathetic components of the heart rate range and gain of the baroreceptor-heart rate reflex in conscious rats. J Auton Nerv Syst 21, 203–213.[CrossRef][Medline]
Herring N, Zaman JA & Paterson DJ (2001). Natriuretic peptides like NO facilitate cardiac vagal neurotransmission and bradycardia via a cGMP pathway. Am J Physiol 281, H2318–H2327.
Hirooka
Y, Takeshita
A, Imaizumi
T, Nakamura
N, Tomoike
H
&
Nakamura
M (1988). Effects of -human atrial natriuretic peptide on the interrelationship of arterial pressure, aortic nerve activity, and aortic diameter. Circ Res
63, 987–996.
Korner PI, West MJ, Shaw J & Uther JB (1974). ‘Steady-state’ properties of the baroreceptor-heart rate reflex in essential hypertension in man. Clin Exp Pharmacol Physiol 1, 65–76.[Medline]
Lumbers
ER, McCloskey
DI
&
Potter
EK (1979). Inhibition by angiotensin II of baroreceptor-evoked activity in cardiac vagal efferent nerves in the dog. J Physiol
294, 69–80.
Minami N, Imai Y, Hashimoto J & Abe K (1995). The role of nitric oxide in the baroreceptor-cardiac reflex in conscious Wistar rats. Am J Physiol 269, H851–H855.
Morita H, Nishida Y, Motochigawa H, Kangawa K, Minamino N, Matsuo H & Hosomi H (1989). Effects of brain natriuretic peptide on renal nerve activity in conscious rabbits. Am J Physiol 256, R792–R796.
Nicholls MG (1994). Minisymposium: the natriuretic peptide hormones. Introduction. Editorial and historical review. J Intern Med 235, 507–514.[Medline]
Parkes DG, Coghlan JP, McDougall JG & Scoggins BA (1990). Hemodynamic interactions of atrial natriuretic factor with the sympathetic nervous system in sheep. Clin Exp Hypertens 12, 383–398.
Richards AM, Nicholls MG & Espiner EA (1995). Natriuretic peptides. Clin Sci 88, 18–21.[Medline]
Sagnella GA (1998). Measurement and significance of circulating natriuretic peptides in cardiovascular disease. Clin Sci 95, 519–529.[Medline]
Solomon LR, Atherton JC, Bobinski H & Green R (1986). Effect of posture on plasma immunoreactive atrial natriuretic peptide concentrations in man. Clin Sci 71, 299–305.[Medline]
Sudhir K, Friberg P, Meredith IT, Woods RL, Esler MD & Jennings GL (1989). Cardiac secretion and renal clearance of atrial natriuretic peptide in normal man: effect of salt restriction. Clin Sci 77, 605–610.[Medline]
Sudhir K, Meredith IT, Jennings GL, Friberg P, Woods RL & Esler MD (1990). Effect of endurance exercise on cardiac secretion and renal clearance of atrial natriuretic peptide in normal humans. Clin Exp Hypertens A 12, 1223–1235.[Medline]
Suga S, Nakao K, Hosoda K, Mukoyama M, Ogawa Y, Shirakami G, Arai H, Saito Y, Kambayashi Y, Inouye K & Imura H (1992). Receptor selectivity of natriuretic peptide family, atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide. Endocrinology 130, 229–239.[Abstract]
Tanabe K, Yamamoto A, Suzuki N, Akashi Y, Seki A, Samejima H, Oya M, Murabayashi T, Nakayama T, Yokoyama Y, Osada N, Omiya K, Itoh H, Miyake F & Murayama M (1999). Exercise-induced changes in plasma atrial natriuretic peptide and brain natriuretic peptide concentrations in healthy subjects with chronic sleep deprivation. Jpn Circ 63, 447–452.[CrossRef]
Thomas CJ, Allen AM, McAllen RM & Woods RL (2002). ANP potentiates nonarterial baroreflex bradycardia: evidence from sinoaortic denervation in rats. Auton Neurosci 97, 89–98.[CrossRef][Medline]
Thomas
CJ, Head
GA
&
Woods
RL (1998). ANP and bradycardic reflexes in hypertensive rats: influence of cardiac hypertrophy. Hypertension
32, 548–555.
Thomas CJ, Head GA & Woods RL (1999). Similar baroreflex bradycardic actions of atrial natriuretic peptide and B and C types of natriuretic peptides in conscious rats. J Hypertens 17, 801–806.[CrossRef][Medline]
Thomas CJ, May CN, Sharma AD & Woods RL (2001). ANP, BNP, and CNP enhance bradycardic responses to cardiopulmonary chemoreceptor activation in conscious sheep. Am J Physiol 280, R282–R288.
Thomas
CJ, Rankin
AJ, Head
GA
&
Woods
RL (1997). ANP enhances bradycardic reflexes in normotensive but not spontaneously hypertensive rats. Hypertension
29, 1126–1132.
Thomas
CJ
&
Woods
RL (2003). Guanylyl cyclase receptors mediate cardiopulmonary vagal reflex actions of ANP. Hypertension
41, 279–285.
Thoren P, Mark AL, Morgan DA, O'Neill TP, Needleman P & Brody MJ (1986). Activation of vagal depressor reflexes by atriopeptins inhibits renal sympathetic nerve activity. Am J Physiol 251, H1252–H1259.
Volpe
M, Cuocolo
A, Vecchione
F, Mele
AF, Condorelli
M
&
Trimarco
B (1987a). Vagal mediation of the effects of atrial natriuretic factor on blood pressure and arterial baroreflexes in the rabbit. Circ Res
60, 747–755.
Volpe M, Mele AF, Indolfi C, De Luca N, Lembo G, Foccacio A, Condorelli M & Trimarco B (1987b). Hemodynamic and hormonal effects of atrial natriuretic factor in patients with essential hypertension. J Am Coll Cardiol 10, 787–793.[Abstract]
Woods RL, Courneya CA & Head GA (1994). Nonuniform enhancement of baroreflex sensitivity by atrial natriuretic peptide in conscious rats and dogs. Am J Physiol 267, R678–R686.
Woods RL & Jones MJ (1999). Atrial, B-type, and C-type natriuretic peptides cause mesenteric vasoconstriction in conscious dogs. Am J Physiol 276, R1443–R1452.
Yang
MY
&
Andresen
MC (1990). Peptidergic modulation of mechanotransduction in rat arterial baroreceptors. Circ Res
66, 804–813.
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Acknowledgements |
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) and natriuretic peptide (
) infusions. Error bars at the end of the curves represent ±
S.E.M. of upper and lower plateaux, respectively. The histograms depict mean arterial baroreflex gain in the absence (Veh, open bars) and presence (filled bars) of natriuretic peptide infusion.