Chronic regulation of arterial blood pressure in ANP transgenic and knockout mice: Role of cardiovascular sympathetic tone

Abstract

Objective: Atrial natriuretic peptide (ANP) lowers arterial blood pressure (ABP) chronically, in association with vasodilation of the resistance vasculature. The mechanism mediating the chronic relaxant effect of ANP is likely indirectly mediated by interactions with tonic vasoeffector mechanisms, inasmuch as the resistance vasculature is relatively insensitive to direct cGMP-mediated relaxation by ANP. On the basis of evidence that ANP has widespread sympatholytic activity, the current study investigated whether the chronic hypotensive effect of ANP is mediated by attenuation of tonic cardiovascular sympathetic tone. Methods: Total plasma catecholamine concentration and changes in basal ABP and heart rate (HR) following autonomic ganglionic blockade were measured as indices of underlying sympathetic nerve activity in hypotensive ANP-overexpressing transgenic mice (TTR–ANP), hypertensive ANP knockout mice (−/−) and the genetically-matched wild type (NT and +/+, respectively) control mice. Pressor and chronotropic responses to norepinephrine infusion were measured in ganglion-blocked mice of all genotypes, and norepinephrine receptor binding was assessed in representative tissues of −/− and +/+ mice, in order to determine whether peripheral adrenergic receptor responsiveness is altered by ANP-genotype. Results: Basal ABP was significantly lower in TTR–ANP and higher in −/− compared to their wild-type controls. Basal HR did not differ significantly between mutant and control mice. Autonomic ganglionic blockade reduced ABP and HR in all genotypes, however, the relative decrease in ABP was significantly smaller in TTR–ANP and greater in −/− mice than in their respective controls. Total plasma catecholamine was significantly higher in −/− than in +/+ mice but did not differ significantly between TTR–ANP and NT mice. Norepinephrine infusion during ganglionic blockade elicited quantitatively similar pressor and chronotropic responses in mutant and control mice. Tissue norepinephrine binding did not differ significantly between −/− and +/+ mice. Conclusions: The present study shows that differences in endogenous ANP activity in mice, resulting in chronic alterations in ABP are accompanied by directional changes in underlying cardiovascular sympathetic tone, and suggests that the chronic vasodilator effect of ANP is, at least partially, dependent on attenuation of vascular sympathetic tone, possibly at a prejunctional site(s).

Autonomic nervous system, Blood pressure, Hypertension, Mice, Natriuretic peptide

Time for primary review 31 days.

1 Introduction

There is evidence that Atrial Natriuretic Peptide (ANP), in addition to its well-known hypotensive effect [1–3], also reduces arterial blood pressure (ABP) chronically. Long term-infusion of ANP into conscious animals causes a sustained fall in ABP [4,5], independently of the actions of the hormone on blood volume and renal salt and water excretion. These findings have recently been corroborated in genetic mouse models expressing lifelong alterations in endogenous ANP activity. Transgenic mice overexpressing a transthyretin-ANP fusion gene (TTR–ANP) constitutively in the liver are markedly hypotensive relative to their genetically-matched nontransgenic (NT) counterparts, in association with lifelong eight- to ten-fold elevation in plasma ANP concentration [6]. In contrast, ‘knockout’ mice harboring a functional deletion of the pro-ANP gene (−/−) [7] or its guanylate cyclase-linked A receptor (GC-A) [8] are hypertensive with respect to their wild type (+/+) siblings, consequent to elimination of ANP or its receptor. The TTR–ANP and the −/− mice do not show evidence of intravascular volume depletion or expansion, respectively [9,10], and maintain salt and fluid balance, even when kept on extremes of dietary salt intake (0.008–8% NaCl) [10,11].

Whereas the acute ANP-dependent fall in ABP is mediated by a reduction in cardiac output [1–3], the primary hemodynamic alteration underlying the chronic hypotensive effect of ANP is a reduction in total peripheral resistance (TPR) [4,5]. TTR–ANP mice have reduced TPR compared to their NT controls, due to vasodilation in most vascular beds [12]. In parallel with this, we have recently shown that the chronic hypertension in −/− mice is determined by an increase in basal TPR, in the absence of significant alterations in other hemodynamic parameters [13]. Thus, these complementary findings indicate that the chronic hypotensive effect of ANP is primarily mediated by vasodilation of the resistance vasculature.

The mechanism by which ANP reduces TPR chronically is not known. Although ANP, at high concentrations, can directly relax vascular smooth muscle in large arteries via GC-A mediated stimulation of cGMP [14,15], the resistance vasculature, with the possible exception of the renal vascular bed [16] has a scarcity of GC-A receptors [17,18] and is insensitive to direct relaxation by ANP [19–21]. Thus, the chronic relaxant effect of ANP in the microvasculature may be indirectly mediated by interactions of the hormone with tonic vasoeffector mechanism(s). We have recently shown that this mechanism is not mediated by differences in synthesis or activity of locally-acting vasoactive endothelial factors NO, CNP or ET-1, inasmuch as the concentration of these substances in the resistance vasculature and their target cardiovascular effects are not altered by chronic differences in endogenous ANP activity [22]. On the other hand, ANP exerts widespread acute inhibition of sympathetic nervous function, both centrally [23,24] and peripherally [25,26]. Furthermore, the hypotensive effect of ANP is significantly attenuated by autonomic ganglionic blockade [27,28], and is exacerbated by chronically elevated sympathetic tone [4,29]. These findings suggest a dependency on inhibition of sympathetic tone for expression of the hypotensive effect of ANP.

It could be inferred from these findings that the sympathoinhibitory activity of ANP, if tonically active, would contribute to the chronic vasodilatory effect of this hormone in the resistance vasculature. The absence of this antagonism in the −/− mice would result in chronic hypertension, in association with an elevation of cardiovascular sympathetic tone, whereas the chronically elevated plasma ANP activity in the TTR–ANP would lead to attenuation of sympathetic tone and manifestation of the hypotensive phenotype characteristic of this model. The objective of the present study was to determine whether alterations in tonic cardiovascular sympathetic tone underlie the differences in ABP associated with chronic differences in endogenous ANP activity. Total plasma catecholamine concentration and changes in ABP and HR following autonomic ganglionic blockade were measured in TTR–ANP, −/− and control mice as indices of sympathetic nerve activity [30,31]. In addition, we measured the pressor and chronotropic responses to exogenous norepinephrine infusion during ganglion blockade in all genotypes and compared norepinephrine receptor binding in representative tissues of −/− and +/+ mice, in order to determine whether any ANP-dependent alteration(s) in cardiovascular sympathetic tone is/are due to differences in peripheral responsiveness to adrenergic receptor stimulation.

2 Methods

2.1 Animals

Production, molecular analysis and hemodynamic characteristics of TTR–ANP, −/− and their respective wild-type controls have been described in detail elsewhere [6,7,12,13]. Male TTR–ANP mice and their NT littermates (DBA/2J background) and −/− and +/+ mice (C57BL/6J background) of both sexes, 8–12 months old, and weighing 24–38 grams were used in this study. Expression of the transgene in the TTR−ANP mice was screened by a modified polymerase chain reaction protocol [6] and the genotypes of the ANP knockout strain were identified by Southern blot analysis of EcoR1-digested genomic DNA from the tail [7] soon after weaning. The animals were housed according to sex and genotype in groups of 2–4 per cage at ambient temperature of 23°C and ≃40% humidity in a room with a 12-h light:dark cycle and maintained on normal rodent chow (0.4% NaCl, Ralston Purina No. 5001). This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, 1996) and with the University of Toronto guidelines governing use of research animals.

2.2 Materials

All materials for the catecholamine radioenzymatic assay were supplied with the kit (Amersham, Oakville, Ontario), with the exception of toluene and isoamyl alcohol which were purchased from Sigma Chemical (St. Louis, MO) and Liquifluor which was purchased from Canberra Packard (Mississauga, Ontario). Pentolinium, hexamethonium and norepinephrine and bacitracin were also from Sigma. PMSF, leupeptin and pepstatin were from ICN (Montreal, Quebec). Scintiverse scintillation cocktail was obtained from Fisher Scientific (Nepean, Ontario) and l-[2,5,6-³H]-norepinephrine was purchased from NEN Dupont (Markham, Ontario). All other chemicals were from VWR (Mississauga, Ontario).

2.3 Surgical preparation

Mice were anesthetized with Inactin (150 μg/g body weight ip) and kept at a body temperature near 38°C with a heat lamp. After tracheostomy, a jugular vein and carotid artery were cannulated with catheters (300–400 μm diameter) fashioned from PE-50 tubing for intravenous infusion and measurement of ABP and heart rate (HR) respectively. Upon completion of surgery, 0.12 ml of isotonic saline containing 2.25% bovine serum albumin and 1% glucose were infused over 15 min as a priming dose, followed by constant infusion of the same solution at 0.12 ml/h for the duration of the experiment. The experiment was begun after an additional 30 min equilibration period.

2.4 Measurement of arterial blood pressure and heart rate

ABP and HR were monitored continuously during the experiment, using a small volume displacement pressure transducer (model RP 1500, Narco Systems, Toronto, Ontario) connected to a MacLab/4e data acquisition system. HR was calculated instantaneously from the pressure pulses.

2.5 Autonomic ganglion blockade (HEX)

Total autonomic ganglion blockade was achieved by intravenous infusion of 0.5 mg min⁻¹ kg⁻¹ hexamethonium: 0.05 mg min⁻¹ kg⁻¹ pentolinium for 30 min. The effectiveness of the blockade was assessed at the end of the experiment by observing reflex changes in ABP and HR subsequent to bilateral carotid artery ligation. The absence of reflex increases in ABP and HR following carotid artery ligation was deemed to indicate physiologically effective autonomic ganglionic blockade.

2.6 Experimental protocol for in vivo studies

All in vivo experiments consisted of a 30-min control period, followed by 30-min autonomic ganglionic blockade and a further 30-min infusion of norepinephrine (±arterenol, 1 μg min⁻¹ kg⁻¹) under continuous ganglionic blockade. Measurements of ABP and HR were recorded every 10 min for the duration of the experiment. The end point of each period is reported.

2.7 Blood sample collection

Blood for catecholamine analysis was collected by exsanguination from freshly decapitated animals into chilled tubes containing a final concentration of 1.8 mg EGTA:1.2 mg glutathione per ml of blood. The tubes were inverted several times to mix the blood with the preservatives and centrifuged at 3000 revolutions per min (rpm) for 15 min at 3°C. The plasma samples were stored at −70°C until assayed.

2.8 Total plasma catecholamines

Total plasma catecholamine (norepinephrine and epinephrine) concentration was measured by a modified radioenzymatic method of Peuler and Johnson [32] with a commercially available kit (Amersham, Oakville, Ont.), according to the instructions provided by the manufacturer. Briefly, 50 μl of plasma were mixed in duplicate with 40 μl of a reaction mixture consisting of Tris–EGTA–MgCl₂ buffer pH 8.5, tritiated S-adenosyl-l-methionine ([³H]-SAM), and catechol-O-methyltransferase (COMT) in disposable glass tubes. A norepinephrine+epinephrine standard was added to one of the plasma samples to a final concentration of 400 pg/ml in a final volume of 100 μl, and an equivalent volume of stabilizing buffer was added to the duplicate sample. All samples were incubated at 37°C for 1 h. At the end of the incubation the contents of each tube were mixed vigorously with 50 μl of a 4 mM mixture of metanephrine and normetanephrine for termination of the methylation reaction. Each sample was mixed with 2 ml of toluene/isoamyl alcohol (3:2 v/v) for catecholamine extraction, centrifuged at 100 rpm and frozen for 15 s in a dry ice:ethanol bath. The upper organic phase was decanted into a second set of tubes and mixed vigorously with 100 μl of 0.1 M acetic acid. The mixture was frozen in the dry-ice–ethanol bath as previously, and the organic phase was aspirated. The acetic acid residue was dried under a stream of air for 2 h and mixed vigorously with 1 ml of 0.05 M ammonium hydroxide. Periodate oxidation of the samples was initiated by addition of 50 μl of 4% (w/v) of sodium metaperiodate and terminated after 5 min by addition of 50 μl of 10% (v/v) glycerol and 1 ml of 0.1 M acetic acid. Each sample was mixed vigorously for 20 s with 10 ml of toluene/Liquifluor (1000:50, v/v) and frozen in dry ice–ethanol. The upper organic phase was decanted in separate scintillation vials containing 2 ml of 0.1 M acetic acid and counted in a liquid scintillation counter for 2 min. The sensitivity of the assay is in the range of 2–5 pg per 50 μl of sample.

2.9 Tissue norepinephrine binding

Membranes for norepinephrine receptor binding were prepared from tissues (kidney, heart, brain, liver and skeletal muscle) of −/− and +/+ mice. Tissues were harvested from anesthetized (sodium pentobarbital, 45 mg/kg) mice, flash-frozen in liquid nitrogen and stored at −80°C until processed. Frozen tissues were thawed and rinsed in 0.9% NaCl. The tissues were then minced and homogenized in a Teflon/glass homogenizer with ten volumes of cold homogenization buffer containing 10 mM MgCl₂, 10 mM Hepes pH 7.5, 10 mM NaCl, 5 mM PMSF, 5 μg/ml leupeptin and 5 μl/ml pepstatin. The suspension was centrifuged at 4000 g for 10 min at 4°C. The pellet was discarded, and the membrane in the supernatant was pelleted by centrifugation at 16 000 g for 15 min at 4°C. The membrane pellet was then resuspended in 10 mM NaHCO₃ at pH 8, containing 5 mM PMSF, 5 μg/ml leupeptin and 0.1% bacitracin and stored at −70°C. Protein concentration was determined by a modified Lowry method using bovine serum albumin (BSA) as a standard. Norepinephrine binding assays were performed at 20°C in a binding buffer containing 50 mM Hepes pH 7.5, 5 mM MgCl₂, 10 mM NaCl, 1 mM PMSF, 2 mg/ml leupeptin, 0.1% BSA and 0.1% bacitracin. The binding reaction was initiated by the addition of 100 000 cpm l-[2,5,6-³H]-norepinephrine, in the presence or absence of competing 100 μM unlabelled norepinephrine, to 100 μg of membrane in a final volume of 300 μl. Duplicate samples were incubated for 30 min and then centrifuged at 16 000 g for 10 min to separate free from bound ligand. The membrane pellets were washed three times with 100 μl of wash buffer (10 mM Hepes, 10% sucrose, pH 7.5). The washed pellet was resuspended in 200 μl of wash buffer and added to 5 ml of ScintiVerse and counted on a Beckman LS 1801 scintillation counter.

2.10 Statistical analysis

All results are presented as means±SE. The unpaired t-test was employed to compare differences between mutant (TTR–ANP, −/−) and their controls (NT, +/+) in plasma catecholamine and the percentage changes in ABP and HR after ganglionic blockade. One-way ANOVA was used to compare ABP and HR responses to the different treatments within each group and between control and mutant mice of each genotype. A P value of <0.05 was considered to indicate statistically significant difference.

3 Results

The effect of autonomic ganglionic blockade (HEX) and norepinephrine infusion on ABP and HR in TTR–ANP and NT mice is shown in Fig. 1. Basal ABP was significantly lower in TTR–ANP than in NT (P<0.0001, Fig. 1A). Basal HR was also lower in TTR–ANP, but this difference did not reach statistical significance (Fig. 1B). Ganglionic blockade significantly decreased ABP (P<0.05, Fig. 1A) and reduced HR (Fig. 1B) in both genotypes. However, the relative decrease in basal ABP following HEX was significantly greater in the NT mice compared to the TTR–ANP mice (P<0.0001) (% change, NT=−34±1, TTR–ANP=−14±3). In contrast, the relative fall in HR was greater in the TTR–ANP mice than in the NT mice (P<0.05) (% change, NT=−8±2, TTR–ANP=−17±3). Norepinephrine infusion during HEX elicited similar pressor (Fig. 1A) and chronotropic (Fig. 1B) responses in both genotypes.

Fig. 1

Average ABP (A) and HR (B) during baseline conditions (basal), after autonomic ganglionic blockade (HEX), and in response to intravenous norepinephrine infusion (HEX+NE) in TTR–ANP (n=6) and NT (n=6) mice. Basal ABP differed significantly between genotypes (* P<0.0001). Significant differences were also found between basal ABP and HEX (#, TTR–ANP, P<0.05; NT, P<0.001) and HEX+NE (+, TTR–ANP, P<0.0001; NT, P<0.05), as well as between HEX and HEX+NE (** P<0.0001). Differences in HR were found between basal and HEX+NE (+, P<0.001) and between HEX and HEX+NE (** P<0.0001).

Fig. 2 shows the effect of HEX and norepinephrine on ABP and HR in −/− and +/+ mice. Basal ABP was significantly elevated in −/− mice (P<0.05, Fig. 2A), whereas HR did not differ significantly between genotypes (Fig. 2B). HEX reduced ABP (−/−, P<0.0001; +/+, P<0.05) (Fig. 2A) and HR (P<0.05) (Fig. 2B) in both genotypes. However, the relative fall in ABP was twice as great in the −/− mice compared to the +/+ mice (P<0.0001) (% change, +/+=−21±2, −/−=−48±2). The pressor (Fig. 2A) and chronotropic (Fig. 2B) responses to norepinephrine during HEX did not differ significantly between the two genotypes

Fig. 2

Average ABP (A) and HR (B) during baseline conditions, after autonomic ganglionic blockade (HEX) and in response to intravenous norepinephrine infusion (HEX+NE) in −/− (n=7) and +/+ (n=7) mice. Basal ABP differed significantly between genotypes (* P<0.05). Significant differences were also found between basal ABP and HEX (# −/−, P<0.0001; +/+, P<0.05) and between HEX and HEX+NE (** P<0.0001). Significant differences in HR were found between basal and HEX (# P<0.05) and HEX+NE (+ P<0.05), as well as between HEX and HEX+NE (** P<0.0001).

Total plasma catecholamine concentration in the four genotypes is shown in Table 1. Plasma catecholamine concentration was lower in the TTR–ANP mice, but this difference did not reach statistical significance. In contrast, −/− mice had significantly higher plasma catecholamine concentration compared to the +/+ mice (P<0.05).

Table 1

Total plasma catecholamine concentration in TTR–ANP, NT, −/− and +/+ micea

Genotype	Catecholamine concentration (pg/ml)
TTR–ANP	5848±514
(n=4)
NT	7475±1124
(n=4)
−/−	13 803±2125b
(n=4)
+/+	8109±852
(n=4)

a

Values are means±SE.

b

Statistical difference between −/− and +/+ (P=0.047 by unpaired t-test).

Table 2 shows adrenergic receptor binding with norepinephrine in representative tissues of −/− and +/+ mice. Kidney and skeletal muscle had the highest and lowest binding respectively. Variable amounts of receptor binding were found in cardiac ventricle, brain and liver. No statistically significant differences in norepinephrine binding were found between tissues of −/− and +/+ mice.

Table 2

Adrenergic receptor binding in tissues of −/− and +/+ micea

	l−[2,5,6− 3H]-Norepinephrine binding (specific cpm/100 μg)
Group	Kidney	Ventricle	Brain	Liver	Skeletal muscle
−/−	1732±405	1244±190	1105±108	1007±178	584±155
(n=3)
+/+	2025±329	1207±230	1140±179	993±163	535±208
(n=4)

a

Values are means±SE.

4 Discussion

The primary finding of this study is that tonic cardiovascular sympathetic tone varies inversely with the chronic level of endogenous ANP activity; this being attenuated in hypotensive ANP-overexpressing transgenic mice and increased in hypertensive ANP ‘knockout’ mice respectively. Furthermore, the magnitude of the fall in ABP and HR to HEX is not correlated with the basal ABP and HR, indicating that the observed differences in cardiovascular sympathetic tone underlie genotype-specific differences in sympathetic nerve activity. These findings suggest that the chronic hypotensive effect of ANP is, at least partially, dependent on attenuation of sympathetic tone.

It is unlikely that the differences in ABP between the mutant mice and their genetically-matched controls are due to differences in intravascular volume. First, hematocrits, which may be viewed as an indirect index of intravascular volume, do not differ between mutant and wild type mice [9,10,22]. Secondly, the mutant mice maintain salt and fluid balance, even when maintained on extremes of low or high dietary salt intake [10,11], indicating that renal function is not compromised in these animals. On the other hand, the differences in basal ABP between the mutant mice and their controls are fully abolished by HEX, indicating that the underlying differences in sympathetic tone per se, account for the difference in cardiovascular phenotype. It would have been expected that if there were a volume component to the differences in ABP between these mice, that this would be manifested, even in the absence of peripheral sympathetic tone, such as during HEX. On the contrary, we have previously shown that the exaggerated hypotensive response of −/− mice to HEX is specifically attributed to a reduction in TPR [13], in the absence of apparent differences in cardiac performance. Thus, these findings imply that the chronic hypotensive effect of ANP requires attenuation of sympathetic tone to the resistance vasculature. The requirement for such an intermediary effector mechanism of ANP-dependent relaxation of the resistance vasculature, is further suggested by the apparent scarcity of GC-A receptors in resistance vessels, and their relative insensitivity to ANP-dependent cGMP synthesis [17,18], thus precluding a direct role of this pathway in mediating the effect of ANP on vascular resistance.

The nature of the neuromodulatory action of ANP on sympathetic nervous activity has not been fully elucidated. When administered acutely, ANP exerts a pervasive sympatholytic effect which is mediated primarily by GC-A receptor activation [18,33]. Centrally, ANP reduces sympathetic outflow from cardiovascular regulatory areas in the brain stem [27,34,35]. Peripherally, ANP inhibits autonomic ganglion neurotransmission [25,36], spontaneous and evoked norepinephrine synthesis and release from post-ganglionic sympathetic nerve fibers [26] and adrenal medulla [37]. In addition, ANP may also interfere with the functional expression of post-synaptic α-1 adrenergic receptors [38,39]. The extent to which these interactions may occur chronically is not known. The present study shows that the genotype-dependent differences in sympathetic tone are not due to changes in responsiveness of target cardiovascular responses to peripheral adrenergic receptor stimulation, given that norepinephrine infusion elicits comparable absolute pressor and chronotropic responses in ganglion-blocked mutant and wild-type control mice. Furthermore, tissue adrenergic receptor binding is not altered by the level of endogenous ANP activity. Although these data do not discount the possibility of regional differences in vascular adrenergic receptor density and/or subtype, the absence of genotype-specific difference in norepinephrine binding in the whole organ membrane preparations, is, nevertheless, in concordance with the observed lack of differences in the in vivo responses to norepinephrine. These findings suggest that the chronic effect of ANP on sympathetic nerve activity is mediated by pre-junctional mechanism(s). Interestingly, we have previously reported that ANP exerts an inhibitory effect on hypotension-induced reflex tachycardia by potentiating parasympathetic-mediated inhibition of sympathetic outflow prejunctionally [40,41], thus, demonstrating the ability of ANP to modulate sympathetic nerve activity at this level. However, it is unlikely that such interaction with autonomic control of heart rate plays a major role in long-term regulation of ABP, since the baroreflex mechanism of ABP and HR regulation quickly resets to the prevailing level of ABP, as exemplified by the different phenotypes of ABP used in the present study.

How does ANP exert its chronic sympatholytic action? In principle, any of the identified neuromodulatory effects of ANP, either singly or in combination, could account for the observed differences in sympathetic tone between the genotypes. For example, the absence of a tonic inhibitory effect on tyrosine hydroxylase activity in postganglionic nerve terminals [42] and in the adrenal medulla [37] could partially account for the elevated plasma catecholamine levels in the −/− mice. It could also be argued that the co-localization of ANP and GC-A in the autonomic ganglia [18,43,44], may function as a tonically active neuromodulatory unit of sympathetic outflow. In this regard, Floras et al. [36] has recently shown that, at least in humans, ANP-mediated sympatholysis is preferentially mediated by inhibition of autonomic ganglionic neurotransmission. Thus, it is conceivable that the lower vascular resistance in ANP-overexpressing transgenic mice [12] could be due to tonic ANP-mediated inhibition of sympathetic ganglionic transmission, whereas the lack of such neuromodulation in −/− mice could account for the high peripheral resistance seen in these animals [13].

The extent to which structural alterations in the resistance vasculature may contribute to the ANP-dependent differences in basal blood pressure is not known. Chronic increases in ABP are usually accompanied by hypertrophy and remodeling of the tunica media of resistance vessels [45,46], and chronically elevated sympathetic nerve activity exerts trophic effects on the resistance vasculature [47]. These changes could increase vascular resistance directly by reducing lumen diameter [46], and indirectly by exacerbating the pressor effects of vasoconstrictors ([48]. However, the pressor responses to norepinephrine and to endothelin-1 [22] do not differ between −/− and +/+ mice. A greater pressor response to these two potent vasoconstrictors by the −/− mice would have been expected if significant hypertrophy of the vasculature had occurred. It is entirely conceivable that media proliferation indeed occurs in the −/− mice, but that the subsequent remodeling of vessel wall leads to redistribution of wall materials, such that there is no net increase in wall thickness or reduction of luminal diameter.

5 Conclusions

In conclusion, the present study shows that chronic differences in endogenous ANP activity in mice, resulting in life-long resistance-dependent alterations in ABP are accompanied by directional changes in underlying cardiovascular sympathetic tone; this being attenuated in hypotensive transgenic mice overexpressing ANP and elevated in hypertensive ANP-gene deleted mice. These findings indicate that ANP exerts a chronic vasodilatory effect leading to hypotension that is, at least partially, dependent on attenuation of vascular sympathetic tone.

Acknowledgments

We thank Judy VanHorne (Queen’s University, Canada) for the norepinephrine binding study. This study was supported by grants from the Heart and Stroke Foundation of Ontario #T-2952 to H. Sonnenberg and #NA-3479 to S.C. Pang and T.G. Flynn, and by a grant from the NIH (HL50873) to M.E. Steinhelper. L.G. Melo was the recipient of a research scholarship from the Heart and Stroke Foundation of Canada.