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Chronic hypertension in ANP knockout mice: contribution of peripheral resistance

Chronic hypertension in ANP knockout mice: contribution of peripheral resistance

Regulatory Peptides 79 (1999) 109–115 Chronic hypertension in ANP knockout mice: contribution of peripheral resistance a, a a b c a L.G. Melo *, A.T...

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Regulatory Peptides 79 (1999) 109–115

Chronic hypertension in ANP knockout mice: contribution of peripheral resistance a, a a b c a L.G. Melo *, A.T. Veress , U. Ackermann , S.C. Pang , T.G. Flynn , H. Sonnenberg a

b

Department of Physiology, University of Toronto, Toronto, Ontario, Canada, M5 S 1 A8 Department of Anatomy and Cell Biology, Queen’ s University, Kingston, Ontario, Canada, K7 L 3 N6 c Department of Biochemistry, Queen’ s University, Kingston, Ontario, Canada, K7 L 3 N6 Received 27 July 1998; received in revised form 20 October 1998; accepted 20 October 1998

Abstract Atrial Natriuretic Peptide (ANP) exerts a chronic hypotensive effect which is mediated by a reduction in total peripheral resistance (TPR). Mice with a homozygous disruption of the pro-ANP gene (2 / 2 ) fail to synthesize ANP and develop chronic hypertension in comparison to their normotensive wild-type ( 1 / 1 ) siblings. In order to determine whether alterations in basal hemodynamics underlie the hypertension associated with lack of endogenous ANP activity, we used anesthetized mice to measure arterial blood pressure (ABP) and heart rate (HR), as well as cardiac output (CO) by thermodilution technique. 2 / 2 (n 5 7) and 1 / 1 (n 5 10) mice of comparable weight and age were used. Stroke volume (SV) and TPR were derived from CO, HR, and ABP by a standard formula. ABP (mm Hg) was significantly higher in 2 / 2 (13264) (P , 0.0001) than in 1 / 1 mice (9562). CO (ml min 21 ), HR (beats min 21 ) and SV (ml beat 21 ) did not differ significantly between 2 / 2 and 1 / 1 mice (CO 2 / 2 5 7.360.5, 1 / 1 5 8.360.6; HR 2 / 2 5 407622, 1 / 1 5 462621; SV 2 / 2 5 17.661.1, 1 / 1 5 17.661.7). However, TPR (mm Hg ml 21 min 21 ) was significantly elevated in 2 / 2 mice (18.460.7) compared to 1 / 1 mice (12.361) (P 5 0.0003). Autonomic ganglion blockade with a mixture of hexamethonium and pentolinium was followed by comparable percent reductions in CO (2 / 2 5 2864, 1 / 1 5 2963), HR (2 / 2 5 964, 1 / 1 5 1664) and SV (2 / 2 5 2164, 1 / 1 5 1566) in both genotypes. However, the concomitant decrease in ABP (%) in 2 / 2 (4162) was significantly greater than in 1 / 1 (2364) mice (P 5 0.0009) and was accompanied by a significant reduction in TPR. We conclude that the hypertension associated with lack of endogenous ANP is due to elevated TPR, which is determined by an increase in cardiovascular autonomic tone.  1999 Elsevier Science B.V. All rights reserved. Keywords: Autonomic; Cardiac output; Heart rate; Hypertension; Stroke volume; Sympathetic tone

1. Introduction Recent findings in genetic mouse models expressing alterations in activity of endogenous Atrial Natriuretic Peptide (ANP) suggest that this hormone, in addition to its well-defined acute hypotensive effect [1,2], also participates in chronic regulation of blood pressure. Overexpression of a transthyretin-ANP fusion gene (TTR-ANP) in mice leads to a marked reduction in arterial blood pressure (ABP), in association with the lifelong elevation of plasma *Corresponding author. Tel.: 11-416-978-4017; fax: 11-416-9784940.

ANP concentration [3]. In contrast, disruption of the native genes for ANP [4] or its guanylate cyclase-A (GC-A) receptor [5] by gene targeting results in hypertension, consequent to elimination of ANP activity. The ANP ‘knockout’ (2 / 2 ) mice develop time-dependent salt sensitivity of ABP after prolonged maintenance on high dietary salt intake [4,6], at least in part, in association with an apparent inability to regulate plasma renin activity [6]. The chronic hypotensive effect of ANP in the TTR-ANP mice is mediated by a reduction in total peripheral resistance (TPR), due to vasodilation of several regional vascular beds [7–9]. The hemodynamic correlates underlying the expression of hypertension in the ANP knockout

0167-0115 / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved. PII: S0167-0115( 98 )00149-9

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mice, however, are not known. On the basis of the hemodynamic characteristics of the TTR-ANP mice [7], it may be expected that the hypertension of 2 / 2 mice is, at least partially, determined by an elevation in baseline TPR. This is indeed implied by the observed increase in baseline diastolic pressures (Melo and Sonnenberg, unpublished observations) in these mice, relative to their normotensive wild-type ( 1 / 1 ) littermates. Elevated diastolic pressure is generally considered to be a manifestation of elevated TPR [10]. The mechanism by which ANP chronically reduces TPR is not known. The insensitivity of the resistance vasculature to ANP [11–13] suggests that the relaxant effect of this hormone in the microvasculature may be mediated by an indirect vasoeffector mechanism. It is not mediated by differences in endogenous activity of C-type natriuretic peptide (CNP), or other endothelium-derived vasoactive factors endothelin-1 (ET-1) and nitric oxide (NO) [14,15]. On the other hand, the autonomic nervous system may play a role as an intermediary effector of ANP-dependent vasodilation, inasmuch as the ability of ANP to reduce ABP is partly determined by its widespread inhibition of sympathetic nervous activity [16–19]. For example, the acute hypotensive effect of ANP is greatly attenuated by autonomic ganglionic blockade [16,19], and is exacerbated by conditions characterized by high sympathetic tone [8]. It may be inferred from these findings that the sympathoinhibitory activity of ANP, if tonically operative, could contribute to the chronic vasodilatory effect of this hormone in the resistance vasculature. Thus, in the absence of this antagonism, the 2 / 2 mice would be expected to develop hypertension, in association with an elevation of cardiovascular sympathetic tone. In addition, the contribution of whole-body autoregulation may not be discounted as a possible compensatory adjustment to an initial disturbance in cardiac output. In the present study, we used the thermodilution method to characterize basal systemic hemodynamics in 2 / 2 and 1 / 1 mice, with the purpose of identifying the hemodynamic alteration(s) underlying the elevation of ABP in 2 / 2 mice. In addition, we measured changes in basal hemodynamics following autonomic ganglionic blockade in both genotypes in order to assess the relative contribution of cardiovascular autonomic tone to the hypertension associated with lack of endogenous ANP.

2. Materials and methods

2.1. Animals The production of ANP knockout mice has been described in detail elsewhere [4]. In brief, a targeting construct was designed to replace 11 base pairs of exon 2 of the mouse pro-ANP gene (Nppa) with the neomycin resistance gene in embryonic stem cells of mouse strain

129. Chimeras harboring the mutation were mated to mice of strain C57BL / 6J (B6). Matings between the resulting 129 3 B6 heterozygotes ( 1 / 2 ) produced F 2 offspring of all three genotypes (2 / 2 , 1 / 2 , 1 / 1 ) in approximately Mendelian proportions. F 2 homozygous mutant (2 / 2 ) and wild-type ( 1 / 1 ) mice of both sexes, 17–22 weeks old and weighing 31–34 g were used in this study. The animals were obtained from our resident colony, which was founded with pathogenfree heterozygous ( 1 / 2 ) breeding pairs. The genotypes were identified by Southern blot analysis of EcoR Idigested genomic DNA from the tail [4] soon after weaning. The animals were housed according to sex in groups of two to four per cage and kept at ambient 238C and 40% humidity in a room with a 12 h:12 h light–dark schedule and maintained on normal rodent chow (0.4% NaCl, Ralston Purina No. 5001).

2.2. Surgical preparation On the day of the experiment the animals were anesthetized with Inactin (150 mg / g body weight i.p.) and kept at a body temperature near 388C with a heat lamp. After tracheostomy, a jugular vein and femoral artery were cannulated with catheters (300–400 mm diameter) fashioned from pulled-out PE-50 polyethylene tubing for intravenous infusion and measurement of mean arterial pressure (ABP, mm Hg) and heart rate (HR, beats min 21 ), respectively. A microprobe thermistor (F[1; Columbus Instruments, Columbus, OH) was inserted into the right common carotid artery and advanced to the junction at the aortic arch for measurement of cardiac output (CO, ml min 21 ) by thermodilution. All catheters were held firmly in place with cotton ligatures and kept patent by prior flushing with heparinized (20 U ml 21 ) isotonic saline. Upon completion of surgery, 0.12 ml of isotonic saline containing 2.25% bovine serum albumin (BSA) and 1% glucose were infused over 15 min and followed by constant infusion of the same solution at 0.12 ml h 21 for the duration of the experiment, except for brief interruptions for measurement of cardiac ouput. The experiment was begun after a 15 min equilibration period.

2.3. Measurement of 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. Measurements of ABP, HR and CO were taken simultaneously.

2.4. Measurement of cardiac output Twenty-five microliters of 0.9% NaCl / 2.25% BSA / 1%

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glucose at room temperature ( ¯ 248C) were rapidly delivered ( ¯ 1 s) into the jugular vein by a microinjector pump (model 500, Columbus Instruments). Injectate and blood temperatures were recorded individually by a Cardiomax II-R CO computer (Columbus Instruments). CO was calculated instantaneously from the thermodilution curve of the injected saline by the Cardiomax II computer using the equation (T blood 2 T inj. ) 3 (vol.) CO 5 ]]]]]]] ` T dt

E

0

where T blood is the temperature of blood prior to injection (8C), T inj. is the temperature of injectate (8C), vol. is the volume of injected bolus (ml), and e0` T dt is the area under the thermodilution curve (8C min 21 ). The thermodilution curve was recorded with an IBMcompatible computer (486 DX2-66) equipped with the Easyest LX (Kithley Asyst, Taunton, MA) data acquisition system. Its area was calculated by a factory installed algorithm that is based on the two sample points method of Williams et al. [20]. The average of four successive CO measurements taken 1 min apart during the control period and following autonomic ganglion blockade is reported in this study. Stroke volume (SV, ml beat 21 ) and total peripheral resistance (TPR, mm Hg ml 21 min 21 ) were derived from the equation ABP5(HR3SV3TPR). We considered the omission of right atrial pressure from the equation to have been a negligible error. At termination of the experiment, the position of the thermocouple microprobe was verified by autopsy.

2.5. Autonomic ganglion blockade Total autonomic ganglion blockade was achieved by intravenous infusion of 0.5 mg min 21 kg 21 hexamethonium / 0.05 mg min 21 kg 21 pentolinium for 30 min. Hemodynamic measurements were performed at the end of the infusion. The effectiveness of the blockade was verified at the end of the experiment by absent or diminished reflex changes in ABP and HR subsequent to bilateral carotid artery ligation.

Fig. 1. Thermodilution curves for four successive cardiac output (CO) measurements taken ¯1 min apart in a 2 / 2 mouse. The differences in peak amplitude were not temporally related to injection sequence. The CO values calculated from the curves differed from one another by less than 10% (n.s.).

2.6. Statistical analysis All results are presented as means 6SE. The unpaired t-test was used to compare differences between mutant and wild-type mice. A two-factor ANOVA followed by the Bonferroni multiple comparison test was used to test separate and combined effects of genotype and ganglion blockade on hemodynamics. A P value of #0.05 was considered to indicate a statistically significant difference.

3. Results The thermodilution curves for four successive measurements of CO in 2 / 2 one mouse is shown in Fig. 1. The individual curves were superimposable, and the calculated CO values in each animal were within 10% of one another, and were highly comparable to those obtained by others using an electromagnetic flow probe [21]. Recirculation was negligible, as suggested by the smoothness of the descending limb of the curves, indicating that the injected bolus of cold (room temperature) saline equilibrated with blood temperature within one circulatory passage. Comparable characteristics were observed in the thermodilution curves of control 1 / 1 mice.

Table 1 Basal systemic hemodynamics in 2 / 2 and 1 / 1 mice

2 / 2 (n57) 1 / 1 (n510)

ABP (mm Hg)

HR (bpm)

CO (ml min 21 )

SV (ml beat 21 )

TPR (mm Hg ml 21 min 21 )

13264 a 9562

407622 462621

7.360.5 8.360.6

17.661.1 17.661.7

18.460.7 a 12.361.0

Values are mean6SE. 2 / 2, 1 / 1: ANP knockout, wild-type, respectively. ABP, arterial blood pressure; HR, heart rate; CO, cardiac output; SV, stroke volume; TPR, total peripheral resistance. a Statistical difference between 2 / 2 and 1 / 1 mice ( a P,0.0003 by unpaired t-test).

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The baseline systemic hemodynamics of 2 / 2 and 1 / 1 mice are summarized in Table 1. ABP and TPR were significantly elevated in the 2 / 2 mice compared to the 1 / 1 control mice. SV did not differ between genotypes. There was a tendency towards lower CO values in the 2 / 2 mice as a result of lower basal HR. The differences in basal CO and HR between 2 / 2 and 1 / 1

mice, however, did not reach statistical significance. The effects of autonomic ganglion blockade on the basal hemodynamics of 2 / 2 and 1 / 1 mice is shown in Fig. 2 and Table 2. The blocker mixture effectively abolished reflex increase in ABP following bilateral carotid artery ligation in both genotypes (Fig. 2), indicating physiologically effective blockade. CO, SV and HR decreased

Fig. 2. Reflex changes in arterial blood pressure (ABP) and heart rate (HR) in response to bilateral carotid artery ligation in a 2 / 2 (A) and a 1 / 1 (B) mouse, following total autonomic ganglion blockade for 30 min. The treatment abolished reflex increases in HR and ABP similarly in both genotypes.

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Table 2 Absolute systemic hemodynamics and changes (%) relative to baseline following autonomic ganglion blockade in 2 / 2 and 1 / 1 mice ABP

HR

mm Hg 2 / 2 (n57) 1 / 1 (n510)

a

7864 7364 a

Two-way ANOVA P, genotypes P, treatment P, interaction

1.7310 26 7.6310 212 1.1310 24

D (%) 24162 22364

CO D (%)

bpm b

a

370619 402612 a

2964 21664

0.0098 0.0051 0.335

ml min

SV 21 a

D (%) 22864 22963

5.360.4 5.660.3 a

0.16 3.6310 25 0.49

ml beat

TPR 21

14.561.2 13.660.8

D (%) 22164 21566

0.92 0.0078 0.90

mm Hg ml 21 min 21 15.561 14.161

c

D (%) 21664 b 1566

4.65310 24 0.57 0.018

Values are mean6SE. 2 / 2, 1 / 1: ANP knockout, wild-type, respectively. ABP, arterial blood pressure; HR, heart rate; CO, cardiac output; SV, stroke volume; TPR, total peripheral resistance. a Statistical difference between 2 / 2 and 1 / 1 mice ( a P,0.0009 by unpaired t-test). b,c Statistical difference between baseline (Table 1) and autonomic ganglion blockade ( a P,0.005, b P,0.05 by Bonferroni multiple comparison test).

significantly and comparably in both genotypes (Table 2). The accompanying fall in ABP, however, was significantly greater in the 2 / 2 mice than in the 1 / 1 mice. This was associated with a significant reduction in TPR in the 2 / 2 mice, whereas in the 1 / 1 mice TPR increased, but this did not reach statistical significance.

4. Discussion Chronic increases in ANP activity, either by administration of exogenous hormone [9,22], or by in vivo overexpression of an ANP transgene in mice [3,7], lead to hypotension, due to a reduction in TPR. The current study shows that mice rendered genetically incapable of synthesizing ANP develop hypertension, in association with an elevation of TPR. These findings thus provide complementary evidence that ANP exerts a chronic hypotensive effect, which is ultimately mediated by vasodilation of the resistance vasculature. The exaggerated vasodepressor response of 2 / 2 mice to acute autonomic ganglionic blockade (GB) suggests that the hypertension in these mice is due, at least in part, to an elevation of cardiovascular sympathetic tone. Indeed, the difference in basal blood pressure between 2 / 2 and 1 / 1 is fully abolished by GB, indicating that the increase in sympathetic tone per se is a sufficient condition for full expression of the hypertensive effect associated with lack of endogenous ANP activity. Furthermore, the elevation of sympathetic tone in the 2 / 2 mice occurs independently of any significant change in cardiac performance, and is manifested preferentially as an increase in peripheral vascular tone. These findings imply that the chronic vasodilatory effect of ANP in the resistance vasculature requires attenuation of sympathetic tone. The requirement for 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 [23]. 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 both centrally and peripherally. Centrally, ANP reduces sympathetic outflow from cardiovascular regulatory areas in the brain stem [24–26] and inhibits autonomic ganglion transmission [27]. Peripherally, ANP inhibits spontaneous and evoked norepinephrine synthesis and release from post-ganglionic sympathetic nerve fibers [28] and adrenal medulla [29,30], and it interferes with the functional expression of post-synaptic a-1 adrenergic receptors [31]. The extent to which these interactions may occur chronically is not known. The present study clearly shows that absence of chronic ANP activity leads to amplification of vascular sympathetic tone. However, we cannot ascertain whether this develops as a direct consequence of lack of neuromodulatory effects of ANP on sympathetic activity, or secondarily to abnormalities in other ANP-dependent actions. It could be argued that the widespread co-localization of ANP and its GC-A receptor in the sympathetic nervous system [32] may function as a tonically active neuromodulatory unit [33,34] participating in local inhibition of sympathetic nervous activity. The lack of such neuromodulation in 2 / 2 mice could account for the high resistance hypertension seen in these animals, and may also explain the hypertension of GC-A knockout mice [5], since the sympatholytic effects of ANP are mediated by the GC-A receptor [35]. The possibility that structural alterations in the resistance vasculature may contribute to the development of hypertension in 2 / 2 mice must also be considered. Chronic increases in ABP are generally accompanied by hypertrophy of the tunica media of resistance vessels [36]. Although this develops initially as an adaptive response to accommodate increases in wall tension, the ensuing expansion of the media could encroach into the lumen and raise vascular resistance [37]. The accompanying increase in

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smooth muscle mass may further elevate resistance by exacerbating the pressor response to vasoconstrictors [38]. The extent to which these alterations may occur in the 2 / 2 mice is not known. ANP has been shown to inhibit proliferation of vascular smooth in vitro [39], and to reduce hyperplasia of the media in response to balloon injury in vivo [40]. These observations raise the possibility that the absence of such effects in the 2 / 2 mice may lead to hypertrophy of the resistance vasculature. The subsequent remodelling of the vessel wall could result in increased wall-to-lumen ratios and reduction of lumen diameter [36]. These structural alterations per se would increase vascular resistance, and may play a role in the development of hypertension in these animals. In conclusion, the present study shows that knockout mice lacking endogenous ANP activity develop high resistance hypertension in association with an elevation of vascular sympathetic tone. These findings, in conjunction with previous observations that ANP exerts a chronic, low-resistance hypotensive effect, thus provide complementary evidence that ANP participates in the longterm regulation of arterial blood pressure, ultimately by affecting the tone of the resistance vasculature.

5. Perspectives The present study shows that chronic deficiency in ANP activity leads to high resistance hypertension, apparently due to an elevation in vascular sympathetic tone. These findings suggest that the long-term hypotensive effect of ANP is predominated by its indirect action on vascular resistance. It remains controversial whether deficiencies in endogenous ANP activity contribute to the pathology of hypertensive disease. A decrease in ANP secretion is seen in the prehypertensive stages of several hypertensive rat models with close resemblance to variants of human essential hypertension [41,42], suggesting that a defect in ANP synthesis / secretion may be an initiating factor in the development of hypertension. Polymorphisms of the ANP gene have recently been reported to occur with significantly greater frequency in humans with essential hypertension [43], and mutations in the GC-A receptor gene cosegregate with ABP in Dahl salt-sensitive rats [44]. The common theme between these and the current findings is that genetic defects in ANP activity may predispose towards development of hypertension.

Acknowledgements 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. L.G. Melo is

the recipient of a research scholarship from the Heart and Stroke Foundation of Canada.

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