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original contributionsnature publishing group

Abnormal circadian rhythm of blood pressure (BP) is linked to increased cardiovascular risk and is characterized by an excessive morning BP surge or lack of normal nocturnal BP fall.1,2 The mechanisms that underlie abnormal circadian BP are yet to be fully clarified. However, given that mental, emotional, and physical activities are critical contributors to diurnal BP variation,2,3 the morning surge is likely to relate primarily to an increase in feed-forward behavioral influences on BP. By contrast, nocturnal nondipping may reflect principally a reduced excretory ability of the kidney4 and subsequent activation of homeostatic feedback mecha-nisms, leading to a compensatory increase in vasomotor tone and BP.5

High-salt diet (HSD) can impair circadian BP rhythm in susceptible subjects both by exacerbating morning BP rise6 and by shifting night-time BP from dipper to nondipper pattern.5,7 The precise mechanisms involved in these dele-terious effects of HSD remain elusive. It is conceivable how-ever, that the salt-induced sensitization of central pressor pathways,8–10 leading to increased autonomic responsiveness to mental/physical activity11,12 can be essential for exacerbat-ing morning BP surge. Conversely, abnormal sodium handling by the kidney may be primarily responsible for the induction or aggravation of nocturnal hypertension by HSD.4

The renin–angiotensin system plays a key role in the regu-lation of sodium and fluid homeostasis.13 AT1A-receptor defi-ciency compromises renal function and promotes salt- and volume-sensitivity of BP.14–16 The renin–angiotensin system is also implicated in central autonomic control,17,18 and AT1A-receptor deficiency enhances the synergistic effects of stress and salt on BP.19 The renin–angiotensin system may thus be important in modulating both feed forward and feedback influences on circadian BP. Emerging evidence supports this possibility, indicating a role of the renin–angiotensin system

1Baker IDI Heart and Diabetes Institute, Melbourne, australia; 2Department of Physiology, University of Melbourne, Melbourne, australia; 3Centre for Biomedical Research, Hull york Medical School, University of Hull, Hull, UK; 4Department of Pharmacology, University of Melbourne, Melbourne, australia. Correspondence: Dmitry N. Mayorov (dmayorov@unimelb.edu.au)

Received 27 September 2009; first decision 21 October 2009; accepted 13 January 2010; advance online publication 18 February 2010. doi:10.1038/ajh.2010.12

© 2010 American Journal of Hypertension, Ltd.

The Day–Night Difference of Blood Pressure Is Increased in AT1A-Receptor Knockout Mice on a High-Sodium DietDaian Chen1,2, Luisa La Greca1, Geoffrey A. Head1, Thomas Walther3 and Dmitry N. Mayorov4

Backgroundabnormal circadian variation of blood pressure (BP) increases cardiovascular risk. In this study, we examined the influence of angiotensin aT1a receptors on circadian BP variation, and specifically on its behavioral activity-related and -unrelated components.

MethodsBP and locomotor activity were recorded by radiotelemetry in aT1a-receptor knockout mice (aT1a

−/−) and their wild-type controls (aT1a

+/+) placed on a normal-salt diet (NSD) or high-salt diet (HSD, 3.1% Na).

resultsThe 24-h BP was lower in aT1a

−/− than aT1a+/+ mice on a NSD

(92 ± 2 and 118 ± 2 mm Hg, respectively), whereas the day–night BP difference (ΔDNBP) was similar between groups (11 ± 2 and 12 ± 1 mm Hg, respectively). HSD increased BP by 20 ± 2 mm Hg and ΔDNBP by 7 ± 1 mm Hg in aT1a

−/− mice, without affecting these parameters much in aT1a

+/+ mice. The ΔDNBP increase in aT1a−/−

mice was caused by nondipping BP during the inactive late-dark

period. Conversely, BP rise associated with circadian behavioral activation during the early dark period was not altered by HSD in aT1a

−/− mice. The BP change associated with spontaneous ultradian activity–inactivity bouts was also similar between strains on HSD as was the BP rise associated with induced (cage-switch) behavioral activity. Ganglionic or α1-adrenergic blockade decreased BP in both strains; HSD did not affect this response in aT1a

−/−, but abolished it in aT1a

+/+ mice.

conclusionsaT1a-receptor deficiency, when combined with HSD, can increase circadian BP difference in mice. This increase is mediated principally by activity-unrelated factors, such as the nonsuppressibility of basal resting sympathetic tone by HSD, thus suggesting a form of salt-/volume-dependent hypertension.

Keywords: angiotensin II; blood pressure; circadian; hypertension; knockout mouse

Am J Hypertens 2010; 23:481-487 © 2010 American Journal of Hypertension, Ltd.

482 May 2010 | VOLUME 23 NUMBER 5 | AMERICAN JOURNAL OF HYPERTENSION

original contributions Salt and Circadian BP in AT1A−/− Mice

in both nocturnal and diurnal BP changes.20–22 In particular, Chen et al. recently reported that the normal day–night BP (DNBP) rhythm is disrupted in AT1A-receptor knockout (AT1A

−/−) mice placed on HSD.22 However, the underlying mechanisms, and specifically the role of activity-dependent and -independent regulatory factors, were not systematically investigated in either this or earlier works.20–22

The present study therefore determined the mechanisms by which AT1A-receptor deficiency may alter circadian BP profile under HSD conditions. We first examined whether the sensi-tivity of circadian BP rhythm to HSD in AT1A

−/− mice reflects their more active behavior, an increased autonomic respon-siveness to activity, or is independent of activity. Given the complex relationship between natural behaviors and BP,23,24 cardiovascular reactivity to a standardized arousing event, cage-switch, was also determined. Spectral analysis of BP variability was employed, as a complementary approach, to estimate autonomic responsiveness without affecting animal behavior. Finally, peripheral autonomic blockade was used to clarify the effects of AT1A-receptor deficiency and HSD on activity-unrelated changes in basal vascular tone.

MethodsAnimals. The experiments were performed using 2–3-month-old male AT1A

−/− mice (n = 7) and AT1A+/+ mice (n = 9) in

accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. AT1A

−/− mice and their own control strain14 were bred in the animal facilities of the Baker IDI Heart and Diabetes Institute (Melbourne, Australia). Mice were originally obtained from breeding stocks of T.W. at the FEM (Charité, Berlin, Germany). Genotypes were determined by PCR as described earlier.25

Telemetry recording of BP and locomotor activity. Under halothane anesthesia, mice were implanted with TA11PA-C10 telemetry probes (Data Science International, St Paul, MS) with catheters placed in the ascending aorta via the carotid artery, as described elsewhere.26 Each mouse was then housed individu-ally. After a 1-week recovery period, mice were placed randomly on a normal-salt diet (NSD, 0.2% Na) or HSD (3.1% Na) for 2 weeks and the diet was rotated for a total of 4 weeks of study. Mice were maintained on a 12:12-h light-dark cycle (lights off at 6 PM) with water and food ad libitum. At the end of each diet regimen, 2-day telemetric BP recordings were performed in mice in home cages. During the recording, pulsatile BP, and locomo-tor activity were continuously monitored and sampled, and the beat-to-beat mean arterial pressure (MAP) and heart rate (HR) were detected online and analyzed as described previously.27 Only animals with the pulse BP amplitude of at least 20 mm Hg were included in the study. Following the 2-day recording ses-sion, mice were randomly allocated to the experimental groups for BP reactivity and pharmacological tests (see below).

Day–night and activity–inactivity BP difference. Apart from MAP, HR, and locomotor activity, the primary outcome measures used in the study included the DNBP difference (ΔDNBP) and the

activity–inactivity BP difference. The ΔDNPB was calculated as the difference between the average levels of 12-h dark and light periods. The activity–inactivity BP difference was calculated as the difference between the average levels of active and inactive ultradian periods (see Figure 4). Active periods were defined as follows: raw locomotor activity counts over 1-min period > 0, and raw activity counts over the preceding or following 1-min period > 0 (to exclude single spikes of activity from the analy-sis). Inactive periods were correspondently defined as follows: raw activity counts over 1-min period = 0 and raw activity counts over the preceding or following 1-min period = 0. In addition, active time, the proportion of samples with raw activity counts > 0, was determined over the 24-h period in each animal.

BP reactivity test. The activity–inactivity BP difference was eval-uated in mice placed in a new clean home cage with a stand-ard wood-chip bedding (n = 4 for each group). This procedure induced a state of behavioral activation in mice, including exploratory locomotion, grooming, and nesting, which nor-mally lasted 30–40 min. To minimize the influence of circadian rhythms on BP, mice were transferred to new cages at the same period of time (between 10 am and 2 pm).

Adrenergic and ganglionic pharmacological blockade. BP was determined before and after intraperitoneal administration of α1-adrenoceptor antagonist prazosin (1 mg/kg), ganglionic blocker hexamethonium (30 mg/kg), or saline (0.2 ml) in AT1A

−/− and AT1A

+/+ mice (n = 4 for each group). Hexamethonium was dis-solved in physiological saline. Prazosin was dissolved in distilled water. All tests were performed following at least a 15-min con-tinuous inactive period, between 10 am and 2 pm. The selected doses of the drugs were based on our preliminary experiments and on previous studies by others.22,28

Spectral analysis of BP and HR. Autonomic nerve activity was assessed by monitoring spontaneous changes in BP and HR as described earlier.27 Briefly, beat-to-beat MAP and HR were analyzed between 7 and 8 pm (active period), and between 4 and 5 am (inactive period) on two consecutive days. The auto- and cross-power spectra were calculated for multiple overlap-ping (by 50%) segments of MAP and HR using fast Fourier transform. The average value of the transfer gain in the fre-quency band in low frequency (LF) band (0.3–0.5 Hz) was used as the estimate of the baroreflex sensitivity. Other fre-quency bands analyzed were very LF (0.08–0.3 Hz) and high frequency (0.5–3 Hz). Normalized power was obtained by dividing the cumulative power within each frequency band by the total power.

BP reactivity to locomotor activity. In order to calculate the reactivity index, average MAP and locomotor activity values were calculated over 1-min intervals, and MAP-activity sam-ples with raw activity counts > 0 were taken for further analy-sis. The activity units were then logarithmically transformed to correct for positive skew.29–31 For each mouse, least-squares regression slopes for the relationships between MAP and

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original contributionsSalt and Circadian BP in AT1A−/− Mice

activity were calculated over 48-h periods (with raw activ-ity counts > 0) using Prism GraphPad software (GraphPad Software, San Diego, CA).

Statistical analysis. All values are expressed as mean ± s.e.m. All data were tested for normality and equal variance and analyzed by a two-way repeated-measures analysis of variance followed by Bonferroni’s multiple comparisons test using GraphPad Prism. The relationships between cardiovascular reactivity and physical activity (regression slopes) were compared by analysis of covariance using GraphPad Prism. Statistical significance was set at a value of P < 0.05 (two-tailed).

resultsΔdnBPThe 24-h MAP was reduced by 26 ± 4 mm Hg in AT1A

−/− mice in comparison with AT1A

+/+ mice on NSD, whereas HR and locomotor activity were not different between groups (Table 1). There was a distinct circadian pattern of MAP, HR, gross loco-motor activity, and time spent active (active time) in both groups, characterized by higher values of these parameters during the night (Figure 1). The ΔDNBP was similar between AT1A

+/+ and AT1A

−/− mice, as was the day–night difference of HR and loco-motor activity (Figure 1, Table 2). The proportion of active time was higher in AT1A

−/− than AT1A+/+ mice (Table 1).

The 24-h MAP was increased by HSD in both groups (Figure 1, Table 1). This increase was relatively small in AT1A

+/+ mice (+5 ± 1 mm Hg), but much more prominent in AT1A

−/− mice (+20 ± 2 mm Hg). The ΔDNBP was not affected by HSD in AT1A

+/+ mice, but was augmented in AT1A−/− mice

by 57 ± 7% (Table 2). The latter change was primarily medi-ated by a greater pressor effect of HSD during the night than the day (+23 ± 2 and +16 ± 2 mm Hg, respectively; repeated-measures analysis of variance with Bonferroni, P = 0.01). HSD did not statistically alter the day–night difference of HR or locomotor activity, or the proportion of time spent active in either group (Tables 1 and 2).

nocturnal BP profile and variabilityNocturnal increase in BP was similar between AT1A

−/− and AT1A

+/+ mice on NSD either during the early (6 pm–8 pm) or

late (2 am–6 am) dark period (Figure 2a,b). Conversely, the late-nocturnal BP rise was markedly higher in AT1A

−/− than in AT1A

+/+ mice on HSD, whereas the early nocturnal rise was similar between groups. HR and locomotor activity did not differ between groups during either period (data not shown). The proportion of time spent active was higher in AT1A

−/− than AT1A

+/+ mice on NSD during the late-dark hours, and was not affected by HSD in either group (Figure 2c).

table 1 | the average 24-h values of MaP, hr, and locomotor activity

Normal-salt diet High-salt diet Diet Genotype Interaction

P

Genotype aT1a+/+ aT1a

−/− aT1a+/+ aT1a

−/−

MaP, mm Hg 118 ± 2 92 ± 2*** 123 ± 3†† 112 ± 3*,††† <0.001 <0.001 < 0.001

HR, bpm 562 ± 11 576 ± 8 563 ± 12 560 ± 9†† 0.018 0.714 0.010

activity, units 1.71 ± 0.35 1.69 ± 0.60 2.07 ± 0.42 2.23 ± 0.75† 0.016 0.635 0.361

active time (%) 44 ± 2 58 ± 4* 51 ± 2 63 ± 5* 0.087 0.002 0.824

Body weight, g 29.8 ± 0.7 30.0 ± 0.7 30.0 ± 0.8 29.4 ± 0.6 0.803 0.892 0.538

Two-way repeated-measures ANOVA determined the effects of diet, genotype, and their interaction on hemodynamic and locomotor parameters (significant P values are given in bold). Bonferroni post hoc testing: *P < 0.05, ***P < 0.001 between genotypes, within the same diet; †P < 0.05, ††P < 0.01, †††P < 0.001 between diets, within the same genotype.ANOVA, analysis of variance; HR, heart rate; MAP, mean arterial pressure.

150

75

700

450

8 AT1A+/+NSD

AT1A–/–NSD

AT1A+/+HSD

AT1A–/–HSD

0

100

0

Act

ive

time

(%)

Act

ivity

(un

its)

HR

(bp

m)

MA

P (

mm

Hg)

6 10 14 18

Time (h)

22 2 6

Figure 1 | The effect of genotype and dietary sodium on circadian variation in MaP, HR, locomotor activity, and proportion of time spent active in aT1a

+/+ and aT1a

−/− mice. Each dot represents an average over a 1-h period. HR, heart rate; MaP, mean arterial pressure.

484 May 2010 | VOLUME 23 NUMBER 5 | AMERICAN JOURNAL OF HYPERTENSION

original contributions Salt and Circadian BP in AT1A−/− Mice

Normalized power of MAP variability in the LF spectral band was similar between AT1A

−/− and AT1A+/+ mice on HSD during

either the early or late-dark period (Figure 3), as was the power

in the very low and high-frequency bands (data not shown). Normalized power of HR variability in the LF band during the early dark period was lower in AT1A

−/− than AT1A+/+ mice on

HSD, and was not further altered during the late-dark period. The spontaneous baroreflex sensitivity was similar between groups on HSD during either period (Figure 3).

There was a weak, but significant correlation between MAP and locomotion in both AT1A

−/− and AT1A+/+ mice on HSD

(r2 = 0.24 and r2 = 0.21, respectively, P < 0.0001). MAP reac-tivity to locomotion was not statistically different between AT1A

−/− and AT1A+/+ mice on HSD (11.5 ± 0.2 mm Hg/log10

activity units and 15.0 ± 0.3 mm Hg/log10 activity units, respectively).

activity–inactivity BP differenceBoth AT1A

−/− and AT1A+/+ mice typically have regular bouts

of behavioral activity during the day and rest bouts during the night (Figure 4). The average change in MAP and HR

table 2 | the average changes in MaP and hr associated with the dark/light cycle and with activity/inactivity periods

Normal-salt diet High-salt diet Diet Genotype Interaction

P

Genotype aT1a+/+ aT1a

−/− aT1a+/+ aT1a

−/−

ΔDN MaP, mm Hg 11 ± 2 12 ± 1 12 ± 2 19 ± 2*,† 0.009 0.063 0.082

ΔDN HR, bpm 60 ± 10 80 ± 9 81 ± 11 74 ± 5 0.262 0.563 0.054

ΔDN activity, units 1.59 ± 0.38 2.06 ± 0.90 2.41 ± 0.54† 2.69 ± 0.79 0.020 0.678 0.732

ΔaI MaP, mm Hg 21 ± 2 18 ± 1 24 ± 2 19 ± 2 0.106 0.075 0.545

ΔaI HR, bpm 116 ± 11 109 ± 13 142 ± 14 105 ± 12 0.357 0.120 0.213

Two-way repeated-measures ANOVA determined the effects of diet, genotype and their interaction on hemodynamic and locomotor parameters (significant P values are given in bold). Bonferroni post-tests: *P < 0.05 between genotypes, within the same diet; †P < 0.05 between diets, within the same genotype.ΔAI, activity/inactivity difference; ANOVA, analysis of variance; ΔDN, day/night difference; HR, heart rate; MAP, mean arterial pressure.

a+30

+0

HSD

NSD

∆MA

P (

mm

Hg)

b c

+30

Early night

Time (h)

–AT1A+/+

–AT1A–/–

Late night Early night Late night

+0

6 10 14 18 22 2 6

∆MA

P (

mm

Hg)

+30 100

0

** ** *

+0NSD HSD NSD HSD NSD HSD NSD HSD

∆MA

P (

mm

Hg)

Act

ive

time

(%)

Figure 2 | Nocturnal BP profile in aT1a+/+ and aT1a

−/− mice. (a) The circadian MaP variation from the average daytime level in aT1a

+/+ and aT1a−/− mice. (b) The

average change in MaP from the daytime level during the early (6–8 pm) and late (2–6 am) dark hours. Two-way aNOVa (early night): influence of diet P = 0.026, influence of genotype NS, interaction NS. Two-way aNOVa (late night): influence of diet NS, influence of genotype P = 0.018, interaction P = 0.011. (c) The propor-tion of time spent active during the early and late dark hours. Two-way aNOVa (early night): influence of diet, genotype, and their interaction NS. Two-way aNOVa (late night): influence of diet NS, influence of genotype P = 0.004, interaction N; *P < 0.05, **P < 0.01 Bonferroni post-tests. aNOVa, analysis of variance; HSD, high-salt diet; MaP, mean arterial pressure; NSD, normal-salt diet.

Early night0

1

0

LF B

P p

ower

(nu

)

LF H

R p

ower

(nu

)LF

gai

n (b

pm/m

m H

g)

LF c

oher

ence

15

0

15

0

15

Late night Early night Late night

–AT1A+/+

–AT1A–/–

* **

##

Figure 3 | Normalized power of MaP and HR variability in the low frequency (LF) spectral band, coherence, and spontaneous baroreflex sensitivity in aT1a

−/− and aT1a+/+ mice on high-salt intake during the early and late dark

hours. Bonferroni post-test: *P < 0.05, **P < 0.01 between genotypes, within the same time; #P < 0.05 between times, within the same genotype. HR, heart rate; MaP, mean arterial pressure.

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original contributionsSalt and Circadian BP in AT1A−/− Mice

associated with these natural activity–inactivity cycles over 24-h was very similar between AT1A

−/− and AT1A+/+ mice on

NSD, and was not further affected by HSD (Table 2). Likewise, the HSD-induced increase in MAP in AT1A

−/− mice, averaged over inactive periods between 2 am and 6 am, was similar to that averaged over active periods during this time (+18 ± 3 and +18 ± 3 mm Hg, respectively). At the same time period, there was little effect of HSD on MAP during either active or inactive states in AT1A

+/+ mice (+3 ± 3 and +3 ± 3 mm Hg, respectively).

Placing a mouse in a novel home cage produced a rapid and sustained increase in MAP, HR, and locomotor activity. The pressor responses were similar in AT1A

−/− and AT1A+/+ mice

on NSD (average change in the first 15 min: +28 ± 4 and +27 ± 2 mm Hg, respectively), and were not affected by HSD (+24 ± 5 and +23 ± 7 mm Hg, respectively). Likewise, the tachycardic and locomotor responses were similar between groups on either diet (data not shown).

BP response to peripheral autonomic blockadeSaline injection (handling) equally increased MAP in AT1A

−/− and AT1A

+/+ mice; HSD did not affect these responses in either group (Figure 5). The α1-adrenoceptor antagonist prazosin decreased MAP by 23 ± 4 and by 40 ± 4 mm Hg in AT1A

+/+ and AT1A

−/− mice on NSD, respectively. This response was not altered by HSD in AT1A

−/− mice (−35 ± 4 mm Hg), but abol-ished in AT1A

+/+ mice (−2 ± 6 mm Hg). Likewise, the depres-sor response to the ganglionic blocker hexamethonium was very similar in AT1A

−/− mice on either diet, but tended to be smaller in AT1A

+/+ mice on HSD, although this did not reach statistical significance (P = 0.13; Figure 5). The maximal reductions in MAP were observed within 5–10 min follow-ing drug administration in both strains on either diet. During

this period, locomotor activity was low and not different from resting levels immediately before injections (data not shown).

discussionThe present study indicates that genetic deficiency of AT1A receptors, when combined with HSD, can substantially increase the ΔDNBP in mice, by shifting BP from a dipper to nondipper pattern during the late-dark, low-activity period.

Given that mental, emotional, and physical activities are critical contributors to circadian BP rise,2,3 one possibility is that this increase was due to the salt-induced augmentation of the intensity and/or duration of these activities. However, our findings that both locomotor activity and the proportion of time spent active were not altered by HSD in either strain do not support this possibility. It should be noted however, that locomotor activity, as measured by DSI telemetry, is primarily an index of gross body movement, and as such may seem less

∆MA

P (

mm

Hg)

+30

Vehicle Prazosin Hexamethonium

NSD HSD

NSD HSD NSD HSD0

–30

–60

+30

0

–30

–60

+30

0

–30

–60

#

**** *

–AT1A+/+

–AT1A–/–

Figure 5 | The effect of genotype and dietary sodium on the depressor response to autonomic blockade. Bonferroni post-test: *P < 0.05, **P < 0.01 between genotypes, within the same diet; #P = 0.04 between diets, within the same genotype. MaP, mean arterial pressure. HSD, high-salt diet; MaP, mean arterial pressure; NSD, normal-salt diet.

MA

P (

mm

Hg)

AT1A+/+ AT1A

–/– AT1A+/+ AT1A

–/–

160

60

HR

(bp

m)

Act

ivity

(un

its)

800

300

15

0

0 6 12 18 24

Time (h) Time (h)

0 6 12 18 24 0 6 12 18 24 0 6 12 18 24

NSD HSD

Figure 4 | The representative recordings of circadian changes in MaP, HR, and locomotor activity in an aT1a+/+ mouse and aT1a

−/− mouse on a normal- (NSD) and high-salt diet (HSD) throughout the 24-h cycle. The gray and white bars at the top represent the 12-h dark and light periods of the day, respectively. The black and white bars at the bottom represent activity and inactivity bouts, respectively. HR, heart rate; MaP, mean arterial pressure.

486 May 2010 | VOLUME 23 NUMBER 5 | AMERICAN JOURNAL OF HYPERTENSION

original contributions Salt and Circadian BP in AT1A−/− Mice

appropriate for detecting finer behaviors, including orienting, grooming, eating, or drinking. Nonetheless, this appears not to be the case for most stereotypic behaviors in mice, as we found recently that in this species not only eating,19 but also groom-ing, orienting-sniffing, rearing, and drinking (D.N. Mayorov, unpublished data) are associated with distinct increases in locomotor activity, as recorded by DSI telemetry.

Another possibility is that the increase in ΔDNBP was due to the salt-induced augmentation of autonomic responsiveness to activity. Several observations, however, do not support this possibility. First, the BP rise did not differ between AT1A

−/− and AT1A

+/+ mice on HSD during their behaviorally most active, early dark period. Second, the BP change associated with spontaneous ultradian activity–inactivity bouts, includ-ing those in the late-dark period, was not altered by HSD in either group. Third, the pressor response to induced behavioral activation by cage-switch in the present study, or food presen-tation and feeding in our recent work19 remained unaffected by HSD in either AT1A

−/− or AT1A+/+ mice. Finally, the power

of spectral LF peaks of BP and HR, which is considered to be a reliable quantifier of autonomic responsiveness,32,33 was simi-lar between groups on HSD, as were spontaneous baroreflex sensitivity and BP reactivity to a given increase in locomotor activity. Taken together, these observations suggest that late-nocturnal nondipping in AT1A

−/− mice cannot be ascribed to the salt-induced increase in autonomic responsiveness to normal behavioral activities.

It is thus conceivable that the nondipping condition in AT1A

−/− mice on HSD was mediated by factors which do not relate directly to mental/physical activity, such as a blunted pressure-natriuresis and associated increase in sodium and volume sensitivity of BP.16,34 These factors are typically manifested by an increase in sympathetic vasomotor tone in salt-sensitive animals or human subjects to ensure adequate sodium and water excretion.11,35 Accordingly, in our AT1A

−/− mice, sympathetic vasomotor tone was elevated even under NSD conditions, in agreement with earlier observations by Chen et al.36 An important new finding of the present study is the lack of suppression of sympathetic vasomotor tone in response to HSD in AT1A

−/− mice. By contrast, sympathetic activity is normally decreased in response to HSD in salt-re-sistant animals or subjects,11,37 a phenomenon which was also observed in our AT1A

+/+ mice. It is plausible that the nondip-ping condition in AT1A

−/− mice reached its peak in the late-dark hours due to sodium and water overload in the preceding early dark period, when eating and drinking activity of this nocturnal species is known to be at the highest level.38 Thus, late-nocturnal nondipping in AT1A

−/− mice is likely to reflect a salt-/volume- dependent hypertensive condition. However, further studies are clearly necessary to identify the precise mechanisms underlying this abnormal condition.

In contrast to our findings, a recent study by Chen and colleagues22 indicates that the light-dark BP rhythm was sta-tistically abolished in AT1A

−/− mice on HSD. This dispar-ity is unlikely to be due to differences in surgical procedures, recovery time after surgery, salt content in the diet or genetic

background, as these factors were similar between studies. It may however, relate to differences in basal BP. For exam-ple, daytime BP in AT1A

−/− mice on NSD was considerably higher in the study by Chen et al.22 (~100 mm Hg) than in our (~85 mm Hg) and earlier works (~70 mm Hg).14,15 This sub-stantial increase in daytime BP could reflect a higher degree of stress in their mice, which were implanted with heavier and bulkier telemetry probes (TA11PA-C20), and also subjected to blood collection by tail bleeding during the course of the experiment. The disparity between studies may also reflect a difference in the diet regimen which was shorter (8 days) in the previous work. Notably, the reduction in the ΔDNBP in the former study was apparently caused by greater BP increases during the light than dark period. It has been shown however, that HSD can shift circadian BP rhythm toward the inactive phase in salt-sensitive subjects and animals.7,39 It is therefore possible that, in the study by Chen et al.,22 more acute dietary sodium overload, in conjunction with elevated stress lev-els, produced more long-lasting pressor responses that could extend to daytime hours and thereby reduce the ΔDNBP.

In summary, the present results indicate that AT1A-receptor deficiency, when combined with HSD, can increase circadian BP difference in mice by changing BP pattern to a nondipper status during the late-dark hours. This nondipping condition in AT1A

−/− mice cannot be attributed to HSD-induced increase in natural activity or autonomic responsiveness to activity, but may reflect a compensatory increase in sympathetic vasomo-tor tone to maintain adequate sodium balance.

acknowledgments: This work was supported in part by grants from the National Health and Medical Research Council of australia and the High-Blood Pressure Research Foundation of australia.

Disclosure: The authors declared no conflict of interest.

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