Strain-dependent differences of restraint stress-induced hypertension in WKY and SHR

Physiology & Behavior 97 (2009) 341–346

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Strain-dependent differences of restraint stress-induced hypertension in WKY and SHR Alexander Grundt, Christina Grundt, Stefan Gorbey, Martin A. Thomas, Björn Lemmer ⁎ Institute of Experimental and Clinical Pharmacology and Toxicology, Ruprecht-Karls-Universität of Heidelberg, Mannheim, Germany

a r t i c l e

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Article history: Received 22 January 2009 Received in revised form 18 February 2009 Accepted 26 February 2009 Keywords: Restrained stress Tail-cuff Radiotelemetry Hypertension Sympathetic nervous activity

a b s t r a c t The aim of our study was to investigate differences in restraint stress-response between normotensive Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) and the consequences for tail-cuff (TC) blood pressure measurements. We therefore radiotelemetrically collected cardiovascular data from WKY and SHR that underwent TC procedures and measured plasma norepinephrine (NE) and angiotensin II (ATII) levels as well as gene expression of the adrenal and hypothalamic tyrosine-hydroxylase, the rate-limiting enzyme in NE synthesis. Furthermore, we determined the effects of antihypertensive therapy using the beta1-receptor antagonist metoprolol, the alpha1-receptor antagonist doxazosin and the AT1-receptor antagonist telmisartan as mono- or combination therapies during the TC procedure. Results show that the TC procedure induced a stress reaction characterised by greatly increasing heart rate (HR) and blood pressure (BP) and elevating plasma norepinephrine and angiotensin II concentrations. Strain-dependent differences were found concerning stress reactions during rest (more pronounced effects) and activity of the two rat strains. In both strains, metoprolol inhibited the TC-induced increase in HR and doxazosin the TC-induced increase in BP. Telmisartan, in addition, reduced hypertension in SHR, slightly reduced the TC-induced increase of BP in SHR but had no effect in WKY. The cardiovascular data as well as those on NE, ATII and TH expression clearly show that SHR are less able to cope with stress-related mechanisms than the normotensive WKY. Since TC activates both the sympathetic as well as renin–angiotensin system this method is not appropriate to evaluate neither physiological nor drug-induced effects on BP and HR. © 2009 Elsevier Inc. All rights reserved.

1. Introduction The tail-cuff (TC) method is employed to monitor conscious blood pressure (BP) in transgenic and/or knock-out rodents in order to correlate it with molecular modifications and/or drug effects [1,2]. The American Heart Association recommended tail-cuff measurement for high-throughput experimental design [3] but pointed out that the agreement with radiotelemetric data is dependent on the tail-cuff method used [4]. The draw-back of the TC method is that the fixation of animals leads to a stress reaction [5–9] characterised by an increase in cardiovascular parameters such as blood pressure and heart rate. Stress reactions are usually mediated by the sympathetic nervous system, which activity is circadian time-dependent as well as straindependent and dominantly regulates blood pressure and heart rate. Therefore, analyzing and comparing antihypertensive drug effects by the TC device in different rat strains or under antihypertensive therapy require information about the activity and capacity of the sympathetic nervous system in the strains compared. An additional confounding ⁎ Corresponding author. Institute of Experimental and Clinical Pharmacology and Toxicology, Ruprecht-Karls-Universität Heidelberg, Maybachstrasse 14, 68169 Mannheim, Germany. Tel.: +49 621 383 9704. E-mail address: [email protected] (B. Lemmer). 0031-9384/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2009.02.029

factor using the TC method is that circadian rhythms in blood pressure and heart rate in normotensive, hypertensive and knock-out rodents [10–13] are not taken into account. Animal experiments that aim to identify antihypertensive effects of drugs were usually performed in normotensive Wistar Kyoto rats (WKY) as controls and the hypertensive backcross of WKY, the spontaneously hypertensive rats (SHR) [14]. SHR develop an essential hypertension between 12 and 20 weeks of age resulting in left heart hypertrophy and hypertension related end organ damages as seen as in human patients with essential hypertension [15]. As in most patients with essential/primary hypertension the fundamental cause of hypertension in SHR is still far from being solved. Nevertheless, SHR were frequently used to identify antihypertensive drug effects by TC blood pressure measurements and data were compared to normotensive WKY [14]. However, it was not yet tested, whether restraint stress by TC procedure modifies antihypertensive drug effects equally in SHR and WKY. We, therefore, compared antihypertensive drug effects in WKY and SHR in unrestraint freely moving rats by radiotelemetry and under restraint stress conditions during TC procedure. In addition, plasma norepenephrine (NE) and angiotensin II (ATII) levels were determined. The aim of this study was to get more insight into strainspecific differences in response to antihypertensive treatment during

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restraint stress conditions by TC. Our results clearly show that the restraint stress-induced activation of the sympathetic nervous system differs between WKY and SHR and differently masks antihypertensive drug effects with varying effects during rest and activity. 2. Methods 2.1. Animals and materials Experiments were performed in 23 male SHR and 24 WKY, obtained from Charles River Laboratories (Sulzfeld, Germany). Animals were housed individually in cages (25 × 43 × 16 cm) in an isolated, ventilated, light-controlled, sound-isolated animal container [Scantainer®, Type C-110; width × depth × height: 1420 × 640 × 1865 mm, Scanbur, Karlslunde, Denmark] placed within the temperaturecontrolled animal room at 23 ± 1 °C with relative humidity of 60 ± 5%. Food (Altromin, Lage-Lippe, Germany) and tap water were available ad libitum. Animals were synchronized to a 12 h:12 h light–dark regime with lights (100 lx) on at 07:00 h (Zeitgeber Time, ZT0) and lights off at 19:00 h (ZT12). Dusk and dawn were simulated within the container for 30 min starting at ZT0 and ZT12. The animal experiments were performed in adherence to the Guide for the Care and Use of Laboratory Animals as published by the National Institutes of Health and the recommendations for chronobiological research [16] and were approved by the German federal regulations (RP Karlsruhe, AZ: 35-9185.81/91/99).

antihypertensive drug effects [11,12,17,18]. Our study design took into account that the half-life of the drugs tested (e.g. propranolol) is much shorter in rats (40 min) [12] than in humans (4 h) [19] due to a greater cardiac output and higher liver perfusion in rats. Thus, another drug was tested after more than 40 half-lives of the preceding one. Telmisartan was dissolved in 10% ethanol/0.9% sodium chloride solution, metoprolol and doxazosin were dissolved in 0.9% sodium chloride solution. Application of vehicles (10% ethanol/0.9% sodium chloride and 0.9% sodium chloride solution, respectively) did not significantly affect blood pressure and HR (data not shown). For measuring daytime- and drug-dependency of restrained stressinduced hypertension each rat underwent nine tail-cuff measurements within 8 weeks. No stress habituation was observed in additional control runs. To further investigate the activity of the sympathetic nervous system and the RAS, separate groups of age matched rats were placed into a restrainer for 25 min and were afterwards immediately decapitated within 15 s. Unrestrained rats underwent the same decapitation procedure and served as controls. Trunk blood was collected in heparin tubes, centrifuged and stored in aliquots at −80 °C until quantification of plasma NE and ATII concentrations. To analyze gene expression of adrenal and hypothalamic tyrosine-hydroxylase (TH), hypothalami and adrenal glands of restrained and control animals (see above) were dissected, frozen in liquid nitrogen and stored at −80 °C until mRNA extraction. 2.3. Plasma norepinephrine

2.2. Study protocol Telemetric radiotransmitters (TA11PA-C40, Data Sciences, St. Paul, USA) were implanted into the abdominal aorta under enflurane (Ethrane, Abbott GmbH, Wiesbaden, Germany) and Ketamin/Xylazin (Intervet, Schwabenheim, Germany) anesthesia in 6 WKY and 5 SHR at the age of 12 weeks as described elsewhere in detail [10]. SBP, DBP and HR were monitored telemetrically using the Dataquest system (Data Sciences, St. Paul, USA). At the age of 20 weeks, cardiovascular basal values of animals were measured by radiotelemetry. Parameters were taken in 5 minute intervals for 6 s each. TC measurements were performed within the animal room in a restrainer (6 cm in diameter) using the Harvard Rat Tail Blood Pressure Monitor System (Edenbridge, England). To analyze TC-induced changes in cardiovascular parameters, rats were placed into the restrainer for 25 min and cardiovascular data were monitored in 5 minute intervals simultaneously by TC (SBP) and radiotelemetry (SBP, DBP, HR) with the restrainer placed on top of the telemetric receiver. Afterwards, rats were replaced into their cages in the container, and data were telemetrically recorded for another 30 min. To habituate animals to the procedure of TC measurements rats had been placed into the restrainer three times per week for 3 weeks (totally 9 times) before experiments started. Since we could observe more pronounced TCinduced cardiovascular effects during the resting phase (ZT01-03), all further experiments with drug interventions were only performed during rest. Various drugs were used as experimental tools to analyze the contribution of the sympathetic nervous system and renin–angiotensin system (RAS) on blood pressure and HR during TC procedure. Animals were intraperitoneally (i.p.) injected with the AT1-receptor antagonist telmisartan (10 mg/kg; Boehringer Ingelheim, Ingelheim, Germany) 12 h before measurements, the beta1-adrenoceptor antagonist metoprolol (30 mg/kg) or the alpha1-adrenoceptor antagonist doxazosin (1 mg/kg; Sigma Aldrich, Taufkirchen, Germany) 30 min before measurements. The compounds were applied in single dosages either as monotherapy or in different combinations. All dosages of drugs chosen herein have been shown to significantly reduce blood pressure in SHR [11,12,17,18]. After each drug injection a wash-out period of at least 5 days was performed, which has been shown to eliminate all

Plasma NE concentrations were determined using the ClinRep Kit for Plasma Catecholamines (Recipe Chemicals + Instruments, München, Germany) by HPLC analysis according to the manufacturer's instructions. To compare strain-specific differences in NE concentrations in unrestraint and freely moving animals, arterial and venous catheters were implanted into the femoral artery and the jugular vein of eight 7-week-old WKY and SHR [20]. Catheters were connected to a dual-channel fluid swivel system (FMI, Seeheim, Germany) allowing free and unstressed movement of the animals and blood sampling. Blood samples were taken over a time period of 16 days every 3 h with a 2 day recovering phase after 3 samples. 2.4. Plasma angiotensin II For the determination of plasma ATII in freely moving rats (see above), trunk blood was mixed with the angiotensinase inhibitor bestatin (Bühlmann Laboratories, Schönenbuch, Switzerland) immediately after taking of blood samples. Plasma ATII concentrations were determined using the Angiotensin II Radioimmunoassay (Bühlmann Laboratories, Germany) according to the manufacturer's instructions. 2.5. Quantification of tyrosine-hydroxylase mRNA expression The mRNA expression of tyrosine-hydroxylase (TH, GI:126432544) was determined in adrenal glands and hypothalami of WKY and SHR without and with TC procedures. Total RNA was extracted using Tri Reagent (Sigma, Taufkirchen, Germany) and purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany). 10 µg purified total RNA was transcribed into cDNA using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). The cDNA quantification was performed by real time PCR using QuantiTect SYBR Green PCR Kit (Qiagen, Hilden, Germany) and the LightCycler system (Roche, Penzberg, Germany) with the following primers and conditions: TH (forward: 5′CTTTGACCCAGACACAG CAG-3, reverse: 5′-TGGATACGAGAGGCATAGTTCC-3′, annealing: 52 °C, product size: 123 bp), 18S RNA (forward: 5′-GAAAGCATTTGCCAAGAATG-3′, reverse: 5′-AGTCGGCATCGTTTATGGTC-3′, annealing: 55 °C, product size: 101 bp). Quantification of appropriate PCR products was ensured by exemplary sequencing

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and melting point analysis. Semi-quantitative analysis of mRNA expression was performed using an external standard curve and two calibrator probes per run adjusting for inter-run variabilities and calculated using MS Excel. 2.6. Statistical analyses For statistical evaluations, the software BIAS was used [21]. Straindependent differences were tested using the Student's t-test and drug-treated groups were compared by univariate analysis of variance (ANOVA). Differences were considered statistically significant at p ≤ 0.05. 3. Results 3.1. Restraint stress reaction in WKY and SHR during rest and activity phase TC procedure performed during the resting phase (between ZT0103) increased telemetered HR, SBP and DBP both in WKY and SHR (Figs. 1, 2). Immediately after being placed into the restrainer, HR increased by about 200 bpm and SBP by 45 mm Hg in both strains and was significantly elevated during the entire TC procedure (Fig. 1). After releasing rats from the restrainer HR was normalized within 20 min in SHR and partially normalized within 30 min in WKY, whereas SBP normalized within 5 min in both WKY and SHR (Fig. 1). In WKY the restraint stress reactions was significantly more pronounced in the resting phase (ZT01-03) than in the activity phase (ZT13-15) (Fig 2). In SHR, only the TC-induced increase in HR – but not in SBP and DBP – was significantly greater during rest than during activity but less than in WKY controls (Fig. 2). 3.2. Effects of antiadrenergic and AT1-receptor blocking drugs during restraint stress: influence on HR In both strains beta1-adrenoceptor blockade by metoprolol reduced basal HR (Table 1) and significantly reduced the TC-induced increase in HR to values of untreated freely moving rats (Fig. 3). Alpha1-adrenoceptor blockade by doxazosin, however, significantly increased basal values in HR in WKY while having no significant effect

Fig. 2. TC-induced changes in HR, SBP and DBP in WKY (white bars) and SHR (black bars) at ZT01-03 (resting phase) and at ZT13-15 (activity phase) compared to control conditions. TC significantly (p ≤ 0.01) increased HR, SBP and DBP in both, WKY and SHR during resting and activity phase. In WKY, the TC-induced increase in HR and BP was significantly more pronounced during the resting phase than during the activity phase. In SHR, only the TC-induced increase in HR was significantly greater during rest than activity, with no differences concerning SBP and DBP. Shown are mean values ± SEM of 6 WKY and 5 SHR obtained during 25 min of TC procedure. ⁎p ≤ 0.05 activity phase vs. resting phase.

in SHR (Table 1). Doxazosin failed to prevent the TC-induced increase in HR in either group (Fig. 3). In both strains metoprolol plus doxazosin reduced basal HR to values observed with metoprolol alone (Table 1) and also prevented the TC-induced increase in HR (Fig. 3). Telmisartan reduced basal HR both in WKY and SHR (Table 1) with no effect during the TC procedure in either group (Fig. 3). Addition of telmisartan to doxazosin further increased basal HR in both strains (Table 1) and evoked an additional rise in the TC-induced increase in HR (Fig. 3). The combined blockade of the sympathetic nervous system (metoprolol and doxazosin) and the RAS (telmisartan) decreased telemetered basal HR below control values in both strains (Table 1). Under restraint stress conditions this drug combination abolished the increase in HR in both WKY and SHR, but reduction was significantly more pronounced in WKY than in SHR (Fig. 3). 3.3. Effects of antiadrenergic and AT1-receptor blocking drugs during restraint stress: influence on SBP In both strains beta1-adrenoceptor blockade by metoprolol had no significant effect on SBP under control and TC conditions (Table 1,

Fig. 1. Telemetrically measured HR (♦), SBP (▲) and DBP (●) in WKY and SHR during control conditions and during 25 min of TC procedure (highlighted) and 30 min afterwards obtained at ZT01-03 (resting phase). The profiles show an increase in all parameters during TC procedure (empty symbols) compared to control conditions (filled symbols) in both groups that reconstitutes after leaving the restrainer. Shown are mean values ± SEM of 6 WKY and 5 SHR.

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Table 1 Shown are mean values ± SEM of basal heart rate (HR), systolic blood pressure (SBP) and diastolic blood pressure (DBP) measured by radiotelemetry and of SBP measured by tail-cuff device of WKY (n = 6) and SHR (n = 5) with and without antihypertensive treatment at ZT01-03. HR

SBP

SBP (TC)

WKY Control Metoprolol Doxazosin Telmisartan Met + Dox Dox+ Tel Met + Dox + Tel

DBP

289 ± 5 250 ± 10a 386 ± 16a 232 ± 4a 317 ± 18 434 ± 8a 229 ± 7a

110 ± 3 104 ± 9 96 ± 4a 84 ± 9a 102 ± 8 88 ± 5a 60 ± 5a,b

144 ± 19 138 ± 13 124 ± 15 139 ± 14 108 ± 22 99 ± 18a 68 ± 22a

73 ± 1 68 ± 7 71 ± 3 52 ± 6a 65 ± 6 61 ± 3a 34 ± 5a,b

SHR Control Metoprolol Doxazosin Telmisartan Met + Dox Dox+ Tel Met + Dox + Tel

298 ± 14 247 ± 7a 314 ± 10c 246 ± 12a 323 ± 12 395 ± 7a,c 254 ± 3a,c

182 ± 5c 165 ± 5c 135 ± 8a,c 148 ± 7a,c 138 ± 10a,c 118 ± 6a,c 109 ± 7a,c

197 ± 21 226 ± 28 184 ± 29 198 ± 19 199 ± 34 127 ± 13a 145 ± 20a

121 ± 4c 110 ± 2c 96 ± 2a,c 101 ± 2a,c 88 ± 7a,c 80 ± 4a,c 67 ± 5a,c

a

p ≤ 0,05 vs. control. p ≤ 0.05 Met + Dox + Tel vs. Dox + Tel. p ≤ 0.05 SHR vs. WKY.

b c

Fig. 4). Doxazosin significantly reduced basal SBP in both strains (Table 1). The TC-induced increase in SBP was significantly lowered in both strains to values of untreated animals (Fig. 4). Additional pre-treatment with metoprolol had no additive effect on basal values of SBP in WKY and SHR (Table 1), nevertheless under TC conditions an additive effect on SBP reduction was observed in WKY but not in SHR (Fig. 4). Telmisartan significantly reduced basal SBP in both strains (Table 1). However, telmisartan had no significant effect during TC procedure both in WKY and SHR (Fig. 4). Doxazosin plus telmisartan significantly reduced basal values of SBP and reduced the TC-induced increase in SBP in both WKY and SHR (Fig. 4, Table 1). This effect was significantly more pronounced in SHR than in WKY (Fig. 4). In SHR, additional pre-treatment with metoprolol had no additive effect on SBP under control and TC conditions (Fig. 4, Table 1). In WKY the combination of metoprolol, doxazosin and

Fig. 3. TC-induced changes in HR in WKY (white bars) and SHR (black bars) with and without antihypertensive treatment. Tail-cuff procedure (TC) significantly increased HR in both, WKY and SHR which can be antagonized by metoprolol (Met) but not by doxazosin (Dox) or telmisartan (Tel). Shown are mean deltaAUC-values (change in area under the curve) of HR ± SEM of 6 WKY and 5 SHR obtained during 25 min of TC procedure. ⁎p ≤ 0.05 vs. control.

telmisartan resulted in a further significant reduction of SBP during control and TC conditions. In SHR metoprolol had no further additive antihypertensive effect on doxazosin plus telmisation reduced SBP (Fig. 4, Table 1). Interestingly, basal values of BP in SHR pre-treated with metoprolol, doxazosin and telmisartan are almost equal to basal values of untreated normotensive WKY. Changes of DBP paralleled those of SBP (Table 1) and are therefore not described in detail. 3.4. Influence of restraint stress reaction on plasma norepinephrine and angiotensin II When plasma NE was determined in freely moving rats from samples obtained from arterial catheters the 24-hour levels in plasma NE were clearly different between the strains. WKY rats showed a significantly higher 24 h mean plasma concentration of NE (193.1 + 15.6 pg/ml) than SHR (155.5 + 12 pg/ml, p b 0.05), following a circadian rhythm with higher NE levels during activity phase (Fig. 5A). In contrast, basal NE concentrations obtained from decapitated (stress reaction) animals between ZT01-03, i.e. during the early rest hours in which NE are low in both strains, did not significantly differ between strains (Fig. 5B). The TC procedure had divergent effects on NE levels in WKY and SHR: whereas in WKY the TC procedure induced a significant increase, a significant decrease in plasma NE was found in SHR (Fig. 5B). To get more insight in this surprising observation the expression of adrenal and hypothalamic tyrosine-hydroxylase (TH) mRNA expression levels – TH is the ratelimiting enzyme in NE biosynthesis – were analysed. We found that the adrenal TH expression was significantly higher in WKY than in SHR while TH expression in the central nervous system (hypothalamus) was similar in both groups (Fig. 5B). The TC procedure had no effect on adrenal TH gene expressions in WKY, but led to a significantly down-regulation of hypothalamic TH expression (Fig. 5B). Interestingly, in SHR the TC procedure significantly increased adrenal TH expression but hypothalamic TH expression remained unaffected (Fig. 5B). Under unrestraint, freely moving conditions WKY showed significantly lower plasma concentrations of ATII than SHR. The

Fig. 4. TC-induced changes in SBP in WKY (white bars) and SHR (black bars) with and without antihypertensive treatment. Tail-cuff procedure (TC) significantly increased SBP which can be antagonized by doxazosin (Dox) but not by metoprolol (Met) or telmisartan (Tel). Telmisartan significantly enhanced the SBP lowering effect of doxazosin in SHR but not in WKY. Metoprolol significantly enhanced the SBP lowering effect of doxazosin in WKY but not in SHR. Shown are mean deltaAUC-values (change in area under the curve) of SBP ± SEM of 6 WKY and 5 SHR obtained during 25 min of TC procedure. ⁎p ≤ 0.05 vs. control, †p ≤ 0.05 vs. WKYTC + Met + Tel + Dox, ‡p ≤ 0.05 vs. SHRTC + Tel + Dox and vs. SHRTC + Met + Tel + Dox.

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Fig. 5. (A) Circadian variation in the plasma concentration of norepinephrine in WKY and SHR rats under unstressed conditions obtained by catheter method. The shaded area indicates the activity phase from ZT12-00. Shown are mean values ± SEM of 8 animals per strain. (B) TC-induced effects on plasma norepinephrine (NE) and angiotensin II (Ang II) levels and of adrenal and hypothalamic tyrosine-hydroxylase (TH) mRNA expression in WKY (white bars, n = 6) and SHR (black bars, n = 5) at ZT01-03, i.e. during the early rest hours. In WKY, TC induced a significantly increase in Ang II and NE concentrations, decreased hypothalamic but had no effect on adrenal TH expression. In SHR, TC induced a significant increase in Ang II and adrenal TH expression, decreased NE concentration and had no effect on hypothalamic TH expression. Shown are mean values ± SEM for NE and Ang II concentrations as well as mean ratios ± SEM of TH mRNA expressions vs. 18S mRNA expressions. ⁎p ≤ 0.05 vs. control, †p ≤ 0.05 SHR vs. WKY.

TC procedure evoked a significant increase in ATII in both strains but significantly more pronounced in SHR than in WKY (Fig. 5B). 4. Discussion In our study we monitored the effects of antihypertensive drugs on cardiovascular data of normotensive WKY and spontaneously hypertensive SHR simultaneously by radiotelemetry and the indirect tail-cuff method inducing standardized restraint stress. Our results clearly demonstrate that this restraint stress had, despite of 3 weeks of habituation procedures, a pronounced elevating effect on BP and HR in both normotensive WKY and hypertensive SHR. Similar increases in HR and BP were described for a more severe immobilization stress for 1 h/day up to 10 days [7,22]. Most interestingly, a circadian phase-dependency was found in WKY with the TC-induced effects on HR and BP being significantly more pronounced during rest than activity. Conversely, in hypertensive SHR no circadian phase-dependency of the TC stress reaction was observed in SBP and DBP, only reaction on HR was more pronounced during rest. However, the absolute increase in HR and BP due to entrainment was more prominent in SHR than in WKY, indicating that the TC stress reaction is not only strain but also circadian phase-dependent.

Immobilization stress of at least 150 min was shown to result in increased plasma concentrations of NE when obtained by arterial catheter [23]. After a short immobilization stress of 5 min not only NE but also ATII plasma concentrations – also obtained by arterial catheter – increased in normotensive (Sprague–Dawley) as well as in hypertensive transgenic rats (TGR(Ren2)27) [24]. In the present experiments when the blood was withdrawn by decapitation, TC stress leads to an increase in plasma NE in WKY but an NE decrease was observed in SHR, obviously due to a counter regulation of the central nervous system to a further increase in blood pressure. On the other hand, plasma ATII levels were higher in SHR and increased more pronouncedly in SHR than WKY. This data clearly indicate that the NE-inducing effect of a stress reaction is dependent on the duration of the stress and is obviously differently handled by normotensive and spontaneously hypertensive rats. We, therefore, suggested that the activation of predominantly sympathetic nervous system was involved in the TC-induced stress in WKY, whereas the RAS must also play a role in SHR. In support of this assumption beta1adrenoceptor blockade by metoprolol prevented HR increase in WKY, and additional alpha1-adrenoceptor blockade by doxazosin reduced the TC-induced BP increase in this normotensive rat strain. The TC procedure, as already mentioned, induced a more pronounced increase in plasma ATII concentrations in SHR than in WKY.

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Thus, it was not a surprise that in SHR combined treatment with alpha1- and beta1-adrenergic blockade as well as antagonism of the AT1-receptor not only prevented the TC-induced increase in HR and BP but also reduced the hypertensive BP values in SHR. This data clearly demonstrate strain-dependent differences in drug responses between WKY and SHR in restraint stress conditions. In an additional step we studied the effects of TC stress on the gene expression of the tyrosine-hydroxylase (TH), the rate-limiting enzyme in the NE synthesis, in the adrenal glands and in the hypothalami of both rat strains. Under control conditions a less pronounced adrenal gene expression of TH was found in SHR than in WKY which goes well together with the lower plasma NE rhythm values in the hypertensive rat strain. However, after TC-induced stress the adrenal TH expression in SHR was significantly upregulated while having no effect on TH expression in WKY. Under control conditions expression of TH in the hypothalamus was not significantly different between the two strains, though lower in SHR. During TC stress, however, TH expression in the central nervous system was down regulated in WKY, possibly due to stress-induced hypertension in the central nervous system. In SHR, on the other hand, TC stress did not affect hypothalamic TH expression. These findings again indicate that the normotensive WKY rat is able to counterregulate the peripheral-central feedback control of regulating BP, whereas the hypertensive SHR lacks adaptive mechanisms to stress reactions such as the TC procedure. Finally, comparing the two methods to determine SBP in two strains of rats it is obvious that radiotelemetry is clearly superior to the TC method used in our study in that radiotelemetry gives SBP values which represent physiological values in unrestraint freely moving animals. The TC method can create artifacts in SBP due to activating stress-related mechanisms both under control conditions as well as after drug interventions as shown in a normotensive and hypertensive rat strain. The biochemical effects observed during TC-induced stress reaction further support our notion on the superiority of radiotelemetry in blood pressure research. Our conclusion goes in principle well together with the recommendation of the American Heart Association [3] which clearly regards radiotelemetry as gold standard and restricts TC for high-throughput designs. Acknowledgements The assistance of I. Abu-Taha and S. Schiffer is gratefully acknowledged. References [1] Ahn MY, Jung YS, Jee SD, Kim CS, Lee SH, Moon CH, et al. Anti-hypertensive effect of the Dongchunghacho, Isaria sinclairii, in the spontaneously hypertensive rats. Arch Pharm Res 2007;30:493–501. [2] Staudacher T, Pech B, Tappe M, Gross G, Muhlbauer B, Luippold G. Arterial blood pressure and renal sodium excretion in dopamine D3 receptor knockout mice. Hypertens Res 2007;30:93–101.

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