Acta Pharmacologica Sinica 2006 Aug; 27 (8): 1013–1017

Full-length article Effects of nine antihypertensive drugs on blood pressure variability in sinoaortic-denervated rats1

Jin Wang2,3, Fu-ming SHEN2, Min-wei WANG3, Ding-feng SU2,4

2Department of Pharmacology, Second Military Medical University, Shanghai 200433; 3Department of Pharmacology, Shenyang Pharma- ceutical University, Shenyang 110016, China

Key words Abstract antihypertensive drugs; blood pressure; blood Aim: The present work was designed to investigate the effects of nine commonly pressure variability; sinoaortic denervation used antihypertensive drugs on blood pressure (BP) and blood pressure variabil- ity (BPV) in conscious sinoaortic-denervated (SAD) rats. Methods: Seventy-two 1 Project supported by grants from the SAD rats were randomly divided into nine groups. They were respectively given National Natural Science Foundation of 3 mg/kg, 5 mg/kg, 1 mg/kg, 10 µg/kg, China (No 30330650) and the Science and Technology Development Foundation of 0.5 mg/kg, 20 mg/kg, 20 mg/kg, Shanghai (No 03JC14001, 04JC14001). 40 mg/kg or captopril 50 mg/kg. The drugs were given via a catheter previously 4 Correspondence to Prof Ding-feng SU. implanted into the stomach. BP was recorded for 5 h from 1 h before drug admin- Phn 86-21-2507-0323. Fax 86-21-6549-3951. istration to 4 h after drug administration in conscious, freely moving rats. Results: E-mail [email protected] It was found that all these nine drugs significantly decreased BP in SAD rats. Six of these drugs (nifedipine, nitrendipine, amlodipine, clonidine, prazosin and Received 2006-04-26 Accepted 2006-05-04 atenolol) significantly decreased BPV in SAD rats, but the remaining three drugs did not. Clonidine and atenolol increased the heart period and the others did not. doi: 10.1111/j.1745-7254.2006.00381.x No drugs affected the heart period variability. Conclusion: Among nine antihy- pertensive drugs from different classes, calcium antagonists and sympathetic inhibitors decreased BPV in SAD rats.

level is normal in SAD rats. Recently, we found that the Introduction importance of BPV for determining the organ damage was Blood pressure (BP) is not constant and a spontaneous even greater than the BP level[8,9]. Obviously, an antihyper- variation exists. This variation is defined as blood pressure tensive drug with a BP-stabilizing effect would benefit the variability (BPV). The arterial baroreflex acts as an effective hypertensives. Therefore, it is reasonable to take decreas- buffer of short-term BP fluctuations and prevents excessive ing BPV into account in the treatment of and it BP swings[1]. The baroreflex arc may be interrupted by is proposed that BPV reduction may be a new strategy for sinoaortic denervation (SAD) or complete lesion of the the treatment of hypertension[8]. Indeed, it has been found nucleus tractus solitarus[2,3]. In these animals with baroreflex that BPV reduction has an important contribution to organ dysfunction, BPV is dramatically high[4]. Recently, SAD ani- protection in the long-term treatment of nitrendipine or mals have been used as a high-BPV model for studying the in SHR[10,11]. physiology, pathology and pharmacology related to BPV. Clinically, it will be important to know which antihyper- It is reported that BPV is positively related to the sever- tensive drugs possess an effect on BPV reduction among ity of organ damage in hypertensive patients[5,6]. In animal more than 50 agents available. Therefore, the present work studies, we confirmed that BPV was related to the severity of was designed to observe the effects of some representative organ damage in spontaneously hypertensive rats (SHR)[4,7]. antihypertensive drugs on BPV in SAD rats. Nine drugs Furthermore, a positive relationship was also reported to were selected and included in this study: nifedipine, exist in SAD rats[4,7]. This means that BPV alone without nitrendipine, amlodipine (calcium antagonists), clonidine hypertension could induce organ damage as the average BP (centrally acting agent), prazosin (α1 adrenoceptor blocker),

©2006 CPS and SIMM 1013 Wang J et al Acta Pharmacologica Sinica ISSN 1671-4083

atenolol (β1 adrenoceptor blocker), telmisartan (AT1 recep- cally inserted into the lower abdominal aorta via the left femo- tor blocker), captopril ( I converting ral artery for the measurements of BP and heart period (HP), inhibitor) and hydrochlorothiazide (). and another catheter (PE-50) was inserted into the left femo- ral vein for the examination of baroreflex function. The third Materials and methods catheter (PE-50) was inserted into the stomach for drug administration. These catheters were tunneled subcuta- Animals and drugs Male Sprague-Dawley rats were pro- neously, exteriorized between the scapulae, and fixed on vided by Sino-British SIPPR/BK Lab Animals (Shanghai, saddle. After 2 days of recovery, BP was continuously re- China). They were housed in controlled temperatures (22– corded in conscious unrestrained rats with a computerized 25 ºC) and lighting (08:00 to 20:00 light; 20:00 to 08:00 dark), technique (MPA 2000M, Alcott Biotech, Shanghai, China)[13,14] with free access to tap water and rat chow. All procedures . were in accordance with institutional animal care guidelines. The BP signals, transmitted to the electric signals by a Nifedipine, hydrochlorothiazide, prazosin, clonidine, transducer, were digitized and processed on a personal telmisartan and captopril were purchased from Sigma Chemi- computer, which calculated online the BP and HP beat by beat. The average value was used as an index of BP, and the cal Co (St Louis, MO, USA). Amlodipine was provided by standard deviation (SD) of beat-to-beat BP values as an in- Nanjing Pharmaceutical Co (Nanjing, China). Nitrendipine dex of BPV including systolic (SBPV) and diastolic BPV and atenolol were provided by Shanghai Second Pharma- [14] ceutical Co (Shanghai, China). Nifedipine, nitrendipine, (DBPV) . The same method was used for the calculation of telmisartan and hydrochlorothiazide were dissolved in the HP. In SAD rats, arterial baroreflex function for heart-rate 0.5% carboxymethylcellulose sodium (CMC). Amlodipine, control was also assessed by intravenous injection of , 2 to 5 µg/kg. If SAD rats exhibit a bradycar- captopril, atenolol and clonidine were dissolved in distilled dia of less than 20 beats/min, they were considered as suc- water. Prazosin was suspended in polyethylene glycol (PEG) [13,14] and diluted with distilled water for a final concentration of cessful baroreceptor denervation and included in the study . Protocol After approximately 4-h habituation to BP re- 0.2 mg/mL (PEG, 0.5%)[12]. The doses used were as follows: cording environment, the BP signal was recorded for half an nifedipine 3 mg/kg, nitrendipine 5 mg/kg, amlodipine 1 mg/ hour as the basal (pre-drug) value. The drug was then ad- kg, clonidine 10 µg/kg, prazosin 0.5 mg/kg, atenolol 20 mg/ ministered via the catheter of gastric fistula. One and a half kg, telmisartan 20 mg/kg, hydrochlorothiazide 40 mg/kg and hours after administration of nifedipine, nitrendipine, captopril 50 mg/kg. Drugs were administered by intra- clonidine, atenolol, hydrochlorothiazide and captopril, 2.5 h gastric catheter implanted 3 days before the experiment. after administration of prazosin and 3.5 h after administra- Sinoaortic denervation At the age of 10–11 weeks, tion of amlodipine and telmisartan, the BP signal was re- sinoaortic denervation was performed in Sprague-Dawley corded for half an hour as post-drug value. rats according to the method described in a previous study[13]. Statistical analysis Data were expressed as mean±SEM. Rats were anesthetized with a mixture of (50 mg/kg) Comparisons between values obtained in the same group and (5 mg/kg), intraperitoneally. sulfate before and after drug administration were made using the (0.5 mg/kg) was then administered intraperitoneally. Each paired t - test. P<0.05 was considered statistically significant. animal was fixed in a supine position and a 2.5 cm midline incision was made in the neck. After bilateral isolation of the Results neck muscles, aortic baroreceptor denervation was carried out bilaterally by cutting the superior laryngeal nerves near Effects of nine antihypertensive drugs on BP in SAD the vagi, removing the sympathetic trunk, and sectioning rats The effects of nine antihypertensive drugs on BP in aortic depressor nerves. The carotid sinus baroreceptors conscious SAD rats are presented in Figure 1. It was found were denervated bilaterally by stripping the carotid bifurca- that SBP was significantly decreased after the administra- tion and its branches, followed by the application of 10% tion of all of these nine drugs. This decrease in SBP was phenol in 95% ethanol solution to the external, internal, and approximately 20 mmHg (19–25 mmHg) for nifedipine, common carotid arteries and the occipital artery. The inci- nitrendipine, amlodipine, clonidine, prazosin, atenolol, and sion was sutured closed and each rat was injected intramus- telmisartan; and was approximately 16 mmHg for hydrochlo- cularly with 60000 units of penicillin G. rothiazide and captopril. DBP was also found to be signifi- Blood pressure measurement Four weeks after sinoaortic cantly reduced by these nine drugs in SAD rats. denervation, rats were anesthetized as mentioned. A poly- Effects of nine antihypertensive drugs on BPV in SAD ethylene catheter (PE-10 connected to PE-50) was chroni- rats The effects of nine antihypertensive drugs on SBPV in

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Figure 1. Effects of nine antihypertensive drugs on systolic blood pressure (SBP) and diastolic blood pressure (DBP) in sinoaortic-den- ervated rats (SAD, n=8). □, Before drug administration; ■, After drug administration. Nif, nifedipine; Nit, nitrendipine; Aml, amlodipine; Clo, clonidine; Pra, prazosin; Ate, atenolol; Tel, telmisartan; Hyd, hydrochlorothiazide; Cap, captopril. Mean±SEM. cP<0.01 compared with before drug administration.

SAD rats are shown in Figure 2. It was found that nifedipine, nitrendipine, amlodipine, clonidine, prazosin and atenolol all significantly decreased SBPV. Nifedipine decreased SBPV from 11.0±1.2 mmHg to 9.1±0.9 mmHg. Nitrendipine de- creased SBPV from 9.7±0.6 mmHg to 7.7±0.6 mmHg. Amlodi- Figure 2. Systolic blood pressure variability (SBPV) responses to a pine decreased SBPV from 11.8±2.2 mmHg to 10.0±1.8 mmHg. single dose of nine drugs in individual sinoaortic-denervated rats (SAD, Clonidine decreased SBPV from 11.8±1.4 mmHg to 8.2±0.8 n=8). ○, Before drug administration; ●, After drug administration. Nif, nifedipine; Nit, nitrendipine; Aml, amlodipine; Clo, clonidine; mmHg. Prazosin decreased SBPV from 12.4±1.4 mmHg to Pra, prazosin; Ate, atenolol; Tel, telmisartan; Hyd, hydrochloro- 8.9±0.6 mmHg. Atenolol decreased SBPV from 13.1±1.8 ; Cap, captopril. mmHg to 9.5±1.0 mmHg. Although telmisartan, hydrochlo- rothiazide and captopril significantly decreased SBP to a simi- of DBPV were unchanged (Figure 3). lar extent with nifedipine and other drugs as shown in Figure 1, the values of SBPV were unchanged (Figure 2). Effects of nine antihypertensive drugs on HP and HPV Effects of nine antihypertensive drugs on DBPV in SAD after sinoaortic denervation Among these nine antihyper- rats Figure 3 shows the results of 9 antihypertensive drugs tensive drugs, only clonidine and atenolol prolonged HP in SAD rats. There were no significant changes in HPV for any on DBPV in SAD rats. It was found that nifedipine, nitrendi- drugs studied in SAD rats (Figure 4). pine, amlodipine, clonidine, prazosin and atenolol all signifi- cantly decreased DBPV, similar to SBPV. DBPV was de- creased from 9.7±1.1 mmHg to 7.6±0.8 mmHg for nifedipine. Discussion DBPV was decreased from 7.6±0.5 mmHg to 6.2±0.5 mmHg During the past four decades, several classes of antihy- for nitrendipine. DBPV was decreased from 9.3±1.8 mmHg to pertensive agents- blockers, centrally act-

8.1±1.6 mmHg for amlodipine. DBPV was decreased from ing antihypertensive agents, α1-adrenoceptor antagonist, β-

9.4±1.2 mmHg to 6.5±0.7 mmHg for clonidine. DBPV was blockers, AT1 blockers, and angiotensin- decreased from 8.1±0.8 mmHg to 6.6±0.6 mmHg for prazosin. converting enzyme inhibitors have been shown to effectively DBPV was decreased from 12.8±2.0 mmHg to 10.1±1.2 mmHg reduce elevated blood pressure and associated cardiovas- for atenolol. Although telmisartan, hydrochlorothiazide and cular events[15–18]. The present work for the first time sys- captopril significantly decreased DBP to a similar extent with tematically studied and clearly demonstrated the effects of nifedipine and other drugs as shown in Figure 1, the values these representative drugs on reducing BP and BPV in SAD

1015 Wang J et al Acta Pharmacologica Sinica ISSN 1671-4083

Figure 4. Effects of nine antihypertensive drugs on heart period (HP) and heart period variability (HPV) in sinoaortic-denervated rats (SAD, n=8). □, Before drug administration; ■, After drug administration. Nif, nifedipine; Nit, nitrendipine; Aml, amlodipine; Clo, clonidine; Pra, prazosin; Ate, atenolol; Tel, telmisartan; Hyd, hydrochlorothiazide; Cap, captopril. Mean±SEM. cP<0.01 com- pared with before drug administration.

antagonism. Generally speaking, the stability of BP is main- tained by arterial baroreflex in physiological conditions. He- modynamic studies in conscious rats with neonatal chemi- cal sympathectomy, SAD, and acute neurohumoral block- ade have revealed that one major source of hemodynamic Figure 3. Diastolic blood pressure variability (DBPV) responses to perturbations is the myogenic response of vascular smooth a single dose of nine drugs in individual sinoaortic-denervated rats muscle cells[19–21]. Therefore, it is not surprising that all three (SAD, n=8). ○, Before drug administration; ●, After drug administration. Nif, nifedipine; Nit, nitrendipine; Aml, amlodipine; calcium antagonists used decreased BPV in SAD rats by [19] Clo, clonidine; Pra, prazosin; Ate, atenolol; Tel, telmisartan; Hyd, preventing this myogenic response . hydrochlorothiazide; Cap, captopril. Antihypertensive drugs inhibiting the sympathetic ner- vous system used in the present work were prazosin, atenolol rats. The main finding of the present work may be summa- and clonidine. All of these three drugs significantly de- rized as follows: (1) calcium antagonists (nifedipine, creased BPV in SAD rats. These results suggest that activa- nitrendipineand amlodipine) significantly decreased BPV in tion of the sympathetic nervous system is involved in the SAD rats; (2) antihypertensive drugs inhibiting sympathetic increased BPV after SAD. As prazosin and atenolol repre- nervous system (clonidine, prazosin and atenolol) signifi- sent α- and β-receptor antagonists, respectively, it seems cantly decreased BPV in SAD rats; (3) other antihyperten- that both α- and β-receptors are involved in the SAD-in- sive drugs, such as telmisartan, hydrochlorothiazide and duced high BPV. It is well known that arterial baroreflex captopril, did not significantly influence BPV in SAD rats. modulates sympathetic and parasympathetic nervous sys- Dihydropyridine calcium antagonists are the most widely tems and keeps the balance between these two autonomic used antihypertensive drugs. According to their action nervous systems. The loss of this balance after SAD opera- duration, they are divided into three subclasses. Nifedipine, tion is often characterized by an over-activation of sympa- nitrendipine and amlodipine are the representative drugs for thetic nervous systems and a deactivation of parasympa- the short-, medium- and long- lasting drugs, respectively. In thetic nervous systems probably contributing to the eleva- the present work, it was found that all three drugs signifi- tion of BPV. cantly decreased BPV in SAD rats. This means that the The present work also revealed that captopril and effects of calcium antagonists on BPV does not relate to the telmisartan did not influence BPV in SAD rats. This means action duration but relate to the intrinsic property of calcium that the activation of the -angiotensin system is not

1016 Http://www.chinaphar.com Wang J et al important in the maintenance of high BPV induced by SAD 8 Su DF, Miao CY. Reduction of blood pressure variability: a new in rats. In addition, hydrochlorothiazide did not modify BPV strategy for the treatment of hypertension. Trends Pharmacol in SAD rats. This suggests that elevated BPV may mainly be Sci 2005; 26: 388–90. 9 Miao CY, Xie HH, Zhan LS, Su DF. Blood pressure variability is due to the variation of peripheric resistance and not to the more important than blood pressure level in determination of variation of blood volume. end-organ damage in rats. J Hypertens 2006; 24: 1137–46. These results may be helpful in the selection of antihy- 10 Liu JG, Xu LP, Chu ZX, Miao CY, Su DF. Contribution of blood pertensive drugs. For a hypertensive patient with high BPV, pressure variability to the effect of nitrendipine on end-organ a calcium antagonist, a drug inhibiting sympathetic nervous damage in spontaneously hypertensive rats. J Hypertens 2003; 21: 1961–7. system, or the combination of two drugs from these classes 11 Xie HH, Shen FM, Cao YB, Li HL, Su DF. Effect of low-dose would be proposed. However, it should be noted that the ketanserin on blood pressure variability, baroreflex sensitivity mechanisms for increased BPV in SAD rats and in hyperten- and end-organ damage in spontaneously hypertensive rats. Clin sive patients or rats may be different. In SAD rats, the in- Sci 2005; 108: 547–52. creased BPV is induced purely by the loss of baroreflex 12 Gabor R, Marta M, Mark K, Emil M, Laszlo D. Experimental control, while the mechanism for increased BPV seen in hy- orthostasis elicits sustained hypertension, which can be prevented by sympathetic blockade in the rats. J Cardiovasc Pharmacol pertension is more complicated. 2005; 45: 354–61. We conclude that calcium antagonists and drugs inhibit- 13 Fu YJ, Shu H, Miao CY, Wang MW, Su DF. Restoration of ing the sympathetic nervous system significantly decreased baroreflex function by ketanserin is not blood pressure depen- BPV in SAD rats. This suggests the involvement of the dent in conscious freely moving rats. J Hypertens 2004; 22: myogenic response of vascular smooth muscle cells and ac- 1165–72. 14 Xie HH, Miao CY, Jiang YY, Su DF. Synergism of atenolol and tivation of sympathetic nervous system in SAD-induced high nitrendipine on hemodynamic amelioration and organ protec- BPV. tion in hypertensive rats. J Hypertens 2005; 23: 193–201. 15 Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, References Izzo JL Jr, et al. The seventh report of the joint national commit- tee on prevention, detection, evaluation, and treatment of high 1 Jordan J, Tank J, Shannon JR, Diedrich A, Lipp A, Schroeder C, et blood pressure: the JNC 7 report. JAMA 2003; 289: 2560–71. al. Baroreflex buffering and susceptibility to vasoactive drugs. 16 Dahlof B, Devereux RB, Kjeldsen SE, Julius S, Beevers G, de Faire Circulation 2002; 105: 1459–64. U, et al. Cardiovascular morbidity and mortality in the 2 Norman RA Jr, Coleman TG, Dent AC. Continuous monitoring intervention for endpoint reduction in hypertension study (LIFE): of arterial pressure indicates sinoaortic denervated rats are not a randomised trial against atenolol. Lancet 2002; 359: 995– hypertensive. Hypertension 1981; 3: 119–25. 1003. 3 Buchholtz RA, Nathan NA. Chronic lability of the arterial pres- 17 William C. Are there benefits to specific sure produced by electrolytic lesions of the nucleus tractus solitarii therapy? AM J Hypertens 2003; 16: 315–55. in the rat. Circ Res 1984; 54: 227–38. 18 Liu L, Zhang Y, Liu G, Li W, Zhang X, Zanchetti A, et al. The 4 Su DF, Miao CY. Blood pressure variability and organ damage. event reduction (FEVER) study: a randomized long- Clin Exp Pharmacol Physiol 2001; 28: 709–15. term placebo-controlled trial in Chinese hypertensive patients. 5 Parati G, Pomidossi G, Albini F, Malaspina D, Mancia G. Rela- J Hypertens 2005; 23: 2157–72. tionship of 24-hour blood pressure mean and variability to sever- 19 Juline C. Pharmacological attenuation of blood pressure ity of target-organ damage in hypertension. J Hypertens 1987; variability. Acta Pharmacol Sin 2005; 26: 1288–9. 5: 93–8. 20 Burattini R, Borgdorff P, Westerhof N. The baroreflex is coun- 6 Mancia G, Frattola A, Parati G, Santucciu C, Ulian L. Blood teracted by autoregulation, thereby preventing circulatory pressure variability and organ damage. J Cardiovasc Pharmacol instability. Exp Physiol 2004; 89: 397–405. 1994; 24: S6–11. 2 1 Kornet L, Hoeks APG, Janssen BJA, Houben AJ, De Leeuw PW, 7 Miao CY, Su DF. The importance of blood pressure variability in Reneman RS. Neural activity of the cardiac baroreflex decreases rat aortic and left ventricular hypertrophy produced by sinoaortic with age in normotensive and hypertensive subjects. J Hypertens denervation. J Hypertens 2002; 20: 1865–72. 2005; 23: 815–23.

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