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Chronic losartan treatment decreases angiotensin II-mediated facilitation of noradrenaline release in the caudal artery of spontaneously hypertensive rats
Life Sciences 67 (2000) 3153–3162
Chronic losartan treatment decreases angiotensin II-mediated facilitation of noradrenaline release in the caudal artery of spontaneously hypertensive rats Mariano Ruiz-Gayoa,1, Beatriz Somozab, Rosario Bravob, M. Soledad Fernández-Alfonsob, Carmen Gonzáleza,* a
Departamento de Fisiología, Facultad de Medicina, Universidad Autónoma de Madrid, C/ Arzobispo Morcillo s.n., 28029 Madrid, Spain b Departamento de Farmacología, Facultad de Farmacia, Universidad Complutense, Madrid, Spain Received 7 January 2000; accepted 9 May 2000
Abstract Sympathetic activity is modulated by angiotensin II (AII), both at pre- and postsynaptic level in the rat caudal artery. In the spontaneously hypertensive rat (SHR), this artery receives more dense sympathetic innervation than blood vessels of normotensive strains. This fact seems to be linked to the enhanced pressor responses elicited by noradrenaline in SHR. In this work we describe, in the SHR, the effect of a chronic treatment with the angiotensin II AT1-receptor antagonist, losartan, in modulating noradrenergic mechanisms involved in caudal artery contraction. The effect of losartan is compared to that of captopril, given at doses leading to a similar decrease of both arterial blood pressure and left ventricular hypertrophy. The contractile response of caudal artery rings induced by endogenous noradrenaline released by low frequency transmural nerve stimulation (TNS) has been studied. Under our conditions, TNS (0.5–1 Hz) induced higher contractile responses in SHR treated with losartan than in the control and captopril-treated groups. This difference seems to be due to an increase of the postsynaptic effect of noradrenaline (NA) rather than to an increase of noradrenaline release from sympathetic endings, since i) DE50 value for NA was lower in losartan-treated SHR than in the other groups, and ii) AII induced a dose-dependent increase of TNS-evoked release of radioactivity from caudal artery segments loaded with [3H]-NA, in both control and captopril-treated groups but had no effect in the losartan-treated group. These results show that chronic treatment with losartan, although slightly enhancing the pressor effect of NA at postsynaptic level, fully supresses the facilitatory role of AII on NA release. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Losartan; Noradrenaline; Spontaneously hypertensive rat; Transmural nerve stimulation; Chronic; Angiotensin II; AT1 receptor * Corresponding author. E-mail address: [email protected] (C. González) 1 Present address: Departamento de Ciencias Biomédicas. Facultad de Ciencias Experimentales y Técnicas. Universidad San Pablo CEU. Madrid 0024-3205/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 9 0 0 -0
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Introduction Angiotensin II (AII) regulates arterial blood pressure by acting mainly on AT1 angiotensin receptors. At the peripheral level, AII elicits vasoconstriction directely through AT1 smooth muscle receptors [1]and indirectely by facilitating sympathetic activity [2, 3]. AT1 receptors are located both at pre- and postsynaptic level in sympathetic nerve endings. Presynaptic AT1 receptors facilitate noradrenaline release in several tissues and organs, such as atria [4], vas deferens [5], heart [6], blood vessels [7] and kidney [8]. In humans, exogenous AII has been shown to facilitate adrenergic neurotransmission in normo- and hypertensive patients [9] and to facilitate noradrenaline release from renal sympathetic nerves [10]. At postsynaptic level, AII also seems to modulate noradrenergic activity [2, 11], since exogenous subpressor doses of AII enhance the pressor effects of a-adrenergic agonists [12] and induce expression of a-adrenergic receptors in vascular smooth muscle cells [13]. In the spontaneously hypertensive rat (SHR), smooth muscle responsiveness to exogenous AII is enhanced [14, 15]. The increase of the contractile action of AII has been related to the enhancement of the facilitatory effect of AII on noradrenaline release in sympathetic endings. This has been proposed as a factor involved in the hyperesponsiveness of SHR to the pressor effects of AII [15, 16]. Additionally, it has been demonstrated that noradrenergic innervation of the caudal artery of SHR is more dense than in blood vessels of normotensive strains [17]. Moreover, the angiotensin AT1-receptor antagonist, losartan, elicits a sympathoinhibitory effect on SHR [18]. Angiotensin AT1-receptor antagonists, like losartan, are useful drugs for essential hypertension management and are now considered as alternatives to angiotensin-converting enzyme (ACE) inhibitors [19]. Treatment with losartan elicits similar lowering of blood pressure and left ventricular hypertrophy than ACE inhibitors [20, 21], lacking adverse effects of ACE inhibitors, like cough [22, 23]. In this work, we have investigated the effect of a 16-week treatment with losartan on the vascular response to transmural sympathetic nerve stimulation (TNS) in the caudal artery of SHR. The aim of our study has been to analyze the effect of a chronic blockade of AT1 receptors on the sympathetic regulation of vascular activity in SHR, both at pre- and postsynaptic level. We have compared the effect of losartan to that of captopril, a selective ACE inhibitor, used at doses leading to similar decrease of arterial blood pressure.
Methods Experiments were conducted in twelve-week old male spontaneously hypertensive rats (SHR), weighing 250–350 g, obtained from CRIFA (France). At this age, rats are considered adults and arterial hypertension is established. Rats were housed in groups of four under controlled dark-light cycles (12h/12h) and temperature conditions. Food (normal rat chow A0.4 Panlab) and water were available ad libitum. All animal procedures were approved by the Animal Care and Use Committee according to the guidelines for ethical care of experimental animals of the European Community. Systolic arterial blood pressure was measured by the tail-cuff method before the beginning of the treatment. The average of 3 measurements was taken as initial mean systolic blood pressure.
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Treatment Either losartan (15 mg/kg/day; n512 rats) or captopril (60 mg/kg/day; n512 rats) were administered in the drinking water during 16 weeks. Control group (n512 rats) received tap water. Similar doses of losartan and captopril as used in this study have previously been shown to be equipotent in decreasing systolic blood pressure [21]. Drugs were dissolved daily in tap water at a final concentration calculated in function of the body weight and the volume of water consumed the day before. After 16 weeks of treatment, rats were anesthetized with sodium pentobarbital (50 mg/kg) and exsanguinated by intracardiac punction. The tail artery was removed, cleaned of residual blood and placed in oxygenated ice cold Krebs-Henseleit modified solution (KHMS) with the following composition: 115 mM NaCl, 4.6 mM KCl, 2.5 mM CaCl2, 25 mM NaHCO3, 1.2 mM KH2PO4, 1.2 mM MgSO4, 0.01 mM EDTA and 11 mM glucose. From each tail artery several rings were taken for presynaptic and other for postsynaptic studies. Measurement of isometric tension in tail artery rings Arterial rings (2 mm length) were suspended on two intraluminal parallel wires, introduced in an organ bath containing KHMS at 378C, and connected to a Piodem strain gauge for isometric tension recording. All rings were equilibrated at a passive tension of 1 g. Experiments started after an equilibration period of 90 minutes. 75 mM KCl was given in order to evaluate the contractile response of the artery. Transmural nerve stimulation (TNS, 200 mA, 0.2 ms, 0.5–4 Hz, 10 s) was applied by using two parallel platinum electrodes, one at each side of the vessel, connected to a CS-20 stimulator (Cibertec, Spain). Under these conditions, the effect of TNS was inhibited by tetrodotoxin and phentolamine (1 mM) [24]. Tritium release experiments Measurement of tritium release by TNS was performed as previously described [25]. Briefly, caudal artery segments (4 mm approx) were placed into a nylon net (4 segments/net) and incubated at 378C for 1 h in 1 ml of oxygenated KHMS containing 5 mCi of [3H]-noradrenaline. After this period, nets were placed separately into a superfusion chamber with two parallel platinum electrodes, one at each side of the vessel, 0.5 cm apart, connected to a stimulator Cibertec CS-9 (Cibertec, Spain) for TNS. Before TNS application, arteries were washed at a rate of 1 ml/min with oxygenated KHMS containing both cocaine hydrochloride (1025 M) and corticosterone acetate (1025 M) for 120 min. At this time, radioactivity contained in the effluate was constant. TNS (200 mA, 0.3 ms, 5 Hz) was applied during 60 s. One 6 min superfusate fraction was collected before TNS (basal release). Another 6 min fraction was collected from starting the TNS. Radioactivity was measured in a scintillation counter in polypropylene vials containing 15 ml of scintillation liquid (Scharlau Biogreen 2). The rate of radioactivity overflow was normalized by calculating the % of radiaoactivity released during/ after TNS over basal release. Arteries were washed for 30 min previously to the application of a new stimulus. A total of four stimuli were applied. No changes in % of radioactivity release was detected between consecutive estimuli. For drugs testing, arteries were washed for 30 min, before TNS, with superfusion liquid containing the drug to be tested. No differences
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in uptake of [3H]-noradrenaline due to pharmacological treatment were detected, since remaining radiactivity was similar in tissue from the three experimental groups. Drugs and solutions Losartan was a gift of Merck Sharp & Dohme (Spain). Captopril was from RBI (USA). [3H]-noradrenaline (30–50 Ci/mmol) was purchased from Amersham (England). Cocaine hydrochloride was provided by Ministerio de Sanidad y Consumo (Spain). Other drugs, reagents and solvents were from Sigma (USA). Statistics Comparisons between groups were made by using a one-way analysis of variance (ANOVA). Post-hoc comparisons were made by using the Newman-Keuls test. EC50 and their 95% confidence limits were calculated by log-probit analysis according to the method of Litchfield and Wilcoxon [26]. Statistical significance was set at P, 0.05. Results Effect of treatment on systolic arterial blood pressure and left ventricular hypertrophy Systolic blood pressure was measured every two weeks during treatment. Table 1 indicates the values of blood pressure determined at the end of treatment. Initial blood pressure was 240615 mm Hg (n536). Both losartan and captopril significantly reduced the ratio between left ventricular weight and body weight (Table 1). Systolic blood pressure correlated (r50.736; P , 0.01) with the ratio left ventricular weight/body weight. Effect of transmural nerve stimulation (TNS) TNS-induced contractions were completely blocked by 1 mM tetrodotoxin as well as by 1 mM phentolamine (data not shown). This indicates that contractions were mediated by stimulation of perivascular noradrenergic nerves and that a2adrenergic receptors were involved [24]. TNS produced frequency-dependent contractions of the caudal artery between 0.5 and 4 Hz. As illustrated in figure 1, contractions induced by both 0.5 Hz and 1 Hz were significantly higher in the losartan than in the control group. In contrast, at these frequencies, treat-
Table 1 Effect of treatment on systolic arterial blood pressure and left ventricular hypertrophy
Control Captopril Losartan
Systolic arterial pressure (mm Hg)
Left ventricle weight/ body weight (mg/g)
249 6 28 203 6 20* 214 6 11*
3.00 6 0.10 2.57 6 0.09* 2.56 6 0.06*
Either captopril or losartan groups were compared to their respective control by using the Student’s t test. * P , 0.05.
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Fig. 1. Effect of transmural nerve stimulation (0.5 to 4 Hz) on the contraction of caudal artery rings in untreated and treated SHR. Contraction is expressed as % of a previous contraction elicited by 75 mM KCl. Data are expressed as means 6 S.E.M. n5 indicates the number of vascular rings used. * p,0.05 compared to the control group.
ment with captopril did not modify the response to TNS. Neither losartan nor captopril modified the response to TNS at frequencies higher than 1 Hz. Postsynaptic Effects The contractile effect of noradrenaline was tested in caudal artery rings. Contraction is expressed as % of the response induced by 75 mM KCl, which was 1546675 mg, 1680675 mg and 1357652 mg for control, losartan and captopril-treated groups, respectivelly. Fig 2 shows the concentration-response curves for noradrenaline on caudal arteries from control
Fig. 2. Effect of noradrenaline in the contractility of the caudal artery. Contraction is expressed as % of a previous contraction elicited by 75 mM KCl. Data are expressed as means 6 S.E.M. n5 indicates the number of vascular rings used. * p,0.05 compared to the control group.
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(EC5051.58 (1, 2.5) 3 1027 M), losartan (EC5050.56 (0.32, 1) 3 1027 M) and captopriltreated (EC5051.26 (1, 1.99) 3 1027 M) SHR. The concentration-response plot for noradrenaline was similar in control and captopril groups. However, in arteries from the losartan treated group, the concentration-effect curve of noradrenaline was significantly shifted to the left. Presynaptic effects The effect of AII, tested in a concentration range from 1029 to 1026 M, in modulating the effect of TNS (5 Hz) on the release of radioactivity is illustrated in figure 3. In the control group, one-way ANOVA revealed a significant effect of AII on radioactivity release evoked by TNS (FANOVA(4,36)5 9.937; P,0.001). In the captopril treated group, the effect of AII was similar to that observed in the control group (FANOVA(4,28)53.010; P,0.05). However, AII was without effect in the losartan-treated group (FANOVA(4,38)50.927; NS). The effect of TNS (5 Hz) on the release of radioactivity from caudal artery segments loaded with [3H]-noradrenaline was not significantly different on control, losartan and captopriltreated groups (Figure 3). The facilitatory effect of AII was fully suppressed by preincubating the arteries with 1027 M losartan, both in the control (FANOVA(3,12)50.89; P.0.05) (figure 4) and in the captopril-treated group (FANOVA(3,12)50.51; P.0.05) (results not shown). In contrast, the AT2 receptor antagonist, PD-123,319, also at 1027 M, was without effect in both groups (results not shown).
Fig. 3. Effect of angiotensin II on radiactivity release evoked by TNS (5 Hz) from caudal artery segments of control, captopril and losartan-treated SHR loaded with [3H]-noradrenaline. Radiactivity release is expressed as % of basal release. Data are expressed as means 6 S.E.M. n 5 8–11 rats/group. * p , 0.05 compared to the control group.
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Fig. 4. Effect of losartan (0.1 mM) on angiotensin II-induced radioactivity release evoked by TNS (5 Hz) from caudal artery segments of control SHR loaded with [3H]-noradrenaline. Radiactivity release is expressed as % of basal release. Data are expressed as means 6 S.E.M. n 5 11 rats. * p , 0.05 compared to the control group.
Discussion In this work we describe the effect of chronic treatment with the angiotensin AT1-receptor antagonist, losartan, on sympathetic mechanisms regulating the vascular tone of the SHR caudal artery. The effect of losartan was compared to the effect of captopril, given at doses leading to an equivalent decrease of both systolic blood pressure and left ventricular hypertrophy. The response initially studied was the contractility of caudal artery rings induced by TNS. Under our conditions, low frequency TNS (0.5 and 1 Hz), which stimulates the release of noradrenaline by sympathetic endings [25], induced higher contractile responses in losartantreated SHR than in the control and captopril-treated groups. This difference might be due either to an increase of the postsynaptic effect of noradrenaline or/and to a higher release of the neurotransmitter from noradrenergic endings. The first possibility was tested by comparing the contractile effect of noradrenaline in the three experimental groups. The concentrationresponse curve for noradrenaline was slightly, although significantly, shifted to the left in the losartan-treated group, compared to the control group. In contrast, treatment with captopril did not modify the contractile effect of noradrenaline. Although a similar treatment with losartan decreases the vasoconstriction induced by noradrenaline in aorta of SHR [27], in a densely innervated vessel, as the caudal artery, the enhancement of the sensitivity of noradrenergic receptors to noradrenaline could reflect a compensatory mechanism caused by a decrease of noradrenaline release, induced by chronic losartan treatment. In order to test this hypothesis we examined the release of radioactivity induced by TNS from caudal artery segments loaded with [3H]-noradrenaline. For these experiments TNS frequency was 5 Hz instead 0.5–1 Hz, since, in our hands, lower frequencies than 5 Hz led to
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too low radioactivity release. Under these conditions, we found that radioactivity release induced by 5 Hz was similar in the three groups, which is in agreement with the results obtained in 2 and 4 Hz TNS-induced contractility. However, the effect of AII on the release of radioactivity evoked by 5 Hz TNS was deeply modified in the losartan treated group. AII elicited a concentration-dependent enhancement of TNS-induced efflux of radioactivity both in the control and in the captopril-treated SHR. In both groups, the effect of AII was blocked by preincubation of arteries with losartan, and not by PD-123,319 indicating the involvement of AT1 angiotensin receptors in this response. In contrast, in the losartan treated group, the modulatory role of AII on radioactivity release was suppressed. Similar results have been reported by Foucart et al. [28], who described that a twelve-day treatment with losartan, but not with enalaprilat, abolished the effect of AT1 on the release of [3H]-noradrenaline from atria of SHR, under similar experimental conditions. A loss of effect of AII would be consistent with a down-regulation of prejunctional AT1 receptors, which is rather unexpected from a chronic treatment with an AT1-receptor antagonist. However, it has been previously described that losartan down-regulates AT1 receptors in chronic treatment [29]. Interestingly, it has been reported that chronic losartan reduces blood pressure by increasing availability of NO [30, 31], which has been shown to down-regulate AT1 receptors expression [32, 33]. Another possibility would be an eventual partial-agonist character of losartan or of some losartan metabolite. Althought this possibility cannot be discarded, such a partial agonist character of losartan has been only detected in in vitro experiments in rat isolated glomeruli and in human mesangial cells by using losartan concentrations higher than 1 mM [34]. However, most work in this field show a lack of agonist character for losartan [19]. Concerning metabolites, EXP 3174, an active metabolite of losartan, endowed with non-competitive antagonist properties, has been identified [35]. EXP 3174 has a slow dissociation kinetic from AT1 receptors [36] and could theoretically account for the results obtained. Similar to our results, a permanent inhibition of the potentiating effect of AII on sympathetic nerve function, at least 10 weeks after the end of a 12-week treatment with AT1 antagonists, has been also described [37]. In conclusion, chronic treatement with the the AT1 receptor antagonist, losartan i) slightly enhances the pressor effect of noradrenaline at postsynaptic level, and ii) suppresses the facilitatory role of AII on noradrenaline release. This could be an important feature in regulating blood pressure since noradrenergic innervation is increased in SHR vessels. Acknowledgments This work has been supported by a Medical School Grant of Merck Sharp & Dohme, Spain and by a Grant of the Comunidad de Madrid (CAM 08.4/0003/1997), Spain. References 1. Ferrario CM, Brosnihan KB, Diz DI, Jaiswal N, Khosla MC and Milsted A. Angiotensin-(1-7): a new hormone of the angiotensin system. Hypertension 1991; 18: 26–33. 2. Zimmerman RG. Action of angiotensin on vascular adrenergic nerve endings. Facilitation of norepinephrine response. Fed. Proc. 1972; 31: 1344–1350.
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Chronic losartan treatment decreases angiotensin II-mediated facilitation of noradrenaline release in the caudal artery of spontaneously hypertensive rats Mariano Ruiz-Gayoa,1, Beatriz Somozab, Rosario Bravob, M. Soledad Fernández-Alfonsob, Carmen Gonzáleza,* a
Departamento de Fisiología, Facultad de Medicina, Universidad Autónoma de Madrid, C/ Arzobispo Morcillo s.n., 28029 Madrid, Spain b Departamento de Farmacología, Facultad de Farmacia, Universidad Complutense, Madrid, Spain Received 7 January 2000; accepted 9 May 2000
Abstract Sympathetic activity is modulated by angiotensin II (AII), both at pre- and postsynaptic level in the rat caudal artery. In the spontaneously hypertensive rat (SHR), this artery receives more dense sympathetic innervation than blood vessels of normotensive strains. This fact seems to be linked to the enhanced pressor responses elicited by noradrenaline in SHR. In this work we describe, in the SHR, the effect of a chronic treatment with the angiotensin II AT1-receptor antagonist, losartan, in modulating noradrenergic mechanisms involved in caudal artery contraction. The effect of losartan is compared to that of captopril, given at doses leading to a similar decrease of both arterial blood pressure and left ventricular hypertrophy. The contractile response of caudal artery rings induced by endogenous noradrenaline released by low frequency transmural nerve stimulation (TNS) has been studied. Under our conditions, TNS (0.5–1 Hz) induced higher contractile responses in SHR treated with losartan than in the control and captopril-treated groups. This difference seems to be due to an increase of the postsynaptic effect of noradrenaline (NA) rather than to an increase of noradrenaline release from sympathetic endings, since i) DE50 value for NA was lower in losartan-treated SHR than in the other groups, and ii) AII induced a dose-dependent increase of TNS-evoked release of radioactivity from caudal artery segments loaded with [3H]-NA, in both control and captopril-treated groups but had no effect in the losartan-treated group. These results show that chronic treatment with losartan, although slightly enhancing the pressor effect of NA at postsynaptic level, fully supresses the facilitatory role of AII on NA release. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Losartan; Noradrenaline; Spontaneously hypertensive rat; Transmural nerve stimulation; Chronic; Angiotensin II; AT1 receptor * Corresponding author. E-mail address: [email protected] (C. González) 1 Present address: Departamento de Ciencias Biomédicas. Facultad de Ciencias Experimentales y Técnicas. Universidad San Pablo CEU. Madrid 0024-3205/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 9 0 0 -0
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Introduction Angiotensin II (AII) regulates arterial blood pressure by acting mainly on AT1 angiotensin receptors. At the peripheral level, AII elicits vasoconstriction directely through AT1 smooth muscle receptors [1]and indirectely by facilitating sympathetic activity [2, 3]. AT1 receptors are located both at pre- and postsynaptic level in sympathetic nerve endings. Presynaptic AT1 receptors facilitate noradrenaline release in several tissues and organs, such as atria [4], vas deferens [5], heart [6], blood vessels [7] and kidney [8]. In humans, exogenous AII has been shown to facilitate adrenergic neurotransmission in normo- and hypertensive patients [9] and to facilitate noradrenaline release from renal sympathetic nerves [10]. At postsynaptic level, AII also seems to modulate noradrenergic activity [2, 11], since exogenous subpressor doses of AII enhance the pressor effects of a-adrenergic agonists [12] and induce expression of a-adrenergic receptors in vascular smooth muscle cells [13]. In the spontaneously hypertensive rat (SHR), smooth muscle responsiveness to exogenous AII is enhanced [14, 15]. The increase of the contractile action of AII has been related to the enhancement of the facilitatory effect of AII on noradrenaline release in sympathetic endings. This has been proposed as a factor involved in the hyperesponsiveness of SHR to the pressor effects of AII [15, 16]. Additionally, it has been demonstrated that noradrenergic innervation of the caudal artery of SHR is more dense than in blood vessels of normotensive strains [17]. Moreover, the angiotensin AT1-receptor antagonist, losartan, elicits a sympathoinhibitory effect on SHR [18]. Angiotensin AT1-receptor antagonists, like losartan, are useful drugs for essential hypertension management and are now considered as alternatives to angiotensin-converting enzyme (ACE) inhibitors [19]. Treatment with losartan elicits similar lowering of blood pressure and left ventricular hypertrophy than ACE inhibitors [20, 21], lacking adverse effects of ACE inhibitors, like cough [22, 23]. In this work, we have investigated the effect of a 16-week treatment with losartan on the vascular response to transmural sympathetic nerve stimulation (TNS) in the caudal artery of SHR. The aim of our study has been to analyze the effect of a chronic blockade of AT1 receptors on the sympathetic regulation of vascular activity in SHR, both at pre- and postsynaptic level. We have compared the effect of losartan to that of captopril, a selective ACE inhibitor, used at doses leading to similar decrease of arterial blood pressure.
Methods Experiments were conducted in twelve-week old male spontaneously hypertensive rats (SHR), weighing 250–350 g, obtained from CRIFA (France). At this age, rats are considered adults and arterial hypertension is established. Rats were housed in groups of four under controlled dark-light cycles (12h/12h) and temperature conditions. Food (normal rat chow A0.4 Panlab) and water were available ad libitum. All animal procedures were approved by the Animal Care and Use Committee according to the guidelines for ethical care of experimental animals of the European Community. Systolic arterial blood pressure was measured by the tail-cuff method before the beginning of the treatment. The average of 3 measurements was taken as initial mean systolic blood pressure.
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Treatment Either losartan (15 mg/kg/day; n512 rats) or captopril (60 mg/kg/day; n512 rats) were administered in the drinking water during 16 weeks. Control group (n512 rats) received tap water. Similar doses of losartan and captopril as used in this study have previously been shown to be equipotent in decreasing systolic blood pressure [21]. Drugs were dissolved daily in tap water at a final concentration calculated in function of the body weight and the volume of water consumed the day before. After 16 weeks of treatment, rats were anesthetized with sodium pentobarbital (50 mg/kg) and exsanguinated by intracardiac punction. The tail artery was removed, cleaned of residual blood and placed in oxygenated ice cold Krebs-Henseleit modified solution (KHMS) with the following composition: 115 mM NaCl, 4.6 mM KCl, 2.5 mM CaCl2, 25 mM NaHCO3, 1.2 mM KH2PO4, 1.2 mM MgSO4, 0.01 mM EDTA and 11 mM glucose. From each tail artery several rings were taken for presynaptic and other for postsynaptic studies. Measurement of isometric tension in tail artery rings Arterial rings (2 mm length) were suspended on two intraluminal parallel wires, introduced in an organ bath containing KHMS at 378C, and connected to a Piodem strain gauge for isometric tension recording. All rings were equilibrated at a passive tension of 1 g. Experiments started after an equilibration period of 90 minutes. 75 mM KCl was given in order to evaluate the contractile response of the artery. Transmural nerve stimulation (TNS, 200 mA, 0.2 ms, 0.5–4 Hz, 10 s) was applied by using two parallel platinum electrodes, one at each side of the vessel, connected to a CS-20 stimulator (Cibertec, Spain). Under these conditions, the effect of TNS was inhibited by tetrodotoxin and phentolamine (1 mM) [24]. Tritium release experiments Measurement of tritium release by TNS was performed as previously described [25]. Briefly, caudal artery segments (4 mm approx) were placed into a nylon net (4 segments/net) and incubated at 378C for 1 h in 1 ml of oxygenated KHMS containing 5 mCi of [3H]-noradrenaline. After this period, nets were placed separately into a superfusion chamber with two parallel platinum electrodes, one at each side of the vessel, 0.5 cm apart, connected to a stimulator Cibertec CS-9 (Cibertec, Spain) for TNS. Before TNS application, arteries were washed at a rate of 1 ml/min with oxygenated KHMS containing both cocaine hydrochloride (1025 M) and corticosterone acetate (1025 M) for 120 min. At this time, radioactivity contained in the effluate was constant. TNS (200 mA, 0.3 ms, 5 Hz) was applied during 60 s. One 6 min superfusate fraction was collected before TNS (basal release). Another 6 min fraction was collected from starting the TNS. Radioactivity was measured in a scintillation counter in polypropylene vials containing 15 ml of scintillation liquid (Scharlau Biogreen 2). The rate of radioactivity overflow was normalized by calculating the % of radiaoactivity released during/ after TNS over basal release. Arteries were washed for 30 min previously to the application of a new stimulus. A total of four stimuli were applied. No changes in % of radioactivity release was detected between consecutive estimuli. For drugs testing, arteries were washed for 30 min, before TNS, with superfusion liquid containing the drug to be tested. No differences
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in uptake of [3H]-noradrenaline due to pharmacological treatment were detected, since remaining radiactivity was similar in tissue from the three experimental groups. Drugs and solutions Losartan was a gift of Merck Sharp & Dohme (Spain). Captopril was from RBI (USA). [3H]-noradrenaline (30–50 Ci/mmol) was purchased from Amersham (England). Cocaine hydrochloride was provided by Ministerio de Sanidad y Consumo (Spain). Other drugs, reagents and solvents were from Sigma (USA). Statistics Comparisons between groups were made by using a one-way analysis of variance (ANOVA). Post-hoc comparisons were made by using the Newman-Keuls test. EC50 and their 95% confidence limits were calculated by log-probit analysis according to the method of Litchfield and Wilcoxon [26]. Statistical significance was set at P, 0.05. Results Effect of treatment on systolic arterial blood pressure and left ventricular hypertrophy Systolic blood pressure was measured every two weeks during treatment. Table 1 indicates the values of blood pressure determined at the end of treatment. Initial blood pressure was 240615 mm Hg (n536). Both losartan and captopril significantly reduced the ratio between left ventricular weight and body weight (Table 1). Systolic blood pressure correlated (r50.736; P , 0.01) with the ratio left ventricular weight/body weight. Effect of transmural nerve stimulation (TNS) TNS-induced contractions were completely blocked by 1 mM tetrodotoxin as well as by 1 mM phentolamine (data not shown). This indicates that contractions were mediated by stimulation of perivascular noradrenergic nerves and that a2adrenergic receptors were involved [24]. TNS produced frequency-dependent contractions of the caudal artery between 0.5 and 4 Hz. As illustrated in figure 1, contractions induced by both 0.5 Hz and 1 Hz were significantly higher in the losartan than in the control group. In contrast, at these frequencies, treat-
Table 1 Effect of treatment on systolic arterial blood pressure and left ventricular hypertrophy
Control Captopril Losartan
Systolic arterial pressure (mm Hg)
Left ventricle weight/ body weight (mg/g)
249 6 28 203 6 20* 214 6 11*
3.00 6 0.10 2.57 6 0.09* 2.56 6 0.06*
Either captopril or losartan groups were compared to their respective control by using the Student’s t test. * P , 0.05.
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Fig. 1. Effect of transmural nerve stimulation (0.5 to 4 Hz) on the contraction of caudal artery rings in untreated and treated SHR. Contraction is expressed as % of a previous contraction elicited by 75 mM KCl. Data are expressed as means 6 S.E.M. n5 indicates the number of vascular rings used. * p,0.05 compared to the control group.
ment with captopril did not modify the response to TNS. Neither losartan nor captopril modified the response to TNS at frequencies higher than 1 Hz. Postsynaptic Effects The contractile effect of noradrenaline was tested in caudal artery rings. Contraction is expressed as % of the response induced by 75 mM KCl, which was 1546675 mg, 1680675 mg and 1357652 mg for control, losartan and captopril-treated groups, respectivelly. Fig 2 shows the concentration-response curves for noradrenaline on caudal arteries from control
Fig. 2. Effect of noradrenaline in the contractility of the caudal artery. Contraction is expressed as % of a previous contraction elicited by 75 mM KCl. Data are expressed as means 6 S.E.M. n5 indicates the number of vascular rings used. * p,0.05 compared to the control group.
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(EC5051.58 (1, 2.5) 3 1027 M), losartan (EC5050.56 (0.32, 1) 3 1027 M) and captopriltreated (EC5051.26 (1, 1.99) 3 1027 M) SHR. The concentration-response plot for noradrenaline was similar in control and captopril groups. However, in arteries from the losartan treated group, the concentration-effect curve of noradrenaline was significantly shifted to the left. Presynaptic effects The effect of AII, tested in a concentration range from 1029 to 1026 M, in modulating the effect of TNS (5 Hz) on the release of radioactivity is illustrated in figure 3. In the control group, one-way ANOVA revealed a significant effect of AII on radioactivity release evoked by TNS (FANOVA(4,36)5 9.937; P,0.001). In the captopril treated group, the effect of AII was similar to that observed in the control group (FANOVA(4,28)53.010; P,0.05). However, AII was without effect in the losartan-treated group (FANOVA(4,38)50.927; NS). The effect of TNS (5 Hz) on the release of radioactivity from caudal artery segments loaded with [3H]-noradrenaline was not significantly different on control, losartan and captopriltreated groups (Figure 3). The facilitatory effect of AII was fully suppressed by preincubating the arteries with 1027 M losartan, both in the control (FANOVA(3,12)50.89; P.0.05) (figure 4) and in the captopril-treated group (FANOVA(3,12)50.51; P.0.05) (results not shown). In contrast, the AT2 receptor antagonist, PD-123,319, also at 1027 M, was without effect in both groups (results not shown).
Fig. 3. Effect of angiotensin II on radiactivity release evoked by TNS (5 Hz) from caudal artery segments of control, captopril and losartan-treated SHR loaded with [3H]-noradrenaline. Radiactivity release is expressed as % of basal release. Data are expressed as means 6 S.E.M. n 5 8–11 rats/group. * p , 0.05 compared to the control group.
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Fig. 4. Effect of losartan (0.1 mM) on angiotensin II-induced radioactivity release evoked by TNS (5 Hz) from caudal artery segments of control SHR loaded with [3H]-noradrenaline. Radiactivity release is expressed as % of basal release. Data are expressed as means 6 S.E.M. n 5 11 rats. * p , 0.05 compared to the control group.
Discussion In this work we describe the effect of chronic treatment with the angiotensin AT1-receptor antagonist, losartan, on sympathetic mechanisms regulating the vascular tone of the SHR caudal artery. The effect of losartan was compared to the effect of captopril, given at doses leading to an equivalent decrease of both systolic blood pressure and left ventricular hypertrophy. The response initially studied was the contractility of caudal artery rings induced by TNS. Under our conditions, low frequency TNS (0.5 and 1 Hz), which stimulates the release of noradrenaline by sympathetic endings [25], induced higher contractile responses in losartantreated SHR than in the control and captopril-treated groups. This difference might be due either to an increase of the postsynaptic effect of noradrenaline or/and to a higher release of the neurotransmitter from noradrenergic endings. The first possibility was tested by comparing the contractile effect of noradrenaline in the three experimental groups. The concentrationresponse curve for noradrenaline was slightly, although significantly, shifted to the left in the losartan-treated group, compared to the control group. In contrast, treatment with captopril did not modify the contractile effect of noradrenaline. Although a similar treatment with losartan decreases the vasoconstriction induced by noradrenaline in aorta of SHR [27], in a densely innervated vessel, as the caudal artery, the enhancement of the sensitivity of noradrenergic receptors to noradrenaline could reflect a compensatory mechanism caused by a decrease of noradrenaline release, induced by chronic losartan treatment. In order to test this hypothesis we examined the release of radioactivity induced by TNS from caudal artery segments loaded with [3H]-noradrenaline. For these experiments TNS frequency was 5 Hz instead 0.5–1 Hz, since, in our hands, lower frequencies than 5 Hz led to
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too low radioactivity release. Under these conditions, we found that radioactivity release induced by 5 Hz was similar in the three groups, which is in agreement with the results obtained in 2 and 4 Hz TNS-induced contractility. However, the effect of AII on the release of radioactivity evoked by 5 Hz TNS was deeply modified in the losartan treated group. AII elicited a concentration-dependent enhancement of TNS-induced efflux of radioactivity both in the control and in the captopril-treated SHR. In both groups, the effect of AII was blocked by preincubation of arteries with losartan, and not by PD-123,319 indicating the involvement of AT1 angiotensin receptors in this response. In contrast, in the losartan treated group, the modulatory role of AII on radioactivity release was suppressed. Similar results have been reported by Foucart et al. [28], who described that a twelve-day treatment with losartan, but not with enalaprilat, abolished the effect of AT1 on the release of [3H]-noradrenaline from atria of SHR, under similar experimental conditions. A loss of effect of AII would be consistent with a down-regulation of prejunctional AT1 receptors, which is rather unexpected from a chronic treatment with an AT1-receptor antagonist. However, it has been previously described that losartan down-regulates AT1 receptors in chronic treatment [29]. Interestingly, it has been reported that chronic losartan reduces blood pressure by increasing availability of NO [30, 31], which has been shown to down-regulate AT1 receptors expression [32, 33]. Another possibility would be an eventual partial-agonist character of losartan or of some losartan metabolite. Althought this possibility cannot be discarded, such a partial agonist character of losartan has been only detected in in vitro experiments in rat isolated glomeruli and in human mesangial cells by using losartan concentrations higher than 1 mM [34]. However, most work in this field show a lack of agonist character for losartan [19]. Concerning metabolites, EXP 3174, an active metabolite of losartan, endowed with non-competitive antagonist properties, has been identified [35]. EXP 3174 has a slow dissociation kinetic from AT1 receptors [36] and could theoretically account for the results obtained. Similar to our results, a permanent inhibition of the potentiating effect of AII on sympathetic nerve function, at least 10 weeks after the end of a 12-week treatment with AT1 antagonists, has been also described [37]. In conclusion, chronic treatement with the the AT1 receptor antagonist, losartan i) slightly enhances the pressor effect of noradrenaline at postsynaptic level, and ii) suppresses the facilitatory role of AII on noradrenaline release. This could be an important feature in regulating blood pressure since noradrenergic innervation is increased in SHR vessels. Acknowledgments This work has been supported by a Medical School Grant of Merck Sharp & Dohme, Spain and by a Grant of the Comunidad de Madrid (CAM 08.4/0003/1997), Spain. References 1. Ferrario CM, Brosnihan KB, Diz DI, Jaiswal N, Khosla MC and Milsted A. Angiotensin-(1-7): a new hormone of the angiotensin system. Hypertension 1991; 18: 26–33. 2. Zimmerman RG. Action of angiotensin on vascular adrenergic nerve endings. Facilitation of norepinephrine response. Fed. Proc. 1972; 31: 1344–1350.
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