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Endothelium-dependent vasomotor effects of telmisartan in isolated rat femoral arteries
Pharmacological Research 63 (2011) 199–206
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Endothelium-dependent vasomotor effects of telmisartan in isolated rat femoral arteries Ilias Siarkos, Vincenzo Urso, Tiziana Sinagra, Filippo Drago, Salvatore Salomone ∗ Department of Experimental and Clinical Pharmacology, University of Catania, Viale Andrea Doria 6, 95125 Catania, Italy
a r t i c l e
i n f o
Article history: Received 17 September 2010 Received in revised form 6 October 2010 Accepted 7 October 2010 Key words: Endothelium Telmisartan Isolated femoral arteries
a b s t r a c t AT1 receptor antagonists (ARBs) are drugs widely used for preventing and/or treating major cardiovascular diseases. Some of these drugs also show AT1 receptor-independent effects that may have patho-physiological significance, such as Peroxisome Proliferator-Activated Receptors gamma (PPAR␥) stimulation. Here we investigated the effect of telmisartan (that also stimulates PPAR␥) on vasomotor responses of femoral arteries isolated from rat, in comparison to losartan. Femoral artery segments were mounted in a wire myograph and challenged with cumulative concentrations of phenylephrine (PE) and acetylcholine (ACh) after 30-min incubation in the absence or presence of 30 M telmisartan or 30 M losartan. Vasomotor responses were not significantly changed by losartan, whereas telmisartan reduced vasoconstriction to PE and increased vasodilatation to ACh. Incubation with 0.1 mM NG -nitrol-arginine abolished relaxation to ACh in untreated controls as well as in losartan-treated preparations, but did not in telmisartan-treated preparations (were 20% relaxation subsisted); this residual relaxing effect was abolished by indomethacin and by endothelium removal. Incubation with 30 M GW9662 (PPAR␥ antagonist), 10 M PD123319 (AT2 antagonist) or 30 M A779 (angiotensin(1-7)/Mas antagonist) did not change the effect of telmisartan on vasomotor responses in preparations with intact endothelium. We conclude that telmisartan modifies constriction and dilatation of isolated arteries in an endothelium-dependent manner, involving both nitric oxide and prostanoid production. The present effect of telmisartan, however, does not seem to involve PPAR␥, AT2 or angiotensin(1-7)/Mas. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Renin-angiotensin-aldosterone system (RAAS) is essential in controlling blood pressure and water and salt homeostasis and contributes to patho-physiological processes such as cardio-vascular hypertrophy and remodeling [1]. The main component, angiotensin II (ANGII), stimulates at least two different G-protein coupled receptors in the cardiovascular system, termed AT1 and AT2 . AT1 receptor antagonists (ARBs, also known as sartans) are widely used to treat hypertension, heart failure and diabetic kidney disease [1]. In contrast with angiotensin converting enzyme (ACE) inhibitors, ARBs have the advantage of selectively and completely block AT1 receptor [2]. Indeed, because of the existence of alternative pathways, such as chymase [3], some ANGII production still takes place during treatment with ACE inhibitors. Furthermore, ARBs do not induce cough as side effect (cough is related to kinin accumulation following ACE inhibition) and maintain ANGII levels sufficient to stimulate AT2 receptors which potentially produces beneficial effects [2]. Evidence from both pre-clinical and
∗ Corresponding author. Tel.: +39 095 7384085; fax: +39 095 7384228. E-mail address: [email protected] (S. Salomone). 1043-6618/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2010.10.010
clinical studies indicates that in vivo treatment with ARBs prevents and/or slows down development of endothelial dysfunction associated with cardio-vascular risk [4,5]. Furthermore, ARBs offer additional cardiovascular protection in type 2 diabetic patients [6–8]. Because the pharmacological properties mentioned above are shared by all ARBs, the question may be risen on whether or not they should be considered interchangeable (i.e. to display a “class effect”) and, for example, if their impact on endothelial function may or may not be identical. In this respect, recent studies show for some ARBs additional properties that are compound-related rather than class-related, i.e. all ARBs do not have an identical pharmacological profile. Telmisartan, for example, is capable of inducing Peroxisome Proliferator-Activated Receptor ␥ (PPAR␥)-dependent gene expression [9,10], an AT1 receptor-unrelated effect. PPAR␥ is a nuclear receptor involved in regulation of insulin sensitivity and glucose metabolism [11], through induction of adiponectin, an insulin-sensitizing protein [10,12]. Worthy of note, telmisartan in vitro stimulates PPAR␥ activity at concentrations comparable to those achieved in plasma in humans [9,10,13]. Despite the considerable amount of in vivo data on the impact of ARBs on endothelial dysfunction [5,14], very limited data are available on impact of in vitro ARB application on endothelium-dependent vasomotor responses.
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In this study we tested the hypothesis that ARBs directly affect endothelium-dependent vasomotor responses of isolated vessels, and try to determine if compound-related effect are distinguishable from class effect, by comparing losartan and telmisartan applied to isolated femoral artery of rat. Femoral artery was chosen because it is widely used for studying endothelial injury and repair and because, in our previous experience [15–17], it is damaged by dissection, manipulation and mounting less than other rodent small vessels. The results show that short-term in vitro treatment with telmisartan, but not with losartan, enhances endotheliumdependent relaxation, an effect involving both NO-dependent and prostanoid-dependent vasodilatation.
2. Materials and methods 2.1. Preparation of femoral artery and analysis of vasomotor responses Animal use was approved by the subcommittee for research and animal care at the University of Catania according to guidelines from Italian Ministry of Health. Male Sprague–Dawley rats (Stefano Morini, San Polo d’Enza, Italy; 200–250 g) were killed by CO2 asphyxiation. Femoral arteries were removed, immersed in physiological salt solution (composition, mM: NaCl, 118; KCl, 4.6; NaHCO3 , 25; MgSO4 , 1.2; KH2 PO4 , 1.2; CaCl2 , 1.2; glucose, 10; EDTA, 0.025; pH 7.4 at 37 ◦ C) and cut in segments (2 mm length). In some experiments, in order to remove endothelium, arteries were cannulated and perfused with 200 L of 0.03% triton-x 100, followed by 500 L physiological salt solution. Each arterial segment was subsequently mounted in a wire myograph (610 M, Danish Myo Technology, Aarhus, Denmark), by using 40 m-diameter stainless steel wire, for isometric record of contractile force. After mounting, each preparation was equilibrated unstretched, for 30 min, in physiologic salt solution, maintained at 37 ◦ C and aerated with a gas mixture 95% O2 –5% CO2 . The normalized passive resting force and the corresponding diameter were then determined for each preparation from its own length–pressure curve [18]. Contractile responses were recorded into a computer, by using a data acquisition and recording software (Myodaq and Myodata, Danish Myo Technology). After normalization and 30-min equilibration in physiological solution, the preparations were stimulated with isotonic depolarizing KCl solution, in which part of NaCl had been replaced by an equimolar amount of KCl (composition, mM: NaCl, 22.6; KCl, 98.8; NaHCO3 , 25; MgSO4 , 1.2; KH2 PO4 , 1.2; CaCl2 , 1.2; glucose, 10; EDTA, 0.025, pH 7.4 at 37 ◦ C). After washout and 30-min recovery, the preparations were exposed to 30 M telmisartan or losartan for 30 min; experimental conditions were selected based on preliminary experiments, where lower concentration of telmisartan did not exert detectable effects and incubation longer than 30 min did not increase the effect of 30 M telmisartan (not shown). After incubation with ARBs, cumulative concentrations of phenylephrine (PE, 10 nM to 10 M) or serotonin (5-HT, 1 nM to 1 M) were added to the organ chamber; once the vasoconstriction to PE or 5-HT had reached steady state, cumulative concentrations of acetylcholine (ACh, 1 nM to 10 M) or sodium nitroprusside (SNP, 1 nM to 10 M) were added to the organ chamber. Relaxing responses were expressed in percentage of pre-existing contractile tone, induced by PE or 5-HT. Some experiments were carried out in the presence of 100 M NG -nitro-l-arginine (LNNA) and/or 10 M indomethacin, preincubated for 30 min before challenging with PE, 5-HT, ACh, SNP, to inhibit NO and prostanoid synthesis, respectively. In other experiments, when used, 30 M GW9662, 10 M PD123319 or 30 M A779 were added together with telmisartan for 30 min, before challenging with PE and ACh.
2.2. Drugs and reagents PE, ACh, 5-HT, SNP, LNNA, indomethacin were from Sigma (St. Louis, MO, U.S.A.). These drugs were dissolved at 10 mM in aqueous stock solutions, except indomethacin, that was dissolved in ethanol. Stock solutions were further diluted in water or directly in physiological salt solution, as required to reach the final concentration. Telmisartan was obtained from Boehringer-Ingelheim (Ridgefield, CT, U.S.A.); losartan and GW9662, were from Sigma; PD123319 was from Tocris (Ellsville, MO, U.S.A.); stock solutions of telmisartan, losartan, GW9662 and PD123319 were prepared at 10 mM in dimethyl sulfoxide (DMSO). A779 [DRVYIH-(d-Ala)] was synthesized by Biomatik (Markham, Ontario, Canada) and showed a purity > 90%; a 10 mM stock solution of A779 was prepared in water. 2.3. Statistical analysis Concentration–response curves to agonists, carried out in the presence of vehicle (control), telmisartan or losartan, were plotted as a percentage of the previous vasoconstriction induced by KCl in the same arteries in the absence of ARB against log molar concentration of drug. Each set of data points was curve-fitted by a non-linear regression, best-fit, sigmoidal dose-response curve with no constraints, with the use of GraphPad Prism (GraphPad Software, San Diego, CA). Pharmacological parameters (concentration producing 50% of maximum effect or EC50 and maximum effect or Emax ) were calculated from these non-linear fits. Each curve represents preparations from at least 7 different animals. Whenever it was possible, arterial segments from the same animal were represented in the different experimental conditions. Whole curves were compared by two-way analysis of variance (ANOVA), with significance set at P < 0.05. 3. Results Femoral artery segments mounted in a wire myograph were first constricted by exposing to a 100 mM KCl-depolarizing solution. Arterial segments were subsequently incubated with or without losartan (30 M) or telmisartan (30 M) before being challenged with cumulative concentrations of PE and eventually (on top of PE-induced vasoconstriction) with ACh, to assess endotheliumdependent vasodilatation. As shown in Fig. 1 and Table 1, 30-min incubation with losartan did not change vasoconstriction to PE or endothelium-dependent vasodilatation to ACh. In contrast, incubation with telmisartan produced a 20% decrease in maximum vasoconstriction to PE (P < 0.01) and a 15% increase in maximum vasodilatation to ACh (P < 0.01). These effects on vasomotor responses to PE and ACh were further confirmed in a different experimental paradigm, by analyzing two consecutive runs in the same preparations, in the absence and in the presence of losartan or telmisartan. As shown in Fig. 2, in run 2, in the absence of treatment (control) and in losartantreated preparation, vasoconstriction to PE and vasodilatation to ACh was similar to that observed in run 1 (both without drugtreatment); in contrast, run 2 in telmisartan-treated preparation showed a decrease of vasoconstriction to PE and an increase of vasodilatation to ACh as compared to run 1 without drugtreatment. Because vasodilatation to ACh is known to be endotheliumdependent, mostly NO-mediated, we hypothesized that stimulation by telmisartan of endothelial NO production may also occur in the absence of ACh (i.e. telmisartan may stimulate basal NO production) which could account for the observed reduction of vasoconstriction to PE. To test this hypothesis we challenged iso-
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Fig. 1. Effects of losartan or telmisartan on vasomotor responses of rat femoral arteries to cumulative concentrations of phenylephrine (PE, A) and acetylcholine (ACh, B). Data are mean ± SEM of 12 independent measurements. Pharmacological parameters are given in Table 1. **P < 0.01 versus control, †† P < 0.01 versus losartan, by two-way ANOVA. Table 1 Pharmacological parameters of vasomotor responses to PE and ACh in femoral arteries: effects of telmisartan. PE pD2 Control (n = 12) Losartan (n = 12) Telmisartan (n = 12) Telmisartan + GW9662 (n = 8) Telmisartan + PD123319 (n = 8) Telmisartan + A779 (n = 8)
5.68 5.63 5.57 5.63 5.93 5.90
± ± ± ± ± ±
0.08 0.12 0.11 0.08 0.18 0.07
EC50 (M)
Emax (% K+ )
2.10 (1.47–3.00) 2.33 (1.31–4.14) 2.69 (1.65–4.40) 2.35 (1.61–3.44) 1.16 (0.51–2.64) 1.24 (0.90–1.73)
135.5 134.1 102.9 99.6 81.3 86.5
± ± ± ± ± ±
7.6 12.5 8.8** 6.1** 8.9** 3.8**
ACh
Control (n = 12) Losartan (n = 12) Telmisartan (n = 12) Telmisartan + GW9662 (n = 8) Telmisartan + PD123319 (n = 8) Telmisartan + A779 (n = 8)
pD2
EC50 (M)
Emax (% tone)
6.41 ± 0.15 6.16 ± 0.21 6.63 ± 0.10 6.74 ± 0.12 6.76 ± 0.12 6.68 ± 0.14
0.39 (0.20–0.77) 0.69 (0.27–1.80) 0.24 (0.15–0.37) 0.18 (0.11–0.31) 0.17 (0.10–0.30) 0.21 (0.11–0.40)
39.7 ± 4.2 39.1 ± 6.7 26.5 ± 3.2** 23.6 ± 3.7** 24.7 ± 3.8** 21.6 ± 4.9**
Pharmacological parameters are from non-linear regression; pD2 is the negative logarithm of the concentration producing 50% of the maximum effect, EC50 is the concentration producing 50% of maximum effect (95% confidence limits are given in parenthesis), Emax is the maximum effect (vaconstriction to PE in % of vasoconstriction evoked in the same preparations by 100 mM K+ ; vasodilatation to ACh as % of residual tone). PD123319 was 10 M; losartan, telmisartan, GW9662, A779 were 30 M; all drugs were incubated for 30 min before exposing to PE and/or ACh and maintained in the organ chamber during the vasomotor challenge. Statistical comparisons were made on the whole curves by two-way ANOVA. ** P < 0.01 versus control.
lated femoral arteries with PE and ACh either after removing endothelium or in the presence of LNNA to inhibit NO. As shown in Fig. 3 and Table 2, both removal of endothelium or LNNAtreatment produced a leftward shift of concentration-contraction curve to PE, which reached the curve obtained in LNNA-treated controls, suggesting that reduced vasoconstriction to PE by telmisartan (as illustrated in Fig. 1) was likely related to increased basal NO production. As expected, removal of endothelium abol-
ished the relaxing effect of ACh, either in untreated controls (not shown) or in telmisartan treated preparations (Fig. 4D). However, in telmisartan-treated preparations with intact endothelium, when LNNA had been included in incubation medium to inhibit NO synthesis, vasorelaxation to ACh was reduced but not abolished (Fig. 4B and E). Further incubation with indomethacin was necessary to abolish relaxation to ACh in the presence of telmisartan (Fig. 4C and E).
Table 2 Pharmacological parameters of concentration-contraction curve to PE in the presence of telmisartan: effects of LNNA and endothelium removal. PE pD2 Control E(+) (n = 12) Control E(+) (n = 8) + LNNA Telmisartan E(+) (n = 12) Telmisartan E(+) (n = 8) + LNNA Control E (−) (n = 8) Telmisartan E(−) (n = 8)
5.68 6.31 5.57 6.04 6.38 5.95
± ± ± ± ± ±
0.08 0.06 0.11 0.07 0.11 0.13
EC50 (M)
Emax (% K+ )
2.10 (1.47–3.00) 0.50 (0.38–0.64)** 2.69 (1.65–4.40) 0.91 (0.67–1.23)a 0.43 (0.25–0.73)** 1.13 (0.61–2.08)a
135.5 120.1 102.9 130.2 123.8 125.6
± ± ± ± ± ±
7.6 3.1 8.8** 4.8a 3.5 9.6a
Pharmacological parameters are from non-linear regression; pD2 is the negative logarithm of the concentration producing 50% of the maximum effect, EC50 is the concentration producing 50% of maximum effect (95% confidence limits are given in parenthesis), Emax is the maximum effect (vasoconstriction to phenylephrine, PE, in % of vasoconstriction evoked in the same preparations by 100 mM K+ ). NG -nitro-l-arginine (LNNA) 100 M was preincubated for 30 min before exposing to PE. E(+), intact endothelium, E(−), endothelium removed (see also Section 2). Data for control E(+) and telmisartan E(+) are those from Table 1. Statistical comparisons were made on the whole curves by two-way ANOVA. ** P < 0.01 versus control E(+). a P < 0.01 versus telmisartan E(+).
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Fig. 2. Effects of losartan or telmisartan on vasomotor responses to phenylephrine (PE) and acetylcholine (ACh) in isolated femoral arteries. (A) Control; (B) losartan; (C) telmisartan. Cumulative concentrations of PE (10 nM to 10 M) or ACh (1 nM to 10 M), added to the organ chamber by half log increase, are indicated by black dots on the tracing. Two consecutive runs were carried out in each preparation. Notice in run 2, in telmisartan-treated preparation (C), a decrease of vasoconstriction to PE and an increase of vasodilatation to ACh as compared to run 1 without drug-treatment.
To exclude that telmisartan acted directly on vascular smooth muscle cells, for ex. by inhibiting contractile proteins, and to rule out the possibility that increase in ACh-induced vasorelaxation was due to weaker preconstricting force by PE, experiments were repeated using 5-HT as vasoconstrictor and reaching equal preconstricting force before challenge with ACh. As shown in Fig. 5 and Table 3, concentration contraction curve to 5-HT was poorly changed by telmisartan pre-treatment. After reaching a similar level of contractile plateau in both groups (in % of 100 mM K+ : control, 107.6 ± 4.5, n = 8; telmisartan, 108.5 ± 3.0, n = 8) ACh-induced
vasodilatation was still more pronounced in telmisartan-treated preparations (P < 0.01) than in control (Fig. 5B). Endotheliumindependent vasodilation to the SNP was unchanged (Fig. 5C). PPAR␥ ligands increase NO release from endothelial cells [19]; because telmisartan has been shown to activate PPAR␥ in endothelial cells [20,21], we investigated the effect of GW9662, a PPAR␥ antagonist [22,23], on vasomotor effects of telmisartan. As shown in Fig. 6 and Table 1, 30 M GW9662 did not change PE-induced vasoconstriction and ACh-induced vasodilatation in the presence of telmisartan, suggesting that the vasomotor effects of telmisartan,
Table 3 Pharmacological parameters of vasomotor responses to 5-HT, ACh and SNP in femoral arteries: effects of telmisartan. 5-HT
Control (n = 8) Telmisartan (n = 8)
pD2
EC50 (M)
Emax (% K+ )
6.99 ± 0.09 6.94 ± 0.06
0.10 (0.07–0.16) 0.12 (0.09–0.15)
135.7 ± 7.3 139.4 ± 5.0
pD2
EC50 (M)
Emax (% tone)
ACh Control (n = 8) Telmisartan (n = 8)
6.48 ± 0.16 6.66 ± 0.16
0.33 (0.15–0.70) 0.22 (0.11–0.45)
76.9 ± 1.9 59.9 ± 2.9**
SNP Control (n = 8) Telmisartan (n = 8)
6.99 ± 0.06 7.14 ± 0.04
0.10 (0.08–0.13) 0.07 (0.06–0.09)
26.7 ± 1.8 26.5 ± 1.1
Notice that vasodilatation to ACh reported in this Table has been obtained using 1 M 5-HT as pre-contraction, whereas data in Table 1 have been obtained using 10 M PE as pre-contraction. Vasodilatation to SNP was assessed on the plateau of contraction to 1 M 5-HT, in preparations were endothelial NO production had been blocked by 100 M LNNA. Pharmacological parameters are from non-linear regression; pD2 is the negative logarithm of the concentration producing 50% of the maximum effect, EC50 is the concentration producing 50% of maximum effect (95% confidence limits are given in parenthesis), Emax is the maximum effect (vaconstriction to 5-HT in % of vasoconstriction evoked in the same preparations by 100 mM K+ ; vasodilatation to ACh and SNP as % of residual tone). Statistical comparisons were made on the whole curves by two-way ANOVA. ** P < 0.01 versus control.
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has been shown to stimulate production of NO and prostanoids through a G-protein coupled receptor termed Mas [26], abundantly expressed in vascular endothelium [27]. We therefore tested the effect of PD123319, a specific AT2 antagonist [28], and of A779, a specific Mas antagonist [29], on vasomotor effects of telmisartan in isolated femoral arteries. As shown in Fig. 7 and Table 1, neither 10 M PD123319 nor 30 M A779 induced any detectable change in PE-induced vasoconstriction and in ACh-induced vasodilatation in the presence of telmisartan. 4. Discussion
Fig. 3. Effect of nitric oxide inhibition and endothelium removal in telmisartantreated rat femoral arteries. Preparations with [E(+)] or without [E(−)] endothelium were preincubated with telmisartan and/or NG -nitro-l-arginine (LNNA, 100 M) before challenging with phenylephrine (PE). Data are mean ± SEM of 8 independent measurements. Data for telmisartan E(+) are the same reported in Fig. 1 (n = 12). Pharmacological parameters are given in Table 2. **P < 0.01 versus telmisartan E(+) by two-way ANOVA.
after the short term (30-min) exposure used here, did not involve PPAR␥ stimulation. ARBs have been claimed to induce AT2 receptor stimulation [24,25], possibly because blockade of AT1 receptors increases local bioavailability of ANGII, which might increase endothelial NO release; furthermore, the heptapeptide angiotensin-(1-7) (AT(1-7) )
This work was undertaken to determine whether ARBs directly affect vasomotor responses of isolated vessels in a short-term paradigm (30-min incubation), and try to determine if compoundrelated effects are distinguishable from class effects. Our results show that telmisartan, but not losartan, decreased the vasoconstriction to PE and enhanced the vasodilatation to ACh. Constriction of isolated vessels to ␣-adrenergic agonists, such as PE, is well known to be enhanced in the absence of NO or functional endothelium [30,31]; consistently we observed a half-log shift to the left of concentration–contraction curve to PE in control preparations, following incubation with LNNA or endothelium removal (Fig. 3 and Table 2); in telmisartan-treated preparations, LNNA or endothelium removal restored the vasoconstriction to PE back to the level of control preparations (Fig. 3 and Table 2). Because the inhibition of constriction to PE by telmisartan was reversed by NO synthase inhibition or endothelium removal, it appears to be related to enhanced basal NO production.
Fig. 4. Effect of nitric oxide and prostanoid inhibition on vasomotor effects of telmisartan. (A and B) Nitric oxide synthase inhibition by NG -nitro-l-arginine (LNNA, 100 M); C nitric oxide synthase inhibition by LNNA + cyclooxygenase inhibition by indomethacin (Indo, 10 M); D, Endothelium removal; E, average vasodilatation to acetylcholine (ACh). Cumulative concentrations of phenylephrine (PE, 10 nM to 10 M) or ACh (1 nM to 10 M), added to the organ chamber by half log increase, are indicated by black dots on the tracing. Notice in run 2, in (A) (control telmisartan-untreated) vasodilatation to ACh is abolished by l-NNA, whereas in (B) (telmisartan-treated) some vasodilatation to ACh is still observed in the presence of LNNA. **P < 0.01 versus telmisartan + LNNA by two-way ANOVA.
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Fig. 5. Effects of telmisartan on vasomotor responses of rat femoral arteries to cumulative concentrations of serotonin (5-HT, A), acetylcholine (ACh, B) and sodium nitroprusside (SNP, C). Vasodilatation to ACh was assessed on the plateau of contraction to 1 M 5-HT. Vasodilatation to SNP was assessed on the plateau of contraction to 1 M 5-HT, in preparations were endothelial NO production had been blocked by NG -nitro-l-arginine (LNNA, 100 M). Data are mean ± SEM of 8 independent measurements. Pharmacological parameters are given in Table 3. **P < 0.01 versus control, by two-way ANOVA.
In addition to basal NO synthesis, 30-min incubation with telmisartan increased endothelium-dependent vasodilatation to ACh, again, an effect not reproduced with losartan. Worthy of note, LNNA, which effectively blocked relaxation to ACh in control (telmisartan-untreated) preparations, was unable to completely abolish relaxation to ACh in telmisartan-treated preparations. The residual relaxation to ACh (about 20%) may be related to prostanoid synthesis, enhanced by telmisartan, because it disappeared when cyclooxygenases had been blocked by indomethacin. Several compounds belonging to ARBs have been reported to exert endothelial effects [32,33] that are considered as protective or beneficial for cardiovascular diseases [34]. In general, however, the impact of ARBs on endothelial function requires in vivo chronic treatments or in vitro long-lasting (>10 h) incubation (see also below). For example, in vivo treatment with telmisartan in animal models of cardiovascular disease improves endothelial function and increases NO bioavailability, but both effects are reversed by GW9662, a PPAR␥ antagonist [35,36]. These findings have been therefore interpreted on the basis of a PPAR␥ agonist activity of telmisartan [9,10]. Consistently, PPAR␥ agonists have been shown to increase NO release, an effect that requires 24 h treatment [19], and to upregulate endothe-
lial NO synthase [37]. The effects of telmisartan or PPAR␥ activators on endothelium occur either following in vivo treatment or after several hours of in vitro incubation, consistent with the latency of PPAR␥-activated gene expression, which requires at least 2 h for significantly changing mRNAs [38]. Therefore, in our system, the short latency (30–60 min) of telmisartan effect on vasomotor responses makes unlikely PPAR␥ stimulation as the underlying mechanism; furthermore, the lack of effect of 30 M GW9662, a PPAR␥ antagonist [23] that we used at a concentration much higher than the reported IC50 s (3.8 nM [39]), rules out the involvement of PPAR␥. PPAR␥-independent effects of telmisartan on endothelium have been attributed to radical scavenging [40]; protection of endothelial function from oxidant radicals has also been repeatedly reported for losartan [41,42]; losartan, however, in our hands, did not enhance ACh-induced vasodilatation as telmisartan did, suggesting that the radical scavenging effect, supposedly exerted by both sartans, is not sufficient to produce a detectable endothelial outcome in our in vitro system. Furthermore, telmisartan did not change the vasodilatation to exogenous NO (given as SNP, Fig. 5C), indicating that a scavenging effect by telmisartan, if any, does not increase bioavailability of exogenous NO. An increased phospho-
Fig. 6. Effect of GW9662 in telmisartan-treated rat femoral arteries. Preparations were preincubated with telmisartan and with or without GW9662, before exposing to phenylephrine (PE, A) and acetylcholine (ACh, B). Data are mean ± SEM of 8 independent measurements. Pharmacological parameters are given in Table 1.
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Fig. 7. Effect of PD123319 (A and C) and A779 (B and D) in telmisartan-treated rat femoral arteries. Preparations were preincubated with telmisartan and with or without PD123319 or A779, before exposing to phenylephrine (PE) and acetylcholine (ACh). Data are mean ± SEM of 8 independent measurements. Pharmacological parameters are given in Table 1.
rylation of endothelial NO synthase (eNOS) following >7 day in vivo treatment with ARBs, in Dahl rats, has been recently reported [43,44]; because eNOS phosphorylation increases enzymatic activity and endothelium-dependent vasodilatation [45], if occurring in vitro it might be responsible for at least part of vasomotor effects of telmisartan reported here. However, at least an additional mechanism, acting on prostanoid synthesis, must be hypothesized to explain the indomethacin-sensitive vasodilation observed here in the presence of telmisartan. In this respect, an increased prostanoid release from endothelium of rat aorta has been reported following in vivo treatment with ARBs [46,47], but the precise mechanism responsible for this phenomenon remains to be determined. Local expression and/or processing of angiotensin peptides has been proposed, since long time, as an important mechanism contributing to vascular and cardiac remodeling [48], which may account for at least part of long term beneficial effects of ACE inhibitors and ARBs in patients with hypertension, heart failure, myocardial infarction [49,50]. Recently, attention has been focused on the heptapeptide AT(1-7) , which is produced mainly locally from ANGII by ACE2 [51] and stimulates a G-protein coupled receptor termed Mas [26], abundantly expressed in vascular endothelium [27]. Because either AT2 or Mas are expected to increase NO production [52–54], we hypothesized that telmisartan increased NO release in isolated femoral arteries by effectively opposing AT1 receptor and thereby increasing AT2 and/or Mas stimulation by locally produced ANGII and/or AT(1-7) . To block AT2 we used PD123319 at 10 M (IC50 30 nM [55]); to block Mas we used A779 at 30 M (IC50 20 nM [56]). None of these drugs induced any detectable change in PE-induced vasoconstriction and in AChinduced vasodilatation in the presence of telmisartan, suggesting that neither AT2 nor Mas are involved in the vasomotor effects of telmisartan in isolated arteries, as we observed after short-term incubation. Finally, we believe that the effects of a 30 M concentration of losartan or telmisartan we report in this in vitro study potentially have in vivo clinical significance; in fact, after single oral adminis-
tration of 100 mg losartan or 80 mg telmisartan in humans (doses commonly prescribed), plasma Cmax values of about 1300 ng/ml are attained for both compounds (for losartan an active metabolite is generated, with a Cmax of 650 ng/ml, similar to that of losartan itself, even though delayed) [57,58], corresponding to concentrations of approximately 3 M. Furthermore, after repeated administrations, at steady state, average plasma concentration of telmisartan in humans is 4.6 g/ml [58], i.e. about 10 M, a value compatible with the concentration used in the present study. In conclusion, telmisartan modifies constriction and dilatation of isolated arteries in an endothelium-dependent manner, involving both nitric oxide and prostanoid production. These effects cannot be considered as “class effect”, because they are not shared by losartan, the ARB prototype. Furthermore, these vasomotor effects of telmisartan, as observed after 30-min incubation, do not seem to involve PPAR␥, AT2 or AT(1-7) /Mas. Acknowledgement This work was supported by a grant from the University of Catania and by a grant from Boehringer-Ingelheim Italia S.p.A.
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Endothelium-dependent vasomotor effects of telmisartan in isolated rat femoral arteries Ilias Siarkos, Vincenzo Urso, Tiziana Sinagra, Filippo Drago, Salvatore Salomone ∗ Department of Experimental and Clinical Pharmacology, University of Catania, Viale Andrea Doria 6, 95125 Catania, Italy
a r t i c l e
i n f o
Article history: Received 17 September 2010 Received in revised form 6 October 2010 Accepted 7 October 2010 Key words: Endothelium Telmisartan Isolated femoral arteries
a b s t r a c t AT1 receptor antagonists (ARBs) are drugs widely used for preventing and/or treating major cardiovascular diseases. Some of these drugs also show AT1 receptor-independent effects that may have patho-physiological significance, such as Peroxisome Proliferator-Activated Receptors gamma (PPAR␥) stimulation. Here we investigated the effect of telmisartan (that also stimulates PPAR␥) on vasomotor responses of femoral arteries isolated from rat, in comparison to losartan. Femoral artery segments were mounted in a wire myograph and challenged with cumulative concentrations of phenylephrine (PE) and acetylcholine (ACh) after 30-min incubation in the absence or presence of 30 M telmisartan or 30 M losartan. Vasomotor responses were not significantly changed by losartan, whereas telmisartan reduced vasoconstriction to PE and increased vasodilatation to ACh. Incubation with 0.1 mM NG -nitrol-arginine abolished relaxation to ACh in untreated controls as well as in losartan-treated preparations, but did not in telmisartan-treated preparations (were 20% relaxation subsisted); this residual relaxing effect was abolished by indomethacin and by endothelium removal. Incubation with 30 M GW9662 (PPAR␥ antagonist), 10 M PD123319 (AT2 antagonist) or 30 M A779 (angiotensin(1-7)/Mas antagonist) did not change the effect of telmisartan on vasomotor responses in preparations with intact endothelium. We conclude that telmisartan modifies constriction and dilatation of isolated arteries in an endothelium-dependent manner, involving both nitric oxide and prostanoid production. The present effect of telmisartan, however, does not seem to involve PPAR␥, AT2 or angiotensin(1-7)/Mas. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Renin-angiotensin-aldosterone system (RAAS) is essential in controlling blood pressure and water and salt homeostasis and contributes to patho-physiological processes such as cardio-vascular hypertrophy and remodeling [1]. The main component, angiotensin II (ANGII), stimulates at least two different G-protein coupled receptors in the cardiovascular system, termed AT1 and AT2 . AT1 receptor antagonists (ARBs, also known as sartans) are widely used to treat hypertension, heart failure and diabetic kidney disease [1]. In contrast with angiotensin converting enzyme (ACE) inhibitors, ARBs have the advantage of selectively and completely block AT1 receptor [2]. Indeed, because of the existence of alternative pathways, such as chymase [3], some ANGII production still takes place during treatment with ACE inhibitors. Furthermore, ARBs do not induce cough as side effect (cough is related to kinin accumulation following ACE inhibition) and maintain ANGII levels sufficient to stimulate AT2 receptors which potentially produces beneficial effects [2]. Evidence from both pre-clinical and
∗ Corresponding author. Tel.: +39 095 7384085; fax: +39 095 7384228. E-mail address: [email protected] (S. Salomone). 1043-6618/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2010.10.010
clinical studies indicates that in vivo treatment with ARBs prevents and/or slows down development of endothelial dysfunction associated with cardio-vascular risk [4,5]. Furthermore, ARBs offer additional cardiovascular protection in type 2 diabetic patients [6–8]. Because the pharmacological properties mentioned above are shared by all ARBs, the question may be risen on whether or not they should be considered interchangeable (i.e. to display a “class effect”) and, for example, if their impact on endothelial function may or may not be identical. In this respect, recent studies show for some ARBs additional properties that are compound-related rather than class-related, i.e. all ARBs do not have an identical pharmacological profile. Telmisartan, for example, is capable of inducing Peroxisome Proliferator-Activated Receptor ␥ (PPAR␥)-dependent gene expression [9,10], an AT1 receptor-unrelated effect. PPAR␥ is a nuclear receptor involved in regulation of insulin sensitivity and glucose metabolism [11], through induction of adiponectin, an insulin-sensitizing protein [10,12]. Worthy of note, telmisartan in vitro stimulates PPAR␥ activity at concentrations comparable to those achieved in plasma in humans [9,10,13]. Despite the considerable amount of in vivo data on the impact of ARBs on endothelial dysfunction [5,14], very limited data are available on impact of in vitro ARB application on endothelium-dependent vasomotor responses.
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In this study we tested the hypothesis that ARBs directly affect endothelium-dependent vasomotor responses of isolated vessels, and try to determine if compound-related effect are distinguishable from class effect, by comparing losartan and telmisartan applied to isolated femoral artery of rat. Femoral artery was chosen because it is widely used for studying endothelial injury and repair and because, in our previous experience [15–17], it is damaged by dissection, manipulation and mounting less than other rodent small vessels. The results show that short-term in vitro treatment with telmisartan, but not with losartan, enhances endotheliumdependent relaxation, an effect involving both NO-dependent and prostanoid-dependent vasodilatation.
2. Materials and methods 2.1. Preparation of femoral artery and analysis of vasomotor responses Animal use was approved by the subcommittee for research and animal care at the University of Catania according to guidelines from Italian Ministry of Health. Male Sprague–Dawley rats (Stefano Morini, San Polo d’Enza, Italy; 200–250 g) were killed by CO2 asphyxiation. Femoral arteries were removed, immersed in physiological salt solution (composition, mM: NaCl, 118; KCl, 4.6; NaHCO3 , 25; MgSO4 , 1.2; KH2 PO4 , 1.2; CaCl2 , 1.2; glucose, 10; EDTA, 0.025; pH 7.4 at 37 ◦ C) and cut in segments (2 mm length). In some experiments, in order to remove endothelium, arteries were cannulated and perfused with 200 L of 0.03% triton-x 100, followed by 500 L physiological salt solution. Each arterial segment was subsequently mounted in a wire myograph (610 M, Danish Myo Technology, Aarhus, Denmark), by using 40 m-diameter stainless steel wire, for isometric record of contractile force. After mounting, each preparation was equilibrated unstretched, for 30 min, in physiologic salt solution, maintained at 37 ◦ C and aerated with a gas mixture 95% O2 –5% CO2 . The normalized passive resting force and the corresponding diameter were then determined for each preparation from its own length–pressure curve [18]. Contractile responses were recorded into a computer, by using a data acquisition and recording software (Myodaq and Myodata, Danish Myo Technology). After normalization and 30-min equilibration in physiological solution, the preparations were stimulated with isotonic depolarizing KCl solution, in which part of NaCl had been replaced by an equimolar amount of KCl (composition, mM: NaCl, 22.6; KCl, 98.8; NaHCO3 , 25; MgSO4 , 1.2; KH2 PO4 , 1.2; CaCl2 , 1.2; glucose, 10; EDTA, 0.025, pH 7.4 at 37 ◦ C). After washout and 30-min recovery, the preparations were exposed to 30 M telmisartan or losartan for 30 min; experimental conditions were selected based on preliminary experiments, where lower concentration of telmisartan did not exert detectable effects and incubation longer than 30 min did not increase the effect of 30 M telmisartan (not shown). After incubation with ARBs, cumulative concentrations of phenylephrine (PE, 10 nM to 10 M) or serotonin (5-HT, 1 nM to 1 M) were added to the organ chamber; once the vasoconstriction to PE or 5-HT had reached steady state, cumulative concentrations of acetylcholine (ACh, 1 nM to 10 M) or sodium nitroprusside (SNP, 1 nM to 10 M) were added to the organ chamber. Relaxing responses were expressed in percentage of pre-existing contractile tone, induced by PE or 5-HT. Some experiments were carried out in the presence of 100 M NG -nitro-l-arginine (LNNA) and/or 10 M indomethacin, preincubated for 30 min before challenging with PE, 5-HT, ACh, SNP, to inhibit NO and prostanoid synthesis, respectively. In other experiments, when used, 30 M GW9662, 10 M PD123319 or 30 M A779 were added together with telmisartan for 30 min, before challenging with PE and ACh.
2.2. Drugs and reagents PE, ACh, 5-HT, SNP, LNNA, indomethacin were from Sigma (St. Louis, MO, U.S.A.). These drugs were dissolved at 10 mM in aqueous stock solutions, except indomethacin, that was dissolved in ethanol. Stock solutions were further diluted in water or directly in physiological salt solution, as required to reach the final concentration. Telmisartan was obtained from Boehringer-Ingelheim (Ridgefield, CT, U.S.A.); losartan and GW9662, were from Sigma; PD123319 was from Tocris (Ellsville, MO, U.S.A.); stock solutions of telmisartan, losartan, GW9662 and PD123319 were prepared at 10 mM in dimethyl sulfoxide (DMSO). A779 [DRVYIH-(d-Ala)] was synthesized by Biomatik (Markham, Ontario, Canada) and showed a purity > 90%; a 10 mM stock solution of A779 was prepared in water. 2.3. Statistical analysis Concentration–response curves to agonists, carried out in the presence of vehicle (control), telmisartan or losartan, were plotted as a percentage of the previous vasoconstriction induced by KCl in the same arteries in the absence of ARB against log molar concentration of drug. Each set of data points was curve-fitted by a non-linear regression, best-fit, sigmoidal dose-response curve with no constraints, with the use of GraphPad Prism (GraphPad Software, San Diego, CA). Pharmacological parameters (concentration producing 50% of maximum effect or EC50 and maximum effect or Emax ) were calculated from these non-linear fits. Each curve represents preparations from at least 7 different animals. Whenever it was possible, arterial segments from the same animal were represented in the different experimental conditions. Whole curves were compared by two-way analysis of variance (ANOVA), with significance set at P < 0.05. 3. Results Femoral artery segments mounted in a wire myograph were first constricted by exposing to a 100 mM KCl-depolarizing solution. Arterial segments were subsequently incubated with or without losartan (30 M) or telmisartan (30 M) before being challenged with cumulative concentrations of PE and eventually (on top of PE-induced vasoconstriction) with ACh, to assess endotheliumdependent vasodilatation. As shown in Fig. 1 and Table 1, 30-min incubation with losartan did not change vasoconstriction to PE or endothelium-dependent vasodilatation to ACh. In contrast, incubation with telmisartan produced a 20% decrease in maximum vasoconstriction to PE (P < 0.01) and a 15% increase in maximum vasodilatation to ACh (P < 0.01). These effects on vasomotor responses to PE and ACh were further confirmed in a different experimental paradigm, by analyzing two consecutive runs in the same preparations, in the absence and in the presence of losartan or telmisartan. As shown in Fig. 2, in run 2, in the absence of treatment (control) and in losartantreated preparation, vasoconstriction to PE and vasodilatation to ACh was similar to that observed in run 1 (both without drugtreatment); in contrast, run 2 in telmisartan-treated preparation showed a decrease of vasoconstriction to PE and an increase of vasodilatation to ACh as compared to run 1 without drugtreatment. Because vasodilatation to ACh is known to be endotheliumdependent, mostly NO-mediated, we hypothesized that stimulation by telmisartan of endothelial NO production may also occur in the absence of ACh (i.e. telmisartan may stimulate basal NO production) which could account for the observed reduction of vasoconstriction to PE. To test this hypothesis we challenged iso-
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Fig. 1. Effects of losartan or telmisartan on vasomotor responses of rat femoral arteries to cumulative concentrations of phenylephrine (PE, A) and acetylcholine (ACh, B). Data are mean ± SEM of 12 independent measurements. Pharmacological parameters are given in Table 1. **P < 0.01 versus control, †† P < 0.01 versus losartan, by two-way ANOVA. Table 1 Pharmacological parameters of vasomotor responses to PE and ACh in femoral arteries: effects of telmisartan. PE pD2 Control (n = 12) Losartan (n = 12) Telmisartan (n = 12) Telmisartan + GW9662 (n = 8) Telmisartan + PD123319 (n = 8) Telmisartan + A779 (n = 8)
5.68 5.63 5.57 5.63 5.93 5.90
± ± ± ± ± ±
0.08 0.12 0.11 0.08 0.18 0.07
EC50 (M)
Emax (% K+ )
2.10 (1.47–3.00) 2.33 (1.31–4.14) 2.69 (1.65–4.40) 2.35 (1.61–3.44) 1.16 (0.51–2.64) 1.24 (0.90–1.73)
135.5 134.1 102.9 99.6 81.3 86.5
± ± ± ± ± ±
7.6 12.5 8.8** 6.1** 8.9** 3.8**
ACh
Control (n = 12) Losartan (n = 12) Telmisartan (n = 12) Telmisartan + GW9662 (n = 8) Telmisartan + PD123319 (n = 8) Telmisartan + A779 (n = 8)
pD2
EC50 (M)
Emax (% tone)
6.41 ± 0.15 6.16 ± 0.21 6.63 ± 0.10 6.74 ± 0.12 6.76 ± 0.12 6.68 ± 0.14
0.39 (0.20–0.77) 0.69 (0.27–1.80) 0.24 (0.15–0.37) 0.18 (0.11–0.31) 0.17 (0.10–0.30) 0.21 (0.11–0.40)
39.7 ± 4.2 39.1 ± 6.7 26.5 ± 3.2** 23.6 ± 3.7** 24.7 ± 3.8** 21.6 ± 4.9**
Pharmacological parameters are from non-linear regression; pD2 is the negative logarithm of the concentration producing 50% of the maximum effect, EC50 is the concentration producing 50% of maximum effect (95% confidence limits are given in parenthesis), Emax is the maximum effect (vaconstriction to PE in % of vasoconstriction evoked in the same preparations by 100 mM K+ ; vasodilatation to ACh as % of residual tone). PD123319 was 10 M; losartan, telmisartan, GW9662, A779 were 30 M; all drugs were incubated for 30 min before exposing to PE and/or ACh and maintained in the organ chamber during the vasomotor challenge. Statistical comparisons were made on the whole curves by two-way ANOVA. ** P < 0.01 versus control.
lated femoral arteries with PE and ACh either after removing endothelium or in the presence of LNNA to inhibit NO. As shown in Fig. 3 and Table 2, both removal of endothelium or LNNAtreatment produced a leftward shift of concentration-contraction curve to PE, which reached the curve obtained in LNNA-treated controls, suggesting that reduced vasoconstriction to PE by telmisartan (as illustrated in Fig. 1) was likely related to increased basal NO production. As expected, removal of endothelium abol-
ished the relaxing effect of ACh, either in untreated controls (not shown) or in telmisartan treated preparations (Fig. 4D). However, in telmisartan-treated preparations with intact endothelium, when LNNA had been included in incubation medium to inhibit NO synthesis, vasorelaxation to ACh was reduced but not abolished (Fig. 4B and E). Further incubation with indomethacin was necessary to abolish relaxation to ACh in the presence of telmisartan (Fig. 4C and E).
Table 2 Pharmacological parameters of concentration-contraction curve to PE in the presence of telmisartan: effects of LNNA and endothelium removal. PE pD2 Control E(+) (n = 12) Control E(+) (n = 8) + LNNA Telmisartan E(+) (n = 12) Telmisartan E(+) (n = 8) + LNNA Control E (−) (n = 8) Telmisartan E(−) (n = 8)
5.68 6.31 5.57 6.04 6.38 5.95
± ± ± ± ± ±
0.08 0.06 0.11 0.07 0.11 0.13
EC50 (M)
Emax (% K+ )
2.10 (1.47–3.00) 0.50 (0.38–0.64)** 2.69 (1.65–4.40) 0.91 (0.67–1.23)a 0.43 (0.25–0.73)** 1.13 (0.61–2.08)a
135.5 120.1 102.9 130.2 123.8 125.6
± ± ± ± ± ±
7.6 3.1 8.8** 4.8a 3.5 9.6a
Pharmacological parameters are from non-linear regression; pD2 is the negative logarithm of the concentration producing 50% of the maximum effect, EC50 is the concentration producing 50% of maximum effect (95% confidence limits are given in parenthesis), Emax is the maximum effect (vasoconstriction to phenylephrine, PE, in % of vasoconstriction evoked in the same preparations by 100 mM K+ ). NG -nitro-l-arginine (LNNA) 100 M was preincubated for 30 min before exposing to PE. E(+), intact endothelium, E(−), endothelium removed (see also Section 2). Data for control E(+) and telmisartan E(+) are those from Table 1. Statistical comparisons were made on the whole curves by two-way ANOVA. ** P < 0.01 versus control E(+). a P < 0.01 versus telmisartan E(+).
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Fig. 2. Effects of losartan or telmisartan on vasomotor responses to phenylephrine (PE) and acetylcholine (ACh) in isolated femoral arteries. (A) Control; (B) losartan; (C) telmisartan. Cumulative concentrations of PE (10 nM to 10 M) or ACh (1 nM to 10 M), added to the organ chamber by half log increase, are indicated by black dots on the tracing. Two consecutive runs were carried out in each preparation. Notice in run 2, in telmisartan-treated preparation (C), a decrease of vasoconstriction to PE and an increase of vasodilatation to ACh as compared to run 1 without drug-treatment.
To exclude that telmisartan acted directly on vascular smooth muscle cells, for ex. by inhibiting contractile proteins, and to rule out the possibility that increase in ACh-induced vasorelaxation was due to weaker preconstricting force by PE, experiments were repeated using 5-HT as vasoconstrictor and reaching equal preconstricting force before challenge with ACh. As shown in Fig. 5 and Table 3, concentration contraction curve to 5-HT was poorly changed by telmisartan pre-treatment. After reaching a similar level of contractile plateau in both groups (in % of 100 mM K+ : control, 107.6 ± 4.5, n = 8; telmisartan, 108.5 ± 3.0, n = 8) ACh-induced
vasodilatation was still more pronounced in telmisartan-treated preparations (P < 0.01) than in control (Fig. 5B). Endotheliumindependent vasodilation to the SNP was unchanged (Fig. 5C). PPAR␥ ligands increase NO release from endothelial cells [19]; because telmisartan has been shown to activate PPAR␥ in endothelial cells [20,21], we investigated the effect of GW9662, a PPAR␥ antagonist [22,23], on vasomotor effects of telmisartan. As shown in Fig. 6 and Table 1, 30 M GW9662 did not change PE-induced vasoconstriction and ACh-induced vasodilatation in the presence of telmisartan, suggesting that the vasomotor effects of telmisartan,
Table 3 Pharmacological parameters of vasomotor responses to 5-HT, ACh and SNP in femoral arteries: effects of telmisartan. 5-HT
Control (n = 8) Telmisartan (n = 8)
pD2
EC50 (M)
Emax (% K+ )
6.99 ± 0.09 6.94 ± 0.06
0.10 (0.07–0.16) 0.12 (0.09–0.15)
135.7 ± 7.3 139.4 ± 5.0
pD2
EC50 (M)
Emax (% tone)
ACh Control (n = 8) Telmisartan (n = 8)
6.48 ± 0.16 6.66 ± 0.16
0.33 (0.15–0.70) 0.22 (0.11–0.45)
76.9 ± 1.9 59.9 ± 2.9**
SNP Control (n = 8) Telmisartan (n = 8)
6.99 ± 0.06 7.14 ± 0.04
0.10 (0.08–0.13) 0.07 (0.06–0.09)
26.7 ± 1.8 26.5 ± 1.1
Notice that vasodilatation to ACh reported in this Table has been obtained using 1 M 5-HT as pre-contraction, whereas data in Table 1 have been obtained using 10 M PE as pre-contraction. Vasodilatation to SNP was assessed on the plateau of contraction to 1 M 5-HT, in preparations were endothelial NO production had been blocked by 100 M LNNA. Pharmacological parameters are from non-linear regression; pD2 is the negative logarithm of the concentration producing 50% of the maximum effect, EC50 is the concentration producing 50% of maximum effect (95% confidence limits are given in parenthesis), Emax is the maximum effect (vaconstriction to 5-HT in % of vasoconstriction evoked in the same preparations by 100 mM K+ ; vasodilatation to ACh and SNP as % of residual tone). Statistical comparisons were made on the whole curves by two-way ANOVA. ** P < 0.01 versus control.
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has been shown to stimulate production of NO and prostanoids through a G-protein coupled receptor termed Mas [26], abundantly expressed in vascular endothelium [27]. We therefore tested the effect of PD123319, a specific AT2 antagonist [28], and of A779, a specific Mas antagonist [29], on vasomotor effects of telmisartan in isolated femoral arteries. As shown in Fig. 7 and Table 1, neither 10 M PD123319 nor 30 M A779 induced any detectable change in PE-induced vasoconstriction and in ACh-induced vasodilatation in the presence of telmisartan. 4. Discussion
Fig. 3. Effect of nitric oxide inhibition and endothelium removal in telmisartantreated rat femoral arteries. Preparations with [E(+)] or without [E(−)] endothelium were preincubated with telmisartan and/or NG -nitro-l-arginine (LNNA, 100 M) before challenging with phenylephrine (PE). Data are mean ± SEM of 8 independent measurements. Data for telmisartan E(+) are the same reported in Fig. 1 (n = 12). Pharmacological parameters are given in Table 2. **P < 0.01 versus telmisartan E(+) by two-way ANOVA.
after the short term (30-min) exposure used here, did not involve PPAR␥ stimulation. ARBs have been claimed to induce AT2 receptor stimulation [24,25], possibly because blockade of AT1 receptors increases local bioavailability of ANGII, which might increase endothelial NO release; furthermore, the heptapeptide angiotensin-(1-7) (AT(1-7) )
This work was undertaken to determine whether ARBs directly affect vasomotor responses of isolated vessels in a short-term paradigm (30-min incubation), and try to determine if compoundrelated effects are distinguishable from class effects. Our results show that telmisartan, but not losartan, decreased the vasoconstriction to PE and enhanced the vasodilatation to ACh. Constriction of isolated vessels to ␣-adrenergic agonists, such as PE, is well known to be enhanced in the absence of NO or functional endothelium [30,31]; consistently we observed a half-log shift to the left of concentration–contraction curve to PE in control preparations, following incubation with LNNA or endothelium removal (Fig. 3 and Table 2); in telmisartan-treated preparations, LNNA or endothelium removal restored the vasoconstriction to PE back to the level of control preparations (Fig. 3 and Table 2). Because the inhibition of constriction to PE by telmisartan was reversed by NO synthase inhibition or endothelium removal, it appears to be related to enhanced basal NO production.
Fig. 4. Effect of nitric oxide and prostanoid inhibition on vasomotor effects of telmisartan. (A and B) Nitric oxide synthase inhibition by NG -nitro-l-arginine (LNNA, 100 M); C nitric oxide synthase inhibition by LNNA + cyclooxygenase inhibition by indomethacin (Indo, 10 M); D, Endothelium removal; E, average vasodilatation to acetylcholine (ACh). Cumulative concentrations of phenylephrine (PE, 10 nM to 10 M) or ACh (1 nM to 10 M), added to the organ chamber by half log increase, are indicated by black dots on the tracing. Notice in run 2, in (A) (control telmisartan-untreated) vasodilatation to ACh is abolished by l-NNA, whereas in (B) (telmisartan-treated) some vasodilatation to ACh is still observed in the presence of LNNA. **P < 0.01 versus telmisartan + LNNA by two-way ANOVA.
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Fig. 5. Effects of telmisartan on vasomotor responses of rat femoral arteries to cumulative concentrations of serotonin (5-HT, A), acetylcholine (ACh, B) and sodium nitroprusside (SNP, C). Vasodilatation to ACh was assessed on the plateau of contraction to 1 M 5-HT. Vasodilatation to SNP was assessed on the plateau of contraction to 1 M 5-HT, in preparations were endothelial NO production had been blocked by NG -nitro-l-arginine (LNNA, 100 M). Data are mean ± SEM of 8 independent measurements. Pharmacological parameters are given in Table 3. **P < 0.01 versus control, by two-way ANOVA.
In addition to basal NO synthesis, 30-min incubation with telmisartan increased endothelium-dependent vasodilatation to ACh, again, an effect not reproduced with losartan. Worthy of note, LNNA, which effectively blocked relaxation to ACh in control (telmisartan-untreated) preparations, was unable to completely abolish relaxation to ACh in telmisartan-treated preparations. The residual relaxation to ACh (about 20%) may be related to prostanoid synthesis, enhanced by telmisartan, because it disappeared when cyclooxygenases had been blocked by indomethacin. Several compounds belonging to ARBs have been reported to exert endothelial effects [32,33] that are considered as protective or beneficial for cardiovascular diseases [34]. In general, however, the impact of ARBs on endothelial function requires in vivo chronic treatments or in vitro long-lasting (>10 h) incubation (see also below). For example, in vivo treatment with telmisartan in animal models of cardiovascular disease improves endothelial function and increases NO bioavailability, but both effects are reversed by GW9662, a PPAR␥ antagonist [35,36]. These findings have been therefore interpreted on the basis of a PPAR␥ agonist activity of telmisartan [9,10]. Consistently, PPAR␥ agonists have been shown to increase NO release, an effect that requires 24 h treatment [19], and to upregulate endothe-
lial NO synthase [37]. The effects of telmisartan or PPAR␥ activators on endothelium occur either following in vivo treatment or after several hours of in vitro incubation, consistent with the latency of PPAR␥-activated gene expression, which requires at least 2 h for significantly changing mRNAs [38]. Therefore, in our system, the short latency (30–60 min) of telmisartan effect on vasomotor responses makes unlikely PPAR␥ stimulation as the underlying mechanism; furthermore, the lack of effect of 30 M GW9662, a PPAR␥ antagonist [23] that we used at a concentration much higher than the reported IC50 s (3.8 nM [39]), rules out the involvement of PPAR␥. PPAR␥-independent effects of telmisartan on endothelium have been attributed to radical scavenging [40]; protection of endothelial function from oxidant radicals has also been repeatedly reported for losartan [41,42]; losartan, however, in our hands, did not enhance ACh-induced vasodilatation as telmisartan did, suggesting that the radical scavenging effect, supposedly exerted by both sartans, is not sufficient to produce a detectable endothelial outcome in our in vitro system. Furthermore, telmisartan did not change the vasodilatation to exogenous NO (given as SNP, Fig. 5C), indicating that a scavenging effect by telmisartan, if any, does not increase bioavailability of exogenous NO. An increased phospho-
Fig. 6. Effect of GW9662 in telmisartan-treated rat femoral arteries. Preparations were preincubated with telmisartan and with or without GW9662, before exposing to phenylephrine (PE, A) and acetylcholine (ACh, B). Data are mean ± SEM of 8 independent measurements. Pharmacological parameters are given in Table 1.
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Fig. 7. Effect of PD123319 (A and C) and A779 (B and D) in telmisartan-treated rat femoral arteries. Preparations were preincubated with telmisartan and with or without PD123319 or A779, before exposing to phenylephrine (PE) and acetylcholine (ACh). Data are mean ± SEM of 8 independent measurements. Pharmacological parameters are given in Table 1.
rylation of endothelial NO synthase (eNOS) following >7 day in vivo treatment with ARBs, in Dahl rats, has been recently reported [43,44]; because eNOS phosphorylation increases enzymatic activity and endothelium-dependent vasodilatation [45], if occurring in vitro it might be responsible for at least part of vasomotor effects of telmisartan reported here. However, at least an additional mechanism, acting on prostanoid synthesis, must be hypothesized to explain the indomethacin-sensitive vasodilation observed here in the presence of telmisartan. In this respect, an increased prostanoid release from endothelium of rat aorta has been reported following in vivo treatment with ARBs [46,47], but the precise mechanism responsible for this phenomenon remains to be determined. Local expression and/or processing of angiotensin peptides has been proposed, since long time, as an important mechanism contributing to vascular and cardiac remodeling [48], which may account for at least part of long term beneficial effects of ACE inhibitors and ARBs in patients with hypertension, heart failure, myocardial infarction [49,50]. Recently, attention has been focused on the heptapeptide AT(1-7) , which is produced mainly locally from ANGII by ACE2 [51] and stimulates a G-protein coupled receptor termed Mas [26], abundantly expressed in vascular endothelium [27]. Because either AT2 or Mas are expected to increase NO production [52–54], we hypothesized that telmisartan increased NO release in isolated femoral arteries by effectively opposing AT1 receptor and thereby increasing AT2 and/or Mas stimulation by locally produced ANGII and/or AT(1-7) . To block AT2 we used PD123319 at 10 M (IC50 30 nM [55]); to block Mas we used A779 at 30 M (IC50 20 nM [56]). None of these drugs induced any detectable change in PE-induced vasoconstriction and in AChinduced vasodilatation in the presence of telmisartan, suggesting that neither AT2 nor Mas are involved in the vasomotor effects of telmisartan in isolated arteries, as we observed after short-term incubation. Finally, we believe that the effects of a 30 M concentration of losartan or telmisartan we report in this in vitro study potentially have in vivo clinical significance; in fact, after single oral adminis-
tration of 100 mg losartan or 80 mg telmisartan in humans (doses commonly prescribed), plasma Cmax values of about 1300 ng/ml are attained for both compounds (for losartan an active metabolite is generated, with a Cmax of 650 ng/ml, similar to that of losartan itself, even though delayed) [57,58], corresponding to concentrations of approximately 3 M. Furthermore, after repeated administrations, at steady state, average plasma concentration of telmisartan in humans is 4.6 g/ml [58], i.e. about 10 M, a value compatible with the concentration used in the present study. In conclusion, telmisartan modifies constriction and dilatation of isolated arteries in an endothelium-dependent manner, involving both nitric oxide and prostanoid production. These effects cannot be considered as “class effect”, because they are not shared by losartan, the ARB prototype. Furthermore, these vasomotor effects of telmisartan, as observed after 30-min incubation, do not seem to involve PPAR␥, AT2 or AT(1-7) /Mas. Acknowledgement This work was supported by a grant from the University of Catania and by a grant from Boehringer-Ingelheim Italia S.p.A.
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