Tranilast antagonizes angiotensin II and inhibits its biological effects in vascular smooth muscle cells

atherosclerosis Atherosclerosis 121 (1996) 167-173

Tranilast antagonizes angiotensin II and inhibits its biological effects in vascular smooth muscle cells Keiji Miyazawa *, Juichi Fukuyama, Phormacolog~cal Laboratories,

Keiko Misawa, Shuichiro Hamano, Arao Ujiie

Kissei Pharmaceutical

Co. Ltd., Hotaka, Nagano 399-83, Japan

Received 7 June 1995; revision received 28 August 1995; accepted 31 August 1995

Abstract Recent studies have been reported indicating that angiotensin II may potentiate neointimal formation. In the present study, we examined the antagonistic effect of tranilast on angiotensin-II. Losartan was used as the reference compound. First, tranilast inhibited the angiotensin II-induced contraction of rabbit aortic strips in a noncompetitive manner (pD; = 3.7), whereas it had little effect on the contraction induced by noradrenaline or endothelin-1 . Second, tranilast inhibited the binding of iz51-labeled angiotensin II to angiotensin ATI receptors in rat liver membranes with an IC,, value of 289 PM. Finally, functional antagonism of tranilast (100 and 300 ,uM) was demonstrated by its from human vascular smooth muscle cells (VSMC). blockade of angiotensin II (10 - *M)-induced ?a2+-efflux However, tranilast (30-300 ,uM) exerted no influence on PDGF-induced formation of inositol triphosphates which cause an increase in [Ca2+li in human VSMC. The antagonistic activity of tranilast towards angiotensin II may be involved in part in preventing restenosis after percutaneous transluminal coronary angioplasty (PTCA). Keywords: Tranilast;

Angiotensin

II receptor; Muscle contraction;

1. Introductiou Recently, the potential role of autocrine/ paracrine mediators such as angiotensin II in the vascular wall has been suggested based on advances in our knowledge of the molecular biology of vascular physiology [l--4]. An increase in the number of angiotensin II AT1 subtype receptor

sites and elevation of the angiotensin-converting enzyme level has been demonstrated in ballooninjured arteries [5-71. Furthermore, angiotensin * Corresponding author. Tel.: + 81 263 82 8820; Fax: + 81 263 82 8826.

Restenosis; PTCA

converting enzyme (ACE) inhibitors [8] and angiotensin II receptor antagonists [9,10] prevent intimal thickening after vascular injury. Therefore, the intravascular renin-angiotensin system is considered to play a critical role in contributing to vascular hyperplasia or hypertrophy [l 1,121. Tranilast, an anti-allergic drug, has been clinically used for the treatment of not only patients with bronchial asthma, allergic rhinitis, and atopic dermatitis [13], but also those with keloids and hypertrophic scars [14]. Recently, a doubleblind, large-scale, multicenter trial demonstrated the potent effect of tranilast (600 mg/day for 3

0021-9150/96/$15.000 1996 Elsevier Science Ireland Ltd. All rights reserved SSDI 002 I .-9150(95)05709-9

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et al. 1 Atherosclerosis

months) in preventing restenosis (restenosis rate 14.7%, Tranilast 600 mg/day, n = 68, P < 0.001 vs. 46.5%, placebo, n = 71) after percutaneous transluminal coronary angioplasty (PTCA) [ 151. Also, our previous study indicated the inhibitory effects of tranilast on platelet-derived growth factor (PDGF)- and transforming growth factor-p1 (TGF-P 1)-induced collagen synthesis, migration, and proliferation in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) [16]. The aim of the present study was to evaluate the effect of tranilast on angiotensin AT1 receptor antagonism. We further determined the functional effect of the antagonist on angiotensin II-induced Ca2+ -efflux which reflects an increase in [Ca* +]iin human VSMC. 2. Materials and methods 2.1. Materials

anTranilast, N-(3,4-dimethoxycinnamoyl) thranilic acid, and losartan (Dup753) were synthesized in our laboratories. Fetal bovine serum (FBS) was purchased from Gibco Laboratories (Grand Island, NY). Angiotensin II was obtained from Peptide Research Laboratories (Osaka, Japan). ‘251-labeled angiotensin II (human) and 45Ca-CaC1, were obtained from Du Pont-NEN (Boston, MA). 2.2. Rabbit aorta contraction studies

The thoracic aorta from Japanese White male rabbits (2-3 kg) (SLC Japan Inc., Shizuoka) was isolated and cut into helical strips 3-4 mm wide and 45 mm long. The strips were mounted in lo-ml Magnus’ baths containing Krebs-Henseleit solution (118 mM NaCl, 4.7 mM KCl, 1.2 mM KH,PO,, 1.2 mM MgS04.7H,0, 2.5 mM CaCl,, 25 mM NaHCO,, 11 mM glucose) under a resting tension of 1 g. The Krebs-Henseleit solution was kept at 37°C and bubbled continuously with 5% CO, in oxygen. A control cumulative concentration-response curve for angiotensin II (lo- *O10-’ M) was obtained for each strip. Tranilast or

121 (1996) 167-173

losartan was added 15 min prior to a challenge with angiotensin II. The analog contractile signal was recorded with a force-displacement transducer (NEC San-ei Instruments, Tokyo, Japan) connected to a polygraph (NEC San-ei Instruments) via a strain amplifier (NEC San-ei Instruments). Data were expressed as a percentage of the angiotensin II (3 x 10- 8 M)-induced maximum response. Similarly, contraction studies for noradrenaline (3 x 10-9-10-4 M) and endothelin-1 (3 x lo-“-3 x lop8 M) were performed as described above. Data for noradrenaline and endothelin-1 were expressedas a percentage of the noradrenaline (3 x 10- 5 M)-induced maximum response. 2.3. Angiotensin II binding assay

Male Sprague-Dawley rats (250-350 g) were obtained from SLC Japan. Angiotensin II receptors from rat liver plasma membrane were prepared according to the method described in [17], with slight modifications. The livers were homogenized in 3 volumes of ice-cold homogenizing buffer (50 mM Tris, 5 mM EDTA, 0.1 mM PMSF; pH 7.4) in an Ultradisperser (Yamato Scientific Co., Tokyo) and Astrason ultrasonic processor (Wakenyaku Co., Kyoto, Japan) and centrifuged at 1000 x g for 10 min at 4°C. The supernatant was filtered through a nylon membrane and centrifuged at 48 000 x g for 20 min at 4°C. The pellet was resuspended in homogenizing buffer by use of the Astrason ultrasonic processor and then centrifuged as before. The final pellet was resuspendedin Tris buffer (50 mM Tris, 5 mM MgCl,; pH 7.4) at a protein concentration of 85 mg/ml and stored at - 80°C until required. Binding assays were performed by incubating the membranes (0.1 mg protein/tube) in 150 ~1 of assay buffer (50 mM Tris, 5 mM MgC&, 0.25% BSA; pH 7.4) with 50 pM lz51-labeledangiotensin II in the presence or absenceof tranilast or losartan. After incubation for 120 min at room temperature, bound and free radioactivity were separated by rapid filtration through Whatman GF/B glass fiber filters. The filters were washed with ice-cold washing buffer (50 mM Tris, 5 mM MgCl,), and the trapped radioactivity was mea-

K. Miyazawa

et al. 1 Atherosclerosis

sured with ;I y-counter. Specific binding was defined as tha!t displaceable by 10 ,uM angiotensin II. In saturation experiments, rat liver membranes were incubateld with each concentration of 1251-labeled angiotensin 11 (0.02-l nM). Bound radioactivity, in the presence or absence of 10 PM angiotensin II for each concentration of 1251-labeled angiotensin II, was determined by filtration as described above. For determination of the effects of tranilast on saturation binding, membranes were incubated with 300 PM tranilast and ‘251-labeledangiotensin II. 2.4. Cell cultwe

Human aortic smooth muscle cells at the fourth passage in culture were provided by Sanko-junyaku (Tokyo). Confluent VSMC were subcultured at 1:5 split ratio in DMEM supplemented with 10% FBS. WMC were used within passages 510, and were characterized as smooth muscle by morphologic criteria and by expression of smooth muscle a-actin (clone lA4; Dako Japan Co., Kyoto) [18].. The cells were negative in mycoplasma assays. 2.5. Measurement of 45Ca.‘i eflux The measurement of 45Ca2+ efflux was performed as des’cribed by Chiu et al. [19]. Briefly, human VSMC were grown to confluence in 6-well tissue culture ,dishes,with each well containing 2 ml of DMEM plus 10% FBS. One milliliter of fresh DMEM containing 10% FBS plus 1 PCi of 45Ca2+ was added to each well on the day before experiment. Tranilast or losartan was added 20 min before the start of the assay. For initiation of the assay, culture dishes were washed 3 times in balanced salt solution (BSS) containing 130 mM NaCl, 5 mM KCl, 1 mM MgCl,, 1.5 mM CaCl, (buffered to pH 7.4 with Tris base), 10 mM glucose, and 1 mg/ml BSA. After the third wash, tranilast or losartan was again added to the cells in 1 ml of DMEM containing glucose and BSA. The dishes were then placed in 37°C water bath and angiotensin II (10 - ’ M) was added to initiate the efflux. The assaywas terminated after 150 s by

121 (1996) 167-I 73

169

washing the cells rapidly 4 times in ice-cold BSS containing LaCl, instead of CaCl,. Cell-associated 45Ca2+ was determined by solubilizing the cells with 1 ml of 0.1 N nitric acid and counting the radioactivity. 2.6. Measurement of inositol triphosphate (IP,) A suspension of human VSMC in DMEM containing 1 mg/ml BSA was warmed at 37°C for 10 min. Then PDGF-BB (50 rig/ml) was added and the mixture was promptly vortexed. After 20 s, 0.2 volume of ice-cold 20% perchloric acid was added and the mixture was kept on ice for 20 min. Protein was removed by centrifugation at 2000 x g for 15 min at 4”C, and the supernatant was titrated to pH 7.5 with 10 N KOH and kept on ice for 60 min. After removal of insoluble KCIO, by centrifugation, IP, was measured with a radioimmunoassay kit (TRKlOOO, Amersham, UK). 2.7. Data analysis Data are shown as means +_ S.E. Statistical analysis was performed by ANOVA and Scheffe’s F test on a Stat View 4.0 software program (Abacus Concepts, Berkeley, CA). A P-value of < 0.05 was considered to be significant.

3. Results

3.1. Efect of tranilast on agonist-induced contraction of rabbit aorta The antagonistic effect of tranilast on angiotensin II-induced vasoconstriction was evaluated in isolated rabbit aortic strips. Tranilast (30-300 PM) concentration-dependently covered the concentration-response curve for angiotensin II, indicating that the mode of inhibition is noncompetitive (pD2 = 3.7) (Fig. 1A). However, tranilast had little effect on the contraction-response curves induced by noradrenaline and endothelin-1 (Fig. 2). On the other hand, losartan (100 nM) shifted to the right the concentrationresponse curve for angiotensin II (Fig. 1B).

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3.2. Efect of tranilast binding to angiotensin II receptor

121 (1996) 167-l 73 120

1

100

The ability of tranilast to interact with angiotensin II type 1 receptors in rat liver membranes was evaluated. As shown in Fig. 3, both tranilast and losartan inhibited the binding of ‘251-labeled angiotensin II to high-affinity angiotensin II receptors in rat liver membranes, with IC,, values of 289 PM and 11 nM, respectively. To establish the characteristics of the antagonism exerted by tranilast, we determined its effect on the saturation curve of 1251-labeledangiotensin II binding. The results for tranilast (300 ,uM) are

A ‘2

*

Control

80

El ‘e g

60

Control 30 pM Tranilast 100fiM

x

s

20 0

400I 1 1@10 lo-9

10-s

10-7

IO.5

10-5

l&4

Endothelin or Noradrenaline (M) Fig. 2. Effects of tranilast on the concentration-contractile response curves to noradrenaline and endothelin-1 in isolated helical strips of the rabbit aorta. Data are shown as means _+ S.E. of 4-8 experiments.

shown in Fig. 4 in the form of a Scatchard analysis. Tranilast (300 PM) decreased the total number of binding sites B,,, from 105 fmol/mg protein to 82 fmol/mg protein and increased the dissociation constant Kd of 1251-labeled angiotensin II binding from 0.37 nM to 0.61 nM.

100 ‘;; @ 80

0’

St

* t -

3

'3 60 L

3.3. Effect of tranilast on angiotensin II-induced 4’Ca2+ eflux in human VSMC To determine whether tranilast was in fact a functional antagonist, we examined its inhibition

1 o-s :‘-6 Angiotensin

B

II (M)

120100-

-et

g 80E 'fi 60!! 5 40-

1w

10-a Angiotensin

1 o-7

1W

II (M)

Fig. 1. Effects of tranilast (A) and losartan (B) on the concentration-response curves for angiotensin II in isolated helical strips of the rabbit aorta. Data are shown as means + S.E. of 4-5 experiments.

Control 30

160 360 1000 Tranilast (FM)

i

ib

160

Losartan (nM)

Fig. 3. Inhibitory effects of tranilast and losartan on the specific binding of 1251-labeledangiotensin II (50 PM) to rat liver membranes. Data are shown as means k SE. of 4 experiments. **P < 0.01, **P < 0.001 as compared with the control.

K. Miyazawa

0.25

121 (1996) 167- 173

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4. Discussion

1

0 Control 0

0

et al. / Atherosclerosis

20

40

Tranilast (300 PM)

60

80

100

120

Bound ( fmol / mg protein ) Fig. 4. Scatchard analysis of the effect of tranilast on the binding characteristics of ‘251-labeledangiotensin II in rat liver membranes. Sym’Sols represent means of two determinations. Specific binding of ‘251-labeled angiotensin II in control (Kd 0.37 nM, B,,,,, 105 fmol/mg protein) and that in the presence of 300 PM tranilast (K,, 0.61 nM, B,,, 82 fmoljmg protein)

of angiotensin II-induced 45Ca2+ efflux, an event subsequent to receptor activation, from human VSMC. In human VSMC, angiotensin II produced a submaximal efflux at IO- ’ M (data not shown). Angiotensin II (10 - ’ M)-induced response was blocked effectively by tranilast (100 and 300 PM) in a concentration-dependent manner (Fig. 5). Similarly, losartan (10 and 100 nM) exerted an effective blockade of angiotensin II-induced 45(Ya2 i + efflux (Fig. 5).

In this study, we investigated whether tranilast has an antagonistic effect on angiotensin AT1 receptors. Tranilast antagonized angiotensin II-induced contraction in rabbit isolated aortic strips competed with ‘251-labeled angiotensin II for binding sites in rat liver membrane, and blocked angiotensin II-induced 45Ca2+ efflux in human VSMC. These preparations were chosen because it is well established that the angiotensin II receptors present in them belong to the angiotensin AT1 subtype [20-241, that AT1 receptors are the only subtype that, thus far, are known to mediate the major physiological effects of angiotensin II, and that there is little significant speciesdifference in the ATI receptor structure and its signal transduction pathway [25-271. Losartan caused a parallel displacement to the right without significantly affecting the maximum response to angiotensin II in rabbit aortic strips. As shown by several investigators [25], losartan was a potent competitive, surmountable antagonist of angiotensin AT1 receptors. In contrast, tranilast caused a concentration-dependent shift to the right and lowered the concentration-response curve to angiotensin II in rabbit aortic strips. The 50% suppression of the maximum response of angiotensin II was caused by 212 ,uM 1251

3.4. Effect of lranilast on PDGF-BB-induced inositol tripho.sphate (IP,) .formation in human VSMC The effect of tranilast on PDGF-BB (50 ng/ml)induced inositsl triphosphate (IP,) formation, a PDGF-BB-induced early response event, in human VSMC was examined. Ten seconds after the PDGF-BB (50 rig/ml) stimulation, the IP, level was significantly elevated from 9.7 pmol/5 x lo5 cells to 15.8 pmol/5 x lo5 cells. However, there was no inhibitory effect of tranilast (30-300 PM) on this PDGF-BB-induced IP, formation.

0

30

100

300

Tranilast (PM)

1

1-o

100

Losartan (nM)

Fig. 5. Effects of tranilast and losartan on angiotensin II (10 nM)-induced 45Ca2+ efflux in human VSMC. Data are shown as means F SE. of 4 experiments. *P < 0.05, **P < 0.01, ***P < 0.001 as compared with the control.

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tranilast (pD; = 3.7). From these results, we conclude tranilast to be a weak, non-competitive, insurmountable antagonist of angiotensin AT1 receptors. Several theories have been proposed to explain insurmountable but saturable antagonism [28]. However, the mechanism responsible for the suppression of the maximum response to angiotensin II remains unclear. The antagonistic profile of tranilast in vitro suggeststhat the drug might be a nonspecific antagonist, which implies that it blocks the cascade of events leading to signal transduction. However, the following experimental facts suggest that this is unlikely: tranilast did not affect contractile responses induced by endothelin or noradrenaline in rabbit aortic strips. This indicates that tranilast does not interfere with the signal transduction process that mediates the contraction in response to angiotensin II, since angiotensin II [29], endothelin [30] and noradrenaline [31] all induce VSMC contraction by receptor-mediated IP, formation leading to increased [Ca2+&. Moreover, the specificity of action of tranilast is supported by the finding that tranilast had no effect on the PDGF-induced IP, formation, which increases [Ca2+] in human VSMC. Therefore, these data suggest that the site of action of tranilast is the angiotensin AT1 receptor macromolecule or a closely related site. The results obtained from the radioligand binding studies support those from the contraction studies using rabbit aortic strips. In rat liver membranes, the IC,, for tranilast was 289 PM, and the nature of the binding of tranilast to angiotensin ATI receptors was characteristic of neither a simple nor a competitive antagonist. This indicates a mode of action of tranilast similar to that shown in rabbit aorta as described above. Thus, the effective concentrations of tranilast on antagonism of angiotensin AT1 receptors are higher than those of losartan. Furthermore, tranilast inhibited the angiotensin II-induced 45Ca2+ efflux, which reflects an increase in [Ca2‘1 in human VSMC. In human VSMC, it is known that 1251-labeledangiotensin II essentially binds to angiotensin AT1 receptors [24] and that angiotensin II induces an increase in [Ca2+] that is mediated through angiotensin AT1 subtype receptors [24]. Therefore,

121 (1996) 167-l 73

tranilast may be functionally effective towards antagonism of angiotensin AT1 receptors in humans as well as in rabbits and rats. On the other hand, our previous studies showed that tranilast, at concentrations of more than 30 PM, inhibited PDGF-induced migration and proliferation of VSMC from SHR [16]. In the present investigations, tranilast, at concentrations greater than 100 ,uM, antagonized angiotensin II at the angiotensin AT1 receptor level. However, tranilast concentrations of more than 100 ,uM are attainable in plasma during therapeutic dosing by oral administration of 600 mg/day tranilast. These observations suggest that tranilast may prevent restenosis after PTCA mainly through its inhibitory effect on PDGF-induced migration and proliferation, but also that the antagonistic effect of tranilast toward angiotensin II may be involved in part. In conclusion, the present studies show that tranilast is a weak angiotensin AT1 receptor antagonist. Moreover, we demonstrated that tranilast, at the physiologically relevant concentrations, functionally acts on human VSMC as an angiotensin AT1 receptor antagonist. The antagonistic activity of tranilast towards angiotensin II may be associated in part with preventing the development of restenosis after PTCA. References PI Dzau VJ. Vascular renin-angiotensin: a possible au-

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