Role of endothelin ETB receptors in bronchoconstrictor and vasoconstrictor responses in guinea pigs

European Journal of Pharmacology, 233 (1993) 47-51

47

© 1993 Elsevier Science Publishers B V All rights reserved 0014-2999/93/$06 00

EJP 52964

Role of endothelin ET B receptors in bronchoconstrictor and vasoconstrictor responses in guinea-pigs Kazuh~to N o g u c h l , Yuk~ko N o g u c h l , H l r o y a s u H t r o s e , M a s a r u N~shlklbe, M a s a k l I h a r a , Klyofuml Ishlkawa and Mltsuo Yano New Drug Discovery Research Laboratories, Tsukuba Research Institute, Banyu Pharmaceutical Co, Ltd , Tsukuba Techno-Park Oho, Okubo 3, Tsukuba 300-33. Japan

Recewed 13 August 1992, revised MS received 27 November1992, accepted 15 December 1992

In anesthetized and ventilated guinea-pigs, intravenous rejections of endothehn (ET)-I (0 5 nmol/kg), ET-3 (0 5 nmol/kg), and [Alal'a'xl'lS]ET-1 (20 nmol/kg), an ETa-selectwe receptor agomst, reduced bronchoconstrlctor and transient vasoconstrictor responses Only the ET-l-mduced transient vasoconstrlctmn was followed by a secondary sustained pressor response The ETA-selectwe receptor antagomst, BQ-123 (1 mg/kg 1v ), attenuated only the sustained pressor response These results m&cate that the bronchoconstnctor and transient vasoconstrictor responses to endothehns m gumea-p~gs are medmted by ET a receptors, whdst the sustained pressor response is medmted by ETA receptors The thromboxane A 2 receptor antagomst, L-670,596 (0 5 mg/kg, i v ) and a high dose of BQ-123 (30 mg/kg i v ) abohshed the bronchoconstncUon only without affecting the transient pressor response to endothehn lsopeptldes These results suggest that the ETa-medmted bronchoconstnct~on depends on thromboxane A 2 formation The different sensitivity of these ETB-medmted transient responses to BQ-123 suggests the possible exastence of distinct ET a receptor subtypes

Endothehn, [Ala 1'3'1115]endothehn-1, BQ-123, Bronchoconstnctlon, Hypertension, (Receptor subtype)

1. Introduction Endothehn (ET), originally identified in the culture media of porcine aortic endothelial cells, is one of the most potent vasoconstrictor substances discovered so far (Yanaglsawa et al, 1988) In humans and other species the endothehns are a family of three lsopeptides, termed ET-1, ET-2, ET-3 (Inoue et al, 1989, Matsumoto et al, 1989) Endothehns exhibit various biological actions via at least two distinct E T receptors, namely the E T A and ET a receptors ET A is highly sensltwe to ET-1 and ET-2, whereas ET B is highly sensitive to all endotheln tsopepUdes E T A (AraI et al, 1990) and ET a (Sakurai et al, 1990) were first cloned from the c D N A hbrarles of bovine and rat lungs, respectwely Recently, we demonstrated the different distribution of ET A and E T B In porcine pulmonary tissues, using specific hgands for ET A and E T a (Nakamlchl et al., 1992) However, the functional roles of the two distinct ET receptors m the lung remain to be elucidated

Correspondence to K Nogucht,Department of Pharmacology,New Drug DiscoveryResearch Laboratories, Tsukuba Research Institute, Banyu Pharmaceutical Co, Ltd, Tsukuba Techno-Park Oho, Okubo 3, Tsukuba 300-33, Japan Tel 81 298 77 2000, fax 81 298 77 2028

In guinea-pigs, an Intravenous (1 V ) rejection of ET-1 ehclts vasoconstrictor responses accompanied by bronchoconstrlctlon (Whittle et al, 1989, Macquin-Mavler et al, 1989, Touvay et al, 1990) ET-1, ET-2 and ET-3 have a similar binding affinity for lung membranes in vitro and are nearly equipotent in inducing transient broncho- and vasoconstrictor responses in vwo (Pons et al, 1991a) These findings suggest that ET B receptors are Involved, at least in part, in the responses of guinea-pigs to endothehns The present study was designed to identify functional ET receptor subtypes in the guinea-pig, using selective ET A and ET B hgands We demonstrate here that two distinct ET receptors are responsible for the different responses to ET observed in guinea-pigs, ET A receptors mediate the sustained pressor response, whilst ET a receptors mediate the initial transient bronchoconstrlctor and vasoconstrictor responses

2. Materials and methods 2 1 Assessment o f bronchoconstnctor and vasoconstrictor responses

Male Hartley guinea-pigs (380-400 g) were anesthetized with urethane (750 m g / k g 1 p ) and a-chlo-

48

1°°t

ET-1

[Ala1'Za1'lS]ET - I

ET-3

m

~.~ ¢:: ~

l.k---

30 20

cO

-10

~

i

E

0

10

20

0

10

J 20

J

0

10

20

Ttme after admlnmtratlon (mm) Fig 1 Changes m bronchoconstnctor response and m e a n arterml blood pressure (MAP) after ~v rejection of ET-1 (0 5 n m o l / k g ) , ET-3 (0 5 n m o l / k g ) and [Ala 1'3 n 15lET_ 1 (20 n m o l / k g ) m guinea-pigs The values are m e a n s ± S E for five animals

ralose (37 5 mg/kg i p ) A catheter was inserted into the right carotid artery for measurement of systemic blood pressure (Polygraph RM-6000, Nlhon Kohden, Japan), and another catheter was inserted into the left jugular vein for drug admmlstratlon The ammals were connected to a respirator (Model 7025, Ugo Basde, Italy) through a tracheal cannula and ventilated at 10 ml alr/kg, 60 strokes/mln Spontaneous breathing was suppressed with succmylchohne (5 mg/kg s c ) Pulmonary inflation pressure was recorded w~th a bronchospasm transducer (Model 7020, Ugo Basde, Italy) connected to a s~de arm of the tracheal cannula, according to the method of Konzett Rossler (Konzett et al, 1940) At the end of the experiment the tracheal cannula was clamped to obtain a maximum pulmonary inflation pressure reading, and the bronchoconstnctor responses are expressed as a percentage of this max~mum

1), BQ-123 (cyclo(-D-Trp-D-Asp-L-Pro-D-Val-L-Leu-)) and nlcardlplne were synthesized in our laboratories L-670,596 ((-)6,8-dlfluoro-9-p-methylsulfonyl benzyl1,2,3,4-tetra-hydrocarbazol-l-yl-acetlc acid) was a gift from Merck Frosst (Montreal, Canada) Endothehn lsopeptldes were dissolved in 001 M phosphatebuffered saline (pH 7 4) containing 0 05% bovine serum albumin Indomethacln (Sigma, US) was dissolved m 3% NaHCO 3 Nlcardlpme and U-46619 were dissolved m 0 1 and 0 02% ethanol sahne, respectively

2 2 Drugs

3 1 Bronchoconstrtctor and vasoconstrictor responses to ET-1, ET-3 and [Ala1'3"11'15]ET-1

ET-1 and ET-3 were purchased from the Peptlde InsUtute Inc (Osaka, Japan) and U-46619 was purchased from Iwal Chemicals (Tokyo, Japan) A linear and alanyl substituted ET-1 analogue ([Alal'3'lt'IS]ET -

2 3 Stattsttcal analysts Statistical analysis was done with Mann-Wh~tney's U-test

3. Results

A bolus intravenous 0 v ) injection of ET-I, ET-3 (both 0 5 nmol/kg) or [AIaI'3m'IS]ET-1 (20 nmol/kg) reduced bronchoconstrlctlon and increased mean arte-

TABLE 1 Effect of BQ-123 on ET-1- and ET-3-evoked bronchoconstrlctor and transient vasoconsmctor responses m guinea-pigs T h e values are the m e a n s ± S E for five animals BQ-123 was gwen i v 5 m m before ET-1 or ET-3 (0 5 n m o l / k g 1 v ) Drug

Control BQ-123

Dose

ET-1

(mg/kg)

Bronchoconstrlctor response (%)

MAP (mmHg)

Bronchoconstnctor response (%)

MAP (mmHg)

86±21 86±69 77±66 62±48 a 0±00 b

30±35 28±32 30±47 32±34 29±49

86±19 84±27 76±54 13±57 b 0±00 b

25±43 25±30 24±40 25±24 20±32

10 30 100 300

Significant difference from the control, a p < 0 05 or b p < 0 01

ET-3

49

"~ .IE

100

40

< o

o

50 x

0

o

x

m

~

2'0

1;0

o

2'o

5;0

2'o

Ioo

soo

Dose ( p m o l / k g )

Fig 2 Dose-response curves of ET-1 (o) and ET-3 (e) for bronchoconstnctlon (left panel), transient vasoconstriction (middle panel), and the sustained pressor response (right panel) Pressor responses are expressed as the values obtained after subtraction of the average change (6 3 mmHg) in vehicle (0 1% BSA PBS)-treated animals from the maximum change in MAP in ET-1- or ET-3-treated animals The values are means 5: S E for five animals The values are means 5: S E for five animals

rml blood pressure (MAP) m anesthettzed gumea pigs (fig 1) The three lsopeptldes caused transient mcreases m the bronchoconstrlctor response and MAP A secondary sustamed mcrease m MAP was observed with ET-1 only (figs 1, 2) ET-1 at doses m excess of 1 nmol/kg i v was lethal (LD100 = 2 nmol/kg) In the range of 0.02-0 5 nmol/kg 1 v , ET-1 and ET-3 were eqmpotent m reducing transxent bronchoconstncUon and vasoconstriction (fig 2)

[Alal'3jlaS]ET-1 evoked transient bronchoconstrlctor and pressor responses at doses higher than 0 5 nmol/kg i v , the responses bemg mayamal at 20 nmol/kg Even at a higher dose (50 nmol/kg), [Alal'a'n'15]ET-1 &d not ehclt a sustamed pressor response [Alal'3'nJS]ET-1 was approxamately 40 Umes less potent than either ET-1 and ET-3 m ellotmg the mmal transient responses (fig 1)

32 Effects of BQ-123 and L-670,596 on the bronchoconstrtctor and vasoconstrictor responses to ET-1, ET-3 and [Aial'3"ll"lS]ET-1

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. 10

15

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Pretreatment with BQ-123 (1 m g / k g 1.v ) 5 mm before ET-1 mjecUon s~gmficantly attenuated the sustained pressor response to ET-1 without affectmg the transient bronchoconstnctor and vasoconstrictor responses (fig 3) Higher doses of BQ-123 (10-30 mg/kg) antagonized the bronchoconstnetor, but not the transient vasoconstrictor, response to ET-1, ET-3 and [Alal'3'n'15]ET-1 (tables 1, 2) The thromboxane A 2 (TXA 2) mlmeuc, U-46619 (1 /~g/kg iv.), reduced transient bronchoconstnctor and vasoconstrictor responses, and these responses were TABLE 2

60

Effects of L-670,596 and BQ-123 on [Alal'3'naS]ET-l-evoked bronchoconstrletor and pressor responses m guinea-pigs

P=

The values are means+S E for five animals L-670,596 and BQ-123 were gwen i v 5 mm before [Ala1'3,1115]ET-1 (20 nmol/kg)

g 20

Drug o 0

5

10

15

20

Dose (mg/kg)

T,me after ET-1 admin,stratlon (re,n)

Fig 3 Effects of BQ-123 on ET-l-evoked bronchoeonstnctlon, transient vasoconstriction, and the sustained pressor response in guineapigs BQ-123 (1 mg/kg e) and the vehicle (o) were given 1v 5 nun before ET-1 rejection (0 5 nmol/kg) The values are means + S E for five animals

Maximal increase m bronchoconstnctor response (%)

Maximal change m MAP

(mmHg) Control ~6~,5~ BQ-I~

05 300

~±39 6±24 b 2±05 b

bSlgmficant&fferen~ ~ o m t h e ~ n t r o l ( P < 0 0 1 )

30±21 27±24 29±21

50 ET-3

ET-1

U-46619 4O

100

• ~

80

b ~,

6o

E = ~o

40

0

20

~L

n

r

i

30

E

20

~ ~

lo

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13 Cont 0 0 5

0 1

0 2

}

0 5

Cont 0 0 5

0 1

0 2

F L-670,596

L-670,596

0 5

Cont

0 05 }

0 1

0 2

0 5 (mg / kg) I

L-670,596

Fig 4 Effects of L-670,596 on ET-1 (left panel)-, ET-3 (middle panel)- and U-46619 (right panel)-evoked bronchoconstncUon and transient vasoconstrictor responses m guinea-pigs Closed and open columns represent bronchoconstnctor and vasoconstrictor responses, respectwely The values are m e a n s + S E for fwe ammals

dose dependently inhibited by the TXA 2 receptor antagomst, L-670,596 (0 05-0 5 m g / k g ) (fig 4) L-670,596 also inhibited the transient bronchoconstrlCtlOn elicited by ET-I, ET-3 (0 5 n m o l / k g ) or [Ala 13'11'lS]ET-1 (20 n m o l / k g ) In contrast, the transient vasoconstrictor response was not affected by L-670-596 (fig 4, table 2)

4. Discussion Endothehn lsopeptldes exert their physiological functions by Interacting with their speofic receptors, E T A, E T a or with other unknown subtypes We have shown previously that the pressor response to ET-1 in rats is mediated by E T A receptors, since it was selectively inhibited by the E T A receptor antagonists, BE18257B (50 m g / k g I p ) and BQ-123 (1 m g / k g i v ), in vivo (Ihara et al, 1991, 1992) The present study revealed that E T A medmtes, at least in part, the pressor response to ET-1 in the guinea-pig, because the sustained pressor response was evoked by ET-1, but not ET-3 (0 5 nmol/kg), and was attenuated by BQ-123 (1 m g / k g i v ) BQ-123, however, had no significant effect on the transient vasoconstrictor or bronchoconstrlctor response to the endothehn isopeptldes [Ala1'3'11'X5]ET-1 is a potent and selective E T a receptor agonlst (Hiley et al, 1991, Saekl et al, 1991) with a more than 1700-fold higher bmdlng affinity for E T B receptors than for ET A receptors [Ala 1'3'1115]ET-1 also evokes endothehum-dependent vasorelaxation in isolated porcine pulmonary arteries, which suggests that the vasorelaxatlon is an ETa-mediated response (Saekl et al, 1991) In the present study, [Ala l'3,I115]ET-1 (20 n m o l / k g i v ) evoked transient vasoconstrictor and bronehoconstrlctor responses in guinea-pigs ET-3 (0 02-0 5 n m o l / k g i v ) was approximately equipotent with ET-1 in inducing the initial transient responses These transient responses were resistant to a low dose (1 m g / k g i v ) of BQ-123 Taken together, these results Indicate that E T a receptors are largely

responsible for the initial transient responses to endothehns in guinea-pigs The bronchoconstrlctor response to ET-1 (l v ) in guinea-pigs is reported to be mediated by cyclooxygenase metabohtes (Payne and Whittle, 1988, Braquet et al, 1989, Macqmn-Mavler et al, 1989, Touvay et al, 1990) The present study reveals that cyclooxygenase metabohtes are also involved in the bronchoconstrlctor response to ET-3 and [Ala 13,1115]ET_1, because bronchoconstrlctlon was inhibited by L-670,596, a thromboxane A2/prostaglandln endoperoxlde receptor antagonist (Ford-Hutchinson et al, 1989) In isolated and perfused lungs, lntra-arterlally injected ET-1 increased pulmonary inflation pressure accompanied by the release of thromboxane A 2 Into the perfusate, both effects being inhibited by lndomethacln (Pons et al, 1991b) Taken together, these findings suggest that the bronchoconstrlctor response to systemic administration of endothehns is mediated by the release of thromboxane A z into the pulmonary orculatlon In contrast to the bronchoconstrlctor response, the initial transient pressor response to either ET-1, ET-3 or [Ala1311'15]ET-1 was not affected by L-670,596, which suggest that a mechanism(s) Independent of thromboxane A2 formation is involved because the pressor response to U-46619 was inhibited by L-670,596 tn the same dose range This result is supported by the observation that lndomethacln does not have a significant effect on the pressor responses to ET-1, ET-2 and ET-3 in guinea-pigs (Pons et al, 1991a) Thus, the pressor responses to the endothehn lsopeptides are clearly dissociated from the bronchoconstnctor effects in guinea-pigs Besides L-670,596, higher doses of BQ-123 (10-30 m g / k g i v ) also inhibited the bronchoconstrictor responses, but not the lnltml transient vasoconstriction, ehclted by ET-1, ET-3 or [AIa1'a'll'15]ET-1 Treatment of guinea-pigs with BQ-123 in doses up to 50 m g / k g (i v ) did not inhibit the bronchoconstrictor response to either histamine, acetylchohne, U-46619, PAF or

51 l e u k o t r l e n e D 4 in VlVO (data n o t shown) C o n s e q u e n t l y , it is u n h k e l y that a nonspeclfiC action of BQ-123 Is responsible for the l n h l b m o n of e n d o t h e h n lSOp e p t l d e - e h c l t e d b r o n c h o c o n s t r l c t l o n T h e different sensltwlty of E T B - m e d l a t e d responses to BQ-123 suggests the possible extstence of at least two E T B receptor subtypes, o n e of which is sensltwe to BQ-123 I n d e e d , high c o n c e n t r a t i o n s of BQ-123 inhibit [1251] ET-1 b i n d i n g to E T a - r i c h p o r c i n e c e r e b e l l u m m e m b r a n e s (IC50 1 8 / x M ) ( I h a r a et a l , 1992) Moreover, it has b e e n suggested that a high-affinity site for [Alal'3'xl'lS]ET-1 in p o r c i n e l u n g tissue m e m b r a n e s is also a low-affimty site for BQ-123 (Nakamlchl et a l , 1992) Thus, it is c o n c e w a b l e that a BQ-123-sensttwe E T a r e c e p t o r subtype may exast in the g u i n e a - p i g pulm o n a r y system However, th~s site n e e d s to b e characterized, using specific E T a r e c e p t o r antagomsts, to s u b s t a n t m t e this posslbdlty T h e p r e s e n t study estabhshes that E T B receptors are r e s p o n s i b l e for e n d o t h e h n l s o p e p t l d e - e h c l t e d b r o n choconstnct~on a n d the mltlal t r a n s i e n t vasoconstrictor r e s p o n s e m g u i n e a - p i g s Moreover, the secondary long-lasting vascular effect of ET-1 seems to b e m a i n l y m e d i a t e d by E T A receptors C o n s e q u e n t l y , the results of this study hlghhght the fact that the e n d o t h e h n famdy has m u l t i p l e actions by sUmulatmg different E T r e c e p t o r subtypes T h e d e v e l o p m e n t of specific E T B r e c e p t o r a n t a g o m s t s a n d f u r t h e r c h a r a c t e r i z a t i o n of E T r e c e p t o r subtypes is essentml to elucidate the pathophyslologlcal role(s) of the e n d o t h e h n s

Acknowledgements We thank Dr A W Ford-Hutchinson (Merck Frosst, Canada) for the generous gift of L-670,596 and his helpful discussion We also thank Dr I W Rodger (Merck Frosst, Canada) for his crmcai reading of the manuscript

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