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In vivo potency and selectivity of the first β3-adrenoceptor antagonist, SR 59230A, in rats
USE O F THE OPERATIONAL M O D E L O F AGONISM TO DESCRIBE THE oq-ADRENOCEPTOR ALTERED VAS DEFERENS RESPONSIVENESS O F SPONTANEOUSLY HYPERTENSIVE RATS. A Badia,J. Giraldo I and NM Vivas. Dept de Farmacologia and ~Lab. Med. Computacional. Univ Autbnoma de Barcelona, Spain
CLONING AND TISSUE DISTRIBUTION OF THE MOUSE ALPHAIB-ADRENERGIC RECEPTOR. Cavalli A., M o s t a r d i n i M. a n d C o t e c c h i a S. - I n s t i t u t de P h a r m a c o l o g i e e t Toxicologie, Rue d u B u g n o n 27, 1005 Lausanne, Switzerland.
E p i n e p h r i n e a n d n o r e p i n e p h r i n e elicit a variety of f u n c t i o n a l effects b y b i n d i n g to d i f f e r e n t a d r e n e r g i c r e c e p t o r s (AR) w h i c h b e l o n g to the s u p e r f a m i l y of Gp r o t e i n - c o u p l e d receptors. T h r e e m a j o r classes of ARs h a v e b e e n d e s c r i b e d (~1, ix2 a n d I~) a n d each clas s is f o r m e d by d i s t i n c t r e c e p t o r subtypes. T h r e e cd-AR s u b t y p e s (cdA, etlB, a l C ) h a v e b e e n i d e n t i f i e d in d i f f e r e n t species i n c l u d i n g r a t a n d m a n . On the o t h e r h a n d , v e r y little is k n o w n a b o u t ~ I - A R h e t e r o g e n e i t y in t h e m o u s e . T h u s , we h a v e u n d e r t a k e n the c l o n i n g a n d c h a r a c t e r i z a t i o n of d i f f e r e n t ~I-AR s u b t y p e s in the mouse. A cDNA e n c o d i n g the m o u s e cdB-AR was i s o l a t e d f r o m a cDNA l i b r a r y d e r i v e d f r o m m o u s e B a l b / c brain. C o m p a r i s o n of its d e d u c e d a m i n o acid s e q u e n c e w i t h t h a t o f the previously c l o n e d h a m s t e r cDNA s h o w e d 98% identity. The m o u s e ctlB-AR expressed in COS-7 cells r e v e a l e d p h a r m a c o l o g i c a l a n d s i g n a l - t r a n s d u c t i o n p r o p e r t i e s similar to those of the h a m s t e r receptor. We also isolated a g e n o m i c c l o n e c o n t a i n i n g t h e first exon of the r e c e p t o r , w h i c h was i n t e r r u p t e d b y a n i n t r o n at n u c l e o d d e 9 4 8 of its o p e n r e a d i n g frame. The position of this i n t r o n is identical to t h a t previously r e p o r t e d for the h a m s t e r a n d h u m a n ~IB-AR genes. The mRNA d i s t r i b u t i o n of the t h r e e cd-AR s u b t y p e s was assessed in selected mouse tissues by N o t h e r n analysis as well as RT-PCR. Our findings i n d i c a t e t h a t the cdB-AR is e x p r e s s e d in heart, brain, liver, k i d n e y a n d s p e e n . T h e ctlC-AR is also e x p r e s s e d in t h e s a m e tissues, b u t only weakly in the spleen. On the o t h e r h a n d , e x p r e s s i o n of the ctlA-AR c o u l d be d e t e c t e d only in the h e a r t , b r a i n a n d spleen. The c h a r a c t e r i z a t i o n of the m o u s e ctl-AR s u b t y p e s as well as cloning of t h e i r genes will allow to a l t e r the expression of specific ctl-AR genes in mice by h o m o l o g o u s r e c o m b i n a t i o n to b e t t e r u n d e r s t a n d t h e functional correlates of different r e c e p t o r subtypes.
It has been found that vasa deferentia from age-matched spontaneously hypertensive rats (SHR) exhibit an altered postjunctional ~l-adrenoceptor mediated responsiveness respect to control WKY. To clarify the contradictory existing results radioligand binding and funeional studies were performed. An increased steadystate density of [3H]prazosin binding sites was observed in the vas deferens of SHR ( B ~ , = 148 4-18 frnol.mg-1 protein) vs WKY controls (B~x=71 + 9 fmol-mg 1 protein) with no changes in the dissociation constant value. Concentration-response (E/[A]) curves to noradrenaline showed a higher maximal effect in vasa deferentia from SHR compared to WKY (25.3 ___.96 mN vs 15.64- .52 raN) but pECso were not modified. Partial alkylation with phenoxybenzamine (0.1 #M) was performed to apply the operational model of Black and Left (1983) on E/[A] curves. The affinity (KA) and efficacy (r) estimates for noradrenaline, and the parameters E m and n in each experimental group were tested for similarity by analysis of variance. The results of this analysis showed that the model could fit both sets of data by assuming the same pK A value (4.90;4.9~), different r values (logrv,xv=l.63+__0.23 and logrsnR=l.74+0.29) and a significant change in E~ (Em wrv=15.82 mN vs E~ s ~ = 2 5 . 8 6 mN) for noradrenaline. The results also show that the altered responsiveness could not be only explained as an increase in cq-adrenoceptors. Black and Left, Proc R Soc Lend, B220:141-62 (1983)
IN VIVO POTENCY AND SELECTIVITY OF THE FIRST B3ADRENOCEPTOR ANTAGONIST, SR 59230A, IN RATS T, Croci, A. Giudice, L. Manara and G. Le Fur*. SANOFI-MIDY S.p.A. Research Center, Via Piranesi 38, 20137 Milan, Italy. *SANOFI-RECHERCHE, 32/34 rue Marbeuf, 75008 Paris, France
FUNCTIONAL DISTRIBUTION OF ALPHA-1 ADRENOCEPTOR SUBTYPES IN MESENTERIC SMALL ARTERIES OF THE RAT M. Ipsen, °N. Dragsted and M.J. Mulvany Department of Pharmacology, 8000 Aarhus, and °H. Lundbeck A/S, 2500 Valby, Denmark.
We presented the aryloxypropanolaminotetralin SR 59230A, 3-(2-ethylphenoxy)-1-1(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-(2S)-2-propanoloxalate, as the first potent and selective antagonist for atypical (133) 13-adrenoceptors (PA2: 113,8.8; 132,6.6; 13b 7.1), thereby providing final unambiguous evidence of the distinctive functional features of those abundant in the rat proximal colon (2). In this study SR 59230A and alprenolol were compared in vivo in animal models designed for scoring responses presumably elicited at conventional (B1 and 132,increase in heart rate and bronchodilation) and atypical (133,inhibition of colonic motility) 13-adrenoceptors.Antagonists were given p.o. to Crl(CD)BR fasted male rats 60 rain before a submaximallyeffective dose of the appropriate agonist: SR 58611A, 45 ~tg/kg i.v., inhibiting myoelectricaUy assessed colon motility scored for 60 min (1); isoprenaline, 5 I.tg/kg s.c., increasing heart rate scored for 15 rain (1); salbutamol, 40/ag/kg i.v., producing bronchodilation scored for 10 min by measuring specific airway resistance with a two-chamber plethysmograph connected to a respiratory analyzer (3) in metacholine responsive (6% in H20 nebulized for 10 sec) animals.
131 Alprenolol SR 59230A
132
As cq-blockers are used in the treatment of essential hypertension, it is of interest to know which receptors are involved in the resistance vasculature. To examine which cq-adrenoceptor subtypes are present in mesenteric small arteries of male Wistar rats, vessels (diameter 188-279 Ixm) were mounted as endothelium-free ring preparations on a microvascular myograph. Concentration-response curves for the cq-agonist phenylephrine (PE) were obtained, the first as control and the following curves with increasing concentrations of the cqA-antagonists a) 5-methylurapidil (5MU): 10, 30, 100 nM (n=4), or b) WB 4101 # (WB): 1, 3, 10 nM (n=6); or the cqB-antagonist c) chlorethylclonidine (CEC): 10, 30, 100 ~tM (n=6). Results of reversible antagonists were analysed by Schild plots: pA2 values and slopes were for WB 9.47 ± 0.14 (mean _+SEM) and 1.07 _+ 0.11 , and for 5MU 9.21 _+ 0.30 and 1.07 _+ 0.11, respectively. For the irreversible antagonist CEC, -log EChoand % of max. contraction were calculated as follows. Control: 6.57 -+ 0.11,100 %; 10 ~tM: 5.64 + 0.06 (a 9-fold change), 93 + 11%; 30 IxM: 4.64 +_ 0.13 (a 85-fold change), 70 _+ 6 %; 100 ~M: 3.81 + 0.08 (a 575-fold change) and 46 _+8 %. The experiments show that both cqA and cqB are present in these small arteries. In six experiments ~A- and ebB-antagonists were both added in the highest concentrations, 24 % of max. contraction still remained. This indicates that other cq-subtypes are present, which remain to be identified.
133
IDs0 , mg/kg, p.o. (95% confidence limits) - 3 (2-4) 7.6 (2.8-22) 11 (7-18) 56 (25-125) 48 (28-91) 3.6 (2-6.5)
*IDso = dose of antagonist inhibiting agonist response by 50%. Unlike alprenolol SR 59230A was over ten times less effective in preventing either 131or 132responses than as an antagonist of the putative 133response. The potency, oral effectiveness and selectivity of SR 59230A support its utility tbr investigating the functional role of 133-adren°ceptorsin vivo, also in view of the possible therapeutic potential of new compounds like aryloxypropanolaminotetralins. 1. Croci T, Giudice A, Bianchetti Aet al (1991) J Gastrointest Motil 3,273 2. Manara L, Badone D, Baroni Met al (1995) Pharmacol Commun 6, 240 3. Pennock BE, Cox CP, Rogers RM et al (1979) J Appl Physiol 46, 399
"WB 4101: 2-(2,6-Dimethoxyphenoxyethyl)aminemethyl-1,4-benzodioxane hydrochloride.
--
192--
CLONING AND TISSUE DISTRIBUTION OF THE MOUSE ALPHAIB-ADRENERGIC RECEPTOR. Cavalli A., M o s t a r d i n i M. a n d C o t e c c h i a S. - I n s t i t u t de P h a r m a c o l o g i e e t Toxicologie, Rue d u B u g n o n 27, 1005 Lausanne, Switzerland.
E p i n e p h r i n e a n d n o r e p i n e p h r i n e elicit a variety of f u n c t i o n a l effects b y b i n d i n g to d i f f e r e n t a d r e n e r g i c r e c e p t o r s (AR) w h i c h b e l o n g to the s u p e r f a m i l y of Gp r o t e i n - c o u p l e d receptors. T h r e e m a j o r classes of ARs h a v e b e e n d e s c r i b e d (~1, ix2 a n d I~) a n d each clas s is f o r m e d by d i s t i n c t r e c e p t o r subtypes. T h r e e cd-AR s u b t y p e s (cdA, etlB, a l C ) h a v e b e e n i d e n t i f i e d in d i f f e r e n t species i n c l u d i n g r a t a n d m a n . On the o t h e r h a n d , v e r y little is k n o w n a b o u t ~ I - A R h e t e r o g e n e i t y in t h e m o u s e . T h u s , we h a v e u n d e r t a k e n the c l o n i n g a n d c h a r a c t e r i z a t i o n of d i f f e r e n t ~I-AR s u b t y p e s in the mouse. A cDNA e n c o d i n g the m o u s e cdB-AR was i s o l a t e d f r o m a cDNA l i b r a r y d e r i v e d f r o m m o u s e B a l b / c brain. C o m p a r i s o n of its d e d u c e d a m i n o acid s e q u e n c e w i t h t h a t o f the previously c l o n e d h a m s t e r cDNA s h o w e d 98% identity. The m o u s e ctlB-AR expressed in COS-7 cells r e v e a l e d p h a r m a c o l o g i c a l a n d s i g n a l - t r a n s d u c t i o n p r o p e r t i e s similar to those of the h a m s t e r receptor. We also isolated a g e n o m i c c l o n e c o n t a i n i n g t h e first exon of the r e c e p t o r , w h i c h was i n t e r r u p t e d b y a n i n t r o n at n u c l e o d d e 9 4 8 of its o p e n r e a d i n g frame. The position of this i n t r o n is identical to t h a t previously r e p o r t e d for the h a m s t e r a n d h u m a n ~IB-AR genes. The mRNA d i s t r i b u t i o n of the t h r e e cd-AR s u b t y p e s was assessed in selected mouse tissues by N o t h e r n analysis as well as RT-PCR. Our findings i n d i c a t e t h a t the cdB-AR is e x p r e s s e d in heart, brain, liver, k i d n e y a n d s p e e n . T h e ctlC-AR is also e x p r e s s e d in t h e s a m e tissues, b u t only weakly in the spleen. On the o t h e r h a n d , e x p r e s s i o n of the ctlA-AR c o u l d be d e t e c t e d only in the h e a r t , b r a i n a n d spleen. The c h a r a c t e r i z a t i o n of the m o u s e ctl-AR s u b t y p e s as well as cloning of t h e i r genes will allow to a l t e r the expression of specific ctl-AR genes in mice by h o m o l o g o u s r e c o m b i n a t i o n to b e t t e r u n d e r s t a n d t h e functional correlates of different r e c e p t o r subtypes.
It has been found that vasa deferentia from age-matched spontaneously hypertensive rats (SHR) exhibit an altered postjunctional ~l-adrenoceptor mediated responsiveness respect to control WKY. To clarify the contradictory existing results radioligand binding and funeional studies were performed. An increased steadystate density of [3H]prazosin binding sites was observed in the vas deferens of SHR ( B ~ , = 148 4-18 frnol.mg-1 protein) vs WKY controls (B~x=71 + 9 fmol-mg 1 protein) with no changes in the dissociation constant value. Concentration-response (E/[A]) curves to noradrenaline showed a higher maximal effect in vasa deferentia from SHR compared to WKY (25.3 ___.96 mN vs 15.64- .52 raN) but pECso were not modified. Partial alkylation with phenoxybenzamine (0.1 #M) was performed to apply the operational model of Black and Left (1983) on E/[A] curves. The affinity (KA) and efficacy (r) estimates for noradrenaline, and the parameters E m and n in each experimental group were tested for similarity by analysis of variance. The results of this analysis showed that the model could fit both sets of data by assuming the same pK A value (4.90;4.9~), different r values (logrv,xv=l.63+__0.23 and logrsnR=l.74+0.29) and a significant change in E~ (Em wrv=15.82 mN vs E~ s ~ = 2 5 . 8 6 mN) for noradrenaline. The results also show that the altered responsiveness could not be only explained as an increase in cq-adrenoceptors. Black and Left, Proc R Soc Lend, B220:141-62 (1983)
IN VIVO POTENCY AND SELECTIVITY OF THE FIRST B3ADRENOCEPTOR ANTAGONIST, SR 59230A, IN RATS T, Croci, A. Giudice, L. Manara and G. Le Fur*. SANOFI-MIDY S.p.A. Research Center, Via Piranesi 38, 20137 Milan, Italy. *SANOFI-RECHERCHE, 32/34 rue Marbeuf, 75008 Paris, France
FUNCTIONAL DISTRIBUTION OF ALPHA-1 ADRENOCEPTOR SUBTYPES IN MESENTERIC SMALL ARTERIES OF THE RAT M. Ipsen, °N. Dragsted and M.J. Mulvany Department of Pharmacology, 8000 Aarhus, and °H. Lundbeck A/S, 2500 Valby, Denmark.
We presented the aryloxypropanolaminotetralin SR 59230A, 3-(2-ethylphenoxy)-1-1(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-(2S)-2-propanoloxalate, as the first potent and selective antagonist for atypical (133) 13-adrenoceptors (PA2: 113,8.8; 132,6.6; 13b 7.1), thereby providing final unambiguous evidence of the distinctive functional features of those abundant in the rat proximal colon (2). In this study SR 59230A and alprenolol were compared in vivo in animal models designed for scoring responses presumably elicited at conventional (B1 and 132,increase in heart rate and bronchodilation) and atypical (133,inhibition of colonic motility) 13-adrenoceptors.Antagonists were given p.o. to Crl(CD)BR fasted male rats 60 rain before a submaximallyeffective dose of the appropriate agonist: SR 58611A, 45 ~tg/kg i.v., inhibiting myoelectricaUy assessed colon motility scored for 60 min (1); isoprenaline, 5 I.tg/kg s.c., increasing heart rate scored for 15 rain (1); salbutamol, 40/ag/kg i.v., producing bronchodilation scored for 10 min by measuring specific airway resistance with a two-chamber plethysmograph connected to a respiratory analyzer (3) in metacholine responsive (6% in H20 nebulized for 10 sec) animals.
131 Alprenolol SR 59230A
132
As cq-blockers are used in the treatment of essential hypertension, it is of interest to know which receptors are involved in the resistance vasculature. To examine which cq-adrenoceptor subtypes are present in mesenteric small arteries of male Wistar rats, vessels (diameter 188-279 Ixm) were mounted as endothelium-free ring preparations on a microvascular myograph. Concentration-response curves for the cq-agonist phenylephrine (PE) were obtained, the first as control and the following curves with increasing concentrations of the cqA-antagonists a) 5-methylurapidil (5MU): 10, 30, 100 nM (n=4), or b) WB 4101 # (WB): 1, 3, 10 nM (n=6); or the cqB-antagonist c) chlorethylclonidine (CEC): 10, 30, 100 ~tM (n=6). Results of reversible antagonists were analysed by Schild plots: pA2 values and slopes were for WB 9.47 ± 0.14 (mean _+SEM) and 1.07 _+ 0.11 , and for 5MU 9.21 _+ 0.30 and 1.07 _+ 0.11, respectively. For the irreversible antagonist CEC, -log EChoand % of max. contraction were calculated as follows. Control: 6.57 -+ 0.11,100 %; 10 ~tM: 5.64 + 0.06 (a 9-fold change), 93 + 11%; 30 IxM: 4.64 +_ 0.13 (a 85-fold change), 70 _+ 6 %; 100 ~M: 3.81 + 0.08 (a 575-fold change) and 46 _+8 %. The experiments show that both cqA and cqB are present in these small arteries. In six experiments ~A- and ebB-antagonists were both added in the highest concentrations, 24 % of max. contraction still remained. This indicates that other cq-subtypes are present, which remain to be identified.
133
IDs0 , mg/kg, p.o. (95% confidence limits) - 3 (2-4) 7.6 (2.8-22) 11 (7-18) 56 (25-125) 48 (28-91) 3.6 (2-6.5)
*IDso = dose of antagonist inhibiting agonist response by 50%. Unlike alprenolol SR 59230A was over ten times less effective in preventing either 131or 132responses than as an antagonist of the putative 133response. The potency, oral effectiveness and selectivity of SR 59230A support its utility tbr investigating the functional role of 133-adren°ceptorsin vivo, also in view of the possible therapeutic potential of new compounds like aryloxypropanolaminotetralins. 1. Croci T, Giudice A, Bianchetti Aet al (1991) J Gastrointest Motil 3,273 2. Manara L, Badone D, Baroni Met al (1995) Pharmacol Commun 6, 240 3. Pennock BE, Cox CP, Rogers RM et al (1979) J Appl Physiol 46, 399
"WB 4101: 2-(2,6-Dimethoxyphenoxyethyl)aminemethyl-1,4-benzodioxane hydrochloride.
--
192--