I N G U I N E A P I G G A L L B L A D D E R A h m e t A k ı c ı , M . D . * / M u r a d i y e N a c a k , M . D . * * A r t h u r C h r is to p o u lo s , P h . D . * * * / Ş u le O k t a y , M . D . , P h .D .* * D e p a r tm e n t o f P h a rm a c o lo g y a n d C lin ic a l P h a rm a c o lo g y , M a r m a r a U n iv e rs ity , S c h o o l o f M e d ic in e , Is ta n b u l, T u rk e y . * ' D e p a r tm e n t o f P h a rm a c o lo g y , G a z ia n te p U n iv e rs ity , S c h o o l o f M e d ic in e , G a z ia n te p , T u rk e y . * " D e p a r tm e n t o f P h a rm a c o lo g y , F a c u lty o f M e d ic in e , D e n tis tr y a n d H e a lth S c ie n c e s , U n iv e rs ity o f M e lb o u rn e , M e lb o u rn e , A u s tra lia .
A B STR A C T
O b je ctive : Previous studies have revealed the presence of M1 to M4 m uscarinic receptors in guinea pig gallbladder, with M3 and M4 receptors being claimed to mediate contractions.
M e th o d s: In the p resent study, cum ulative concentration-response cu rve s to carbachol were constructed in the absence and presence of darifenacin, which has greater selectivity for M3 over M4 receptors, in guinea-pig gallbladder. R e s u lt s : D arifenacin ca u se d concentration- dependent dextral shifts of the carbachol curve, however, at 1 pM, it also caused a degree of insurm ountable antagonism . Using a novel analytical approach, we obtained a p K B estimate for darifenacin (7.51 ± 0.14) that is in excellent agreement with its affinity for M2/M4, but not M3 receptors.
C o n c lu s io n : Given our previous demonstration that M2 receptors are unlikely to contribute to m uscarinic contractions in this tissue, our current
findings provide pharm acological evidence for a predominant role of M4 m uscarinic receptors in guinea pig gallbladder contractions.
K e y W o r d s : M uscarinic receptor, Guinea-pig, Gallbladder, Contraction, Darifenacin.
IN T R O D U C T IO N
M uscarin ic a ce tylch o lin e recep to rs play an important role in regulating sm ooth m uscle
contractility (1). Although abundant evidence
e xists to suggest that the m uscarinic M3 receptor is the predominant subtype causing a contraction in most smooth m uscle preparations (2), there are exceptions to this general observation. One notable exam ple is the guinea pig gallbladder, w here m u scarin ic receptor-m ediated smooth m uscle contraction helps to control bile outflow in response to feeding; despite previous claim s of functional M3 receptors being present in this tissue (3-6), a combination of biochem ical and organ bath e xp e rim e n ts h ave re ve a le d the
(Accepted 4 August, 2002)
Marmara Medical Journal 2002;15(4):248-252
Correspondence to: Şule Oktay, M.D., Ph.D., - Department of Pharmacology and Clinical Pharmacology, School of Medicine, Marmara University, Haydarpaşa 81326 Istanbul, Turkey,
presence of M ,, M2> M3 and M4 m uscarinic receptor subtypes (7-11).
A major difficulty in ascribing functional roles to subtypes of m uscarinic receptors has long been the lack of se le c tiv e an tag o n ists that can differentiate between receptors mediating the sam e response (1). N evertheless, a comparison of the potencies of a group of antagonists may
still be used to determ ine the possible
contribution of different subtypes to a particular re sp o n se . U sing this stra te g y, w e provided evidence for a functional role of both M3 and M4 receptors in mediating guinea-pig gall bladder co ntractio ns (1 1 ). H o w ever, that study w as unable to determ ine which of the two subtypes played the predominant functional role. In the present study, w e have utilized the antagonist, darifenacin, which is at least greater than 50 times more se lective for the M3 (p K B approx. 9) over the M4 receptor (p K B approx. 7.5) (12,13), and in vestig ated its effe cts on carbachol- mediated contractions in guinea-pig gallbladder.
M A T E R IA L A N D M E T H O D S
Guinea-pigs of either se x (300-350 g) were killed by C 0 2 asphyxiation. G allbladders were then removed and longitudinal strips w ere prepared and mounted in an organ bath containing Krebs solution (composition in mmol/l; N aCI, 118.4; K C I, 4 .7 ; C a C I2, 2 .5 ; M g S 0 4, 1.2; N a H C 0 3, 25.0; KH 2P 0 4, 1.2; glucose, 11.1) at 37 °C bubbled with a mixture of 95 % 0 2 and 5 % C 0 2. T issu e s w ere allowed to equilibrate for 2 h under a resting tension of 0.5 g., and isometric contractions were recorded on a polygraph (G ra ss Model 7) via a force-displacem ent transducer (G ra ss FT0 3 ). Control cu m u lative co ncen tratio n -resp o n se curves w ere constructed to carbachol (Sigm a, St. Louis, U S A ). An interval of 45 min w a s then allowed during which the tissu e s w ere washed with Kreb s solution. A second concentration- re sp o n se cu rve to ca rb a ch o l w a s then constructed in the a b se n c e or p re se n ce of darifenacin. O ne or two concentrations of the antagonist w ere used for each strip.
Normalized concentration-response data were fitted to a three-param eter Hill equation using a p re-release version of Prism 4 .0 (G rap hPad
Softw are, S an Diego, C A ). B e ca u se the highest concentration of darifenacin (1 pM) caused a significant reduction in the m aximal response to carbachpl (se e R e su lts), w e determ ined eq uieffective agonist co ncen tratio n s, in the ab se n ce or p re se n ce of antagonist, at a response level close to the tissue minimum (15% ) in order to derive antagonist potency estim ates. The validity of this analytical approach has been demonstrated previously (14-16). The agonist p E C 15o/o v a lu e s w ere fitted to the following equation:
p E C is% = -log ([B]s + 1 0-PK) - logo (1) where [B] denotes antagonist concentration, pK and logc are fitting constants, and s is equivalent to the Schild slope factor; when s=1, pK=pKB (15). For presentation purposes, the relationship between the estimated p K B and the shift of the agonist co ncen tratio n -resp on se c u rv e s w a s displayed a s a Clark plot (17).
Data are shown a s mean ± s.e .m . Estim ates of agonist concentration-response curve maxima were compared by one-way AN O VA. P < 0.05 w as taken a s significant.
RESULTS
C a rb a ch o l ca u se d concentration-dependent
co ntractio ns in guinea-pig gallbladder
longitudinal m uscle strips, with a p E C 5 0 value of 5.63 0.18 (n = 7). Th e addition of darifenacin caused dextral shifts of the carbachol curve (Fig.
1A). However, the highest concentration of
darifenacin also caused a significant reduction in the m axim al re sp o n se to carb ach o l, an observation not consistent with the expectations of simple competitive antagonism.
B e c a u se the insurmountable effect of 1 pM darifenacin meant that the carbachol p E C 50 valu es do not all represent equieffective agonist concentrations, we fitted the data to equation (1) utilizing equieffective agonist concentrations that w ere determined at the p E C 15°/o response level (Fig. 1 A ). The a n alysis using this method yielded a p K B value of 7.51 0.14 (n = 25). In this a n a lysis, the Schild slope param eter w as not sig nificantly different from unity and w as constrained a s such for the estimation of the antagonist p KB value.
Log [C arbachol]
F i g - 1 : (A) C oncentration-response curves of carbachol in the absence (□ ) or presence of darifenacin 0.01 (■ ), 0.1 (O) or 1 pM ( A ) in guinea-pig gallbladder. Also indicated on the Figure is the 15% response level (dashed line) that was used fo r the derivation of equieffective agonist co n ce ntra tio n s fo r s u b se qu e n t d e term in a tion of the antagonist p K B from Equation (1) of the M aterials and M ethods. D ata points rep re se n t the m eans of 4-8 experim ents. ' S ignificantly d ifferent (P < 0.05) than the control m axim al response to carbachol. (B) C lark plots of the interaction betw een increasing concentrations of darifenacin w ith carbachol. The a ntagonist p K B estim ate w as firs t de rive d by n o n lin e a r re g re ssio n a n a lysis according to equation (1) in the M aterials and M ethods, and w as subsequently used in the construction of the Clark plot.
D IS C U S S IO N
Studies on most smooth m uscle preparations have demonstrated a major role for functional
m u scarinic M3 receptors in mediating
contractions, even though th ese tissu e s
invariably contain a m ixture of m u scarinic receptor subtypes (2). In a few smooth m uscle preparations, how ever, contractile resp o n ses
may be mediated by m u sca rin ic receptor subtypes other than the M3 receptor (18-22). In the guinea-pig gallbladder, Von S ch re n ck et al. (6) reported that carbachol-induced inhibition in adenylate c y c la s e , but not stim ulation of phosphoinositides, w a s pertussis toxin sensitive, indicating the presence of more than one subtype of m u scarin ic recep to rs. S u b se q u e n tly , biochemical and functional studies have revealed the presence of M ,, M2, M3 and M4 m uscarinic receptor subtypes in this tissue (2 3 ,2 4 ,8 ,9 ). Of these subtypes, a predominant functional role in guinea pig gallbladder sm ooth m u scle w a s suggested for the M3 receptors (3-6), but we recently provided evidence for the co existen ce of both functional m uscarinic M3 and M4 receptor subtypes mediating C a 2+ mobilization in this tissue (11).
The present study extended our previous work by
utilizing d arifen acin to differentiate the
m u scarin ic receptor subtype involved in
carbachol-mediated contractions of the guinea pig gallbladder. Darifenacin is cla sse d a s an M3- se le ctive m u scarin ic receptor antag on ist, showing 100-fold selectivity for M3 receptors over M2 receptors in atria and 30 fold over M, receptors in rabbit v a s deferens (12). The pK, v a lu e s ag ain st hum an cloned m u sca rin ic receptors have been reported a s : M ,, 8 .1 5 ; M2, 7.3 5 ; M3, 9.1 2 ; M4, 7.3 4 ; M5, 8.03 (13).
Although d arifen acin w a s able to inhibit
carbachol-m ediated co ntractio n s in our
preparation, the highest concentration used here resulted in a significant degree of insurmountable
antagonism (Fig . 1A ). T h is property of
darifenacin has been reported previously at pig, rat and dog bladder smooth m uscle, but not in rabbit and m ouse bladder (25-29). Although the reason for the apparent insurm ountability of d arifen acin antagonism is not know n, it is possibly related to slow binding kinetics at the m uscarinic receptors, relative to the time sc a le of the m easured resp o n ses, a s previously noted with other m uscarinic receptor antagonists (14).
Irre sp e ctive of m e ch a n ism , apparent
insurm ountable antag onism in valid a te s the cla ssic approaches for quantifying competitive antagonism that rely on the com parison of agonist E C 50 valu e s, b e ca u se these va lu e s no
concentrations and hence the a n alysis is no
longer response-null. A s a consequence, the
use of E C 50 v a lu e s in the determination of antagonist potency under such conditions leads to an underestimation of the true antagonist potency (14-16). H ow ever, we have previously d em onstrated in different cell and tissu e
p re p aratio n s how the im pact of this
underestimation can be minimized by choosing truly equieffective agonist concentrations at an appropriate re sp o n se level (14-16). In the present study, w e utilized the 15% response level in equation (1).
Using our analytical method, we derived a p K B estim ate for darifenacin of 7.51 that is more than an order of magnitude lower than any previous estim ates of the affinity of darifenacin for the M3 receptor, but in excellent agreem ent with its affinity for either the M2 receptor or the M4 receptor (13). B e c a u se we have previously used a se rie s of se lective antagonists to rule out a functional role for m uscarinic M2 receptors in guinea pig gallbladder (11), our current findings with darifenacin lead us to conclude that the major functional m uscarinic receptor subtype in this preparation is the M4 receptor.
A c k n o w le d g m e n t s
The authors wish to thank Pfizer, U K for the generous gift of darifenacin.
REFERENCES
1. C aufield M l’, Birdsall HJM. In ternatio n al Union o f P harm aco lo g y. X V II: C lassificatio n o f m u s c a rin ic a c e ty lc h o lin e recep to rs. P harm acol Rev 19 9 8 :5 0 :2 7 9 -2 9 0 .
2. Eglen RM, Reddy fl, Watson hi, Challiss RAJ. M uscarinic acetylcho lin e re c e p to r subtypes in s m o o th m u s c le . Trends P h a rm a c o l Sci
1 9 9 4 ,1 5 : 1 14-1 19.
3. E ltze M, R onig fl, U llrich B, G re b e T. C o n tra c tio n o f g u in e a -p ig g a llb la d d e r: m u s c a rin ic M3 o r M 4 re c e p to rs ? E ur J Pharm acol 1 9 9 7 ;3 3 2 :7 7 -8 7 .
4. T a k a h a s h i T, K urosaw a S, O w yang C. R eg u latio n o f PI h ydrolysis a n d cAMP fo rm a tio n by m u s c a rin ic M 3 re c e p to r in guinea-pig g allbladder. Am J Physiol 1 9 9 4 ; 2 6 7 .G 5 2 3 -G 5 2 8 .
5. Von Schrenck T, Sievers J, Mirau S, Raedler A. G reten fl. C ha ra c te riza tio n o f m uscarinic receptors on guinea-pig g allb la d d e r sm ooth m uscle. G astro entero lo g y 1 9 9 3 ; 1 0 5 : 13 4 1-
1349.
6. Von Schrenck T, Mackensen B, M ende U, et at. Signal transduction p athw ay o f the m uscarinic receptors m ed iatin g g allb la d d e r contraction. Haunyn-Schm ied. Arch P harm acol 1994.-349: 3 4 6 -3 5 4 .
7. Özkutlu U, Alican İ, Karahan E, et al. Are m- c h o lin o c e p to rs o f g u in e a -p ig g a llb la d d e r sm ooth m uscle o f m 4 subtype? Pharmacology
1 9 9 3 ;4 6 :3 0 8 -3 1 4 .
8. O ktay Ş, C abad ak fl, İs k e n d e r E, et al. Evidence fo r the p resence o f m uscarinic M2 an d M4 receptors in guinea-pig g allblad der sm o o th m u scle. J A u to n P h arm aco l
1 9 9 8 ,1 8 :1 9 5 -2 0 4 .
9. Parkman HP, Pagano AP, Ryan JP. Subtypes o f m uscarinic receptors regulating gallblad der c h o lin erg ic co n tra c tio n s . Am J Physiol 1 9 9 9 ;8 4 :7 1 -7 6 .
10. Karaalp A, A kıcı A, A kbu lu t tl, Ulusoy FIB, O k ta y Ş. D is tin c t fu n c tio n a l m u scarinic receptors in guinea-pig gallbladder, ileum and atria. Pharm acol Res 19 9 9 :3 9 :3 8 9 -3 9 5 . 11. Akıcı A, Karaalp, A, İs k e n d e r E, Christopoulus
A, El-Eakahany EE, O ktay Ş. Further evidence fo r the h etero g eneity o f fun ctio n al m uscarinic receptors in guinea-pig gallbladder. Eur J Pharm acol 2 0 0 0 : 3 8 8 : 1 15-123.
12. A labaster VA. Discovery an d develo p m en t o f selective M 3 antagonists fo r clinical use. Life Sci 1 9 9 7 :6 0 :10 5 3 -1 0 6 0 .
13. Wallis RM, Fiapier CM. Muscarinic antagonists in d e v elo p m en t fo r disorders o f sm oothe m uscle function. Life Sci 1 9 9 9 :6 4 :3 9 5 -4 0 1. 14. Christopoulos A, Parsons AM, Lew MJ, El-
Fakahany EE. The assessm ent o f antagonist p o te n c y u n d e r c o n d itio n s o f transient response kinetics. E ur J Pharm acol 1 999; 3 8 2 :2 1 7 -2 2 7 .
15. Christopoulos A, Coles P, Lay L, Lew MJ, Angus JA. Pharmacological analysis o f cannabinoid receptor activity in the rat vas deferens. Br J Pharmac 2 0 0 1 ;1 3 2 :1 2 8 1 -1 2 9 1 .
16. Christopoulos A. From captive agonism to in su rm ountable antagonism : Dem onstrating the p o w e r o f an alytical pharm acology. Clin Exp Pharm acol Physiol 2 0 0 1,-28 :2 2 3 -2 2 9 . 17. Lew MJ, Angus. Analysis o f c o m p e titiv e
agonist-antagonist interactions by n onlinear regression. Trends P harm aco l Sci
1 9 9 5 ,1 6 :3 2 8 -3 3 7 .
18. Van C h arld o rp KJ, Van Z w ie te n PA. Com parison o f the m uscarinic receptors in the coronary artery, cerebral artery and atrium o f the pig, riaunyn-Schm ied. Arch Pharmacol
1 9 8 9 ;3 3 9 :4 0 3 -4 0 8 .
19. D oods fin , W illim KD, B o d d e k e HWGM, Entzeroth M. Characterization o f m uscarinic re c e p to rs in g u in e a -p ig uterus. E u r J Pharmacol 1 9 9 3 ;2 5 0 :2 2 3 -2 3 0 .
20. Eglen RM, M ic h e l AD, W hiting RL. Characterization o f the m uscarinic recep to r subtype m ediating contractions o f the guinea- pig uterus. Br J Pharm acol 1 9 8 9 ; 9 6 :4 9 7 -4 9 9 . 2 1 . Eglen RM, M ichel AD, Levine WB, Whiting RL.
Analysis o f m uscarinic receptors in guinea-pig estrogen-dom inated uterus, in vitro. Br J Pharmacol 1991 ; 104.-44P.
2 2 . Eglen RM, W hiting RL. H e te ro g e n e ity o f vascular m uscarinic receptors. J A uton Pharmacol 1 9 9 0 ;1 0 :2 3 3 -2 4 5 .
2 3 . Chen Q, Yu P, De Petris G, B iancani P, Behar J. Distinct m uscarinic receptors an d signal transduction pathways in g allblad der muscle. J Pharm acol Exp Ther 19 9 5 ;2 7 3 :6 5 0 - 6 5 5 . 2 4 . Heinig T, Von Schrenck T, De Weerth A,
Kölsch K, S c h u ltz M, Iz b ic k i JR. C haracterization o f g a llb la d d e r m uscarinic recep to r subtypes: Signal transduction and
RT-PCR stu d ies. G a s tro e n te ro lo g y 1 9 9 7 ; 1 1 2 :7 4 6 .
2 5 . C ho p pin A, Eglen RM. P h a rm a c o lo g ic a l characterization o f m uscarinic receptors in dog isolated ciliary a n d urinary b la d d e r s m o o th m u scle. B r J P h a rm a c o l 2 0 0 1 ;1 3 2 :8 3 5 -8 4 2 .
2 6 . C ho p p in A, Eglen RM, P h a rm a c o lo g ic a l characterization o f m uscarinic receptors in m o u s e is o la te d u rin ary b la d d e r s m o o th m uscle. B r J P h arm aco l 2 0 0 1 ; 1 3 3 :1 0 3 5 -
10 4 0 .
2 7 . C h o p p in A, Eglen RM, H e d g e SS. P h a rm a c o lo g ic a l c h a ra c te ris a tio n o f m uscarinic receptors in rab b it iso lated iris s p hin cter m uscle an d urinary b la d d e r sm ooth muscle. Br J Pharm acol 1 9 9 8 ;1 2 4 :8 8 3 -8 8 8 . 2 8 . Hedge SS, Choppin A, Bonhaus D, e t al.
Functional role o f M 2 a n d M 3 m uscarinic receptors in the urinary b la d d e r o f rats in vitro and in vivo. Br J Pharm acol 1 9 9 7 ; 1 2 0 :1 4 0 9
1418.
2 9 . Yam anishi T, C hap p ie CR, Yasuda K, Chess- W illiam s R. The ro le o f M 2 -m u s c a rin ic receptors in m ed iatin g contraction o f th e pig urinary b la d d e r in vitro. Br J P harm acol 2 0 0 0 ;1 3 1 :1 4 8 2 - 1 4 8 8 .
