Multidisciplinary analysis of muscarinic receptors in guinea-pig isolated ileum, atria and uterus in vitro

1 The pharmacological characteristics of muscarinic receptors in rat isolated uterus were studied in ovariectomized (ov.) and sham operated (sh.) animals. 2 Competition radioligand binding studies, using uterine membranes and [ 3 H]-NMS, were undertaken with several muscarinic receptor antagonists. Most of the antagonists indicated a onesite ®t with apparent a nity estimates (pK i ) unchanged by ovariectomy. The selective M 2 antagonist, tripitramine revealed high (representing 33+8 and 38+2%) and low (67+8 and 62+2%) a nity binding sites in both sh. and ov. rat uterus, respectively. These sites likely represented muscarinic M 2 and M 3 receptors and the proportions were not signi®cantly di erent in the two conditions. 3 Carbachol induced concentration-dependent contractions which were surmountably antagonized by several muscarinic receptor antagonists (pK B , sh.; ov.): zamifenacin (9.19; 9.18), p-F-HHSiD (8.50; 9.06), tripitramine (7.23; 7.54), himbacine (7.21; 7.41), methoctramine (6.79; 7.49), pirenzepine (6.48; 7.21), AF DX 116 (6.26; 6.61), MTx 3 (57.00; 57.00) and PD 102807 (57.00; 57.00). 4 The apparent a nity values obtained in functional studies using the uteri from both sh. and ov. animals correlated most closely with values reported at human recombinant muscarinic M 3 receptors. This suggests that the muscarinic M 3 receptor mediates contraction under both conditions. 5 Radioligand binding experiments indicate the presence of M 2 receptors, in addition to M 3 receptors, which probably explains the discrepancies between functional and binding a nities. These data further suggest that the pharmacological pro®le and proportions of the two populations of muscarinic receptors are una ected by ovariectomy.

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Characterization of the muscarinic receptor in isolated uterus of sham operated and ovariectomized rats

Choppin¹,

Stepan²,

Loury³

et al. 1999

“…The Hill slopes of competition curves of antagonists to [ 3 H]‐NMS binding would suggest that only one muscarinic receptor subtype is expressed in this tissue. However, it has been shown by immunological methods and Northern blotting techniques that both M 2 ‐ and M 4 ‐mACh receptors can coexist in uterine tissues (Dörje et al , 1991; Eglen et al , 1992), so it may be that these antagonists are still not sufficiently selective between mACh receptor subtypes to detect coexpression, particularly if expression levels of one receptor subtype are low. These data however support previous evidence from Eglen et al (1989) and Doods et al (1993), in which radioligand binding and functional data suggested that M 2 ‐mACh receptors alone were responsible for contraction in this tissue.…”

Section: Discussionmentioning

confidence: 99%

“…Alternative suggestions that M 2 ‐ and M 3 ‐mACh receptors may be jointly responsible (Leiber et al , 1990), or M 4 ‐mACh receptors may mediate this response (Dörje et al , 1990) have also been proposed. Although immunological methods have demonstrated the presence of M 2 ‐ and M 4 ‐mACh receptors in rabbit uterus (Dörje et al , 1991) and Northern blot analysis of guinea‐pig uterine tissue revealed evidence for m2 and m4 mRNA transcripts (Eglen et al , 1992) it is unclear whether such receptors can mediate a contractile response in myometrial tissue. Inhibition of adenylyl cyclase and stimulation of phosphoinositide‐specific phospholipase C by mACh receptor agonists both can occur in guinea‐pig isolated uterus, with the former being attributed to M 2 ‐ and the latter to M 3 ‐mACh receptor activation (Marc et al , 1988; Leiber et al , 1990).…”

Section: Introductionmentioning

confidence: 99%

Characterization of an atypical muscarinic cholinoceptor mediating contraction of the guinea‐pig isolated uterus

Boxall

Ford

Choppin

et al. 1998

1 In many smooth muscle tissues a minor M 3 -muscarinic acetylcholine (mACh) receptor population mediates contraction, despite the presence of a larger M 2 -mACh receptor population. However, this is not the case for guinea-pig uterus where radioligand binding and functional studies exclude a dominant role for M 3 -mACh receptors. 2 Using tissue from animals pre-treated with diethylstilboestrol, estimates of antagonist a nity were made before and after selective alkylation procedures, together with estimates of agonist a nity to characterise the mACh receptor population mediating carbachol-induced contraction of guinea-pig isolated uterus. 3 Antagonist a nity estimates made at`protected' receptors were not signi®cantly di erent from those made in untreated tissues. However all estimations were signi®cantly di erent from those reported in guinea-pig ileum and atria. The rank order of a nities were atropine4zamifenacin=tripitramine4 methoctramine. Carbachol-induced contractions were insensitive to the M 4 -selective muscarinic toxin MTx-3, or PD102807 (0.1 mM) ruling out a role for M 4 -mACh receptors. 4 The agonist a nity value for L-660,863, a putative`M 2 -selective' agonist of 5.44+0.30 (n=6) was signi®cantly di erent from that reported in guinea-pig atria. In contrast, the pK A value for carbachol (4.22+0.17; n=8) agrees with that reported for guinea-pig ileum. 5 Carbachol-induced contractions were insensitive to pertussis toxin although carbachol-induced inhibition of forskolin-stimulated cyclic AMP production was attenuated, ruling out the involvement of G i -proteins in contraction. 6 Radioligand binding studies revealed a K D for N-[ 3 H]-methylscopolamine of 0.12+0.05 nM and a B max of 147+18 fmol mg protein 71 . Antagonist a nity estimates made using competition binding studies supported previous data suggesting the presence of a homogenous population of M 2 -mACh receptors. 7 These data suggest a small population of mACh receptors with an atypical operational pro®le which can not be distinguished using radioligand binding studies may mediate carbachol-induced contraction of guinea-pig isolated uterus.

“…(1990) studied the effects of specific agonists and antagonists on carbachol‐induced contractions of guinea‐pig uterine tissue and measured accumulation of inositol phosphates and cyclic AMP inhibition, and concluded that in the myometrium of the guinea‐pig the contractile event is triggered by the M 3 receptor subtype and may be modulated by the M 2 receptor subtype. However, other studies with the guinea‐pig uterus have indicated that the M 2 , M 3 or M 4 receptors are actively involved in mediating uterine contraction (Eglen et al ., 1989; 1991; Dörje et al ., 1990a,b; Doods et al ., 1993). More recent studies by the latter group indicate that the muscarinic receptor present in the guinea‐pig uterus may be a novel subtype (Boxall et al ., 1997).…”

Section: Introductionmentioning

confidence: 99%

Pharmacological characterization of muscarinic receptors in the uterus of oestrogen‐primed and pregnant rats

Munns

Pennefather

1998

1 Radioligand binding and contractility studies were undertaken to determine the subtype/s of muscarinic receptors present in uteri of oestrogen-treated and late pregnant rats. 2 Competition binding studies with uterine membrane preparations and [ 3 H]-QNB (quinuclidinyl benzilate) provided negative log dissociation constants (pK i ) for each antagonist as follows; oestrogentreated ± atropine (7.98)5himbacine (7.83)4methoctramine (7.52)5hexahydrosiladiphenidol (HHSiD; 7.32)55,11]benzodiazepin-6-one (AF-DX 116, 6.77)4pirenzepine (6.17); late pregnant ± atropine (8.05)5methoctramine (7.95)5himbacine (7.71)5HHSiD (7.52)5AF-DX 384 (7.34)4AF-DX 116 (6.72)4pirenzepine (6.18). 3 The potency of carbachol in causing uterine contraction was similar in preparations from pregnant and non-pregnant animals (pD 2 =5.57 and 5.46, respectively). Each muscarinic antagonist caused parallel, rightward shifts of carbachol concentration-response curves. The pA 2 estimates were: oestrogentreated ± atropine (9.42)4himbacine (8.73)5HHSiD (8.68)5methoctramine (8.49)5AF-DX 384 (7.91)5AF-DX 116 (7.36)5pirenzepine (7.26); late pregnant ± atropine (9.48)4himbacine (8.37)5HHSiD (8.22)5methoctramine (8.01)5AF-DX 116 (7.73)5AF-DX 384 (7.44)5pirenzepine (6.92). 4 The relative pK i estimates for antagonists obtained in membrane preparations from oestrogen-treated rats suggest the presence of muscarinic M 2 subtypes. In functional studies pA 2 values indicated the additional presence of muscarinic M 3 receptor or, possibly an atypical receptor subtype. The similarity between pK i and pA 2 estimates obtained in uteri from oestrogen-treated and pregnant animals, respectively, indicates that pregnancy does not a ect myometrial muscarinic receptors in the rat.