Agnes Choppin

Functional role of M2 and M3 muscarinic receptors in the urinary bladder of rats in vitro and in vivo

Urinary bladder smooth muscle is enriched with muscarinic receptors, the majority of which are of the M2 subtype whereas the remaining minority belong to the M3 subtype. The objective of the present study was to assess the functional role of M2 and M3 receptors in the urinary bladder of rat in vitro and in vivo by use of key discriminatory antagonists. In the isolated bladder of rat, (+)‐cis‐dioxolane produced concentration‐dependent contractions (pEC50=6.3) which were unaffected by tetrodotoxin (0.1 μm). These contractions were antagonized by muscarinic antagonists with the following rank order of affinity (pA2) estimates: atropine (9.1) > 4‐diphenyl acetoxy‐methyl piperidine methiodide (4‐DAMP) (8.9) > darifenacin (8.5) > para fluoro hexahydrosiladifenidol (p‐F‐HHSiD) (7.4) > pirenzepine (6.8) > methoctramine (5.9). These pA2 estimates correlated most favourably (r=0.99, P<0.001) with the binding affinity (pKi) estimates of these compounds at human recombinant muscarinic m3 receptors expressed in Chinese hamster ovary cells, suggesting that the receptor mediating the direct contractile responses to (+)‐cis‐dioxolane equates with the pharmacologically defined M3 receptor. As M2 receptors in smooth muscle are negatively coupled to adenylyl cyclase, we sought to determine whether a functional role of M2 receptors could be unmasked under conditions of elevated adenylyl cyclase activity (i.e., isoprenaline‐induced relaxation of KCl pre‐contracted tissues). Muscarinic M3 receptors were preferentially alkylated by exposing tissues to 4‐DAMP mustard (40 nm, 1 h) in the presence of methoctramine (0.3 μm) to protect M2 receptors. Under these conditions, (+)‐cis‐dioxolane produced concentration‐dependent reversal (re‐contraction) of isoprenaline‐induced relaxation (pEC50=5.8) but had marginal effects on pinacidil‐induced, adenosine 3′:5′‐cyclic monophosphate (cyclic AMP)‐independent, relaxation. The re‐contractions were antagonized by methoctramine and darifenacin, yielding pA2 estimates of 6.8 and 7.6, respectively. These values are intermediate between those expected for these compounds at M2 and M3 receptors and were consistent with the involvement of both of these subtypes. In urethane‐anaesthetized rats, the cholinergic component (∼55%) of volume‐induced bladder contractions was inhibited by muscarinic antagonists with the following rank order of potency (ID35%inh, nmol kg−1, i.v.): 4‐DAMP (8.1) > atropine (20.7) > methoctramine (119.9) > darifenacin (283.3) > pirenzepine (369.1) > p‐F‐HHSiD (1053.8). These potency estimates correlated most favourably (r=0.89, P=0.04) with the pKi estimates of these compounds at human recombinant muscarinic m2 receptors. This is consistent with a major contribution of M2 receptors in the generation of volume‐induced bladder contractions, although the modest potency of darifenacin does not exclude a role of M3 receptors. Pretreatment with propranolol (1 mg kg−1, i.v.) increased the ID35%inh of methoctramine significantly from 95.9 to 404.5 nmol kg−1 but had no significant effects on the inhibitory responses to darifenacin. These data suggest an obligatory role of β‐adrenoceptors in M2 receptor‐mediated bladder contractions in vivo. The findings of the present study suggest that both M2 and M3 receptors can cause contraction of the rat bladder in vitro and may also mediate reflex bladder contractions in vivo. It is proposed that muscarinic M3 receptor activation primarily causes direct contraction of the detrusor whereas M2 receptor activation can contract the bladder indirectly by reversing sympathetically (i.e. β‐adrenoceptor)‐mediated relaxation. This dual mechanism may allow the parasympathetic nervous system, which is activated during voiding, to cause more efficient and complete emptying of the bladder.

Therapeutic opportunities from muscarinic receptor research

Muscarinic acetylcholine receptor subtypes have been the subjects of research for at least a quarter of a century. Nonetheless, there are few selective muscarinic receptor ligands presently used as therapeutics. The extensive development of muscarinic M(1) receptor agonists for the treatment of cognitive dysfunction has culminated in a series of unsuccessful drug candidates, which reflects a lack of understanding of the disease and the role played by muscarinic cholinergic transmission. Paradoxically, the most successful antagonist approved for use in urinary incontinence is the nonselective muscarinic receptor antagonist tolterodine. This deficit in subtype-selective ligands could be circumvented by the development of transgenic mice, each lacking functional M(1), M(2), M(3), M(4) or M(5) receptors. In this article, the current status of muscarinic receptor research is critically assessed.

Muscarinic receptor ligands and their therapeutic potential

Over the past year, the introduction of novel ligands has accelerated the classification of muscarinic receptor subtypes and has led to a better understanding of their physiological role. Important in this respect is the recent recognition of the exquisite selectivity of a series of snake toxins, enabling better definition of the muscarinic subtype 4 receptor. Moreover, several compounds, both agonists and antagonists, are progressing in advanced clinical trials for the treatment of several conditions, including Alzheimers disease, pain, urinary incontinence and chronic obstructive pulmonary disease.

Pharmacological characterization of muscarinic receptors in rabbit isolated iris sphincter muscle and urinary bladder smooth muscle

The pharmacological characteristics of muscarinic receptors in the rabbit iris sphincter muscle were studied and compared to M3 receptors in rabbit urinary bladder smooth muscle. (+)‐Cis‐dioxolane induced concentration‐dependent contractions of the iris sphincter muscle (pEC50=6.41±0.10, Emax=181±17 mg, n=38) and urinary bladder smooth muscle (pEC50=6.97±0.04, Emax=4.28±0.25 g, n=54). These contractions were competitively antagonized by a range of muscarinic receptor antagonists (pKB values are given for the iris sphincter muscle and the bladder smooth muscle, respectively): atropine (9.30±0.07 and 9.40±0.04), AQ‐RA 741 (6.35±0.04 and 6.88±0.03), darifenacin (9.56±0.05 and 9.12±0.05), methoctramine (5.75±0.07 and 5.81+0.06), oxybutynin (8.10±0.09 and 8.59±0.06), pirenzepine (6.79±0.05 and 6.89±0.04), secoverine (7.54±0.05 and 7.66±0.05), p‐F‐HHSiD (7.55±0.09 and 7.50±0.05) and zamifenacin (8.69±0.10 and 8.36±0.06). A significant correlation between the pKB values in the bladder and the pKB values in the iris was obtained. In both tissues, the pKB values correlated most favorably with pKi values for these compounds at human recombinant muscarinic m3 receptors. A reasonable correlation was also noted at human recombinant muscarinic m5 receptors given the poor discriminative ability of ligands between m3 and m5 receptors. Overall, the data from this study suggest that the muscarinic receptors mediating contraction of the rabbit iris sphincter muscle and urinary bladder smooth muscle are similar and equate most closely with the pharmacologically‐defined muscarinic M3 receptor.

Pharmacological characterization of muscarinic receptors in mouse isolated urinary bladder smooth muscle

The pharmacological characteristics of muscarinic receptors in the male mice urinary bladder smooth muscle were studied. (+)‐Cis‐dioxolane, oxotremorine‐M, acetylcholine, carbachol and pilocarpine induced concentration‐dependent contractions of the urinary bladder smooth muscle (pEC50=6.6±0.1, 6.9±0.1, 6.7±0.1, 5.8±0.1 and 5.8±0.1, EMax=3.2±0.8 g, 2.7±0.4 g, 1.0±0.1 g, 2.7±0.3 and 0.9±0.2 g, respectively, n=4). These contractions were competitively antagonized by a range of muscarinic receptor antagonists (pKB values): atropine (9.22±0.09), pirenzepine (6.85±0.08), 4‐DAMP (8.42±0.14), methoctramine (5.96±0.05), p‐F‐HHSiD (7.48±0.09), tolterodine (8.89±0.13), AQ‐RA 741 (7.04±0.12), s‐secoverine (8.21±0.09), zamifenacin (8.30±0.17) and darifenacin (8.70±0.09). In this tissue, the pKB values correlated most favourably with pKi values for these compounds at human recombinant muscarinic M3 receptors. A significant correlation was also noted at human recombinant muscarinic m5 receptors given the poor discriminative ability of ligands between M3 and m5 receptors. In recontraction studies, in which the muscarinic M3 receptor population was decreased, and conditions optimized to study M2 receptor activation, methoctramine exhibited an affinity estimate consistent with muscarinic M3 receptors (pKB=6.23±0.14; pA2=6.16±0.03). Overall, these data study suggest that muscarinic M3 receptors are the predominant, if not the exclusive, subtype mediating contractile responses to muscarinic agonists in male mouse urinary bladder smooth muscle.

Pharmacological characterization of muscarinic receptors in dog isolated ciliary and urinary bladder smooth muscle

The pharmacological characteristics of muscarinic receptors mediating contraction of dog isolated ciliary muscle were determined and compared to those mediating contraction of dog urinary bladder smooth muscle. (+)‐Cis‐dioxolane induced concentration‐dependent contractions of ciliary muscle (pEC50=7.18±0.07, Emax=453±64 mg, n=19) and urinary bladder isolated smooth muscle (pEC50=6.55±0.07, Emax=11±1 g, n=19). These responses were antagonized by several muscarinic receptor antagonists (pKb values for the ciliary muscle and the bladder smooth muscle, respectively): atropine (8.25±0.14 and 9.21±0.09), pirenzepine (6.31±0.13 and 6.70±0.25), tolterodine (7.97±0.14 and 8.68±0.12), oxybutynin (7.40±0.08 and 7.88±0.12), zamifenacin (6.46±0.19 and 7.69±0.11), S‐secoverine (6.66±0.14 and 8.13±0.07), AQ‐RA 741 (6.16±0.15 and 7.08±0.23), p‐F‐HHSiD (7.10±0.27 and 7.35±0.07) and responses were not antagonized by PD 102807 (up to 100 nM). In urinary bladder smooth muscle, the profile of antagonist pKB values correlated significantly with pKi values at human recombinant m3 muscarinic receptors, suggesting that M3 muscarinic receptors mediated the response. In the ciliary muscle, a significant (P<0.01) correlation was obtained with human recombinant m3 and m5 receptors. Darifenacin displayed insurmountable antagonism at receptors in the bladder. At receptors in the ciliary muscle, it exhibited two phases of antagonism, comprising an initial low affinity (pKB<6) component and a high affinity phase (pKB>8). The role of pigmentation in the atypical behaviour of darifenacin was examined. In blue coloured eyes, darifenacin produced apparent surmountable, competitive antagonism of the responses to (+)‐cis‐dioxolane (pKB=8.76±0.07). The antagonist profile obtained in this tissue suggested the involvement of a site which has the pharmacological attributes of the M5 receptor. We suggest that the dog urinary bladder contracts in response to M3 muscarinic receptor activation. Contraction of the brown‐eyed dog ciliary muscle is more complex and may include involvement of at least two receptors, possibly the M5 and M3 receptor, whereas blue‐eyed dog ciliary muscle may involve a single population of M5 muscarinic receptors.

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

The pharmacological characteristics of muscarinic receptors in rat isolated uterus were studied in ovariectomized (ov.) and sham operated (sh.) animals. Competition radioligand binding studies, using uterine membranes and [3H]‐NMS, were undertaken with several muscarinic receptor antagonists. Most of the antagonists indicated a one‐site fit with apparent affinity estimates (pKi) unchanged by ovariectomy. The selective M2 antagonist, tripitramine revealed high (representing 33±8 and 38±2%) and low (67±8 and 62±2%) affinity binding sites in both sh. and ov. rat uterus, respectively. These sites likely represented muscarinic M2 and M3 receptors and the proportions were not significantly different in the two conditions. Carbachol induced concentration‐dependent contractions which were surmountably antagonized by several muscarinic receptor antagonists (pKB, 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 (<7.00; <7.00) and PD 102807 (<7.00; <7.00). The apparent affinity values obtained in functional studies using the uteri from both sh. and ov. animals correlated most closely with values reported at human recombinant muscarinic M3 receptors. This suggests that the muscarinic M3 receptor mediates contraction under both conditions. Radioligand binding experiments indicate the presence of M2 receptors, in addition to M3 receptors, which probably explains the discrepancies between functional and binding affinities. These data further suggest that the pharmacological profile and proportions of the two populations of muscarinic receptors are unaffected by ovariectomy.

Effect of YM-44781, YM-44778 and YM-49598, Novel Tachykinin Antagonists, in a Drug-Induced Bladder Contraction Model

The radioligand binding profiles and in vivo pharmacological characteristics of YM-44781, YM-44778 and YM-49598, novel non-peptide tachykinin receptor antagonists, were examined and compared to those of FK-888 and GR-159897. Since no functional NK₃ receptors were found in the rat bladder, the emphasis will be on the other two subtypes. YM-44781 and YM-49598 exhibited high binding affinities at NK₂ (pK_i = 9.94 ± 0.03) and NK₁ (pK_i = 9.09 ± 0.02) receptors, respectively, whereas YM-44778 exhibited high binding affinities at both NK₁ (pK_i = 8.08 ± 0.07) and NK₂ (pK_i = 8.55 + 0.04) receptors stably transfected in CHO-K1 cells (Chinese hamster ovary cells). In an in vivo rat model, a drug-induced bladder contraction model, antagonism of the contractions produced by the selective NK₂ receptor agonist, [βAla⁸]neurokinin A (4–10) (10 µg·kg^–1 i.v.) was observed after intravenous administration (dose range 0.001–1 mg·kg^–1) of YM-44781 and YM-44778 (IC₅₀ = 27 ± 8 and 100 ± 44 µg· kg^–1, respectively). YM-44781 was more potent (about 3-fold) than YM-44778. YM-49598 was almost inactive but produced a potent inhibition (IC₅₀ = 11 ± 7 µg·kg^–1) of the contraction of the rat urinary bladder induced by challenge with the NK₁-selective receptor agonist [Sar⁹,Met(O₂)¹¹]substance P sulphone (0.3 µg·kg^–1). YM-44781 and YM-44778 did not produce major inhibition of [Sar⁹,Met(O₂)¹¹]substance P-induced bladder contraction. These findings indicate that YM-44781 and YM-49598 are potent NK₂ and NK₁ receptor antagonists, respectively, whereas YM-44778 is a nonselective NK₂/NK₁ receptor antagonist in the drug-induced bladder contraction model.

In Vitro Isolated Tissue Functional Muscarinic Receptor Assays

Muscarinic receptor (mAChRs) subtypes are viable targets for the design of novel agents for use in a number of central and peripheral disorders. In vitro isolated tissue functional assays for muscarinic receptor subtypes have played an invaluable role in basic research and drug discovery. The availability of biological assays for generation of quantitative estimates of affinity and potency of ligands allows evaluation of the contribution of a given mAChR to the functional end organ response and also enables drug discovery by facilitating the iterative process of screening and optimization of chemical leads. This unit describes isolated tissue functional assays for the quantification of ligand affinity and efficacy at the M1, M2, M3, M4, and M5 muscarinic receptor subtypes in tissues expressing the native receptor using organ bath techniques. Curr. Protoc. Pharmacol. 48:4.15.1‐4.15.29.