Muscarinic Acetylcholine Receptor M3 Mutation Causes Urinary Bladder Disease and a Prune-Belly-like Syndrome

Urinary bladder malformations associated with bladder outlet obstruction are a frequent cause of progressive renal failure in children. We here describe a muscarinic acetylcholine receptor M3 (CHRM3) (1q41-q44) homozygous frameshift mutation in familial congenital bladder malformation associated with a prune-belly-like syndrome, defining an isolated gene defect underlying this sometimes devastating disease. CHRM3 encodes the M3 muscarinic acetylcholine receptor, which we show is present in developing renal epithelia and bladder muscle. These observations may imply that M3 has a role beyond its known contribution to detrusor contractions. This Mendelian disease caused by a muscarinic acetylcholine receptor mutation strikingly phenocopies Chrm3 null mutant mice.     

Hypertrophy changes the muscarinic receptor subtype mediating bladder contraction from M3 toward M2

Major pelvic ganglion electrocautery (MPGE) and spinal cord injury in the rat induce bladder hypertrophy and a change in muscarinic receptor subtypes mediating bladder contraction from predominantly M3 to a combination of M2 and M3. To determine whether this is a result of bladder hypertrophy or denervation, we studied the following groups: sham-operated controls, urinary diversion (DIV), MPGE together with urinary diversion (DIV-DEN), bilateral MPGE (DEN), bladder outlet obstruction (BOO), and MPG decentralization (MPGDEC). The degree of bladder denervation was determined by the maximal carbachol response normalized to the response to electric field stimulation. Receptor subtype density was determined by immunoprecipitation. The affinity of subtype-selective muscarinic antagonists for inhibition of carbachol-induced contractions was used to determine the subtype-mediating contraction. DEN, MPG-DEC, and BOO bladders were hypertrophic whereas DIV bladders were atrophic compared with sham operated. Bladder contraction in sham-operated, DIV, and DIV-DEN was mediated by the M3 receptor subtype, whereas the M2 subtype participated in contraction in the DEN, MPG-DEC, and BOO groups. The hypertrophied bladders had an increase in total and M2 receptor density while all experimental groups showed a reduction in M3 receptor density. Thus bladder hypertrophy, independent from bladder denervation, causes a shift in the muscarinic receptor subtype mediating bladder contraction from M3 toward M2.     

Muscarinic receptor subtypes modulating smooth muscle contractility in the urinary bladder

Normal physiological voiding as well as generation of abnormal bladder contractions in diseased states is critically dependent on acetylcholine-induced stimulation of contractile muscarinic receptors on the smooth muscle (detrusor) of the urinary bladder. Muscarinic receptor antagonists are efficacious in treating the symptoms of bladder hyperactivity, such as urge incontinence, although the usefulness of available drugs is limited by undesirable side-effects. Detrusor smooth muscle is endowed principally with M2 and M3 muscarinic receptors with the former predominating in number. M3 muscarinic receptors, coupled to stimulation of phosphoinositide turnover, mediate the direct contractile effects of acetylcholine in the detrusor. Emerging evidence suggests that M2 muscarinic receptors, via inhibition of adenylyl cyclase, cause smooth muscle contraction indirectly by inhibiting sympathetically (beta-adrenoceptor)-mediated relaxation. In certain diseased states, M2 receptors may also contribute to direct smooth muscle contraction. Other contractile mechanisms involving M2 muscarinic receptors, such as activation of a non-specific cationic channel and inactivation of potassium channels, may also be operative in the bladder and requires further investigation. From a therapeutic standpoint, combined blockade of M2 and M3 muscarinic receptors would seem to be ideal since this approach would evoke complete inhibition of cholinergically-evoked smooth muscle contractions. However, if either the M2 or M3 receptor assumes a greater pathophysiological role in disease states, then selective antagonism of only one of the two receptors may be the more rational approach. The ultimate therapeutic strategy is also influenced by the extent to which pre-junctional M1 facilitatory and M2 inhibitory muscarinic receptors regulate acetylcholine release and also which subtypes mediate the undesirable effects of muscarinic receptor blockade such as dry mouth. Finally, the consequence of muscarinic receptor blockade in the central nervous system on the micturition reflex, an issue which is poorly studied and seldom taken into consideration, should not be ignored.     

In vivo demonstration of M3 muscarinic receptor subtype selectivity of darifenacin in mice

A novel muscarinic receptor antagonist, darifenacin, inhibited specific binding of [N-methyl-(3)H]scopolamine ([(3)H]NMS) in the mouse bladder, submaxillary gland and heart in a concentration-dependent manner. The inhibitory effect was most potent in the submaxillary gland, followed by the bladder and heart. In addition, darifenacin inhibited specific [(3)H]NMS binding in the membranes of CHO-K1 cell lines expressing muscarinic M(2) and M(3) receptor subtypes, and the potency was significantly (22-fold) greater at the M(3) than at the M(2) subtype. At 0.5 to 12 h after oral administration of darifenacin, a significant increase in K(d) values for specific [(3)H]NMS binding was seen in the bladder, submaxillary gland and lung of mice, compared with control values. Also, there was a sustained decrease in the B(max) values in the submaxillary gland. These data suggest that muscarinic receptor binding of oral darifenacin is rapid in onset and of a long duration. On the other hand, oral darifenacin exerted only temporary or little binding of muscarinic receptors in the heart and colon. Pilocarpine-induced salivary secretion in mice was continuously suppressed by oral darifenacin. The time-course of suppression coincided well with that for the muscarinic receptor binding in the submaxillary gland. The antagonistic effect of darifenacin against the dose-response curves for pilocarpine appeared to be insurmountable. In conclusion, the present study has shown that oral darifenacin may exert a pronounced and long-lasting binding of muscarinic receptors in tissues expressing the M(3) subtype.     

The M2 muscarinic receptor mediates in vitro bladder contractions from patients with neurogenic bladder dysfunction

Bladder muscle specimens from seven patients with neurogenic bladder dysfunction were analyzed to determine whether the muscarinic receptor subtype mediating contraction shifts from M(3) to the M(2) subtype as found in the denervated, hypertrophied rat bladder. Seven bladder specimens were analyzed from six female and one male patients. Six of the patients had traumatic cervical spinal cord injuries (C(4)-C(7)), and the other patient had an L(1) congenital myelomeningocele. This was compared with results from bladder specimens obtained from eight organ transplant donors. The affinities of three subtype-selective muscarinic receptor antagonists for inhibition of carbachol-induced contractions were determined. The affinity of the M(3) selective antagonists darifenacin or p-fluoro-hexahydrosiladifenadol (p-F-HHSiD) was determined in six of the seven spinal injury patient specimens. The affinity was consistent with M(2)-mediated contractions in four of these six specimens, intermediate between M(2) and M(3) in one specimen, and within the M(3) range in one specimen. The other specimen, tested only with the M(2) selective antagonist methoctramine, showed an M(3) affinity. In the organ donors, the affinity of p-F-HHSiD was within the M(2) range for six of seven specimens, whereas the affinity of darifenacin was within the M(3) range for five of six and intermediate between M(2) and M(3) for the other specimen tested. The affinity of methoctramine in both organ donor specimens tested was within the M(3) range. Whereas normal detrusor contractions are mediated by the M(3) receptor subtype, in patients with neurogenic bladder dysfunction as well as certain organ transplant donors, contractions can be mediated by the M(2) muscarinic receptor subtype.     

Pharmacological profile of enantiomerically pure chiral muscarinic agonists: desoxymuscarines

The enantiomers desoxymuscarine 6 were tested in vitro on guinea pig tissues, and their muscarinic potency was evaluated at M2 (heart force and rate) and M3 (ileum and bladder) receptor subtypes together with the enantiomers of the parent compound muscarine 1. The eutomers (+)-1 and (+)-6 and distomers (-)-1 and (-)-6 were also assayed in vivo on pithed rat. Affinity, relative efficacy and enantio-selectivity were also determined for the compounds under study at M2 (heart force and rate) and M3 (ileum and bladder), in order to investigate muscarinic receptor heterogeneity. The results of this study have been discussed in comparison with the data previously reported for the structurally related fluoromuscarine (+)-4 and difluoromuscarines (+)-5 and (-)-5.     

Quinuclidin-2-ene-based muscarinic antagonists

A series of achiral 3-heteroaryl substituted quinuclidin-2-ene derivatives and related compounds have been synthesized by facile methods. The compounds were evaluated for muscarinic and antimuscarinic properties in receptor binding studies using (-)-[3H]-QNB as the radioligand and in a functional assay using isolated guinea pig urinary bladder. 3-(2-Benzofuranyl)-quinuclidin-2-ene (15) displayed the highest M1-receptor affinity in the present series (Ki = 9.6 nM).     

Ca2+ mobilization and activation of extracellular acidification by carbachol in acutely dispersed cells from guinea pig detrusor: Fura 2 fluorometry and microphysiometry using the cytosensor

The study aim was to develop a simple in vitro model for pharmacophysiological investigation of urinary bladder smooth muscles. Smooth muscle cells from guinea pig detrusor were dissociated, and the suspended cells were stimulated with carbachol (CCh), an acetylcholine receptor agonist. Cytosolic Ca2+ levels were determined using Fura 2 fluorescence and extracellular acidification rates were monitored by the Cytosensor microphysiometer. CCh dose-dependently increased cytosolic Ca2+ levels and extracellular acidification rates, with EC50 values of approximately 1 microM. Both the acetylcholine muscarinic receptor antagonist atropine and the M3 muscarinic receptor-preferring antagonist 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) inhibited the effects of CCh, three orders of magnitude more potently than the selective M2 muscarinic receptor antagonist, methoctramine. These data indicate the dominant role of M3 receptors in guinea-pig bladder but fail to show clear evidence of any functional role for M2 receptors. Since this finding agrees with a number of other studies using in vivo and in vitro models (1), cell suspensions such as these may prove to be simple tools for the pharmacological study of urinary bladder smooth muscle tissue.     

Synthesis and antimuscarinic activity of a series of 4-(1-Imidazolyl)-2,2-diphenylbutyramides: discovery of potent and subtype-selective antimuscarinic agents.

In a study directed toward the development of new, selective agents with potential utility in the treatment of altered smooth muscle contractility and tone, for example, as seen in urinary incontinence associated with bladder muscle instability, a series of 4-(1-imidazolyl)-2,2-diphenylbutyramide derivatives was prepared. These compounds were examined for M1, M2, and M3 muscarinic receptor subtype selectivity in isolated tissue assays. The compounds that showed potency and/or selectivity in these tests were further evaluated for in vivo anticholinergic effects on various organs and tissues, including urinary bladder, salivary gland, and eye in rats. The structure activity relationships for the series of 4-(1-imidazolyl)-2,2-diphenylbutyramide derivatives are also discussed. This study led to the identification of 4-(2-methyl-1-imidazolyl)-2,2-diphenylbutyramide (KRP-197) as a candidate drug for the treatment of urinary bladder dysfunction.     

M(3)-receptor knockout mice: muscarinic receptor function in atria, stomach fundus, urinary bladder, and trachea

Negative chronotropic and smooth muscle contractile responses to the nonselective muscarinic agonist carbamylcholine were compared in isolated tissues from M(3)-muscarinic receptor knockout and wild-type mice. Carbamylcholine (10(-8)-3.0 x 10(-5) M) induced a concentration-dependent decrease in atrial rate that was similar in atria from M(3)-receptor knockout and wild-type mice, indicating that M(3) receptors were not involved in muscarinic receptor-mediated atrial rate decreases. In contrast, the M(3) receptor was a major muscarinic receptor involved in smooth muscle contraction of stomach fundus, urinary bladder, and trachea, although differences existed in the extent of M(3)-receptor involvement among the tissues. Contraction to carbamylcholine was virtually abolished in urinary bladder from M(3)-receptor knockout mice, suggesting that contraction was predominantly due to M(3)-receptor activation. However, approximately 50-60% maximal contraction to carbamylcholine occurred in stomach fundus and trachea from M(3)-receptor knockout mice, indicating that contraction in these tissues was also due to M(2)-receptor activation. High concentrations of carbamylcholine relaxed the stomach fundus from M(3)-receptor knockout mice by M(1)-receptor activation. Thus M(3)-receptor knockout mice provided unambiguous evidence that M(3) receptors 1) play no role in carbamylcholine-induced atrial rate reduction, 2) are the predominant receptor mediating carbamylcholine-induced urinary bladder contractility, and 3) share contractile responsibility with M(2) receptors in mouse stomach fundus and trachea.     

Design, synthesis, and biological evaluation of pirenzepine analogs bearing a 1,2-cyclohexanediamine and perhydroquinoxaline units in exchange for the piperazine ring as antimuscarinics

Pirenzepine (2) is one of the most selective muscarinic M(1) versus M(2) receptor antagonists known. A series of 2 analogs, in which the piperazyl moiety was replaced by a cis- and trans-cyclohexane-1,2-diamine (3-6) or a trans- and cis-perhydroquinoxaline rings (7 and 8) were prepared, with the aim to investigate the role of the piperazine ring of 2 in the interaction with the muscarinic receptors. The structural change leading to compounds 3-6 abolished in binding assays the muscarinic M(1)/M(2) selectivity of 2, due to an increased M(2) affinity. Rather, compounds 3-6 displayed a reversed selectivity showing more affinity at the muscarinic M(2) receptor than at all the other subtypes tested.     

Muscarinic antagonists in development for disorders of smooth muscle function

Compounds with high affinity for muscarinic M3 receptors have been used for many years to treat conditions associated with altered smooth muscle tone or contractility such as urinary urge incontinence, irritable bowel syndrome or chronic obstructive airways disease. M3 selective antagonists have the potential for improved toleration when compared with non-selective compounds. Darifenacin has high affinity (pKi 9.12) and selectivity (9 to 74-fold) for the human cloned muscarinic M3 receptor. Consistent with this profile, the compound potently inhibited M3 receptor mediated responses of smooth muscle preparations (guinea pig ileum, trachea and bladder, pA2 8.66 to 9.4) with selectivity over responses mediated through the M1 (pA2 7.9) and M2 receptors (pA2 7.48). Interestingly, darifenacin also exhibited functional tissue selectivity for intestinal smooth muscle over the salivary gland. The M3 over M1 and M2 selectivity of darifenacin was confirmed in a range of animal models. In particular, in the conscious dog darifenacin inhibited intestinal motility at doses lower than those which inhibit gastric acid secretion (M1 response), increase heart rate (M2 response) or inhibit salivary secretion. Clinical studies are ongoing to determine if darifenacin has improved efficacy and or toleration when compared with non-selective agents.     

In vitro and in vivo antimuscarinic effects of (-)cis 2,3-dihydro-3-(4-methylpiperazinylmethyl)-2-phenyl-1,5 benzothiazepin-4-(5H)one HCl (BTM-1086) in guinea pig peripheral tissues

The potency and selectivity of (-)cis-2,3-dihydro-3-(4-methylpiperazinylmethyl)-2-phenyl-1,5 benzothiazepin-4-(5H)one HCl (BTM-1086) for muscarinic receptor subtypes was compared in functional assay systems, in guinea pig peripheral tissues, to known reference drugs: atropine (nonselective), pirenzepine (M1), AF-DX 116 (M2) and HHSiD (M3). Like atropine, BTM-1086 was a potent, nonselective, competitive muscarinic antagonist with no detectable antispasmodic activity in urinary bladder or ileal muscle. In vivo, in the guinea pig cystometrogram, BTM-1086 depressed intravesical bladder pressure (PvesP) with the same efficacy and potency as oxybutynin, a drug used clinically for the treatment of urinary incontinence. The pharmacological profile of BTM-1086, however, suggests that it may not be suitable for development for bladder dysfunction disorders.     

New analogues of oxotremorine and oxotremorine-M: estimation of their in vitro affinity and efficacy at muscarinic receptor subtypes

Two subsets of tertiary amines (1a-6a) and methiodides (1b-6b) with a structural resemblance to oxotremorine and oxotremorine-M were tested at rabbit vas deferens (M1), guinea pig left atrium (M2), guinea pig ileum and urinary bladder (M3) muscarinic receptor subtypes. The pharmacological profile of the derivatives under study has been discussed by evaluating their potency, affinity and efficacy as well as the regional differences in muscarinic receptor occupancy.     

4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]-trimethylammonium (McN-A-343)-related compounds. Effect of the butynyl chain inclusion into an aromatic unit on the potency for muscarinic receptors

A series of derivatives of the known M1 selective muscarinic receptor agonist McN-A-343 (1) was designed with the aim of investigating the effects of structural variations on both the butynyl chain and the phenyl ring of 1. The butynyl chain was replaced with an aromatic spacer, and the effects of such a modification on the stereoelectronic properties of the molecules were theoretically studied and considered compatible with muscarinic receptor affinity. Substituents on the phenyl ring of 1 were selected so as to vary their electronic and hydrophobic properties. This design strategy did not produce muscarinic M1 receptor agonists more potent than the prototype 1, even if some analogues displayed functional selectivity for different muscarinic receptor subtypes. Compounds 3 and 7 were selective agonists towards muscarinic M3 receptors, while compounds 14, 16 and 18 were selective muscarinic M2 receptor agonists. The most interesting derivative was 8, a full agonist at muscarinic M3 receptors devoid of activity at both muscarinic M1 and M2 subtypes. The pharmacological profile of the series was further characterized by studying the anticholinesterase and miotic activities of some representative compounds. Compounds 3-8 turned out to be weak acetylcholinesterase inhibitors, while derivatives 4, 6, 8 and 11 were able to significantly reduce the pupillary diameter in rabbit, indicating 8 as an effective miotic agent.     

Clinical experiences with tolterodine

Tolterodine is the first muscarinic receptor antagonist that has been specifically developed for the treatment of overactive bladder. The objectives in the discovery program were to design a potent muscarinic receptor antagonist that is equipotent to oxybutynin in the bladder, but less potent in salivary glands, with the aim of improving tolerability (less dry mouth) in patients with overactive bladder. Tolterodine is non-selective with respect to the muscarinic M1-M5 receptor subtypes, but has a greater effect on the bladder than on salivary glands in vivo, in both animals and humans. Clinical results show that the efficacy and safety of tolterodine in overactive bladder is equal to that of oxybutynin, but that tolterodine is significantly better tolerated by the patients.     

Synthesis and optimization of novel and selective muscarinic M(3) receptor antagonists

A series of constrained piperidine analogues were synthesized as novel muscarinic M(3) receptor antagonists. Evaluation of these compounds in binding assays revealed that they not only have high affinity for the M(3) receptor but also have high selectivity over the M(2) receptor.     

Muscarinic receptors and gastrointestinal tract smooth muscle function

Over the last decade, several lines of evidence have shown that both muscarinic M2 and M3 receptors are postjunctionally expressed in many smooth muscles, including the gastrointestinal tract. Although in vitro data suggests that both receptors are functional in that they inhibit adenylate cyclase activity and activate non-selective cation channels, few studies support a role in vivo. Thus, data from procedures that ablate the signaling pathway of the muscarinic M2 receptor, including receptor antagonism, pertussis toxin pretreatment reveal little effect on gastrointestinal smooth muscle responsiveness to muscarinic agonists. Recently, information from knockout mice, lacking either M2 or M3 receptor, indicate reveal a role for both subtypes. However, the contribution of the M2 receptor appears greater in the ileum than in the urinary bladder. Therapeutically, non-selective, as well as selective M3 receptor antagonists are being clinically studied, although it remains to be shown which is the optimal approach to disorders of smooth muscle motility.     

Selective alkylation of rat urinary bladder muscarinic receptors with 4-DAMP mustard reveals a contractile function for the M2 muscarinic receptor.

Our previous data indicate that M3 muscarinic receptors mediate carbachol induced bladder contractions. The data presented here were obtained by selective alkylation of M3 receptors with 4-DAMP mustard and suggest that the M2 receptor subtype may be involved in inhibition of beta-adrenergic receptor induced relaxation, therefore, allowing recontraction. Alkylation resulted in 85% of M3 receptors and 65% of M2 receptors unable to bind radioligand as demonstrated by subtype selective immunoprecipitation. Rat bladder strips subjected to our alkylation procedure contracted submaximally, and direct carbachol contractions were inhibited by antagonists with affinities consistent with M3 receptor mediated contraction. In contrast, the affinities of antagonists for inhibition of carbachol induced recontractions following isoproterenol stimulated relaxation in the presence of 90 mM KCl, indicated a contractile function for the M2 receptor that was not observed in control strips. In conclusion, these studies demonstrate a possible role for the M2 subtype in bladder smooth muscle contraction.     

M2 receptors in genito-urinary smooth muscle pathology

In vitro bladder contractions in response to cumulative carbachol doses were measured in the presence of selective muscarinic antagonists from rats which had their major pelvic ganglion bilaterally removed (denervation, DEN) or from rats in which the spinal cord was injured (SCI) via compression. DEN induced both hypertrophy (505+/-51 mg bladder weight) and a supersensitivity of the bladders to carbachol (EC50=0.7+/-0.1 uM). Some of the SCI rats regained the ability to void spontaneously (SPV). The bladders of these animals weighed 184+/-17 mg, significantly less than the bladders of non voiding rats (NV, 644+/-92 mg). The potency of carbachol was greater in bladder strips from NV SCI animals (EC50=0.54+/-0.1 uM) than either bladder strips from SPV SCI (EC50=0.93+/-0.3 microM), DEN or control (EC50=1.2+/-0.1 microM) animals. Antagonist affinities in control bladders for antagonism of carbachol induced contractions were consistent with M3 mediated contractions. Antagonist affinities in DEN bladders for 4-diphenlacetoxy-N-methylpiperidine methiodide (4-DAMP, 8.5) and para fluoro hexahydrosilodifenidol (p-F-HHSiD, 6.6); were consistent with M2 mediated contractions, although the methoctramine affinity (6.5) was consistent with M3 mediated contractions. p-F-HHSiD inhibited carbachol induced contraction with an affinity consistent with M2 receptors in bladders from NV SCI (pKb=6.4) animals and M3 receptors in bladders from SPV SCI animals (pKb=7.9). Subtype selective immunoprecipitation of muscarinic receptors revealed an increase in total and an increase in M2 receptor density with no change in M3 receptor density in bladders from DEN and NV SCI animals compared to normal or sham operated controls. M3 receptor density was lower in bladders from SPV SCI animals while the M2 receptor density was not different from control. This increase in M2 receptor density is consistent with the change in affinity of the antagonists for inhibition of carbachol induced contractions and may indicate that M2 receptors or a combination of M2 and M3 receptors directly mediate smooth muscle contraction in bladders from DEN and NV SCI rats.