Muscarinic receptor subtypes and signalling involved in the attenuation of isoprenaline-induced rat urinary bladder relaxation.

Depts. of Pharmacology and Pharmacotherapy and of Urology, Academic Medical Center, Amsterdam, The Netherlands.
Archiv für Experimentelle Pathologie und Pharmakologie (Impact Factor: 2.15). 09/2011; 384(6):555-63. DOI: 10.1007/s00210-011-0689-8
Source: PubMed

ABSTRACT β-Adrenoceptors are important mediators of smooth muscle relaxation in the urinary bladder, but the concomitant presence of a muscarinic agonist, e.g., carbachol, can attenuate relaxation responses by reducing potency and/or efficacy of β-adrenoceptor agonists such as isoprenaline. Therefore, the present study was designed to explore the subtypes and signalling pathways of muscarinic receptors involved in the attenuation of isoprenaline-induced isolated rat detrusor preparations using novel subtype-selective receptor ligands. In radioligand binding studies, we characterized BZI to be a M(3)-sparing muscarinic agonist, providing selective M(2) stimulation in rat bladder, and THRX-182087 as a highly M(2)-selective antagonist. The use of BZI and of THRX-182087 in the presence of carbachol enabled experimental conditions with a selective stimulation of only M(2) or M(3) receptors, respectively. Confirming previous findings, carbachol attenuated isoprenaline-induced detrusor relaxation. M(2)-selective stimulation partly mimicked this attenuation, indicating that both M(2) and M(3) receptors are involved. During M(3)-selective stimulation, the attenuation of isoprenaline responses was reduced by the phospholipase C inhibitor U 73,122 but not by the protein kinase C inhibitor chelerythrine. We conclude that both M(2) and M(3) receptors contribute to attenuation of β-adrenoceptor-mediated relaxation of rat urinary bladder; the signal transduction pathway involved in the M(3) component of this attenuation differs from that mediating direct contractile effects of M(3) receptors.

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    ABSTRACT: While acetylcholine (ACh) and muscarinic receptors in the bladder are mainly known for their role in the regulation of smooth muscle contractility, in other tissues they are involved in tissue remodelling and promote cell growth and proliferation.In the present study we have used primary cultures of human detrusor smooth muscle cells (HDSMCs), in order to investigate the role of muscarinic receptors in HDSMC proliferation. Samples were obtained as discarded tissue from men >65 years undergoing radical cystectomy for bladder cancer and cut in pieces that were either immediately frozen or placed in culture medium for the cell culture establishment. HDSMCs were isolated from samples, propagated and maintained in culture. [(3)H]-QNB radioligand binding on biopsies revealed the presence of muscarinic receptors, with a Kd of 0.10±0.02nM and a Bmax of 72.8±0.1 fmol/mg protein. The relative expression of muscarinic receptor subtypes, based on Q-RT-PCR, was similar in biopsies and HDSMC with a rank order of M2≥M3>M1>M4>M5. The cholinergic agonist carbachol (CCh, 1-100μM) concentration-dependently increased [(3)H]-thymidine incorporation (up to 46±4%). This was concentration-dependently inhibited by the general muscarinic receptor antagonist atropine and by subtype-preferring antagonists with an order of potency of darifenacin >4-DAMP>AF-DX 116. The CCh-induced cell proliferation was blocked by selective PI-3 kinase and ERK activation inhibitors, strongly suggesting that these intracellular pathways mediate, at least in part, the muscarinic receptor-mediated cell proliferation. This work shows that M2 and M3 receptors can mediate not only HDSM contraction but also proliferation; they may also contribute bladder remodelling including detrusor hypertrophy.
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    ABSTRACT: Activation of β3-adrenoceptors has been shown to have a direct relaxant effect on urinary bladder smooth muscle from both rats and humans, however there are very few studies investigating the effects of β3-adrenoceptor agonists on nerve-evoked bladder contractions. Therefore in the current study, the role of β3-adrenoceptors in modulating efferent neurotransmission was evaluated. The effects of β3-adrenoceptor agonism on neurogenic contractions induced by electrical field stimulation (EFS) were compared with effects on contractions induced by exogenous acetylcholine (Ach) and αβ-methylene adenosine triphosphate (αβ-meATP) in order to determine the site of action. Isoproterenol inhibited EFS-induced neurogenic contractions of human bladder (pD2=6.79; Emax=65%). The effect of isoproterenol was selectively inhibited by the β3-adrenoceptor antagonist L-748,337 (pKB=7.34). Contractions induced by exogenous Ach (0.5-1μM) were inhibited 25% by isoproterenol (3μM) while contractions to 10Hz in the same strip were inhibited 67%. The selective β3-adrenoceptor agonist CL-316,243 inhibited EFS-induced neurogenic contractions of rat bladder (pD2=7.83; Emax=65%). The effects of CL-316,243 were inhibited in a concentration dependent manner by L-748,337 (pA2=6.42). Contractions induced by exogenous Ach and αβ-meATP were significantly inhibited by CL-316,243, 29% and 40%, respectively. These results demonstrate that the activation of β3-adrenoceptors inhibits neurogenic contractions of both rat and human urinary bladder. Contractions induced by exogenously applied parasympathetic neurotransmitters are also inhibited by β3-agonism however the effect is clearly less than on neurogenic contractions (particularly in human), suggesting that in addition to a direct effect on smooth muscle, activation of prejunctional β3-adrenoceptors may inhibit neurotransmitter release.
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    ABSTRACT: Muscarinic receptor antagonists and β-adrenoceptor agonists are used in the treatment of obstructive airway disease and overactive bladder syndrome. Here we review the pharmacological rationale for their combination. Muscarinic receptors and β-adrenoceptors are physiological antagonists for smooth muscle tone in airways and bladder. Muscarinic agonism may attenuate β-adrenoceptor-mediated relaxation more than other contractile stimuli. Chronic treatment with one drug class may regulate expression of the target receptor but also that of the opposing receptor. Prejunctional β2-adrenoceptors can enhance neuronal acetylcholine release. Moreover, at least in the airways, muscarinic receptors and β-adrenoceptors are expressed in different locations, indicating that only a combined modulation of both systems may cause dilatation along the entire bronchial tree. While all of these factors contribute to a rationale for a combination of muscarinic receptor antagonists and β-adrenoceptor agonists, the full value of such combination as compared to monotherapy can only be determined in clinical studies.
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