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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±0.1fmol/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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Introduction: The purpose of the study is to understand whether the cholinergic stimulation is important, not only in inducing contraction of the detrusor muscle, but also in modulating the proliferation of smooth muscle cells. These results could help to better understand the role of antimuscarinic drugs, which are currently used for the treatment of many urological diseases., Patients and Methods: Primary cultures were prepared from biopsies of human detrusor muscle of subjects >65 years. From the cell culture set-up for each patient, mRNA was extracted and both the gene expression and the influence of increasing passages on the expression of muscarinic receptor subtypes were evaluated by semi-quantitative and quantitative PCR (RT-PCR and Q-RT-PCR). The rate of cell proliferation induced by cholinergic drugs was assessed by the evaluation of the [3H]-thymidine incorporation., Results: The gene expression analysis demonstrated that the range of expression of muscarinic subtypes in human detrusor smooth muscle cells (HDSMCs) is M2 > M3 > M1 > M4 >> M5. The exposure to the cholinergic agonist carbachol induced a concentration-dependent increase in cell proliferation rate. The pharmacological characterization indicated that this effect was mainly mediated by the receptor subtypes M3 and M2., Discussion: The cholinergic stimulation led to an increase in HDSMC proliferation, suggesting that this phenomenon might be involved in the pathogenic mechanism through which the cervico-urethral obstruction causes a detrusor hypertrophy, followed by a loss of function. These results could then provide an indication of the use of antimuscarinic drugs in the treatment of lower urinary tract disorders.

Introduction: In the last years, tissue engineering has attracted lots of researchers, in urology too. This is due to the possibility to use this technique in several pathologies' therapies, which generally require reconstructive surgical solutions. Our work's aim is to evaluate morphological and functional aspects of cultivated urothelial and detrusorial tissues, both in "monolayer growth" and on scaffolds, in order to understand the chance of using them in reconstructive surgery., Materials and Methods: Tissue cultures of detrusorial and urothelial cells have been obtained from animals (pigs, rabbits) and men. The urothelial nature of obtained cells has been demonstrated by traditional histological observation (Hematoxylin - Eosin), by immuno-fluorescence assay (specific for cyto-keratins antibodies), by immuno-histo-chemistry techniques (using specific cyto-keratins 7, 17, and 20 antibodies). Detrusorial tissue has been studied by using antibodies specific for alpha-actin., Results: Urothelial and smooth muscle cells, when isolated and expanded in vitro, keep the typical characteristics of original tissue, as showed by classical histological observation (H-E), immuno-histo-chemistry and immuno-fluorescence assays. These results were positive both in monolayer colonies and on scaffolds. In vitro results are encouraging and they demonstrate that it is possible to obtain in vitro vesical tissue that could have analogous characteristics to the original organ; even though clinical utilisation of this technique must be more investigated, both in vitro and in vivo.

Aims: Muscarinic receptor subtypes expressed in the human urinary bladder mucosa were characterized, investigating whether there were gender-dependent differences and if aging could induce changes in their expression., Methods: The study was carried out on 34 subjects, 22 men and 12 women, divided in four groups, based on gender and age. Gene expression was evaluated by quantitative RT-PCR. The Western blot was performed using the 4-12% NuPAGE Bis-Tris Gel System., Results: The molecular expression of each subtype of the M(1) receptor family was observed and it was not influenced either by gender or age. M(2) receptor family transcripts revealed that both M(2) and M(4) were detected and that the M(2) transcripts were modified by both gender and age. Indeed, M(2) mRNA was lower in old rather than adult men (P < 0.05), but higher in rather old than adult women (P < 0.05). Further, adult men expressed more M(2) mRNA than adult women (P < 0.05), while the opposite was detected in old age (P < 0.05). The Western blot followed by quantification confirmed that the mRNAs were translated into proteins, and that the M(2) subtype showed similar modifications found at molecular level., Discussion: The selective modification of M(2) receptors observed at the urinary bladder mucosa levels indicates that this anatomical structure could play an active role in the pathophysiology of micturition and supports evidence suggesting an effect of antimuscarinic drugs at this level. Whether these results may influence the age-dependent development of micturition disorders remains to be determined.