670 © 2 0 0 5 B J U I N T E R N A T I O N A L | 9 6 , 6 7 0 – 6 7 6 | doi:10.1111/j.1464-410X.2005.05703.x Original Article CONNEXIN EXPRESSION IN THE BLADDER NEUHAUS et al. Alterations in connexin expression in the bladder of patients with urge symptoms JOCHEN NEUHAUS, FRIEDERIKE PFEIFFER*, HARTWIG WOLBURG*, LARS-CHRISTIAN HORN† and WOLFGANG DORSCHNER Departments of Urology and †Pathology, University of Leipzig, Leipzig, and *Department of Pathology, University of Tubingen, Tubingen, Germany Accepted for publication 21 March 2005 transurethral tumour resection for bladder cancer were used as controls. Tissue samples from patients with severe idiopathic urge symptoms were taken for exclusion diagnostics of interstitial cystitis (IC) and carcinoma in situ. The formation of functional syncytia between detrusor smooth muscle cells were examined in dye-coupling experiments by injecting with Lucifer Yellow. The morphology and structure of gap junctions were assessed by transmission electron microscopy and immunogold labelling of Cx43 and Cx45. The expression of connexin subtypes Cx40, Cx43 and Cx45 was compared by indirect immunofluorescence, and confocal laser scanning microscopy used for semiquantitative analysis. RESULTS There was dye coupling between smooth muscle cells of the detrusor in situ. Electron microscopy and immunogold labelling showed very small gap junctional plaques. These findings were confirmed by confocal immunofluorescence. Semiquantitative analyses showed significantly higher Cx43 expression in the detrusor muscle, and a tendency to higher Cx45 expression in the suburothelial layer associated with urge symptoms, whereas Cx40 expression was unaffected. CONCLUSIONS Smooth muscle cells of the human detrusor muscle are coupled by classical gap junctions, forming limited local functional syncytia. Both Cx43 and Cx45 are expressed at low levels in normal detrusor. Up-regulation of Cx43 in patients with urge incontinence supports the possibility of functional changes in the syncytial properties of detrusor smooth muscle cells in this condition. In addition, the observed increase of Cx45 in the myofibroblast cell layer supports the idea that alterations in sensory signalling are also involved. Comparison with previous reports implies that the pathophysiology of urgency is distinct from that of the unstable bladder and other forms of incontinence. KEYWORDS gap junction, electron microscopy, functional syncytium, myofibroblast, smooth muscle cell, urge incontinence OBJECTIVE To compare the formation of gap junctions between detrusor smooth muscle cells in situ and the distribution of connexin (Cx)40, Cx43 and Cx45 expressions in bladder biopsies from a control group (with bladder tumour) and from patients with urge symptoms, as smooth muscle cells of the human detrusor muscle communicate via gap junctions and express several connexin subtypes, alterations of which may be involved in the causes of lower urinary tract symptoms. MATERIALS AND METHODS Connexin expression is prominent in myofibroblast-like cells, supposedly involved in afferent signalling pathways of the bladder. Their strategic position directly beneath the urothelium suggests they are a link between urothelial ATP signalling during bladder filling and afferent Ad-fibre stimulation for co- ordination of bladder tonus and initialization of the micturition reflex. Modification of their coupling characteristics may have profound impact on bladder sensation. Bladder tissue probes of patients undergoing cystectomy or INTRODUCTION Coupling between smooth muscle cells of the detrusor muscle is evident in normal human urinary bladder [1–4]. Connexin (Cx) 43 and Cx45 are the predominant connexins expressed in human urinary bladder, as shown by RT-PCR [2], immunohistochemistry and Western blot analysis [3,4]. While connexin expression in normal bladder is usually low in detrusor muscle, strong Cx43 expression was found in a suburothelial cell layer [4]. These cells stained positively for vimentin and a-actin and negative for desmin on immunofluorescence, and had the ultrastructural characteristics of myofibroblasts [4]. The suburothelial myofibroblasts are supposedly a major component of the afferent signalling system of the urinary bladder, serving as an amplifier and a relay between urothelium and Ad sensory nerve fibres [5]. ATP release on stretch activation by urothelium can be detected by the suburothelial myofibroblasts, leading to higher tonus or contraction [6]. Recently ATP activation of suburothelial myofibroblasts via metabotropic P2Y receptors, possibly of the P2Y2 or P2Y4 subtype, was reported in an electrophysiological study in guinea pigs [7]. The functional myofibroblast syncytium might not only serve to amplify the urothelial signal, but modulating the coupling strength between the cells would influence the travel distance of the signal within the syncytium, and consequently the number of Ad-fibres stimulated. As the myofibroblasts are susceptible to various other chemical activators they are an ideal candidate for signal integration [5]. The importance of ATP signalling under pathological conditions has been implicated in idiopathic detrusor instability [8,9] and in patients with BOO [10]. Recently, Birder et al. [11] found down-regulation of P2X1 and P2Y2 purinergic receptors in cats with feline interstitial cystitis, in which ATP release from urothelium is increased; Wada et al. [12] reported an increase in bladder sensitivity to purinergic stimulation in rats after experimental BOO. These results suggest