Altered muscarinic receptor subtype expression and functional responses in cyclophosphamide induced cystitis in rats
Introduction
The muscarinic receptor population of the urinary bladder is heterogeneous and different bladder tissue structures express different subtypes. On the detrusor cell muscarinic M2 receptors occur in vast majority over the muscarinic M3 receptors (Longhurst et al., 1995, Wang et al., 1995). In spite of its low number, and the fact that the functional role of the numerous muscarinic M2 receptors is only partly understood, the muscarinic M3 receptors are considered to mediate all, or at least a major part, of the cholinergic contractile response (Giglio et al., 2001, Hegde et al., 1997). Nerve terminals exhibit facilitator and inhibitory muscarinic receptors classified as muscarinic M1 and M2/M4 receptors, respectively, which regulate the release of acetylcholine (D'Agostino et al., 2000, Somogyi et al., 1994, Tobin and Sjogren, 1995, Tobin and Sjogren, 1998). Muscarinic receptors have also been described within the urothelium and urothelial factors may indirectly modify the bladder cholinergic responses (Fovaeus et al., 1999, Hawthorn et al., 2000). Under pathological conditions, the expression and functional roles of the different muscarinic receptor subtypes may be altered in the bladder (Braverman et al., 1999, Somogyi and de Groat, 1999) and during bladder inflammation, in patients as well as experimentally, the cholinergic response is substantially affected resulting in a decrement of the contractile response (Mok et al., 2000, Palea et al., 1993).
In patients with interstitial cystitis, it has been reported that afferent pathways are sensitised and the release of several substances, such as adenosine triphosphate, prostanoids and nitric oxide, has been associated to the condition (Andersson, 2002). Of these mediators, nitric oxide seems to have a pivotal role as a signalling molecule, mediating many of the pathophysiological events in haemorrhagic cystitis (Logadottir et al., 2004, Oter et al., 2004). However, nitric oxide synthase has been demonstrated in nerve fibres in the bladder and nitric oxide has therefore been suggested to influence efferent as well as afferent neurotransmission also in the normal bladder (Andersson and Persson, 1995).
In the present study, we addressed the issue whether or not experimentally induced cystitis in the rat may affect the cholinergic efferent pathways that elicit the detrusor contractile responses, and if the expression of the detrusor muscarinic receptors is altered concomitantly. In view of the pivotal role of nitric oxide in some forms of cystitis, any muscarinic effects via nitric oxide production was also looked for. In order to induce experimental cystitis, we pretreated rats with cyclophosphamide, a cytostatic widely used in the treatment of neoplastic diseases that is bladder toxic and induces haemorrhagic cystitis (deVries and Freiha, 1990). In the in vitro functional studies of carbachol-evoked responses of control and cyclophosphamide-treated bladders, various muscarinic antagonists were employed (see Eglen et al., 1996): pirenzepine (“M1-selective”), methoctramine (“M2-selective”), 4-diphenylacetoxy-N-methylpiperidine (4-DAMP; “M3/M1/M5-selective”) as well as the non-selective nitric oxide synthase inhibitor N-ω-nitro-l-arginine (Dubbin et al., 1990). Furthermore, semi-quantification Western blot analysis of muscarinic M1–M5 receptors was performed, and in order to determine which type of muscarinic receptor that occurs in the different structures within the bladder, immunohistochemistry employing antisera specific to the muscarinic receptor subtypes (M1–M5) was performed. A preliminary report of some of these results has been published previously (Giglio et al., 2004).
Section snippets
Methods and materials
The ethics committee of the Göteborg University approved the study design, in which 73 male rats (300–350 g) of the Sprague–Dawley strain were used. Chemical cystitis was induced by a single intraperitoneal injection of cyclophosphamide (100 mg/kg) and controls received saline (9 mg/ml). Both types of injection were conducted in the presence of the analgetic buprenorphinum (10 μg/kg IM). At 36, 48, 60 or 100 h subsequent to the pretreatment, the rats were anaesthetized with pentobarbitone (40
Characteristics of the cyclophosphamide bladder
The cyclophosphamide-treated rats exhibited macroscopical signs of bladder inflammation, i.e. redness, oedema and wall thickening. However, no sign of deterioration of the animals' general condition were observed, and neither could any sign of bleeding be traced in the urine. The macroscopical observations were also confirmed under light microscopy (see below; all bladders of the cyclophosphamide-treated rats showed signs of inflammation). The inflammation resulted in an increase of the wet
Discussion
In the current study, immunostaining revealed muscarinic M2 and M3 receptors to be located on the smooth muscle cells similarly in controls and cyclophosphamide-treated animals. The immunostaining also seems to confirm the occurrence of muscarinic M1 and M4 on nerve fibres in the rat urinary bladder, since they were visualized in the suburothelial connective tissue of the animals. Muscarinic receptors were also demonstrated in the urothelium although the staining in the control specimens for
Conclusions
The current study shows that in the cyclophosphamide-induced inflamed bladder, the expression of muscarinic receptors is markedly altered in the urothelium but not in other parts of the bladder. The treatment results in the appearance of muscarinic M2, M4 and M5 receptors in the urothelium, of which the extent of expression of the latter increased substantially. The cholinergic response may thus be substantially affected by a general deterioration of the muscle function of the bladder as well
Acknowledgements
The study was supported by grants from Ferrings och Svenska Enures Akademin, Wilhelm och Martina Lundgrens Vetenskapsfond, Magn. Bergvall's Foundation and the Sahlgrenska LUA agreement (Delbro). We are indebted to Mr. Simon Pinnock for valuable comments on the manuscript and grateful for expert technical assistance to Histo-Center, Va. Frolunda, Sweden (immunohistochemistry).
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