Altered muscarinic receptor subtype expression and functional responses in cyclophosphamide induced cystitis in rats

Abstract

In the in vitro study, it was investigated whether the expression of muscarinic receptors and cholinergic responses were altered in the situation of experimental cystitis. Rats were treated with cyclophosphamide intraperitoneally and the bladders were excised 36–100 h later. Immunohistochemistry and immunoblotting showed all subtypes of the muscarinic receptor (M1–M5) to be present in the specimens from inflamed urinary bladders and controls. In the cyclophosphamide-treated rats, the expression of muscarinic M5 receptors was increased by more than 40 times (p < 0.01; n = 8) both in the smooth muscle and the urothelium. Both the maximal contractile response to carbachol and to a high potassium concentration was approximately halved in cyclophosphamide-treated tissues, whereas the reduction was substantially greater in response to low carbachol concentrations (< EC₅₀). The administration of 4-DAMP inhibited the carbachol-induced contractile responses of inflamed strips less potently than of controls, whereas pirenzepine and methoctramine showed equipotency in the two groups. The nitric oxide synthase inhibitor l-NNA increased the contractile effect of carbachol in inflamed detrusor strips, while it had no effect in controls. Immunoblotting showed endothelial nitric oxide synthase (eNOS) to be up-regulated in cystitis, and immunohistochemistry revealed the change to occur in the urothelium and in the suburothelial layer. The alteration of cholinergic detrusor responses in cyclophosphamide-treated rats depends mainly on a general detriment of contractility but also on indirect effects possibly via nitric oxide synthesis. The most prominent histological alterations occurred in the urothelium in which muscarinic M5 receptors increased in particular. The study further underlines that the urothelium may play significant roles in the pathogenesis of urinary bladder disorders such as interstitial cystitis.

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).