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

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 (<EC(50)). 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.