Expression of inducible nitric oxide synthase in primary culture of rat bladder smooth muscle cells by plasma from cyclophosphamide-treated rats

Intraperitoneal administration of cyclophosphamide (50-150 mg/kg) for 6 or 12 h induced edema and hemorrhagic changes in rat bladder, which were both dose and time-dependent. Pretreatment with nitric oxide synthase (NOS) inhibitors NG-nitro-L-arginine methyl ester (L-NAME, 40 mg/kg) or with s-methylisothiourea (40 mg/kg) ameliorated the cyclophosphamide-induced cystitis. Cyclophosphamide administration also produced increases in NO-metabolite levels (nitrate+nitrite) in the urine and plasma of rats. Greater increases in NO metabolites were observed with 150 than with 50 mg/kg of cyclophosphamide, and at 12 than at 6 h after cyclophosphamide injection. Pretreatment with l-NAME and s-methylisothiourea significantly reduced cyclophosphamide-induced increases in urine and plasma NO-metabolite levels. To explore the mechanism by which cyclophosphamide increases the expression of inducible NOS (iNOS), primary cultures of rat bladder smooth muscle were developed. Exposure to tumor necrosis factor α (TNF-α) plus interferon γ, produced a marked increase in the expression of iNOS and in NO production in the culture medium. However, exposure to cyclophosphamide or to its metabolite acrolein (10-6-10-4 M for 24 h) did not increase iNOS or NO-metabolite levels. On the other hand, incubation of primary cell cultures with plasma from rats treated with cyclophosphamide (150 mg/kg, 12 h) produced a marked increase in iNOS expression and NO production. Taken together, our results indicate that NO plays an important role in the pathogenesis of cyclophosphamide-induced cystitis in rats, and some factors may be released in cyclophosphamide-treated rat plasma which stimulate iNOS expression in primary culture of rat bladder smooth muscle cells.