title: Altered neuronal and endothelial nitric oxide synthase expression in the bladder and urethra of cyclophosphamide-treated rats.
abstract: Increased nitric oxide (NO) production seems to play a key role in cyclophosphamide (CYP)-induced cystitis, although the underlying mechanisms and the relative involvement of the different NO synthase (NOS) isoforms remain to be elucidated. Moreover, the role of the urethra in this process is also unclear. In this study, we have analyzed the changes in the expression and distribution of the inducible (iNOS), endothelial (eNOS) and neuronal (nNOS) isoforms of NOS, and the alterations in nerve-mediated contractility in the bladder and urethra of CYP-treated rats. Accordingly, Wistar rats were treated with 150 mg kg(-1) CYP for 4 (acute treatment) or 48 h (intermediate treatment), or with 70 mg kg(-1) CYP every 3 days for 10 days (chronic treatment), and the changes in protein expression were assessed by immunohistofluorescence and in Western blots, while mRNA expression was assessed by conventional and quantitative PCR. Similarly, nerve-mediated contractility was analyzed in vitro. Unexpectedly, no iNOS expression was detected in CYP-treated animals, while a transient downregulation of nNOS expression and a progressive upregulation of eNOS was observed, although the eNOS accumulated was not in the active phosphorylated form. Qualitative changes in mRNA expression were also observed in the bladder and urethra, although contractility only diminished in the bladder and this change was not dependent on NOS activity. These findings suggest that spatiotemporal alterations in NO production by constitutive NOS may be involved in the pathogenicity of CYP. Further studies will be necessary to understand the contribution of eNOS to the increases in NO associated with bladder inflammation, or that of free radicals.
authors.affiliation: Department of Physiology, School of Veterinary Medicine, Complutense University, Madrid, Spain; Instituto Investigación Sanitaria Hospital Clínico San Carlos (IdISSC), Madrid, Spain.
authors.forename: Maria
authors.initials: M
authors.lastname: Sancho
authors.affiliation: Department of Physiology, School of Veterinary Medicine, Complutense University, Madrid, Spain; Instituto Investigación Sanitaria Hospital Clínico San Carlos (IdISSC), Madrid, Spain.
authors.forename: Jose J
authors.initials: JJ
authors.lastname: Ferrero
authors.affiliation: Department of Physiology, School of Veterinary Medicine, Complutense University, Madrid, Spain; Instituto Investigación Sanitaria Hospital Clínico San Carlos (IdISSC), Madrid, Spain.
authors.forename: Domingo
authors.initials: D
authors.lastname: Triguero
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Complutense University, Madrid, Spain; Instituto Investigación Sanitaria Hospital Clínico San Carlos (IdISSC), Madrid, Spain.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Torres
authors.affiliation: Department of Physiology, School of Veterinary Medicine, Complutense University, Madrid, Spain; Instituto Investigación Sanitaria Hospital Clínico San Carlos (IdISSC), Madrid, Spain. Electronic address: angarcia@vet.ucm.es.
authors.forename: Angeles
authors.initials: A
authors.lastname: Garcia-Pascual
mesh_terms: D000818:Animals; D003520:Cyclophosphamide / Q000009:adverse effects*; D003556:Cystitis / Q000139:chemically induced; D005260:Female; D015870:Gene Expression / Q000187:drug effects*; D052248:Nitric Oxide Synthase Type I / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D014521:Urethra / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D001743:Urinary Bladder / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism
chemical_list: D003520:Cyclophosphamide; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; C496322:Nos1 protein, rat; C496316:Nos3 protein, rat
keywords: Cyclophosphamide; Cystitis; Urethra; eNOS; iNOS; nNOS