Nitric Oxide Production Within Rat Urothelial Cells

Abstract

Recent studies have suggested that nitric oxide (NO) synthase (NOS) may be localized in the urothelium of the proximal part of the mammalian ureter. We investigated endogenous NO production in the proximal half of the rat ureter, localized its cellular source, characterized the NOS isoforms involved and assessed the impact of NO on ureteral motility. Direct detection of NO production was performed on primary cultures of living rat ureteral cells with the fluorescent indicator diaminofluorescein. Cultures were incubated with the NO precursor L-arginine or the NOS inhibitors L-NAME (N-nitro-L-arginine-methyl ester) and 1400W. NOS expression was determined by immunofluorescence and Western blot analysis. The functional effects of NO donors were assessed on isolated ureters. Significant basal NO production was demonstrated by the high fluorescence level detected in diaminofluorescein treated cell cultures. NO production was strictly limited to urothelial cells since no fluorescence was seen in smooth muscle cells. Pretreatment with L-NAME or 1400W resulted in a significant decrease in fluorescence. Constitutive and inducible NOS isoforms were detected in urothelial cultured cells and in lysates of the urothelial layer. NO donors inhibited in a concentration dependent manner the agonist induced contractile activity of isolated ureters. These results suggest that NO production stems from the urothelium and the NO pathway inhibits contractile activity in the proximal half of the rat ureter. Hence, the nitrergic pathway may be an important target for drugs producing relaxation of the mammalian ureter.