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Journal of Histochemistry and Cytochemistry, Vol. 47, 739-750, June 1999, Copyright © 1999, The Histochemical Society, Inc.

ARTICLE

Morphological and Biochemical Investigation of Nitric Oxide Synthase and Related Enzymes in the Rat and Pig Urothelium

Katarina Perssona, Mirjana Poljakovica, Kjell Johanssonb, and Bengt Larssona
a Departments of Clinical Pharmacology, Lund University, Lund, Sweden
b Zoology, Lund University, Lund, Sweden

Correspondence to: Katarina Persson, Dept. of Clinical Pharmacology, Lund University Hospital, S-221 85 Lund, Sweden.

We investigated the enzymes involved in the NADPH-diaphorase (d) reaction in the rat and pig bladder urothelium. The urothelial cell layer displayed intense and uniform NADPH-d activity. Preincubation with the flavoprotein inhibitor diphenyleneiodionium chloride (DPI) and the alkaline phosphatase inhibitor levamisole concentration-dependently decreased the urothelial NADPH-d activity. Immunoreactivities to neuronal (n), endothelial (e), or inducible (i) nitric oxide synthase (NOS) were not detected in rat or pig urothelial cells. In rats, the urothelium was uniformly immunoreactive for NADPH cytochrome P450 reductase, whereas the pig urothelium displayed inconsistent labeling. In lipopolysaccharide (LPS)-treated rats, the bladder urothelium showed positive iNOS immunoreactivity. The iNOS labeling was found predominantly in cells located in the basal layer of the urothelium. In the pig bladder mucosa, a Ca2+-dependent NOS activity was evident in cytosolic and particulate fractions that was quantitatively comparable to the NOS activity found in the smooth muscle. In ultrastructural studies of urothelial cells, NADPH-d reaction products were found predominantly on membranes of the nuclear envelope, endoplasmatic reticulum and mitochondria. In conclusion, NADPH-d staining of the urothelium cannot be taken as an indicator for the presence of constitutively expressed NOS. Activity of alkaline phosphatase and cytochrome P450 reductase may account for part of the NADPH-d reaction in urothelial cells. However, LPS treatment of rats caused expression of iNOS in urothelial cells. (J Histochem Cytochem 47:739–749, 1999)

Key Words: NADPH diaphorase histochemistry, nitric oxide synthase, bladder, urothelium, LPS, NADPH cytochrome P450 reductase


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