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

Abstract

SUMMARYWe 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 Ca 2 ϩ -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.

Distribution of Inducible Nitric Oxide Synthase, Interleukin-1.BETA., and Interleukin-1 Receptor in the Temporomandibular Joint of Normal Rats.

“…In type-A cells, these products were localized to the cytoplasm and vescicles, and on the plasma membrane and rER. It has been reported that these products are also localized to the cytoplasm in smooth muscle cells [39] and urothelial cells [30]. In addition, iNOS is expressed in macrophages and fibroblasts [33].…”

Cascade Bioassay Evidence for the Existence of Urothelium-Derived Inhibitory Factor in Guinea Pig Urinary Bladder

“…After experiments the whole preparation of urothelium-intact and -denuded ureters or urinary bladders were incubated in Tris-HCl buffer solution (50 mM, pH 8) containing 1 mM β-NADPH, 0.5 mM nitroblue tetrazolium and 0.2% Triton X-100 at 37°C for 10 min [23], [24]. After wash in saline tissues were immediately subjected to microscopic observation in reflective light.…”

Direct single cell determination of nitric oxide synthase related metabolites in identified nitrergic neurons

“…Although these methods present the advantage of relative simplicity and routinely produce preliminary anatomical maps, there remains the possibility that such protocols generate some false positive identifications of the localization of NOS activity. For example, NADPH-d staining has been associated with several different enzymes [57][58][59][60][61][62][63][64][65][66][67], and some published accounts of NADPH-d staining in Lymnaea, including CGC [54] have used lower concentrations of fixative compared to more established protocols [40,52,59,60,[68][69][70] designed to reveal specific fixative resistant NADPH-d activity as a putative marker for NOS activity. Furthermore, current NO-sensitive electrodes are constrained by both their large tip size (2 mm diameter in [55]) and sensitivity [56] such that reliable NO measurements in the low nanomolar range in complex biological tissues, and especially from single neurons, are difficult to interpret.…”