Expression of neuronal nitric oxide synthase (nNOS) and nitric-oxide-induced changes in cGMP in the urothelial layer of the guinea pig bladder

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Abstract
The urothelium plays a sensory role responding to deformation of the bladder wall; this involves the release of adenosine trisphosphate (ATP) and nitric oxide (NO), which affect afferent nerve discharge and bladder sensation. The urothelial cells responsible for producing ATP and NO and the cellular targets, other than afferent nerves, for ATP and NO remain largely unexplored. Sub-urothelial interstitial cells (SU-ICs) lie immediately below the urothelium and respond to NO with a rise in cGMP. To determine which cells might target SU-ICs by producing NO, areas of dome, lateral wall and base wall were treated with isobutyl-methyl-xanthine, exposed to the NO donor diethylamino NONOate and then fixed for immunohistochemistry. Surface urothelial cells (SUCs) in the base and dome expressed neuronal nitric oxide synthase (nNOS), whereas those in the lateral wall did not. Distinct populations of SUCs were present in the bladder base. SUCs with significant amounts of nNOS lay adjacent to cells with low levels of nNOS. In specific base regions, the few SUCs present contained nNOS within discrete intracellular particles. In the basal urothelial cell (BUC) layer of the lateral wall, nNOS-positive (NOS+) BUCs neither showed an elevation in cGMP in response to NO, nor expressed the β1 sub-unit of soluble guanylate cyclase, protein kinase I or protein kinase II. Thus, they produced but did not respond to NO. The BUC layer also stained for the stem cell factor c-Kit suggesting its involvement in urothelial cell development. No NOS+ BUCs were present in the SUC-sparse region in the bladder base. Exogenous NO produced an elevation in cGMP in SUCs and SU-ICs. The distribution and proportion of these target cells varied between the dome, lateral wall and base. cGMP+ SU-ICs were present as a dense layer in the bladder base but were rarely seen in the lateral wall, which contained nNOS+ BUCs. No nNOS+ BUCs and cGMP+ SU-ICs were apparent in the dome. The degree of complexity in nNOS distribution and NO target cells is therefore greater than has previously been described and may reflect distinct physiological functions that have yet to be identified.