Autonomic Receptor‐mediated Regulation of Production and Release of Nitric Oxide in Normal and Malignant Human Urothelial Cells

Abstract

In the urinary bladder, the main source of NO seems to be the urothelium and the underlying suburothelium. In this study, we aimed to characterize how receptors in the human urothelium regulate the production and release of NO. For this, we cultured two human urothelial cell lines - the normal immortalized cell line UROtsa and the malignant cell line T24. These were treated with an array of agonists and antagonists with affinity for adrenergic, muscarinic and purinergic receptors. The production of NO and expression of nitric oxide synthase (NOS) was studied by immunocytochemistry and Western blotting. The amount of released NO was measured indirectly by detecting nitrite using amperometry and a Griess reaction kit. The results showed that NO, endothelial NOS and inducible NOS were predominantly produced and expressed in the close vicinity of the nucleus in untreated human urothelial cells. Upon treatment with a beta-adrenoceptor agonist, but not any of the other agonists or antagonists, the pattern of NO production changed, showing a more even production throughout the cytosol. The pattern of expression of endothelial NOS changed in a similar way upon dobutamine treatment. The release of nitrite, as a measurement of NO, increased after treatment with dobutamine from 0.31 ± 0.029 to 1.97 ± 0.18 nmol and 0.80 ± 0.12 to 3.27 ± 0.24 nmol in UROtsa and T24, respectively. In conclusion, our results show that the expression of NOS and production of NO as well as the release of NO from human urothelial cells is regulated by beta-adrenoceptor activation.

Effects of caveolae depletion and urothelial denudation on purinergic and cholinergic signaling in healthy and cyclophosphamide-induced cystitis in the rat bladder

“…However, NO also affects both the detrusor smooth muscle and afferent nerve fibres (Ozawa et al, 1999;Yoshimura et al, 2001) and may play a role in the development of cystitis (Aronsson et al, 2014b). Another source of NO in the bladder is the urothelium, mainly released upon activation of adrenergic receptors (Birder et al, 1998;Winder et al, 2017). Furthermore, stimulation of urothelial muscarinic receptors has been shown to mediate urothelial release of NO, primarily in a state of inflammation (Andersson et al, 2008).…”

Combination drug therapy against OAB normalizes micturition parameters and increases the release of nitric oxide during chemically induced cystitis

“…The group sizes were calculated using the G*Power software ver 3.1.9.4 . The calculation was based on an expected effect size of 2.5 (estimated from data from a previous study ), α = .05, 1‐ β = .95, and an allocation ratio of 1. To avoid underpowering, that is, to compensate for biological variation, a sample size of n = 8 was chosen for the current study.…”

“…Part of the efficacy of mirabegron has been suggested to depend on induced release of nitric oxide (NO), tentatively from the urothelium . Even though it has been shown that β‐adrenoceptor activation causes the release of NO from human urothelial cells, the actual effect of mirabegron on NO release in the bladder remains to be determined in conclusive studies. Considering previous findings that antimuscarinics could potentially decrease the release of NO, mainly in a state of cystitis, and that this could be part of the reason for their lack of efficacy in a state of concomitant bladder overactivity and inflammation, the exact role that mirabegron has on NO release is highly interesting.…”

Immunohistochemistry as a paramount tool in research of normal urothelium, bladder cancer and bladder pain syndrome

“…Other receptors and channels involved in urothelial signalling have been reported, 9 including adrenoreceptors, muscarinic and nicotinic receptors. 103 However, with all of them there is the same problem of conflicting IHC results. The problem of inconclusive localization results can be resolved in two ways.…”