The negative impact of aging on the urinary system is particularly common, severe and significantly contributes to decreased quality of life and increased health care costs. In this regard, the prevalence of lower urinary tract disorders (LUTDs) significantly increases in both men and women with age and are due to multiple causations. The urinary bladder urothelium functions as an integral part of a ‘sensory web’ whereby release of urothelially-derived mediators can communicate changes in the uroepithelial milieu to underlying bladder nerves and smooth muscle, altering their function.  The consequence of this sensory web is the coordinated function of the bladder during cycles of filling and voiding and disruption of this web is likely to lead to bladder dysfunction. A common characteristic change that occurs with advanced age is an augmentation of sympathetic activity and increased sensitivity to norepinephrine.  Here we evaluated our hypothesis that aging-related LUT dysfunction is mediated in part by augmented release of urothelially-derived norepinephrine, and this aberrant release may contribute to age-associated LUTDs.

This study employed female young (3 mo) and aged (24-26 mo) Fischer 344 (F344) rats.  This investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).  Norepinephrine was measured in the bladder instillate from anesthetized (5% isoflurane) rats. In all cases, bladders were filled to a consistent pressure (40cm2) with sterile Krebs solution, removed after 30 min and analyzed using ultra-performance liquid chromatography-tandem mass spectrometry. All samples were provided with d6-norepinephrine as an internal standard for quantitation of norepinephrine.  Bladder and urothelial-cell preparations (mucosa, isolated urothelial cells and smooth muscle) were homogenized, and protein lysates from each were prepared using published methods and imaged using a ChemiDoc MP for enzymes that are involved in norepinephrine synthesis (enzyme dopamine beta-hydroxylase, DBH) and metabolism (catechol-O-methyltransferase, COMT); and the alpha 1D- adrenoceptor.  Data was quantified and analyzed using Image Lab software and volume (intensity) of each protein species was determined and normalized to total protein imaging of the membrane (Bio-Rad Stain Free SDS-PAGE gel technology).  Cryosections of the urinary bladder were used to visualize norepinephrine positive bladder nerves and cells by immunofluorescence using STAINperfect immunostaining kit using a rabbit polyclonal anti-L-noradrenaline antibody (Immusmol France). Immunofluorescence was imaged on a BX63 Olympus fluorescent microscope and analyzed with Olympus cellSens software. Data were analyzed in GraphPad Prism 10 (GraphPad, La Jolla, CA) by unpaired student’s t-test (2-tailed) was used to evaluate significance. P<0.05 was considered significant.

We found positive expression of the noradrenergic enzymes DBH and COMT in both bladder mucosa and isolated urothelial cells using western immunoblotting. In addition to visualizing noradrenergic-positive sympathetic nerves, we also found positive norepinephrine-like immunoreactivity throughout the urothelial layer.   Norepinephrine was released into the bladder lumen during filling and this release was augmented in aging.  Aging also increased expression of alpha 1D adrenoceptor.

Our findings reveal that the bladder urothelium expresses the enzymatic machinery for the synthesis and metabolism of the neurotransmitter norepinephrine. The bladder urothelium responds to mechanical stresses that result in norepinephrine release.  Aging significantly increases norepinephrine release as well as the bladder expression of alpha-adrenergic receptors (whereas the expression of beta-adrenoceptors decreases with age which in turn can enhance the effects of alpha-adrenoceptor stimulation).

Many cases of LUTDs in older adults may be associated with bladder ischemia, which may be due in part to increased norepinephrine that vasoconstricts the bladder microcirculation leading to decreased blood flow. The increased release of urothelially-derived norepinephrine with increased age could also modulate the activity of underlying smooth muscle and nearby sensory neurons.  Augmented norepinephrine release (in addition to increased alpha-adrenoceptor expression) may cause increased afferent activity and hypercontractility of smooth muscle. These findings suggest this type of noradrenergic hypersensitivity may be an important mechanism in geriatric voiding dysfunction which may include increased detrusor overactivity.

Continence 12S (2024) 101467
DOI: 10.1016/j.cont.2024.101467