Maintaining and regulating water balance is fundamental to survival.  The “perceived view” is that: 1) fine-tuning of water homeostasis along the collecting duct of the kidney where arginine vasopressin (AVP) type 2 receptors (V2Rs) are found in high density; and 2) the urinary bladder urothelium (UT), due to its low permeability to water and urea and high electrical resistance, only serves as an impermeable blood-urine barrier. 
Several reports now challenge the traditional view that the UT is an impermeable barrier and suggest that the UT exhibits a transport function that participates in adjusting the composition of urine by affecting net transport of solutes. Thus, the UT may be an active participant in normal physiological fluid and solute reabsorption. In support of this concept, we recently obtained novel preliminary findings that the urinary bladder mucosa expresses AVP type 2 receptors (V2Rs).  Further, our findings also show that UT cells strongly express V2Rs; this suggests that UT cells are the major cell type in the bladder mucosa that expresses V2Rs.
A number of factors and conditions can modulate V2R signaling including aging as well as autocoids such as adenosine. For example, in the kidney adenosine can inhibit AVP-induced effects via adenosine type 1 receptors (A1Rs), effectively acting as a ‘brake’ on V2R-mediated signaling and fluid reabsorption. The main purpose of this study was to test the hypothesis that V2Rs are expressed within the apical UT and that the adenosine/A1R system in the UT might potentially regulate V2R signaling in the urinary bladder.  An additional goal was to determine whether the expression and regulation of the apical UT V2R system is altered with aging.

Urinary bladders (excised from anesthetized young versus aged Fischer 344 rats) were used to examine expression of V2Rs using fluorescent immunocytochemistry (measures total V2R expression) and surface biotinylation/western blot methodology (measures specifically apical surface V2R expression). In addition, we also utilized a recently validated methodology that utilizes N6-etheno-bridge chemistry to examine adenosine formation (i.e., metabolism of etheno-adenine nucleotides by ecto-nucleotidases) in aged versus young bladders. In brief, bladders from young versus aged rats were filled with either vehicle (Krebs buffer) or 100 nM desmopressin for one hour, followed by instillation of 10 uM N6-etheno-ATP which is a substrate for ecto-nucleotidases (Axxora, USA). The solution was removed and the metabolism of etheno-bridged adenine nucleotides by ecto-nucleotidases was monitored by high pressure liquid chromatography with fluorescence detection. We used western immunoblotting using the bladders from these animals to assess the expression of ectonucleotidases (nucleotide metabolizing enzymes) in young versus aged bladder.

We observed V2Rs localized to the UT including basolateral surfaces of umbrella cells and surfaces of intermediate and basal UT cells. V2Rs were also found in small vesicles scattered throughout the apical cytoplasm of umbrella cells.  In addition, surface biotinylation/western blots in rat bladder showed evidence for a large apical surface fraction of uroplakin 3A (apical surface protein used as positive control) and V2Rs.  Western immunoblots revealed an age-related increase in CD39, which is a major ecto-nucleotidase that regulates extracellular purine levels by mediating the metabolism of ATP to ADP and ADP to AMP (adenosine precursor).  In addition, in the aged bladder we observed a significant increase in the metabolism of N6-etheno-ATP to N6-etheno-adenosine, a finding consistent with the increased expression of CD39.  These age-associated increases in ecto-nucleotidase expression and corresponding increases in purine metabolism to adenosine are normalized to levels in that of a younger state by treatment with desmopressin.

The metabolism of ATP to adenosine is regulated in large part by activities of two enzymes, ENTPD1 (aka, CD39) and CD73. Our findings that aging upregulates CD39 (2-fold increase) in the urinary bladder suggests that adenosine (via A1R stimulation) may act as a “brake” on V2R function; thus, similar to the kidney, adenosine may inhibit water absorption by the UT. Indeed, we find significantly more rapid metabolism of ATP to adenosine in the aging bladder UT that is normalized to younger levels with desmopressin (there was no change in young bladder mucosa). These findings point to an augmented braking mechanism on bladder V2R signaling via an interaction between bladder V2Rs and A1Rs at adenylate cyclase.

Dysregulation of the kidney V2R system results in abnormalities of water homeostasis, which may lead to urinary disturbances in patients, for example nocturia, hyponatremia or lower urinary tract symptoms (LUTS). Whether and how bladder V2Rs affect bladder function and whether and how this might impact LUTS and hyponatremia remain open questions.  Nonetheless, our initial findings demonstrate that adenosine production by the bladder mucosa is increased with age due to upregulation of one of the enzymes in the adenosine biosynthesis pathway. This would likely brake V2R signaling and tend to cause nocturia in the elderly because there would be more “adenosine brake” and impairment of urinary concentrating ability.  Notably, agents such as desmopressin could reduce nocturia/nocturnal polyuria in the older patient both by stimulating V2Rs and by inhibiting the putative “adenosine brake”. Further studies are warranted to determine the role and significance of V2R/A1R interactions in the UT.