β-NAD is a novel nucleotide released on stimulation of nerve terminals in human urinary bladder detrusor muscle

Abstract

Endogenous nucleotides with extracellular functions may be involved in the complex neural control of human urinary bladder (HUB). Using HPLC techniques with fluorescence detection, we observed that in addition to ATP and its metabolites ADP, AMP and adenosine, electrical field stimulation (EFS; 4–16 Hz, 0.1 ms, 15 V, 60 s) of HUB detrusor smooth muscle coreleases novel nucleotide factors, which produce etheno-1 N6-ADP-ribose (eADPR) on etheno-derivatization at high temperature. A detailed HPLC fraction analysis determined that nicotinamide adenine dinucleotide (β-NAD+; 7.0 ± 0.7 fmol/mg tissue) is the primary nucleotide that contributes to the formation of eADPR. The tissue superfusates collected during EFS also contained the β-NAD+metabolite ADPR (0.35 ± 0.2 fmol/mg tissue) but not cyclic ADPR (cADPR). HUB failed to degrade nicotinamide guanine dinucleotide (NGD+), a specific substrate of ADP ribosyl cyclase, suggesting that the activity of this enzyme in the HUB is negligible. The EFS-evoked release of β-NAD+was frequency dependent and is reduced in the presence of tetrodotoxin (TTX; 0.3 μmol/l), ω-conotoxin GVIA (50 nmol/l), and botulinum neurotoxin A (BoNT/A; 100 nmol/l), but remained unchanged in the presence of guanethidine (3 μmol/l), ω-agatoxin IVA (50 nmol/l), or charbachol (1 μmol/l). Capsaicin (10 μmol/l) increased both the resting and EFS-evoked overflow of β-NAD+. Exogenous β-NAD+(1 μmol/l) reduced both the frequency and amplitude of spontaneous contractions. In conclusion, we detected nerve-evoked overflow of β-NAD+and ADPR in HUB. The β-NAD+/ADPR system may constitute a novel inhibitory extracellular nucleotide mechanism of neural control of the human bladder.