beta-NAD is a novel nucleotide released on stimulation of nerve terminals in human urinary bladder detrusor muscle.
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-1N(6)-ADP-ribose (eADPR) on etheno-derivatization at high temperature. A detailed HPLC fraction analysis determined that nicotinamide adenine dinucleotide (beta-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 beta-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 beta-NAD+ was frequency dependent and is reduced in the presence of tetrodotoxin (TTX; 0.3 micromol/l), omega-conotoxin GVIA (50 nmol/l), and botulinum neurotoxin A (BoNT/A; 100 nmol/l), but remained unchanged in the presence of guanethidine (3 micromol/l), omega-agatoxin IVA (50 nmol/l), or charbachol (1 micromol/l). Capsaicin (10 micromol/l) increased both the resting and EFS-evoked overflow of beta-NAD+. Exogenous beta-NAD+ (1 micromol/l) reduced both the frequency and amplitude of spontaneous contractions. In conclusion, we detected nerve-evoked overflow of beta-NAD+ and ADPR in HUB. The beta-NAD(+)/ADPR system may constitute a novel inhibitory extracellular nucleotide mechanism of neural control of the human bladder.
Breen
Leanne T
LT
Department of Physiology and Cell Biology, Anderson Medical Sciences Bldg./MS 352, University of Nevada School of Medicine, Reno, NV 89557-0271, USA.
Smyth
Lisa M
LM
Yamboliev
Ilia A
IA
Mutafova-Yambolieva
Violeta N
VN
D000241:Adenosine / Q000378:metabolism; D018663:Adrenergic Agents / Q000494:pharmacology; D000818:Animals; D019274:Botulinum Toxins, Type A / Q000494:pharmacology; D002211:Capsaicin / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D020916:Conotoxins / Q000494:pharmacology; D004558:Electric Stimulation; D006145:Guanethidine / Q000494:pharmacology; D006150:Guanine Nucleotides / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth / Q000378:metabolism / Q000502:physiology*; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism*; D013237:Stereoisomerism; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism; D013779:Tetrodotoxin / Q000494:pharmacology; D001743:Urinary Bladder / Q000378:metabolism / Q000502:physiology*; D020867:omega-Agatoxin IVA / Q000494:pharmacology
D018663:Adrenergic Agents; D020916:Conotoxins; D006150:Guanine Nucleotides; D050825:Synaptosomal-Associated Protein 25; D020867:omega-Agatoxin IVA; D009243:NAD; D013779:Tetrodotoxin; C102728:nicotinamide guanine dinucleotide; D019274:Botulinum Toxins, Type A; D000241:Adenosine; D002211:Capsaicin; D006145:Guanethidine