Storage and secretion of beta-NAD, ATP and dopamine in NGF-differentiated rat pheochromocytoma PC12 cells.

In nerve-smooth muscle preparations beta-nicotinamide adenine dinucleotide (beta-NAD) has emerged as a novel extracellular substance with putative neurotransmitter and neuromodulator functions. beta-NAD is released, along with noradrenaline and adenosine 5'-triphosphate (ATP), upon firing of action potentials in blood vessels, urinary bladder and large intestine. At present it is unclear whether noradrenaline, ATP and beta-NAD are stored in and released from common populations of synaptic vesicles. The answer is unattainable in complex systems such as nerve-smooth muscle preparations. Adrenal chromaffin cells are thus used here as a single-cell model to examine mechanisms of concomitant neurosecretion. Using high-performance liquid chromatography techniques with electrochemical and fluorescence detection we simultaneously evaluated secretion of dopamine (DA), ATP, adenosine 5'-diphosphate, adenosine 5'-monophosphate, adenosine, beta-NAD and its immediate metabolites ADP-ribose and cyclic ADP-ribose in superfused nerve growth factor-differentiated rat pheochromocytoma PC12 cells. beta-NAD, DA and ATP were released constitutively and upon stimulation with high-K(+) solution or nicotine. Botulinum neurotoxin A tended to increase the spontaneous secretion of all substances and abolished the high-K(+)-evoked release of beta-NAD and DA but not of ATP. Subcellular fractionation by continuous glycerol and sucrose gradients along with immunoblot analysis of the vesicular marker proteins synaptophysin and secretogranin II revealed that beta-NAD, ATP and DA are stored in both small synaptic-like vesicles and large dense-core-like vesicles. However, the three substances appear to have different preferential sites of release upon membrane depolarization including sites associated with SNAP-25 and sites not associated with SNAP-25.

N-type and P/Q-type calcium channels regulate differentially the release of noradrenaline, ATP and beta-NAD in blood vessels.

Using HPLC techniques we evaluated the electrical field stimulation-evoked overflow of noradrenaline (NA), adenosine 5'-triphosphate (ATP), and beta-nicotinamide adenine dinucleotide (beta-NAD) in the presence of low nanomolar concentrations of omega-conotoxin GVIA or omega-agatoxin IVA in the canine mesenteric arteries and veins. omega-conotoxin GVIA abolished the evoked overflow of NA and beta-NAD in artery and vein, whereas the evoked overflow of ATP remained unchanged in the presence of omega-conotoxin GVIA. omega-agatoxin IVA significantly reduced the evoked overflow of ATP and beta-NAD. The overflow of NA remained largely unaffected by omega-agatoxin IVA, except at 16Hz in the vein where the overflow of NA was reduced by about 50%. Artery and vein exhibited similar expression levels of the alpha(1B) (CaV2.2, N-type) subunit, whereas the vein showed greater levels of the alpha(1A) (CaV2.1, P/Q-type) subunit than artery. Therefore, there are at least two release sites for NA, beta-NAD and ATP in the canine mesenteric artery and vein: an N-type-associated site releasing primarily NA, beta-NAD and some ATP, and a P/Q-type-associated site releasing ATP, beta-NAD and some NA. The N-type-mediated mechanisms are equally expressed in artery and vein, whereas the P/Q-type-mediated mechanisms are more pronounced in the vein and may ensure additional neurotransmitter release at higher levels of neural activity. In artery, beta-NAD caused a dual effect consisting of vasodilatation or vasoconstriction depending on concentrations, whereas vein responded with vasodilatation only. In contrast, ATP caused vasoconstriction in both vessels. beta-NAD and ATP may mediate disparate functions in the canine mesenteric resistive and capacitative circulations.

Nicotinamide adenine dinucleotide is released from sympathetic nerve terminals via a botulinum neurotoxin A-mediated mechanism in canine mesenteric artery.

Using high-performance liquid chromatography techniques with fluorescence and electrochemical detection, we found that beta-nicotinamide adenine dinucleotide (beta-NAD) is released in response to electrical field stimulation (4-16 Hz, 0.3 ms, 15 V, 120 s) along with ATP and norepinephrine (NE) in the canine isolated mesenteric arteries. The release of beta-NAD increases with number of pulses/stimulation frequencies. Immunohistochemistry analysis showed dense distribution of tyrosine hydroxylase-like immunoreactivity (TH-LI) and sparse distribution of TH-LI-negative nerve processes, suggesting that these blood vessels are primarily under sympathetic nervous system control with some contribution of other (e.g., sensory) neurons. Exogenous NE (3 micromol/l), alpha,beta-methylene ATP (1 micromol/l), neuropeptide Y (NPY, 0.1 micromol/l), CGRP (0.1 micromol/l), vasoactive intestinal peptide (VIP, 0.1 micromol/l), and substance P (SP, 0.1 micromol/l) had no effect on the basal release of beta-NAD, suggesting that the overflow of beta-NAD is evoked by neither the sympathetic neurotransmitters NE, ATP, and NPY, nor the neuropeptides CGRP, VIP, and SP. Botulinum neurotoxin A (BoNTA, 0.1 micromol/l) abolished the evoked release of NE, ATP, and beta-NAD at 4 Hz, suggesting that at low levels of neural activity, release of these neurotransmitters results from N-ethylmaleimide-sensitive factor attachment protein receptor/synaptosomal-associated protein of 25 kDa-mediated exocytosis. At 16 Hz, however, the evoked release of NE, ATP, and beta-NAD was reduced by BoNTA by approximately 90, 60, and 80%, respectively, suggesting that at higher levels of neural activity, beta-NAD is likely to be released from different populations of synaptic vesicles or different populations of nerve terminals (i.e., sympathetic and sensory terminals).

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.

Release of beta-nicotinamide adenine dinucleotide upon stimulation of postganglionic nerve terminals in blood vessels and urinary bladder.

Chemical signaling in autonomic neuromuscular transmission involves agents that function as neurotransmitters and/or neuromodulators. Using high performance liquid chromatography techniques with fluorescence and electrochemical detection we observed that, in addition to ATP and norepinephrine (NE), electrical field stimulation (EFS, 4-16 Hz, 0.1-0.3 ms, 15 V, 60-120 s) of isolated vascular and non-vascular preparations co-releases a previously unidentified compound with apparent nucleotide or nucleoside structure. Extensive screening of more than 25 nucleotides and nucleosides followed by detailed peak identification revealed that beta-nicotinamide adenine dinucleotide (beta-NAD) is released in tissue superfusates upon EFS of canine mesenteric artery (CMA), canine urinary bladder, and murine urinary bladder in the amounts of 7.1 +/- 0.7, 26.5 +/- 4.5, and 15.1 +/- 3.2 fmol/mg of tissue, respectively. Smaller amounts of the beta-NAD metabolites cyclic adenosine 5'-diphosphoribose (cADPR) and ADPR were also present in the superfusates collected during EFS of CMA (2.5 +/- 0.9 and 5.8 +/- 0.8 fmol/mg of tissue, respectively), canine urinary bladder (1.8 +/- 0.5 and 9.0 +/- 6.0 fmol/mg of tissue, respectively), and murine urinary bladder (1.4 +/- 0.1 and 6.2 +/- 2.4 fmol/mg of tissue, respectively). The three nucleotides were also detected in the samples collected before EFS (0.2-1.6 fmol/mg of tissue). Exogenous beta-NAD, cADPR, and ADPR (all 100 nm) reduced the release of NE in CMA at 16 Hz from 27.8 +/- 6.0 fmol/mg of tissue to 15.5 +/- 5.0, 12 +/- 3.0, and 10.0 +/- 4.0 fmol/mg of tissue, respectively. In conclusion, we detected constitutive and nerve-evoked overflow of beta-NAD, cADPR, and ADPR in vascular and non-vascular smooth muscles, beta-NAD being the prevailing compound. These substances modulate the release of NE, implicating novel nucleotide mechanisms of autonomic nervous system control of smooth muscle.