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

Abstract

Using high-performance liquid chromatography techniques with fluorescence and electrochemical detection, we found that β-nicotinamide adenine dinucleotide (β-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 β-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 μmol/l), α,β-methylene ATP (1 μmol/l), neuropeptide Y (NPY, 0.1 μmol/l), CGRP (0.1 μmol/l), vasoactive intestinal peptide (VIP, 0.1 μmol/l), and substance P (SP, 0.1 μmol/l) had no effect on the basal release of β-NAD, suggesting that the overflow of β-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 μmol/l) abolished the evoked release of NE, ATP, and β-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 β-NAD was reduced by BoNTA by ∼90, 60, and 80%, respectively, suggesting that at higher levels of neural activity, β-NAD is likely to be released from different populations of synaptic vesicles or different populations of nerve terminals (i.e., sympathetic and sensory terminals).