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 -nicotinamide adenine dinucleotide (-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 -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 -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 -NAD, cADPR, and ADPR in vascular and non-vascular smooth muscles, -NAD being the prevailing compound. These substances modulate the release of NE, implicating novel nucleotide mechanisms of autonomic nervous system control of smooth muscle.Chemical signaling constitutes a major mechanism in neuroeffector transmission in the central and peripheral nervous systems. Postganglionic nerve terminals, in particular, characteristically release multiple factors upon action potential, a process referred to as plurichemical neurotransmission (1) or co-transmission (2). The evidence is particularly strong for postganglionic sympathetic nerves, which have been shown to co-release ATP, NE, 1 and neuropeptide Y, factors that presently fulfill transmitter criteria (3-6). Postganglionic parasympathetic nerve terminals, on the other hand, release acetylcholine and ATP (2). In addition, neural control of effector cells involves molecules that implement potent excitatory or inhibitory actions at the neuroeffector junction in the absence of stringent evidence for transmitter function. These effects, collectively referred to as neuromodulation, are usually poorly understood, often due to lack of potent and selective antagonists (6); nonetheless, neuromodulation is an important mechanism, as is neurotransmission (7, 8).We report here that postganglionic nerve terminals in several smooth muscle preparatio...
Breen, Leanne T., Lisa M. Smyth, Ilia A. Yamboliev, and Violeta N. Mutafova-Yambolieva. -NAD is a novel nucleotide released on stimulation of nerve terminals in human urinary bladder detrusor muscle.
In nerve-smooth muscle preparations β-nicotinamide adenine dinucleotide (β-NAD) has emerged as a novel extracellular substance with putative neurotransmitter and neuromodulator functions. Thus, β-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 β-NAD are stored in and released from common populations of synaptic vesicles. This matter is unattainable to study in complex systems such as nerve-smooth muscle preparations. Adrenal chromaffin cells are used here as a single-cell model to examine mechanisms of concomitant neurosecretion. Using high-performance liquid chromatography techniques with electrochemical and fluorescence detections we simultaneously evaluated secretion of dopamine (DA), ATP, adenosine 5'-diphosphate, adenosine 5'-monophosphate, adenosine, and β-NAD and its immediate metabolites ADP-ribose and cyclic ADP-ribose in superfused nerve growth factor-differentiated rat pheochromocytoma PC12 cells. β-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 β-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 β-NAD, ATP and DA are stored in both small synaptic-like vesicles and large dense-core-like vesicles. Yet, the three substances appear to have different preferential sites of release upon membrane depolarization including sites associated with SNAP-25 and sites non-associated with SNAP-25.
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).
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.