Storage and secretion of β‐NAD, ATP and dopamine in NGF‐differentiated rat pheochromocytoma PC12 cells

Abstract

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.

ATP synthesis and storage

“…It was found co-stored with acetylcholine in guinea pig cortex, calf superior cervical ganglion, and motor nerve terminals of rat diaphragm [76]; with noradrenaline, it was found in human blood vessels, smooth muscles, and endothelial cells [80]; it was also found in adrenal chromaffin cells together with serotonin, with neuropeptide Y and glutamate in astrocytes, and with dopamine in neuron-differentiated PC12 [81][82][83][84]. Moreover, in other cell types in the nervous tissue, particularly astrocytes, ATP was found also in small synaptic-like vesicles [85].…”

The impact of commercially available purinergic ligands on purinergic signalling research

“…Asc-403-β-NAD Interestingly, it has recently been suggested that another molecule, β-nicotinamide adenine dinucleotide (β-NAD) like ATP, may also be a co-transmitter and may likely represent a novel extracellular signalling molecule [57,58]. Smyth et al [58] revealed that β-NAD is released from sympathetic nerve terminals and as such suggested that as with ATP, β-NAD has putative neurotransmitter or neuromodulator functions.…”

“…Smyth et al [58] revealed that β-NAD is released from sympathetic nerve terminals and as such suggested that as with ATP, β-NAD has putative neurotransmitter or neuromodulator functions. Furthermore, β-NAD has also recently been shown to be a P2Y 1 and P2Y 11 receptor agonist (EC 50 =6.1 at P2Y 1 ) [10,59].…”

Neuronal and extraneuronal release of ATP and NAD+ in smooth muscle

“…It is suggested that ATP in vesicles is important for acidification of the vesicle lumen, for creating a proton gradient driving vesicular neurotransmitter uptake and for steps of exocytosis itself (15) in addition to serving neurotransmission (5). ATP is present in large dense-cored (LDCV) and small synaptic vesicles (SSV) as well as in chromaffin vesicles (13,27). Loading of ATP occurs through all stages of vesicle formation and recycling and ATP is taken up by vesicles of both reserved and readily releasable pools (6).…”

“…The studies on cellular sources, mechanisms of release, postjunctional targets and functions of extracellular purines in smooth muscle, in particular, are hampered by the complex nature of smooth muscle tissues and the many possible roles of extracellular purines. Nevertheless, continual work has, during the last decade, expanded our view of the role of ATP and other purines (i.e., NAD + and ADPribose, ADPR) in neurotransmission, and this analysis suggests that multiple purines likely contribute to the complex neural regulation of smooth muscle (11,12,13,14), moving the field of purine neurotransmission beyond the sovereign role of ATP.…”