Appropriate experimental approach is critical for identifying neurotransmitter substances: application to enteric purinergic neurotransmission

to the editor: The identity of the purinergic neurotransmitter in the gastrointestinal (GI) tract is a controversial issue (12). For several decades adenosine 5′-triphosphate (ATP) was assumed to be the enteric inhibitory purinergic neurotransmitter (1–3). However, recent studies have shown that β-nicotinamide adenine dinucleotide (β-NAD), ADP-ribose, and uridine adenosine tetraphosphate (Up4A) satisfy both pre- and postjunctional requirements for a neurotransmitter in GI muscles of human, monkey, and mouse better than ATP (4, 5, 10, 13). The study by Wang et al. (14) investigated whether β-NAD might be a neurotransmitter in the enteric nervous system (ENS) or a prejunctional modulator of neurotransmitter release in muscles of guinea pig small intestine and colon and human jejunum. We believe that there are errors in the interpretation of the data in this study resulting from the experimental approach. In this study, β-NAD was applied to solutions bathing muscles in organ baths. Applied in this manner, β-NAD did not affect membrane potentials in smooth muscles of guinea pig colon or human jejunum. However, β-NAD suppressed inhibitory junction potentials (IJPs) and the contractile responses to nerve stimulation. The latter effect was inhibited by an adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine. From these observations the authors concluded that “the results do not support an inhibitory neurotransmitter role for β-NAD at intestinal neuromuscular junctions.” Instead, the authors suggested that “β-NAD is a ligand for adenosine A1 receptors expressed by enteric neurons.”

Our own results suggest that such pharmacological characterizations of the postjunctional effects of bath-applied purines are difficult to evaluate because the responses to bath-applied purines amount to integrated responses to the primary substance and to downstream metabolites. We reported that brief contact of β-NAD with GI muscles (e.g., 1–5 s) causes formation of several metabolites, including adenosine (5). Thus effects attributed to A1 receptors by Wang et al. (14) may have been caused by adenosine generated by metabolism of β-NAD.

Neurotransmitters from enteric inhibitory nerves are released from nerve varicosities in a relatively confined volume (e.g., the neuroeffector junction, NEJ). The purine transmitter activates a specific class of receptors on target cells within the NEJ. In the large intestine purinergic neurotransmission is mediated by P2Y1 purinoceptors located in the NEJ (4, 5, 7, 9, 10, 13). In contrast, bath-applied purines and their metabolites can bind to a variety of purinergic receptors that appear not to be restricted to the NEJ and can elicit effects not mediated by P2Y1 receptors that are even contradictory to purinergic inhibitory neurotransmission (11). Compounds added to bath solutions may not even reach NEJ receptors in effective concentrations. This is the problem for β-NAD, which has been shown to have little or no effect when applied in bath solutions but causes hyperpolarization, appropriate for the postjunctional purinergic response, when applied directly via a picospritz pipet (4, 5, 10, 13). Loss of β-NAD effects, as described in Wang and coworkers (14) and in other pharmacological studies employing bath application of drugs (8), is likely to represent deactivation of β-NAD through metabolism. The localized responses to β-NAD, ADP-ribose (a primary metabolite of β-NAD) and Up4A applied by picospritzing, but not responses to ATP and ADP, are inhibited by specific P2Y1 receptor antagonists, such as MRS 2179 or MRS 2500, and in colonic muscles of p2ry1^−/− mice (4, 5, 9, 10, 13). Thus the responses of β-NAD, ADP-ribose, and Up4A mimic the authentic purinergic neurotransmitter.

Nerve-evoked release of β-NAD, ADP-ribose, and Up4A, but not of ATP, was shown to be inhibited by tetrodotoxin (TTX) and ω-conotoxin GVIA (4, 5, 10, 13), suggesting that ATP is released from sites other than nerve terminals in colonic muscles. This was further emphasized by stimulating enteric motor neurons with agonists for nicotinic cholinergic or serotonergic (5HT3) receptors. These agonists caused release of ATP and β-NAD in muscle strips containing myenteric ganglia but no response in muscle strips with ganglia removed. Release of β-NAD was blocked by neurotoxins (TTX and ω-conotoxin GVIA) but release of ATP was unaffected, suggesting that ATP is most likely released from ganglionic sources but not motor neurons (6).

There is much to learn about the dynamics of neurotransmitter release, concentration profiles of neurotransmitters in the NEJ, receptor binding, and postjunctional transduction of neurotransmitter signals in the gastrointestinal tract. At present, several experimental approaches (combining measurements of neurotransmitter release, analyzing the kinetics of neurotransmitter metabolism and fate, comparing the postjunctional effects of exogenous substances reasoned to be neurotransmitters with substances released from nerve terminals, and direct analysis of cell-specific responses of cells in and near the NEJ) support roles for β-NAD, ADPR, and Up4A as enteric inhibitory motor neurotransmitters in the large intestine.

GRANTS

Research of the authors was sponsored by an NIH grant P01 DK41315.