Mechanisms of release, metabolism and action of purines in the enteric and central nervous systems

In Chapter 2 of this dissertation we utilized a single cell model to examine storage and release of NAD+ from vesicles in nerve-growth factor (NGF)-differentiated rat pheochromocytoma PC12 cells which phenotypically resemble sympathetic neurons. In this study we verified the presence of NAD + in vesicles along with ATP and catecholamines (dopamine). Interestingly, we revealed differential mechanisms of release of these three substances from vesicles: release of NAD+ and dopamine required intact SNAP-25-mediated exocytosis whereas ATP was released largely via SNAP-25-independent mechanisms. These observations in conjunction with a previous finding demonstrating &ohgr;-conotoxin GVIA-insensitive ATP release in blood vessels led us to question the true identity of the NANC neurotransmitter in GI muscles where purinergic neurotransmission was first described.;In Chapter 3 we demonstrated that electrical field stimulation (EFS) evoked release of NAD+ that was dependent on the level of nerve stimulation and was significantly attenuated by blockers of neural activity. We also demonstrated that postsynaptic hyperpolarizations to exogenous NAD + were abolished by factors inhibiting the endogenous purine-mediated IJP. On the other hand, release and postsynaptic effects of ATP remained largely intact by inhibitors of enteric purine neurotransmission. Therefore our evidence suggests that NAD+ is a better candidate than ATP as the purinergic inhibitory motor transmitter in colons from humans and non-human primates.;In Chapter 4 we examined postjunctional effects of direct metabolites of ATP and NAD+, adenosine 5'-diphosphate (ADP) and ADP-ribose (ADPR), respectively, in colonic muscles. First, we demonstrated that these metabolites are produced very rapidly in murine and primate colons (within 1 sec). Next, we found that membrane hyperpolarizations to ADPR, but not to ADP, mimicked the pharmacology of endogenous purine response; this is the first study demonstrating a bioactive role of ADPR in enteric smooth muscles. Our evidence indicates that rapid metabolism cannot explain the failure of ATP to match the endogenous transmitter in colon. Moreover our evidence suggests that multiple purines might contribute to enteric inhibitory responses produced during NANC neurotransmission. Thus purinergic inhibitory regulation of enteric smooth muscle is more complex than originally believed.;In Chapter 5 we attempted to clarify the sites of release of ATP and NAD+ in GI smooth muscles by utilizing an alternative approach to stimulate purine release. Here we chemically activated the neuronal ligand-gated ion channel receptors, nicotinic acetylcholine receptors and serotonin 5-HT3 receptors, which are localized on cell bodies and dendrites of inhibitory motor neurons. We demonstrated that the release of ATP and NAD+ upon activation of these receptors originated from different sites within neurons and via different mechanisms. The release of NAD+ appeared to originate exclusively from nerve terminals and was abolished by neural inhibitors. However the release of ATP remained intact in the presence of neural inhibitors suggesting that ATP release may have originated primarily from the nerve cell bodies. Therefore in agreement with our previous studies release of NAD+ in the gut occurs by mechanisms consistent for a neurotransmitter.;In Chapter 6 we examined release, metabolism and postjunctional effects of NAD+ in the rat brain. We demonstrated that in isolated rat forebrain synaptosomes NAD+ is released by mechanisms requiring intact vesicle exocytosis machinery. We also found that localized application of NAD+ (and ADPR) elicited Ca2+ transients in cultured cortical neurons suggesting that endogenous NAD+ could participate in neuronal-neuronal communication in the brain. Finally, we demonstrated that mechanisms involved in terminating the extracellular action of NAD+ exist in the brain. This is the first study suggesting that NAD+ qualifies as a putative neurotransmitter in the CNS. (Abstract shortened by UMI.).