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Endogenous adenosine inhibits CNS terminal Ca(2+) currents and exocytosisBursts of action potentials (APs) are crucial for the release of neurotransmitters from dense core granules. This has been most definitively shown for neuropeptide release in the hypothalamic neurohypophysial system (HNS). Why such bursts are necessary, however, is not well understood. Thus far, biophysical characterization of channels involved in depolarization-secretion coupling cannot completely explain this phenomenon at HNS terminals, so purinergic feedback mechanisms have been proposed. We have previously shown that ATP, acting via P2X receptors, potentiates release from HNS terminals, but that its metabolite adenosine, via A(1) receptors acting on transient Ca(2+) currents, inhibit neuropeptide secretion. We now show that endogenous adenosine levels are sufficient to cause tonic inhibition of transient Ca(2+) currents and of stimulated exocytosis in HNS terminals. Initial non-detectable adenosine levels in the static bath increased to 2.9 microM after 40 min. These terminals exhibit an inhibition (39%) of their transient inward Ca(2+) current in a static bath when compared to a constant perfusion stream. CPT, an A(1) adenosine receptor antagonist, greatly reduced this tonic inhibition. An ecto-ATPase antagonist, ARL-67156, similarly reduced tonic inhibition, but CPT had no further effect, suggesting that endogenous adenosine is due to breakdown of released ATP. Finally, stimulated capacitance changes were greatly enhanced (600%) by adding CPT to the static bath. Thus, endogenous adenosine functions at terminals in a negative-feedback mechanism and, therefore, could help terminate peptide release by bursts of APs initiated in HNS cell bodies. This could be a general mechanism for controlling transmitter release in these and other CNS terminals.
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Nicotinamide adenine dinucleotide (NAD) and its metabolites inhibit T lymphocyte proliferation: role of cell surface NAD glycohydrolase and pyrophosphatase activitiesThe presence of NAD-metabolizing enzymes (e.g., ADP-ribosyltransferase (ART)2) on the surface of immune cells suggests a potential immunomodulatory activity for ecto-NAD or its metabolites at sites of inflammation and cell lysis where extracellular levels of NAD may be high. In vitro, NAD inhibits mitogen-stimulated rat T cell proliferation. To investigate the mechanism of inhibition, the effects of NAD and its metabolites on T cell proliferation were studied using ART2a+ and ART2b+ rat T cells. NAD and ADP-ribose, but not nicotinamide, inhibited proliferation of mitogen-activated T cells independent of ART2 allele-specific expression. Inhibition by P2 purinergic receptor agonists was comparable to that induced by NAD and ADP-ribose; these compounds were more potent than P1 agonists. Analysis of the NAD-metabolizing activity of intact rat T cells demonstrated that ADP-ribose was the predominant metabolite, consistent with the presence of cell surface NAD glycohydrolase (NADase) activities. Treatment of T cells with phosphatidylinositol-specific phospholipase C removed much of the NADase activity, consistent with at least one NADase having a GPI anchor; ART2- T cell subsets contained NADase activity that was not releasable by phosphatidylinositol-specific phospholipase C treatment. Formation of AMP from NAD and ADP-ribose also occurred, a result of cell surface pyrophosphatase activity. Because AMP and its metabolite, adenosine, were less inhibitory to rat T cell proliferation than was NAD or ADP-ribose, pyrophosphatases may serve a regulatory role in modifying the inhibitory effect of ecto-NAD on T cell activation. These data suggest that T cells express multiple NAD and adenine nucleotide-metabolizing activities that together modulate immune function.
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Regulation of Contractility by Adenosine A<sub>1</sub> and A<sub>2A</sub> Receptors in the Murine Heart: Role of Protein Phosphatase 2A: A DissertationAdenosine is a nucleoside that plays an important role in the regulation of contractility in the heart. Adenosine receptors are G-protein coupled and those implicated in regulation of contractility are presumed to act via modulating the activity of adenylyl cyclase and cAMP content of cardiomyocytes. Adenosine A1 receptors (A1R) reduce the contractile response of the myocardium to β-adrenergic stimulation. This is known as anti adrenergic action. The A2A adenosine receptor (A2AR) has the opposite effect of increasing contractile responsiveness of the myocardium. The A2AR also appears to attenuate the effects of A1R. The effects of these receptors have been primarily studied in the rat heart and with the utilization of cardiomyocyte preparations. With the increasing use of receptor knockout murine models and murine models of various pathological states, it is of importance to comprehensively study the effects of adenosine receptors on regulation of contractility in the murine heart. The following studies examine the adenosinergic regulation of myocardial contractility in isolated murine hearts. In addition, adenosinergic control of contractility is examined in hearts isolated from A2AR knockout animals. Responses to adenosinergic stimulation in murine isolated hearts are found to be comparable to those observed in the rat, with A1R exhibiting an anti adrenergic action and A2AR conversely enhancing contractility. A significant part of the A2AR effect was found to occur via inhibition of the A1R antiadrenergic action. A part of the anti adrenergic action of A1R has previously been shown to be the result of protein phosphatase 2A activation and localization to membranes. Additional experiments in the present study examine the effect of adenosinergic signaling on PP2A in myocardial extracts from wild type and A2AR knockout hearts. A2AR activation was found to decrease the activity of PP2A and enhance localization of the active enzyme to the cytosol; away from its presumed sites of action. In the A2AR knockout the response to A1R activation was enhanced compared with the wild type and basal PP2A activity was reduced. It is concluded that A2AR modulation of PP2A activity may account for the attenuation of the A1R effect by A2AR observed in the contractile studies.
