Physiologically, there is evidence for presynaptic P2 receptors controlling the release of dopamine (Trendelenburg & Bültmann, 2000), serotonin (Von Kügelgen et al, 1997), noradrenaline (Koch et al, 1997;Von Kügelgen et al, 1994), glycine (Rhee et al, 2000), GABA (Hugel & Schlichter, 2000) and glutamate in the CNS (see below for references).
Some of these studies need to be interpreted cautiously because, although the results point to the involvement of a presynaptic P2 receptor, it is not necessarily located on the afferent terminals or the axons. However, there are studies where the presynaptic receptor is clearly located on the
terminals or the axon. One such study is that of Gu and MacDermott (Gu & MacDermott, 1997) on a preparation of cultured dorsal horn neurons in which the somata had been removed. In this study the P2X selective agonist a,(3-methyleneATP increased the frequency of spontaneous, TTX- insensitive glutamate currents by two mechanisms; the facilitation was partly action potential dependent presumably indicating that P2X receptors depolarized the axons to firing threshold; the remainder of the facilitation was action potential independent and resulted from C a^ influx through the P2X receptors per se. This work has been extended to acute slices of the spinal cord with similar results, except that the C a^ influx was solely through P2X receptors (Nakatsuka & Gu, 2001). Khakh and Henderson (Khakh & Henderson, 1998) reported presynaptic P2X receptors on terminals of trigeminal neurons. Facilitation of glutamate release was greatly reduced by TTX and abolished by C d^ indicating that, unlike primary afferents in the spinal cord, the C a^ influx depended on voltage- dependent C a^ channels (VDCCs). In contrast, in the nucleus tractus solitarius, where ATP and a,(3-methyleneATP reversibly increased the frequency of spontaneous, TTX-insensitive glutamate currents, the effect was completely insensitive to Cd^, indicating that the C a^ influx was entirely through P2X receptors (Kato & Shigetomi, 2001). Similarly, in a preparation of cultured sympathetic ganglion neurites (no somata present) ATP stimulated noradrenaline release was also Cd^-insensitive (Boehm,
1999). It appears, therefore, that P2X receptors can facilitate transmitter release by two distinct mechanism, possibly depending on the position of the receptors relative to the release site. To trigger release independently of VDCCs, the P2X receptors presumably have to be very close to the Ca^- sensing release machinery in order to achieve the local high concentration of C a^ required (Heidelberger et al, 1994). In contrast, distant P2X receptors, perhaps located on the axons, are unlikely to produce the
necessary C a^ signal without the opening of VDCCs. In addition, P2X receptors producing a Ca"^ signal below the release threshold could still enhance action-potential dependent release. The relative physiological importance of axonal versus terminal P2X receptors is not known, although clearly there is a difference between a receptor which enhances action-potential dependent release and one which induces release
independently of cell firing; with respect to the latter effect, it is interesting to note that the application of ATP in the NTS released sufficient
glutamate from presynaptic terminals to fire the postsynaptic cell (Kato, F, unpublished observation). Furthermore, axonal and terminal receptors might be exposed to different and independent sources of ATP.
One noteworthy study is that of Deuchars and colleagues (Deuchars et al, 2 0 0 1) in which they combined immunohistochemistry and
electrophysiology to show functional presynaptic P2X? receptors on vagal afferent terminals; immunopositive terminals were clearly visible under the electron microscope and the application of the P2Xy agonist BzATP depolarized the postsynaptic cell, an affect completely blocked by
glutamate antagonists; this was interpreted as due to a release of glutamate following activation of the P2X? on presynaptic terminals. The electron microscope also identified presynaptic P2X? receptors at the
neuromuscular junction and these were shown to be functional by
destaining FM I-43 loaded terminals with BzATP. This identification of presynaptic P2X? receptors was unexpected not only because they had previously gone undetected in CNS neurons (Collo et al, 1997) but also because the P2X? receptor can form a wide pore permeable to large molecules and associated with cell lysis (Rassendren et al,
1997;Surprenant et al, 1996). However, the presynaptic P2X? receptor appeared not to be forming such a pore, as there was no loading of terminals by either YO-PRO or 6carboxyfluorescein. Very recently, the
P2Xy receptor has also been detected on glutamate terminals in the hippocampal CAl and CA3 regions (Sperlagh et al, 2002) raising the possibility that presynaptic P2X? receptors are widespread.
The C a^ permeability of the P2X receptors predicts that they would facilitate transmitter release at terminals where they are expressed, exactly in the manner described for glutamate in the spinal cord and the NTS. In contrast, inhibitory presynaptic P2X receptors have yet to be described. There is evidence for release-inhibiting G-protein coupled receptors on glutamate terminals; however, the evidence seems less clear cut than that for P2X receptors, perhaps unavoidably so because the data is complicated by concomitant, and well documented, inhibitory effects of adenosine (Dolphin & Prestwich, 1985;Prestwich et al, 1987;Lupica et al, 1992;Fredholm & Dunwiddie, 1988). For example, at the Schaffer collateral-commissural fibre synapses onto hippocampal CAl neurons, Mendoza-Femandez and colleagues found evidence for ATP-mediated presynaptic inhibition of glutamate release (Mendoza-Femandez et al, 2000); while some of the data were consistent with the inhibition being due to breakdown to
adenosine (eg lack of effect of P2 antagonists but competitive antagonism by adenosine antagonists), other data was inconsistent with this
interpretation (eg ATP was a more potent inhibitor than adenosine, and its effects were not blocked by adenosine deaminase). Evidence that the ATP effect was via a G-protein coupled receptor came from its sensitivity to pertussis toxin. The authors concluded that there were inhibitory effects of ATP per se, acting at a P2Y receptor sensitive to theophylline-derivatives (possibly the P3 receptor proposed by others (Shinozuka et al,
1988;Todorov et al, 1994)). In contrast, in a similar study by Cunha et al (Cunha et al, 1998), the inhibitory effects of ATP were reduced by adenosine deaminase and ectonucleotidase inhibitors and potentiated by adenosine uptake inhibitors, leading them to conclude that the effects were subsequent to breakdown to adenosine. (In the same study, they were able to inhibit the effects of y-substituted ATP analogs with ectonucleotidase inhibitors whilst being unable to detect any hydrolysis of these
compounds; consequently they proposed that there is highly localized catabolism of ATP at the synapse, “channelling” the adenosine so
produced directly towards the adenosine receptors. They further proposed that this could explain the higher potency of ATP compared to adenosine -
as reported by Mendoza-Femandez - because the ATP, unlike adenosine, could slip past the adenosine uptake sites to deliver adenosine to the receptors.) It is interesting to note that the Mendoza-Femandez study reported both P2X receptors facilitating release and P2Y receptors inhibiting release at the same glutamate synapses. Why might ATP have bidirectional effects on the same terminals? As the P2 receptors have different sensitivities to ATP, one hypothesis is that the effect of
extracellular ATP is concentration dependent. A second possibility is again the idea that the receptors might be spatially separated on the terminals and therefore selectively activated by ATP from different sources; application of exogenous ATP would be unable to make this distinction.
In summary, there is good evidence for presynaptic facilitatory P2X receptors on glutamate terminals in the CNS. There is also evidence for P2X receptor-mediated presynaptic facilitation of glycine (Rhee et al,
2000), GABA (Hugel & Schlichter, 2000;Gômez-Villafuertes et al, 2001) and noradrenaline (Boehm, 1999) release in the CNS, suggesting that this mechanism of facilitation may be widespread. In the cases of
noradrenaline and GABA in the spinal cord, these are examples of positive feedback at an ATP autoreceptor.