Thomas D. White

Classification of adenosine receptors mediating antinociception in the rat spinal cord

1 Analogues of adenosine were injected intrathecally into rats implanted with chronic indwelling cannulae in order to determine a rank order of potency and hence characterize adenosine receptors involved in spinal antinociception. 2 In the tail flick test l‐N6‐phenylisopropyl adenosine (l‐PIA), cyclohexyladenosine (CHA) and 5′‐N‐ethylcarboxamide adenosine (NECA) produced dose‐related antinociception which attained a plateau level. NECA and CHA also produced an additional distinct second phase of antinociception. d‐N6‐Phenylisopropyl adenosine (d‐PIA) and 2‐chloroadenosine (CADO) had very little antinociceptive activity in this test. The rank order of potency in producing the plateau effect was l‐PIA > CHA > NECA > d‐PIA = CADO, while that for the second phase of antinociception was NECA >‐CHA. 3 Pretreatment with both theophylline and 8‐phenyltheophylline (8‐PT) antagonized antinociception produced by CHA, with 8‐PT being at least an order of magnitude more potent than theophylline. Both antagonists produced a significant hyperalgesia in the tail flick test. l‐PIA and CHA also produced methylxanthine‐sensitive antinociception in the hot plate test. 4 These results suggest that activation of A1‐receptors in the spinal cord can produce antinociception. Activation of A2‐receptors may produce an additional effect, but the relative activity of CHA in this component of activity is unusual.

RELEASE OF ATP FROM A SYNAPTOSOMAL PREPARATION BY ELEVATED EXTRACELLULAR K+ AND BY VERATRIDINE

A technique was developed which permitted the release of ATP from synaptosomes by elevated extracellular K+ or by veratridine to be directly and continuously monitored. The released ATP interacted with firefly luciferin and luciferase in the incubation medium to produce light which could be detected by a photomultiplier. The assay system was specific for ATP, in that similar concentrations of adenosine, AMP or ADP did not produce chemiluminescence. Moreover, the maximum peak of light emission correlated linearly with the concentrations of ATP present in the medium, so that semiquantitative estimates of ATP release could be made.

N-methyl-D-aspartate- and non-N-methyl-D-aspartate-evoked adenosine release from rat cortical slices: distinct purinergic sources and mechanisms of release.

Abstract: Excitatory amino acids, acting at both Nmethyl‐d‐aspartate (NMDA) and non‐NMDA receptors, release the inhibitory neuromodulator adenosine from superfused rat cortical slices. This study was initiated to investigate the possible purinergic sources and mechanisms of release for the adenosine release evoked by NMDA and non‐NMDA receptor activation. Inhibition of the bidirectional nucleo‐side transporter with dipyridamole greatly enhanced adenosine release evoked by glutamate, NMDA, kainate, and (RS‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA). Inhibition of ecto‐5′‐nucleotidase with α,β‐methylene ADP and GMP had no effect on either kainateor AMPA‐evoked adenosine release, but it decreased glutamate‐ and NMDA‐evoked adenosine release by 23 and 68%, respectively. A similar inhibition of NMDA‐evoked adenosine release was observed with α,β‐methylene ADP alone, indicating that the inhibitory effect was not due to the reported competitive inhibition of NMDA receptors by GMP. Finally, NMDA‐evoked adenosine release, but not kainate‐ or AMPA‐evoked release, was Ca2+ dependent. These results indicate that activation of non‐NMDA receptors releases adenosine per se in a Ca2+‐independent manner. In contrast, NMDA receptor activation releases primarily a nucleotide that is subsequently converted extracellularly to adenosine; in this case, release is Ca2+ dependent. Although neither NMDA‐ nor non‐NMDA‐evoked adenosine release occurs via the nucleoside transporter, this transporter does appear to be a major route for removal of adenosine from the extracellular space.

Role of excitatory amino acid receptors in K+- and glutamate-evoked release of endogenous adenosine from rat cortical slices

Abstract: : K+ and glutamate released endogenous adenosine from superfused slices of rat parietal cortex. The absence of Ca2+ markedly diminished K+‐ but not glutamate‐evoked adenosine release. Tetrodotoxin decreased K+‐ and glutamate‐evoked adenosine release by 40 and 20%, respectively, indicating that release was mediated in part by propagated action potentials in the slices. Inhibition of ecto‐5′‐nucleo‐tidase by α,β‐methylene ADP and GMP decreased basal release of adenosine by 40%, indicating that part of the adenosine was derived from the extracellular metabolism of released nucleotide. In contrast, inhibition of ecto‐5′‐nucle‐otidase did not affect release evoked by K+ or glutamate, suggesting that adenosine was released as such. Inhibition of glutamate uptake by dihydrokainate potentiated glutamate‐evoked release of adenosine. Glutamate‐evoked adenosine release was diminished 50 and 55% by the TV‐methyl‐D‐aspartate (NMDA) receptor antagonists, DL‐2‐amino‐5‐phosphonovaleric acid and (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine hydrogen maleate (MK‐801), respectively. The remaining release in the presence of MK‐801 was diminished a further 66% by the non‐NMDA receptor antagonist, 6,7‐dinitroquinoxaline‐2,3‐dione, suggesting that both NMDA and non‐NMDA receptors were involved in glutamate‐evoked adenosine release. Surprisingly, K+‐evoked adenosine release was also diminished about 30% by NMDA antagonists, suggesting that K+‐evoked adenosine release may be partly mediated indirectly through the release of an excitatory amino acid acting at NMDA receptors.

Adenosine release may mediate spinal analgesia by morphine

Spinal analgesia produced by morphine is blocked by methylxanthine adenosine receptor antagonists. In biochemical studies, morphine releases adenosine from spinal cord synaptosomes prepared from the dorsal spinal cord, as well as from the intact spinal cord in vivo. Adenosine release is reduced by intrathecal and neonatal pretreatment with capsaicin but not by intrathecal pretreatment with 6-hydroxydopamine or 5,7-dihydroxytryptamine, indicating that adenosine originates from small-diameter primary afferent neurons but not descending monoaminergic pathways. In this Viewpoint Jana Sawynok and colleagues review the evidence supporting the hypothesis that the spinal analgesic action of morphine is due to the release of adenosine from primary afferent nerve terminals and subsequent activation of A1 and A2 adenosine receptors.

Release of adenosine 5′-triphosphate from synaptosomes from different regions of rat brain

Abstract The release of adenosine 5′-triphosphate by elevated extracellular concentrations of KC1 and by veratridine was determined in synaptosomal fractions prepared from different regions of rat brain. Following correction for yields of synaptosomes from the various regions, the relative distribution of K + -induced release was corpus striatum > cerebral cortex > medulla > hypothalamus > cerebellum. In contrast, the relative distribution of veratridine-induced release of adenosine 5′-triphosphate was medulla > corpus striatum > hypothalamus > cerebral cortex > cerebellum. From these findings, it was concluded that (1) depolarization-induced release of adenosine 5′-triphosphate was not distributed uniformly throughout the brain but varied from region to region, (2) the K + -induced release of adenosine 5′-triphosphate which is Ca 2+ -dependent, had a different regional distribution than the veratridine-induced release, which is greatest in Ca 2+ -free medium, and (3) the distribution of K + -induced release of adenosiae 5′-triphosphate did not correlate well with the known distribution of noradrenaline concentrations in rat brain, but did correlate to some extent with the distributions of 5-hydroxytryptamine, dopamine and especially acetylcholine, so that co-release of adenosine 5′-triphosphate with these transmitters may possibly occur.

Nature of extrasynaptosomal accumulation of endogenous adenosine evoked by K+ and veratridine.

Abstract: When rat brain synaptosomes were incubated for 10 min at 37°C, basal accumulation of adenosine in the medium was 66 pmol/mg of protein. An elevated K+ level (24 mM) evoked an additional accumulation of 200 pmol/mg of protein, and 50 μM veratridine evoked 583 pmol of adenosine accumulation/mg of protein. K+‐ and veratridine‐evoked accumulation of adenosine did not arise from microsomal or mitochondrial contaminants of the synaptosomal preparation, because purified microsomes and mitochondria did not exhibit evoked accumulation of adenosine in the medium. K+‐evoked accumulation of extrasynaptosomal adenosine was Ca2+‐dependent, whereas veratridine‐evoked accumulation of adenosine was increased in Ca2+‐free medium. In the presence of α,β‐methylene ADP and GMP, which inhibit ecto‐5′‐nucleotidase, conversion of added ATP and AMP to adenosine was inhibited by 90% in synaptosomal suspensions. However, inhibition of ecto‐5′‐nucleotidase only reduced basal extrasynaptosomal accumulation of adenosine by 74%, veratridine‐evoked accumulation of adenosine by 46%, and K+‐evoked accumulation by 33%. Most of the basal accumulation of extrasynaptosomal adenosine appears to be derived from released nucleotide, probably ATP, but about half of the veratridine‐evoked accumulation of adenosine and most of the K+‐evoked accumulation may arise from adenosine released in its own right, rather than from a released nucleotide.

N-methyl-d-aspartate, kainate and quisqualate release endogenous adenosine from rat cortical slices

N-Methyl-D-aspartate, kainate, and quisqualate released endogenous adenosine from superfused slices of rat parietal cortex. N-Methyl-D-aspartate-evoked adenosine release was blocked by D,L-2-amino-5-phosphono-valeric acid and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801), indicating that it was receptor-mediated, although it did not show the expected potentiation in the absence of Mg2+. In contrast, N-methyl-D-aspartate-evoked release of [3H]noradrenaline from the same slices was markedly potentiated in Mg2(+)-free medium. Therefore, the lack of Mg2+ modulation of N-methyl-D-aspartate-evoked adenosine release was not due to depolarization-induced alleviation of the Mg2+ block in the slices. Kainate-evoked adenosine release was diminished by the non-specific excitatory amino acid antagonist, gamma-D-glutamyl-glycine, and kainate- and quisqualate-evoked adenosine release was diminished by 6,7-dinitroquinoxaline-2,3-dione, indicating that these agonists release adenosine by acting at non-N-methyl-D-aspartate receptors. Tetrodotoxin decreased N-methyl-D-aspartate- and kainate-evoked adenosine release by 40% and 19% respectively, indicating that release was mediated in part by propagated action potentials in the slices. Total release of adenosine by N-methyl-D-aspartate, kainate or quisqualate was not diminished in the absence of Ca2+. A second exposure to kainate following restoration of Ca2+ to slices previously depolarized in the absence of Ca2+ resulted in an amount of adenosine release equal to an initial release by slices in the presence of Ca2+, a result suggesting the presence of separate Ca2(+)-dependent and Ca2(+)-independent pools of adenosine. The present experiments demonstrate that activation of all three major subtypes of excitatory amino acid receptors in the cortex releases adenosine, possibly from separate Ca2(+)-dependent and -independent pools. Adenosine released from the cortex following excitatory amino acid stimulation may, by acting at inhibitory P1 purinoceptors, diminish excitatory neurotransmission and protect against excitotoxicity.

Glutamate-Evoked Release of Endogenous Adenosine from Rat Cortical Synaptosomes Is Mediated by Glutamate Uptake and Not by Receptors

Abstract: L‐Glutamate (10 μM–1 mM) released endogenous adenosine from rat cortical synaptosomes. Studies with excitatory amino acid antagonists, (+)‐5‐methyl‐16, 11,dihydro‐5H‐dibenzo[a,d]cyclohepten‐5, 10‐imine maleate (MK‐801), 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX), Mg2+, and agonists N‐methyl‐D‐aspartate (NMDA), kainate, and quisqualate, indicated that this release was not receptor mediated. D,L‐2‐Amino‐4‐phosphonobutanoic acid (APB) also did not affect glutamate‐evoked adenosine release. Inhibition of glutamate uptake by dihydrokainate or replacement of extracellular Na+ blocked glutamate‐evoked adenosine release. D‐aspartate, which is a substrate for the glutamate transporter but is not metabolized, also released adenosine, suggesting that release was due to amino acid transport and not to its subsequent metabolism. D‐Glutamate, a relatively poor substrate for the transporter, was correspondingly less potent than L‐glutamate at releasing adenosine. Glutamate‐evoked adenosine release was not Ca2+ dependent or tetrodotoxin sensitive and did not appear to occur on the bidirectional nucleoside transporter. Inhibition of ecto‐5′‐nucleotidase virtually abolished glutamate‐evoked adenosine release, indicating that adenosine was derived from extracellular metabolism of released nucleotide(s). However, L‐glutamate did not release ATP and did not appear to release cyclic AMP. Therefore, transport of glutamate into presynaptic terminals releases some other nucleotide which is converted extracellularly to adenosine. This adenosine could act at P1‐purinoceptors to modulate glutamatergic neurotransmission.

Neural Release of ATP and Adenosinea

Release of ATP can be evoked from noradrenergic nerve varicosities isolated from guinea pig ileal myenteric plexus by depolarization with K+ and veratridine and during exposure to acetylcholine or 5-HT. Clonidine, however, modulates the release of [3H]noradrenaline without affecting the release of ATP. ATP is also released from noradrenergic sympathetic nerves in the vas deferens, where it mediates the initial depolarization and contraction in the smooth muscle. Factors that apparently modulate the release of noradrenaline do not produce corresponding effects on ATP release. The above results are best explained by the hypothesis that ATP and noradrenaline are stored in separate populations of vesicles within sympathetic nerves and that these pools are subject to differential presynaptic modulation. Depolarization of rat brain synaptosomes releases adenosine by a process that is mediated, at least in part, by efflux on the nucleoside transporter. Drugs that block the nucleoside transport (such as dipyridamole) reduce evoked adenosine release and may thereby diminish, rather than augment, the actions of adenosine at its receptors. Release of adenosine does not appear to be uniformly distributed throughout the brain insofar as release varies from synaptosomes prepared from different regions. Although the distribution of several markers for possible adenosine pathways in the brain, including adenosine release, do not show any consistent correlations, the non-uniform distribution for these markers suggests that adenosine may have differential functions in various brain regions.