Prejunctional Adenosine and ATP Receptors" E. M. SILINSKY, J. M. HUNT, C. S. SOLSONA, AND J. K. HIRSH Department of Pharmacology Northwestern University Medical School Chicago, Iiiinois 6061I

INTRODUCTION ATP and adenosine are potent inhibitors of neurotransmitter secretion in the vertebrate nervous system.'" At the majority of synapses investigated, the prejunctional inhibitory effects of ATP are mediated by the hydrolysis product adenosine acting on adenosine receptors.' At some synaptic loci, however, unhydrolyzed ATP exerts the presynaptic inhibitory The first purpose of this paper is to discuss the likely mechanism by which adenosine, via adenosine receptors, inhibits the release of acetylcholine (ACh) from peripheral nerve endings in frog and electric fish. The second purpose is to provide a discussion of the direct inhibitory action of ATP on certain prejunctional purinoceptors. The processes by which adenosine inhibits transmitter release are controversial at present and may involve different cellular mechanisms at different presynaptic membranes.6 We will focus on the effects of adenosine at presynaptic motor nerve endings in frog and electric fish, where a more cohesive picture of the action of adenosine is emerging. The presynaptic receptor responsible for the inhibitory effects of adenosine is a P , - p ~ r i n o c e p t o rThe . ~ ~ potential ~~ mechanisms underlying this inhibition are depicted in FIGURE 1, sites 1-4. First, adenosine could inhibit transmitter release by effects on the Na+ and Kf currents underlying the nerve terminal action potential (ntp) (site 1). Next, adenosine could inhibit transmitter release by reducing Ca2+entry through voltage-gated Ca2+channels, thus impairing the delivery of Ca2' to strategic regions associated with neurotransmitter secretion (site 2). Based upon studies of ionic currents in cell soma, two particularly favored explanations for the inhibitory action of adenosine are 1) that adenosine decreases Caz+entry and/or 2) that adenosine increases "This research was supported by Grant NS 12782 from the National Institutes of Health. J. K. H. was supported by a predoctoral fellowship from the Lucille P. Markey Charitable Trust Foundation (and by NS 12782 for postdoctoral work); J. M. H. was supported by a predoctoral training grant from the National Institute of Neurological and Communicative Disorders and Stroke. 324

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K + conductance, thus hyperpolarizing the membrane and preventing activation of Ca2+channels (for a discussion, see Silinskyll). In contrast to the potential regulation of Ca2+availability by effects on membrane ionic channels, adenosine could also regulate Ca2+availability via intracellular processes. For example, adenosine could inhibit ACh release by increasing the uptake of Ca2+ into storage sites, thereby reducing free intracellular Ca2+ concentrations (site 3). Finally, adenosine could act directly on the secretory process by decreasing the ability of Ca2+ to promote ACh release, for example, by decreasing the apparent affinity for Ca2+ or decreasing the number of activatable release sites (site 4). Earlier evidence from this laboratory suggested that the inhibitory action of adenosine was at the level of the secretory apparatus (site 4) in frog motor n e r ~ e . + ' ~ * ~ ~ Further evidence in support of this viewpoint from our laboratory and from otherd4 will be presented in this paper.

FIGURE 1. Possible target sites for the inhibitory effects of adenosine on neurotransmitter secretion. Asterisks: Ca2+;ntp: nerve terminal action potential; V: synaptic vesicle; az: active zone of secretion.

METHODS Conventional electrophysiologicalmethods for intracellular and focal (loose patch) recording were employed in this study on frog cutaneous pectoris nerve-muscle preparation~.'~.'~ Effects of adenosine derivatives on quanta1 ACh release were assessed by changes in 1) the mean number of ACh quanta released synchronously (E) as determined from the ratio of the mean end-plate potential (epp) to miniature endplate potential (mepp) amplitudes, 2) the epp or end-plate current (epc) amplitude (as adenosine does not affect the amplitude of the mepp), or 3) the frequency of occurrence of mepps.

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RESULTS

Adenosine Derivatives Do Not Alter Na+ and K + Currents Associated with the Action Potential in Frog Motor Nerve Endings

Using focal (loose patch) recording, which measures both the prejunctional action currents and the epc with the same recording electrode, it was found that neither Na+ nor K+ currents were impaired under conditions in which adenosine derivatives inhibits ACh release4 (FIGS.2a & 2b). In recent experiments, we found that concentrations of tetraethylammonium (TEA, 1 mM) and 3,4-diaminopyridine (DAP, i00 pM) that eliminate all K+ currents in motor nerve endings did not prevent the inhibitory effects of adenosine (FIG.2c). It thus appears that adenosine does not affect the Na+ or K+ currents associated with the presecretory nerve terminal action potential.

Blockade of Ca2+Entry b Unlikely to Be Responsible for the Inhibitory Effects of Adenosine in Frog and Electric Fish

Measurements of Ca2+ Entry

Direct measurements of the entry of radiolabeled Caz+ (45Ca2+)into Torpedo nerve terminals and the concomitant release of ACh were made by Muller et aI.14 These workers found that under conditions in which adenosine reduced ACh release (FIG. 3a), this nucleoside did not reduce Ca2+ entry. In contrast, Caz+ entry and evoked ACh release were blocked competitively by Ca2+ channel blockers such as Cd2+ (FIG.3b). In preliminary experiments from our laboratory, Ca2+currents were measured directly using loose patch recordings from the heminode or from the nerve ending of frog motor nerve after complete blockade of K+ channels with TEA and DAP.” Neither adenosine (50 pM) nor 2-chloroadenosine (2.5 pM) inhibited the Ca2+ currents under conditions in which the Ca2+ channel blocker Co2+ (2.5-5.0 mM) blocked such currents. Indirect measurements of Ca2+ currents were made using the reverse gradient approach. By this method, a depolarizing stimulus applied in Caz+-freeEGTA Ringer decreases rather than increases ACh release. This is because Ca2+-freeEGTA Ringer generates a reverse concentration gradient whereby Ca2+ is in higher concentration inside the cell than outside. Depolarization under reverse gradient conditions thus elicits the efflux of Ca2* from the cell through voltage-gated Ca2+ channels, reduces the Ca2+concentrations at release sites, and reduces mepp frequency.” If adenosine blocks Ca2+ channels, then adenosine should increase ACh release under reverse gradient conditions by blocking the egress of Ca2+.This is not the case, however. Adenosine derivatives continue to inhibit ACh secretion under reverse gradient conditions-conditions in which the Ca2+ channel blocker Co2+increases release ( N = 8). The results of Muller et al. l 4 and our own data thus suggest that Ca2+ channel blockade is unlikely to be responsible for the inhibitory effect of adenosine.

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Effects of Adenosine on ACh Release Evoked Independently of Ca2+Channels Two methods that evoke CaZ -dependent ACh release by mechanisms bypassing active Ca2+ channels were employed: CaZ'-containing liposomes4 and the Caz+ ionophore ionomycin." It was found previously that adenosine derivatives inhibit ACh +

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FIGURE 2. Evidence that an increased K + conductance is not responsible for the inhibitory effects of adenosine. (a) Control response recorded with a focal microelectrode. (b) Responses after an 8-min superfusion with 10 pM 2-chloroadenosine. Note the decline in the end-plate current (epc) without an effect on the currents underlying the nerve terminal action potential (ntp). The upward-going phase of the ntp reflects the Na+ component of the action potential whereas the downward-going phase represents the K ' currents recorded at the frog nerve ending (for details, see reference 15). Ringer contained low Ca" and high M g 2 ' . Traces in a and b are the averaged responses to 128 stimuli ( 1 Hz). Reproduced with permission from reference 4. (c) Adenosine still inhibits ACh release after treatment with sufficient concentrations of K ' channel blockers (TEA, 1 mM; 3,4-diaminopyridine, 100 pM) to block completely K' channels at frog motor nerve endings. Each record is the response to a single stimulus. As thcsc K + channel blockers produce large increases in evoked ACh release, the Ringer contained reduced Ca", elevated Mg2+, and tubocurarine to reduce the ACh release to levels at which the immediately available store of quanta would not be depleted and to maintain subthreshold epps.

release produced by Ca2' -containing lipo~omes.~ In two preliminary experiments using ionomycin (1.3-50 pM), increases in mepp frequency produced by this ionophore were reversibly inhibited by adenosine. It thus appears that active CaZ' channels are not required for inhibitory effects of adenosine to be observed on CaZ' -evoked ACh secretion.

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The Cellular Ca2+Buffer BAPTA neither Mimics the Effect of Adenosine nor Occludes the Inhibitory Effects of Adenosine At frog motor nerve endings, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, delivered to the cytoplasm by using extracellular BAPTA-AM) was found to reduce mepp frequency and to inhibit facilitation; yet it did not impair

FIGURE 3. Noncompetitive blockade of evoked ACh release by adenosine (a) and competitive blockade of ACh release by the Caz+channel blocker Cd 2 + (25 p M ) (b). Under these conditions, adenosine (0.5 mM) had no effect on Ca2+ entry into nerve endings whereas Cd2' inhibited both evoked Ca2+ entry and ACh release. Reprinted with permission from reference 14.

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synchronous evoked ACh release in response to one nerve impulse (hi)." Apart from the similar decrease in mepp frequency, the other effects of BAPTA differ from those of adenosine. Specifically,adenosine produces a decrease in both mepp frequency and m without inhibiting facilitation. It is thus unlikely that evoked ACh release is inhibited by a decrease in the free intracellular Caz+ concentration. If adenosine inhibited spontaneous ACh release by decreasing free Ca2+levels in the nerve ending, then the similar reductions in mepp frequency reported for BAPTAI9 and for adenosine1*2,4

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suggest that BAPTA has a buffering capacity near that of the putative adenosinesensitive Ca2+ translocation mechanism. It would thus be predicted that BAPTA should occlude the inhibitory effects of adenosine on ACh release. In nine experiments on BAPTized preparations, we found2’ that BAPTA did not alter the inhibitory effects of adenosine on mepp frequency (mean of 41% inhibition 2 5 % standard error of the mean, which is in the range of control values found in other studies in our laboratory). Evidence that BAPTA was actually buffering Ca2’ is based upon the observations that 1) BAPTA blocked the excitatory effects of caffeine and that 2) BAPTA delayed that excitatory effects of high K+ and ionomycin on ACh release (the delay being due to the time required to saturate the intracellular BAPTA with Ca2’). These results? when taken in conjunction with earlier results: suggest that adenosine is unlikely to inhibit ACh release by decreasing free cytoplasmic Ca2+ concentrations.

Adenosine Decreases the Ability of Ca2+to Promote ACh Secretion Previous results from this laboratory have found that dose-response relationships for extracellular Ca2+and Sr2+in the process of evoked ACh release are incompatible with simple competitive block of alkaline earth cation entry by adenosinePrl2Computer modeling demonstrated that a reduction in the apparent affinity of Ca2+for a strategic component of the secretory apparatus accurately simulates the actual effect of adenosine analogues on evoked ACh r e l e a ~ e . ~ FIGURE ’.~~ 3 shows results from the work of Muller et aZ.I4 demonstrating that adenosine acts very differently from Ca2+channel blockers in electric fish. The effects observed in FIGURE 3 are thus incompatible with competitive inhibition of alkaline earth cation entry by adenosine derivatives. Muller et a l l 4 suggested that adenosine reduces the number of active release sites. These results from frog and electric fish suggest that the inhibition of ACh release by adenosine is primarily due to the decreased ability of a fixed cellular concentration of Ca2+to promote the secretory process.

What Type of Adenosine Receptor Is Responsible for the Inhibition of ACh Release? 8-Cyclopentyltheophylline is a potent inhibitor of ACh release.6.22This observation, when coupled with the results to be described below, suggests that the receptor responsible for the inhibition of neurotransmitter release is a PI-purinoceptor of the A, flavor. (Considerable controversy does exist, however, and the reader is referred to references 5 , 11, and 21 for discussion.) At rat2’ and mousez4motor nerve endings, pertussis toxin (PTX) consistently blocked the effects of adenosine in inhibiting ACh release. In some experiments on frog, PTX blocked the inhibitory effects of adenosine on evoked ACh release.2’ Unfortunately, the effects of PTX in frog were inconsistent?’ The successful experiments from both species suggest, however, that a PTX-sensitive guanine nucleotide-binding protein (G protein) is responsible for the inhibitory effect of adenosine. Specific PTX-sensitive G proteins include GI, which links adenosine to the inhibition of adenylyl cyclase, and Go, which may directly link adenosine to a cellular

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effector without the need for pho~phorylation.2~ To determine whether phosphorylation via the cyclic AMP system is involved in the inhibitory effects of adenosine, we treated preparations with a nonspecific isoquinoline sulfonamide inhibitor of protein kinases, H-7.26In the presence of concentrations of H-7 sufficient to block the effects of the cyclic AMP analogue 8 4 4-~hlorophenylthio)-cAMP*~ and of phorbol esters:* aden2~ thus appears osine still exerted its usual inhibitory effects in frog or ~ a t . Adenosine to inhibit ACh release independently of phosphorylation via CAMP-dependent protein kinase or protein kinase C.

ATP and the Inhibition of Transmitter Release As discussed above, the prejunctional effects of ATP at most synapses are due to the hydrolysis product, adenosine. There are a number of instances, however, where ATP directly inhibits ACh release. FIGURE 4 shows that in frog sympathetic chain ganglia, ATP but not adenosine inhibits ACh release.3 2-Chloroadenosine did not significantly inhibit ACh release from these synapses at concentrations two orders of magnitude greater than that necessary to produce maximal inhibition of ACh release FIGURE 5 shows that a$-methylenefrom motor nerve endings of the same ~pecies.~ ATP was without effect (suggesting that a P,,-purinoceptor was not involved in inhibition at preganglionic nerve endings), but that theophylline did block the effect of ATP. Because of such results, it has been suggested that a new purinoceptor subtype (P,) may be necessary to explain some of these effects of ATP.3' In bullfrog sympathetic ganglia, the potency order of purines was ATP > ADP > AMP > adenosine for the inhibition of ACh release. In bullfrog, the effect has been attributed to a depolarization of the nerve ending.32Interestingly, in guinea pig ileum, ATP, in a theophylline-sensitive manner, exerted a direct action to inhibit ACh release? This effect of ATP was not due to degradation to adenosine, and was not mimicked by a,& methylene-ATP. The effects of ATP derivatives at cholinergic nerve endings in frog sympathetic ganglia and in guinea pig ileum are thus remarkably similar. Adenosine was also an effective inhibitor of ileal ACh release, however, through the same purinoceptor as ATP.

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FIGURE 4. Inhibition of ACh release from frog sympathetic chain ganglion by ATP but not adenosine. The electrophysiological methods were similar to those described in the text for frog neuromuscular junctions. Ringer contained 1 mM Ca2+and 18 mM Mg2+.Each point represents m calculated by the method of failures'6 in response to 128 preganglionic nerve stimuli delivered at a frequency of I Hz. Open circles: control m. Reprinted with permission from reference 3.

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FIGURE 5. Effects of ATP (200 pM), a,P-methylene-ATP (200 pM), and theophylline ( 2 mM) on evoked ACh release (K) in frog sympathetic chain ganglia. Evoked ACh release is expressed relative to the control level, which is taken as 1; values below 1 indicate inhibition. Ringer solution contained 1 mM Ca" and 17 mM Mg*+.Modified with permission from reference 3.

DISCUSSION AND CONCLUSIONS The results suggest that activation of PI-purinoceptors by adenosine inhibits ACh release by impairing the ability of Ca2+ to promote secretion at frog and electric fish motor nerve endings. Inhibition of ACh secretion by adenosine is unlikely to be mediated by A or C kinases. It is possible that adenosine receptor sites may be directly coupled to the secretory apparatus via G proteins. The independent suggestions of a decreased Caz+ affinity?" and of a decreased number of activatable release are compatible as it has been found that a release site is likely to require a certain concentration of bound Caz+ to be in an activatable The generality of these results to other synapses is unknown at present, although a case may be made for a similar mechanism for the inhibition of transmitter release by adenosine from the CA1 region of the hippocampus (as argued in reference 11, but see references 6 and 30 for other possibilities). It also appears that in some systems ATP is capable of inhibiting ACh release in a theophylline-sensitive manner, and that it does not do so by degradation to adenosine. Although such results may be due to a P, receptor, it is plausible to suggest that PIpurinoceptors be further divided into a P,, subtype-the conventional adenosine receptor-and a PI, subtype-the theophylline-sensitive site at which ATP is more potent than the other naturally occurring adenosine derivatives. Such a subdivision would be consistent with the traditional approach of defining receptors by selective competitive antagonists.

REFERENCES 1. GINSBORG, B. L. & G. D. S. HIRST.1972. The effect of adenosine on the release of the transmitter from the phrenic nerve of the rat. J. Physiol. 224 629-645. 2. SILINSKY, E. M. 1980. Evidence for specific adenosine receptors at cholinergic nerve endings. Br. J. Pharmacol. 71: 191-194. 3. SILINSKY, E. M. & B. L. GINSBORG.1983. Inhibition of acetylcholine release from preganglionic frog nerves by ATP but not adenosine. Nature 305: 327-328.

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4. SILINSKY,E. M. 1984.On the mechanism by which adenosine receptor activation inhibits the release of acetylcholine from motor nerve endings. J. Physiol. 346: 243-256. 1986. Adenosine receptors and calcium: Basis for 5. RIBEIRO,J. A. & A. M. SEBASTIAO. proposing a third (A,) adenosine receptor. Prog. Neurobiol. 26: 279-309. B. B. & T. V. DUNWIDDIE. 1988. How does adenosine inhibit transmitter 6. FREDHOLM, release? Trends Pharmacol. Sci. 9: 130-134. 1987.On the role, inactivation and origin of endogenous 7. RIBEIRO,J. A. & A. M. SEBASTIAO. adenosine at the frog neuromuscular junction. J. Physiol. 385: 571-585. N. P., L. E. GUSTAFSSON & J. LUNDIN.1985.Pre- and postjunctional modulaton 8. WIKLUND, of cholinergic neuroeffector transmission by adenine nucleotides: Experiments with agonist and antagonist. Acta Physiol. Scand. 125: 681-691. G. 1990. Overview: Purinergic mechanisms. Ann. N.Y. Acad. Sci. This 9. BURNSTOCK, volume. G. 1990.Dual control of local blood flow by purines. Ann. N.Y. Acad. Sci. 10. BURNSTOCK, This volume. 11. SILINSKY,E. M. 1989.Adenosine derivatives and neuronal function. Semin. Neurosci.: 1: in press. 12. SILINSKY,E. M. I981. On the calcium receptor that mediates depolarization-secretion coupling at cholinergic motor nerve terminals. Br. J. Pharmacol. 7 3 413-429. 13. SILINSKY, E. M. 1986.Inhibition of transmitter release by adenosine: Are calcium currents depressed or are the intracellular effects of calcium impaired? Trends Pharmacol. Sci. 7: 180-185. 14. MULLER,D., F. LOCTIN& Y. DUNANT.1987. Inhibition of evoked acetylcholine release: Two different mechanisms in the Torpedo electric organ. Eur. J. Pharmacol. 133 225-234. 15. MALLART,A. 1984.Presynaptic currents in frog motor endings. Pfluegers Arch. (Eur. J. Physiol.) 400: 8-13. 16. SILINSKY,E. M. 1987.Electrophysiological methods for studying acetylcholine secretion. I n The Secretory Process. Vol. 3:In Vim Methods for Studying Secretion. A. M. Poisner & J. T. Trifaro, Eds.: 255-271.Elsevier. Amsterdam. 17. SHIMONI, Y., E. ALNAES& R.RAHAMIMOFF. 1977.IS hyperosmotic neurosecretion from motor nerve endings a calcium-dependent process? Nature 267: 170-172. 18. BEELER,T. J., I. JONA & A. MARTONOSI. 1979.The effect of ionomycin on calcium fluxes in sarcoplasmic reticulum vesicles and liposomes. J . Biol. Chem. 254: 6229-623I . 19. KIJIMA,H. & N. TANABE.1988.Calcium-independent increase of transmitter release at frog end-plate by trinitrobenzene sulphonic acid. J. Physiol. 403 135- 149. 20. HUNT J. M. & E. M. SILINSKY. 1989.BAPTism of frog motor nerve terminals does not impair the inhibitory actions of adenosine on acetylcholine release. SOC.Neurosci. Abstr. 15: 484. 21. SILINSKY,E. M., C. S. SOLSONA,J. K. HIRSH& J. M. HUNT. 1989. Calcium-dependent acetylcholine secretion: Influence of adenosine. In Adenosine Receptors in the Nervous System. J. A. Ribeiro, Ed.: 141-149.Taylor & Francis. London. A. M. & J. A. RIBEIRO.1989. 1,3,8-and 1,3,7-Substituted xanthines: Relative 22. SEBASTIAO, potency as adenosine receptor antagonists at the frog neuromuscular junction. Br. J. Phannacol. 96 211-219. E. M. C. S. SOLSONA & J. K. HIRSH.1989.Pertussis toxin prevents the inhibitory 23. SILINSKY, effect of adenosine and unmasks adenosine-induced excitation at mammalian motor nerve endings. Br. J. Pharmacol. 97: 16-18. 24. CHEN,H., Y. N. SINGH& W. F. DRYDEN.1989.Transduction mechanism involving the presynaptic adenosine receptor at mouse motor nerve terminals. Neurosci. Lett. 96: 3 18-322. 25. KURACHI,Y., T. NAKAJIMA& T. SUGIMOTO. 1986. On the mechanism of activation of muscarinic K + channels by adenosine in isolated atrial cells: Involvement of GTP-binding proteins. Pfluegers Arch. (Eur. J. Physiol.) 407: 264-274. S. KAWAMOTO & Y. SASAKI.1984.Isoquinolinesulfonamides: 26. HIDAKA,H., M. INAGAKI, Novel and potent inhibitors of cyclic nucleotide-dependent protein kinase and protein kinase C. Biochemistry 23 5036-5041. 27. HIRSH,J. K. & E. M. SILINSKY.1989. Signal transduction and the adenosine receptor

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inhibitory to acetylcholine release in frog motor nerve endings. SOC.Neurosci. Abstr. 15 484. CARATSCH, C. G., S. SCHUMACHER, F. GRASSI& F. EUSEBI.1988. Influence of protein kinase C stimulation by a phorbol ester on neurotransmitter release at frog end-plates. Naunyn-Schmeideberg’s Arch. Pharmacol. 337: 9- 12. SILINSKY, E. M. 1985. The biophysical pharmacology of calcium-dependent acetylcholine secretion. Pharmacol. Rev. 37: 81 - 132. HAMILTON,B. R., Z. Lu & D. 0. SMITH. 1988. Modulation of calcium currents in mammalian motor nerve terminals. SOC.Neurosci. Abstr. 14: 69. STONE,T. 1985. Some unresolved problems. In Purines: Pharmacological and Physiological Roles. T. W. Stone, Ed.: 245-251. Macmillan. London. AKASU,T., P. SHINNICK-GALLAGHER & P. GALLAGHER. 1985. Actions of purines in autonomic ganglia. In Purines: Pharmacological and Physiological Roles. T. W. Stone, Ed.: 57-66. Macmillan. London.

DISCUSSION OF THE PAPER

E. W. WESTHEAD(University of Massachusetts, Amherst, MA): Have you any way of determining whether adenosine or ATP reduces the Ca2+ transient produced by stimulation? In chromaffin cells, we were surprised to find that even ionomycinstimulated Caz+ transients are reduced by adenosine, suggesting that adenosine increases the rate of sequestration or extrusion of Caz+. SILINSKY:This would indeed be an ideal experiment, but at this stage fura-2 injection into the very small presynaptic motor neuron endings in frog and subsequent quantitative measurements of Caz+ transients is technically impossible for mere mortals. Your result with chromaffin cells is most interesting. It suggests that active membrane ionic channels are not necessary for the inhibitory effects of adenosine in chromaffin cells. We need to do another series of experiments with BAPTA-AM and ionomycin before direct comparison between your results and ours may be made, especially if adenosine is capable of reducing [ C a l l , below that buffered by BAPTA. For example, if adenosine is decreasing mepp frequency by decreasing [ Ca*+],,then in the presence of BAPTA, after adenosine has produced its inhibitory effect, ionomycin should produce rapid restoration of mepp frequency to the pre-adenosine level (as this Caz+ would not be buffered by BAPTA). If adenosine works by another mechanism, then ionomycin would have its usual delay in BAPTA before producing any increase in mepp frequency. These experiments are currently in progress by JimBob Hunt. N. P. WIKLUND(Karolinska Institutet, Stockholm, Sweden): Have you tried determining whether inhibitors of ATP degradation have any effect on the proposed prejunctional ATP effects? In the guinea pig ileum, inhibition of 5’-nucleotidases with such inhibitors as a,P-Me-ADP and IMP enhances the inhibitory effects of ATP or ADP on contractile responses to nerve stimulation, suggesting that ATP and ADP can act per se on prejunctional P,-purinoceptors (Wiklund et. al., Acta Physiol. Scand. 126 217-223, 1986). Furthermore Schild analysis on inhibition of contractile responses to nerve stimulation by adenine nucleotides in guinea pig ileum, using the selective adenosine receptor antagonist 8-p-sulfophenyltheophylline,showed slopes of unity for several nucleotides, including ATP, ADP, AMPPNP, and j3,y-Me-ATP. The nucleotides had pA2-values not significantly different from the pA2-value of 2-chlo-

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roadenosine, strongly suggesting action by adenine nucleotides per se on prejunctional adenosine receptors ( c j Acta Physiol. Scund. 1 2 5 681 -691, 1985). SILINSKY: We have not tried the experiment you described. Akasu and colleagues have found in bullfrog that ADP and AMP have slight effects, with the following sequence: ATP > ADP > AMP > adenosine. We definitely need to pursue a more extensive series of analogues, including adenosine deaminase inhibitors and 5’-nucleotidases. We should also explore the possible antagonism of ATP effects by a,PMe-ATP and other nonhydrolyzable analogues. Based upon 1 ) the Wiklund et ul. reference cited by you and in our paper, 2) the work of Collis and Pettinger, 3) the poster from Dr. Westfall’s laboratory at this meeting, and 4) our results, it is predicted that the putative P,,-purinoceptor would not be activated by a,P-Me-ATP (which might be antagonistic), P,y-Me-ATP (which should be an agonist), and blockade by 8-phenyltheophylline. The stimulating presentations and discussions at this meeting have provided an impetus for us to resume work on the inhibitory purine receptor at frog preganglionic nerve endings. Y. H. EHRLICH (College of Stuten Island, New York, NY): Could inhibition of the ATP effect by theophylline be mediated by a mechanism other than blockade of a P,-type receptor? SILINSKY: Theoretically, many of the secondary effects reported for theophylline would certainly suggest that your question is a valid one. My feeling, however, is that theophylline is indeed working as a blocker of P, (P,,?) sites at frog preganglionic nerves. At PI sites in peripheral cholinergic nerves of the same species, phosphodiesterase inhibitors (including caffeine) increase the level of inhibition by P, agonists, rather than blocking their effects. Our now rather dated 1983 experiments on ganglion definitely need to be repeated with newer, more selective alkyl xanthines. C. LONDOS(National Institutes of Health, Bethesda, MD): Perhaps another explanation for theophylline reversal of an ATP effect is that the ATP effect is being facilitated by adenosine arising from ATP metabolism. One example of facilitation is found in PC12 pheochromicytoma cells in which adenosine receptor agonists, which have no effect of neurite outgrowth, greatly enhance the stimulation of neurite outgrowth by nerve factor. SILINSKY: Your suggestion is intriguing. My belief is that adenosine deaminase would not alter the effects of ATP, but we have not done these experiments. From work we have done, 1 mM adenosine does not influence the action of concurrently superfused ATP, so at least exogenous adenosine is not influencing the action of ATP.