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How does adenosine inhibit transmitter release?
TIPS - April 1988 [Vol. 91
B. B. Fredholm and T. V. Dunwiddie Adenosine is able to decrease the release of most neurotransmitters. This is consistent with its general role in adjusting the rate of energy consumption to the metabolic supply in a tissue. Bertil Fredholm and Tom Dunwiddie discuss the mechanisms behind the presynaptic inhibitory action of adenosine. By stimulating receptors similar to AI-receptors, adenosine can inhibit adenylate cyclase, open K’ channels and reduce flux through Ca2+ channels.. it is suggested that adenosine may depress transmitter release in several ways and that the relative imvortance of these presynaptic actions may vary between nerve terminals. ’ ’ The depressant effects of adenine nucleotides and nucleosides on synaptic transmission were first observed at the rat neuromuscular junction by Ginsborg and Hirst using electrophysiological techSubsequently, it was niquesl. shown that the overflow of labelled noradrenaline is reduced by adenosine derivatives in several different tissues’. It soon became apparent that such presynaptic inhibition is the rule rather than the exception, and that it occurs both in peripheral nerves and in the CNS (see Refs 3-5). In the peripheral sympathetic nervous system, inhibition due to endogenous adenosine becomes significant under conditions of hypoxia and/or prolonged nerve activation4. In the CNS, it seems to contribute significantly to the depressant effects of adenosine and adenosine derivatives and to the stimulatory effect of xanthine adenosine receptor antagonists including caffeines5. Adenosine receptors are present in many types of nerves (see Table 1 and Fig. 1). Since they are so widespread, they may be more suitable for biochemical studies of the mechanisms underlying presynaptic inhibition than many other types of presynaptic receptor, which often have a more Berti1Fredholmis professor al the Department of Pharmacology, Karolinska Institutet, Box 60 400, S-104 01, Stockholm, Sweden, and Tom Dunwiddie is associate professor at the Department of Phnrmacology, University of Colorado, Denver, CO 80262, USA, 8 1988. Elaevier Publlcallone, Cnmbridgc
restricted distribution. Because of similarities with other presynaptic receptor systems, the adenosine receptors may serve as a prototype for understanding the mechanisms by which a whole range of modulators (including noradrenaline, dopamine, opiates, acetylcholine and GABA) may act. The identification of mechanisms by which transmitter release may be modulated is also important in clarifying which steps are ratelimiting in the transmitter release process.
Adenosine receptors There is now general agreement that there are at least two types of adenosine receptor which can be
differentiated on the basis of the relative potency of a series of agonist@. Stimulation of Al receptors, at which several Nosubstituted derivatives (including R-PIA and CHA) are the most is often linked to a potent, decrease in the formation of CAMP. Conversely, stimulation of adenosine As receptors (at which NECA is more potent than R-PIA or CHA), often causes the levels of CAMP to rise in the cell or tissue studied. There is some evidence that there may be sub pes of adenosine As receptors 3. However, the evidence that there are subtypes of adenosine Ar receptors is not compelling. Binding curves may be biphasic, but this seems to reflect the fact that the binding of agonists, v&ich are the most commonly used ligands in adenosine receptor studies, is strongly influenced by the association of the receptor to the relevant guanine nucleotidebinding (G) proteins (see for example Ref. 5). Recently developed receptor selective antagonists may prove the most useful in classifying adenosine receptor subtypes. Presynaptic adenosine effects can be demonstrated by electrophysiological methods and by biochemical techniques, particularly after prelabelling of the transmitter stores. Irrespective of the technique used and of whether peripheral or central nerve endings are studied, the effect appears to be mediated by adenosine receptors of the Al subtype-.
TABLE I. Transmitter systems known to be affected by presynapticadenosine Transmitter aMbstaglcs
Tlswe, commenYs
receptors Ret.
Peripheral nervous system Noradrenaline most tissues and species, except cat Acetylcholine parasympathetic, motor nerve, ganglion
t
Central nervous system Acetylcholine Glutamate Noradrenaline Dopamine 5-HT GABA
d.e f*g ah a a,i
cortex, striatum, hippocampus cortex, hippocampus cortex, hippocampus striatum striatum striatum, cortex (but also negative results)
a&c
i
Systems where there is biochemical evidence for an inhibition by adenosine 91 fransmitter release. (There are also several examples of speciftc neuronal pathways where electrophysiological evidence indicates that adenosine inhibits transmitter release, but the transmitter may not be conclusively Identified.) ‘Harms, H. l-l. et al. (1979) Neuropharmacokrgy18, 577-580; bPedata, F. et al. (1983) Neuropharmacology 22.609-814; CCorradetti, R. et al. (1984) Eur.J. Pharmacol. 104,l Q-26: Dolphin, A. C. and Archer, E. R. (1983) Neuroscl. Leti. 43,4Q-54; ‘Jonzon, B. and Fredholm, B. B. (1984) Life Sci. 35, 1971-lb79; sJacklsch, R. ef al. (1984) Neuropharmacology 23, 1363-1371; “MichaelIs, M. L. ef al. (1979) Lila Sci, 24, 2083-2092; ‘Holllns, C. and Stone, T. W. (1980) Br. J. Pharmacol. 69, 107-112: tlimberger, N. ef al. (1986) J. Neumchem. 46, 1104-1117
0165 - 6107~BB/Jo~.~
TIPS - April 1988 [Vol. 91
fig. 1. The wide distribution of adenosine A, receptors. The receptors were visuakad by Selective binding of [3H]cyctohexytadenosibe. Brain slices fnnn (a) mouse, (b) rat, (c) guinea ptg, (d) human and(e) cat. Scalebar,3 mm. For furtt?er details see Ref. 28. Photographs kindly aupplled by J. Fastbom and J. M. Palacios.
Role of GTP-binding proteins
There is only circumstantial evidence that the presynaptic adenosine Al receptors are linked to G proteins. The prototype actions of Al and A2 receptors, namely inhibition and stimulation respectively of adenylate cyclase, are clearly dependent upon G proteins. Adenosine Al receptors, including receptors on nerve terminals, are modified by GTP and by procedures that alter GTPbinding proteins, both in binding assays and in autoradiographic experiments (cf. Ref. 5). It was also shown by Dolphin and Prestwichg that pertussis toxin was able to reduce the inhibitory effect of a&cnosine analogues on glutamfite release from cultured cerebellar cells, and N-ethylmaleimide treat-
ment as well as pertussis toxin can selectively eliminate the presynaptic effects of different agonists includin adenosine in the hippocampus g‘,ll. However, recent electrophysiological studies using the latter preparation (T-D., unpublished) indicate that at least part of the presynaptic component of the inhibition by adenosine of the excitatory transmission to CA1 pyramid cells is not mediated by a pertussis toxin-sensitive GTPbinding protein. It is not known whether they are linked to a GTPbinding protein that is insensitive to pertussis toxin, or if another mechanism is involved. Biochemical studies have shown that even quite closely related GTP-binding proteins can differ markedly in their sensitivity to inactivation
by pertussis toxin (Ref. 12, for example). Relation to adenylate cyclase
In a TiFS Viewpoint, Silinsky13 suggested that adenosine inhibits transmitter release secondarily to an increase in CAMP which caused a change in intracellular Ca*+ utilization. However, the A2 receptors mediating increases in CAMP and the receptors mediatneuroinhibition of ing transmitter and transmission are clearly different release entitiesa. Furthermore, increases in CAMP in nerve endings usually cause a fairly marked increase in transmitter release, rather than an inhibition14. It is therefore unlikely that stimulation of adenylate cyclase is responsible
TIPS -April
132 for the inhibition of transmitter release caused by adenosine. Conversely, a decrease in CAMP might be the signal to inhibit transmitter release. Adenosine A1 receptors often decrease CAMP accumulatioI+, and many receptors linked to inhibition of transmitter release, including LYZadrenergic, opiate, GABAs, muscarinic cholinergic, dopamine D2 and NPY are all able to decrease adenylate cyclase and lower CAMP levels. However, there are problems also with the hypothesis that inhibition of CAMP accumulation is the cause of the presynaptic adenosine effects. It is occasionally possible to completely inhibit transmitter release by adenosine analogs. If lowering of CAMP was indeed the mechanism behind this effect it would imply that CAMP is required for transmitter is unlikelf. which release, Moreover, direct tests have failed to demonstrate that inhibitors of adenylate cyclase, including 2’,5’dideoxyadenosine, inhibit transmitter release, and in electrophysiological as well as biochemical studies it has proved difficult to block the presynaptic inhibition by adenosine anaioiues by increasing CAMP levels . Thus, neither increases nor decreases in CAMP can entirely account for the presynaptic effect of adenosine. However, it is possible that inhibition of CAMP formation may contribute to the presynaptic effect of adenosine, particularly under conditions when CAMP has been raised in the nerve terminal - for example when a facilitatory, CAMPelevating modulator is simultaneousiy present. Other effector systems coupled to adenosine receptors In addition to inhibiting and activating adenylate cyclase, adenosine affects at least two types of ionic currents, both of which would be expected to limit entry of Ca*+ and thus to limit transmitter release through mechanisms which are apparently cyclic nucleotide independent. These different mechanisms are summarized in Table II. In those instances when the adenosine receptor subtype responsible has been determined these effects are mediated by receptors that appear more closely related to A1 than to AZ.
1988 [Vol. 91
TABLE II. Effects of adenosine ion conductance mechanisms Type of channel
Ref.
TISSUB,receptor, etc.
a b,c,d
K+
Hippocampal neurons, pertussis toxin sensitive Atrial myocyte, mediated via pertussis toxin-sensitive G protein, probably A, Cultured strlatal neurons, GTP required, pertussis toxin sensitive e,f
Caz+
Ca*’ spikes in hippocampus Ca2+ currents in superior cervical ganglion Ca*+ currents in dorsal root ganglion cells Ca*+ currents in hippocampal neurons
: t,i 16,16
*Andrade, R. et a/. (1986) Sot. Neumsci. Absk 12, 15; ‘B6hm, M. et al. (1986) NaunynSchmiedeberg’sArch. Pharmacol.332,403-405; =Kurachl, Y. et al. (1966) PflUgers Arch. 407. 264-274; “West 0. A. and Belardinelli, L. (1965) pncisers Arch. 403, 66-74; TrusselI, L. 0. and Jackson. Pmt. Nat/ Acad. Sci: USA 62. 4657461: ‘TrusselI. L. 0. and -..-___ ..__..,M. 0. 119851 . ___,.._~ Jackson, M. B. (1967) J. Neurosci. 7,3306-3316; *Proctor, h. R. and Dinwiddie, ?. V. (1963) Neumsci.Lelt. 35197-201; hHenon, 8. K. and McAfee, D. A. (1963) in RegulatoryFunctionof Adenosine (Beme, R. et a/., eds), pp. 465-466, Martinus Nijhoff; ‘Dolphin, p. C. et al. (1966) J. Physiol. (London) 373, 47-61; Macdonald, R. L. e1 a/. (1966) J. Phys~ol.(London) 370, 75-90
Probably the best characterized ionic conductance linked to adenosine receptors is the K* conductance identified in whole-cell clamp studies of atrial cells and in hippocampal neurons (see Table II). The receptor appears to be coupled to the channel via a pertussis toxin-sensitive G protein. Intracellular GTP is required, but CAMP is not. The non-hydrolysable GTP analogue GTP-yS irreversibly activates this conductance in the absence of adenosine receptor agonist. Adenosine can also inhibit voltage sensitive Ca2+ currents in dorsal root ganglion cells, superior cervical ganglion cells and in hippocampal pyramidal neurons (see Ref. 35 and Table II). This current may be carried via the Nchannel16, which is the oconotoxin sensitive channel that is suggested to be responsible for the Ca2+ influx that triggers transmitter release. The relationship of the N-channel to G proteins is unclear even though a similar current in neuroblastoma x glioma cells that is inhibitable by opiates was found to be linked to a G proteinI’. The existence of an adenosine-sensitive N-channel on nerve terminals has not been demonstrated, but this would clearly provide a very direct mechanism by which adenosine could Jimit transmitter release. It has not proved possible to consistently block or reduce the presynaptic adenosine effects by Ca*+ channel agonists (for example Bay K-8644) or antagonists (for example nifedipine, diltiazem). A functional antagonism between adenosine and Bay K-3644 can sometimes be demonstrated’s
perhaps because the dihydropyridine-sensitive L-type Ca*+ channels” which are normally not involved in the electrically evoked transmitter release, may become important if other ion channels, for example the N-channels”, are blocked by adenosine. A role for calcium In an attempt to determine whether Ca*+ and/or CAMP is altered in a nerve ending by A1 receptor agonists, one of us (B.F.) examined the effects of the adenosine A1 agonist CHA on phosphorylation of synapsin I during a visit in Paul Greengard’s laboratory. Synapsin I is phospho lated on distinct sites by Ca*r and CAMP-dependent protein kinases. Because of the unique localization of the protein, its phosphorylation ~~~~ge~ai~ cb~~s~~d “,a2~~~$~ in nerve endings. Furthermore, the state of phosphorylation of .synapsin I, and the activity of Ca”icaimoduiin-dependent protein kinase II, has been shown to control transmitter release*O. CHA was able to reduce the Ca2+dependent phosphorylation induced by very brief, but not by sustained, K+ depolarization. This is compatible with an effect of adenosine to limit Ca*+ entry into the nerve terminal by inhibiting an N-channel, but many other possibilities also exist. P. role for protein kinase @? Phorbol esters that stimulate protein kinase C increased the release of transmitters from many tissues, including the rat hippocampus 21#22.In the case of nor-
adrenaline
this increase,
which
TIPS - April 1988 [Vol. 91
amounted to between two and three-fold, could not be blocked by nifedipine or by the ar2-receptor antagonist clonidine (unpublished). Whereas the cuz-adrenoceptor effects were reduced or even eliminated by phorbol esters, the effect of R-PIA was not. This suggests that different presynaptic agonists may rely on different intracellular mechanisms for inhibiting transmitter release, as recognized earlier. Whereas stimulation of protein kinase C blocked the inhibitory effect of or2-agonists on noradrenaline release in our experiments in rat Wppocampus, phorbol esters have been shown not to significantly affect cifzmechanisms in the rabbit hippocampus”. This suggests the possibility of species differences. Although not studied directly, it seems possible that there could also be differences in the way that protein kinase C activation alters the effect of, say, Al and LX*receptors regulating the release of different transmitter substances in the same tissue. A tentative model It seems impossible to accommodate all the available evidence on the mechanism(s) by which adenosine inhibits transmitter release into one simple scheme. The fact that good experimental support is available for several types of mechanism might be explained in one of several ways (see Fig. 2): l There may be different sub-
types of adenosine AI-receptors (AI*, A1s, A& each of which is coupled via a GTP-binding protein (GA, Gs, Gc) to its respective effecters (Fig. 2a). 0 There is only one type of adenosine receptor (similar, if not identical to the Al receptor), which can couple with different types of G protein that have specificities for the three types of effector system (Fig. 2b). 0 There is only one type of adenosine Al receptor, which couples to one G protein, but the G protein may interact with several types of effector (Fig. 2~).Thus this single G protein, here called G,, may either interact with a K+ channel, or with adenylate cyclase or with a Ca*+ channel. The three possibilities are not mutually exclusive. It should also be pointed out that GA may be
133
a
K+
Ca2+
-c Ca2+
ATP
CAMP
ATP
CAMP
K+
Ca*+ C
Ca*+
Ca2+
K+
Fig 2. Modelsof coupling of adenosine receptors to mu/tip/e ektors. (a) Subtypes of adenosine receptor, each of which Is coupled via a different G pmtein to its eflector system. (b) One type of mephv, which couples with difierent G pmteins, eachof which interactswith a different effector pmtein. (c) One type of receptor and one G pmtein which interacts with several types of effector.
simi!ar
to G, according to pub-
lished studies lg. Gs could either be G, or G, (or any other type of G
protein that produces a pysubunit that may inactivate G,,). Gc has been variously characterized and has been associated with ir$h;; Q-su~;~;; and with a $yspeculative schemes have only indicated G protein-dependent effecters in the cell membranes. However, effects that are independent of G proteins have not been ruled out. Furthermore, there are many possible intracellular sites where presynaptic modulators could act (see Refs 13, 14 and 35). Since there is little independent evidence for the presence of mul-
tiple A&pe receptors we do not favor the krst possibility (Fig. 2a). On the other hand, it has been shown that one and the same receptor can interact with several effectors26 which is compatible with the second model (Fig. 2b). The finding that there is a different pertussis toxin sensitivity for the pre- and the postsynapticA1 receptor effects in the hippocampus is difficult to reconcile with the third possibility (Fig. 2c), and we therefore favor a scheme similar to that shown in Fig. 2b. In such a scheme,
the adenosine receptor-ligand complex might itself be envisioned as a kind of ‘messenger’, interacting in the membrane with a variety
of substrates
to elicit
a
TIPS - April 1988 [Vol. 9J
134 coordinated cdlular response involving multiple mechanisms. The relative importance of these different effector mechanisms might vary from nerve terminal to nerve terminal, from species to species and with age and disease. It is also possible that these different mechanisms co-exist in one and the same nerve terminal and that the relative importance of these mechanisms wih vary depending on, for example, how the transmitter release is induced. This type of scheme could explain published differences in the inhibition of electrically evoked and potassium evoked transmitter release*‘. 0
El
0
The apparent contradictions in the literature concerning the mechanism of action of a presynaptic modulator, such as adenosine, may therefore reflect the fact that such studies have only characterized specific aspects of a more complicated cellular system. As suggested also by Starker4 it may be futile to search for one mechanism that is the unique cause of presynaptic inhibition for all types of terminals, all types of evoked release and all types of presynaptic receptors. References
1 Ginsborg, 5. L. and Hirst, C. D. S. (1972) J. Physiol (London) 224, 629-645 2 Hedqvist, P. and Fredholm, 8.8. (1976) Naunyn-Schmiedeberg’s Arch. Pharmacot. 293,217-223 3 Fredholm, B. B. and Hedqvist, P. (1980) 3iachem. ~ha~aco~. 29,X351643 4 Fredhohn, 8. B., Gustafsson, L. E., Hedqviat, P. and Sollevi, A. (1983) in Regulatory Function of Adenosine (Berne, R. M., RaB, T. W. and Rubio, R.. eds), pp. 479-495, Martinus Nijhoff 5 Snyder, S. H. (1985) Annu. Rev. Neurosci. 8,103-124 6 Londos, C., Cooper, D. M. F. and Wolff, J. (1980) Proc. Not2Acad. Sci. USA 77, 2551-2.554 7 Bruns, R. F., Lu, G H. and Pugsley, T. A, (1987) in Topics and Perspectives of Adsnosine Research (Gerlach, E. and Becker, 8. F., eds), pp. 5957, SpringerVerlag 8 Dunwiddie, T. V. and Fredholm, B. B. (1985) AC. Cycfic Nuc~eottde Res. 19, 259-272 9 Dolphin, A. C. and Prestwich, S. A. 0985) Nature 316,148-150 10 Fredholm, B. B. and Lindgren, E. (1987) Acta Physiol. Stand. 130,9%105 11 A&aier, C., Feurstein, T. J., Jackisch, R. and Hertting, G. (1985) . Schmtedeberg’s Arch. P~arrnu~~~‘~~, 235-239 12 Hoff, R. M. and Neer, E. J. (1986) J, Biot. Chem. 261,1X&1110
13 Silinsky, E. M. (1986) Trends Pharmacol. Sci. 7, 180-185 14 Starke, K. (1987) Rev. Physiol. Biochem. Pks~ucoi. 167,~146 15 Dunwiddie, T. V. (1985) ht. Rev. Neurubiol. 27, 63-139 16 Madison, D. V., Fox, A. P. and Tsien, R. W. (1987) Proc. Biophys. 1. 51, 30 17 Hescheler, J., Rosenthal, W., Trautwein, W. and Schultz, G. (1987) Nature 325, 445-447 18 Fredholm, B. B., Hu, P. S. and Lindgren, E. (1986) Acfn Physiof. Stand. 128, 659668 19 Miller, R. J. (1987) Science 235,46-52 20 Llinas, R., McGuinness, T. L., Leonard, C. S., Sugimori, M. and Greengard, P. (1985) Proc. Nat2 Acad. Sci. USA 82, 3035-3039 21 Wakade, A. R., Mafhofra, R. K. and T. D. (1985) NaunynWakade, Schmiedeberg’s Arch. Pharmacol. 331, 122-124 22 Nichols, R. A., Haycock, J. W., Wang,
23
24 25
26 27 28
J. K. T. and Greengard, P. (1987) J. Neurochem. 48,6X5-621 Malder, A. H. and Schoffehneer, A. N. M. (1985) A&. Cyctic Nucreoftde Res, 19,273-286 Allgaier, C., Hertting, G. and Kugelen, 0. V. (1987) Br. 1. Pharmacol. 90,403-412 Augustine, G. J.” Charlton, M. P. and Smith, S. J. (1987) Anuu. Rev. Neurosci. 10,633-693 Haga, K., Haga, T. and Ichyama, A. (1986) J. B&J. Chem. 261,lOl~lO~~ Alberts, P., Bartfai, P. and Stj&ne, L. (1981) J. Phystol. Ilondon) 312,297-334 Fastbom, J., Pazos, A. and Palacios, J. M. (1987) Neuroscience 22,813-826
R-PI& Nb-phenylisopropyl adenosine CHA: N6-cyciohexyl adenosine NECA: 5’-N-ethyl~boxam~do adenosine Bay K-8644r methyl-1,4-dihydro-2,6-dimethyl-3-nitro4-(2-tfluoromethylphenyI)pyridine5-carboxylate
Can supraspinal 6-opioid receptors mediate antinociception? Julius S. ~ey~an, Jeffry L. Vau~~t, Robert 8. Raffa and Frank Porreca The antinociception mediated by supraspinal opioid receptors has traditionally been thought to arise solely from activation of the p-opioid receptor subtype. There is a growing body of evidence, however, which suggests that supraspinal ~-opio~d receptors may also be able to directly initiate antinociception. Frank Porreca and colleagues review the evidence favoring exclusive involvement of’ p-opioid receptors (as well as studies implicating b-opioid receptors) in supraspinal antinociceptors. They conclude that S-opioid receptors can be pharmacologically activated to initiate antinociception, suggesting the existence of an alternative mechanism for pain relief which may be of significant clinical importance. A great deal of controversy currently exists regarding the role of brain 8-opioid receptors in the mediation of antinociception in rodents, particularly in tests utilizing heat as the noxious stimuhas. Traditional thought has associated antinociceptive activity with receptors activated by prototype agonists such as morphine, i.e. popioid receptors. The question of involvement of other receptor subtypes in the mediation of antinociception is one of considerable sigIulius Heyman is a Research Associate atui Frank Porreca is Assistant Professor at the Depurtment ofPharmacology, Health Sciences Center, University of Arizona, Tucs~ax, AZ 85724, USA. feffry Vaught is Head and Robert Ru~a is a Research A~ociate in fhe Department of Biotogicd Research, Janssern Research Foundation, Spring House, PA 1947, USA. 0 198B.Elscvler Publlcatlons,
Comhddgc
nificance as we strive to develop analgesics which are free of the undesirable properties associated with morphine-like drugs. The recent discovery of ligands with a high degree of selectivity for pand &receptor& has made exploration of this issue possible (Fig. 1). Conflicting results and interpretations have appeared, however, even though investigators have studied the same ligands and employed similar techniques. Thus, it seems timely to evaluate the evidence and attempt to determine possible reasons for these divergent conclusions. In-vitro support for p-recepOors Shortly after the discovery of the enkephalins, endogenous figands of the b-receptor, one of the first
0169 - 6147/88/$02.00
B. B. Fredholm and T. V. Dunwiddie Adenosine is able to decrease the release of most neurotransmitters. This is consistent with its general role in adjusting the rate of energy consumption to the metabolic supply in a tissue. Bertil Fredholm and Tom Dunwiddie discuss the mechanisms behind the presynaptic inhibitory action of adenosine. By stimulating receptors similar to AI-receptors, adenosine can inhibit adenylate cyclase, open K’ channels and reduce flux through Ca2+ channels.. it is suggested that adenosine may depress transmitter release in several ways and that the relative imvortance of these presynaptic actions may vary between nerve terminals. ’ ’ The depressant effects of adenine nucleotides and nucleosides on synaptic transmission were first observed at the rat neuromuscular junction by Ginsborg and Hirst using electrophysiological techSubsequently, it was niquesl. shown that the overflow of labelled noradrenaline is reduced by adenosine derivatives in several different tissues’. It soon became apparent that such presynaptic inhibition is the rule rather than the exception, and that it occurs both in peripheral nerves and in the CNS (see Refs 3-5). In the peripheral sympathetic nervous system, inhibition due to endogenous adenosine becomes significant under conditions of hypoxia and/or prolonged nerve activation4. In the CNS, it seems to contribute significantly to the depressant effects of adenosine and adenosine derivatives and to the stimulatory effect of xanthine adenosine receptor antagonists including caffeines5. Adenosine receptors are present in many types of nerves (see Table 1 and Fig. 1). Since they are so widespread, they may be more suitable for biochemical studies of the mechanisms underlying presynaptic inhibition than many other types of presynaptic receptor, which often have a more Berti1Fredholmis professor al the Department of Pharmacology, Karolinska Institutet, Box 60 400, S-104 01, Stockholm, Sweden, and Tom Dunwiddie is associate professor at the Department of Phnrmacology, University of Colorado, Denver, CO 80262, USA, 8 1988. Elaevier Publlcallone, Cnmbridgc
restricted distribution. Because of similarities with other presynaptic receptor systems, the adenosine receptors may serve as a prototype for understanding the mechanisms by which a whole range of modulators (including noradrenaline, dopamine, opiates, acetylcholine and GABA) may act. The identification of mechanisms by which transmitter release may be modulated is also important in clarifying which steps are ratelimiting in the transmitter release process.
Adenosine receptors There is now general agreement that there are at least two types of adenosine receptor which can be
differentiated on the basis of the relative potency of a series of agonist@. Stimulation of Al receptors, at which several Nosubstituted derivatives (including R-PIA and CHA) are the most is often linked to a potent, decrease in the formation of CAMP. Conversely, stimulation of adenosine As receptors (at which NECA is more potent than R-PIA or CHA), often causes the levels of CAMP to rise in the cell or tissue studied. There is some evidence that there may be sub pes of adenosine As receptors 3. However, the evidence that there are subtypes of adenosine Ar receptors is not compelling. Binding curves may be biphasic, but this seems to reflect the fact that the binding of agonists, v&ich are the most commonly used ligands in adenosine receptor studies, is strongly influenced by the association of the receptor to the relevant guanine nucleotidebinding (G) proteins (see for example Ref. 5). Recently developed receptor selective antagonists may prove the most useful in classifying adenosine receptor subtypes. Presynaptic adenosine effects can be demonstrated by electrophysiological methods and by biochemical techniques, particularly after prelabelling of the transmitter stores. Irrespective of the technique used and of whether peripheral or central nerve endings are studied, the effect appears to be mediated by adenosine receptors of the Al subtype-.
TABLE I. Transmitter systems known to be affected by presynapticadenosine Transmitter aMbstaglcs
Tlswe, commenYs
receptors Ret.
Peripheral nervous system Noradrenaline most tissues and species, except cat Acetylcholine parasympathetic, motor nerve, ganglion
t
Central nervous system Acetylcholine Glutamate Noradrenaline Dopamine 5-HT GABA
d.e f*g ah a a,i
cortex, striatum, hippocampus cortex, hippocampus cortex, hippocampus striatum striatum striatum, cortex (but also negative results)
a&c
i
Systems where there is biochemical evidence for an inhibition by adenosine 91 fransmitter release. (There are also several examples of speciftc neuronal pathways where electrophysiological evidence indicates that adenosine inhibits transmitter release, but the transmitter may not be conclusively Identified.) ‘Harms, H. l-l. et al. (1979) Neuropharmacokrgy18, 577-580; bPedata, F. et al. (1983) Neuropharmacology 22.609-814; CCorradetti, R. et al. (1984) Eur.J. Pharmacol. 104,l Q-26: Dolphin, A. C. and Archer, E. R. (1983) Neuroscl. Leti. 43,4Q-54; ‘Jonzon, B. and Fredholm, B. B. (1984) Life Sci. 35, 1971-lb79; sJacklsch, R. ef al. (1984) Neuropharmacology 23, 1363-1371; “MichaelIs, M. L. ef al. (1979) Lila Sci, 24, 2083-2092; ‘Holllns, C. and Stone, T. W. (1980) Br. J. Pharmacol. 69, 107-112: tlimberger, N. ef al. (1986) J. Neumchem. 46, 1104-1117
0165 - 6107~BB/Jo~.~
TIPS - April 1988 [Vol. 91
fig. 1. The wide distribution of adenosine A, receptors. The receptors were visuakad by Selective binding of [3H]cyctohexytadenosibe. Brain slices fnnn (a) mouse, (b) rat, (c) guinea ptg, (d) human and(e) cat. Scalebar,3 mm. For furtt?er details see Ref. 28. Photographs kindly aupplled by J. Fastbom and J. M. Palacios.
Role of GTP-binding proteins
There is only circumstantial evidence that the presynaptic adenosine Al receptors are linked to G proteins. The prototype actions of Al and A2 receptors, namely inhibition and stimulation respectively of adenylate cyclase, are clearly dependent upon G proteins. Adenosine Al receptors, including receptors on nerve terminals, are modified by GTP and by procedures that alter GTPbinding proteins, both in binding assays and in autoradiographic experiments (cf. Ref. 5). It was also shown by Dolphin and Prestwichg that pertussis toxin was able to reduce the inhibitory effect of a&cnosine analogues on glutamfite release from cultured cerebellar cells, and N-ethylmaleimide treat-
ment as well as pertussis toxin can selectively eliminate the presynaptic effects of different agonists includin adenosine in the hippocampus g‘,ll. However, recent electrophysiological studies using the latter preparation (T-D., unpublished) indicate that at least part of the presynaptic component of the inhibition by adenosine of the excitatory transmission to CA1 pyramid cells is not mediated by a pertussis toxin-sensitive GTPbinding protein. It is not known whether they are linked to a GTPbinding protein that is insensitive to pertussis toxin, or if another mechanism is involved. Biochemical studies have shown that even quite closely related GTP-binding proteins can differ markedly in their sensitivity to inactivation
by pertussis toxin (Ref. 12, for example). Relation to adenylate cyclase
In a TiFS Viewpoint, Silinsky13 suggested that adenosine inhibits transmitter release secondarily to an increase in CAMP which caused a change in intracellular Ca*+ utilization. However, the A2 receptors mediating increases in CAMP and the receptors mediatneuroinhibition of ing transmitter and transmission are clearly different release entitiesa. Furthermore, increases in CAMP in nerve endings usually cause a fairly marked increase in transmitter release, rather than an inhibition14. It is therefore unlikely that stimulation of adenylate cyclase is responsible
TIPS -April
132 for the inhibition of transmitter release caused by adenosine. Conversely, a decrease in CAMP might be the signal to inhibit transmitter release. Adenosine A1 receptors often decrease CAMP accumulatioI+, and many receptors linked to inhibition of transmitter release, including LYZadrenergic, opiate, GABAs, muscarinic cholinergic, dopamine D2 and NPY are all able to decrease adenylate cyclase and lower CAMP levels. However, there are problems also with the hypothesis that inhibition of CAMP accumulation is the cause of the presynaptic adenosine effects. It is occasionally possible to completely inhibit transmitter release by adenosine analogs. If lowering of CAMP was indeed the mechanism behind this effect it would imply that CAMP is required for transmitter is unlikelf. which release, Moreover, direct tests have failed to demonstrate that inhibitors of adenylate cyclase, including 2’,5’dideoxyadenosine, inhibit transmitter release, and in electrophysiological as well as biochemical studies it has proved difficult to block the presynaptic inhibition by adenosine anaioiues by increasing CAMP levels . Thus, neither increases nor decreases in CAMP can entirely account for the presynaptic effect of adenosine. However, it is possible that inhibition of CAMP formation may contribute to the presynaptic effect of adenosine, particularly under conditions when CAMP has been raised in the nerve terminal - for example when a facilitatory, CAMPelevating modulator is simultaneousiy present. Other effector systems coupled to adenosine receptors In addition to inhibiting and activating adenylate cyclase, adenosine affects at least two types of ionic currents, both of which would be expected to limit entry of Ca*+ and thus to limit transmitter release through mechanisms which are apparently cyclic nucleotide independent. These different mechanisms are summarized in Table II. In those instances when the adenosine receptor subtype responsible has been determined these effects are mediated by receptors that appear more closely related to A1 than to AZ.
1988 [Vol. 91
TABLE II. Effects of adenosine ion conductance mechanisms Type of channel
Ref.
TISSUB,receptor, etc.
a b,c,d
K+
Hippocampal neurons, pertussis toxin sensitive Atrial myocyte, mediated via pertussis toxin-sensitive G protein, probably A, Cultured strlatal neurons, GTP required, pertussis toxin sensitive e,f
Caz+
Ca*’ spikes in hippocampus Ca2+ currents in superior cervical ganglion Ca*+ currents in dorsal root ganglion cells Ca*+ currents in hippocampal neurons
: t,i 16,16
*Andrade, R. et a/. (1986) Sot. Neumsci. Absk 12, 15; ‘B6hm, M. et al. (1986) NaunynSchmiedeberg’sArch. Pharmacol.332,403-405; =Kurachl, Y. et al. (1966) PflUgers Arch. 407. 264-274; “West 0. A. and Belardinelli, L. (1965) pncisers Arch. 403, 66-74; TrusselI, L. 0. and Jackson. Pmt. Nat/ Acad. Sci: USA 62. 4657461: ‘TrusselI. L. 0. and -..-___ ..__..,M. 0. 119851 . ___,.._~ Jackson, M. B. (1967) J. Neurosci. 7,3306-3316; *Proctor, h. R. and Dinwiddie, ?. V. (1963) Neumsci.Lelt. 35197-201; hHenon, 8. K. and McAfee, D. A. (1963) in RegulatoryFunctionof Adenosine (Beme, R. et a/., eds), pp. 465-466, Martinus Nijhoff; ‘Dolphin, p. C. et al. (1966) J. Physiol. (London) 373, 47-61; Macdonald, R. L. e1 a/. (1966) J. Phys~ol.(London) 370, 75-90
Probably the best characterized ionic conductance linked to adenosine receptors is the K* conductance identified in whole-cell clamp studies of atrial cells and in hippocampal neurons (see Table II). The receptor appears to be coupled to the channel via a pertussis toxin-sensitive G protein. Intracellular GTP is required, but CAMP is not. The non-hydrolysable GTP analogue GTP-yS irreversibly activates this conductance in the absence of adenosine receptor agonist. Adenosine can also inhibit voltage sensitive Ca2+ currents in dorsal root ganglion cells, superior cervical ganglion cells and in hippocampal pyramidal neurons (see Ref. 35 and Table II). This current may be carried via the Nchannel16, which is the oconotoxin sensitive channel that is suggested to be responsible for the Ca2+ influx that triggers transmitter release. The relationship of the N-channel to G proteins is unclear even though a similar current in neuroblastoma x glioma cells that is inhibitable by opiates was found to be linked to a G proteinI’. The existence of an adenosine-sensitive N-channel on nerve terminals has not been demonstrated, but this would clearly provide a very direct mechanism by which adenosine could Jimit transmitter release. It has not proved possible to consistently block or reduce the presynaptic adenosine effects by Ca*+ channel agonists (for example Bay K-8644) or antagonists (for example nifedipine, diltiazem). A functional antagonism between adenosine and Bay K-3644 can sometimes be demonstrated’s
perhaps because the dihydropyridine-sensitive L-type Ca*+ channels” which are normally not involved in the electrically evoked transmitter release, may become important if other ion channels, for example the N-channels”, are blocked by adenosine. A role for calcium In an attempt to determine whether Ca*+ and/or CAMP is altered in a nerve ending by A1 receptor agonists, one of us (B.F.) examined the effects of the adenosine A1 agonist CHA on phosphorylation of synapsin I during a visit in Paul Greengard’s laboratory. Synapsin I is phospho lated on distinct sites by Ca*r and CAMP-dependent protein kinases. Because of the unique localization of the protein, its phosphorylation ~~~~ge~ai~ cb~~s~~d “,a2~~~$~ in nerve endings. Furthermore, the state of phosphorylation of .synapsin I, and the activity of Ca”icaimoduiin-dependent protein kinase II, has been shown to control transmitter release*O. CHA was able to reduce the Ca2+dependent phosphorylation induced by very brief, but not by sustained, K+ depolarization. This is compatible with an effect of adenosine to limit Ca*+ entry into the nerve terminal by inhibiting an N-channel, but many other possibilities also exist. P. role for protein kinase @? Phorbol esters that stimulate protein kinase C increased the release of transmitters from many tissues, including the rat hippocampus 21#22.In the case of nor-
adrenaline
this increase,
which
TIPS - April 1988 [Vol. 91
amounted to between two and three-fold, could not be blocked by nifedipine or by the ar2-receptor antagonist clonidine (unpublished). Whereas the cuz-adrenoceptor effects were reduced or even eliminated by phorbol esters, the effect of R-PIA was not. This suggests that different presynaptic agonists may rely on different intracellular mechanisms for inhibiting transmitter release, as recognized earlier. Whereas stimulation of protein kinase C blocked the inhibitory effect of or2-agonists on noradrenaline release in our experiments in rat Wppocampus, phorbol esters have been shown not to significantly affect cifzmechanisms in the rabbit hippocampus”. This suggests the possibility of species differences. Although not studied directly, it seems possible that there could also be differences in the way that protein kinase C activation alters the effect of, say, Al and LX*receptors regulating the release of different transmitter substances in the same tissue. A tentative model It seems impossible to accommodate all the available evidence on the mechanism(s) by which adenosine inhibits transmitter release into one simple scheme. The fact that good experimental support is available for several types of mechanism might be explained in one of several ways (see Fig. 2): l There may be different sub-
types of adenosine AI-receptors (AI*, A1s, A& each of which is coupled via a GTP-binding protein (GA, Gs, Gc) to its respective effecters (Fig. 2a). 0 There is only one type of adenosine receptor (similar, if not identical to the Al receptor), which can couple with different types of G protein that have specificities for the three types of effector system (Fig. 2b). 0 There is only one type of adenosine Al receptor, which couples to one G protein, but the G protein may interact with several types of effector (Fig. 2~).Thus this single G protein, here called G,, may either interact with a K+ channel, or with adenylate cyclase or with a Ca*+ channel. The three possibilities are not mutually exclusive. It should also be pointed out that GA may be
133
a
K+
Ca2+
-c Ca2+
ATP
CAMP
ATP
CAMP
K+
Ca*+ C
Ca*+
Ca2+
K+
Fig 2. Modelsof coupling of adenosine receptors to mu/tip/e ektors. (a) Subtypes of adenosine receptor, each of which Is coupled via a different G pmtein to its eflector system. (b) One type of mephv, which couples with difierent G pmteins, eachof which interactswith a different effector pmtein. (c) One type of receptor and one G pmtein which interacts with several types of effector.
simi!ar
to G, according to pub-
lished studies lg. Gs could either be G, or G, (or any other type of G
protein that produces a pysubunit that may inactivate G,,). Gc has been variously characterized and has been associated with ir$h;; Q-su~;~;; and with a $yspeculative schemes have only indicated G protein-dependent effecters in the cell membranes. However, effects that are independent of G proteins have not been ruled out. Furthermore, there are many possible intracellular sites where presynaptic modulators could act (see Refs 13, 14 and 35). Since there is little independent evidence for the presence of mul-
tiple A&pe receptors we do not favor the krst possibility (Fig. 2a). On the other hand, it has been shown that one and the same receptor can interact with several effectors26 which is compatible with the second model (Fig. 2b). The finding that there is a different pertussis toxin sensitivity for the pre- and the postsynapticA1 receptor effects in the hippocampus is difficult to reconcile with the third possibility (Fig. 2c), and we therefore favor a scheme similar to that shown in Fig. 2b. In such a scheme,
the adenosine receptor-ligand complex might itself be envisioned as a kind of ‘messenger’, interacting in the membrane with a variety
of substrates
to elicit
a
TIPS - April 1988 [Vol. 9J
134 coordinated cdlular response involving multiple mechanisms. The relative importance of these different effector mechanisms might vary from nerve terminal to nerve terminal, from species to species and with age and disease. It is also possible that these different mechanisms co-exist in one and the same nerve terminal and that the relative importance of these mechanisms wih vary depending on, for example, how the transmitter release is induced. This type of scheme could explain published differences in the inhibition of electrically evoked and potassium evoked transmitter release*‘. 0
El
0
The apparent contradictions in the literature concerning the mechanism of action of a presynaptic modulator, such as adenosine, may therefore reflect the fact that such studies have only characterized specific aspects of a more complicated cellular system. As suggested also by Starker4 it may be futile to search for one mechanism that is the unique cause of presynaptic inhibition for all types of terminals, all types of evoked release and all types of presynaptic receptors. References
1 Ginsborg, 5. L. and Hirst, C. D. S. (1972) J. Physiol (London) 224, 629-645 2 Hedqvist, P. and Fredholm, 8.8. (1976) Naunyn-Schmiedeberg’s Arch. Pharmacot. 293,217-223 3 Fredholm, B. B. and Hedqvist, P. (1980) 3iachem. ~ha~aco~. 29,X351643 4 Fredhohn, 8. B., Gustafsson, L. E., Hedqviat, P. and Sollevi, A. (1983) in Regulatory Function of Adenosine (Berne, R. M., RaB, T. W. and Rubio, R.. eds), pp. 479-495, Martinus Nijhoff 5 Snyder, S. H. (1985) Annu. Rev. Neurosci. 8,103-124 6 Londos, C., Cooper, D. M. F. and Wolff, J. (1980) Proc. Not2Acad. Sci. USA 77, 2551-2.554 7 Bruns, R. F., Lu, G H. and Pugsley, T. A, (1987) in Topics and Perspectives of Adsnosine Research (Gerlach, E. and Becker, 8. F., eds), pp. 5957, SpringerVerlag 8 Dunwiddie, T. V. and Fredholm, B. B. (1985) AC. Cycfic Nuc~eottde Res. 19, 259-272 9 Dolphin, A. C. and Prestwich, S. A. 0985) Nature 316,148-150 10 Fredholm, B. B. and Lindgren, E. (1987) Acta Physiol. Stand. 130,9%105 11 A&aier, C., Feurstein, T. J., Jackisch, R. and Hertting, G. (1985) . Schmtedeberg’s Arch. P~arrnu~~~‘~~, 235-239 12 Hoff, R. M. and Neer, E. J. (1986) J, Biot. Chem. 261,1X&1110
13 Silinsky, E. M. (1986) Trends Pharmacol. Sci. 7, 180-185 14 Starke, K. (1987) Rev. Physiol. Biochem. Pks~ucoi. 167,~146 15 Dunwiddie, T. V. (1985) ht. Rev. Neurubiol. 27, 63-139 16 Madison, D. V., Fox, A. P. and Tsien, R. W. (1987) Proc. Biophys. 1. 51, 30 17 Hescheler, J., Rosenthal, W., Trautwein, W. and Schultz, G. (1987) Nature 325, 445-447 18 Fredholm, B. B., Hu, P. S. and Lindgren, E. (1986) Acfn Physiof. Stand. 128, 659668 19 Miller, R. J. (1987) Science 235,46-52 20 Llinas, R., McGuinness, T. L., Leonard, C. S., Sugimori, M. and Greengard, P. (1985) Proc. Nat2 Acad. Sci. USA 82, 3035-3039 21 Wakade, A. R., Mafhofra, R. K. and T. D. (1985) NaunynWakade, Schmiedeberg’s Arch. Pharmacol. 331, 122-124 22 Nichols, R. A., Haycock, J. W., Wang,
23
24 25
26 27 28
J. K. T. and Greengard, P. (1987) J. Neurochem. 48,6X5-621 Malder, A. H. and Schoffehneer, A. N. M. (1985) A&. Cyctic Nucreoftde Res, 19,273-286 Allgaier, C., Hertting, G. and Kugelen, 0. V. (1987) Br. 1. Pharmacol. 90,403-412 Augustine, G. J.” Charlton, M. P. and Smith, S. J. (1987) Anuu. Rev. Neurosci. 10,633-693 Haga, K., Haga, T. and Ichyama, A. (1986) J. B&J. Chem. 261,lOl~lO~~ Alberts, P., Bartfai, P. and Stj&ne, L. (1981) J. Phystol. Ilondon) 312,297-334 Fastbom, J., Pazos, A. and Palacios, J. M. (1987) Neuroscience 22,813-826
R-PI& Nb-phenylisopropyl adenosine CHA: N6-cyciohexyl adenosine NECA: 5’-N-ethyl~boxam~do adenosine Bay K-8644r methyl-1,4-dihydro-2,6-dimethyl-3-nitro4-(2-tfluoromethylphenyI)pyridine5-carboxylate
Can supraspinal 6-opioid receptors mediate antinociception? Julius S. ~ey~an, Jeffry L. Vau~~t, Robert 8. Raffa and Frank Porreca The antinociception mediated by supraspinal opioid receptors has traditionally been thought to arise solely from activation of the p-opioid receptor subtype. There is a growing body of evidence, however, which suggests that supraspinal ~-opio~d receptors may also be able to directly initiate antinociception. Frank Porreca and colleagues review the evidence favoring exclusive involvement of’ p-opioid receptors (as well as studies implicating b-opioid receptors) in supraspinal antinociceptors. They conclude that S-opioid receptors can be pharmacologically activated to initiate antinociception, suggesting the existence of an alternative mechanism for pain relief which may be of significant clinical importance. A great deal of controversy currently exists regarding the role of brain 8-opioid receptors in the mediation of antinociception in rodents, particularly in tests utilizing heat as the noxious stimuhas. Traditional thought has associated antinociceptive activity with receptors activated by prototype agonists such as morphine, i.e. popioid receptors. The question of involvement of other receptor subtypes in the mediation of antinociception is one of considerable sigIulius Heyman is a Research Associate atui Frank Porreca is Assistant Professor at the Depurtment ofPharmacology, Health Sciences Center, University of Arizona, Tucs~ax, AZ 85724, USA. feffry Vaught is Head and Robert Ru~a is a Research A~ociate in fhe Department of Biotogicd Research, Janssern Research Foundation, Spring House, PA 1947, USA. 0 198B.Elscvler Publlcatlons,
Comhddgc
nificance as we strive to develop analgesics which are free of the undesirable properties associated with morphine-like drugs. The recent discovery of ligands with a high degree of selectivity for pand &receptor& has made exploration of this issue possible (Fig. 1). Conflicting results and interpretations have appeared, however, even though investigators have studied the same ligands and employed similar techniques. Thus, it seems timely to evaluate the evidence and attempt to determine possible reasons for these divergent conclusions. In-vitro support for p-recepOors Shortly after the discovery of the enkephalins, endogenous figands of the b-receptor, one of the first
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