Effects of adenosine and adenine nucleotides on synaptic transmission in the cerebral cortex.

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Effects of adenosine and adenine nucleotides on synaptic transmission in the cerebral cortex Bepurtrnerat of Pl~ysiology,College oj' Medicirze, L7tz6~*ers6'f;l' oj'Si.zsk~ztcItewa~z, Snskatoon, Susk., Canada Received June 11, 1999 PPIILLIS, J. W., EDSTIPOM, J. P., KOSTOPOULOS, G. K., and KIRKPATRICK. J. R. 1979. Effects of adenosine and adenine nucleotides on synaptic transmission in the cerebral cortex. Can. J. Physiol. Pharmacol. 57, 1289-13 12. Adenosine and the adenine nucleotides have a potent depressant action on cerebral cortical neurons, including identified corticospinal cells. Other p ~ ~ r i nand e pyrimidine nucleotides were either weakly depressant (inosine and guanosine derivatives) or largely inactive (xanthine, cytidine, thymidine. uridine derivatives). The 5'-triphosphates and to a lesser extent the 5'diphosphates of all the purine and pyrimidines tested had excitant actions on cortical. neurons. Adenosine transport blockers and deamllnase inhibitors depressed the firing of cortical neurons and potentiated the depressant actions of adenosine and the adenine nucleotides. Methylxanthines (theophylline, caffeine, and isobutylmethy$xanthine) antagonized the depressant efyects of adenosine and the adenine nucleotides and enhanced the spontaneous firing rate of cerebral cortical neurons. lintracellular recordings showed that adenosine 5'-monc~phosphate hyperpolarizes cerebral cortical neurons and suppresses spontaneous and evoked excitatory postsynaptic potentials in the absence of any pronounced alterations in membrane resistance or of the threshold for action potential generation. lit is suggested that adenosine depresses spontaneous and evoked activity by inhibiting the release of transmitter from presynaptic nerve terminals. Furthermore, the depressant effects of potentiators and excitant efl'ects of antagonists of adenosine on neuronal firing are consistent with the hypothesis that cortical neurons are subject to control by endogenously released purines. PHILLIS,J. W., EDSTROM, J. P., KOSTOPOULOS, G. K.,et KIRKPATRICK, 9.R. 1979. Effects of adenosine and adenine nucleotides on sy~laptictrans~nissionin the cerebral cortex. Can. J. Physiol. Pharmacol. 57, 1289-1312. Les nuclkotides d'adknosine et d'adknine exercent une action dkpressive puissante sur les neurones du cortex cerkbral, ceci incluant des cellules corticospinales bien identifikes. Les nuclkotides de purine et de pyrimidine n'ont qu9un effet faiblemeiat dkpresseur (derives de l'inosine et de 19uridine)ou sont totalement inactifs (dCrivCs de la xanthine, de la cytidine, de la thymidine, de B'uridine). Les triphosphates-5' et au5si un degrk moindre, les diphosyhates-5' de purine et de pyrimidine qui gtnt CtC testes. exercent une action excitratrice sur les neurones corticaux. Les bloqueurs du transport d'adknosine ainsi que les inhilsiteurs des desaminases diminuent le taux de dkcharge des neuroiles corticaux et accentuent les actions dkpressives des nuclkotides d'adenosine et d'ad6raine. L e b mithylxanthines (thkophylline, cafkine et isobutylmethylxanthine) antagonisent les en'ets dkpresseurs des nuclCotides d9adinosine et d'adknine et intensifient le taux de dkharge spontanbe des neurones du cortex ckrkbral. Des enregistrements intracellulaires nnontrent que le rnonophosphate-5' d'adk~~osine hyperpolaris e les neurones du cortex cCrCbral et supprime les potentiels postsynaptiques spontanes et 6v o quks en l'absence d'une modification marquee de la rksistance membranaire ou du seuil requi s pour qu'il y ait gkneration d'un potentiel d'action. On suggkre clue l'adknosine diminue 19activitespontanie et kvoquee en inhibant la liberation d'un transmetteur par ies terminaisons nerveuses prksynaptiques. B e plus, les effets dkpresseurs dej potentiateurs ainsi que les effets excitateurs des antagonistes de l'adenosine sur la dkcharge des neurones sont compatibles avec l'hypothkse selon laquelle les neurones corticaux sont sujets a un contrhie par des purines libkrkes de f a ~ o nendoghne. [Traduit par le journal]

Introduction A role for 5'-ATp as the transmitter released by primary afferent fibers was initially proposed by Holton and Holtoll ( 1954). Subsequently evidence 5'-AMP, adenosine 5'-monophosphate; ABBREVIATIONS: 5'-ADP, adenosine 5'-dipkosphatc; 5'-A'TP, adenosine 5'triphosphate; GABA, y-arninobutyric acid; ACh, acetyl-

was presented that purines could function as transmitters at autoIl0mic llervoUs System j U n ~ t i o n ~ (Burnstock 1972 1, and the concept of central

c h u l i n e ; G i , adenosine 2'-monophosphate; AM AMP, adenosine 3'-monophssphate; cyclic 2',3'-AMP. adenosine 2',3'-cyclic monophosphate; cyclic 3',5'-AMP, adenosine 3',5'-cyclic monophosphate; AMP-PNP, adenosine 5'-irnidcsdiphosphate; EPSB, excitatory postsynaptic potential.

0008-4212/79/111289-24$01.00/0 @ 1979 National Research Council of Canada/ConseiB national de recherches du Canada

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purinergic transmission has recently been resurrected (Pull and McIlwain 1972; Shirnizu 1973). Adenosine and its nucleotides are intricately interwoven into brain intermediary metabolism as substrates, cofactors, and regulators, making it dificult to relate the results of biochemical studies on these compounds to an individual physiological role, especially neurotransrnission or modulation. Brain concentrations of the adenine nucleotides are high, exceeding 2 pmal/g of fresh brain (Mandel 1971 ; Mahler 1992; Kleihues et al. 1974). Adenosine levels in brain tissue rise rapidly during periods of brain ischemia (Kleihues et al. 1974) and under such conditions may reach levels in excess of 1 pM in cerebrospinal fluid (Berne et al. 1974). Tlae release C B ~adenosine and adenine nucleotides from cerebral tisstaes has been studied both in situ and from brain slices or synaptosonsal preparations (Pull and McIBwain 1972; Heller and McIlwain 11973; Kuroda and hkllwain 2974; Sulakhe and Phillis 1975; White 1998; Wu and Phialis 1998). Release is calcium dependent and can be enhanced by procedures that dcpalarize the tissues. A role for adenine derivatives ill the regulation of ce~ntralnervous function is also suggested by the observations that adenosine and 5'-ATP have sedative or hypnogenic actions when administered intraver~tricullarly (Feldberg and Shemood 1954; Hauliea et al. 1973) and by the demonstration of an antiepileptic action of adenosine (hlaitre et al. 1974). Moreover, adenosine levels in the brain were found to be correlated with changes in the level of physiological activity (Haulica et al. 1973). Early reports 011 the effects of iontophoretically applied adenine ilucleotides or1 central neurons indicated that 5'-ATP was either without effect (on neurons in the spinal cord (Curtis et al. 1961 ) ), or had excitatory actions (an cclls in the brainstem and on cerebellar Purkinje cells (Galindo et al. 1967; Hoffer et al, 1971 ) ). As 5'-AMP and 5'-ABP were ineffective, this excitant action was tentatively attributed to the calcium chelating action of 5'-ATP. The disco~7e1-y that adenosine and various adenine nucleotidcs were potent depressants of the spontaneous firing of rat cerebral cortical neurons (Phillis et al. 1974, 1975) led to a series of iontophoretic studies of the actions sf purine and pyrimidine nucleotides in various regions of the brain. These studies were subsequently extended to include agents which purportedly inhibit the enzymatic degradation of adennosine, block the uptake of adcr~osine. or act as antagonists at the adenosine receptor. The objective of this paper is

to present a comprehensive account of the results of these studies. The experinlents reported here were conducted on 224 adult male Sprague-Dawley rats (250-350 g body weight). These animals werc anaesthetized with halothane and, after insertion of a tracheal cannalla, anaesthesia was naaintained with a mixture of methoxyflurane, nitrous oxide (80%). and oxygen ( 2 0 % ) . The animaIs were placed in a stereotaxic frame and body tempcorature was maintained at 37 -+ 0.5"C aiia an electric heating pad co~~trolled by a rectal thermal probe. Forty-eight of these rats had a cannula placed in the right femorai vein and 87 also had an arterial cannula inserted into the femoral artery to record arterial blood pressure. Three of this latter group had a cranialIy directed cannula in the right carotid artery. Large (600 g ) rat5 were used in all the arterial cannulation experiments to facilitate cannula placement. After reflection of the skin overlying the dorsal sku11, a small hole was drilled through the parietal bone 2 rnm lateral to the sagittal suture and 1.5 mm posterior to the coronal mture line. In many experiments a further hole was made in the interparietal bone over the ipsilateral cerebellar cortex 0.5 mm lateral to the midline and 10.5 nam po5terior to bregma. A bipolar coaxial stimrnlating electrode was placed in the ipsilateral pyramidal tract through this hole, The anterior hole was used for access to neurons in the sensorimotor coltex and in some experiments a fine coaxial stin-nulritingelectrode was placed on the cortical surface adjacent to the site of penetration of the microelectrode. The exposed skin, malscle, and bone were covered with a thin Iayer of 4% agar in Ringer's sola~tionto prevent drying and to stabilize the corticai s~arface. Seven-barrelled micropipettes were used to record extracellular spike potentials and iontophorese drugs onto cerebral cortical nea~rons.The central recording barrel and one side barrel were filled with 2 l t l NaCl and the remaining barrels were dilled by centrifugation with various cornbinations of substances (see below). Each drug was tested on deep (800-1400 ptm) spontaneously firing neurons in the sensorimotor cortices of a rninin-num of two animals and applied from at least three different electrode barrels. Neurons were identified as corticospinal cells if they responded to ipsilatcral pyramidal tract stimulation with a short. constant-latency spike that followed stianulation freq~~encies of at least 100/s. Daug efFccts were evahtated in most instances by observing the alterations in the rate of spontaneous firing. although on occasion, ACh or 1,-glutamate werc used to excite cells that were quiescent or discharging infl-equently and irregularly. The relative depressant potencies of individual compounds were subjectively evaluated on the basis of the magnitude and timc course sf the neuroanal responses to equivalent "doses" of each compound and either adenosine or 5'-AMP. Two sclledules of drug applications were used during these pote~lcyevaluations. In one schedule, adenosine (or 5'-AMP) and the compound being evaluated were applied for identical periods alternately at regular intervals and the application current passed through the barrel containing the latter was adjusted until both substances had comparable depressant actions. In the other schedule, adenosine (or 5'-AMP) was applied for constant periods at regular intervals until uniform responses were obtained.


PHILLPS ET AL.

The test substance was then applied on the same schednle and its application current adjusted aititil a dep~ebsiou equivalent to that evoked by aclenosine (or 5'-AMP) was observed, Adenosine ( 0 1 5'-AblPj was thcn applied at its original strength on its original schedule to enstarc that the r.esponsivenes\ of the ~ l e ~ i t o\+'as n unaltered. In testing substances which potentiated or ztntagoniacd the action of adenosine, a staiidard le5ting sequence as used. Identical adenosine (or 5'-AMP) pulses were ,~ppIied at regular intervals. The agent being tested was ttaen applied conc~arrentlyand changes in the response of the cell to adenosine cdbserved. 'The adenosine applications were col~tin~aed until conti'cal responses returned whenever possible. When the potentiators o r antagonists haat a depressing action (e.g.. the deouyadenosines). these were applied with currents which were either tfmreshold o r subthresholtl for depressing the neraronal firing rate. With twc? exceptions, 8(benzylthio)adenosine 3',5'-cyclic phosphate and 8-(chlolophenylthiobadenosine 3',5'-cyclic pliosphalc, which were applied with currents of u p to 200 nia, the maximalm drng application current rased was 100 nA. Eficcts Mere considersct genuine drug effects if they vderr: not rlgirnicked by similar current pulses passed through the NaCl-containing b a ~ r e l . In mnany experiments, the sotiium chioride barrel was used for current neutraliration at the electlode tip so that all effects were deemed to be genuine drug tespcmses. Intracell~alarrecording microelectrodes were mack from Pyrex capillary tubing, fiber-filled with 5 ICI potassialn-n acetate and beveled with a Sutter type (D.K.1.) micropipette heveler so that they had a final resistance of between 20 ancl 50 MR. Double-barrel extracellular ionlopl~oreliselectrodes were glued to the recording mic~oelectrode with Styrofoam dissc~lvedin chloroform near their tips and with Pyseal at the shoulders. Thiq was done whiIc both wele held in the Narashige (HMT-3) electrode holder. 'The tip of the recording microelectrode projected 20-50 pm beyond that of the double electrode. A Mentor N-950 pwan~plifier was employed to record intracellular potentials :inel generate transmembrane current pailses. T h e following compounds were tested during the course of these experirnents: sodium a_-glutamate (0.2,49; p H 8.0), GABA (0.1 M, pH 5.0, Sigma), ACh chloride (0.1 A f , pH 5.81, adenine (saturated solution. pH 4.5, Calbiocllem) , aclenil-te hydrochloride (0.1 IM, p H 4.8, Sigma), adenosine (saturated solution, pH 4.0, Sigma), ;idealosine hernisulphate (0.2 lI4, pH 4.8, SignnaB, 2'-4MP (8.2 h!. pH 5.6, Ntatritional Biochemicals), 3'-1\hfP (0.2 '34, pH 5.0, Sigma), cyclic 2',3'-AMP (0.2 M , pH 5.0, Sigma). 5'-.;IMP ( 0 . L and 0.2 ,M, pH 5.0. Sigma. and P-k Biochemicals), 5'-ADP (0.1 and 0.2 p H 4.5, Sigma and ZB-1, Riochen-~icals), adenosine 2',5'-diphosphate (0.1 M , pH 5.0, Sigma), adenosine 3'3'-diphosphate (0.1 11.1, pH 5.0, Sigma). 5'-ATP (0.1 and 0.2 ,W, pH 4.5, Sigma), adenosine 5'-tetraphosphate (8.1 Af, p H 5.0, Sigma), cyclic 3'3'-AMP (0.2 M ,p H 6.0, Sign-ma and P-L Biochemicals), AMP-PNP 60.1 '2.1, pH 6.0, ICN Pharmaceuticals), adenosine 5'-mc~nonisotinate (saturated solution, pH 8, Niatritional Biochemicals), adealosine N1-oxide (saturated, p H 5.0, Nutritional Biochemicals ) adenosine 5'-sulphate (0.025 M , p H 4.5, P-1, Biochemicals), adenmine xylofurnnoside' (0.1, pH 6.0), 2-arninoadenosine' (0.002LW, pH 4.5)- S-(benzylthio)adenosine 3',5'-cyclic monophosphate (8.1 M , pH 7.5, ICN Pharmaceuticals),

w,

.

'Gifts of these compounds are gratefully acknowledged.

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2-hrornoadenssine' (0.1 114, pH 6 . 8 ) , 8-bromoadewosiale (satiaratcd solution. pH 7.2, Sigma), caffeine (0.1 ill, pH 6.0, B.D.H.), caffeine citrate (0.2 /kg, p H 4 5 . K. M. Laborato! ies), 2-chlcsroadenosine (0.04 A f , pH 7.0, Sigma), 8 - ( p chlorophenyltt~io)adenoiine 3',5'-cyclic phosphate (0.04 M , pbI 6.5, I61N Pharn~aceuticals), 7-dca~aaafcnosine (ambercidin. 0.01 IW, pH 6.0. Sigma), 2'-decaxyadenosine (0.05 IW. pH 7.5. Sigma b. 3'-~leoxyadenosisne (cordycepin, saturated s01utiun, pH6.0, Sigma), 2'-cieoxya~lenosine 3'-monophosphate (0.1 M. p H 5.5, Sigma), 2'-deoxyadenosine 5'-monophosphate (0.2 hf, pH 9.0, Sigma), 5'-deoxyadenosine (saturated solution. pH 6.0, P-L Biochemicals), cieoxycssforrnycin' (0.5 M , pH 6.5), pl,y'-diadenosine 5'pentaphospkate ( 0. l iW, pH 6.5, Sigma), pl.p"diadenosine 5'-pyrophosphk~te (0.1, p H 7.0, Sigma), diazepam' (Valium, saturated solution in 165 m M NaCB, Hoffnlan - La Roche Ltd.), dipyriclamole (0.1 M in DMSO solvent, C. H . Boehringer Sohw ) , cryrlaro-9- (2-h ydroxy-3-nonyl) adenine (0.1 M , pH 6.5. Burroughs Wellcome), fiavira mononucleotide (0.1 M , plt3 4.8, Sigma), %-f-laloa-oadcnosine'(0.05 A4, pH 8.0), l~exobendinedihyd~ochloride~ (0.1 !If, p H 6.0, Cbemie 1-inz, A.G.), homoadenosine 6'-phosphonic :tcidl (0.1 M. pH 6.4, Syntexb, honloadenosine triphosphate' (0.1 M , pH 6.5, Syntcx), 2-hvdroxyadenosine' (0.82 M, pH 4.5), 2-hydrox~~-5-nitrobenzy1thiog~i,anosine~ (saturated solution in 165 m M NaCl, pH 6.5), inosine (0.1 '44,pH 5._gl Signli:~), inosine 5'-mor10phosphate (8.1 114, pH 6.0, Sigma), inosinc 5'-triphosphate (0.2 '4.9, p H 5.6, Sigma), 2'-deoxj'inosine (0. 1 i%I, pH 4.5, Sigma), 3-isobntyl- l methylxanthine (saturated solution in 0.1 WaCl, p H 5.6, Aldrich Chemical Co. 9, lidoflazine' (0.1 IW, p H 3.5. Jansscn Phai maceutical) , alp-mcthyleraeadenosim tl iphssphate (0. I, pH 7.0, Milcs Laboratories), +,y-n~ethyleneadenosine lriphosphate (0.1 M, p H 7 . 0 , Miles Laboratories), cr,pmethygene-adenosine diphosphate (0.1 '34, pH 7.0, Miles Laborntorics), 2-p-methoxyphenyIadenusinel(CV 1674, saturated solution, pH 3.5, Takeds Chemical Indaastries Ltd.), nicolinamide adenine dintrcleotide phosphate (0.1 i14, p H 5.0. Sigma), pupaverine hydrochloride (0.05 hf, p H 4.5, Sigma), Wi-phenylader~osine'(0.1 ,34 in DhlSO .oIvent), purine riboside (8.1 Its in DMSO solvernt, Sigma), 2,2'pridylisatogen tosylate' (0.01 M , pH 3.0), 8-(p-sulphop21erryl)theophyllinel ( 0 . 0 '44, p H S . 5 ) , c~tidirie (0.2 IW, pH 7.0, Sigma), cytidine 5'-n~onophosphate(0.2 M , pH 7.2. Sigma), cytidine 5'-triphosphate (0.2 ha, pH 4.6, Sigma), cytidine 3'62') -monophosphate (0.1 M , p H 6.0, Calbiochem) , guanosine (saturated solution, pH 4-0, Sigma and Calbiochem) , guanosine 5'-monophosphate (0.1 ,W. p H 6.0, Sigma), guanosine 5'-diphosphate (0.2 M , pH 6.5, Sigma), guanosine 5'-triphosphale (0.2 M , p H 6.0, P-E Biochemicals and Sigrna ) , guanosine 3',5'-cjcIic mono phosphate (0.2 IV. pH 6.5. Sigma), 2'-deoxyguanosine (0.1 h9. pH 5.6, Sigma). thymidine (0.2 LW, pH 6.8, Sigma), thymidine 5'-monophosphate (0.2 IV, p H 8.0, Sigma), thymidine 5'-triphosphate (0.2 114, p H 6.2, Sigma), uaidine (0.2 ,W, pH 5.0, Calbiochem), uridine 5'-n~onaphosphilte(0.2 A4, p H 7.8, Sigma), uridine 5'-triphosphate (0. B M , p H 6.2, Sigma), uridine 3'(2') -monophosphate (0.2 44, pH 6.3, Calbischem), xanthine (Na' salt, 0.1 M , pH 6.5, Sigma), xanthosine (0.005 M, p H 4.0, Sigma), xanthosine 5'-monophosphate (0.2 M , p H 7.0, Sigma), xanthosine 5'-triphosphate (0.2 M , 19H 3.8, Sigma), clonidiwe hydrocl-tloridel ( 1 0 ,and 0 kf, pH 5.8, Boehringer Ingelheim), nicotinamide (0.1 M , p H 6.2, and in 100 m M NaCl, Signla), sodium phosphate (0.1 M , p H I 1.O), sodium pyrophosphale (0.2 M , p H 10.5).

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Results


( 1 b Actions o f Adenosine and Adenine Nucbeotides on Cerebral Cortical Ne~rrons ( a ) Depressant Actions of Adeno,~ineand 5'-Ark!$ Adenosine and 5'-AMP have both been tested on several hundred neurons in the rat cerebral cortex. Most of these neurons were spontaneously active and located at depths of 800-1400 p m in the sensorimotor cortex; many were identified as corticospinal neurons. Both substances (at application currents of 10-50 nA) produced similar responses on all of the neurons tested, namely a powerful depression of spontaneous firing which became apparent within a few seconds of the onset of the application and terminated within 38-60 s of the end of the application. The depressant action of these compounds on spontaneous activity is exceptionally potent and on some neurons removal of the "braking" current on she adenosine- or 5'-AkIPcontaining barrel was sufficient to depress firing. When the depressant potency of 5'-AMP was cornpared with that of the inhibiiory rncsimocarboxylic amino acid, GABA, the two depressants were observed to be equipotent, although the rapidity of onset of depression was greater with GABA. The effects of adenosine and 5'-AMP on various types of chemically evoked firing were less clear cut. Many of the deep spontaneously active cortical neurons are excited by ACh and a consistent finding was that adenosine or 5'-AMP, at currents that were able to depress spontaneous firing, had less of an effect on the ACh-elicited discharge. Larger applications of the purine usually depressed the ACh-evoked activity as well. Applicatio~nsof Lglutamate were frequently used to excite more superficjal, quiescent, cortical neurons. Such excitations were similar to the acetylcholine excitations of decper cc11s in that they were relatively insensitive to depression by adenosine or 5'-AMP; larger ejection currents were needed to depress glutamateevoked excitation than to suppress the spontaneous firing of deeper neurons. Unlike GABA, ader~osineand 5'-AMP were unable to prevent the invasion of antidromically propagated action potentials into the somas sf corticospinal neurons. Hn some instances, the passage of large (100-280 nA) currents through the purine barrel did saappress spike invasion, but such effects were observed itnfrequeratly and were difficult to reproduce consistently, even on the same neuron. l%7hernadministered intravenously, adenosine was able to depress tlme firing of cerebral cortical neurons, but only when relatively large doses were

administered. The threshold dose of adenosine required to elicit a discernible fall in blood pressrase was betwecn 5-10pg/kg, and at this dosage adenosine did not alter the firing frequency of cortical neurons. Larger doses (200-1 000 [&kg) of adenosine caused more marked, but transient, falls in blood presstare ( > 50 mmH-fg) which were frequently associated with a brief reductiotn in the firing frequency of cortical neurons. To resolve the issue of whether thcse depressant effects of intravenously administered adenosine on neuronaal firing frequency were either direct or a result of the alterations in arterial blood pressure, three rats were prepared for close intraartcrial administration sf adenosine into the right carotid artery. Adenosine was injected via this route in doses of 15-500 pg/kg. A very clear relationship between a fall in blood pressure and a reduction in cell firing frequency emerged from these experiments. Unless the arterial blood pressure fell by 50 rnnnHg or more there was from its control level of 95-1 10 rn~ng%g~ no clear alteration in neuronal firing frequency. The short-term effects of intravenous%y administered adenosine are, therefore, a result of the changes in blood pressure. An effective blood-brain barrier apparently prevents the entry of adenosine into the tissues of the brain (Berne et al. 1974). ( b ) Effects of Modificafiosas to the Purine and Ribose Ring,r on Inhibitory Actkli~dty Results from testing a series of ade110sB11edcrivatives and related compounds are presented in Table 1. Depressant activity requires the presence of both the ribose moiety and an amino group on the 6 position of the purine ring. Adenine (6aminopurine), which 'lacks the ribose group? has only weak depressant activity and inosine, the deaminated metabolite of adenosine, is also a weak depressant. Hypoxanthine (6-oxypurine), lacking both the 6-amino group and the ribose, is inactive as is purine riboside with its unsubstitaated purine ring. S~~bstitutions in the purine l-inag of adenosine can affect its depressant activity. Substitution of a halogen atom in the 2 position gave analogs that were either significantly more potent and long lasting than adenosine (2-chloro- and 2-fluoroadenosine) or which had a very prolonged a@tion (2-bromoadenosine). 2-Aminoadenosine (2,6diaminopurine riboside) , 2-hydroxyadenosine, and 2-p-methoxyphemyladePlosi~le were also relatively potent depressants. With bromine substituted in the 8 position there was a marked loss of depressant activity. Addition of a phenyH group at the 6 position

TABLE 1 , Depressant effects of adelao3ine analogs on cerebral cortical neurons


Substance

Neurons tested*

Relativet depressant activity

Adenine Adenosine Inosi~ae Hy poxanthine Purine derivatives sf adenosine 2-Chloroadenosine 2.-Fluorsadenosine 2-Bromoadenosine 2-Arninoadenosine 2=Hydroxyadenosine 2-p-Methc~xyj~he~~yladenosine N6-Phenyladenosine 7-Deazaadenosinc (tubercidin) 8-Bromoadenosine Adenosine Nl-oxide Purine ribosade Ribose derivatives of adenosine 2'-Deoxyadenosine 3'-Beoxy adenosine 5'-Deoxyadenosine Adenosine xylofuranoside Adenosine 5'-sulphate Adenosine 5'-mononicotinate Mononucleotides and dinucleotides Adeno\ine 5'-monophosphate Adenosinne 5'-diphosphate Adenosine 5'-triphosphate Adenosine 5'-tetraphosphate Adenosine 2'-monophosphate Adenosine %',5'-diphosphate Adenosine 3'-rnonophosphate Adenosiile 3',5'-diphosphate Adenosine 3',5'-cyclic monophosphate Adenosine 2',3'-cyclic monophosphate Adenosine 5'-imidod~phosphate Homoadenosine 6'-phosphonie acid I-Iomoadenosine eraphosphate a,@-Methylene5'-ATBZ @,?-Methylene 5'-ATP a,@-Methylene5'-ADP 2'-lkoxyadenosine 5'- non no phosphate 2'-Deoxyaclenosine 3'-monophosphate Nieotinaanide adenine dinucleotide phosphate Coen~ymeA pl.p"Diade~losine 5'-pentaphosphate p1,y2-Biarie~aosinc5'-pyrophosphate ]Flakin mononucleotide *She ratios indicate the number of cells depressed over t h e number of cells tested, and include data from both identified corticosyinnl neurons and uriidcntified deep spontaneously active cells. fAdenosine slid 5'-AMP were used as controls in the comparisons I'or potency. These substances were giver) a ---- rating. Substances with a very weak tiepressant actiori were rated -,and a "0" means no effect.

(Ns-phenyIade~aosine) yielded a compound with weak depressant activity. Alterations to the ribose ring aIso affccted potency. The prcsenace of 2'-, 3'-, aaad 5'-kydroxy groups are necessary for the manifestation of fulI agonist yo-

tency, although substitution of a phosphate group or chain at these positions is acccgtable. Thus, the 2'-, 3'-, and 5'-deoxyadenosines. although pc~ssessing depressant activity, were not as potent as the parent co~aapound. The 2'-, 3'-, and 5'-adenosi~ae mono-

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phosphates were all equipoten~twith adenosine, as were the 2',5'- and 3',5'-adenosine diphosphates. Coenzyme '4, which conatairns adenosine 3',5'-diphosphate, was also a potent depressant. Potency was not sigtmificantty affected by exterlsion of the poll phc~sphateside chain at the 5' j~ositjon (5'-ABY" 5'ATP, 5'-adea~osinetetraphosphate) although the onset of depression was delayed by an increased tendcncy for excitation. Substitution o f a siltlpb-late group at the S'position abolished depressant activity. Cyclic 3',5'-AMP Inad only weak deprcssant activity, whercas cyclic 2',3'-AhfP as as active as adenosine. The 8-(benzylthio) and 8-(parachIorophenylthio) analogs of cyclic 3',5'-AMP were tested on a number sf cells with ejection currents of up t s 208 nA. The 8- (parachHoropherayli thio) ctrrnpound depressed only 2 06 the 20 sells tested and the 8(benzylthio) compoua~dhael no effect a n the 28 neurons s n avhicla it was tested. %ever,,'l rnetllylene isosteres of the adenssitle polyphusphates were compared nritIl adenosine for depressant potency. Replacement sf an anhydride oxygen by a rnethylene group in tlme cu,p position of 5'-AD]$ and 5'-ATP (AOPCP. AOPCPOP, respectivcly ) greatly reduced agot~istpotency whereas substitution of the methylcne group at the P,y positic311 (AOPOPCP) o11Py slightly iaa~paired potency in comparison with 5'-ATP. Honnoadenosine 6'phosphsnic acid, the rnethylene isostere of 5'-AMP A >

ACP 100

(ACP, Fig. 1 ) , and homoadenosine triphosphate (ACPOPOP) were without depressant activity. The AMP-PNP (AOPOPNP), in which an irnido group substitutes for the P,y oxygen, possessed quite pronouraccd deprcssant activity. Nicotinarnide adenine dinusleokide phosphate suppressed spontaneous firing of cerebral cortical neurons, but glicotinamide itself had only a weakly excitant action (on 7 c~fthe 32 neurons tested). (c) Excitant Actions 68 was founatf that the excitant actions of 5'-ATB were ir~verselyrelated to the spontaneous firing rate of cortical neurons. For this reason tine pronounced spontaneous activity sf the majority of cerebral cortical incurons otn which they were tested precluded a comprchernsive study of the excitant actions of the adenine nucleotides. Excitant responses to 5'-ATP were especially prominent in a few aanirnals, in which virtually all of the more superficial quiescent neurons tested could be induced to fire during applications of the nucleotide. I n other aninla15 it was dificult to elicit excitation even when quiescent cells were tested. The excitant action of 5'-ATP had a short latency and declined rapidly once the application had ceased. Its characteristics were, therefore, rather comparable to the excitant acticrn of I--glutamate. On many spontaneoalsly firing neurons it was possible to demonstrate a biphasic action of 5'-ATP, ACP 400

-

A 20

FIG. 1. The: spontaneous firing of this cerebral cortical neuron wnq deprebscd by applications s f atlenosirae (A, 20 nAB, but rnot by laomoadcnosine 6'-phssphsnic acid (ACP, 100 raA). 'Fkis is a rate meter record of ne~irorralfiring with the number of action potentials per second on the ordinate. W-lc>si~ontal bar:: indicate periods of drug application. Aj~plib'atiolt currents are shown ii-a waiaca Amperes.


ET AL.

a brief initial excitant action being followed by a longer phase of depression of firing. This apparent duality of action of 5'-ATP frequently resulted in its appearing to have a slower onset and weaker depressant action than 5'-AMP and adenosine. Comparable excitant actions were observed with adenosine %'-tetraphosphate and itaomoadenosine triphosphate which excited 25 of the 36 neurons tested, but a reduction in the length of the 5'-phosphate chain reduced excitant activity. Thus. 5'-ADP had only an irlfreyuerlt weak excitant action on quiescent cells and excitation was rarely observed with 5'-AMP. Excitations were also observed during some applications of cyclic 3',5'-AMP. The excitant effects of 5'-ATP on 22 of 24 neurons werc replicated by applications of POg3from an electrode barrel containing Na:,PO,. Pyrophosphate ( P 2 0 7 4 - )applied from a barrel containing Na+P207excited 14 of the 15 neurons tested. Excitations elicited by both phosphate groups had a similar time course tc9 thoqe evoked by 5'-ATP. These observations suggested that the excitant actions of 5'-ATP might result from chelation and removal of Ca from the extracellular space by the phosphate groups. (It must be borne in mind that the very process of iontophoretic ejection will separate the 5'-ATP from its accompanying cation (Nat) in the electrode barrel.) In an attempt to test this hypothesis, Mg" or Ca" ions were ejected from another barrel simultaneously with 5'-ATP. At cor~centraticrns that did not affect glutamateevoked excitation of cortical neurons. both cations were able to prevent 5'-ATP-elicited responses. Results of this type support the suggestion that 5'-ATP may excite, at least in part, by chelating extracelIular calciu~nand that if Ca" or Mg2* ions are provided for it to chelate, such excitation does not occur. (d) Intrcl.c-ellrr?arlg:Recorded Actions of 5'-AMP Acceptable intracellular records were obtained from nine ncurolss in eight rats. The criteria for acceptability were that the neuron should have a resting potential of not less than 40 mV, be capable of generatinmg action potentials. and that the duration of stable intracellular recording should have been long enough to obtain at least cpne rcsponse to extraeellularly applied 5"AMP with subsequent recovery. The nine cells reported on here satisfied these: conditions for periods of 5 min to over 1.5 h, and were tested a total of 81 times with 5'-AMP. The nucleotide was applied to each neuron an average of eight times. The 5'-AMP-elicited inhibitory effects were evident with all of the neurons tested and 110 exeitatio~mswere observed. The ionto-

1295

phoretic currents used ranged between 20 and 200 nA, with inhibition evident to varying degrees with both low and high application currents. The nine cells had input resistarlces ranging between 4 and 36 Mil with a mean resistance of 15 1. 3.2 (SEM) MR. The most striking effect sf %'-AMP was a decrease in spontaneous and evoked synaptic potentials accompanied by a hyperpolarization of the cell membrane potential. The magnitude of the hyperpolarization was not always constant, nor was it directly related to the current strength used to eject the 5'-AMP. Rather, the hyperpolarization was more dependent on the amount of spontaneous synaptic drive onto the cell at the time of application. In the absence of spontaneous synaptic EPSP's only small or no hyperpolarizations were observed. Another typical feature of the %'-AMP inhibition, shown in Fig. 2 A and B, was the lack of a change in the cell's input resistance, measured in this instance with a depolarizing current pulse of 8.2 nA. Tn five cells where resistance was measured with de- or hyper-polarizing pulses before, during, and after repeated 5'-AMP applications, no significant change in input resistance was detected. Measurements of the threshold currents required to elicit action potentials demonstrated that 5'-AMP did not affect the excitability of the spike-generating meehanism (Fig. 2 A and B ) , although as shown in Fig. 2, there was often a small decrease inn the peak amplitude of the action potential (independent of any alteration in membrane potential ) . The EPSP's were evoked in cortical neurons by stimulation of the adjacent cortex with a small bipolar stimulating electrode. The stimulation pulse which evoked the EPSP showin in Fig. 2A was adjusted so that the synaptic potential was threshold for spike generation. During the application of 5'AMP the rate of rise of the EPSP was decreased with a resultant failure to generate an action potential (Fig. 2B). A reduction in the rate of rise of EPSP's was observed in other trials. The upper trace in Fig. 3A is a firing frequency record from a cortical neuron whose intracellular potential record is shown in the lower trace (the polygraph was unable to follow the individual intracellularly recorded spike potentials). Pulses of 1,-glutamate (40 nA) applied repetitively to this neuron elicited a depolarization accompanied by a burst of spikes. The 5'-AMP (100 nA) hyperpolarized the neuron by several millivolts and there was a marked reduction in the ongoing excitatory synaptic drive. The amplitudes of the glutamateevoked depolarizations werc reduced to about 50%

1296

CAN. J. PHYSIOL. PHARMACBL. VOL. 57, 1979

A

8 8.2

irQe

nA %'-AMP

nA


pulse

FIG.2. Intracellular potentials recorded fro111 a cerebral cortical neuron. (A) Action potentials were evoked by an EPSP elicited by stinaulation of the adjacent cortex and then by a depolari~ingcurrent pulse (1.2 nA) passed through the cell membrane. (MI Recorded during the applicntion of 5'-AMP (108 nA). The EI'SP evoked by cortical stimulation now fails to elicit an action poteimtial, but the klnreshold for action potential generation by a current pulse is unaltered. Voltage calibration, 20 mV; time calibration, 5 ms.

of the control aanplitude a~nd glutamate-evoked firing was virtually abolished. Similar results were obtained in other trials of the interactio~~ between glutan] ate and 5'-AMP.

(2) Potentiators of Adenosine or ~"ALWPBDe~ I ~ X S ~ O P E

Inhibitors Of Adenosine Uptake Removal of extracellular adenosine as a factor in

(Q)

FIG.3. A chart recording of the firing frequency (upper trace) and intracellular membrane potential of a cerebral cortical neuron. The resting potential of this neuron was 65 mBr. Repeated applications of 1.-glutamate (GLUT) cdcpolarized the neuron and initiated a burst of spikes. During the application of 5'-AMP (100 nA), the neuron was hyperpolarized by an apparent 18 mV. However, the record in (I%), recorded extracellularly, shows that about BO mV of this apparent hqperpolari~ationcould be accounted for by electrode polarizatioal. During and after the application of 5'-AMP, spontaneous degolarizing syanaptic actikiky decreased to barely perceptible levels and the L-glutamate-evoked depolasizations were reduced in anaplitude. The L-glutamate-evoked firing was nearly abolished by 5'-AMP.

PHILLIS ET AL.

DIPY RIDAMOLE

-


AMP

A

25

-

-

0.1

mg/kg

B

-

-

-

-

-

u

I

rnin

FIG.4. The 5'-AMP-evoked depression of this cerebral cortical neuron was potentiated in magnitude and duratlorm by an intravenc~usadministration of dipyridamole (DIPYR, 0.1 mglkg). A second injection of dipyridamole (1.0 mg/kg) further potentiated 5'-AMP and caused a reduction in the spontaneous firing frequency. There was a gap of4 rnin between recordings (Aj and (B) and a gap s f 2 min between recordings (B) and (C).

the termination of adenosine-induced depression of spontaneous firing has bzen studied using a series of compounds which inhibit the uptake of adenosine by brain and other tissues, including hexobendine, papaverine, lidoflazine, dipyridamole, diazepam, and 2-hydroxy-5-nitrobcnzylthioguanosine (Wuang and Daly 1974; Mah and Daly 1976). Dipyridamole was administered both by intravenous injection and by iontophoretic application from barrels containing dipyridamole solubilized in dimethyl sulphoxide. Applied by either technique, it potentiated the effects of adenosine or 5'-AMP on the 14 neurons tested and with larger applications depressed the frequency of neuronal firing. Tontoghoretically applied dipyridamole (20-30 nA) caused a rapid potentiation of purine-elicited depressions which persisted for several minutes after each application. Examples of the effects of intravenously administered dipyridamole are shown in Fig. 4. The 5'-AMP-evoked depression of the spontaneous activity sf this neuron was enhanced in both

magnitude and duration by an initial intravenous dose of dipyridamole (0.1 mg/kg). A second injection of dipyridamole ( 1.0 mg/kg) 12 min later resulted in a marked depression of the rate of spontaneous firing associated with a further prolongation of the duration of 5'-AMP-evoked depression. Recovery of the firing rate to near control levels occurred within about 8 min, but potentiation of 5'-AMP depressions contiilued for the remaining 40 rnin during which recording was continued. Papaverine was tested on 14 neurons and on 10 of these, it was possible to demonstrate a potentiation of adenosine- or 5'-AMP-evoked depressions with papaverine applications which by themselves did not depress cell firing. The responses of one of these neurons is shown in Fig. 5. A 30-nA dose of papaverine, which had no effect on firing rate, greatly prolonged the depressant actions of adenosine (20 nA) . When larger currents (40-80 nA) were passed through the papaverine barrels, a depression of ~leuronalfiring was frequently observed.

CAN. J. PHYSIBL. PHARMACOL. \'OL. 57, 1979


PAPAVERINE 30

FIG.5. Potentiation of adenosine (20 mA) depression of a cerebral cortical neuron by papaverine (30 nA). With this application current papaverille had no direct effect on cell excitability, but did enhance the amplitude and duration of adenosine-evoked depressions. The adenosine effect returned to control levels within 5 min of the termination of papaverine application.

This effect proved to be rather weak and ineonsistent. Hcxobendine was extremely potent. It was tested on 20 cortical neurons and often reduced the firing rate when applied with currents as low as 5 nA. This depressant action of hexobendine complicated assessment of its potzntiating action on purine depression. However, when the currents used to apply hexobendine were reduced to below threshold Ievels for overt depressiora, a potentiation of the effects of adenosine or 5'-AMP could be observed. Recovery from this depression did not occur upon terrninatiorl of the application of the purine; rather the cell remained depressed until the hexobendine current was stopped. Findings of this type suggested that the purines had a synergistic action with hexobcndine as well as being potentiated by it. In order to determine whether hexobendine depresses neurons by a direct action on thc adenosine receptor, by potentiating the actions sf endogenously released adenosine, or by some other rnechanism, experiments were performed with the adenosine antagonist, thesphylline. When injected intravenously into six rats at dose levels of 50-100 rng/'kg, theophylline invariably antagonized the depressant actions of adenosine, but not those of hexobendine. Hexobendine depression of cortical neurons is, therefore, likely to be mediated by some mechanisms other than a direct or indirect activation of the adenosine receptor. Nucleoside transport across erythrocyte plasma membranes is inhibited by 2-hydroxy-5-nitroberazylthioguanosine (Paul et al. 1975). This substance was tested on 17 neurons and potentiated 5-AMP

depression in each instance. Typically, as illustrated in Fig. 6, this substance increased the magnitude and duration of purine-evoked inhibition, and in many instances depressed the rate of spontaneous firing of the neuron. This depression is clearly evident in the record shown in Fig. 6. Recovery was slow, usually requiring 20-60 min (firing of the neuron shown in Fig. 4 is still depressed at 46 min after the 2-hydroxy-5-nitrobenzylthioguanosine application). kidoglazine potentiates the actions of adenosine on vascular ant1 intestinal smooth inemscle by inhibiting its uptake (see Hulme and Weston 1974). When tested iontophoretically on 13 cortical neurons, lidoflazine (4-30 nA) potentiated both the magnitude and duration of adenosine- or 5'AMP-evoked depression (Fig. 7 ) . With larger ejection currents (30-60 nA), lidoflazine caused a long-lasting (up to 40 min ) depression of firing, even if adenosine was not applied. Isobutylmetl~ylxanthineinhibits adenosine uptake and antagoilizes adenosine-elicited accumulation of cyclic 3',5'-AMP by brain slices (Huang and Daly 1974; Mah and Daly 1976). When applied iontophoretically onto 14 cortical neurons, its most frequent effect was to potentiate 5'-AMP- and 5'-ATPelicited depressions, which it did on nine cells (Fig. 8 ) . Potentiation of purinergic depression continued for several minutes after the termination of isobutylmethylxanthine applications. On another cell it antagonized 5'-ATP-elicited depression and on four neurons it had no clear effect on purinergic depression. When administered intravenously, isobutylmethylxanthine had only antagonistic effects on 5'-


PHILLIS ET AL.

35 min

I

u 2 min

FIG.6. Potentiation of the depressant effects of 5'-AMP (I0 nA) by the adenosine uptake inhibitor 2-hydroxy-5-nitrobenzylthioguanosine (2-OH-NBTC, 60 nA). The 5'-AMP applications were continued until recovery was nearly complete some 40 rnin later.

AMP- and 5'-ATP-elicited depressions. Diazepam was applied iontophoretically from a saturated solution in 165 mil4 NaCl onto 13 cortical neurons, and consistently potentiated the depressant effects of 5'-AMP. Potentiation became apparent within 90-120 s of the onset of diazepam administrations, and slowly increased in magnitude. The potentiation contiilued for several minutes after the cessation of each diazepam application. Larger application currents passed through the diazepam barrel usuaily resulted in a direct depression of the firing of cortical neurons, which lasted for several minutes. Diazepam was administered intravenously to five animals (in doses of 0.01-1 rng/kg). It caused a variable amount of depression of the firing rate of cerebral cortical neurons, which began

-

AMP

30

-

-

-

-

about 30 s after administration. The duration of depression varied with the magnitude of the dose, lasting for some 5-1 0 min with the lowest doses and for over 60 rnin after 1 mg/kg. The effects of 5'AMP were clearly enhanced in magnitude and duration after diazepam administration at all three dose levels. Potentiation was most clearly evident when the spontaneous firing rate had returned to control levels. ( b ) Inhibitors of Adc.nosirze Dearninase The role of adellosine inactivation by deamination to its relatively inert metabolite, inosine, has been studied with two potent inhibitors of the enzyme adenosine deaminase, deoxycoformycin and erythro-9- (2-hydroxy-3-nonyl ) adenine (Woo et al. 1974; Schaeffer and Schwender 1974; Skolnick

-

-

-

-

-

u

I

min

FIG.7. Lidoflazine (20 nA), an adenosine uptake inhibitor, potentiated the effects of 5'-AMP (30 nA) on this corticospinal neuron. Recovery was complete 10 min after the lidoflazine application.

CAN. 9. PHYSIOL. PHARMACOL. VBL. 57. 1979

ALP


30

-

-

AT,

AT P

ALP

ALP

IBMX

FIG.8. 3-Ysobu~yl-1-methylxanthine (IBMX, 200 nA), applied iontophoretically, potentiated the depressant effects of 5'-ATP (30 nA) on this cerebral cortical neuron. There was a 3-min break between traces (A) and (B).

et al. 1978). Erythro-9-(2-hydroxy-3-nonyl) adenine potentiated the depressant actions of 5'-AMP on the 15 neurons tested and frequently caused a longlasting reduction in the rate of firing of cortical neurons. With lower application currents, it was possible to potentiate the action of 5'-AMP without causing any accoinpanying alterations in the basal firing frequency of the neuron. Similar observatioi~swere made with deoxycoformycin which was tested on 16 neurons. This inhibitor potentiated the actions of 5'-AMP (Fig. 9 ) and 5'-ATP and frequently caused a long-lasting ( 15-30 min) reduction in the spontaneous discharge frequency. ( 3 ) Antagonists of Purinergic Depressions The methylxanthines, theophylline, caffeine, and

isobutylmethylxanthine, antagonize the stimulatory effects of adenosine on cyclic 3',5'-AMP formation in brain slices (Sattin and Rall 1970; Mah and Daly 1976). Attempts to test these agents by iontophoretic appIication were generalIy unsucccssful due to their limited solubility in water, and in most of the experiments reported in this section, the antagonists were administered by intravellous injection. Theophylline was tested as an adenosine or adenine nucleotide antagonist in 25 animals. Administered in doscs ranging from 10 to 100 mg/kg, it invariably blocked or reduced the depressant effects of adenosine (10 animals), 5'-AMP (14 animals), 5'-ATP ( 5 animals), and cyclic 3'3'AMP ( 2 animals). The immediate effect of theophylline administration was to increase the spon-

2 rnin

Fro. 9. The adenosine deaminase inhibitor deoxycoforrnycin (40 nA) potentiated the actions of 5'-AMP (10 nA) and caused a depression of spontaneous firing which lasted for over 20 min.

PHILLIS ET AL.

-


AMP

LLINE B THEOPHY mg/k9 60

-

AMP

I

-

AT P

AT P

30

AMP -

AT P -

AT P -

AMP

-

AT P -

AMP -

-

AT P -

AMP

I

min

FIG. 10. Records from a corticospinal neuron. The 5'-AMP (30 nA) and 5'-ATP (30 nA) depressed spontaneous firing (A). Fourteen minutes after an intravenous injection of theophylline (60 r n g / k g ) the effects of bath compounds were blacked (B). Trace (C), recorded 160 min after the administration of theophylline, shows partial recovery from the antagonism.

taneous firing rate of cortical neurons, lasting 10-15 min, after which the firing rate slowly returned to near control levels. Within a few minutes of theophylline administration, the effects of iontophoreticaIIy applied purines were noticeably reduced and maximal blocking activity was attained 8-12 min after the theophylliile administration (Fig. 10). The antagonism could invariably be overcome if higher purine ejection currents were used. Noradrenaline-, 5-hydroxytryptamine-, and GABAevoked inhibitions of cell firing were unaffected by

theophylline. Partial recovery from the antagonism became apparent after 2-3 h. The prolonged duration of the antagonism made it difficult to evaluate the effects of different doses of theophylline on the same animals, but it was clear that low doses ( 10-20 mg/kg) could only antagonize effects of the purines when these were applied with low currents. Caffeine was administered by intravenous injection to seven animals in doses of 10-60 mg/kg. Caffeine injection resulted in a pronounced increase

CAN. J. PHYSIOL. PHARMACOL. VQL. 57, 1979


AMP 25

AT P 25

IBM X

20

mg/kg

AMP

AT P -

-

AMP

-

AMP

ATP -

AT P -

25

2

min

0

-

AMP

AT P -

AMP

ATP -

L I rnin

FIG,11. The 5'-AMP (25 nA) and 5'-ATP (25 nA) depressed the firing of this cerebral cortical neuron. Two minutes after an intravenous injection of 3-isobutyl-1-methylxailahine(IBMX, 20 mgjkg) the effects of both purines were greatly reduced. The lower trace, recorded 110 mill after the IBMX injection, illustrates recovery from the antagonism.

in the rate of spontaneous firing of cerebral cortical neurons and it antagonized the depressant effects of iontophoretically applied adenosine (two animals) and 5'-AMP (seven animals). The antagonism could again be overcome by larger doses of the agonist and the rate of onset and recovery to caffeine antagonism were comparable to those observed with theophylline. Arterial blood pressure recordings were made during these tests of theophylline and caffeine, and the effect of both drugs was to cause an initial transient fall in pressure followed by a small but long-lasting elevation of 4-10 rnmHg. hnt~phsretically applied caffeine was

tested on 24 neurons with application currents sf up to 150 nA. On five of these neurons caffeine enhanced the spontaneous firing rate, and in four instances it antagonized the depressant actions of adenosine or 5'-AMP. Isobutylmethylxanthine (20 mg/kg) was tested by intravenous administration on three animals. In each test it rapidly (within 2 min) antagonized the depressant actions sf 5'-AMP and 5'-ATP (Fig. 1 1 ) . Isobutylmethylxanthine also caused an increase in the firing rate of the neurons tested. Recovery was apparent 90-120 min after its administration.


PHILLIS ET A%.

I 2 min

FIG. 12. The 2,2'-pyridylisatogen tosylate (2-2'-P, 15 nA) depressed the firing of this cortical neuron, but did not antagonize the depressant action of 5'-ATI' (20 nA).

We have recently obtained a water-soluble analog of theophylline, the sodium salt of 8- (p-sulphophenyl) theophylline. This coalpound has flow been tested as a purine antagonist on 38 neurons in the cerebral cortex of five rats and antagonized adenosine-evoked depressions in every instance. The most striking observation made with it to date when applied with currents of 5-20 nA is its rapid (latency 1-2 min) and potent antagonism of adenosine. Recovery of the adenosine response occurred within 6-12 min after termination of the $-(p-sulphophenyl) theophylline application. Another characteristic response to its application has been a pronounced increase in the spontaneous firing rate, ~rhichwas observed with 32 of the 38 neurons tested. The 8- (p-sulphophenyl) theophylline did not antagonize noradrenaline-elicited depressions of 1 1 neurons, but it did reduce GABA inhibition of 4 of the 15 neurons tested. On each of these four cells the recovery of respoilses to the amino acid preceded that to adenosine by several minutes. 2?2'-Pyridylisatogen tosylate has been identified as a selective antagonist of 5'-ATP, but not of adenosine, on the guinea pig caecum (Spedding and Weetman 1976). This compound was tested on five cells on which it had a potent depressant action, but no antagonism of adenosine or 5'-ATP depressions was observed (Fig. 12). Adenosine-evoked stimulation of cyclic 3'3'AMP formation in brain slices is antagonized by the 2'-, 3'-, and 5'-deoxy derivatives of adenosine and by adenosine xylofuranoside (Mah and Daly 1976). These compounds, as well as 2'-deoxy-

adenosine 3'-monophosphate, an inhibitor of adenylate cyclase (Sahyoun et al. 19761, and 2'deoxyadenosine 5'-monophosphate were, therefore, tested as adenosine antagonists. The deoxyadenosine derivatives all had depressant activity on the spontaneous firing of cerebral cortical neurons and with none of them was it possible to antagonize the actions of adenosine or 5'-AMP. Adenosine xylofuranoside was tested on 25 neurons. It had a very weak depressant action on the spontaneous activity of six of these and a weak excitant action on nine neurons. On 7 of the 25 neurons, it partially antagonized the depressant action of adenosine or 5'-AMP. Inosine ( 2 1 cells), 2'-deoxyinosine ( 14 cells), inosine 5'-monophosphate ( 11 cells), adenine ( 10 cells), and guanosine ( 3 3 cells) were tested for adenosine antagonism using application currents of 20-1 20 nA. These compounds had depressant actions on many of the neurons tested and in some instances there appeared to be a summation with adenosine-elicited depressions. There were no instances of antagonism in these trials (Fig. 1 3 ) . Clonidine has been reported to be able to antagonize the depressant actions of adenosine and 5'-AMP on the firing of rat cerebral cortical neurons (Stone and Taylor 1978). When applied with currents of 20-50 nA, clonidine usually depressed the spontai~eousfiring of cortical neurons, especially if the application was prolonged for several minutes, or when the larger currents were used. Tested as an adenosine or 5'-AMP antagonist on 39 neurons in six methoxyflurane - nitrous oxide anaesthetized


CAN. J. PHYSIOL. PHARMACBL. VOL. 57. 1949

FIG.13. Continuous records of the firing of a corticospinal neuron. The 2'-deoxyinosine (40 nA) and inosine (40 nA) depressed firing, but did not antagonize the effects of adenosine (AD, 40 nA).

rats, clonidine either had no eRect on (with small application currents) or potentiated (with larger currents) the puriilergic depressions. Enhancement of the purine response by clonidine was observed with every neuron in this series, but in none of the trials was any antagonism evident. Elevated levels of calcium in the superfusion fluid (2-6 inrM) prevent the depression by adenosine of postsynaptic potentials in guinea pig olfactory cortex slices (Kuroda et al. 1976a). Attempts were, therefore, made to determine whether iontophoretically applied calcium ions antagonized the depressant effects of adenosine or 5'-AMP on rat cerebral cortical neurons. Tests condlicted on 27 neurons in seven rats, using CaH application currents of 5-20 nA, failed to reveal any antagonism of purinergic depressions; rather when usii~gthe larger Ca" application currents a potentiation of the inhibitory effects of adenosine and 5'-AMP was observed. The use of even larger CaH applications was precluded by its own depressant action on action potential generation.

(4) Studies with OtBzer Purines and Pyrimiclines (a) Depressant Eflc.cts These are summarized in Table 2. The p a n i n e nucleotides, 5'-guanosine mono-, di-, and tri-phosphate, and guanosine 3',5'-cyclic monophosphate had weak depressant actions on many of the cerebral cortical neurons tested. Guanosine itself had a weak depressant action on only a few neurons. Guanosinc 3',5'-cyclic inonophosphatc is of so~ame interest because of its postuIated role as a second messenger for the ~nuscasialicactions of ACh. This nucleotide depressed 17 of the 21 unidentified neurons and 37 of the 59 corticospinal neurons tcstcd. Of these cclls 42 were tested with, and excited by, ACh and 27 sf the 42 were depressed by quanosine 3',5'-cyclic monophosphate (Fig. 14). None of the ACh-excited neurons were excited by the nucleotide althougl~it did weakly excite some of the more superficial quiescent neurons on which it was tested. Xanthine, xanthosine, and xantl~osinemono- and tri-phosphate had a very weak depressant action on

PHILLIS ET AL.


3,s-eSMP 80

GUANOSINE M

3-AYP 20

I

rnin

FIG, 14. Thc 3'-AMP (20 HIA]and guanosine 3',5'-cyclic monophosphate (3',5'-cGMP, 80 nA), but not guanosine, depressed the Biring of this corticospinal neuron. The ACh (20 nA) excited the neuron.

a few neurons and no effect on the rest. The cytidine, thymidine, and uridine derivatives likewise had only very weak depressant activity. In many instances, it TABLE2. %'urine and pyrimidine derivatives with eithcr weak depressant activity or lacking in depressant activity Substance

Neurons tested"

(A) Weak depressant activity Inissine %nosine5'-monophorphate Inosine 5'-triphosphate Ciuanosine 5'-monophosphate Guanosine 5'-diphosphate Guanosine 5'-triphosphate Ciuanosine 3',5'-cyclic monsphosphatc (B) Very weak or no depressant actikity Guanusine Cytidine Cytidine 3'(2']-anonophosphate Cytidine 5'-monophosphate Cytidinc 5'-triphosphate 2'-Deoxyinosine Thymidine Thymidine 5'-monophosphatc Thymidine 5'-triphosphate Uridine Uridine 3'(2')-monnphosphate Uridine 5'-msnsphosphate Uridine 5'-triphasphate Xanthine Xanthosine Xanthosine 5'-moa~ophosphate Xanthsline 5'-triphosphate *The ratios indicate the number of cells depressed over the number of cells tested, and include data f r o n ~both identified corticospinal neurons and unidentified deep spontaneously active cells.

is possible that the small reductions in firing frequency observed after application of these nucleotides were actually "post-cathodal depressions" following the depolarizing actions of these compounds. (b) Excitant Actions The triphosphates of all the purines and pyrimidines tested had excitant actions on cerebral cortical neurons. This effect was quite pronounced on many Geurons and, as observed with the adenine nucleotides, was more apparent in some animals than in others, and on neurons which were either quiescent or firing slowly. When tested on such neurons, xanthosine 5'-triphosphate excited 11 of the 33 neurons tested, uridinc 5'-triphosphate 20 of the 57 neurons, cytidine 5'-triphosphate 7 of the 2 1 neurons, and thymidine 5'-triphosphate 7 of 24 neurons. Examples of the excitant actions of cytidine 5'-triphosphate and uridine 5'-triphosphate on a spontaneously active neuron which was depressed by 5'-ATP are illustrated in Fig. 15. Diphosphates of these purines and pyrimidines had less pronounced excitant effects and the monophosphates were weakly excitant on a few neurons.

Discussion The discovery of a potent depressant action of adenosine, its analogs and nucleotides, was a surprising one. There had not been any previous studies on the n-nicroiontophoretic effects of adenosine itself, and studies with 5'-ATP applied iontophoretically

CAN. J. PHYSIBL. PHARMACOL. VOL. 57, 1979

-


ATP 40

- - UTP 100

UPP 60

UPP 40

UPP 20

_ I

CTP 80

CTP I

100

u I min FIG. 15. Uridine 5'-tripkosphate QUTP,PO, 40, 60, and 100 nA) and cytidiwe 5'-triphosphate (CTP,$0 and 100 nA) excited this neuron. The 5'-ATP (40 mA) depressed its spontaneous firing.

onto spinal and cerebral cortical neurons of cats the firing of cerebral cortical neurons that can be under barbiturate anaesthesia had failed to reveal opposed in an apparently competitive manner by the any action of this compound (Curtis et al. 1961; methylxanthines (thesphylline, caffeine, and isoKrnjeviC and Phillis 1963 ) . On the other hand, 5'- butylnsethylxanthine). There is presently some ATP had excitatory effects in the cuneate nucleus, doubt, however, regarding the specificity of action 5'-ADf being ineffective (Galindo et al. 1967), while of the methylxanthines, as these compounds can, in both 5'-ATP and 5'-AMP have been reported to addition to antagonizing adenosine, inhibit phosexcite neurons in the cerebellum (Siggins et al. phodiesterases, mobilize calcium, inhibit 5'-nucleo197 1 ), an action which has been attributed to cal- tidases, release catecholamines and ACh, and cium chelation (KrnjeviC 1974). On cerebellar antagonize some of the actions of opiates and Purkinje cells cyclic 3',5'-AMP was observed to benzodiazepines ( J hamandas and Sawynok 1976; have both excitatory and depressant effects which Phillis 1977; Phillis et al. 1979). If, as has been were interpreted in terms of two mechanisms: one suggested, the opiates and benzodiazepines exert nonspecific and excitatory, the other being a specific their methylxanthine-reversible effects by releasing depressant action. Our original finding of a potent or preventing the reuptake of the elsdogenous purines, depressant action of adenosine and its nucleotides on it is possible that ~nethylxanthineswill be recognized cerebral cortical neurons has since been confirmed in as selective antagonists of the adenosine receptor. The concept of an adenosine receptor has been other laboratories (Stone and Taylor 1977 ) . The rather weaker depressant actions of guanosine-5'- accepted in a number of peripheral smooth muscle inonophosphate and guanosine-5'-triphosphate on tissues (including the ileum and vas deferens), where rat cortical neurons described by Phillis et al. ( 1974) purines have dose-dependent depressant actions on have also been confirmed (Stone and Taylor 1978). coiltractile activity that are competitively inhibited Key questions that arise from the observations by theophylline (Ally and Nakatsu 1976; Okwe~apresented in this paper include the following. ( a ) Is saba et al. 1977; McKenzie et al. 1977; Clanachan there an adenosine receptor? ( b ) What are the struc- et al. 1977). An adenosine receptor has also been tural requirements for agonist activity at the re- proposed as the site at which adenosine elicits an ceptor? ( c ) Where are these receptors located (pre- accumulation of cyclic 3',5'-AMP in brain slices or post-synaptic, neuronal and (or) glial)? ( d ) (Huang and Daly 1974; Mah and Daly 1976: What is the mechanism by which adenosine de- Kuroda 1978 ) . Evidence from experiments on a variety of tissues presses the firing of central neurons? ( e ) Is adenosine a neurotransmitter or nlodulator in the brain? suggests that the adenosine receptor is extracellularly A receptor may be assumed to be present when a located. Results obtained with adenosine uptake indrug elicits a dose-dependent response that can be hibitors both in the present study and in experiments opposed competitively by an appropriate antagonist. on cyclic 3',5'-AMP accumulation in brain slices Our results with adenosine and its analogs provide (Huang and D d y 1974), and with the coi~ipound clear evidence for a dose-dependent depression of 2-chloroadenosine (which is not taken u p to any sig-


PHILLIS ET AL.

nificant extent by brain tissues (Sturgill et al. 1975; Wilkening and Makman 1975)) provide strong evidence for an extracellular location of the adenosine receptor. Comparable results with uptake inhibitors and 2-chloroadenosine have been reported for peripheral tissues (Satchel1 and Maguire 1975; Okwuasaba et al. 1977; McKenzie et al. 1977; Clanachan et al. 1977). Further evidence for a cell surface locus of the reccptor in intestinal smooth muscle has been gained by coupling adenosine and theophylline to an oxidized oligosaccharide to produce large molecular weight compounds. Although confined to the extraccllular space. these exerted typical agonist and antagonist activity, respectively (Okwuasaba et al. 1978). Burnstock (1978) has proposed that there are two types of purincrgic receptors on the basis of tht., selective actions of agonists and antagonists; a P I receptor activated equipotsntly by adenosine and its nucleotides and antagonized by the methylxanthines and a P, receptor which is activated preferentially by 5'-ATP, with quinidine, 2-substituted imidazolines, and 2,Z'-pyridylisatogen acting as antagonists. No evidence was obtained in the present experiments of the existence of a P2 receptor mediating depressant actions of 5'-ATP on cerebral cortical neurons. The 5'-ATP was approximateIy equipotent with adenosine and 5'-AMP as a depressant and its actions were potentiated by adenosine uptake blockers and deaminase inhibitors. Moreover, 2,2'pyridylisatogeil failed to antagonize 5'-ATP-elicited inhibition of the firing of cortical neurons. Although further investigation will be required to establish conclusively the absence of a Py purinergic receptor mediating depressant actions of 5'-ATP on cerebral cortical neurons, the existing data strongly suggest that this is indeed thc case. The excitant actions of 5'-ATP, which are neither mimicked by adenosine nor antagonized by theophylline, may yet prove to be mediated through a P2 receptor. Uptake inhibitors (dipyridamole, lidoflazine, papaverine, hexobendine, 2-hydroxy-5-nitrobenzylthioguanosine, and diazepam) potentiated the actions of both adenosine and the adenine nucleotides. This effect on the adenine nucleotides is at first surprising as these compounds, unlike adenosine, do not readily traverse cell membranes (Hattori et al. 1969). A plausible explanation is that the nucleotides must first be hydrolyzed by extracellular nucleotidases to adenosine in order to cause depression. Potentiation of the actions of 5'-AMP and 5'ATP by adenosine deaminase inhibitors, erythro-9(2-hydroxy-3 -nonyl ) adenine and deoxycoform ycin, is consistent with this interpretation. Further evi-

1387

dence that the adenine nucleotides act via a common metabolite, adenosine, comes from the experiments with the methylene isosteres of these substances. The phosphorous methylene bond is stable and resistant to metabolic transformations involving either hydrolysis or phosphate transfer (Yount 1975). The C Y , / ~ isosteres of 5'-ABP and 5'-ATP were very weak depressants of cortical neurons, in coiltrast with the P,y- analog of 5'-ATP which possessed quite pronounced depressant activity (as did AMP-PNP). The latter finding suggests that even though the terminal bond in the 5'-ATP analog may have been stabilized: cleavage could still occur in the a,/3 position, allowing further degradation to adenosine. The methylene isostere of 5'-AMP, homoadenosine 6'-phosphonic acid ( ACP) , and homoadenosine triphosphate (ACPBPBP) both lacked depressant activity on cortical neurons, indicating an absolute requirement for dephosphorylation to adenosine for expression of depressant activity. Examination of Tables 1 and 2 suggests that the adenosine receptor exhibits specificity for both the base and ribose moieties of the purine rnolecule. The structure-activity relationships displayed in these tables are remarkably consistent with those observed in biochemical investigations on stimulation of cyclic AMP formation by purinergic drugs in brain slices (Daly 1977, pp. 110-1 11 ), implying the presence of a very similar adenosine reccptor in both instances. Further support for this concept is found in the effects of adenine derivatives on the depression of the amplitude of evoked postsynaptic potentials and concurrent formation of cyclic 3',5'-AMP in guinea pig olfactory cortex slices (Kuroda et al. 1976b; Kuroda 1978). Here the pharmacological similarities between the two responses suggest the involvement of a common receptor. The structure-activity relationships observed with peripheral tissues are also remarkably similar to those obtained with cortical neurons. Among the purine derivatives only adenosine, its analogs, and the adenine nucleotides shared a potent depressant activity. Inosine and its nucleotides were n~oderately effective, while hypoxanthine and the xanthine derivatives were nearly inactive. Guanine nucleotides also depressed cortical cells, but were considerably less potent than adenosine. (Attempts to antagonize the action of guanosine 5'-monophosphate and guanosine 5'-triphosphate with theophylline yielded equivocal results. In some trials, their depressant effects seemed to be reduced, but in others they were unaected. ) Pyrimidine nucleotides (cytidines, thymidines, and uridines) were essentially void of depressant activity. Inosine is very

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CAW. J. PHYSIOL. PHARMACOL. V8L. 57, 1979


much weaker than adenosine, and guanosine, which has an amino group at the 2 position, was inactive suggesting that an amino group at the 6 position of the purine ring is necessary for full agonist activity. Purine riboside was also inactive. Adenosine analogs with a halogen at the 2 position are either very potent (2-chlorn- and 2-Auoro-adenosine) or have a long duration of action (2-bromoadenosine), The 8bronlo derivative was, however, oi-lly very weakly depressant. A high apparent potency and long duration of action of the two halogenated adenosines has been shown in other systems (Satchel and Maguire 1975; Muller and Paton 6979) and may result from the resistance of these compounds to deamination and uptake. Modification of the ribose ring also affected agonist potency. Phosphate grc)up(s) at the %', 4', or 5' positions do not affect potency possibly because, as mentioned above, thcse compounds are rapidly hydrolysed to adenosine by extrace~lular ATPases and nucleotidases (Mandel et al. 1977). Cyclic 2',3'-AMP was consistently a more potent depressant of cortical neurons than cyclic 3',5'AMP. Evidence frona bischeniical studies suggests that the 3'3' compound must be dephosphorylated to adenosine before activating the adenosine receptor, as its action is absent in the presence of adenosine deaminase. Conversion to adenosine is probably necessary for the depressant action of both the %',a'and 3',5'-cyclic nucleotides and the greater potency of the 2',3' compound implies that its hydrolysis by phosphodiesterases must proceed at a considerably higher rate than that of cyclic 3',5'-AMP (Drumrnond and Yanlamoto 1 97 1 ; Phillis 1977 ). Unsvbstituted hydroxy groups at the 2', 3', and 5' positions of the ribose ring are necessary for full agsnlist potency. An interesting difference hetween pharmacological and biochemical responses of cerebral cortical tissue to purines has been revealed by our finding that 2'- and 3'-deoxyadenosine have agonist, but not antagonist, activity. In cerebral cortical slices, 2'- and 3'-deoxyadenosine antagonize adenosine-stinmulated formation of cyclic AMP while 5'-deoxyadenosine acts as a partial agonist (Mah and Daly 1976). The antagonism of adenosine-evoked cyclic AMP formation by these compounds is thought to be due to a direct action at an inhibitory site on the adenylate cyclase. It is possible that this inhibitory action of the 2'- and 3'-cteoxyadenosines prevents expression of adenosine receptor activation in the biochemical studies. A11 three co~npoundsas well as 2'-deoxyadenosine 5'-monophosphate and 2'-deoxyadenosine 3'-tnonopl-losphate had agonist and not antagonist activity in the present experiments. Our

interpretation of these observations is that activation of the adenylate cyclase studied by Mah and Daly ( 1976) is not necessary for manifestation of the depressant activity of adenosine. Intracellular recordings from cerebral cortical neurons show that adenosine-elicited depression is associated with a hyperpolarization which occurs in the absencc of any significant alteration in tnembrane resistance. There is a decrease in the size and rate of rise of evoked excitatory postsynaptic potentials and a decrease or disappearance of spsntaneously occurring synaptic potentials. Action potentials generated by direct depolarizing pulses were unaffected by purine applications. Sinlilar results have been obtained by intracellular recordings from neurons in guinea pig olfactory cortex slices (Scholfield 1978 ) . Topically applied adenosine ( 1 pM to 1 mM) had no effect on ~lae~nbrane potential, resting resistance, or action potentials, but did depress EPSP's generated by lateral olfactory tract stimulation. Presynaptic spikes in the lateral olfactory tract fibers were also unaffected. The failure of adenosine to alter the naembrane resistance or threshold for action potential generation implies ssnle form of interference with the synaptic mechanism. It may involve an inhibition of the presynaptic excitation - transmitter release coupling as has been shown at peripheral synaptic junctions (Ribeiro 1978 ) . Another possibility is that it may block postsynaptic receptors or inhibit the coupling between the receptors and the mechanism? controlled by the receptor. Tl-le relative lack of depression by adenosine of ACh- and, in particular, L-glutamate-evoked excitations of cerebral cortical neurons would bc consistent with a presyrmaptic locus of action (L-glutamate excitation is prinaariiy a direct effect on the postsynaptic neuron (Shapovalov et aI. 1978) ) . LckiC ( 1974) has reported evidence favoring a presynaptic site of action of 5'-AMP at motor axon collateral - Renshaw cell synapses in the spinal cord. Activation of Renshaw cells by afferent hampulses in cholinergic motor axon collaterals is suppressed by 5'-AMP while Wenshaw cell responses to iontophoretically applied acetylcholine are not affected. Also consistent with a presynaptic locus of action of adenosine are reports of purinergic depression of ACh and noradrenaline release from cerebral cortical preparations (Jhamandas and Sawynok 1976; Harms et al. 1978; Vizi and Knoll 1976), the neuromuscular junction (Giwsborg and Mirst 2 972; Wibeiro and Walker 1973) , and the peripheral sympathetic junction in kidney (Hcdqvist and Fredholna 1976). 'If the adenosine-elicited hyperpolarization does


PHIELIS ET AL.

result froin a withdrawal of the summed effects of ongoing excitatory synaptic potentials shouId it be accoinpanied by an increase in membrane resistance? This depends on the identity of the excitatory transmitter generating these synaptic potentials. If it is ACh, which seems likely as the spontaneous activity of these cortical neurons is greatly reduced after administration of atropine (KrnjeviC and Phillis 1963 ), the withdrawal of synaptic activity would not be expected to increase membrane resistance. The AChevoked depolarizations of these cortical neurons are associated either with a small increase in their membvarae resistance (KrnjeviC et al. 197 1 ) or occur without a change in resistance (Phillis 1977; Zieglgailsberger and Reiter 1974). Because some transmitters increase conductance while others decrease conductance, it is also possible that the effects of simultaneous removal of two or more synaptic influences might offset each other yielding no significant change in resistance. A presynaptic action of adenosine, including a reduction in transmitter output, is the most plausible explanation for our observations, although it is not possible to rule out an additional postsynaptic action with the present data. It is uncertain at this point if adenosine reduces transmitter release from all nerve endings. The evidence suggests that only nerve endings containing certain transmitters, such as ACh and noradrenaline, may be affected. Perhaps a hint as to the identity of the susceptible transmitters can be deduced from the presence or absence of 5'-ATF copackaged in the synaptic vesicles together with the transmitter. It is co~lceivablethat locally released adenosine suppresses transn~issionat a few specific synapses without affecting adjacent junctions, thus exerting circumscribed control over the transfer of information. Furthern~ore,if adenine nuclecstides are released together with certain transmitter substances, they may scrve an autoregulatory role so that especially active nerve termi~lalswould be prevented from inonopotizing the attention of the postsynaptic neuron. On the other hand. if there are purinergic nerves, as Burnstock ( 1972) proposes, thcn it is possible that the adenosine compounds may act as more conventional inhibitory transmitters. The inechanisnns involved in excitation by purine and pyrimidine polyphosphates are presently unclear. When applied iontophoretically at least part of their action is probably due to chelation of calcium ions. However. on an isolated amphibian spinal cord preparation, 5'-ATP elicited an excitation which was apparently unrelated to calcium chelation as perfusion of this preparation with a Ca" salt of 5'ATP caused excitant effects coillparable to the Na'

2 309

5'-ATP salt (Phillis and Kirkpatrick 1978). Purine and pyrimidine nucleotides also depolarize ileurons of explailted sympathetic ganglia, usually with no measurable change in membrane resistance (Siggins et al. 1977). This preparation may serve as a useful model on which to test the hypotheses of the nature of the excitant response. Present evidence from both central and ganglionic neurons suggests that the response is dependent on the phosphate side chain rather than the purine or pyrimidine base itself, and does not, therefore, have the specificity of the adenosine-evoked depressant action. For example, Feldberg and Webb ( 1948) showed that the excitatory action of ATP on cat sympathetic ganglia could be mimicked by phospl~ate.When injected close arterially into rabbit ear arteries, 5'-ATP excites sensory afferents, but adenosine does not (Juan and Lembeck 1974), again suggesting that the excitatory effects of adenine nucleotides have little to do with the central inhibitory effects of adenosine. The depressant responses to adenosine and the adenine nucleotides described in this paper are not restricted to the cerebral cortex. Similar effects hav;: been observed in the hippocampus, caudate nucleus, thalamus, superior colliculus, -olfactory bulb, and cerebellar cortex (Kostopoulos and Phillis 1977; Kostopoulos et al. 1975 ) . The final question to be asked, therefore, concerns the role that endogenously released adenosine or adenine nucleotides play in brain function. Some light has been shed on this problem by the observations reported in this paper. Applications of a nuinber of adenosine uptake inhibitors and two adenosine deaminase inhibitors resulted in a depression of the spontaneous firing of cerebral cortical neurons. Although further studies using adenosine antagoi~ists will be required to establish that these depressions were due to an accumulation of endogenously released adenosine, this appears to be the most logical explanation for our findings at present. Complementarily, administration of three inethylxanthine adenosine antagonists enhanced the spontaneous firing rate of cortical neurons. This pl~endmenonoccurred even when theophylline and caffeine were applied iontophoretically, thus assuring a local site of action. Excitant responses to the methylxanthines would be expected if cortical neurons are persistently suppressed by endogenously released adenosine or adenine nucleotides. n this context, it is understandable that theophylliile enhances the resting release of ACh from the cerebral cortex (Vizi and Knoll 1976) as well as antagonizing the depression of ACh release by adenosine ( J hamandas and Sawynok 1976). When considered together, the effects of ap-

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CAN. J. PHYSIOL. PHARMACOL. VOL. 57, 1979


plication of adenosirle potentiators and antagonists strongly suggest that cerebral cortical neurons are subject to continuous rnodulatioil by locally released purines. Adenosine and adenine nucleotides are released from the intact cerebral cortex (Berne et al. 1974; Sulakhe and Phillis 1975; Lewin 1977; Wu and Phillis 1978) and hippocampus (Schubert et al. 1976), and the rate of release is enhanced by electrical stimulation of these structures. The actual site of release remains to be determined. Adenine nucleotides are released from isolated brain synaptosoma1 preparations (Kuroda and McIlwain 1974; White 1978) indicating that their release froin intact brain may originate from presynaptic nerve terminals, possibly including the terminals of hypothesized gurinergic nerve cells. The 5'-ATP is packaged in vesicles with ACh and ~loradrenaliale in cholinergic and sympathetic nerve terminals. Release from such structures could accompany the release of the primary transmitter as sugg&ted by Silinsky ( 1975 ) . A negative feedback role has been postulated for adenine nucleotides at peripheral cholinergic and adrenergic nerve terminals (Ribeiro 197 8 ) and may be a common feature at many other typcs of central synapses. Adenosine or adenine a~ucleotide release from postsynaptic sites may also occur during intense activity as a result of 5'-ATP utilization (Israel et al. 1976) and could feed back to depress release of transmitters from nervc terminals. 117 conclusion, we would like to suggest that purinergic modulation sf synaptic transnlission in the central nervous system prescnts exciting new possibilities in the field of neuropharmacology. Antagonism of adenosine's depressant actions at central synapses offers a plausible explanation for the wellknown central stimulant and convulsant actions of caffeine and theophylline. Potentiation of the actions of adenosine by inhibiting its uptake may be a significant factor in the aetiology of the anticonvulsant and anxiolytic actions of the-benz~diaze~ines. Manipulation of extracellular adenosine levels or use of synthetic blockers or agonists should ultimately offer effective methods for the control of central nervous system excitability.

Acknswledgments The assistance of Dr. J. J. Limacher and Mr. S. W. Ellis in some of the experiments described in this manuscript is appreciated. Financial support for the investigation came from the Medical Research Council of Canada. It is a pleasure to acknowledge the generous donation of sanbstances used in the research

by Dr. C. Chester Stock of the Memorial SloanKettering Cancer Centre, Dr. J. A. Montgomery of the Southern Research Institute, Drs. J. W. Daly and R. F. Bruns of the National Institute of Arthritis and Metabolic Diseases, Dr. A. R. P. Paterson of the University of Alberta Cancer Research Unit, the Roswell Park Memorial Institute, and Dr. M. Spedding of Sunderland Polytechnic. ALLY, A. I., and NAKATSU,K. 1976. Adenosine inhibition of isolated rabbit ileum and antagonism by theophylline. J. Pharmacol. Exp. Ther. 199, 208-215. BERNE,R. M., RUBIO,R., and CURNISH,R. R. 1974. Release of adenosine from ischemic brain. Effect of cerebrbtl vascular resistance and incorporation into cerebral adenine nucleotides. Circ. Res. 35, 262-271. BURNSTOCK, G. 11972. Purinergic nerves. Pharmacol. Rev. 24, 509-581. 1978. A basis for distinguishing two types of purinergic receptor. I n Cell membrane receptors for drugs and hormones: A multidisciplinary approach. Edited by L. Bolis and R. W. Straub. Raven Press, New York. pp. f 07-1 118. CIANAHAN,A. S., JOHNS, A., and PATON,D . M. 1977. Presynaptie inhibitory actions of adenine nucleotides and adenosine on laeurotransmission in the rat vas deferens. Neuroscience, 2, 597-602. CURTIS,D. R., PHIILLIS. J. W., and WATKTNS, J. C. 1962. Cholinergic and non-cholincrgic transmission in the mammalian spinal cord. J. Physiol. (London), 158, 296-323. DALY,J. 1977. Cyclic i ~ ~ ~ c l e o t i dine sthe nervous system. Plenum Publishing Corporation, New York, DRUMMOND, 6. I., and YAMAMOTO, M. 1971. Nucleoside cyclic phosphate diesterases. Enzyme, 4, 355-37 1 . FELDERG, W., and MEHB,C. 0. 1948. The stimulating action of phosphate compounds on the perfused superior cervical ganglion of the cat. J. Physiol. (London), 107, 23 0-221. FELDBEWG. W., and SHERWOOD, %. L. 1954. Injections of drugs into the lateral ventricle of the cat. J. Physiol. (London), 123, 148-1 67. GALINBO,A,, KRNJEVI~',K., and SCHWARTZ,S. 1967. Micro-iontophoretic studies on neurones in the cuneate nucleus. J. Physiol. (London), 192, 359-377. GIKSBORG, B. L,, and HIRST, G. D. S. 1972. The effect of adenosine on the release of transmitter from the phrenic nerve of the rat. J. Physiol. (London), 224, 629-645. HARMS,H. H., WARDEH,G., and MULDER,A. H. 1978. Adenosine modulates depolarization-induced release of ,)El-noradrenalinefrom slices of rat brain neocortex. Eur. J . Pharmacol. 49, 305-308. HiirTowr, E., MIYAZAKI, T., and NAKAMURA, M. 1969. Incorporation of adenosine and adenosine triphosphate into rat myocardium. Jpn. Heart J. 10, 47-52. HAULICA,I., ABABEI,L., BRXNISTEANW, D., and TOPOI,JCEANU,F. 1973. Preliminary data on the possible hypnogenic role of adenosine. 3. Neurochem. 21, 10191020. HI~DQVIST, P.. and FREDHOLM, B. W. 1976. Effects s f adenosine on adrenergic neurotransmission; grejunctional inhibition and postjunctional enhancement. NaunynSchmeideberg's Arch. Pharmacol. 293, 217-223.


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Inhibitory actions of adenine nucleotides and adenosine on transmission in rat vas deferens [proceedings].

Receptors for adenosine and adenine nucleotides.

The effects of cyclic adenosine 3',5'-monophosphate and other adenine nucleotides on body temperature.

Cholinergic Mechanisms in the Cerebral Cortex: Beyond Synaptic Transmission.

Effects of adenine nucleotides and phosphate on adenosine triphosphate sulphurylase from Anabaena cylindrica.

Effects of adenine nucleotides on rice-root adenosine triphosphate sulphurylase activity in vitro.

Adenosine and adenine nucleotides stimulation of skin (epidermal) adenylate cyclase.

The effects of adenine nucleotides on cutaneous afferent nerve activity.

Adenosine effects on inhibitory synaptic transmission and excitation-inhibition balance in the rat neocortex.

Effects of adenine nucleotides on oxidation of phenothiazine tranquilizers.

Stable adenine nucleotides inhibit [3H]-noradrenaline release in rabbit brain cortex slices by direct action at presynaptic adenosine A1-receptors.

Effects of allopurinol on hepatic adenosine nucleotides in hemorrhagic shock.

Turnover of adenine nucleotides in cholinergic synaptic vesicles of the Torpedo electric organ.

Effects of cyclobenzaprine on spinal synaptic transmission.

Biphasic effects of polarizing current on adenosine-sensitive generation of cyclic AMP in rat cerebral cortex.

The effects of adenine nucleotides and guanine nucleotides on urate synthesis de novo by isolated chick liver and kidney cells.

Microiontophoretic studies of the effects of cylic nucleotides on excitability of neurones in the rat cerebral cortex.

The action of ether and methoxyflurane on synaptic transmission in isolated preparations of the mammalian cortex.

Comparison of the inhibitory effects on the guinea-pig taenia coli of adenine nucleotides and adenosine in the presence and absence of dipyridamole.

Effects of chronic stress in adolescence on learned fear, anxiety, and synaptic transmission in the rat prelimbic cortex.

Inhibition of sympathetic neurotransmission in canine blood vessels by adenosine and adenine nucleotides.

Catecholamine-sensitive adenylate cyclase of caudate nucleus and cerebral cortex. Effects of guanine nucleotides.

Fluorimetric determination of adenine and adenosine and its nucleotides by high-performance liquid chromatography.

ATP; related nucleotides and adenosine on neurotransmission.