European Journal of Pharmacology 30 (1975) 125-129
© North-Holland Publishing Company Short communication
A POTENT DEPRESSANT ACTION OF ADENINE DERIVATIVES ON CEREBRAL CORTICAL NEURONES John W. PHILLIS, George K. KOSTOPOULOS and James J. LIMACHER Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Canada
Received 7 October 1974, accepted 28 November 1974 J.W. PHILLIS, G.K. KOSTOPOULOS and J.J. LIMACHER,A potent depressant action of adenine derivatives on cerebral cortical neurones, European J. Pharmacol. 30 (1975) 125-129. Adenosine and several adenine nucleotides depress the excitability of cerebral cortical neurones, including identified Betz cells. Cyclic 3 ',5 '-adenosine monophosphate was a less effective depressant than various other adenine nucleotides, including cyclic 2 ',3 '-adenosine monophosphate. Adenine and inosine had only weak depressant activity. An initial excitant action of ATP was observed on several neurones. This was succeeded by a depressant effect when the application of ATP was terminated. Adenine nucleotides
Cerebral cortical neurones
1. Introduction Adenosine derivatives have been proposed as synaptic transmitters at junctions in both the peripheral and central nervous systems. The possibility of peripheral junctional transmission by the release of adenosine triphosphate (5'-ATP) or related purines from 'purinergic' nerves has recently been reviewed by Bumstock (1972). 5'-ATP is released from a variety of tissues and has marked excitatory or inhibitory actions on several kinds of smooth muscle. Holton and Holton (1954) suggested that 5'-ATP might be released as a neurotransmitter from the central (and peripheral) ends of dorsal root fibres. Tests of iontophoretically applied ATP on central neurones, however, often failed to reveal any pronounced action of this compound (Curtis et al., 1961; Krnjevid and Phillis, 1963) although many cells in the cuneate nucleus were strongly excited by 5 '-ATP (Galindo et al., 1967). Since 5'-ADP was ineffective, this excitant action was attributed to the calcium-chelating action of 5 '-ATP. Studies in this laboratory on the actions of the adenine derivatives were initiated to explore the potential link between membrane calcium mobilization
Depression
by the biogenic amines and cyclic 3',5'-adenosine monophosphate (cyclic 3',5 '-AMP) (Phillis, 1974). Cyclic 3',5 '-AMP has proven to be a rather weak depressant of cerebral cortical neurones when applied iontophoretically (Lake et al., 1973), a finding that has been difficult to reconcile with its proposed role as a mediator of the inhibitory actions of noradrenaline (NA). Noradrenaline does, however, elicit accumulations of cyclic AMP in brain cortical slices (Perkins and Moore, 19.73). Sattin and Rail (1970) have demonstrated that adenosine and various adenine nucleotides also stimulate cyclic 3',5 '-AMP formation in cerebral cortical slices. Application of these adenine derivatives directly onto cerebral cortical neurones therefore offered another opportunity for evaluating the effects of agents known to stimalate cyclic AMP formation on the activity of cerebral cortical neurones.
2. Materials and methods 15 male Sprague-Dawley rats (250-350 g) were used in these experiments. Anaesthesia was induced by halothane inhalation and maintained, after inser-
126
J.W. Phillis e t al., A d e n i n e derivatives a n d cerebral cortical n e u r o n e s
tion of a tracheal cannula, with a mixture of methoxyflurane, nitrous oxide (80%) and oxygen (20%). The animals were placed in a stereotaxic frame and a rectal probe was inserted which maintained body temperature at 37-38°C via an electric heating pad. After reflection of the overlying skin, small holes were drilled through the bones overlying the somatosensory cortex and ipsilateral cerebellar vermis near the midline. A bipolar coaxial stimulating electrode was inserted through the posterior opening and advanced until its tip was in the ipsilateral pyramidal tract. The anterior hole provided access to the cortex for the recording microelectrode. The exposed skin, muscle, dura and cortex were covered with a thin layer of 4% agar in Ringer solution to prevent drying. The recording of neuronal activity and iontophoresis of drug solutions were accomplished with sevenbarrelled micropipettes with overall tip diameters of 6 - 1 0 ~m. The central recording barrel was filled with 2 M NaC1 and the remaining barrels were filled by centrifugation with various combinations of the following solutions: NA bitartrate (0.2 M, pH 5.0), L-glutamic acid, sodium salt (0.2 M), NaC1 (2 M), adenine (saturated solution, pH 4.5, Calbiochem), adenine hydrochloride (0.1 M, pH 4, Signa), adenosine (saturated solution, pH 4, Sigma), adenosine hemisulphate (0.2 M, pH 4, Sigma), adenosine 5'-sulphate (0.05 M, pH 4.5, P-L Biochemicals), 5'-adenosine monophosphate (5'-AMP) (0.2 M, pH 5, P-L Biochemicals), 5'-adenosine diphosphate (5'-ADP) (0.2 M, pH 4.5, P-L Biochemicals), 5'-adenosine triphosphate (5 '-ATP) (0.2 M, pH 4.5, Sigma), cyclic 3 ',5 'adenosine monophosphate (cyclic 3',5 '-AMP) (0.2 M, pH 6, Calbiochem and P-L Biochemicals), 3 '(2')-adenosine monophosphate [3'(2')-AMP] (0.2 M, pH 5, Calbiochem), 3 '-adenosine monophosphate (3 '-AMP) (0.2 M, pH 5, Sigma), cyclic 2',3 '-adenosine monophosphate (cyclic 2',3 '-AMP) (0.2 M, pH 5, Sigma), inosine (0.1 M, pH 5.5, Sigma), acetylcholine chloride (ACh) (0.2 M), 7-aminobutyric acid (GABA) (0.2 M, pH 5). Since adenosine and adenine are relatively insoluble at room temperature, the solutions were warmed and loaded into the micropipettes immediately prior to centrifugation. Substances were applied by passing currents of the appropriate polarity through the desired barrel of the micropipette. Adenine and adenosine were applied by anodal current; the various nucleotides, which had been prepared as
the sodium salts, were passed by cathod',d current. The maximum current employed in this survey was 80 hA. Large currents tend to cause complications when the neurones respond electrically rather than chemically and were therefore avoided. Although it is possible that the use of larger currents would have resulted in higher proportions of responsive neurones, it is likely that spurious effects would also have been included in the analysis. After initial studies revealed that the adenine derivatives had a pronounced depressant action on deep, spontaneously firing cortical neurones, the majority of tests were conducted on such neurones. Neurones were identified as Betz cells if they responded to an antidromic volley in the ipsilateral pyramidal tract with a constant latency spike which would follow stimulation frequencies of 100 per sec or higher. Non-spontaneously active neurones were excited by a series of uniform pulses of L-glutamate. A response was considered to be genuine if a neurone responded consistently to a substance, and if the response was not mimicked by applications of equivalent amounts of current of the same polarity passed through another barrel of the micropipette.
3. Results
The results are summarized in table 1, and illustrate the remarkable consistency with which the adenine nucleotides depressed cerebral cortical neurones. The relative potency of individual nucleotides varied slightly from electrode to electrode, but when all the results were examined it became evident that, with the exception of cyclic 3 ',5 '-AMP, there was little difference in the depressant potency of the various adenine nucleotides. In partial confirmation of earlier observations, cyclic 3 ',5 '-AMP was found to have little or no depressant action on non-spontaneously active, glutamate-excited cells or on non-identified spontaneously active cortical neurones. Cyclic 3',5 '-AMP did depress 75% of the identified Betz cells on which it was tested; however even on these its depressant potency was considerably lower than that of the other adenine nucteotides. Adenine had a weak depressant action on a few of the neurones tested. Adenosine, with one exception, depressed all the neurones tested but was invariably
127
J. W. Phillis et al., Adenine derivatives and cerebral cortical neurones
Table 1 Depressant effects of adenine derivatives on cerebral cortical neurones. Substance
Glutamatedriven
Adenine (2) Adenosine (3) Adenosine 5'-sulphate 5°-AMP 5'-ADP 5'-ATP 3' (2')-AMP
Cell type
2/3 (1) 14/14 27/27 21/21 17/17 1/1 0/5
3'-AMP
5'-cAMP 2', T-cAMP Inosine 3',
Spontaneously active (Not identified)
Bctz cell
Depressant potency
4/22 46/47 0/17 133/133 50/50 80/80 1/1 16/16 7/46 6/6 0/1
2/12 45/46 0/39 133/133 36/36 35/35 25/25 48/48 24/32 20/20 7/23
-0 -
-
(1) The ratios indicate the number of cells that were depressed over the number of cells tested. The maximum current employed was 80nA. (2) Pooled data for adenine and adenine hydrocldoride. (3) Pooled data for adenosine and adenosine hemisulphate.
A
ATP
t__oo
ADP
1.0
AMP
somewhat less p o t e n t than the adenine nucleotides. This could have been a result o f its relative insolubility but adenosine hemisulphate, which is quite soluble, had a comparable depressant action. The depressant potency o f the adenine derivatives was tested on several forms o f excitation o f cortical neurones, including glutamate excitation, ACh excitation as well as spontaneous firing. Examples o f some o f these results are illustrated b y figs. 1 and 2. The adenine derivatives appeared to have their most pronounced action on rapidly firing spontaneously active neurones. A comparison o f the actions o f three nucleotides and NA is presented in fig. 1A. 5'-ATP, 5'-ADP and 5 '-AMP (all applied b y currents o f 10 hA) had pronounced depressant actions on this neurone. The typical time course o f nucleotide action is well displayed in this trace; the depression had a rapid
NA
I_o
20
4O
0 t
j 1 mln
e
ATP 40
A M P 40
ADP 40
N A 40
60r
0
.
t
I
1rain
Fig. 1. Depression of the excitability of spontaneously firing (A) and glutamate-driven (B) neurones in the somatosensory cortex by adenine nucleotides and noradrenaline (NA). Ratemeter records of neuronal firing with number of action potentials per second on the ordinate. Bars above and below records indicate periods of drug application. Application currents shown in nA. Glutamate application currents 35 nA.
128
J.W. Phillis et al., Adenine derivatives and cerebral cortical neurones
A 10
ATP 20 40
B 60 A T P 10
50
A T P 20
ATP 40
ATP 80
S0
oL2
0
1 rain
Fig. 2. (A) Excitation and (B) depression of two cerebral cortical neurones by incrementing amounts of 5 '-ATP. onset and a duration which was to some extent dependen t on the magnitude of the initial depression. NA, in contrast, was a less potent depressant and this recording indicates the long duration of the inhibition of firing frequently observed when this amine was tested on deep, spontaneously active cortical neurones and Betz cells. The adenine nucleotides and adenosine were usually less effective on L-glutamate-driven neurones. Currents of 40 nA were required for 5'-ATP, 5 '-ADP and 5'-AMP to depress the glutamate-excited neurones in fig. lB. Typically the depressant action of the adenine derivatives outlasted the period o f application and recovery of excitability is rapid after a NA application. The depressant potency of 5'-AMP was compared with that of the potent inhibitory amino acid, GABA. When tested on 21 unidentified spontaneously active neurones, applications of 5'-AMP by currents of 15 nA were found to cause similar depressions to those evoked by currents of l0 nA through the GABA barrel. Several examples of an initial 5 '-ATP induced excitation of cerebral cortical neurones were observed. One of these is presented in fig. 2A. This neurone was exceptionally sensitive to the excitant action of 5'ATP, responding to application currents as low as 10 nA. More pronounced excitations were observed with progressively larger currents. However, even with this cell is was possible to demonstrate that the period of excitation was succeeded by a phase of depression when the neurone was retested using pulses of Lglutamate. 5'-ATP excitation was most clearly dem-
onstrable on quiescent or slowly Firing neurones and was not observed when neurones were already firing rapidly. Examples of the depressant effects of a series of incrementing 5'-ATP applications on spontaneously firing neurones are shown in fig. 2B. 4. Discussion The present finding demonstrates that a series of adenine derivative has a potent depressant action on cerebral cortical neurones. In the tight of biochemical studies on the stimulation of cyclic 3 ',5 '-AMP formation by adenine derivatives, it is conceivable that the depressant action of these compounds on nerve cells may also involve stimulation of cyclic 3 ',5 '-AMP formation. Sattin and Rall (1970) have shown that there is tittle difference between the cyclic 3',5'-AMP formation stimulating properties of a range of adenine derivatives including adenosine, 5'-AMP, 5'oADP, 5 '-ATP, 2 '-AMP and 3 '-AMP, and that inosine and adenosine 5 '-sulphate do not stimulate cyclic 3 ',5 'AMP formation. These activities are reflected in the present results in that adenosine, the 5'-nucleotides, 3 '-AMP, 3 '(2')-AMP and cyclic 2 ',3 '-AMP all had pronounced depressant actions on cortical neurones whereas adenine and inosine were only weakly active on those neurones that they depressed. Adenosine 5 '-sulphate was inactive. Cyclic 3 ',5 '-AMP itself was less active than the other adenine nucleotides as a neuronal depressant. It is also less effective than 5'ATP, 5'-ADP and 5'-AMP on a variety of different peripheral structures (Bumstock, 1972).
J. w. Phillis et al., A d e n i n e derivatives and cerebral cortical neurones
The actual mechanism by which the adenine derivatives :depress neuronal excitability remains unelucidated. Studies on the taenia coil o f the intestine indicate that their action is accompanied b y a hyperpolarization which may result from an increase in membrane potassium conductance (Burnstock, 1972). Whether this is their action on cerebral cortical neurones remains to be established. Recently pubfished data showing that adenosine stimulation of cyclic 3 "5 '-AMP formation in cerebral cortical slices is potentiated b y agents which inhibit the uptake o f adenosine, suggests that the adenine derivatives may act on an extracellularly located receptor (Huang and Daly, 1974). An alternative possibility that adenine derivatives depress neuronal excitability by stimulating the activity of Na/K-activated ATPase must also be considered. Kuroda and Mcllwain (1974) have shown that incubation with adenosine causes an elevation in brain 5'-ATP and the increased availability of this compound might enhance the activity of the membrane sodium pump. However, experiments on gut and cardiac muscle indicating that ouabain, a selective inhibitor o f this enzyme, does not affect the relaxation induced by 5'-ATP provide strong evidence against this hypothesis, at least for peripheral tissues (Burnstock, 1972). Our observation o f a potent depressant action of the adenine nucleotides on cerebral cortical neurones raises the question of the existence of purinergic nerves in the central nervous system. A calcium-dependent release of adenosine and adenine 5 '-nucleotides from synaptosome beds prepared from cerebral cortical tissue by electrical pulses or the addition o f potassium has been demonstrated (Kuroda and Mcllwain, 1974). Adenine nucleotides are also known to be released from peripheral adrenergic and cholinergic nerve terminals and might reach effective concentrations at such synaptic sites. If an analogous situation occurs at noradrenergic and cholinergic synapses in the central nervous system, it is possible that the postsynaptic response to depolarization of an aminergic nerve ter-
129
minal would depend on the composite actions o f the various agents released.
Acknowledgements It is a pleasure to acknowledge our helpful discussions with Dr. P.V. Sulakhe during the course of these experiments. The support of the Canadian Medical Research Council and the University of Saskatchewan is gratefully acknowledged. J.J.L. is a postdoctoral fellow of the C.M.R.C.
References Burnstock, G., 1972, Purinergic nerves, Pharmacol. Rev. 24, 509. Curtis, D.R., J.W. Phillis and J.C. Watkins, 1961, Cholinergic and non-cholinergic transmission in the mammalian spinal cord, J. Physiol. (London) 158, 296. Galindo, A., K. Krnjevi6 and S. Schwartz, 1967, Micro-iontophoretic studies on neurones in the cuneate nucleus, J. Physiol. (London) 192, 359. Holton, F.A. ~md P. Holton, 1954, The capillary dilator substances in dry powders of spinal roots; a possible role of adenosine triphosphate in chemical transmission from nerve endings, J. Physiol. (London) 126, 124. Huang, M. and J.W. Daly, 1974, Adenosine-elicited accumulation of cyclic AMP in brain slices: Potentiation by agents which inhibit uptake of adenosine, Life Sci. 14,489. Krnjevi6, K. and J.W. Phillis, 1963, Actions of certain amines on cerebral cortical neurones, Brit. J. Pharmacol. 20,471. Kuroda, Y. and H. Mcllwain, 1974, Uptake and release of [14C]-adenine derivatives at beds of mammalian cortical synaptosomes in a superfusion system, J. Neurochem. 22, 691. Lake, N., L.M. Jordan and J.W. Phillis, 1973, Evidence against cyclic adenosine 3 ',5 '-monophosphate (AMP) mediation of noradrenaline depression of cerebral cortical neurones, Brain Res. 60, 411. Perkins, J.P. and M.M. Moore, 1973, Characterization of the adrenergic receptors mediating a rise in cyclic 3 ',5 "adenosine monophosphate in rat cerebral cortex, J. Pharmacol. Exptl. Therap. 185,371. Phillis, J.W., 1974, The role of calcium in the central effects of biogenic amines, Life Sci. 14, 1189. Sattin, A. and T.W. Rail, 1970, The effect of adenosine and adenine nucleotides on the cyclic adenosine 3',5'-phosphate content of guinea pig cerebral cortex slices, Mol. Pharmacol. 6, 13.
© North-Holland Publishing Company Short communication
A POTENT DEPRESSANT ACTION OF ADENINE DERIVATIVES ON CEREBRAL CORTICAL NEURONES John W. PHILLIS, George K. KOSTOPOULOS and James J. LIMACHER Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Canada
Received 7 October 1974, accepted 28 November 1974 J.W. PHILLIS, G.K. KOSTOPOULOS and J.J. LIMACHER,A potent depressant action of adenine derivatives on cerebral cortical neurones, European J. Pharmacol. 30 (1975) 125-129. Adenosine and several adenine nucleotides depress the excitability of cerebral cortical neurones, including identified Betz cells. Cyclic 3 ',5 '-adenosine monophosphate was a less effective depressant than various other adenine nucleotides, including cyclic 2 ',3 '-adenosine monophosphate. Adenine and inosine had only weak depressant activity. An initial excitant action of ATP was observed on several neurones. This was succeeded by a depressant effect when the application of ATP was terminated. Adenine nucleotides
Cerebral cortical neurones
1. Introduction Adenosine derivatives have been proposed as synaptic transmitters at junctions in both the peripheral and central nervous systems. The possibility of peripheral junctional transmission by the release of adenosine triphosphate (5'-ATP) or related purines from 'purinergic' nerves has recently been reviewed by Bumstock (1972). 5'-ATP is released from a variety of tissues and has marked excitatory or inhibitory actions on several kinds of smooth muscle. Holton and Holton (1954) suggested that 5'-ATP might be released as a neurotransmitter from the central (and peripheral) ends of dorsal root fibres. Tests of iontophoretically applied ATP on central neurones, however, often failed to reveal any pronounced action of this compound (Curtis et al., 1961; Krnjevid and Phillis, 1963) although many cells in the cuneate nucleus were strongly excited by 5 '-ATP (Galindo et al., 1967). Since 5'-ADP was ineffective, this excitant action was attributed to the calcium-chelating action of 5 '-ATP. Studies in this laboratory on the actions of the adenine derivatives were initiated to explore the potential link between membrane calcium mobilization
Depression
by the biogenic amines and cyclic 3',5'-adenosine monophosphate (cyclic 3',5 '-AMP) (Phillis, 1974). Cyclic 3',5 '-AMP has proven to be a rather weak depressant of cerebral cortical neurones when applied iontophoretically (Lake et al., 1973), a finding that has been difficult to reconcile with its proposed role as a mediator of the inhibitory actions of noradrenaline (NA). Noradrenaline does, however, elicit accumulations of cyclic AMP in brain cortical slices (Perkins and Moore, 19.73). Sattin and Rail (1970) have demonstrated that adenosine and various adenine nucleotides also stimulate cyclic 3',5 '-AMP formation in cerebral cortical slices. Application of these adenine derivatives directly onto cerebral cortical neurones therefore offered another opportunity for evaluating the effects of agents known to stimalate cyclic AMP formation on the activity of cerebral cortical neurones.
2. Materials and methods 15 male Sprague-Dawley rats (250-350 g) were used in these experiments. Anaesthesia was induced by halothane inhalation and maintained, after inser-
126
J.W. Phillis e t al., A d e n i n e derivatives a n d cerebral cortical n e u r o n e s
tion of a tracheal cannula, with a mixture of methoxyflurane, nitrous oxide (80%) and oxygen (20%). The animals were placed in a stereotaxic frame and a rectal probe was inserted which maintained body temperature at 37-38°C via an electric heating pad. After reflection of the overlying skin, small holes were drilled through the bones overlying the somatosensory cortex and ipsilateral cerebellar vermis near the midline. A bipolar coaxial stimulating electrode was inserted through the posterior opening and advanced until its tip was in the ipsilateral pyramidal tract. The anterior hole provided access to the cortex for the recording microelectrode. The exposed skin, muscle, dura and cortex were covered with a thin layer of 4% agar in Ringer solution to prevent drying. The recording of neuronal activity and iontophoresis of drug solutions were accomplished with sevenbarrelled micropipettes with overall tip diameters of 6 - 1 0 ~m. The central recording barrel was filled with 2 M NaC1 and the remaining barrels were filled by centrifugation with various combinations of the following solutions: NA bitartrate (0.2 M, pH 5.0), L-glutamic acid, sodium salt (0.2 M), NaC1 (2 M), adenine (saturated solution, pH 4.5, Calbiochem), adenine hydrochloride (0.1 M, pH 4, Signa), adenosine (saturated solution, pH 4, Sigma), adenosine hemisulphate (0.2 M, pH 4, Sigma), adenosine 5'-sulphate (0.05 M, pH 4.5, P-L Biochemicals), 5'-adenosine monophosphate (5'-AMP) (0.2 M, pH 5, P-L Biochemicals), 5'-adenosine diphosphate (5'-ADP) (0.2 M, pH 4.5, P-L Biochemicals), 5'-adenosine triphosphate (5 '-ATP) (0.2 M, pH 4.5, Sigma), cyclic 3 ',5 'adenosine monophosphate (cyclic 3',5 '-AMP) (0.2 M, pH 6, Calbiochem and P-L Biochemicals), 3 '(2')-adenosine monophosphate [3'(2')-AMP] (0.2 M, pH 5, Calbiochem), 3 '-adenosine monophosphate (3 '-AMP) (0.2 M, pH 5, Sigma), cyclic 2',3 '-adenosine monophosphate (cyclic 2',3 '-AMP) (0.2 M, pH 5, Sigma), inosine (0.1 M, pH 5.5, Sigma), acetylcholine chloride (ACh) (0.2 M), 7-aminobutyric acid (GABA) (0.2 M, pH 5). Since adenosine and adenine are relatively insoluble at room temperature, the solutions were warmed and loaded into the micropipettes immediately prior to centrifugation. Substances were applied by passing currents of the appropriate polarity through the desired barrel of the micropipette. Adenine and adenosine were applied by anodal current; the various nucleotides, which had been prepared as
the sodium salts, were passed by cathod',d current. The maximum current employed in this survey was 80 hA. Large currents tend to cause complications when the neurones respond electrically rather than chemically and were therefore avoided. Although it is possible that the use of larger currents would have resulted in higher proportions of responsive neurones, it is likely that spurious effects would also have been included in the analysis. After initial studies revealed that the adenine derivatives had a pronounced depressant action on deep, spontaneously firing cortical neurones, the majority of tests were conducted on such neurones. Neurones were identified as Betz cells if they responded to an antidromic volley in the ipsilateral pyramidal tract with a constant latency spike which would follow stimulation frequencies of 100 per sec or higher. Non-spontaneously active neurones were excited by a series of uniform pulses of L-glutamate. A response was considered to be genuine if a neurone responded consistently to a substance, and if the response was not mimicked by applications of equivalent amounts of current of the same polarity passed through another barrel of the micropipette.
3. Results
The results are summarized in table 1, and illustrate the remarkable consistency with which the adenine nucleotides depressed cerebral cortical neurones. The relative potency of individual nucleotides varied slightly from electrode to electrode, but when all the results were examined it became evident that, with the exception of cyclic 3 ',5 '-AMP, there was little difference in the depressant potency of the various adenine nucleotides. In partial confirmation of earlier observations, cyclic 3 ',5 '-AMP was found to have little or no depressant action on non-spontaneously active, glutamate-excited cells or on non-identified spontaneously active cortical neurones. Cyclic 3',5 '-AMP did depress 75% of the identified Betz cells on which it was tested; however even on these its depressant potency was considerably lower than that of the other adenine nucteotides. Adenine had a weak depressant action on a few of the neurones tested. Adenosine, with one exception, depressed all the neurones tested but was invariably
127
J. W. Phillis et al., Adenine derivatives and cerebral cortical neurones
Table 1 Depressant effects of adenine derivatives on cerebral cortical neurones. Substance
Glutamatedriven
Adenine (2) Adenosine (3) Adenosine 5'-sulphate 5°-AMP 5'-ADP 5'-ATP 3' (2')-AMP
Cell type
2/3 (1) 14/14 27/27 21/21 17/17 1/1 0/5
3'-AMP
5'-cAMP 2', T-cAMP Inosine 3',
Spontaneously active (Not identified)
Bctz cell
Depressant potency
4/22 46/47 0/17 133/133 50/50 80/80 1/1 16/16 7/46 6/6 0/1
2/12 45/46 0/39 133/133 36/36 35/35 25/25 48/48 24/32 20/20 7/23
-0 -
-
(1) The ratios indicate the number of cells that were depressed over the number of cells tested. The maximum current employed was 80nA. (2) Pooled data for adenine and adenine hydrocldoride. (3) Pooled data for adenosine and adenosine hemisulphate.
A
ATP
t__oo
ADP
1.0
AMP
somewhat less p o t e n t than the adenine nucleotides. This could have been a result o f its relative insolubility but adenosine hemisulphate, which is quite soluble, had a comparable depressant action. The depressant potency o f the adenine derivatives was tested on several forms o f excitation o f cortical neurones, including glutamate excitation, ACh excitation as well as spontaneous firing. Examples o f some o f these results are illustrated b y figs. 1 and 2. The adenine derivatives appeared to have their most pronounced action on rapidly firing spontaneously active neurones. A comparison o f the actions o f three nucleotides and NA is presented in fig. 1A. 5'-ATP, 5'-ADP and 5 '-AMP (all applied b y currents o f 10 hA) had pronounced depressant actions on this neurone. The typical time course o f nucleotide action is well displayed in this trace; the depression had a rapid
NA
I_o
20
4O
0 t
j 1 mln
e
ATP 40
A M P 40
ADP 40
N A 40
60r
0
.
t
I
1rain
Fig. 1. Depression of the excitability of spontaneously firing (A) and glutamate-driven (B) neurones in the somatosensory cortex by adenine nucleotides and noradrenaline (NA). Ratemeter records of neuronal firing with number of action potentials per second on the ordinate. Bars above and below records indicate periods of drug application. Application currents shown in nA. Glutamate application currents 35 nA.
128
J.W. Phillis et al., Adenine derivatives and cerebral cortical neurones
A 10
ATP 20 40
B 60 A T P 10
50
A T P 20
ATP 40
ATP 80
S0
oL2
0
1 rain
Fig. 2. (A) Excitation and (B) depression of two cerebral cortical neurones by incrementing amounts of 5 '-ATP. onset and a duration which was to some extent dependen t on the magnitude of the initial depression. NA, in contrast, was a less potent depressant and this recording indicates the long duration of the inhibition of firing frequently observed when this amine was tested on deep, spontaneously active cortical neurones and Betz cells. The adenine nucleotides and adenosine were usually less effective on L-glutamate-driven neurones. Currents of 40 nA were required for 5'-ATP, 5 '-ADP and 5'-AMP to depress the glutamate-excited neurones in fig. lB. Typically the depressant action of the adenine derivatives outlasted the period o f application and recovery of excitability is rapid after a NA application. The depressant potency of 5'-AMP was compared with that of the potent inhibitory amino acid, GABA. When tested on 21 unidentified spontaneously active neurones, applications of 5'-AMP by currents of 15 nA were found to cause similar depressions to those evoked by currents of l0 nA through the GABA barrel. Several examples of an initial 5 '-ATP induced excitation of cerebral cortical neurones were observed. One of these is presented in fig. 2A. This neurone was exceptionally sensitive to the excitant action of 5'ATP, responding to application currents as low as 10 nA. More pronounced excitations were observed with progressively larger currents. However, even with this cell is was possible to demonstrate that the period of excitation was succeeded by a phase of depression when the neurone was retested using pulses of Lglutamate. 5'-ATP excitation was most clearly dem-
onstrable on quiescent or slowly Firing neurones and was not observed when neurones were already firing rapidly. Examples of the depressant effects of a series of incrementing 5'-ATP applications on spontaneously firing neurones are shown in fig. 2B. 4. Discussion The present finding demonstrates that a series of adenine derivative has a potent depressant action on cerebral cortical neurones. In the tight of biochemical studies on the stimulation of cyclic 3 ',5 '-AMP formation by adenine derivatives, it is conceivable that the depressant action of these compounds on nerve cells may also involve stimulation of cyclic 3 ',5 '-AMP formation. Sattin and Rall (1970) have shown that there is tittle difference between the cyclic 3',5'-AMP formation stimulating properties of a range of adenine derivatives including adenosine, 5'-AMP, 5'oADP, 5 '-ATP, 2 '-AMP and 3 '-AMP, and that inosine and adenosine 5 '-sulphate do not stimulate cyclic 3 ',5 'AMP formation. These activities are reflected in the present results in that adenosine, the 5'-nucleotides, 3 '-AMP, 3 '(2')-AMP and cyclic 2 ',3 '-AMP all had pronounced depressant actions on cortical neurones whereas adenine and inosine were only weakly active on those neurones that they depressed. Adenosine 5 '-sulphate was inactive. Cyclic 3 ',5 '-AMP itself was less active than the other adenine nucleotides as a neuronal depressant. It is also less effective than 5'ATP, 5'-ADP and 5'-AMP on a variety of different peripheral structures (Bumstock, 1972).
J. w. Phillis et al., A d e n i n e derivatives and cerebral cortical neurones
The actual mechanism by which the adenine derivatives :depress neuronal excitability remains unelucidated. Studies on the taenia coil o f the intestine indicate that their action is accompanied b y a hyperpolarization which may result from an increase in membrane potassium conductance (Burnstock, 1972). Whether this is their action on cerebral cortical neurones remains to be established. Recently pubfished data showing that adenosine stimulation of cyclic 3 "5 '-AMP formation in cerebral cortical slices is potentiated b y agents which inhibit the uptake o f adenosine, suggests that the adenine derivatives may act on an extracellularly located receptor (Huang and Daly, 1974). An alternative possibility that adenine derivatives depress neuronal excitability by stimulating the activity of Na/K-activated ATPase must also be considered. Kuroda and Mcllwain (1974) have shown that incubation with adenosine causes an elevation in brain 5'-ATP and the increased availability of this compound might enhance the activity of the membrane sodium pump. However, experiments on gut and cardiac muscle indicating that ouabain, a selective inhibitor o f this enzyme, does not affect the relaxation induced by 5'-ATP provide strong evidence against this hypothesis, at least for peripheral tissues (Burnstock, 1972). Our observation o f a potent depressant action of the adenine nucleotides on cerebral cortical neurones raises the question of the existence of purinergic nerves in the central nervous system. A calcium-dependent release of adenosine and adenine 5 '-nucleotides from synaptosome beds prepared from cerebral cortical tissue by electrical pulses or the addition o f potassium has been demonstrated (Kuroda and Mcllwain, 1974). Adenine nucleotides are also known to be released from peripheral adrenergic and cholinergic nerve terminals and might reach effective concentrations at such synaptic sites. If an analogous situation occurs at noradrenergic and cholinergic synapses in the central nervous system, it is possible that the postsynaptic response to depolarization of an aminergic nerve ter-
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minal would depend on the composite actions o f the various agents released.
Acknowledgements It is a pleasure to acknowledge our helpful discussions with Dr. P.V. Sulakhe during the course of these experiments. The support of the Canadian Medical Research Council and the University of Saskatchewan is gratefully acknowledged. J.J.L. is a postdoctoral fellow of the C.M.R.C.
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