Neuropharmacology,
1978, 17, 13-19
Pergamon
Press.
Prmted
I” Great
Britam
A COMPARISON OF THE EFFECTS OF GABA, 3-AMINOPROPANESULPHONIC ACID AND IMIDAZOLEACETIC ACID ON THE FROG SPINAL CORD A. NISTRI* and R. CORRADETTI Institute of Pharmacology, University of Florence, Florence, Italy (Accepted 19 Maq’ 1977) Summary-The effects of GABA, 3-aminopropanesulphonic acid (3APS) or imidazoleacetic acid (IMA) on the isolated spinal cord of the frog were studied by means of extracellular recording techniques. In the presence of tetrodotoxin (6.2~~), GABA, 3APS or IMA produced dose-dependent dorsal root depolarizations. From the analysis of the doseeresponse relationships the order of depolarizing potency for these substances was 3APS 9 GABA > IMA. The effects of IMA were antagonized by either strychnine or picrotoxin whereas those of GABA or 3APS were only antagonized by picrotoxin. None of the observed antagonisms appeared competitive. The action of GABA was dependent on the Na+ and Cl- content of the bathing solution while that of 3APS and IMA was dependent only on external Cl-. By stepwise increases in the extracellular K+ content it was found that the polarity of 3APS or IMA responses could be inverted at a d.c. level where GABA still produced depolarizations. It is suggested that the mode of action of 3APS and IMA on the frog spinal cord is somewhat different from that of GABA; therefore 3APS and IMA could not be regarded as specific GABA receptor agonists on this preparation.
The availability of potent agonists at the 1;-aminobutyric acid (GABA) receptor would be of considerable help in the study of GABA-operated synapses in the central nervous system; furthermore such agonists might represent prototypes of drugs capable of ameliorating neurological diseases such as Huntington’s chorea where a deficit of GABA and its synthetic enzyme has been found in the basal ganglia (Perry, Hansen and Kloster, 1973; Bird and Iversen, 1974). 3-Aminopropanesulphonic acid (3APS; homotaurine) has been reported to have GABA-mimetic activity more potent than GABA on the cerebral cortex (Krnjevic’ and Puil, 1976), spinal cord (Curtis, Phillis and Watkins, 1961; Eccles, Schmidt and Willis, 1963; Schmidt, 1963) and sympathetic ganglion (Bowery and Brown, 1974; Bowery, Brown, Collins, Galvan, Marsh and Yamini, 1976). Enna and Snyder (1975) observed that in rat brain homogenates 3APS competes with GABA for binding sites; these findings would thus suggest that 3APS is an agonist at GABA receptors. Imidazoleacetic acid (IMA) also produces some GABA-like effects on the cerebral cortex (Krnjevic’ and Phillis, 1963; Godfraind, Krnjevic’, Maretic’ and Pumain, 1973; Enna and Snyder, 1975), spinal cord (Curtis, Duggan, Felix and Johnston, 1971; Barker, Nicoll and Padjen, 1975a) and sympathetic ganglion (Bowery and Jones, 1976). In these studies (with the exception of that by Barker et al., 1975a) IMA was somewhat less potent than GABA. However, despite
the above evidence that 3APS and IMA might interact with GABA receptors, there has been no quantitative study of the effects of 3APS and IMA on a central nervous system preparation under in vitro controlled conditions. We therefore decided to study dose-response relationships for dorsal root responses of the isolated frog spinal cord to bath-application of GABA, 3APS or IMA. In this preparation GABA is supposed to play a transmitter role in the presynaptic inhibition of primary afferent terminals (Barker, Nicoll and Padjen, 1975b). Some of our results have been presented in a preliminary form (Nistri and Corradetti, 1976). METHODS All experiments were carried out on frogs (Rana rsculenta). The procedure for setting up and recording from the isolated frog spinal cord has been recently described in detail (Constanti and Nistri, 1976; Nistri and Constanti, 1976). In brief, the hemisected spinal cord was bathed in a Ringer solution (NaCl, 109 mM; KCl, 4m~; CaCl, (titrated), 1.5 mM; NaHCo,, 1Om~; glucose, 4 mu, pH 7.2-7.3 with O&O,95/5%) maintained at 12°C. Electrical stimuli (0.25 Hz; 0.05 msec; supramaximal voltage) applied to the VIII or XIth ventral root produced dorsal root potentials (V-DRPs) in the corresponding dorsal root. These potentials and changes in polarization level of the dorsal root were recorded differentially using two extracellular Ag/AgCl electrodes (one placed on a dorsal root and the other placed in the bathing medium) and monitored on a conventional oscilloscope as well as on a storage oscilloscope and a chartrecorder. The bath was grounded via a third Ag/AgCl electrode.
* Present address: Department of Research in Anaesthesia, McGill University, Montreal, PQ, Canada H3G lY6. Key words: GABA, 3-aminopropanesulphonic acid, imidazoleacetic
acid, spinal
cord, receptors. 13
14
A. NISTRIand R. CORKADE~TI
_-b-r----
-
a
b
-
C
_--
--
J
0.5 mV
100 msec Fig. 1. Effects of GABA or 3APS on V-DRPs. a: control V-DRP; b: 45 set after bath application of GABA (2 mM); c: after 2 min wash; d: control V-DRP; e: 45 set after bath application of 3APS (0.2 mM); f: after 3 min wash. For abbreviations see text. Negativity upwards. All these responses were recorded from the same cord.
RESULTS Bath-application of GABA (2m~) or 3APS (0.2 mM) rapidly depressed the V-DRPs, this effect being quickly reversible on washing (Fig. I). Similar effects were also produced by IMA (4 ITIM; not shown). The depression of V-DRPs after the administration of GABA, 3APS or IMA was associated with a reversible dorsal root depolarization. In order to block propagated interneuronal activity, tetrodotoxin (TTX; 6.2 PM) was added to the bathing solution. This toxin rapidly ( IMA as judged on the basis of their ED,, values. The slopes for 3APS and GABA were < 1 whereas that for IMA was 1.72. Picrotoxin is a seemingly non-competitive antagonist of GABA in the frog spinal cord (Constanti and
Nistri, 1976). In the present study a low dose (5 PM) of this convulsant depressed the apparent maximum of the GABA dose-depolarization curve without a displacement to the right along the abscissa (see Fig. 4); however, higher concentrations of picrotoxin (3 10,~~) shifted the GABA curve to the right in an apparently non-parallel fashion as previously reported (Constanti and Nistri, 1976). Similar findings were obtained when 3APS responses were measured in the presence of 5 PM picrotoxin (Fig. 4); it should be noted that in the presence of picrotoxin the fading of GABA as well as 3APS responses was increased (cf. top tracings of Fig. 4). Recovery from the action of picrotoxin was observed after 2-3 hr washing. Responses to IMA were also antagonized by picrotoxin (not shown). In order to obtain some information about the activity of picrotoxin as a blocker of aminoacid effects, the picrotoxin concentrations which halved the maximal response to each agonist were calculated (Table 1). It appears that picrotoxin was a more active inhibitor of the maximal effects of 3APS and IMA than of those of GABA. Strychnine (100 PM) did not affect GABA or 3APS responses but at a relatively low dose (10 PM) antagonized IMA responses as shown,by the non-parallel shift to the right of the IMA dose-response curve (Fig. 3B). Recovery Table
GABA 3APS IMA
I. Estimated kinetic values for GABA, IMA responses of frog dorsal root
3APS
and
Picrotoxin required to halve maximal response
ED,, (m@
Slope (ii)
(PM)
1.1 0.1 2.3
0.73 0.83 1.72
16.0 8.2 6.0
ED,, values were obtained as described by Bowery and Brown (I 974); slope values refer to log-log transformations of the dose-response curves for GABA, 3APS and IMA (see Werman, 1969).
A. NISTRI and R. CORRADETTI
16
-h -c n
n
l-
0.5
05
7
10
20 log
0 0.2
1
05 log
[GABA]
100
[3APS] (pm)
10
5
2
50
(mM)
Fig. 4. Effect of picrotoxin on GABA or 3APS log dose-response curves obtained from dorsal roots of tetrodotoxin-treated spinal cords. On the left: control GABA curve (0) and GABA curve obtained in the presence of 5 PM picrotoxin (0); the tracings above these curves show on the left a control GABA (2m~) response and on the right the response to the same dose in the presence of 10 PM picrotoxin. On the right: control 3APS curve (0) and 3APS curve obtained in the presence of 5 PM picrotoxin (0); the tracings above these curves show on the left the response to 50~~ 3APS and on the right the response to the same dose in the presence of 10~~ picrotoxin. Calibration marks (applicable to both GABA and 3APS responses) were: I mV; 1 min.
mV
2 mV
0.2
0.5 log
1 [GABA]
2
5 (mM)
10
0.02
0.05 log
0.1
0.2
[3 APS]
0.5
1.0
(mM)
Fig. 5. Interactions between GABA, 3APS and IMA at dorsal root level. The two graphs show some “combination” curves obtained by adding a fixed dose of one agonist to increasing doses of another. A: (0) control GABA curve; (0) “combination” curve where 0.2 mM 3APS was added to varying doses of GABA; (A) “combination” curve where 2rn~ IMA was added to varying doses of GABA; (A) and (Cl) indicate the depolarizations produced by 0.2 mM 3APS and 2 mM IMA respectively. B: (0) control 3APS curve; (0) “combination” curve where 2rn~ IMA was added to varying doses of 3APS; (A) shows the depolarization produced by 2rn~ IMA alone. Same cord as in A.
200
Spinal effects of GABA-like drugs from strychnine block required at least 3-4 hr washing. In order to obtain an indication of whether GABA, 3APS and IMA were acting through a similar mechanism, GABA/3APS or GABA/IMA “combination” dose-response curves were obtained; in these experiments a fixed dose of 3APS or IMA was added to increasing concentrations of GABA. Figure 5A shows that equi-effective doses of IMA or 3APS produced an upward shift of the initial part of the GABA doseeresponse curve suggesting a synergistic action (Ariens and Simonis, 1964a; Constanti, 1976); however. when higher concentrations of GABA were used some mutual antagonism between GABA and 3APS or IMA became apparent since the responses to the combination of GABA and one of these substances was smaller than would be expected if a dose of GABA equipotent with 3APS or IMA was added to the actual dose of GABA applied. These “combination” curves had an intersection with the control GABA curve near the apparent maximum. Conversely, if a fixed dose of IMA was combined with increasing concentrations of 3APS. the “combination” curve so obtained did not cross over the control 3APS curve (Fig. 5B); this effect is usually produced by drugs acting on different receptors but producing their effects by a common effector system (Ariens and Simonis. 1964b). Fixed doses of GABA combined
A
GABA
3APS
IMA control
GLU
TAU
17
with increasing doses of 3APS (not shown) had a synergistic action and the “combination” curve so obtained crossed the control 3APS curve near the apparent maximum. Finally, the ionic dependency of root responses to GABA. 3APS and IMA was studied. Responses to taurine and glutamate, two other neuroactive aminoacids, were also tested for comparison. After changeover from control Ringer to a Cll or Na+deficient medium about 30min were allowed for stabilization of possible electrode junction potential changes and for an effective decrease in the tissue concentration of the ionic species investigated. Figure 6A shows that 50% substitution of the Cl- content of the bathing solution with the presumably impermeant isethionate ion produced a partial reduction of GABA or taurine responses whereas 3APS or IMA were more markedly depressed; responses to glutamate were little affected. All these effects were reversible l&l5 min after return to control Ringer solution; at this time responses to GABA or glutamate were enhanced. Figure 6B shows the effects of stepwise increases in the K’ content of the Ringer solution (K,SO, was added without any further change in ionic composition), these changes being associated with graded depolarizations of the dorsal root. Raising K’ con-
B
K+OmM
I
K*
I
-I
.?
K+
4mM
n
4 mM
Fig. 6. Effect of changes in the ionic content of the Ringer solution on amino acid-evoked responses from dorsal roots of TTX-treated spinal cords. A: top row shows control responses to GABA, 3APS, IMA, glutamate (GLU) or taurine (TAU), all substances being applied at the dose of 1 mM for the periods indicated by the black bars; the middle row shows responses to the same substances in a medium containing 50% Cll (substituted by equimolar amounts of isethionate); the bottom row shows responses to the same substances after 10min return to control Ringer. B: Effect of increasing the K+ concentration of the Ringer on the responses to 1 mM GABA (0) 1 mM 3APS (W) and 1 mM IMA (H). Different preparation from A. Changes from 0 to 4m~ K+ were associated with a 3 mV dorsal root depolarization, whereas changeover from 4 to 20 IIIM K+ produced 8 mV depolarization and a further 2 mV depolarization was brought about by changeover from 20 to 40 mM K+; these effects were completely reversible on return to control Ringer (4 mM K’). Calibration marks: 1 mV; 1 min for both A and B.
A.
NISTRI and
control I
I
low
Nat I
I
AhA Cl
w
EA
recovery I
I
Fig. 7. Effect of Na+ substitution on the dorsal root responses of a TTX-treated spinal cord. Top row shows control responses to GABA (2 mM, q), 3APS (0.5 mM, n) or IMA (I mM, a). The middle row depicts responses to the same substances after 30min exposure to low Na+ (98% substitution with equimolar choline) Ringer. The bottom row shows recovery responses after 30min return to control Ringer. Calibration marks: I mV; I min. tent from 0 to 4 mM (K’ level of the control Ringer) had little effect on the responses to GABA, 3APS or IMA; however, further increase in the K+ content of the Ringer (up to 20mM) was associated with a progressive reduction of GABA, 3APS or IMA responses. In the presence of 40m~ K+, 3APS and IMA responses were inverted in their polarity while the GABA response was still in the depolarizing direction. All these effects were reversible on return to control Ringer (4m~ K+). In other experiments the Naf content of the bathing medium was reduced by 98% and substituted with an equimolar amount of lithium or choline; Figure 7 shows that this procedure brought about a reduction of the effect of GABA but not of 3APS or IMA. It is interesting to note that the fading of GABA response in a 2%Na+ medium was still quite remarkable. DISCUSSION
The GABA-evoked responses of the dorsal root of the frog Rana esculenta were very similar to those obtained in a previous study with Ram temporariu (Constanti and Nistri, 1976). The observed effects of GABA or 3APS, a GABA-related agent, on the
R.
CORRADE~I
V-DRPs and the root d.c. levels were similar although the effects of 3APS appeared to be about IO times more potent and less susceptible to fading than the effects of GABA. It is pertinent to mention that the fading of GABA responses was unlikely to be caused by a rapid uptake of GABA by spinal cells since it was maintained in 2”j,Na’ medium where uptake mechanisms are strongly reduced (Davidoff and Adair, 1975); therefore, the fading phenomenon might reflect receptor desensitization rather than drug removal during prolonged application of GABA. On the other hand IMA, a GABA-mimetic compound, was slightly less potent than GABA. The question arises as to whether the effects of 3APS or IMA are due to an agonistic action on GABA receptors. Indirect contributions from interneuronal activity to the observed responses was probably eliminated by TTX; furthermore 3APS and IMA seem to have little effect on the uptake and release of GABA (Iversen and Johnston, 1971; Balcar and Johnston, 1973; Olsen, Bayless and Ban, 1975; Bowery et ~1..1976). Although there were similarities between the effects of 3APS and GABA as shown by their sensitivity to picrotoxin, the similar shape of their log dose-response curves and their similar slope values, there were also some differences. In particular the effects of GABA seemed to depend on the Na+ and Cl- content of the bathing medium, while those of 3APS were found to be dependent on Cl- only; moreover in a 40m~ K’ medium the polarity of 3APS responses was inverted whereas that of GABA responses was not, a result which indirectly suggests that the reversal potential for GABA might be different from that for 3APS. Furthermore, the effects of 3APS (and also of IMA) were more sensitive than those of GABA to the blocking action of picrotoxin, a drug that on invertebrate preparations antagonizes the effects of GABA presumably by blocking the receptor-linked Cl- channels (Takeuchi and Takeuchi, 1969). Finally the IMA,/3APS “combination” curve did not cross over the dose-response curve to 3APS whereas the GABA/3APS “combination” curve did; this suggests that by the use of IMA differences between the mode of action of GABA and 3APS may become apparent. The mechanisms of action of IMA also seemed to differ from that of GABA. For example, these two substances had different slope values of their log-log plots. Since the slope value for IMA was near 2 and that for GABA was 0.73 we propose that two molecules of IMA (but only one of GABA) were needed to activate a single sensitive site. Another feature of the action of IMA was its sensitivity to strychnine as well as picrotoxin, a fact already noted by Barker et al. (1975a); this finding shows that the effect of IMA on the frog spinal cord resembles more closely that of taurine (Nistri and Constanti. 1976) than that of GABA. The action of IMA, like that of 3APS but not that of GABA was dependent on Cl- only and inverted at a d.c. level where GABA still produced depolarizations.
Spinal effects of GABA-like Constanti and Quilliam (1974)suggested that at the lobster neuromuscular junction IMA might act partly on extrasynaptic membrane areas and partly on GABA receptors. It is possible that in the frog spinal cord IMA and 3APS have effects on synaptic and extrasynaptic GABA receptors. If extrasynaptic GABA receptors exist. they might have a different nature from synaptic ones and be sensitive to IMA and also to 3APS. Another hypothesis to account for our results stems from the proposal of Godfraind et ul. (1973) and Constanti (1976) who suggested that IMA may act as a Cll ionophore able to increase directly membrane conductance to this ion. In the frog spinal cord IMA and 3APS might directly increase Cl- permeability; in this case these agents would produce effects similar to those of taurine (Nistri and Constanti. 1976). These hypotheses are clearly speculative and will have to be tested experimentally with intracellular recordings. Nonetheless, the present results indicate that in the frog spinal cord 3APS and IMA mimic only in part the effects of GABA and that they should not be considered mere GABA agonists.
AcknoM,lrdqomenTs-We thank Dr K. Krnievic for a critical reading of this paper and Dr D. A: Brown for his kind gift of 3APS. This work was supported by a grant from CNR (Rome) and by an equipment grant from the University of Florence. Part of this work was submitted by R. Corradetti as an M.D. thesis to the University of Florence.
REFERENCES Ariens, E. J. and Simonis. A. M. (1964a). A molecular basis for drug action. J. Pharm. Plzurmcrc. 16: 137-157. Arilns, E. J. and Simonis, A. M. (1964b). A molecular basis for drug action. The interaction of one or more drugs with different receptors. J. Pharm. Pharmac. 16: 2899312. Balcar, V. J. and Johnston, G.A.R. (1973). High affinity uptake of transmitters: studies on the uptake of L-aspartale, GABA, L-glutamate and glycine in cat spinal cord. J. Neurochern. 20: 5299539. Barker. J. L., Nicoll. R. A. and Padjen, A. (1975a). Studies on convulsants in the isolated frog spinal cord. I. Antagonism of amino acid responses. J. Physiol., Lond. 245: 521-536. Barker. J. L., Nicoll, R. A. and Padjen, A. (1975b). Studies on convulsants in the isolated frog spinal cord, II. Effects on root potentials. J. Physiol., Land. 245: 537-548. Bird. E. D. and Iversen, L. L. (1974). Huntington’s chorea: post-mortem measurement of glutamic acid decarboxylase, choline acetyltransferase and dopamine in basal ganglia Brclin 97: 4577472. Bowery. N. G. and Brown, D. A. (1974). Depolarizing actions of y-aminobutyric acid and related compounds on rat superior ccervical ganglia in vitro. Br. J. Pharmac. 50: 205-2 1X. Bowcry, N. G. and Jones, G. P. (1976). A comparison of y-aminocrotonic acid and imidazole-4-acetic acid on the isolated superior cervical ganglion of the rat. Br. J. Pharmu. 56: 323-330.
Bowery,
19
drugs N. G., Brown,
D. A., Collins,
G. G. S., Galvan, effects of amino-acids on sympathetic ganglion cells mediated through the release of y-aminobutyric acid from glial cells. Br. J. Pharmac. 57: 73-91. Constanti. A. (1976) A quantitative study of the actions of gamma-aminobutyric acid (GABA), GABA analogues and antagonists on the membrane conductance of lobster muscle fibres. Ph.D. thesis, University of London. Constanti, A. and Nistri. A. (1976). A comparative study of the action of y-aminobutyric acid and piperazine on the lobster muscle fibre and the frog spinal cord. Br. J. Pharmac. 57: 347-358. Constanti, A. and Quilliam, J. P. (1974). A comparison of the effects of GABA and imidazoleacetic acid on the membrane conductance of lobster muscle fibres. Bruin Rrs. 79: 30663 10. Curtis, D. R., Duggan, A. W., Felix, D. and Johnston, G. A. R. (1971). Bicuculline an antagonist of GABA and synaptic inhibition in the spinal cord. Brain Rex 32: 69996. Curtis, D. R.. Phillis, J. W. and Watkins, J. C. (1961). Actions of amino acids on the isolated hemisected spinal cord of the toad. Br. J. Pharmuc. Chernoth. 16: 2621283. Davidoff. R. A. and Adair. R. (1975). High affinitv amino acid transport by frog spinal cord slices. J. Neurochem. 24: 545-552. Eccles. J. C., Schmidt, R. and Willis, W. D. (1963). Pharmacological studies on presynaptic inhibition, J. Physiol., Lond. 168: SOS530. Enna, S. J. and Snyder, S. H. (1975). Properties of y-aminobutyric acid (GABA) receptor binding in rat brain synaptic membrane fractions. Brain Res. 100: 81-97. Godfraind. J. M., Krnjevic, K., Maretic, H. and Pumain, R. (1973). Inhibition of cortical neurones by imidazole and some derivatives. Can. J. Physiol. Pharmac. 51: 790~~ 797 Iversen. L. L. and Johnston, G. A. R. (1971). GABA uptake in rat central nervous system: comparison of uptake in slices and homogenates and the effects of some inhibitors. J. Yeurochem. 18: 193991950. Krnjevic, K. and Phillis, J. W. (1963). Actions of certain amines on cerebral cortical neurones. Br. J, Pharmac. Chemoth. 20: 47 l-490. Krnjevic’, K. and Puil, E. (1976). Electrophysiological studies on actions of taurine. In: Taurine (Huxtable, R. and Barbeau, A., Eds), pp. 1799189. Raven Press, New York. Nistri. A. and Constanti, A. (1976). The action of taurine on the lobster muscle fibre and the frog spinal cord. Neuropharmacology 15: 6355641. Nistri, A. and Corradetti, R. (1976). A quantitative study of the neuronal actions of 3-aminopropanesulphonic acid (3APS) and GABA, Proceedings ofthe First Meeting of the European Society of Neurochemistry, Bath, England. Abstract No. 16. Olsen, R. W., Bayless, J. D. and Ban, M. (1975). Potency of inhibitors for y-aminobutyric acid uptake by mouse brain subcellular particles at 0”. MO&. Pharmac. 11: 558-565. Perry, T. L., Hansen, S. and Kloster, M. (1973). Huntington’s chorea: deficiency of y-aminobutyric acid in brain, New Enyl. J. Med. 288: 337-342. Schmidt, R. F. (1963). Pharmacological studies on the primary afferent depolarization of the toad spinal cord. Pfliigers Arch. ges. Physiol. 277: 325-346. Takeuchi, A. and Takeuchi, N. (1969). A study of the action of picrotoxin on the inhibitory neuromuscular junction of the crayfish. J. Physiol., Lond. 205: 377-391. Werman, R. (1969). An electrophysiological approach to drug-receptor mechanisms. Comp. Biochem. Physiol. 30: 997-1017.
M.. Marsh, S. and Yamini, G. (1976). Indirect
1978, 17, 13-19
Pergamon
Press.
Prmted
I” Great
Britam
A COMPARISON OF THE EFFECTS OF GABA, 3-AMINOPROPANESULPHONIC ACID AND IMIDAZOLEACETIC ACID ON THE FROG SPINAL CORD A. NISTRI* and R. CORRADETTI Institute of Pharmacology, University of Florence, Florence, Italy (Accepted 19 Maq’ 1977) Summary-The effects of GABA, 3-aminopropanesulphonic acid (3APS) or imidazoleacetic acid (IMA) on the isolated spinal cord of the frog were studied by means of extracellular recording techniques. In the presence of tetrodotoxin (6.2~~), GABA, 3APS or IMA produced dose-dependent dorsal root depolarizations. From the analysis of the doseeresponse relationships the order of depolarizing potency for these substances was 3APS 9 GABA > IMA. The effects of IMA were antagonized by either strychnine or picrotoxin whereas those of GABA or 3APS were only antagonized by picrotoxin. None of the observed antagonisms appeared competitive. The action of GABA was dependent on the Na+ and Cl- content of the bathing solution while that of 3APS and IMA was dependent only on external Cl-. By stepwise increases in the extracellular K+ content it was found that the polarity of 3APS or IMA responses could be inverted at a d.c. level where GABA still produced depolarizations. It is suggested that the mode of action of 3APS and IMA on the frog spinal cord is somewhat different from that of GABA; therefore 3APS and IMA could not be regarded as specific GABA receptor agonists on this preparation.
The availability of potent agonists at the 1;-aminobutyric acid (GABA) receptor would be of considerable help in the study of GABA-operated synapses in the central nervous system; furthermore such agonists might represent prototypes of drugs capable of ameliorating neurological diseases such as Huntington’s chorea where a deficit of GABA and its synthetic enzyme has been found in the basal ganglia (Perry, Hansen and Kloster, 1973; Bird and Iversen, 1974). 3-Aminopropanesulphonic acid (3APS; homotaurine) has been reported to have GABA-mimetic activity more potent than GABA on the cerebral cortex (Krnjevic’ and Puil, 1976), spinal cord (Curtis, Phillis and Watkins, 1961; Eccles, Schmidt and Willis, 1963; Schmidt, 1963) and sympathetic ganglion (Bowery and Brown, 1974; Bowery, Brown, Collins, Galvan, Marsh and Yamini, 1976). Enna and Snyder (1975) observed that in rat brain homogenates 3APS competes with GABA for binding sites; these findings would thus suggest that 3APS is an agonist at GABA receptors. Imidazoleacetic acid (IMA) also produces some GABA-like effects on the cerebral cortex (Krnjevic’ and Phillis, 1963; Godfraind, Krnjevic’, Maretic’ and Pumain, 1973; Enna and Snyder, 1975), spinal cord (Curtis, Duggan, Felix and Johnston, 1971; Barker, Nicoll and Padjen, 1975a) and sympathetic ganglion (Bowery and Jones, 1976). In these studies (with the exception of that by Barker et al., 1975a) IMA was somewhat less potent than GABA. However, despite
the above evidence that 3APS and IMA might interact with GABA receptors, there has been no quantitative study of the effects of 3APS and IMA on a central nervous system preparation under in vitro controlled conditions. We therefore decided to study dose-response relationships for dorsal root responses of the isolated frog spinal cord to bath-application of GABA, 3APS or IMA. In this preparation GABA is supposed to play a transmitter role in the presynaptic inhibition of primary afferent terminals (Barker, Nicoll and Padjen, 1975b). Some of our results have been presented in a preliminary form (Nistri and Corradetti, 1976). METHODS All experiments were carried out on frogs (Rana rsculenta). The procedure for setting up and recording from the isolated frog spinal cord has been recently described in detail (Constanti and Nistri, 1976; Nistri and Constanti, 1976). In brief, the hemisected spinal cord was bathed in a Ringer solution (NaCl, 109 mM; KCl, 4m~; CaCl, (titrated), 1.5 mM; NaHCo,, 1Om~; glucose, 4 mu, pH 7.2-7.3 with O&O,95/5%) maintained at 12°C. Electrical stimuli (0.25 Hz; 0.05 msec; supramaximal voltage) applied to the VIII or XIth ventral root produced dorsal root potentials (V-DRPs) in the corresponding dorsal root. These potentials and changes in polarization level of the dorsal root were recorded differentially using two extracellular Ag/AgCl electrodes (one placed on a dorsal root and the other placed in the bathing medium) and monitored on a conventional oscilloscope as well as on a storage oscilloscope and a chartrecorder. The bath was grounded via a third Ag/AgCl electrode.
* Present address: Department of Research in Anaesthesia, McGill University, Montreal, PQ, Canada H3G lY6. Key words: GABA, 3-aminopropanesulphonic acid, imidazoleacetic
acid, spinal
cord, receptors. 13
14
A. NISTRIand R. CORKADE~TI
_-b-r----
-
a
b
-
C
_--
--
J
0.5 mV
100 msec Fig. 1. Effects of GABA or 3APS on V-DRPs. a: control V-DRP; b: 45 set after bath application of GABA (2 mM); c: after 2 min wash; d: control V-DRP; e: 45 set after bath application of 3APS (0.2 mM); f: after 3 min wash. For abbreviations see text. Negativity upwards. All these responses were recorded from the same cord.
RESULTS Bath-application of GABA (2m~) or 3APS (0.2 mM) rapidly depressed the V-DRPs, this effect being quickly reversible on washing (Fig. I). Similar effects were also produced by IMA (4 ITIM; not shown). The depression of V-DRPs after the administration of GABA, 3APS or IMA was associated with a reversible dorsal root depolarization. In order to block propagated interneuronal activity, tetrodotoxin (TTX; 6.2 PM) was added to the bathing solution. This toxin rapidly ( IMA as judged on the basis of their ED,, values. The slopes for 3APS and GABA were < 1 whereas that for IMA was 1.72. Picrotoxin is a seemingly non-competitive antagonist of GABA in the frog spinal cord (Constanti and
Nistri, 1976). In the present study a low dose (5 PM) of this convulsant depressed the apparent maximum of the GABA dose-depolarization curve without a displacement to the right along the abscissa (see Fig. 4); however, higher concentrations of picrotoxin (3 10,~~) shifted the GABA curve to the right in an apparently non-parallel fashion as previously reported (Constanti and Nistri, 1976). Similar findings were obtained when 3APS responses were measured in the presence of 5 PM picrotoxin (Fig. 4); it should be noted that in the presence of picrotoxin the fading of GABA as well as 3APS responses was increased (cf. top tracings of Fig. 4). Recovery from the action of picrotoxin was observed after 2-3 hr washing. Responses to IMA were also antagonized by picrotoxin (not shown). In order to obtain some information about the activity of picrotoxin as a blocker of aminoacid effects, the picrotoxin concentrations which halved the maximal response to each agonist were calculated (Table 1). It appears that picrotoxin was a more active inhibitor of the maximal effects of 3APS and IMA than of those of GABA. Strychnine (100 PM) did not affect GABA or 3APS responses but at a relatively low dose (10 PM) antagonized IMA responses as shown,by the non-parallel shift to the right of the IMA dose-response curve (Fig. 3B). Recovery Table
GABA 3APS IMA
I. Estimated kinetic values for GABA, IMA responses of frog dorsal root
3APS
and
Picrotoxin required to halve maximal response
ED,, (m@
Slope (ii)
(PM)
1.1 0.1 2.3
0.73 0.83 1.72
16.0 8.2 6.0
ED,, values were obtained as described by Bowery and Brown (I 974); slope values refer to log-log transformations of the dose-response curves for GABA, 3APS and IMA (see Werman, 1969).
A. NISTRI and R. CORRADETTI
16
-h -c n
n
l-
0.5
05
7
10
20 log
0 0.2
1
05 log
[GABA]
100
[3APS] (pm)
10
5
2
50
(mM)
Fig. 4. Effect of picrotoxin on GABA or 3APS log dose-response curves obtained from dorsal roots of tetrodotoxin-treated spinal cords. On the left: control GABA curve (0) and GABA curve obtained in the presence of 5 PM picrotoxin (0); the tracings above these curves show on the left a control GABA (2m~) response and on the right the response to the same dose in the presence of 10 PM picrotoxin. On the right: control 3APS curve (0) and 3APS curve obtained in the presence of 5 PM picrotoxin (0); the tracings above these curves show on the left the response to 50~~ 3APS and on the right the response to the same dose in the presence of 10~~ picrotoxin. Calibration marks (applicable to both GABA and 3APS responses) were: I mV; 1 min.
mV
2 mV
0.2
0.5 log
1 [GABA]
2
5 (mM)
10
0.02
0.05 log
0.1
0.2
[3 APS]
0.5
1.0
(mM)
Fig. 5. Interactions between GABA, 3APS and IMA at dorsal root level. The two graphs show some “combination” curves obtained by adding a fixed dose of one agonist to increasing doses of another. A: (0) control GABA curve; (0) “combination” curve where 0.2 mM 3APS was added to varying doses of GABA; (A) “combination” curve where 2rn~ IMA was added to varying doses of GABA; (A) and (Cl) indicate the depolarizations produced by 0.2 mM 3APS and 2 mM IMA respectively. B: (0) control 3APS curve; (0) “combination” curve where 2rn~ IMA was added to varying doses of 3APS; (A) shows the depolarization produced by 2rn~ IMA alone. Same cord as in A.
200
Spinal effects of GABA-like drugs from strychnine block required at least 3-4 hr washing. In order to obtain an indication of whether GABA, 3APS and IMA were acting through a similar mechanism, GABA/3APS or GABA/IMA “combination” dose-response curves were obtained; in these experiments a fixed dose of 3APS or IMA was added to increasing concentrations of GABA. Figure 5A shows that equi-effective doses of IMA or 3APS produced an upward shift of the initial part of the GABA doseeresponse curve suggesting a synergistic action (Ariens and Simonis, 1964a; Constanti, 1976); however. when higher concentrations of GABA were used some mutual antagonism between GABA and 3APS or IMA became apparent since the responses to the combination of GABA and one of these substances was smaller than would be expected if a dose of GABA equipotent with 3APS or IMA was added to the actual dose of GABA applied. These “combination” curves had an intersection with the control GABA curve near the apparent maximum. Conversely, if a fixed dose of IMA was combined with increasing concentrations of 3APS. the “combination” curve so obtained did not cross over the control 3APS curve (Fig. 5B); this effect is usually produced by drugs acting on different receptors but producing their effects by a common effector system (Ariens and Simonis. 1964b). Fixed doses of GABA combined
A
GABA
3APS
IMA control
GLU
TAU
17
with increasing doses of 3APS (not shown) had a synergistic action and the “combination” curve so obtained crossed the control 3APS curve near the apparent maximum. Finally, the ionic dependency of root responses to GABA. 3APS and IMA was studied. Responses to taurine and glutamate, two other neuroactive aminoacids, were also tested for comparison. After changeover from control Ringer to a Cll or Na+deficient medium about 30min were allowed for stabilization of possible electrode junction potential changes and for an effective decrease in the tissue concentration of the ionic species investigated. Figure 6A shows that 50% substitution of the Cl- content of the bathing solution with the presumably impermeant isethionate ion produced a partial reduction of GABA or taurine responses whereas 3APS or IMA were more markedly depressed; responses to glutamate were little affected. All these effects were reversible l&l5 min after return to control Ringer solution; at this time responses to GABA or glutamate were enhanced. Figure 6B shows the effects of stepwise increases in the K’ content of the Ringer solution (K,SO, was added without any further change in ionic composition), these changes being associated with graded depolarizations of the dorsal root. Raising K’ con-
B
K+OmM
I
K*
I
-I
.?
K+
4mM
n
4 mM
Fig. 6. Effect of changes in the ionic content of the Ringer solution on amino acid-evoked responses from dorsal roots of TTX-treated spinal cords. A: top row shows control responses to GABA, 3APS, IMA, glutamate (GLU) or taurine (TAU), all substances being applied at the dose of 1 mM for the periods indicated by the black bars; the middle row shows responses to the same substances in a medium containing 50% Cll (substituted by equimolar amounts of isethionate); the bottom row shows responses to the same substances after 10min return to control Ringer. B: Effect of increasing the K+ concentration of the Ringer on the responses to 1 mM GABA (0) 1 mM 3APS (W) and 1 mM IMA (H). Different preparation from A. Changes from 0 to 4m~ K+ were associated with a 3 mV dorsal root depolarization, whereas changeover from 4 to 20 IIIM K+ produced 8 mV depolarization and a further 2 mV depolarization was brought about by changeover from 20 to 40 mM K+; these effects were completely reversible on return to control Ringer (4 mM K’). Calibration marks: 1 mV; 1 min for both A and B.
A.
NISTRI and
control I
I
low
Nat I
I
AhA Cl
w
EA
recovery I
I
Fig. 7. Effect of Na+ substitution on the dorsal root responses of a TTX-treated spinal cord. Top row shows control responses to GABA (2 mM, q), 3APS (0.5 mM, n) or IMA (I mM, a). The middle row depicts responses to the same substances after 30min exposure to low Na+ (98% substitution with equimolar choline) Ringer. The bottom row shows recovery responses after 30min return to control Ringer. Calibration marks: I mV; I min. tent from 0 to 4 mM (K’ level of the control Ringer) had little effect on the responses to GABA, 3APS or IMA; however, further increase in the K+ content of the Ringer (up to 20mM) was associated with a progressive reduction of GABA, 3APS or IMA responses. In the presence of 40m~ K+, 3APS and IMA responses were inverted in their polarity while the GABA response was still in the depolarizing direction. All these effects were reversible on return to control Ringer (4m~ K+). In other experiments the Naf content of the bathing medium was reduced by 98% and substituted with an equimolar amount of lithium or choline; Figure 7 shows that this procedure brought about a reduction of the effect of GABA but not of 3APS or IMA. It is interesting to note that the fading of GABA response in a 2%Na+ medium was still quite remarkable. DISCUSSION
The GABA-evoked responses of the dorsal root of the frog Rana esculenta were very similar to those obtained in a previous study with Ram temporariu (Constanti and Nistri, 1976). The observed effects of GABA or 3APS, a GABA-related agent, on the
R.
CORRADE~I
V-DRPs and the root d.c. levels were similar although the effects of 3APS appeared to be about IO times more potent and less susceptible to fading than the effects of GABA. It is pertinent to mention that the fading of GABA responses was unlikely to be caused by a rapid uptake of GABA by spinal cells since it was maintained in 2”j,Na’ medium where uptake mechanisms are strongly reduced (Davidoff and Adair, 1975); therefore, the fading phenomenon might reflect receptor desensitization rather than drug removal during prolonged application of GABA. On the other hand IMA, a GABA-mimetic compound, was slightly less potent than GABA. The question arises as to whether the effects of 3APS or IMA are due to an agonistic action on GABA receptors. Indirect contributions from interneuronal activity to the observed responses was probably eliminated by TTX; furthermore 3APS and IMA seem to have little effect on the uptake and release of GABA (Iversen and Johnston, 1971; Balcar and Johnston, 1973; Olsen, Bayless and Ban, 1975; Bowery et ~1..1976). Although there were similarities between the effects of 3APS and GABA as shown by their sensitivity to picrotoxin, the similar shape of their log dose-response curves and their similar slope values, there were also some differences. In particular the effects of GABA seemed to depend on the Na+ and Cl- content of the bathing medium, while those of 3APS were found to be dependent on Cl- only; moreover in a 40m~ K’ medium the polarity of 3APS responses was inverted whereas that of GABA responses was not, a result which indirectly suggests that the reversal potential for GABA might be different from that for 3APS. Furthermore, the effects of 3APS (and also of IMA) were more sensitive than those of GABA to the blocking action of picrotoxin, a drug that on invertebrate preparations antagonizes the effects of GABA presumably by blocking the receptor-linked Cl- channels (Takeuchi and Takeuchi, 1969). Finally the IMA,/3APS “combination” curve did not cross over the dose-response curve to 3APS whereas the GABA/3APS “combination” curve did; this suggests that by the use of IMA differences between the mode of action of GABA and 3APS may become apparent. The mechanisms of action of IMA also seemed to differ from that of GABA. For example, these two substances had different slope values of their log-log plots. Since the slope value for IMA was near 2 and that for GABA was 0.73 we propose that two molecules of IMA (but only one of GABA) were needed to activate a single sensitive site. Another feature of the action of IMA was its sensitivity to strychnine as well as picrotoxin, a fact already noted by Barker et al. (1975a); this finding shows that the effect of IMA on the frog spinal cord resembles more closely that of taurine (Nistri and Constanti. 1976) than that of GABA. The action of IMA, like that of 3APS but not that of GABA was dependent on Cl- only and inverted at a d.c. level where GABA still produced depolarizations.
Spinal effects of GABA-like Constanti and Quilliam (1974)suggested that at the lobster neuromuscular junction IMA might act partly on extrasynaptic membrane areas and partly on GABA receptors. It is possible that in the frog spinal cord IMA and 3APS have effects on synaptic and extrasynaptic GABA receptors. If extrasynaptic GABA receptors exist. they might have a different nature from synaptic ones and be sensitive to IMA and also to 3APS. Another hypothesis to account for our results stems from the proposal of Godfraind et ul. (1973) and Constanti (1976) who suggested that IMA may act as a Cll ionophore able to increase directly membrane conductance to this ion. In the frog spinal cord IMA and 3APS might directly increase Cl- permeability; in this case these agents would produce effects similar to those of taurine (Nistri and Constanti. 1976). These hypotheses are clearly speculative and will have to be tested experimentally with intracellular recordings. Nonetheless, the present results indicate that in the frog spinal cord 3APS and IMA mimic only in part the effects of GABA and that they should not be considered mere GABA agonists.
AcknoM,lrdqomenTs-We thank Dr K. Krnievic for a critical reading of this paper and Dr D. A: Brown for his kind gift of 3APS. This work was supported by a grant from CNR (Rome) and by an equipment grant from the University of Florence. Part of this work was submitted by R. Corradetti as an M.D. thesis to the University of Florence.
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