- Email: [email protected]
Potent inhibitory GABAB receptors in stimulated guinea-pig taenia coli
Neuropharmocology
Vol. 26, No.
11, pp. 1622-1627,
0028s3908/87 $3.00 + 0.00 Copyright Q 1987 PergamonJournals Ltd
1987
Printed in Great Britain. All rights reserved
POTENT INHIBITORY GABA, RECEPTORS IN STIMULATED GUINEA-PIG TAENIA COLI A. Z. R~NAI~, JULIANNAKARDOS’*and M. SIMONYI’ ‘Central Research Institute for Chemistry, Hungarian Academy of Sciences, Budapest, Hungary and 2Department of General Psychology, Eotvos University, Budapest, Hungary (Accepted 18 May 1987)
Scan-~-Aminobutyric acid (GABA, half-maxims inhibitory concentration, IC, = 9.9 PI%%) and (-)-baclofen (IC, =4.5pM) but not 10m4M muscimol, exerted a presynaptic inhibitory effect on choline+ transmission in field-stimulated guinea-pig taenia coli preparations, in virro. The antagonism by 10e5M (IS, 9R)-bicuculline methiodide was not specific for GABA agonists. The results suggest that the GABA receptors involved were of type B. Key words: GABA, receptors, guinea-pig taenia-coli, (-)-baclofen, muscimol, (IS, 9R)-bicuculline methiodide.
There are several lines of evidence that y-aminobutyric acid (GABA) may serve as a neuroregulator (transmitter or modulator) in the mammalian enteric nervous system (Jessen, Hills, Dennison and Mirsky, 1983; Ong and Kerr, 1984). ~-Aminobutyric acid can be demonstrated in enteric neuronal elements; there is a high affinity neuronal uptake mechanism for GABA, a GABA-synthesizing mechanism and Caz+dependent release of GABA upon appropriate stimulation (Jessen et al., 1983; Krantis, Kerr and Dennis, 1986). Though there are several neural networks sensitive to GABA in the peripheral autonomic nervous system (Bowery, Doble, Hill, Hudson, Shaw, Turnbull and Warrington, 1981), the actions of this amino acid transmitter have been studied most extensively in the gut (Krantis, Costa, Furness and Orbach, 1980; Kaplita, Waters and Triggle, 1982; Giotti, Luzzi, Spagnese and Zilletti, 1983; Ong and Kerr, 1984; Krantis ef al., 1986). y-Aminobutyric acid exerts both excitatory and inhibitory actions on the choline@ innervation of intestinal smooth muscle (Bowery et al., 1981; Kaplita et al., 1982; Giotti et al., 1983; Kleinrok and Kilbinger, 1983), it also activates certain noncholinergic inhibitory neurones (Krantis et al., 1980; Ong and Kerr, 1984) and appears to have a physiological role in the control of intestinal motility (Kleinrok and Kilbinget, 1983; Ong and Kerr, 1984). The stimulatory effects in the myenteric plexus of the guinea-pig are mediated by GABA, receptors (Kaplita et al., 1982; Giotti et al., 1983), whereas the relaxant actions are due to the activation of GABA, receptor sites (Bowery et al., 1981; Ong and Kerr, 1984). Although GABA has been shown to exert a *Present address: Fidia-Georgetown Institute for the Neurosciences, 3900 Reservoir Road, Washington D.C. 20007, U.S.A.
potent inhibitory action on spontaneously-working peripheral autonomic nerve-muscle preparations (Giotti et al., 1983; Ong and Kerr, 1984), the effects on the twitch responses or release of transmitter elicited by nerve stimulation did not show maximal inhibition (Bowery et al., 1981; Kaplita et al., 1982). The present investigations were aimed at analysing the effects of various GABAergic agents in nerve (field) stimulated guinea-pig taenia coli preparations. METHODS The chemicals used were: GABA, muscimol, atropine sulphate (Sigma); ascorbic acid (Merck); (-)-baclofen (Ciba-Geigy); acetylcholine chloride, noradrenaline bitarta~te (Serva). (1S, 9R)-bi~uculline methiodide was prepared from (1 S, 9R)-bicuculline (Sigma). All other substances used were of analytical grade. Solutions of noradrenaline contained also 10e4M ascorbic acid; solutions of noradrenaline and (1S, 9R)-bicuculline methiodide (BMI) were kept protected from light. Male guinea-pigs, weighing 250-350 g, were used. For the purpose of isolated organ experiments, S-8 mm long taenia coli segments were prepared. The preparations were set up in Krebs solution aerated with carbogen (O,:CO, = 95: 5) at 37”C, under a resting tension of 0.5 g. The composition of the Krebs solution was as follows (in mM): NaCl 118.0, NaHCO, 25.0, KC1 4.7, KH,PO, 1.2, CaCi, 2.5, MgSO, 0.6 and glucose 11.0, pH 7.4. The taeniae were equilibrated for 45_60min, washed for 10min and the resting tension was readjusted at the end of the first 5 min. The contractions were measured under non-isometric conditions by means of strain-gaugepreamplifier (Rolitron, Hungary), compenzograph recorder (Radelkis, Hungary) system. Field electrical stimulation was applied; 3 pulses were delivered at
1623
1624
A. Z. R&W
0.5 Hz frequency every 3 min. The parameters of individual pulses were as follows: square-wave pulses of 0.5 msec duration, 9 V/cm voltage-drop. Stimulation was started at the beginning of the equilibration period. Whenever the results are given numerically, mean + SEM values are listed with the indication of the number of experiments. RESULTS
y-Aminobutyric acid, in the concentration range of 10-6-10~’ M, affected moderately the tone of taeniacoli preparations as compared either to acetylcholine or electrical stimulation. No relaxation was observed; the contractile responses, if they occurred at all, were smaller than 0.4 g. The field stimulation, using the parameters indicated above, elicited contractions through the activation of cholinergic neurones since the muscle completely abolished by responses were almost 2 x lo-’ M atropine sulphate (Fig. 1). Applied in this concentration, atropine reduced the contractions elicited by low-frequency, short-train electrical stimulation by 96.0 + 2.4% (n = 4, P < 0.005) whereas the contractions produced by exogenously administered 3.2 x IO-‘M acetylcholine were reduced by 99.3 + 0.3% (n = 4, P < 0.005). y-Aminobutyric acid and (-)-baclofen, but not muscimol(6 experiments), when given at an interval of 15-20 min to avoid tachyphylaxis (5 min contact time, 3-5 washes between doses), produced a dose-dependent inhibitory effect on contractions elicited by field stimulation (Fig. 2 lower panel). The half-maximal inhibitory concentration for GABA was 9.9 -f 3.8 nM (n = 7).
ef a/.
In one of seven experiments, 50% inhibition was not attained; in the rest, the maximum values were well above 50%. The IC,, for (-)-baclofen was as high as 4.5 + 1.0 PM (n = 5). The dose-response curves for (-)-baclofen were considerably steeper than those for GABA. Whilst both GABA-agonists depressed nerve-
mediated contractions, they did not affect the responses to exogenously-administered acetylcholine (Fig. 2 upper and middle panels). The standard concentrations of acetylcholine were chosen such as to detect even moderate modifications in the response; under these conditions, concentrations of GABA and (-)-baclofen (1Om4 and lo-‘M, respectively) that reduced electrically-evoked twitches by 70.1 -t_5.1% (n =4, PcO.025) and 78.2t_h.l% (n =4, P < 0.001) did not change significantly the
acetylcholine-induced contractions (110.8 rt: 11.6%, n =4 and 100.3 f 3.5%. n = 4 of control, respectively, see Fig. 2). Bicuculline methiodide (10-5P2 x lo-’ M) immediately restored the contractions depressed by 10e4 M GABA (69.7 + 9.5% inhibition, n = 4, P < 0.01) or 10e5 M (-)-baclofen (75.9 + 5.9% inhibition, n = 4, P < O.OOS), see also Figure 2 and Table I. The inhibitory effect of 10-6M noradrenaline (96.0 I_+2.3% inhibition, n = 4, P < 0.001) was also fully reversed by bicuculline (see Table 1). The antagonism of the effect of GABA by bicuculline, assessed after 30 or 60 min exposure to the antagonist, was apparently non-competitive. The control response to 2.9 x lo-‘M exogenous acetylcholine was as large as 0.48 It 0.08 g (n = 3); after 5 min exposure to 10e5 M bicuculline the
10 Ku”
if&)-+ 8
0.52 1
4
_ 32
ACH
ACH
16x10-ebl
ACETYLCHOLINE i ATROPINE
2 ~10‘~
M
Fig. 1. The inhibitory eff=t of 2 x IO-’ M atropine sulphate on contractions of the guinea-pig taenia-coli elicited by field electrical stimulation and exogenous acetylcholine, respectively. Symbols: 0 contractions elicited by low-frequency (0.5 Hz), short-train (3 shocks) field electrical stimuli (square wave pulses of OSmsec duration, 9V/cm voltage drop); A contractions produced by exogenously administered acetylcholine. Between individual doses single washes were used. The standard dose of acetylcholine is indicated by underlining in the initial dose-response curve.
GABA,
1625
receptors in taenia coli
*CETYLCWO‘IWE
GABA
BICUCULLINE
10-4M
10-5M
t + 10-5M
.
M
. . . . . . . l. .
. . . . ..d...
..*.bb&hA***.&
f4.4
111 x
7.2
dY
t
K”
BACLOFEN
10
ACETYLCNOUNE
BACLOFEN
10-5M
lO+M
BICUCULLINE + lo+
1O’5 M
M
Fig. 2. The inhibitory effect of GABA and (-)-baclofen on field stimulated guinea-pig taenia coli. Upper and middle panek the contractions of 5-4 mm long proximal taenia segments were elicited either by field electrical stimulation (dark circles, short trains, 3 shocks at 0.5 Hz, of square wave pulses of 0.5 msec duration, 9 V/cm voltage drop) or by exogenous acetylcholine (open triangles). Between individual doses of a~tylcholine single washes were used; repeated (5 x ) washout was applied after GABA [upper panel) and (-)-baclofen (middle panel). The standard dose of acetylcholine is indicated by underlining in the initial dose-response curve. Bicuculline was applied as (lS, 9R)-bicuculline methiodide. Lower panel: the inhibitor dose-response curve for GABA and (-)-baclofen. The points represent the mean, vertical lines the SEM, values; number of experiments appears in parenthesis. Symbols: l (-)-baclofen, 0 GABA. same dose produced a contraction amounting to 1.82 f 0.11 g (II = 3, P < 0.05) which was close to the maximal attainable effect of exogenous acetylcholine in these preparations (see also Discussion).
DISCUSSION
Using electrical field- or transmura~ stimulation, either relaxation or contraction (or both) can be
A. 2.
I626 Table
&hAI
et al.
I. The effects of GABA agonists, noradrenaline and (IS, 9R)-bicuculline methiodide in field-stimulated guinea-pig taenia coii preparations
Treatment I. EABA 2. k&w* 3. CABA i BMf 4. ( - )-Baclofen 5.
f - )-Baclofen + EM1
6. Muscimol 7. Noradrenaline
8. Noradrenaline + BMI
Concentrations (W 10-J i&i 10~4-+lo
4
30.3 & 9.5 139.3 * 16.6 138.2 & 7.4
4
24. I k 5.9
4 4
5
10 s 10. %+ lwl lO-5 IO b
10-6-l” IO
No.
5
Response to stimulations (percentage of control)
4
1245t33.6
6 4
114.0 f 4.8 4.0 + 2.3
4
148.5 i 21.4
Statistics* (vs control and each other) P (YScontrol) < 0.01 0.1 > P (YS control) > 0.05 P (I vs 3) c 0.005; P (2 vs 3) n.s. P (vs control) < 0.005; P (I vs 4) n.s. P (4 vs 5) < 0.05; P (2 “S 5) n.s. P (vs control) < 0.05 P (vs control) < 0.001; P (1 vs 7) < 0.05: P i4 vs 7j < 0.02; P 17 vs’8)
*Paired ‘“r”-test was used where comparisons were made with control (100%) values (non-transformed and also in 1 YS3, 4 YS S and 7 vs 8; otherwise, non-paired “f”-test was applied. **BMI = (IS, 9R)-bicuculline methiodide.
obtained in guinea-pig taenia coli preparations (Shim, and Ishi, 1978; Cocks and Burnstock, 1979). The character of the response depends on the general experimental conditions but most of all it appears to be governed by the “intrinsic” tone of the taeniae (Cocks and Burnstock, 1979). With the stimulation parameters and general experimental conditions used in the present experiments, the relaxation response was present in less than one third of cases (8 out of 28 experiments). Even in these cases, the magnitude of the relaxation was always less than l/6 of the contractions. The assumption that the preparations were “low tone” ones, as defined by Cocks and Burnstock (19791, would not offer a full explanation for the low incidence of the relaxation response, since Shimo and Ishi (1978) using stimulation parameters similar to those used here, regularly obtained relaxation. Two further factors can be pointed out as possible explanations. First, although the recording system used was a non-isometric one, it permitted much smaller displacement of the muscle than a real isotonic transducer (Shim0 and Ishi, 1978), or even a soft spring of 1 cm/g compliance (Cocks and Burnstock, 1979). Second, the physiological solution contained twice as much potassium than the one used by Shimo and Ishi (1978); the Ca: Mg ratios were also higher (4.17 vs 3.0). The guinea-pig taenia coli-myenteric plexus appeared to differ from the ileal longitudinal musclemyenteric plexus preparations in two aspects, as far as the GABAergic mechanisms are concerned. Firstly, the GABA-mediated stimulatory mechanisms (Kaplita et al., 1982; Giotti et al., 1983) were much less apparent if they occurred at all. On the other hand, the excitatory transmission upon field stimulation, in contrast to the ileal longitudinal muscle (Kaplita et at., 1982) was potently influenced by inhibitory GABAergic mechanisms, as revealed by the pawerful effect of GABA and (- )-baclofen on contractions induced by nerve stimulation. The GABA receptors involved in this inhibitory action,
data were used)
assessed from the agonist-selectivity, appear to be of type B. The reduction of nerve-mediated contractions, at least under the experimental conditions used, appeared to be due to an inhibitory effect on the liberation of acetylcholine from nerve terminals. These results seem to be at variance with the ones reported by Cocks and Burnstock (1979); they could elicit contractions by field stimulation even in the presence of 10F6 g/ml atropine. However, they used considerably longer trains of stimuli and, although their experiments covered the frequency range of 0.5-5 Hz, it appears from demonstrations that in the majority of cases they used a stimulation frequency of 5 Hz or close to it. It is possible that the longer train of stimuli (especially at relatively higher frequencies) would recruit further excitatory network(s), other than the cholinergic ones, stimulated here by short-train (3 pulses) low-frequency (0.5 Hz) stimuli. The stimulation parameters used here were quite similar to the ones described by Shimo and Ishi (1978); neither these authors nor the present ones were able to detect contractile responses upon transmural- or field-stimulation, in the presence of 2 x 10-‘M atropine, Since it is known that bicuculline in other cholinergic systems is capable of potentiating the effect of acetylchoIine (Miller and McLennan, 19741, possibly, at least in part, due to its cholinesterase-inhibiting action (Svenneby and Roberts, 1973), it is likely that the reversal of the inhibition produced either by GABA agonists or by noradrenaline is related to the potentiation of the effect of endogenously-released acetylcholine by an increase in effective concentrations of acetylcholine (rather than to GAElA* receptor antagonism). It is to be noted, however, that &antis and Kerr (1981) demonstrated that the responses to acetylcholine after the addition of bicuculline did not change signi~cantly. The ineffectiveness of b~cuculline might be due to the instability of bicuculline (as contrasted to bicuculline) in aqueous solutions. The antagonism
GABA, receptors in taenia coli
of the effect of GABA by bicuculline was apparently non-competitive, which is in accord with the nonselectivity of the antagonism. Morphological studies on the neuromuscular junction of the gut (Jessen et ai., 1983) are consistent with the possibility that GABA may serve as a presynaptic modulator in the intestine (Jessen et al., 1983). Neurally-organized relaxation, mediated by GABA, receptors, has been demonstrated in small intestine of the guinea-pig (Giotti et al., 1983), whereas contractions of cholinergic elements elicited by electrical stimulation were only slightly modified by GABA agonists (Kaplita et al., 1982). It is known that GABA is a potent inhibitor of propulsive activity in the colon of the guinea-pig (Krantis et al., 1980); it was demonstrated here that in guinea-pig caecum, GABA, receptors exert a strong inhibitory presynaptic control on cholinergic transmission. Acknowledgement-The
skilled technical assistance of MS Maria Simon is gratefully acknowledged.
REFERENCES
Bowery N. G., Doble A., Hill D. R., Hudson A. L., Shaw J. S., Turnbull M. J. and Warrington R. (1981) Bicuculline-insensitive GABA receptors on autonomic nerve terminals. Eur. J. Pharmac. 71: 53-70. Cocks Th. and Burnstock G. (1979) Effects of neuronal polypeptides on intestinal smooth muscle; a comparison with noradrenergic, noncholinergic nerve stimulation and ATP. Eur. J. Pharmac. 54: 251-259. Giotti A., Luzzi S., Spagnese S. and Zilletti L. (1983)
1621
GABA, and GABAs receptor mediated effects in guineapig ileum. Br. J. Pharmac. 78: 469478. Jessen K. R., Hills J. M., Dennison M. E. and Mirsky R. (1983) y-Am.inobutyrate in guinea-pig large intestine and cultured enteric neurones using pharmacological methods and electron microscopic autoradiography. Neuroscience IO: 1427-1442. Kaplita P. V., Waters D. H. and Triggle D. J. (1982) y-Aminobutyri~ acid action in guinea-pig ileal myenteric plexus. Ear. J. Pharmac. 79: 43-5 1. Kleinrok A. and Kilbinger H. (1983) y-Aminobutyric acid and chohnergic transmission in the guinea-pig ileum. Naunyn-Schmiedebergs
Arch. Pharmac. 322: 216-220.
Krantis A., Costa M., Furness J. B. and Orbach J. (1980) Gamma-aminobutyric acid stimulates inhibitory and excitatory nerves in the guinea-pig intestine. Eur. J. Pharmac. 67: 461-468.
Krantis A. and Kerr D. I. B. (1981) GABA induced excitatory responses in the guinea-pig small intestine are antagonized by bicuculline, picrotoxinin and chloride ion blockers. Naunyn-Schmiedebergs Arch. Pharmac. 317: 257-26
1.
Krantis A., Kerr D. I. 8. and Dennis B. J. (1986) Autoradiographic study of the dist~bution of iH-y-aminobutyrate-a~umulating neural elements in guinea-pig intestine: evidence for a transmitter function of y-aminobutyrate. Neuroscience 17: 1243-1255. Miller J. J. and McLennan H. (1974) The action of bicuculline upon acetylcholine induced excitations of central neurones. Neuropharmacology 13: 785-787. Ong J. and Kerr D. I. B. (1984) Evidence for a physiological role of GABA in the control of guinea-pig intestinal motility. Neurosci. Lett. 50: 339-343. Shimo Y. and Ishi T. (1978) Effects of morphine on non-adrenergic inhibitory responses of the guinea-pig taenia coli. J. Pharm. Pharmac. 30: 596-597. Svenneby G. and Roberts E. (1973) Bicuculline and Nmethylbicu~ulline-com~titive inhibitors of brain acetylchohnesterase. J. Neurochem. 21: 102st026.
I
Vol. 26, No.
11, pp. 1622-1627,
0028s3908/87 $3.00 + 0.00 Copyright Q 1987 PergamonJournals Ltd
1987
Printed in Great Britain. All rights reserved
POTENT INHIBITORY GABA, RECEPTORS IN STIMULATED GUINEA-PIG TAENIA COLI A. Z. R~NAI~, JULIANNAKARDOS’*and M. SIMONYI’ ‘Central Research Institute for Chemistry, Hungarian Academy of Sciences, Budapest, Hungary and 2Department of General Psychology, Eotvos University, Budapest, Hungary (Accepted 18 May 1987)
Scan-~-Aminobutyric acid (GABA, half-maxims inhibitory concentration, IC, = 9.9 PI%%) and (-)-baclofen (IC, =4.5pM) but not 10m4M muscimol, exerted a presynaptic inhibitory effect on choline+ transmission in field-stimulated guinea-pig taenia coli preparations, in virro. The antagonism by 10e5M (IS, 9R)-bicuculline methiodide was not specific for GABA agonists. The results suggest that the GABA receptors involved were of type B. Key words: GABA, receptors, guinea-pig taenia-coli, (-)-baclofen, muscimol, (IS, 9R)-bicuculline methiodide.
There are several lines of evidence that y-aminobutyric acid (GABA) may serve as a neuroregulator (transmitter or modulator) in the mammalian enteric nervous system (Jessen, Hills, Dennison and Mirsky, 1983; Ong and Kerr, 1984). ~-Aminobutyric acid can be demonstrated in enteric neuronal elements; there is a high affinity neuronal uptake mechanism for GABA, a GABA-synthesizing mechanism and Caz+dependent release of GABA upon appropriate stimulation (Jessen et al., 1983; Krantis, Kerr and Dennis, 1986). Though there are several neural networks sensitive to GABA in the peripheral autonomic nervous system (Bowery, Doble, Hill, Hudson, Shaw, Turnbull and Warrington, 1981), the actions of this amino acid transmitter have been studied most extensively in the gut (Krantis, Costa, Furness and Orbach, 1980; Kaplita, Waters and Triggle, 1982; Giotti, Luzzi, Spagnese and Zilletti, 1983; Ong and Kerr, 1984; Krantis ef al., 1986). y-Aminobutyric acid exerts both excitatory and inhibitory actions on the choline@ innervation of intestinal smooth muscle (Bowery et al., 1981; Kaplita et al., 1982; Giotti et al., 1983; Kleinrok and Kilbinger, 1983), it also activates certain noncholinergic inhibitory neurones (Krantis et al., 1980; Ong and Kerr, 1984) and appears to have a physiological role in the control of intestinal motility (Kleinrok and Kilbinget, 1983; Ong and Kerr, 1984). The stimulatory effects in the myenteric plexus of the guinea-pig are mediated by GABA, receptors (Kaplita et al., 1982; Giotti et al., 1983), whereas the relaxant actions are due to the activation of GABA, receptor sites (Bowery et al., 1981; Ong and Kerr, 1984). Although GABA has been shown to exert a *Present address: Fidia-Georgetown Institute for the Neurosciences, 3900 Reservoir Road, Washington D.C. 20007, U.S.A.
potent inhibitory action on spontaneously-working peripheral autonomic nerve-muscle preparations (Giotti et al., 1983; Ong and Kerr, 1984), the effects on the twitch responses or release of transmitter elicited by nerve stimulation did not show maximal inhibition (Bowery et al., 1981; Kaplita et al., 1982). The present investigations were aimed at analysing the effects of various GABAergic agents in nerve (field) stimulated guinea-pig taenia coli preparations. METHODS The chemicals used were: GABA, muscimol, atropine sulphate (Sigma); ascorbic acid (Merck); (-)-baclofen (Ciba-Geigy); acetylcholine chloride, noradrenaline bitarta~te (Serva). (1S, 9R)-bi~uculline methiodide was prepared from (1 S, 9R)-bicuculline (Sigma). All other substances used were of analytical grade. Solutions of noradrenaline contained also 10e4M ascorbic acid; solutions of noradrenaline and (1S, 9R)-bicuculline methiodide (BMI) were kept protected from light. Male guinea-pigs, weighing 250-350 g, were used. For the purpose of isolated organ experiments, S-8 mm long taenia coli segments were prepared. The preparations were set up in Krebs solution aerated with carbogen (O,:CO, = 95: 5) at 37”C, under a resting tension of 0.5 g. The composition of the Krebs solution was as follows (in mM): NaCl 118.0, NaHCO, 25.0, KC1 4.7, KH,PO, 1.2, CaCi, 2.5, MgSO, 0.6 and glucose 11.0, pH 7.4. The taeniae were equilibrated for 45_60min, washed for 10min and the resting tension was readjusted at the end of the first 5 min. The contractions were measured under non-isometric conditions by means of strain-gaugepreamplifier (Rolitron, Hungary), compenzograph recorder (Radelkis, Hungary) system. Field electrical stimulation was applied; 3 pulses were delivered at
1623
1624
A. Z. R&W
0.5 Hz frequency every 3 min. The parameters of individual pulses were as follows: square-wave pulses of 0.5 msec duration, 9 V/cm voltage-drop. Stimulation was started at the beginning of the equilibration period. Whenever the results are given numerically, mean + SEM values are listed with the indication of the number of experiments. RESULTS
y-Aminobutyric acid, in the concentration range of 10-6-10~’ M, affected moderately the tone of taeniacoli preparations as compared either to acetylcholine or electrical stimulation. No relaxation was observed; the contractile responses, if they occurred at all, were smaller than 0.4 g. The field stimulation, using the parameters indicated above, elicited contractions through the activation of cholinergic neurones since the muscle completely abolished by responses were almost 2 x lo-’ M atropine sulphate (Fig. 1). Applied in this concentration, atropine reduced the contractions elicited by low-frequency, short-train electrical stimulation by 96.0 + 2.4% (n = 4, P < 0.005) whereas the contractions produced by exogenously administered 3.2 x IO-‘M acetylcholine were reduced by 99.3 + 0.3% (n = 4, P < 0.005). y-Aminobutyric acid and (-)-baclofen, but not muscimol(6 experiments), when given at an interval of 15-20 min to avoid tachyphylaxis (5 min contact time, 3-5 washes between doses), produced a dose-dependent inhibitory effect on contractions elicited by field stimulation (Fig. 2 lower panel). The half-maximal inhibitory concentration for GABA was 9.9 -f 3.8 nM (n = 7).
ef a/.
In one of seven experiments, 50% inhibition was not attained; in the rest, the maximum values were well above 50%. The IC,, for (-)-baclofen was as high as 4.5 + 1.0 PM (n = 5). The dose-response curves for (-)-baclofen were considerably steeper than those for GABA. Whilst both GABA-agonists depressed nerve-
mediated contractions, they did not affect the responses to exogenously-administered acetylcholine (Fig. 2 upper and middle panels). The standard concentrations of acetylcholine were chosen such as to detect even moderate modifications in the response; under these conditions, concentrations of GABA and (-)-baclofen (1Om4 and lo-‘M, respectively) that reduced electrically-evoked twitches by 70.1 -t_5.1% (n =4, PcO.025) and 78.2t_h.l% (n =4, P < 0.001) did not change significantly the
acetylcholine-induced contractions (110.8 rt: 11.6%, n =4 and 100.3 f 3.5%. n = 4 of control, respectively, see Fig. 2). Bicuculline methiodide (10-5P2 x lo-’ M) immediately restored the contractions depressed by 10e4 M GABA (69.7 + 9.5% inhibition, n = 4, P < 0.01) or 10e5 M (-)-baclofen (75.9 + 5.9% inhibition, n = 4, P < O.OOS), see also Figure 2 and Table I. The inhibitory effect of 10-6M noradrenaline (96.0 I_+2.3% inhibition, n = 4, P < 0.001) was also fully reversed by bicuculline (see Table 1). The antagonism of the effect of GABA by bicuculline, assessed after 30 or 60 min exposure to the antagonist, was apparently non-competitive. The control response to 2.9 x lo-‘M exogenous acetylcholine was as large as 0.48 It 0.08 g (n = 3); after 5 min exposure to 10e5 M bicuculline the
10 Ku”
if&)-+ 8
0.52 1
4
_ 32
ACH
ACH
16x10-ebl
ACETYLCHOLINE i ATROPINE
2 ~10‘~
M
Fig. 1. The inhibitory eff=t of 2 x IO-’ M atropine sulphate on contractions of the guinea-pig taenia-coli elicited by field electrical stimulation and exogenous acetylcholine, respectively. Symbols: 0 contractions elicited by low-frequency (0.5 Hz), short-train (3 shocks) field electrical stimuli (square wave pulses of OSmsec duration, 9V/cm voltage drop); A contractions produced by exogenously administered acetylcholine. Between individual doses single washes were used. The standard dose of acetylcholine is indicated by underlining in the initial dose-response curve.
GABA,
1625
receptors in taenia coli
*CETYLCWO‘IWE
GABA
BICUCULLINE
10-4M
10-5M
t + 10-5M
.
M
. . . . . . . l. .
. . . . ..d...
..*.bb&hA***.&
f4.4
111 x
7.2
dY
t
K”
BACLOFEN
10
ACETYLCNOUNE
BACLOFEN
10-5M
lO+M
BICUCULLINE + lo+
1O’5 M
M
Fig. 2. The inhibitory effect of GABA and (-)-baclofen on field stimulated guinea-pig taenia coli. Upper and middle panek the contractions of 5-4 mm long proximal taenia segments were elicited either by field electrical stimulation (dark circles, short trains, 3 shocks at 0.5 Hz, of square wave pulses of 0.5 msec duration, 9 V/cm voltage drop) or by exogenous acetylcholine (open triangles). Between individual doses of a~tylcholine single washes were used; repeated (5 x ) washout was applied after GABA [upper panel) and (-)-baclofen (middle panel). The standard dose of acetylcholine is indicated by underlining in the initial dose-response curve. Bicuculline was applied as (lS, 9R)-bicuculline methiodide. Lower panel: the inhibitor dose-response curve for GABA and (-)-baclofen. The points represent the mean, vertical lines the SEM, values; number of experiments appears in parenthesis. Symbols: l (-)-baclofen, 0 GABA. same dose produced a contraction amounting to 1.82 f 0.11 g (II = 3, P < 0.05) which was close to the maximal attainable effect of exogenous acetylcholine in these preparations (see also Discussion).
DISCUSSION
Using electrical field- or transmura~ stimulation, either relaxation or contraction (or both) can be
A. 2.
I626 Table
&hAI
et al.
I. The effects of GABA agonists, noradrenaline and (IS, 9R)-bicuculline methiodide in field-stimulated guinea-pig taenia coii preparations
Treatment I. EABA 2. k&w* 3. CABA i BMf 4. ( - )-Baclofen 5.
f - )-Baclofen + EM1
6. Muscimol 7. Noradrenaline
8. Noradrenaline + BMI
Concentrations (W 10-J i&i 10~4-+lo
4
30.3 & 9.5 139.3 * 16.6 138.2 & 7.4
4
24. I k 5.9
4 4
5
10 s 10. %+ lwl lO-5 IO b
10-6-l” IO
No.
5
Response to stimulations (percentage of control)
4
1245t33.6
6 4
114.0 f 4.8 4.0 + 2.3
4
148.5 i 21.4
Statistics* (vs control and each other) P (YScontrol) < 0.01 0.1 > P (YS control) > 0.05 P (I vs 3) c 0.005; P (2 vs 3) n.s. P (vs control) < 0.005; P (I vs 4) n.s. P (4 vs 5) < 0.05; P (2 “S 5) n.s. P (vs control) < 0.05 P (vs control) < 0.001; P (1 vs 7) < 0.05: P i4 vs 7j < 0.02; P 17 vs’8)
*Paired ‘“r”-test was used where comparisons were made with control (100%) values (non-transformed and also in 1 YS3, 4 YS S and 7 vs 8; otherwise, non-paired “f”-test was applied. **BMI = (IS, 9R)-bicuculline methiodide.
obtained in guinea-pig taenia coli preparations (Shim, and Ishi, 1978; Cocks and Burnstock, 1979). The character of the response depends on the general experimental conditions but most of all it appears to be governed by the “intrinsic” tone of the taeniae (Cocks and Burnstock, 1979). With the stimulation parameters and general experimental conditions used in the present experiments, the relaxation response was present in less than one third of cases (8 out of 28 experiments). Even in these cases, the magnitude of the relaxation was always less than l/6 of the contractions. The assumption that the preparations were “low tone” ones, as defined by Cocks and Burnstock (19791, would not offer a full explanation for the low incidence of the relaxation response, since Shimo and Ishi (1978) using stimulation parameters similar to those used here, regularly obtained relaxation. Two further factors can be pointed out as possible explanations. First, although the recording system used was a non-isometric one, it permitted much smaller displacement of the muscle than a real isotonic transducer (Shim0 and Ishi, 1978), or even a soft spring of 1 cm/g compliance (Cocks and Burnstock, 1979). Second, the physiological solution contained twice as much potassium than the one used by Shimo and Ishi (1978); the Ca: Mg ratios were also higher (4.17 vs 3.0). The guinea-pig taenia coli-myenteric plexus appeared to differ from the ileal longitudinal musclemyenteric plexus preparations in two aspects, as far as the GABAergic mechanisms are concerned. Firstly, the GABA-mediated stimulatory mechanisms (Kaplita et al., 1982; Giotti et al., 1983) were much less apparent if they occurred at all. On the other hand, the excitatory transmission upon field stimulation, in contrast to the ileal longitudinal muscle (Kaplita et at., 1982) was potently influenced by inhibitory GABAergic mechanisms, as revealed by the pawerful effect of GABA and (- )-baclofen on contractions induced by nerve stimulation. The GABA receptors involved in this inhibitory action,
data were used)
assessed from the agonist-selectivity, appear to be of type B. The reduction of nerve-mediated contractions, at least under the experimental conditions used, appeared to be due to an inhibitory effect on the liberation of acetylcholine from nerve terminals. These results seem to be at variance with the ones reported by Cocks and Burnstock (1979); they could elicit contractions by field stimulation even in the presence of 10F6 g/ml atropine. However, they used considerably longer trains of stimuli and, although their experiments covered the frequency range of 0.5-5 Hz, it appears from demonstrations that in the majority of cases they used a stimulation frequency of 5 Hz or close to it. It is possible that the longer train of stimuli (especially at relatively higher frequencies) would recruit further excitatory network(s), other than the cholinergic ones, stimulated here by short-train (3 pulses) low-frequency (0.5 Hz) stimuli. The stimulation parameters used here were quite similar to the ones described by Shimo and Ishi (1978); neither these authors nor the present ones were able to detect contractile responses upon transmural- or field-stimulation, in the presence of 2 x 10-‘M atropine, Since it is known that bicuculline in other cholinergic systems is capable of potentiating the effect of acetylchoIine (Miller and McLennan, 19741, possibly, at least in part, due to its cholinesterase-inhibiting action (Svenneby and Roberts, 1973), it is likely that the reversal of the inhibition produced either by GABA agonists or by noradrenaline is related to the potentiation of the effect of endogenously-released acetylcholine by an increase in effective concentrations of acetylcholine (rather than to GAElA* receptor antagonism). It is to be noted, however, that &antis and Kerr (1981) demonstrated that the responses to acetylcholine after the addition of bicuculline did not change signi~cantly. The ineffectiveness of b~cuculline might be due to the instability of bicuculline (as contrasted to bicuculline) in aqueous solutions. The antagonism
GABA, receptors in taenia coli
of the effect of GABA by bicuculline was apparently non-competitive, which is in accord with the nonselectivity of the antagonism. Morphological studies on the neuromuscular junction of the gut (Jessen et ai., 1983) are consistent with the possibility that GABA may serve as a presynaptic modulator in the intestine (Jessen et al., 1983). Neurally-organized relaxation, mediated by GABA, receptors, has been demonstrated in small intestine of the guinea-pig (Giotti et al., 1983), whereas contractions of cholinergic elements elicited by electrical stimulation were only slightly modified by GABA agonists (Kaplita et al., 1982). It is known that GABA is a potent inhibitor of propulsive activity in the colon of the guinea-pig (Krantis et al., 1980); it was demonstrated here that in guinea-pig caecum, GABA, receptors exert a strong inhibitory presynaptic control on cholinergic transmission. Acknowledgement-The
skilled technical assistance of MS Maria Simon is gratefully acknowledged.
REFERENCES
Bowery N. G., Doble A., Hill D. R., Hudson A. L., Shaw J. S., Turnbull M. J. and Warrington R. (1981) Bicuculline-insensitive GABA receptors on autonomic nerve terminals. Eur. J. Pharmac. 71: 53-70. Cocks Th. and Burnstock G. (1979) Effects of neuronal polypeptides on intestinal smooth muscle; a comparison with noradrenergic, noncholinergic nerve stimulation and ATP. Eur. J. Pharmac. 54: 251-259. Giotti A., Luzzi S., Spagnese S. and Zilletti L. (1983)
1621
GABA, and GABAs receptor mediated effects in guineapig ileum. Br. J. Pharmac. 78: 469478. Jessen K. R., Hills J. M., Dennison M. E. and Mirsky R. (1983) y-Am.inobutyrate in guinea-pig large intestine and cultured enteric neurones using pharmacological methods and electron microscopic autoradiography. Neuroscience IO: 1427-1442. Kaplita P. V., Waters D. H. and Triggle D. J. (1982) y-Aminobutyri~ acid action in guinea-pig ileal myenteric plexus. Ear. J. Pharmac. 79: 43-5 1. Kleinrok A. and Kilbinger H. (1983) y-Aminobutyric acid and chohnergic transmission in the guinea-pig ileum. Naunyn-Schmiedebergs
Arch. Pharmac. 322: 216-220.
Krantis A., Costa M., Furness J. B. and Orbach J. (1980) Gamma-aminobutyric acid stimulates inhibitory and excitatory nerves in the guinea-pig intestine. Eur. J. Pharmac. 67: 461-468.
Krantis A. and Kerr D. I. B. (1981) GABA induced excitatory responses in the guinea-pig small intestine are antagonized by bicuculline, picrotoxinin and chloride ion blockers. Naunyn-Schmiedebergs Arch. Pharmac. 317: 257-26
1.
Krantis A., Kerr D. I. 8. and Dennis B. J. (1986) Autoradiographic study of the dist~bution of iH-y-aminobutyrate-a~umulating neural elements in guinea-pig intestine: evidence for a transmitter function of y-aminobutyrate. Neuroscience 17: 1243-1255. Miller J. J. and McLennan H. (1974) The action of bicuculline upon acetylcholine induced excitations of central neurones. Neuropharmacology 13: 785-787. Ong J. and Kerr D. I. B. (1984) Evidence for a physiological role of GABA in the control of guinea-pig intestinal motility. Neurosci. Lett. 50: 339-343. Shimo Y. and Ishi T. (1978) Effects of morphine on non-adrenergic inhibitory responses of the guinea-pig taenia coli. J. Pharm. Pharmac. 30: 596-597. Svenneby G. and Roberts E. (1973) Bicuculline and Nmethylbicu~ulline-com~titive inhibitors of brain acetylchohnesterase. J. Neurochem. 21: 102st026.
I