332
Brain Research, 516 (1990) 332-334 Elsevier
BRES 24085
Is picolinic acid a glycine agonist at strychnine-sensitive receptors? Toshiyuki Tonohiro, Mitsuo Tanabe, Tsugio Kaneko and Nobuyoshi Iwata Biological Research Laboratories, Sankyo Co., Tokyo (Japan) (Accepted 30 January 1990) Key words: Picolinic acid; Glycine; Strychnine; Electrophoresis; Feline spinal interneuron By means of unit recording and electrophoretic application, the effect of picolinic acid on feline spinal interneurons in situ was studied in comparison with glycine. Picolinic acid inhibited neuronal firing in 60% of neurons and in some cases the inhibitory actions were antagonized by strychnine. Inhibition of firing by glycine, which was also strychnine-sensitive,was reduced in case of concomitant administration of picolinic acid. These results suggest that picolinic acid might act as a glycine agonist at strychnine-sensitive receptors. Glycine has been thought to be an inhibitory neurotransmitter in the mammalian central nervous system, especially in the spinal cord and in the brainstem 5'6'15 and it is well known that its inhibitory effects are selectively antagonized by strychnine 7. Recently it has been shown that glycine potentiates N-methyl-D-aspartate (NMDA) receptor responses evoked by N M D A analogs through strychnine-insensitive glycin e receptors 11, which are thought to exist in the cerebral cortex 2'9'11"17 and hippocampus 4'13 as well as in the spinal cord 3'1°. But these receptors are thought to be saturated by endogenous levels of glycine 3,H. So any exogenously applied agonist at this receptor is unlikely to affect N M D A responses. In contrast, glycine agonists at strychnine-sensitive receptors, if any, are expected to be useful as muscle relaxants with little influence upon wakefulness because few receptors of this kind are in the upper part of neuraxis 15. Indeed, glycine itself has been reported to ameliorate spasticity in a human trial, although as much as 3-4 g was administered daily 16. Picolinic acid (2-pyridinecarboxylic acid) is a naturally occurring compound and it can be synthesized from tryptophan in the liver 14. Lapin ~2 reported that picolinic acid inhibited convulsions induced by kynurenine, pentylenetetrazole and strychnine in mice and suggested that it acted on the periphery or on the central nervous system remote from cerebral ventricles. In addition, we observed in preliminary experiments that picolinic acid suppressed cat decerebrate rigidity. So in the present study, effects of picolinic acid on activities of feline spinal interneurons were investigated to elucidate its site of action. The experiments were performed on 12 adult male cats. Surgery was performed during ether anesthesia. The animals were anaemically decerebrated, immobilized
with pancuronium bromide and artificially respired. A bilateral pneumothorax was routinely made. Rectal temperature was monitored and maintained between 36 and 39 °C by a heating pad. Blood pressure was monitored continuously. The laminectomy was performed between L 5 and S3 of the spinal cord, which was covered with a pool of paraffin oil kept at body temperature. For ventral root (cut end of L 7 V R ) and peripheral nerve (great sciatic nerve) stimulation, bipolar silver electrodes were used. Stimulation current varied between 0.1 and 1 mA (duration 0.05-0.1 ms). Recordings began more than 4 h after cessation of ether inhalation. The spinal cord was penetrated at the entry zone of the dorsal roots. Extracellular recordings from single neurons in L7-S 1 segments were obtained with a central barrel (3 M NaC1) of a 7-barrelled micropipette. In some cases, a carbon filament (7/~m in diameter) was inserted into this central barrel before pulling the electrode to improve the S/N ratio. In the other cases, the tip was broken to make the overall tip diameter between 5 and 10/~m. The recorded neurons were identified as interneurons because they showed no response to stimulation of ventral roots or they responded repetitively to peripheral stimulation. The extracellular action potentials were displayed on an oscilloscope and the firing rate was detected by a pulse ratemeter (time constant, 0.25 s) and recorded on an ink writing oscillograph. The surrounding barrels were filled with the following solutions: L-glutamic acid monosodium salt (1 or 2 M, pH 7-8), glycine (0.5 M, pH 4.5), strychnine HC1 (2 mM in 165 mM NaCI, pH 3.4) and picolinic acid (0.1 M, pH 10 or 4.5). Glycine, strychnine and picolinic acid (pH 4.5) were ejected electrophoretically as a cation, and glutamic acid and picolinic acid (pH
Correspondence: T. Tonohiro, Biological Research Laboratories, Sankyo Co., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140, Japan. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
333 TABLE I
Inhibitory effects of picolinic acid and glycine on spinal interneurons More than 20% reduction of firing rates was taken as effective inhibition. Data are expressed as mean ± S.E.M.
Gly20nA PA 50nA
Percentageinhibition of firing rates in inhibited cells (number of cells inhibited~tested)
Picolinic acid a Glycine b
Glutamate-evoked firing
Spontaneous firing
53 ± 6 (20/30) 94 ± 5 (13/14)
82 ± 9 (11/26) 86 ± 5 (27/27)
a Ejection current 50-100 nA; 87 + 2 nA (mean + S.E.M.); b Ejection current 5-100 nA; 44 + 5 nA (mean + S.E.M.).
10.0) as an anion. There was no appreciable difference between the effects of picolinic acid of either pH, so for convenience the results of this compound have been combined. Fifty-four spinal interneurons from 12 cats were recorded from extracellularly, and effects of electrophoretically applied picolinic acid and/or glycine on their firing, which was spontaneous or chemically activated by periodically applied L-glutamate (10-50 nA), were investigated. More than 20% reduction of firing rates was taken as effective inhibition. Percent inhibition of firing rates in inhibited cells are summarized in Table I. Electrophoretically administered picolinic acid (87 + 2 nA; mean ___ S.E.M., for 1-5 min) inhibited chemically evoked firing in 67% of neurons (20/30 neurons tested, for example see Fig. 1). In contrast, similarly applied glycine (44 + 5 nA, up to 1 min) inhibited chemically evoked firing in 93% of neurons (13/14). As for spontaneous firing, picolinic acid inhibited 42% of neurons
11oo
_
--
,,..
~
__/'%
,
410
Gly:O.5M, PA:O.IM
Fig. 2. Inhibitory effect of picolinic acid on glycine inhibitory responses of a spinal interneuron. This neuron fired rather constantly at about 16 spikes/s. Glycine (Gly, 20 nA) was periodically applied as indicated by the shorter bars. Picolinic acid (PA, 50 nA) was applied for the longer period as indicated. Upper and lower traces are continuous recordings of the firing rate.
(11/26), whereas glycine inhibited 100% of neurons (27/27). In total, neuronal discharges, which were spontaneous or chemically evoked, were inhibited in 60% of neurons (28/47) by picolinic acid and in 97% of neurons (33/34) by glycine. Unlike instantaneous responses to glycine (Fig. 2), picolinic acid developed inhibition rather slowly and its inhibitory effects remained after cessation of application (Fig. 1) except in a few cases (Fig. 3). Moreover, a larger current was required for picolinic acid to inhibit neuronal firing than for glycine (85 + 2 vs 43 + 5 nA, mean + S.E.M.). In 4 spontaneously firing cells, effects of picolinic acid on inhibitory responses to periodically applied glycine were studied. In 2 out of these neurons, inhibitory responses to glycine were remarkably decreased by prolonged application of picolinic acid (40-50 nA), which by itself caused slight or little inhibition of spontaneous firing (Fig. 2). In another neuron, where picolinic acid (80 nA) inhibited spontaneous firing almost completely, inhibitory responses to glycine were found to be decreased when tested after spontaneous firing was almost recovered. In the remaining one neuron, picolinic acid
1
..... ~
.t"°°
' i
1-oO,,
PA lOOnA r',, ~, ....
r-
lmin
G 50nA __~.
' ,
,,~,___~.
']
mifl
Hz
t
;I
PA 70nA StrychlOOnA
G:2M,
PA:O.1M
lmin
Fig. 1. Inhibitory effect of picolinic acid on the glutamate-activated firing rate of a spinal interneuron. Glutamate (G, 50 nA) was periodically applied as indicated by the shorter bars. Picolinic acid (PA, 100 nA) was applied for the longer period as indicated. Upper, middle and lower traces are continuous recordings of the firing rates.
PA: 0.1M,
Strych: 2mM
Fig. 3. Antagonistic action of strychnine on picolinic acid inhibitory responses of a spinal interneuron. Picolinic acid (PA, 70 nA) was applied for the period of time indicated by the solid bars. Strychnine (Strych, 100 nA) was applied for the period of time indicated by the broken bar.
334 (80-100 nA) failed to affect spontaneous discharges or responses to glycine. Inhibitory actions of picolinic acid on neuronal discharges were challenged by strychnine and antagonism was successfully observed in 3 out of 6 neurons tested, where 80-100 n A of strychnine was used. A n example of this antagonism is shown in Fig. 3. In this and another neuron, inhibitory effects of picolinic acid on spontaneous firing were reduced. In the third, inhibition of glutamate responses by picolinic acid was antagonized by strychnine. In contrast, inhibitory responses to glycine were abolished by strychnine in 8 out of 12 neurons tested. Electrophoretically applied picolinic acid inhibited neuronal discharges in 60% of spinal interneurons. Picolinic acid, although applied at a larger current, resulted in a smaller reduction of firing than glycine (Table I). Therefore, inhibitory effects of picolinic acid seem to be weaker than those of glycine. In addition, out of 27 neurons where glycine and picolinic acid were tested on the same neurons, 26 cells were inhibited by glycine, whereas only 10 cells were inhibited by picolinic acid. So the possibility cannot be ruled out that picolinic acid predominantly inhibits certain types of spinal interneurons. Picolinic acid reduced inhibitory responses to glycine in 3 out of 4 neurons tested, which suggests that it interacts with glycine receptor complexes. There are reportedly two types of glycine receptors, strychninesensitive 5'6'15 and -insensitive ones 3'1°, in the spinal cord. The fact that the inhibitory action was antagonized by strychnine indicates that this action is mediated by
activation of strychnine-sensitive inhibitory glycine receptors. Strychnine has been recently reported to block NMDA-activated cationic channels in rat cortical neurons, sparing glycine potentiation of N M D A responses 1. But such an action of strychnine can be ruled out because blocking of the cationic channel leads to inhibition of neurons. In neurons where strychnine failed to affect the inhibitory action of picolinic acid, the amount of strychnine around the recorded neuron was probably insufficient. This is possible because in 33% of neurons, similarly applied strychnine failed to antagonize glycine responses. Taken altogether, picolinic acid is likely to act as an agonist at strychnine-sensitive glycine receptors in spinal interneurons with relatively lower intrinsic activity than glycine. Apart from strychnine-sensitive glycine receptors, antagonism of glycine potentiation of N M D A receptors is conceivably able to explain inhibitory responses of picolinic acid in this study, because these glycine receptors may be saturated by endogenous levels of glycine in the cerebrospinal fluid TM. But Huettner 1° has reported, using primary cultures of rat neurons, that indole2-carboxylic acid, but not picolinic acid, inhibits potentiation of N M D A responses by glycine. So the inhibitory effect of picolinic acid, which was antagonized by strychnine in this study, is unlikely to be mediated by inactivation of strychnine-insensitive glycine receptors. Further studies are necessary to clarify the actual mechanism of the action of picolinic acid. However, the present experiments provide a possibility that picolinic acid might act as a glycine agonist, although weak, at strychnine-sensitive receptors in the spinal cord.
1 Bertolino, M. and Vicini, S., Voltage-dependent block by strychnine of N-methyl-D-aspartic acid-induced cationic channels in rat cortical neurons in culture, Mol. Pharmacol., 34 (1988) 98-103. 2 Bertolino, M., Vicini, S., Mazetta, J. and Costa, E., Phencyclidine and glycine modulates NMDA-activated high conductance cationic channels by acting at different sites, Neurosci. Lea., 84 (1988) 351-355. 3 Birch, P.J., Grossman, C.J. and Hayes, A.G., Kynurenic acid antagonises responses to NMDA via an action at the strychnineinsensitive glycine receptor, Eur. J. Pharmacol., 154 (1988) 85-87. 4 Bonhaus, D.W., Burge, B.C. and McNamara, J.O., Biochemical evidence that glycine allosterically regulates an NMDA receptorcoupled ion channel, Eur. J. Pharmacol., 142 (1987) 489-490. 5 Curtis, D.R., Hosli, L. and Johnston, G.A.R., A pharmacological study of the depression of spinal neurones by glycine and related amino acids, Exp. Brain Res., 6 (1968) 1-18. 6 Curtis, D.R., Hosli, L., Johnston, G.A.R. and Johnston, I.H., The hyperpolarization of spinal motoneurones by glycine and related amino acids, Exp. Brain Res., 5 (1968) 235-258. 7 Curtis, D.R., Duggan, A.W. and Johnston, G.A.R., The specificity of strychnine as a glycine antagonist in the mammalian spinal cord, Exp. Brain Res., 12 (1971) 547-565. 8 Ferrano, T.N. and Hare, T.A., Free and conjugated amino acids in human CSF: influence of age and sex, Brain Research, 388 (1985) 53-60.
9 Fletcher, E.J. and Lodge, D., Glycine reverses antagonism of N-methyl-D-aspartate (NMDA) by 1-hydroxy-3-aminopyrrolidone-2 (HA-966) but not by D-2-amino-5-phosphonovalerate (D-AP5) on rat cortical slices, Eur. J. Pharmacol., 151 (1988) 161-162. 10 Huettner, J.E., Indole-2-carboxylic acid: a competitive antagonist of potentiation by glycine at the NMDA receptor, Science, 243 (1989) 1611-1613. 11 Johnson, J.W. and Ascher, P., Glycine potentiates the NMDA response in cultured mouse brain neurons, Nature (Lond.), 325 (1987) 529-531. 12 Lapin, I.P., Antagonism of kynurenine-induced seizures by picolinic, kynurenic and xanthurenic acids, J. Neural Transm., 56 (1983) 177-185. 13 Mayer, M.L., Vyklicky, L. and Clements, J., Regulation of NMDA receptor desensitization in mouse hippocampal neurons by glycine, Nature (Lond.), 338 (1989) 425-427. 14 Mehler, A.H. and Everette, L.M., Studies with carboxy-labeled 3-hydroxy-anthranilic and picolinic acids in vivo and in vitro, J. Biol. Chem., 223 (1956) 449-455. 15 Snyder, S.H., The glycine synaptic receptor in the mammalian central nervous system, Br. J. Pharmacol., 53 (1975) 473-484. 16 Stern, P. and Bokonji6, R., Glycine therapy in 7 cases of spasticity, Pharmacology, 12 (1974) 117-119. 17 Wong, E.H.E, Knight, A.R. and Ranson, R., Glycine modulates [3H]MK-801 binding to the NMDA receptor in rat brain, Eur. J. Pharmacol., 142 (1987) 487-488.
Brain Research, 516 (1990) 332-334 Elsevier
BRES 24085
Is picolinic acid a glycine agonist at strychnine-sensitive receptors? Toshiyuki Tonohiro, Mitsuo Tanabe, Tsugio Kaneko and Nobuyoshi Iwata Biological Research Laboratories, Sankyo Co., Tokyo (Japan) (Accepted 30 January 1990) Key words: Picolinic acid; Glycine; Strychnine; Electrophoresis; Feline spinal interneuron By means of unit recording and electrophoretic application, the effect of picolinic acid on feline spinal interneurons in situ was studied in comparison with glycine. Picolinic acid inhibited neuronal firing in 60% of neurons and in some cases the inhibitory actions were antagonized by strychnine. Inhibition of firing by glycine, which was also strychnine-sensitive,was reduced in case of concomitant administration of picolinic acid. These results suggest that picolinic acid might act as a glycine agonist at strychnine-sensitive receptors. Glycine has been thought to be an inhibitory neurotransmitter in the mammalian central nervous system, especially in the spinal cord and in the brainstem 5'6'15 and it is well known that its inhibitory effects are selectively antagonized by strychnine 7. Recently it has been shown that glycine potentiates N-methyl-D-aspartate (NMDA) receptor responses evoked by N M D A analogs through strychnine-insensitive glycin e receptors 11, which are thought to exist in the cerebral cortex 2'9'11"17 and hippocampus 4'13 as well as in the spinal cord 3'1°. But these receptors are thought to be saturated by endogenous levels of glycine 3,H. So any exogenously applied agonist at this receptor is unlikely to affect N M D A responses. In contrast, glycine agonists at strychnine-sensitive receptors, if any, are expected to be useful as muscle relaxants with little influence upon wakefulness because few receptors of this kind are in the upper part of neuraxis 15. Indeed, glycine itself has been reported to ameliorate spasticity in a human trial, although as much as 3-4 g was administered daily 16. Picolinic acid (2-pyridinecarboxylic acid) is a naturally occurring compound and it can be synthesized from tryptophan in the liver 14. Lapin ~2 reported that picolinic acid inhibited convulsions induced by kynurenine, pentylenetetrazole and strychnine in mice and suggested that it acted on the periphery or on the central nervous system remote from cerebral ventricles. In addition, we observed in preliminary experiments that picolinic acid suppressed cat decerebrate rigidity. So in the present study, effects of picolinic acid on activities of feline spinal interneurons were investigated to elucidate its site of action. The experiments were performed on 12 adult male cats. Surgery was performed during ether anesthesia. The animals were anaemically decerebrated, immobilized
with pancuronium bromide and artificially respired. A bilateral pneumothorax was routinely made. Rectal temperature was monitored and maintained between 36 and 39 °C by a heating pad. Blood pressure was monitored continuously. The laminectomy was performed between L 5 and S3 of the spinal cord, which was covered with a pool of paraffin oil kept at body temperature. For ventral root (cut end of L 7 V R ) and peripheral nerve (great sciatic nerve) stimulation, bipolar silver electrodes were used. Stimulation current varied between 0.1 and 1 mA (duration 0.05-0.1 ms). Recordings began more than 4 h after cessation of ether inhalation. The spinal cord was penetrated at the entry zone of the dorsal roots. Extracellular recordings from single neurons in L7-S 1 segments were obtained with a central barrel (3 M NaC1) of a 7-barrelled micropipette. In some cases, a carbon filament (7/~m in diameter) was inserted into this central barrel before pulling the electrode to improve the S/N ratio. In the other cases, the tip was broken to make the overall tip diameter between 5 and 10/~m. The recorded neurons were identified as interneurons because they showed no response to stimulation of ventral roots or they responded repetitively to peripheral stimulation. The extracellular action potentials were displayed on an oscilloscope and the firing rate was detected by a pulse ratemeter (time constant, 0.25 s) and recorded on an ink writing oscillograph. The surrounding barrels were filled with the following solutions: L-glutamic acid monosodium salt (1 or 2 M, pH 7-8), glycine (0.5 M, pH 4.5), strychnine HC1 (2 mM in 165 mM NaCI, pH 3.4) and picolinic acid (0.1 M, pH 10 or 4.5). Glycine, strychnine and picolinic acid (pH 4.5) were ejected electrophoretically as a cation, and glutamic acid and picolinic acid (pH
Correspondence: T. Tonohiro, Biological Research Laboratories, Sankyo Co., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140, Japan. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
333 TABLE I
Inhibitory effects of picolinic acid and glycine on spinal interneurons More than 20% reduction of firing rates was taken as effective inhibition. Data are expressed as mean ± S.E.M.
Gly20nA PA 50nA
Percentageinhibition of firing rates in inhibited cells (number of cells inhibited~tested)
Picolinic acid a Glycine b
Glutamate-evoked firing
Spontaneous firing
53 ± 6 (20/30) 94 ± 5 (13/14)
82 ± 9 (11/26) 86 ± 5 (27/27)
a Ejection current 50-100 nA; 87 + 2 nA (mean + S.E.M.); b Ejection current 5-100 nA; 44 + 5 nA (mean + S.E.M.).
10.0) as an anion. There was no appreciable difference between the effects of picolinic acid of either pH, so for convenience the results of this compound have been combined. Fifty-four spinal interneurons from 12 cats were recorded from extracellularly, and effects of electrophoretically applied picolinic acid and/or glycine on their firing, which was spontaneous or chemically activated by periodically applied L-glutamate (10-50 nA), were investigated. More than 20% reduction of firing rates was taken as effective inhibition. Percent inhibition of firing rates in inhibited cells are summarized in Table I. Electrophoretically administered picolinic acid (87 + 2 nA; mean ___ S.E.M., for 1-5 min) inhibited chemically evoked firing in 67% of neurons (20/30 neurons tested, for example see Fig. 1). In contrast, similarly applied glycine (44 + 5 nA, up to 1 min) inhibited chemically evoked firing in 93% of neurons (13/14). As for spontaneous firing, picolinic acid inhibited 42% of neurons
11oo
_
--
,,..
~
__/'%
,
410
Gly:O.5M, PA:O.IM
Fig. 2. Inhibitory effect of picolinic acid on glycine inhibitory responses of a spinal interneuron. This neuron fired rather constantly at about 16 spikes/s. Glycine (Gly, 20 nA) was periodically applied as indicated by the shorter bars. Picolinic acid (PA, 50 nA) was applied for the longer period as indicated. Upper and lower traces are continuous recordings of the firing rate.
(11/26), whereas glycine inhibited 100% of neurons (27/27). In total, neuronal discharges, which were spontaneous or chemically evoked, were inhibited in 60% of neurons (28/47) by picolinic acid and in 97% of neurons (33/34) by glycine. Unlike instantaneous responses to glycine (Fig. 2), picolinic acid developed inhibition rather slowly and its inhibitory effects remained after cessation of application (Fig. 1) except in a few cases (Fig. 3). Moreover, a larger current was required for picolinic acid to inhibit neuronal firing than for glycine (85 + 2 vs 43 + 5 nA, mean + S.E.M.). In 4 spontaneously firing cells, effects of picolinic acid on inhibitory responses to periodically applied glycine were studied. In 2 out of these neurons, inhibitory responses to glycine were remarkably decreased by prolonged application of picolinic acid (40-50 nA), which by itself caused slight or little inhibition of spontaneous firing (Fig. 2). In another neuron, where picolinic acid (80 nA) inhibited spontaneous firing almost completely, inhibitory responses to glycine were found to be decreased when tested after spontaneous firing was almost recovered. In the remaining one neuron, picolinic acid
1
..... ~
.t"°°
' i
1-oO,,
PA lOOnA r',, ~, ....
r-
lmin
G 50nA __~.
' ,
,,~,___~.
']
mifl
Hz
t
;I
PA 70nA StrychlOOnA
G:2M,
PA:O.1M
lmin
Fig. 1. Inhibitory effect of picolinic acid on the glutamate-activated firing rate of a spinal interneuron. Glutamate (G, 50 nA) was periodically applied as indicated by the shorter bars. Picolinic acid (PA, 100 nA) was applied for the longer period as indicated. Upper, middle and lower traces are continuous recordings of the firing rates.
PA: 0.1M,
Strych: 2mM
Fig. 3. Antagonistic action of strychnine on picolinic acid inhibitory responses of a spinal interneuron. Picolinic acid (PA, 70 nA) was applied for the period of time indicated by the solid bars. Strychnine (Strych, 100 nA) was applied for the period of time indicated by the broken bar.
334 (80-100 nA) failed to affect spontaneous discharges or responses to glycine. Inhibitory actions of picolinic acid on neuronal discharges were challenged by strychnine and antagonism was successfully observed in 3 out of 6 neurons tested, where 80-100 n A of strychnine was used. A n example of this antagonism is shown in Fig. 3. In this and another neuron, inhibitory effects of picolinic acid on spontaneous firing were reduced. In the third, inhibition of glutamate responses by picolinic acid was antagonized by strychnine. In contrast, inhibitory responses to glycine were abolished by strychnine in 8 out of 12 neurons tested. Electrophoretically applied picolinic acid inhibited neuronal discharges in 60% of spinal interneurons. Picolinic acid, although applied at a larger current, resulted in a smaller reduction of firing than glycine (Table I). Therefore, inhibitory effects of picolinic acid seem to be weaker than those of glycine. In addition, out of 27 neurons where glycine and picolinic acid were tested on the same neurons, 26 cells were inhibited by glycine, whereas only 10 cells were inhibited by picolinic acid. So the possibility cannot be ruled out that picolinic acid predominantly inhibits certain types of spinal interneurons. Picolinic acid reduced inhibitory responses to glycine in 3 out of 4 neurons tested, which suggests that it interacts with glycine receptor complexes. There are reportedly two types of glycine receptors, strychninesensitive 5'6'15 and -insensitive ones 3'1°, in the spinal cord. The fact that the inhibitory action was antagonized by strychnine indicates that this action is mediated by
activation of strychnine-sensitive inhibitory glycine receptors. Strychnine has been recently reported to block NMDA-activated cationic channels in rat cortical neurons, sparing glycine potentiation of N M D A responses 1. But such an action of strychnine can be ruled out because blocking of the cationic channel leads to inhibition of neurons. In neurons where strychnine failed to affect the inhibitory action of picolinic acid, the amount of strychnine around the recorded neuron was probably insufficient. This is possible because in 33% of neurons, similarly applied strychnine failed to antagonize glycine responses. Taken altogether, picolinic acid is likely to act as an agonist at strychnine-sensitive glycine receptors in spinal interneurons with relatively lower intrinsic activity than glycine. Apart from strychnine-sensitive glycine receptors, antagonism of glycine potentiation of N M D A receptors is conceivably able to explain inhibitory responses of picolinic acid in this study, because these glycine receptors may be saturated by endogenous levels of glycine in the cerebrospinal fluid TM. But Huettner 1° has reported, using primary cultures of rat neurons, that indole2-carboxylic acid, but not picolinic acid, inhibits potentiation of N M D A responses by glycine. So the inhibitory effect of picolinic acid, which was antagonized by strychnine in this study, is unlikely to be mediated by inactivation of strychnine-insensitive glycine receptors. Further studies are necessary to clarify the actual mechanism of the action of picolinic acid. However, the present experiments provide a possibility that picolinic acid might act as a glycine agonist, although weak, at strychnine-sensitive receptors in the spinal cord.
1 Bertolino, M. and Vicini, S., Voltage-dependent block by strychnine of N-methyl-D-aspartic acid-induced cationic channels in rat cortical neurons in culture, Mol. Pharmacol., 34 (1988) 98-103. 2 Bertolino, M., Vicini, S., Mazetta, J. and Costa, E., Phencyclidine and glycine modulates NMDA-activated high conductance cationic channels by acting at different sites, Neurosci. Lea., 84 (1988) 351-355. 3 Birch, P.J., Grossman, C.J. and Hayes, A.G., Kynurenic acid antagonises responses to NMDA via an action at the strychnineinsensitive glycine receptor, Eur. J. Pharmacol., 154 (1988) 85-87. 4 Bonhaus, D.W., Burge, B.C. and McNamara, J.O., Biochemical evidence that glycine allosterically regulates an NMDA receptorcoupled ion channel, Eur. J. Pharmacol., 142 (1987) 489-490. 5 Curtis, D.R., Hosli, L. and Johnston, G.A.R., A pharmacological study of the depression of spinal neurones by glycine and related amino acids, Exp. Brain Res., 6 (1968) 1-18. 6 Curtis, D.R., Hosli, L., Johnston, G.A.R. and Johnston, I.H., The hyperpolarization of spinal motoneurones by glycine and related amino acids, Exp. Brain Res., 5 (1968) 235-258. 7 Curtis, D.R., Duggan, A.W. and Johnston, G.A.R., The specificity of strychnine as a glycine antagonist in the mammalian spinal cord, Exp. Brain Res., 12 (1971) 547-565. 8 Ferrano, T.N. and Hare, T.A., Free and conjugated amino acids in human CSF: influence of age and sex, Brain Research, 388 (1985) 53-60.
9 Fletcher, E.J. and Lodge, D., Glycine reverses antagonism of N-methyl-D-aspartate (NMDA) by 1-hydroxy-3-aminopyrrolidone-2 (HA-966) but not by D-2-amino-5-phosphonovalerate (D-AP5) on rat cortical slices, Eur. J. Pharmacol., 151 (1988) 161-162. 10 Huettner, J.E., Indole-2-carboxylic acid: a competitive antagonist of potentiation by glycine at the NMDA receptor, Science, 243 (1989) 1611-1613. 11 Johnson, J.W. and Ascher, P., Glycine potentiates the NMDA response in cultured mouse brain neurons, Nature (Lond.), 325 (1987) 529-531. 12 Lapin, I.P., Antagonism of kynurenine-induced seizures by picolinic, kynurenic and xanthurenic acids, J. Neural Transm., 56 (1983) 177-185. 13 Mayer, M.L., Vyklicky, L. and Clements, J., Regulation of NMDA receptor desensitization in mouse hippocampal neurons by glycine, Nature (Lond.), 338 (1989) 425-427. 14 Mehler, A.H. and Everette, L.M., Studies with carboxy-labeled 3-hydroxy-anthranilic and picolinic acids in vivo and in vitro, J. Biol. Chem., 223 (1956) 449-455. 15 Snyder, S.H., The glycine synaptic receptor in the mammalian central nervous system, Br. J. Pharmacol., 53 (1975) 473-484. 16 Stern, P. and Bokonji6, R., Glycine therapy in 7 cases of spasticity, Pharmacology, 12 (1974) 117-119. 17 Wong, E.H.E, Knight, A.R. and Ranson, R., Glycine modulates [3H]MK-801 binding to the NMDA receptor in rat brain, Eur. J. Pharmacol., 142 (1987) 487-488.
