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Stizolobic acid, a competitive antagonist of the quisqualate-type receptor at the crayfish neuromuscular junction
Brain Research, 451 (1988) 353-356 Elsevier
353
BRE 22927
Stizolobic acid, a competitive antagonist of the quisqualate-type receptor at the crayfish neuromuscular junction H. Shinozaki and M. Ishida Tokyo MetropolitanInstituteof MedicalScience, Tokyo (Japan) (Accepted 23 February 1988)
Key words: Glutamate; Quisqualate; Stizolobic acid; Competitive inhibitor; Crayfish neuromuscular junction
Stizolobic acid and stizolobinic acid are amino acids isolated from a plant, Stizolobium hassjoo. Both amino acids reduced responses to glutamate and quisqualate in a competitive manner at the crayfish neuromuscular junction, without affecting responses to GABA and acromelic acid. Excitatory junctional potentials were decreased in the presence of stizolobic or stizolobinic acid in a concentration dependent manner. Stizolobinicacid was about 5 times less potent than stizolobic acid.
At present, highly specific antagonists for kainateand quisqualate-type receptors are not yet available, although antagonists for N-methyl-D-aspartate (NMDA)-type receptors have been extensively reported 1. In order to elucidate physiological functions of glutamate in the invertebrates and vertebrates, it is necessary to find various types of glutamate antagonists ll. Stizolobic acid (2-amino-3-(6-carboxy-2oxo-2H-pyran-4~yl)propanoic acid) and stizolobinic acid (2-amino-3-(6-carboxy-2-oxo-2H-pyran-3-yl)propanoic acid) are amino acids isolated from a plant, Stizolobium hassjoo 4, which may be structurally envisaged as an excitatory amino acid related compound (Fig. 1). Acromelic acid, which is one of the most potent kainoids (kainate analogs) at the crayfish neuromuscular junction 11A3, possesses a similar structure to stizolobinic acid 7,8. When we examined the action of acromelic acid on the crayfish neuromuscular junction t3 and the rat central nervous system, the chemical structure of stizolobic acid and stizolobinic acid prompted us to examine their possible actions on the glutamatergic system, expecting that these amino acids might be a glutamate inhibitor. The methods used for the electrophysiological ex-
periment at the crayfish neuromuscular junction were similar to those previously described 5. The solution used was a modified van Harreveld's solution containing (mM): NaCI 195, CaCI 2 18, KCI 5.4, Trismaleate buffer (pH 7.4) 10 and glucose 11. Sometimes, the membrane voltage was clamped in order to determine the synaptic current induced by glutamate agonists. The glutamate- or quisqualate-filled micropipette was critically adjusted to a glutamate-sensitive spot showing the greatest amplitude of the glutamate or quisqualate potential, which was induced by a constant amount of glutamate or quisqualate. Excitatory junctional potentials (e.j.p.s) induced by 9 trains of pulses at an interval of 12 ms were also evoked. When the responses were found to be sufficiently stable, stizolobic acid or stizolobinic acid was
HOOC.~O L,~COOH H2~NOCOOH HOOC
NH 2
St~o~b~
acid
S t i z o l o b i n i c acid
Fig. 1. Chemical structure of stizolobic acid and stizolobinic acid.
Correspondence: H. Shinozaki, Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113, Japan. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
354
B
A -~Lr~ Stizolobic acid
-
ir
"~"
~.I
_~.-
_
120nA
_,
~150
2 mV
m c
100 m s e c ~
Stizolobic
0.5
acid Stizolobinic acid
j'-d ..._] 2mV
Kynurenic acid
~
.~j-~
mM
50 maec
1
i o
o
[
,,
0.01
0.1 Dose (nC)
I
1
Fig. 2. A: depression of glutamate potentials and e.j .p.s (induced by 9 trains of pulses at an interval of 12 ms) by stizolobic acid, stizolobinic acid and kynurenate. Traces above the glutamate responses indicate the monitored injection current of glutamate. Kynurenate (1 mM) did not reduce the amplitude of e.j.p.s so much. The numerals represent the concentration of test samples (mM). B: doseresponse curves for quisqualate in the presence and absence of stizolobic acid. Ordinate: amplitudes of synaptic currents induced by
quisqualate in the voltage clamped muscle. Abscissa: logarithmic amounts of iontophoretic quisqualate, The continuous curves were drawn by the method of least-squares accordingto the equation (A = agonist concentration;t = inhibitor concentration,K a andKb = equilibrium constant, }'max= maximal response; m and n = the number of antagonistand agonistmolecule.s, respeetively);y = Ym~[1 + Ka (1 + I~/Kb)/A"], assuming Ka = 0. 0187, Kb = 0.0056, n = 1.62, m = 0.69, and Ymax= 149.6, ©, control; 0, sfizolobicacid 0.3 mM; I , stizolobic acid 1 raM.
added to the bathing solution at various concentrations (0.1-1 mM). Both amino acids did not cause any depolarization of the muscle fiber in the concentration range used. In the presence of these amino acids e.j.p.s and responses to glutamate and quisqualate were reduced in a concentration-dependent manner (Fig. 2) and recovery from the drug action was very rapid after washing the preparation. Stizolobinic acid seemed to be about 5 times less potent than stizolobic acid: the degree of reduction caused by stizolobinic acid (1 mM) of responses to glutamate and quisqualate was almost equivalent to that caused by 0.2 mM stizolobic acid. The input resistance of the muscle fiber, inhibitory junctional potentials and G A B A actions were not affected by stizolobic acid. The synaptic current induced by iontophoretically applied quisqualate was determined in the voltageclamped muscle fiber. Fig. 2B represents dose-response curves for quisqualate in the presence of various doses of stizolobic acid. The dose-response curve shifted in parallel to the higher dose side in the presence of stizolobic acid, suggesting that stizolobic acid reduced the response to quisqualate in a competitive manner. To explore the possibility that stizolobic acid is a competitive antagonist for the glutamate receptor at the crayfish neuromuscular junction, we ex-
amined whether the action of stizolobic acid is usedependent or not. If stizolobic acid would bind with the glutamate receptor as a competitive antagonist, stizolobic acid should not demonstrate a use-dependent action, although, like matrine 6, some non-competitive antagonists did not exhibit a use-dependent action. When stizolobic acid was added to the perfusmg solution, the amplitude of glutamate potentials, evoked every 5 s, was decreased to a steady-state level of about 35% of their control value relatively rapidly (Fig. 3). After a wash in stizolobic acid-free solution the response was restored to its control level very rapidly. After confirming the response to be sufficiently stable, solution containing stizotobic acid (0.5 mM) was reintroduced, but the iontophoretic pulses of glutamate were discontinued until the muscle fiber had been exposed to stizolobic acid for 1 min. The amplitude for the first glutamate,induced potential after resuming the iontophoretic pulses was already reduced to a steady-state level of about 35% of the control, indicating that the action ofstizolobic acid was not use-dependent. Glutamate, quisqualate and some kainoids cause a large depolarization of the crayfish muscle fiber and both glutamate and quisqualate bind with a common receptor at the crayfish neuromuscular junction t4
355
_ ___r~X~_ i / • . . ___~.._ II]t,, , , ,LIIIlnlllll , , , ILI I L /IlL , , ,ILI I I,,my I I I]I J
t
¢
1
2mV
SOmsec
I I [ r l t II q I HI) ~H . I I I I I H ,,111.11; [ l l l ; l l l l l l r f ] r I I I I I , I H III1(111 H I l l H H H
IIIIrl]]l
IIIIIIIIIllllllllh,................,llll!llllllllllllllllllllltlllllllllHIIII Stiz
-6O my Iltllllll ,m,,,~llllllllllllIIIIllllllllI-a5 Stlz I rain
0.5raM
IIIIII H I I I I T H I H I I I f H r I I[I IIII
0.5ram
Fig. 3. Depression of glutamate potentials by stizolobic acid. Glutamate was applied by iontophoresis with pulses at intervals of 5 s. Stizolobic acid (Stiz) was added to the bathing solution for a period indicated by the horizontal bar. Upper traces represent the glutamate potential and the monitored injection current at various times indicated by arrows, and the bottom trace is the membrane potential change recorded with a pen writer using a DC amplifier, demonstrating that stizoiobic acid did not affect the resting membrane potential of the muscle fiber.
but NMDA does not cause a depolarization at all even in higher concentrations than 1 mM, suggesting that the NMDA-type receptor does not exist or function at the crayfish neuromuscular junction. In order to examine the specificity of stizolobic acid to quisqualate-type receptors, actions of stizolobic acid on responses to kainoids were examined. At the crayfish neuromuscular junction, acromelic acid possesses common pharmacological properties t o kainoids and is one of the most potent kainoids 13. Kainic acid is less active than other kainoids 1~ and causes only a slight depolarization of the crayfish muscle fiber 14.
Stizolobic acid 0
10
--
A
GIu
--J ~ --50
--
--
lmin
~
-
L
__] 2 . 5 m V 1rain
Ouis lpM --
Fig. 4. Specific depression of quisqualate or glutamate responses by stizolobic acid. Responses to acromelic acid (Acro; 10/~M), glutamate (Glu; 50/~M) and quisqualate (Quis; 1/zM) were determined in the presence of 0.5 or 1 mM stizolobic acid. Agonists and stizolobic acid were added to the bathing solution at the same time for a period indicated by bars.
Therefore, we tested the action of stizolobic acid on responses to acromelic acid. Fig. 4 represents the action of stizolobic acid on the response to bath-applied acromelic acid, glutamate and quisqualate. Stizolobic acid slightly augmented the sub-maximal response to acromelic acid rather than reduced it, in spite of the fact that stizolobic acid significantly reduced responses to glutamate and quisqualate. To explore the possibility that stizolobic acid may decrease the unit size of e.j.p., a quantum analysis was performed. When the muscle fiber was treated with stizolobic acid, some apparent changes in the extracellular e.j.p, was observed. The size of the average unit size markedly reduced (control 0.24 mV; stizolobic acid (0.5 mM) 0.14 mV) while the quantum content was slightly affected (control 0.47; stizolobic acid (0.5 mM) 0.33). The decay time constant of the tail of extracellular e.j.p.s was slightly decreased from 0.67 + 0.01 (n = 94) to 0.56 + 0.01 ms (n -97). In the invertebrate glutamatergic system, glutamate methyl ester 9 and APB (2-amino-4-phosphonobutyric acid) 2,3 are known as glutamate blockers, and kynurenate and 7-DGG are also expected to be effective. However, their potency is fairly low; even at a concentration of 1 mM, e.j.p.s were not decreased by kynurenate at the crayfish neuromuscular junction (see Fig. 2). It has been known that at this junction responses to quisqualate are reduced by kainoids such as domoic acid and kainic acid 12, which markedly potentiate the glutamate response 15J6. Stizolobic acid did not potentiate the responses to glutamate at all at the crayfish neuromuscular junction. Antagonists of neurotransmitters would act against the transmitter in common manner to all animal species. Therefore, we examined the action of stizolobic acid on the mammalian central nervous system. Contrary to our expectations, stizolobic acid caused a marked depolarizing response extracellularly recorded from the new-born rat spinal cord ventral roots. This depolarizing response was not affected by the existence of Mg 2+ ions or APV (2-amino-5-phosphonovaleric acid) (unpublished observation), suggesting that stizolobic acid is a non-NMDA-type agonist. In the preliminary binding-assay examinations, stizolobic acid seems to block the binding of [3H]kainate rather than [3H]glutamate to the receptor in the frog spinal cord cell membranes (Maruyama et al., unpublished ob-
356 servation). A neurotoxic amino acid, fl-N-oxalyl-L-
dertaken to characterize the neuropharmacological
a,fl-diaminopropionate (fl-ODAP), found in seeds of
profile of stizolobic acid. The purpose of this paper is
Lathyrus sativus, was equipotent with kainic acid as a
to encourage the utilization of this new and potentially valuable compound.
depolarizing agent on flog spinal cord ventral roots, kainic acid and f l - O D A P appearing to act on a common receptor 1°. It is of great interest that some compounds other than kainoids bind with the kainatetype receptor. Further experiments need to be un-
1 Cotman, C.W. and Iversen, L.L., Excitatory amino acids in the brain - - focus on NMDA receptors, Trends Neurosci., 10 (1987) 263-265. 2 Cull-Candy, S.G., Donnellan, J.F., James, R.W. and Lunt, G.G., 2-Amino-4-phosphonobutyric acid as a glutamate antagonist on locust muscle, Nature (Lond.), 262 (1976) 408-409. 3 Dudel, J., Aspartate and other inhibitors of excitatory synaptic transmission in crayfish muscle, Plfiigers Arch. Ges. Physiol., 369 (1977) 7-16. 4 Hattori, S. and Komamine, A., Stizolobic acid: a new amino acid in Stizolobium hassjoo, Nature (Lond.), 183 (1959) 1116-1117. 5 Ishida, M. and Shinozaki, H., Differential effects of diltiazem on glutamate potentials and excitatory junctional potentials at the crayfish neuromuscular junction, J. Physiol. (Lond.), 298 (1980) 301-319. 6 Ishida, M. and Shinozaki, H., Glutamate inhibitory action of matrine at the crayfish neuromuscular junction, Br. J. Pharmacol., 82 (1984) 523-531. 7 Konno, K., Hashimoto, K., Ohfune, Y., Shirahama, H. and Matsumoto, T., Synthesis of acromelic acid A, a toxic principle of Clitocybe acromelalga, Tetrahedron Lett., 27 (1986) 607-610. 8 Konno, K., Shirahama, H. and Matsumoto, T., Isolation and structure of acromelic acid A and B: New kainoids of Clitocybe acromelalga, Tetrahedron Lett., 24 (1983)
The authors wish to thank Prof. H. Shirahama for a generous gift of acromelic acid.
939-942. 9 Lowagie, C. and Gerschenteld, H.M., Glutamate antagonists at a crayfish neuromuscular junction, Nature (Lond.), 248 (1974) 533-535. 10 Pearson, S. and Nunn, P.B, The neurolathyrogen, fl-NoxalyI-L-a,fl-diaminopropionic acid, is a potent agonist at glutamate preferring receptors in the frog spinal cord, Brain Research, 206 (1981) 178-182. 11 Shinozaki, H., Pharmacology of the glutamate receptor, Prog. Neurobiot., 30 (1988)399-435. 12 Shinozaki, H. and tshida. M.. Inhibition of quisqualate responses by domoic or kainic acid in crayfish opener muscle. Brain Research, 109 (1976)435-439. 13 Shinozaki, H.. Ishida, M. and Okamoto, T.. Acrometic acid. a novel excitatory amino acid from a poisonous mushroom: effects on the crayfish neuromuscular junction. Brain Research, 399 (1986) 395-398. 14 Shinozaki, H. and Shibuya, I., A new potent excitant, quisqualic acid: effects on crayfish neuromuscular junction. Neuropharmacology, 13 (1974) 665-672. 15 Shinozaki, H. and Shibuya, I., Potentiation of glutamate induced depolarization by kainic acid in the crayfish opener muscle, Neuropharmacology, 13 (19743 1057-1065. 16 Shinozaki, H. and Shibuya, I.. Effects of kainic acid analogues on crayfish opener muscle. Neuropharmacology, 15 (1976) 145-147.
353
BRE 22927
Stizolobic acid, a competitive antagonist of the quisqualate-type receptor at the crayfish neuromuscular junction H. Shinozaki and M. Ishida Tokyo MetropolitanInstituteof MedicalScience, Tokyo (Japan) (Accepted 23 February 1988)
Key words: Glutamate; Quisqualate; Stizolobic acid; Competitive inhibitor; Crayfish neuromuscular junction
Stizolobic acid and stizolobinic acid are amino acids isolated from a plant, Stizolobium hassjoo. Both amino acids reduced responses to glutamate and quisqualate in a competitive manner at the crayfish neuromuscular junction, without affecting responses to GABA and acromelic acid. Excitatory junctional potentials were decreased in the presence of stizolobic or stizolobinic acid in a concentration dependent manner. Stizolobinicacid was about 5 times less potent than stizolobic acid.
At present, highly specific antagonists for kainateand quisqualate-type receptors are not yet available, although antagonists for N-methyl-D-aspartate (NMDA)-type receptors have been extensively reported 1. In order to elucidate physiological functions of glutamate in the invertebrates and vertebrates, it is necessary to find various types of glutamate antagonists ll. Stizolobic acid (2-amino-3-(6-carboxy-2oxo-2H-pyran-4~yl)propanoic acid) and stizolobinic acid (2-amino-3-(6-carboxy-2-oxo-2H-pyran-3-yl)propanoic acid) are amino acids isolated from a plant, Stizolobium hassjoo 4, which may be structurally envisaged as an excitatory amino acid related compound (Fig. 1). Acromelic acid, which is one of the most potent kainoids (kainate analogs) at the crayfish neuromuscular junction 11A3, possesses a similar structure to stizolobinic acid 7,8. When we examined the action of acromelic acid on the crayfish neuromuscular junction t3 and the rat central nervous system, the chemical structure of stizolobic acid and stizolobinic acid prompted us to examine their possible actions on the glutamatergic system, expecting that these amino acids might be a glutamate inhibitor. The methods used for the electrophysiological ex-
periment at the crayfish neuromuscular junction were similar to those previously described 5. The solution used was a modified van Harreveld's solution containing (mM): NaCI 195, CaCI 2 18, KCI 5.4, Trismaleate buffer (pH 7.4) 10 and glucose 11. Sometimes, the membrane voltage was clamped in order to determine the synaptic current induced by glutamate agonists. The glutamate- or quisqualate-filled micropipette was critically adjusted to a glutamate-sensitive spot showing the greatest amplitude of the glutamate or quisqualate potential, which was induced by a constant amount of glutamate or quisqualate. Excitatory junctional potentials (e.j.p.s) induced by 9 trains of pulses at an interval of 12 ms were also evoked. When the responses were found to be sufficiently stable, stizolobic acid or stizolobinic acid was
HOOC.~O L,~COOH H2~NOCOOH HOOC
NH 2
St~o~b~
acid
S t i z o l o b i n i c acid
Fig. 1. Chemical structure of stizolobic acid and stizolobinic acid.
Correspondence: H. Shinozaki, Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113, Japan. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
354
B
A -~Lr~ Stizolobic acid
-
ir
"~"
~.I
_~.-
_
120nA
_,
~150
2 mV
m c
100 m s e c ~
Stizolobic
0.5
acid Stizolobinic acid
j'-d ..._] 2mV
Kynurenic acid
~
.~j-~
mM
50 maec
1
i o
o
[
,,
0.01
0.1 Dose (nC)
I
1
Fig. 2. A: depression of glutamate potentials and e.j .p.s (induced by 9 trains of pulses at an interval of 12 ms) by stizolobic acid, stizolobinic acid and kynurenate. Traces above the glutamate responses indicate the monitored injection current of glutamate. Kynurenate (1 mM) did not reduce the amplitude of e.j.p.s so much. The numerals represent the concentration of test samples (mM). B: doseresponse curves for quisqualate in the presence and absence of stizolobic acid. Ordinate: amplitudes of synaptic currents induced by
quisqualate in the voltage clamped muscle. Abscissa: logarithmic amounts of iontophoretic quisqualate, The continuous curves were drawn by the method of least-squares accordingto the equation (A = agonist concentration;t = inhibitor concentration,K a andKb = equilibrium constant, }'max= maximal response; m and n = the number of antagonistand agonistmolecule.s, respeetively);y = Ym~[1 + Ka (1 + I~/Kb)/A"], assuming Ka = 0. 0187, Kb = 0.0056, n = 1.62, m = 0.69, and Ymax= 149.6, ©, control; 0, sfizolobicacid 0.3 mM; I , stizolobic acid 1 raM.
added to the bathing solution at various concentrations (0.1-1 mM). Both amino acids did not cause any depolarization of the muscle fiber in the concentration range used. In the presence of these amino acids e.j.p.s and responses to glutamate and quisqualate were reduced in a concentration-dependent manner (Fig. 2) and recovery from the drug action was very rapid after washing the preparation. Stizolobinic acid seemed to be about 5 times less potent than stizolobic acid: the degree of reduction caused by stizolobinic acid (1 mM) of responses to glutamate and quisqualate was almost equivalent to that caused by 0.2 mM stizolobic acid. The input resistance of the muscle fiber, inhibitory junctional potentials and G A B A actions were not affected by stizolobic acid. The synaptic current induced by iontophoretically applied quisqualate was determined in the voltageclamped muscle fiber. Fig. 2B represents dose-response curves for quisqualate in the presence of various doses of stizolobic acid. The dose-response curve shifted in parallel to the higher dose side in the presence of stizolobic acid, suggesting that stizolobic acid reduced the response to quisqualate in a competitive manner. To explore the possibility that stizolobic acid is a competitive antagonist for the glutamate receptor at the crayfish neuromuscular junction, we ex-
amined whether the action of stizolobic acid is usedependent or not. If stizolobic acid would bind with the glutamate receptor as a competitive antagonist, stizolobic acid should not demonstrate a use-dependent action, although, like matrine 6, some non-competitive antagonists did not exhibit a use-dependent action. When stizolobic acid was added to the perfusmg solution, the amplitude of glutamate potentials, evoked every 5 s, was decreased to a steady-state level of about 35% of their control value relatively rapidly (Fig. 3). After a wash in stizolobic acid-free solution the response was restored to its control level very rapidly. After confirming the response to be sufficiently stable, solution containing stizotobic acid (0.5 mM) was reintroduced, but the iontophoretic pulses of glutamate were discontinued until the muscle fiber had been exposed to stizolobic acid for 1 min. The amplitude for the first glutamate,induced potential after resuming the iontophoretic pulses was already reduced to a steady-state level of about 35% of the control, indicating that the action ofstizolobic acid was not use-dependent. Glutamate, quisqualate and some kainoids cause a large depolarization of the crayfish muscle fiber and both glutamate and quisqualate bind with a common receptor at the crayfish neuromuscular junction t4
355
_ ___r~X~_ i / • . . ___~.._ II]t,, , , ,LIIIlnlllll , , , ILI I L /IlL , , ,ILI I I,,my I I I]I J
t
¢
1
2mV
SOmsec
I I [ r l t II q I HI) ~H . I I I I I H ,,111.11; [ l l l ; l l l l l l r f ] r I I I I I , I H III1(111 H I l l H H H
IIIIrl]]l
IIIIIIIIIllllllllh,................,llll!llllllllllllllllllllltlllllllllHIIII Stiz
-6O my Iltllllll ,m,,,~llllllllllllIIIIllllllllI-a5 Stlz I rain
0.5raM
IIIIII H I I I I T H I H I I I f H r I I[I IIII
0.5ram
Fig. 3. Depression of glutamate potentials by stizolobic acid. Glutamate was applied by iontophoresis with pulses at intervals of 5 s. Stizolobic acid (Stiz) was added to the bathing solution for a period indicated by the horizontal bar. Upper traces represent the glutamate potential and the monitored injection current at various times indicated by arrows, and the bottom trace is the membrane potential change recorded with a pen writer using a DC amplifier, demonstrating that stizoiobic acid did not affect the resting membrane potential of the muscle fiber.
but NMDA does not cause a depolarization at all even in higher concentrations than 1 mM, suggesting that the NMDA-type receptor does not exist or function at the crayfish neuromuscular junction. In order to examine the specificity of stizolobic acid to quisqualate-type receptors, actions of stizolobic acid on responses to kainoids were examined. At the crayfish neuromuscular junction, acromelic acid possesses common pharmacological properties t o kainoids and is one of the most potent kainoids 13. Kainic acid is less active than other kainoids 1~ and causes only a slight depolarization of the crayfish muscle fiber 14.
Stizolobic acid 0
10
--
A
GIu
--J ~ --50
--
--
lmin
~
-
L
__] 2 . 5 m V 1rain
Ouis lpM --
Fig. 4. Specific depression of quisqualate or glutamate responses by stizolobic acid. Responses to acromelic acid (Acro; 10/~M), glutamate (Glu; 50/~M) and quisqualate (Quis; 1/zM) were determined in the presence of 0.5 or 1 mM stizolobic acid. Agonists and stizolobic acid were added to the bathing solution at the same time for a period indicated by bars.
Therefore, we tested the action of stizolobic acid on responses to acromelic acid. Fig. 4 represents the action of stizolobic acid on the response to bath-applied acromelic acid, glutamate and quisqualate. Stizolobic acid slightly augmented the sub-maximal response to acromelic acid rather than reduced it, in spite of the fact that stizolobic acid significantly reduced responses to glutamate and quisqualate. To explore the possibility that stizolobic acid may decrease the unit size of e.j.p., a quantum analysis was performed. When the muscle fiber was treated with stizolobic acid, some apparent changes in the extracellular e.j.p, was observed. The size of the average unit size markedly reduced (control 0.24 mV; stizolobic acid (0.5 mM) 0.14 mV) while the quantum content was slightly affected (control 0.47; stizolobic acid (0.5 mM) 0.33). The decay time constant of the tail of extracellular e.j.p.s was slightly decreased from 0.67 + 0.01 (n = 94) to 0.56 + 0.01 ms (n -97). In the invertebrate glutamatergic system, glutamate methyl ester 9 and APB (2-amino-4-phosphonobutyric acid) 2,3 are known as glutamate blockers, and kynurenate and 7-DGG are also expected to be effective. However, their potency is fairly low; even at a concentration of 1 mM, e.j.p.s were not decreased by kynurenate at the crayfish neuromuscular junction (see Fig. 2). It has been known that at this junction responses to quisqualate are reduced by kainoids such as domoic acid and kainic acid 12, which markedly potentiate the glutamate response 15J6. Stizolobic acid did not potentiate the responses to glutamate at all at the crayfish neuromuscular junction. Antagonists of neurotransmitters would act against the transmitter in common manner to all animal species. Therefore, we examined the action of stizolobic acid on the mammalian central nervous system. Contrary to our expectations, stizolobic acid caused a marked depolarizing response extracellularly recorded from the new-born rat spinal cord ventral roots. This depolarizing response was not affected by the existence of Mg 2+ ions or APV (2-amino-5-phosphonovaleric acid) (unpublished observation), suggesting that stizolobic acid is a non-NMDA-type agonist. In the preliminary binding-assay examinations, stizolobic acid seems to block the binding of [3H]kainate rather than [3H]glutamate to the receptor in the frog spinal cord cell membranes (Maruyama et al., unpublished ob-
356 servation). A neurotoxic amino acid, fl-N-oxalyl-L-
dertaken to characterize the neuropharmacological
a,fl-diaminopropionate (fl-ODAP), found in seeds of
profile of stizolobic acid. The purpose of this paper is
Lathyrus sativus, was equipotent with kainic acid as a
to encourage the utilization of this new and potentially valuable compound.
depolarizing agent on flog spinal cord ventral roots, kainic acid and f l - O D A P appearing to act on a common receptor 1°. It is of great interest that some compounds other than kainoids bind with the kainatetype receptor. Further experiments need to be un-
1 Cotman, C.W. and Iversen, L.L., Excitatory amino acids in the brain - - focus on NMDA receptors, Trends Neurosci., 10 (1987) 263-265. 2 Cull-Candy, S.G., Donnellan, J.F., James, R.W. and Lunt, G.G., 2-Amino-4-phosphonobutyric acid as a glutamate antagonist on locust muscle, Nature (Lond.), 262 (1976) 408-409. 3 Dudel, J., Aspartate and other inhibitors of excitatory synaptic transmission in crayfish muscle, Plfiigers Arch. Ges. Physiol., 369 (1977) 7-16. 4 Hattori, S. and Komamine, A., Stizolobic acid: a new amino acid in Stizolobium hassjoo, Nature (Lond.), 183 (1959) 1116-1117. 5 Ishida, M. and Shinozaki, H., Differential effects of diltiazem on glutamate potentials and excitatory junctional potentials at the crayfish neuromuscular junction, J. Physiol. (Lond.), 298 (1980) 301-319. 6 Ishida, M. and Shinozaki, H., Glutamate inhibitory action of matrine at the crayfish neuromuscular junction, Br. J. Pharmacol., 82 (1984) 523-531. 7 Konno, K., Hashimoto, K., Ohfune, Y., Shirahama, H. and Matsumoto, T., Synthesis of acromelic acid A, a toxic principle of Clitocybe acromelalga, Tetrahedron Lett., 27 (1986) 607-610. 8 Konno, K., Shirahama, H. and Matsumoto, T., Isolation and structure of acromelic acid A and B: New kainoids of Clitocybe acromelalga, Tetrahedron Lett., 24 (1983)
The authors wish to thank Prof. H. Shirahama for a generous gift of acromelic acid.
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