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Effects of kainic acid analogues on crayfish opener muscle
EFFECTS
OF KAINIC ACID CRAYFISH OPENER H.
Research Laboratory, Pharmaceutical
SHINOZAKI
and IZUMI
ANALOGUES MUSCLE
ON
SHIBUYA
Division. Nippon Kayaku Co.. Ltd.. Shimo. Kita-ku. Tokyo 115.Japan
Summary---The depolarizing action of ten analogues of kainic acid was investigated using the crayfish neuromuscular preparation. The most potent excitant of these compounds was domoic acid. Domoic acid is a heterocyclic amino acid obtained from a seaweed, Chor~driu ~rtwtn. The depolarizing action of bath-applied domoic acid was 5-10 times more potent than that of I.-glutamate. The elcctrophoretic injection of domoic acid produced a siow and small depolarization and the sensitive area roughly overlapped the glutamate sensitive area. Domoic acid will hc available as a useful ph~irm~~colog~~l tool in addition to kainic acid
Since it has been shown that kainic acid has a neuroexcitatory property and a potentiating action on the glu~mate-induced discharges in rat cortical neurones (SHINOZAKI and KOWSHI. 1970), some experimental works showing noticeable neuropharmacological actions of kainic acid have been reported (JOHNSTON. CURTIS,DAVIESand MCCULLOH,1974; OLNEY.RHEE and Ho. 1974; SHINOZAKI and SHIBUYA. 1974b). Although kainic acid had a powerful excitatory action on the mammalian central neurones, the de~larjzing action on crayfish opener muscle fibres was very weak. Recently we found a potent depolarizing action of domoic acid, an analogue of kainic acid (SHINOZAKI and SHIBUYA.1974~). It occurred to us to search for more powerful excitants than the parent compound from among the analogues of kainic acid in the crayfish opener muscle. In crayfish neuromuscular junction. quisqualic acid so far has the highest potency (SHINOZAKIand SHIRUYA,1974a). The crayfish neuromuscular preparation can be used to provide convenient and accurate bioassays of excitatory activity of glutamate related compounds.
RESL;LTSAiVD Stmctur+actit,$ty
The methods used are similar to those reported previously (S~I~~~AKI and SHIBLWA. 1974a). The opener muscle of a walking leg of crayfish was used in all studies. Drugs were either administered by bath application or injected electrophoretically. In some cases a double-barrelled micropipette was used for comparing the effect of the drugs. The test compounds were domoic acid (see below), r-dihydro kainic acid. r-kainic acid lactone. r-N-acetyl kainic acid, ct-kainic acid dimethyl ester, 2-kainic acid methyl ketone, cc-allo-kainic acid, P-kainic acid, /j-Nacetyl kainic acid and fi-N-acetyl kainic acid anhydride, and, as control substances, L-a-kainic acid. quisqualic acid and L-glutamate were used. 14s
of kainic acid wuloguc’s
The most potent exe&ant of the ten com~o~lnds tested was domoic acid. The comparison of the potencies of kainic acid analogues to depolarize the muscle fibre are shown in Table 1. Esterification and N-acetylation of kainic acid caused the loss of depolarizing activity and the allo- or /&isomers of the amino acid had no influence on the crayfish n~uromus~ul~~r junction at concentrations less than IO-%. r-Kainic acid methyl ketone and domoic acid were potent excitants and exerted a potentiating action on the glutamnteinduced depolarization. The fact that z-dihydro kainic acid possesses no depolarizing action but z-kainic acid methyl ketone and domoic acid have a potent action shows that a 4-substituent in kainic acid. particuiarly the double bond. must play an important role as an excitant (JOHNSTON t’r al.. 1974). Kainic acid so Far is known to be the most potent neuroexcitant in the mammalian central nervous system. Domoic acid will bc available as a useful pharmacological tool as well as kainic acid.
Ejkt of dornoic
METHODS
wlatiomhip
DlSCUSSlOlV
acid 011 tlw cra$sh
opr~er musrlr
Domoic acid is an analogue of kainic acid (~IAICXJ. 1959) (Fig. I) and a heterocyclic amino acid obtained from a seaweed, C~z~~~z~~ju ~~~~~~f~z (Kut~ing~ Okamura (Rl?o~lonzrluc~~~~~~). The entire kainic acid molecule is included in the domoic acid molecule and there is a structural similarity to glutamate. A decoction of the seaweed had been used as a folklore drug of the anthelmintics in a solitary island in Japan. It was noticed during drying of the collected seaweed that many flies swarmed round to taste it and al1 the flies that tasted it died of poison. Afterwards the fly-killing principle of the seaweed was proved to be domoic acid itself and the fly-killing action of domoic acid was more potent than that of kainic acid (TAWMOTO, personal communication).
146
H. Table
1.
Depolarizing
and
SHINOZAKI
and
IZLMI
glutamate-potentiating in crayfish opener
SHIBUYA
actions muscle.
of kainic
acid
analogues
Minimum effective concentration Depolarization Potentiation r_-r-kainic acid Domoic acid
(M)
3 x lo‘4
5 x
6 x lo-”
3 x 10-O
lo-’
r-dihydro kainic acid r-kainic acid lactonc -
r-N-acetyl kainic acid r-kainic acid dimethyl ester z-kainic acid methyl ketone r-allo-kainic acid /I-kainic acid P-N-acetyl kainic acid P-N-acetyl kainic acid anhydride quisqualic acid t_-glutamic acid
~C no effect at concentrations
10m5
-. lo-’ 3 x 10-5
less than
Bath application of domoic acid produced a large depolarization of the muscle fibre. The responses of domoic acid were relatively slower than the glutamate response. Domoic acid increased the amplitude of the electrotonic potential produced by constant, hyperpolarizing current pulses in low concentrations which could not depolarize the muscle fibre. When the approximate potency to cause depolarization was compared in terms of the minimum effective dose, domoic acid was 5-10 times more effective than L-glutamate on a molar basis (Fig. 2A), but less powerful than quisqualic acid. In previous experiments (SHINOZAKI and SHIBUYA. 1974a). it was shown that quisqualic acid did not depolarize the muscle fibre which was desensitized by prolonged application of L-glutamate. On the other hand. domoic acid still depolarized the desensitized muscle fibre. This suggests that there are some differences in the mode of action between quisqualic acid and domoic acid. A double barrelled micropipette filled with glutamate and domoic acid was moved along the surface of the muscle fibrc. The injection of domoic acid produced a slow and small depolarization (Fig. 2C). This is in contrast with a faster and larger depolarization produced by iontophoretically applied L-glutamate at a critically adjusted glutamate sensitive spot (Fig. 2D). The domoic acid sensitive area roughly overlapped the glutamate sensitive area (Fig. 2B). Sometimes a small and long-lasting depolarization induced by iontophoretically applied domoic acid was observed near A /
c% ..._. i,
:I H3C
N H
H2CJ’&COOH N H
COOH
COOH
COOH
Domoic
Fig.
-CH,COOH
acid
I. Chemical
~-a-
Kolnlc
structure of domoic kainic acid (B).
acid
3 x 10-6
acid
(A) and L-Z-
1om3M.
the glutamate sensitive spot (Fig. 2E). Similar depolarization was obtained all over the surface of the muscle fibre when domoic acid was iontophoretically applied by long and large current pulses (Fig. 2F). LEA and USHERWOOD(1973) suggested the existence of non-synaptic glutamate receptors with different pharmacological properties in the locust muscle fibre. FELTZ and MALLART (1971) examined the extra-junctional receptors of frog muscle fibres. It was mentioned previously (SHINOZAKI and SHIBUYA, 1974b) that there might be two pharmacologically different receptors responding to L-glutamate in the crayfish neuromuscular junction. It seems possible that both c _,-
50nA
I IrnV
,50nA
F-
_-I
‘m” IOTmsec
Fig. 2. Effects of domoic acid on crayfish opener muscle. A: Dose-response curves of r_-glutamic acid and domoic acid. Abscissa, log concentration. Ordinate. amplitude of the depolarization of the muscle fibre. 0: Domoic acid; 0: L-glutamic acid. B: Distribution of glutamate and domoate potentials along a muscle fibre. 0: Amplitude of glutamate potential obtained by injecting same doses at spots on the surface. 0: Amplitude of domoate potential obtained by injecting same doses from the same spot where the glutamate potential was obtained. Abscissa, distance along the muscle surface. C: Domoate potential at the glutamate sensitive spot. D: Glutamate potential. E: Domoate potential near the glutamate sensitive area. F: A potential induced by a long current pulse of domoic acid at a spot where no glutamate potential could be observed.
Kainic
acid analogues
acid and domoic acid interact with the same receptor. Further investigations on mechanisms of the action of these compounds, including domoic acid may afford new clues to the pharmacological separation or the identification of the population of glutamate receptors. kainic
A~k/loM?I~,dl!~~~ents-TThe authors wish to express their thanks to Professor T. TAKEMOTO for supplying domoic acid. They are indebted to Mr. K. NIIJIMA for supplying test compounds.
JOHNSTON, G. A. R., CURTIS. D. R.. DAVIIS. J. and MCCLLLOH, R. M. (1974). Excitation of spinal interneurones by some conformationally restricted analogues of L-glutamic acid. Nature, Land. 248: 804 805. LEA, T. J. and USHERWOOD, P. N. R. (1973). The site of action of ibotenic acid and the identification of two populations of glutamate receptors on insect muscle fibres. Comp. Gen. Pharmac. 4: 333 350. OLNEY, J. W.. RHW, V. and Ho. 0. L. (1974). Kainic acid: a powerful neurotoxic analogue of glutamate. Bruin Rrs. 77: 507-512. SHINOZAKI, H. and KONISHI, S. (1970). Actions of several anthelmintics and insecticides on rat cortical neuroncs. Brain
REFERENCES
DAIGO. K. (1959). Studies arm&a.
J. Pharmacrut.
on the cbnstituents Sot. Japan
of Chondria
79: 350-368.
FELTZ, A. and MALLART. A. (1971). An analysis of acetylcholine responses of junctional and extra-junctional receptors of frog muscle fibres. J. Physiol. 218: 85-100.
147
Rrs. 24: 368-371.
SHINOZAKI, H. and SHIBUYA. 1. (1974a). A new potent excitant. quisqualic acid: effects on crayfish neuromuscular junction. Nwropharmacoloyy 13: 665-672. SHINOZAKI, H. and SH~WYA, I. (1974b). Potentiation of glutamate induced depolarization by kainic acid in the crayfish opener muscle. Nruropharmacolo~~~. 13: IO57 1065.
SHINOZAKI, H. and SHIRUYA. 1. (1974~). Effects of amino acids on crayfish neuromuscular junction. Folio phurmtrc. ,jap. 70: 178P.
OF KAINIC ACID CRAYFISH OPENER H.
Research Laboratory, Pharmaceutical
SHINOZAKI
and IZUMI
ANALOGUES MUSCLE
ON
SHIBUYA
Division. Nippon Kayaku Co.. Ltd.. Shimo. Kita-ku. Tokyo 115.Japan
Summary---The depolarizing action of ten analogues of kainic acid was investigated using the crayfish neuromuscular preparation. The most potent excitant of these compounds was domoic acid. Domoic acid is a heterocyclic amino acid obtained from a seaweed, Chor~driu ~rtwtn. The depolarizing action of bath-applied domoic acid was 5-10 times more potent than that of I.-glutamate. The elcctrophoretic injection of domoic acid produced a siow and small depolarization and the sensitive area roughly overlapped the glutamate sensitive area. Domoic acid will hc available as a useful ph~irm~~colog~~l tool in addition to kainic acid
Since it has been shown that kainic acid has a neuroexcitatory property and a potentiating action on the glu~mate-induced discharges in rat cortical neurones (SHINOZAKI and KOWSHI. 1970), some experimental works showing noticeable neuropharmacological actions of kainic acid have been reported (JOHNSTON. CURTIS,DAVIESand MCCULLOH,1974; OLNEY.RHEE and Ho. 1974; SHINOZAKI and SHIBUYA. 1974b). Although kainic acid had a powerful excitatory action on the mammalian central neurones, the de~larjzing action on crayfish opener muscle fibres was very weak. Recently we found a potent depolarizing action of domoic acid, an analogue of kainic acid (SHINOZAKI and SHIBUYA.1974~). It occurred to us to search for more powerful excitants than the parent compound from among the analogues of kainic acid in the crayfish opener muscle. In crayfish neuromuscular junction. quisqualic acid so far has the highest potency (SHINOZAKIand SHIRUYA,1974a). The crayfish neuromuscular preparation can be used to provide convenient and accurate bioassays of excitatory activity of glutamate related compounds.
RESL;LTSAiVD Stmctur+actit,$ty
The methods used are similar to those reported previously (S~I~~~AKI and SHIBLWA. 1974a). The opener muscle of a walking leg of crayfish was used in all studies. Drugs were either administered by bath application or injected electrophoretically. In some cases a double-barrelled micropipette was used for comparing the effect of the drugs. The test compounds were domoic acid (see below), r-dihydro kainic acid. r-kainic acid lactone. r-N-acetyl kainic acid, ct-kainic acid dimethyl ester, 2-kainic acid methyl ketone, cc-allo-kainic acid, P-kainic acid, /j-Nacetyl kainic acid and fi-N-acetyl kainic acid anhydride, and, as control substances, L-a-kainic acid. quisqualic acid and L-glutamate were used. 14s
of kainic acid wuloguc’s
The most potent exe&ant of the ten com~o~lnds tested was domoic acid. The comparison of the potencies of kainic acid analogues to depolarize the muscle fibre are shown in Table 1. Esterification and N-acetylation of kainic acid caused the loss of depolarizing activity and the allo- or /&isomers of the amino acid had no influence on the crayfish n~uromus~ul~~r junction at concentrations less than IO-%. r-Kainic acid methyl ketone and domoic acid were potent excitants and exerted a potentiating action on the glutamnteinduced depolarization. The fact that z-dihydro kainic acid possesses no depolarizing action but z-kainic acid methyl ketone and domoic acid have a potent action shows that a 4-substituent in kainic acid. particuiarly the double bond. must play an important role as an excitant (JOHNSTON t’r al.. 1974). Kainic acid so Far is known to be the most potent neuroexcitant in the mammalian central nervous system. Domoic acid will bc available as a useful pharmacological tool as well as kainic acid.
Ejkt of dornoic
METHODS
wlatiomhip
DlSCUSSlOlV
acid 011 tlw cra$sh
opr~er musrlr
Domoic acid is an analogue of kainic acid (~IAICXJ. 1959) (Fig. I) and a heterocyclic amino acid obtained from a seaweed, C~z~~~z~~ju ~~~~~~f~z (Kut~ing~ Okamura (Rl?o~lonzrluc~~~~~~). The entire kainic acid molecule is included in the domoic acid molecule and there is a structural similarity to glutamate. A decoction of the seaweed had been used as a folklore drug of the anthelmintics in a solitary island in Japan. It was noticed during drying of the collected seaweed that many flies swarmed round to taste it and al1 the flies that tasted it died of poison. Afterwards the fly-killing principle of the seaweed was proved to be domoic acid itself and the fly-killing action of domoic acid was more potent than that of kainic acid (TAWMOTO, personal communication).
146
H. Table
1.
Depolarizing
and
SHINOZAKI
and
IZLMI
glutamate-potentiating in crayfish opener
SHIBUYA
actions muscle.
of kainic
acid
analogues
Minimum effective concentration Depolarization Potentiation r_-r-kainic acid Domoic acid
(M)
3 x lo‘4
5 x
6 x lo-”
3 x 10-O
lo-’
r-dihydro kainic acid r-kainic acid lactonc -
r-N-acetyl kainic acid r-kainic acid dimethyl ester z-kainic acid methyl ketone r-allo-kainic acid /I-kainic acid P-N-acetyl kainic acid P-N-acetyl kainic acid anhydride quisqualic acid t_-glutamic acid
~C no effect at concentrations
10m5
-. lo-’ 3 x 10-5
less than
Bath application of domoic acid produced a large depolarization of the muscle fibre. The responses of domoic acid were relatively slower than the glutamate response. Domoic acid increased the amplitude of the electrotonic potential produced by constant, hyperpolarizing current pulses in low concentrations which could not depolarize the muscle fibre. When the approximate potency to cause depolarization was compared in terms of the minimum effective dose, domoic acid was 5-10 times more effective than L-glutamate on a molar basis (Fig. 2A), but less powerful than quisqualic acid. In previous experiments (SHINOZAKI and SHIBUYA. 1974a). it was shown that quisqualic acid did not depolarize the muscle fibre which was desensitized by prolonged application of L-glutamate. On the other hand. domoic acid still depolarized the desensitized muscle fibre. This suggests that there are some differences in the mode of action between quisqualic acid and domoic acid. A double barrelled micropipette filled with glutamate and domoic acid was moved along the surface of the muscle fibrc. The injection of domoic acid produced a slow and small depolarization (Fig. 2C). This is in contrast with a faster and larger depolarization produced by iontophoretically applied L-glutamate at a critically adjusted glutamate sensitive spot (Fig. 2D). The domoic acid sensitive area roughly overlapped the glutamate sensitive area (Fig. 2B). Sometimes a small and long-lasting depolarization induced by iontophoretically applied domoic acid was observed near A /
c% ..._. i,
:I H3C
N H
H2CJ’&COOH N H
COOH
COOH
COOH
Domoic
Fig.
-CH,COOH
acid
I. Chemical
~-a-
Kolnlc
structure of domoic kainic acid (B).
acid
3 x 10-6
acid
(A) and L-Z-
1om3M.
the glutamate sensitive spot (Fig. 2E). Similar depolarization was obtained all over the surface of the muscle fibre when domoic acid was iontophoretically applied by long and large current pulses (Fig. 2F). LEA and USHERWOOD(1973) suggested the existence of non-synaptic glutamate receptors with different pharmacological properties in the locust muscle fibre. FELTZ and MALLART (1971) examined the extra-junctional receptors of frog muscle fibres. It was mentioned previously (SHINOZAKI and SHIBUYA, 1974b) that there might be two pharmacologically different receptors responding to L-glutamate in the crayfish neuromuscular junction. It seems possible that both c _,-
50nA
I IrnV
,50nA
F-
_-I
‘m” IOTmsec
Fig. 2. Effects of domoic acid on crayfish opener muscle. A: Dose-response curves of r_-glutamic acid and domoic acid. Abscissa, log concentration. Ordinate. amplitude of the depolarization of the muscle fibre. 0: Domoic acid; 0: L-glutamic acid. B: Distribution of glutamate and domoate potentials along a muscle fibre. 0: Amplitude of glutamate potential obtained by injecting same doses at spots on the surface. 0: Amplitude of domoate potential obtained by injecting same doses from the same spot where the glutamate potential was obtained. Abscissa, distance along the muscle surface. C: Domoate potential at the glutamate sensitive spot. D: Glutamate potential. E: Domoate potential near the glutamate sensitive area. F: A potential induced by a long current pulse of domoic acid at a spot where no glutamate potential could be observed.
Kainic
acid analogues
acid and domoic acid interact with the same receptor. Further investigations on mechanisms of the action of these compounds, including domoic acid may afford new clues to the pharmacological separation or the identification of the population of glutamate receptors. kainic
A~k/loM?I~,dl!~~~ents-TThe authors wish to express their thanks to Professor T. TAKEMOTO for supplying domoic acid. They are indebted to Mr. K. NIIJIMA for supplying test compounds.
JOHNSTON, G. A. R., CURTIS. D. R.. DAVIIS. J. and MCCLLLOH, R. M. (1974). Excitation of spinal interneurones by some conformationally restricted analogues of L-glutamic acid. Nature, Land. 248: 804 805. LEA, T. J. and USHERWOOD, P. N. R. (1973). The site of action of ibotenic acid and the identification of two populations of glutamate receptors on insect muscle fibres. Comp. Gen. Pharmac. 4: 333 350. OLNEY, J. W.. RHW, V. and Ho. 0. L. (1974). Kainic acid: a powerful neurotoxic analogue of glutamate. Bruin Rrs. 77: 507-512. SHINOZAKI, H. and KONISHI, S. (1970). Actions of several anthelmintics and insecticides on rat cortical neuroncs. Brain
REFERENCES
DAIGO. K. (1959). Studies arm&a.
J. Pharmacrut.
on the cbnstituents Sot. Japan
of Chondria
79: 350-368.
FELTZ, A. and MALLART. A. (1971). An analysis of acetylcholine responses of junctional and extra-junctional receptors of frog muscle fibres. J. Physiol. 218: 85-100.
147
Rrs. 24: 368-371.
SHINOZAKI, H. and SHIBUYA. 1. (1974a). A new potent excitant. quisqualic acid: effects on crayfish neuromuscular junction. Nwropharmacoloyy 13: 665-672. SHINOZAKI, H. and SH~WYA, I. (1974b). Potentiation of glutamate induced depolarization by kainic acid in the crayfish opener muscle. Nruropharmacolo~~~. 13: IO57 1065.
SHINOZAKI, H. and SHIRUYA. 1. (1974~). Effects of amino acids on crayfish neuromuscular junction. Folio phurmtrc. ,jap. 70: 178P.