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Biogenic monoamine system detected simultaneously in the neural complex of the ascidian, Ciona intestinalis
Camp. Biochem.Physiof. Vol. 103C,No. 3, pp. 489-493, 1992 Printedin Great Britain
0306~4492/92 $5.00+ 0.00 0 1992PergamonPressLtd
BIOGENIC MONOAMINE SYSTEM DETECTED SIMULTANEOUSLY IN THE NEURAL COMPLEX OF THE ASCIDIAN, CIONA INTESTINALIS NAOKUNI TAKEDA Department of Biot~hnology, Research and Development Center, COSMO Research Institute, Satte, Saitama, 340-01, Japan (Tel. 0480-42-2211; Fax. 0480-42-3790) (Received 21 April 1992; accepted for publication 5 June 1992)
Abstract-l. By using a three dimension coulometric HPLC system, a wide range of monoamines and related metabolites were quantified simultaneously in the neural complex of the ascidian, Ciona intestinalis. 2. The main biogenic monoamine metabolic pathways were shown to be catecholamines: Tyrosine-4L-Dopa-Dopamine-Norepinephrine-Normetanephrine and Dopamine-Dihydroxyphenylacetic acid, and indolalkylamines: Tryptophan-5-Hydroxytryptophan-5-Hydroxytryp~mine. 3. In addition to these, Tyramine and Vanillic acid were suggested to be present.
Since its first description by Hancock (1868), the neural complex of ascidians has attracted considerable interest, because of its proposed homology to the vertebrate pituitary (e.g. Dodd and Dodd, 1966; Elwyn, 1937; Willey, 1893). At the present time,
however, the endocrine function of the neural complex has not been established, although vertebratelike neuropeptides have been demonstrated immunohistochemically (Fritsch et al., 1979, 1982; Mizuno and Takeda, 1988a,b; O’Neil et al., 1987; Pestarino, 1982, 1984). On the other hand, the classical monoamine neurotransmitters that are derived from precursor amino acids such as tyrosine and tryptophan and form the basis of this study, have been reported in the neural complex of these animals only occassionally (Erspamer, 1946; Osborne et al., 1979; Welsh and Loveland, 1968). Three dimensional high performance liquid chromatography (HPLC), with coulometric electrochemical detection @CD), has been developed to the field of clinical chemistry only relatively recently (Langlaris et al., 1985; Matson et al., 1984). In contrast to conventional two-dimensional HPLC, three dimensional HPLC system can separate many compounds simultaneousiy with the peak purity by ratio accuracies. The present HPLC system has been applied to neurobiology for the research of the dynamics of biogenic monoamines in the central nervous system of invertebrates (Shimizu and Takeda, 1991; Shimizu et al., 1991; Takeda, 1991; Takeda and Svendsen, 1991; Takeda et al., 1991) and lower vertebrates (Takeda and Takaoka, 1991). The aim of this study was to use three dimensional HPLC to biogenic monoamines and their metabolites in the neural complex of the ascidian, Ciona intestinalis. MATERIALS AND
METHODS
Sample preparation
Specimens of adult Ciona ~~esr~QIis, averaging 10 cm in length, were obtained from the Misaki Marine Biological
Station of Tokyo University. Neural complexes were extirpated (Fig. 1) from 16 specimens and put into the solution consisted of 300 )rl of 0.4 N perchioric acid, 20 ~1 of 50 mM ethylenediaminetetraacetic acid (EDTA) and 10 &L of 0.2 N sodium metabisulphite and homogenized with a Physcotron (Niti-On, Tokyo, Japan). The homogenates and the supernatant collected were then centrifuged at 3000 rpm for 10 min and filtered. Eighteen ~1 samples were injected into the column. Totally, four samples including 16 specimens were used for analysis. HPLC
with ECD
The analytical apparatus used was a three dimensional HPLC system (Coulochem Electrode Array System; ESA, Bedford, MA, U.S.A.). The system consists of a gradient HPLC system and 16 high ~nsitivity coulometric ECD coupled to a compatible computer. The concept and inherent advantage of the multi-electrode HPLC system has been described elsewhere (Matson ef al., 1984). In this study, a reverse phase C,s column (4.6 x 150 mm, NBS column: NIKO Biosci., Tokyo, Japan) was used. Two mobile phases, A and B, were employed in a solvent profile that contained both linear and step gradients. Solvent system A consisted of 0.1 M sodium phosphate and 10 mg/ml of sodium dodecyl sulfate at a pH of 3.35, while mobile phase B consisted of methanol-water (1: 1, V/V) and 50 mg/l sodium dodecylsulfate at a pH 3.45. Together, this protocol permitted simultaneous separation of 24 compounds. The 16 serial electrodes were set in an incremental 60mV array that ranged from 0 to 900 mV. The column and electrodes were maint~ned at a tem~rat~e of 34°C throu~out the runs. Data from each electrode were collected in the computer and stored on the hard disk for later analyses. Typically, each compound would be detected at three e&&odes, with an average ratio in peak height between the electrodes of 1:6: 1. However, the exact ratio was specific for each compound and could be used to establish the purity of the compound present in unknown peaks in the sample that eluted from the column at the same time as a known standard. Unknown peaks found in the sample were matched with standards using both retention time and the oxidation electrode. As nearly all compounds would spread over at least two electrodes, ratio could be calculated and used to compare standards against unknown peaks giving a “ratio accuracy” measurement. A ratio accuracy of 100% meant that the unknown compound detected at a certain retention time spread over the array of electrodes in exactly
489
490
NAOKUNI TAKEDA RESULTS
Fig. 1. Schematic drawing of the ascidian, Ciona intestinalis. Arrow indicates the neural complex. the same way as the standard and was therefore pure. In biological samples there are always some other ions and trace metabolites which shift this ratio slightly and in this study a ratio accuracy of over 70% was taken as being sufficiently close to the pure standard. Final concentration data was calculated by comparing the dominant peak of the standard to the dominant peak of the unknown in the sample. Chemicals The chemicals for analyses were all analytical reagent grade. All compounds were purchased from Sigma (U.S.A.). These compounds are shown in Table 1 along with the specific oxidation potentials and abbreviations. A HPLC of these standards, obtained in the manner outlined above, is presented in Fig. 2.
AND DISCUSSION
The neural complex of Ciona intestinalis consists of the cerebral ganglion and neural gland. The latter is situated ventral to the former and, consequently. is called the hypoganglionic gland. A representative HPLC chromatogram of an extract of the neural complex is shown in Fig. 3. It consists of many peaks which correlate closely with known standard. In addition, however, many unknown peaks were found in higher voltages of the early retention times. Although 24 compounds were analysed (Fig. 1), surely detected compounds were few in number. Levels of detected biogenic monoamines are shown in Table 2. The predominant biogenic monoamines were dopamine (DA), norepinephrine (NE) and 5-hydroxytryptamine (5-HT) with DA being the most abundant biogenic monoamine in the neural complex. NE and dihydroxyphenylacetic acid (DOPAC) were present at comparatively high levels. Vanillic acid (VA) was also suggested to be present. Based on the detected monoamines, their metabolic pathways derived from the precursor amino acid of Tyrosine-4 (TYR4) and Tryptophan (TRP) in the neural complex of Ciona intestinalis were suggested to be as follows: TYR-4-L-DOPA-DA-NE, DA-DOPAC and TYR-4-Tyramine in catecholamines and TRP-5-Hydroxytryptophan (5-HTP)-5HT in indolalkylamines (Fig. 3). In a coulometric system, which differs from an ampelometric system, 100% of the target compounds react with the detector electrodes (Langlaris et al., 1985), resulting in a very sensitive detection. Most biologically-derived samples separated by HPLC system exist in combination with coeluting compounds, and compounds of no immediate importance to the present research. Conventional HPLC systems show the separated mixture in two dimensions, current uersus time. With these systems, what frequently appears as a single resolved chromatographic peak
Table I. List of standard used
Compound
Abbreviation
Oxidation potential (mV) First Second
A: 8: C: D: E: F: G: H: I: J: K: L: M: N: 0: P: Q: R: S: T: U: V: W: X:
VMA NE L-DOPA MHPG OCT E TY R-4 NMN DOPAC DA 30MD 5-HTP MN EPN 5-HIAA NACETS-HT VA TYRA HVA 5-HT 3-MT N-MET MEL TRP
300 180 150 450 620 180 650 480 150 150 450 300 480 120 180 I80 480 620 450 I80 450 300 600 600
Vanillylmandelic acid Norepinephrine L-dopa 3-Methoxy-4-hydroxyphenylethylene glycol Cktopamine Epinephrine Tyrosine-4 Normetanephrine 3,4-dihydroxyphenylacetic acid Dopamine 3-O-methyldopa 5-hydroxytryptophan Metanephrine Epinine S-hydroxyindoleacetic acid N-acetyl-5-hydroxytryptamine Vanillic acid Tyramine Homovanillic acid 5-hydroxytryptamine 3-methoxytyramine N-methyl-5-hydroxytryptamine Melatonin Trvotoehan
600
650
750 700 -
700 700 -
E&genie monoamines in Ciona
5
18
Retention
Time
Fig. 2. A standard chromatogram
491
15
( Min. )
including 24 compounds (see Table 1 for abbreviations).
may actually be composed of two or more coeluting compounds. The HPLC system used in this study employes multiple electrodes in series to provide better resolution in complex samples. Therefore,
it was possible to represent the peak purity by ratio accuracies. The values obtained, therefore, can be decided whether it is acceptable or not by considering ratio accuracies. Although the levels may be
--.__l___n”
FJ
Retention
----___l__
as
12
Time
(
Min.
>
Fig. 3. A representative chromatogram of the neural complex in the ascidian, Cionu intestinalis (fun scale; IOflm).
492
NAOKUNI TAKEDA
Table 2. Biogenic
monoamine levels in the neural ascidian, Ciona inkxinah Concentration (ng/neural complex)
Conmounds (TYR-4) L-DOPA DA NE NMN DOPAC VA TYRA (TRP) 5-HTP 5-HT
the
Ratio
I
2
(0.77:0.80) (0.85:0.76) (0.98: 0.94) (0.82:0.98) (0.85:0.27) (0.90:0.45) (0.98:0.27) (0.25:0.88) (0.90: 0.871 io.91 :o.ooj (0.86:0.73)
125.07 +_ 0.09 0.71 f 0.30 14.46 f 10.85 3.05 i 2.85 0.28 + 0.19 1.40 + 0.67 0.12 + 0.07 0.20? 0.11 24.56 + 16.35 0.07 I 0.03 0.38 k 0.31
(Mean + SD.; N = 4, each contained
complexof
4 neural complexes)
somewhat lower than those measured by conventional HPLC, the values are highly acceptable in accuracy. Monoamines are widespread neuroactive substances in the animal kingdom. In particular, DA and 5-HT have been shown to occur in the nervous systems of all higher animals examined, although their levels and distribution within phyla have varied in a species from each phylum (e.g. Fujii and Takeda, 1988) and with developmental stages or physiological conditions (e.g. Klemm, 1985). As is the case with other invertebrate species, DA and S-HT were the main biogenic monoamines detected in Ciona intestinalis. In ascidians, the presence of 5-HT was first suggested by Erspamer (1946). It is known to be also present in the endostyle of Styelu, Molgulu (De la Torre and Surgeon, 1976; Sakharov and Salimova, 1982) Ciona, Ascidia, Ascidiella and Phallusia (Georges, 1985) and digestive tract of Ciona (Fritsch et al., 1982) and Styela (Pestarino, 1982) and a weak paraformaldehyde-induced fluorescence has been reported in the periphery of the ganglion in swimming tadpoles of Ciona but not in adults (Georges, 1985). In an other ascidian species, Halocynthia roretzi, 5-HT immunoreactivity has been detected in cells in the central nervous system (Fujii and Takeda, 1988). In the present analysis, 5-HT was quantitatively shown to be present in the neural complex of Ciona intestinalis.
The current study was also designed to investigate biogenic monoamines from phylogenetic stand point. TYR-4 3 L-DOPA
TRP * TYRA
+ 5-HTP
+
+
DA 3 NE
5-HT L DOPAC
l
3 VA
Fig. 4. Schematic drawing of biogenic monoamine metabolism
in the neural complex of the ascidian, intestinalis (see Table 1 for abbreviations).
Ciona
Ciona is a representative protochordate which is thought to be a phylogenetic offshot from the deuterostomian line leading from invertebrates to vertebrates. So, it was desirable to make a more thorough research of the possible occurrence of neurotransmitters. Compared to the compounds detected in protostomian species, the metabolic pathways involved in monoamine synthesis in Ciona intestinalis as a deuterostome seem to be simple. For example, epinephrine which is usually present in higher ranking Protostomian species was absent. 5-HIAA. a well known metabolite of 5-HT was also lacking. This may be due to its fixed life without motility in adults. From the view point of ontogeny, analyses of biogenie amines in tadpole stages will be necessary to clarify this problem. Neural control has been suggested in some species such as Diplosoma macdonaldi (Cavey and Cloney, 1976) and Halocynthia roretzi (Ohmori and Sasaki, 1977). As the biogenic monoamines were demonstrated to be present in the neural complex, the role of aminergic neural control in ascidians should be clarified. Acknowledgements-The author wishes to express his thanks to the Misaki Marine Biological Station of Tokyo University for Supplying Ciona. Special thanks are also made to Mr H. Takaoka, head of the present Department, COSMO Research Institute. for his encouragement throughout the course of the present work.
REFERENCES
Cavey M. J. and Cloney R. A. (1976) Ultrastructure and differentiation of ascidian muscle. I. Caudal musculature of the larvae of Diplosoma macdonaldi. Cell Tissue Res. 174, 289-313. De la Torre J. C. and Surgeon J. W. (1976) Histochemical fluorescence of tissue and brain monoamines: results in 18 minutes using the sucrose-phosphateeglyoxylic acid (SPG) method. Neuroscience i, 451-453. _ Dodd J. M. and Dodd M. H. I. (1966) An exoerimental investigation of the supposed pituitary affiniiies of the ascidian neural complex. In Some Contemporary Studies in Marine Science (Edited by Barnes J.). pp. 433-448. Allen and Unwin, London. Elwyn A. (1937) Some stages in the development of the neural complex in Ecteinascidia rurbinata. Bull. Neurol. Inst. N. Y. 6, 1633167. Erspamer V. (1946) Presenza di enteramina o di une sostanza enteraminosimile negli estratti gastrointestinali e splenici dei pesci e negli estratti gastrointerici delle Ascidie. Experientia 2, 369-371. Fritsch H. A. R., Van Noorden S. and Pearse A. D. E. (1979) Localization of somatostatin-, substance P-. and calcitonin-like immunoreactivity in the neural ganglia of Ciona intestinalis L. Cell Tissue Res. 202, 2633274. Fritsch H. A. R., Van Noorden S. and Pearse A. D. E. (1982) Gastro-intestinal and neurohormonal peptides in the alimentary tract and cerebral complex of Ciona intestinalis (Ascidiaceae). Cell Tissue Res..223, 369-402. Fuiii K. and Takeda N. (1988) Phvloeenetic detection of serotonin immunoreactive cells in the central nervous system of invertebrates. Comp. Biochem. Physiol. 89C, 233-239. Georges D. (1985) Presence of cells resembling serotonergic elements in four species of tunicates. Cell Tissue Res. 242, 341-348. Hancock A. (1868) On the anatomy and physiology of the Tunicata. J. Linn. Sot. 9, 309-346.
Biogenic mono amines in Ciona Klemm N. (1985) The distribution of biogenic monoamines in invertebrates. In Neurobiology (Edited by Gilles R. and Balthazart J.), pp. 433-448. Springer, Berlin. Langlaris P. J., Matson W. R. and Bird E. D. (1985) An automated HPLC series coulometric electrochemical detection method for the simultaneous assay of monoamines and metabolites in crude tissue extracts of CSF. In Advances in Neurochemistry (Edited by Chichester J.), pp. 2244232. London Press, London. Matson W. R., Langlais P. J., Volicer L., Gamache. P. H., Bird E. and Mark K. A. (1984) n-Electrode three-dimensional liquid chromatography with electrochemical detection for the determination of neurotransmitters. Clin. Chem. 30, 1477-1488.
Mizuno J. and Takeda N. (1988a) Phylogenetic study of the oxytocin-like immunoreactive system in invertebrates. Comp. Biochem. Physiol. 91A, 733-738.
Mizuno J. and Takeda N. (1988b) Phylogenetic study of the arginine-vasotocin/arginine-vasopressin-like immunoreactive system in invertebrates. Comp. Biochem. Physiol. 91A, 739-747.
Ohmori H. and Sasaki S. (1977) Development of neuromuscular transmission in a larval tunicate. J. Physiol. 269, 221-254.
O’Neil, G. S., Conlon J. M., Deacon C. F. and Thorndyke M. C. (1987) Tachykinins in the cerebral and peripheral nervous system of the ascidian, Ciona intestinalis. Gen. Comp. Endocrinol. 66, 3 14-322.
Osborne N. N , Neuhoff V., Ewers E. and Robertson H. A. (I 979) Putative neurotransmitters in the cerebral ganglia of the tunicate Ciona intestinalis. Camp. Biochem. Physiol. 63C, 209-213.
Pestarino M. (1982) Occurrence of different secretinlike cells in the digestive tract of the ascidian Sfyela plicata (Urochordata, Ascidiacea). Cell Tissue Res. 226, 231-235.
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Pestarino M. (1984) Immunocytochemical demonstration of prolactin-like activity in the neural gland of the ascidian Styela plicaia. Gen. Comp. Endocrinol. 54, 444449. Sakharov D. A. and Salimova N. B. (1982) Serotonincontaining cells in the ascidian endostyle. Experientia 38, 802-803.
Shimizu T., Mihara M. and Takeda N. (1991) High performance liquid chromatography of biogenic amines in the corpus cardiacum of the American cockroach, Peliplaneta americana. J. Chromarogr. 539, 193-197.
Shimizu T. and Takeda N. (1991) Biogenic amine levels in the haemolymph of the cabbage armyworm larvae (Mamesfra brassicae) following injection of octoperminergic insecticides. Z. Naturforsch. 46C, 127-132. Takeda N. (1991) Dynamics of monoamine metabolism in the central nervous system of some terrestrial isopods. In The Bioloav of Terrestrial Isooods (Edited bv Juchault P. and Mocquard J. P.), pp. 195-200. University of Poitiers Press, Poitier, France. Takeda N. and Svendsen C. N. (1991) Monoamine concentrations in Hydra magnipapillata. J. Hydrobiol. 2161217, Y.
_
-
.
549-554.
Takeda N. and Takaoka H. (1991) Dynamics of neurotransmitter metabolism in the central nervous system of frogs. Sot. for Neuroscience 17, 745. Takeda N., Takaoka H., Shimizu T.. Yazawa M. and Yagi S. (1991) Biogenic amine levels in the central nervous system and haemolymph of the silkworm, Bombyx mori. Comp. Biochem. Physiol. lOOC, 677-684.
Welsh J. H. and Loveland R. E. (1968) 5Hydroxytryptamine in the ascidian, Ciona imestinalis L. Comp. Biochem. Physiol. 27, 7199722.
Willey A. (1893) Studies on the Protochordata. II. The development of the neurohypophysial system in Ciona imestinalis and Clavelina lepadiformis. with an account of the origin of the sense-organs in Ascidia men&/a. Quart. J. micr. Sci. 35, 295-316.
0306~4492/92 $5.00+ 0.00 0 1992PergamonPressLtd
BIOGENIC MONOAMINE SYSTEM DETECTED SIMULTANEOUSLY IN THE NEURAL COMPLEX OF THE ASCIDIAN, CIONA INTESTINALIS NAOKUNI TAKEDA Department of Biot~hnology, Research and Development Center, COSMO Research Institute, Satte, Saitama, 340-01, Japan (Tel. 0480-42-2211; Fax. 0480-42-3790) (Received 21 April 1992; accepted for publication 5 June 1992)
Abstract-l. By using a three dimension coulometric HPLC system, a wide range of monoamines and related metabolites were quantified simultaneously in the neural complex of the ascidian, Ciona intestinalis. 2. The main biogenic monoamine metabolic pathways were shown to be catecholamines: Tyrosine-4L-Dopa-Dopamine-Norepinephrine-Normetanephrine and Dopamine-Dihydroxyphenylacetic acid, and indolalkylamines: Tryptophan-5-Hydroxytryptophan-5-Hydroxytryp~mine. 3. In addition to these, Tyramine and Vanillic acid were suggested to be present.
Since its first description by Hancock (1868), the neural complex of ascidians has attracted considerable interest, because of its proposed homology to the vertebrate pituitary (e.g. Dodd and Dodd, 1966; Elwyn, 1937; Willey, 1893). At the present time,
however, the endocrine function of the neural complex has not been established, although vertebratelike neuropeptides have been demonstrated immunohistochemically (Fritsch et al., 1979, 1982; Mizuno and Takeda, 1988a,b; O’Neil et al., 1987; Pestarino, 1982, 1984). On the other hand, the classical monoamine neurotransmitters that are derived from precursor amino acids such as tyrosine and tryptophan and form the basis of this study, have been reported in the neural complex of these animals only occassionally (Erspamer, 1946; Osborne et al., 1979; Welsh and Loveland, 1968). Three dimensional high performance liquid chromatography (HPLC), with coulometric electrochemical detection @CD), has been developed to the field of clinical chemistry only relatively recently (Langlaris et al., 1985; Matson et al., 1984). In contrast to conventional two-dimensional HPLC, three dimensional HPLC system can separate many compounds simultaneousiy with the peak purity by ratio accuracies. The present HPLC system has been applied to neurobiology for the research of the dynamics of biogenic monoamines in the central nervous system of invertebrates (Shimizu and Takeda, 1991; Shimizu et al., 1991; Takeda, 1991; Takeda and Svendsen, 1991; Takeda et al., 1991) and lower vertebrates (Takeda and Takaoka, 1991). The aim of this study was to use three dimensional HPLC to biogenic monoamines and their metabolites in the neural complex of the ascidian, Ciona intestinalis. MATERIALS AND
METHODS
Sample preparation
Specimens of adult Ciona ~~esr~QIis, averaging 10 cm in length, were obtained from the Misaki Marine Biological
Station of Tokyo University. Neural complexes were extirpated (Fig. 1) from 16 specimens and put into the solution consisted of 300 )rl of 0.4 N perchioric acid, 20 ~1 of 50 mM ethylenediaminetetraacetic acid (EDTA) and 10 &L of 0.2 N sodium metabisulphite and homogenized with a Physcotron (Niti-On, Tokyo, Japan). The homogenates and the supernatant collected were then centrifuged at 3000 rpm for 10 min and filtered. Eighteen ~1 samples were injected into the column. Totally, four samples including 16 specimens were used for analysis. HPLC
with ECD
The analytical apparatus used was a three dimensional HPLC system (Coulochem Electrode Array System; ESA, Bedford, MA, U.S.A.). The system consists of a gradient HPLC system and 16 high ~nsitivity coulometric ECD coupled to a compatible computer. The concept and inherent advantage of the multi-electrode HPLC system has been described elsewhere (Matson ef al., 1984). In this study, a reverse phase C,s column (4.6 x 150 mm, NBS column: NIKO Biosci., Tokyo, Japan) was used. Two mobile phases, A and B, were employed in a solvent profile that contained both linear and step gradients. Solvent system A consisted of 0.1 M sodium phosphate and 10 mg/ml of sodium dodecyl sulfate at a pH of 3.35, while mobile phase B consisted of methanol-water (1: 1, V/V) and 50 mg/l sodium dodecylsulfate at a pH 3.45. Together, this protocol permitted simultaneous separation of 24 compounds. The 16 serial electrodes were set in an incremental 60mV array that ranged from 0 to 900 mV. The column and electrodes were maint~ned at a tem~rat~e of 34°C throu~out the runs. Data from each electrode were collected in the computer and stored on the hard disk for later analyses. Typically, each compound would be detected at three e&&odes, with an average ratio in peak height between the electrodes of 1:6: 1. However, the exact ratio was specific for each compound and could be used to establish the purity of the compound present in unknown peaks in the sample that eluted from the column at the same time as a known standard. Unknown peaks found in the sample were matched with standards using both retention time and the oxidation electrode. As nearly all compounds would spread over at least two electrodes, ratio could be calculated and used to compare standards against unknown peaks giving a “ratio accuracy” measurement. A ratio accuracy of 100% meant that the unknown compound detected at a certain retention time spread over the array of electrodes in exactly
489
490
NAOKUNI TAKEDA RESULTS
Fig. 1. Schematic drawing of the ascidian, Ciona intestinalis. Arrow indicates the neural complex. the same way as the standard and was therefore pure. In biological samples there are always some other ions and trace metabolites which shift this ratio slightly and in this study a ratio accuracy of over 70% was taken as being sufficiently close to the pure standard. Final concentration data was calculated by comparing the dominant peak of the standard to the dominant peak of the unknown in the sample. Chemicals The chemicals for analyses were all analytical reagent grade. All compounds were purchased from Sigma (U.S.A.). These compounds are shown in Table 1 along with the specific oxidation potentials and abbreviations. A HPLC of these standards, obtained in the manner outlined above, is presented in Fig. 2.
AND DISCUSSION
The neural complex of Ciona intestinalis consists of the cerebral ganglion and neural gland. The latter is situated ventral to the former and, consequently. is called the hypoganglionic gland. A representative HPLC chromatogram of an extract of the neural complex is shown in Fig. 3. It consists of many peaks which correlate closely with known standard. In addition, however, many unknown peaks were found in higher voltages of the early retention times. Although 24 compounds were analysed (Fig. 1), surely detected compounds were few in number. Levels of detected biogenic monoamines are shown in Table 2. The predominant biogenic monoamines were dopamine (DA), norepinephrine (NE) and 5-hydroxytryptamine (5-HT) with DA being the most abundant biogenic monoamine in the neural complex. NE and dihydroxyphenylacetic acid (DOPAC) were present at comparatively high levels. Vanillic acid (VA) was also suggested to be present. Based on the detected monoamines, their metabolic pathways derived from the precursor amino acid of Tyrosine-4 (TYR4) and Tryptophan (TRP) in the neural complex of Ciona intestinalis were suggested to be as follows: TYR-4-L-DOPA-DA-NE, DA-DOPAC and TYR-4-Tyramine in catecholamines and TRP-5-Hydroxytryptophan (5-HTP)-5HT in indolalkylamines (Fig. 3). In a coulometric system, which differs from an ampelometric system, 100% of the target compounds react with the detector electrodes (Langlaris et al., 1985), resulting in a very sensitive detection. Most biologically-derived samples separated by HPLC system exist in combination with coeluting compounds, and compounds of no immediate importance to the present research. Conventional HPLC systems show the separated mixture in two dimensions, current uersus time. With these systems, what frequently appears as a single resolved chromatographic peak
Table I. List of standard used
Compound
Abbreviation
Oxidation potential (mV) First Second
A: 8: C: D: E: F: G: H: I: J: K: L: M: N: 0: P: Q: R: S: T: U: V: W: X:
VMA NE L-DOPA MHPG OCT E TY R-4 NMN DOPAC DA 30MD 5-HTP MN EPN 5-HIAA NACETS-HT VA TYRA HVA 5-HT 3-MT N-MET MEL TRP
300 180 150 450 620 180 650 480 150 150 450 300 480 120 180 I80 480 620 450 I80 450 300 600 600
Vanillylmandelic acid Norepinephrine L-dopa 3-Methoxy-4-hydroxyphenylethylene glycol Cktopamine Epinephrine Tyrosine-4 Normetanephrine 3,4-dihydroxyphenylacetic acid Dopamine 3-O-methyldopa 5-hydroxytryptophan Metanephrine Epinine S-hydroxyindoleacetic acid N-acetyl-5-hydroxytryptamine Vanillic acid Tyramine Homovanillic acid 5-hydroxytryptamine 3-methoxytyramine N-methyl-5-hydroxytryptamine Melatonin Trvotoehan
600
650
750 700 -
700 700 -
E&genie monoamines in Ciona
5
18
Retention
Time
Fig. 2. A standard chromatogram
491
15
( Min. )
including 24 compounds (see Table 1 for abbreviations).
may actually be composed of two or more coeluting compounds. The HPLC system used in this study employes multiple electrodes in series to provide better resolution in complex samples. Therefore,
it was possible to represent the peak purity by ratio accuracies. The values obtained, therefore, can be decided whether it is acceptable or not by considering ratio accuracies. Although the levels may be
--.__l___n”
FJ
Retention
----___l__
as
12
Time
(
Min.
>
Fig. 3. A representative chromatogram of the neural complex in the ascidian, Cionu intestinalis (fun scale; IOflm).
492
NAOKUNI TAKEDA
Table 2. Biogenic
monoamine levels in the neural ascidian, Ciona inkxinah Concentration (ng/neural complex)
Conmounds (TYR-4) L-DOPA DA NE NMN DOPAC VA TYRA (TRP) 5-HTP 5-HT
the
Ratio
I
2
(0.77:0.80) (0.85:0.76) (0.98: 0.94) (0.82:0.98) (0.85:0.27) (0.90:0.45) (0.98:0.27) (0.25:0.88) (0.90: 0.871 io.91 :o.ooj (0.86:0.73)
125.07 +_ 0.09 0.71 f 0.30 14.46 f 10.85 3.05 i 2.85 0.28 + 0.19 1.40 + 0.67 0.12 + 0.07 0.20? 0.11 24.56 + 16.35 0.07 I 0.03 0.38 k 0.31
(Mean + SD.; N = 4, each contained
complexof
4 neural complexes)
somewhat lower than those measured by conventional HPLC, the values are highly acceptable in accuracy. Monoamines are widespread neuroactive substances in the animal kingdom. In particular, DA and 5-HT have been shown to occur in the nervous systems of all higher animals examined, although their levels and distribution within phyla have varied in a species from each phylum (e.g. Fujii and Takeda, 1988) and with developmental stages or physiological conditions (e.g. Klemm, 1985). As is the case with other invertebrate species, DA and S-HT were the main biogenic monoamines detected in Ciona intestinalis. In ascidians, the presence of 5-HT was first suggested by Erspamer (1946). It is known to be also present in the endostyle of Styelu, Molgulu (De la Torre and Surgeon, 1976; Sakharov and Salimova, 1982) Ciona, Ascidia, Ascidiella and Phallusia (Georges, 1985) and digestive tract of Ciona (Fritsch et al., 1982) and Styela (Pestarino, 1982) and a weak paraformaldehyde-induced fluorescence has been reported in the periphery of the ganglion in swimming tadpoles of Ciona but not in adults (Georges, 1985). In an other ascidian species, Halocynthia roretzi, 5-HT immunoreactivity has been detected in cells in the central nervous system (Fujii and Takeda, 1988). In the present analysis, 5-HT was quantitatively shown to be present in the neural complex of Ciona intestinalis.
The current study was also designed to investigate biogenic monoamines from phylogenetic stand point. TYR-4 3 L-DOPA
TRP * TYRA
+ 5-HTP
+
+
DA 3 NE
5-HT L DOPAC
l
3 VA
Fig. 4. Schematic drawing of biogenic monoamine metabolism
in the neural complex of the ascidian, intestinalis (see Table 1 for abbreviations).
Ciona
Ciona is a representative protochordate which is thought to be a phylogenetic offshot from the deuterostomian line leading from invertebrates to vertebrates. So, it was desirable to make a more thorough research of the possible occurrence of neurotransmitters. Compared to the compounds detected in protostomian species, the metabolic pathways involved in monoamine synthesis in Ciona intestinalis as a deuterostome seem to be simple. For example, epinephrine which is usually present in higher ranking Protostomian species was absent. 5-HIAA. a well known metabolite of 5-HT was also lacking. This may be due to its fixed life without motility in adults. From the view point of ontogeny, analyses of biogenie amines in tadpole stages will be necessary to clarify this problem. Neural control has been suggested in some species such as Diplosoma macdonaldi (Cavey and Cloney, 1976) and Halocynthia roretzi (Ohmori and Sasaki, 1977). As the biogenic monoamines were demonstrated to be present in the neural complex, the role of aminergic neural control in ascidians should be clarified. Acknowledgements-The author wishes to express his thanks to the Misaki Marine Biological Station of Tokyo University for Supplying Ciona. Special thanks are also made to Mr H. Takaoka, head of the present Department, COSMO Research Institute. for his encouragement throughout the course of the present work.
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