Camp. Biochem. Physiol. Vol. 102A, No. 4, pp. 703-707, 1992 Printed in Great Britain

N-ACETYLTRANSFERASE AND MELATONIN LEVELS IN THE OPTIC LOBE OF GIANT FRESHWATER PRAWNS,

MACROBRACHIUM ROSENBERGII DE MAN

0300~9629/92 $5.00 + 0.00 Q 1992 Pergamon Press Ltd

BOONSIRM WITHYACHUMNARNKUL, KANOKBHAN BUPPANIROJ and AXHALEE PONGSA-ASAWAPAIB~N

department of Anatomy, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand

(Received 23 December I991)

Abstract-l. N-Acetyltransferase (NAT), and melatonin were determined at 3-hour intervals in the optic lobe of the giant freshwater prawn, Mucrobrachium rosenbergii de Man.

2. A relatively high activity of NAT, compared to that in rat pineal, was found in the tissue. The enzyme did not show a significant diurnal rhythm although a slight suppressive level was detected at night.

3. Melatonin level varied considerably and showed a peak during the day (1500 hr) and a nadir during the night (2400 hr).

4. The results suggest that the optic lobe has certain biochemical activities similar to those found in pineal glands of vertebrates.

INTRODUCTION

The crnstacean optic lobe is a part of the nervous tissue that extends into the eyestalk. It contains several groups of cells that synthesize and secrete peptide hormones to regulate various physiological activities such as molting, reproduction, locomotion, color changing, blood osmolarity and blood sugar level (Adiyodi and Adiyodi, 1970). Some of these activities have been shown to be under the influence of photoperiodic environment. For instance, dark- ness increases locomotive activity of the lobster, ~e~~~~~~ ~~r~eg~e~~ (Chapman and Rice, 1971) and stimulates growth of giant freshwater prawns, Macrobrachium rosenbergii de Man (Withyachum- narnkul et al., 1990), blood sugar level of the crayfish Orconectes limosus showed a diurnal rhythm with a high level at night (Kallen et al., 1990) and seasonal rhythm of reproductive function has been observed in a number of crustacean species (Cheung, 1969; O’Connor, 1979; Sastry, 1983). This phenomenon of photoperiodic influence on activities has been well- documented in several vertebrate species especially in mammals. In mammals, the pineal gland is the organ that senses photoperiodic information, indirectly from the eyes and via sympathetic innervation of the gland (Ariens Kappers, 1971). The gland responds to neural signals by synthesis and secretion of its hormone, melatonin, which serves as a hormonal message to mediate rhythmic activities of several internal organs (Reiter, 1991). For most species, the hormone is high at night and low during the day (Reiter, 1991). The pineal gland is found in both mammalian and non-mammalian vertebrates such as birds, reptiles, amphibians and fishes (Ariens Kappers, 1971; Gundy and Wurst, 1976; Kikuchi and Aoki, 1984; McNulty, 1982; McNulty and Mafpaktitis, 1976; Oksche et al., 1972; Vivien-Reels and Arendt, 1983); it has not, however, been de-

scribed in invertebrates. In crustaceans, a structure located in the optic lobe called the organ of Bellonci is composed of pinealocyte-like cells and contains a very high concentration of 5-hydroxytryptamine (5HT) (Elofsson and Lake, 1971; Bellon-Humbert and Van Herp, 1988), the substance found abun- dantly in mammalian pineal glands (Reiter, 1991). To investigate whether the optic lobe contains a pineal- equivalent structure, we determine the activity of the enzyme N-acetyltransferase (NAT), which is the rate- limiting enzyme in melatonin synthesis (Klein et af., 1971), and melatonin contents in the optic lobe of M. rosenbergi~.

MATERIALS AND METHODS

Animals and tissue preparation

Adult female M. rosenbergii were obtained from a local farm and kept in laboratory aquarium for 5 days prior to the experiment. The aquarium was in a light-controlled room with light on between 0600 and 1800 hr. Light inten- sity on the water surface was approximately 600 Iux. The prawns were fed nd lib. with fresh mussels. At the time of the experiment, prawns were randomly sampled in groups of eight to IO at 3-hour intervals, starting from 0900 hr. At night, they were sampled under a dim red light. The eyestalks were cut off at the base and the optic lobes were immediately isolated and individually placed in microfuge tubes which were kept frozen with solid CO,. The isolated optic lobes included the lamina ganglionalis, medulla ex- terna, medulla interna and medulla terminalis (Fig. 1). A variable length of the optic peduncle was usually attached to the optic Iobe. The tissue samples were transferred to a deep freezer (-WC) pending assays for NAT and mela- tonin.

To determine the distribution of NAT in the optic lobe, the tissue sample from a separate set of prawns was divided into three parts and each part was determined for NAT activity. Part I consisted of the lamina ganglionalis, medulla extema and medulla interna; Part II was the medulla terminalis and Part III was the optic peduncle.

703

704 BOONSIRMWITHYACHUMNARNKUL et al.

Compound eye

optic peduncle

Fig. 1. A diagram showing the optic lobe within the eyestalk of M. rosenbergii. The optic lobe situated behind the compound eye comprises the lamina ganglionalis, medulla externa, medulla interna and

medulla terminalis.

For comparison, pineal glands taken from control and isoproterenol (ISO)-treated rats were simultaneously deter- mined for NAT and melatonin levels. Adult male albino rats, weighed between 120-150 g, were injected intraperitoneally with either O.Sml saline or IS0 (4mg/kg body weight) at 1100 hr and decapitated at 1300 hr (light was on between 0600-1800 hr). The pineal glands were immediately isolated and kept frozen (-75°C) pending NAT and melatonin dete~inations.

N-Acetyitransferase assay

NAT activity was determined according to a method described by Champney ef al. (1984). Tissue samples were sonicated with 100~1 phosphate buffered saline (PBS), 0.1 M, pH 6.8. Twenty microliters of the sonicate were incubated with 30~1 of a mixture containing tryptamine (0.6pmol), acetyl CoA (0.2 nmol) and [‘Hlacetyl CoA (0.5 @i, specific activity 3.84 Ci/mmol, Amersham, U.K.). The reaction mixture was incubated for 20 min at 37X. At the end of the period the acetylated [3H]tryptamine was extracted into 1 ml of water-saturated chloroform and washed with 200 ~1 of the buffer. A 500~1 aliquot of chloroform was placed into a scintillation vial and allowed to evaporate. Ten milliliters of scintillant were added and the samples were counted in a liquid scintillation counter (Beckman LS6OOOTA, U.S.A.). Blanks, not containing tissue, were simultaneously carried through the same pro- C&llZ.

Melatonin antibody was purchased from Stockgrand Ltd., U.K. and melatonin radioimmunoassay was performed according to a technique slightly modified from that rec- ommended by the company. Thirty microliters of the soni- cate were aliquoted and diluted with tricine buffer, 0.1 M, pH 5.5, to make 500~1. Two hundred microliters of diluted melatonin antibody were added to the sample and the mix- ture was incubated at room temperature for 30 min. One hun- dred microliters of [‘H] melatonin (specific activity 86Cij mmol, Amersham, U.K.) were then added and the mixture incubated at 4°C for 72 hr. Bound and free ligands were sep arated by incubation with 500 ~1 of dextran-coated charcoal for 15 min at 4°C and centrifuged at 1500 g, 4°C. for 30 min. Seven hundred microliters of the supemate were aliquoted into a scintillation vial containing 10 ml of the scintillant, the mixture was shaken for one hour before count.

The standard curve was simultaneously carried through the same procedure but using 500 ~1 of melatonin standard,

containing 0 to 250 pg of melatonin in melatonin-free optic lobe sonicate, instead of the unknown. The melatonin-free optic lobe sonicate was prepared by using charcoal-stripped technique as described in the protocol.

Protein &termination and statistical analysis

Protein contents were determined in individual samples using Lowry’s method (Lowry et al., 1951). N-Acetyltrans- ferase activities and melatonin contents were expressed as nmol product~hr~~~ protein and pg/gg protein, respectively. Data were analysed by ANOVA followed by Newman-Keuls test.

RESULTS

The NAT activity in the optic lobes of M. rosen- bergii was five to eight times higher than that of pineal glands from the ISO-treated rats (Fig. 2). In the optic lobe, the NAT activities during daytime and night-time did not differ, although the values seemed to decrease at night-time.

N-ACfTY!Jf?ANSERASE AcTlVlTY

2.0

f *P(o.ol, vs.coN

J

kpgT&i I-OPTIC LOES OFMmnkrpii -I

nrm Izd NGHT

Fig. 2. N-Acetyltransferase activities in rat pineal glands and optic lobes of M. rosenbergii. The pineal glands were taken from control (CON) and isoproterenol-treated (ISO) rats. The optic lobes were taken at 3-hour intervals at times

indicated.

NAT and melatonin in prawn optic lobe 705

N- ACETYLTRANSFERASE ACTIVITY

rP(oa,vs PartIan

1

1 * cl

Fig. 3. N-Acetyltransferase activities in Parts I, II and III of the tissue of M. rosenbergii. Part I comprised the lamina ganglionalis, medulla externa and medulla interna; Part II,

the medulla terminalis; Part III, the optic peduncle.

When NAT levels of Parts I, II and III of the optic lobe were compared, it appeared that the activities in Parts I and II were more than five times that of Part III (Fig. 3). The levels in Parts I and II were comparable to those found in the whole optic lobe.

Melatonin concentrations in the optic lobes varied considerably among individual prawns. In general, the daytime values were higher than the night time values. The peak occurred at 1500 hr and was signifi- cantly different from the nadir which occurred at 2400 hr (Fig. 4). Melatonin contents in the rat pineal glands were, in contrast, very consistent and much higher than those in the optic lobes. The pineal glands from the control and ISO-treated rats contained 9.8 + 1.2 and 98.3 + 13.4 pg melatonin/pg protein (P < O.Ol), respectively. Melatonin levels in the optic lobes were about half of those in the pineal glands of the control rats.

DISCUSSION

The results indicate that the optic lobe of M. rosenbergii contains much higher NAT activity than that found in the pineal glands of rats stimulated by isoproterenol, a b-adrenergic stimulator known to stimulate NAT activity in the pineal (Klein et nl., 1971). The NAT is localized mainly in the optic lobe since high activity was found in Parts I and II which represent the four parts of the optic lobe, but not in Part III which is the optic peduncle. In contrast to several species which have a remarkable diurnal rhythm with a night time increase (Reiter, 1991; Rudeen et al., 1975), the NAT activity in M. rosen- bergii is probably not diurnally rhythmic. If there is any rhythm at all, it seems to be opposite to that found in most species since a possible decrease in night-time NAT was detected. This suppression is supported by decreased night-time levels of mela- tonin in the optic lobe, assuming that the optic lobe NAT is also a rate-limiting enzyme in melatonin synthetic pathway of the optic lobe.

This assumption may not be justified when consid- ering that while NAT activity in the optic lobe was more than five times that in the stimulated rat pineal, yet melatonin concentrations were less than half of those from the non-stimulated rat pineal. Had

the optic lobe NAT been responsible mainly for melatonin production, higher and more consistent melatonin levels would be expected. This raises a possibility that the optic lobe NAT might also be responsible for production of other acetylated compound(s). In mammals, NAT converts SHT to N-acetylserotonin (NAS) which is further methylated by the enzyme hydroxyindole-O-methyltransferase (HIOMT) to melatonin (Reiter, 1991). Therefore, the low levels of melatonin in the optic lobe are possibly due to a low HIOMT activity in the tissue. In that case the main final product might be NAS instead of melatonin. A complete analysis of indoleamine levels in the optic lobe would probably shed light on this interesting question.

To reveal the physiological significance of NAT in the optic lobe, it is necessary to broaden the view to include the enzyme’s actions in other species. Besides acetylating arylalkylamines (tryptamine and SHT, for example) the enzyme can also acetylate arylamines (aniline and phenetidine, for example). The arylamine NAT is mainly localized in the mammalian liver and its function is to detoxify chemicals or drugs (Deguchi et al., 1988; Ebisawa and Deguchi, 1991; Sasaki et al., 1991; Vatsis et al., 1991); whereas arylalkylamine NAT is mainly localized in the pineal gland and has been shown to play a key role in generation of circadian rhythmicity of melatonin synthesis (Reiter, 1991; Rudeen et al., 1975). In rat and sheep, both arylamine and arylalkylamine NAT are found in the pineal glands (Voisin et al., 1984); the arylalkylamine NAT is responsible for N-acetyl serotonin and melatonin synthesis whereas the aryl- amine NAT is responsible for a synthesis of yet unknown pineal product (Heim et al., 1991). Since the NAT in the optic lobe used tryptamine as sub- strate, it is apparently the arylalkylamine NAT. However, it needs to be shown if the enzyme can also acetylate arylamines or whether the enzyme prefers arylamines to arylalkylamines as substrates.

MELATONH CONCENTRATION

* P<O.OS,vr 600

Fig. 4. Melatonin concentrations in the optic lobe of M. rosenbergii taken at 3-hour intervals at times indicated.

CBP !KL4,4-H

706 B~~NSIRM WITHYACHUMNARNKUL et al.

Melatonin, in most mammals, has a characteristic diurnal rhythm with a high value at night-time and low value in daytime (Reiter, 1991). The hormone and its circadian rhythm have also been demon- strated in birds and chickens (Axelrod et al., 1964; Binkley et al., 1978; Deguchi, 1979; Quay, 1966). Melatonin has been detected in reptiles, fishes and amphibians but its circadian rhythm has never been reported (Ariens Kappers, 1971). We have not been aware of any report concerning melatonin levels and its rhythms in invertebrates. In this study, melatonin concentrations in the optic lobe varied considerably among prawns. This wide variation could be due to differences in physiological stages, such as the molt- ing cycle, of individual prawns. The rhythm which showed a peak in daytime and a nadir at night-time is also opposite to what has been found in the majority of species tested thus far. While explanation to this phenomenon requires further investigations, it seems likely that the hormone rhythm is under photo- periodic influence.

In this study, the NAT activity was found mainly in Parts I and II of the tissue which comprised the optic lobe but not in Part III, the optic peduncle. This indicates that NAT is localized in all but not in any particular part of the optic lobe. Since the organ of Bellonci is in the medulla terminalis (Elofsson and Lake, 1971) it is unlikely that the structure is the only source of NAT. The exact location of the enzyme in the optic lobe and its function and kinetics are interesting questions that may lead to a disclosure of its function in the optic lobe; these problems are currently under investigation.

Acknowledgements-The study was supported by the grant from the Science and Technology Development Board (STDB) of Thailand, grant #DSN 88A-I-05-117.

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