Characteristics of high affinity binding of [3H]N-acetylserotonin in rat brain

NeurtJpharmacolog.vVol. 22, No. I 1, pp. 1311 1314, 1983 Prin,ed in Great Britain

0028-3908/83$3.00+ 0.00 Pergamon Press Ltd

CHARACTERISTICS OF HIGH AFFINITY BINDING OF [3H]N-ACETYLSEROTONIN IN RAT BRAIN L. P. NILES, G. M. BROWN and R, K. MISHRA Department of Neurosciences, McMaster University, Faculty of Health Sciences, Hamilton, Ontario, Canada L8N 3Z5 (Accepted 31 March 1983) Summary--Evidence that the putative pineal hormone, N-acetylserotonin (NAS), is present in diverse areas of the brain of the rat and that this compound is biologicallyactive, prompted a study of its central binding characteristics. [3H]N-acetylserotonin ([3H]-NAS) exhibits specific, saturable and high affinity binding in synaptosomal fractions from the brain of the rat. In flesh CNS membranes, [~H]N-acetylserotonin appeared to bind to a single population of high affinity sites with a dissociation constant (K,~) of 3 5 nM and a maximal binding site concentration (B~) of 250~00 fmol mg 1protein. A comparison of the regional distribution of the binding of [3H]N-acetylserotonin and [~H]-5-hydroxytryptamine([~H]-5-HT)indicated that both radioligands exhibited the greatest binding in the striatum and frontal cortex, while relatively more [3H]N-acetylserotoninwas bound in the cerebellum and brainstem. The structural specificity of the binding suggests that [3H]N-acetylserotonin labelled serotonergic receptors in the frontal cortex. Key words: N-acetylserotonin, serotonin, receptors, pineal, hormone.

There are reports that the pineal gland modulates brain and endocrine function: however, the mechanisms involved await clarification. The N-acetylindoleamine, melatonin, is generally assumed to be the major pineal hormone although increasing evidence indicates that other pineal compounds may also play a role in neuroendocrine regulation. One such pineal agent is the melatonin precursor, Nacetylserotonin (NAS), which, like melatonin, is present in serum (Yu, Pang, Tang and Brown, 1981). N-acetylserotonin is biologically active as indicated by it:~ effects on endocrine function (Porter, Mical and Cramer, 1971; Vaughan, Vaughan, Reiter and Benson, 1972) and its ability to depress significantly glutamate-induced neuronal firing in the rat hippocampus when applied iontophoretically (Porietis and Brown, personal communication). Immunohistological studies have demonstrated the presence of N-acetylserotonin and/or melatonin in the r~t brain (Bubenik, Brown and Grota, 1976). A differential distribution of N-acetylserotonin and melatonin has been reported, with certain regions, such as the cerebellum, pons and hippocampus appearing to contain N-acetylserotonin exclusively (Bubenik et al., 1976: Pulido, Brown and Grota, 1981). The N-acetylserotonin-synthesizing enzyme, N-acetyltransferase, is present in the brain of the rat (Paul, Hsu and Mandell, 1974) and N-acetylserotonin in brain has been measured by radioimmunoassay (Pang, Tang, Yu and Yip, 1983) and gas chromatography-mass spectrometry (Brown, Porietis and Narasimhachari, 1981). Moreover, levels of N-

acetylserotonin in brain are not affected by pinealectomy (Pang et al., 1983). These findings suggest that N-acetylserotonin is produced not only in the pineal but also in the brain of the rat where it may play a role in neurophysiology. The central binding characteristics ot" N-acetylserotonin were therefore investigated in order to determine whether specific binding sites for this putative neurohormone, are present in the brain. METHODS

Animals and tissue preparation Adult male or female Sprague-Dawley rats (Canadian Breeding Labs) were decapitated and selected brain areas immediately dissected on a chilled glass plate and then prepared for binding assays. Tissues were homogenized by a Brinkman Polytron (setting 6) in 10 vol of ice-cold 0.32 M sucrose for 20 sec and centrifuged at 1000g for 10 rain at 4 C . The crude nuclear pellet (P1) was discarded and the supernatant was centrifuged at 20,000g for 20 rain to yield a crude mitochondrial-synaptosomal pellet (P2) which was resuspended in 20 vol of distilled water and left on ice for 30 min to allow lysis of synaptosomes. Following centrifugation at 48,000g lbr 10 min the pellet was resuspended in 20 vol of distilled water and incubated at 3 7 C for 10 rain to remove endogenous serotonin (Nelson, Herbet, Bourgoin, Glowinski and Hamon, 1978). The tissue suspension was then spun at 48,000g for 10 rain and the pellet washed three times by resuspension in distilled water and centrifugation

1311

I ?12

L.P. NlLESet al.

at 48,000 g f o r I 0 min. The final pellet was suspended in 80 100 vol of 0.05 M Tris HCI buffer (pH 7.4) and used in binding assays.

Radio/(eamlv Tritiated N-acetylserotonin (pH]-NAS, 35 Ci/ retool) and tritiated serotonin ([3H]-5-HT, 26.4Ci/ rnmol) were obtained from Amersham Corp. and New England Nuclear respectively. In order to determine whether the radioligand was altered during the binding assay, [3H]N-acetylserotonin (20riM) was incubated with cerebellar membranes in 0.05 M Tris HCI buffer (pH 7.4) at 37 C for 20 min. Membranes were separated by centrifugation at 48,000g for 10rain and radioactivity was then eluted from the membranes by the addition of 3ml of absolute ethanol and incubating at 37 C for 20rain. The suspension was spun at 48,000g for 10rain and the supernatant was reduced under nitrogen. Aliquots 1201~I) o f the supernatant were spotted with 221tg of cold N-acetylserotonin on silica gel-coated, thinlayer chromatography plates (Brinkman Instruments Inc., Westbury) and developed for 90rain in a Gelman TLC chamber (Gelman Instrument Co., Ann Arbor) using chloroform methanol glacial-acetic acid (80: 10: 10). A chromatogram was obtained by counting I cm samples of the silica gel plate after the elution of radioactivity in t00'J; ethanol and the addition of scintillation fluid. The chromatogram re\,ealed a single peak of radioactivity with an R~ value of 0.45 which was identical to that of cold N-acetylserotonin and unbound [~H]N-acetylserotonin, indicating that the radioligand was not metabolized during incubation at 37 C. Drut,.~ N-acetylserotonin, serotonin, 5-methoxytryptamine and melatonin were obtained from Sigma. N-acetyltryptamine was supplied by Dr V. Snieckus. Other drugs used were: quipazine (Miles), methysergide (Sandoz), mianserine (Organon), ketanserine (Janssen), cyproheptadine (Merck Frosst), and spiperone (Janssen). Drugs were dissolved in distilled water or in a minimal amount of absolute ethanol or 5",, ascorbic acid followed by dilution in distilled water.

Bimtin~ as,~'ay.~ Binding assays for N-acetylserotonin (NAS) or serotonin (5-HT) were performed by incubating l ml aliquots of tissue suspension in triplicate with either [~H]N-acetylserotonin or [3H].5_HT. Incubations were carried out in a shaking water-bath at 3 7 C for 30 min for ['H]N-acetylserotonin or for 15min for [~H]-5-HT, as time-course experiments indicated that equilibrium was achieved within these incubation times. Samples were then vacuum-filtered over Whatman GF/B filters, and incubation tubes and filters were rapidly rinsed with 4 x 3 ml aliquots of ice-cold 0.05 M Tris HCI buffer (pH 7.4). Filters

were placed in minivials, 5 ml of PCS (Amersham) counting cocktail was added and after overnight equilibration the radioactivity was measured by liquid scintillation spectrometry. Specific binding of [~H]N-acetylserotonin or [~H]-5-HT was defined as the total bound radioactivity minus that not displaced by 10 #M of nonradioactive N-acetylserotonin or 5-HT. For both radioligands, specific binding ranged from 70 to 75'], of total binding. Less than 0.2°11 of total added [~H]N-acetylserotonin or [~H]-5-HT was nonspecifically bound to filters in the absence of tissue. Protein concentrations were determined by the method of Lowry, Rosebrough, Farr and Randall (1951), using bovine serum albumin as standard. RESULTS

Binding of [~H]N-acet3%erotonin Specific and saturable binding in female ratcerebellar membranes as a function of increasing concentrations of [3H]N-acetylserotonin is presented in Fig. 1. A Scatchard plot of the binding isotherm is shown in the inset indicating the presence of a single class of high affinity sites with an apparent dissociation constant (K,~) of 3.58 nM and a binding site concentration (Bin,,) o f 381.55frnol/mg protein. Saturation binding experiments conducted 3 4 times in triplicate indicated similar affinities and binding site maxima in membranes from brains of both males and females. In males, the values lk)r binding parameters were K j - 3.80 ± 0.59 nM and .c_

Rat

cerebellum

o. 300 ' ii~ c c ~ l O 0

o ~ 3= 538155 8 nM fm01/mg protein 8K MAX • =098

250

200

.c

150

I00 o

o

50

40

/

o

-- 20

i

1

b

-7

I

pc)

5

[3HIN

X~L

LL

I

I

100 200 300 4 0 0 [3 H ] - N A S bound ( fmol / mg ) £ I I IO

- acetylserotonin

15

20

conc ( n M )

Fig. I. Specific binding of [~H]N-acetylserotonin m syn+ aptosomal membranes from the cerebellum o f the rat. The inset shows a Scatchard plot of data with a dissociation constant (K,~),a maximal number of binding sites (Bm~~ ) and a correlation coefficient (r) ['or regression analysis of Scab chard plot data as indicated. Data shown are the means of triplicate values from a representative experiment which was repeated three times.

N-acetylserotonin binding Table 1. Regional distribution of specific binding of [~H]N-acetylserotonin (NAS) and [3H]-5-HT in the CNS of the rat

CNS region Stria~ um Cortex Hypothalamus Cerebellum Pons-medulla Hippocampus Midbrain

Specific binding [~HINAS [3H]-5-HT (fmol/mg protein) ....... 34.12 ± 0.86 197.91±2.34 32.83 + 4.94 185.13+8.10 167.92 ±8.89 22.69 ++_2.30 22.43 + 1.73 91.39 +_27.07 15.69 + 2.00 48.54_+ 5.78 13.93 + 0.34 141.49±12.05 12.55 + 4.36 50.01 _+4.38

Syna3tosomal membranes were incubated in triplicate with 5 nM of[~H]N-acetylserotonin or [3H]-5-HT at 37 'C for 30 rain. Non-specitic binding was measured in the presence of I()/,M non-radioactive N-acetylserotonin or 5 FIT. The correlation coefficient (r) between the binding of [3H]N-acetylserotonin and [3H]-5-HT is 0.79. Means ± SEM of triplicate determinations are presented. B ...... = 239.84 + 42.88 fmol/mg protein. Corresponding values in females were Kj = 3.12 _+ 0.46 nM and B...... = 306.45 + 75.32.

Regumal distribution of binding A comparison of the binding of [~H]N-acetylsero:onin and [~H]-5-HT in various areas of the rat brain is shown in Table 1. Binding of [3H]-5-HT was 3 l(-li~ld greater than binding of [3H]N-acetylserotonin in all the CNS areas examined; however, the relative binding levels of both radioligands were simiiar in the striatum and frontal cortex which showed the greatest binding. Relatively more binding of [H]N-acetylserotonin was present in the cerebellum and brainstem, whereas more binding of [~H]-5-HT occurred in the hypothalamus and hippocam3us. As a result of the relative differences in binding in certain brain regions, a low correlation coefficient (r) of 0.79 was obtained for binding between the two radioligands. Omission of binding data for the cerebellum, midbrain and pons-medulla restated in a significant binding correlation (r = 0.98) further indicating the divergence between the binding of [~H]N-acetylserotonin and [~H]-5-HT in these regions.

Pharmacological characteristics of binding The inhibitory effects of a variety of tryptamines and other drugs on the binding of [3H]N-acetylsero:onin in fresh cortical membranes from rat brain are shown in Table 2. Clearly, N-acetylserotonin and its precursor, 5-HT displayed the greatest binding atfinity and they were also equipotent in displacing binding of [~H]N-acetylserotonin. The slopes of the dispIacement curves for N-acetylserotonin and 5-HT were similar with Hill coefficients approximately equal to unity, suggesting a single class of binding sites. Replacement of the 5-hydroxyl group possessed by N-acetylserotonin and 5-HT by a methoxy group caused a greater than 30-fold decrease in affinity, as

1313

shown by 5-methoxytryptamine. Melatonin, which possesses a 5-methoxy group and an N-acetyl group, exhibited very low affinity which was about 700-fold less than that of N-acetylserotonin. N-acetyltryplamine, which lacks an indole substituent but has an N-acetyl group, showed no binding affinity. The 5-HT agonist, quipazine, the 5-HT antagonists, methysergide, mianserine and ketanserine, and the neuroleptic, spiperone, all showed moderate binding affinity and were about 7 15 times less potent than N-acetylserotonin. However, the anti-serotonergic agent, cyproheptadine showed very low binding affinity, being more than 400 times weaker than N-acetylserotonin. DISCUSSION The major finding of the present study was that N-acetylserotonin and 5-HT are approximately equipotent in displacing the high affinity binding of pH]N-acetylserotonin. Moreover, the 5-hydroxyl group possessed by both 5-HT and N-acetylserotonin appears to be essential for high affinity binding, as shown by the marked decrease in affinity when this group was replaced by a 5-methoxy substituent and by the absence of binding activity when a C-5 substituent was lacking. This, coupled with previous reports that the 5-hydroxyl group is necessary for high affinity binding of the putative neurotransmitter, 5-HT, to its own receptors in the brain of the rat (Bennett and Snyder, 1976) suggests that [3H]N-acetylserotonin labels serotonergic receptors. This view is supported by the intermediate potency displayed by the 5-HT agonist, quipazine, and by various anti-serotonergic agents against binding of [3 H]N-acetylserotonin. Table 2. Inhibition of the binding of pH]N-acetylserotonin (NAS) to synaptosomal membranes fi'om frontal cortex of the rat Drug N-acetylserotonin Serotonin Quipazine 5-Methoxytryptamine Melatonin N-acetyltryptamine Methysergide Mianserine Ketanserine Spiperone Cyproheptadine

(nM)

nH

6.70 + 1.72 12.08 ± 1.14 110 250 > 5000 inactive 49 62 69 92 3130

1.20 + 0.08 1.13 +0.08 0.49 0.82 0.39 0.86 1.0 I 0.98 {/.64 {).64

Membranes were incubated with 2.5 nM p H]N-acetylserotonin and at least five concentrations (10 "' I0 " M) of each drug. Apparent K, values were calculated from the equation K, = IC50/(1 + L/Kjt where ICs0 is equal to the concentration of drug which inhibits 5t)~!,, of binding, L is the concentration of radioligand and Kj is the dissociation constant. Hill coefficients (nil) are equal to the slopes of inhibition plots. Non-specilic binding was measured in the presence of 10 It M N-acetylserolonin. Means ± SEM of 2-4 experiments are presented.

1314

L.P. Nn,ES et al.

On the basis of the pharmacological characteristics of the binding of [3H]-5-HT and [3H]spiroperidol, Peroutka and Snyder (1979) suggested that these radioligands respectively label 5-HTt and 5-HT: receptor classes in the frontal cortex of the rat. The high potency of 5-HT in competing for [3 H]N-acetytserotonin binding sites and the structural specificity of this binding suggests that this radioligand binds to 5-HT~ receptors. However, the intermediate potency of the highly selective 5-HT2 antagonist, ketanserine (Leysen, Awouters, Kennis, Laduron, Vandenberk and Janssen, 1981) and also that of mianserine and spiperone, which preferentially bind to 5-HT: sites in the frontal cortex of the rat (Leysen et al., 1981), suggests that [3H]N-acetylserotonin also labels 5-HT~ binding sites. The similarity in the relative binding levels of [3H]N-acetylserotonin and [3H]-5-HT in the frontal cortex of the rat supports the above suggestion that [3H]N-acetylserotonin binds to serotonergic sites in this region of the brain. Nonetheless, a comparison of the pharmacological features of the binding of [3H]-5-HT (Nelson et al., 1978; Peroutka and Snyder, t979) and [3H]N-acetylserotonin indicates certain discrepancies. Thus, 5-HT was 2-7 times more efficient in displacing [3 H]-5-HT than [~H]N-acetylserotonin. Similarly, 5-methoxytryptamine, which displays moderate affinity for [3H]-5-HT binding sites, was up to 20 times less potent against [3H]N-acetylserotonin. A m o n g the antagonists tested, methysergide exhibited comparable affinity against the two radioligands, while mianserine and spiperone were 8-14 times more effective in displacing bound [3H]N-acetylserotonin. Conversely, cyproheptadine was about twice as potent against the binding of [~H]-5-HT as against [3H]N-acetylserotonin. Additional work will be required to determine whether the above discrepancies might be related to the presence of other nonserotonergic binding sites for [3H]N-acetylserotonin in the frontal cortex. The relatively higher binding levels of [3H]N-acetylserotonin as compared with [3H]-5-HT in the cerebellum and brainstem, also suggest that non-serotonergic binding sites for [3H]N.acetylserotonin are present in these areas of the brain. The reported presence of an aryl acylamidase in the brain of the rat (Paul, Halaris, Freedman and Hsu, 1976) raises the question of whether [3H]N-acetylserotonin undergoes deacetylation to produce [3H]5-HT during binding experiments. This appears unlikely, however, as radioactivity eluted from pelleted membranes following incubation at 37~C with [3H]N-acetylserotonin co-migrated with authentic Nacetylserotonin when tested by thin layer chromatography as described above. In summary, saturable, high affinity binding of [~H]N-acetylserotonin has been demonstrated in membranes from rat brain. This binding, like that of [3H]-5-HT, appeared to be greatest in forebrain regions, although relatively more binding of [~H]-

N-acetylserotonin was present in the cerebellum and brainstem. More importantly, the pharmacological profile of the binding of [3 H]N-acetylserotonin in the frontal cortex suggests that this radioligand labels 5-HT binding sites. These data suggest that the biological effects of the putative neurohormone, Nacetylserotonin, may involve its interaction with central serotonergic receptors. Acknowledgements This work was supported in part by the Ontario Mental Health Foundation and the Medical Research Council of Canada (MA-7741). We thank Mrs Lorraine Koutalos for typing the manuscript and Mrs Micheline Viau-Strankowski for preparation of tissue. REFERENCES

Bennett Jr J. P. and Snyder S. H. (1976) Serotonin and lysergic acid diethylamide binding in rat brain membranes: Relationship to postsynaptic serotonin i'eceptors. Molec. Pharmac. 12: 373-389. Brown G. M., Porietis A. and Narasimhachari N. (198t) Identification and quantitation of N-acetylserotonin in rat brain regions by GCMS. Soc. Neurosci. Ahstr. 7: 8. Bubenik G. A., Brown G. M. and Grota L. J. (1976) Differential localization of N-acetylated indolealkylamines in CNS and the Harderian gland using immunhistology. Bra#2 Res. 118: 417M27. Leysen J. E., Awouters F.. Kennis L., Laduron P. M., Vandenberk J. and Janssen P. A. J. (1981) Receptor binding profile of R41 468, a novel antagonist at 5-HT, receptors. L(~, Sci. 28:1015 1022. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193:265 275. Nelson D. L., Herbet A., Bourgoin S., Glowinski J. and Hamon M. (1978) Characteristics of central 5-HT receptors and their adaptive changes following intracerebral 5,7-dihydroxytryptamine administration in the rat. Molec. Pharmac. 1 4 : 9 8 3 995. Pang S. F., Tang H. S.. Yu P. L. and Yip M. K. (1982) The level of N-acetylserotonin and melatonin in the brain of male rats: Diurnal variations and effects of pinealectomy. J. exp. Zool. 219: 271-276. Paul S. M., Halaris A. E., Freedman D. X. and Hsu L. L. (1976) Rat brain aryl acylamidase: stereospecific inhibition by LSD and serotonin-related compounds. J. Neurochem. 27:625 627. Paul S. M., Hsu L. L. and Mandell A. J. (1974) Extrapineal N-acetyltransferase activity in rat brain. L!/b Sci. 15: 2135-2143. Peroutka S. J. and Snyder S. H. (1979) Multiple serotonin receptors: Differential binding of [3H]5-hydroxytryptamine, [3H]lysergic acid diethylamide and [~H]spiroperidot. Molec. Pharmac. 16: 687-699. Porter J. C., Mical R. S. and Cramer O. M. (1971) Effect of serotonin and other indoles on the release of LH, FSH and prolactin. Gynec. Ira,est. 2:t3 22. Putido O. M., Brown G. M. and Grota L. J. (1981) Localization of N-acetylserotonin (NAS) in the rat hindbrain by immunohistology. Prog. Neuropsychopharmac. 5 : 5 7 3 576. Vaughan M. K., Vaughan G. M., Reiter R. J. and Benson B. (1972) Effect of melatonin and other pineal indoles on adrenal enlargement produced in male and female mice by pinealectomy, unilateral adrenalectomy, castration and cold stress. Neuroendocrmolog) 1 0 : 1 3 9 154. Yu H. S., Pang S. F., Tang P. L. and Brown G. M. (1981) Persistence of circadian rhythms of melatonin and Nacetylserotonin in the serum of rats after pinealeetomy. Neuroendocrinolgy 32: 262-265.