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Melatonin synthesis by rabbit platelets
Life Sciences, Vol. 31, pp. 1487-1494 Printed in the U.S.A.
Pergamon Press
MELATONIN SYNTHESIS BY RABBIT PLATELETS J.M. Launay I, B.J. Lemaitre 2, H.P. Husson 3, C. Dreux I, L. Hartmann 2 and M. Da Prada 4. iLaboratoire de Biochimie, H6pital Saint-Louis, Paris, France. 2Laboratoire de Chimie Clinique et de Biologie Mol4culaire, Institut Biom4dical des Cordeliers, Paris, France. 31nstitut de Chimie des Substances Naturelles, CNRS, Gif-sur-Yvette, France. 4pharmaceutical Research Department, F. Hoffmann-La Roche & Co., Ltd., CH-4OO2 Basle, Switzerland (Received in final form July 9, 1982) Summary Platelets of reserpinized rabbits, incubated in buffer containing tritiated 5-hydroxytryptamine (3H-SHT), have the ability to convert 3H-5HT into labelled compound(s) extractable with alkalinechloroform. The bulk of the chloroform-extractable radioactivity showed a Rf value similar to authentic melatonin on silicagel thinlayer chromatography. The labelled product eluted with ethanol from the silicagel area where melatonin was suspected to reside was identified as melatonin by mass spectrometry and radioimmunoassay. Our in vitro experiments demonstrate that rabbit platelets are capable of converting 5-HT into melatonin apparently because they possess the enzymatic equipment necessary for this biosynthesis i.e. serotonin N-acetyltransferase and hydroxyindole-O-methyltransferase. Melatonin (N-acetyl-5-methoxytryptamine) biosynthesis takes place in the pineal gland as well as in other extra-pineal tissues (for review see i) including retina (2,3), Harderian gland (3,4) and enterochromaffin cells (5). The enzymes involved in the formation of melatonin from 5-hydroxytryptamine (5-HT, serotonin) (i.e. serotonin acetyl coenzyme-A N-acetyltransferase, NAT and hydroxyindole-O-methyltransferase, HIOMT) show circadian rhythms of activity with higher melatonin biosynthesis at night than during the day (6,7,8). Melatonin production in a rhythmic pattern occurs in the pineal gland and in the retina but not in other extra-pineal tissues (i). In spite of the presence of melatonin in human and animal plasma, nothing is known about melatonin formation in platelets, and only few investigators have studied the effects of this 5-HT derivative on the platelet physiology. Nevertheless, as demonstrated for the pineal
1
To whom reprint requests should be addressed.
0024-3205/82/141487-08503.00/0 Copyright
(c) 1982 Pergamon Press Ltd.
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Melatonin Synthesis by Rabbit Platelets
Vol. 31, No. 14, 1982
gland, some platelet functions show rhythmic patterns which could be governed by environmental lighting. Thus, diurnal and seasonal variations have been reported for 5-HT uptake and content in human platelets (9,10). Moreover, it has been shown that phototherapy reduces both production and lifespan of platelets (II). On the other hand, it was reported that melatonin increases platelet production (12) and that it inhibits platelet aggregation as well as collagen-induced thromboxane B 2 release (13). The aim of the present study was to assess whether rabbit blood platelets, which store relatively high amounts of 5-HT, are able to synthesize melatonin. This paper provides the first direct evidence that rabbit platelets, loaded in vitro with tritiated 5-HT, have the ability to convert 5-HT into melatonin. Methods .D.ru~s and animals - Chemicals used and their respective sources were : melatonin (Fluka, Switzerland), serotonin binoxalate (Sigma, USA), reserpine (Serpasil, Ciba-Geigy, Switzerland), 3H serotonin binoxalate (43.6 Ci.mmol-l, New England Nuclear). All other reagents used were analytical grade and obtained from commercial sources. 'Fauve de Bourgogne' rabbits weighing 2.5 to 3.2 kg were used. All experiments were performed in dim light. Solutions not specified to be sterile were filtered through sterile Millipore filters (0.22 ~m pore size~ Millipore Corp., USA). Blood collection, ~latelet isolation and loading with tritiated 5-HT - To release endogenous 5-BT from the platelets, 49 rabbits were injected with reserpine (5 mg.kg -I i.p., 16 h) and then bled under ether anaesthesia (14). Whole blood was collected (between 8 and 9 a.m.) into tubes containing citrate (9:1 volume 3,8 % trisodium citrate.2 H20). Two liters of platelet-rich plasma (PRP) were prepared by blood centrifugation (400 g, iO min, 15°C). To prevent 5-HT deamination and accumulation into the 5-HT organelles PRP was incubated at 37°C for 30 min with pargyline and reserpine, both at the final concentration of 2.10 -6 M. Thereafter, platelets were isolated on dextran T-10 gradients (15). The gradient layer rich in platelets was then removed and platelet counts were performed by phase contrast microscopy. The platelet-rich layer, diluted with 4 vol modified Tyrode buffer (16) containing 5-HT (final conc. 5 x 10 -5 M, 5 ~Ci 3H-5-HT), was incubated in aliquots of 50 ml, for 1 h at 37°C. Platelet 5-HT uptake and bacterial contamination were measured in small aliquots of the platelet suspension. The amount of 5-HT taken up by the platelets (estimated by subtracting the residual radioactivity of 0.5 ml platelet-free supernatant from the radioactivity present in 0.5 ml whole platelet suspension) corresponded to about 50 % of the radioactivity added to the incubation medium. The bacteriological analysis showed no detectable contamination of the platelet suspension with aerobic and/ or anaerobic bacteria. Preparation of the ~latelet extracts - After 5-HT loading the platelets were sedlmented and washed twice by centrifugation (2.300 g, IO min, 4°C) with modified ice-cold Tyrode buffer (16). All supernatants were discarded and after the tubes were wiped dry with tissue paper, each platelet pellet was suspended in
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500 U1 borate buffer (0.5 M, pH 12.4), homogenized by sonication and the radioactivity extracted from the aqueous phase twice with 5 ml of chloroform. Purification of the ~latelet extracts b~ one dimensional thin-la~er chromatography - The alkaline-chloroform extracts were pooled and evaporated to dryness under partial vacuum and nitrogen atmosphere. The residue, carefully dissolved in HCI (10 -3 M)-methanol (1:4 v/v) was subjected to thin-layer chromatography (TLC), in parallel with melatonin standard (200 ~g), on analytical precoated plates (silicagel 20 x 20 cm, 240 ~m thickness, LK 5F, Whatman). The TLC was developed with toluene-ethylacetate-glacial acetic acid (27:68:5) since with this solvent melatonin was separated more rapidly than with previously proposed solvent mixtures (17). A peak of radioactivity (whose Rf value was very close to that of authentic melatonin) was observed on the radiochromatogram (Berthold LB 283 TLC linear analyzer). The silicagel corresponding to this peak was scraped out, and suspended in 1 ml absolute ethanol, and after sonication, the silicagel fragments sedimented by centrifugation (2.000 g, 15 min, 4°C). This extraction procedure was repeated 4 times and no radioactivity was detectable in the last ethanolic extract. The three ethanolic extracts were pooled and evaporated under nitrogen stream to a final volume of 1 ml. An aliquot (50 ~i) of this ethanol solution (final platelet extract) was then counted by liquid scintillation spectrometry (SL 3000, Intertechnique, France). On the basis of this measurement it was estimated that following TLC the whole radioactivity present in the final platelet extract c o ~ responded to i.ii ~Ci. Characterization of the final ~latelet extract %~_o_d~mens~ona!_th~n[!azs£_ch{omeSogrepbz_(18) It was performed on IO ul of the final platelet extract. After localization under UV light the spots corresponding to the different standards were scraped off and radioactivity was counted after ethanol elution as previously described for one dimensional TLC. ~ _ £ ~ £ ~ { Z (direct introduction of the samples without derivatization) was performed by electron impact on authentic melatonin and on the dry residue of iOO ~i of the final platelet extract using a AEI mass spectrometer, type 50. Mass spectrometer conditions were : ionization chamber temperature 18OOC, electron energy, 70 eV. Rad~o~m~__tu/_oassa[ - Aliquots of a solution of melatonin standard and of the final platelet extract were measured by radioimmunoassay as previously described (19,20) using a specific antimelatonin immunserum (19). The standard curve was established using (in duplicate) 8 different concentrations of cold melatonin (from 108 to 1.7 x 10 -2 pmoles in two-fold dilution steps). The immunological behaviour of the final platelet extract was assessed and compared to that of authentic melatonin starting from a minimal iO.OOO-fold dilution of the platelet extract. At this dilution the platelet extract (similarly to 108 x 10 -2 pmoles of authentic melatonin), displaced about 85 % of the antibody-bound 3Hmelatonin (fig. 4). It should be noted that the radioactivity of the final platelet extract after iO.OOO-folddiluticn was never higher than the background of the scintillation counter. Results Figure 1 shows the radiochromatogram obtained by subjecting to TLC the alkaline-chloroform extract of rabbit platelets, previously incubated with tritiated 5-HT.
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Melatonln Synthesis by Rabbit Platelets
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Vol. 31, No. 14, 1982
dpm (xlO3)
20 16 12,
:start
front
4 2OFIG. 1 Radiochromatogram of the alkaline-chloroform extract of rabbit platelets incubated in modified Tyrode for 30 min at 37oc in the presence of 3H-5HT. The main peak of radioactivity had the same Rf value as authentic melatonin (spot localization by UV light, 254 nm wavelength). Solvent system used : toluene, ethylacetate, glacial acetic acid (27:68:5). Development time : 60 min.
~-'Kt'~ %zl,~
2200
dpn
.l
5-HTOL
NAS
5-HT S-MT Trp
S-WI'ID
A
( ~
FIG. 2 Two dimensional thin-layer radiochromatogram of the final platelet extract. Solvent systems : A - chloroform, methanol, acetic acid (93:7:1) } B - ethylacetate} tryptophan (Trp), 5-hydroxytryptophan (5-HTP), 5-methoxytryptamine (5-MT), 5-hydroxyindoleacetic acid (5-HIAA), 5-hydroxytryptophol (5-HTOL), 5-methoxyindoleacetic acid (5-MIAA), 5-methoxytryptophol (5-MTOL), N-acetylserotonin (NAS), melatonin (MEL).
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Melatonin
Synthesis
i00 - A 80>. t.t') ¢(-. (D > 4.-,
160
6040200
I
I
1491
173
-,,,o I
!
145
']°
I
160
I !
173
232
117
41
0
Platelets
232 117 ,
100 B 806040200
by Rabbit
40
145
89,90
I
I
80
120 FIG.
I
160
200
I
240 m/z
3
Mass spectra of melatonin standard (A) and platelet extract (B). The instrumental conditions are d e s c r i b e d in the text. m/z = m a s s - t o - c h a r g e ratio The relative intensities are e x p r e s s e d in percentage of the highest yield of the melatonin fragment at m/z 173 (= iOO %). The m o l e c u l a r peak of melatonin (M +) is shown at m/z = 232. This r a d i o c h r o m a t o g r a m i n d i c a t e s that the radioactivity of the silicagel plate is mainly localized on a large peak with a Rf value (=.62) identical to the m e l a t o n i n standard. To confirm that the 3H-labelled product(s) with a c h r o m a t o g r a p h i c m o b i l i t y identical to melatonin consists essentially of this amine, the ethanol extract of the silicagel area where m e l a t o n i n is suspected to reside,was subjected to a two-dimensional TLC, mass spectrometry and radioimmunoassay. The o n l y radioactive spot on the two dimensional thin-layer radiochromatogram of the final e t h a n o l i c platelet extract corresponds to melatonin as shown in Fig. 2. The mass spectra of authentic m e l a t o n i n and of the product removed from the TLC by the e t h a n o l i c extraction are shown in Fig. 3. Both authentic m e l a t o n i n (Fig. 3 A) and the silicagel e t h a n o l i c extract (Fig. 3 B) p r o d u c e d characteristic ions o c c u r r i n g at m a s s - t o - c h a r g e (m/z 232 (M+), 173 (loss of C2NOH5), 160 (loss of C3NOH6) , 145 and 117. The identification of m e l a t o n i n is based upon the coincident occurrence of the 5 ions (m/z 232, 173, 160, 145 and 117) in the appropriate relative intensities. E s s e n t i a l l y all fragments g e n e r a t e d by the compound isolated by TLC from the p l a t e l e t s are also produced by authentic melatonin with similar relative intensities (Fig. 3). The two fragments (m/z 41 and 149) shown only in the mass s p e c t r u m of the platelet extracts are p r o b a b l y due to TLC impurities.
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The presence of m e l a t o n i n in the ethanolic platelet extract was also confirmed by radioimmunoassay. The findings in Fig. 3 show that at various dilutions, the ethanol p l a t e l e t extract displaces 3H-melatonin from its specific immunserum exactly as authentic melatonin. As a consequence the two displacement curves obtained are e s s e n t i a l l y superposable (Fig. 4).
10090-
•"•"•
• Standardcurve
' ~
o Platelet extract
807060~-508 40:3020100
I 085
1 1.70
I I I I 3 4 0 6.75 13.5 27 Melatonin (pmoles x 10 -2)
FIG.
I 54
I 108
4
D i s p l a c e m e n t of 3H-melatonin from a specific a n t i m e l a t o n i n immunserum by platelet extract (O) and m e l a t o n i n standard (@). Inhibition of 3H-melatonin fixation on the immunserum (expressed as %) and m e l a t o n i n standard concentrations are shown on the y and x (logscale) axes, respectively. Discussion The present study provides evidence that platelet of r e s e r p i n i z e d rabbits are able to synthesize m e l a t o n i n when incubated in the presence of 5-HT. A first indication that p l a t e l e t s may convert 5-HT into m e l a t o n i n was obtained by TLC experiments. Indeed, the a l k a l i n e - c h l o r o f o r m extract of 3H-5-HT loaded platelets c o n t a i n e d a labelled compound which m i g r a t e d on TLC as authentic melatonin. After elution from the silicagel of the spot suspected to be melatonin, the identity of the compound was further assessed by subjecting to two-dimensional TLC, mass s p e c t r o m e t r y and r a d i o i m m u n o a s s a y aliquots of this ethanol extract. The two-dimensional radiochromatogram, the fragmentation pattern as well as the d i s p l a c e m e n t curve o b t a i n e d strongly indicate that the compound present in the ethanol extract b e h a v e s as authentic melatonin. It is well known that after intravenous injection, m e l a t o n i n is rapidly c o n v e r t e d into 6 - h y d r o x y m e l a t o n i n and thereafter excreted in the urine as unconjugated as well as sulpho- and g l u c u r o n o c o n J u g a t e d d e r i v a t i v e s (21,22). In our hands, both free and c o n j u g a t e d 6 - h y d r o x y m e l a t o n i n have been separated by TLC from the urine of rats injected intravenously with either the ethanol extract formed here or a l t e r n a t i v e l y w i t h labelled m e l a t o n i n (data not reported). On the whole, these findings lend support to the notion that p l a t e l e t s can convert 5-HT into melatonin. The lack of bacterial contamination allows us to exclude a bacterial origin (23) for the m e l a t o n i n present in the p l a t e l e % ex-
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Melatonin Synthesis by Rabbit Platelets
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tracts. Since platelet incubation was performed in artificial buffer it can be inferred that platelets are specifically endowed with the enzymatic machinery involved in the conversion of 5-HT into melatonin. In fact another work has shown that N-acetyl-serotonin as well as NAT and HIOMT activity could be measured in platelets but not in plasma of rabbits by highly specific and sensitive radioenzymatic techniques (24). Previous experiments have also shown that rabbit platelets contain melatonin (185 pg/mg platelet protein) and that they have the ability to accumulate melatonin in vitro by active transport mechanism(s) (25). It is reasonable to assume that in the present experimental conditions (reserpinization, monoamine oxldase inhibition and relatively high concentration of 5-HT in the incubation medium) platelet 5-HT can attain high concentrations within the cytoplasmic (extragranular) compartment (26,27,28) resulting in a significant melatonin synthesis. We have estimated which percentage of the 5-HT taken up by the platelets was converted to melatonin. In our experimental conditions about 50 % of the exogenous 5-HT accumulated by the platelets was transformed into melatonin : 44.4 % and 55.6 % according to TLC and to the radioimmunoassay experiment, respectively. This difference probably corresponds to the melatonin originally present in the platelets, prior to incubation with 3H-5-HT. Taking into account platelet number and volume it was possible to estimate the platelet/plasma concentration ratio for 5-HT and catecholamines (14). As far as platelet endogenous melatonin is concerned, we have calculated that in rabbits the platelet/plasma concentration ratio for melatonin corresponds to 430. Thus, similarly to 5-HT, histamine and catecholamines (29) melatonin is several times more concentrated in platelets than in plasma. However, the fact that the concentration ratio for melatonin is about 60 times lower than for 5-HT (14,29) indicates that at physiological extracellular concentrations, platelets have a much more efficient transport and storage mechanism for 5-HT than for melatonin. It ks well established that the platelet monoamines are mainly accumulated into specific subcellular storage sites, namely into the 5-HT organelles (30). Preliminary density gradient experiments suggest that rabbit platelet melatonin, at variance with other platelet monoamines, is not accumulated into dense bodies but rather into Q-granules (LemaTtre et al., in preparation). The present study has shown that rabbit platelets, similarly to other e x t r ~ pineal tissues, are able to convert part of the 5-HT taken up from the incubation medium into melatonin. Even though it has been reported that exogenous melatonin inhibits platelet aggregation and thromboxane B2 release (13), the physiological role of the endogenous platelet melatonin remains to be entirely worked out (as e.g. a possible modulation of its synthesis by the light-darkness cycles). Given the proposed similarity between platelets and serotoninergic neurons (31), a byproduct of the present results ks the inference that a fraction of 5-HT might be converted to melatonin also An serotoninergic n e u r o n ~ Acknowledgements We thank Dr W.E. Haefely and Dr L. Pieri for critical reading of the manuscripL
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References I. C.L. RALPH, in: Melatonin - current status and ~ers~ectives, (eds. N. Birau and W. Schloot), pp 35-46, Pergamon Press, New York (1981). 2. D. CARDXNALI and R.J. WURTMAN, Endocrinology 91 247-252 (1972). 3. G.A. BUBENIK, R.A. PURTILL, G.A. BROWN and L.J. GROTA, Exp. Eye Res. 27 323-333 (1978). 4. S.F. PANG, G.M. BROWN, L.J. GROTA, J.W. CHAMBERS, R.L. RODMAN, Neuroendocrinology.23 1-13 (1977). 5. G. BUBENIK, G. BROWN and L. GROTA, Experientia 3 3 6 6 2 - 6 6 3 (1977). 6. J. AXELROD, R.J. WURTMAN and S.H. SNYDER, J. Biol. Chem. 240 949-954 (1965). 7. D.C. KLEIN and J.L. WELLER, Science 169 1093-1095 (1970). 8. T. DEGUCHI, Mol. Cell Biochem. 27 57-66 (1979). 9. A. WIRZ-JUSTICE and E. CHAPPUIS-ARNDT, Eur. J. Pharmacol. 40 21-25 (1976). IO.A. WIRZ-JUSTICE, M. LICHTSTEINER and H. FEER, J. Neural Transm. 41 7-15 (1977). II.M.H. MAURER, M. FRATKLIN, N.B. McWILLIAMS, B. KIRKPATRICK, D. DRAPER, J.C. HAGGINS and C.R. HUNTER, Pediatrics 57 506-512 (1976). 12.L. DI BELLA, L. GUALANO, M.T. ROSSI and G. SCALERA, Boll. Soc. It. Biol. Sper. 55 389-393 (1979). 13.C.M. LEACH and G.D. THORBURN, Prostaglandins 20 51-56 (1980). 14.M. DA PRADA and G.B. PICOTTI, Brit. J. Pharmacol. 65 653-662 (1979). 15.M. GRAF, A. LAUBSCHER, J.G. RICHARDS and A. PLETSCHER, J. Lab. Clin. Med. 93 257-265 (1979). 16.M. DA PRADA, G. BARTHOLINI and A. PLETSCHER, Biochem. Pharmacol. 14 17211726 (1965). 17.~M. SAAVEDRA, M. BROWNSTEIN and J. AXELROD, J. Pharmacol. Exp. Ther. 186 508-515 (1973). 18.D.C. KLEIN and A. NOTIDES, Anal. Biochem. 31 480-483 (1969). 19.B.J. LEMAITRE and L. HARTMANN, J. Immunol. Meth. 32 339-347 (1980). 20.R. BESSELIEVRE, B.J. LEMAITRE, H.P. HUSSON and L. HARTMANN, Biomedicine 33 226-228 (1980). 21.S. KVEDER and W.M. McISAAC, J. Biol. Chem. 236 3214-3220 (1980). 22.M.E. SISAl<, S.P. MARKEY, R.W. COLBURN, A.P. ZAVADIL and I.J. KOPIN, Life Sci. 25 803-806 (1979). 23.Y. OZAKI and H.J. LYNCH, Endocrinology 99 641-644 (1976). 24.N. BARRE, Etude sur les hydroxy et m4thoxyindoles plaquettaires: mise en 4vidence d'une voie de blosynth~se de la m~latonine, Th~se de Doctorat de 3~me cycle, Paris VII (1981). 25.B.J. LEMAITRE, J.M. LAUNAY, C. DREUX, L. HARTMANN and M. DA PRADA, 2nd International Colloquium of the European Pineal Study Group, Giessen (1981). 26.H.J. REIMERS, D.J. ALLEN, I.A. FEUERSTEIN and J.F. MUSTARD, J. Cell Biol. 65 359-372 (1975). 27.A. PLETSCHER, G. BARTHOLINI and M. DA PRADA, Mechanisms of release of biogenic amines (eds. U.S. Von Euler, S. Rosell and B. Uvnas), pp 165-174, Pergamon Press, London (1966). 28.M. DA PRADA and A. PLETSCHER, Brit. J. Pharmacol. 34 591-597 (1968). 29.M. DA PRADA, G.B. PICOTTI, R. KETTLER and J.M. LAUNAY, Platelets: cellular response mechanisms and their biological significance (eds. A. Rot/nan, F.A. Meyer, C. Gitler and A. Silberberg), pp 277-288, J. Wiley & Sons, London (1980). 30.M. DA PRADA, J.G. RICHARDS and R. KEI~fLER, Platelets in Biology and Pathology (ed. J.L. Gordon) ~ 107-145, North-Holland Biomedical Press, Amsterdam (1981). 31.A.PLETSCHER,Essays in Neurochemistry and Neuropharmacology (eds.M.B.H. Youdim, D.F.Sharman,W.Lovenberg and J.R.Lagnado)~ 49-1Oi,J.Wiley & Sons,London (1978) .
Pergamon Press
MELATONIN SYNTHESIS BY RABBIT PLATELETS J.M. Launay I, B.J. Lemaitre 2, H.P. Husson 3, C. Dreux I, L. Hartmann 2 and M. Da Prada 4. iLaboratoire de Biochimie, H6pital Saint-Louis, Paris, France. 2Laboratoire de Chimie Clinique et de Biologie Mol4culaire, Institut Biom4dical des Cordeliers, Paris, France. 31nstitut de Chimie des Substances Naturelles, CNRS, Gif-sur-Yvette, France. 4pharmaceutical Research Department, F. Hoffmann-La Roche & Co., Ltd., CH-4OO2 Basle, Switzerland (Received in final form July 9, 1982) Summary Platelets of reserpinized rabbits, incubated in buffer containing tritiated 5-hydroxytryptamine (3H-SHT), have the ability to convert 3H-5HT into labelled compound(s) extractable with alkalinechloroform. The bulk of the chloroform-extractable radioactivity showed a Rf value similar to authentic melatonin on silicagel thinlayer chromatography. The labelled product eluted with ethanol from the silicagel area where melatonin was suspected to reside was identified as melatonin by mass spectrometry and radioimmunoassay. Our in vitro experiments demonstrate that rabbit platelets are capable of converting 5-HT into melatonin apparently because they possess the enzymatic equipment necessary for this biosynthesis i.e. serotonin N-acetyltransferase and hydroxyindole-O-methyltransferase. Melatonin (N-acetyl-5-methoxytryptamine) biosynthesis takes place in the pineal gland as well as in other extra-pineal tissues (for review see i) including retina (2,3), Harderian gland (3,4) and enterochromaffin cells (5). The enzymes involved in the formation of melatonin from 5-hydroxytryptamine (5-HT, serotonin) (i.e. serotonin acetyl coenzyme-A N-acetyltransferase, NAT and hydroxyindole-O-methyltransferase, HIOMT) show circadian rhythms of activity with higher melatonin biosynthesis at night than during the day (6,7,8). Melatonin production in a rhythmic pattern occurs in the pineal gland and in the retina but not in other extra-pineal tissues (i). In spite of the presence of melatonin in human and animal plasma, nothing is known about melatonin formation in platelets, and only few investigators have studied the effects of this 5-HT derivative on the platelet physiology. Nevertheless, as demonstrated for the pineal
1
To whom reprint requests should be addressed.
0024-3205/82/141487-08503.00/0 Copyright
(c) 1982 Pergamon Press Ltd.
1488
Melatonin Synthesis by Rabbit Platelets
Vol. 31, No. 14, 1982
gland, some platelet functions show rhythmic patterns which could be governed by environmental lighting. Thus, diurnal and seasonal variations have been reported for 5-HT uptake and content in human platelets (9,10). Moreover, it has been shown that phototherapy reduces both production and lifespan of platelets (II). On the other hand, it was reported that melatonin increases platelet production (12) and that it inhibits platelet aggregation as well as collagen-induced thromboxane B 2 release (13). The aim of the present study was to assess whether rabbit blood platelets, which store relatively high amounts of 5-HT, are able to synthesize melatonin. This paper provides the first direct evidence that rabbit platelets, loaded in vitro with tritiated 5-HT, have the ability to convert 5-HT into melatonin. Methods .D.ru~s and animals - Chemicals used and their respective sources were : melatonin (Fluka, Switzerland), serotonin binoxalate (Sigma, USA), reserpine (Serpasil, Ciba-Geigy, Switzerland), 3H serotonin binoxalate (43.6 Ci.mmol-l, New England Nuclear). All other reagents used were analytical grade and obtained from commercial sources. 'Fauve de Bourgogne' rabbits weighing 2.5 to 3.2 kg were used. All experiments were performed in dim light. Solutions not specified to be sterile were filtered through sterile Millipore filters (0.22 ~m pore size~ Millipore Corp., USA). Blood collection, ~latelet isolation and loading with tritiated 5-HT - To release endogenous 5-BT from the platelets, 49 rabbits were injected with reserpine (5 mg.kg -I i.p., 16 h) and then bled under ether anaesthesia (14). Whole blood was collected (between 8 and 9 a.m.) into tubes containing citrate (9:1 volume 3,8 % trisodium citrate.2 H20). Two liters of platelet-rich plasma (PRP) were prepared by blood centrifugation (400 g, iO min, 15°C). To prevent 5-HT deamination and accumulation into the 5-HT organelles PRP was incubated at 37°C for 30 min with pargyline and reserpine, both at the final concentration of 2.10 -6 M. Thereafter, platelets were isolated on dextran T-10 gradients (15). The gradient layer rich in platelets was then removed and platelet counts were performed by phase contrast microscopy. The platelet-rich layer, diluted with 4 vol modified Tyrode buffer (16) containing 5-HT (final conc. 5 x 10 -5 M, 5 ~Ci 3H-5-HT), was incubated in aliquots of 50 ml, for 1 h at 37°C. Platelet 5-HT uptake and bacterial contamination were measured in small aliquots of the platelet suspension. The amount of 5-HT taken up by the platelets (estimated by subtracting the residual radioactivity of 0.5 ml platelet-free supernatant from the radioactivity present in 0.5 ml whole platelet suspension) corresponded to about 50 % of the radioactivity added to the incubation medium. The bacteriological analysis showed no detectable contamination of the platelet suspension with aerobic and/ or anaerobic bacteria. Preparation of the ~latelet extracts - After 5-HT loading the platelets were sedlmented and washed twice by centrifugation (2.300 g, IO min, 4°C) with modified ice-cold Tyrode buffer (16). All supernatants were discarded and after the tubes were wiped dry with tissue paper, each platelet pellet was suspended in
Vol.
31, No.
14, 1982
Melatonln
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Platelets
1489
500 U1 borate buffer (0.5 M, pH 12.4), homogenized by sonication and the radioactivity extracted from the aqueous phase twice with 5 ml of chloroform. Purification of the ~latelet extracts b~ one dimensional thin-la~er chromatography - The alkaline-chloroform extracts were pooled and evaporated to dryness under partial vacuum and nitrogen atmosphere. The residue, carefully dissolved in HCI (10 -3 M)-methanol (1:4 v/v) was subjected to thin-layer chromatography (TLC), in parallel with melatonin standard (200 ~g), on analytical precoated plates (silicagel 20 x 20 cm, 240 ~m thickness, LK 5F, Whatman). The TLC was developed with toluene-ethylacetate-glacial acetic acid (27:68:5) since with this solvent melatonin was separated more rapidly than with previously proposed solvent mixtures (17). A peak of radioactivity (whose Rf value was very close to that of authentic melatonin) was observed on the radiochromatogram (Berthold LB 283 TLC linear analyzer). The silicagel corresponding to this peak was scraped out, and suspended in 1 ml absolute ethanol, and after sonication, the silicagel fragments sedimented by centrifugation (2.000 g, 15 min, 4°C). This extraction procedure was repeated 4 times and no radioactivity was detectable in the last ethanolic extract. The three ethanolic extracts were pooled and evaporated under nitrogen stream to a final volume of 1 ml. An aliquot (50 ~i) of this ethanol solution (final platelet extract) was then counted by liquid scintillation spectrometry (SL 3000, Intertechnique, France). On the basis of this measurement it was estimated that following TLC the whole radioactivity present in the final platelet extract c o ~ responded to i.ii ~Ci. Characterization of the final ~latelet extract %~_o_d~mens~ona!_th~n[!azs£_ch{omeSogrepbz_(18) It was performed on IO ul of the final platelet extract. After localization under UV light the spots corresponding to the different standards were scraped off and radioactivity was counted after ethanol elution as previously described for one dimensional TLC. ~ _ £ ~ £ ~ { Z (direct introduction of the samples without derivatization) was performed by electron impact on authentic melatonin and on the dry residue of iOO ~i of the final platelet extract using a AEI mass spectrometer, type 50. Mass spectrometer conditions were : ionization chamber temperature 18OOC, electron energy, 70 eV. Rad~o~m~__tu/_oassa[ - Aliquots of a solution of melatonin standard and of the final platelet extract were measured by radioimmunoassay as previously described (19,20) using a specific antimelatonin immunserum (19). The standard curve was established using (in duplicate) 8 different concentrations of cold melatonin (from 108 to 1.7 x 10 -2 pmoles in two-fold dilution steps). The immunological behaviour of the final platelet extract was assessed and compared to that of authentic melatonin starting from a minimal iO.OOO-fold dilution of the platelet extract. At this dilution the platelet extract (similarly to 108 x 10 -2 pmoles of authentic melatonin), displaced about 85 % of the antibody-bound 3Hmelatonin (fig. 4). It should be noted that the radioactivity of the final platelet extract after iO.OOO-folddiluticn was never higher than the background of the scintillation counter. Results Figure 1 shows the radiochromatogram obtained by subjecting to TLC the alkaline-chloroform extract of rabbit platelets, previously incubated with tritiated 5-HT.
1490
Melatonln Synthesis by Rabbit Platelets
24-
Vol. 31, No. 14, 1982
dpm (xlO3)
20 16 12,
:start
front
4 2OFIG. 1 Radiochromatogram of the alkaline-chloroform extract of rabbit platelets incubated in modified Tyrode for 30 min at 37oc in the presence of 3H-5HT. The main peak of radioactivity had the same Rf value as authentic melatonin (spot localization by UV light, 254 nm wavelength). Solvent system used : toluene, ethylacetate, glacial acetic acid (27:68:5). Development time : 60 min.
~-'Kt'~ %zl,~
2200
dpn
.l
5-HTOL
NAS
5-HT S-MT Trp
S-WI'ID
A
( ~
FIG. 2 Two dimensional thin-layer radiochromatogram of the final platelet extract. Solvent systems : A - chloroform, methanol, acetic acid (93:7:1) } B - ethylacetate} tryptophan (Trp), 5-hydroxytryptophan (5-HTP), 5-methoxytryptamine (5-MT), 5-hydroxyindoleacetic acid (5-HIAA), 5-hydroxytryptophol (5-HTOL), 5-methoxyindoleacetic acid (5-MIAA), 5-methoxytryptophol (5-MTOL), N-acetylserotonin (NAS), melatonin (MEL).
Vol.
31, No.
14,
1982
Melatonin
Synthesis
i00 - A 80>. t.t') ¢(-. (D > 4.-,
160
6040200
I
I
1491
173
-,,,o I
!
145
']°
I
160
I !
173
232
117
41
0
Platelets
232 117 ,
100 B 806040200
by Rabbit
40
145
89,90
I
I
80
120 FIG.
I
160
200
I
240 m/z
3
Mass spectra of melatonin standard (A) and platelet extract (B). The instrumental conditions are d e s c r i b e d in the text. m/z = m a s s - t o - c h a r g e ratio The relative intensities are e x p r e s s e d in percentage of the highest yield of the melatonin fragment at m/z 173 (= iOO %). The m o l e c u l a r peak of melatonin (M +) is shown at m/z = 232. This r a d i o c h r o m a t o g r a m i n d i c a t e s that the radioactivity of the silicagel plate is mainly localized on a large peak with a Rf value (=.62) identical to the m e l a t o n i n standard. To confirm that the 3H-labelled product(s) with a c h r o m a t o g r a p h i c m o b i l i t y identical to melatonin consists essentially of this amine, the ethanol extract of the silicagel area where m e l a t o n i n is suspected to reside,was subjected to a two-dimensional TLC, mass spectrometry and radioimmunoassay. The o n l y radioactive spot on the two dimensional thin-layer radiochromatogram of the final e t h a n o l i c platelet extract corresponds to melatonin as shown in Fig. 2. The mass spectra of authentic m e l a t o n i n and of the product removed from the TLC by the e t h a n o l i c extraction are shown in Fig. 3. Both authentic m e l a t o n i n (Fig. 3 A) and the silicagel e t h a n o l i c extract (Fig. 3 B) p r o d u c e d characteristic ions o c c u r r i n g at m a s s - t o - c h a r g e (m/z 232 (M+), 173 (loss of C2NOH5), 160 (loss of C3NOH6) , 145 and 117. The identification of m e l a t o n i n is based upon the coincident occurrence of the 5 ions (m/z 232, 173, 160, 145 and 117) in the appropriate relative intensities. E s s e n t i a l l y all fragments g e n e r a t e d by the compound isolated by TLC from the p l a t e l e t s are also produced by authentic melatonin with similar relative intensities (Fig. 3). The two fragments (m/z 41 and 149) shown only in the mass s p e c t r u m of the platelet extracts are p r o b a b l y due to TLC impurities.
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Melatonin
Synthesis
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Platelets
Vol.
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14, 1982
The presence of m e l a t o n i n in the ethanolic platelet extract was also confirmed by radioimmunoassay. The findings in Fig. 3 show that at various dilutions, the ethanol p l a t e l e t extract displaces 3H-melatonin from its specific immunserum exactly as authentic melatonin. As a consequence the two displacement curves obtained are e s s e n t i a l l y superposable (Fig. 4).
10090-
•"•"•
• Standardcurve
' ~
o Platelet extract
807060~-508 40:3020100
I 085
1 1.70
I I I I 3 4 0 6.75 13.5 27 Melatonin (pmoles x 10 -2)
FIG.
I 54
I 108
4
D i s p l a c e m e n t of 3H-melatonin from a specific a n t i m e l a t o n i n immunserum by platelet extract (O) and m e l a t o n i n standard (@). Inhibition of 3H-melatonin fixation on the immunserum (expressed as %) and m e l a t o n i n standard concentrations are shown on the y and x (logscale) axes, respectively. Discussion The present study provides evidence that platelet of r e s e r p i n i z e d rabbits are able to synthesize m e l a t o n i n when incubated in the presence of 5-HT. A first indication that p l a t e l e t s may convert 5-HT into m e l a t o n i n was obtained by TLC experiments. Indeed, the a l k a l i n e - c h l o r o f o r m extract of 3H-5-HT loaded platelets c o n t a i n e d a labelled compound which m i g r a t e d on TLC as authentic melatonin. After elution from the silicagel of the spot suspected to be melatonin, the identity of the compound was further assessed by subjecting to two-dimensional TLC, mass s p e c t r o m e t r y and r a d i o i m m u n o a s s a y aliquots of this ethanol extract. The two-dimensional radiochromatogram, the fragmentation pattern as well as the d i s p l a c e m e n t curve o b t a i n e d strongly indicate that the compound present in the ethanol extract b e h a v e s as authentic melatonin. It is well known that after intravenous injection, m e l a t o n i n is rapidly c o n v e r t e d into 6 - h y d r o x y m e l a t o n i n and thereafter excreted in the urine as unconjugated as well as sulpho- and g l u c u r o n o c o n J u g a t e d d e r i v a t i v e s (21,22). In our hands, both free and c o n j u g a t e d 6 - h y d r o x y m e l a t o n i n have been separated by TLC from the urine of rats injected intravenously with either the ethanol extract formed here or a l t e r n a t i v e l y w i t h labelled m e l a t o n i n (data not reported). On the whole, these findings lend support to the notion that p l a t e l e t s can convert 5-HT into melatonin. The lack of bacterial contamination allows us to exclude a bacterial origin (23) for the m e l a t o n i n present in the p l a t e l e % ex-
Vol. 31, No. 14, 1982
Melatonin Synthesis by Rabbit Platelets
1493
tracts. Since platelet incubation was performed in artificial buffer it can be inferred that platelets are specifically endowed with the enzymatic machinery involved in the conversion of 5-HT into melatonin. In fact another work has shown that N-acetyl-serotonin as well as NAT and HIOMT activity could be measured in platelets but not in plasma of rabbits by highly specific and sensitive radioenzymatic techniques (24). Previous experiments have also shown that rabbit platelets contain melatonin (185 pg/mg platelet protein) and that they have the ability to accumulate melatonin in vitro by active transport mechanism(s) (25). It is reasonable to assume that in the present experimental conditions (reserpinization, monoamine oxldase inhibition and relatively high concentration of 5-HT in the incubation medium) platelet 5-HT can attain high concentrations within the cytoplasmic (extragranular) compartment (26,27,28) resulting in a significant melatonin synthesis. We have estimated which percentage of the 5-HT taken up by the platelets was converted to melatonin. In our experimental conditions about 50 % of the exogenous 5-HT accumulated by the platelets was transformed into melatonin : 44.4 % and 55.6 % according to TLC and to the radioimmunoassay experiment, respectively. This difference probably corresponds to the melatonin originally present in the platelets, prior to incubation with 3H-5-HT. Taking into account platelet number and volume it was possible to estimate the platelet/plasma concentration ratio for 5-HT and catecholamines (14). As far as platelet endogenous melatonin is concerned, we have calculated that in rabbits the platelet/plasma concentration ratio for melatonin corresponds to 430. Thus, similarly to 5-HT, histamine and catecholamines (29) melatonin is several times more concentrated in platelets than in plasma. However, the fact that the concentration ratio for melatonin is about 60 times lower than for 5-HT (14,29) indicates that at physiological extracellular concentrations, platelets have a much more efficient transport and storage mechanism for 5-HT than for melatonin. It ks well established that the platelet monoamines are mainly accumulated into specific subcellular storage sites, namely into the 5-HT organelles (30). Preliminary density gradient experiments suggest that rabbit platelet melatonin, at variance with other platelet monoamines, is not accumulated into dense bodies but rather into Q-granules (LemaTtre et al., in preparation). The present study has shown that rabbit platelets, similarly to other e x t r ~ pineal tissues, are able to convert part of the 5-HT taken up from the incubation medium into melatonin. Even though it has been reported that exogenous melatonin inhibits platelet aggregation and thromboxane B2 release (13), the physiological role of the endogenous platelet melatonin remains to be entirely worked out (as e.g. a possible modulation of its synthesis by the light-darkness cycles). Given the proposed similarity between platelets and serotoninergic neurons (31), a byproduct of the present results ks the inference that a fraction of 5-HT might be converted to melatonin also An serotoninergic n e u r o n ~ Acknowledgements We thank Dr W.E. Haefely and Dr L. Pieri for critical reading of the manuscripL
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Melatonin
Synthesis
by Rabbit Platelets
Vol.
31, No.
14, 1982
References I. C.L. RALPH, in: Melatonin - current status and ~ers~ectives, (eds. N. Birau and W. Schloot), pp 35-46, Pergamon Press, New York (1981). 2. D. CARDXNALI and R.J. WURTMAN, Endocrinology 91 247-252 (1972). 3. G.A. BUBENIK, R.A. PURTILL, G.A. BROWN and L.J. GROTA, Exp. Eye Res. 27 323-333 (1978). 4. S.F. PANG, G.M. BROWN, L.J. GROTA, J.W. CHAMBERS, R.L. RODMAN, Neuroendocrinology.23 1-13 (1977). 5. G. BUBENIK, G. BROWN and L. GROTA, Experientia 3 3 6 6 2 - 6 6 3 (1977). 6. J. AXELROD, R.J. WURTMAN and S.H. SNYDER, J. Biol. Chem. 240 949-954 (1965). 7. D.C. KLEIN and J.L. WELLER, Science 169 1093-1095 (1970). 8. T. DEGUCHI, Mol. Cell Biochem. 27 57-66 (1979). 9. A. WIRZ-JUSTICE and E. CHAPPUIS-ARNDT, Eur. J. Pharmacol. 40 21-25 (1976). IO.A. WIRZ-JUSTICE, M. LICHTSTEINER and H. FEER, J. Neural Transm. 41 7-15 (1977). II.M.H. MAURER, M. FRATKLIN, N.B. McWILLIAMS, B. KIRKPATRICK, D. DRAPER, J.C. HAGGINS and C.R. HUNTER, Pediatrics 57 506-512 (1976). 12.L. DI BELLA, L. GUALANO, M.T. ROSSI and G. SCALERA, Boll. Soc. It. Biol. Sper. 55 389-393 (1979). 13.C.M. LEACH and G.D. THORBURN, Prostaglandins 20 51-56 (1980). 14.M. DA PRADA and G.B. PICOTTI, Brit. J. Pharmacol. 65 653-662 (1979). 15.M. GRAF, A. LAUBSCHER, J.G. RICHARDS and A. PLETSCHER, J. Lab. Clin. Med. 93 257-265 (1979). 16.M. DA PRADA, G. BARTHOLINI and A. PLETSCHER, Biochem. Pharmacol. 14 17211726 (1965). 17.~M. SAAVEDRA, M. BROWNSTEIN and J. AXELROD, J. Pharmacol. Exp. Ther. 186 508-515 (1973). 18.D.C. KLEIN and A. NOTIDES, Anal. Biochem. 31 480-483 (1969). 19.B.J. LEMAITRE and L. HARTMANN, J. Immunol. Meth. 32 339-347 (1980). 20.R. BESSELIEVRE, B.J. LEMAITRE, H.P. HUSSON and L. HARTMANN, Biomedicine 33 226-228 (1980). 21.S. KVEDER and W.M. McISAAC, J. Biol. Chem. 236 3214-3220 (1980). 22.M.E. SISAl<, S.P. MARKEY, R.W. COLBURN, A.P. ZAVADIL and I.J. KOPIN, Life Sci. 25 803-806 (1979). 23.Y. OZAKI and H.J. LYNCH, Endocrinology 99 641-644 (1976). 24.N. BARRE, Etude sur les hydroxy et m4thoxyindoles plaquettaires: mise en 4vidence d'une voie de blosynth~se de la m~latonine, Th~se de Doctorat de 3~me cycle, Paris VII (1981). 25.B.J. LEMAITRE, J.M. LAUNAY, C. DREUX, L. HARTMANN and M. DA PRADA, 2nd International Colloquium of the European Pineal Study Group, Giessen (1981). 26.H.J. REIMERS, D.J. ALLEN, I.A. FEUERSTEIN and J.F. MUSTARD, J. Cell Biol. 65 359-372 (1975). 27.A. PLETSCHER, G. BARTHOLINI and M. DA PRADA, Mechanisms of release of biogenic amines (eds. U.S. Von Euler, S. Rosell and B. Uvnas), pp 165-174, Pergamon Press, London (1966). 28.M. DA PRADA and A. PLETSCHER, Brit. J. Pharmacol. 34 591-597 (1968). 29.M. DA PRADA, G.B. PICOTTI, R. KETTLER and J.M. LAUNAY, Platelets: cellular response mechanisms and their biological significance (eds. A. Rot/nan, F.A. Meyer, C. Gitler and A. Silberberg), pp 277-288, J. Wiley & Sons, London (1980). 30.M. DA PRADA, J.G. RICHARDS and R. KEI~fLER, Platelets in Biology and Pathology (ed. J.L. Gordon) ~ 107-145, North-Holland Biomedical Press, Amsterdam (1981). 31.A.PLETSCHER,Essays in Neurochemistry and Neuropharmacology (eds.M.B.H. Youdim, D.F.Sharman,W.Lovenberg and J.R.Lagnado)~ 49-1Oi,J.Wiley & Sons,London (1978) .