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Melatonin effect on serotonin uptake and release in rat platelets: diurnal variation in responsiveness
Life Sciences, Vol. 53, pp. 1079-1087 Printed in the USA
Pergamon Press
MELATONIN EFFECT ON SEROTONIN UPTAKE AND RELEASE IN RAT PLATELETS: DIURNAL VARIATION IN RESPONSIVENESS. F.J. Martin, G. Atienza, M. Aldegunde and J.M. Miguez 1. Departamento de Fisioloxia, Facultade de Bioloxia Universidade de Santiago de Compostela 15706-Santiago de Compostela, Spain. (Received in final form July 16, 1993)
Summarv The present study was conducted to examine whether melatonin impairs serotonin (5HT) release and uptake in rat platelets. Exposure of platelet-rich plasma samples (PRP) to melatonin induced a concentration-dependent inhibition of 5HT uptake and the value of ICso was 1.3x10-3M. We have also investigated the melatonin effect on the kinetic parameters of platelet 5HT uptake. Transport capacity was inhibited (Vmax; Control: 2.28+0.52, Melatonin: 0.74+0.13 pmol/107 platelet.min; p<0.05) while the affinity of 5HT for its uptake carriers remained unaltered, thus indicating a non-competitive effect. Studies carried out to determine the existence of a differential morning (8:00h)-evening (21:00h) melatonin effect showed a higher platelet uptake sensitivity at 8:00h (two-way ANOVA, p<0.001). Spontaneous 5HT release was not impared by the hormone and no daily variation in sensitivity was detected. The possible mechanism of action of melatonin on platelet transport is dicussed, and the results support the suitability of the platelet model for studying sensitivity changes in target cells to the hormone. The pineal hormone melatonin is synthesized in the pineal gland in response to environmental light information. Its secretion to the blood thereby displays a very marked circadian rhythm resulting in high circulating levels during the night and low levels during light hours (1,2). The physiological effect of the hormone not only depends on its blood levels but also the sensitivity of the target tissues. Circadian changes in the central and peripheral responsiveness to the hormone have been observed (3-8), some of which were related to daily variations in binding sites (9).
1 T o w h o m correspondence should be addressed 0024-3205/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.
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Melatonin and Platelet 5HT Transport
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Melatonin treatments induced changes in several platelet parameters (i.e., aggregation evoked by ADP and arachidonic acid and ATP and 5HT release) showing a lower responsiveness in the early morning hours than in the evening (7,8,10). The hemostatic role of plasma 5HT its also well known and the availability of this amine is mainly regulated by platelet uptake (11,12). This amine has been implicated in the ethiology of cardiovascular diseases, many of which show a higher incidence in the morning (13). However, the existence of sensitivity windows to melatonin in 5HT platelet uptake, as well as its relationship with the circadian changes in platelet aggregability, remains to be established. Structural and functional similarities between the platelet and the brain 5HT neurons led to the use of platelets as a peripheral model for the study of transport processes (i.e., uptake, release), which determine 5HT function (14). Recent works have demonstrated an inhibitory effect of melatonin on human platelet 5HT release (7) similar to those found in 5HT uptake and release of rodent synaptosomes (15). Nevertheless, there are not data relating to the effect of melatonin on platelet 5HT uptake process. As a result of the aforementioned, we have undertaken a study to examine the melatonin effect on rat platelet 5HT uptake. We also studied the presence of daily fluctuations in the hormone activity in platelet 5HT release and uptake processes. Material and methods Reaoents. Melatonin was purchased from Sigma Co. (St. Louis, MO). [14C]-Serotonin (55mCi/mmol) was obtained from Amersham Co. (UK). Paroxetine-HCI was donated by Beecham Pharm. (UK). The water used was of HPLC grade and all other reagents were of analytical grade and commercially available. v
Animals and sample oreoaration. Male Sprague-Dawley rats of 350-450 g were kept in a controlled environment (21 _+1°C) with a light:dark periodicity of 14:10 h (light on at 7:00 h). They were housed four per cage with water and food ad libitum. The rats were anaesthetized with 2,2,2-Tribromoethanol (2,5% in saline, 25 mg/kg), and ten mililiters of blood was obtained according to Ortiz et al. (16) with minor modifications. The carotid artery was cannulated with a polythene tube (0.58mm i.d., 0.96 mm o.d.; Portex, U.K.), cut beveled and filled with 1% EDTA-K 2. Blood was collected into two polypropylene tubes (5ml/tube) containing 200 /JI EDTA-I~ each (25%, w/v). Immediately after sampling, the blood was centrifuged (400 g, 15 min) at room temperature to obtain platelet rich plasma samples (PRP). PRPs from each individual animal were mixed together (final volume approx. 3-4ml). PRP platelets were counted in a Technicon H6 and the number varied between 1.2x10 s and 1.8x106 platelets/pl. Uotake assavs conditions. PRP aliquots (100pl) were preincubated for 10 minutes at 37°C in a shaking water bath (30 cycles/min) with 25 pl of melatonin diluted in buffer (50mM Tris, 20 mM EDTA-Na 2, 120 mM NaCI, pH 7,4) or with 25 pl of buffer. Passive transport and
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Melatonin and Platelet 5HT Transport
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nonspecific platelet membrane binding were assesed by the addition of 25 pl 70pM paroxetine to samples assayed in parallel. After exactly 1 minute of incubation with [14C]-5HT, uptake was stopped by the addition of 4 ml of ice cold buffer (4°C) and immediately filtered through GF/B (Whatman) glass fibre filters (Millipore sampling manifold). The tubes were rinsed twice with buffer and decanted on filters. The filters were transferred to scintillation vials and 10 ml of liquid scintillator added (toluene: triton X-100, 2:1; 4g/I 2,5-diphenyloxazole). Radioactivity was measured using a Beckman liquid scintillation counter (LS 3801). Uptake assays were always carried out within two hours of sampling. Release assay conditions. PRP samples prepared as described above were preloaded with [14C]-5HT by incubating them for 30 minutes at 37°C in a shaking water bath. The [~4C]-5HT final concentration was 0.5pM. PRP was centrifuged at 10,000 g for 10 minutes at 4°C and resuspended in Krebs-Ringer phosphate buffer (KRP) containing (final concentration, raM): 131 NaCI, 5.9 KCI, 1.14 ascorbic acid, 11.1 D-glucose, 0.16 Na2-EDTA, 0.1 pargyline, and 15.8 de Na2HPO4. This buffer was preequilibrated with a mixture of O2-CO 2 (95:5) by continuous bubbling at room temperature and then the pH was adjusted to 7.4 with 1N NaOH. A series of polypropylene tubes, each containing 1.1 ml of KRP with paroxetine (10pM) and melatonin (0, 2x10 -6, or 2x10-4M), was preincubated at 37°C in a shaking water bath. An aliquot (0.1 ml) of preloaded platelet suspension was added to each tube and incubated for exactly 30 minutes. Release was stopped rapidly by adding 3ml of KRP, followed by filtering and washing twice with KRP. Three parallel assayed tubes were stopped after 0 minutes of incubation to quantify the radioactive serotonin uptake in the platelet suspension. The filter radioactivity was measured using the method described above. Percentage release was calculated as follows: % release = 100 (1 - platelet [14C]-5HT3omtn/ platelet [1--~C]-5HT-~-om,n) Soecific exoerimental design, Melatonin effect on platelet 5HT uptake was established in a pilot experiment using 0.5 pM [14C]-5HT and two melatonin concentrations (2x10-gM and 2x10-4M) each assayed in triplicate. In order to calculate the IC5ovalue platelets were incubated with 0.5pM [~4C]-5HT in the presence of various concentrations of melatonin (from 2x10-4M to 2x10-gM) assayed in triplicate. The IC5o value was the mean of four separate experiments, and was determined by linear regression analysis of transformed sigmoidal curve-response with the probit transformation Iogit (P)-Iog melatonin concentration [M], where Iogit (P)=ln(p/(1 00-p)) and p=pmol/10~platelets.min. Melatonin effect on kinetic parameters of platelet 5HT uptake was examined with six [14C]-serotonin concentrations, ranging from 0.08 to 2pM, for each experiment. Preincubation and incubation was carried out in the absence (control) or presence of 2x10-4M melatonin. Each 5HT concentration was triplicate assayed and the results presented are the mean of two independent experiments. The aparent value of Vmax and Km were fitted with the aid of a non-linear regression programme
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Melatonin and Platelet 5HT Transport
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(Enzfitter, Elsevier-BIOSOFT, UK), using Lineweaver-Burk data transformations for initial estimates of kinetic parameters. In experiments 1 and 2, the samples were obtained and processed between 18:00h and 20:00h. Diurnal variations of melatonin effect uptake and release were assesed in samples of PRP Different concentrations of melatonin (0, 2x10-6M, triplicate samples and two independent experiments
on spontaneous platelet 5HT obtained at 8:00h and 21:00h. 2x10-4M) were conducted in were carried out.
Statistical analysis. Results were statistically analysed by Student's t-test for paired samples when the data was from the same animal (Experiment 1). The significance of differences between the means of kinetic variables was evaluated by a non-paired t-test (Experiment 2). The data from experiment 3 was analyzed by a two-way ANOVA. The significance between the the concentrations examined at the same time of day was calculated by a one-way ANOVA followed by Student-Newman-Keuls' test. All analyses were performed with the SPSS-PC+ (V 2.3) statistical package. 5F
T
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i -9
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i -7
i -6
i -5
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i -3
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i 0
Log melatonin [M] FIG. 1 Linear regression of transformed data for [14C]-5HT uptake inhibiting action of melatonin. IC~o, melatonin concentration when Iogit(P)=0, was estimated from the following regression equation: Iogit(P)= -1.25411 0.43517 log melatonin [M]. Iogit(P)=ln(p/(100-p)). IC~ovalue was 1.3x103M. Results E~2~J[]~d3EL In a pilot experiment we observed a concentration-dependent inhibitory effect of melatonin on platelet uptake (control: 9.14 _+ 0.88 pmol/min; melatonin 2x10-4M:-46% versus control, p<0.05; melatonin 2x10-gM: -17% versus control, not significant). The analysis of the concentration-effects curve relating % inhibition of uptake to melatonin concentrations gave a value of ICso=l.3xl0-3M (concentration producing half the maximun inhibition, Fig. 1).
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.E,~2P,IJm~ztJ~ Kinetic paremeters (Vmax and Km) for platelet 5HT uptake in the control group were essentially similar to those described by other authors (17,18). Melatonin inhibition was non-competitive, i.e. it decreased Vmax but did not impair the affinity constant (Fig. 2). Samples obtained at 8:00h from control animals showed a higher 5HT uptake than those sampled in the evening (aprox. +100%, p<0.003). An ANOVA directed to time and melatonin concentration factors showed a clear dependence on both factors (Time: F=86.5, d.f.=l, p<0.001; Melatonin concentrations: F=45.9, d.f.=2, p<0.001). The highly significant interaction factor (time x melatonin concentrations; F= 9.3, d.f.=2, p<0.001) shows that the inhibiting effect was greater in the morning, but the inhibiting effect on ['4C]-5HT uptake only attained significance at 2x10-4M melatonin (one-way ANOVA followed by Student-Newman-Keuls' test, p<0.05). Similar melatonin concentrations did not impair spontaneous platelet 5HT release in either of the testing times (Fig. 3). t-
3O
Melatonin (2x10 -4 M)j / j ~
.D
25
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J
J
10
E ~
5
f
J
Control
i
0
Group Control Melatonin
J
/
~
O
J
J
2
4 6 8 1/SHT pM
Vmax pmol/lO 7 platelet.min 2.28 t 0.52 0.74 ± 0.13"
i
i
10
12 Km gM 1.26 ± 0.15 1.55 t 0 . 0 3
FIG. 2 Graphic representation of the double reciprocal of ['4C]-5HT concentrations and its acumulation in rat platelets, in absence (control) or presence of melatonin (2x10-4M). [14C]-5HT concentration ranged from 0.08 to 2pM. Significant diferences for kinetic parameters were estimated by a two-tail Student's t-test (*, p<0.05). Two animals were included for each group and kinetic values are the mean of two independent experiments. Discussion The results reported herein demonstrate for the first time an in vitro melatonin
1084
Melatonin and Platelet 5HT Transport
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inhibitory effect upon platelet 5HT uptake that is also concentration-dependent. The melatonin concentration needed to elicit a 50% inhibition is in the mM range, indicating a weak effect of the pineal hormone on amine uptake. In a recent study, melatonin in/JM concentration showed no inhibitory effect on rabbit platelet 5HT uptake (19). However, the authors did not examine more effective melatonin concentrations. The low inhibitory effect of melatonin found was not unexpected since methylation of the hydroxy group of tryptamine results in a considerable drop in the inhibitory potency on platelet 5HT transport (19). Moreover, the acethylation of the terminal NH2 of melatonin could be responsible for its lesser inhibitory potency on platelet 5HT uptake with respect to the methylated tryptamines. A similar hypothesis has been postulated to explain the weak in vitro melatonin effect on synaptosomal 5HT uptake (15). 30
1.6
8:00h
1.4
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1.2
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20 1to
6 ~
15
I
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1.0 I-1- 0.8 to I o ',d" 0.6
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21:00h
0.2 i
i
0 2x10 - 6
i
2x10 - 4
Melatonin [M]
i
h
0 2x10 - 6
i
2x10 - 4
Melatonin [M]
FIG. 3 Platelet 5HT uptake and release responsiveness to melatonin of PRP obtained at 8:00h and 21:00h. A 0.5/JM concentration of [;4C]-5HT was used. Points represents mean + SEM from two separate experiments where samples were conducted in triplicate. Units: uptake is expressed in pmol/10Zplatelets.min; release in percentage (see Material and methods). Melatonin inhibition appears to be due to a non-competitive modification of the uptake process since it only decreases the transport capacity of its uptake carriers (Vmax). The mechanism of melatonin action on platelet transport remains to be elucidated. The platelet membrane carrier involved in 5HT uptake is energy dependent (ATP) and requires sodium and chloride in order to function well (17). There are substantial similarities between platelets and presynaptic nerve endings, such as those referred to in 5HT uptake, storage and metabolism. Thus, many drugs inhibit the specific 5HT uptake in both platelets and neurons, and the potencies are of similar order in both cells (14, 20). For all those mentioned it is not surprising that melatonin's non-competitive inhibition of 5HT uptake in platelets agrees with the melatonin effect described in synaptosome-rich homogenates of the rat hypothalamus (15). Melatonin effect on 5HT uptake may depend upon a variety of mechanisms such
Vol. 53, No. 13, 1993
Melatonin and Platelet 5HT Transport
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as changes in cell membranes or interference in ATP production. Melatonin interacts with brain microtubule or actin-like proteins (21), which have also been implicated in the process of neurotransmitter uptake and release by the nerve endings. Agents such as Vinca alkaloids are known to interact with microtubular proteins and have also been found to inhibit non-competitively the synaptosomal uptake (22-24). Moreover, vinblastine inhibits both synaptosomal and platelets secretory processes interacting with microtubular proteins (22, 25). At present no data is available about either the effect of such drugs on platelet uptake or the effect of melatonin on platelet microtubules. Therefore, the hypothesis that melatonin influences platelet 5HT uptake by interacting with microtubular protein requieres further study. Results show that platelet 5HT uptake presents diurnal variations with higher values in the early morning hours (controls 8:00h=1.29_+0.13; 21:00h=0.63_+0.05 pmol/107platelets.min), similar to those changes observed in humans (26, 27). However, platelet 5HT release did not vary during the day. Both results agree with the role that transport processes play in mantaining plasma free serotonin levels (the active fraction of 5HT in blood). Therefore, it seems reasonable that platelet uptake, which acts tonically in regulating plasmatic 5HT levels, should present daily rhythmic variations while amine release -commonly associated with platelet acute responses, e.g., aggregation- does not present such variations. In fact, a lesser uptake in the evening could partially explain the simultaneous plasma 5HT increment found in humans (16). Melatonin inhibited the 5HT release evoked by thrombin in washed human platelet preparations, with a higher effect ocurring in the evening (7). Surprisingly, spontaneous rat platelet release of amine was not sensitive to melatonin. The following reasons could be suggested to explain such discrepancies: 1. Species-specific differences; 2. Methodological aspects: results reported herein were obtained without platelet exposition to proaggregant agents. In any case, our data indicates an improbable direct melatonin action on the exocitotic process. Moreover, our results do not contradict the idea that melatonin exerts an inhibitory effect on platelet release through partial depression of the arachidonic acid pathway, as has been proposed by other authors (7, 28). Melatonin inhibition of platelet 5HT uptake was dependent on the time of day at which the experiments were performed, displaying a higher sensitivity in the early morning (8:00h). Nighttime rodent blood melatonin concentrations are in the pM and low nM range (29). However, melatonin nM concentrations only elicited a slight decrease of in vitro platelet uptake thus raising doubts about the physiological significance of the present findings and its contribution to the morning peak in platelet aggregability found in humans (13). The origin of the diurnal variation in the hormone activity on platelet uptake remains unknown. The morning sensitivity peak may not be explained by a down-regulation of the receptor sites by exposure to high circulating levels of the hormone at night, and support the idea of a non-mediated receptor platelet action for melatonin. This proposal is compatible with the recently reported occurrence of [3H]-melatonin binding sites in human platelets -Kd within the nM range- with any morning-evening difference (30). In addition, it must be noted that the greater responsiveness to 5HT uptake in the morning was detected in PRP and
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Melatonin and Platelet 5HT Transport
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a platelet interaction with other plasma components could modify the cell sensitivity to the hormone. In summary, the results described herein support the use of platelets as a suitable model for monitoring peripheral organ responsiveness to the pineal hormone. Acknowledaements The authors would like to thank Dr. Joaqufn Espinosa for his kind collaboration. This work was carried out with a research grant from the Fondo de Investigaciones Sanitarias de la Seguridad Social (FISS 91/0471). J.M.M. and F.M.C. are recipients of predoctoral fellowships from the Xunta de Galicia. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
M. EBADI, The Pineal Gland. R.J. Reiter (Ed), pp. 1-37, Raven Press, New York (1984). R.J. REITER, Endocrine Rev. 12 151-180 (1991). L. TAMARKIN, W.K. WESTROM, A.I. HAMILL and B.D. GOLDMAN, Endocrinology 99 1534-1541 (1976). M.H. STETSON and D.E. TAY, Biol. Reprod. 24 432-438 (1983). N. ZISAPEL, Y. EGOZI and M. LAUDON, Neuroendocrinology 40 102-108 (1985). R.J. REITER, Life Sci. 40 2119-2131 (1987). M . M . DEL ZAR, M. MARTINUZZO, D.P. CARDINALI, L.O. CARRERAS and M.I. VACAS, Acta Endocrinol. (Copenh) 123 453-458 (1990). M . M . DEL ZAR, M. MARTINUZZO, C. FALCON, D.P. CARDINALI, L.O. CARRERAS and M.I. VACAS, J. Clin. Endocrinol. Metab. 70 246-251 (1990). M.I. VACAS and D.P. CARDINALI, Neurosci. Lett. 15 259-263 (1979). M.I. VACAS, M.M. DEL ZAR, M. MARTINUZZO, C. FALCON, L.O. CARRERAS and D.P. CARDINALI, J. Pineal Res. 11 135-139 (1991). G.M. TYCE, J. Cardiovasc. Pharmacol. 16 $1-$7 (1990). P.M. VANHOUTTE, J. Cardiovasc. Pharmacol. 17 $6-$12 (1991). G. TOFLER, D. BREZNISKI, A. SCHAFFER, C.A. CZEISLER, J.D. RUTHERFORD, S.N. WILLICH, R.E. GLEASON, G.H. WILLIAMS and J.E. MULLER, N. Engl. J. Med. 316 1514-1518 (1987). A. PLETSCHER, Experientia 44 152-155 (1988). D.P. CARDINALI, C.A. NAGLE, F. FREIRE and J.M. ROSNER, Neuroendocrinology 18 72-85 (1975). J. ORTIZ, F. ARTIGAS and E. GELPI, Life Sci. 43 983-990 (1988). J.M. SNEDDON, Br. J. Pharmac. 37 680-688 (1969). D.T. CHOU, H. CUZZONE and K.R. HIRSH, Life Sci. 33 1149-1156 (1983). R. WOLFEL and K. GRAEFE, Naunyn-Schmiedeberg's Arch. Pharmacol. 345 129-136 (1992). S.M. STAHL, The olatelets: Phvsiology and Pharmacology, G.L. Longenecker (Ed), pp. 307-340, Academic Press, London (1985). F.FREIRE and D.P. CARDINALI, J. Neural Transm. 37 237-257 (1975). W.J. NICKLAS, S. PUSZKIN and S. BERL, J. Neurochem. 20 109-121 (1973). W.J. NICKLAS, S. PUSZKIN and S. BERL, Nature (Lond) 247 471-473 (1974).
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24. 25. 26. 27. 28. 29. 30.
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T. SEGAWA, H. MURAKAMI, A. INOUYE and Y. TANAKA, J. Neurochem. 30 175-180 (1978). J.P. BRAMMER, L. KERECSEN and M.H. MAGUIRE, Eur. J. Pharmacol. 81 577-585 (1982). R.C.ARORA, L. KREGEL and M.Y. MELTZER, Biol. Psychiatry. 19 1579-1584 (1984). I. MODAl, R. MALMGREN, M. ,~SBERG and H. BEVING, Psychopharmacology 88 493-495 (1986). M. MARTINUZZO, M.M. DEL ZAR, D.P. CARDINALI, L.O. CARRERAS and M.I. VACAS, J. Pineal Res. 11 111-115 (1991). R.J. REITER, J. Neural Transm. 21 35-54 (1986). M.I. VACAS, M.M. DEL ZAR, M. MARTINUZZO and D.P. CARDINALI, J. Pineal Res. 13 60-65 (1992).
Pergamon Press
MELATONIN EFFECT ON SEROTONIN UPTAKE AND RELEASE IN RAT PLATELETS: DIURNAL VARIATION IN RESPONSIVENESS. F.J. Martin, G. Atienza, M. Aldegunde and J.M. Miguez 1. Departamento de Fisioloxia, Facultade de Bioloxia Universidade de Santiago de Compostela 15706-Santiago de Compostela, Spain. (Received in final form July 16, 1993)
Summarv The present study was conducted to examine whether melatonin impairs serotonin (5HT) release and uptake in rat platelets. Exposure of platelet-rich plasma samples (PRP) to melatonin induced a concentration-dependent inhibition of 5HT uptake and the value of ICso was 1.3x10-3M. We have also investigated the melatonin effect on the kinetic parameters of platelet 5HT uptake. Transport capacity was inhibited (Vmax; Control: 2.28+0.52, Melatonin: 0.74+0.13 pmol/107 platelet.min; p<0.05) while the affinity of 5HT for its uptake carriers remained unaltered, thus indicating a non-competitive effect. Studies carried out to determine the existence of a differential morning (8:00h)-evening (21:00h) melatonin effect showed a higher platelet uptake sensitivity at 8:00h (two-way ANOVA, p<0.001). Spontaneous 5HT release was not impared by the hormone and no daily variation in sensitivity was detected. The possible mechanism of action of melatonin on platelet transport is dicussed, and the results support the suitability of the platelet model for studying sensitivity changes in target cells to the hormone. The pineal hormone melatonin is synthesized in the pineal gland in response to environmental light information. Its secretion to the blood thereby displays a very marked circadian rhythm resulting in high circulating levels during the night and low levels during light hours (1,2). The physiological effect of the hormone not only depends on its blood levels but also the sensitivity of the target tissues. Circadian changes in the central and peripheral responsiveness to the hormone have been observed (3-8), some of which were related to daily variations in binding sites (9).
1 T o w h o m correspondence should be addressed 0024-3205/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.
1080
Melatonin and Platelet 5HT Transport
Vol. 53, No. 13, 1993
Melatonin treatments induced changes in several platelet parameters (i.e., aggregation evoked by ADP and arachidonic acid and ATP and 5HT release) showing a lower responsiveness in the early morning hours than in the evening (7,8,10). The hemostatic role of plasma 5HT its also well known and the availability of this amine is mainly regulated by platelet uptake (11,12). This amine has been implicated in the ethiology of cardiovascular diseases, many of which show a higher incidence in the morning (13). However, the existence of sensitivity windows to melatonin in 5HT platelet uptake, as well as its relationship with the circadian changes in platelet aggregability, remains to be established. Structural and functional similarities between the platelet and the brain 5HT neurons led to the use of platelets as a peripheral model for the study of transport processes (i.e., uptake, release), which determine 5HT function (14). Recent works have demonstrated an inhibitory effect of melatonin on human platelet 5HT release (7) similar to those found in 5HT uptake and release of rodent synaptosomes (15). Nevertheless, there are not data relating to the effect of melatonin on platelet 5HT uptake process. As a result of the aforementioned, we have undertaken a study to examine the melatonin effect on rat platelet 5HT uptake. We also studied the presence of daily fluctuations in the hormone activity in platelet 5HT release and uptake processes. Material and methods Reaoents. Melatonin was purchased from Sigma Co. (St. Louis, MO). [14C]-Serotonin (55mCi/mmol) was obtained from Amersham Co. (UK). Paroxetine-HCI was donated by Beecham Pharm. (UK). The water used was of HPLC grade and all other reagents were of analytical grade and commercially available. v
Animals and sample oreoaration. Male Sprague-Dawley rats of 350-450 g were kept in a controlled environment (21 _+1°C) with a light:dark periodicity of 14:10 h (light on at 7:00 h). They were housed four per cage with water and food ad libitum. The rats were anaesthetized with 2,2,2-Tribromoethanol (2,5% in saline, 25 mg/kg), and ten mililiters of blood was obtained according to Ortiz et al. (16) with minor modifications. The carotid artery was cannulated with a polythene tube (0.58mm i.d., 0.96 mm o.d.; Portex, U.K.), cut beveled and filled with 1% EDTA-K 2. Blood was collected into two polypropylene tubes (5ml/tube) containing 200 /JI EDTA-I~ each (25%, w/v). Immediately after sampling, the blood was centrifuged (400 g, 15 min) at room temperature to obtain platelet rich plasma samples (PRP). PRPs from each individual animal were mixed together (final volume approx. 3-4ml). PRP platelets were counted in a Technicon H6 and the number varied between 1.2x10 s and 1.8x106 platelets/pl. Uotake assavs conditions. PRP aliquots (100pl) were preincubated for 10 minutes at 37°C in a shaking water bath (30 cycles/min) with 25 pl of melatonin diluted in buffer (50mM Tris, 20 mM EDTA-Na 2, 120 mM NaCI, pH 7,4) or with 25 pl of buffer. Passive transport and
Vol. 53, No. 13, 1993
Melatonin and Platelet 5HT Transport
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nonspecific platelet membrane binding were assesed by the addition of 25 pl 70pM paroxetine to samples assayed in parallel. After exactly 1 minute of incubation with [14C]-5HT, uptake was stopped by the addition of 4 ml of ice cold buffer (4°C) and immediately filtered through GF/B (Whatman) glass fibre filters (Millipore sampling manifold). The tubes were rinsed twice with buffer and decanted on filters. The filters were transferred to scintillation vials and 10 ml of liquid scintillator added (toluene: triton X-100, 2:1; 4g/I 2,5-diphenyloxazole). Radioactivity was measured using a Beckman liquid scintillation counter (LS 3801). Uptake assays were always carried out within two hours of sampling. Release assay conditions. PRP samples prepared as described above were preloaded with [14C]-5HT by incubating them for 30 minutes at 37°C in a shaking water bath. The [~4C]-5HT final concentration was 0.5pM. PRP was centrifuged at 10,000 g for 10 minutes at 4°C and resuspended in Krebs-Ringer phosphate buffer (KRP) containing (final concentration, raM): 131 NaCI, 5.9 KCI, 1.14 ascorbic acid, 11.1 D-glucose, 0.16 Na2-EDTA, 0.1 pargyline, and 15.8 de Na2HPO4. This buffer was preequilibrated with a mixture of O2-CO 2 (95:5) by continuous bubbling at room temperature and then the pH was adjusted to 7.4 with 1N NaOH. A series of polypropylene tubes, each containing 1.1 ml of KRP with paroxetine (10pM) and melatonin (0, 2x10 -6, or 2x10-4M), was preincubated at 37°C in a shaking water bath. An aliquot (0.1 ml) of preloaded platelet suspension was added to each tube and incubated for exactly 30 minutes. Release was stopped rapidly by adding 3ml of KRP, followed by filtering and washing twice with KRP. Three parallel assayed tubes were stopped after 0 minutes of incubation to quantify the radioactive serotonin uptake in the platelet suspension. The filter radioactivity was measured using the method described above. Percentage release was calculated as follows: % release = 100 (1 - platelet [14C]-5HT3omtn/ platelet [1--~C]-5HT-~-om,n) Soecific exoerimental design, Melatonin effect on platelet 5HT uptake was established in a pilot experiment using 0.5 pM [14C]-5HT and two melatonin concentrations (2x10-gM and 2x10-4M) each assayed in triplicate. In order to calculate the IC5ovalue platelets were incubated with 0.5pM [~4C]-5HT in the presence of various concentrations of melatonin (from 2x10-4M to 2x10-gM) assayed in triplicate. The IC5o value was the mean of four separate experiments, and was determined by linear regression analysis of transformed sigmoidal curve-response with the probit transformation Iogit (P)-Iog melatonin concentration [M], where Iogit (P)=ln(p/(1 00-p)) and p=pmol/10~platelets.min. Melatonin effect on kinetic parameters of platelet 5HT uptake was examined with six [14C]-serotonin concentrations, ranging from 0.08 to 2pM, for each experiment. Preincubation and incubation was carried out in the absence (control) or presence of 2x10-4M melatonin. Each 5HT concentration was triplicate assayed and the results presented are the mean of two independent experiments. The aparent value of Vmax and Km were fitted with the aid of a non-linear regression programme
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(Enzfitter, Elsevier-BIOSOFT, UK), using Lineweaver-Burk data transformations for initial estimates of kinetic parameters. In experiments 1 and 2, the samples were obtained and processed between 18:00h and 20:00h. Diurnal variations of melatonin effect uptake and release were assesed in samples of PRP Different concentrations of melatonin (0, 2x10-6M, triplicate samples and two independent experiments
on spontaneous platelet 5HT obtained at 8:00h and 21:00h. 2x10-4M) were conducted in were carried out.
Statistical analysis. Results were statistically analysed by Student's t-test for paired samples when the data was from the same animal (Experiment 1). The significance of differences between the means of kinetic variables was evaluated by a non-paired t-test (Experiment 2). The data from experiment 3 was analyzed by a two-way ANOVA. The significance between the the concentrations examined at the same time of day was calculated by a one-way ANOVA followed by Student-Newman-Keuls' test. All analyses were performed with the SPSS-PC+ (V 2.3) statistical package. 5F
T
3 n
2
0
1 \ -1
i -9
i -8
i -7
i -6
i -5
k -4
\
i -3
i -2
~ -1
i 0
Log melatonin [M] FIG. 1 Linear regression of transformed data for [14C]-5HT uptake inhibiting action of melatonin. IC~o, melatonin concentration when Iogit(P)=0, was estimated from the following regression equation: Iogit(P)= -1.25411 0.43517 log melatonin [M]. Iogit(P)=ln(p/(100-p)). IC~ovalue was 1.3x103M. Results E~2~J[]~d3EL In a pilot experiment we observed a concentration-dependent inhibitory effect of melatonin on platelet uptake (control: 9.14 _+ 0.88 pmol/min; melatonin 2x10-4M:-46% versus control, p<0.05; melatonin 2x10-gM: -17% versus control, not significant). The analysis of the concentration-effects curve relating % inhibition of uptake to melatonin concentrations gave a value of ICso=l.3xl0-3M (concentration producing half the maximun inhibition, Fig. 1).
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Melatonin and Platelet 5 H T Transport
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.E,~2P,IJm~ztJ~ Kinetic paremeters (Vmax and Km) for platelet 5HT uptake in the control group were essentially similar to those described by other authors (17,18). Melatonin inhibition was non-competitive, i.e. it decreased Vmax but did not impair the affinity constant (Fig. 2). Samples obtained at 8:00h from control animals showed a higher 5HT uptake than those sampled in the evening (aprox. +100%, p<0.003). An ANOVA directed to time and melatonin concentration factors showed a clear dependence on both factors (Time: F=86.5, d.f.=l, p<0.001; Melatonin concentrations: F=45.9, d.f.=2, p<0.001). The highly significant interaction factor (time x melatonin concentrations; F= 9.3, d.f.=2, p<0.001) shows that the inhibiting effect was greater in the morning, but the inhibiting effect on ['4C]-5HT uptake only attained significance at 2x10-4M melatonin (one-way ANOVA followed by Student-Newman-Keuls' test, p<0.05). Similar melatonin concentrations did not impair spontaneous platelet 5HT release in either of the testing times (Fig. 3). t-
3O
Melatonin (2x10 -4 M)j / j ~
.D
25
J
N 2O b,. O
J
J
10
E ~
5
f
J
Control
i
0
Group Control Melatonin
J
/
~
O
J
J
2
4 6 8 1/SHT pM
Vmax pmol/lO 7 platelet.min 2.28 t 0.52 0.74 ± 0.13"
i
i
10
12 Km gM 1.26 ± 0.15 1.55 t 0 . 0 3
FIG. 2 Graphic representation of the double reciprocal of ['4C]-5HT concentrations and its acumulation in rat platelets, in absence (control) or presence of melatonin (2x10-4M). [14C]-5HT concentration ranged from 0.08 to 2pM. Significant diferences for kinetic parameters were estimated by a two-tail Student's t-test (*, p<0.05). Two animals were included for each group and kinetic values are the mean of two independent experiments. Discussion The results reported herein demonstrate for the first time an in vitro melatonin
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inhibitory effect upon platelet 5HT uptake that is also concentration-dependent. The melatonin concentration needed to elicit a 50% inhibition is in the mM range, indicating a weak effect of the pineal hormone on amine uptake. In a recent study, melatonin in/JM concentration showed no inhibitory effect on rabbit platelet 5HT uptake (19). However, the authors did not examine more effective melatonin concentrations. The low inhibitory effect of melatonin found was not unexpected since methylation of the hydroxy group of tryptamine results in a considerable drop in the inhibitory potency on platelet 5HT transport (19). Moreover, the acethylation of the terminal NH2 of melatonin could be responsible for its lesser inhibitory potency on platelet 5HT uptake with respect to the methylated tryptamines. A similar hypothesis has been postulated to explain the weak in vitro melatonin effect on synaptosomal 5HT uptake (15). 30
1.6
8:00h
1.4
25 cO
1.2
_¢
20 1to
6 ~
15
I
8:00h
Q.
1.0 I-1- 0.8 to I o ',d" 0.6
21:00h
lO
\.
0.4
21:00h
0.2 i
i
0 2x10 - 6
i
2x10 - 4
Melatonin [M]
i
h
0 2x10 - 6
i
2x10 - 4
Melatonin [M]
FIG. 3 Platelet 5HT uptake and release responsiveness to melatonin of PRP obtained at 8:00h and 21:00h. A 0.5/JM concentration of [;4C]-5HT was used. Points represents mean + SEM from two separate experiments where samples were conducted in triplicate. Units: uptake is expressed in pmol/10Zplatelets.min; release in percentage (see Material and methods). Melatonin inhibition appears to be due to a non-competitive modification of the uptake process since it only decreases the transport capacity of its uptake carriers (Vmax). The mechanism of melatonin action on platelet transport remains to be elucidated. The platelet membrane carrier involved in 5HT uptake is energy dependent (ATP) and requires sodium and chloride in order to function well (17). There are substantial similarities between platelets and presynaptic nerve endings, such as those referred to in 5HT uptake, storage and metabolism. Thus, many drugs inhibit the specific 5HT uptake in both platelets and neurons, and the potencies are of similar order in both cells (14, 20). For all those mentioned it is not surprising that melatonin's non-competitive inhibition of 5HT uptake in platelets agrees with the melatonin effect described in synaptosome-rich homogenates of the rat hypothalamus (15). Melatonin effect on 5HT uptake may depend upon a variety of mechanisms such
Vol. 53, No. 13, 1993
Melatonin and Platelet 5HT Transport
1085
as changes in cell membranes or interference in ATP production. Melatonin interacts with brain microtubule or actin-like proteins (21), which have also been implicated in the process of neurotransmitter uptake and release by the nerve endings. Agents such as Vinca alkaloids are known to interact with microtubular proteins and have also been found to inhibit non-competitively the synaptosomal uptake (22-24). Moreover, vinblastine inhibits both synaptosomal and platelets secretory processes interacting with microtubular proteins (22, 25). At present no data is available about either the effect of such drugs on platelet uptake or the effect of melatonin on platelet microtubules. Therefore, the hypothesis that melatonin influences platelet 5HT uptake by interacting with microtubular protein requieres further study. Results show that platelet 5HT uptake presents diurnal variations with higher values in the early morning hours (controls 8:00h=1.29_+0.13; 21:00h=0.63_+0.05 pmol/107platelets.min), similar to those changes observed in humans (26, 27). However, platelet 5HT release did not vary during the day. Both results agree with the role that transport processes play in mantaining plasma free serotonin levels (the active fraction of 5HT in blood). Therefore, it seems reasonable that platelet uptake, which acts tonically in regulating plasmatic 5HT levels, should present daily rhythmic variations while amine release -commonly associated with platelet acute responses, e.g., aggregation- does not present such variations. In fact, a lesser uptake in the evening could partially explain the simultaneous plasma 5HT increment found in humans (16). Melatonin inhibited the 5HT release evoked by thrombin in washed human platelet preparations, with a higher effect ocurring in the evening (7). Surprisingly, spontaneous rat platelet release of amine was not sensitive to melatonin. The following reasons could be suggested to explain such discrepancies: 1. Species-specific differences; 2. Methodological aspects: results reported herein were obtained without platelet exposition to proaggregant agents. In any case, our data indicates an improbable direct melatonin action on the exocitotic process. Moreover, our results do not contradict the idea that melatonin exerts an inhibitory effect on platelet release through partial depression of the arachidonic acid pathway, as has been proposed by other authors (7, 28). Melatonin inhibition of platelet 5HT uptake was dependent on the time of day at which the experiments were performed, displaying a higher sensitivity in the early morning (8:00h). Nighttime rodent blood melatonin concentrations are in the pM and low nM range (29). However, melatonin nM concentrations only elicited a slight decrease of in vitro platelet uptake thus raising doubts about the physiological significance of the present findings and its contribution to the morning peak in platelet aggregability found in humans (13). The origin of the diurnal variation in the hormone activity on platelet uptake remains unknown. The morning sensitivity peak may not be explained by a down-regulation of the receptor sites by exposure to high circulating levels of the hormone at night, and support the idea of a non-mediated receptor platelet action for melatonin. This proposal is compatible with the recently reported occurrence of [3H]-melatonin binding sites in human platelets -Kd within the nM range- with any morning-evening difference (30). In addition, it must be noted that the greater responsiveness to 5HT uptake in the morning was detected in PRP and
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a platelet interaction with other plasma components could modify the cell sensitivity to the hormone. In summary, the results described herein support the use of platelets as a suitable model for monitoring peripheral organ responsiveness to the pineal hormone. Acknowledaements The authors would like to thank Dr. Joaqufn Espinosa for his kind collaboration. This work was carried out with a research grant from the Fondo de Investigaciones Sanitarias de la Seguridad Social (FISS 91/0471). J.M.M. and F.M.C. are recipients of predoctoral fellowships from the Xunta de Galicia. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
M. EBADI, The Pineal Gland. R.J. Reiter (Ed), pp. 1-37, Raven Press, New York (1984). R.J. REITER, Endocrine Rev. 12 151-180 (1991). L. TAMARKIN, W.K. WESTROM, A.I. HAMILL and B.D. GOLDMAN, Endocrinology 99 1534-1541 (1976). M.H. STETSON and D.E. TAY, Biol. Reprod. 24 432-438 (1983). N. ZISAPEL, Y. EGOZI and M. LAUDON, Neuroendocrinology 40 102-108 (1985). R.J. REITER, Life Sci. 40 2119-2131 (1987). M . M . DEL ZAR, M. MARTINUZZO, D.P. CARDINALI, L.O. CARRERAS and M.I. VACAS, Acta Endocrinol. (Copenh) 123 453-458 (1990). M . M . DEL ZAR, M. MARTINUZZO, C. FALCON, D.P. CARDINALI, L.O. CARRERAS and M.I. VACAS, J. Clin. Endocrinol. Metab. 70 246-251 (1990). M.I. VACAS and D.P. CARDINALI, Neurosci. Lett. 15 259-263 (1979). M.I. VACAS, M.M. DEL ZAR, M. MARTINUZZO, C. FALCON, L.O. CARRERAS and D.P. CARDINALI, J. Pineal Res. 11 135-139 (1991). G.M. TYCE, J. Cardiovasc. Pharmacol. 16 $1-$7 (1990). P.M. VANHOUTTE, J. Cardiovasc. Pharmacol. 17 $6-$12 (1991). G. TOFLER, D. BREZNISKI, A. SCHAFFER, C.A. CZEISLER, J.D. RUTHERFORD, S.N. WILLICH, R.E. GLEASON, G.H. WILLIAMS and J.E. MULLER, N. Engl. J. Med. 316 1514-1518 (1987). A. PLETSCHER, Experientia 44 152-155 (1988). D.P. CARDINALI, C.A. NAGLE, F. FREIRE and J.M. ROSNER, Neuroendocrinology 18 72-85 (1975). J. ORTIZ, F. ARTIGAS and E. GELPI, Life Sci. 43 983-990 (1988). J.M. SNEDDON, Br. J. Pharmac. 37 680-688 (1969). D.T. CHOU, H. CUZZONE and K.R. HIRSH, Life Sci. 33 1149-1156 (1983). R. WOLFEL and K. GRAEFE, Naunyn-Schmiedeberg's Arch. Pharmacol. 345 129-136 (1992). S.M. STAHL, The olatelets: Phvsiology and Pharmacology, G.L. Longenecker (Ed), pp. 307-340, Academic Press, London (1985). F.FREIRE and D.P. CARDINALI, J. Neural Transm. 37 237-257 (1975). W.J. NICKLAS, S. PUSZKIN and S. BERL, J. Neurochem. 20 109-121 (1973). W.J. NICKLAS, S. PUSZKIN and S. BERL, Nature (Lond) 247 471-473 (1974).
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24. 25. 26. 27. 28. 29. 30.
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T. SEGAWA, H. MURAKAMI, A. INOUYE and Y. TANAKA, J. Neurochem. 30 175-180 (1978). J.P. BRAMMER, L. KERECSEN and M.H. MAGUIRE, Eur. J. Pharmacol. 81 577-585 (1982). R.C.ARORA, L. KREGEL and M.Y. MELTZER, Biol. Psychiatry. 19 1579-1584 (1984). I. MODAl, R. MALMGREN, M. ,~SBERG and H. BEVING, Psychopharmacology 88 493-495 (1986). M. MARTINUZZO, M.M. DEL ZAR, D.P. CARDINALI, L.O. CARRERAS and M.I. VACAS, J. Pineal Res. 11 111-115 (1991). R.J. REITER, J. Neural Transm. 21 35-54 (1986). M.I. VACAS, M.M. DEL ZAR, M. MARTINUZZO and D.P. CARDINALI, J. Pineal Res. 13 60-65 (1992).