0 1992 MUNKSGAARD
Binding sites for ['H]-melatonin in human platelets Vacas MI, Del Zar MM, Martinuzzo M, Cardinali DP. Binding sites for ['HI-melatonin in human platelets. J. Pineal Res. 1992:13:60-65.
Abstract: A number of in vitro effects of melatonin on human platelets were revealed in previous studies. In order to examine whether high affinity binding sites for ['HI-melatonin were present in membrane preparations of human platelets, a rapid filtration procedure through Whatman GFB paper was employed. Maximal melatonin binding was attained within 3 hr at 0°C. Scatchard analysis indicated a single population of binding sites with a dissociation constant (Kd) = 4.1 & 0.5 nM and maximal number of binding sites (Bmax) = 24.2 1.9 fmolimg protein (mean SEM of five experiments). When various indole analogs were tested for their ability to inhibit [3H]-melatoninbinding, the following Ki (nM) were obtained: 6-chloromelatonin (1 1.4), 2-iodomelatonin (22.0), melatonin (24.7), 5-methoxytryptophol (49.9), N-acetylserotonin (68.9), 6-hydroxymelatonin (78.2), 5-methoxytryptamine (184). Serotonin was a potent inhibitor of ['H]-melatonin binding with a Ki = 20.6 nM. Except for 2-methylserotonin and a-methylserotonin, a number of serotonin agonists and antagonists tested did not affect melatonin binding to platelet membranes. Binding experiments carried out at either 0800 or 2000 did not reveal time-dependent differences in Kd or Bmax. The results suggest that high affinity melatonin acceptors are present in human platelets.
*
'Departamento de Fisiologia, Facultad de Medicina and 2Seccion Hernostasia y Trombosis. Divisidn Hernatologia, Hospital de Clinicas "Jose de San Martin", Universidad de Buenos Aires, Buenos Aires, Argentina
*
Introduction
Melatonin has been implicated in the control of several physiological processes including circadian rhythmicity and the photoperiodic control of seasonal breeding [Arendt, 1988; Reiter, 19911. Presumably, melatonin acts on the brain to affect biologic rhythms, as suggested by the description of putative receptors for the hormone in cerebral structures [Dubocovich, 1988; Zisapel, 1988; Stankov and Reiter, 1990; Cardinali et al., 19911. In addition, a number of studies have indicated the existence of direct effects of melatonin in the periphery [for ref. see Reiter, 19911. Among putative peripheral targets for melatonin, the human platelets were postulated as possible peripheral markers for clinical studies on tissue responsiveness to the hormone [Del Zar et al., 1990a,b]. Melatonin interferes with several physiological processes in platelets including the aggregation phenomenon, the release of ATP and serotonin (indexes of the platelet secretory mechanism), and the production of thromboxane B, (TxB,). A generally greater, and dose-dependent, effect of nanomolar melatonin concentrations in the evening
60
Maria I. Vacas,' Maria de las M. Del Zar,' Marta Martinuzzo,* and Daniel P. Cardinali'
Key words: [3H]-rnelatonin binding-human platelets-diurnal changes in melatonin response
Address reprint requests to Or. M.I. Vacas, Departamento de Fisiologia, Facultad de Medicina, UBA. CC 243. 1425 Buenos Aires, Argentina Received October 1, 1991; accepted April 15, 1992.
(1830-2000) as compared to morning samples (0800) was observed for several of the parameters examined [Del Zar et al., 1990a,b; Martinuzzo et al., 1991; Vacas et al., 19911. Such a diurnal variability in human platelet sensitivity to melatonin is compatible with the increased evening response to melatonin found in vivo in humans [Webley et al., 19881. The present report describes the occurrence of [3H]-melatonin binding sites in membrane preparations of human platelets. Additionally, morningevening studies on platelet melatonin binding were carried out in order to assess whether a daily rhythm in platelet responsiveness to melatonin depended on changes in binding site concentration or affinity. Materials and methods Experimental subjects and sample preparation
Blood was obtained by venipuncture from healthy volunteers of both sexes (age range 20-50 years) who had taken no medication for 2 weeks at least; the volunteers gave informed consent to the study.
Melatonin binding in human platelets
There was no restriction in the activity, sleep, or diet prior to the study. The experiments were performed between April and July 1990. The blood samples were collected in 1:10 vol/vol of 0.11 M sodium citrate. A platelet-rich plasma (PRP) was prepared by centrifugation at 150g for 10 min at room temperature. PRP was centrifuged at 2,500g for 10 min at 0°C and the platelets were decanted. The pellet was washed twice with a pH 7.4, 5 mM Tris-HC1 solution containing 20 mM EDTA and 150 mM NaCl, at 0°C. Platelets were resuspended in pH 7.4, 5 mM Tris-HC1 solution containing 4.75 mM EDTA, and were homogenized by using a glass-teflon homogenizer (ten strokes of 5 sec each) at 0°C. The homogenates were centrifuged at 18,OOOg for 10 min and the platelet membrane fraction was finally resuspended in pH 7.4, 50 mM Tris-HC1 buffer containing 4 mM CaCI, at 0°C. Membrane binding assay
Binding experiments were carried out in triplicate by incubating 200 pl of membrane fraction with 200 p1 of drug or vehicle and 200 pl of [3H]-melatonin (0.2-8 mM in saturation studies and 3.5-5.0 nM in competition studies). After an incubation period of 3 hr (unless stated) the binding reaction was terminated by adding 4 ml of ice-cold 50 mM Tris-HC1 buffer and by filtering the samples through Whatman GFB filters soaked priorly in a 0.5% polyethyleneimine solution. Tubes and filters were washed thrice with 4 ml aliquots of ice-cold Tris-HC1 buffer, and the filters were dried and counted by liquid scintillation spectrometry. Nonspecific binding was assessed in the presence of 10 pM melatonin. Specific [3H]-melatonin binding was calculated by subtracting nonspecific binding from total binding. Protein concentration was measured colonmetrically by the procedure of Lowry et al. [1951] employing bovine serum albumin as a standard. Competition experiments for [3H]-melatonin binding were carried out by using a radioligand concentration within the range of concentration of the Kd. The inhibition constants for the competitors were calculated from the equation of Cheng and Prusoff [ 19731. IC,,s were calculated from logit-log :oncentration response curves (four to nine concenrations of the competitor) by the method of the least iquares. The Kd value for [3H]-melatonin was istimated independently from saturation studies. Assessment of the identity of bound radioligand
The filters employed in a typical binding assay were removed and placed in 1 ml of methanol. After 3 hr,
the solvent was evaporated under N, and the residue was resuspended in 50 pl of methanol. Authentic melatonin was then added as a tracer (10 pg), and the extract was applied onto a silica gel TLC plate and developed in the system chloroformlmethanol , 9:1, vol/vol. Melatonin was localized on the plate under a UV light. The TLC plates were then cut in 0.5 cm segments and the radioactivity was measured by liquid scintillation spectrometry. Radioligands and other chemicals
Melatonin [meth~xy-~H], specific activity 85 Ci/ mmol, was purchased from Amersham International, Buckinghamshire, U.K. The radioligand was purified by the chromatographic procedure above described not longer than 20 days before the assays. 1-(3-~hlorophenyl)piperazine HCl (mCPP), 2-methylserotonin maleate, a-methylserotonin maleate, 3-tropanyl-indole-3-carboxylate(ICS205-930), 3-tropanyl-3,5-dichlorobenzoate(MDL- 1-(2,5-dimethoxy-4-iodo pheny1)-272222), amino propane HCl ( * DOI), spiperone, and ketanserin were purchased from Research Biochemical Incorporated, Natick, MA, U . S . A . 6-Chloromelatonin was kindly donated by Eli Lilly Research Laboratories, Indianapolis, IN, U.S.A. All other chemicals were obtained from Sigma Chemical Co., St. Louis, MO, U.S.A.
*
Results
The time course of [3H]-melatonin binding to a platelet membrane preparation is shown in Figure 1. Maximum binding was attained at 0°C within 3 hr and remained constant for at least 2 hr more. Therefore, all subsequent binding studies were carried out at 0°C for 3 hr. Specific melatonin binding was linear for up to 420 pg of protein in the incubation tubes; specific binding represented 3550% of total binding (Fig. 2). There was no significant [3H]-melatonin metabolism (less than 5%) during the assay. Saturation kinetics of melatonin binding to platelet membranes was examined in incubations employing increasing concentrations of [3H]-melatonin. The Scatchard analysis of five independent experiments using different pools from donors revealed a dissociation constant, Kd, of 4.1 0.5 nM, mean k SEM, and a maximum number of binding sites, Bmax, of 24.2 k 1.9 fmol/mg protein (Fig. 3). The pharmacological profile of platelet melatonin binding sites is summarized in Table 1 . Melatonin, serotonin, and the synthetic melatonin analogues 6-chloromelatonin and 2-iodomelatonin
*
61
Vacas et al.
/
U
BINDING
c
3
mo
.-t
M
0 2 4 6 Incubation Time ( h )
Fig. 1 . [3H]-Melatonin association to human platelet membranes as a function of time of incubation at 0°C. Membranes were incubated with 4 nM [3H]-melatonin in the presence or absence of 10 p M melatonin. Each point is the mean of triplicate samples.
were potent competitors for [3H]-melatonin binding sites. In competition studies melatonin displaced [3H]-melatonin with a Ki about six times higher than the Kd found in Figure 3 . A similar discrepwas observed by Duncan et al. [1988] for [ I]-iodomelatonin binding in hamster brain membranes, and was probably due to the high nonspecific binding found in the assay (Fig. 2). As far as
SPECIFIC BINDING
"0
25 50 [3H-MELATONINl(nM1
Fig. 2 . ['HI-Melatonin binding to human platelet membranes as a function of increasing concentration of ['HI-melatonin in medium. Specific binding ( 0 ) was defined as total binding ( A ) minus non-specific binding (0). Each point is the mean of triplicate samples.
several other naturally occurring melatonin analogues, the following order of affinity was found 5-methoxytryptophol > N-acetylserotonin > 6-hydroxymelatonin > 5-methoxytryptamine (Table 1). It is known that serotonin has a low-to-moderate affinity for 5HT, binding sites, the only serotonin receptor sub-type described in human platelets [Hartig, 19891. Thus, we considered it worthwhile
Bmax 24 6 f mol/mg prot
m
0
1
2
3 4 5 6 [3H-MELATONIN]( nM)
7
Fig. 3. ['HI-Melatonin binding to human platelet membranes as a function of increasing concentration of ['HI-melatonin in medium. Each point is the mean of triplicate samples. The inset shows data in a Scatchard plot. In this experiment: Kd = 3.1 nM; Bmax = 24.6 fmol/mg protein.
62
Melatonin binding in human platelets TABLE 1. Pharmacological profile of [3H]-melatonin binding sites in human platelets: Affinity for various indoles and serotonergic drugs Compound 6-chloromelatonin 2-iodomelatonin melatonin 5-methoxytryptophol N-acetylserotonin I-hydroxymelatonin 6-methoxytryptamine serotonin 2-methylserotonin a-methylserotonin spiperone m-CPP ICS-205-930 MDL-72222 propranolol ketanserin ( r )DO1
TABLE 2. [3H]-Melatonin binding to human platelet membranes prepared from blood obtained from normal volunteers at 0800 or 2000a
Ki (nM) for [3H]melatonin bindinga 11.4 2 3.2 22.0 t 4.6 24.7 t 6.7 49.9 2 0.9 68.9 t 16.5 78.2 t 6.0 184.0 t 30.5 20.6 t 6.6 27.0 t 9.9 34.2 t 13.2 2,100 t 410 > 10,000 > 10,000 > 10,000 > 10,000 > 10,000 > 10,000
'Ki values, representing the mean t SEM of four independent experiments, were calculated as described by Cheng and Prusoff [1973] from the C I , values obtained from analysis of competition curves using four to nine different concentrations of competitor and 3.5-5.0 nM [3H]-melatonin.
to assess whether the activity of serotonin to inhibit melatonin binding to platelet membranes could be ascribed to the indolic moiety. As shown in Table 1 , the competition experiments indicated that while the SHT, analogue a-methylserotonin was a potent inhibitor of ['HI-melatonin binding, (k)DOI, a specific nonindolic 5HT2 agonist , displayed no effect. Additionally, the 5HT, antagonists spiperone and ketanserin (also having a nonindolic structure) did not compete for ['HI-melatonin binding. 2-Methylserotonin maleate, an indolic 5HT, agonist, displayed high affinity for melatonin binding while two non-indolic 5HT, antagonists, MDL72222 and ICS-205-930, were without effect. As far as 5HT, receptor agonists and antagonists, neither the selective agonist m-CPP nor the antagonists spiperone or propranolol (all non-indolic) exhibited any significant activity to displace [,HImelatonin binding. Table 2 shows [3H]-melatonin binding data in platelet membranes prepared from normal volunteers at 0800 or 2000. Neither Kd nor Bmax values exhibited time-related changes in four independent studies. Discussion
The foregoing results constitute the first description of [,H]-rnelatonin binding sites in human platelet membranes, with a Kd within the nanomolar range.
Kd (nM) Bmax (fmolimg protein)
0800
2000
5.7 t 0.8 25.6 t 1.9
5.4 t 0.6 23.5 t 8.7
aSpecific [3H]-melatonin binding was determined at equilibrium (3 hr at 0°C) as described in "Materials and Methods." Shown are the means 2 SEM of four independent experiments.
In competition experiments, 6-chloromelatonin, 2-iodomelatonin, and serotonin were potent inhibitors of [,H]-melatonin binding, while 5-methoxytryptophol, N-acetylserotonin, 6-hydroxymelatonin, and 5-methoxytryptamine exhibited less activity for the melatonin binding sites. It is interesting that several peripheral melatonin metabolites, such as the 6-hydroxymelatonin formed in the liver or the demethylated (N-acetylserotonin) or deacetylated (5-methoxytryptamine) metabolites 'of melatonin, did compete for ['HImelatonin binding to platelets, in view of the potential capacity of these circulating compounds to affect platelet physiology. In this sense, we recently observed that the major melatonin peripheral metabolite, 6-hydroxymelatonin, inhibited, with a potency similar to melatonin, ADP-induced aggregation and arachidonate-induced TxB, release in human platelets [M.I. Vacas, M. del Zar, M. Martinuzzo, D.P. Cardinali, unpublished observations]. There is abundant information on the significance of serotonin in platelet physiology [Arora et al., 1984; Fuster et al., 1987; Haslam, 1987; Holmsen, 1987; Arora and Meltzer, 1988; De Clerck et al., 1988; Siess, 19891. Moreover, serotonin binding sites are present in human platelets [Hartig, 19891, and a possible interpretation for the high affinity [3H]-melatonin binding hereby described is that it represents the binding of melatonin to platelet serotonin receptors. Indeed, in the present study serotonin exhibited a high affinity for [3H]-melatonin binding sites among the several competitors tested. However, the activity of serotonin contrasted with the very low affinities displayed by several 5HT,, 5HT2, and 5HT, agonists and antagonists, except by those closely related to the serotonin structure, i.e., a-methylserotonin or 2-methoxyserotonin, which share the indole moiety. Since the typical pharmacological profile of platelet 5HT2 binding sites was not found in the present study, the results suggest that the binding sites labeled by [3H]-melatonin in platelets are not serotonergic binding sites. Perhaps, serotonin is
63
Vacas et al.
competing for another melatonin acceptor site of a lower affinity, although the linearity of the Scatchard plots for ['HI-melatonin binding indicated a single type of binding site for the radioligand in the concentration range tested. A similar displacement by serotonin of [3Hj-melatonin binding was described in competition studies for melatonin binding in bovine [Cardinali et al., 19791 and rat brain membranes [Niles, 19871. Since the introduction of 2-1251-melatoninas a probe for biochemical and autoradiographic determination of melatonin binding sites [Laudon and Zisapel, 1986; Niles et al., 1987; Vanecek et al., 1987; Dubocovich, 1988; Dubocovich et al., 19891, ['HI-labeled melatonin has been rarely employed in binding experiments. The rationale to use [3H]melatonin as a radioligand in the present study (allowing us to examine only binding sites of a nanomolar or higher Kd) was that all the effects previously observed in vitro with melatonin in platelets were obtained at melatonin concentrations higher than lop8 M [Del Zar et al., 1990a,b; Martinuzzoet al., 1991; Vacas et al., 19911. It must be noted, however, that in test-tube binding studies using the 1251-melatonin probe, both nanomolar [Laudon and Zisapel, 1986; Niles et al., 1987; Zisapel et al., 19881 as well as subnanomolar binding sites were revealed in the brain [Vanecek et al., 1987; Duncan et al., 1988; Dubocovich et al., 1989; Morgan et al., 19891. Therefore, additional studies employing '251-melatonin would be helpful to further analyze the melatonin binding capacity of human platelets. In the present study we did not identify any morning-evening difference in the nanomolar [3H]melatonin binding to platelets. Hence, the previously reported increase in the in vitro response to melatonin in human platelets found at the evening [Del Zar et al., 1990a,b] could not be attributed to modification in the nanomolar type of melatonin binding sites. Further studies employing '251-melatonin as a ligand will be useful to assess the feasibility of employing platelets as peripheral "windows" of central melatonin activity in humans.
Acknowledgments This work was supported in part by grants from the Consejo Nacional de Investigaciones Cientificas y Tecnicas, Argentina, and the Fundacion Antorchas, Buenos Aires. We are deeply indebted to the volunteers for their collaboration.
Literature cited ARENDT, J. (1988) Melatonin. Clin. Endocrinol. 29:205-229. ARORA,R.C., L. KREGEL,M.Y. MELTZER(1984) Circadian
64
rhythms of serotonin uptake in the blood platelets of normal controls. Biol. Psychiatry 19:1579-1584. ARORA,R.C., H.Y. MELTZER(1988) Seasonal variation of imipramine binding in blood platelets of normal and depressed patients. Biol. Psychiatry 23:217-226. H.E. CARDINALI, D.P., R.E. ROSENSTEIN, D.A. GOLOMBEK, CHULUYAN, B. KANTEREWICZ, M.M. DELZAR,M.I. VACAS (1991) Melatonin binding sites in brain: Single or multiple?In: Advances in Pineal Research, vol. 5 . J. Arendt, ed. John Libbey, London, pp. 159-165. CARDINALI, D.P., M.I. VACAS,E.E. BOYER(1979) Specific binding of melatonin in bovine brain. Endocrinology 105:437441. CHENG,Y., W.H. PRUSOFF(1973) Relationship between the inhibition constant (Ki) and the concentration of inhibition which causes 50 percent inhibition (150) of an enzymatic reaction. Biochem. Pharmacol. 22:3099-3 108. DE CLERCK, F., B. XHONNEUX, D. DE CHAFFOY DE COURCELLES (1988) Functional expression of the amplification reaction between serotonin and epinephrine on platelets. J. Cardiovasc. Pharmacol. 1 I(suppl):SI-S5. L.O. DEL ZAR, M.M., M. MARTINUZZO, D.P. CARDINALI, CARRERAS, M.I. VACAS(1990a) Diurnal variation in melatonin effect on adenosine triphosphate and serotonin release by human platelets. Acta Endocrinol. 123:453458. DELZAR, M.M., M. MARTINUZZO, C. FALCON,D.P. CARDINALI,L.O. CARRERAS, M.I. VACAS(1990b) Inhibition of human platelet aggregation and thromboxane B, production by melatonin. Evidence for a diurnal variation. J. Clin. Endocrinol. Metab. 70:246-25 I . DUBOCOVICH, M.L. (1988) Pharmacology and function of melatonin receptors. FASEB J . 2:2765-2773. DUBOCOVICH, M.L., G . SHANKAR, M. MICKEL(1989) 2-Iz5Imelatonin labels sites with identical pharmacological characteristics in chicken brain and chicken retina. Eur. J. PharmaCOI.1621289-299. DUNCAN,M.J., J.S. TAKAHASHI, M.L. DUBOCOVICH (1988) 2-'2sI-Iodomelatonin binding sites in hamster brain membranes: Pharmacological characteristics and regional distribution. Endocrinology 122:1825-1833. J. BADIMAN, V. TURITTO, J.H. FUSTER,V., L. BADIMAN, CHESEBRO (1 987) Drugs interfering with platelet function: Mechanisms and clinical relevance. In: Thrombosis and Haemostasis 1987. M. Verstraete, J. Vermylen, R. Lijnen, J. Arnout, eds. Leuven University Press, Leuven, pp. 349418. HARTIG,P.H. (1989) Molecular biology of 5-HT receptors. Trends Pharmacol. Sci. 10:64-69. HASLAM, R.J. (1987) Signal transduction in platelet activation. In: Thrombosis and Haemostasis 1987. M. Verstraete, J. Vermylen, R. Lijnen, J. Arnout, eds. Leuven University Press, Leuven, pp. 147-174. HOLMSEN,H. (1987) Platelet secretion. In: Hemostasis and Thrombosis: Basic Principles and Clinical Practice. R.W. Colman, J. Hisch, V. Marder, E.W. Salzman, eds. J.B. Lippincott, Philadelphia, pp. 606-617. LAUDON,M., N. ZISAPEL(1986) Characterisation of central melatonin receptors using '251-melatonin. FEBS Lett. 197:912. LOWRY,O.H., N.J. ROSEBROUGH, A . L . FARR,R.J. RANDALL (1951) Protein measurement with the Fohn phenol reagent. J. Biol. Chem. 193:265-275. L.O. MARTINUZZO, M., M.M. DEL ZAR, D.P. CARDINALI, CARRERAS, M.I. VACAS(1991) Melatonin effect on arachidonic acid metabolism to cyclooxygenase derivatives in human platelets. J. Pineal Res. 11:111-I 15. MORGAN, P.J., L.M. WILLIAMS, G. DAVIDSON, W. LAWSON, E. HOWELL(1989) Melatonin receptors on ovine pars tuber-
Melatonin binding in human platelets ah: Characterization and autoradiographical localization. J. Neuroendocrinol. 1:1 4 . NILES, L.P. (1987) 3H-melatonin binding in membrane and cytosol fractions from rat and calf brain. J. Pineal Res. 4:89-98. NILES,L.P., D.S. PICKERING, B.G. SAYER(1987) HPLCPurified 2- '2sI-iodomelatonin labels multiple binding sites in hamster brain. Biochem. Biophys. Res. Commun. 147:949956. REITER, R.J. (1991) Pineal melatonin: Cell biology of its synthesis and of its physiological interactions. Endocr Rev. 12:151-180. SIESS, W. ( 1989) Molecular mechanism of platelet activation. Physiol. Rev. 6958-178. STANKOV,B., R.J. REITER(1990)Melatonin receptors: Current status, facts, and hypotheses. Life Sci. 46:971-982. VACAS, M.I., M.M. DEL ZAR,M. MARTINUZZO, C. FALCON,
L.O. CARRERAS, D.P. CARDINALI (1991) Inhibition of human platelet aggregation and thromboxane B2 production by melatonin during night. Correlation with plasma melatonin levels. J. Pineal Res. 11:135-139. J., A . PAVLIK, H. ILLNEROVA(1987) Hypothalamic VANECEK, melatonin receptor sites revealed by autoradiography . Brain Res. 435:359-362. WEBLEY,G.E., A . BOHLE,F.A. LEIDENBERGER (1988) Positive relationship between the nocturnal concentrations of melatonin and prolactin, and a stimulation of prolactin after melatonin administration in young men. J . Pineal Res. 5:1933. ZISAPEL,N. ( 1988) Melatonin receptors revisited. J. Neural Transm. 73: 1-5. ZISAPEL,N., 1. NIR,M. LAUDON (1988) Circadian variations in melatonin-binding sites in discrete areas of the male rat brain. FEES Lett. 232:172-176.
65
Binding sites for ['H]-melatonin in human platelets Vacas MI, Del Zar MM, Martinuzzo M, Cardinali DP. Binding sites for ['HI-melatonin in human platelets. J. Pineal Res. 1992:13:60-65.
Abstract: A number of in vitro effects of melatonin on human platelets were revealed in previous studies. In order to examine whether high affinity binding sites for ['HI-melatonin were present in membrane preparations of human platelets, a rapid filtration procedure through Whatman GFB paper was employed. Maximal melatonin binding was attained within 3 hr at 0°C. Scatchard analysis indicated a single population of binding sites with a dissociation constant (Kd) = 4.1 & 0.5 nM and maximal number of binding sites (Bmax) = 24.2 1.9 fmolimg protein (mean SEM of five experiments). When various indole analogs were tested for their ability to inhibit [3H]-melatoninbinding, the following Ki (nM) were obtained: 6-chloromelatonin (1 1.4), 2-iodomelatonin (22.0), melatonin (24.7), 5-methoxytryptophol (49.9), N-acetylserotonin (68.9), 6-hydroxymelatonin (78.2), 5-methoxytryptamine (184). Serotonin was a potent inhibitor of ['H]-melatonin binding with a Ki = 20.6 nM. Except for 2-methylserotonin and a-methylserotonin, a number of serotonin agonists and antagonists tested did not affect melatonin binding to platelet membranes. Binding experiments carried out at either 0800 or 2000 did not reveal time-dependent differences in Kd or Bmax. The results suggest that high affinity melatonin acceptors are present in human platelets.
*
'Departamento de Fisiologia, Facultad de Medicina and 2Seccion Hernostasia y Trombosis. Divisidn Hernatologia, Hospital de Clinicas "Jose de San Martin", Universidad de Buenos Aires, Buenos Aires, Argentina
*
Introduction
Melatonin has been implicated in the control of several physiological processes including circadian rhythmicity and the photoperiodic control of seasonal breeding [Arendt, 1988; Reiter, 19911. Presumably, melatonin acts on the brain to affect biologic rhythms, as suggested by the description of putative receptors for the hormone in cerebral structures [Dubocovich, 1988; Zisapel, 1988; Stankov and Reiter, 1990; Cardinali et al., 19911. In addition, a number of studies have indicated the existence of direct effects of melatonin in the periphery [for ref. see Reiter, 19911. Among putative peripheral targets for melatonin, the human platelets were postulated as possible peripheral markers for clinical studies on tissue responsiveness to the hormone [Del Zar et al., 1990a,b]. Melatonin interferes with several physiological processes in platelets including the aggregation phenomenon, the release of ATP and serotonin (indexes of the platelet secretory mechanism), and the production of thromboxane B, (TxB,). A generally greater, and dose-dependent, effect of nanomolar melatonin concentrations in the evening
60
Maria I. Vacas,' Maria de las M. Del Zar,' Marta Martinuzzo,* and Daniel P. Cardinali'
Key words: [3H]-rnelatonin binding-human platelets-diurnal changes in melatonin response
Address reprint requests to Or. M.I. Vacas, Departamento de Fisiologia, Facultad de Medicina, UBA. CC 243. 1425 Buenos Aires, Argentina Received October 1, 1991; accepted April 15, 1992.
(1830-2000) as compared to morning samples (0800) was observed for several of the parameters examined [Del Zar et al., 1990a,b; Martinuzzo et al., 1991; Vacas et al., 19911. Such a diurnal variability in human platelet sensitivity to melatonin is compatible with the increased evening response to melatonin found in vivo in humans [Webley et al., 19881. The present report describes the occurrence of [3H]-melatonin binding sites in membrane preparations of human platelets. Additionally, morningevening studies on platelet melatonin binding were carried out in order to assess whether a daily rhythm in platelet responsiveness to melatonin depended on changes in binding site concentration or affinity. Materials and methods Experimental subjects and sample preparation
Blood was obtained by venipuncture from healthy volunteers of both sexes (age range 20-50 years) who had taken no medication for 2 weeks at least; the volunteers gave informed consent to the study.
Melatonin binding in human platelets
There was no restriction in the activity, sleep, or diet prior to the study. The experiments were performed between April and July 1990. The blood samples were collected in 1:10 vol/vol of 0.11 M sodium citrate. A platelet-rich plasma (PRP) was prepared by centrifugation at 150g for 10 min at room temperature. PRP was centrifuged at 2,500g for 10 min at 0°C and the platelets were decanted. The pellet was washed twice with a pH 7.4, 5 mM Tris-HC1 solution containing 20 mM EDTA and 150 mM NaCl, at 0°C. Platelets were resuspended in pH 7.4, 5 mM Tris-HC1 solution containing 4.75 mM EDTA, and were homogenized by using a glass-teflon homogenizer (ten strokes of 5 sec each) at 0°C. The homogenates were centrifuged at 18,OOOg for 10 min and the platelet membrane fraction was finally resuspended in pH 7.4, 50 mM Tris-HC1 buffer containing 4 mM CaCI, at 0°C. Membrane binding assay
Binding experiments were carried out in triplicate by incubating 200 pl of membrane fraction with 200 p1 of drug or vehicle and 200 pl of [3H]-melatonin (0.2-8 mM in saturation studies and 3.5-5.0 nM in competition studies). After an incubation period of 3 hr (unless stated) the binding reaction was terminated by adding 4 ml of ice-cold 50 mM Tris-HC1 buffer and by filtering the samples through Whatman GFB filters soaked priorly in a 0.5% polyethyleneimine solution. Tubes and filters were washed thrice with 4 ml aliquots of ice-cold Tris-HC1 buffer, and the filters were dried and counted by liquid scintillation spectrometry. Nonspecific binding was assessed in the presence of 10 pM melatonin. Specific [3H]-melatonin binding was calculated by subtracting nonspecific binding from total binding. Protein concentration was measured colonmetrically by the procedure of Lowry et al. [1951] employing bovine serum albumin as a standard. Competition experiments for [3H]-melatonin binding were carried out by using a radioligand concentration within the range of concentration of the Kd. The inhibition constants for the competitors were calculated from the equation of Cheng and Prusoff [ 19731. IC,,s were calculated from logit-log :oncentration response curves (four to nine concenrations of the competitor) by the method of the least iquares. The Kd value for [3H]-melatonin was istimated independently from saturation studies. Assessment of the identity of bound radioligand
The filters employed in a typical binding assay were removed and placed in 1 ml of methanol. After 3 hr,
the solvent was evaporated under N, and the residue was resuspended in 50 pl of methanol. Authentic melatonin was then added as a tracer (10 pg), and the extract was applied onto a silica gel TLC plate and developed in the system chloroformlmethanol , 9:1, vol/vol. Melatonin was localized on the plate under a UV light. The TLC plates were then cut in 0.5 cm segments and the radioactivity was measured by liquid scintillation spectrometry. Radioligands and other chemicals
Melatonin [meth~xy-~H], specific activity 85 Ci/ mmol, was purchased from Amersham International, Buckinghamshire, U.K. The radioligand was purified by the chromatographic procedure above described not longer than 20 days before the assays. 1-(3-~hlorophenyl)piperazine HCl (mCPP), 2-methylserotonin maleate, a-methylserotonin maleate, 3-tropanyl-indole-3-carboxylate(ICS205-930), 3-tropanyl-3,5-dichlorobenzoate(MDL- 1-(2,5-dimethoxy-4-iodo pheny1)-272222), amino propane HCl ( * DOI), spiperone, and ketanserin were purchased from Research Biochemical Incorporated, Natick, MA, U . S . A . 6-Chloromelatonin was kindly donated by Eli Lilly Research Laboratories, Indianapolis, IN, U.S.A. All other chemicals were obtained from Sigma Chemical Co., St. Louis, MO, U.S.A.
*
Results
The time course of [3H]-melatonin binding to a platelet membrane preparation is shown in Figure 1. Maximum binding was attained at 0°C within 3 hr and remained constant for at least 2 hr more. Therefore, all subsequent binding studies were carried out at 0°C for 3 hr. Specific melatonin binding was linear for up to 420 pg of protein in the incubation tubes; specific binding represented 3550% of total binding (Fig. 2). There was no significant [3H]-melatonin metabolism (less than 5%) during the assay. Saturation kinetics of melatonin binding to platelet membranes was examined in incubations employing increasing concentrations of [3H]-melatonin. The Scatchard analysis of five independent experiments using different pools from donors revealed a dissociation constant, Kd, of 4.1 0.5 nM, mean k SEM, and a maximum number of binding sites, Bmax, of 24.2 k 1.9 fmol/mg protein (Fig. 3). The pharmacological profile of platelet melatonin binding sites is summarized in Table 1 . Melatonin, serotonin, and the synthetic melatonin analogues 6-chloromelatonin and 2-iodomelatonin
*
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Vacas et al.
/
U
BINDING
c
3
mo
.-t
M
0 2 4 6 Incubation Time ( h )
Fig. 1 . [3H]-Melatonin association to human platelet membranes as a function of time of incubation at 0°C. Membranes were incubated with 4 nM [3H]-melatonin in the presence or absence of 10 p M melatonin. Each point is the mean of triplicate samples.
were potent competitors for [3H]-melatonin binding sites. In competition studies melatonin displaced [3H]-melatonin with a Ki about six times higher than the Kd found in Figure 3 . A similar discrepwas observed by Duncan et al. [1988] for [ I]-iodomelatonin binding in hamster brain membranes, and was probably due to the high nonspecific binding found in the assay (Fig. 2). As far as
SPECIFIC BINDING
"0
25 50 [3H-MELATONINl(nM1
Fig. 2 . ['HI-Melatonin binding to human platelet membranes as a function of increasing concentration of ['HI-melatonin in medium. Specific binding ( 0 ) was defined as total binding ( A ) minus non-specific binding (0). Each point is the mean of triplicate samples.
several other naturally occurring melatonin analogues, the following order of affinity was found 5-methoxytryptophol > N-acetylserotonin > 6-hydroxymelatonin > 5-methoxytryptamine (Table 1). It is known that serotonin has a low-to-moderate affinity for 5HT, binding sites, the only serotonin receptor sub-type described in human platelets [Hartig, 19891. Thus, we considered it worthwhile
Bmax 24 6 f mol/mg prot
m
0
1
2
3 4 5 6 [3H-MELATONIN]( nM)
7
Fig. 3. ['HI-Melatonin binding to human platelet membranes as a function of increasing concentration of ['HI-melatonin in medium. Each point is the mean of triplicate samples. The inset shows data in a Scatchard plot. In this experiment: Kd = 3.1 nM; Bmax = 24.6 fmol/mg protein.
62
Melatonin binding in human platelets TABLE 1. Pharmacological profile of [3H]-melatonin binding sites in human platelets: Affinity for various indoles and serotonergic drugs Compound 6-chloromelatonin 2-iodomelatonin melatonin 5-methoxytryptophol N-acetylserotonin I-hydroxymelatonin 6-methoxytryptamine serotonin 2-methylserotonin a-methylserotonin spiperone m-CPP ICS-205-930 MDL-72222 propranolol ketanserin ( r )DO1
TABLE 2. [3H]-Melatonin binding to human platelet membranes prepared from blood obtained from normal volunteers at 0800 or 2000a
Ki (nM) for [3H]melatonin bindinga 11.4 2 3.2 22.0 t 4.6 24.7 t 6.7 49.9 2 0.9 68.9 t 16.5 78.2 t 6.0 184.0 t 30.5 20.6 t 6.6 27.0 t 9.9 34.2 t 13.2 2,100 t 410 > 10,000 > 10,000 > 10,000 > 10,000 > 10,000 > 10,000
'Ki values, representing the mean t SEM of four independent experiments, were calculated as described by Cheng and Prusoff [1973] from the C I , values obtained from analysis of competition curves using four to nine different concentrations of competitor and 3.5-5.0 nM [3H]-melatonin.
to assess whether the activity of serotonin to inhibit melatonin binding to platelet membranes could be ascribed to the indolic moiety. As shown in Table 1 , the competition experiments indicated that while the SHT, analogue a-methylserotonin was a potent inhibitor of ['HI-melatonin binding, (k)DOI, a specific nonindolic 5HT2 agonist , displayed no effect. Additionally, the 5HT, antagonists spiperone and ketanserin (also having a nonindolic structure) did not compete for ['HI-melatonin binding. 2-Methylserotonin maleate, an indolic 5HT, agonist, displayed high affinity for melatonin binding while two non-indolic 5HT, antagonists, MDL72222 and ICS-205-930, were without effect. As far as 5HT, receptor agonists and antagonists, neither the selective agonist m-CPP nor the antagonists spiperone or propranolol (all non-indolic) exhibited any significant activity to displace [,HImelatonin binding. Table 2 shows [3H]-melatonin binding data in platelet membranes prepared from normal volunteers at 0800 or 2000. Neither Kd nor Bmax values exhibited time-related changes in four independent studies. Discussion
The foregoing results constitute the first description of [,H]-rnelatonin binding sites in human platelet membranes, with a Kd within the nanomolar range.
Kd (nM) Bmax (fmolimg protein)
0800
2000
5.7 t 0.8 25.6 t 1.9
5.4 t 0.6 23.5 t 8.7
aSpecific [3H]-melatonin binding was determined at equilibrium (3 hr at 0°C) as described in "Materials and Methods." Shown are the means 2 SEM of four independent experiments.
In competition experiments, 6-chloromelatonin, 2-iodomelatonin, and serotonin were potent inhibitors of [,H]-melatonin binding, while 5-methoxytryptophol, N-acetylserotonin, 6-hydroxymelatonin, and 5-methoxytryptamine exhibited less activity for the melatonin binding sites. It is interesting that several peripheral melatonin metabolites, such as the 6-hydroxymelatonin formed in the liver or the demethylated (N-acetylserotonin) or deacetylated (5-methoxytryptamine) metabolites 'of melatonin, did compete for ['HImelatonin binding to platelets, in view of the potential capacity of these circulating compounds to affect platelet physiology. In this sense, we recently observed that the major melatonin peripheral metabolite, 6-hydroxymelatonin, inhibited, with a potency similar to melatonin, ADP-induced aggregation and arachidonate-induced TxB, release in human platelets [M.I. Vacas, M. del Zar, M. Martinuzzo, D.P. Cardinali, unpublished observations]. There is abundant information on the significance of serotonin in platelet physiology [Arora et al., 1984; Fuster et al., 1987; Haslam, 1987; Holmsen, 1987; Arora and Meltzer, 1988; De Clerck et al., 1988; Siess, 19891. Moreover, serotonin binding sites are present in human platelets [Hartig, 19891, and a possible interpretation for the high affinity [3H]-melatonin binding hereby described is that it represents the binding of melatonin to platelet serotonin receptors. Indeed, in the present study serotonin exhibited a high affinity for [3H]-melatonin binding sites among the several competitors tested. However, the activity of serotonin contrasted with the very low affinities displayed by several 5HT,, 5HT2, and 5HT, agonists and antagonists, except by those closely related to the serotonin structure, i.e., a-methylserotonin or 2-methoxyserotonin, which share the indole moiety. Since the typical pharmacological profile of platelet 5HT2 binding sites was not found in the present study, the results suggest that the binding sites labeled by [3H]-melatonin in platelets are not serotonergic binding sites. Perhaps, serotonin is
63
Vacas et al.
competing for another melatonin acceptor site of a lower affinity, although the linearity of the Scatchard plots for ['HI-melatonin binding indicated a single type of binding site for the radioligand in the concentration range tested. A similar displacement by serotonin of [3Hj-melatonin binding was described in competition studies for melatonin binding in bovine [Cardinali et al., 19791 and rat brain membranes [Niles, 19871. Since the introduction of 2-1251-melatoninas a probe for biochemical and autoradiographic determination of melatonin binding sites [Laudon and Zisapel, 1986; Niles et al., 1987; Vanecek et al., 1987; Dubocovich, 1988; Dubocovich et al., 19891, ['HI-labeled melatonin has been rarely employed in binding experiments. The rationale to use [3H]melatonin as a radioligand in the present study (allowing us to examine only binding sites of a nanomolar or higher Kd) was that all the effects previously observed in vitro with melatonin in platelets were obtained at melatonin concentrations higher than lop8 M [Del Zar et al., 1990a,b; Martinuzzoet al., 1991; Vacas et al., 19911. It must be noted, however, that in test-tube binding studies using the 1251-melatonin probe, both nanomolar [Laudon and Zisapel, 1986; Niles et al., 1987; Zisapel et al., 19881 as well as subnanomolar binding sites were revealed in the brain [Vanecek et al., 1987; Duncan et al., 1988; Dubocovich et al., 1989; Morgan et al., 19891. Therefore, additional studies employing '251-melatonin would be helpful to further analyze the melatonin binding capacity of human platelets. In the present study we did not identify any morning-evening difference in the nanomolar [3H]melatonin binding to platelets. Hence, the previously reported increase in the in vitro response to melatonin in human platelets found at the evening [Del Zar et al., 1990a,b] could not be attributed to modification in the nanomolar type of melatonin binding sites. Further studies employing '251-melatonin as a ligand will be useful to assess the feasibility of employing platelets as peripheral "windows" of central melatonin activity in humans.
Acknowledgments This work was supported in part by grants from the Consejo Nacional de Investigaciones Cientificas y Tecnicas, Argentina, and the Fundacion Antorchas, Buenos Aires. We are deeply indebted to the volunteers for their collaboration.
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