Platelet α2-adrenoceptor binding and function during the menstrual cycle

J. psychial. Rex, Vol. 21, No. 2, pp. 163-169, Printed in Great Britain.

PLATELET

1987. 0

a2-ADRENOCEPTOR DURING

002?.-3956/87 $3.00 + 00 1987 Pergamon Journals Ltd.

BINDING AND FUNCTION

THE MENSTRUAL

CYCLE

ANDREAS E. THEODOROU, HAMANT MISTRY, SHARON L. DAVIES, YUKIHARU YAMAGUCHI

and ROGER W. HORTON Department of Pharmacology and Clinical Pharmacology, St. George’s Hospital Medical School, Cranmer Terrace, London SW17 ORE, U.K. (Received

2 October

1986; revised 11 November

1986)

Summary-Blood platelet receptors are widely used as peripheral models of central nervous system receptors, particularly in attempts to understand the biological basis of a number of neurological and psychiatric disorders. It is important to differentiate factors other than the primary disease process which may influence platelet receptors. One such potential factor is the menstrual cycle. In this study we have determined platelet high-affinity cr,-adrenoceptor binding, using the agonist ligand ‘H-UK-14,304 and platelet aggregatory responses to adrenaline in 14 healthy young women, sampled on four occasions at weekly intervals. Our results indicate that, within the limits of individual variation, neither the KD or B,, of high-affinity 3H- UK-14,304 binding or the aggregatory responses to adrenaline differed significantly between various stages of the menstrual cycle.

INTRODUCTION

sex steroids have been shown to modify a,-adrenoceptors in uterus, blood platelets and brain. For example, oestrogen administration to immature rabbits leads to a marked increase in the number of uterine a,-adrenoceptor binding sites and to increased contractile responses to noradrenaline, compared with control animals or those treated with oestrogen and progesterone (ROBERTS et al., 1977; 1981; WILLIAMS and LEFKOWITZ, 1977; HOFFMAN et al., 1981). Studies with human myometrial membranes reveal a similar picture with increased cqadrenoceptor binding sites concomitant with high circulating plasma oestrogen concentrations during the menstrual cycle and low values in postmenopausal women (BOTTARI et al., 1983). In contrast to the effects on the uterus, oestrogen administration to rabbits resulted in a reduced number of platelet a,-adrenoceptors and reduced aggregatory responses to adrenaline (ELLIOTT et al., 1980; MISHRA et al., 1985). The effects of sex steroids on human platelet cz,-adrenoceptors appear to differ from effects in the rabbit. Platelet a,-adrenoceptor binding capacity is higher and platelets are more sensitive to noradrenaline-mediated aggregation in women taking combined oral contraceptive preparations at 21 days into the cycle (last day of pill taking) than 7 days later (start of next cycle) (PETERS et al., 1979). A decrease in platelet a,-adrenoceptor number after childbirth corresponds to the decline in concentrations of circulating sex steroids from the high levels in pregnancy (METZ et al., 1983). Human platelets, because of their accessibility and the possibility of repeat sampling, are widely used as models of central nervous system receptors. Alterations in platelet FEMALE

163

164

ANDREAS E. THEODOROU et al.

a,-adrenoceptors have been claimed in a number of disorders, such as depression, anorexia nervosa, bulimia and Parkinson’s disease (GARCIA-SEVILLA et al., 1981; HEUFELDER et al., 1985; BERLANet al., 1986). It is critical when examining platelets in such conditions to distinguish factors other than the primary disease process which influence platelet receptors. A prime example is the effects of sex hormones during the normal menstrual cycle. Several groups have addressed this issue. However, studies to date have used either the selective a,-antagonist, 3H-yohimbine, or the less specific antagonist, 3H-dihydroergocryptine (PETERSet al., 1979; JONESet al., 1983; BARNETTet al., 1984). These antagonist radioligands label platelet a,-adrenoceptor binding sites with a single affinity, whereas agonist ligands identify two affinity states of platelet a,-adrenoceptor binding sites. The importance of using agonist radioligands is suggested from work related to platelet a,-adrenoceptors in depression. Three such studies, using agonist radioligands have shown increased numbers of a,-adrenoceptor binding sites in this disorder (GARCIA-SEVILLA et al., 1981, 1986; DOYLEet al., 1985) whereas no differences between depressed patients and controls were found using antagonist radioligands (P~MOULE et al., 1983; STAHLet al., 1983; CAMPBELL et al., 1985; THEODOROU et al., 1985). We have recently used the selective agonist radioligand, 3H-UK-14,304 (5-bromo-6-[2 imidazolin-2-yl-amino] quinoxaline), to label platelet c+adrenoceptors and believe it to be a particularly useful ligand since it has a 50-fold difference in affinity for the high and low affinity agonist sites and a high ratio of specific to non-specific binding (GIBSON et al., 1986). With careful choice of radioligand concentrations, 3H-UK-14,304 can be used selectively to quantify high affinity agonist sites, with minimal inteference from low affinity sites. In this study, we report platelet high affinity a,-adrenoceptor binding using 3H-UK-14,304 in a group of subjects sampled throughout the normal menstrual cycle. Concurrent measurements of platelet function (adrenaline-induced aggregation) and of plasma oestrogen and progesterone were performed. METHODS 1. Subjects Fourteen healthy female volunteers (age 18-33 yr) with regular menstrual cycles, who were not currently taking any medication and had never received oral contraceptives, participated in the study. Blood samples (60 ml) were taken between 0830 and 0915 h by antecubital venipuncture at weekly intervals for four weeks. Blood was immediately mixed with acid-citrate-dextrose (9 volumes blood: 1 volume anticoagulant). Platelet rich plasma (PRP) was obtained by centrifugation of the blood at 190g for 20 min at room temperature. Residual red and white cells were removed by further centrifugation of the PRP at 190 g for 5 min. Aliquots of the PRP were taken for platelet aggregation and platelet counting (Coulter model ZM). The remainder was used to prepare platelet membranes for the binding studies. 2. Binding studies Well-washed platelet membranes were obtained from the PRP by the method of DA~GUJI

165

PLATELET (Ye-ADRENOCEPTORSAND THE MENSTRUAL CYCLE

et al. (1981) and were finally

resuspended in ice-cold 50 mM Tris-HCl buffer, pH 7.5 at 25°C. Saturation binding of 3H-UK-14,304 (Amersham International, specific activity 77-88 Ci/mmol) was determined using eight concentrations (0.14-3.3 nM) on freshly prepared platelet membranes. All assay tubes contained manganese chloride (0.1 mM final concentration). Following incubation for 60 min at 25”C, the samples were filtered through GF/C glass fibre filters under vacuum using a cell harvester (Brandel; Gaithesburg MD). The tubes and filters were rinsed with 16 ml of ice-cold 50 mM Tris-HCl buffer and filters transferred to vials containing 6 ml of ES299 scintillation fluid (Packard Ltd) and counted at an efficiency of 40-45%. Non-specific binding was defined as the radioactivity bound in the presence of 10 PM phentolamine. The maximal number of binding sites (B,,) and equilibrium dissociation constant (KJ were determined by weighted least squares nonlinear regression analysis. Aliquots of platelet membranes were stored at -20°C subsequent to protein determination by the method of LOWRY et al. (195 l), using bovine serum albumin as standard. 3. Platelet aggregation PRP was placed in a tightly capped container at room temperature and aggregation studies completed within 3 h of blood sampling. Aggregation responses to adrenaline (seven concentrations, 3 x 1O-8 -lO-SM made up in normal saline) were determined using a dualchannel aggregometer. Autologous platelet poor plasma was used as a blank. The response to adrenaline was calculated as the initial slope of the primary aggregation and plotted as a function of the adrenaline concentration. The concentration of adrenaline which produced 50% of the maximal response (EC,,) was determined graphically. 4. Plasma hormones Plasma oestradiol 17/3 and progesterone were determined commercially available kits (R.I.A. UK Ltd).

by radioimmunoassay

using

5. Statistical analysis All statistical analyses were performed using Statistical Analytical System (SAS, SAS users guide, 1985). Moving averages were calculated with f 2 day intervals based on 28-day menstrual cycles. For example, the moving average on day 28 of the cycle is the mean of the data obtained on 26th, 27th, 28, 1st and 2nd days of cycle. Differences between means within the cycle stages were compared using Student’s t-test and correlations were performed using Pearson product-moment correlation. RESULTS

All the subjects studied claimed to have regular menstrual cycles (mean 28.3 ho.7 days, n = 14). Blood samples were taken from each subject on four occasions at weekly intervals, the initial sample being taken at the the subject and thus at a random stage of the cycle. Changes in plasma sex hormones during the menstrual cycle calculated average technique (see methods) are shown in Fig. 1. The expected changes

cycle duration a minimum of convenience of by the moving were observed.

166

ANDREAS E. Stage

I

Stage 2

--Stage 3

Day

et al.

THEODOROU

Stage 4

Stage 5 500

of menstrual

FIG. 1. Plasma oestrogen (M) and progesterone (&-0) are moving averages ( f 2 days) of four samples from

cycle

concentrations during the menstrual cycle. Values 14 subjects. Cycle stages are described in the text.

There was a sharp rise in plasma oestrogen starting at day 7 and reaching a maximum at day 14, with somewhat decreased but rather variable values up to day 24 and a second maximum around day 26. Plasma progesterone concentrations showed a single sharp peak beginning to rise at about day 14 and peaking at 21-22 days. On the basis of these variations, we divided the cycle into 5 stages, corresponding to the major hormonal changes. Stage 1, menses (2-5 days); stage 2, early rising phase of plasma oestrogen (6-9 days); stage 3, late rising phase of plasma oestrogen (10-l 5 days); stage 4, rising phase of plasma progesterone (16-21 days) and stage 5, falling phases of plasma oestrogen and progesterone (22-29 days). Binding and platelet aggregation data and platelet characteristics in these 5 stages of the menstrual cycle are presented in Table 1. All saturation binding curves were well fitted to a single binding site model in all subjects. Maximum receptor occupancy ranged from TABLE 1. CY+~JRENOCEPTORBINDING, ADRENALINE(Ad)-INDUCED AGGREGATION,PLASMAHORMONESAND PLATELET CHARACTERISTICSDURING DIFFERENT STAGESOF THE MENSTRUALCYCLE (IN DAYS) 16-21

2-5

6-9

10-15

Oestrogen (pmol/l) Progesterone (nmol/l) B max 3H-UK KD 3H-UK (nM) EC,, Ad (x lo-‘M) Platelet number/ ml PRP (x 106) Median platelet volume (f 1)

130*28 1.0*0.4 16Ozt 19 0.9ztO.l 4.4zko.7 350*31

178 +48 0.9zto.4 133+20 0.7ztO.l 4.1 zko.4 314+42

455+101 0.6ztO.3 143*14 0.9*0.1 3.8zt0.5 332zt26

434+59 20.4iz7.2 148*19 0.7+0.1 4.2hO.6 310+27

7.5~0.2

7.2zkO.3

7.3 +0.2

7.4ztO.2

B max = fmol/mg

Values are means*SEM

Variable/Cycle

stage

protein

for 6-14 estimations

22-29 470 l 73 24.7~5.0 125ztl7 1.1 l 0.2 4.0*0.5 322+16 7.4+0.3

PLATELETCQ-ADRENOCEPTORS AND

THE

MENSTRUALCYCLE

167

62-91 Vo of total receptor number (mean f SEM 80 f 1, n = 48). There were no significant differences in the B,, or KD of 3H-UK-14,304 binding between the various stages of the cycle (Student’s t-test), although B,, values tended to be lower and KD values higher at the end of the cycle than at other stages. The EC,, for adrenaline-induced aggregation, platelet number and median platelet volumes remained stable throughout these stages of the cycle. At each cycle stage, we examined the relationships between the measured variables. The strongest correlations were between platelet volume and KD (stage 3, r= 0.69, p ~0.05; stage 4, r= 0.70, p < 0.05), platelet volume and plasma oestrogen (r= 0.62, p < 0.05) and plasma progesterone (r= 0.69, p < 0.01) in stage 4 and platelet volume and progesterone (r=0.57, p CO.05) in stage 5. We also looked for relationships between plasma hormones and other variables with lag phases of one or two cycle stages. No significant correlations were found with a lag of one stage but correlations were found between plasma hormones and KD of 3HUK-14,304 binding with a lag of two stages. Plasma oestrogen during stages 5 and 1 correlated positively with KD during stages 2 and 3, respectively (r=0.81, p ~0.05; r = 0.66, p < 0.05) while plasma progesterone during stage 4 correlated negatively with KD during stage 1 (r= 0.64, p < 0.05).

DISCUSSION

In this study of 14 healthy young females, we found some variability of platelet a,-adrenoceptor binding and platelet aggregation between various stages of the menstrual cycle. The variability in all platelet parameters was small in comparison to the inter-subject variability and none of the differences reached statistical significance. Thus the normal cyclical changes in sex hormones throughout the menstrual cycle do not induce changes in platelet a,-adrenoceptor binding of similar magnitude to those seen in the rabbit following oestrogen or progesterone administration (ELLIOTT et al., 1980). Two previous studies using antagonist radioligands have reported small variations in a,-adrenoceptor binding throughout the human menstrual cycle (JONES et al., 1983; BARNETT et al., 1984). JONES et al. (1983) found highest binding capacity at day l-2 which fell maximally by 26% in mid-cycle. BARNETT et af. (1984) noted lowest binding on day 7 and higher and stable values at days 14, 21 and 28. Thus there are detailed differences both between previous studies of cY,-adrenoceptors in the menstrual cycle and between previous studies and the present results. These may relate to different methodologies and different techniques of analysis. The present use of an agonist radioligand did not reveal changes in platelet high affinity a,-adrenoceptor binding of greater magnitude than those detected with antagonist radioligands. The possibility of changes in the low affinity sites labelled by agonists during the menstrual cycle has yet to be studied. In view of the extensive literature on sex steroids and czy,-adrenoceptors in platelets and other body organs (see Introduction) we looked carefully at our own data to attempt to identify similar relationships. We found no direct correlation between plasma sex steroids and binding or functional parameters. This was not too surprising since the known actions of steroid hormones are via the cell nucleus and platelets are anucleate. The most likely

ANDREAS E. THEODOROU et al.

168

site of action would be the nucleated platelet stem cell, the megakaryocyte. One would predict some time delay between the action of steroid hormones on the megakaryocyte before this was reflected in circulating platelets since the platelet has a life-time of about 9 days in the circulation. We thus examined the relationship of platelet parameters and plasma sex steroids with various lag phases. We found no relationship between plasma sex steroids and binding capacity but noted several correlations between sex steroids and KD of 3H-UK-14,304 binding. During two consecutive phases of the cycle, there was a positive correlation between plasma oestrogen and K,, with a lag of two cycle stages and, during one phase, a negative correlation between plasma progesterone and KD with a similar lag of two cycle stages. Such time delay is compatible with an effect of sex steroids at the level of the megakaryocyte and the life-time of circulating platelets. A possible relationship between sex steroids and affinity of a,-adrenoceptors is in contrast to effects upon binding capacity which have been reported in most animal studies. However, a combination of progesterone and oestrogen has been reported to increase the ZCDof 3Hdihydroergocryptine binding in rabbit platelets, an effect not found with either steroid alone (ELLIOTT et al., 1980). In conclusion, the effects of variation in sex steroids during the normal menstrual cycle do not markedly influence the binding or function of platelet a,-adrenoceptors and thus studies of platelet a-adrenoceptors in disease states are unlikely to be adversely influenced by studying patients at different cycle stages. A similar conclusion has recently been reached in respect of platelet 3H-imipramine binding during the menstrual cycle (POIRIER et al., 1986). In man, the ability of the c+ agonist, clonidine, to stimulate the release of growth hormone has been used as an in vivo test of central a,-adrenoceptor function. The magnitude of this response is cycle-stage dependent, being greater at ovulation than during menses (~~ATUSSEK, 1984). The consistency of platelet aggregatory responses to adrenaline, measured ex vivo, at least suggests a differential regulation by sex,steroids of platelet and brain cz,-adrenoceptor function. Acknowledgements-We

thank

the Nuffield

Foundation

and the Wellcome

Trust

for financial

support.

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Psychopharmacology 79, 308-312. POIRER, M.-F., BENKELFAT, C., GALZIN, A.-M. and LANCER, S. Z. (1986) Platelet 3H-imipramine binding and steroid hormone serum concentrations during the menstrual cycle. Psychopharmacology 88, 86-89. ROBERTS, J. M., INSEL, P. A. and GOLDFIEN, A. (1981) Regulation of myometrial adrenoreceptors and adrenergic response by sex steroids. Mol. Pharmac. 20, 52-58. ROBERTS, J. M., INSEL, P. A., GOLDWN, R. D. and GOLDFIEN, A. (1977) cY-Adrenoceptors but not fi-adrenoceptors increase in rabbit uterus with oestrogen. Nature 270, 624-625. SAS User’s guide (1985) Version 5 edition. SAS Institute Inc. Cary, NC 27511-8000. STAHL, S. M., LEMOINE, P. M., CIAIIANELLO, R. D. and BERGER, P. A. (1983) Platelet a*-adrenergic receptor sensitivity in major depressive disorder. Psychiat. Res. 10, 157-164. THEODOROU, A. E., KATONA, C. L. E., DAVIES, S. L., TUNNICLIFFE, C., HALE, A. S., HORTON, R. W., PAYKEL, E. S.and KELLY, J. S. (1985) Platelet cYz-adrenergic and imipramine binding in depressed patients, before and during treatment. Br. J. din. Pharmac. 20, 528P. WILLIAMS, L. T. and LEFKOWITZ, R. J. (1977) Regulation of rabbit myometrial alpha adrenergic receptors by estrogen and progesterone. J. c/in. Invest. 60, 815-818.