Platelet peripheral benzodiazepine receptors in repeated stress

Life Sciences, Vol. 48, pp. 341-346 Printed in the U.S.A.

Pergamon Press

PLATELET PERIPHERAL BENZODIAZEPINE RECEPTORS IN REPEATED STRESS Dalit E. Dar 1), Abraham Weizman 2), Leon Karp 2), Alexander Grinshpoon 3), Miri Bidder 1), Moshe Kotler 3), Sam Tyano 2), Avraham Bleich 3), and Moshe Gavish 1)* I ) Department of Pharmacology, Faculty of Medicine and Rappaport Family I n s t i t u t e for Research in the Medical Sciences, Technion-Israel I n s t i t u t e of Technology, P.O.B. 9649, 31096 Haifa, Israel; 2) Geha Psychiatric Hospital, Beilinson Medical Center, Petah Tiqva and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 3) Mental Health Department, Israel Defence Forces (Received in final form November 19, 1990)

Summary [3H]PK 11195 binding to platelet~mbranes and plasma stress hormones were studied in soldiers at the beginning of a parachute training course, following 6 days of preparatory exercises, and after the fourth actual parachute Jump. A s l i g h t reduction (15%; NS) in the number of peripheral benzodiazepine receptors (PBR) was detected at the end of the exercise period, prior to the f i r s t jump. Reduced (26%; P < 0.05) density of PBR was observed inTnediately after the repeated actual jumps. Equilibrium dissociation constants were not affected by the stressful situation. Plasma cortisol and prolactin levels remained unaltered during the entire study period. Specific h i g h - a f f i n i t y benzodiazepine receptors (PBR) have been identified in a variety of peripheral organs (1-4) as well as in the brain (3,5,6), where the highest densities are in the olfactory bulb (3,6) and pineal gland (7,8). The distribution of PBR has been demonstrated using autoradiographic techniques and has been shown to d i f f e r from that of the central benzodiazepine receptors (CBR) (3,4). PBR are located mainly in non-neuronal tissues of the brain, such as glia, ependyma, and choroid plexus (3,6,9), while the CBR are confined to tissues derived from the neural crest (3). CBR are coupled to the y-aminobutyric acid (GABA) recognition site and to the chloride ion channel and bind with high a f f i n i t y the ligands clonazepam, B-carboline-3-carboxylic acid ethyl ester, and flumazenil (Ro 15-1788). The muscle-relaxant, sedative, hypnotic, and anxiolytic effects of the benzodiazepines (BZs) are mediated via the central GABA/BZ receptors. PBR bind with high a f f i n i t y the ligands Ro 5-4864 (a nonanxiolytic BZ) and PK 11195 (a non-BZ isoquinoline carboxami~e derivative), which are inactive at the CBR. I t has been suggested that [°H]PK 11195 binds to sites coupled to the voltage-regulated calcium channel complex of the cell membrane (10). However, the relationship between PBR and the calcium channels is not clear (11,12). The physiological and pharmacological functions of PBR are not as yet clear, although i t has been demonstrated that these sites are sensitive to hormonal changes (13-17) and environmental stress (18,19). Ro 5-4864 possesses proconflict and proconvulsant effects (20),

* To whom correspondence and requests for reprints should be addressed. 0024-3205/91 $3.00 +.00 Copyright (c) 1991 Pergamon Press plc

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while PK 11195, which is believed to be a specific antagonist of PBR, shows a n t i c o n f l i c t a c t i v i t y in animals (21) and was reported in a p i l o t study to possess anxiolytic properties in man (22). In previous studies we demonstrated opposite effects of acute and chronic stress on platelet PBR, i . e . , up-regulation of the binding site following examination stress (23) and down-regulation in chronically anxious patients (19). However, i t was not clear i f the decrease in [~H]PK 11195 binding detected in generalized anxiety disorder reflected the pathology of c l i n i c a l anxiety or i f i t was a normal, physiological adaptive response to continuous stress. The aim of the present study was to investigate whether similar changes in platelet PBR would occur after repeated stress (parachuting) in normal, healthy volunteers and whether such alterations would correlate with modulations in plasma levels of stress hormones (cortisol and prolactin). Materials and Methods Subjects. Six male soldiers aged 18-19 years agreed to participate in the study. All the participants were physically healthy, nonaddicted, nonobese, and with no past history of psychiatric disorder. All the subjects were selected for a parachute training course consisting of 6 days of preparatory exercises followed by once-daily actual parachute jumps. A blood sample for determination of platelet PBR and hormonal levels was collected from each subject three times, between 9:00 and 10:00 a.m.: 1) baseline, on the f i r s t day of the parachute training course (6 days before actual parachuting); 2) 30 min before the f i r s t parachute jump following the training course; and 3) 30 min after the fourth daily parachute jump. Concomitantly with blood sampling, blood pressure was measured, and the Hamilton anxiety rating scale (HARS) (24) as well as the Spielberger State-Trait Anxiety Inventory (STAI) (25) were completed. Binding assay. Preparation of platelet membranes and [3H]PK 11195 binding a@say Were performed as previously described (26) Six concentrations of [aH]PK 11195 (final concentration, 0.2-6 nM) in the absence (total binding) or presence (nonspecific binding) of 10~M unlabeled PK 11195 were used to examine the binding to the platelets of each subject. The data were evaluated by Scatchard analysis 5o obtain the maximal number of binding sites (BmaX) and their a f f i n i t y to [~H]PK 11195 as expressed by the equilibrium diss6~Tation constant (KD). Hormonal determination. Plasma cortisol levels were measured by solid-phase radioimmunoassay (RIA) using materials provided by Diagnostic Products Corporation (Los Angeles, CA); the antibody was covalently bound to the inner surface of a polypropylene tube. The s e n s i t i v i t y of the assay was 0.5 pg/dl, and the mean intra-assay coefficient of variation was 7%. Plasma prolactin (PRL) levels were determined by double-antibody RIA using materials provided by Diagnostic Products Corporation. The s e n s i t i v i t y of the assay was 2.5 ng/ml, and the mean intra-assay coefficient of variation was 6.7%. All the hormonal assays were run in the same batch. Statistical

evaluation.

Statistical evaluation of the data was performed

using paired t - t e s t for intragroup variations. mean + SEM.

All results are expressed as

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Resul ts The density of [3H]PK 11195 binding sites on platelet membranes was significantly decreased (26%) immediately after the fourth parachute jump, in comparison to basal levels (2852 + 410 vs. 3846 + 297 fmol/mg protein; t = 2.55, df = 5, P < 0.05). A slight reduction-of 15% was observed following the training course, immediately prior to actual parachuting, when compared to basal levels (3265 + 420 vs. 3846 + 297 fmol/mg protein; NS) (Fig. 1). The KD values remained unchanged (baseline, 7.6 + 0.6 nM; before f i r s t jump, 7.8 + 0.5 nM; after fourth jump, 8.2 + O.T nM; NS).

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FIG. 1 Maximal binding capacity of [3H]PK 11195 to platelet membranes of the study population. Platelet membraqes (70-100 ~g protein) were incubated with six concentrations of [°H]PK 11195 Xfinal concentration, 0.2-6 nM) in the absence (total binding) or presence (nonspecific binding) of 10 ~M unlabeled PK 11195. Scatchard analysis was performed to obtain Bmax and KD values. Results are expressed as mean + SEM. Baseline,-6 days before actual parachuting; Before, 30 min before the f i r s t actual jump; After, 30 min after the fourth daily jump. *P < 0.05, vs. baseline values (paired t - t e s t ) . Plasma cortisol and PRL levels were not affected either by the training course or by the actual parachuting, as shown in Fig. 2. In contrast to our expectations, no alterations in the anxiety scores were reported by the soldiers (HARS--baseline, before f i r s t jump, and after fourth jump: 17.7 + 2.7, 15.5 + 0.8, and 17.5 + 3.1 points, respectively; STAI-state score: 35.3 +--2.5, 38.5 T 4 . 2 , and 32.3 ¥ 2.2 points, respectively).

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FIG. 2 Plasma cortisol (pg/dl) and PRL (ng/ml) levels in the study population. Hormonal levels were determined by RIA: baseline, before f i r s t jump, after fourth jump. Results are expressed as mean + SEM. No correlation was found between [3H]PK 11195 maximal binding capacity and hormonal levels in the three conditions ( c o r t i s o l : r = 0.49, NS; PRL: r = 0.37, NS), nor was any correlation observed between the density of p l a t e l e t PBR and the anxiety scores (HARS: r = 0.01, NS; STAI-state score: r = 0.38, NS). A significant decrease in systolic blood pressure was observed after the fourth jump when compared to baseline values (117 + 4 vs. 143 + 8 mm Hg; P < 0.05) but not prior to the f i r s t actual jump X123 + 5-vs. 143 + 8-mm Hg; NS).

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Discussion Repeated parachuting ~tress (a naturalistic paradigm of stress) induced a down-regulation (26%) of [ H]PK 11195 binding sites in platelets of young soldiers, while the training course per se did not s i g n i f i c a n t l y affect platelet PBR density. These results are similar to our previous observation that long-term anxiety is associated with decreased platelet PBR (19). In contrast, acute stress is accompanied by an increase in PBR density (23,27). Repeated inescapable tail-shock sessions in rats were reported to induce a decrease in renal PBR (18), an observation which is in accordance with our findings in humans (19), while single forced-swimming stress and surgical stress were accompanied by an increase in renal PBR (27,28). This line of evidence indicates a bidirectional effect of stress on PBR, i . e . , up-regulation following acute stress and down-regulation after exposure to repeated stress. The baseline binding values of the subjects in the present study were in the range of normal control values (3000-4000 fmol/mg protein) previously reported by us (19,23,26) and others (29). The h a l f - l i f e of platelets is approximately 3 days; thus, the binding measured following the fourth parachute jump (8 days after the baseline measurement) was due to platelets not present at the time of the f i r s t sampling. I t is possible that the stress-induced receptoral changes occur at the stem cells (megakaryocytes) and are not confined to the peripheral blood platelets. The decrease in PBR density was accompanied by a parallel decrease in systolic blood pressure. These effects may be related to a habituation process which involves an adaptive reduction in sympathetic responsivity. The role of the stress hormones cortisol and PRL in the regulation of PBR is unclear. In the present study, the receptoral changes in PBR were not accompanied by hormonal alterations. However, i t is possible that in our study the peak release of stress hormones occurred at an earlier time point (during jumping from the tower mock-training apparatus or during actual parachuting) and were undetectable later. On the other hand, the p r e d i c t a b i l i t y of the stressor may blunt the hormonal responses to the stressful external challenge (30). A previous study showed increased plasma cortisol and PRL levels immediately after m i l i t a r y parachute-training jumps (31), while repeated jumps resulted in a gradual decrease in fear score and in the plasma levels of stress hormones (32). In our study, the anxiety rating scales (HARS and STAI) did not show any differences in the anxiety levels throughout the entire study period. This finding may be attributable either to a denial mechanism or to a normal coping response of young volunteers to a stressful situation, in an effort to control (deemphasize) their emotional reactions (30). In conclusion, alterations in the density of PBR seem to be a sensitive indicator of stress. The receptoral modulations are in opposite directions in single and repeated stress, even with no concomitant alterations in stress hormones. The physiological and pharmacological implications for the pathophysiology and treatment of stress need further investigation. Acknowledgments This study was supported in part by Grant 87-00234 from the U.S.-Israel Binational Science Foundation and by Grant 184-131 from the Technion V.P.R. Fund - Hedson Fund for Medical Research. We thank Drs. Ronnie Ziv and Jeff Asaacs of the Israel Defence Forces for their kind assistance in the organization of the study and Miss Ruth Singer for editorial and typing assistance.

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