J Neural Transm [-GenSect] (1992) 88:25-36

__ Journal o f Neural Transmission 9 Springer-Verlag 1992 Printed in Austria

Heterologous supersensitization between serotonin2 and alpha2-adrenergic receptor-mediated intracellular calcium mobilization in human platelets A. Kagaya 1' 2, M. Mikuni 1, H. Yamamoto 1., S. Muraoka 1, S. Yamawaki 2, and K. Takahashi 1

1Division of Mental Disorder Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, and 2Department of Neurology and Psychiatry, Hiroshima University School of Medicine, Hiroshima, Japan Accepted November 11, 1991

Summary. Recent reports suggest that serotonin (5-HT)2 receptor-mediated

second messenger systems are enhanced in platelets of affective disorders. To make the mechanism of the enhanced response clear, we investigated 5-HT2 and alpha (u)2-adrenergic receptor-induced intracellular calcium (Ca 2+) mobilization in platelets of healthy volunteers, using fura-2. 5-HT2 and a2-adrenergic receptor-mediated Ca 2+ mobilization was enhanced by prior exposure to the other type of agonist, so called "heterologous supersensitization." The supersensitization was due to the enhancement of maximal response without change in agonist affinity. Chelating extracellular Ca 2+ did not diminish the supersensitization. This enhancement of Ca 2+ mobilization was not inhibited by H-7, an inhibitor of protein kinase C. However, this supersensitization was inhibited by pretreatment with sodium fluoride which directly activates guanine nucleotide binding regulatory proteins (G proteins). These results suggest that the supersensitization was caused from intracellular Ca 2+ storage sites through a G protein-coupled pathway. Keywords: Serotonin2 receptor, a2-adrenergic receptor, calcium mobilization,

heterologous supersensitization, human platelets. Abbreviations: fura-2/AM 1-(2-(5"-carboxyoxazol-2"-yl)-6-aminobenzofuran-5-oxy)-2(2'-amino-5'-methylphenoxy)-ethane-N,N,N',N'-tetraaceticacid, pentaacetoxymethyl ester; H-7 1-(5-isoquinolinesulfonyl)-2-methylpiperazinedihydrochloride; EGTA ethylenedioxyhis(ethylamine)-N,N,N',N'-tetraacetic acid; HEPES 4-(2-hydroxyethyl)-l-piperazi* Present address: Department of Psychopharmacology,PsychiatricResearch Institute of Tokyo, Tokyo, Japan

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A. Kagaya et al.

neethanesulfonic acid; N a F sodium fluoride; Fmax maximal fluorescence intensity; Fmin minimal fluorescence intensity. Introduction

It has been widely proposed that the alteration of monoaminergic receptor function is involved in the pathophysiology of affective disorders including manic-depressive disorder. Several investigators have reported dysfunction of serotonin (5-HT)2 receptor in depressed patients in view of hormonal response (Meltzer etal., 1984) or by means of receptor binding assay using platelets (Biegon et al., 1987; Arora and Meltzer, 1989; Pandey etal., 1990), supporting the hypothesis of hypersensitivity of 5-HT receptors in brain (Aprison et al., 1978). Another abnormality about alpha (a)2-adrenergic receptor in platelets has been reported from several investigators (Mitrius etal., 1983; Kafka and Paul, 1986; Garcia-Sevilla et al., 1987). Various monoaminergic receptors are well-known to couple to transmembrane effectors mediated by guanine nucleotide binding regulatory proteins (G proteins). The effector systems include adenylyl cyclase, phospholipase C, phospholipase A2 or ion channels, leading to intracellular changes in the levels of cyclic AMP (cAMP), intracellular calcium concentration ([Ca 2+ ]i), diacyl glycerol etc. 5-HT2 receptor, one ofmonoaminergic receptors, is known to stimulate phosphoinositide (PI) hydrolysis (de Chaffoy de Courcelles etal., 1985), increasing [Ca 2+ Ji (Affolter et al., 1984; Kagaya et al., 1990). Another group of monoaminergic receptors, a2-adrenergic receptor inhibits adenylyl cyclase activity, however, this receptor is also reported to be able to couple to other second messengers, for instance, Na+/H + exchange (Sweatt etal., 1986), PI hydrolysis (Mori etal., 1989; Cotecchia etal., 1990) or calcium (Ca 2+) movement (McFadzean etal., 1989; Michel etal., 1989; Siess and Lapetina, 1989). Thus, it appears to be important to investigate 5-HT 2 and a2-adrenergic receptor-mediated second messenger systems to understand monoaminergic receptor dysfunction in affective disorders. We, therefore, have attempted to investigate 5-HT2 receptor-mediated second messenger system in platelets of depressed patients, and recently found that the receptor-mediated inositol monophosphate accumulation (Mikuni et al., 1991) and intracellular Ca 2+ mobilization (Mikuni etal., 1992) were enhanced. Another group also reported the enhanced response to 5-HT in platelets of depressive illness recently (Kusumi etal., 1992). These results seem to be in agreement with the hypothesis of hypersensitivity of 5-HT receptor in affective disorders (Aprison et al., 1978). However, the precise mechanism of the facilitation of 5-HT2 receptor-stimulated intracellular response is still obscure. It is of great interest to investigate what mechanism is involved in the enhanced response. Some kinds of receptors are controlled with one another, causing various responses. For example, epinephrine enhances an effect of 5-HT on phosphatidic acid metabolism in human platelets (de Chaffoy de Courcelles et al., 1987).

Sensitization between 5-HT2 and %-receptors in platelets

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A l o n g these lines, we have studied 5-HT 2 a n d a2-adrenergic receptor-mediated intracellular Ca 2+ m o v e m e n t , a n d have investigated the relationship between these receptors in intact h u m a n platelets. We, further, d e m o n s t r a t e d in this study an e n h a n c e m e n t o f 5 - H T 2 and ~2-adrenergic r e c e p t o r - m e d i a t e d Ca 2+ mobilization i n d u c e d by prior exposure to the other agonist, n a m e l y " h e t e r o l o g o u s supersensitization" via a m e c h a n i s m involving G protein-mediated p a t h w a y .

Materials and methods

Materials 5-HT creatinine sulfate, (-)norepinephrine(NE) hydrochloride, mezerein, prazosin hydrochloride and yohimbine hydrochloride were purchased from Sigma Chemical Co., fura-2/ acetoxymethylester (AM) from Dojindo Lab., NaF from Nakarai Chemicals LTD., and H-7 from Seikagaku Kogyo Co.

Preparation of human platelets Platelets were prepared from freshly drawn human blood with ACD (5.0 mM citric acid monohydrate, 11 mM trisodium citrate dihydrate and 8.7 mM dextrose in final concentration). Platelet-rich plasma (PRP) was obtained by centrifugation of whole blood at 400 • g for 5 rain at room temperature.

Loading of fura-2 to human platelets The PRP was then incubated with 3~M fura-2/AM for 15rain at 37~ Thereafter, the PRP was centrifuged at 700 • g for 10 rain. The platelet pellet was resuspended in KrebsRinger HEPES buffer consisting of 145 mM NaC1, 5 mM KC1, 0.5 mM Na2HPO4, 1 mM MgSO4, 10raM HEPES, 10raM dextrose and 0.2raM CaCI2,at pH7.4.

Measurement of intracellular C a 2+ concentration in human platelets The fluorescence of fura-2 was measured in a Hitachi F-2000 fluorescence spectrophotometer with excitations at 340 and 380 nm and with emission at 510nm using a Ca2+-dye dissociation constant, Kd, of 224nM as described before (Grynkiewicz etal., 1985) with minor modification. The platelets were prewarmed in a cuvette at 37 ~ with gently stirring and then ligands were added. Calibration of the fura-2 signals was calculated after the experimental treatment with 0.1% Triton X to obtain Fmax and with 4 mM EGTA to obtain Fmin by individually calibrating each run as previously described (Kagaya et al., 1990). When extracellular CaCI2 was free in the presence of 2 mM EGTA, change in [Ca2+]i was calculated as the difference of the ratio of fluorescent intensities which were obtained with the two excitation wave length.

Results

Effect of 5 - H T and norepinephrine (NE) on [Ca 2+ ]i in human platelets The m e a n resting [-Ca2+]i was 79 + 3 n M (n = 12). 5-HT, at the c o n c e n t r a t i o n o f 10 I~M, caused a rapid increase in [-Ca2+]i to 190 + 8 n M and the response u n d e r w e n t loss (Fig. 1 A). The response was completely abolished by pretreatm e n t o f platelets with 1 laM ketanserin (data n o t shown), indicating t h a t the

A. Kagaya et al.

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were applied at the arrows. Each trace represents a typical example from 12 experiments increase induced by 5-HT was mediated by 5-HT 2 receptor. NE, at the concentration of 100 gM, induced smaller than that of 5-HT, but significant increase in [Ca2+]i to 112 4- 4nM (The increase above resting level was 33 4- 3nM) (Fig. 1 B). The response to NE was fully antagonized by pretreatment with 100nM yohimbine, a potent blocker of %-adrenergic receptor but was not antagonized by ketanserin or (tl-adrenergic blocker prazosin, indicating that stimulation of %-adrenergic receptor increased [Ca 2+ ]i in human platelets. As shown in Fig. 2, and increase in [Ca 2+]i induced by 5-HT or NE was in their dose dependent manner, and the maximal increase in [Ca2+]i was obtained with 10 gM 5-HT or 100 and 300 gM NE, respectively.

Heterologous supersensitization between 5-HT2 and a2-adrenergic receptors As shown in Fig. 1 C and D, when 5-HT or NE was first applied, the subsequent addition of the other agonist induced an enhanced Ca 2 + mobilization, so called "heterologous supersensitization". The increase in I-Ca2+ ]i stimulated by 10 ~tM 5-HT was 108 4- 8 nM in the absence, and 128 4- 9 nM in the presence of 100 ~tM NE, respectively (n = 12). The increase in [Ca2+]i stimulated by 100 ~tM NE was 33 4- 3 nM in the absence and 52 4- 3 nM in the presence of 10 ~tM 5-HT, respectively (n = 12). To study the effect of supersensitization on agonist affinity, we investigated 0.3~M 5-HT- or 3 ~tM NE-induced Ca 2+ mobilization. 5-HT at the concen-

Sensitization between 5-HT 2 and a2-receptors in platelets

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-Io9]-agonist](M) Fig. 2. Dose response curves for 5-HT and NE on intracellular C a 2 + mobilization in human platelets. Values show [Ca2+]i increase from resting level. The data are mean of 5 separate experiments. The vertical bars represent S.E.M. open circle: 5-HT; closed circle: NE

Table 1. Effect of NE on 5-HT-induced intracellular Ca 2+ mobilization in human platelets Pretreatment

- (control) 100gM NE

Increase in [Ca2+]i(nM ) stimulated

Ratio

0.3 gM 5-HT

10 gM 5-HT

0.3 gM/10 gM

53 4- 7 74 4- 14 n.s.

103 4- 12 131 4- 13"*

0.52 4- 0.02 0.56 4- 0.08 n.s.

Values show increase in [Ca2+]i(nM ) or ratio of the response to 0.3 gM and 10 ~tM 5-HT in human platelets. Each value represents the mean 4- S.E.M. of 5 separate experiments. Statistical analysis was performed by paired Student's t test. n.s. = not significant difference, **p < 0.01 compared with control values

tration o f 0.31~M increased [Ca2+]i by 53 + 7 n M in the absence and 74 4- 1 4 n M in the presence o f 1 0 0 g M N E . As shown in Table 1, the ratio o f 0.3 g M a n d 10 g M 5-HT-stimulated Ca 2+ mobilization was not different w h e n platelets were p r e t r e a t e d w i t h o u t or with 100 g M NE. Similarly, the ratio o f 3 g M a n d 100 laM N E - i n d u c e d response was n o t significantly different in the absence or absesence o f 10 g M 5-HT (Table 2). Thus, the ECs0 value for agonists was n o t c h a n g e d after developing heterologous supersensitization.

Effect of extracellular calcium concentration on the supersensitization To determine the source o f e n h a n c e d response, the response induced by 5-HT or N E was investigated in the absence o f extracellular Ca 2+ (in the presence o f 2 m M E G T A ) . As shown in Table 3, b o t h 5-HT- a n d N E - i n d u c e d e n h a n c e d response were observed even in the absence o f extracellular Ca 2+ . Thus, the

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Table 2. Effect of 5-HT on NE-induced intracellular Ca 2+ mobilization in human platelets Pretreatment

- (control) 10gM 5-HT

Increase in [Ca 2+ ]i(nM) stimulated

Ratio

3 ~tM NE

100 gM NE

3 gM/100 l.tM

17 4- 4 31 4- 5*

28 4- 4 55 4- 9*

0.59 4- 0.04 0.56 4- 0.03 n.s.

Values show increase in [Ca2+]i (nM) or ratio of the response to 3 gM and 100gM NE in human platelets. Each value represents the mean 4- S.E.M. of 5 separate experiments. Statistical analysis was performed by paired Student's t test. n.s. = not significant difference, *p < 0.05 compared with control values

Table 3. Supersensitization in the absence of extracellular Ca 2+ in human platelets Pretreatment/stimulation

Response (%)

1001.tM NE/10gM 5-HT 10gM 5-HT/1001xM NE

130 + 1'1"* 152 + 12"*

Values show a percentage against the response which was obtained without pretreatment. Data are mean + S.E.M of 4 separate experiments. Statistical analysis was performed by Mann-Whitney U test. **p < 0.01 compared with the response with no pretreatment (100%)

supersensitization o f Ca 2 + m o v e m e n t occurred f r o m intracellular Ca 2 + storage sites.

Effect of protein kinase C on the supersensitization Because several studies suggest that the desensitization o f PI t u r n o v e r is mediated by an activation o f protein kinase C ( W a t s o n and Lapetina, 1985; K a g a y a etal., 1990), we investigated the effect o f protein kinase C on the supersensitization. Mezerein, one o f activators o f protein kinase C, reduced 5-HT a n d N E - i n d u c e d Ca 2 + m o v e m e n t r a t h e r t h a n p o t e n t i a t e d it (Fig. 3 A a n d B). In the presence o f 1 0 n M mezerein, the response to 1 0 g M 5-HT or 1 0 0 g M N E was 18 + 4 n M or 2 -4- 2 n M , respectively (n = 5). M o r e o v e r , H-7, an inhibitor o f protein kinase C, did n o t reduce either 10 g M 5 - H T - i n d u c e d Ca 2 + mobilization p o t e n t i a t e d by 100 g M N E , or 100 laM N E - i n d u c e d Ca 2 + m o v e m e n t p o t e n t i a t e d by 10 g M 5-HT (Fig. 3 C a n d D, T a b l e 4 a n d 5). These results suggest that the p a t h w a y which activates protein kinase C does n o t induce the heterologous supersensitization.

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Fig. 3. Effect of protein kinase C on Ca 2+ movement in human platelets. Indicated drugs (101aM 5-HT, 100gM NE, 10nM mezerein or 300gM H-7) were applied at the arrows. Each trace represents a typical example from 5 experiments

Table 4. Effect of various pretreatment on 5-HT2 receptor-mediated Ca 2+ mobilization in human platelets Pretreatment

10 gM 5-HT-induced Ca 2+ mobilization

- (control) 100laM NE 300l.tM H-7/100gM NE 1 mM NaF 3 mM NaF 1 mM NaF/100 I~M NE 3 mM NaF/100 ~tM NE

106 + 10 132 + 7** 136 + 11"* 105 + 15 104 4- 7 119 4- 14" 100 Jr 6

Values show increase in [Ca2+]i(nM ) in human platelets. Each value represents the mean + S.E.M. of 5 separate experiments. Statistical analysis was performed by paired Student's t test. *p