(;en Pharmac. 1977. t,-d 8. pp 201 to 206 Pergamon Pre.~.s Printed in Great Brltatn

TRANSPORT OF 5-HYDROXYTRYPTAMINE (SEROTONIN) BY CANINE BLOOD PLATELETS WILLIAM W. KAY* ANt) GARYEN (_~IONG Departments of Biochemistry and Surgery, University of Saskatchewan, Saskatoon. Saskatchewan, Canada (Received 17 Not,ember 1976) Abstract 1. 5-Hydroxytryptaminc (5-HT) was transported into canine bh)od platclcts by a system characterized by distinct temperature (Q~c~ 2.3) and pH optima (37(7. 7.0 respectively) and ~lturation kinetics (K,, 1.6 × 10-TM). 2. Competitive inhibition studies with 14 structural analogues indicated a high degree of stercospecificity. 3. N-acctyl-5-hydroxytryptaminc (NAHT) was not actively translx-,rted by the same system but was an effective competitive inhibitor of 5-HT uptake. 4. Canine platclets, when immobilized on membrane filters, did not release labelled 5-HT unless washed with adenosincdiphosphate (ADP) or thrombin, however tilter immobilized platclets were still able to undergo shape changes. Under these conditions accumulatcd 5-HT only slowly exchanged with exogenous 5-1tT or NAHT. 5. Of several metabolic inhibitors tested only the respiratory poisons potassium ferricyanide and sodium amytal were exceptionally strong inhibitors of 5-HT transport and caused the release of 5-HT from pre-loaded platelets. N-ethylmaleimide (NEM) was similarly inhibitory.

INTRODI.:C'r ION

MATERIALS AND METItODS

Platelets of many m a m m a l i a n species including h u m a n s contain 5-HT in comparatively high concentrations (Rand & Reid, 1951). Although 5-HT is secreted from the gastrointestinal tract into the blood it is accumulated by circulating platelets (Weissbach & Redfield, 1961). The cummulative evidence from studies on a variety of species indicates that 5-HT is actively transported through the outer m e m b r a n e (Born 8,: Gillson, 1959; Weissbach & Redfield, 1960; O k u d a & Nemcrson, 1971), by a system showing some structural specificity (Born et ~1., 1972). The subsequent accumulation of 5-HT in cytoplasmic o.ganelles in association with adenosine triphosphate (ATP) occurs by a separate mechanism (Da Prada & Pletscher, 1966, 1969; Pletscher et al., 1969; Baker et al., 1972). Once accumulated the 5-HT pool appears to be extremely stable, apparently maintained at the expense of energy generated via both glycolysis and respiration (Weissback & Redfield, 1961). Finally the release of 5-HT from platelets can be affected by thrombin, adenosine diphosphate (ADP), or collagen as well as a great variety of agents (Mustard & Packham, 1970). In the present paper we describe the general properties of the serotonin transport system of canine platelets, introduce a new technique of studying immobilized platelets and compare the system to that of other species prior to the investigation of the energetics of serotonin transport.

Platelet~

Blood was drawn from the jt,gular vein directly into a plastic syringe and mixed immediately with acid citratedextrose (ACD) (ACD:blood, 1:6). Larger blood cells were removed by centrifugation at 120g for 20rain leaving the top la3cr of pk, tclct-rich plasma (PRP). When required platclets were sedimented from the PRP at 200g for 30 rain and resuspended in an equal w)lumc of ACD or Ringers buffer. Platelet numbers were assayed dircctl~ either with a hcmocytomcler or by particle counting (Hcmolab. Celloscopc Platclet Counter). Platclet preparations which showed signs of aggregation prior to or during an experiment wcrc discarded. 5- tl 7" Tran.sport

Platelet suspensions (150.000 300,000:ram ~) were incubated for 5 rain at 37 C for temperature equilibration prior to the addition of labelled 5-HT. 5-HT (0.1 ml) was added to 5 ml suspensions and aliqt, ots of platclcts were harvested intermittently either by ccntrifugation (Eppendorf Microfuge 3200 centrifuge) or b', suction filtration (~irtorius membrane tilters. 0.45/~m). Aliquots of the supernarants from centrifuged cells were assayed for radioactivity. Filtered cells were washed with 5 ml of ACD and the tilters assayed directly for radioactivity. All radioactive as~tys were conducted in a dioxane-base scintillation fluid (PCS: Amcrsham/~arle Corp.) with a Nuclear Chicago Mark 11 liquid-scintillation spectrometer. In kinetic studies at variable substratc or inhibitor concentrations the initial rotes of uptake v~ere calct, lated from the linear uptake rates obtained by 30 sec sampling for 2 min. Platelets were inct, bated with metabolic inhibitors for 15 rain prior to the addition of labelled serotonin.

*To whom reprint requests should be sent: Department of Bacteriology and Biochemistry University of Victoria. P.O. Box 17(~). Victoria. B.C. VSW 2Y2. Canada. 201

5-H T release

Platelcts were incubated with labelled 5-ttT lor 20 rain to ensure steady-state conditions. 5-HT release was

~,~:11.1 IA\I ~,l,:. KA', AND (.La.R'~IN ('II()NG

2(12

PRP

llleastlrcd b,. t'o,o m e t h o d s : b\ the lit'st, tile inhibitor was added to the labelled suspcnsiorl alld irnnlcdiatcl 3 followed b,. intermittant sampling, b,. tile second method tile htbellcd i-,latclcts v, crc ln)mobili.,'cd on membrane tilters and ~aashcd continuousl.~ v~ith A('[) containing the appropriate inhJbitors. At '.arious interxals membranes were telllO~,cd altd :.tssa.,,cd for radioaclixitL

60 ~ /e '''~'e'-'--e

ReD •

/

~, 4o

t'at(' ol intraccllulm" 5-111 Platclets ~crc incubated ~,ith t).l tiM 5-HT for 60 min, harxcstcd b 3 filtration, v, ashcd, immersed in 10 ml of ACD containing 0.2 ml toluene at 0 C and mixed intermittently for 10 ram. The mixture ~as tiltcrcd again ,,nd the filtrate I,,ophilized arid chromatographcd b', two-dimensional l L ( on silica gel in sol',cnt systems of propionic acid. isopropanol 25",, NH4OIt (9:7:4. v x ,.'t and chloroform acetic acid 119:1. v,,). Thc dried plates were exposed to X-ray lilm (Kodak Medical X-ray tilm no screenl for two w e e k s p r i o r to ale',eloping.

"~

20

i

0

0

20

T,

Sttttltt'~ix O/ ~l I- N-acetyl-5-hydro.xytryptamine (N,4 II T) and "~ll-diacet.vl-5-hwh'oxytrvptaminc (DAtlT) Acctxlation was carried out as h)llows: 0.25g 5-HT (crcatininc sulfate) ;',as mixed ;vith 3 ml glacial acetic acid and brought to a gentle boil. After cooling 0.122 ml of acetic anhydride ~as added and the mixture again ~armed. ()n cooling for several hours {d (') a brown gum tortncd and ,aas SCl',arated and rinsed v,ith 10",, acetic acid and dissol,,ed m 3 ml of the .,,ame solution. Tile products of the reaction. NAItT and DAttT. v, cre twice separated b) thin-la,,cr chromatography on silica gel with the solvcn! s3stcm ('I1CI 3 McO]l ItOAc 196:3:1 (v v)] (Rr's 0.16 and 0.066. respcctivcl',), and wcrc scraped off and cluted with ether, r.()r the preparation of labelled N-acetyl-serotonin the procedure v, as scaled down 250 times and I mCi of 3tt-5-HT v, as used.

.S't'(tnnin¢l t'l('clron inicroscop.v Platelct suspensions wcrc tixcd with 1.7", glutaraldehyde m Ol M phosphate buffer, ptl 7.4 for 60mm at 37 C, then collected onto naembranc tilters (Nt,cleopore 0.4 it). deh,,dratcd stcpwise m an ethanol gradient (50%-absolutc). then immersed m absolute alcohol..amyl acetate (50 50. v ',1. then m amvl acetate and finally critical point dried. The spccitncns v,.crc then plated with gold and vist,alizcd by scanning electron microscopy (Cambridge Stereo Scannnlg t'I.M.i.

Fig. 1. ~H-5-HT transport by canine platelets in PRP and in Ringers buffer. Platelets were either assa.~ed dtrcctl) ill PRP

(I × I() ~ p l a t e l e t s m m ,

1.2 x 10"~,u("iimmle)

5 - f t T transport kinetics This 5-HT t r a n s p o r t system r e s p o n d e d to t e m p e r a turc a n d p H with characteristic t r a n s p o r t protiles (Fig. 2) d e m o n s t r a t i n g pH a n d t e m p e r a t u r e o p t i m a of 7.0 a n d 35 4 ( ) ( ' . respectively, a n d a Q~o of 2.3. 5-HT was t r a n s p o r t e d into canine platclets b ) a saturable system described by linear kinetics on a d o u b l e - r e c i p r o c a l plot (Fig. 3). This system was characterized b', a particular high affinity for 5-HT (K,, 1.56 × 1 0 : M) similar to o t h e r m a m m a l i a n systems. a n d a I',,~, of 5.35 p m o l e s : m i n per [0 s platelcts.

Competitire inhihitimt of 5 - H ? ' tran~port C o m p e t i t i v e inhibition studies with various analogues (Table 1) indicated both a substituted indole nucleus and a specific side chain are requisite for

u IO (o)

311-5-HI' v, as obtained as the creatinmc .,.adt from Amcrsham Scarle ( o r p . All structural amdogues and metabolic inhibitors ~ct'e obtained commercially from either Sigma (hcntical ('o. or Aldrich Chemicals.

S.A.

(It .... O/ or were assayed after resuspension in Ringers buffer (O -OL

%

.'datcrials

30

rain

i~ *

I

(b)

g o.~ Q. 04t

RESt I.TS

c

o2

Incorporation a n d / a t e O! ~1 {-5-1t 7 C a n i n e platelets were t o u n d to incorrx)rate labelled 5 - l I T mpidl.~ and a p p a r e n t l y actively, either in P R P or ~ h e n w a s h e d with a n d r e s u s p c n d e d m Ringerscitrate d e x t r o s e buffer ( R ( ' I ) ) (Fig. 1). a l t h o u g h in the latter case total uptake was only half as much as in the former. In either case the label was present in the platelets in a form which could be quantitatively released and recovered either bx treatment with either 5",, trichloracctic acid (T('A). toluene water [1:20 Ix' v)]. or by sonication. Thin-layer c h r o m a t o g r a p h y and a t , t o r a d i o g r a p h y of the released serotonin yielded o n N one spot which c o r r e s p o n d e d to a u t h e n t i c 5-1 I T .

I

O3 i

20 pH

40

Temperoture,

60

°C

Fig. 2. The temperature and pll dependent transport of 3H-5-HT by washed canine platelets. Platelets from PRP (2.5 × l0 s platclcts:mm 3) were washed twice and rcst, spendcd ill Ringers buffer prior to the assay for 3H-5-11"I uptake (SA. 1.2 × ItP I~Ci l~mole). In (a) platelets were equilibrated to temperatt, rc for 5 rain prior to the addition of 5-HT. In (b) the pH of the resuspension buffer v, as adjusted prior to platclet resuspension. The pH before and after the experiment ~as checked. The platelets were preincubated at the desired pll for 5 rain.

Platelet 5-hydroxytryptamine transport

203

25 v) ¢g

o

IO

///"

2.0

% ~,.

I.$

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E

o.

. //

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8

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/1

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I

I

I

I

I

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4

6

8

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S -I 1 0 - 7 M

I

Fig. 3. Kinetics of 3H-5-HT transport into canine platclcts. PRP (2 × 105 platelets.mm3] was preincubated at 37 C for 5 min prior to the addition of labelled 5-HT at various concentrations. Kinetic data in the form of a double-reciprocal plot was calculatcd from linear rates of incorporation determined from 30 see tiltration data for 2 min.

recognition by the transport system. Furthermore. a side chain amine is required but the system still recognized N-substituted amines such as NAHT, but with reduced affinity. These inhibitions were indeed competitive as dcmonstrated by the kinetics of Fig. 4 for NAHT. The inhibition constants for several ana• logucs (Table 2) indicated however that none of thc above analogues wcre strong competitive inhibitors of 5-HT transport since K~ values were two orders of magnitude greater than the K,~ for 5-HT transport. Indeed, platelcts were unable to accumulate significant quantities of 3H-NAHT as compared to 5-HT (Fig. 5). suggesting that perhaps events subsequent to initial substrate binding did not respond to 3H-NAHT.

-5

1

1

I

0

5

I0

NAHT

x IO-SM

Fig. 4. Kinetics of competitive inhibition of 5-HT transIx~rt in canine platelets. The platclets were incubated with labelled 5-tIT and unlabclled NAHT as the competitive inhibitor and samples wcrc tiltercd at 30 sec intervals for 2 rain to obtain linear rate data. K, values were derived from the intercepts on l)ixon plots. The 5-HT concentrations wcrc 2 x 1 0 - ' M (O ...... OJ and 6 x 1 0 - ' M Ie

--e).

Table 2. 5-liT transport inhibition constants of selected analogues of 5-lIT in canine platelets Analogue

K

7-Mcthyltryptaminc 5-Flurotr)ptamine N-acct~l-5-hydroxytryptamine 6-Fluorotryptaminc

2.6 3.6 5.7 7.0

x x × ×

10 ~ M 10 SM 10 s M 10- ~ M

Initial rates of 5-HT transport were measured as functions of inhibitor concentration and K, values determined from Dixon plots of the inhibition data.

Stabilit v and e.xchan~te o] accumulated 5-HT Table 1. Inhibition of 5-HT transport in canine platelets by structural analogues Analogue (20 ;~M)

Inhibition (",,)

None 5-Hydroxytryptamine 7-Methyltryptamine 6-Fluorotryptaminc N-acetyl-5-hydroxytrypt amine 5-Fluorotryptamine L-Tryptophan Tyramine 5- H ydroxyindoleacetic acid 5-Hydroxy-DL-tryptophan lndole-3-butyric acid 3-Hydroxytryptamine Tryptamine lndoleacetaldehyde 5-Hydroxyindole L-Epinephrine

0 96.8 97.6 89.2 76.9 74.4 46.8 32.8 32.5 30.7 23.1 20.2 18.2 17.8 12.5 7.0

Initial rates of 5-HT (1 x I 0 - " M ) uptake were measured from the linear uptake rates obtained by 30 sec sampling for 2 min. Inhibitors at 1 × 10 ~ M were added simultaneously.

In order to determine the stability of the 5-HT intracellular pool 5-1IT loaded platelcts were immobilized t)n membrane filters and wcrc continuously washed with Ringers buffer to minimize the recapture of leaking 5-HT. Plalclets did not readily release

03

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0.2

Z~

z= ~

#

0 0

o.~

g'l

2

4

6

T,

rain

8

iO

Fig. 5. Transport of ~H-5-HT and 3H-NAHT by canine platclets. PRP (3 x 10s platclet:mm 3) was incubated with 2 x I ( ) - ' M ~H-5-HT (S.A. 401~Ci/~mole} (O. . . . O) or with 1 x 10 " M 3t|-NAHT (S.A. 38.5 ,uCi. ltmolej at 37 C (O----O) or tit () C (A . A) for various time intervals indicated.

~'II.LIAM W'. KA'f AND (iAR~I'x; (_'H(}N(,

204 I00

E(lect of metaholic inhihilor.s

80 _2 o

Q.

60

.£

~ 4o g, i

T m

20

I

I

20

4O 1",

I

6O

rain

Fig. 6. Release or exchange of ~H-5-HT from canine platelets. P R P (3 x I05 platelet:mm) was pre-incubatcd with 3H-5-HT (S.A. 1.2 × 1041~Ci/~mole)for 30 rain prior to collection of several equal samples on membrane filters. The vacuum was released and Ringers buffer with the indicated additions was allowed to wash through slowly. Filters were removed at intervals and assayed for radioactivity. Control (0 --II), NAHT (1 mM) (OO). 5-HT (lmM} (AA), ADP (l x 10-4My ([7 ---Ui), or thrombin (4 unit/rely (11- II).

labelled 5-HT when washed with N A H T or tryptophan (Fig. 6) but slowly released the label in exchange for 5-HT. Platelets were found to be relatively stable when trapped on membrane filters and did not readily release labelled 5-HT unless washed with A D P or thrombin (Fig. 6). Morphological examination of these platelets by ,scanning electron microscopy compared to platelets in fluid suspension indicated that although "immobilized' platelets were still able to undergo characteristic shape changes commensurate with the release reaction, pronounced aggregation was of course prevented (Fig. 7).

Table 3. Inhibition of 5-H-I transport in canine platelets by metabolic inhibitors Inhibitor Iodoacetamide NEM PCMB Arsenite Fluoride CCCP DNP DNP DCCD Oligomycin NaN 3 KsFe(CN)~, KyFe(CN)~, Amytal Amytal

Concentration (raM)

Inhibition C,,)

5 5 l 5 20 5 5 5O 5 O1 50 5 50 50 500

100.0 95.3 16.7 47.7 2.6 49.6 18.1 60.1 26.2 5.6 1.2 22.3 98.0 86.9 93.2

Initial rates of 5-HT (1 x 10 ~ M) transport were measured after a 15 rain prc-incubation with the various inhibitors.

on

5-1t 7mltu.\- and ell/u\

Platclet transport of 5-lIT v,as so,torch inhibited by sult'hydryl group reagents, iodoacclamide and NEM ',','ere the most potent. Surprisingl.,,. P ( ' M B was only slightly inhibitor 3 even at high concentrations (Table 3). Fluoride. even at 20raM. v,as inerfectivc. The energy uncouplers, carbon 3lcya,fidc-m-chlorophenylhydrazone (('('('P) or dinitrophcnol (I)NP) which apparentl 5 act as proton c o n d u c t o r s , w e r e not greatly inhibitory. Dicyclohexylcarbodiimidc (D('CD} and oligomycin, the well known mitochondrial ATPase inhibitors were ;.list) n o t inhibitory. Sodium azidc was ineffective, howevcr K.~Fe(CN),, and amytal were potent mhibitors but only at unusuall.~ high concentrations. N(me o f these inhibitors caused platclet aggregation. In parallel experiments these .~tme inhibitors caused preloaded platelets to release ~I'-I-5-HT. Figure 8 demonstrates the time coursc of 5-tlT release by' K.~Fe(('Nb, and am,,tal- the other mhibitors were essentially without effect. Thus a correlation exists between 5 - | I T inllux and ellh, x with respect to these metabolic inhibitors. I ) I SC t..l'L~;I0%,

]'he transport of 5-HT i,ll(_) canine platclets appears to be mediated by system(s) similar to other metabolic active transport systems: that is it is temperature and pH-dependent, has structural specificity and interfered with by metabolic mhibitors. However this partict, lar system has an added complication. Once transported through the cytoplasmic membrane 5-HT is accumulated in specific organdies apparently as a 5-HT..ATP complex {Baker eta/.. 1959: Tranzer et al.. 1969). The questions of stereospecificity and mobilization of energy at both the cytoplasmic and organelle membrane have to be considered Studies here on the stereospecificity of 5-HT transport are considered to bc at the level of the cytoplasmic membrane and the results agree generally with studies of others with human and rabbit platelcts. Of several analogues investigated ~,,ith human platelets o n h six were competitivc inhibitors and onh one. z(-melhyltryptamine, was taken t,p (Born c t a / . . 1972). The 5-HT uptake mechanism was shoran to be specific requiring both the 5-OH group and an unsubstitutcd amino group. 5.6-Dihydroxytr) ptanfinc v, as shown to be a competitive inhibitor and was also taken up by rabbit and guinea-pig platclcts (Da Prada et ah, 1973). The canine transport s',stem was highly specific for 5-HT also, a specificity apparently manifested at the level of the cytoplasmic membrane. Of the four analogues which were similarly competitive. N A H T was still not taken up by platclets 5-HT organelles have been shown to take up 5-HT at a much greater rate than other biogenic amines suggesting that similar structural speciticity exists at the organelle membrane {Murphy & Kopm. 1972: Da Prada & Pletscher. 1969). Oncc accumulated, the Jntracclluku 5-11"I pool appears to be remarkably stable. It is completely retained in our experiments at t ) C (unpublished observation} and only slowly exchanges at 3 7 ( ' . An equilibrium between exogenous and intracellular 5 - H r apparently exists since the exchange-washing

Platelet 5-hydroxytryptamine transport

205

Fig. 7. Scanning electron microscopy of platelets either in solution or trapped upon membrane tilters. Platelcts [approx 2 × 10~:mm ~) in PRP were fixed either (a) before the addition of thrombin. 10.4 units, mll or (bl after the exposure to thrombin, with 1.7",, glutaraldehydc for 60 min. The fixed platelcts were then collected on Nucleopore membrane filters (0.4 !~M). Similarly platelets were first collected on membrane filters to). then exposed to thrombin in Ringers wash solution (d). These trapped platelets were tixcd on the filters with glutaraldchyde, then alcohol washed, immersed in amyl acetate, critical point dried, and finally plated with gold (magnitication× 10.000). procedure used hcrc rcmovcs intraccllular 5-HT .... albeit slowly--and pcrh~lps exchange from both the cytoplasm and granules may bc occurring sequentially. The behavior of platelets entrapped on membrane filters is of interest. Although the platclets are at least partially immobilized they appear to bc completely stable. Furthermorc. they exhibit the relcasc reaction when washed with ADP or thrombin. Scanning electron microscopy rcvczds that shape changes and pseudopodia-likc projections characteristic of platclcts aggregating in ~)lution occur on the filter. The inhibition of platclct 5-HT transport by various metabolic poisons is in keeping with the classical definition of an active transport system; that in this system is particularly .sensitive to K3Fe[('N)¢, and amytal. The poor inhibition by other classical uncouplers may possibly bc due to their inability to

permeate the platelet and effect the mitochondria. however the failure of either C C C P or D N P to inhibit 5-HT transport suggests that transport is not energized ~lt the exr,cn.~ of a proton gradient. We have recently found that various ionophores cannot illicit their inhibitory effects past the level of the cytoplasmic membrane IChong & Kay, 1977). There is somc controversy in the literature concerning the effects of inhibitors on 5-HT transport (Sano et al., 1958; Staccy. 1961: Weissbach & Redfield, 1961; Hughes & Brodic, 1969 ; Murphy & K opin. 1972), most of which appears to arise from the use of different platelet sources and different conditions of treatment. We conclude here that the canine platelet 5-HT transport system is simply dependent on metabolic cncrgy and inhibited by some sulfhydryl group reagents. The questions as to where the energy is directed has never bccn ~ttisfactorily rcsolved. If 5-HT is accumulated

206

tll,"ll I IAM \V. KA'~ AND (.IAR'~I:\ ('I'I(IN(;

io o

o°

R. l'. (1959) %tLIdics ()It the uptake of 5-h~drox)tr)ptanmlc b', blood platelcis. ,/. Physiol.. Lend. 146. 472 491. BORN G. V. R.. Jt/IN(;JAR()IN K. & Michal R. (1972) Relative activities on and uptake b,, human blood pkltelels of 5-h)droxytryptamine and sc',eral analogues. Br. ,I. Pl!amnuc 44. 117 139 ('lioN(; ( i . & KA5 W. ~'. {1977) Sodiunl-depetldent trarlsport of 5-hydroxytr~ptamine IScrotonml b,, canine blood platclets..4robs Biochem Biophy.~. 179. 6(X/ 607. DA [)RAI')AM..O'BRIIN R..A..TRANZIR J. P. & PI.IIY,(IIIR A. (19731 The effect of 5.6-dihydroxytamine on uptake, storage and metabolism o[ 5-lDdr(~x.'.tr}ptamme b~ blood platelets. J. Pharmac. e.',p, 7her. 186, 213 219. BORX (;. V. R. & ( } l l . l . s o x

i-..~..,............~Control

60

c_

m

I)A PRAI)A M. & PIIIS('IIIR A. 11969)I)iltcrential uptake

2O

of biogenic anlin¢s b) isolated 5-h)drox.',tr'.ptaminc organelles of blood platelets. Li/c Sci. 8, 65 72. HI(;IIt.S B. R &, BR()I)II B, B. (1969)"rhc nlechanism e l

o

II 0

l

20

I

I

40 rain

60

T, Fig. S. Release of q t - 5 - H T from canine platclets b) nletabelie inhibilors PRP ,aas incubated with 3H-5-HT (S.A. 1.2 x Ill"~pC)pmole, for 3llmm prior to tile addition of K3t'e(("N) . (50 mM), ( I I ) , Nt!M (5 mM), (,'k A). or amytal (50mM) IF] i it. Platclcts were harvested at the indicated times b~ filtration. (.'ontrol ( 0 O).

free in the c y t o p l a s m then energy must be e x p e n d e d at the level of the c y t o p l a s m i c m e m b r a n e . O n the o t h e r h a n d if the inlracellular 5-l-IT is c o m p l e t e l y t r a p p e d in 5-HT granules then energy may be e x p e n d e d at the granule m e m b , ' a n e level. Either or both of these allernatives may prevail. •tckmm'h,&lCmC,t.~ ()no of us. \~. W. K. is indebted to tile (alls.ldlan ] t c a i l totllld~illoll lof stlpporl, and to I)r.

F. (ihadiallv for the list" of Ihc E'leclron Micro.~opc.

REFERENCES

B A K I r R V.. BIAs('iiK() 1t. & B o r n (i. V. R 11959)The isokition from blood platelets of particles containing 5-lIT alld ATP..l. Physiol.. l,oml. 149. 55 56. BI:R:',ilS K. I1.. DA I"R.,,I),X M. & P t l : r s ( H i r A. 119691 Micelle h)rmation between 5-h)droxytryptammc and adenosine lriphosphatc in platelct storage organelles. Science 196, 913 914.

serotonJn and C,:liceholanlJllc tlpt:.lkc b} platelets. J. l~harma( ". c\p. Thor. 12'7. 96 1(/2.

MIRPII', D. L. & Kol'lX I. J. 119721 .'~h'taholi~ "l)'anspor! (Egdited b) llol,:Ix L. li.I \%1. 4. p. 504. Academic Press, Nee York. MtSIARI) ,I. F. & P*X('l,;liaM M. A. (19701 I'aclors Inllucnc-

iilg platelet fUllCtion, adhesion, release and aggregation. Pharmac. Rer. 22. 98 187 OKII)A M. (~ NI.MtRS(iN Y. 11971) T r a n s p o r l of ~,¢rolonin by blood plaiclcts: a pump-leak system..lm. J Phvsi,l.

2211. 283 288. PLI:rs('Ht:R A., I)A PrAi)A M. & T r a x z i r J. P. (19691 Transfer and storage of biogeni¢ monoalniil¢s in subcellui:.lr organellcs of blood platclcts. Prog. IJrai, Rcs. 31. 47 52. Raxl) M. & Ri!ii) (i. 11951) Source of scrotonm m serum. \:ature. Lond. 168, 385 386. SAY() 1.. KAKiXI()I,) Y. & "|'AM(it 1'1tl K. (19581 Binding and llallspori of scrotonm in rabbit blood pkitcicts and action of reserpine...lm. J. PJiy.~iof 195. 495 4(t8. .~'IA('I:'I R. S. (19611 Uptake of 5-h',droxbtr)pt,imme b,, platclets. Br J. Ph,.'m.( 16. 284 295 I'RAN/IR J. P.. DA PRAI)A M. & PIIiTS(III:R A. 11966) [;ntrastructural localization of 5-HT in blood platelets. :\:~ttllre. #.,rod. 212. 1574 1575. WI![ssB~.('II H. & Rtil)FIH.D I]. (i. (1961) Hh,nl l~htteh'ts 1Edited by .IO|[NS()N ,~. :\.. R|BI.'('K R. V~:. & HORN R.

C. Jrl p. 393. Little Broan. Boston. MA. WlilSSBA( II 11. & RI[)I.III.I) B G. Ilg6(ll [-'actors affecting

the uptake of 5-hydro',;}tryptanline by hUnlall blood in an inorganic medium. J. biol. Chem. 235, 3287 3291.