Differential uptake of biogenic amines by isolated 5-hydroxytryptamine organelles of blood platelets

Life Sciences Vol . 8, Part 1, pp . 65-72, 1969 . Printed in Great Britain .

Perga"mon Press

DIFFERENTIAL UPTAKE OF B10GENIC AMINES BY ISOLATED 5-HYllROXYTRYPTAMINE ORGANELLES OF BLOOll PLATELETS ht .

Da. Prada and A . Pletscher

Research Department, F . Hoffncann-La Roche irC Co . Ltd ., Basle, Switzerland (Received 7 October 1968) The uptake of 5-hydroxytr "yptamirte platelets

is generally more pronounced

to be

of

energy

(_',

4-12) .

transport ma.y be

this amine

port mechanisms

system cts well

platelet membrane

less

important

or virtually" non-c " ~ci,tertt

(histamine artd

therefore

prt,sive diffusion appear,

to

(?-~9,

the

be

511T has bean

plrtelut

gether with adenosine-triphospha.te

(ATP)

vesicle-like organelles wlriclr can be which t" nke up the hl~ttc " lets,

type

(11,13) . For

t-ryptamine), and

rclativel "y more pro-

1 "1,15) .

It.ecuntly,

terusi . t o

as on meta-

(norepinephrine and epi-

nephrine) nounced

organelles

t.ha.rt 511T, carrier artcf energy-delrertdent transto be

soem

into the

Evidence has been provided that this

Located in the

biogenic: ct.mine" s other

thought

In platelets incubated with low con-

of SPIT, the entry of

seems to depend on a yet unknown carrier bolic

that of other biogenic

The transport mechanisms for these amines are

of different nature .

centrations

than

by isolated blood

epinephrine, trylrtamine and hist

amines such as norepinelrltrine, amine (1-4) .

(511T)

amine

in vitro

investi~arte whether atre able

amines with regard

shown

bc" stored to-

in specific

isolated

(1(i-18) .

to

.intracellular

in pure form and

Therefore,

isolatedi organelles,

it was of

in-

such as intact

to differentitrte between various biogenic to

their uptake . Experimental

lhctbbi t.s weighing ~ - 3

kg, fasted for 1G hours,

were bled

under ether anesthesia through a polyethylene cannula in the carotid artery .

The blood was

ethylenedia .mine tetraacetate agulation .

After

supplemented with 1/10 vol disodium (EDTA)

separation of

the 65

(5 j^)

in order to prevent co-

platelets from the blood by

ßß

PLATELET AMINE UPTAKE

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centrifugation (19), the 5HT organelles were isolated as previously described using a continuous urografin gradient (18) . In each experiment, the isolated organelles from the blood of 3 - 4 animals

were suspended in their plasma . This pool was divided into equal parts of 0,9 ml each which were incubated with 5-hydroxy-3-indolyl 14 ) cteatinine sulfate monohydrate*(specific (ethyl-2-amine-1-C radioactivity 40 me/mM) or with the following amines or their precursors : tryptamine-2-C 14 -bisuccinate** (10,3 me/mM) ; tyramine-l'-

C 14 hydrochloride* (10,3 me/mM) ; 3,4-dihydroxyphenyl (ethylamine-2C 14 ) hydrochloride* (= dopamine) (40 me/mM) ; DL-norepinephrine 14 ) bitartrate* (31 (carbinol-C .5 me/mM) ; DL-epinephrine (carbinolC 14 ) bitartrate* (9 .0 me/mM) ; 3-4-5 trihydroxyphenethylamine a,ß14 ) 3H2 hydrobromide*** (675 me/mM) ; histamine (ring-2-C dihydrochloride* (54 me/mM) ; and L-histamine-C14 (uniformly labelled)** (248 me/mM), The amines were dissolved in modified Tyrode buffer (with glucose, see below), and 0,1 ml of this solution was added to 0 .9 ml of the plasma suspension of the organelles to yield the final concentrations indicated in the Figure and Tables . In some other experiments with 14 C-SHT, modified Tyrode with ôr without glucose**** was used as incubation medium . Furthermore, organelles

were incubated in plasma containing 0 .57 yM 14 C-5HT in the presence or absence of ouabain or of metabolic inhibitors, such as monoiodoacetate and NaF .

After incubation, the organelles were separated from the media by centrifugation (44,000 x g, 10 min .), taken up in the scintilla-

Radiochemical Centre, Amersham New England Nuclear Corporation Synthetized by Dr . J . Würsch, Physical Department of F . Hoffmann-La Roche & Co . Ltd ., Basle, Switzerland **** Modified Tyrode : NaCl KC1 Disodium ethylenediamine tetraacetate (EDTA) NaH2P04 . 2H20 NaHC03 Albumin Glucose Saccharose

with sugars : 0 .760 ~ 0,042 ~~ 0,080 0,014 0,210 0 , 500 0,200 0,450

~ ~ y~ 9~ ~ ~

without sugars : 0,837 0,042 jo 0,080 O,Olh 0,210 0 . 500 qb -

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PLATELET AMINE UPTAKE

ß7

tion liquid, and the radioactivity was measured in a Packard liquid scintillation counter . A blank was obtained from organelles that

had been resuspended in the incubation fluid containing the radioactive amines and which had been separated from the medium immediately after their resuspension . Quantitative measurements of the added radioactive amines in the medium before and 30 minutes after

incubation indicated that no major metabolism or degradation of the radioactive amines takes place during the incubation period .

In the experiments with 14 C-histidine, the organelles were

dissolved in a few drops of distilled water and subjected to paper

chromatography (Whatmatn No . 3) using isopropanol : concentrated ammonia : H2O (20 : 1 : 2) as a solvent system'. The radioactivity of the chromatograms was measured with a Packard radiochromatogram scanner .

Deter~pinations of endogenous 5HT of the organelles were carried out as previously described (20) . Results 1 . Isolated 5HT organelles incubated for 30 minutes in plasma with 0 .57 yM of various biogenic monoamines take up different amounts of the amines . The uptake of 5HT is most marked, whereas that of histamine is lowest ; catecholamines ; tyramine and tryptamine enter the organelles in intermediate amounts (Table I) .

2 . The differences in the uptake of 5HT, norepinephrine and hist-

amine exist over a wide concentration range of the amines (0 .57 - 57 yM) and for various incubation times (Fig . 1) .

3 . The uptake of 5HT by organelles incubated in artificial media devoid of metabolic substrates (modified Tyrode without glucose) is not influenced by glucose and is even higher than in experi-

ments with plasma as an incubation medium . Furthermore, ouabain, monoiodoacetate and NaF in the relatively high concentrations used do not inhibit the uptake of 5HT by isolated organelles in plasma (Table II) .

4 . Incubation of isolated 5HT organelles with 14 C-histidine in plasma does not result in any appreciable accumulation of 14Chistamine in the organelles .

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TABLE I Uptake of Radioactive Amines by Isolated 5-Hydroxytryptamine (5HT) Organelles Incubated in Plasma at 37 °C for 30 Minutes . Concentration of amines in incubation fluid 0 .57 yM . The values represent averages with S .E . and are expressed in percent of the 1 4C5HT taken up by the organelles in the same experiment . Absolute uptake of 14C-5HT in myg per yg endogenous 5HT : 0 .91 ± 0 .08 (14 ex periments) .

AMIN Tryptamne

H HUl'

trptamlne

Nr, of e

UpTAKE

.3 ~

10±2

1<

100±9

Hi~~F:

NH2

Tyromirc

2

13±3

HD~r

~2

Doponiro

3

88±2

Wi H

^H2

Histamine

3

2t1

EPi~Pfrlne

<

36 * i

NoreplnepMM

B

2!i f 3

~~y d

3

13±1

OH

H H

H0~1, H OH

H3

~

Discussion The present results demonstrate that the 5HT organelles are capable of discriminating with regard to the uptake of biogenic

monoamines . 5HT enters the organelles to a most marked degree, whereas catec~olamines and especially histamine, tryptamine, tyr-

amine and 5-hydroxydopamine are taken up less extensively . Hence, the differences in the uptake of monoamines by platelets which have been described earlier (see above) may at least in part be due to specific properties of the organelles rather than to mechanisms lo-

cated exclusively in the platelet membrane . Isolated 5HT organelles of platelets seem therefore to differ from splenic nerve granules which, in contrast to the axon membrane, do not possess discriminat-

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89

100 -

s 0

â

0-

-

v

v

r

r

0

5

15

30

minutes FIG . 1 Uptake of 14 C-5-hydroxytryptamine (5HT), 14C-DL-norepinephrine (NE) and 14 C-histamine by isolated 5HT organelles incubated at 37°C in plasma . Left : influence of incubation time ; amine concentration 0 .5 yM . Right : influence of the amine concentration ; incubation time 30 minutes . The values represent averages with S .E . of 3 experiments and are expressed in percent of the 5HT taken up after 30 minutes (right hand graph : values at the external 5HT concentration of 57 yM = 100 ~) . For absolute uptake of 5HT see Table I . TABLE II Effect of Ouabain, Glucose and Metabolic Inhibitors on the Uptake of 14 C-5-Hydroxytryptamine (5HT) by Isolated 5HT Organelles Incubated for 30 Minutes at 37 ° C . The values represent averages with S .E . of 2 experiments and are expressed in percent of the 14C-5HT taken up by organelles in normal plasma . Absolute u take of 5HT by organelles incubated in plasma (per experiment : 9 .5 + 0 .1 myg . )tedium -_____________Plasma Plasma

Supplement ~ 14 C-5HT uptake -______________=====T________________ None 100 + 1 i

Plasma Plasma

~

Synthetic* Synthetic*

~

Ouabain

10 -3 M

96 ± 2

Indoacetate 10 -2 M

122 + 7

NaF 10 -2 M

145 + 5

Glucose

180 ± 8

tione

175 ± 17

Synthetic medium : Tyrode buffer devoid of CaC12 and sugars, but added EDTA 0 .80 g/1 .

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ittg properties for the uptake of amines such as norepinephrine, epinephrine and isoprenaline (21) . Some structural features of the amines may influence their uptake by the organelles . An OH-group in m-position of the phenyl ring seems to be of importance, since 5HT and dopamine enter the organelles to a more marked extent than tryptamine and tyramine respectively . On the other hand, additional OH-groups may diminish the uptake . Thus, epinephrine and norepinephrine (OH in 2-position of the side-chain) as well as 5-hydroxydopamine enter the organelles less markedly than dopamine . Possible differences itt the uptake of the 1- and d-isomers of norepinephrine and epittephrittc retttain to be investigated . The mechanism by which the various monoamines eater the 5HT organelles has still to be clat" iPied . The fact that certain amines (especially 5HT) are accumulated more markedly than others as well as the high ATP content of the organelles suggests t}te participaLion of a specific, active (i .e . energy-dependent) process . Ttte present experiments do not, however, provide evidence for such a mechanism . Thus, ouabain which in other systems (c .g . of electrolytes) is thought to inhibit the "transport A'fY-ttse" ('_') and which markedly diminishes the 5HT uptake in intact. ttltttelc" ts (e .g . of guinea pigs and man) (11-13) has no effect oft ttte ettt,ry of the amine into the isolated organelles . The presence of metabolic" enzymes, e .g . those of carbohydrate metabolism in pure frac" t.iutts of 5HT organelles (devoid of mitochondria, microsumes etnd platelet supernatant), is unlike l,y . In Pact, absence uF metabolic substrates such as glucose does no t influence the tretttsfer of 5HT into isolated organelles (compare Tyrode with and without glucose tts well as Tyrode and plasma ; Tabl e II) . Furthermore, metctbulic inhibitors such as monoiodoacetate attd NaF cause no inhibition of the 5HT uptake in plasma . Therefo re, the discriminating properties of the organelles are possibly c ottnected with some physic " u-chemical qualities of the amines attd the organelles which might influence the extra- versus intravesicular distribution of the amines, their penetration through the ntentbrtttte of the organelles, their binding to iut.ratvc" siculur constituents, etc 'Phew" i~hysiru-c"hemicttl proparties may be estm" ciull)' important for thr furnatt iuu of cumt>tcxes betwc" c~tt tttc" amint" s nn .l APf within tttr or;"anrlles .

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PLATELET AMINE UPTAKE

71

The considerable difference between the uptake of 5HT and that of histamine is of particular interest . Thus, compared to 5HT, only a smrr,ll fraction (about 2 ~) of histamine is accumulated in plate

lets . As previously shown, 5HT organelles of rabbits contain considerable amounts of endogenous histamine (about 1/3 that of 5HT on a molar base) (17,1t;) . It is therefore conceivable that in vivo a precursor of histamine, e .g . histidine" , penetrates into the or-

ganelles where transformation into histamine occurs . In the present experiments, no evi
since no 14 C-histamine accumulated in isolated 5HT

organelles inc".rrbtrte " d wi th 14 C-histidine . The mechanism o£ accumulation of endogenous tristaminc in the 5HT organelles remains therefore to be eluciderte~d .

Summary The entry of biogc" nic" morrvu .mines into isolated 5-hydroxytryptamine (5HT) orgernc" IIc" s of blood platelets of rabbits decreases in the following order : 511'1', dopamine, I)L-epinephrine, DL-nor-epi nephrine~, tyrermirrc " i" r"yhtecmine == 5-hydroxydopamine, histamine . Glucose, oun.bvin, monoivdoacei"ate and NaF do not influence the uptake of 511T . No n.ccumuln.tion of 1^C-histamine takes place in isolated organclle~s inanbvtod with h- 14 ( ;-histidine .

It is c:vnc " Inrlod i.lrcet the discriminating properties of the platelets wi th regu.rcl to tlrc " crlrtake oP various monoaminea might at least partly trey due" to c;c " rt+r.in Irhysico-chemical properties of the intracellular 511'1' crrger.nelle " s .

Retcr~nces 1{ . WF~ISSIIACII, II .F . Il0(il)ANSKI rr.nei S . UUENFRIEND, Arch . Biochem . 492 (1958) .

F .ß . I1l1GHES and ß .1l, ßll0i)IE, "J . Pharmacol . exp . Ther . 1~, 96 (1y59) . 3 . fI . WEISSBACH and B .G . REDFIELll, J, biol . Chem . 2~, 3287 (1960) 4, R .S . STACEY, Brit . J . Pharmacol . 1~6 , 284 (1961) . 5, J .F I . Iil1MPHREY eLncl C .C . TOH, J . Physiol . (Lond .) 1~, 300 (1954)

6 . R .M . IIARUISTY an
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PLATELET AMIIdE UPTAKE

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9 . I . SANO, Y . KAKIMOTO and K . TANIGUCHI, Amer . J . Physiol . ~, 495 (1958) . 10 . G .Y .R . BORN and R .E . GILSON, J . Physiol . (Loud .) 146, 472 (1959) . 11 . A PLETSCHER W P BURKARD, J .P . TRANZER and K .F . GEY, Liße Sci . 6, 273 1967) . 12 . H . WEISSBACH, B .G . REDFIELD and E . TITUS, Nature (Lond .) 185, 99 (1960) . 13 . A . PLETSCHER, Brit . J . Pharmacol . 32, 1 (1968) . 14 . I . SANO, Y . KAKIMOTO, K . TANIGUCHI and M . TAKESADA, Amer . J . Physiol . ~, 81 (1959) . 15 . G .Y .R . BORN, 0 . HORNYKIEWICZ and A . STAFFORD, Brit . J Pharmacol . 13, 411 (1958) . 16 . J.P . TRANZER, M . DA PRADA and A . PLETSCHER, Nature (Lond .) _212, 1574 (1966) . 17 . M . DA PRADA, A . PLETSCHER, J .P . TRANZER and H . KNUCHEL, Nature 216, 1315 (1967) . Lond . 18 . M . DA PRADA and A . PLETSCHER, Brit . J . Pharmacol . , in press . 19 . G . BARTHOLINI, A . PLETSCHER and K .F . GEY, Experientia _17, 541 (1961) . 20 . D .F . BOGDANSKI, A . PLETSCHER, B .B . BRODIE and S . UDENFRIEND, J . Pharmacol . exp . Ther . 1 1 , 82 (1956) . 21 . U .S . Von EULER and F . LISHAJKO, Int . J . Neuropharmacol . 6, 431 (1967) . 22 . T .Z . CZÂKY, Ann . Rev . Physiol . ~, 415 (1965) .