Sugar derivatives of indolamines. Part I: Aggregation induced by desoxyfructo-serotonin and its active transport through the platelet membrane

SUGAR

Inqtitut l:niti!

Centre

DERIVATlVESOFINDOLA~INES,PARTI:AGGREGATIONINDUCED BY DESOXYFRUCTO-SEROTONIN ANDITS ACTIVETRANSPORT THROUGHTHE PLATELET MEMBRANE IA.6 “ester and :I.!!ester de

de

Chimie

des

Recherches Hapita 2uroni;en (Received

dn

Substances F.Rendu de Thrombose Lariboisiere, C.Labrid and “.echerches

28.10.1977;

Accepted

Naturelles,

Cif-sur-Vivcttc,

Experimentale ?aris 10e, G.Duren;:,

L’auverna::,

et and

?rance

i!6r?ostase

I: i 0 I? 63ZQl

~,rznc2

in revised form 23.2.1978. by Editor W.H. Seegers)

ABSTRACT I-Desoxy-I--(5-hydroxytryptamino)-D-cructose shows a serotonrn-like activity on both D and !! tyne tryptaminergic 100 times receptors of smooth muscles, but-its aifinity is less. Its intrinsic activity for the g type receptors is unchanged, while for the M type receptors is only 70 7. At a final concentration of 5x10-5 Moles, the sugar derivative shows a nearly unchanged activity in inducing platelet aggregation, but its uptake by the platelets is very limited. At lower concentrations desoxyfructoserotonin has little direct activity on platelets, whilst it can still depress the activity of serotonin on the platelets.

INTRODUCTION Desoxyfructo-serotonin is a new synthetic sugar derivative of serotonin (l), however, its formation in physiological conditions can also be expected (2). Recent evidence (3) suggested that the receptor which mediates platelet aggregation by 5hydroxytryptamine is different from that which mediates uptake of the amine by platelets. The dissociation of the intrinsic activitisz of desoxyfructo-serotonin towards D and M type receptors, reported in this paper, led us to investigate the-validity of this theory, using the sugar derivative instead of serotonin.

MATERIAL Desoxyfructo-serotonin

AND METHODS

oxalate

783

salt

(m.ri.

428)

and

PLATELET

784

I4 C-labelled desoxyfructo-serotonin prepared from serotonin formiate of the side chain (CEA, Saclay, Platelet-rich blood collected on to a concentration

AGGREGATION

Vo1.12,No.5

(sp.a. 0.5 mCi/mM) were labelled with 14C on the a carbon France) as reported (1).

plasma (PRP) at a pH = 6.5 hypercitrated anticoagulant of 3x10* platelets/ml.

was obtained from (4) and adjusted

Platelet aggregation induced by serotonin oxalate and by desox fructoserotonin oxalate at a final concentration of 5x10- X Mol, has been investigated using a “Labintec” (Montpellier, France) aggregometer. To 0.3 ml titrated PRP a pH = 7.4 Michaelis buffer solution was added to make up a final volume of 0.4 ml, after addition of the test solutions. 14 C-labelled serotonin oxalate and of The uptake of 14 C-labelled desoxyfructo-serotonin oxalate at a final concentration of 5x10-5 Mol was measured by the increase with time of 14C in the platelets during incubation at 37°C. The incubation was stopped by one minute centrifugation at 15,000 g. The radioactivity scintillation counter measured in the pellet in a liquid Intertechnique SL 4000. For studying of desoxyfructo-serotonin towards _ - the activity male Wistar rats (2OOt2Og) were D type tryptaminergic receptors, sacrificed after 18 hours fasting and stomach fundus preparations The contraction of smooth muscles was were isolated (5,6,7). technique (8) for making investigated following van Rossum’s cumulative dose response curves. tracheal receptors

Tricolored guinea pigs (600+100 g) were sacrificed and the muscle was used for investigation of M type tryptaminergic following Van Rossurn’s (8) technique. RESULTS AND DISCUSSION

a serotonin-lik,e activity I-Desoxyfructo-serotonin shows towards g and _M type tryptaminergic receptors, but its affinity than that of serotonin. Its intrinsic is one hundred times less is identical with that of serotonin, affinity for D type receptors but its intrinsic activity towards M type receptors is only 70 % The crossed curves on Fig.3 and 4 clearly show (9) I and 2). (Fig. that serotonin and desoxyfructo-serotonin act on the same receptor This observation is validated by the fact that sites. antagonizes the activity of desoxyfructo-serotonin “dimetotiazin” towards 1 and J! type receptors in the same manner as in the case of serotonin (Fig.5 and 6) (IO). No significant difference has been observed in the intensity induced by serotonin oxalate and velocity of platelet aggregation oxalate (Fig.7) at a final concentration and desoxyfructo-serotonin of 5x10-5 Mol. At lower concentrations of the amines, the difference pre-incubation of the platelets with is more marked. After 3 min. serotonin loses completely its ability to desoxyfructo-serotonin, aggregate platelets, and _-c__-____ vice versa. From this experiment it has been concluded that desoxyfructo-serotonin occupy the same receptor sites on the platelet membrane as serotonin, in respect of platelet aggregation.

PLATELET

Vol.12,No.j

AGGREGXTIOS

p& ~4.67

p&:6.67

SEROTONIN-LIKE ACTION OF DESOXYFRUCTO - SEROTONIN ON Q TYPE TRYPTAMINERGIC RECEPTORS

FIG.

EA’Ernax

1

S-Hi

0.5

0

p&=6.23 SEROTONIN-LIKE TRYPTAMINERCIC

pD2=4.50

ACTION OF DESOXYFRUCTO

-SEROTONIN

RECEPTORS

FIG. 2

ON M TYPE

786

E

PLATELET

AGGREGATION

~01.12,.'0.5

/E AB mrx.A l-

0

I

I

1O-s

I

1O-8

8

10"

,

I

1o'6

10-4M

1o-5

[A]=SHT

COMPETITIVE INTERACTION OF DESOXYFRUCTOSEROTONIN (DF-5HT) AND SEROTONIN (S-HT ) ON Q TYPE TRYPTAMINERGIC RECEPTORS FIG.

3

EAB/E mrx.A l-

0.5 -

0

, 10-O

1 10"

t

I

1o-6

1o-s

1 10"M

[A]=5--HT

COWETITIVE INTERACTION OF DESOXYFRUCTO-SEROTONIN (DF-5HT) AND SEROTDNIN (S-HT) ON&l TYPE TRYPTAMINERGIC RECEPTORS FIG.

4

PLATELET

L-o1.17,xo.j

E

AB

/E

XGGREGXTIOS

max.A =DIMETOTIAZiNE

1

0.5

10x10-‘M 0

t

1

1o-6

1o-5

i

1

lo-’

1O-3

I

1O-2 M [A]= OF-5HT

ANTAGONIST ACTION OF OIMETOTIAZINE ON THE SEROTONINLIKE EFFECT OF OESOXYFRUCTO-SEROTONIN (OF-5HT) ON 9 TYPE TRYPTAMINERGIC RECEPTORS FIG.

5

[B]z OlMETOTlAZlNE

EAB’Emax.A

10-6 ANTAGONIST EFFECT

10-S

10’ ACTION OF OIMETOTIAZINE

OF OESOXYFRUCTO-SEROTONIN(OF-5HT)

TRYPTAMINERGIC

RECEPTORS FIG.

6

i0-%1

[A]=oF-

ON THE SEROTONIN-LIKE ON M

TYPE

5HT

788

PLATELET

AGGREGATION

Vo1.12,No.5

1. 5-HT 2. DF-5HT

1.

2.

3

FIG.

Min

7

Platelet aggregation induced by serotonin (S-HT) and by desoxyfructo-serotonin (DF-5HT) at a final concentration of 5X1O-5 Mol in a titrated PRP (3x10* platelets/ml) -5 Mol, the at a final concentration of 5x10 In contrast, is about three times less uptake of desoxyfructo-serotonin oxalate than for serotonin oxalate (TABLE I). The difference in the uptake of serotonin oxalate and of i) to a decreased desoxyfructo-serotonin oxalate may be due groups affinity of the sugar derivative for those receptor ii) to a decreased transport of the responsible for uptake, or, sugar derivative through the platelet membrane. -5 At final concentrations less than IO Moles, desoxyfructoserotonin shows very limited direct activity on platelets, whilst it still inhibits platelet aggregation induced by serotonin, without lowering significantly the activity on smooth muscle receptors.

PLATELET

Uptake platelets

of

I 1 Time I

“C-5-HT and in a titrated

of in

;a?

AGG~EGxTI*~Z

TASLE I I&C-desoxyfructo 5-HT PRP (3x108 platelets/ml) the amines of ?ianomoles

incubation

of

in 5 -HT

minutes

after

incubation with 5x10-5

amine incorporated IO9 platelets Desoxyf rut to-5-HT

+

of X01

/ 1 1

!/ I I

IO 20

IO I$

3.3 5

30

17

5.3

40

20

7

50

23

6

60

25

7

I

I

CONCLUSION Desoxyfructo-serotonin, when compared with serotonin, shows a nearly unchanged activity in inducing platelet aggregation at a final concentration of 5r10e5 Yol, but its uptake by the platelets is very limited. This is due probably to the observed dissociation of the intrinsic activities of the sugar derivative towards g and E type tryptaminergic receptors. to obtain receptors through

Using other sugar derivatives of serotonin, it is hoped further information concerning the nature of the which mediate platelet aggregation and active transport the platelet membrane, respectively.

REFERENCES (I)

(2)

(3)

XESTER,L. D-fructose J.Carbohydr.

and

MESTER, XI., I-Desoxy-I-(5-hydroxy-tryptamino)reducing sugar derivative of Nucleos. Nucleot. 2, ISI, 1975.

: A new

MESTER, L., KRASKA, B., CRISBA, Amine Interactions in the Blood Effects on Haemostatis. Proc.Sth Haemostatis, Paris, 1975, p.233.

J. and MESTER, Clotting System Congr.Intern.

M., Sugarand their Thrombosis

BORN, G.V.R., JUENGJAROEN, K. and MICHAL, F., Relative activities on and uptake by human blood platelets of tryptamine and several analogs. Br.J.Pharmacol., %,

(4)

BORN, G. V. R. and HLJME, t-t., Effects of of Platelet Aggregates on the Optical Nature (London), 215, 1027, 1967.

(5)

VANE, J.R., tryptamine

(6)

serotonin.

VANE, J.R., analogues J.Pharmacol.,

A sensitive Brit.J.Pharmacol., on

The relative the isolated 14, 87-98,

method

for 2,

the Numbers Density of

the assay 344-349,

activities rat stomach 1959.

of

some strips

of 1957.

and

5-hydroxy 1972 117,

and Sizes Plasma. 5-hydroxy-

tryptamine preparation,

Brit

790

(7) FRANKHUIJZEN, A.L. action of 5-HT and fundus preparation.

PLATELET

AGGREGATION

vo1.12,xo.5

et BONTA, J.L., Receptors involved in the tryptamine on the isolated rat stomach Eur.J.Pharmacol., 26, 220-230, 1974.

(8) VAN ROSSUM, J.M., Cumulative dose response curves. II : Technique for the making of dose response curves in isolated and the evaluation of drugs parameters, Arch.Int. organs, Pharmacodyn., 143, 299-330, 1963. (9) ARIENS, E.J., SIMONIS, A.M. et VAN ROSSUM, J.M., Drug interaction : A - Interaction of one or more drugs receptors with one receptor systems, Med.Chem. (Molecular Pharmacology), I_, 119-393, 1964. (lO)JlJLOU, L., DUCROT, E., BARDONNE, M.C., DETAILLE, J.. FEO, GUYONNET, J.C'., LOISEAU, G. et PASQUET, J., Pharmacological properties of 3-dimethyl-sulfamide-IO (2-dimethyl-aminopropyl) phenothiazine (8599 R.P.), Arch.Int.Pharmacodyn., 70-86, 1966.

C.,

159,