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Sugar derivatives of indolamines, part III: Effect of desoxyfructo-serotonin on the uptake and release of serotonin by human platelets
THROMBOSIS RESEMCH 13; 805-810 @ Pergamon Press Ltd.1978. Printed in Great Britain 0049--3848/78/1101-08Cj $O2.!30/0
SUGARDERIVATIVES OF INDOLAMINES, PART III:EFFECTOF DESOXYFRUCTO-SEROTONIN ON THE UPTAKEAND RELEASEOF SEROTONIN BY HUMANPLATELETS J,-Ma
LAUNAY
AND
Ls
MESTER+
Institut de Chimie des Substances Naturelles, Gif-sur-Yvette-91 France (Received 18.7.1978; in revised form 10.8.1978. Accepted by Editor W.H. Seegers) ABSTRACT -5 Moles, desoxyfructoAt concentrations less than 10 serotonin oxalate has very little direct effect on human blood platelets, but still interferes with the active transport and release of serotonin.
INTRODUCTION 1-Desoxyfructo-serotonin oxalate shows a serotonin-like activity towards g and E type tryptaminergic receptors (l), but its affinity is a hundred times less. Its intrinsic affinity for 2 type receptors is identical with that of serotonin, but only 70% for E type receptors. -5 Moles, desoxyAt final concentrations of less than 10 fructo-serotonin has very little direct effect on human blood Platelets, but still active on smooth muscles. In order to elucidate the mechanism which suppress platelet functions while still support vascular smooth muscle contraction, the effect of desoxyfructo-serotonin oxalate on the uptake and release of serotonin by platelets have been investigated.
MATERIAL AND METHODS S-Hydroxytryptamine binoxalate Li,2-3H(N)T, spec. act. 24 Ci/mmol, supplied by New England Nuclear, has 6een used in the uptake and release experiments. +
To whom requests should be addressed. 805
806
DESOXYFRUCTO-SEROTONIX
Vo1.13,No.5
Platelet rich plasma (2) (PRP) was obtained from blood collected on Acid citric-Citrate-Dextrose Anticoagulant (1 vol ACDA/ 9 vol blood) and centrifugated 5 min. at 200 g and 15'C. The number of platelets was measured optically in a Malassez cell. Shape-change of rabbit platelets (3) in titrated plasma produced by desoxyfructo-serotonin oxalate was measured as the maximum rate of decrease in light transmission. Inhibition of the uptake of 5-hydroxytryptamine-(3H) (4,5,6). 25 ,ul of 0.9% NaCl (control) or 25 ~1 of 0.9% NaCl containing the inhibitor was added to 75 ul of ( H)-5-HT in 0.9% NaCl to have a final concentration of 10" Moles of labelled Serotonin. After mixing, the solution was heated to 37°C for 5 min. Then 100 ~1 of PRP, preincubated 10 min. at 37'C with shaking, was added. After agitation in a Vortex agitator, the mixture was incubated for 1 min. at 37'C with shaking. To stop the reaction, 2 ml of 0.9% NaCl-0.4% EDTA were added at +4'C, the platelets were filtered on nitrated cellulose filter ("Millipore" Type GS 0.22 p), washed 6 times with 5 ml of 0.9% NaCl-0.4%EDTA, and the radioactivity on the filter was counted in a liquidscintillation counter (Intertechnic, Plaisir, France). Inhibition of the release of 5-hydroxytryptamine-(3H) induced by 5,6_dihydroxytryptamine. After uptake of labelled serotonin by the above method, the platelets were isolated on "Sepharose 2B" column (7,8). 175 ~1 samples of GFP (gel filtered platelets), labelled with serotonin, and 25 1 of 0.9% NaCl solution containing the inhibitor and/or 5,Z-dihydroxytryptamine at a final concentration of lOa Moles, were agitated in a Vortex agitator, and then incubated for 60 min. at 37'C with shaking. The incubated mixtures were centrifuged for 10 min. at 1200 g and +4'C, and the radioactivity was counted in 50 /ul of supernatant. RESULTS AND DISCUSSION Study of the shape-change of rabbit platelets in buffer solution shows a very low activity for desoxyfructo-serotonin ox.alate : the maximal effect is only 33% of the effect of serotonin. The concentration requirement for the sugar derivative to produce 50% of the maximal effect (ECsO) is 500 times more than for serotonin. TABLE I. Concentration required to produce 50% of the maximal shape-change of rabbit platelets
I I 5-HT
oxalate
I DF-5-HT I
oxalate
Maximal effect
SE 95%
EC50 1
1.9x10-7 1 (1.8-2.0) 1 100 %
I
7.2~10-~ 1 (4.2-12.3)1
i
I
I
33 %
1 1 I
-6 At low concentration (10 4101es) desoxyfructo-serotonin oxalate inhibits only moderately,on the extent of 45%, the uptake of serotonin by the platelets (Figure l).At higher concentrations (lo-4 Moles) the compound becomes a powerful inhibitor, showing 86% inhibition. The presence of the free hydroxyl group in position 5 seems to be important for this inhibition, because of the low effect of desoxyfructo-S-methoxytryptamine oxalate : 40% inhibition at the concentration of 10m4 Holes, but only 14% at 10-6 Moles. Table II shows the concentrations required for the sugar derivatives to obtain 50% of the total inhibition (ICso) : TABLE
II.
Concentrations required to produce 50 4 inhibition of the uptake of serotonin
Desoxyfructo-5-methoxytryptamine oxalate 1
5.56x10-4Moles I
I
In the concentration ranges corresponding to the IC50 values for inhibition of the uptake, the sugar derivatives show very little effect on the "release" phenomenon. With increasing concentrations, the sugar derivative of serotonin shows an increased release of serotonin, but not the su ar derivative of 5-methoxytryptamine. At a concentration of lo- 8 Moles, the release induced by desoxyfructo-serotonin oxalate is about 30% of the release produced by 5,6_dihydroxytryptamine (Figure 2), but only 5% in the case of desoxyfructo-5-methoxytryptamine (Figure 3) At high concentrations desoxyfructo-serotonin oxalate is a good inhibitor of the release of serotonin induced by 5,6dihydryxytryptamine : the inhibition is 70% at the concentration of lo- Moles and 43% at 10e5 Moles. At lower concentrations, in contrast, the sugar derivative becames an activator of the release of serotonin, with a value of +116% at the concentration range from 10m6 to 10-4 of 10m6 Moles. Over the concentration was found to be an Moles, desoxyfructo-5-methoxytryptamine inhibitor of the release of serotonin with values of 59.4, 73.4 and 36.55, respectively (Table III). % inhibition The by 5,6-dihydroxytryptamine 1 _ 5-HT released
by
or activation of the release was calculated as follows :
(DF-5HT+5,6-DHT) - 5HT released 5HT released by 5,6-DHT
induced
by DF-5HT
DESOXYFRUCTO-SEROTONIN
808
Vo1.13,No.5
FIG. 1 Inhibition by desoxyfructo-serotonin oxalate and by desoxyfructo-5-methoxytryptamine oxalate of the uptake of the serotonin by human platelets % inhibitian
FIG. 3
FIG. 2
Inhibition by desoxyfructo-5Inhibition by desoxyfructo-5hydroxytryptamine oxalate of the methoxytryptamine oxalate of the release of serotonin induced by release of serotonin induced by 5,6_dihydroxytryptamine 5,6_dihydroxytryptamine
,I
20. Rhmd (%)S-HT , ?? *lrr /IO9
/
5,6-h OH-l+dF-SHT ( Tbnr4r*l rah
I
.z.lr:.
)
/’ )h
,t’ I
IS. I
,#’
t!’
;,t 5,6 -diOH -1 /..
5,6-diOH-f+dF-5HTarf~tt (Obwwd *.I11 1
!%6-diOH-T+dF-5MaT (Obwrad wluoc)
Cow
in lndolrminr
DESOXYFRUCTO-SEROTONIN
809
TABLE III Inhibition or activation of the uptake or release of S-HT observed with the sugar derivative of 5-hydroxy- and S-methoxy-tryptamine
esoxy-
fructo5-HT i xalate
Inhibition (-) of uptake of 5-HT Inhibition (-) or activation (+) of release of 5-HT
+119j9*1%
1 -43.573.83 1 -70.4jl.7%
t
DesoxyfructoS-MeOT oxalate
Inhibition (-) of uptake of 5-HT Inhibition (-) or activation (+) of release of S-HT
-59.4+10.3%
-73.473.4%
-36.5+4.2%
CONCLUSION -5 Moles, In the concentration range from 10-6 to 10 desoxyfructo-serotonin oxalate shows very little if any direct effect on platelet function : no aggregation is observed, the direct transport of the compound into the platelets is nearly zero, and its effect on the release of serotonin is limited. Even the concentration required to produce as small an effect as 50% maximal shape-change on rabbit platelets, is more than 10e5 Moles.
In contrast, at about the same concentration, the sugar derivative is still active on g and g type tryptaminergic receptors and interferes with the uptake and release of serotonin. ACKNOWLEDGEMENT The authors are grateful to Prof. A. Pletscher and Dr.M. Graf (Hoffmann-La Roche Co., Basel) for measurement of shapechange and valuable interpretation of the results.
810
DESOXYFRUCTO-SEROTONIN
Vo1.13,No.j
REFERENCES 1. MESTER, L., MESTER, M., RENDU, F., LABRID, C. and DURENG, G., Sugar Derivatives of Indolamines, Part I : Aggregation Induced by Desoxyfructo-serotonin and its Active Transport Through the Platelet Membrane. Thromb. Res., -12, 783, 1978. 2. ASTER, R.H. and JANDL, J.H., Platelet Sequestration in Man. I: Methods.,J. Clin. Invest., -43, 843, 1964. 3. GRAF, M. and PLETSCHER, A., Shape Change of Blood Platelets : a Model for Cerebral 5-Hydroxytryptamine Receptors? Br. J. Pharm., submitted for publ. 4.
DRUMMOND, A.H. and GORDON, J.L., Specific binding sites for 5-HT on rat blood platelets. Biochem. J., 150,129, 1975.
5. TUOMISTO, J. and TUKIAINEN, E., Decreased uptake of 5-HT in blood platelets from depressed patients. Nature,262,596, 1976. 6. RUDNICK, G., Active transport of 5-hydroxy-tryptamine by plasma membrane vesicles isolated from human blood platelets. J. Biol. Chem., =,2170, 1977. 7. HAWIGER, J., HAWIGER, A., STECKLEY, S., TIMMONS, S. and CHENG, C * I Membrane changes in human platelets induced by lipopolysaccharide endotoxin.,Brit. J. Haematol., 35,285, 1977. J., 8. LEVY-TOLEDANO, s., RENDU, F., BESSON, P. and mm, Aggregation of human gel-filtered platelets.- Requirement of apyrose and proteins. Rev. Europ. Et. Clin. Biol., =,513,1972.
SUGARDERIVATIVES OF INDOLAMINES, PART III:EFFECTOF DESOXYFRUCTO-SEROTONIN ON THE UPTAKEAND RELEASEOF SEROTONIN BY HUMANPLATELETS J,-Ma
LAUNAY
AND
Ls
MESTER+
Institut de Chimie des Substances Naturelles, Gif-sur-Yvette-91 France (Received 18.7.1978; in revised form 10.8.1978. Accepted by Editor W.H. Seegers) ABSTRACT -5 Moles, desoxyfructoAt concentrations less than 10 serotonin oxalate has very little direct effect on human blood platelets, but still interferes with the active transport and release of serotonin.
INTRODUCTION 1-Desoxyfructo-serotonin oxalate shows a serotonin-like activity towards g and E type tryptaminergic receptors (l), but its affinity is a hundred times less. Its intrinsic affinity for 2 type receptors is identical with that of serotonin, but only 70% for E type receptors. -5 Moles, desoxyAt final concentrations of less than 10 fructo-serotonin has very little direct effect on human blood Platelets, but still active on smooth muscles. In order to elucidate the mechanism which suppress platelet functions while still support vascular smooth muscle contraction, the effect of desoxyfructo-serotonin oxalate on the uptake and release of serotonin by platelets have been investigated.
MATERIAL AND METHODS S-Hydroxytryptamine binoxalate Li,2-3H(N)T, spec. act. 24 Ci/mmol, supplied by New England Nuclear, has 6een used in the uptake and release experiments. +
To whom requests should be addressed. 805
806
DESOXYFRUCTO-SEROTONIX
Vo1.13,No.5
Platelet rich plasma (2) (PRP) was obtained from blood collected on Acid citric-Citrate-Dextrose Anticoagulant (1 vol ACDA/ 9 vol blood) and centrifugated 5 min. at 200 g and 15'C. The number of platelets was measured optically in a Malassez cell. Shape-change of rabbit platelets (3) in titrated plasma produced by desoxyfructo-serotonin oxalate was measured as the maximum rate of decrease in light transmission. Inhibition of the uptake of 5-hydroxytryptamine-(3H) (4,5,6). 25 ,ul of 0.9% NaCl (control) or 25 ~1 of 0.9% NaCl containing the inhibitor was added to 75 ul of ( H)-5-HT in 0.9% NaCl to have a final concentration of 10" Moles of labelled Serotonin. After mixing, the solution was heated to 37°C for 5 min. Then 100 ~1 of PRP, preincubated 10 min. at 37'C with shaking, was added. After agitation in a Vortex agitator, the mixture was incubated for 1 min. at 37'C with shaking. To stop the reaction, 2 ml of 0.9% NaCl-0.4% EDTA were added at +4'C, the platelets were filtered on nitrated cellulose filter ("Millipore" Type GS 0.22 p), washed 6 times with 5 ml of 0.9% NaCl-0.4%EDTA, and the radioactivity on the filter was counted in a liquidscintillation counter (Intertechnic, Plaisir, France). Inhibition of the release of 5-hydroxytryptamine-(3H) induced by 5,6_dihydroxytryptamine. After uptake of labelled serotonin by the above method, the platelets were isolated on "Sepharose 2B" column (7,8). 175 ~1 samples of GFP (gel filtered platelets), labelled with serotonin, and 25 1 of 0.9% NaCl solution containing the inhibitor and/or 5,Z-dihydroxytryptamine at a final concentration of lOa Moles, were agitated in a Vortex agitator, and then incubated for 60 min. at 37'C with shaking. The incubated mixtures were centrifuged for 10 min. at 1200 g and +4'C, and the radioactivity was counted in 50 /ul of supernatant. RESULTS AND DISCUSSION Study of the shape-change of rabbit platelets in buffer solution shows a very low activity for desoxyfructo-serotonin ox.alate : the maximal effect is only 33% of the effect of serotonin. The concentration requirement for the sugar derivative to produce 50% of the maximal effect (ECsO) is 500 times more than for serotonin. TABLE I. Concentration required to produce 50% of the maximal shape-change of rabbit platelets
I I 5-HT
oxalate
I DF-5-HT I
oxalate
Maximal effect
SE 95%
EC50 1
1.9x10-7 1 (1.8-2.0) 1 100 %
I
7.2~10-~ 1 (4.2-12.3)1
i
I
I
33 %
1 1 I
-6 At low concentration (10 4101es) desoxyfructo-serotonin oxalate inhibits only moderately,on the extent of 45%, the uptake of serotonin by the platelets (Figure l).At higher concentrations (lo-4 Moles) the compound becomes a powerful inhibitor, showing 86% inhibition. The presence of the free hydroxyl group in position 5 seems to be important for this inhibition, because of the low effect of desoxyfructo-S-methoxytryptamine oxalate : 40% inhibition at the concentration of 10m4 Holes, but only 14% at 10-6 Moles. Table II shows the concentrations required for the sugar derivatives to obtain 50% of the total inhibition (ICso) : TABLE
II.
Concentrations required to produce 50 4 inhibition of the uptake of serotonin
Desoxyfructo-5-methoxytryptamine oxalate 1
5.56x10-4Moles I
I
In the concentration ranges corresponding to the IC50 values for inhibition of the uptake, the sugar derivatives show very little effect on the "release" phenomenon. With increasing concentrations, the sugar derivative of serotonin shows an increased release of serotonin, but not the su ar derivative of 5-methoxytryptamine. At a concentration of lo- 8 Moles, the release induced by desoxyfructo-serotonin oxalate is about 30% of the release produced by 5,6_dihydroxytryptamine (Figure 2), but only 5% in the case of desoxyfructo-5-methoxytryptamine (Figure 3) At high concentrations desoxyfructo-serotonin oxalate is a good inhibitor of the release of serotonin induced by 5,6dihydryxytryptamine : the inhibition is 70% at the concentration of lo- Moles and 43% at 10e5 Moles. At lower concentrations, in contrast, the sugar derivative becames an activator of the release of serotonin, with a value of +116% at the concentration range from 10m6 to 10-4 of 10m6 Moles. Over the concentration was found to be an Moles, desoxyfructo-5-methoxytryptamine inhibitor of the release of serotonin with values of 59.4, 73.4 and 36.55, respectively (Table III). % inhibition The by 5,6-dihydroxytryptamine 1 _ 5-HT released
by
or activation of the release was calculated as follows :
(DF-5HT+5,6-DHT) - 5HT released 5HT released by 5,6-DHT
induced
by DF-5HT
DESOXYFRUCTO-SEROTONIN
808
Vo1.13,No.5
FIG. 1 Inhibition by desoxyfructo-serotonin oxalate and by desoxyfructo-5-methoxytryptamine oxalate of the uptake of the serotonin by human platelets % inhibitian
FIG. 3
FIG. 2
Inhibition by desoxyfructo-5Inhibition by desoxyfructo-5hydroxytryptamine oxalate of the methoxytryptamine oxalate of the release of serotonin induced by release of serotonin induced by 5,6_dihydroxytryptamine 5,6_dihydroxytryptamine
,I
20. Rhmd (%)S-HT , ?? *lrr /IO9
/
5,6-h OH-l+dF-SHT ( Tbnr4r*l rah
I
.z.lr:.
)
/’ )h
,t’ I
IS. I
,#’
t!’
;,t 5,6 -diOH -1 /..
5,6-diOH-f+dF-5HTarf~tt (Obwwd *.I11 1
!%6-diOH-T+dF-5MaT (Obwrad wluoc)
Cow
in lndolrminr
DESOXYFRUCTO-SEROTONIN
809
TABLE III Inhibition or activation of the uptake or release of S-HT observed with the sugar derivative of 5-hydroxy- and S-methoxy-tryptamine
esoxy-
fructo5-HT i xalate
Inhibition (-) of uptake of 5-HT Inhibition (-) or activation (+) of release of 5-HT
+119j9*1%
1 -43.573.83 1 -70.4jl.7%
t
DesoxyfructoS-MeOT oxalate
Inhibition (-) of uptake of 5-HT Inhibition (-) or activation (+) of release of S-HT
-59.4+10.3%
-73.473.4%
-36.5+4.2%
CONCLUSION -5 Moles, In the concentration range from 10-6 to 10 desoxyfructo-serotonin oxalate shows very little if any direct effect on platelet function : no aggregation is observed, the direct transport of the compound into the platelets is nearly zero, and its effect on the release of serotonin is limited. Even the concentration required to produce as small an effect as 50% maximal shape-change on rabbit platelets, is more than 10e5 Moles.
In contrast, at about the same concentration, the sugar derivative is still active on g and g type tryptaminergic receptors and interferes with the uptake and release of serotonin. ACKNOWLEDGEMENT The authors are grateful to Prof. A. Pletscher and Dr.M. Graf (Hoffmann-La Roche Co., Basel) for measurement of shapechange and valuable interpretation of the results.
810
DESOXYFRUCTO-SEROTONIN
Vo1.13,No.j
REFERENCES 1. MESTER, L., MESTER, M., RENDU, F., LABRID, C. and DURENG, G., Sugar Derivatives of Indolamines, Part I : Aggregation Induced by Desoxyfructo-serotonin and its Active Transport Through the Platelet Membrane. Thromb. Res., -12, 783, 1978. 2. ASTER, R.H. and JANDL, J.H., Platelet Sequestration in Man. I: Methods.,J. Clin. Invest., -43, 843, 1964. 3. GRAF, M. and PLETSCHER, A., Shape Change of Blood Platelets : a Model for Cerebral 5-Hydroxytryptamine Receptors? Br. J. Pharm., submitted for publ. 4.
DRUMMOND, A.H. and GORDON, J.L., Specific binding sites for 5-HT on rat blood platelets. Biochem. J., 150,129, 1975.
5. TUOMISTO, J. and TUKIAINEN, E., Decreased uptake of 5-HT in blood platelets from depressed patients. Nature,262,596, 1976. 6. RUDNICK, G., Active transport of 5-hydroxy-tryptamine by plasma membrane vesicles isolated from human blood platelets. J. Biol. Chem., =,2170, 1977. 7. HAWIGER, J., HAWIGER, A., STECKLEY, S., TIMMONS, S. and CHENG, C * I Membrane changes in human platelets induced by lipopolysaccharide endotoxin.,Brit. J. Haematol., 35,285, 1977. J., 8. LEVY-TOLEDANO, s., RENDU, F., BESSON, P. and mm, Aggregation of human gel-filtered platelets.- Requirement of apyrose and proteins. Rev. Europ. Et. Clin. Biol., =,513,1972.