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The effect of betaadrenoceptor blocking drugs on the integrity of rat platelets as measured by 51Cr liberation
THRI?%OSISRESWRCH 23; 187-191,1981 0049-38&8/81/130187-05$02.00/O Copvright(c) 1981 ?erganon Press Ltd. Al.1rights reserved. Brief
Communication
THE EFFECT OF BETAADRENOCEPTOR
BLOCKING DRUGS
ON THE INTEGRITY OF RAT PLATELETS AS NEASURED 51 Cr LIBEMTION BY
PETER TURCANI and RADO NOSAL
Institute of Experimental Pharmacology, Centre of Physiological Sciences, Slovak Academy of Sciences, 881 05 Bratislava 1, Czechoslovakia (Received 17.2.1981; in revised form 30.4.1981.
Acceptedby Editor F. Kornalik. Receivedin final foxm by ExecutiveEditorialOffice 6.7.1981) INTRODUCTION Betaadrenoceptor blocking drugs release and/or block the uptake of serotonin from platelets in a dose-dependent way by a mechanism other than beta-blockade (1,2,3). Electron microscopic observations showed ultrastructural changes and platelet lysis after treatment with betaadrenoceptor blocking drugs (4) whereas no release of the cytoplasmic marker lactate dehydrogenase (LDH) was demonstrated (5). The liberation of 51Cr from platelets is equally suitable as an indicator of platelet lysis (6). In this study the effect of 10 betaadrenoceptor blocking drugs was inves51 Cr from rat platelet rich tigated by measuring the release of plasma (PRP).
METHODS Blood vss collected from male '&star albino rats (350-400 g) in ether anesthesia via polyethylene cannula from common carotid artery (9 ml of blood + 1 ml of 3.8% trisodium citrate). Platelet rich plasma (PRP) was obtained by centrifugation 20 min 250 G at 22'. To label platelets PRP was centrifuged 1000 G 20 min at 22'. The supernatant was used as PPP (platelet poor plasma) and the pelet was resuspended in RCD solution (7 parts of Ringer solution 2 parts of 0.1 mol.l-' Na3-citrate,
1 part of 5% dextrose).
Reprint requests: Rado Nossl,MD,PhD, Institute of Experimental Pharmacology, Slovak Academy of sciences, 881 05 Bratislava 1 POB 1041, Czechoslovakia 51 Cr-liKey :iords: Platelets, Betaadrenoceptor blocking drugs, beration 187
Na2 %04
(ROTOD
DDR,
?57C
G3q/g
,Cr) in the amcuzt of 3/,uCi (=
=0.185 NBq) per iOg platelets was Yaed a-d incubated under shaking 30 pin at 22"+ After two centrifugal xashes in RCD platelets and incubawere resuspended ir. PPP (5 x lo5 platelets ?er l,,;~) ted with betaadrenoceptor blocking drugs at 37'. fliter incubation samples were cooled to 0' and centrifuged quickly at 14 000 C 1 min at 4". From the supernatant 900 cl were taken for gaaa coun/ 51 ting (Tesla gama-automat) at 2.1 kV. The 2 release of Cr in the supernatant was calculated from the total acti.city in the sample. Results were evaluated by Student-s t-test.
RESULTS and DISCUSSIO?J c 51 The percent liberation of Cr in the control group was 2.5 0.5 after 1 min and 2.9 + 0.5 after 15 min. The effect of 10 betaadrenoceptor blocking drugs (BAB-drugs) in concentration 10-3 -1 51 mol.1 on the liberation of Cr accumulated in platelets after 15 min incubation is demonstrated in Table I. One min incubation with alprenolol (ALP:HZssle) and propranoiol (PRO:ICI) signifi51 cantly decreased the amount of liberated Cr as compared with control and other drugs. After 5 min incubation ALP, doberol (DOB: Boehringer), metipranol (MET=trimepranol Spofa), oxprenolol (OXP: TABLE
I
Percent 5'Cr release from rat PRP after BAB-drugs x + S.E.M.
n=
6
.P zGo.05
1 min
5 min
-3 -1 (10 mol.1 )
?? eP~O.ol 10 min
15 min
??
Alprenolol
0.9 + 0.2
1:: + 0.2
2:: + 0.2
2.8 + 0.1
Atenolol
2.1 + 0.2
2.8 z 0.3
3.3 I 0.1
Doberol
1.7 + 0.2
l?8 + 0.1
3.2 + 0.2 00 1.8 + 0.1
Ko 1124
1.4 f 0.2
2.6 + 0.2
3.6 z 0.3
Metipranol
1.6 + 0.2
2to + 0.3
2+i 2 0.2 mo 1.9 2 0.1
Oxprenolol
1.9 + 0.1
I?9
2:: + 0.2
3.6 2 0.1
Practolol
1.8
f 0.1
2?0 + 0.2
2.9 2 0.1
Pricoron
1.8 + 0.1
4:s + 0.3
2f?l + 1.8
Pronethalol
1.8 z 0.1
1:9 + 0.2
2::: 2 0.1 00 5.8 + 0.3 .a 1.8 I 0.2
Propranolol
1:2 2 0.2
It8 + 0.1
2:': 2 0.1
3.0 2 0.2
Control
2.5 1 0.5
3.1 2 0.4
3.6 i 0.i
2.9 2 0.3
+ 0.1
2?3 + 0.2
2.9 + 0.3
2f5 1 0.1
Ciba),
PraCtOlOl
(PRA:ICI) and pronethalol (PRON:ICI) signigican51 tly reduced the liberation of Cr. As evident pricoron (PRI:VULM) 51 was the only one drug that increased significantly the Cr liberation to 23.1 % after 15 min incubation. Table II demonstrates the dose-dependent liberation of 51Cr after -4 -5 10 min incubation with BAB-drugs. In concentration 10 and 10 -1 mol.1 all drugs significantly decreased the liberation of 51Cr as compared with control. TABLE II Percent
51
Cr liberation
IO min at 37..
from rat PRP treated with BAB-drugs
x I S.E.M. 1o-5
n = 6 mol.1
-1
??p
??eP~0.01
<0.05
-4 -1 10 mol.1
Alprenolol
I'? + 0.1
1::
Atenolol
2:: + 0.1
Doberol
2:tl + 0.1 ?? * 1.5 + 0.2
Ko 1124
1:s + 0.2
1:: + 0.2
Metipranol
2%
1::
+ 0.2
1::
+ 0.1
+ 0.1
2
0.1
?? o
Oxprenolol
I?: 2 0.2
1.6 + 0.2
Practolol
1:: + 0.1
Pricoron
1:: 2 0.1
1:: + 0.1 ?? * 2.0 + 0.1
Pronethalol
1:: + 0.1
1:: + 0.2
Propranolol
1?4 + 0.2
1%
Control
3.6 f 0.3
3.6 + 0.3
2 0.1
Betaadrenoceptor blocking drugs possess a high membrane affinity resulting in different non-specific activities (7). The mechanism of action of BAB-drugs on platelets remains unexplained similar to that described for fenfluoramine or suloctidil (8,9). Conformational changes in membrane phospholipids (101, changes in membrane transport of Ca 2+ , Na+ and K+ ions induced by these drugs (11,121, moreover possible intracellular exchange for serotonin (13) must be taken into consideration. The exposure of platelets to BAB-drugs resulted in a dose- and time-dependent liberation of serotonin (1). Such release seems to be different to that demonstrated during the release reaction (14). Some investigators concerned electronmicroscopic changes in platelet membranes treated with BAB-drugs as cell lysis (4). On the other hand any lac'
act ivity was measured extracelluary (5). 5i Cr w-e ha71e shown that B-AS-drugs Using prelabeled platelets with differ in the effect on platelet integrity. Most of the used drugs even in high concentration decreased spontaneous liberation of 51 Cr. It suggests that the liberation of serotonin from platelets by most of BAB-drugs is not a result of platelet lysis as presumed previously (1). Pricoron was the only one drug in our 51 experiments that increased the activity of Cr outside platelets as a result of platelet lysis. We assume this effect as a result of high lipid solubility and membrane affinity of this drug. tatedehydrogenase
REFERENCES 1.
LEM.MER,B., WIETHOLD,G., BAK,I.J., HELLENBRECHT,D. and GROBECKER,H. Human blood platelets as cellular models for investigation of membrane active drugs:Beta adrenergic blocking agents. Naunyn-Schmiedebergs Arch.Pharmacol. 275, 299-313, 1972
2. NOSAL,R. and MENYHARDTOVA,Z. The effect of trimepranol on thrombocyte functions and histamine release in the rat. Agents Act. 2, 9-14, 1975 3. WEKSLER,B.B., GILLIK,M. and PINK,J. Effect of propranolol platelet function. ___Blood 49, 185-196, 1977
on
4. LEMMER,B., WIETHOLD,G., BAK,I.J., HELLENBRECHT,D. and GROBECKER,H. Human blood platelets as cellular models for ivestigation of membrane active drugs. In: Erythrocytes, Thrombocytes, Leukocytes. Recent advances in membrane and metabolic research Ed. E.Gerlach, K.Moser, E.Deutsch, W.Wilmanns. Georg Thieme Publ., Stuttgart 1973, pp. 220-223 5. NATHAN,I., DVILANSKY,A., SAGE,J. and KORCZYN,A.D. Effect of propranolol and pindolol on platelet aggregation and serotonin release. Life Sci. -20, 669-673, 1977 6. KINLOUGH-RATHBONE,R.L., PACKHAM,M.A. and MUSTARD,J.F. Comparison of methods of measuring loss of plasmatic constituents from platelets. Throm.Res. -9, 669-673, 1976 7. LANGSLET,A. Membrane stabilization and cardiac effect of D,Lpropranolol, D-propranolol and chlorpromazine. Europ.J.Pharm. 13. 6-14. 1970 8. WIELOSZ,M., RONCAGLIONI,M.C., DE GAETANO G. and GARATTINI,S. Interference by fenfluoramine with the storage of 14C-S-hydroxytryptamine in rat platelets. Arch Int.Pharmacodyn.Therap. 225, 232-239, 1977 9. MILLS,D.C.B. and MCFARLANE D-E. Depletion of platelet amine storage granules by the antithrombotic agent suloctidil. Thromb. Haemostas. 38, 1010-1017, 1977
191
10. GODIN,D.V., WANG NG,T. and TUCHER,J.M. Studies on the interaction of propranolol with erythrocyte membranes. Biochim.Biophys. Acta 436, 757-773, 1976 11. MANNINEN,V. Movements of sodium and potassium ions and their tracers in propranolol-treated red cells and diaphragm muscle. Acta physiol.scand. 80, ~~~~1.355, l-76, 1970 12. GLYNN,J.M. and WARNER,A.E. Nature of the calcium dependent potassium leak induced by (+I propranolol and its possible relevance to the drug-s antiarrhytmic effect. Brit.J.Pharmacol. 44, 271-278, 1972 13. DA PRADA,M. and PLETSCHER,A. Accumulation of basic drugs in 5-hydroxytryptamine storage organelles of rabbit blood platelets. Europ.J.Pharmacol. -32, 179-185, 1975 14. MACINTYRE,D.E. The platelet release reaction: Association with adhesion and aggregation, and comparison with secretory responses in other cells. In: Platelets in biology and pathology- Ed. J.L.Gordon, Elsevier 1976, pp.61-85
Communication
THE EFFECT OF BETAADRENOCEPTOR
BLOCKING DRUGS
ON THE INTEGRITY OF RAT PLATELETS AS NEASURED 51 Cr LIBEMTION BY
PETER TURCANI and RADO NOSAL
Institute of Experimental Pharmacology, Centre of Physiological Sciences, Slovak Academy of Sciences, 881 05 Bratislava 1, Czechoslovakia (Received 17.2.1981; in revised form 30.4.1981.
Acceptedby Editor F. Kornalik. Receivedin final foxm by ExecutiveEditorialOffice 6.7.1981) INTRODUCTION Betaadrenoceptor blocking drugs release and/or block the uptake of serotonin from platelets in a dose-dependent way by a mechanism other than beta-blockade (1,2,3). Electron microscopic observations showed ultrastructural changes and platelet lysis after treatment with betaadrenoceptor blocking drugs (4) whereas no release of the cytoplasmic marker lactate dehydrogenase (LDH) was demonstrated (5). The liberation of 51Cr from platelets is equally suitable as an indicator of platelet lysis (6). In this study the effect of 10 betaadrenoceptor blocking drugs was inves51 Cr from rat platelet rich tigated by measuring the release of plasma (PRP).
METHODS Blood vss collected from male '&star albino rats (350-400 g) in ether anesthesia via polyethylene cannula from common carotid artery (9 ml of blood + 1 ml of 3.8% trisodium citrate). Platelet rich plasma (PRP) was obtained by centrifugation 20 min 250 G at 22'. To label platelets PRP was centrifuged 1000 G 20 min at 22'. The supernatant was used as PPP (platelet poor plasma) and the pelet was resuspended in RCD solution (7 parts of Ringer solution 2 parts of 0.1 mol.l-' Na3-citrate,
1 part of 5% dextrose).
Reprint requests: Rado Nossl,MD,PhD, Institute of Experimental Pharmacology, Slovak Academy of sciences, 881 05 Bratislava 1 POB 1041, Czechoslovakia 51 Cr-liKey :iords: Platelets, Betaadrenoceptor blocking drugs, beration 187
Na2 %04
(ROTOD
DDR,
?57C
G3q/g
,Cr) in the amcuzt of 3/,uCi (=
=0.185 NBq) per iOg platelets was Yaed a-d incubated under shaking 30 pin at 22"+ After two centrifugal xashes in RCD platelets and incubawere resuspended ir. PPP (5 x lo5 platelets ?er l,,;~) ted with betaadrenoceptor blocking drugs at 37'. fliter incubation samples were cooled to 0' and centrifuged quickly at 14 000 C 1 min at 4". From the supernatant 900 cl were taken for gaaa coun/ 51 ting (Tesla gama-automat) at 2.1 kV. The 2 release of Cr in the supernatant was calculated from the total acti.city in the sample. Results were evaluated by Student-s t-test.
RESULTS and DISCUSSIO?J c 51 The percent liberation of Cr in the control group was 2.5 0.5 after 1 min and 2.9 + 0.5 after 15 min. The effect of 10 betaadrenoceptor blocking drugs (BAB-drugs) in concentration 10-3 -1 51 mol.1 on the liberation of Cr accumulated in platelets after 15 min incubation is demonstrated in Table I. One min incubation with alprenolol (ALP:HZssle) and propranoiol (PRO:ICI) signifi51 cantly decreased the amount of liberated Cr as compared with control and other drugs. After 5 min incubation ALP, doberol (DOB: Boehringer), metipranol (MET=trimepranol Spofa), oxprenolol (OXP: TABLE
I
Percent 5'Cr release from rat PRP after BAB-drugs x + S.E.M.
n=
6
.P zGo.05
1 min
5 min
-3 -1 (10 mol.1 )
?? eP~O.ol 10 min
15 min
??
Alprenolol
0.9 + 0.2
1:: + 0.2
2:: + 0.2
2.8 + 0.1
Atenolol
2.1 + 0.2
2.8 z 0.3
3.3 I 0.1
Doberol
1.7 + 0.2
l?8 + 0.1
3.2 + 0.2 00 1.8 + 0.1
Ko 1124
1.4 f 0.2
2.6 + 0.2
3.6 z 0.3
Metipranol
1.6 + 0.2
2to + 0.3
2+i 2 0.2 mo 1.9 2 0.1
Oxprenolol
1.9 + 0.1
I?9
2:: + 0.2
3.6 2 0.1
Practolol
1.8
f 0.1
2?0 + 0.2
2.9 2 0.1
Pricoron
1.8 + 0.1
4:s + 0.3
2f?l + 1.8
Pronethalol
1.8 z 0.1
1:9 + 0.2
2::: 2 0.1 00 5.8 + 0.3 .a 1.8 I 0.2
Propranolol
1:2 2 0.2
It8 + 0.1
2:': 2 0.1
3.0 2 0.2
Control
2.5 1 0.5
3.1 2 0.4
3.6 i 0.i
2.9 2 0.3
+ 0.1
2?3 + 0.2
2.9 + 0.3
2f5 1 0.1
Ciba),
PraCtOlOl
(PRA:ICI) and pronethalol (PRON:ICI) signigican51 tly reduced the liberation of Cr. As evident pricoron (PRI:VULM) 51 was the only one drug that increased significantly the Cr liberation to 23.1 % after 15 min incubation. Table II demonstrates the dose-dependent liberation of 51Cr after -4 -5 10 min incubation with BAB-drugs. In concentration 10 and 10 -1 mol.1 all drugs significantly decreased the liberation of 51Cr as compared with control. TABLE II Percent
51
Cr liberation
IO min at 37..
from rat PRP treated with BAB-drugs
x I S.E.M. 1o-5
n = 6 mol.1
-1
??p
??eP~0.01
<0.05
-4 -1 10 mol.1
Alprenolol
I'? + 0.1
1::
Atenolol
2:: + 0.1
Doberol
2:tl + 0.1 ?? * 1.5 + 0.2
Ko 1124
1:s + 0.2
1:: + 0.2
Metipranol
2%
1::
+ 0.2
1::
+ 0.1
+ 0.1
2
0.1
?? o
Oxprenolol
I?: 2 0.2
1.6 + 0.2
Practolol
1:: + 0.1
Pricoron
1:: 2 0.1
1:: + 0.1 ?? * 2.0 + 0.1
Pronethalol
1:: + 0.1
1:: + 0.2
Propranolol
1?4 + 0.2
1%
Control
3.6 f 0.3
3.6 + 0.3
2 0.1
Betaadrenoceptor blocking drugs possess a high membrane affinity resulting in different non-specific activities (7). The mechanism of action of BAB-drugs on platelets remains unexplained similar to that described for fenfluoramine or suloctidil (8,9). Conformational changes in membrane phospholipids (101, changes in membrane transport of Ca 2+ , Na+ and K+ ions induced by these drugs (11,121, moreover possible intracellular exchange for serotonin (13) must be taken into consideration. The exposure of platelets to BAB-drugs resulted in a dose- and time-dependent liberation of serotonin (1). Such release seems to be different to that demonstrated during the release reaction (14). Some investigators concerned electronmicroscopic changes in platelet membranes treated with BAB-drugs as cell lysis (4). On the other hand any lac'
act ivity was measured extracelluary (5). 5i Cr w-e ha71e shown that B-AS-drugs Using prelabeled platelets with differ in the effect on platelet integrity. Most of the used drugs even in high concentration decreased spontaneous liberation of 51 Cr. It suggests that the liberation of serotonin from platelets by most of BAB-drugs is not a result of platelet lysis as presumed previously (1). Pricoron was the only one drug in our 51 experiments that increased the activity of Cr outside platelets as a result of platelet lysis. We assume this effect as a result of high lipid solubility and membrane affinity of this drug. tatedehydrogenase
REFERENCES 1.
LEM.MER,B., WIETHOLD,G., BAK,I.J., HELLENBRECHT,D. and GROBECKER,H. Human blood platelets as cellular models for investigation of membrane active drugs:Beta adrenergic blocking agents. Naunyn-Schmiedebergs Arch.Pharmacol. 275, 299-313, 1972
2. NOSAL,R. and MENYHARDTOVA,Z. The effect of trimepranol on thrombocyte functions and histamine release in the rat. Agents Act. 2, 9-14, 1975 3. WEKSLER,B.B., GILLIK,M. and PINK,J. Effect of propranolol platelet function. ___Blood 49, 185-196, 1977
on
4. LEMMER,B., WIETHOLD,G., BAK,I.J., HELLENBRECHT,D. and GROBECKER,H. Human blood platelets as cellular models for ivestigation of membrane active drugs. In: Erythrocytes, Thrombocytes, Leukocytes. Recent advances in membrane and metabolic research Ed. E.Gerlach, K.Moser, E.Deutsch, W.Wilmanns. Georg Thieme Publ., Stuttgart 1973, pp. 220-223 5. NATHAN,I., DVILANSKY,A., SAGE,J. and KORCZYN,A.D. Effect of propranolol and pindolol on platelet aggregation and serotonin release. Life Sci. -20, 669-673, 1977 6. KINLOUGH-RATHBONE,R.L., PACKHAM,M.A. and MUSTARD,J.F. Comparison of methods of measuring loss of plasmatic constituents from platelets. Throm.Res. -9, 669-673, 1976 7. LANGSLET,A. Membrane stabilization and cardiac effect of D,Lpropranolol, D-propranolol and chlorpromazine. Europ.J.Pharm. 13. 6-14. 1970 8. WIELOSZ,M., RONCAGLIONI,M.C., DE GAETANO G. and GARATTINI,S. Interference by fenfluoramine with the storage of 14C-S-hydroxytryptamine in rat platelets. Arch Int.Pharmacodyn.Therap. 225, 232-239, 1977 9. MILLS,D.C.B. and MCFARLANE D-E. Depletion of platelet amine storage granules by the antithrombotic agent suloctidil. Thromb. Haemostas. 38, 1010-1017, 1977
191
10. GODIN,D.V., WANG NG,T. and TUCHER,J.M. Studies on the interaction of propranolol with erythrocyte membranes. Biochim.Biophys. Acta 436, 757-773, 1976 11. MANNINEN,V. Movements of sodium and potassium ions and their tracers in propranolol-treated red cells and diaphragm muscle. Acta physiol.scand. 80, ~~~~1.355, l-76, 1970 12. GLYNN,J.M. and WARNER,A.E. Nature of the calcium dependent potassium leak induced by (+I propranolol and its possible relevance to the drug-s antiarrhytmic effect. Brit.J.Pharmacol. 44, 271-278, 1972 13. DA PRADA,M. and PLETSCHER,A. Accumulation of basic drugs in 5-hydroxytryptamine storage organelles of rabbit blood platelets. Europ.J.Pharmacol. -32, 179-185, 1975 14. MACINTYRE,D.E. The platelet release reaction: Association with adhesion and aggregation, and comparison with secretory responses in other cells. In: Platelets in biology and pathology- Ed. J.L.Gordon, Elsevier 1976, pp.61-85