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Effects of labetalol on the arachidonic acid metabolism in human blood platelets, and in lung and aorta of the rat
Prostaglandins Leukotrienes and Medicine 12: l-9, 1983
EFFECTS OF LABETALOL ON THE ARACHIDONIC ACID METABOLISM IN HUMAN BLOOD PLATELETS, AND IN LUNG AND AORTA OF THE RAT K.C. Srivastava and K.K. Awasthi, Department of Environmental Medicine, Institute of Community Health, Odense University, J.B. WinslBws vej 19, DK-5000 Odense, Denmark (reprint requests to KCS). ABSTRACT Effects of labetalol on the arachidonic acid metabolism in washed human blood platelets, and in lung and aorta of the rat was studied. Effects of this drug on the aggregation induced by usual aggregation agents, and on the prostacyclin generating capacity of rat aorta as assessed by its antiaggregation effect were also studied. The following results were obtained. 1. Reduced generation of thromboxane and HHT in platelets was observed at 1 mM cont. of the drug. 2. This drug inhibited ADP-, collagen-, and epinephrine-induced aggregation in a dose dependent manner. 3. Arachidonate-induced aggregation was only slightly inhibited at I mM cont. of labetalol. 4. Aorta and lung synthesized more prostacyclin in its presence from exogenous labelled arachidonate. 5. Aorta synthesized more prostacyclin from its endogenous AA pool in the presence of labetalol as assessed by inhibition of platelet aggregation. INTRODUCTION Non-selective beta-adrenoreceptor antagonists show some unwanted effects. For modifying or removing such effects, newer agents with cardioselective activity or an added alpha-adrenoreceptor blocking activity have been developed. Labetalol possesses a combined alpha- and beta-adrenoreceptor activity. It shows potential benefits in hypertensive patients suffering from angina, particularly in situations where there is also present peripheral vascular disease or coronary artery spasm (1,2). Because of its two modes of action, this drug could be useful in a wide spectrum of hypertensive patients. It has been used in the management of severe hypertension in pregnancy (3). Control of raised blood pressure improves foetal survival and reduces material complications (4-6). It has been difficult to obtain adequate information on the precise mode of action of labetalol in patients suffering from hypertension. This has probably been due to (i) apparent change in ratio of the strength of alpha- and beta-adrenoreceptor blocking action, and (ii) varying doses and different routes of drug admninis-
1
tration (7). With the discovery of two new classes of compounds, the thromboxanes and prostacyclins, showing opposite effects in the cardiovascular system, it is not surprising that mechanism of action of cardiovascular drugs is being evaluated in the light of the effects of such drugs on the biosynthesis of these two biologically potent compounds of physiological origin. In this report effects of labetalol has been examined on the metabolism of labelled arachidonic acid (AA) in human platelets, and also in the lung and aorta of the rat. Besides, effects of this drug on the aggregation induced by usual aggregation agents, and on the prostacyclin generating capacity of rat aorta as assessed by its anti-aggregation effect have also been studied. MATERIALS AND METHODS Labetalol hydrochloride (Trandate) was obtained from Glaxo, Glaxo Group Research Limited, Ware, Hertfordshire. Labelled (l-14C) arachidonic acid, specific activity 58.4 mCi/mmol, was purchased from the Radiochemical Centre (Amersham, England). Labelled TxB2 (8) and prostaglandins (9) were prepared as described earlier. Unlabelled prostaglandins E2, -D2, -F2o, 6-keto-Flu and TxB2 were obtained as generous gift from ON0 Pharmaceutical Company Limited, Higashiku, Osala, Japan. Hydroxy fatty acid (HHT) was kindly provided as generous gift by Dr. D.H. Nugteren, Unilever Research, Vlaardingen, The Netherlands. Preparation
of platelet suspension blood from healthy donors who had not taken aspirin for at least 10 days was collected into acid-citrate-dextrose (ACD) and platelet suspensions were prepared by the method of Schmidt et al. (10). Venous
Preapration of drug solution Solutions of this drug in appropriate concentrations for various experiments were prepared in alcohol-water (I:1 v/v) and stored at -2O*C until use. Incubation Platelet suspensions (300 ul, 1 x 108 platelets) were first incubated with the drug for 10 min at room temperature in final concentrations as indicated in Table 1. This was followed by a further incubation with labelled arachidonate (25 uM) for 10 min at 37OC. Platelet suspensions serving as controls were incubated with an appropriate volume of alcohol-water (l:l, v/v) mixture for 10 min at room temperature to be followed by incubation with labelled arachidonate as above. Extraction and TLC separation of labelled AA-metabolites Extraction of the labelled AA-met&o&es from the incubation medium and their separation by a two dimensional TLC were achieved as described earlie (8). Preparation of lung homogenate and aorta The tissues were prepared as described (ca. 0.5 cm, wet wt. ca. 6.5 mg> were prepared.
by us earlier
(11). Pieces of aorta
Incubation Incubations of lung homogenates and aorta pieces with the drug followed by labelled arachidonate (lung 8 u&i, aorta 15 uM) were done as described earlier (11). Appropriate controls were run simultaneously.
2
Extraction of labelled AA-metabolites and their separation by two-dimensional TLC were done exactly as described by us (8).
II.
ect of this drug on platelet aggregation induced by ADP, collagen, epinephrine and arachidonate was examined. All aggregation studies were carried out using human platelet-rich plasma (PRP). Venous blood from healthy donors, who had not taken any medication for about a week before giving blood, was collected in silicon&d tubes ‘containing l/l0 volume of 3.1 %, trisodium citrate and l/20 volume saline. Platelet-rich plasma and platelet poor plasma (PPP) were prepared in the usual way. Platelet aggregation was studied in a Bryston aggregometer using PRP (ca. 2.5-3.0 x 108 platelets). Platelet-rich plasma was preincubated with a desired concentration of the drug at room temperature for 10 min and then for 30 set in the aggregometer at 370C under stirring. This was followed by addition of the aggregating agent. Control PRP samples were treated with the corresponding volume of the solvent (alcohol-water, (1:l) v/v) prior to inducing aggregation. The total volume of PRP and drug/solvent was maintained constant to 1.0 ml. on the In another set of aggregation experiment, the effect of labetalol ability of rat aorta in inhibiting collagen-induced aggregation of human blood platelets (PRP) was examined. Prior to aggregation step, incubation of aorta pieces was done in the following way. a) The incubation medium consisted of 200 ul Tris-HCI (50 mM, pH 8.41, a piece of rat aorta (0.5 cm, ca. 6.5 mg) and 10 ul of the solvent (alcoholwater l:l, v/v) (control) or 10 ul of the drug solution (experimental) in a final cont. of 0.85 mM. The control and the experimental tubes were kept in ice-cold water for 30 min. Each tube (one at a time) was then incubated at 35OC for 5 min. Ten microliters of the aorta incubate were transferred to the aggregation tube, the contents allowed to mix for 30 set under stirring followed by addition of collagen (2 ug/ml) to induce aggregation. The incubation tubes were placed in ice-cold water (-3oC) immediately after pipetting of the aorta incubate. Sequence of incubation: control, experimental. b) The incubation tubes from above, one at a time, were incubated again at 35OC for 5 min. Aorta incubate (10 ul) was added to the aggregation tube, mixed for 30 set followed by addition of collagen. The incubation tubes were placed in ice-cold water (-3oC) immediately as above. Sequence of incubation: experimental, control. c) The incubation tubes from above, one at a time, were further incubated at 35OC for 5 min. Aorta incubate (10 ul) was added to the aggregation tube, the contents mixed for 30 set followed by addition of collagen. The incubation tubes were placed in ice-cold water as above. Sequence of incubation: control, experimental. d) The incubation tubes from above, one at a time, were finally incubated at 35OC for 5 min. Aorta incubate (10 ml) was added to the aggregation tube, the contents mixed for 30 set followed by addition of collagen to induce aggregation. Sequence of incubation: control, experimental.
The effect of labetalol on the aorta prostacyclin synthesis was examined using aorta pieces from 5 rats. Aorta pieces were stored at -200C immediately and used later in incubation experiments. In the aorta from three rats, labetalol was
3
found to induce an increased synthesis of prostacyclin; in one rat there was observed no effect, while in another this drug showed an opposite effect, that is, apparently a reduced formation of prostacyclin compared to control took place. Malondialdehyde (MDA) formation in platelet suspension in the presence of labetalol Malondialdehyde formation by platelets in the presence of labetalol was examined with washed platelet preparations in a way similar to that described earlier (12). RESULTS Table 1 gives data on the effect of labetalol on the metabolism of labelled AA by washed human platelets. The amount of TxB2 was found to be reduced at 1.0 mM cont. of this drug. HHT was also found to be reduced. There was observed no effect of this drug on other AA-metabolites. Table 2 gives data on the effect of this drug on the metabolism of labelled AA in the aorta and lung (homogenate) of the rat. In lung this drug seems to increase the synthesis of prostaglandins, TxB2 and prostacyclin; this effect being more pronounced on PC12 synthesis. In aorta the drug increases the formation of PG12. Figure 1 shows the effect of labetalol on collagen-induced aggregation: (A) and (8); ADP-induced aggregation: (Cl; arachidonate-induced aggregation: (D). As is obvious labetalol shows inhibitory effect on collagen induced aggregation in a dose-dependent manner. As low as at 31 uM labetalol shows inhibition of aggregation induced by 1 ug/ml collagen. Inhibition of ADP-induced aggregation was observed only at higher labetalol concentrations (0.5, 1.0 mM). A slight inhibition of arachidonate-induced aggregation was observed at 1.0 mM cone of iabetalol. Figure 2 shows the inhibitory effect of this drug on epinephrine-induced biphasic aggregation. Labetalol showed inhibitory effect in a dose-dependent manner in a dose range from 25 uM to 1.0 mM. Figure 3 shows the effect of labetalol on the ability of rat aorta in inhibiting collagen-induced aggregation of human blood platelets. As is obvious incubate from the labetalol treated aorta was more effective compared to control in inhibiting collagen induced platelet aggregation. This was found to be consistent irrespective of the sequence of incubation, i.e., first control followed by experimental or the other way. DISCUSSION Beta-adrenoreceptor blocking drugs alter platelet function by mechanisms other than beta-blockade (13). These drugs have been shown to release serotonin from washed platelets or PRP in a dose-dependent manner and inhibit platelet aggregation induced by several aggregation agents (14-16). When stimulated by thrombin and during ADP-involved aggregation, platelets synthesize prostaglandins and MDA, the latter is thought to be an indicator of prostaglandin (17) and thromboxane synthesis (18). Malondialdehyde formation is decreased in platelets by
4
Tabk 1. Effect of labetalol on the biosynthesis of AA metabolites blood platelets
in washed human
Amounta (picomoks/lo% platekts) formed after platelet suspension with labelled AA for 10 min
incubation of
PGF2 ci
T*2
PGE2
PGD2
HHT
Control
19 +6
715 + 237
31+ 12
292 11
561 f 228
+Labetalol (0.5 mM, n=8)
18 25
709 f 262
282 5
24-+ 3
563 f 220
Control
2024
682 * 259
29+ 8
262 8
4242 128
+Labetalol (1.0 mM, n=6)
18 24
606 f 234 it
26+ 9
21+ 4
398* 144
* p < 0.01 (t-test
for paired data)
a Mean *SD
Table 2. Effect of labetalol on the biosynthesis homogenate and aorta of the rat
of AA metabolites
Amounta (picomoles/IiOO ul) formed labelled AAX for 15 min
after
incubation
PCF2 c1
TxB2
PCE2
PGI2XX
Control
26?7
77 2 19
25* 4
1142 46
+LabetaIol (2mM, n=4)
3329
83 + 35
312 4
166* 48
a Mea&SD
x Final cone 8 uM
xx Measured as 6-keto-PGFl
a
6-keto-PGFl values for aorta picomoles, 1$e talol43 picomoles
5
from
in lung
two rats;
control
with
22
Figure 1. Tracings showing the effect of labetabl (Lb) on the human blood platelet aggr ation induced by some aggregating agents. A. collagen (1 ug/ml ‘g, 1. collagen; 2. Lb 31 uM; 3. Lb 62 uM; 4. Lb 125 uM; 5. Lb 250 uM. B. collagen (2 ug/ml) 1. collagen; 2. Lb 62 uM; 3. Lb 125 mM; 4 Lb 250 uM; 5. Lb 500 uM; 6. Lb 1 mM. C. ADP (3 uM) 1. ADP; 2. aggregation by ADP (3 uM) of PRP pretreated with 10 ul of alcohol-water (l:l, v/v); 3. Lb 500 uM; 4. Lb 1 mM. D. 1. arachidonate; 2. Lb 500 uM; 3. Lb 1 mM.
Epinphrmr(SpH)
EpicuphrmallOpHl
1
I
+
0
\
“l”
A \
1
Figure 2. Tracings showing the effect of labetalol (Lb on the human blood platelet aggregation induced by epinephrine. A. epinephrine (5 uM) 1. epinephrine; 2. Lb 100 uM; 3. Lb 250 uM; (10 uM) 1. 4. Lb 500 uM; 5. Lb 1 mM. B. epinephrine epinephrine; la.. epinephrine (5 uM); 2. Lb 25 uM; 3. Lb 50 uM; 4. Lb 250 uM; 5. Lb 500 uM (with the exception of la, other tracings were obtained with 10 uM epinephrine).
6
x Addition of on
Figure 3. Tracings showing the effect of labetalol (Lb) on the ability of aorta in inhibiting collagen-induced aggregation of human blood platelets. A. 1. collagen; 2. in the presence of incubate from the aorta piece not pre-treated with Lb (control); 3. in the presence of incubate from the aorta piece pre-treated with Lb (experimental). B. 1. collagen; 2. experimental; 3. control. C. 1. collagen; 2. control; 3. experimental; 4. control; 5. experimental. membrane stabilizing drugs including propranolol meaning that they inhibit phosphalipase A2 activity with the result a reduced formation of prostaglandin endoperoxides and TxA2 takes place (19). Several other beta-adrenoreceptor blocking drugs have been shown to inhibit platelet production of MDA on activation with thrombin (20). Labetalol did not reduce the generation of MDA in washed platelets from exogenous arachidonate. Results of our experiments on the metabolism of labelled AA in the presence of labetalol show that this drug inhibits platelet Tx-synthetase activity at 1 mM cone while showing no such effect at 0.5 mM cone in washed platelet preparations. But this drug inhibits ADP-, collagen- and epinephrine-induced aggregation of PRP at lower concentrations; and it shows only a slight inhibition of aggregation induced by arachidonate at 1 mM cont. These observations may suggest that labetalol may exert its effect on the AA cascade at the platelet phospholipase A2 level. Aggregation results obtained with the aorta incubates suggest that this drug causes an increased formation of prostacyclin. The experiments were carried out using aorta preparations from three rats. Aorta pieces (on at a time) were incubated for 5 min at 370C four times with intermittent breaks in incubation by placing the tubes in ice-cold water (-300. The sequence of incubation was also changed, i.e., first incubation of control followed by experimental, and then experimental followed by control and so on. This was necessary to see if synthesis and thus accumulation of PC12 during the periods the tubes were placed in ice-cold water took place. This was not found to be so.
7
In conclusion one may suggest that one of the mechanisms by which labetalol may show its beneficial effect in the cardiovascular system is by modifying AA metabolism, that is, by inhibiting platelet phospholipase A2 and by inducing an increased formation of prostacyclin both in the lung and blood vessels. ACKNOWLEDGEMENTS The authors wish to thank the Danish International Development Agency (DANIDA) for award of fellowship to KKA. Blood Bank, Odense University Hospital arranged for the blood samples. Mrs. Ruth 8. Alexandersen provided technical assistance and Mr. Erik Gstergaard did the animal experiments. Mrs. Yrsa Kildeberg and Mrs. Inge B0gelund typed the manuscript. REFERENCES 1. Gilmore, E., Weil, J., Chidsey, C. Treatment of essential hypertension with a new vasodilator in combination with beta adrenergic blockade. New England Journal of Medicine 282: 521, 1970. 2. Halprin, S., Fisherman, W., Kirchner, M., Strom, J. Labetalol therapy in angina pectoris and systemic hypertension: effects of combined alpha-beta adrenergic blockade. American Journal of Cardiology 47: 430, 1980. 3. Michael, C.A. Use of labetalol in the treatment of severe hypertension pregnancy. British Journal of Clinical Pharmacology 8: 211, 1979.
during
4. Leather, H.M., Humphreys, D.M., Baker, P.B., Chadd, M.A. A controlled trial of hypotensive agents in hypertension in pregnancy. Lancet 1: 488, 1968. 5. Michael, C. The use of bethanidine in severe hypertension in pregnancy. Australian and New Zealand Journal of Obstetrics and Cynacology 15, 75a. 6. Redman, C.W.G., Bielin, L.J., Bonnar, J., Ounsted, M.K. Foetal outcome in trial of antihypertensive treatment in pregnancy. Lancet 2: 753, 1976. 7. Richards, D.A., Prichard, B.N.C., Boakes, A.J., Tuckman, J., Knight, E.J. Pharmacological basis for antihypertensive effects of intravenous labetalol. British Heart Journal 39: 99, 1977. 8. Srivastava, K.C., Awasthi, K.K. Separation and quantitative determination of radiolabeled prostaglandins, thromboxanes, 6-keto-prostaglandin Fl and other arachidonic acid metabolites produced in the biol ical material. Journal of Chromatography, Biomedical Applications (in press. 7 9. Srivastava, K.C. Prostaglandin and prostacyclin synthesizing systems: their distribution in different organs of the rat. South African Journal of Science 76: 182, 1980. 10. Schmidt, K.G., Rasmussen, J.W. Preparation of platelet suspensions from whole blood in buffer. Description of a method which gives a large platelet yield. Scandinavian Journal of Haematology 23: 88, 1979.
8
11. Srivastava, K.C., Awasthi, K.K. Arachidonic acid metabolism in isolatad aorta and lung of the rat: Effects of dipyridamole, nifedipinc, prsprarrcrtol, hydralazine and verapamil. Prostaglandins Leukotrienes and Medicine (in press). 12. Srivastava, K.C., Awasthi, K.K., Lindegaard, P., Tiwari, K.P. Effect of some saturated and unsaturated fatty acids on prostaglandin biosynthesis in washed human blood platelets from (I- 14C) arachidonic acid. Prostaglandin Leukotrienes and Medicine 8: 219, 1982. 13. Weksler, B.B., Gillick, M., Pink, J. Effect of propranolol on platelet Blood 49: 185, 1977.
function.
14. Lemmer, B., Wiethold, G., Hellenbrecht, D., Bak, I.J., Grobecker, H. Human blood platelets as cellular models for investigation of membrane active drugs: Beta-adrenergic blocking agents. Naunyn-SchmiedebergS Archives of Pharmacology 275 229, 1972. 15. Rubegni, M., Provedi, D., Bellini, P.G. Beta-blocking agents and platelet aggregation. The Journal of the American Medical Association 228: 465, 1974. 16. Nosal, R., Menyhardtov& 2. The effect of trimepranol on thrombocyte tions and histamine release in the rat. Agents Actions 5: 9, 1975.
func-
17. Smith, J.B., Ingerman, C.M., Silver, M.J. Malondialdehyde formation as an indicator of prostaglandin production by human platelets. Journal of Laboratory and Clinical Medicine 88~167, 1976. 18. Diczfalusy, U., Falardeau, P., Hammarstriim, S. Conversion of prostaglandin endoperoxides to Cl7-hydroxy acids catalyzed by human platelet thromboxane synthetase. FEBS Letters 84: 271, 1977. 19. Vanderhoek, J.Y., Feinstein, M.B. Local anesthetics, chlorpromazine and propranolol inhibit stimulus-activation of phospholipase A2 in human platelets. Molecular Pharmacology 16: 171, 1979. 20. Turcani, P., Nosal, R. The effect of betaadrenoreceptor blocking drugs on malondialdehyde production in the rat platelets. Thrombosis Research 513, 1981.
9
21:
EFFECTS OF LABETALOL ON THE ARACHIDONIC ACID METABOLISM IN HUMAN BLOOD PLATELETS, AND IN LUNG AND AORTA OF THE RAT K.C. Srivastava and K.K. Awasthi, Department of Environmental Medicine, Institute of Community Health, Odense University, J.B. WinslBws vej 19, DK-5000 Odense, Denmark (reprint requests to KCS). ABSTRACT Effects of labetalol on the arachidonic acid metabolism in washed human blood platelets, and in lung and aorta of the rat was studied. Effects of this drug on the aggregation induced by usual aggregation agents, and on the prostacyclin generating capacity of rat aorta as assessed by its antiaggregation effect were also studied. The following results were obtained. 1. Reduced generation of thromboxane and HHT in platelets was observed at 1 mM cont. of the drug. 2. This drug inhibited ADP-, collagen-, and epinephrine-induced aggregation in a dose dependent manner. 3. Arachidonate-induced aggregation was only slightly inhibited at I mM cont. of labetalol. 4. Aorta and lung synthesized more prostacyclin in its presence from exogenous labelled arachidonate. 5. Aorta synthesized more prostacyclin from its endogenous AA pool in the presence of labetalol as assessed by inhibition of platelet aggregation. INTRODUCTION Non-selective beta-adrenoreceptor antagonists show some unwanted effects. For modifying or removing such effects, newer agents with cardioselective activity or an added alpha-adrenoreceptor blocking activity have been developed. Labetalol possesses a combined alpha- and beta-adrenoreceptor activity. It shows potential benefits in hypertensive patients suffering from angina, particularly in situations where there is also present peripheral vascular disease or coronary artery spasm (1,2). Because of its two modes of action, this drug could be useful in a wide spectrum of hypertensive patients. It has been used in the management of severe hypertension in pregnancy (3). Control of raised blood pressure improves foetal survival and reduces material complications (4-6). It has been difficult to obtain adequate information on the precise mode of action of labetalol in patients suffering from hypertension. This has probably been due to (i) apparent change in ratio of the strength of alpha- and beta-adrenoreceptor blocking action, and (ii) varying doses and different routes of drug admninis-
1
tration (7). With the discovery of two new classes of compounds, the thromboxanes and prostacyclins, showing opposite effects in the cardiovascular system, it is not surprising that mechanism of action of cardiovascular drugs is being evaluated in the light of the effects of such drugs on the biosynthesis of these two biologically potent compounds of physiological origin. In this report effects of labetalol has been examined on the metabolism of labelled arachidonic acid (AA) in human platelets, and also in the lung and aorta of the rat. Besides, effects of this drug on the aggregation induced by usual aggregation agents, and on the prostacyclin generating capacity of rat aorta as assessed by its anti-aggregation effect have also been studied. MATERIALS AND METHODS Labetalol hydrochloride (Trandate) was obtained from Glaxo, Glaxo Group Research Limited, Ware, Hertfordshire. Labelled (l-14C) arachidonic acid, specific activity 58.4 mCi/mmol, was purchased from the Radiochemical Centre (Amersham, England). Labelled TxB2 (8) and prostaglandins (9) were prepared as described earlier. Unlabelled prostaglandins E2, -D2, -F2o, 6-keto-Flu and TxB2 were obtained as generous gift from ON0 Pharmaceutical Company Limited, Higashiku, Osala, Japan. Hydroxy fatty acid (HHT) was kindly provided as generous gift by Dr. D.H. Nugteren, Unilever Research, Vlaardingen, The Netherlands. Preparation
of platelet suspension blood from healthy donors who had not taken aspirin for at least 10 days was collected into acid-citrate-dextrose (ACD) and platelet suspensions were prepared by the method of Schmidt et al. (10). Venous
Preapration of drug solution Solutions of this drug in appropriate concentrations for various experiments were prepared in alcohol-water (I:1 v/v) and stored at -2O*C until use. Incubation Platelet suspensions (300 ul, 1 x 108 platelets) were first incubated with the drug for 10 min at room temperature in final concentrations as indicated in Table 1. This was followed by a further incubation with labelled arachidonate (25 uM) for 10 min at 37OC. Platelet suspensions serving as controls were incubated with an appropriate volume of alcohol-water (l:l, v/v) mixture for 10 min at room temperature to be followed by incubation with labelled arachidonate as above. Extraction and TLC separation of labelled AA-metabolites Extraction of the labelled AA-met&o&es from the incubation medium and their separation by a two dimensional TLC were achieved as described earlie (8). Preparation of lung homogenate and aorta The tissues were prepared as described (ca. 0.5 cm, wet wt. ca. 6.5 mg> were prepared.
by us earlier
(11). Pieces of aorta
Incubation Incubations of lung homogenates and aorta pieces with the drug followed by labelled arachidonate (lung 8 u&i, aorta 15 uM) were done as described earlier (11). Appropriate controls were run simultaneously.
2
Extraction of labelled AA-metabolites and their separation by two-dimensional TLC were done exactly as described by us (8).
II.
ect of this drug on platelet aggregation induced by ADP, collagen, epinephrine and arachidonate was examined. All aggregation studies were carried out using human platelet-rich plasma (PRP). Venous blood from healthy donors, who had not taken any medication for about a week before giving blood, was collected in silicon&d tubes ‘containing l/l0 volume of 3.1 %, trisodium citrate and l/20 volume saline. Platelet-rich plasma and platelet poor plasma (PPP) were prepared in the usual way. Platelet aggregation was studied in a Bryston aggregometer using PRP (ca. 2.5-3.0 x 108 platelets). Platelet-rich plasma was preincubated with a desired concentration of the drug at room temperature for 10 min and then for 30 set in the aggregometer at 370C under stirring. This was followed by addition of the aggregating agent. Control PRP samples were treated with the corresponding volume of the solvent (alcohol-water, (1:l) v/v) prior to inducing aggregation. The total volume of PRP and drug/solvent was maintained constant to 1.0 ml. on the In another set of aggregation experiment, the effect of labetalol ability of rat aorta in inhibiting collagen-induced aggregation of human blood platelets (PRP) was examined. Prior to aggregation step, incubation of aorta pieces was done in the following way. a) The incubation medium consisted of 200 ul Tris-HCI (50 mM, pH 8.41, a piece of rat aorta (0.5 cm, ca. 6.5 mg) and 10 ul of the solvent (alcoholwater l:l, v/v) (control) or 10 ul of the drug solution (experimental) in a final cont. of 0.85 mM. The control and the experimental tubes were kept in ice-cold water for 30 min. Each tube (one at a time) was then incubated at 35OC for 5 min. Ten microliters of the aorta incubate were transferred to the aggregation tube, the contents allowed to mix for 30 set under stirring followed by addition of collagen (2 ug/ml) to induce aggregation. The incubation tubes were placed in ice-cold water (-3oC) immediately after pipetting of the aorta incubate. Sequence of incubation: control, experimental. b) The incubation tubes from above, one at a time, were incubated again at 35OC for 5 min. Aorta incubate (10 ul) was added to the aggregation tube, mixed for 30 set followed by addition of collagen. The incubation tubes were placed in ice-cold water (-3oC) immediately as above. Sequence of incubation: experimental, control. c) The incubation tubes from above, one at a time, were further incubated at 35OC for 5 min. Aorta incubate (10 ul) was added to the aggregation tube, the contents mixed for 30 set followed by addition of collagen. The incubation tubes were placed in ice-cold water as above. Sequence of incubation: control, experimental. d) The incubation tubes from above, one at a time, were finally incubated at 35OC for 5 min. Aorta incubate (10 ml) was added to the aggregation tube, the contents mixed for 30 set followed by addition of collagen to induce aggregation. Sequence of incubation: control, experimental.
The effect of labetalol on the aorta prostacyclin synthesis was examined using aorta pieces from 5 rats. Aorta pieces were stored at -200C immediately and used later in incubation experiments. In the aorta from three rats, labetalol was
3
found to induce an increased synthesis of prostacyclin; in one rat there was observed no effect, while in another this drug showed an opposite effect, that is, apparently a reduced formation of prostacyclin compared to control took place. Malondialdehyde (MDA) formation in platelet suspension in the presence of labetalol Malondialdehyde formation by platelets in the presence of labetalol was examined with washed platelet preparations in a way similar to that described earlier (12). RESULTS Table 1 gives data on the effect of labetalol on the metabolism of labelled AA by washed human platelets. The amount of TxB2 was found to be reduced at 1.0 mM cont. of this drug. HHT was also found to be reduced. There was observed no effect of this drug on other AA-metabolites. Table 2 gives data on the effect of this drug on the metabolism of labelled AA in the aorta and lung (homogenate) of the rat. In lung this drug seems to increase the synthesis of prostaglandins, TxB2 and prostacyclin; this effect being more pronounced on PC12 synthesis. In aorta the drug increases the formation of PG12. Figure 1 shows the effect of labetalol on collagen-induced aggregation: (A) and (8); ADP-induced aggregation: (Cl; arachidonate-induced aggregation: (D). As is obvious labetalol shows inhibitory effect on collagen induced aggregation in a dose-dependent manner. As low as at 31 uM labetalol shows inhibition of aggregation induced by 1 ug/ml collagen. Inhibition of ADP-induced aggregation was observed only at higher labetalol concentrations (0.5, 1.0 mM). A slight inhibition of arachidonate-induced aggregation was observed at 1.0 mM cone of iabetalol. Figure 2 shows the inhibitory effect of this drug on epinephrine-induced biphasic aggregation. Labetalol showed inhibitory effect in a dose-dependent manner in a dose range from 25 uM to 1.0 mM. Figure 3 shows the effect of labetalol on the ability of rat aorta in inhibiting collagen-induced aggregation of human blood platelets. As is obvious incubate from the labetalol treated aorta was more effective compared to control in inhibiting collagen induced platelet aggregation. This was found to be consistent irrespective of the sequence of incubation, i.e., first control followed by experimental or the other way. DISCUSSION Beta-adrenoreceptor blocking drugs alter platelet function by mechanisms other than beta-blockade (13). These drugs have been shown to release serotonin from washed platelets or PRP in a dose-dependent manner and inhibit platelet aggregation induced by several aggregation agents (14-16). When stimulated by thrombin and during ADP-involved aggregation, platelets synthesize prostaglandins and MDA, the latter is thought to be an indicator of prostaglandin (17) and thromboxane synthesis (18). Malondialdehyde formation is decreased in platelets by
4
Tabk 1. Effect of labetalol on the biosynthesis of AA metabolites blood platelets
in washed human
Amounta (picomoks/lo% platekts) formed after platelet suspension with labelled AA for 10 min
incubation of
PGF2 ci
T*2
PGE2
PGD2
HHT
Control
19 +6
715 + 237
31+ 12
292 11
561 f 228
+Labetalol (0.5 mM, n=8)
18 25
709 f 262
282 5
24-+ 3
563 f 220
Control
2024
682 * 259
29+ 8
262 8
4242 128
+Labetalol (1.0 mM, n=6)
18 24
606 f 234 it
26+ 9
21+ 4
398* 144
* p < 0.01 (t-test
for paired data)
a Mean *SD
Table 2. Effect of labetalol on the biosynthesis homogenate and aorta of the rat
of AA metabolites
Amounta (picomoles/IiOO ul) formed labelled AAX for 15 min
after
incubation
PCF2 c1
TxB2
PCE2
PGI2XX
Control
26?7
77 2 19
25* 4
1142 46
+LabetaIol (2mM, n=4)
3329
83 + 35
312 4
166* 48
a Mea&SD
x Final cone 8 uM
xx Measured as 6-keto-PGFl
a
6-keto-PGFl values for aorta picomoles, 1$e talol43 picomoles
5
from
in lung
two rats;
control
with
22
Figure 1. Tracings showing the effect of labetabl (Lb) on the human blood platelet aggr ation induced by some aggregating agents. A. collagen (1 ug/ml ‘g, 1. collagen; 2. Lb 31 uM; 3. Lb 62 uM; 4. Lb 125 uM; 5. Lb 250 uM. B. collagen (2 ug/ml) 1. collagen; 2. Lb 62 uM; 3. Lb 125 mM; 4 Lb 250 uM; 5. Lb 500 uM; 6. Lb 1 mM. C. ADP (3 uM) 1. ADP; 2. aggregation by ADP (3 uM) of PRP pretreated with 10 ul of alcohol-water (l:l, v/v); 3. Lb 500 uM; 4. Lb 1 mM. D. 1. arachidonate; 2. Lb 500 uM; 3. Lb 1 mM.
Epinphrmr(SpH)
EpicuphrmallOpHl
1
I
+
0
\
“l”
A \
1
Figure 2. Tracings showing the effect of labetalol (Lb on the human blood platelet aggregation induced by epinephrine. A. epinephrine (5 uM) 1. epinephrine; 2. Lb 100 uM; 3. Lb 250 uM; (10 uM) 1. 4. Lb 500 uM; 5. Lb 1 mM. B. epinephrine epinephrine; la.. epinephrine (5 uM); 2. Lb 25 uM; 3. Lb 50 uM; 4. Lb 250 uM; 5. Lb 500 uM (with the exception of la, other tracings were obtained with 10 uM epinephrine).
6
x Addition of on
Figure 3. Tracings showing the effect of labetalol (Lb) on the ability of aorta in inhibiting collagen-induced aggregation of human blood platelets. A. 1. collagen; 2. in the presence of incubate from the aorta piece not pre-treated with Lb (control); 3. in the presence of incubate from the aorta piece pre-treated with Lb (experimental). B. 1. collagen; 2. experimental; 3. control. C. 1. collagen; 2. control; 3. experimental; 4. control; 5. experimental. membrane stabilizing drugs including propranolol meaning that they inhibit phosphalipase A2 activity with the result a reduced formation of prostaglandin endoperoxides and TxA2 takes place (19). Several other beta-adrenoreceptor blocking drugs have been shown to inhibit platelet production of MDA on activation with thrombin (20). Labetalol did not reduce the generation of MDA in washed platelets from exogenous arachidonate. Results of our experiments on the metabolism of labelled AA in the presence of labetalol show that this drug inhibits platelet Tx-synthetase activity at 1 mM cone while showing no such effect at 0.5 mM cone in washed platelet preparations. But this drug inhibits ADP-, collagen- and epinephrine-induced aggregation of PRP at lower concentrations; and it shows only a slight inhibition of aggregation induced by arachidonate at 1 mM cont. These observations may suggest that labetalol may exert its effect on the AA cascade at the platelet phospholipase A2 level. Aggregation results obtained with the aorta incubates suggest that this drug causes an increased formation of prostacyclin. The experiments were carried out using aorta preparations from three rats. Aorta pieces (on at a time) were incubated for 5 min at 370C four times with intermittent breaks in incubation by placing the tubes in ice-cold water (-300. The sequence of incubation was also changed, i.e., first incubation of control followed by experimental, and then experimental followed by control and so on. This was necessary to see if synthesis and thus accumulation of PC12 during the periods the tubes were placed in ice-cold water took place. This was not found to be so.
7
In conclusion one may suggest that one of the mechanisms by which labetalol may show its beneficial effect in the cardiovascular system is by modifying AA metabolism, that is, by inhibiting platelet phospholipase A2 and by inducing an increased formation of prostacyclin both in the lung and blood vessels. ACKNOWLEDGEMENTS The authors wish to thank the Danish International Development Agency (DANIDA) for award of fellowship to KKA. Blood Bank, Odense University Hospital arranged for the blood samples. Mrs. Ruth 8. Alexandersen provided technical assistance and Mr. Erik Gstergaard did the animal experiments. Mrs. Yrsa Kildeberg and Mrs. Inge B0gelund typed the manuscript. REFERENCES 1. Gilmore, E., Weil, J., Chidsey, C. Treatment of essential hypertension with a new vasodilator in combination with beta adrenergic blockade. New England Journal of Medicine 282: 521, 1970. 2. Halprin, S., Fisherman, W., Kirchner, M., Strom, J. Labetalol therapy in angina pectoris and systemic hypertension: effects of combined alpha-beta adrenergic blockade. American Journal of Cardiology 47: 430, 1980. 3. Michael, C.A. Use of labetalol in the treatment of severe hypertension pregnancy. British Journal of Clinical Pharmacology 8: 211, 1979.
during
4. Leather, H.M., Humphreys, D.M., Baker, P.B., Chadd, M.A. A controlled trial of hypotensive agents in hypertension in pregnancy. Lancet 1: 488, 1968. 5. Michael, C. The use of bethanidine in severe hypertension in pregnancy. Australian and New Zealand Journal of Obstetrics and Cynacology 15, 75a. 6. Redman, C.W.G., Bielin, L.J., Bonnar, J., Ounsted, M.K. Foetal outcome in trial of antihypertensive treatment in pregnancy. Lancet 2: 753, 1976. 7. Richards, D.A., Prichard, B.N.C., Boakes, A.J., Tuckman, J., Knight, E.J. Pharmacological basis for antihypertensive effects of intravenous labetalol. British Heart Journal 39: 99, 1977. 8. Srivastava, K.C., Awasthi, K.K. Separation and quantitative determination of radiolabeled prostaglandins, thromboxanes, 6-keto-prostaglandin Fl and other arachidonic acid metabolites produced in the biol ical material. Journal of Chromatography, Biomedical Applications (in press. 7 9. Srivastava, K.C. Prostaglandin and prostacyclin synthesizing systems: their distribution in different organs of the rat. South African Journal of Science 76: 182, 1980. 10. Schmidt, K.G., Rasmussen, J.W. Preparation of platelet suspensions from whole blood in buffer. Description of a method which gives a large platelet yield. Scandinavian Journal of Haematology 23: 88, 1979.
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11. Srivastava, K.C., Awasthi, K.K. Arachidonic acid metabolism in isolatad aorta and lung of the rat: Effects of dipyridamole, nifedipinc, prsprarrcrtol, hydralazine and verapamil. Prostaglandins Leukotrienes and Medicine (in press). 12. Srivastava, K.C., Awasthi, K.K., Lindegaard, P., Tiwari, K.P. Effect of some saturated and unsaturated fatty acids on prostaglandin biosynthesis in washed human blood platelets from (I- 14C) arachidonic acid. Prostaglandin Leukotrienes and Medicine 8: 219, 1982. 13. Weksler, B.B., Gillick, M., Pink, J. Effect of propranolol on platelet Blood 49: 185, 1977.
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14. Lemmer, B., Wiethold, G., Hellenbrecht, D., Bak, I.J., Grobecker, H. Human blood platelets as cellular models for investigation of membrane active drugs: Beta-adrenergic blocking agents. Naunyn-SchmiedebergS Archives of Pharmacology 275 229, 1972. 15. Rubegni, M., Provedi, D., Bellini, P.G. Beta-blocking agents and platelet aggregation. The Journal of the American Medical Association 228: 465, 1974. 16. Nosal, R., Menyhardtov& 2. The effect of trimepranol on thrombocyte tions and histamine release in the rat. Agents Actions 5: 9, 1975.
func-
17. Smith, J.B., Ingerman, C.M., Silver, M.J. Malondialdehyde formation as an indicator of prostaglandin production by human platelets. Journal of Laboratory and Clinical Medicine 88~167, 1976. 18. Diczfalusy, U., Falardeau, P., Hammarstriim, S. Conversion of prostaglandin endoperoxides to Cl7-hydroxy acids catalyzed by human platelet thromboxane synthetase. FEBS Letters 84: 271, 1977. 19. Vanderhoek, J.Y., Feinstein, M.B. Local anesthetics, chlorpromazine and propranolol inhibit stimulus-activation of phospholipase A2 in human platelets. Molecular Pharmacology 16: 171, 1979. 20. Turcani, P., Nosal, R. The effect of betaadrenoreceptor blocking drugs on malondialdehyde production in the rat platelets. Thrombosis Research 513, 1981.
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