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Arachidonic acid metabolism in isolated aorta and lung of the rat: Effects of dipyridamole, nifedipine, propranolol, hydralazine and verapamil
Prostaglandins
Leulcotrienes
and Medicine
10r 411-421,
1983
ARACHIDONIC ACID METABOLISM IN ISOLATED AORTA AND LUNG OF THE RAT: EFFECTS OF DIPYRIDAMOLE, NIFEDIPINE, PROPRANOLOL, HYDRALAZINE AND VERAPAMIL K. C. Srivastava and K. K. Awasthi, Department of Environmental Medicine, Institute of Community Health, Odense University, J. B. WinsMws Vej 19, DK-5000 Odense, Denmark. ABSTRACT Effects of some vasodilating~ole, nifedipine and verapamil) and antihypertensive (propranolol, hydralazine) drugs on arachidonic acid metabolism in isolated rat aorta and lung have been studied. Dipyridamole significantly increased the formation of PC12 in aorta and lung. Nifedipine and verapamil decreased the formation of PC12 in aorta, these drugs though significantly increased the formation of PC12 in lung. Nifedipine showed no appreciable effect on the generation of TxA2 in rat aorta but in lung both nifedipine and verapamii reduced TxA2 formation though significantly only in the latter case. Dipyridamole showed no effect. The beneficial effect of dipyridamole, seems, at least in part, to be due to its ability to enhance the production of PGI2 both in the aorta and lung, and probably in other tissues as well. Nifedipine and verapamil may show their antianginal effect by a combined effect of enhanced PG12 and reduced TxA2 formation in lung. In lung, whereas hydralazine reduced the formation of both PC12 and TxA2, propranolol increased the formation of PG12. Hydralazine reduced the formation of TxA2 and increased PC12 formation in aorta. The effect of the drugs on the ability of rat aorta to inhibit collagen induced platelet aggregation of human blood platelets was also examined. INTRODUCTION Several vasodilating and antihypertensive drugs have been used in the treatment of cardiovascular diseases without a clear understanding of the mechanism(s) involved in their action. The vasodiiating drugs produce a diIatation of the coronary arteries and an increase of the coronary blood blow thus increasing oxygen saturation of the coronary sinus blood (1). These drugs also possess antithrombotic properties, thus decreasing aggregation, adhesion and release reaction of platelets, as is observed with dipyridamoie and several other drugs (2,3). Dipyridamole at clinically achieved low plasma levels blocks adenosine transport into cells, a mechanism some consider to be the basis for its cardiovascular actions (4). Whether such a mechanism is also operative for other antianginal drugs remains to be elucidated.
411
Of the several mechanisms considered to be involved in the action of these drugs, one may be their effect on the prostaglandin (PC) biosynthesis in the cardiovascular system and other organs. Focus on the relative production of thromboxane A2 (TxA2) and prostacyclin (PG12) in the presence of such drugs would be of relevance as TxA2 is platelet aggregatory (5), and contracts aorta and other vessel preparations (5,6), whereas PG12 is antiaggregatory, antihypertensive (7), and relaxes the strips of coronary artery (8). Thus the discovery of TxA2 and PG12, and recognition of the roles played by these substances in platelet aggregation and coronary regulation is of great significance. In the present study, therefore, an attempt has been made to examine the effects of some antianginal (vasodilating) (dipyridamole, nifedipine and verapamil) and antihypertensive (propranolol and hydralazine) drugs on the biosynthesis of prostaglandins (including PGI2) and TxA2 in the aorta and lung of the rat in order to explain their mechanism of action. MATERIALS AND METHODS Arachidonic acid (I-IQC) (specific activity 58.4 mCi/mmol) was purchased from the Radiochemical Centre, Amersham, England. Verapamil hydrochloride (Isoptin) was obtained as a gift from Meda A/S, dipyridamole (Persantine) from Boehringer-Ingelheim A/S, hydralazine (Apresoline) from Ciba-Geigy A/S, propranolo1 hydrochloride (Inderal) from ICI-Pharma A/S and nifedipine (Adalat) from Baeyer Kemi A/S, all in Denmark. Standard prostaglandins Fa, E2, D2, thromboxane B2 and 6-keto-prostaglandin F c1were kindly provided by ON0 Pharmaceutical Co., Ltd., Higashiku, Osaka (Japan f. The animals used were male albino SPS Wistar rats weighing 504 + 22 g. Preparation of solutions of drugs The solutions of dipyridamole and nifedipine were prepared in 99% alcohol, whereas those of propranolol hydrochloride, hydralazine and verapamil were prepared in acetone:water (l:l, v/v) mixture. Preparation of lung homogenate and aorta Lung was freed from the surrounding tissue. cut into pieces washed with ice-cold saline several times and finally with phosphate but& (0.06 M pH 7.10 containing 2 m&l EDTA. This was then homogenized with phosphate buffer (I g lung in 4 ml buffer). The rat aorta was perfused witn saline, and extra tissue was removed from it. A total length of 6 cm aorta was removed, blotted on filter paper and then cut into eight equal parts ka. 13.6 mg each). Finally the pieces were pfaceo In separate tubes each contammg 250 ul phosphate buffer. lncuoaflon one Incubation medium for lung homogenate consisted of 500 ul of the homogenate. This was first incubated with 10 ul of a drug solution for 15 min at room temperature in a final concentration indicated in tables 1 and 2. This was followed by a further incubation with labelled AA for 15 min at 370C. The controls were incubated with 10 ul of the solvent (in which the solution of the corresponding drug was prepared) for 15 min at room temperature followed by a further incubation with labelled AA for 15 min at 37OC. The incubation medium for aorta consisted of aorta and 250 ul phosphate buffer which was incubated first with 5 ul of a drug solution for 15 min at room temperature foltowed by incubation with labelled AA for 15 min at 37OC. Corresponding blanks were incubated simultaneously with appropriate solvent for 15 min at room temperature followed by labelled AA for 15 min at 370C.
412
te with labelled AA, 1 ml saline was added to each tube followed by addition of 50 ul N HCI to adjust the pH of the medium to 3.0. The AA metabolites were extracted with ethyl time with 2 ml of this solvent. In the case of aorta 1 ml saline by addition of 60 ul N HCI to adjust the pH to 3.0. This mixture was extractad with 2 ml ethyl acetate twice. The combined extract was evaporated to dryness in a stream of nitrogen. Separation of AA metabolites Separation of AA metabolites was done by a modified procedure (9) of our earlier TLC separation schedule (IO). The incubation extract of the lung homogenate was dissolved in 250 ul ethyl acetate:methanol mixture (1:4,v/v). 25 ul of this solution were applied on the left hand bottom corner of a silica gel G TLC plate (0.25 mm, 20 x 20 cm). Prior to the application of the incubation extract containing the AA metabolites, reference compounds, 6-keto-PGFla, PGFz(~ PGE2, PGD2 and TxB2 were placed on the plate. The plates were first developed in solvent II: chloroform:methanol:acetic acid (90:8:6, v/v) followed by development in solvent III: benzene:dioxane:acetic acid (40:20:2, v/v) in a direction perpendicular to the first development. The spots were shown in iodine, marked, scraped off and the radioactivity contained therein measured. The incubation extract of aorta was dissolved in 200 ul methanohethyl acetate (2~1, v/v) and chromatograhic separation of AA metabolites and the measurement of radioactivity contained in them were done as described for the lung homogenate. Separation of AA metabolites produced in aorta by a double two-dimensional TLC: Formation of TxA7 in rat aorta This exoerRnent was done with aorta from two rats. Incubation extracts from 8 aorta- pieces (each 0.7 cm, weight 13.6 mg) were pooled together, evaporated to dryness and reconstituted in 300 ul methanol:ethyl acetate (2:1, v/v) mixture. 25 ul of this were applied on each plate (in all four TLC plates were used). The plates were run successively in solvents II: chloroformtmethanohacetic acid (90:8:6, v/v) and III: benzene:dioxane:acetic acid (40~20~2, v/v). After development the plates were dried in open air, ex,posed to iodine, prostaglandin spots marked and scraped off. The AA metabolites (PGF2, PGE2, PGD2, TxB2, 6-keto PGFld scrapings of one plate were counted individually for their radioactivity whereas those of the remaining three plates were scraped off and pooled together individually. Silica gel scrapings were extracted three times, twice with methanohethyl acetate (3:2, v/v) and once with methanohethyl acetate (2:1, v/v) using I ml of the solvent in each extraction. The combined extract was evaporated to dryness under nitrogen and redissolved in 300 ul methanol:ethyl acetate (2:1, v/v) mixture. A second two-dimensional TLC was performed for individual prostaglandins using authentic samples successively using solvents IV: ether:methanol:acetic acid (90:1:2, v/v) and V: ethyl acetate:acetic acid:isooctane: water (110:2O:50:100, v/v, using the upper organic phase). After development, plates were dried in open air, exposed to iodine, PC spots marked, scraped off and the radioactivity counted. The amounts of various PCs and TxB2 were calculated after taking into account the losses during extraction and TLC, and compared with those obtained after the first two-dimensional TLC. Aggregation experiment Rat aorta was prepared as described above. It was cut into small pieces, ca. 6 and 3 mm, weighing about 8.76 and 4.38 mg respectively. Prior to aggregation experiment, incubation of aorta pieces was done in the following way. The 413
incubation medium consisted of 100 ul Tris-HCI (50 mM, pH 8.4), a piece of aorta and an appropriate volume (5 or 10 ul) of the solvent (99% ethanol or acetonewater I:& v/v (control), whereas the experimental tubes contained a corresponding volume of the drug solution to give the desired concentration. The volume of platelet rich plasma (3.2 x log platelets) was maintained to 1 ml in each case. The control and experimental tubes were kept at OoC for 30 min. Each tube (one at a time) was then incubated at 300C for 4 min, and immediately 5 or 10 ul aliquot from the aorta incubate were added to a PRP sample maintained at 370C under constant stirring in the aggregometer. Exactly after 30 set of mixing of the contents, collagen was added to induce aggregation. Siliconized glass tubes were used both in aorta incubation and platelet aggregation. Statistics Statistical paired data.
significance
was evaluated
by the two-tailed
Student t-test
for
RESULTS Table I gives data on the effects of antianginal (dipyridamole, nifedipine and verapamil) and antihypertensive (propranolol and hydralazine) drugs on the biosynthesis of AA metabolites in rat aorta. As can be seen, dipyridamole significantly increased the formation of PCI2, PCFa and PCE2. Thromboxane B2 and PGD2 formation was reduced but not significantly. In the presence of nifedipine PC12 was reduced though not significantly, other AA metabolites were also reduced to a small extent with the exception of PGFa, which increased slightly. Verapamil reduced the formation of PGI2. Propranolol reduced the formation of all AA metabolites. In the presence of hydralazine, formation of PC12 was enhanced, thromboxane B2 and PGD2 were reduced, the latter significantly. However, there was seen no effect of this drug on the formation of PGF2o and PGE2. Table 2 presents data on the effects of antianginal and antihypertensive drugs on the generation of AA metabolites in rat lung homogenate. Dipyridamole significantly enhanced the formation of PC12 and PGE2. There was no change in other AA metabolites. Nifedipine significantly increased the generation of PGI2, whereas other AA metabolites were reduced but not significantly. Verapamil increased the formation of PG12 and decreased the formation of TxB2 both changes being significant; PGF2o and PGD2 remained unchanged and PGE2 increased but not significantly. Propranolol increased the formation of PG12 and TxB2. In the presence of hydralazine, all the AA metabolites were reduced. Table 3 gives data on the ratio of PC12 and TxB2 formed in the presence of the antianginal and antihypertensive drugs in the aorta and lung homogenates of the rat. In the case of the aorta the ratio significantly increased in the presence of dipyridamole and hydralazine. This ratio was enhanced also in the presence of nifedipine and propranolol though not significantly. The ratio of PC12 and TxB2 was increased in the presence of all the drugs in rat lung homogenate. Table 4 gives data on the production of the classical PCs, TxB2 and 6-ketoPGFlo in the aorta of rat. As can be seen measurable quantities of these AA metabolites can be obtained after the second two-dimensional TLC; their amounts were comparable to those found after the first two-dimensional TLC. That blood vessels also produce TxA2 besides other PGs was further demonstrated by performing similar experiments with aorta from 6 rats. In each 414
Table
I.
Effect of antia”glnal and antihypemsaive AA metabolita in rat aorta.
drugs on the biosynthesis
Amounta (picomoleslaorta piece “/ formed with labelled AAX for 15 min
Drug fconc.)
after
incubation
PC12 ”
PGFz a
T*2
PGE2
PGD2
Control
102 f46
5 c3
7*4
II?
6+4
+ Dipyridamole (0.2 mhl) “~6
158 *75
9 r2”
5*3
19? 7**
5+3
+ Nifedipine (0.2 mhi) n:6
81 %8
6 t2
5*2
IO? 3
4+ I
Control
80 +I5
7 t1
Sk3
8*3
6*2
+ Propranolol (1.0 mM) “~3
73 *36
5 +I
6? I
4*1
5t I
??
6
Control
88 + 56
6 i3
7?6
6+2
6*3
+ Hydralazine (0.75 mhi) n=5
113+ 73
6 +2
3k1
6*1
2+ 1*
a Mean r SD ?? p < 0.05
??*
b Values for verapamil(1.0 X Final concentration
Table 2.
p < 0.01 mh4, n=6) 64 f 33 agaiwt
25 uM kt.
control
values 89 * 43
13.60 mg
Effect of antiangina and antihypertemive AA metabolites in rat lung homogenate.
Drug konc.)
of
drugs on the biosynthesis
Amounta (picomolesI500 ul) formed labelled AAX for 15 min
after
incubation
with
PC12
P=F2,
T*2
PCE2
PGD2
Control
145 +46
75* 30
103k 50
49t: 13
59*29
+ Dipyridamole (0.2 mM) n=7
274 *76”
75* 17
101+ 37
72+ 23’
55i27
Control
147 +50
84k 21
II5*
53*
66224
+ Nifedi Pine (0.2 mM “~6
215 *67**
59+ 21
76 +38
Control
184 236
35+ 18
+ Propra”olol (1.0 mM) “~3
229 +63
44?
14
+ Hydralark (0.75 mM) n=3
117 +6
23+
9
Control
174 260
+ Verapamil (1.0 mM) “~7
266t120”*
a Mean‘SD
42
9
32*21
42 231
143 + 10
36t
14
40*20
161+37
40+ 15
37 cl6
65 +I1
16*
8
20 *II
77+36
119*53
57 * 20
69 ?28
76k34
118 *43’*
68 + 33
71 t32
?? p
< 0.05
X Final concentration
415
??*
12.5 uM
p-C 0.01
of
Table 3.
Influence of antianginal and antihypertensive ratio in rat aorta and lung homogenate.
Aorta Drug (cont.)
drugs on PGI2:TxB2
Lung homogenate ratio
PGI2/TxB2
Drug (cont.)
6.09
ratio
PC12/TxB2
Control
16.48
+
Control
1.81
+
1.25
+ Dipyridamole (0.2 mM) n-6
33.60
t 14.60+
+ Dipyridamole (0.2 mM) n-6
2.95
+
0.971’
Control
16.46
t
6.12
Control
1.40
+
0.56
+ Nifedi ine (0.2 mM P n=6
17.46
+
8.70
+ Nifedipine (0.2 mM) ~6
3.18
t
1.24’
Control
10.83
*
I.78
Control
1.27
?
0.15
+ Propranolol (1.0 mM) n=3
11.88
+
6.45
??Propranolol (1.0 mhi) n=3
I.41
+
0.11
4.71
Control
14.46
+
+ Hydrakzine (0.75 mM) n=5
30.38
t 15.38’
Control
1.27
+
0.15
+ Hydralazine (0.75 mM) n.3
1.83
+
0.40
Control
2.01
+
1.14
+ Verapamil (1.0 mM) n.6
3.72
+
2.07”
L ?? p
Table 4.
< 0.05
?? *
P < OJJI
Formation of various arachidonic acid metabolitn arachldonic acid in the aorta of rat.
Amounta
of AA-metabolites
from (I-146)
(pIcomoles/aorta
piece”)
AA-metaboUte6 1st tw-dimenaionaI
6-keto-PCFI
2nd twodimemIona1
79.7
66.8
PCE2
8.1
7.8
PGF2a
8.2
6.4
PCD2
4.2
2.5
12.7
9.7
Ta2
cL
TLC
* Mean of 2 rats3
X might
TLC
(‘#at) 13.6 mg
A slmlhr experiment was performed with aorta from 6 rats. 1x62 was wqwated and mawad aCt8r ht and 2nd twodlmewlonbl TLC. Amounts ~amaWlO.40 m aorta) found afta the two TLC separations were 6.lSf 2.13 and 6.14* 5.36 &a” isa mpcctive1y.
416
Figure 1. Representative tracings showing the effects of various drugs on the ability of rat aorta to inhibit collagen induced aggregation of human blood pJateJets. a) A. only collagen; B. aggregation of PRP pretreated with aorta incubate without dipyridamoie (control); C. same as 8. in the presence of dipyridamole (0.2 mM). b) A. only colJagen; B. aggregation of PRP pretreated with aorta incubate without nifedipine (control); C. in the presence of nifedJpJne (0.2 m&i). c) A. only collagen; B. in the presence of hydraJazine (0.75 mM); C. in the presence of verapamil (1.0 mM); D. in the presence of propranolol (1.0 mM); E. without any drug (control). d) A. on1 colJagen; B. in the presence of verapamil(0.5 mM); C. without verapamil (control. r For a) and d) the aorta incubation medium cudsted of an aorta piece (ca 8.76 mg) + 100 uJ Tris-HCJ (50 mM, pH 8.4) + 10 ul solvent or drug soiution from which 5 uJ were used in regation. For b) and c) the aorta incubation medium consisted of an aorta pieceY ca. 4.38 mg>.+ 100 ul TrisHCI + 5 ul solvent or drug solution from which 10 ul were used in aggregation.
417
rat, 4 pieces (each piece: length 0.7 cm, wet wt. 10.4 mg) were used for incubation. A double two-dimensional TLC of the incubation extract gave TxB2 value 6.14 2 5.36 (mean + SD) in picomoles/aorta piece. Figure 1 shows the effects of the above drugs on the ability of rat aorta in inhibiting collagen induced aggregation of human blood platelets. As is obvious dipyridamole potentiates the inhibiting action of aorta (a). Nifedipine seems also to potentiate this action, though slightly (b). Other drugs (propranolol, verapamil and hydralazine) seem to reduce the platelet aggregation inhibiting capability of aorta (c,d). DISCUSSION The effects of three antianginal drugs (dipyridamole, nifedipine and verapamill and two antihypertensive drugs (propranolol and hydralazine) on the formation of various AA metabolites in the aorta and lung of rats were examined. Both the tissues generate prostacyclin (8,111 a naturally occurring potent vasodilating substance, which also exhibits a strong antiaggregation effect. Lungs generate also TxA2 besides other prostaglandins (12,131. Recently, evidence has been produced indicating that TxA2 is also synthesized in blood vessels (14). Thus when mechanisms of hemostasis or thrombosis are examined, one should also take into account the ability of blood vessels to produce TxA2 besides PGI2. However, there is no denying of the fact that in circulation a substantially large portion of TxA2 is produced by blood platelets and that a balance between these two unstable arachidonic acid metabolites has tremendous significance for the state of health or disease in the cardiovascular system. Antianginal drugs work by dilating the blood vessels and thus are essentially vasodilating in nature. With the discovery of PGI2 formation in blood vessels, it is pertinent to examine the effect of a vasodilating drug on the formation of PC12 in blood vessels and for that matter any other organ which produces it. If a certain drug induces an enhanced production of PGI2, one may presume that at least one of the ways by which the drug shows antianginal activity is through its effect on PGI2 formation. This was found to be so in the case of dipyridamole which induced an enhanced production of PC12 in the aorta and lung of rat - an observation confirming the earlier reports (15,161. As nifedipine and verapamil reduced, though not significantly, the formation of PC12 in aorta, their antianginal action cannot be explained by way of PC12 formation in aorta/blood vessels unless one takes into consideration their effect on the production of this substance in lungs which have been shown to generate prostacyclin in vivo and release it into the arterial circulation (11). Both nifedipine and Era-i1 were found to enhance PG12 generation significantly in the lung homogenates. As no marked effect was observed on the aorta TxA2 generation in the presence of dipyridamole and nifedipine, while only nifedipine and verapamil causing reduced formation of TxA2 in lungs, one may explain the vasodilating action of these drugs in the following way. Dipyridamole is effective mainly by its action on enhanced production of PGI2, while nifedipine and verapamil show their antianginal effect by a combined effect of enhanced PG12 production and reduced TxA2 formation in the lungs. Of the two antihypertensive drugs studied, in aorta hydralazine increased the formation of PC12 though not significantly. In lung, propranolol enhanced the formation of PC12 while hydralazine reduced its synthesis. Hydralazine also reduced the formation of TxA2 in lungs. These observations may suggest that the antihypertensive effect of propranolol is probably by its action on the increased formation of PC12 and that of hydralazine is by its ability to reduce the formation 418
of TxA2 in lungs. The effects of these drugs can be understood in a better way by looking at the PGI2/TxA2 ratios in aorta and more so in lungs. In aorta PGI2/TxA2 ratios increased in the presence of dipyridamole and hydralazine; in the presence of nifedipine and propranolol the ratios were slightly elevated. However, for lungs, with all the drugs PGI2/TxA2 ratios were increased. Thus the effects of these drugs (with the exception of dipyridamole) may be readily explained by considering their effect on the formation of TxA2 in aorta and especially in lungs. These results find support from our recent report in which it has been shown that several antianginal and antihypertensive drugs (including those examined in this report) reduced the formation of TxA2 in human blood platelets in vitro. Also HHT formation was reduced in the presence of these drugs and t&observation was supported by a reduced MDA formation in the platelets. These drugs inhibited also ADP-induced platelet aggregation (3). Initial reports (17, 18) on the ability of a blood vessel to synthesize TxA2 were confined only to umbilical artery, observations supported by another recent study (19). Later studies produced evidence showing that arterial vessels could also be synthesizing TxA2 (14,20-24). Our studies (based on a double two-dimensional TLC separation of the incubation extract containing a mixture of labelted AA metabolites) also provide evidence that rat aorta synthesize TxA2 from exogenous arachidonate though in amounts much less than those of PGI2. Platelet aggregation study shows that the five drugs can be classified in two categories; one which potentiate the ability of aorta in inhibiting platelet aggregation, and the other which do not do so, rather they reduce the ability of aorta to inhibit platelet aggregation. The potentiating effect of a drug (e.g., dipyridamole) could either be by inducing an increased synthesis of prostacyclin in aorta, or by potentiating the action of PGI2, or by a combination of both. For drugs showing an effect in the opposite direction, their action could be either by reducing the formation of PC12 in aorta, or by inhibiting the ability of PGI 2 to inhibit platelet aggregation, or by a combination of both. The drugs inhibit thromboxane synthetase of platelets (washed) in concentrations used in the aorta incubation (3). As their concentrations are reduced in the aggregation medium (PRP, containing plasma proteins) by a factor of 100, it is unlikely that they would have any effect on the platelet thromboxane synthetase. Thus the difference in aggregation observed in the presence of a drug compared to its control, should mainly be due to the effect of the drug on the aorta prostacyclin synthesis. In such a situation, however, one must take into consideration that prostacyclin synthesis from endogenous arachidonic acid is mainly dependent on one step prior to PGI2 synthetase, i.e ., on the liberation of arachidonic acid by the action of phospholipase on which the drug may also show its effect. Aorta prostacyclin synthesis from labelled arachidonic acid in the presence of the drugs partly supports the aggregation results. ACKNOWLEDGEMENTS We wish to thank the Danish International Development Agency (DANIDA) for the award of fellowship to KKA. Mrs. Ruth Alexandersen and Mrs. Marianne Hansen provided technical assistance. Our thanks are also due to Mr. Erik (bstergaard for the animal experiments. Mrs. Inge Bggelund and Mrs. Yrsa Kiideberg typed the manuscript.
419
1.
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and hypertension.
Prosta-
15. Mentz, P., Piinicke, K., Block, H.-U., Blass, K.-E.,Giessler, Ch., F&ster, W. Stimulation of the prostacyclin biosynthesis as a possible me&w&m of the cardiovascular effects of dipyridamole. p. 289 in:Prostaglandins and Thromboxanes in the Cardiovascular System and in Gynaecology and Obstetrics (W. FiSrster ed.) VEB Gustav Fischer Verlag Jena 1981. Proceedings of the 3rd International Symposium on Prostaglandins and Thromboxanes in the Cardiovascular System, Halle/Saale, CDR, May 5-7, 1980. 16. Moncada, S., Korbut R. Dipyridamole and other phosphodiesterase inhibitors act as anti-thrombotic agents by potentiating endogenous prostacyclin. Lancet 1:1286, 1978. 17. Tuvemo, T. Action of prostaglandins and blockers of prostaglandin synthesis on the isolated human umbilical artery. p. 271 in: Advances in Prostaglandin and Thromboxane Research, Volume 4 (F. Coceani and P.M. Olley eds.) Raven Press, New York 1978. 18. Samuelsson, B., Folco, C., Cranstriim, E. et al. Prostaglandins and thromboxanes. Biochemical and physiological considerations. p. 1 in: Advances in Prostaglandin and Thromboxane Research, Volume 4 (F. Coceani and P.M. Olley eds.) Raven Press, New York, 1978. 19. Mlkill, U.M., Wahlberg, L., Viinikka, L., Ylikorkala, 0. Regulation of prostacyclin and thromboxane production by human umbilical vessels: the effect of estradiol and progesterone in a superfusion model. Prostaglandins Leukotrienes and Medicine 8~115, 1982. 20. Hagen, A.A., White, R.P., Robertson, J.T. Synthesis of prostaglandins thromboxanes by cerebral arteries. Stroke lOr306, 1979.
and
21. Maurer, P., Moskovitz, M.A., Levine, L. et al. The synthesis of prostaglandins by bovine cerebral microvessels. Prostaglandins and Medicine 4: 153, 1980. 22. Levine, L., Alam, I. Arachidonic acid metabolism by cells in culture: analyses of culture fluids for cyclooxygenase products by radioimmunoassay before and after separation by high pressure liquid chromatography. Prostaglandins and Medicine 3:295, 1979. 23. Ingerman-Wojenski, C., Silver, M.J., Smith, J.B., Macarak, E. Bovine endotheha1 cells in culture produce thromboxane as well as prostacyclin. The Journal of Clinical Investigation 67:1292, 1981. 24. Salzman, P.M., Salmon, J.A., Moncada, S. Prostacyclin and thromboxane A2 synthesis by rabbit pulmonary artery. The Journal of Pharmacology and Experimental Therapeutics 215:240, 1980.
421
Leulcotrienes
and Medicine
10r 411-421,
1983
ARACHIDONIC ACID METABOLISM IN ISOLATED AORTA AND LUNG OF THE RAT: EFFECTS OF DIPYRIDAMOLE, NIFEDIPINE, PROPRANOLOL, HYDRALAZINE AND VERAPAMIL K. C. Srivastava and K. K. Awasthi, Department of Environmental Medicine, Institute of Community Health, Odense University, J. B. WinsMws Vej 19, DK-5000 Odense, Denmark. ABSTRACT Effects of some vasodilating~ole, nifedipine and verapamil) and antihypertensive (propranolol, hydralazine) drugs on arachidonic acid metabolism in isolated rat aorta and lung have been studied. Dipyridamole significantly increased the formation of PC12 in aorta and lung. Nifedipine and verapamil decreased the formation of PC12 in aorta, these drugs though significantly increased the formation of PC12 in lung. Nifedipine showed no appreciable effect on the generation of TxA2 in rat aorta but in lung both nifedipine and verapamii reduced TxA2 formation though significantly only in the latter case. Dipyridamole showed no effect. The beneficial effect of dipyridamole, seems, at least in part, to be due to its ability to enhance the production of PGI2 both in the aorta and lung, and probably in other tissues as well. Nifedipine and verapamil may show their antianginal effect by a combined effect of enhanced PG12 and reduced TxA2 formation in lung. In lung, whereas hydralazine reduced the formation of both PC12 and TxA2, propranolol increased the formation of PG12. Hydralazine reduced the formation of TxA2 and increased PC12 formation in aorta. The effect of the drugs on the ability of rat aorta to inhibit collagen induced platelet aggregation of human blood platelets was also examined. INTRODUCTION Several vasodilating and antihypertensive drugs have been used in the treatment of cardiovascular diseases without a clear understanding of the mechanism(s) involved in their action. The vasodiiating drugs produce a diIatation of the coronary arteries and an increase of the coronary blood blow thus increasing oxygen saturation of the coronary sinus blood (1). These drugs also possess antithrombotic properties, thus decreasing aggregation, adhesion and release reaction of platelets, as is observed with dipyridamoie and several other drugs (2,3). Dipyridamole at clinically achieved low plasma levels blocks adenosine transport into cells, a mechanism some consider to be the basis for its cardiovascular actions (4). Whether such a mechanism is also operative for other antianginal drugs remains to be elucidated.
411
Of the several mechanisms considered to be involved in the action of these drugs, one may be their effect on the prostaglandin (PC) biosynthesis in the cardiovascular system and other organs. Focus on the relative production of thromboxane A2 (TxA2) and prostacyclin (PG12) in the presence of such drugs would be of relevance as TxA2 is platelet aggregatory (5), and contracts aorta and other vessel preparations (5,6), whereas PG12 is antiaggregatory, antihypertensive (7), and relaxes the strips of coronary artery (8). Thus the discovery of TxA2 and PG12, and recognition of the roles played by these substances in platelet aggregation and coronary regulation is of great significance. In the present study, therefore, an attempt has been made to examine the effects of some antianginal (vasodilating) (dipyridamole, nifedipine and verapamil) and antihypertensive (propranolol and hydralazine) drugs on the biosynthesis of prostaglandins (including PGI2) and TxA2 in the aorta and lung of the rat in order to explain their mechanism of action. MATERIALS AND METHODS Arachidonic acid (I-IQC) (specific activity 58.4 mCi/mmol) was purchased from the Radiochemical Centre, Amersham, England. Verapamil hydrochloride (Isoptin) was obtained as a gift from Meda A/S, dipyridamole (Persantine) from Boehringer-Ingelheim A/S, hydralazine (Apresoline) from Ciba-Geigy A/S, propranolo1 hydrochloride (Inderal) from ICI-Pharma A/S and nifedipine (Adalat) from Baeyer Kemi A/S, all in Denmark. Standard prostaglandins Fa, E2, D2, thromboxane B2 and 6-keto-prostaglandin F c1were kindly provided by ON0 Pharmaceutical Co., Ltd., Higashiku, Osaka (Japan f. The animals used were male albino SPS Wistar rats weighing 504 + 22 g. Preparation of solutions of drugs The solutions of dipyridamole and nifedipine were prepared in 99% alcohol, whereas those of propranolol hydrochloride, hydralazine and verapamil were prepared in acetone:water (l:l, v/v) mixture. Preparation of lung homogenate and aorta Lung was freed from the surrounding tissue. cut into pieces washed with ice-cold saline several times and finally with phosphate but& (0.06 M pH 7.10 containing 2 m&l EDTA. This was then homogenized with phosphate buffer (I g lung in 4 ml buffer). The rat aorta was perfused witn saline, and extra tissue was removed from it. A total length of 6 cm aorta was removed, blotted on filter paper and then cut into eight equal parts ka. 13.6 mg each). Finally the pieces were pfaceo In separate tubes each contammg 250 ul phosphate buffer. lncuoaflon one Incubation medium for lung homogenate consisted of 500 ul of the homogenate. This was first incubated with 10 ul of a drug solution for 15 min at room temperature in a final concentration indicated in tables 1 and 2. This was followed by a further incubation with labelled AA for 15 min at 370C. The controls were incubated with 10 ul of the solvent (in which the solution of the corresponding drug was prepared) for 15 min at room temperature followed by a further incubation with labelled AA for 15 min at 37OC. The incubation medium for aorta consisted of aorta and 250 ul phosphate buffer which was incubated first with 5 ul of a drug solution for 15 min at room temperature foltowed by incubation with labelled AA for 15 min at 37OC. Corresponding blanks were incubated simultaneously with appropriate solvent for 15 min at room temperature followed by labelled AA for 15 min at 370C.
412
te with labelled AA, 1 ml saline was added to each tube followed by addition of 50 ul N HCI to adjust the pH of the medium to 3.0. The AA metabolites were extracted with ethyl time with 2 ml of this solvent. In the case of aorta 1 ml saline by addition of 60 ul N HCI to adjust the pH to 3.0. This mixture was extractad with 2 ml ethyl acetate twice. The combined extract was evaporated to dryness in a stream of nitrogen. Separation of AA metabolites Separation of AA metabolites was done by a modified procedure (9) of our earlier TLC separation schedule (IO). The incubation extract of the lung homogenate was dissolved in 250 ul ethyl acetate:methanol mixture (1:4,v/v). 25 ul of this solution were applied on the left hand bottom corner of a silica gel G TLC plate (0.25 mm, 20 x 20 cm). Prior to the application of the incubation extract containing the AA metabolites, reference compounds, 6-keto-PGFla, PGFz(~ PGE2, PGD2 and TxB2 were placed on the plate. The plates were first developed in solvent II: chloroform:methanol:acetic acid (90:8:6, v/v) followed by development in solvent III: benzene:dioxane:acetic acid (40:20:2, v/v) in a direction perpendicular to the first development. The spots were shown in iodine, marked, scraped off and the radioactivity contained therein measured. The incubation extract of aorta was dissolved in 200 ul methanohethyl acetate (2~1, v/v) and chromatograhic separation of AA metabolites and the measurement of radioactivity contained in them were done as described for the lung homogenate. Separation of AA metabolites produced in aorta by a double two-dimensional TLC: Formation of TxA7 in rat aorta This exoerRnent was done with aorta from two rats. Incubation extracts from 8 aorta- pieces (each 0.7 cm, weight 13.6 mg) were pooled together, evaporated to dryness and reconstituted in 300 ul methanol:ethyl acetate (2:1, v/v) mixture. 25 ul of this were applied on each plate (in all four TLC plates were used). The plates were run successively in solvents II: chloroformtmethanohacetic acid (90:8:6, v/v) and III: benzene:dioxane:acetic acid (40~20~2, v/v). After development the plates were dried in open air, ex,posed to iodine, prostaglandin spots marked and scraped off. The AA metabolites (PGF2, PGE2, PGD2, TxB2, 6-keto PGFld scrapings of one plate were counted individually for their radioactivity whereas those of the remaining three plates were scraped off and pooled together individually. Silica gel scrapings were extracted three times, twice with methanohethyl acetate (3:2, v/v) and once with methanohethyl acetate (2:1, v/v) using I ml of the solvent in each extraction. The combined extract was evaporated to dryness under nitrogen and redissolved in 300 ul methanol:ethyl acetate (2:1, v/v) mixture. A second two-dimensional TLC was performed for individual prostaglandins using authentic samples successively using solvents IV: ether:methanol:acetic acid (90:1:2, v/v) and V: ethyl acetate:acetic acid:isooctane: water (110:2O:50:100, v/v, using the upper organic phase). After development, plates were dried in open air, exposed to iodine, PC spots marked, scraped off and the radioactivity counted. The amounts of various PCs and TxB2 were calculated after taking into account the losses during extraction and TLC, and compared with those obtained after the first two-dimensional TLC. Aggregation experiment Rat aorta was prepared as described above. It was cut into small pieces, ca. 6 and 3 mm, weighing about 8.76 and 4.38 mg respectively. Prior to aggregation experiment, incubation of aorta pieces was done in the following way. The 413
incubation medium consisted of 100 ul Tris-HCI (50 mM, pH 8.4), a piece of aorta and an appropriate volume (5 or 10 ul) of the solvent (99% ethanol or acetonewater I:& v/v (control), whereas the experimental tubes contained a corresponding volume of the drug solution to give the desired concentration. The volume of platelet rich plasma (3.2 x log platelets) was maintained to 1 ml in each case. The control and experimental tubes were kept at OoC for 30 min. Each tube (one at a time) was then incubated at 300C for 4 min, and immediately 5 or 10 ul aliquot from the aorta incubate were added to a PRP sample maintained at 370C under constant stirring in the aggregometer. Exactly after 30 set of mixing of the contents, collagen was added to induce aggregation. Siliconized glass tubes were used both in aorta incubation and platelet aggregation. Statistics Statistical paired data.
significance
was evaluated
by the two-tailed
Student t-test
for
RESULTS Table I gives data on the effects of antianginal (dipyridamole, nifedipine and verapamil) and antihypertensive (propranolol and hydralazine) drugs on the biosynthesis of AA metabolites in rat aorta. As can be seen, dipyridamole significantly increased the formation of PCI2, PCFa and PCE2. Thromboxane B2 and PGD2 formation was reduced but not significantly. In the presence of nifedipine PC12 was reduced though not significantly, other AA metabolites were also reduced to a small extent with the exception of PGFa, which increased slightly. Verapamil reduced the formation of PGI2. Propranolol reduced the formation of all AA metabolites. In the presence of hydralazine, formation of PC12 was enhanced, thromboxane B2 and PGD2 were reduced, the latter significantly. However, there was seen no effect of this drug on the formation of PGF2o and PGE2. Table 2 presents data on the effects of antianginal and antihypertensive drugs on the generation of AA metabolites in rat lung homogenate. Dipyridamole significantly enhanced the formation of PC12 and PGE2. There was no change in other AA metabolites. Nifedipine significantly increased the generation of PGI2, whereas other AA metabolites were reduced but not significantly. Verapamil increased the formation of PG12 and decreased the formation of TxB2 both changes being significant; PGF2o and PGD2 remained unchanged and PGE2 increased but not significantly. Propranolol increased the formation of PG12 and TxB2. In the presence of hydralazine, all the AA metabolites were reduced. Table 3 gives data on the ratio of PC12 and TxB2 formed in the presence of the antianginal and antihypertensive drugs in the aorta and lung homogenates of the rat. In the case of the aorta the ratio significantly increased in the presence of dipyridamole and hydralazine. This ratio was enhanced also in the presence of nifedipine and propranolol though not significantly. The ratio of PC12 and TxB2 was increased in the presence of all the drugs in rat lung homogenate. Table 4 gives data on the production of the classical PCs, TxB2 and 6-ketoPGFlo in the aorta of rat. As can be seen measurable quantities of these AA metabolites can be obtained after the second two-dimensional TLC; their amounts were comparable to those found after the first two-dimensional TLC. That blood vessels also produce TxA2 besides other PGs was further demonstrated by performing similar experiments with aorta from 6 rats. In each 414
Table
I.
Effect of antia”glnal and antihypemsaive AA metabolita in rat aorta.
drugs on the biosynthesis
Amounta (picomoleslaorta piece “/ formed with labelled AAX for 15 min
Drug fconc.)
after
incubation
PC12 ”
PGFz a
T*2
PGE2
PGD2
Control
102 f46
5 c3
7*4
II?
6+4
+ Dipyridamole (0.2 mhl) “~6
158 *75
9 r2”
5*3
19? 7**
5+3
+ Nifedipine (0.2 mhi) n:6
81 %8
6 t2
5*2
IO? 3
4+ I
Control
80 +I5
7 t1
Sk3
8*3
6*2
+ Propranolol (1.0 mM) “~3
73 *36
5 +I
6? I
4*1
5t I
??
6
Control
88 + 56
6 i3
7?6
6+2
6*3
+ Hydralazine (0.75 mhi) n=5
113+ 73
6 +2
3k1
6*1
2+ 1*
a Mean r SD ?? p < 0.05
??*
b Values for verapamil(1.0 X Final concentration
Table 2.
p < 0.01 mh4, n=6) 64 f 33 agaiwt
25 uM kt.
control
values 89 * 43
13.60 mg
Effect of antiangina and antihypertemive AA metabolites in rat lung homogenate.
Drug konc.)
of
drugs on the biosynthesis
Amounta (picomolesI500 ul) formed labelled AAX for 15 min
after
incubation
with
PC12
P=F2,
T*2
PCE2
PGD2
Control
145 +46
75* 30
103k 50
49t: 13
59*29
+ Dipyridamole (0.2 mM) n=7
274 *76”
75* 17
101+ 37
72+ 23’
55i27
Control
147 +50
84k 21
II5*
53*
66224
+ Nifedi Pine (0.2 mM “~6
215 *67**
59+ 21
76 +38
Control
184 236
35+ 18
+ Propra”olol (1.0 mM) “~3
229 +63
44?
14
+ Hydralark (0.75 mM) n=3
117 +6
23+
9
Control
174 260
+ Verapamil (1.0 mM) “~7
266t120”*
a Mean‘SD
42
9
32*21
42 231
143 + 10
36t
14
40*20
161+37
40+ 15
37 cl6
65 +I1
16*
8
20 *II
77+36
119*53
57 * 20
69 ?28
76k34
118 *43’*
68 + 33
71 t32
?? p
< 0.05
X Final concentration
415
??*
12.5 uM
p-C 0.01
of
Table 3.
Influence of antianginal and antihypertensive ratio in rat aorta and lung homogenate.
Aorta Drug (cont.)
drugs on PGI2:TxB2
Lung homogenate ratio
PGI2/TxB2
Drug (cont.)
6.09
ratio
PC12/TxB2
Control
16.48
+
Control
1.81
+
1.25
+ Dipyridamole (0.2 mM) n-6
33.60
t 14.60+
+ Dipyridamole (0.2 mM) n-6
2.95
+
0.971’
Control
16.46
t
6.12
Control
1.40
+
0.56
+ Nifedi ine (0.2 mM P n=6
17.46
+
8.70
+ Nifedipine (0.2 mM) ~6
3.18
t
1.24’
Control
10.83
*
I.78
Control
1.27
?
0.15
+ Propranolol (1.0 mM) n=3
11.88
+
6.45
??Propranolol (1.0 mhi) n=3
I.41
+
0.11
4.71
Control
14.46
+
+ Hydrakzine (0.75 mM) n=5
30.38
t 15.38’
Control
1.27
+
0.15
+ Hydralazine (0.75 mM) n.3
1.83
+
0.40
Control
2.01
+
1.14
+ Verapamil (1.0 mM) n.6
3.72
+
2.07”
L ?? p
Table 4.
< 0.05
?? *
P < OJJI
Formation of various arachidonic acid metabolitn arachldonic acid in the aorta of rat.
Amounta
of AA-metabolites
from (I-146)
(pIcomoles/aorta
piece”)
AA-metaboUte6 1st tw-dimenaionaI
6-keto-PCFI
2nd twodimemIona1
79.7
66.8
PCE2
8.1
7.8
PGF2a
8.2
6.4
PCD2
4.2
2.5
12.7
9.7
Ta2
cL
TLC
* Mean of 2 rats3
X might
TLC
(‘#at) 13.6 mg
A slmlhr experiment was performed with aorta from 6 rats. 1x62 was wqwated and mawad aCt8r ht and 2nd twodlmewlonbl TLC. Amounts ~amaWlO.40 m aorta) found afta the two TLC separations were 6.lSf 2.13 and 6.14* 5.36 &a” isa mpcctive1y.
416
Figure 1. Representative tracings showing the effects of various drugs on the ability of rat aorta to inhibit collagen induced aggregation of human blood pJateJets. a) A. only collagen; B. aggregation of PRP pretreated with aorta incubate without dipyridamoie (control); C. same as 8. in the presence of dipyridamole (0.2 mM). b) A. only colJagen; B. aggregation of PRP pretreated with aorta incubate without nifedipine (control); C. in the presence of nifedJpJne (0.2 m&i). c) A. only collagen; B. in the presence of hydraJazine (0.75 mM); C. in the presence of verapamil (1.0 mM); D. in the presence of propranolol (1.0 mM); E. without any drug (control). d) A. on1 colJagen; B. in the presence of verapamil(0.5 mM); C. without verapamil (control. r For a) and d) the aorta incubation medium cudsted of an aorta piece (ca 8.76 mg) + 100 uJ Tris-HCJ (50 mM, pH 8.4) + 10 ul solvent or drug soiution from which 5 uJ were used in regation. For b) and c) the aorta incubation medium consisted of an aorta pieceY ca. 4.38 mg>.+ 100 ul TrisHCI + 5 ul solvent or drug solution from which 10 ul were used in aggregation.
417
rat, 4 pieces (each piece: length 0.7 cm, wet wt. 10.4 mg) were used for incubation. A double two-dimensional TLC of the incubation extract gave TxB2 value 6.14 2 5.36 (mean + SD) in picomoles/aorta piece. Figure 1 shows the effects of the above drugs on the ability of rat aorta in inhibiting collagen induced aggregation of human blood platelets. As is obvious dipyridamole potentiates the inhibiting action of aorta (a). Nifedipine seems also to potentiate this action, though slightly (b). Other drugs (propranolol, verapamil and hydralazine) seem to reduce the platelet aggregation inhibiting capability of aorta (c,d). DISCUSSION The effects of three antianginal drugs (dipyridamole, nifedipine and verapamill and two antihypertensive drugs (propranolol and hydralazine) on the formation of various AA metabolites in the aorta and lung of rats were examined. Both the tissues generate prostacyclin (8,111 a naturally occurring potent vasodilating substance, which also exhibits a strong antiaggregation effect. Lungs generate also TxA2 besides other prostaglandins (12,131. Recently, evidence has been produced indicating that TxA2 is also synthesized in blood vessels (14). Thus when mechanisms of hemostasis or thrombosis are examined, one should also take into account the ability of blood vessels to produce TxA2 besides PGI2. However, there is no denying of the fact that in circulation a substantially large portion of TxA2 is produced by blood platelets and that a balance between these two unstable arachidonic acid metabolites has tremendous significance for the state of health or disease in the cardiovascular system. Antianginal drugs work by dilating the blood vessels and thus are essentially vasodilating in nature. With the discovery of PGI2 formation in blood vessels, it is pertinent to examine the effect of a vasodilating drug on the formation of PC12 in blood vessels and for that matter any other organ which produces it. If a certain drug induces an enhanced production of PGI2, one may presume that at least one of the ways by which the drug shows antianginal activity is through its effect on PGI2 formation. This was found to be so in the case of dipyridamole which induced an enhanced production of PC12 in the aorta and lung of rat - an observation confirming the earlier reports (15,161. As nifedipine and verapamil reduced, though not significantly, the formation of PC12 in aorta, their antianginal action cannot be explained by way of PC12 formation in aorta/blood vessels unless one takes into consideration their effect on the production of this substance in lungs which have been shown to generate prostacyclin in vivo and release it into the arterial circulation (11). Both nifedipine and Era-i1 were found to enhance PG12 generation significantly in the lung homogenates. As no marked effect was observed on the aorta TxA2 generation in the presence of dipyridamole and nifedipine, while only nifedipine and verapamil causing reduced formation of TxA2 in lungs, one may explain the vasodilating action of these drugs in the following way. Dipyridamole is effective mainly by its action on enhanced production of PGI2, while nifedipine and verapamil show their antianginal effect by a combined effect of enhanced PG12 production and reduced TxA2 formation in the lungs. Of the two antihypertensive drugs studied, in aorta hydralazine increased the formation of PC12 though not significantly. In lung, propranolol enhanced the formation of PC12 while hydralazine reduced its synthesis. Hydralazine also reduced the formation of TxA2 in lungs. These observations may suggest that the antihypertensive effect of propranolol is probably by its action on the increased formation of PC12 and that of hydralazine is by its ability to reduce the formation 418
of TxA2 in lungs. The effects of these drugs can be understood in a better way by looking at the PGI2/TxA2 ratios in aorta and more so in lungs. In aorta PGI2/TxA2 ratios increased in the presence of dipyridamole and hydralazine; in the presence of nifedipine and propranolol the ratios were slightly elevated. However, for lungs, with all the drugs PGI2/TxA2 ratios were increased. Thus the effects of these drugs (with the exception of dipyridamole) may be readily explained by considering their effect on the formation of TxA2 in aorta and especially in lungs. These results find support from our recent report in which it has been shown that several antianginal and antihypertensive drugs (including those examined in this report) reduced the formation of TxA2 in human blood platelets in vitro. Also HHT formation was reduced in the presence of these drugs and t&observation was supported by a reduced MDA formation in the platelets. These drugs inhibited also ADP-induced platelet aggregation (3). Initial reports (17, 18) on the ability of a blood vessel to synthesize TxA2 were confined only to umbilical artery, observations supported by another recent study (19). Later studies produced evidence showing that arterial vessels could also be synthesizing TxA2 (14,20-24). Our studies (based on a double two-dimensional TLC separation of the incubation extract containing a mixture of labelted AA metabolites) also provide evidence that rat aorta synthesize TxA2 from exogenous arachidonate though in amounts much less than those of PGI2. Platelet aggregation study shows that the five drugs can be classified in two categories; one which potentiate the ability of aorta in inhibiting platelet aggregation, and the other which do not do so, rather they reduce the ability of aorta to inhibit platelet aggregation. The potentiating effect of a drug (e.g., dipyridamole) could either be by inducing an increased synthesis of prostacyclin in aorta, or by potentiating the action of PGI2, or by a combination of both. For drugs showing an effect in the opposite direction, their action could be either by reducing the formation of PC12 in aorta, or by inhibiting the ability of PGI 2 to inhibit platelet aggregation, or by a combination of both. The drugs inhibit thromboxane synthetase of platelets (washed) in concentrations used in the aorta incubation (3). As their concentrations are reduced in the aggregation medium (PRP, containing plasma proteins) by a factor of 100, it is unlikely that they would have any effect on the platelet thromboxane synthetase. Thus the difference in aggregation observed in the presence of a drug compared to its control, should mainly be due to the effect of the drug on the aorta prostacyclin synthesis. In such a situation, however, one must take into consideration that prostacyclin synthesis from endogenous arachidonic acid is mainly dependent on one step prior to PGI2 synthetase, i.e ., on the liberation of arachidonic acid by the action of phospholipase on which the drug may also show its effect. Aorta prostacyclin synthesis from labelled arachidonic acid in the presence of the drugs partly supports the aggregation results. ACKNOWLEDGEMENTS We wish to thank the Danish International Development Agency (DANIDA) for the award of fellowship to KKA. Mrs. Ruth Alexandersen and Mrs. Marianne Hansen provided technical assistance. Our thanks are also due to Mr. Erik (bstergaard for the animal experiments. Mrs. Inge Bggelund and Mrs. Yrsa Kiideberg typed the manuscript.
419
1.
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on the
2.
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3.
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4.
Pearson, J.D., Carleton, J.S., Hutchings, A., Gordon, J.L. Uptake and metabolism of adenosine by pig aortic endothelial and smooth muscle cells in culture. Biochemical Journal 170:265, 1978.
5.
Hamberg, M., Svensson, J., Samuelsson, B. Thromboxanes: A new group of biologically active compounds derived from prostaglandin endoperoxides. Proceedings of the National Academy of Sciences, U.S.A. 72:2994, 1975.
6.
Needleman, P., Moncada, S., Bunting, S., Vane, J.R., Hamberg, M., Samuelsson, B. Identification of an enzyme in platelet microsomes which generates thromboxane A2 from prostaglandin endoperoxides. Nature 261:558, 1976.
7.
Pace-Asciak, C., Carrara, M.C. Evidence suggesting a systemic antihypertensive role for prostacyclin. Prostaglandins 15:407, 1978 (Abstract).
8.
Dusting, G.J., Moncada, S., Vane, J.R. Prostacyclin is the endogenous metabolite responsible for relaxation of coronary arteries induced by arachidonic acid. Prostaglandins 13:3, 1977.
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