PROSTAGLANDINS

PROSTACYCLIN PRODUCTION BY RAT AORTA "IN VITRO" IS INCREASED BY THE COMBINED ACTION OF DIPYRIDAMOLE PLUS PENTOXIFYLLINE

M. Teresa Santos 1, Vic~nta M ~ t i n e z - S a l e s 1, Juana Val l~s 1, Justo Aznar 2 , Ricardo Yaya 3 , A~paro Vay~ 2 , Piedad Villa 2 .

iResearch Center, 2Department of Clinical Pathology and 3Neurology Service, Hospital "La Fe". Valencia. Spain.

ABSTRACT

The present study evaluates the effect of dipyridamole and pen- toxifylline, individually and in combination, on PGI2-1ike pro- duction and arachidonic acid metabolism of rat aorta "in vitro". Pentoxifylline 100 ~M and dipyridamole 92 and 184 ~M increased PGI2-1ike activity, as measured by the platelet aggregation inhibitory capacity of the aortic ring incubates, by 71%,46% and 60% respectively; a greater increase in PGI2-1ike activity was obser- ved with the combination of the drugs than when they were used separately. This effect was observed even at the lowest doses assayed. In fact, dipyridamole 9.2 ~M plus pentoxifylline 1 ~M increased the PGI2-1ike activity by 30% while the individual increase was 4.5% and 10.6% respectively. To obtain more infor- mation on the effect of the dipyridamole-pentoxifylline combina- tion on arachidonic acid metabolism, arteries were incubated with (l-14C)arachidonic acid, and the 6-keto-PGFl~ and PGE 2 quantified. Dipyridamole 92 #M plus pentoxifylline 1 and i0 #M increased 6-keto-PGFl~ and PGE 2 production by about 30% and 48% respectively while the combination with pentoxifylline i00 ~M increased the 6-keto-PGFle 76.5% and the PGE 2 50%. The possible biological effect and therapeutic implications of increased PGI~ production by the arteries due to the dlpyrldamole-pentoxlfylll ne combination remains to be ascertained.

INTRODUCTION

It is generally thought that platelets can be one of the most important pathogenic factors in ischemic cerebrovascular disease (ICD) (i). For this reason several drugs with anti- platelet activity are being used in the prophylaxis and treatment of these processes, especially aspirin (2),dipyrida- mole (3), sulfinpyrazone (4), and pentoxifylline (5,6).

Dipyridamole in combination with aspirin gives better results in the treatment of ICD patients than aspirin alone (7). We are, therefore, using a combination of dipyridamole (300 mg/day) and pentoxifylline (1200 mg/day) in a clinical study of ICD patients. Since the preliminary data suggested a favourable effect, it seemed of interest to study more

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deeply the pathways by which these drugs work, especially when they act together.

Among the suggested mechanisms of dipyridamole and pentoxifylline action is stimulation of PGI 2 synthesis (8,9). We have therefore evaluated their effect on the production "in vitro" of PGI 2 by rat aortic rings.

MATERIALS AND METHODS

Chemicals Dipyridamole and pentoxifylline were obtained from

Boehringer Ingelheim and Hoechst, respectively.(l-14C)- Arachidonic acid (Sp. act. 60 Ci / Mol) was purchased from Amersham International. Standards of arachidonic acid PGE2, PGF2~ , PGD 2 and 6-keto-PGFl~ were purchased from Sigma. Chemical Co (St.Louis, MO). The ADP for the aggregation tests was purchased from Diagnostica Stago (France). Other reagents, all of analytical grade, were obtained from Merck (Darmstadt, West Germany).

Animals Male Sprague-Dawley rats, weighing 200-250 g were

anesthetized with ether, opened by midline incision and arterial blood collected in 129 mM sodium citrate. The descending thoracic and abdominal aortas were rapidly re- moved, washed in cold tris buffer (0.01 M,0.15 M NaCl, pH 7.4) and carefully freed from adjacent tissue. Rat platelet rich plasma (PRP) was obtained by whole blood centrifugation 200 x g for i0 min, and PRP from various animals was pooled.

PGI2-1ike activity assay

The PGI2-1ik e activity of the aortic incubates was evaluated by their inhibitory effect on platelet aggrega- tion. a) Incubations: The dissected aortas were kept in 0.2 ml of ice-cold tris buffer (0.05 M, pH 7.4) for 60 min befo- re the assay. The rings were then transferred to 0.6 ml of tris buffer (0.05 M, pH 9) and incubated for 7 min at 37oc; they were then transferred to 0.4 ml of an incubation medium containing either the drug being assayed plus tris buffer 0.05 M, pH 9, or tris buffer alone (control experi- ments) and incubated for 15 min at 37°C. b) Platelet aggregation tests: The platelet aggregation tests were performed in an aggregometer Chrono-long (Chrono- log Corporation, U.S.A.)on siliconized aggregometer cuve- ttes containing 150 #i of rat PRP, diluted with CaCl 2 at the same final concentration as that described by Hornstra et al. (i0); I min before the stimulation with ADP, 50 ~i of an aortic ring incubate or 50 ~i of tris buffer 0.05 M, pH 9 (blank) were added. The concentration of ADP was cho-

114 JANUARY 1985 VOL. 29 NO. 1

PROSTAGLANDINS

sen so that the maximum intensity of aggregation (Imax) in the blank samples were 70-80% (generaly 5 #M ADP). The ra- tio of Imaxl/Imax obtained by the addition of 50 ~i of the 2 two consecutive incubations of each set of aortic rings was taken as an index of the PGI2-1ike activity produced by the aortic rings.

Analysis of metabolites of arachidonic acid

Aortic tissue (30 mg) cut into small pieces were incu- bated, within 40 min of the animal's death, for 90 min at 37°C in one of the following: i) 1 ml tris buffer 0.05 M, 1 mM EDTA, 0.15 M NaCl, pH 7.5 with 15 nmols of (l-14C)sodi - um arachidonate formed by mixing arachidonic acid with 200 ~i of 0.15 M Na2CO 3 (ii) control experiments; 2) the above medium plus i, i0 or 100 #M pentoxifylline, or 92 ~M dipyridamole, or a mixture of the two drugs.

The incubation mixture was acidified to pH 3 with 1 M HCI, extracted three times with 3 ml ethyl acetate and the organic phase was evaporated to dryness under a N 2 stream.

The residue, dissolved in 200 #i of methanol and a mix- ture of standards of arachidonic acid, PGD2, PGE2, PGF2~ and 6-keto-PGFl~ were applied to thin layer chromatoplates (250 #m silica gel 60 F254, Merck). The plates were develo- ped twice in the organic phase of the mixture of ethyl acetate/iso-octane/acetic acid/water (110:50:20:100: v/v) (12).

The standards were visualized by iodine vapor and the unknown samples were either scanned for radioactivity with a Berthold DOnnschicht Scanner II or visualized by autora- diography using MAFE RP-Xl x-ray film. The radioactive zo- nes were scraped off and counted in a Packard Tri-carb liquid scintillation spectrometer model 3390.

The products of (l-14C)arachidonate conversion are expressed as a percentage of the total radioactivity reco- vered on the thin layer chromatographic plates.

Student's t-test and the linear correlation coeffi- cient were used for statistical analysis.

RESULTS

Dipyridamole and pentoxifylline, assayed separately, increased the PGI2-1ike activity generated by rat aortic rings as measured by inhibition of platelet aggregation. This increase was statistically significant with 92 and 184 ~M dipyridamole or 100 #M pentoxifylline (Table i). When the two drugs were used in combination more PGI2-1ike activity was released by rat aortas than when the drugs were used individually (Table 2).

The drugs alone, or their combinations, in tris buffer 0.05 M, pH 9, at the same concentrations as those used for the incubation of the aortic ring did not produce any inhi- bitory effect on the platelet aggregation induced by ADP

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PROSTAGLANDINS

(pm0.05 in all cases; n = 5).

After incubation of rat aortas with (l-14C)arachidonate and pentoxifylline i, 10 or 100 ~M or dipyridamole 92 ~M a small increase in 6-keto-PGFla formation with pentoxifylli- ne (6-15%) (p~ 0.05) or dipyridamole (21%) (p~ 0.05) was observed. Dipyridamole 92 ~M plus pentoxifylline (i, 10 or 100 ~M),however, increased the yield of 6-keto-PGFl~ by 31% (p< 0.05), 33% (p<0.05) and 76% (p<0.001), respecti- vely (Table 3). The transformation of (l-14C)arachidonate into PGE 2 was not affected by pentoxifylline, whereas dipy- ridamole 92 ~M tended to cause an increase (34%)(p> 0.05). Dipyridamole 92 ~M plus pentoxifylline i, i0 or 100 ~M increased PGE 2 formation by 48% (p~ 0.02), 48% (p< 0.02 and 50% (pc0.01), respectively (Table 4).

DISCUSSION

This study shows that both dipyridamole 92 and 184 ~M and pentoxifylline (100 ~M) increase the PGI2-1ike activity in rat aortic ring incubates as measured by their inhibition of platelet aggregation. Other authors found that dipyrida- mole increase the release of PGI2-1ike activity from human umbilical veins (13) and rat aortas (14) and pentoxifylline increase this activity from rat aortas (15), bovine coronary arteries and veins,and human umbilical arteries and veins (16) .

When dipyridamole and pentoxifylline acted in combina- tion on rat aortas, a greater increase in PGI2-1ike activity was observed than when the drugs were used separately. The potentiation might be due to an increased production of PGI 2 by rat aortas (17,9) and/or to an increased inhibition of the platelet phosphodiesterase (18,19). The latter effect may favour the inhibiting action of PGI 2 on the platelet aggregation by maintaining the increased platelet cAMP levels (20).

This experimental finding may be of interest in rela- tion to the posible antithrombotic effect of the combina- tion of these drugs "in vivo".

Experiments carried out to obtain more information on the mechanism by which the dipyridamole-pentoxifylline com- bination influences the arachidonate metabolism, show that pentoxifylline alone had little or no effect on the trans- formation of exogenous (l-14C)arachidonate to 6-keto-PGF~ or PGE 2 by the rat aorta. On the other hand dipyridamole z~ showed a tendency to increase the release of 6-keto-PGFl~ (p = 0.i) and PGE 2 (0.05L p<0.1).The latter,is agree- ment with Srivastava et al (21) who found a higher trans- formation of (l-14C)arachidonate to 6-keto-PGF~and PGE 2 by rat aorta when incubated with dipyridamole.

116 JANUARY 1985 VOL. 29 NO. 1

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It is generally thought that the synthesis of PGI 2 by vascular tissue is a process controlled by the enzymatic activities of phospholipase A2, ciclooxygenase and PGI 2- synthetase (22).

Dipyridamole has been reported to influence arachido- nate metabolism increasing the release of arachidonic acid (23), activating the cyclooxygenase-peroxidase system (24), stimulating the PGI2-synthetase activity (25), enhancing the formation of PGE 2 and PGF~(23) and by its possible antioxidant action (26). Data-on the effect of pentoxify- lline on the arachidonate metabolism are scarce, but it has been suggested that this drug stimulates the PGI2-synthetase activity (15).

The biochemical mechanism by which the dipyridamole- pentoxifylline combination increases the PGI2-1ike activi- ty and the 6-keto-PGF~production by rat aorta has not yet been defined. However,one possible explanation is that the selective activation of the PGI2-synthetase step of the arachidonate metabolism by pentoxifylline together with the general activation of the arachidonate cascade by dipy- ridamole and its protective effect on the cyclooxygenase and PGI2-synthetase from free radical intermediates and lipid peroxides could lead to the greater 6-keto-PGFl~ release and PGI2-1ike activity found in our results.

The possible biological effect of the dipyridamole- pentoxifylline combination on PGI 2 synthesis and its thera- peutic implications remain to be ascertained. However, the clinical improvements observed in our preliminary study of ICD patients treated with this combination are encouraging and suggest that the antithrombotic effect of this combi- nation should be further investigated.

ACKNOWLEDGEMENTS

We greatly appreciate the techical assistance of M.D. Lopez, M. Aragon,s, R. Ferrer, P. Llorens and M.C. Insa.

JANUARY 1985 VOL. 29 NO. 1 117

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Editor: A. Bennett Received: 8-9-84 Accepted: 10-24-84

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