Atherosclerosis decreased prostacyclin formation in rabbit lungs and kidneys

PROSTAGLANDINS

ATHEROSCLEROSIS DECREASED PROSTACYCLIN FORMATION IN RABBIT LUNGS AND KIDNEYS Aldona Dembifiska-KieC', Werner Riicker and Peter S. Schonhijfer Department fiirPharmakologie und Toxikologie, Abt. II, Medizinische Hochschule Hannover, Karl-Wiechert-Allee 9, D-3000 Hannover 61, GFR

ABSTRACT Metabolism of arachidonic acid (AA) was studied in perfused lungs and kidneys of normal and atherosclerotic rabbits by determination of PGE2, PGF and the stable metabolites of PG12 (6-keto-PGF,,) anAaTXA2 (TXB2). PG12 was the main AA metabolite formed by normal lungs and kidneys. Atherosclerosis reduced the formation of PG12 by about 50 % in both organs. TXA2 formation was similarily decreased in lungs. In kidneys, the decrease in PG12 formation was accompanied by an increase in PGE2 formation. INTRODUCTION An imbalance in the homeostasis between prostacyclin (PGI2) formed by arterial walls and possessing antiaggregatory and vasodilatatory properties (1, 2) and thromboxane A2 (TXA2) formed by platelets and possessing proaggregatory and vasoconstrictory properties (3, 4) may play a role in atherosclerosis, since enhanced platelet aggregation has been linked to the progression of atherosclerotic plaque formation (5). A decrease in PG12 formation by arterial walls was reported for experimental as well as human atherosclerosis (6, 7, 8). Recently it was found that the lungs may be an essential source for PG12 in vivo (9, 10). PG12 is also a major AA metabolite intheidney cortex and released

1

A. Dembinska-Kiec is fellow of the Alexander von Humboldt-Stiftung from the Department of Pharmacology, Copernicus Medical Academy, Cracow/Poland.

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during perfusion (11, 12). Released PG12 has been postulated to participate in the protection of circulating platelets from aggregation (9, 10). Since a reduction in PG12 release from the lungs and other tissues such as the kidney may play a role in the increased platelet aggregability observed in atherosclerosis, alterations of PG12 formation and release were studied in perfused lungs and kidneys of normal and atherosclerotic rabbits. MATERIALS AND METHODS Atherosclerosis was induced in male bastard rabbits (3-4 kg) by feeding a standard rabbit pellet food supplemented with 1 % cholesterol and 3 % olive oil for 5 month. Control rabbits were kept under identical conditions receiving the same food without cholesterol and olive oil for 5 month. Normal and atherosclerotic rabbits were anaesthetised with sodium pentobarbital (30 mg/kg i.v.). Pulmonary and renal arteries were cannulated in situ. Lungs and kidneys were caref_ully dissected and washed with saline followed by perfusion with Krebs solution gased with carbogen (95 % 02 + 5 % CO2) for 10 min at 37o C for removing the residual blood. The isolated organs were reperfused with 20 ml regased Krebs solution containing 0.3 I.IM(0.35 mCi) [l-?4C)arachidonic acid (AA) at a rate of 5 ml/min for 20 min after which time the perfusate was collected. The organs were washed with AA-free Krebs solution perfused for 5 min at a rate of 5 ml/min. The effluents were combined, acidified to pH 3.0 by 0.1 N HCl and filtered three times through a column filled with 5 g amberlite XAD-2 (Serva, Heidelberg). The columns were washed with destilled water. The acidic lipids were eluted with 15 ml .ethanol. The ethanol was evaporated under a stream of nitrogen. The residues were redissolved in chloroform-methanol (2:l v/v) and developed by TLC (solvent: ethylacetate-isooctane-acetic acid-water, 11:5:2:10) accordingtoCottee et al (13). Radioactivity was localised by a thin layer scanner (Berthold, Wildbad) and analysed by use of prostaglandin standards (PGE2, PGF2,, 6-keto-PGF,, and AA) run on the same TLC plate and visualised by iodine vapor. The Rf values for TXB2 (14) were checked by incubating washed rabbit platelets with labeled AA and by developing the extract on the same TLC system.

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The radioactive AA metabolites were quantified by scrapping off the corresponding spots from the TLC plates and determining the radioactivity by liquid scintillation counting. The recovery was 70-80 % for extraction of radioactive material from perfusates. The results are expressed as percentage of the total radioactivity recovered from the TLC plate. The values represent the means + SEM of 7-11 organ perfusions. Student's t-test was used for statistical analysis. RESULTS Most of the labeled AA was taken up by the organs during the perfusion. The effluents contained about 20-30 % of the added radioactivity in the lungs and about 40-70% in the kidneys. The radioactivity released consisted mainly of non metabolized 14C-AA, the perfusates from lungs containing 74 + 6 % (controls) and 81 f 2 % (atherosclerotic) unchanged AA, the perfusates from kidneys containing 86 2 4 % (controls) and 89 f 1 % (atherosclerotic). The main AA metabolite observed on the TLC plates corresponded to 6-keto-PGFlc in both perfused organs. PGE2 and PGF2, formation was less than half that of 6-keto-PGFlc. In controls, the lungs were twice more active in converting AA into 6-keto-PGF1, than the kidneys (Fig. 1). In atherosclerotic rabbits, 6-keto-PGFlo formation was reduced by 65 % in the lungs (~~0.005) and by 51 % in the kidneys (~(0.005). In the kidneys, but not in the lungs, the inhibition of 6-keto-PGFlc formation was associated with a 46 % increase in PGE2 formation (~(0.005). The kidneys showed only a minute TXB2 formation. In the lungs, the average TXB2 formation was also lowered by about 60 % in atherosclerosis. However, this reduction was of low statistical significance (~(0.02).

DISCUSSION The lungs have been demonstrated to be an essential source for PG12 which has been claimed to play a role in maintaining the stability of circulating platelets (9, 10). Therefore, the observed reduction of PG12 formation by about 66 % in the lungs from atherosclerotic rabbits may add to the understanding of the increased platelet aggregability and platelet loss in atheroscle-

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LUNG

6-Keto PG5a

Fig.

TX02

KIDNEY

PGE2

6 -Keto

TXB2

0

Normal

63

Atherosclerotic

PGE2

PGF2.x

PG%l

1:

Formation of 6-keto-PGFlc, TXB2, PGE2 and PGF2, in perfused normal and atherosclerotic lungs (left panel) and kidneys (right panel) from rabbits. Results are expressed as the percentage of the total radioactivity converted into the respective AA metabolite. The total radioactivity recovered from the TLC plates is set as 100 %. Each value represents the mean + SEM of 7-11 organ Significance: 'p <0.02, **p <0.005 (atheperfusions. rosclerotic vs normal organs).

rosis, especially since it has been shown that local PG12 formation was also reduced in peripheral as well as coronary vascular bed in experimental atherosclerosis of rabbits (5, 7).

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TXA2 formation in the lungs has been described to occur in this organ only under special conditions sech as in vitro perfusion with AA or challenge with histamine (15, 16). The decrease in TXA2 formation observed in atherosclerosis was not accompanied by a similar decree se in PGE or PGF2, formation and may, therefore, suggest a re3uction in the acti.vity of the synthesising enzyme in atherosclerosis, especially since a similar reduction in enzyme activity was found in homogenates of platelets from atherosclerotic rabbits (17). The synthesis of the stable PGI2-metabolite 6-ketoPGFl, is well documented for microsomes of the kidney cortex (11). The physiological function of PG12 in the kidney has been linked to an increase in renin release and an elevation of renal blood flow, especially in the inner cortex, since both PG12 and PGE2 were found to increase natriuresis and diuresis, accompanied by an increase in the excretion of potassium (11, 18). PaceAsciak and coworkers reported that PGI2 exerted blood pressure lowering effects in experimental hypertension (19, 20). In view of these findings, the reduction in PG12 formation in the kidneys reported in this study for atherosclerotic rabbits may add to the understanding of events associating atherosclerosis with alterations of sodium excretion and blood pressure. The increase of PGE2 formation found in the kidneys of atherosclerotic rabbits may be understood as a compensatory mechanism for the maintenance of diuresis (18) and may result from the observed inhibition of PG12 formation, since a higher conversion of prostaglandin endoperoxides to prostaglandins has also been reported for atherosclerotic arteries due to aselective inhibition of PG12 formation (21).

Acknowledgements: This study was supported by grant Scho 169/7 of the The authors wish to Deutsche Forschungsgemeinschaft. thank Miss I. Krause and Mrs. I. Klappstein for their eminently skilled technical assistance.

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