Inhibition by ethanol and mepacrine of phospholipase-dependent prostaglandin release from the isolated perfused rat lung

European Journal of Pharmacology, 120 (1986) 145-150

145

Elsevier

I N H I B I T I O N BY E T H A N O L A N D M E P A C R I N E OF P H O S P H O L I P A S E - D E P E N D E N T P R O S T A G L A N D I N RELEASE FROM T H E ISOLATED PERFUSED RAT LUNG SUSAN

H. P E E R S * and J.R.S.

HOULT **

Department of Pharmacology, King's College. Strand, London WC2R 2LS, United Kingdom Received 24 July 1985, revised MS received 24 September 1985, accepted 15 October 1985

S.H. P E E R S a n d J.R.S. H O U L T , Inhibition t~v ethanol and mepacrine of phospholipase-dependent prostaglandin release from the isolated perfused rat lung, European J. Pharmacol. 120 (1986) 145-150. To investigate the possibility that millimolar concentrations of ethanol have a m e m b r a n e - d i r e c t e d inhibitory effect on phospholipase A 2 and prostanoid generation (suggested from previous platelet experiments), we studied the release of prostacyclin, t h r o m b o x a n e A z a n d prostaglandin E 2 from isolated perfused rat lung. Prostanoid release was evoked by arachidonic acid, b r a d y k i n i n and ionophore A23187 and was measured after extraction by radioimmunoassay. In these experiments, prostanoid release is d e p e n d e n t upon biosynthesis from fatty acid precursors as there is no endogenous prostanoid storage pool. Arachidonic acid and bradykinin caused e n h a n c e d release of more prostacyclin than t h r o m b o x a n e A 2 with much less prostaglandin E 2 and no detectable prostaglandin ~,,, whereas A23187 released equal p r o p o r t i o n s of prostacyclin and t h r o m b o x a n e A 2 with less prostaglandin E 2. Ethanol at 50 m M resembled mepacrine (46 /zM) in that prostanoid release in response to bradykinin and A23187 was highly significantly reduced with little effect on release induced by arachidonic acid. We suggest that ethanol, like mepacrine, interferes with prosta.glandin generation by an action at the phospholipase step. This may be secondary to a physical effect on m e m b r a n e configuration.

Lung Prostaglandins Inhibition

Prostacyclin

Thromboxane

1. Introduction

The biosynthesis of prostaglandins requires the cleavage from membrane phospholipids of their polyunsaturated precursor fatty acids, notably arachidonic acid. This fatty acid is esterified primarily at the 2'-acyl position of phospholipids in biological membranes, and phospholipase A 2 has been proposed to be responsible for its liberation, although other enzymes may also be involved (see Irvine, 1982). The mechanism by which phospholipase A~ activity is controlled has not been firmly estab-

* Present address: School of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, U.K. ** To whom all correspondence should be addressed. 0014-2999/86/$03.50 :,5, 1986 Elsevier Science Publishers B.V.

Ethanol

Mepacrine

Phospholipase A

lished, but there are several possibilities (see Isakson et al., 1978; Van den Bosch, 1980). The activity of all phospholipase A~ enzymes except those in lysozomes is Ca2+-dependent, and prostaglandin synthesis does occur in response to increased intracellular Ca 2+ levels (e.g. Forstermann and Hertting, 1979; Juan, 1979; Thomas et al., 1981). Regulation of phospholipase A~ by means of interaction with an inhibitory protein has been suggested (Hirata, 1981), and the further possibility of control by means of modulation of the membrane microviscosity also exists. Phospholipase A, is membrane-bound, and thus the membrane phospholipids provide both its environment and its substrate. The activity of many membrane-located enzymes can be affected by the thermodynamic state of their environment (Farias et al., 1975: Sandermann, 1978; Gordon et al., 1980). Indeed,

146

the activity of exogenous soluble phospholipase A : enzymes such as those from pig pancreas and bee or snake venom is markedly affected by the physical state of their substrate, although this may be related to the penetration of the enzyme into the substrate prior to the catalytic step (Jain and Cordes, 1973; Jain et al., 1982) and may not be entirely relevant to those phospholipases which are already membrane-bound. Ethanol, a membrane fluidising agent (Chin and Goldstein, 1977), inhibits platelet aggregation to a variety of agents such as thrombin and collagen but not to arachidonic acid {Haut and Cowan, 1974; Pennington and Smith, 1979; Fenn and Littleton, 1982). This may be due to inhibition of phospholipase A 2, for, in addition, ethanol inhibits the loss of radiolabelled arachidonic acid from phosphatidylcholine but not from phosphatidylinositol which occurs upon platelet activation (Fenn and Littleton, 1984; Hoult and Peers, 1985). The experiments reported here were performed to investigate whether ethanol inhibits phospholipase A~ in another tissue, and to compare its actions with those of a drug with established phospholipase A e inhibitory activity, namely mepacrine. The isolated perfused lung was chosen since it has been used to demonstrate the presence of phospholipase A 2 activity, as shown by hydrolysis of infused [~H]phosphatidylcholine (Flower and Blackwell, 1976), and drugs which inhibit phospholipase A : such as mepacrine or the corticosteroids (via the steroid-induced protein lipocortin, Flower, 1984) inhibit pulmonary prostaglandin synthesis in response to several stimuli (Vargaftig and Dao Hal, 1972; Blackwell et al., 1978; Robinson and Hoult, 1980).

2. Materials and methods

Lungs from male Wistar rats of 150 to 200 g were removed, cannulated via the pulmonary artery and perfused at 6 m l / m i n with warmed (37°C) Krebs' solution gassed with 95% O 2, 5% C02. Potential inhibitor drugs were dissolved in Krebs' solution and perfused continuously throughout the experiment; 'challenge' substances were dissolved

in Krebs' solution and infused over 1 min via a side line after an initial perfusion period necessary to obtain a steady basal efflux of prostaglandins. Effluent from the lungs was collected in 5 min fractions (20 ml), acidified to pH 3 with hydrochloric acid and the prostaglandins extracted by passing the effluent over Sep-Pak C18 reverse phase cartridges (Waters) prepared by prewetting with acetonitrile and distilled water, and eluting with 5 ml ethyl acetate. Afer removing solvent, the residues were taken up in 1 ml distilled water and the prostaglandin content was measured by double antibody radioimmunoassay of suitable aliquots (2 to 15 #1) using rabbit antibodies to 6-keto PGF~,, (for prostacyclin), thromboxane B, (for thromboxane A2), and in some experiments, prostaglandins E~ and F2,. The antibodies to 6-keto PGF~,~ and T X B 2 w e r e used at final titres of 1 : 5200 and 1:4000 giving a sensitivity of approximately 10 pg, and had cross-reactivity to PGE 2, PGF2,~, P G D 2, TXB 2, 6-keto PGE 1 and 6-keto PGFI, of (in percentages): 3.2, 0.91, 0.2, 1.36, 9.2 and 100, and 0.26, 0.14, 1.13, 100, 0.12 and 0.02, respectively, with inter-assay coefficients of variation for the two assays of, respectively, 12.7 and 13.6%. The ~H,-tracers were purchased from Amersham International, U.K., at specific activities of 140-160 Ci/mmol. Arachidonic acid, bradykinin and mepacrine were purchased from Sigma Chemical Co. (Poole, U.K.) and the ionophore A23187 was purchased from Calbiochem (Bishops Stortford, U.K.).

3. Results

An initial perfusion period of 30 min was necessary to stabilise the tissue and to establish a steady rate of basal release of prostaglandins. Once established, this remained constant over the time period of the experiment, and was not affected by small alterations (_+15%) in the perfusion rate. The basal efflux varied between lungs, and was therefore measured for each lung prior to infusion of the challenge substance. Each lung was also used for one test only, since preliminary results showed that subsequent responses of the tissue

147

6 ketoPGFl(o

TABLE 1 Basal release of prostaglandins from the perfused rat lung, and its inhibition by mepacrine and ethanol. Data shown in the form of ng released/5 rain mean _+S.E.M. for (n) lungs. Results are shown from one batch of animals. Perfusion conditions Control Mepacrine 46/zM Ethanol 50raM

TXB2

= 6 0 " L ~ 40-

40

20-

20

ii1

Release ( n g / 5 min) of ~

TXB 2

6-keto PGFI,

11.3_+0.6(6)

12.3_+0.8(6)

PGE 2 2.5_+0.6(6)

lO l lO

2.8+0.1(6)"

4.9+0.6(6)"

<0.5

(6) ~'

2.8+0.3(5)"

4.4+0.6(5)"

<0.5

(5) ~'

P < 0.001 compared to control value.

were not reproducible. Thus perfusion experiments were completed within 60 rain. Under these conditions, isolated perfused rat lungs continuously released small amounts of TXB 2, 6-keto P G F I , and P G E : , as shown in table 1 and fig. 1. PGF2, could not be detected. The inhibitor substances mepacrine and ethanol substantially and highly significantly reduced the basal efflux of all three prostaglandins (fig. 1, table 1): consequently 'evoked' release in response to challenge substances was calculated by deducting basal release values obtained prior to infusion from the release following infusion. Ionophore A23187 (fig. 1), bradykinin and arachidonic acid all released TXB2 and 6-keto

PGE2

30

-10 A/ 10 30 Time,rain

-,; t ,;

3;

Fig. 1. Release of 6-keto PGFh,, TXB 2 and PGE 2 from the isolated perfused rat lung in response to 10 /tg ionophore A23187 injected at the arrow. Results show mean values+ S.E.M. in 6 control lungs (O) perfused with normal Krebs' solution and in 5 lungs perfused with 50 mM ethanol ('*'). All points with ethanol were significantly different from control, P < 0.05 by S t u d e n t s unpaired t-test. Note that in this experiment analysis of prostanoids was continued for 30 rain postinjection, cf. table 2.

PGFh~ from the perfused rat lungs, as shown in table 2. PGE 2 release was less, and was not measured in every experiment; P G ~ , was not detectable. It should be noted that direct comparisons of the potency of the three challenge substances cannot be made because of variations between different batches of animals in the absolute amounts of prostanoids released under basal or challenge conditions. The evoked release in response to bradykinin and arachidonic acid consisted of approxi-

TABLE 2 Release of prostaglandins from isolated perfused rat lung following infusion of ionophore A23187, bradykinin and arachidonic acid, and the effects of mepacrine and ethanol. Data shown in the form of ng released, mean_+ S.E.M. for (n) lungs. * Evoked release is calculated as ng released over 20 min following infusion of challenge substance, corrected for basal release measured immediately prior to infusion of challenge substance in the same lung. Perfusion conditions

Control Mepacrine 46 p,M Ethanol 50 mM

Evoked release * of TXB 2 and 6-keto PGF]. in response to A23187 10 ~g

Bradykinin 10 ,ug

Arachidonic acid 10 ,ttg

TXB 2

6-kPGF l,,

TX B~

6-kPGF I.

TX B2

6-k PG F I,,

146.5 + 16.4 (6) 14.4+ 6.6 ~ (5) 13.8_+ 7.8 ~ (5)

140.5 _-4-22.6 (5) 32.3_+ 7.5b (5) 9.9_+ 3.7 ~ (5)

13.5 4- 2.6 (4) 3.0_+1.5 ~ (5) 3.5_+1.1 b (5)

27.7 + 3.1 (4) 4.7_+1.6" (5) 14.3_+3.8" (5)

! 9.7 + 5.7 (5) 9.1 _+3.8 (4) 16.5+7.4 (6)

57.6 + 18.9 (5) 18.7+ 3.4 (4) 31.3+ 7.4 (6)

a p < 0.05, b p < 0.01, c p < 0.001, compared to control value.

148 mately twice as much 6-keto PGF~,, as TXB, (table 2); however, similar amounts of the two prostaglandins were released in response to infusion of ionophore A23187 (table 2, fig. 1). Experiments with the inhibitor substances were therefore performed using the same batch of animals as the control experiments, and table 2 shows that mepacrine and ethanol significantly inhibited the evoked release of 6-keto PGF1, ~ and TXB: following infusion of ionophore and bradykinin but not that due to arachidonic acid. Figure 1 illustrates the results for A23187-induced release of 6-keto PGF~,~, TXB~ and PGE 2 and shows the substantial inhibition of this and of the basal rate of release of the three prostaglandins in the presence of 50 mM ethanol. Similar results were obtained with 46 /xM mepacrine. Mepacrine also reduced the prostaglandin release in response to arachidonic acid, but this was not significant (see table 2).

4. Discussion

Basal prostaglandin release from the perfused rat lung consisted approximately of equal amounts of TXB~ and 6-keto PGF~, with smaller amounts of PGE 2, but PGF2, ~was not detected. These findings agree in general with results from lung fragments (Ally et al., 1982). It should be noted that in this context, release of prostanoids does not imply release from an endogenous storage pool; rather it indicates enzyme-catalysed biosynthesis from unsaturated fatty acid precursors. The difference in the ratio of 6-keto P G F ~ and TXB~ in the evoked release following infusion of bradykinin and arachidonic acid vs. A23187 might be due to differences in the cellular location of synthesis of these prostaglandins, since 6-keto PGF~,, may originate largely from the vascular endothelium, whereas TXB 2 is thought to be released from other cell populations (Bakhle, 1981). The ratio of released substances may thus vary according to the route of administration and cellular targets of the challenge drug, as well as the accessibility to the drug of different cell populations and the mechanism by which prostaglandin synthesis is stimulated. For example, the evoked

release following infusion of ionophore A23187 was more prolonged than that following the other challenge substances, remaining elevated until 2030 rain after infusion of the ionophore (fig. I), whereas release following arachidonic acid and bradykinin had returned to basal levels within 15 min. The results suggest that ethanol acts to inhibit prostaglandin synthesis in the perfused rat lung at the phospholipase step, since it substantially inhibits the evoked release caused by ionophore A23187 and bradykinin, but has little effect on that following infusion of arachidonic acid. In addition, we have previously shown (Hoult and Peers, 1983) that cyclo-oxygenase prepared from sheep seminal vesicles is unaffected by low concentrations of ethanol, and indeed that supra-molar concentrations are necessary to inhibit enzymatic activity. Two other possibilities for the mode of action of ethanol can be considered, but must be regarded as improbable. Firstly, it is unlikely that ethanol acts to reduce the access of the stimulating agents to the prostanoid-releasing cells since it is well known that ethanol is a vasodilator. Secondly, any effect of ethanol to enhance prostaglandin degradation (thereby reducing apparent biosynthesis) may be discounted because we have shown that ethanol inhibits prostaglandin metabolism via prostaglandin 15-hydroxydehydrogenase (Hoult and Peers, unpublished experinaents). The effects of ethanol on phospholipase resemble those of mepacrine (used in our experiments as a reference inhibitor), which are considered to be due to a direct action on phospholipase At (Vargaftig and Dao Hai, 1972; Flower and Blackwell, 1976; Blackwell et al., 1977). However under certain circumstances mepacrine has also been shown to affect cyclo-oxygenase activity (Nijkamp et al., 1976; Blackwell et al., 1977), an action which might explain the modest but not significant reduction in prostaglandin release in response to arachidonic acid seen here with mepacrine (table 2). Both ethanol and mepacrine reduced the basal efflux of prostaglandins from the lung, suggesting a continuous low level of phospholipase activity. It has been suggested that there are several pools of phospholipase A~ in the lung, in that

149 treatment with anti-inflammatory

steroids reduces

p r o s t a g l a n d i n r e l e a s e in r e s p o n s e to h i s t a m i n e a n d o v a l b u m i n b u t n o t to b r a d y k i n i n in t h e g u i n e a - p i g l u n g ( B l a c k w e l l et al., 1 9 7 8 ; R o b i n s o n a n d H o u l t , 1980). T h i s m i g h t b e d u e to t h e p r e s e n c e o f m a n y d i f f e r e n t cell t y p e s p r e s e n t w i t h i n t h e l u n g , e a c h p o s s i b l y p r o d u c i n g d i f f e r e n t p r o s t a g l a n d i n s in res p o n s e to v a r i o u s s t i m u l i a n d w i t h d i f f e r e n t i a l s e n s i t i v i t y to a n t i - p h o s p h o l i p a s e f a c t o r s . E t h a n o l d o e s n o t a p p e a r to d i f f e r e n t i a t e b e t w e e n t h e s e p o s t u l a t e d p o o l s , a l t h o u g h m e a s u r e m e n t s o f its e f f e c t s u p o n p r o s t a g l a n d i n r e l e a s e in r e s p o n s e to other agents, and a comparison with the effects of a n t i - i n f l a m m a t o r y s t e r o i d s in t h e r a t l u n g s h o u l d be made. The mechanism by which ethanol inhibits phosp h o l i p a s e A 2 is n o t clear, b u t its l o w p o t e n c y c o m p a r e d w i t h m e p a c r i n e s u g g e s t s t h a t it m a y b e a n o n - s p e c i f i c effect. E t h a n o l is a m e m b r a n e - f l u i d i s i n g a g e n t ( C h i n a n d G o l d s t e i n , 1977), a n d t h i s a c t i o n m a y b e r e s p o n s i b l e for t h e i n h i b i t o r y a c t i o n o n p h o s p h o l i p a s e A 2, e i t h e r b y a f f e c t i n g e n z y m e conformation within the membrane or indirectly b y a l t e r i n g p h o s p h o l i p i d c o n f o r m a t i o n to p r e v e n t t h e i r u s e as s u b s t r a t e m o l e c u l e s . A l t e r n a t i v e l y , e t h a n o l a n d o t h e r a n a e s t h e t i c s h a v e b e e n s h o w n to i n c r e a s e b i n d i n g o f C a 2 ÷ to b i o l o g i c a l m e m b r a n e s ( S e e m a n , 1972; O h n i s h i et al., 1980; H a r r i s a n d F e n n e r , 1982), a n d t h i s r e d i s t r i b u t i o n o f i n t r a c e l l u lar Ca 2+ might affect the activation of phospholip a s e A 2. T h e p o s s i b i l i t y t h a t o t h e r e n z y m e s c a p a b l e o f a r a c h i d o n a t e r e l e a s e s u c h as p h o s p h o l i p a s e C or diglyceride lipase may also be affected by ethanol also requires investigation.

Acknowledgement S.H.P. thanks the M.R.C. for the award of a studentship.

References Ally, A.I., R. Boucher, M.R. Knowles and T.E. Eling, 1982, The metabolism of prostaglandin endoperoxides by microsomes from human lung parenchyma and comparison with metabolites produced by pig, bovine, rat, mouse and guinea-pig, Prostaglandins 24, 575. Bakhle, Y.S., 1981, Biosynthesis of prostaglandins and thromboxanes in lung, Clin. Resp. Physiol. 17, 491.

Blackwell, G.J., W.G. Duncombe, R.J. Flower, M.F. Parsons and J.R. Vane, 1977, The distribution and metabolism of arachidonic acid in rabbit platelets during aggregation and its inhibition by drugs, Br. J. Pharmacol. 59. 353. Blackwell, G.J., R..I. Flower, F.P. Nijkamp and J.R. Vane, 1978, Phospholipase A 2 activity of guinea-pig isolated perfused lungs: stimulation and inhibition by anti-inflammatory steroids, Br. J. Pharmacol. 62, 79. Chin, J.H. and D.B. Goldstein, 1977. Effects of low concentrations of ethanol on the fluidity of spinlabelled erythrocyte and brain membranes, Mol. Pharmacol. 13. 435. Farias, R.N,, B. Blos, R.D. Moren, F. Sineriz and R.E. Toniea, 1975, Regulation of allosteric membrane-bound enzymes through changes in lipid composition, Bioehim. Biophys. Acta 415, 235. Fenn, C.G. and J.M. Linleton, 1982, Inhibition of platelet aggregation by ethanol in vitro shows specificity for aggregating agent used and is influenced by platelet lipid composition, Thromb. Haemostas. 48, 49. Fenn, C.G. and J.M. Littleton, 1984, Ethanol inhibits thrombin-stimulated breakdown of platelet membrane phospholipids, Br. J. Pharmacol. 81, 175P. Flower, R.J., 1984, Macrocortin and the antiphospholipase proteins, in: Advances in Inflammation Research, Vol. 8, ed. G, Weissmann (Raven Press, New York) p. 1. Flower, R.J. and G.J. Blackwell, 1976, Importance of phospholipase A, in prostaglandin biosynthesis, Biochem. Pharmacol. 25, 285. Forstermann, U. and G. Hertting, 1979, The importance of Cae+-mediated phospholipase A,-activation for stimulusevoked PGE, release from rabbit splenic capsular strips, Naunyn-Schmiedeb. Arch. Pharmacol. 307, 243. Gordon, L.M., R.D. Saverheber, J.A. Esgate, I. Dipple, R.J. Marchmont and M.D. Houslay, 1980, The increase in bilayer fluidity of rat liver plasma membranes achieved by the local anaesthetic benzyl alcohol affects the activity of intrinsic membrane enzymes, J. Biol. Chem, 255, 4519. Harris, R.A. and D. Fenner, 1982, Ethanol and synaptosomal calcium binding, Biochem. Pharmacol, 3l, 1790. Haut, M. and D. Cowan, 1974, The effect of ethanol on hemostatic properties of human blood platelets, Am. J, Med. 56, 22. Hirata, F., 198l, The regulation of lipomodulin, a phospholipase inhibitory protein, in rabbit neutrophils by phosphorylation, J. Biol. Chem. 256, 77301 Hoult, J.R.S. and S.H. Peers, 1983, Effect of lipid bilayer fluidising agents on cyclo-oxygenase activity, Br. J. Pharmacol. 78, 52P. Houh, J.R.S. and S.H. Peers, 1985, Effects of ethanol and mepacrine on arachidonate mobilisation in thrombin- and A23187-stimulated platelet phospholipids, Br. J. Pharmacol. 84, 153P. lrvine, R.F.. 1982, How is the level of free arachidonic acid controlled in mammalian cells? Biochem. J. 204, 3. lsakson, P.C., A. Raz, W. Hsueh and P. Needleman, 1978, Lipases and prostaglandin biosynthesis, in: Advances in Prostaglandin and Thromboxane Research, Vol. 3, eds. C. Galli, G. Galli and G. Porcellati (Raven Press, New York) p. 113.

150

Jain, M.K. and E.H. Cordes. 1973, Phospholipases II. Enzymatic hydrolysis of lecithin: effects of structure, cholesterol content and sonication, J. Membr. Biol. 14, 119. Jain, M.K., M.R. Egmond, H.M. Verheij, R. Apitz-Castro, R. I)ijkman and G.H. De Haas, 1982, Interaction of phospholipase A , and phospholipid bilayers, Biochim. Biophys. Acta 688, 341. Juan, H., 1979, Role of calcium in prostaglandin E release induced by bradykinin and the ionophore A23187, Naunyn-Schmiedeb. Arch. Pharmacol. 307, 177. Nijkamp, F.P., R.J. Flower, S. Moncada and J.R. Vane, 1976, Partial purification of rabbit aorta contracting substance-releasing factor and inhibition of its activity by antiinflammatory steroids. Nature 263,479. Ohnishi, S.T., D.M. Obzansky and H.L. Price, 1980, Increase in ('a -~' binding of cardiac plasma membrane lipoprotein caused by general anaesthetics and alcohol, Can. J. Physiol. Pharmacol. 58, 525. Pennington, S.N. and C.P. Smith, 1979, The effect of ethanol on thromboxane synthesis by blood platelets, Prostaglandins Med. 2, 43.

Robinson, C. and J.R.S. Hoult, 1980, Evidence for functionally distinct pools of phospholipase responsible for prostaglandin release from the perfused guinea-pig lung, European J. Pharmacol. 64, 333. Sandermann, H., 1978, Regulation of membrane enzymes by lipids. Biochim, Biophys. Acta 515, 209. Seeman, P., 1972, The membrane actions of anaesthetics and tranquillizers, Pharmacol. Revs. 24, 583. Thomas, J.M.F., H. Chap and L. Douste-Blazy, 1981, Calcium ionophore A23187 induces arachidonic acid release from phosphatidylcholine in cultured h u m a n endothelial cells, Biochem. Biophys. Res. Commun. 103, 819. Vargaftig, B.B. and N. Dao Hal, 1972, Selective inhibition by mepacrine of the release of 'rabbit aorta contracting substance' evoked by the administration of bradykinin, J. Pharm. Pharmacol. 24, 159. Van den Bosch, H., 1980, Intracellular phospholipase A, Biochim. Biophys. Acta 604, 191.