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Evidence for functionally distinct pools of phospholipase responsible for prostaglandin release from the perfused guinea-pig lung
European Journal of Pharmacology, 64 (1980) 333--339
333
© Elsevier/North-Holland Biomedical Press
EVIDENCE FOR FUNCTIONALLY DISTINCT POOLS OF PHOSPHOLIPASE RESPONSIBLE FOR PROSTAGLANDIN RELEASE FROM THE PERFUSED GUINEA-PIG LUNG CLIVE ROBINSON * and J.R.S. HOULT
Department of Pharmacology, King's College, Strand, London WC2R 2LS, U.K. Received 29 January 1980, revisedM S received 25 March 1980, accepted 31 March 1980
C. ROBINSON and J.R.S. HOULT, Evidence for functionally distinct pools of phospholipase responsible for prostaglandin release from the perfused guinea-pig lung, European J. Pharmacol. 64 (1980) 333--339. The release of prostaglandin- and thromboxane-like material from the isolated perfused guinea-pig lung was elicited by challenge with arachidonic acid, bradykinin, histamine and ovalbumin (in ovalbumin-sensitised animals), and detected by superfusion cascade bioassay. Mepacrine inhibited release to all agents except arachidonic acid. Dexamethasone and fludrocortisone inhibited release induced by histamine and anaphylactic challenge but not by bradykinin or arachidonic acid. These results suggest two functionally distinct pools of phospholipase responsible for initiating prostaglandin biosynthesis in the guinea-pig lung. PG biosynthesis
Guinea-pig lungs
Phospholipase pools
1. Introduction The synthesis and release of prostaglandins and related substances from cells requires the cleavage from membrane phospholipids of their polyunsaturated fatty acid precursors (e.g. arachidonic acid). This is followed by transformations catalysed by the lipoxygenase and cyclo-oxygenase pathways (reviewed by Samuelsson et al., 1978; Vane, 1978; Lands, 1979). However, two factors limit our understanding of how the signals for synthesis are recognised and coupled to membrane events: neither the phospholipase(s) presumed to be responsible for releasing the fatty acids nor the precise phospholipid pools from which they derive have yet been conclusively identified. Phospholipase A2 is generally regarded as
* Present address: Department of Clinical Pharmacology, Royal Postgraduate Medical School, Ducane Rd, London W12 OHS, U.K.
Inhibitors
the prime candidate (Kunze and Vogt, 1971; Flower and Blackwell, 1976; Vogt, 1978) since it selectively hydrolyses phospholipids at the 2'-acyl position (Van den Bosch, 1974) and the largest amounts of eicosapolyeneoic acids are attached at this position (Flower, 1978). Furthermore, phospholipase A2 causes the release of prostaglandins from intact and isolated tissues (Bartels et al., 1970; Juan, 1979; FSrstermann and Hertting, 1979). However, recent studies have shown that other types of phospholipase enzymes as well as other more complex interactions between phospholipids may effect the release of arachidonic acid, and they imply that the phospholipase step in prostaglandin biosynthesis is not as straightforward as first thought (Rittenhouse-Sirnmons, 1979; Bell et al., 1979; Lapetina and Cuatrecasas, 1979; Hirata et al., 1979). The use of mepacrine (quinacrine), an inhibitor of phospholipase A2 (Markus and Ball, 1969), and of anti-inflammatory steroids -- which are thought to reduce prostaglandin synthesis and release from intact cells by
334
inhibiting phospholipase A2 (Flower, 1978) -- affords a pharmacological method whereby the existence of different functional pools of phospholipase and their relationships to triggers of prostaglandin synthesis may be investigated experimentally. Such a study requires an intact cell system in which prostaglandin biosynthesis can be stimulated by a variety of different effectors both before and after application of inhibitors. We describe here experiments in which prostaglandin and thromboxane release from the perfused guinea-pig lung were stimulated in four different ways and monitored by immediate superfusion cascade bioassay. The results suggest that there are two functionally distinct pools of phospholipase.
2. Materials and methods Lungs from male guinea pigs (Dunkin-Hartley, 250--350 g) were removed, cannulated at the pulmonary artery and perfused at 6 ml/ min with warmed and well~xygenated Krebs solution containing antagonists (atropine 1.8 #M, methysergide 0.48 pM, phentolamine 0.6 pM and practolol 5.6 ~zM). Effluent from the lungs was passed over a superfusion cascade bioassay which generally comprised two rabbit aorta strips, a rat fundus strip and a rat colon. A separate line was used to perfuse Krebs solution containing indomethacin 8.0 pM, hexamethonium 7.0 pM and mepyramine 0.60 pM at 1.0--1.5 ml/min directly over the assay tissues. Responses of the tissues to prostaglandins and other agonists were evoked by injection either at the top of the cascade or into the cannulated pulmonary artery. The stable prostaglandin endoperoxide analogue U46619 (15S-hydroxy-ll~, 9a-epoxymethanoprosta-5Z, 13E
C. ROBINSON, J.R.S. HOULT
after checking by direct superfusion over the cascade that they did not inhibit the responsiveness of the assay tissues. Sensitised guinea pigs were prepared according to the method of Math~ et al. (1977) (50 mg ovalbumin in 0.9% saline injected s.c. and i.p. on day 1, 50 mg i.p. on day 3) and used after 14 days. Control unsensitised animals were similarly injected with vehicle only. Prostaglandin E2 and the endoperoxide analogue U46619 were kind gifts from Dr. J.E. Pike, The Upjohn Company, U.S.A. and mepacrine hydrochloride was a gift from Dr. J.M. Young, University of Cambridge. Dexamethasone acetate, fludrocortisone acetate and bradykinin triacetate were from Sigma. The steroids were made up in a small volume of ethanol and diluted into Krebs solution before perfusion. At this concentration the ethanol did not affect pulmonary generation of prostaglandins or thromboxanes.
3. Results
Four substances were used to elicit the release of prostaglandin- and thromboxanelike material from the isolated perfused guinea-pig lung (table 1): histamine, bradykinin, arachidonic acid and ovalbumin (in sensitised lungs). In all cases, injection into the pulmonary artery was followed by strong contractions of the rabbit aorta and rat fundus strip preparations (fig. l ) which could not be matched by injection of the trigger substances over the tissues alone. The responses of the rat colon were both smaller and more variable; generally it showed a small increase in tone associated with bursts of contractile activity. Bradykinin was the only agent which consistently produced marked contractions of the rat stomach strip at the doses used, but sometimes high doses of arachidonic acid also caused it to contract (e.g. fig. 1); however, in both cases the responses were smaller, of shorter duration and had a different tissue profile when injected direct to the tissues. Since similar responses to those elicited by
PHOSPHOLIPASE TABLE
POOLS
AND
PG SYNTHESIS
335
1
Release of prostaglandin- and thromboxane-like material from perfused guinea-pig lung and inhibition by substances which interfere with phospholipase A 2.
Perfusion conditions
Control Mepacrine Dexamethasone Fludrocortisone
Inhibitor concentration (pg/ml)
Release of PG and TX-like material in response to l Bradykinin 2--8 pg
Arachidonate 2--20 pg
Histamine 1--10 pg
Ovalbumin 10 pg 1 mg
-20 4 20
15 0 6 4
14 4 4 2
11 0 0 0
18 (18) 0 (3) d 1 (6) bcd 2 2(12) 2
(18) (5) a (6) cd (4)
(17) (5) a (4) bce (2)
(12) (3) d (4) cd (6)
1 Data shown in the form M(N) where M is the number of times partial or complete release of PG/TX-like material was observed and N the total number of challenges made under these conditions. 2 The 3/18 'failures' of complete inhibition occurred before the steroids' effect was maximal: later challenges in the same lung showed complete inhibition. Superscript letters denote similar results obtained in guinea-pig lung superfusion cascade bioassay experiments by : a Vargaftig and Dao Hal (1972); b Gryglewski et al. (1975), but using hydrocortisone; c Blackwell et al. (1978); d Blackwell et al. (1978), but using radiochemical assay for phospholipase A 2 activity (see Discussion); e Nijkamp et al. (1976).
the four triggers could be obtained by direct i n j e c t i o n o v e r t h e t i s s u e s o f p r o s t a g l a n d i n E2 and the thromboxane A2-1ike a n a l o g u e U46619, we conclude that the trigger substances cause pulmonary synthesis and release
of prostaglandin- and thromboxane-like material ............ Mepacrine abolished the release of prostag. landin- and thromboxane-like m a t e r i a l in r e s p o n s e t o all t r i g g e r s e x c e p t a r a c h i d o n i c
I
I
,
I
I
I
I
0
BK
BK
2pg D
2 }..Ig L
AA lO)Jg D
AA 10 ).Jg L
H 5.pg D
14 5~g L
200 pg L
Fig. 1. Release of prostaglandin- and thromboxane-like material from perfused guinea-pig lung challenged with ovalbumin (O), bradykinin (BK), arachidonic acid (AA) and histamine (H). Spasmogenic substances were detected by superfusion cascade bioassay, routinely using the rat stomach fundus strip (RSS) and rabbit aortic strip (RAS). Injections were made via the lung (L) or direct to the tissues (D). The figure shows representative results from the tests carried out in the absence of inhibitors as summarised in table 1. For clarity, the responses on the rat colon have been omitted (see Results). Time bar indicates 5 min.
336
acid (table 1). Complete inhibition, was observed within 10 min of starting mepacrine infusion. Synthesis of prostaglandins from exogenously applied arachidonic acid is initiated by the action of cyclo-oxygenase and is independent of any phospholipase(s) or their inhibitors. In 4 out of 6 experiments where survival of the lung and assay tissues permitted it was found that inhibition by mepacrine was slowly reversible within 45--85 min of discontinuing its infusion, although judging by the yellow colour of the lung some mepacrine was still retained within the tissue. Dexamethasone and fludrocortisone were also tested as possible inhibitors of the phospholipases involved in pulmonary prostaglandin release (table 1). Histamine- and anaphylactically induced synthesis were inhibited. The inhibition had a slow onset, reaching its maximum 40--70 min after starting the steroid (based on 28 tests in 13 lungs, using both histamine and ovalbumin challenge) and was maintained for at least 140 min after stopping it (2 experiments using ovalbumin challenge). However, the steroids did not inhibit prostaglandin/thromboxane release triggered by bradykinin or by arachidonic acid, even when these challenges were made 70 or more minutes after starting the steroid infusion and when maximal inhibition to histamine and ovalbumin had been achieved.
4. Discussion These experiments provide evidence for the presence of two or more functionally distinct pools of phospholipase responsible for initiating prostaglandin biosynthesis and release. Thus the two anti-inflammatory steroids inhibited prostaglandin and thromboxane release in response to anaphylactic challenge and histamine but not that induced by bradykinin. All three triggers are considered to require activation of phospholipase A2. Inhibition of prostaglandin formation and release from intact cells by anti-inflammatory steroids is well documented (see Flower and
C. ROBINSON, J.R.S. HOULT
Blackwell, 1979; Di Rosa and Persico, 1979), and it is thought that its expression depends upon the synthesis of new protein (Danon and Assouline, 1978; Di Rosa and Persico, 1979; Russo-Marie et al., 1979). This explains why its effects take some time to develop, as observed here. Indeed, Flower and Blackwell (1979) have identified a soluble protein factor with anti-phospholipase properties which was released from perfused guinea-pig lungs after treatment with dexamethasone. The simplest explanation for our findings is that the steroids or the steroid-induced factor(s) selectively inhibit some but not all of the phospholipases involved in the biosynthesis of prostaglandins, and that there are at least two independent mechanisms for the recognition of different triggers for prostaglandin synthesis and subsequent phospholipase activation. In contrast, mepacrine inhibited synthesis and release in response to all three phospholipase triggers, showing it to be a relatively powerful but non-selective inhibitor of phospholipase action. Inhibition was rapid in onset, and this is consistent with a direct inhibitory action on phospholipase A2 as shown by others (Vargaftig and Dao Hai, 1972; Flower and Blackwell, 1976; Vargaftig, 1977; Blackwell et al., 1977; Hirata et al., 1979). The use of mepacrine for investigating the role of phospholipase is therefore limited, and it is further hampered by the fact that it has a number of other pharmacological and biochemical actions, some of which are non-specific (Rollo, 1970; Vargaftig, 1977). Mepacrine may also interfere in other ways with prostaglandins: for example, it inhibits cyclooxygenase action (Nijkamp et al., 1976; Blackwell et al., 1977), although this was not observed in these experiments or by Vargaftig and Dao Hai (1972). Several of these observations concerning pulmonary prostaglandin and thromboxane release and its inhibition by drugs have been made previously (as indicated in the footnote to table 1), but they have not been the object of a single comparative study. However, there
PHOSPHOLIPASE
POOLS AND PG SYNTHESIS
are some discrepancies. Nijkamp et al. (1976) found that both mepacrine and fludrocortisone depressed the release of thromboxane A2 by arachidonate (implying cyclo-oxygenase inhibition), although precise details were not given. In subsequent experiments the same research group (Blackwell et al., 1978) found that bradykinin-induced stimulation of phospholipase A2 activity was not inhibited by mepacrine at 75 pg/ml. However, these latter results relate to two observations only and were made using a radiochemical assay for phospholipase A2 in which the lung is perfused with [3H]-2-oleoyl phosphatidylcholine. The effect of mepacrine on the release of spasmogenic substances by bradykinin was not tested by cascade bioassay. Nevertheless, in reviewing their data these authors concluded, as we do here, that 'it seems necessary to postulate the existence of a steroid-sensitive and a steroid-insensitive population of receptors or phospholipase enzymes . . . . . '. Our suggestion that at least two pools of phospholipase exist to effect coupling between receptor activation and prostaglandin biosynthesis receives further support from studies published while our experiments were in progress. Rittenhouse-Simmons (1979), Lapetina and Cuatrecasas (1979) and Bell et al. (1979) have shown that platelets contain a phosphatidylinositol-specific phospholipase C which has high activity and is capable of the rapid release of arachidonate-containing phosphatidic acid in response to aggregating agents such as thrombin. According to Bell et al. (1979), free arachidonate is released by subsequent hydrolysis of the phosphatidic acid by diglyceride lipase. Since platelets are also known to contain small amounts of phospholipase A2 activity (Derksen and Cohen, 1975; Bell et al., 1979), it is possible that two independent routes for release in platelets of arachidonic acid may coexist. Needleman and coworkers have postulated two pathways for activation of prostaglandin synthesis in rabbit heart, one of which is hormone-sensitive and specifically releases arachidonic acid (Isakson et al., 1978). The other
337
pathway, activated by ischaemia, results in non-specific lipolysis and arachidonate is one among many products. This idea is further supported by the same group's studies on prostaglandin synthesis in the hydronephrotic (ureter~clamped) rabbit kidney (Needleman et al., 1979). When challenged repeatedly with bradykinin or angiotensin II the kidney releases successively more prostaglandin-like substance due to de novo synthesis of cyclooxygenase (and possibly, therefore, of phospholipase); however, this only represents a small proportion of the total cyclo-oxygenase and the total pool does not appreciably increase in amount after each bradykinin challenge. This implies an 'exquisite compartmentation' (sic) of the prostaglandin synthesising system. In the light of these results it would be interesting to see if this bradykinininduced component is sensitive to inhibition by anti-inflammatory steroids or mepacrine, and, conversely, to examine the effects of these drugs on angiotensin II-induced prostaglandin and thromboxane synthesis in the lung. We could not test this because angiotensin is strongly spasmogenic to all the assay tissues used. Taken together, the results of this study and those discussed above suggest that several pools of phospholipase may exist to effect the coupling between receptor activation and prostaglandin synthesis. Certainly, the simplifying hypothesis that synthesis depends upon phospholipase A2 activation may require more detailed elaboration. Acknowledgements We thank Pharmacia AB and the trustees of the Layton Science Research Prize (King's College) for the award of funds for this research and Drs. J.E. Pike and J.M. Young for gifts of prostaglandins and mepacrine respectively.
References
Bartels, J., H. Kunze, W. Vogt and G. Wille, 1970, Prostaglandin: liberation from and formation in
338 perfused frog intestine, Naunyn-Schmiedeb. Arch. Pharmacol. 266, 199. Bell, R.L., D.A. Kennerly, N. Stanford and P.W. Majerus, 1979, Diglyceride lipase: a pathway for arachidonate release from human platelets, Proc. Nat. Acad. Sci. U.S.A. 76, 3238. 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.J. Flower, F.P. Nijkamp and J.R. Vane, 1978, Phospholipase A2 activity of guineapig isolated perfused lungs: stimulation, and inhibition by anti-inflammatory steroids, Br. J. Pharmacol. 62, 79. Danon, A. and G. Assouline, 1978, Inhibition of prostaglandin biosynthesis by corticosteroids requires RNA and protein synthesis, Nature 273,552. Derksen, A. and P. Cohen, 1975, Patterns of fatty acid release from endogenous substrates by human platelet homogenates and membranes, J. Biol. Chem. 250, 9342. Di Rosa, M. and P. Persico, 1979, Mechanism of inhibition of prostaglandin biosynthesis by hydrocortisone in rat leucocytes, Br. J. Pharmacol. 66,161. Flower, R.J., 1978, Steroidal anti-inflammatory drugs as inhibitors of phospholipase A2, in: Advances in Prostaglandin and Thromboxane Research, Vol. 3, eds. C. Galli, G. Galli and G. Porcellati (Raven Press, New York) p. 105. Flower, R.J. and G.J. Blackwell, 1976, The importance of phospholipase A2 in prostaglandin biosynthesis, Biochem. Pharmacol. 25, 285. Flower, R.J. and G.J. BlackweU, 1979, Anti-inflammatory steroids induce biosynthesis of a phospholipase A2 inhibitor which prevents prostaglandin generation, Nature 278,456. F6rstermann, U. and G. Hertting, 1979, The 'importance of Ca2+-mediated phospholipase A2 activation for stimulus-evoked PGE2 release from rabbit splenic capsular strips, Naunyn-Schmiedeb. Arch. Pharmacol. 307,243 . . . . . Gryglewski, R.J., B. Panczenko, R. Korbut, L. Grodzinska and A. Ocetkiewicz, 1975, Corticosteroids inhibit prostaglandin release from perfused mesenteric blood vessels of rabbit and from perfused lung of sensitized guinea-pigs, Prostaglandins 10, 343. Hirata, F., B.A. Corcoran, K. Venkatasubramanian, E. Schiffmann and J. Axelrod, 1979, Chemoattractants stimulate degradation of methylated phospholipids and release of arachidonic acid in rabbit leukocytes, Proc. Nat. Acad. Sci. U.S.A. 76, 2640. Isakson, P.C., A. Raz, W. Hsueh and P. Needleman, 1978, Lipases and prostaglandin biosynthesis, in: Advances in Prostaglandin and Thromboxane
C. ROBINSON, J.R.S. HOULT Research, Vol. 3, eds. C. Galli, G. Galli and G. Porcellati (Raven Press, New York) p. 113. Juan, H., 1979, Role of calcium in prostaglandin E release induced by bradykinin and the ionophore A23187, Naunyn-Schmiedeb. Arch. Pharmacol. 307,177. Kunze, H. and W. Vogt, 1971, Significance of phospholipase A for prostaglandin formation, Annals N. Y. Acad. Sci. 180, 123. Lands, W.E.M., 1979, The biosynthesis and metabolism of prostaglandins, Ann. Rev. Physiol. 41,633. Lapetina, E.G. and P. Cuatrecasas, 1979, Stimulation of phosphatidic acid production in platelets precedes the formation of arachidonate and parallels the release of serotonin, Biochim. Biophys. Acta 573, 394. Malmsten, C., 1976, Some biological effects of prostaglandin endoperoxide analogs, Life Sci. 18,169. Markus, H.B. and E.G. Ball, 1969, Inhibition of lipolytic processes in rat adipose tissue by antimalarial drugs, Biochim. Biophys. Acta 187,486. Mathd, A.A., S-S. Yen, R. Sohn and P. Hedqvist, 1977, Release of prostaglandins and histamine from sensitised and anaphylactic guinea-pig lungs changes in cyclic AMP levels, Biochem. Pharmacol. 26, 181. Needleman, P., A. Wyche, S.D. Bronson, S. Holmberg and A.R. Morrison, 1979, Specific regulation of peptide-induced renal prostaglandin synthesis, J. Biol. Chem. 254, 9772. 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 anti-inflammatory steroids, Nature 263,479. Rittenhouse-Simmons, S., 1979, Production of diglyceride from phosphatidyl inositol in activated human platelets, J. Clin. Invest. 63,580. Rollo, I.M., 1970, Drugs used in the treatment of malaria, in: The Pharmacological Basis of Therapeutics, 4th edn. eds. L.S. Goodman and A. Gilman (Macmillan, London and Toronto) p. 1095. Russo-Marie, F., M. Paing and D. Duval, 1979, Involvement of glucocorticoid receptors in steroidinduced inhibition of prostaglandin secretion, J. Biol. Chem. 254, 8498. Samuelsson, B., M. Goldyne, E. GranstrSm, M. Hamberg, S. HammarstrSm and C. Malmsten, 1978, Prostaglandins and thromboxanes, Ann. Rev. Biochem. 47, 997. Van den Bosch, H., 1974, Phosphoglyceride metabolism, Ann. Rev. Biochem. 43,243. Vane, J.R., 1978, Inhibitors of prostaglandin, prostacyclin and thromboxane synthesis, in: Advances in Prostaglandin and Thromboxane Research, Vol. 4, eds. F. Coceani and P.M. Olley (Raven Press, New York) p. 27. -
-
PHOSPHOLIPASE POOLS AND PG SYNTHESIS Vargaftig, B.B., 1977, Carrageenan and thrombin trigger prostaglandin synthetase-independent aggregation of rabbit platelets: inhibition by phospholipase A2 inhibitors, J. Pharm. Pharmacol. 29, 222. Vargafting, B.B. and N. Dao Hal, 1972, Selective inhibition by mepacrine of the release of 'rabbit aorta
339 contracting substance' evoked by the administration of bradykinin, J. Pharm. Pharmacol. 24,159. Vogt, W., 1978, Role of phospholipase A2 in prostaglandin formation, in: Advances in Prostaglandin and Thromboxane Research, Vol. 3, eds. C. Galli, G. Galli and G. Porcellati (Raven Press, New York) p. 89.
333
© Elsevier/North-Holland Biomedical Press
EVIDENCE FOR FUNCTIONALLY DISTINCT POOLS OF PHOSPHOLIPASE RESPONSIBLE FOR PROSTAGLANDIN RELEASE FROM THE PERFUSED GUINEA-PIG LUNG CLIVE ROBINSON * and J.R.S. HOULT
Department of Pharmacology, King's College, Strand, London WC2R 2LS, U.K. Received 29 January 1980, revisedM S received 25 March 1980, accepted 31 March 1980
C. ROBINSON and J.R.S. HOULT, Evidence for functionally distinct pools of phospholipase responsible for prostaglandin release from the perfused guinea-pig lung, European J. Pharmacol. 64 (1980) 333--339. The release of prostaglandin- and thromboxane-like material from the isolated perfused guinea-pig lung was elicited by challenge with arachidonic acid, bradykinin, histamine and ovalbumin (in ovalbumin-sensitised animals), and detected by superfusion cascade bioassay. Mepacrine inhibited release to all agents except arachidonic acid. Dexamethasone and fludrocortisone inhibited release induced by histamine and anaphylactic challenge but not by bradykinin or arachidonic acid. These results suggest two functionally distinct pools of phospholipase responsible for initiating prostaglandin biosynthesis in the guinea-pig lung. PG biosynthesis
Guinea-pig lungs
Phospholipase pools
1. Introduction The synthesis and release of prostaglandins and related substances from cells requires the cleavage from membrane phospholipids of their polyunsaturated fatty acid precursors (e.g. arachidonic acid). This is followed by transformations catalysed by the lipoxygenase and cyclo-oxygenase pathways (reviewed by Samuelsson et al., 1978; Vane, 1978; Lands, 1979). However, two factors limit our understanding of how the signals for synthesis are recognised and coupled to membrane events: neither the phospholipase(s) presumed to be responsible for releasing the fatty acids nor the precise phospholipid pools from which they derive have yet been conclusively identified. Phospholipase A2 is generally regarded as
* Present address: Department of Clinical Pharmacology, Royal Postgraduate Medical School, Ducane Rd, London W12 OHS, U.K.
Inhibitors
the prime candidate (Kunze and Vogt, 1971; Flower and Blackwell, 1976; Vogt, 1978) since it selectively hydrolyses phospholipids at the 2'-acyl position (Van den Bosch, 1974) and the largest amounts of eicosapolyeneoic acids are attached at this position (Flower, 1978). Furthermore, phospholipase A2 causes the release of prostaglandins from intact and isolated tissues (Bartels et al., 1970; Juan, 1979; FSrstermann and Hertting, 1979). However, recent studies have shown that other types of phospholipase enzymes as well as other more complex interactions between phospholipids may effect the release of arachidonic acid, and they imply that the phospholipase step in prostaglandin biosynthesis is not as straightforward as first thought (Rittenhouse-Sirnmons, 1979; Bell et al., 1979; Lapetina and Cuatrecasas, 1979; Hirata et al., 1979). The use of mepacrine (quinacrine), an inhibitor of phospholipase A2 (Markus and Ball, 1969), and of anti-inflammatory steroids -- which are thought to reduce prostaglandin synthesis and release from intact cells by
334
inhibiting phospholipase A2 (Flower, 1978) -- affords a pharmacological method whereby the existence of different functional pools of phospholipase and their relationships to triggers of prostaglandin synthesis may be investigated experimentally. Such a study requires an intact cell system in which prostaglandin biosynthesis can be stimulated by a variety of different effectors both before and after application of inhibitors. We describe here experiments in which prostaglandin and thromboxane release from the perfused guinea-pig lung were stimulated in four different ways and monitored by immediate superfusion cascade bioassay. The results suggest that there are two functionally distinct pools of phospholipase.
2. Materials and methods Lungs from male guinea pigs (Dunkin-Hartley, 250--350 g) were removed, cannulated at the pulmonary artery and perfused at 6 ml/ min with warmed and well~xygenated Krebs solution containing antagonists (atropine 1.8 #M, methysergide 0.48 pM, phentolamine 0.6 pM and practolol 5.6 ~zM). Effluent from the lungs was passed over a superfusion cascade bioassay which generally comprised two rabbit aorta strips, a rat fundus strip and a rat colon. A separate line was used to perfuse Krebs solution containing indomethacin 8.0 pM, hexamethonium 7.0 pM and mepyramine 0.60 pM at 1.0--1.5 ml/min directly over the assay tissues. Responses of the tissues to prostaglandins and other agonists were evoked by injection either at the top of the cascade or into the cannulated pulmonary artery. The stable prostaglandin endoperoxide analogue U46619 (15S-hydroxy-ll~, 9a-epoxymethanoprosta-5Z, 13E
C. ROBINSON, J.R.S. HOULT
after checking by direct superfusion over the cascade that they did not inhibit the responsiveness of the assay tissues. Sensitised guinea pigs were prepared according to the method of Math~ et al. (1977) (50 mg ovalbumin in 0.9% saline injected s.c. and i.p. on day 1, 50 mg i.p. on day 3) and used after 14 days. Control unsensitised animals were similarly injected with vehicle only. Prostaglandin E2 and the endoperoxide analogue U46619 were kind gifts from Dr. J.E. Pike, The Upjohn Company, U.S.A. and mepacrine hydrochloride was a gift from Dr. J.M. Young, University of Cambridge. Dexamethasone acetate, fludrocortisone acetate and bradykinin triacetate were from Sigma. The steroids were made up in a small volume of ethanol and diluted into Krebs solution before perfusion. At this concentration the ethanol did not affect pulmonary generation of prostaglandins or thromboxanes.
3. Results
Four substances were used to elicit the release of prostaglandin- and thromboxanelike material from the isolated perfused guinea-pig lung (table 1): histamine, bradykinin, arachidonic acid and ovalbumin (in sensitised lungs). In all cases, injection into the pulmonary artery was followed by strong contractions of the rabbit aorta and rat fundus strip preparations (fig. l ) which could not be matched by injection of the trigger substances over the tissues alone. The responses of the rat colon were both smaller and more variable; generally it showed a small increase in tone associated with bursts of contractile activity. Bradykinin was the only agent which consistently produced marked contractions of the rat stomach strip at the doses used, but sometimes high doses of arachidonic acid also caused it to contract (e.g. fig. 1); however, in both cases the responses were smaller, of shorter duration and had a different tissue profile when injected direct to the tissues. Since similar responses to those elicited by
PHOSPHOLIPASE TABLE
POOLS
AND
PG SYNTHESIS
335
1
Release of prostaglandin- and thromboxane-like material from perfused guinea-pig lung and inhibition by substances which interfere with phospholipase A 2.
Perfusion conditions
Control Mepacrine Dexamethasone Fludrocortisone
Inhibitor concentration (pg/ml)
Release of PG and TX-like material in response to l Bradykinin 2--8 pg
Arachidonate 2--20 pg
Histamine 1--10 pg
Ovalbumin 10 pg 1 mg
-20 4 20
15 0 6 4
14 4 4 2
11 0 0 0
18 (18) 0 (3) d 1 (6) bcd 2 2(12) 2
(18) (5) a (6) cd (4)
(17) (5) a (4) bce (2)
(12) (3) d (4) cd (6)
1 Data shown in the form M(N) where M is the number of times partial or complete release of PG/TX-like material was observed and N the total number of challenges made under these conditions. 2 The 3/18 'failures' of complete inhibition occurred before the steroids' effect was maximal: later challenges in the same lung showed complete inhibition. Superscript letters denote similar results obtained in guinea-pig lung superfusion cascade bioassay experiments by : a Vargaftig and Dao Hal (1972); b Gryglewski et al. (1975), but using hydrocortisone; c Blackwell et al. (1978); d Blackwell et al. (1978), but using radiochemical assay for phospholipase A 2 activity (see Discussion); e Nijkamp et al. (1976).
the four triggers could be obtained by direct i n j e c t i o n o v e r t h e t i s s u e s o f p r o s t a g l a n d i n E2 and the thromboxane A2-1ike a n a l o g u e U46619, we conclude that the trigger substances cause pulmonary synthesis and release
of prostaglandin- and thromboxane-like material ............ Mepacrine abolished the release of prostag. landin- and thromboxane-like m a t e r i a l in r e s p o n s e t o all t r i g g e r s e x c e p t a r a c h i d o n i c
I
I
,
I
I
I
I
0
BK
BK
2pg D
2 }..Ig L
AA lO)Jg D
AA 10 ).Jg L
H 5.pg D
14 5~g L
200 pg L
Fig. 1. Release of prostaglandin- and thromboxane-like material from perfused guinea-pig lung challenged with ovalbumin (O), bradykinin (BK), arachidonic acid (AA) and histamine (H). Spasmogenic substances were detected by superfusion cascade bioassay, routinely using the rat stomach fundus strip (RSS) and rabbit aortic strip (RAS). Injections were made via the lung (L) or direct to the tissues (D). The figure shows representative results from the tests carried out in the absence of inhibitors as summarised in table 1. For clarity, the responses on the rat colon have been omitted (see Results). Time bar indicates 5 min.
336
acid (table 1). Complete inhibition, was observed within 10 min of starting mepacrine infusion. Synthesis of prostaglandins from exogenously applied arachidonic acid is initiated by the action of cyclo-oxygenase and is independent of any phospholipase(s) or their inhibitors. In 4 out of 6 experiments where survival of the lung and assay tissues permitted it was found that inhibition by mepacrine was slowly reversible within 45--85 min of discontinuing its infusion, although judging by the yellow colour of the lung some mepacrine was still retained within the tissue. Dexamethasone and fludrocortisone were also tested as possible inhibitors of the phospholipases involved in pulmonary prostaglandin release (table 1). Histamine- and anaphylactically induced synthesis were inhibited. The inhibition had a slow onset, reaching its maximum 40--70 min after starting the steroid (based on 28 tests in 13 lungs, using both histamine and ovalbumin challenge) and was maintained for at least 140 min after stopping it (2 experiments using ovalbumin challenge). However, the steroids did not inhibit prostaglandin/thromboxane release triggered by bradykinin or by arachidonic acid, even when these challenges were made 70 or more minutes after starting the steroid infusion and when maximal inhibition to histamine and ovalbumin had been achieved.
4. Discussion These experiments provide evidence for the presence of two or more functionally distinct pools of phospholipase responsible for initiating prostaglandin biosynthesis and release. Thus the two anti-inflammatory steroids inhibited prostaglandin and thromboxane release in response to anaphylactic challenge and histamine but not that induced by bradykinin. All three triggers are considered to require activation of phospholipase A2. Inhibition of prostaglandin formation and release from intact cells by anti-inflammatory steroids is well documented (see Flower and
C. ROBINSON, J.R.S. HOULT
Blackwell, 1979; Di Rosa and Persico, 1979), and it is thought that its expression depends upon the synthesis of new protein (Danon and Assouline, 1978; Di Rosa and Persico, 1979; Russo-Marie et al., 1979). This explains why its effects take some time to develop, as observed here. Indeed, Flower and Blackwell (1979) have identified a soluble protein factor with anti-phospholipase properties which was released from perfused guinea-pig lungs after treatment with dexamethasone. The simplest explanation for our findings is that the steroids or the steroid-induced factor(s) selectively inhibit some but not all of the phospholipases involved in the biosynthesis of prostaglandins, and that there are at least two independent mechanisms for the recognition of different triggers for prostaglandin synthesis and subsequent phospholipase activation. In contrast, mepacrine inhibited synthesis and release in response to all three phospholipase triggers, showing it to be a relatively powerful but non-selective inhibitor of phospholipase action. Inhibition was rapid in onset, and this is consistent with a direct inhibitory action on phospholipase A2 as shown by others (Vargaftig and Dao Hai, 1972; Flower and Blackwell, 1976; Vargaftig, 1977; Blackwell et al., 1977; Hirata et al., 1979). The use of mepacrine for investigating the role of phospholipase is therefore limited, and it is further hampered by the fact that it has a number of other pharmacological and biochemical actions, some of which are non-specific (Rollo, 1970; Vargaftig, 1977). Mepacrine may also interfere in other ways with prostaglandins: for example, it inhibits cyclooxygenase action (Nijkamp et al., 1976; Blackwell et al., 1977), although this was not observed in these experiments or by Vargaftig and Dao Hai (1972). Several of these observations concerning pulmonary prostaglandin and thromboxane release and its inhibition by drugs have been made previously (as indicated in the footnote to table 1), but they have not been the object of a single comparative study. However, there
PHOSPHOLIPASE
POOLS AND PG SYNTHESIS
are some discrepancies. Nijkamp et al. (1976) found that both mepacrine and fludrocortisone depressed the release of thromboxane A2 by arachidonate (implying cyclo-oxygenase inhibition), although precise details were not given. In subsequent experiments the same research group (Blackwell et al., 1978) found that bradykinin-induced stimulation of phospholipase A2 activity was not inhibited by mepacrine at 75 pg/ml. However, these latter results relate to two observations only and were made using a radiochemical assay for phospholipase A2 in which the lung is perfused with [3H]-2-oleoyl phosphatidylcholine. The effect of mepacrine on the release of spasmogenic substances by bradykinin was not tested by cascade bioassay. Nevertheless, in reviewing their data these authors concluded, as we do here, that 'it seems necessary to postulate the existence of a steroid-sensitive and a steroid-insensitive population of receptors or phospholipase enzymes . . . . . '. Our suggestion that at least two pools of phospholipase exist to effect coupling between receptor activation and prostaglandin biosynthesis receives further support from studies published while our experiments were in progress. Rittenhouse-Simmons (1979), Lapetina and Cuatrecasas (1979) and Bell et al. (1979) have shown that platelets contain a phosphatidylinositol-specific phospholipase C which has high activity and is capable of the rapid release of arachidonate-containing phosphatidic acid in response to aggregating agents such as thrombin. According to Bell et al. (1979), free arachidonate is released by subsequent hydrolysis of the phosphatidic acid by diglyceride lipase. Since platelets are also known to contain small amounts of phospholipase A2 activity (Derksen and Cohen, 1975; Bell et al., 1979), it is possible that two independent routes for release in platelets of arachidonic acid may coexist. Needleman and coworkers have postulated two pathways for activation of prostaglandin synthesis in rabbit heart, one of which is hormone-sensitive and specifically releases arachidonic acid (Isakson et al., 1978). The other
337
pathway, activated by ischaemia, results in non-specific lipolysis and arachidonate is one among many products. This idea is further supported by the same group's studies on prostaglandin synthesis in the hydronephrotic (ureter~clamped) rabbit kidney (Needleman et al., 1979). When challenged repeatedly with bradykinin or angiotensin II the kidney releases successively more prostaglandin-like substance due to de novo synthesis of cyclooxygenase (and possibly, therefore, of phospholipase); however, this only represents a small proportion of the total cyclo-oxygenase and the total pool does not appreciably increase in amount after each bradykinin challenge. This implies an 'exquisite compartmentation' (sic) of the prostaglandin synthesising system. In the light of these results it would be interesting to see if this bradykinininduced component is sensitive to inhibition by anti-inflammatory steroids or mepacrine, and, conversely, to examine the effects of these drugs on angiotensin II-induced prostaglandin and thromboxane synthesis in the lung. We could not test this because angiotensin is strongly spasmogenic to all the assay tissues used. Taken together, the results of this study and those discussed above suggest that several pools of phospholipase may exist to effect the coupling between receptor activation and prostaglandin synthesis. Certainly, the simplifying hypothesis that synthesis depends upon phospholipase A2 activation may require more detailed elaboration. Acknowledgements We thank Pharmacia AB and the trustees of the Layton Science Research Prize (King's College) for the award of funds for this research and Drs. J.E. Pike and J.M. Young for gifts of prostaglandins and mepacrine respectively.
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