Characterization of the reaction sequence involved in phospholipid labeling and deacylation and prostaglandin synthesis and actions Philip Needleman, Ph.D. St. Louis, MO.

The synthesis and release of prostaglandins (PC) primarily involve a series of enzymatic transformations of the fatty acid arachidonic acid. The source of this free fatty acid is thought to be the cleavage by phospholipase AZ of the acyl bond on the 2-position of phospholipids. The liberated arachidonic acid is primarily converted by cycle-oxygenase (PG-synthetase) into the PG-endoperoxides (PGG2 and PGH,). These compounds represent a critical branch point in PG metabolism since they serve as substrates for the enzymatic synthesis of PGE2, PGD2, thromboaane AZ, PGIz (prostacyclin), as well as some likely products yet to be identified. The ultimate fate of arachidonate in a given tissue is determined by the repertoire of enzymes present. The prostaglandins have a very broad sprectrum of identified biological activities and the newly discovered arachidonate metabolites (endoperoxides, thromboxane A2 and PGIJ are labile and extremely potent.

Preformedprostaglandins(PG) are not storedin the body tissuesl* ‘$ thus their presencereflects initiation of de novo synthesis and release. Early studies showed that free fatty acid precursor (primarily arachidonic acid) was necessary for PG biosynthesis.3*4 Since tissue free fatty acid levels are very low, this led to the suggestionthat phospholipaseactivation may be required to liberate the arachidonic acid substrate(from membranebond phospholipids) neededfor PG biosynthesis in whole tissue.5 Vargaftig and Dao Hai found that mepacrine inhibited the PG and thromboxane synthesisby lungs stimulated by bradykinin but not the synthesiscausedby exogenous arachidonic acid. This experiment suggeststhat hormonesreact with tissue receptors, causing the activation of phospholipaseA2 which liberates arachidonic acid that in turn is immediately converted by the From the Department of Pharmacology, Washington University School of Medicine. Supported by National Institutes of Health Grants SCOR-HL17646, HE- 14397, and HL-20787. Presentedat the PostgraduateCourse in Allergy and Clinical Immunology at the meeting of the American Academy of Allergy in Phoenix, Ariz., February, 1978. Received for publication March 23, 1978. Accepted for publication March 28, 1978. Reprint requests to: Philip Needleman, Ph.D., Department of Pharmacology, Washington University School of Medicine, 4577 McKinley Ave., St. Louis, MO. 63110.

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ubiquitous cycle-oxygenase into PG. Administration of exogenous arachidonate bypasses the need for phospholipase activation. However, mepacrine is a relatively nonspecific drug’ which could act by other mechanisms,e.g., membranestabilization. PHOSPHOLIPID LABELING TECHNIQUE In order to study tissue phospholipase stimulation and prostaglandin synthesis in intact organs, several laboratorieshave employed a technique which labeled the precursor phospholipids. Bills, Smith, and Silvers found that [‘“cl arachidonic acid was efficiently incorporated into the phospholipids (but not triglycerides) of platelets. Subsequentaddition of thrombin, which causesplatelet aggregation and stimulates the release of PG endoperoxides and thromboxanes, led to a loss of radioactivity from phospholipids (primarily phosphatidylcholine and phosphatidylinositol) and releaseof labeled PG endoperoxide metabolites. Prelabeling endogenous lipid pools with [‘“cl arachidonic acid thus provides a simple method for studying metabolism of endogenous arachidonate. There is, however, a drawback to this technique which has been pointed out by Jesseand Cohen.s This method only labels rapidly turning over phospholipids, and therefore the pattern of incorporation may not reflect the relative abundanceof arachidonatein phospholipids formed in vivo.g

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Flower and Blackwell1o pre-labeled spleen slices with [ 14C]arachidonate in order to study arachidonate release from this preparation. They found that the spleen slices incorporated [ 14C]arachidonate into endogenous phospholipids and that the label could be released by mechanical vibration and, to a lesser extent, anaphylaxis. Levine and co-workers studied PG biosynthesis by cultured cells, as well as using a line of tiansformed mouse fibroblasts,” fibrosarcoma cells,** and rheumatoid synovia.13 By pre-labeling cells with [ 3H]arachidonate, they found that anti-inflammatory steroids inhibit PG biosynthesis induced by serum, bradykinin, or thrombin by interfering with release of arachidonate from phospholipids.“’ l4 This confirms earlier work by Gryglewski and co-workers’” who found that steroids inhibited PG release from guinea pig lungs induced by norepinephrine and antigen but did not block PG release from exogenous arachidonate.

HORMONE-INDUCED PHOSPHOLIPASE ACTIVATION We found that perfused organs such as isolated perfused hearts or kidneys were found to efficiently incorporate infused [14C]-arachidonate into endogenous lipids, and radioactivity can be released in the form of [l%]PGs by hormones such as bradykinin.16 The [ 14C] arachidonate is incorporated into phospholipids, primarily in the 2-position of phosphatidylcholine and phosphatidylethanolamine. Our initial experiments with pre-labeled kidneys demonstrated that bradykinin and ATP released predominantly PGE2, but little if any release of labeled arachidonate was observed.16 It was apparent from the almost complete incorporation of exogenous [ 14C] arachidonate that the kidney (as well as other tissues) possessed an efficient mechanism for removing exogenous fatty acid. It seemed reasonable, then, that bradykinin could have released arachidonate but that the arachidonate was rapidly reincorporated. Thus, it would be necessary to “trap” the arachidonate by some means in order to detect its release associated with PG biosynthesis. To accomplish this we added fatty acid-free albumin (which avidly binds fatty acids) to the medium perfusing a rabbit kidney and tested bradykinin.17 In the presence of albumin we observed a large release of radioactive arachidonate. Release of arachidonate occurred in response to various stimuli (bradykinin, angiotensin, ATP, ischemia) and was unaffected by indomethacin, which completely inhibited synthesis of PGs. Similar results were obtained with rabbit hearts, suggesting that this phenomenon is not a peculiarity

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of the kidney. Recently Zusman and Keiser” reported that angiotensin, bradykinin, and vasopressin released arachidonate and PGs from cultured renomedullary interstitial cells labeled with [ 3H] arachidonate. The albumin trapping method has allowed us to further investigate two other problems, namely tachyphylaxis (i.e., the rapid loss of tissue responsiveness to an agonist on repeated administration) and specificity of the deacylation reaction. Infusions of hormones through spleenlg and hea@“, ” result in a transient release of PGs (tachyphylaxis) despite continuous presence of the hormone. Tachyphylaxis is specific for each hormone, since tachyphylaxis induced by bradykinin has no effect on PG release induced by angiotensin or ATP. Infusions of arachidonate do not produce tachyphylaxis and do not affect release due to other hormones. This suggests that there is no feedback regulation of the deacylation mechanism by arachidonate or any of its metabolites.20, 21 To further test the above hypothesis we infused bradykinin through pre-labeled kidneys and analyzed the products released in the presence of albumin. We found that arachidonate release exactly paralleled release of PGs. Furthermore, indomethatin, which completely abolished PG release, had no effect on the transient release of arachidonate.‘? These results directly demonstrate that tachyphylaxis occurs at a step prior to PG biosynthesis and that the release of arachidonate is completely independent of its subsequent metabolism. We have also investigated the specificity of the deacylation reaction by pre-labeling hearts with other radioactive fatty acids22 as previously indicated. The synthesis and release of PGs by the isolated perfused rabbit heart upon bradykinin stimulation result from lipase stimulation which liberates arachidonic acid for PG biosynthesis. The [ 14C]-labeled fatty acids, arachidonate, linoleate, and oleate, when infused into the heart preparation, were efficiently incorporated into the phospholipid pool in the heart, mostly in the 2-position of phosphatidylcholine.22 On the other hand, [14C]-palmitate was esterified into both the 1-and the 2-position. Bradykinin released bioassayable PG when injected into the rabbit hearts regardless of which fatty acid label was incorporated into the phospholipid pool. However, only [ “C]-arachidonic acid (but not [ ‘“Cl-linoleate, oleate, or palmitate) was liberated from the variously labeled hearts upon hormone stimulation. This selective bradykinin effect on fatty acid release suggests that hormone stimulation either activates a specific lipase that distinguishes different fatty acids in the 2-position or activates lipase which is selectively compartmented with arachido-

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nate-containing phospholipids. Ischemia, on the other hand, appeared to nonspecifically stimulate tissue lipases, resulting in a nonselective release of oleic as well as arachidonic acid.22 A disproportionally large release of arachidonic acid was observed accompanying a relatively small PG (10: 1 arachidonate : PG ratio) production during ischemia, as compared to bradykinin (3 : 1 ratio), suggesting distinct mechanisms for PG biosynthesis induced by bradykinin and ischemia. The prostaglandins (PG) are synthesized by virtually every tissue in the body23 and have been detected in the venous effluent from numerous mammalian organs. As indicated above, the release of PGs from organs such as kidney and heart can be stimulated through activation of fatty acid release by specific lipases by various means including: hormones (e.g., bradykinin, angiotensin II), nerve stimulation or exogenous neurotransmitters, mechanical damage, and decreased oxygen tension.‘, 2*20*24-28Since there is no evidence for storage of PGs, release reflects de novo biosynthesis. l, 2, 2s However, little if any PG is detected in arterial blood, largely because of the efficient pulmonary destruction by PG-dehydrogenase.‘, 2, 20,25-28Thus, PGs should be considered as local hormones that are synthesized at or near their site of action.

PROSTAGLANDIN BIOSYNTHESIS Prostaglandins are formed by the enzymatic oxygenation of polyunsaturated fatty acids.30 The most abundant precursors are 5,8,11,14-eicosatetraenoic acid (arachidonic acid) and 8,11,14-eicosatetraenoic (dihomo-y-linolenic acid), which form PGs of the 2 and 1 series, respectively.30-32 Lipase-released PG precursor (principally arachidonic acid) is acted upon by an enzyme, cycle-oxygenase, with the incorporation of oxygen,30 resulting in the formation of the cyclic endoperoxides, PGG, and PGH,. Indomethacin and other aspirin-like drugs inhibit this enzyme and therefore abolish biosynthesis of all prostaglandins.33 Arachidonic acid is also converted by a lipooxygenase in platelets to a noncyclized product, 12-hydroxyeicosatetraenoic acid (HETE).34 Lipo-oxygenase as well as the cycle-oxygenase is inhibited by eicosatetraynoic acid (ETYA).35 The endoperoxides possess considerable biological activity (contract smooth muscle and cause platelet aggregation 34*36,37and serve as intermediates for the synthesis of other prostaglandins). The endoperoxides are unstable in aqueous media and spontaneously decompose (t% of 4-6 min) to a mixture of PGE2 and PGD2. The endoperoxides are enzymatically convetted to a variety of products such as PGE2, PGD2,

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thromboxane A2, and PG12(prostacyclin). Each tissue may possess differing synthetic enzymes that require endoperoxide substrate and therefore produce different types and amounts of PGs. Furthermore, the various PGs have different biologic actions (either complementary or antagonistic). Thus, the ultimate response of an organ to activation of PG biosynthesis often represents the algebraic sum of a number of complex interactions. Platelets predominantly synthesize the potent labile vasocontrictor and aggregatory substance thromboxane A2, as well as HETE and HHT ( 12-hydroxy-heptadecatrienoic acid) .34, 38 Heart, blood vessels, and stomach predominantly synthesize the vasodilator PG12.3843Some tissues possess several enzymes that use the endoperoxides as substrate. For example, the rabbit kidney normally synthesizes from endogenous arachidonate the renal vasodilator PGE2 as its primary metabolite; however, with ureter obstruction the kidney unmasks the synthesis of the vasoconstrictor thromboxane A2.44, 45 Furthermore, the kidney has recently been found to have the enzymatic capacity to convert exogenous arachidonate into PG12.46Thus, depending upon the experimental conditions, the isolated perfused rabbit kidney can synthesize PGE2, thromboxane A2, or PG12. Similarly, the lung has been demonstrated to synthesize these three arachidonate metabolites’, 2, 47 and very recently it has been demonstrated that SRS-A also appears to be an arachidonate metabolite.48

CHARACTERIZATION AND FUNCTION OF ENDOGENOUS PROSTAGLANDINS We believe the following parameters should be fulfilled to establish that endogenous PG mediates or modulates a physiologic or pathologic event: (1) concentration of the produced mediator (i.e., PG) should be proportional to the dose of the stimulus (e.g., hormone stimulation with bradykinin or angiotensin, norepinephrine, etc.); (2) there should be temporal and quantitative correlation between changes in concentration of the PG with changes in the functional status of the organ; (3) exogenous administration of the identified mediator should mimic the physiologic response; (4) Abolition of the synthesis of the PG should abolish the physiologic action of the stimulus; (5) exogenous administration of the precursor of the mediator should produce the physiologic response.

THROMBOXANE A2 AND PGlz CHARACTERISTICS The identity of the arachidonate product involved relies specifically on the chemical and biological properties of the metabolite under investigation. The

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identification of thromboxane AZ should requite that the generated substance: (1) contracts isolated blood vessels; (2) is enzymatically synthesized (e.g., by platelet microsomes) from either arachidonic acid (but not dihomo-y-linolenic acid) or PGHz (but not PGH,4g; (3) has an aqueous decay t% of 30 set; (4) aggregates platelets44; (5) spontaneously forms the stable metabolite thromboxane Bz (identified by thin-layer chromatography in several solvent systems, radioimmunoassay, or GC-mass spectrometry). Furthermore, its biosynthesis in vitro should be blocked by a thromboxane synthetase antagonist such as imidazole or 9,1 I -azoprosta-5, I3-dienoic acid,49-“1

The identity of the newly discovered PGIz (prostacyclin) requires that the generated substance: (1) relaxes isolated bovine or canine coronary artery assay gfips43, 46, 52, 53 and contracts rabbit transverse stomach strips; (2) inhibits platelet aggregationJ4; (3) is synthesized from arachidonic acid (but not dihomo-y-linolenic acid) or PGH, (but not PGHJ4’; (4) is unstable in aqueous solution especially in acid conditions; and (5) is degraded to the stable 6keto-PFG,,* .3g-43,j4, 5SFurthermore, its biosynthesis should be blocked in vitro by hydroperoxy fatty acids.Sfi

CONCLUSIONS AND PROJECTIONS The synthesis and release of PGs involve a series of enzymatic transformations of polyunsaturated fatty acids, arachidonate being the predominant and most abundant precursor. 31, 32 The source of free arachidonic acid is thought to be the cleavage of acyl bond on the 2-position of phospholipids, catalyzed by phospholipase AZ. The evidence supporting this theory may be summarized as follows: (1) most endogenous arachidonic acid is found in the 2-position of phospholipidsS7a ss; (2) addition of phospholipase AZ to tissue homogenates leads to formation of PGs3* 4; (3) perfusion of intact organs with phospholipase A leads to a rapid release of PGs in the effluentSs-“‘; (4) mepacrine, a phospholipase inhibitor, blocked the PG-releasing action from guinea pig lungs of bradykinin but not arachidonate$ (5) anti-inflammatory steroids, which presumably decrease phospholipid deacylation, inhibited PG release from cultured fibroblasts by bradykinin and thrombini4; and (6) in both spleen slices10 and platelet? prelabeled with 14C-arachidonic acid, there was a selective loss of radioactivity from tissue phosphatidylcholine upon activation of PG synthesis. In addition, the fact that only arachidonic acid (as opposed to other fatty acids) was liberated after bradykinin stimulationz2 strongly suggests that hormone stimulation either activates a

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specific phospholipase that distinguishes the different fatty acids in position-2, or activates phospholipase AZ which is selectively compartmented with arachidonate-containing phospholipids. Ischemia, on the other hand, exhibits a nonspecific stimulation on the fatty acid release. The initial contact of a hormone with a given organ is probably by means of a specific cell surface receptor prior to phospholipase activation. Evidence for unique receptors derives from the demonstration of tachyphylaxis restricted to the particular hormone. Thus, hormone-stimulated PG release from intact cells or tissues was transient (tachyphylaxis) despite the continuous presence of bradykinin, while sustained PG production could be maintained by arachidonate (the exogenous administration of arachidonate bypasses the phospholipase step) infusion, suggesting that tachyphylaxis occurs at a step prior to the cyclooxygenase. 17, lg. *’ These observations suggest that existence of a hormone-specific receptor. The secondary event following receptor interaction appears to be the activation of a specific phospholipase A2 which selectively liberates only arachidonic acid from phospholipids or alternatively activates phospholipase AZ which is selectively compartmented with arachidonate-containing phospholipids. However, the initial hormone-receptor interaction must be independent of PG production as indicated by the arachidonate release in response to bradykinin infusion even after complete abolition of PG synthesis by indomethatin.” Furthermore, continuous simultaneous infusion of exogenous arachidonate (part of which is converted to PG) does not alter the brief duration of bradykinininduced PG release.“, 21On the other hand, ischemia stimulates nonspecific fatty acid release probably resulting from a nonselective activation of tissue lipases. In addition, ischemia is less efficient in PG production than hormone stimulation, indicated by a large amount of fatty acid liberation accompanying a relatively small PG release compared to bradykinin. One explanation for this phenomenon is that ischemia activates deacylation at sites remote from the PG synthesis complex, while bradykinin stimulates deacylation at sites close to or continous with the cycle-oxygenase and is more tightly coupled to the PG synthesis than is ischemia. There is an analogy between the deacylation and reacylation of tissue phospholipids with neuronal storage, release, and reuptake of norepinephrine from the adrenergic nerve endings. The norepinephrine expelled into the synapse by nerve stimulation has several possible fates: a portion reacts with the receptor, a portion is degraded, and a substantial fraction (about 60%) undergoes selective reuptake in the presynaptic

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nerve terminal. Thus, the essential transmitter is conserved for subsequent reuse. Since there is no storage form of PG or free arachidonic acid, the critical precursor pool actually is in the arachidonate-containing phospholipids. Hormone stimulation leads to the release of arachidonic acid from phospholipids. The fraction that is oxidized to PG is now considerably more polar than the fatty acid, and the expelled PG cannot re-enter the cell. On the other hand, the released fatty acid undergoes very efficient reacylation and undergoes reuptake into the phospholipid pool, thereby preserving the critical precursor for subsequent reuse. A portion of the liberated arachidonic acid is primarily converted by cycio-oxygenase into the PGendoperoxides (PGH2 and PGG2). These compounds represent a critical branch point in PG metabolism since the endoperoxides serve as substrates for the enzymatic synthesis PGE2, PGD2, thromboxane AZ, PGIZ (prostacyclin), as well as some likely products yet to be identified. The ultimate fate of arachidonate in a given tissue is determined in each tissue by the repertoire of enzymes present. In tissues with multiple enzyme pathways other environmental factors, such as cofactors or substrate concentration, apparently influence the preferential biosynthetic direction. Little or no information is yet available about the regulation of the metabolism of the endoperoxides. Such studies would provide useful insight into approaches to pharmacologically manipulate the ultimate products of arachidonate metabolism. At this juncture we stand at a branch point for numerous investigations. The unresolved issues which seem worth focusing on include: (1) determination of the mechanism of activation and specificity of phospholipase activation; (2) continued isolation and identification of the additional arachidonate metabolites which apparently exist; (3) determination of the factors which modulate the downstream metabolism of the endoperoxides; (4) characterization of the biological properties of the arachidonate metabolites-thus little is known about the actions of HETE, HTT, PGD2, “RCS-RF,” SRS-A, etc.; (5) demonstration and quantification of the interactions of arachidonate metabolites and their receptors; and (6) development of synthetic compounds that could function as receptor antagonists both as tools for mechanistic studies as well as potential therapeutic agents. REFERENCES I. Piper, P. J., and Vane, J. R.: The release of prostaglandins from lung and other tissues, Ann. N. Y. Acad. Sci. lSth363, 1971. 2. Piper, P. J., and Vane, J. R.: Release of additional factors in

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anaphylaxis and its antagonism by anti-inflammatory drugs, Nature 223:29, 1969. 3. Lands, W. E. M., and Samuelsson, B.: Phospholipid precursors of prostaglandins, Biochim. Biophys. Acta 164:426, 1968. 4. Vonkeman, H., and Van Dorp, D. A.: The action of prostaglandin synthetase on 2-arachidonyl-lecithin, Biochim. Biophys. Acta 164430, 1968. 5. Vogt, W., Suzuki, T., and Babilli, S.: Prostaglandins in SRS-C and in a darmstoff preparation from frog intestinal dialysates, Memoirs Sot. Endocrinol. 14: 137, 1966. 6. Vargaftig, B. B., and Dao Hai, N.: Selective inhibition by mepacrine of the release of rabbit aorta contracting substance evoked by the administration of bradykinin, J. Pharm. Pharmacol. 24: 159, 1972. 7. Blackwell, Cl. J., Duncombe, W. Cl., Flower, R. J., Parsons, M. F., and Vane, J. R.: The distribution and metabolism of arachidonic acid in rabbit platelets during aggregation and its modification by drugs, Br. J. Pharmacol. 59:353, 1977. 8. Bills, T. K., Smith, J. B., and Silver, M. J.: Metabolism of [‘%]-arachidonic acid by human platelets, Biochim. Biophys. Acta 424: 303, 1976. 9. Jesse, R. L., and Cohen, P.: Arachidonic acid release from diacyl phosphatidylethanolamine by human platelet membranes, Biochem. J. 158:283, 1976. 10 Flower, R. J., and Blackwell, Cl. J.: The importance of phospholipase AZ in prostaglandin biosynthesis, Biochem. Pharmacol. 25:285, 1976. Il. Hong, S. L., and Levine, L.: Inhibition of arachidonic acid release from cells as the biochemical action of antiinflammatory corticosteroids, Proc. Natl. Acad. Sci. 73: 1730, 1976. 12. Tashjian, A. H., Jr., Voelkel;~ E. F., McDonough, J., and Levine, L.: Hydrocortisone ‘Mbits prostaglandin production by mouse fibrosarcoma cells, Nature 258:739, 1975. 13. Kantrowitz, F., Robinson,’ D. R., McGuire, M. B., and Levine, L.: Corticosteroids inhibit prostaglandin production by rheumatoid synovia, Nature 258:737, 1975. 14. Hong, S. L., and Levine, L.: Stimulation of prostaglandin synthesis by bradykinin and thrombin and their mechanisms of action on MC55 fibroblasts, J. Biol. Chem. 251:5814, 1976. 15. Gryglewski, R. J., Panczewko, B., Korbut, R., Grodzinska, L., and Ocetkiewicz, A. : Corticosteroids inhibit prostaglandin release from perfused mesenteric blood vessels of rabbit and from perfused lungs of sensitized guinea pig, Prostaglandins lot343, 1975. 16. Is&on, P. C., Raz, A., and Needleman, P.: Selective incorporation of %arachidonic acid into the phospholipids of intact tissues and subsequent metabolism to **C-prostaglandins, Prostaglandins 12:739, 1976. 17. Isakson, P. C., Raz, A., Denny, S. E., Wyche, A., and Needleman, P. : Hormonal stimulation of arachidonate release from isolated perfused organs. Relationship to prostaglandin synthesis, Prostaglandins 14:853, 1977. 18. Zusman, R. M., and Keiser, H. R.: Prostaglandin Ez biosynthesis by rabbit renomedullary interstitial cells in tissue culture. Mechanism of stimulation of angiotensin II, bradykinin and arginine vasopressin, J. BioI. Chem. 252:2069, 1977. 19 Gihnore, N. J., Vane, J. R., and Wyllie, J. H.: Prostaglandins released by the spleen, Nature 218: 1135, 1968. 20 Needleman, P.: The synthesis and function of prostaglandins in the heart, Fed. Proc. 35:2376, 1976. 21. Needleman, P., Marshall, G. R., and Sobel, B. E.: Hormone interactions in the isolated rabbit heart. Synthesis and coronary

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23.

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25.

26.

27. 28.

29.

30.

31.

32.

33. 34.

35.

36.

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38.

39.

40.

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vasomotor effects of prostaglandins, angiotensin and bradykinin, Circ. Res. 37:802, 1975. Hseuh, W., Isakson, P. C., and Needleman, P.: Hormone selective lipase activation in the isolated rabbit heart, Prostaglandins 13: 1073, 1977. Christ, E. J., and Van Dorp, D. A.: Comparative aspects of prostaglandin biosynthesis in animal tissues, in Raspe, G., editor: Advances in the biosciences, Elmsford, N. Y.. 1972, Pergamon Press, Inc., vol. 9, pp. 35-38. Aiken, J. W., and Vane, J. R.: Blockade of angioten% induced prostaglandin release from dog kidney by indomethatin, J. Pharmacol. Exp. Ther. 184678, 1973. Ferreria. S. H., and Vane, J. R.: Prostaglandins: Their disappearance from and release into the circulation, Nature 216:868. 1967. McGiff. J. C., Terragno, N. A., Colina, J., Terragno, D. A., and Nasjlettl, A.: Prostaglandins and their relationship to the kallikrein-kinin systems, in Pisano, J. J., editor: Chemistry and biology of the kallikrein system in health and disease, Fogarty International Center Proceedings No. 27, pp. 267274, 197s. McGiff, J. C., and Itskovitz, H.: Prostaglandins and the kidney, Circ. Res. 33479, 1973. Needleman, P., Key, S. L., Denny, S. E., Isakson, P. C., and Marshall, G. R.: Mechanism and modification of bradykinininduced coronary vasodilation, Proc. Natl. Acad. Sci. 72: ‘060, 1975. Ramwell, P. W., Shaw, J. E., Douglas, W. W., and Poisner, A. M.: Efflux of prostaglandins from adrenal glands stimulated with acetylcholine, Nature 210:273, 1966. Samuelsson. B.: On the incorporation of oxygen in the conversion of 8, I I, l4-eicosatrienoic acid to PGE,, J. Am. Chem. Sot. 87:301 I, 1965. Bergstrom, S., Danielsson, H., and Samuelsson, B.: The enzymatic formation of prostaglandin E, from arachidonic acid, Biochem. Biophys. Acta 90:207, 1964. Van Dorp. D. A., Beerthuis, R. K., Nugteren, D. A.. and Vonkerman. H.: Enzymatic conversion of all-cis-polyunsaturated fatty acids into prostaglandins, Nature 203:839, 1964. Vane, J. R.: Inhibition of prostaglandin synthesis as a mechanism of action of aspirin-like-drugs, Nature 231:232, I97 I. Hamberg, M., Svensson. V., and Samuelsson, B.: A new concept concerning the mode of action and release of prostaglandins, Proc. Natl. Acad. Sci. 71:3823, 1974. Willis, A. L.. Kuhn, D. C., and Weiss. H. J.: Acetylenic analog of arachidonate that acts like aspirin on platelets, Science 183:327, 1974. Hamberg, M., Svensson, J., Wakabayaski, T., and Samuelsson, B.: Isolation and structure of two prostaglandin endoperoxides that cause platelet aggregation, Proc. Natl. Acad. Sci. 71:345, 1974. Hamberg, M., Hedqvist, P., Strandberg, K., Svensson. J., and Samuelsson, B.: Prostaglandin endoperoxides. IV. Effects on smooth muscle. Life Sci. 16~451, 1975. Hamberg, M., Svensson, J., and Samuelsson, B.: Thromboxanes: A new group of biologically active compounds derived from prostaglandin endoperoxides, Proc. Nat]. Acad. Sci. 72~2994, 1975. Kulkarni, P. S., Roberts, R., and Needleman, P.: Paradoxical endogenous synthesis of a coronary dilating substance from arachidonate. Prostaglandins 12:337, 1976. Rar, A., Isakson, P. C., Minkes, M. S., and Needleman, P.: Characterization of a novel metabolic pathway of arachidonate

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in coronary arteries which generates a potent endogenous coronary vasodilator. J. Biol. Chem. 252: 1123. 1977. 41, Gryglewski, R. J., Bunting, S., Moncada, S.. Flower. R. J.. and Vane, J. R.: Arterial walls are protected against deposition of platelet thrombi by a substance (prostaglandin X) which they make from prostaglandin endoperoxides. Prostaglandins 12:68S, 1976. 42. Isakson, P. C., Raz, A., Denny, S. E.. Pure, E.. and Needleman, P.: A novel prostaglandin is the major product in ardchidonic acid metabolism in rabbit heart. Proc. Natl. Acad. Sci. 74:lOl. 1977. 43. Needleman, P.. Kulkarni, P. S., and Raz. A.: Coronary tone modulation: Formation and actions of prostaglandins, endoperoxides and thromboxanes, Science 195:409. 1977. 44. Nihsikawa, K., Morrison, A. R., and Needleman, P.: Exaggerated prostaglandin biosynthesis and its infuence on renal resistance in the isolated hydronephrotic rabbit kidney. J. Clin. Invest. 59: 1143, 1977. 45. Morrison, A. R., Nihsikawa, K., and Needleman, P.: Unmasking of thromboxane A, synthesis by ureter obstruction in the rabbit kidney, Nature 267:259. 1977. 46. Needleman, P., Bronson. S. D., Wyche, A., Nicolau, K. C., and Sivakoff, M.: Cardiac and renal prostaglandin I,: Biosynthesis and biological effect in isolated perfused rabbit tissues. J. Clin. Invest. 61:839-849, 1978. 47. Dawson, W., Boot. J. R., Cockerill, A. F.. Mallen, D. N. B., and Osborne, D. J.: The release of novel prostaglandins and thromboxaneh after immunological challenge of guinea pig lung, Nature 262:699, 1976. 48. Jakschik. B. A., S. Falkenhein, S., and Parker, C. W.: Role of arachidonic acid in release of slow reacting substance from rat basophilic leukemia cells, Proc. Natl. Acad. Sci. 744577, 1977. 49. Needleman. P., Raz, A., Ferrendelli, J. ‘4.. and Minkes, M. S.: Application of imidazole as a selective inhibitor of thromboxane synthetase in human platelets, Proc. Natl. Acad. Sci. 74~1716, 1977. SO. Moncada, S., Bunting, S., Mullan, K., Thorogood, P.. Vane, J. R., Raz, A., and Needleman, P.: Imidazole: A selective inhibitor of thromboxane qynthetase. Prostaglandins 13:6l I, 1977. 51. German, R., Bundy, G., Peterson, D., Sun, F., Miller, O., and Fitzpatrick, F.: Inhibition of human platelet thromboxane synthetase by 9. I I-azoprosta-5, l3-dienoic acid. Proc. Natl. Acad. Sci. 74:4007, 1977. S2. Needleman, P., Raz, A., Kulkarni. P. S.. Pure. E., Wyche. A., Denny, S. E., and Isakson, P.: Biological and chemical characterization of a unique endogenous vasodilator prostaglandin produced in isolated coronary artery and in intact perfused heart, in Kharasch, N., and Fried, J.: Biochemical aspect of prostaglandins and thromboxanes. New York, 1977, Academic Press, Inc., pp. 199-214. 53. Dusting. G. J.. Moncada, S., and Vane. J. R.: Prostacyclin (PGX) is the endogenous metabolite responsible for relaxation of coronary arteries induced by arachidonic acid. Prostaglandins 13:3, 1977. 54. Moncada, A., Gryglewski, R., Bunting, S., and Vane, J. R.: An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation, Nature 263:663, 1976. 55. Pace-Asciak, C., and Wolfe, L. S.: A novel prostaglandin derivative formed from arachidonic acid by rat stomach homogenates, Biochemistry l&3657, 197 I. 56. Moncada, S., Gryglewski, R. J.. Bunting. S., and Vane, J.

102 Needleman

.I. ALLERGY

CLIN. IMMUNOL. AUGUST

R. : A lipid peroxide inhibits the enzyme in blood vesselmicrosomesthat generatesfrom prostaglandin platelet aggregation, Prostaglandins12:715, 1976. 57. Possmayer,F., Scherphof, G. L., Dubbelman, T. M. A. R., Van Golde, L. M. G., and Van Deenen, L. L. M.: Positional specificity of saturate and unsaturated fatty acids in phosphatidic acid from rat liver, Biochim. Biophys. Acta 176~95, 1969. 58. Hill, E., and Lands, W. E. M.: Phosphotipid metabolism, in Wakel, S. J., editor: Lipid metabolism, New York, 1970, Academic Press, Inc., pp. 258-359.

1978

59. Vogt, W., Meyer, U., Lufft, E., and Babillia, S.: Formation by phospholipase A of SRS-C in perfused guinea-pig lung, identification with prostaglandins, N-S. Arch. Pharmk. 262~124, 1969. 60. Bartels, J., Kunze, H., Voft, W., and Willie, G.: Prostaglandin: Liberation from and formation in perfused frog intestine, N-S. Arch. Pharmk. 266:199, 1970. 61. Fredholm, B., and Strandberg, K.: Releaseof histamine and formation of smooth-muscle stimulating principles in guinea-pig lung tissue induced by antigen and phospholipase A., Acta Physiol. Stand. 76:446, 1969.

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