Prostaglandins and slow-reacting substance

THE JOURNAL OF

ALLERGY AND

NUMBER 1

VOLUME 63

Original

articles

Prostaglandins substance

and slow-reacting

Charles W. Parker, M.D.* St. Louis, MO.

The prostaglandins are generated de novo at the time of a physiologic stimulus. Once formed they are rapidly inactivated by a variety of enzymatic mechanisms. They exhibit a variety of physiologic actions involving changes in muscle contractility, secretion, lipolysis, differentiation, replication, aggregation, and permeability. Many of these effects appear to be mediated through changes in CAMP or cGMP.‘* ’ At the time prostaglandins were first described, interest was centered on their possible role as circulating hormones. Their effects on tissue CAMP concentrations suggested a possible analogy with polypeptide hormones which generally act by this mechanism. Since the usual effect of the E prostaglandins was to dilate blood vessels, their possible involvement in the control of systemic blood pressure received particular attention. In subsequent studies, however, the rapidity with which E and F prostaglandins are inactivated in the circulation became appreciated and the concept of a local modulatory action began to emerge. While an important role in regulating systemic blood pressure From the Washington University School of Medicine, Department of Medicine, Division of Allergy and Immunology. Received for publication June 12, 1978. Accepted for publication Sept. 15, 1978. Reprint requests to: Dr. Charles W. Parker, Department of Medicine, Division of Allergy and Immunology, 660 South Euclid, St. Louis. MO 631 IO. *Dr. Parker is an Investigator, Howard Hughes Medical Institute.

0091-6749/79/010001+14$01.40/0

still seems possible and circulating prostaglandin may be involved, the most important site of prostaglandin action may be locally, within the renal vasculature itself.2 Renal prostaglandin synthesis may also be important in Bartter’s syndrome where there is hyperplasia of the renal juxtaglomerular apparatus, hyperreninemia, hyperaldosteronism, and increased urinary Na+ and E prostaglandin secretion.3 Since the prostaglandins are renin-releasing agents and also increase sodium excretion by the kidney, a local increase in prostaglandin synthesis could be producing the other changes. In support of this possibility, when affected individuals are treated with indomethacin or aspirin their abnormalities are partially reversed. Prostaglandins may also be important in the modulation of local blood vessel tone in the chest, where they are thought to control the patency and subsequent construction of the ductus arteriosus at or near the time of birth.4 Persistence of a patent ductus is common in premature infants, presumably because the prostaglandin control mechanism is not yet fully developed. Indomethacin has recently been used in an attempt to initiate ductus closure in infants, with apparent success in 14 of 15 treatment trials. Another area where an important physiologic role for prostaglandin has been strongly suggested is in the reproductive system where the F prostaglandins stimulate uterine contraction” and induce labor and

0 1979 The C. V. Mosby Co.

Vol. 63, No. 1, pp. 1-14

2

Parker

J. ALLERGY

CLIN. IMMUNOL. JANUARY

1979

FIG. 1. Time-course of SW-A generation and release from passively sensitized isolated human lungs cells challenged with antigen E. ., SRS-A residual; q , SW-A release; A, SRS-Atotal; and ., histamine. (From Lewis, R. A., et al.: J. Exp. Med. 140:1133, 1974.)

TABLE 1. Possible mediators in bronchial

of bronchospasm

asthma

Histamine Acetylcholine Prostaglandins Other arachidonateendoperoxidemetabolites Slow-reactingsubstance Serotonin abortion. In addition, some examples of male infertility appear to be associated with low prostaglandin levels in the semen. Prostaglandins are also being studied in relation to peptic ulcer disease because of the marked ability of prostaglandin Ea to inhibit gastric acid secretion.6 Prostaglandin effects on bone are occasionally implicated in hypercalcemic states secondary to malignancy.7* l8 While this association is unusual, recognition is important since serum calcium levels can be lowered by indomethacin treatment. Judging from studies in experimental animals, prostaglandins are also important in the regulation of body temperature.g During an inflammatory response, prostaglandins synthesized locally in the hypothalamus could stimulate the temperature control center, helping to initiate changes in the skin and other peripheral tissues which contribute to the development of fever. In accord with this possibility, when leukocytic pyrogen is injected into the third ventricle, fever and shivering inhibited by aspirin are produced. Prostaglandins given in the same location bypass the aspirin block and the temperature response is re-

TABLE II. Characterization substance of anaphylaxis

of slow-reacting by differential bioassay* Smooth muscle preparationt

Test material

SRS-A Histamine Serotonin Bradykinin Prostaglandins

Guinea pig ileum

Estrous rat uterus

Gerbil colon

Human bronchi

+ + 0 + +

+ + +

+ +

+ + 0 -$

*From Orange, R. P., and Austen, K. F.: Slow reacting substance of anaphylaxis, in Dixon, F. J., Jr., and Kunkel, H. G., editors: Advances in immunology, New York, 1969, Academic Press, Inc., vol. 10, p. 106. t + = contraction; - = no contraction; @ = tachyphylaxis. $ Except the prostaglandin PGF,,.

stored. Thus at least part of the antipyretic effect of aspirin and indomethacin can be explained by an interfering effect on prostaglandin synthesis in the brain in or near the temperature control center. Very high levels of circulating prostaglandins, particularly E prostaglandins, are seen in association with medullary carcinomas of the thyroid.‘O Since prostaglandins contract intestinal smooth muscle and alter gastrointestinal secretion, they are probably involved in the marked degree of diarrhea that some individuals with this syndrome have. Perhaps the greatest current research in the prostaglandins from a public health point of view relates to a

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Prostaglandins

TABLE Ill. I&,* values range of agonistst

for FPL-55712

and slow-reacting

substance

3

against a

Agonists

IC,, (pglml-1)

5Hydroxytryptamine Histamine Acetylcholine Bradykinin ProstaglandinF,, ProstaglandinE, SRS-A (guineapig)

28.5 2 12.3 21.4 2 5.9 19.9 t: 5.7 9.8 t 5.0 4.9 +- 2.6 4.7 2 1.4 0.005 -r-0.001

*I& is the concentration necessary to produce a 50% reduction of the contractile response induced by the respective agonist. I-From Augstein, J., et al.: Nature New Biol. 245:215, 1973.

0 -20 0 H -LO bY -60

7

0

s -60

possible new approach to the control of coronary artery disease. Evidence is now beginning to accumulate that platelet aggregation is an important factor in the development of atherosclerosis. There is a very interesting recent study in pigs with von Willebrand’s disease where platelet aggregation is markedly impaired. The affected pigs appear to develop atherosclerosis much more slowly than control pigs fed a similar diet.” Aggregation of platelets is controlled to a very considerable extent by products of arachidonic acid metabolism,” particularly the thromboxanes and prostaglandins. Many platelet aggregation responses are markedly inhibited by aspirin and indomethacin. Indeed, the platelet cyclooxygenase system is sufficiently susceptible to aspirin inhibition that even a relatively small single dose of aspirin taken every several days may be sufficient to inhibit platelet aggregation. While definitive data from clinical trials with aspirin are not yet available, another inhibitor of platelet aggregation has recently been reported to markedly decrease the risk of subsequent clinical episodes of coronary artery disease in individuals with previous symptoms. I3 The prostaglandins are also being studied by immunologists. Some years ago we showed that prostaglandins markedly inhibit mitogenic responses in lymphocytes probably through their effects on CAMP biosynthesis.‘l They also interfere with antigenmediated histamine release from mast cells and basophils, lymphocyte-mediated cytotoxicity, and lysosomal enzyme release from phagocytes. More recently PGE, has been shown to induce maturation in immature thymocytes.‘” A role for prostaglandins in the action of suppressor T lymphocytes has also been suggested.I6 In addition, studies in progress in our laboratory suggest that the thromboxanes may be involved in the stimulation of human lymphocytes by mitogenic lectins.17 As far as the lung is concerned, the prostaglandins

q

SRS-A

FIG. 2. The effects of varying concentrations of cytochalasin B on the antigen-induced release of histamine and SRS-A from human lung fragments. In control samples in this experiment, mean histamine release was 30% and mean SW-A release was 1,400 Uigm. (From Orange, R. P.: J. Immunol. 114:182, 1975.) have been known for some time to affect bronchial smooth muscle tone with PGE, and PGAza exerting opposing actions. ‘R-20 In isolated smooth muscle preparations PGE, produces relaxation whereas PGFzu produces contraction. These two substances are also effective via the aerosol route with PGE, decreasing (usually) and PGFp, increasing pulmonary resistance. Thromboxane A2 and breakdown products of PGFB, such as l$keto-PGF,, also contract bronchial smooth muscle.*’ Not only are these various bronchoconstrictors and bronchodilators present in lung, but many of them are also formed in increased amounts during acute antigen challenge in sensitized lung fragments. The release of PGF2, is more prominent in the peripheral parts of the lung whereas more PGEz is released in or near the major bronchi. I9 Interestingly, PGFz, has been shown to raise cyclic GMP2* in human lung fragments whereas PGE2 raises cyclic AMP. I93” These cyclic nucleotides or their lipophylic analogues, respectively, contract and dilate bronchial smooth muscle and are probably involved as secondary intracellular messengers in the opposing actions of the two prostaglandins. The increased release of prostaglandins may well be a secondary event in the acute allergic reaction since the increase in synthesis of PGF*, is blocked by Hl type histamine blocking agents whereas the release of PGEz is blocked by H2 type histamine blocking agents.‘” Math61g has shown that the airway in asthmatic pa-

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FIG. 3. Inactivation of SRS-A by highly purified eosinophil arylsulfatase at pH 5.0 (O-O), 5.7 (O-O), and 8.0 (-ml. (From Wasserman, S. I., Goetzl, E. J., and Austen, K. F.: J. Immunol. 114:645, 1975.)

1

2.5

5

r

FIG. 4. Dose-response curves for SRS-A alone (x) and in the presence of three concentrations of FPL-55712 (20 ng ml-‘, l ; 40 ng ml-‘, n ; 80 ng ml-‘, A.). Standard errors indicated by vertical bars except when smaller than symbol used for mean value. (From Augstein, J., et al.: Nature New Biol. 245:215, 1973.)

15

IO PREINCUBATION

CLIN. IMMUNOL. JANUARY 1979

(mins )

FIG. 5. The effects of preincubating rat peritoneal cells with 5 x 10e3 M cysteine at 37” C for varying times on the subsequent calcium ionophore-induced release of histamine (0~) and SIX-A (xx). (From Orange, FL, and Moore, E. J.: J. Immunol. 116:392, 1976.)

tients is exquisitely sensitive to PGF2, and suggested that PGFzamay be an important mediator of the bronchospasm in this condition. ls On the other hand, there is no evidence that cyclooxygenase inhibitors are effective in the control of asthmatic paroxysms, contrary to expectation if PGFz, is importantly involved.23 While it is possible that lung tissue is less susceptible to cyclooxygenase inhibition than other tissues, there is no evidence that this is true. Indeed, there is a subpopulation of asthmatic patients which develops increased bronchospasm after ordinary therapeutic doses of aspirin or indomethacin. This same argument can be applied to some of the other mediators that have been suspected to be important in bronchospasm such as histamine, serotonin, and ace-

tylcholine (Table I). While there is no doubt that histamine and acetylcholine constrict human bronchial smooth muscle or that they are particularly prone to do so in asthmatic patients, the absence of a welldefined therapeutic effect with pharmacologically effective blocking agents such as the antihistamines argues against a primary role of these substances in the changes in lung function. The one mediator that does not have an effective antagonism for bronchospasm is slow-reacting substance (SRS), making it a prime suspect in the bronchoconstrictive response. Slow-reacting substance or SRS was first described by Feldberg and Kellawayz4 in 1938 as a smooth muscle contracting activity released during the perfusion of cat and guinea pig lungs with cobra venom.

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and slow-reacting

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5

A23187 pg/ml

FIG.6. Dose-response to A23187; RBL-1 cells (5 x 106) were suspended in medium in a final volume of 0.5 ml and incubated with A23187 at the concentrations indicated for 20 min at 37” C. The SRS activity recovered is expressed as percentage of maximum activity which was observed in each experiment (n = 3) at 10 pglml. The units of activity at this concentration were 490,800, and 938 per 1 x IO’cells. The vertical bars denote SEM (From Jakschik, B. A., et al.: J. Immunol. 119:618, 1977.) TABLE

IV. Effect

of fatty

acids

on SW

formation* Units of SRS activity

Fatty acid

None Arachidonic Dihomo-y-linolenic Arachidic Linoleic Linolenic

-A23187

+A23187

2k1 59 k 39

140 674 252 144 42 36

3rtl 10 2 6 .5&l 15 2 8

k 2 2 t

38 301 95 59

k 18 2 21

Percent of response to A23187 alone

P

100 543 t 257 ” 157 t 27 k 26-c

166 151 97

<0.007 t 0.3 0.6

11

CO.001 $

15


RBL- 1 cells, lO’/ml, were incubated in medium with and without fatty acid (25 pg/ml), A23187 (5 pg/ml), or both. Data are mean t SEM of 5 experiments (3 with 0.1% albumin and 2 without). Experiments with and without albumin were pooled because the data

were very similar. The incubation time was 15 min. *From Jakschik, B., et al.: Fed. F’roc.36~1328, 1977. (Abst.) t Statistically higher than A23 187 alone. fstatistically lower than A23187 alone.

and Trethewie*j demonstrated the production of a substance with similar properties during stimulation of perfused sensitized guinea pig lungs with antigen. Brocklehurst26 clearly distinguished the SRS activity from histamine by the failure of the contractile response on guinea pig ileum to be inhibited by antihistamines. Since that time SRS has been intensively studied in a number of laboratories, most notably those of Austen, Uvnas, Orange, Bach, Ishizaka, Piper, Lichtenstein, and ourselves.32-4” This work has led to an appreciation of the circumstances leading to SRS release and a definition of its major physical-chemical properties, even though its precise chemical structure is still not known. Obviously, without pure, chemically defined SRS and effective competitive antagonists of its acTwo years later Kellaway

tion, its true significance

in immediate

hypersensitiv-

ity reactions cannot be fully assessed.Nonetheless, a good deal of progress has been made. There are now two reproducible systems for generating SRS activity in human tissues. The most widely studied,*’ particularly in the work of Austen, Orange, and their colleagues, is the sensitized lung fragment system, where challenge with ragweed antigen generates quite substantial amounts of SRS reactivity. As shown by Lichtenstein4’ and others, mixed human peripheral blood leukocytes also generate SRS activity in response to antigenic challenge as do monkey and human leukocytes recovered by bronchial lavage. 45 All of these responses appear to be correlated with the presence of IgE antibodies. Antigenmediated SRS release is also demonstrable in rats,

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20 MINUTES

30

40

FIG. 7. Time-course of SW release by A23187: RBL-1 cells (5 x10”) were suspended in a final volume of 0.5 ml and incubated with A23187 (10 pglml) for various time periods at 37” C. The SRS activity recovered is expressed as percentage of maximum activity observed in each respective experiment (n = 3). In two experiments the maximum occurred at 20 min and in one at 10 min. The activities at the maximum were 311, 2,088, and 53 U per 1 x IO’ cells. (From Jakschik, B. A., et al.: J. Immunol. 119:618, 1977.)

guinea pigs, rabbits, monkeys, and calves.4fi In addition to specific antigen challenge, a number of other stimuli have been shown to produce SRS activity, including the histamine-releasing agent 48/80,3’, 32 is a divaLilly compound A23 187,30,37942343which lent cation ionophore also capable of stimulating histamine secretion, and anti-IgE antibody.3g Stimulation of SRS release by anti-IgE antibody has been demonstrated in human and monkey lung fragments, human nasal polyps, and human and monkey bronchial lavage cells. In guinea pig lung fragments, on the other hand, anti-IgG, antibodies increase SRS formation, whereas in the rat peritoneal cavity anti-IgGa antibody is an effective stimulus.28 The results with anti-IgE antibody are of particular interest. Taken together with the evidence referred to above that IgE antibodies appear important in the SRS response to antigen in sensitized human tissues, it seems evident that SRS is normally produced during anaphylactic responses and that IgE immunoglobulins are frequently involved in their formation. Judging from work in other species, however, it appears that

CLIN. IMMUNOL. JANUARY 1979

non-IgE immunoglobulins are also capable of initiating an SRS response. Since IgE-mediated allergic responses normally involve mast cells and basophils, the simplest explanation for the generation of SRS during IgE-mediated responses would be if the mast cells (or basophils) themselves were producing SRS. Somewhat surprisingly, this possibility has been difficult to verify. Based on studies with the calcium ionophore A23 187 and various particulate stimuli, it appears that neutrophils and blood mononuclear cells can both generate SRS, raising the possibility that the release of SRS is indirect through some mast cell product capable of acting on other cell types. Most studies attempting to demonstrate that mast cells or basophils themselves can serve as a source of this material have been negative or inconclusive. However, Lewis Yecies47 and Barbara Jakschik48 from our laboratory have recently obtained convincing evidence that highly purified rat peritoneal mast cells do in fact generate significant amounts of an SRS-like activity which by a variety of criteria is indistinguishable from previously described SRSS.~~,4x Thus it appears that the mast cells themselves could be a major source of SRS during IgE-mediated allergic reactions in vivo. SRS is normally measured by bioassay, based on its smooth muscle contracting activity for guinea pig ileum. The typical response is a sustained contraction occurring after a short latent period which is not blocked by antihistamines. Since a number of other substances, including histamine, serotonin, bradykinin, and the prostaglandins, have significant contractile activity in this system, additional criteria are needed to be certain that the activity is really SRS. These include: (1) the use of selective blocking agents such as antihistamines and methylsergide; (2) evaluation of activity in other smooth muscle bioassay systems (Table II); (3) relative susceptibility to proteolytic and arylsulfatase digestion and extremes of pH; and (4) behavior in suitable column and thin-layer chromatography systems. In addition to its activity on guinea pig ileum, SRS contracts tracheal bronchial smooth muscle in vitro, as demonstrated by Collier and James4’ in guinea pig tissues and by Brocklehurst”” in human tissues. In addition, crude aerosolized preparations of SRS have been observed to affect airway function in vivo in asthmatic patients although not in control subjects.“’ When unanesthetized guinea pigs are injected intravenously with crude SRS, there is a marked decrease in compliance with a relatively small change in conductance, suggesting that SRS may be acting primarily at the level of the small airways.28 While

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none of these observations really clarifies the quantitative importance of SRS in airway function, they do indicate an effect on airway smooth muscle contractility both in vivo and in vitro, making further studies highly desirable. SRS also produces permeability changes in monkey, guinea pig, and human skin, with much less marked changes in rat skin,27 raising the possibility that SRS could also be involved in acute cutaneous reactions to antigens or nonspecific stimuli as well as in acute allergic reactions in lung. It should be emphasized, however, that both the lung and skin studies were conducted with crude SRS. The exact nature of the biologic activity of SRS will require further study once completely pure SRS is available, since the existence of contaminating activities which might have enhanced or inhibited SRS activity cannot be excluded.

Even though the structure of SRS is not known, there is considerable information available in regard to its physical-chemical properties and behavior in various chromatographic systems.“. 31*34By column and thin-layer chromatography, it is more polar than the conventional prostaglandins. It can be extracted into nonpolar organic solvents such as ether from acidic but not from alkaline solutions, suggesting that it is an acidic lipid. Marked losses of activity occur within a few minutes in acidic solutions whereas in alkaline solutions the stability is much greater. Oxidizing and halogenating agents inactivate SRS, as might be the case if unsaturated double bonds are present. Susceptibility to oxidation may help explain the lability of SRS during purification. No one has purified SRS to homogeneity although, as discussed below, our laboratory has recently prepared radiolabeled SRS from rat basophilic leukemia cells which appears to be radiochemically pure.44 The kinetics and pharmacologic control of SRS release have been studied in some detail in the sensitized human and guinea pig lung fragment systems.‘“, J2, x Ordinarily no SRS activity is present until after antigen is added even when the cells are disrupted to release any intracellular SRS which might be present. Therefore, in contrast to histamine, SRS is not present preformed in cells. Occasional human lung preparations do contain small amounts of preexisting SRS, presumably due to in vivo synthesis. SRS activity increases within a few minutes after antigen challenge, reaches a maximum after 15 to 30 min, and then remains stable or decreases. Initially most of the SRS is within the cells, indicating a brief lag before the newly synthesized SRS is secreted into the medium, but by S-10 min most of the SRS is

Prostaglandins and slow-reacting substance

TABLE V. Effect of indomethacin formation* lndomethacin bdml)

0.1 0.4 0.7 1.0 10

7

on SRS

% of control t 172 126 106 87 81

t + -t t_ t

54 21 IO 8 15

RBL-1 cells, lO’/ml, were preincubatcd for 15 min with or without indomethacin, at the concentration indicated. This was followed by a 1%min incubation with A23187 at 10 pg/ml. *FromJakschik, B. A., Folkenhein, S., and Parker, C. W.: Proc. Natl. Acad. Sci. USA 74:4577, 1977. ;Mean I SEM (n = 4).

extracellular (Fig. 1). Whether the intracellular SRS is identical to the SRS in the medium remains to be established. The diminution of SRS activity with prolonged incubation raises the possibility of reuptake by cells, inactivation or conversion to a partially active species. As discussed below, lung fragments contain arylsulfatases and quite possibly other enzymes which degrade SRS, so inactivation probably accounts for part of the change in activity with time. The stimuli which release SRS are the same ones that release histamine, and while the kinetics of release reactions for the two substances are somewhat different (histamine appears sooner in the medium than SRS), as a rule chemical manipulations which enhance or inhibit histamine release affect SRS release in the same way. For example, in the sensitized human lung system cholinergic and a-adrenergic stimuli enhance histamine and SRS release whereas AMP agonists and the removal of Ca’+ from the medium inhibit both responses.‘8 However, as shown by Orange, 35the SRS and histamine release reactions are partially dissociated by the cytochalasins. As seen in Fig. 2, cytochalasin B exerts little or no effect on the histamine release response at concentrations where SRS release is markedly inhibited. The cytochalasins appear to produce the majority of their effects on mammalian cells by inhibiting the crosslinking of microfilaments. and depending on the system either enhance or inhibit secretion. While the significance of the cytochalasin effect in mast cells is not presently clear, it is apparent that histamine can be extruded into the medium when SRS biosynthesis and release is markedly impaired. Austen and his colleagues2x. 33-~i3have suggested that the diminution in SRS bioreactivity in sensitized lung fragments stimulated with antigen and maintained beyond 30 min may be due to SRS inactivation

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CLIN. IMMUNOL. JANUARY 1979

12- HETE t 12-Hydroperoxy-ETE

5-Hydroperoxy-ETE a

Phospholipids

Prostocycl in-

-

0

\ Arochidonic

Acid

1

@

@ PGG2/ PGH, (Endoperoxides) @

6-Kelo

I

PGF,,

by arylsulfatases. Arylsulfatases cleave sulfoester linkages from benzene rings and other unsaturated ring systems and are classified as type A or type B depending on their pH optimum and other characteristics. Originally, highly purified type B limpet or mollusk arylsulfatases were shown to inactivate SRS. Later a similar enzyme present in human lung was shown to have this capability. More recently, an arylsulfatase that inactivates SRS has been demonstrated in human eosinophils (Fig. 3). The Boston group has suggested that part of the role of eosinophils in IgEmediated inflammatory reactions may be to provide arylsulfatase to destroy SRS. However, most or all cells contain at least some arylsulfatases, including type II pneumocytes, one of the prominent cells in the lung. Moreover, purified mast cells themselves contain arylsulfatase and degrade SRS. 56Also, since SRS inactivation by the enzyme is slow, even at high enzyme concentrations, alternative mechanisms of inactivation need to be considered. While the significance of arylsulfatase in control of SRS action in vivo requires further study, the inactivation itself is of considerable interest. Based on the known specificity of arylsulfatase, one might suspect that SRS contains an esterified sulfate group. Moreover, since partial degradation of SRS may be necessary before it can be analyzed by mass spectroscopy, this enzyme should be of value in further attempts to determine SRS structure. Finally, the arylsulfatase inactivation reaction itself appears to be useful in distinguishing SRS (or at least certain forms of SRS) from other smooth muscle contracting substances. Another recently suggested criterion for distinguishing SRS from other agents with smooth muscle contracting activity involves use of the selective SRS antagonist FPL-557 12, a product of the Fisons Com-

YHHT

y

I Prostoglondins I PGF,, ,PGE,,PGD,

FIG. 8. Pathways of arachidonic acid metabolism. is 12-hydroxyarachidonic acid.

/

)

Thromboxone

Az

1 Thromboxone

Bz

5-HETE is 5-hydroxyarachidonic

acid; 12-HETE

pany. FPL-557 12 and related chromone-2-carboxylic acids are potent inhibitors of the contractile response to SRS on guinea pig ileum.57* j8 Fig. 4 is a semilogarithmic plot of the contractile response of guinea pig ileum to SRS in the presence and absence of FPL-55712. Increasing amounts of FPL-557 12 shift the SRS dose-responsecurve in a parallel fashion to the right, suggesting that it is acting as a competitive antagonist. The selectivity of FPL-557 12 for SRS is shown in Table III. Inhibitory potency is expressed as the I& (concentration of FPL-55712 needed to inhibit the contractile response by 50%). High concentrations of FPL-55712 are needed to inhibit responses to 5-hydroxytryptamine, histamine, and acetylcholine (ICsOs of greater than 10 pg/ml). Responses to bradykinin, PGE1, and PGFZa are more subject to inhibition by FPL-55712 (IC& concentrations of 4.7 to 9.8 pg/ml) but are still far above the IQ,, concentration for SRS (0.005 pg/ml). Unfortunately , FPL-557 12 is very unstable, making it unsuitable as a potential SRS antagonist in vivo. Nonetheless, its selectivity is very helpful in verifying the presence of SRS. Whether all SRSs are inhibited by FPL-55712 remains to be established. Webster and her colleagues”s have partially purified several fractions with SRS activity from human lung which appear to differ markedly in their susceptibility in FPL55712 inhibition. Another possible clue that SRS is heterogenous comes from the variable ability of cysteine and several other thiols to enhance SRS generation, depending on the tissue. 33, 34 As shown in Fig. 5, SRS release in mixed rat peritoneal leukocytes in response to calcium ionophore A23 187 is markedly potentiated at high (mM) concentrations of cysteine even though histamine release is completely unaffected. Cysteine

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also augments SRS release in human lung but has little or no effect in rat mast cells and rat basophilic leukemia cells (unpublished observations). The basis for the enhancement in susceptible tissues is unclear. The increase in SRS generation by cysteine is highly time-dependent, the response being most marked when the thiol is added 2 to 5 min before the ionophore. Some thiols are much more effective in producing enhancement than others. For example, sodium sulfide and thioglycolate enhance SRS release in human lung but 2-mercaptoethanol and dithiothreitol fail to do so. The failure of these two thiols to enhance SRS release suggests that more than a simple reducing action is involved. Regardless of the mechanism, the apparent tissue specificity of the response suggests the existence of more than one mechanism for generating SRS and, by implication, multiple species of SRS. Based on the above, SRS can be described as an acidic, probably unsaturated lipid (or more probably, family of lipids) stable in base, labile in acid, and distinguishable from the conventional prostaglandins by a variety of criteria including chromatographic behavior, FPL-557 12 blocking, and inactivation by arylsulfatase. Several years ago my colleagues and I considered the possibility that SRS might be derived from arachidonic acid even though it is not a classic prostaglandin. We believe we now have convincing evidence for this. In the work that I will be describing, Barbara Jakschik, Sandra Falkenhein, and Hanna MacDonald all contributed importantly.““, ” The system that we chose was a line of rat basophilic leukemia cells (RBL- 1) obtained originally in England and brought to this country by Metzgar and Kulczycki for studies of the IgE receptor. These cells contain histamine and closely resemble normal mast cells and basophils in their granular structure. Moreover, they can be grown in considerable quantity in tissue culture in the absence of contaminating cells. If these cells could be induced to generate SRS they could be radiolabeled in tissue culture with possible SRS precursors, and studies of SRS metabolism and structure should be considerably facilitated. A series of conventional stimuli such as anti-IgE antibody, concanavalin A, and 48/80 were tried with little or no success. However, the divalent cation ionophore, A23 187, did generate significant amounts of SRS activity in this cell line. It was in this system that the involvement of arachidonic acid as a biosynthetic precursor of SRS was first demonstrated. The reason A23 187 is able to generate SRS-like activity in RBL- 1 cells when other stimuli are inactive is not presently clear. Just after our studies were un-

Prostaglandins

and slow-reacting

substance

9

1 t

2

FIG. 9. Two-dimensional chromatography of SRS. A Ylabeled SRS-containing sample (partially purified on a silicic acid column) containing 15,900 cpm and 3,000 U was applied in methanol to silica gel G 20-cm x 20-cm plates. Chromatography was performed in the dark with 2,6-di-t-butyl-p-cresol present. First solvent: propanoli ammonia/water (6: 3: 1); second solvent: propanollwater (3 : 1). After a brief drying period, the plate was placed together with Kodak X-omat R film in a lead cassette in a nitrogen atmosphere and allowed to develop for 3 days at 24” C. Spot A contained 93% of the recovered bioreactivity and 12% of the recovered radioactivity (the overall recovery of bioreactivity was 65% and of radioactivity 71%). (From Jakschik, B.A., Falkenhein, S., and Parker, C. W.: Proc. Natl. Acad. Sci. USA 74:4577, 1977.)

derway, Bach and Brashler37 independently described the use of A23187 to produce SRS in rat peritoneal leukocytes. A23 187 also stimulates SRS formation in human peripheral blood leukocytes and basophilic leukemia cells. Obviously, then, the rat basophilic leukemia cells are not the only cells that generate SRS in response to this stimulus. An interesting question that has not yet been studied is whether tissues not containing leukocytes also have the capability of producing SRS when stimulated with the ionophore. A typical dose-response curve for SRS release in RBL-I cells is shown in Fig. 6. Maximal release of SRS was seen at A23187 concentrations of 5 to 10 Fg/ml. SRS activity was demonstrable within a few minutes, reached a maximum after 15 to 20 min, and then decreased (Fig. 7), similar to SRS responses in other tissues. Since Austen, Wasserman, and their colleagues‘* have demonstrated the presence of an arylsulfatase in rat basophilic leukemia cells, the inactivation may be due to this enzyme. The generation of SRS is highly temperature-dependent with little or

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FIG. 10. DEAE-cellulose chromatography of purified SK; Y-labeled SRS (purified by silicic acid and two-dimensional thin-layer chromatography) and unlabeled SRS (purified by Amerlite) were mixed, incubated in 5% methanol/O.1 M NaOH for 30 min at 37” C, neutralized, and dried; an aliquot containing 700 to 750 bioassay units and 865 cpm in 1.7 ml of methanol/chloroform (1 : 7) was applied to a 7-ml DEAE-cellulose column packed in chloroform/methanol (7: 1) and maintained at 4” C in the dark. The column was eluted with four solvents: solvent I, 12 ml of chloroform/methanol (7: 1); solvent II, 14 ml of chloroform/methanol (7:3); solvent Ill, 14 ml of methanol; solvent IV, 50 ml of methanol/2 M ammonium carbonate (IO: 1). Atotal of 15 fractions was collected. Points of addition of the different solvents are indicated by the arrows. After drying, samples were divided into equal parts for radioactivity measurement (lo-min counting times) and bioassay. The values given in the figure are corrected (e.g., multiplied by 2, to correct for the use of only one-half the sample). Recovery of both applied radioactivity and bioreactivity was greater than 80%. Considering the inherent variation in the bioassay (?20%), statistical errors in counting, and possible spontaneous inactivation on the column, this represents excellent correspondence between radioactivity and bioreactivity. (From Jakschik, B. A., Falkenhein, S., and Parker, C. W.: Proc. Natl. Acad. Sci USA 74:4577, 1977.)

no response below 15” C, suggesting that an enzymatic process may be involved. By a variety of criteria, the SRS we were studying appeared to be very similar or identical to SRSs in other tissues: (1) behavior in various column and thin-layer chromatography systems; (2) stability to 0.1 NaOH for 30 min at 37” C; (3) extractability into ether at pH 3; (4) lability to acid; (5) inhibition of activity by FPL-557 12 and limpet arylsulfatase; (6) spectrum of reactivity with various smooth muscle preparations. The first evidence suggesting that arachidonic acid is a precursor of RBL-1 SRS was an ability of eicosatetraenoic acid (ETYA) to diminish SRS formation. ETYA is a CZOanalogue of arachidonic acid, which has triple bonds instead of double bonds at the 5, 8, 11, and 14 positions. ETYA affects both of the two major pathways of arachidonic acid metabolism in mammalian cells (Fig. 8). Prostaglandin synthesis is affected through an action on the cyclooxygenase pathway and hydroxyperoxy- and hydroxy-fatty acid synthesis through an action on the lipoxygenase pathway. Marked (up to 90%) inhibition of the SRS response was observed with ETYA over a

broad range of ionophore concentrations. ETYA was inhibitory at concentrations as low as 1 to 2 pg/ml even with albumin in the medium, making it unlikely that the inhibition was nonspecific. The next question we considered was whether the inhibition of SRS synthesis by ETYA was being exerted through the lipoxygenase or cyclooxygenase pathways. One way of attempting to answer this question is by the use of indomethacin, a selective cyclooxygenase inhibitor. As shown in Table IV, indomethacin did not convincingly inhibit SRS formation at any of the concentrations studied. Indeed, at low concentrations of indomethacin the SRS response actually appeared to be enhanced, as might be the case if SRS were a lipoxygenase product and indomethacin was making more arachidonic acid available for utilization by this pathway. While it might be argued that RBL-1 cells are peculiarly resistant to indomethacin, indomethacin markedly inhibited the synthesis of an arachidonic acid metabolite tentatively identified as PGD2, making this explanation unlikely .60 The evidence that arachidonic acid was involved in SRS formation was considerably strengthened when it

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was shown that exogenous arachidonic acid considerably enhanced the response to A23 187. Moreover, arachidonic acid itself was modestly stimulatory , even when A231 87 was absent. The enhancement of SRS formation by arachidonic acid was dose-related, with effects being demonstrable at arachidonic acid concentrations as low as 0.01 pg/ml. Dihomo-ylinolenic acid (8,11,14 eicosatetraenoic acid), a CZO fatty acid with three double bonds instead of 4, was considerably less active in enhancing the ionophore response, and arachidic acid, which is a C2,,fatty acid with no double bonds, had no activity (Table V). Linoleic and linolenic acids, C,, fatty acids with 2 and 3 double bonds, respectively, actually inhibited SRS formation. The rigid requirements in terms of fatty acid structure for stimulation argued against a nonspecific detergent action and suggested that arachidonic acid might indeed be a substrate for an SRS-synthesizing enzyme. In such a scheme the low-grade stimulation with arachidonic acid alone could be explained by assuming that the enzyme(s) synthesizing SRS in RBL- 1 cells is normally only partially active and that part of the action of the ionophore is to activate the enzyme. A23187 is also a phospholipase activator providing substrate for the enzyme, even when exogenous arachidonic acid is not made available. The best evidence that arachidonic acid is indeed a precursor of SRS comes from radiolabel incorporation studies. RBL-1 cells were preincubated either overnight or for much shorter time periods (5 1.5min) with radiolabeled [‘“Cl arachidonic acid and then challenged with A23187. After 5 to 15 min, the SRS in the medium was purified by ethanol extraction, column chromatography, and thin-layer two-dimensional chromatography (Fig. 9). In the experiment shown, the spots designated as A and B contained about 20% of the total radioactivity and essentially all of the recovered bioreactivity. The overall recovery of bioreactivity was quite good, of the order of 65%. with most of it being in spot A. While the possibility had to be considered that the radiolabel and the bioreactivity were fortuitously comigrating, an exact correspondence between bioactivity and radioactivity was subsequently seen in 9 of 9 additional experiments in the two-dimensional system, making this explanation unlikely. In addition, we have studied a number of other thin-layer chromatographic systems where the recovery of bioreactivity is not as good, and there is always a discrete band of radioactivity in the bioreactive area. Just as importantly, if the material in spot A is eluted and applied to a diethylaminoethyl (DEAE) cellulose column, almost all of the radioactivity and bioreactivity elute together (Fig. 10).

Prostaglandins and slow-reacting substance

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With the possible exception of 8,11,14 eicosatetraenoic acid, preincubation of cells with other radioactive long-chain fatty acids (8,l 1,14 eicosatetraenoic acid, palmitic, stearic, linolenic, and linoleic) was not associated with increased amounts of radioactivity in bioreactive areas. Thus it appears that radioactive arachidonic acid is indeed being selectively incorporated into the SRS molecule and that RBL-I cells produce at least two different species of SRS. However, it is possible that one of the species of SRS is a partial degradation product formed during the purification. Despite the success in obtaining what appears to be radiochemically pure SRS, the pathway involved is a quantitatively minor one from the point of view of overall arachidonic acid metabolism in RBL-1 cells. Only 0.1% to 0.5% of original arachidonic acid radioactivity copurifies with SRS and, even allowing for losses during purification, this is considerably less than some of the other arachidonic acid metabolites which are formed. Interestingly, by calculating the specific activity of the original arachidonic acid label and neglecting any dilution of the label with endogenous arachidonic acid, it appears that only low picogram quantities of SRS are needed to induce contraction of guinea pig ileal muscle. While dilution of the radiolabel with endogenous arachidonic acid is undoubtedly occurring, making estimates of SRS specific activity falsely high, SRS is probably considerably more potent on a molar basis than histamine in this bioassay system. More recent studies from our laboratory indicate that 3?S-labeled SO,, methionine, and cysteine are also apparently incorporated into the SRS molecule, giving radioactive spots after purification and 2D thin-layer chromatography in the same two areas that contain “C radioactivity and bioreactivity. On the basis of this and other evidence, it appears that the two SRS molecules contain a CZOfatty acid core together with a sulfur-containing side chain and that the two SRSs probably differ from one another with respect to the nature of the side chains. However, other structural features remain to be elucidated and final proof that these are the major structural elements of SRS will require that a molecule with the appropriate level of biologic reactivity be obtained by organic synthesis. Subsequent to the initial presentation of our work in 1976,61Bach, Brashler, and German”’ and their colleagues at The Upjohn Company provided data consistent with a precursor role of arachidonic acid for SRS in the rat peritoneal leukocytes stimulated with A23187. While their evidence for incorporation of radioactive arachidonic acid into SRS is much less

12

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convincing than our RBL-1 cell data, ETYA does inhibit SRS formation in their system, just as it does in RBL-I cells. Based on the ability of a selective inhibitor of thromboxane synthesis to partially block SRS formation, they have suggested that SRS may be a product of the thromboxane synthetase pathway. While I feel that SRS formation through the lipoxygenase is a more likely explanation, the available data with inhibitors of arachidonic acid metabolism are not sufficiently conclusive to permit a definitive judgment at this time. Since the function of the lipoxygenase pathway is not presently known, resolution of this question is of considerable interest. If SRS is indeed formed through the lipoxygenase pathway, it will be the first lipoxygenase product with potent smooth muscle contracting activity, in contrast to the many known cyclooxygenase products which exhibit this property. Whatever the pathway, the suggested precursor role for arachidonic acid in SRS formation can no longer be seriously questioned. In this connection it is of interest that glucocorticoids have been reported to inhibit phospholipase A2, the enzyme that releases free arachidonic acid from cellular phospholipids,63 so part of the action of corticosteroids in bronchial asthma could be at this level. Alternatively, glucocorticoids may act by inhibiting the formation of a small polypeptide which releases arachidonic acid.64 Apart from the implications of this work for the rapidly growing field of arachidonic acid metabolism, highly purified radiolabeled SRS is available for the first time, which should be of considerable help in further attempts to determine SRS structure. It seems reasonably safe to predict that within the next one to two years the structure of SRS will be known. Once this has been accomplished, selective antagonists that are less labile than FPL-55712 will almost certainly be synthesized and specific immunochemical or biochemical assays for SRS will be developed. At that point it should be possible to better define the role of SRS in acute allergic responses in man. If SRS turns out to be as important as many workers in the field suspect it will be, a whole new approach to therapy involving the use of selective SRS antagonists will then be available. REFERENCES 1. Crossley, N. S.: Prostaglandins, Chemistry and Industry 1976~334, 1976. 2. Russell, P. T., Eberle, A. J., and Cheng, H. S.: The prostaglandins in clinical medicine. A developing role for the clinical chemist, Clin. Chem. 21:653, 1975. 3. Gill, J. R., Fr%lich, J. C., Bowden, R. E., Taylor, A. A., Keiser, H. R., Seyberth, H., Oates, J. A., and Bartter, F. C.: Bartter’s syndrome: A disorder characterized by high urinary

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prostaglandins and a dependence of hyperreninemia on prostaglandin synthesis, Am. J. Med. 61:43, 1976. 4. Heymann, M. A., Rudolph, A. M., and Silverman, N. H.: Closure of the ductus arteriosus in premature infants by inhibition of prostaglandin synthesis, N. Engl. J. Med. 295530, 1976. 5. Horton, E. W.: Prostaglandins-a short review, Scot. Med. J. 20: 155, 1975. 6. Vane, J. R.: The release and fate of vasoactive hormones in the circulation, Br. J. Pharmacol. 35:209, 1969. 7. Seyberth, H. W., Segre, G. V., Morgan, J. L., Sweetman, B. J., Potts, J. T., and Oates, J. A.: Prostaglandins as mediators of hypercalcemia associated with certain types of cancer, N. Engl. J. Med. 293: 1278, 1975. 8. Tashjian, A. H.: Prostaglandins, hypercalcemia and cancer, N. Engl. J. Med. 293:1317, 1975. (Editorial.) 9. Milton, A. S., and Wendlandt, S.: Effects on body temperature of prostaglandins of the A, E, and F series, on injection into the third ventricle of unanaesthetized cats and rabbits, J. Physiol. (Land.) 218:325, 1971. 10. Jaffe, B. M., Behrman, H. R., and Parker, C. W.: Radioimmunoassay measurement of prostaglandin E, A and F in human plasma, J. Clin. Invest. 52:398, 1973. Il. Fuster, V., Bowie, E. J. W., Lewis, J. C., Fass, D. N., Owen, C. A., Jr., and Brown, A. L.: Resistance to arteriosclerosis in pigs with von Willebrand’s disease. Spontaneous and high cholesterol diet-induced arteriosclerosis, J. Clin. Invest. 61:722, 1978. 12. Samuelsson, B., Granstibm, E., Green, K., Hamberg, M., and Hammarstrbm, S.: Prostaglandins, in Snell, E. E., Boyer, P. D., Meister, A., and Richardson, C. C., editors: Annual review of biochemistry, Palo Alto, 1975, Annual Reviews, Inc., vol. 44, p. 669. 13. The Anturane Reinfarction Trial Research Group: Sulfinpyrazone in the prevention of cardiac death after myocardial infarction, N. Engl. J. Med. 298~289, 1978. 14. Parker, C. W., Sullivan, T. J., and Wedner, H. J.: Cyclic AMP and the immune response, in Greengard, P., and Robison, G. A., editors: Advances in cyclic nucleotide research, New York, 1974, Raven Press, vol. 4, p. I. 15. Pelus, L. M., and Straussner, H. R.: Prostaglandins and the immune response, Life Sci. 20:903, 1977. 16. Goodwin, J. S., Bankhurst, A. D., and Messner, R. P.: Suppression of human T-cell mitogenesis by prostaglandin. Existence of a prostaglandin-producing suppressor cell, J. Exp. Med. 146~1719, 1977. 17. Parker, C. W., and Kelly, J. P.: Studies of lymphocyte activation, Proceedings of the Twelfth Leukocyte Culture Conference, June, 1978, Beer-Sheba, Israel. (In press.) 18. Snider, D. E., and Parker, C. W.: Prostaglandins, in Middleton, E., Ellis, E., and Reed, C., editors: Allergy: Principles and practice, St. Louis, 1978, The C. V. Mosby Co. 19. Math&, A. A.: Studies on actions of prostaglandins in the lung, Acta Physiol. Stand., Supp. 441, 1976. 20. Hyman, A. L., Spannhake, E. W., and Kadowitz, P. J.: Prostaglandins and the lung, Am. Rev. Respir. Dis. 117:111, 1978. 21. Dawson, W., Lewis, R. L., McMahon, R. E., and Sweatman, W. J. F.: Potent bronchoconstrictor activity of 15-keto prostaglandin F,,, Nature 250~331, 1974. 22. Kaliner, M.: Human lung tissue and anaphylaxis. I. The role of cyclic GMP as a modulator of the immunologically induced secretory process, J. ALLERGY CLIN. IMMUNOL. 60~204, 1977.

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23. Parker, C. W.: Aspirin sensitive asthma, in Lichtenstein, L. M., Austen, K. F., and Simon, A. S., editors: Asthma: Physiology, immunopharmacology and treatment, New York. 1977, Academic Press, Inc., p. 301. 24. Feldberg, W., and Kellaway, C. H.: Liberation of histamine and formation of lysocithin-like substancesby cobra venom, J. Physiol. 94: 187, 1938. 25. Kellaway, C. H., and Trethewie, E. R.: The liberation of a slow-reacting smooth muscle-stimulating substance in anaphylaxis, Quart. J. Exp. Physiol. 30:121, 1940. 26. Brocklehurst, W. E.: The release of histamine and formation of a slowreacting substance (SRS-A) during anaphylactic shock, J. Physiol. 151:416, 1960. 27. Orange, R. P., and Austen, K. F.: Slow reacting substance of anaphylaxis, in Dixon, F. J., Jr., and Kunkel, H. G., editors: Advances in immunology. New York, 1969, Academic Press, Inc., vol. 10, p. 106. 28. Lewis. R. A., and Austen, K. F.: Nonrespiratory functions of pulmonary cells: The mast cell, Fed. Proc. 36~2676, 1977. 29. Lewis, R. A., Wasserman, S. I., Goetzl, E. J., and Austen, K. F.: Formation of slow-reacting substance of anaphylaxis in human lung tissue and cells before release, J. Exp. Med. 140: I 133, 1974. 30. Lewis, R. A., Goetzl, E. J., Wasserman, S. I., Valone, F. H.. Rubin, R. H., and Austen, K. F.: The release of four mediators of immediate hypersensitivity from human leukemic basophils, J. Immunol. 114:87, 1975. 3 I. Strandberg, K., and Uvn’gs, B.: Purification and properties of the slow reacting substance formed in the cat paw perfused with compound 48/80, Acta Physiol. Stand. 82:358, 1971. 32. Anggard, E., Bergoqvist, U., H(iberg, B., Johansson, K., Thon, I. L., and Uvn%, B.: Biologically active principles occurring on histamine release from cat paw, guinea-pig lung and isolated rat mast cells, Acta Physiol. Stand. 5997, 1963. 33. Orange. R. P., and Chang, P.-L.: The effect of thiols on immunologic release of slow reacting substance of anaphylaxis. I. Human lung, J. Immunol. 115:1072, 1975. 34. Orange, R.. and Moore, E. G.: The effect of thiols on the immunologic release of slow reacting substance of anaphylaxis. II. Other in vitro and in vivo models, J. Immunol. 116:392. 1976. 35. Orange, R. P.: Dissociation of the immunologic release of histamine and slow reacting substance of anaphylaxis from human lung using cytochalasins A and B, J. Immunol. 114:182, 1975. 36. Orange, R. P., Murphy, R. C., Karnovsky, M. L., and Austen. K. F.: The physicochemical characteristics and purification of slow-reacting substance of anaphylaxis, J. Immunol. 110:760, 1973. 37. Bach. M. K., and Brashler, J. R.: In vivo and in vitro production of a slow reacting substance in the rat upon treatment with calcium ionophores, J. lmmunol. 113:2040, 1974. 38. Brashler, J. R., and Bach, M. K.: Production of slow reacting substance of anaphylaxis (SRS-A) in vitro: Involvement of phagocytes in an ionophore A23187 induced reaction, Fed. Proc. 35864, 1976. (Abst.) 39. Ishizaka, T., Ishizaka, K., Orange, R. P., and Austen, K. F.: Pharmacologic inhibition of the antigen-induced release of histamine and slow reacting substance of anaphylaxis (SRS-A) from monkey lung tissues mediated by human IgE, J. Immunol. lOtil267, 1971. 40. Engineer. D. M., Piper, P. J., and Sirois, P.: Interaction between the release of SRS-A and of prostaglandins (proceedings), Br. J. Pharmacol. 57346OP, 1976.

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41. Grant, J. A., and Lichtenstein, L. M.: Release of slow reacting substance of anaphylaxis from human leukocytes, J. Immunol. 112:897, 1974. 42. Conroy, M. C., Orange, R. P., and Lichtenstein. L. M.: Release of slow reacting substance of anaphylaxis (SRS-A) from human leukocytes by the calcium ionophore A23 187, J. Immunol. 116~1677, 1976. 43. Jakschik, B. A., Kulczycki, A., Jr., MacDonald, H. H.. and Parker. C. W.: Release of slow reacting substance (SRS) from rat basophilic leukemia (RBL-I) cells, J. Immunol. 119:618, 1977. 44, Jakschik, B. A., Falkenhein, S., and Parker. C. W.: Precursor role of arachidonic acid in release of slow reacting substance from rat basophilic leukemia cells, Proc. Nat]. Acad. Sci. USA 74:4577, 1971. 45. Patterson, R., and Suszko, 1. M.: Primate respiratory mast cells. Reactions with ascaris antigen and anti-heavy chain sera, J. Immunol. 106:1274, 1971. 46. Burka, J. F., and Eyre, P.: Modulation of the formation and release of bovine SRS-A in r?tro by several anti-anaphylactic drugs. Int. Arch. Allergy Appl. Immunol. 49:774. 1975. 47. Yecies. L. D.. Wedner, H. J., Johnson, S. M., and Parker, C. W.: Polar metabolites of arachidonic acid in rat mast cells, J. ALLERGY CLIN. IMMKJNOL. 61:13 I, 1978. (Abst.) 48. Jakschik, B., Sullivan, T. J., Kulczycki, A., Jr., and Parker. C. W.: Release of slow reacting substance (SRS) from rat mast cells, Fed. Proc. 36: 1328. 1977. (Abst.) 49. Collier. H. 0. J., and James, G. W. L.: Bradykinin and slow reacting substance in anaphylactic bronchoconstriction in guinea pig in vivo, J. Physiol. 185:7lP, 1966. 50. Brocklehurst, W. E.: Slow reacting substance and related compounds, in Kallos, P., editor: Progress in allergy, Base], 1962. S. Karger AG, vol. 6, p. 539. 5 I. Herxheimer, H., and Stresemann, E.: The effect of slow reacting substance (SRS-A) in guinea-pigs and in asthmatic patients. J. Physiol. 165:78P, 1963. 52. Turnbull, L. S., Jones, D. G.. and Kay, A. B.: Slow reacting substance as a preformed mediator from human lung, Immunology 31:813, 1976. 53. Kay, A. B., Roberts, E. M., and Jones, D. G.: Tissue inactivation of slow reacting substanceof anaphylaxis, Immunology 30~83, 1976. 54. Wasserman, S. I., Goetzl, E. J., and Austen. K. F.: Inactivation of slow reacting substance of anaphylaxis by human eosinophil arylsulfatase, J. Immunol. 114645, 1975. 55. Wasserman. S. I., and Austen, K. F.: Arylsulfatase B of human lung, J. Clin. Invest. 57:738, 1976. 56. Orange. R. P., and Moore, E. G.: Functional characterization of rat mast cell arylsulfatase activity. J. Immunol. 117~2191, 1976. 57. Augstein. J., Farmer, J. B., Lee, T. B., Sheard, P., and Tattersall, M. L.: Selective inhibitor of slow reacting substanceof anaphylaxis, Nature New Biol. 245:215, 1973. 58. Appleton. R. A., Bantick, J. R., Chamberlain. T. R., Hardern. D. N., Lee, T. B., and Pratt, A. D.: Antagonists of slow reacting substance of anaphylaxis. Synthesis of a series of chromone-2-carboxylic acids, J. Med. Chem. 20~371. 1977. 59. Takahashi, H., Webster, M. E., and Newball, H. N.: Separation of slow reacting substance of anaphylaxis (SRS-A) from human lung into four biologically active fractions. J. Immuno]. 117:1039, 1976. 60. Jakschik, B. A., Parker, C. W.. and Needleman, P.: Production of prostaglandin (PG) DI by rat basophilic leukemia cells and purified rat mast cells, Fed. Proc. 37:384, 1978. (Abst.)

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61. Jakschik, B., and Parker, C. W.: Probably precursor role for arachidonic acid (AA) in slow reacting substance (SRS) biosynthesis, Clin. Res. 24:575A, 1976. 62. Bach, M. K., Brashler, J. R., and Gorman, R. R.: On the structure of slow reacting substance of anaphylaxis: Evidence from arachidonic acid, Prostaglandins 14:21, 1977. 63. Tashjian, A. H., Jr., Voelkel, E. F., McDonough, J., and

Information

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Levine, L.: Hydrocortisone inhibits prostaglandin production by mouse fibrosarcoma cells, Nature 258:739, 1975. 64. Nijkamp, F. P., Flower, R. J., Moncada, S., and Vane, J. R.: Partial purification of rabbit aorta contracting substancereleasing factor and inhibition of its activity by antiinflammatory steroids, Nature 263:479, 1976.

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Most of the provisions of the Copyright Act of 1976 became effective on January 1, 1978. Therefore, all manuscripts must be accompanied by the following written statement, signed by one author: “The undersigned author transfers all copyright ownership of the manuscript (title of article) to The C. V. Mosby Company in the event the work is published. The undersigned author warrants that the article is original, is not under consideration by another journal, and has not been previously published. I sign for and accept responsibility for releasing this material on behalf of any and all co-authors.” Authors will be consulted, when possible, regarding republication of their material.