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The relationship between prostaglandin release and lung c-AMP levels during anaphylaxis in the guinea-pig
THE RELATIONSHIP BETWEEN PROSTAGLANDINRELEASE LUNG c-AMP LEVELS DURING ANAPHYLAXIS IN THE GUINEA-PIG
AND
K.J. BARRETT-BEE and L.R. GREEN IMPERIAL CHEMICAL INDUSTRIES LIMITED PHARMACEUTICALS DIVISION MERESIDE,ALDERLEY PARK MACCLESFIELD, CHESHIRE ENGLAND.
A four-fold transient rise in c-AMP levels was seen when sensitized guinea-pig lungs were challenged with antigen in vitro. This rise in c-AMP also occurred in vivo and was shown to be due to release of Prostaglandin E 2. This conclusion is supported by the finding that inhibitors of prostaglandin synthesis (Indomethacin and Poly phloretin phosphate) prevent the rise in c-AMP while neither ICI 74,917, an inhibitor of histamine release~nor antihistamines had any effect on the c-AMP levels.
We wish to acknowledge the skilled technical assistance of Miss Gillian Isherwood in this work.
lO-9-75
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VOL. it) NO. 4
589
PROSTAGLANDINS
INTRODUCTION Cyclic nucleotides play an important role in the regulation of anaphylaxis in various species. Using human lung Orange et al. (i) found that agents which increase lung c-AMP levels inhibit the allergic release of histamine and Slow Reacting Substance (SRS-A). Guinea-pig lung was shown to behave in a similar way. (2)The elegant studies of Lichtenstein and DeBernardo (3) showed that in human leukocytes the c-AMP dependent step is at the calcium independent activation stage, rather than the subsequent calcium dependent step. Mathe et al. (4) demonstrated that antigen stimulation of perfused guinea-pig lung elicits an increase in c-AMP levels by a factor of about six, and in similar studies using chopped lungs Schmutzler and Derwall (5) reported a rise of about 20%. The present study examines the in vivo and in vitro effect of antigen challenge on levels of guinea-pig lung c-AMP and correlates these changes with the antigen stimulated release of histamine and prostaglandins F ~ a n d E 2. MATERIALS AND METHODS Polyphloretin Phosphate Standard IV (PPP) was obtained from AB Leo (Helsingborg); Histamine diphosphate from B.D.H.; Carbachol and Serotonin from Koch Light; Mepyramine Maleate from May & Baker; Burimamide from Smith Kline and French. Guinea-pigs (200-300 grams) were actively sensitized by injection of ovalbumin intraperitoneally (60 mgs.) on days, l, 3 and 5, and the animals used on days 21 and 22. Passive sensitization was carried out by intravenous injection of diluted hyperimmune serum (0.5 ml.) taken from actively sensitized animals. Animals used for in vitro experiments were killed by cervical dislocation, the lungs removed, dissected free of bronchial tissue, chopped on a Mcllwain chopper (set at .5 mm.), the pieces washed with Krebs-Ringer saline and filtered through nylon mesh. Aliquots of tissue were then placed in glass vials and incubated at 37°C in gassed (5% CO2, 95% 02 ) saline for 20 minutes. Additions of antigen or compounds were then made and the preparation incubated for various times. For c-AMP determinations samples (I ml.) were removed using a plastic syringe, added to perchloric acid (0.I ml. of 6M) homogenized for 20 seconds with a Polytron homogenizer and centrifuged in a bench centrifuge. The precipitate was assayed for protein by the method of Lowry (6) and the supernatant neutralized by addition of a solution of Tris (0.6M) KCI (0.4M) KOH (IM) (.4 ml.); it was left overnight at 4°C and the potassium perchlorate precipitate removed by centrifugatioe. The resulting supernatant was assayed for c-AMP by the method of Brown et al. (7). For histamine determinations samples of the incubation mixture (2 ml.) were passed through a glass fibre filter to remove the tissue and the filtrate (i ml.) deproteinized by addition of perchloric acid (0.I ml. of 4M). The histamine content of the supernatant was
590
OCTOBER 1975 VOL. 10 NO. 4
PROSTAGLANDINS
FIGURE 1 :
CYCLIC AMP LEVELS AFTER OVALBUMIN CHALLENGE TO PASSIVELY SENSITIZED CHOPPED GUINEA-PIG LUNG. (Ovalbumin 0.I mg./ml.,Mepyramine lO'4M.,Burir~mide lO-4M.)
Ovalbumin o Ovalbumin + Mepyramine Ovalbumin + Burimamide
o
.£ 30-
~2o~-
I0
4 6 Minutes after challenge
TABLE I:
8
THE EFFECT OF VARIOUS TREATI~NTS ON THE LEVEL OF c-AMP IN PASSIVELY SENSITIZED GUINEA-PIG LUNG AFTER IN VIVO CHALLENGE.
Treatment Control
c-A~ (picomo le s/mg. protein) _4- S.E.M. 0.5 +
.2 (I0)
Ovalbumin (0.i rag.)
10.2 +- 1.4
(5)
Ovalbumin (0. i rag.), Carbachol (0.02 rag. )
22.6 + 4.3
(5)
Carbachol
0.55 +- .2
(5)
Ovalbumin (0.I rag.), ICl 74,917 (0.05 rag.)
Ii.i + 1.3
(5)
ICI 74,917 (0.05 rag.)
0.6 +-- .I
(5)
Ovalbumin (0.I rag.), Indomethacin (2 mg. )
3.2 + 1.9
(5)
(0.02 rag.)
Conditions as per text.
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PROSTAGLANDINS
then determined by the method of Evans et al. (8). For prostaglandin determinations samples (2 ml.) of the incubation mixture were added to ice-cold citric acid (.2 ml. pH 3). The tissue was homogenized with a Polytron homogenizer and extracted three times with one volume of ethyl acetate. The pooled organic phases were evaporated to dryness and taken up in buffer. Prostaglandin F2~ was assayed by a method similar to that of Youssefnejadian et al. (9) with antiserum of similar specificity and prostaglandin E 2 by use of a kit (Clinical Assays, Cambridge, Mass.). For in vivo studies guinea-pigs were challenged with ovalbumin by intravenous injection (0.I mg.). The animals were stunned after 90 seconds and then irradiated (20 seconds) in a microwave oven. iangs were removed, placed in Krebs-Ringer saline and treated as described above. The interval of 90 seconds was used, rather than 4 minutes, since it has been shown (Skidmore, unpublished observations) that the level of c-AMP in lung increases markedly post-mortem. RESULTS AND DISCUSSION Figure 1 demonstrates the level of c-AMP in passively sensitized chopped lung after challenge with ovalbumin (0.i mg./ml.), c-AMP increased four-fold at 4 minutes and declined to control levels after approximately i0 minutes. Comparable results with passively sensitized animals are shown in Table I. Experiments utilizing actively sensitized animals gave identical results both in vivo and in vitro. Non-sensitized animals and animals injected with hyperimmune serum which had been heated for 4 hrs. at 56°C showed no change in c-AMP levels when challenged with antigen. These results suggest that the effect is mediated by a reaginic type antibody. Antigen challenge to sensitized guinea-pig lungs has been shown to release various mediators of anaphylaxis (I0). The rise in c-AMP levels could either be in response to one or more of these mediators or alternatively it may be associated with their release. The results of Lichtenstein and DeBernardo (3), however, suggest that the former is a more likely explanation. The next series of experiments were designed to investigate the possible roles of the mediators of anaphylaxis. Chopped lung tissue was treated with several compounds to investigate their affects on the levels of c-AMP. Histamine~ prostaglandins E l and E2, and ~-stimulants were shown to increase the levels of c-AMP in a dose-dependent manner (Figure 2), whereas prostaglandin F2~ and serotonin had no affect on tissue levels of c-AMP, nor did they interfere with the stimulation of the other agonists. The differential inhibition of mepyramine and burimamide on the response to histamine demonstrated that it was due to an H 1 response.(Table II)
592
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PROSTAGLANDINS
FIGURE 2:
DOSE RESPONSE CURVES FOR COMPOUNDS AFFECTING CLAMP LEVELS IN CHOPPED GUINEA-PIG LUNG (DETERMINED 5 MINUTES AFTER CHALLENGE).
40-
0 P~? • Histamine O
.c
PC'E l
8 ~..
1o-
I
i
l
.~1
.01
.1
~
!
f
I
10
1~
i
1,000
Cone.~M)
TABLE II:
THE EFFECT OF VARIOUS TREATMENTS ON THE c-AMP LEVELS IN CHOPPED PASSIVELY SENSITIZED GUINEA-PIG LUNG.
Treatment Nil
c-AMP picomo le s/rag,protein 8
Histamine (10"6M) Histamine (10-6M),Burimamide (10-4M) Histamine (10-6M),Mepyramine (10-4M) Serotonin (10"5M)
+--2
32.5 +--6 35 +---6 iI +-3 9
+2
PGF2~(IO-6M)
i0
+2
PGF2a(IO-6M),PGE2 (3 x IO-6M)
24
PGE 2 (3 x IO-6M)
24
+-5 +5
Ovalbumin
32
-t- 6
Ovalbumin, Propranolol (10"4M)
31
+ 7
Isoproterenol (IO'5M)
28
+ 5
Isoproterenol (10-SM), Propranolol (10-4M)
i0
+- 2
c-AMP values are taken 5 minutes after treatment. Results derived from 3 typical experiments each in triplicate.
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PROSTAGLANDINS
~ p y r a m i n e (10-4M) and Burimamide (10"4M) were added to tissue 3 minutes prior to challenge with antigen, Figure I shows that the c-AMP response was not affected by either. A compound (ICI 74,917) which has anti-allergic properties and prevents the anaphylaetic release of histamine from guinea-pig lung and rat mast cells (ii), does not affect the c-AMP response to antigen in vivo or in vitro (Figure 3 and Table I~ These experiments suggest that histamine release is not an important factor in the rise in c-AMP levels. Assem and Schild (12) have suggested that circulating catecholamines may be important in lung homeostasis and their release has been demonstrated in guinea-pig anaphylaxis (13). Propranolol, a powerful ~-blocking agent, prevented the rise in c - A M P w h i c h occurs in lungs s~imulated by isoproterenol, however it did not alter the rise in c-AMP following antigen challenge (Table II). Consequently the rise is not due to ~-stimulation of the adenyl cyclase. Unpublished experiments have also demonstrated that under these conditions there is no production of catecholamines. Kaliner et al. (14) have demonstrated that carbachol increases the release of mediators of anaphylaxis possibly in response to an increase in tissue c-GMP levels. Carbachol itself has no effect on lung c-AMP levels but when present at antigen challenge there is an additional increase in the levels of e-AMP in vitro and in vivo (Figure 3 and Table I). These results suggest a proportionality between the release of mediators of anaphylaxis and the level of c-AMP measured. Indomethacin, a potent inhibitor of prostaglandin synthetase, prevented the rise in c-AMP on antigen challenge, when given in vivo or in vitro~ a finding which suggests that the agent causing the increase in c-AMP levels may be a prostaglandin. The results in Table III demonstrate that, under these conditions, there is no prostaglandin synthesis, whereas the histamine release on antigen challenge is the same as the untreated preparation. Polyphloretin Phosphate (PPP) which has been claimed to be an antagonist of PGF2~ (15), inhibited the increase in c-AMP levels on antigen challenge (Figure 3). Since it has been shown in uterine tissue that PPP does not compete for binding sites with prostaglandins (A.Wakeling, private communication), its effect on the c-AMP response suggests that it must act at an earlier stage, the data in Figure 4 demonstrate that in chopped guinea-pig lungs it inhibits the synthesis of prostaglandin F2~ subsequent to antigen challenge. Bronchoconstriction, induced by PGF2~ , or carbachol, does not increase lung levels of c-AMP, consequently the rise in c-AMP cannot be in response to a mechanically-stimulated reaction, such as smooth muscle contraction.
594
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PROSTAGLANDINS
FIGURE 3:
THE EFFECT OF COMPOUNDS ON THE 'c-AMP RESPONSE' AFTER OVALBUMIN CHALI~NGE TO CHOPPED PASSIVELY SENSITIZED GUINEA-PIG LUNG. (Ovalhumin 0.i mg./ml., carbachol 10"6M., Polyphloretin phosphate I mg./ml., ICI 74,917 10-2mg./ml.)
= Ovalbumin + Carbachol o (Y/albumin + ICI 74,917 o Ovalbumin
100-
)° i
t
i
|
m
2
4
6
8
I
I0
Minutes after ch~llter~e
FIGURE 4:
THE EFFECT OF POLYPHLORETIN PHOSPHATE ON THE RELEASE OF PGF2a FROM CHOPPED PASSIVELY SENSITIZED I/JNG CHALLENGED WITH OVALBUMIN. (Conditions as Figure 3)
180 -
6 0 v a l b u rain o PPP + Ovalbumin o Contro~ ___~_--..-=
o o
90-
Minutes after Antigen challenge
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PROSTAGLANDINS
TABLE III:
THE EFFECT OF INDOMETHACIN ON THE ANAPHYLACTIC RESPONSE IN PASSIVELY SENSITIZED CHOPPED GUINEA-PIG LUNG
Time after Challenge mins. expt.A
exp t. B
Histamine release % of total
POE2 pg./sample
c-AMP picomoles./mg. protein
0
2
90
7
4
36
500
33
0
2
0
7
4
38
0
Ii
Expt.A Control, Expt. B 50 Bg./ml. Indomethacin in vitro, lungs from dosed animal (5 mg./kg. I hour before death).
The experiments reported indicate that the factor causing the anaphylactic increase in c-AMP is a prostaglandin, probably PGE 2. Piper (i0) has shown that PGE 2 and F2~ are released when sensitized guinea-pig lung is challenged with antigen and the data in Table II demonstrate that PGE 2 increases lung levels of c-AMP, whereas PGF2a has no effect. Compounds inhibiting prostaglandin synthesis prevent the anaphylactic rise in c-AMP whereas neither ICI 74,917, which inhibits histamine release, nor antihistamine agents have any effect. Further support for this hypothesis comes from unpublished data which shows that in perfused guinea-pig lung ICI 74,917 is able to inhibit the anaphylactic release of histamine and PGF2a while it does not inhibit the production of PGE 2. These facts suggest that PGE 2 is a primary mediator of anaphylaxis whereas P G F 2 a m a y be secondary to the production of histamine. Horton (16) has proposed that endogenous prostaglandins released in response to various stimuli regulate the action of the original stimulus by a negative feedback mechanism. Such a feedback system may operate in lung to regulate bronchial tone. A breakdown of such a mechanism caused by either hypersensitivity to bronchospastic agents (17) or a hyposensitivity to bronchodilating agents (18) could be one of the metabolic lesions associated with asthma. Similarly, aspirin hypersensitivity in asthmatics could be explained if the prostaglandin synthetase in lung were particularly sensitive to non-steroidal anti-inflarmnatory agents such as aspirin or indomethacin (19). The rise in c-AMP seen here in response to anaphylaxis may represent such a feedback mechanism acting to inhibit further synthesis of bronchospastic agents or as a bronchodilator itself.
596
OCTOBER 1975 VOL. 10 NO. 4
PROSTAGLANDINS
REFERENCES
I.
2.
3.
4.
5.
6.
7.
8.
9.
i0.
II.
12. 13.
14.
Orange, R.P., Kaliner, M., Laraia, P.J. and Austen, K.F. Iramunological release of histamine and slow reacting substance of anaphylaxis from human lung II. Influence of cellular levels of cyclic AMP. Fed. Proc., 30:1725 (1971). Sorenby, G. On the Immunological Histamine Release from Guineapig Lung and its Inhibition by Isoprenaline. Acta pharmacol, et toxicol., 34:273-283 (1974). Litchenstein, L.M. and DeBernardo, R. The Immediate Allergic Response: In vitro action of Cyclic AMP and other drugs on the two stages of histamine release. J. Irmnunol., 107:1131-1136 (1971). Mathe, A.A., Volicer, L. and Puri, S.K. Effects of anaphylaxis and histamine, pyrilamine and burimamide on levels of cyclic AMP and cyclic GMP in guinea-pig lung. Res. Commun. in Chem.Path. Pharm., 8:635-651 (1974). Schrm/tzler, W. and Derwall, R. Experiments on the role of Cyclic AMP in guinea-pig anaphylaxis. Int.Arch.All., 45:120-122 (1973). Lowry~ O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. Protein measurement with the Folin Phenol Reagent. J. Biol. Chem., 193:265-275 (1951). Brown, B.L., Albano, J.D.M.j Ekins, R.P. and Sgherzi, A.M. A simple and Sensitive saturation assay method for the measurement of Adenosine 3':5'-Cyclic Monophosphate. Biochem.J., 121:561-562 (1971). Evans, D.P., Lewis, J.A. and Thomson~ D.S. An automated fluorimetric assay for the rapid determination of histamine in biological fluids. Life Sciences, 12 Part II 327-336 (1973). Youssefnejadian, E., Walker, E., Sommerville, I.F. and Craft, I. Simple direct radioirmnunoassay of the F prostaglandins. Prostaglandins, 6:23-35 (1974). Piper, P.J. Mediators of anaphylactic hypersensitivity. In Progress in Immunology II, volume 4, eds. Brent and Holborrow. North Holland (1974). Evans, D.P. and Thomson, D.S. Inhibition of Immediate hypersensitivity reactions in laboratory aninmls by a phenanthroline salt (ICI 74,917). Br.J.Pharmac., 54:409-418 (1975). Assem, E.S.K. and Schild, H.O. ~Adrenergic receptors concerned with the anaphylactic mechanism. Int.Arch.All., 45:62-69 (1973). Bernauer, W.~ Hagedorn, M. and Filipowski, P. Catecholamine release during anaphylactic shock in guinea-pigs. NaunynSchmiedebergs Arch. Pharmak., 270:326-334 (1971). Kaliner, M., Orange, R.P. and Austen, K.F. Inmmnological release of histamine and slow reacting substance of Anaphylaxis from human lung. J.Exp. Med., 136:556-567 (1972).
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PROSTAGLANDINS
15.
16. 17.
18. 19.
598
Mathe, A.A., Strandberg, K. and Fredholm, B. Antagonism of prostaglandin F2~ induced bronchoconstriction and blood pressure changes by polyphloretin phosphate in the guinea-pig and cat. J.Pharm.Pharmac., 24:378-382 (1972). Horton, E.W. Hypotheses on physiological roles of prostaglandin~ Physiol.Revs., 49:122-161 (1969). Mathe, A.A., Hedquist, P., Holmgren, A. and Svanborg, N. Bronchial hyperreactivity to prostaglandin F2~ and histamine in patients with asthma. Br.Med.J., 1:193-196 (1973). Szentivanyi, A. The beta adrenergic theory of the atopic abnormality in bronchial asthma. J.Allergy, 42:203-232 (1968). Szczeklik, A., Gryglewski, R.J. and Czerniawaska-Mysik, G. Relationship of inhibition of prostaglandin biosynthesis by analgesics to asthma attacks in aspirin-sensitive patients. Br.Med. J., 1:67-69 (1975).
O C T O B E R 1975
VOL. 10 NO. 4
AND
K.J. BARRETT-BEE and L.R. GREEN IMPERIAL CHEMICAL INDUSTRIES LIMITED PHARMACEUTICALS DIVISION MERESIDE,ALDERLEY PARK MACCLESFIELD, CHESHIRE ENGLAND.
A four-fold transient rise in c-AMP levels was seen when sensitized guinea-pig lungs were challenged with antigen in vitro. This rise in c-AMP also occurred in vivo and was shown to be due to release of Prostaglandin E 2. This conclusion is supported by the finding that inhibitors of prostaglandin synthesis (Indomethacin and Poly phloretin phosphate) prevent the rise in c-AMP while neither ICI 74,917, an inhibitor of histamine release~nor antihistamines had any effect on the c-AMP levels.
We wish to acknowledge the skilled technical assistance of Miss Gillian Isherwood in this work.
lO-9-75
PROSTAGLANDINS O C T O B E R 1975
VOL. it) NO. 4
589
PROSTAGLANDINS
INTRODUCTION Cyclic nucleotides play an important role in the regulation of anaphylaxis in various species. Using human lung Orange et al. (i) found that agents which increase lung c-AMP levels inhibit the allergic release of histamine and Slow Reacting Substance (SRS-A). Guinea-pig lung was shown to behave in a similar way. (2)The elegant studies of Lichtenstein and DeBernardo (3) showed that in human leukocytes the c-AMP dependent step is at the calcium independent activation stage, rather than the subsequent calcium dependent step. Mathe et al. (4) demonstrated that antigen stimulation of perfused guinea-pig lung elicits an increase in c-AMP levels by a factor of about six, and in similar studies using chopped lungs Schmutzler and Derwall (5) reported a rise of about 20%. The present study examines the in vivo and in vitro effect of antigen challenge on levels of guinea-pig lung c-AMP and correlates these changes with the antigen stimulated release of histamine and prostaglandins F ~ a n d E 2. MATERIALS AND METHODS Polyphloretin Phosphate Standard IV (PPP) was obtained from AB Leo (Helsingborg); Histamine diphosphate from B.D.H.; Carbachol and Serotonin from Koch Light; Mepyramine Maleate from May & Baker; Burimamide from Smith Kline and French. Guinea-pigs (200-300 grams) were actively sensitized by injection of ovalbumin intraperitoneally (60 mgs.) on days, l, 3 and 5, and the animals used on days 21 and 22. Passive sensitization was carried out by intravenous injection of diluted hyperimmune serum (0.5 ml.) taken from actively sensitized animals. Animals used for in vitro experiments were killed by cervical dislocation, the lungs removed, dissected free of bronchial tissue, chopped on a Mcllwain chopper (set at .5 mm.), the pieces washed with Krebs-Ringer saline and filtered through nylon mesh. Aliquots of tissue were then placed in glass vials and incubated at 37°C in gassed (5% CO2, 95% 02 ) saline for 20 minutes. Additions of antigen or compounds were then made and the preparation incubated for various times. For c-AMP determinations samples (I ml.) were removed using a plastic syringe, added to perchloric acid (0.I ml. of 6M) homogenized for 20 seconds with a Polytron homogenizer and centrifuged in a bench centrifuge. The precipitate was assayed for protein by the method of Lowry (6) and the supernatant neutralized by addition of a solution of Tris (0.6M) KCI (0.4M) KOH (IM) (.4 ml.); it was left overnight at 4°C and the potassium perchlorate precipitate removed by centrifugatioe. The resulting supernatant was assayed for c-AMP by the method of Brown et al. (7). For histamine determinations samples of the incubation mixture (2 ml.) were passed through a glass fibre filter to remove the tissue and the filtrate (i ml.) deproteinized by addition of perchloric acid (0.I ml. of 4M). The histamine content of the supernatant was
590
OCTOBER 1975 VOL. 10 NO. 4
PROSTAGLANDINS
FIGURE 1 :
CYCLIC AMP LEVELS AFTER OVALBUMIN CHALLENGE TO PASSIVELY SENSITIZED CHOPPED GUINEA-PIG LUNG. (Ovalbumin 0.I mg./ml.,Mepyramine lO'4M.,Burir~mide lO-4M.)
Ovalbumin o Ovalbumin + Mepyramine Ovalbumin + Burimamide
o
.£ 30-
~2o~-
I0
4 6 Minutes after challenge
TABLE I:
8
THE EFFECT OF VARIOUS TREATI~NTS ON THE LEVEL OF c-AMP IN PASSIVELY SENSITIZED GUINEA-PIG LUNG AFTER IN VIVO CHALLENGE.
Treatment Control
c-A~ (picomo le s/mg. protein) _4- S.E.M. 0.5 +
.2 (I0)
Ovalbumin (0.i rag.)
10.2 +- 1.4
(5)
Ovalbumin (0. i rag.), Carbachol (0.02 rag. )
22.6 + 4.3
(5)
Carbachol
0.55 +- .2
(5)
Ovalbumin (0.I rag.), ICl 74,917 (0.05 rag.)
Ii.i + 1.3
(5)
ICI 74,917 (0.05 rag.)
0.6 +-- .I
(5)
Ovalbumin (0.I rag.), Indomethacin (2 mg. )
3.2 + 1.9
(5)
(0.02 rag.)
Conditions as per text.
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PROSTAGLANDINS
then determined by the method of Evans et al. (8). For prostaglandin determinations samples (2 ml.) of the incubation mixture were added to ice-cold citric acid (.2 ml. pH 3). The tissue was homogenized with a Polytron homogenizer and extracted three times with one volume of ethyl acetate. The pooled organic phases were evaporated to dryness and taken up in buffer. Prostaglandin F2~ was assayed by a method similar to that of Youssefnejadian et al. (9) with antiserum of similar specificity and prostaglandin E 2 by use of a kit (Clinical Assays, Cambridge, Mass.). For in vivo studies guinea-pigs were challenged with ovalbumin by intravenous injection (0.I mg.). The animals were stunned after 90 seconds and then irradiated (20 seconds) in a microwave oven. iangs were removed, placed in Krebs-Ringer saline and treated as described above. The interval of 90 seconds was used, rather than 4 minutes, since it has been shown (Skidmore, unpublished observations) that the level of c-AMP in lung increases markedly post-mortem. RESULTS AND DISCUSSION Figure 1 demonstrates the level of c-AMP in passively sensitized chopped lung after challenge with ovalbumin (0.i mg./ml.), c-AMP increased four-fold at 4 minutes and declined to control levels after approximately i0 minutes. Comparable results with passively sensitized animals are shown in Table I. Experiments utilizing actively sensitized animals gave identical results both in vivo and in vitro. Non-sensitized animals and animals injected with hyperimmune serum which had been heated for 4 hrs. at 56°C showed no change in c-AMP levels when challenged with antigen. These results suggest that the effect is mediated by a reaginic type antibody. Antigen challenge to sensitized guinea-pig lungs has been shown to release various mediators of anaphylaxis (I0). The rise in c-AMP levels could either be in response to one or more of these mediators or alternatively it may be associated with their release. The results of Lichtenstein and DeBernardo (3), however, suggest that the former is a more likely explanation. The next series of experiments were designed to investigate the possible roles of the mediators of anaphylaxis. Chopped lung tissue was treated with several compounds to investigate their affects on the levels of c-AMP. Histamine~ prostaglandins E l and E2, and ~-stimulants were shown to increase the levels of c-AMP in a dose-dependent manner (Figure 2), whereas prostaglandin F2~ and serotonin had no affect on tissue levels of c-AMP, nor did they interfere with the stimulation of the other agonists. The differential inhibition of mepyramine and burimamide on the response to histamine demonstrated that it was due to an H 1 response.(Table II)
592
O C T O B E R 1975
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PROSTAGLANDINS
FIGURE 2:
DOSE RESPONSE CURVES FOR COMPOUNDS AFFECTING CLAMP LEVELS IN CHOPPED GUINEA-PIG LUNG (DETERMINED 5 MINUTES AFTER CHALLENGE).
40-
0 P~? • Histamine O
.c
PC'E l
8 ~..
1o-
I
i
l
.~1
.01
.1
~
!
f
I
10
1~
i
1,000
Cone.~M)
TABLE II:
THE EFFECT OF VARIOUS TREATMENTS ON THE c-AMP LEVELS IN CHOPPED PASSIVELY SENSITIZED GUINEA-PIG LUNG.
Treatment Nil
c-AMP picomo le s/rag,protein 8
Histamine (10"6M) Histamine (10-6M),Burimamide (10-4M) Histamine (10-6M),Mepyramine (10-4M) Serotonin (10"5M)
+--2
32.5 +--6 35 +---6 iI +-3 9
+2
PGF2~(IO-6M)
i0
+2
PGF2a(IO-6M),PGE2 (3 x IO-6M)
24
PGE 2 (3 x IO-6M)
24
+-5 +5
Ovalbumin
32
-t- 6
Ovalbumin, Propranolol (10"4M)
31
+ 7
Isoproterenol (IO'5M)
28
+ 5
Isoproterenol (10-SM), Propranolol (10-4M)
i0
+- 2
c-AMP values are taken 5 minutes after treatment. Results derived from 3 typical experiments each in triplicate.
O C T O B E R 1975
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PROSTAGLANDINS
~ p y r a m i n e (10-4M) and Burimamide (10"4M) were added to tissue 3 minutes prior to challenge with antigen, Figure I shows that the c-AMP response was not affected by either. A compound (ICI 74,917) which has anti-allergic properties and prevents the anaphylaetic release of histamine from guinea-pig lung and rat mast cells (ii), does not affect the c-AMP response to antigen in vivo or in vitro (Figure 3 and Table I~ These experiments suggest that histamine release is not an important factor in the rise in c-AMP levels. Assem and Schild (12) have suggested that circulating catecholamines may be important in lung homeostasis and their release has been demonstrated in guinea-pig anaphylaxis (13). Propranolol, a powerful ~-blocking agent, prevented the rise in c - A M P w h i c h occurs in lungs s~imulated by isoproterenol, however it did not alter the rise in c-AMP following antigen challenge (Table II). Consequently the rise is not due to ~-stimulation of the adenyl cyclase. Unpublished experiments have also demonstrated that under these conditions there is no production of catecholamines. Kaliner et al. (14) have demonstrated that carbachol increases the release of mediators of anaphylaxis possibly in response to an increase in tissue c-GMP levels. Carbachol itself has no effect on lung c-AMP levels but when present at antigen challenge there is an additional increase in the levels of e-AMP in vitro and in vivo (Figure 3 and Table I). These results suggest a proportionality between the release of mediators of anaphylaxis and the level of c-AMP measured. Indomethacin, a potent inhibitor of prostaglandin synthetase, prevented the rise in c-AMP on antigen challenge, when given in vivo or in vitro~ a finding which suggests that the agent causing the increase in c-AMP levels may be a prostaglandin. The results in Table III demonstrate that, under these conditions, there is no prostaglandin synthesis, whereas the histamine release on antigen challenge is the same as the untreated preparation. Polyphloretin Phosphate (PPP) which has been claimed to be an antagonist of PGF2~ (15), inhibited the increase in c-AMP levels on antigen challenge (Figure 3). Since it has been shown in uterine tissue that PPP does not compete for binding sites with prostaglandins (A.Wakeling, private communication), its effect on the c-AMP response suggests that it must act at an earlier stage, the data in Figure 4 demonstrate that in chopped guinea-pig lungs it inhibits the synthesis of prostaglandin F2~ subsequent to antigen challenge. Bronchoconstriction, induced by PGF2~ , or carbachol, does not increase lung levels of c-AMP, consequently the rise in c-AMP cannot be in response to a mechanically-stimulated reaction, such as smooth muscle contraction.
594
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FIGURE 3:
THE EFFECT OF COMPOUNDS ON THE 'c-AMP RESPONSE' AFTER OVALBUMIN CHALI~NGE TO CHOPPED PASSIVELY SENSITIZED GUINEA-PIG LUNG. (Ovalhumin 0.i mg./ml., carbachol 10"6M., Polyphloretin phosphate I mg./ml., ICI 74,917 10-2mg./ml.)
= Ovalbumin + Carbachol o (Y/albumin + ICI 74,917 o Ovalbumin
100-
)° i
t
i
|
m
2
4
6
8
I
I0
Minutes after ch~llter~e
FIGURE 4:
THE EFFECT OF POLYPHLORETIN PHOSPHATE ON THE RELEASE OF PGF2a FROM CHOPPED PASSIVELY SENSITIZED I/JNG CHALLENGED WITH OVALBUMIN. (Conditions as Figure 3)
180 -
6 0 v a l b u rain o PPP + Ovalbumin o Contro~ ___~_--..-=
o o
90-
Minutes after Antigen challenge
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TABLE III:
THE EFFECT OF INDOMETHACIN ON THE ANAPHYLACTIC RESPONSE IN PASSIVELY SENSITIZED CHOPPED GUINEA-PIG LUNG
Time after Challenge mins. expt.A
exp t. B
Histamine release % of total
POE2 pg./sample
c-AMP picomoles./mg. protein
0
2
90
7
4
36
500
33
0
2
0
7
4
38
0
Ii
Expt.A Control, Expt. B 50 Bg./ml. Indomethacin in vitro, lungs from dosed animal (5 mg./kg. I hour before death).
The experiments reported indicate that the factor causing the anaphylactic increase in c-AMP is a prostaglandin, probably PGE 2. Piper (i0) has shown that PGE 2 and F2~ are released when sensitized guinea-pig lung is challenged with antigen and the data in Table II demonstrate that PGE 2 increases lung levels of c-AMP, whereas PGF2a has no effect. Compounds inhibiting prostaglandin synthesis prevent the anaphylactic rise in c-AMP whereas neither ICI 74,917, which inhibits histamine release, nor antihistamine agents have any effect. Further support for this hypothesis comes from unpublished data which shows that in perfused guinea-pig lung ICI 74,917 is able to inhibit the anaphylactic release of histamine and PGF2a while it does not inhibit the production of PGE 2. These facts suggest that PGE 2 is a primary mediator of anaphylaxis whereas P G F 2 a m a y be secondary to the production of histamine. Horton (16) has proposed that endogenous prostaglandins released in response to various stimuli regulate the action of the original stimulus by a negative feedback mechanism. Such a feedback system may operate in lung to regulate bronchial tone. A breakdown of such a mechanism caused by either hypersensitivity to bronchospastic agents (17) or a hyposensitivity to bronchodilating agents (18) could be one of the metabolic lesions associated with asthma. Similarly, aspirin hypersensitivity in asthmatics could be explained if the prostaglandin synthetase in lung were particularly sensitive to non-steroidal anti-inflarmnatory agents such as aspirin or indomethacin (19). The rise in c-AMP seen here in response to anaphylaxis may represent such a feedback mechanism acting to inhibit further synthesis of bronchospastic agents or as a bronchodilator itself.
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REFERENCES
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Orange, R.P., Kaliner, M., Laraia, P.J. and Austen, K.F. Iramunological release of histamine and slow reacting substance of anaphylaxis from human lung II. Influence of cellular levels of cyclic AMP. Fed. Proc., 30:1725 (1971). Sorenby, G. On the Immunological Histamine Release from Guineapig Lung and its Inhibition by Isoprenaline. Acta pharmacol, et toxicol., 34:273-283 (1974). Litchenstein, L.M. and DeBernardo, R. The Immediate Allergic Response: In vitro action of Cyclic AMP and other drugs on the two stages of histamine release. J. Irmnunol., 107:1131-1136 (1971). Mathe, A.A., Volicer, L. and Puri, S.K. Effects of anaphylaxis and histamine, pyrilamine and burimamide on levels of cyclic AMP and cyclic GMP in guinea-pig lung. Res. Commun. in Chem.Path. Pharm., 8:635-651 (1974). Schrm/tzler, W. and Derwall, R. Experiments on the role of Cyclic AMP in guinea-pig anaphylaxis. Int.Arch.All., 45:120-122 (1973). Lowry~ O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. Protein measurement with the Folin Phenol Reagent. J. Biol. Chem., 193:265-275 (1951). Brown, B.L., Albano, J.D.M.j Ekins, R.P. and Sgherzi, A.M. A simple and Sensitive saturation assay method for the measurement of Adenosine 3':5'-Cyclic Monophosphate. Biochem.J., 121:561-562 (1971). Evans, D.P., Lewis, J.A. and Thomson~ D.S. An automated fluorimetric assay for the rapid determination of histamine in biological fluids. Life Sciences, 12 Part II 327-336 (1973). Youssefnejadian, E., Walker, E., Sommerville, I.F. and Craft, I. Simple direct radioirmnunoassay of the F prostaglandins. Prostaglandins, 6:23-35 (1974). Piper, P.J. Mediators of anaphylactic hypersensitivity. In Progress in Immunology II, volume 4, eds. Brent and Holborrow. North Holland (1974). Evans, D.P. and Thomson, D.S. Inhibition of Immediate hypersensitivity reactions in laboratory aninmls by a phenanthroline salt (ICI 74,917). Br.J.Pharmac., 54:409-418 (1975). Assem, E.S.K. and Schild, H.O. ~Adrenergic receptors concerned with the anaphylactic mechanism. Int.Arch.All., 45:62-69 (1973). Bernauer, W.~ Hagedorn, M. and Filipowski, P. Catecholamine release during anaphylactic shock in guinea-pigs. NaunynSchmiedebergs Arch. Pharmak., 270:326-334 (1971). Kaliner, M., Orange, R.P. and Austen, K.F. Inmmnological release of histamine and slow reacting substance of Anaphylaxis from human lung. J.Exp. Med., 136:556-567 (1972).
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Mathe, A.A., Strandberg, K. and Fredholm, B. Antagonism of prostaglandin F2~ induced bronchoconstriction and blood pressure changes by polyphloretin phosphate in the guinea-pig and cat. J.Pharm.Pharmac., 24:378-382 (1972). Horton, E.W. Hypotheses on physiological roles of prostaglandin~ Physiol.Revs., 49:122-161 (1969). Mathe, A.A., Hedquist, P., Holmgren, A. and Svanborg, N. Bronchial hyperreactivity to prostaglandin F2~ and histamine in patients with asthma. Br.Med.J., 1:193-196 (1973). Szentivanyi, A. The beta adrenergic theory of the atopic abnormality in bronchial asthma. J.Allergy, 42:203-232 (1968). Szczeklik, A., Gryglewski, R.J. and Czerniawaska-Mysik, G. Relationship of inhibition of prostaglandin biosynthesis by analgesics to asthma attacks in aspirin-sensitive patients. Br.Med. J., 1:67-69 (1975).
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