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Corticosteroids inhibit prostaglandin release from perfused mesenteric blood vessels of rabbit and from perfused lungs of sensitized guinea pig
PROSTAGLANDINS
CORTICOSTEROIDS
INHIBIT PROSTAGLANDIN RELEASE
FROM PERFUSED MESENTERIC BLOOD VESSELS OF RABBIT AND FROM PERFUSED LUNGS OF SENSITIZED GUINEA PIG
Ryszard
3. Gryglewski, Bogumila Panczenko, Ryszard Lilia Grodzirfska and Anna Ocetkiewicz
Department
Korbut ,
of Pharmacology, Copernicus Medical Academy 31-531 Krakow, Poland
ABSTRACT Infusion of norephinephrine (NE) (1 - 3 u g/ml/min) into the isolated mesenteric vascular preparation of rabbit resulted in a rise in perfusion pressure, which was associated with the release of a prostaglandin E-like substance (PGE) at a concentration of 2.81 f 0.65 ng/ml in terms of PGE2. Indomethacin (3 u g/ml) abolished the NE-induced release of PGE . Arachidonic acid (0.2 u g/ml) in the presence of indomethacin did not restore the NE-induced release of PGE . Hydrocortisone (10 - 30 ug/ml) and dexamethasone (2 - 5 ug/ml) also inhibited the NE-induced release of PGE . The inhibitory action of both corticosteroids was abolished by arachidonic acid (0.2 u g/ml). Antigen-induced release of a prostaglandin-like substance (PCs) (43.1 2 3.8 ng/ml in terms of PGE2 and a rabbit aorta contracting substance (RCS) from perfused lungs of sensitized guinea pigs was completeIy abolished by indomethacin (5 u g/ml) or by hydrocortisone (100 u g/ml). Indomethacin, however, increased histamine release up to 280% of the control level, which was 470 5 54 ng/ml, while hydrocortisone diminished histamine release down to 30% of the control level. A superimposed infusion of arachidonic acid (1 u g/ml) into the pulmonary artery reversed the hydrocortisoneinduced blockade of the release of RCS and PGs . It may be concluded that corticosteroids neither inhibit prostaglandin synthetase nor influence prostaglandin transport through the membranes but they do impair the availability of the substrate for the enzyme.
ACKNOWLEDGEMENT The authors would like to express their gratitude to the Wellcome Trust, London, U.K. for the generous gift of the equipment and to Dr. J. Pike, Upjohn Company, Kalamazoo, Michigan, U .S .A. for the kind supply of prostaglsndins E2 and Fz~.
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INTRODUCTION Herbaczynska-Cedro and Barczak-Staszewska (1) have reported that hydrocortisone diminishes the release of a prostaglandin-like substance into the venous effluent from a dog’s hind leg during its muscular work. Recently, Lewis and Piper (2) have observed that corticosteroids inhibit the prostaglandin-mediated vasodilation accompanying ACTH-induced lipolysis in the subcutaneous fat of the rabbit. Here we report that norepinephrine (NE)-induced release of prostaglandins from contracting mesenteric blood vessels of rabbits is inhibited by hydrocortisone and dexamethasone. Furthermore, immunologically triggered prostaglandin release from the perfused lungs of sensitized guinea pigs (3) is also inhibited by hydrocortisone. This inhibitory effect of corticosteroids is restored in both inhibit preparations by arachidonic acid, It is concluded that corticosteroids the supply of the substrate for prostaglandin synthetase system.
METHODS Perfused
mesenteric
artery
of the rabbit
Rabbits of both sexes were killed by a blow on the neck. The superior mesenteric artery was cannulated and cut out together with all branches supporting the ileum at a length of 80 - 100 cm. The vascular mesenteric preparation was placed in a heated chamber 37OC and perfused with Krebs bicarbonate (NaCl 118.0 mM , KC1 4.7 mM , KH2P04 1.2 mM , MgS04 7H20 1.17 mM , CaC12 6H2O 2.5 mivl , NaHC03 25.0 mM , and glucose 8.4 mM) . The perfusion fluid was bubbled with a mixture of 5% carbon dioxide in oxygen and pumped at a steady rate of 2.5 ml/min to maintain the perfusion pressure at a level of 5 - 10 mm Hg, as meassure by a one arm mercury manometer. The effluent from the vascular preparation superfused in cascade the assay organs: a rat stomach strip (RSS) and a rat colon (RC) . Combined antagonists (4) and indomethacin (3 u g/ml) were infused into the effluent to make the assay organs more specific and sensitive to prostaglandins. The initial load on each tissue was 2 - 3 g and their movements were recorded mechanically with Paton’s auxotonic levers (1: 8). Intra-arterial infusions of D (-) norepinephrine hydrochloride (1 - 3 lo g/ml/min duration of an infusion 5 min) , were used to stimulate the reiease of PGE from NE tias removed from the effluent by its passage through a column filled vessels. The column did not absorb prostaglandins (5). with activated aluminium oxide. Other substances infused intra-arterially were hydrocortisone semisuccinate (Polfa) (10 - 30 u g/ml), dexamethasone (Polfa) (2 - 5 pg/ml) and sodium arachidonate (Sigma) (0.2 )1g/ml).
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In the corticosteroid infused vascular preparations a superimposed infusion of arachidonic acid at a concentration of 0.2 u g/ml restored the ability of the vascular bed to produce PGE in the response to the pressor effect of NE. In these preparations the acute tolerance to NE usually reappeared (fig. 2). Guinea
pig lungs
release of PGs as measThe results are summarized in fig. 5. Antigen-induced ured by contractions of RSS was 43.1 ? 3.8 ng/ml in terms of PGE2 (mean + S .E . , of a RCS in the effluent was evaluated qualitatively. n = 24). The appearance Using an improved bioassay technique (7) histamine concentration in the effluent from shocked lungs was found to be 470f 54 ng/ml (n = 10). In all tested preparations indomethacin (5 u g/ml) completely abolished antigen-induced release of PGs and RCS , while the release of histamine was significantly increased (1319 ? 250 ng/ml, n = 8). If into the same indomethacin-treated lungs arachidonic acid was infused (1 u g/ml) the second challenge released neither RCS nor PGs , while the release of histamine came back to a range of the control level (459 + 21 ng/ml, n= 7) (fig. 3). The antigen-induced release of PGs , RCS and histamine was not influenced by hydrocortisone at a concentration of 20 u g/ml (3 experiments ) . An increase of the hydrocortisone concentration up to 50 u g/ml (6 experiments) resulted in a diminution in the release of RCS , (roughly by 80% as measured by the height of RbA contraction), PGs down to 18 2 6.7 ng/ml and histamine down to 283 + 70 ng/ml. Hydrocortisone at a concentration of 100 u g/ml completely abolished the release of RCS and PGs and significantly decreased histamine release (140 f 43 ng/ml, n = 8). When arachidonic acid (1 u g/ml) was infused into the hydrocortisone-treated lungs and the challenge was repeated, then RCS and PGs were released in all experiments. PGs reappeared in the effluent at a concentration of 28.9 ? 4.3 ng/ml (n =‘8). The release of histamine was depressed (70 +‘20 ng/ml, n = 5) (fig. 4).
DISCUSSION Norephinephrine (NE)-induced vasoconstriction in anaesthetized cats (8) or in a perfused rabbit ear (9) was associated with the release of a prostaglandin E-like substance CPGE). Further evidence that PGE formation and release occur in the vascular wall has been obtained in experiments with isolated mesenteric arteries of the rabbit contracted by NE. In this preparation PGE release is inhibited by indomethacin and this inhibition is not reversed by an additional supply with arachidonic acid (fig. 1). Here we report that the release of PGE from contracting mesenteric vessels of the rabbit is abolished by hydrocortisone (10 - 30 u g/ml) and desamethasone (2 - 5 u g/ml) but this corticosteroid-blockade is easily overcome by an infusion of arachidonic acid. The ratio of inhibitory potency of hydrocortisone and dexamethasone on PGE release (1: 6) was lower than the ratio of their anti-inflammatory potency (1: 25) , but still of similar order of magnitude.
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The perfused lungs of sensitized guinea pigs, when challenged with an antigen , release prostaglandins (PGs), a rabbit aorta contracting substance (RCS) , histamine and a slow reacting substance of anaphylaxis (SRS-A) (3, 6, 10). This last substance was not bioassayed by us. The inhibitory action of indomethacin on the release of RCS and PGs from the shocked lungs is well established (3, 6) and it is not reversed by arachidonic acid (fig. 3) . Here we report that hydrocortisone (100 u g/ml) completely inhibits the release of PGs and RCS from the shocked lungs. Hydrocortisone-blockade is reversed by arachidonic acid (fig. 4). There are no intracellular stores of prostaglandins and their release from tissues is considered to be equivalent to their fresh biosynthesis (11). Therefore we have assumed that.corticosteroids interfere somehow with prostaglandin biosynthesis. Corticosteroids , however, do not inhibit microsomal prostaglandin synthetase . Hydrocortisone , dexamethasone , triamcinolone and fludrocortisone at concentrations of 100 u g/ml are inactive as inhibitors of microsomal prostaglandin synthetase of a dog spleen (12). In cell-free homognates of guinea pig lungs hydrocortisone (50 u g/ml) hardly influences prostaglandin biosynthesis (13). In human and rat skin homogenates hydrocortisone and fluocinolone (100 p g/ml) mildly inhibit generation of prostaglandins (14, 15). We suggest that the integrity of the cell structure is necessary for the inhibitory action of corticosteroids on the prostaglandin release from tissues. According to Weissmann (16) the tissue distortion (in our case either vasoconstriction induced by NE or shock reaction in lungs) results in an escape of lysosomal enzymes through channels created by virtue of the incomplete fusion of the lysosomal membrane with the plasma membrane. A direct contact of lysosomal phospholipases with the membrane phospholipids enables their enzymic cleavage and initiates the liberation of C2. poly-unsaturated fatty acids, which are precursors of prostaglandins. Corticosteroids stabilize biomembranes (17, 18) , prevent their fusion, and consequently the supply of the endogenous precursor of the prostaglandin biosynthesis is diminished (fig. 6). An extra supply of exogenous arachidonic acid is a sufficient procedure to stimulate prostaglandin biosynthesis, even in the presence of corticosteroids . Our interpretation of the mode of action of corticosteroids on prostaglandin release differs from that suggested by Lewis and Piper (2). These author have recently described that ACTH-induced lipolysis in the fat pad of rabbits is accompanied by formation of prostaglandins and vasodilatation . When hydrocortisone was infused (300 ug/min) close-arterially to the epigastric fat depot before the infusion of ACTH , the vasodilatation was greatly reduced. Hydrocortisone neither inhibited lipolysis nor antagonised the vasodilatation caused by exogenous PGE2. Furthermore hydrocortisone did not prevent the formation of prostaglandins in fat tissue homogenates. The authors suggest that corticosteroids inhibit the release of prostaglandins by preventing their transport from inside the fat cell to an extracellular space. If the mechanism proposed by Lewis and Piper (2) would work in our experiments then arachidonic acid should not be able to abolish the corticosteroid-blockade. This, however, occurred (fig. 2, fig. 4).
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The inhibitory action of hydrocortisone on prostaglandin release was also reported by Herbaczyliska-Cedro and Barczak-Staszewska (1). An electrical stimulation of sciatic nerve of the hind leg of the anaesthetized dog results in the release of PGE (0.5 - 3.5 ng/ml in terms of PGE2) into the venous outflow (19). This release is inhibited by indomethacin (2 u g/kg/min i .v . or i. a. ) but not by aldosterone (1). Hydrocortisone did not alter the removal of exogenous PGE2 from circulation of the hind leg. The authors suggest that hydrocortisone , although devoid of the inhibitory action on the prostaglandin synthetase system as well as on the prostaglandin uptake, may still interfere with the rate of endogenous prostaglandin formation. Our results support the suggestion of HerbaczyfiskaCedro and Barczak-Staszewska (1). Corticosteroids are used in the treatment of inflammation, anaphylaxis and cardiovascular shock. An increased release of prostaglandins is symptomatic for all three pathological process (3, 11, 20. 21, 22). It might be that stabilization of membrane phospholipids (17, 19) which leads to the shortage of the substrate supply for the prostaglandin synthetase system (fig. 6) is partially responsible for In case of anaphylactic shock hydrocortisone the therapeutic effect of corticosteroids. inhibits also the release of histamine, possibly due to stabilization of the mastocyte granulae membranes. In these experiments, in which an extra supply with arachidonic acid enabled the shocked lungs to produce prostaglandins , the inhibitory action of hydrocortisone on histamine release was potentiated (fig. 4, 5). In the above experiments the direct effect of hydrocortisone on histamine release was probably reinforced by the generation of endogenous prostaglandins of the E ‘series, which are potent inhibitors of histamine release (23, 24). This explanation seems to be valid, since in our hands indomethacin strongly enhanced the release of histamine in anaphylactic lungs (fig. 5) . Several mediators are released during immunological response (3, 23, 24) and hydrocortisone may interfere with their release as well as with their mutual interactions. Therefore it should be stressed that corticosteroids also inhibit prostaglandin release from perfused blood vessels. In this preparation prostaglanwhich are detected in the perdins are the only biologically active substances fusate during norephinephrine-induced vasoconstriction. In both preparations (i . e. in guinea pig lungs and in mesenteric vessels of rabbit) the corticosteroid blockade of prostaglandin release is abolished by arachidonate . These facts give a strong support for our hypothesis on the mechanism of the therapeutical action of corticosteroids (fig. 6) .
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REFERENCES
1.
Herbaczyfiska-Cedro , K. and J . Barczak-Staszewska: cortical hormones and the release of prostaglandin-like (PLS) . Abstracts - Second Congress of the Hungarian Society, Budapest, 1974, p. 19.
2.
Adrenosubstances Pharmacological
Lewis, G .P . and P .J . Piper: Inhibition d release of prostaglandins as an explanation of some of the action of anti-inflammatory corticosteroids. Nature 254: 308, 1975. Piper. P.J., and J .R. Vane: Release of additional factors in anaphylaxis and its antagonism by anti-inflammatory drugs. Nature 223: 29, 1969. Gilmore, N .J , , J .R. Vane, and J.H. the spleen. Nature 218: 1135, 1968.
Wyllie:
Prostaglandin
release
and R. J . Gryglewski: Generation Grodzifmka, L . , B . Panczenko, prostaglandin E-like material by the guinea-pig trachea contracted histamine. J. Pharm. Pharmac. 27: 88, 1975, Piper, P.J.: Release and metabolism of prostaglandins Pol. J. Pharmacol. Pharm. 26: 61, 1974.
by
of by
in lung tissue.
The use of isoIated organs for detecting active substances Vane, J.R.: in the circulating blood. Br . J . Pharmac. Chemother . 23: 360, 1964. Gryglewski, R .J . and A. Ocetkiewicz: may be respcmsible for acute tolerance Prostaglandins 8: 31, 1974.
348
A release of prostaglandins to norepinephrine infusions.
9.
Prostaglandin feedback mechanism Gryglewski, R . J . and R . Korbut: limits vasoconstrictor action of norepinephrine in perfused rabbit ear, Experientia 31: 89, 1975.
10.
Palmer, M.A., P .J . Piper, and J .R. Vane; Release of rabbit aorta contracting substance (RCS) and prostaglandins induced by chemical or mechanical stimulation of guinea-pig lungs. Br . 3. Pharmac. 49: 226, 1973.
11.
Piper, P. J . and J .R. Vane: The release of prostaglandins and other tissues. Ann. N .Y. Acad. Sci. 180: 363, 1971.
12.
K . Herbaczynska-Cedro , and J .R . Vane: Flower, R., R .J . Gryglewski, Effects of anti-inflammatory drugs on prostaglandin biosynthesis. Nature New Biol. 238: 104, 1972.
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1975
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Inhibition of prostaglandin synthesis as a mechanism 13. Vane, J.R.: action for aspririn-like drugs. Nature New Biol. 231: 232, 1971.
of
14.
Inhibition of prostaglandin Greaves , M ,W . and W . McDonald-Gibson: biosynthesis by corticosteroids. Br. Med. J. 2: 83, 1972.
15.
Prostaglandin biosynthesis Greaves , M .W . and W . McDonald-Gibson: by human skin and its inhibition by corticosteroids . Br . J . Pharmac. 46: 172, 1972.
16. Weissmann, G . : Effects of corticosteroids on the stability and fusion of biomembranes , In Asthma (K. Austen , L .J . Lichtenstein , Editors) Academic Press, New York and London, 1973, p. 221. 17.
Pollock, S .H. and J .H. Brow: Studies on the acute inflammatory response. III. Glucocorticosteroids and vitamin E (in viva) attenuate thermal labilization of isolated hepatic lysosomes. J. Pharm. Exp. Ther. 178: 609, 1971.
18.
Lewis, D.A. and E.H. Day: Biochemical factors in the action of steroids on diseased joints in rheumatoid arthritis. Ann, Rheum. Dis 31: 374, 1972.
19, Herbaczynska-Cedro, K. , J. Staszewska-Barczak, and H. Janczewska: The release of prostaglandin-like substances during reactive and functional hyperemia in the hind leg of the dog. Pol. Pharmacol. Pharm. 26: 167, 1974. 20.
A .G. Herman, and J .R. Vane: Appearance of prostaglandins Collier, J.G., in the renal venous blood of dogs in response to acute systemic hype tension produced by bleeding or endotoxin. J. Physiol. (London) , 230: 190, 1973.
21.
Ferreira, S.H., S. Moncada, and J.R. Vane: Prostaglsndins and signs and symptoms of inflammation. In Prostaglandins Synthetase Inhibitors (I-I. J. Robinson and J .R. Vane, Editors) Raven Press, New York 1974, p. 175.
22.
Korbut, R., A. Ocetkiewicz, and R. Gryglewski: Release of a prostaglandin E-like substance into mixed venous blood during endotoxin hypotension in cats. Pol. J . Pharmacol. Pharm. (In press).
23.
Lichtenstein , L. M .: The control of IgE-mediated histamine release. In Asthma (K. Austen , L. J . Lichtenstein , Editors) Academic Press, New York and London, 1973, p. 91.
24.
The regulatory function of prostaglandins in the release Walker, J.L.: of histamine and SRS-A from passively sensitized human lung tissue. In Advances in Biosciences 9, (G . Raspe, Editor) . International Conference on Prostaglandins . Pergamon Press-Viewer, 1973, p . 235.
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(00mm*
3 &ml ARACHWJNIC
ACID C2jqj,hI
DIR
El
FZd
E2
E2
Et
El
Fn
ng/ml
12
12
06
12
06
a3
06
P9 h’
Fig. 1
350
INCCMEMACIN
NE
NE
NE
3
3
3
The blockade of norepinephrine (NE)-induced release of PGE-like substance by indomethacin from vascular mesenteric preparation of the rabbit. A rat stomach strip (RSS) and a rat colon (RC) were superfused in cascade with the perfusate from a mesenteric preparation (2.5 ml/min) . The aluminium oxide column was placed between the vascular mesenteric preparation and the assay organs. Sensitivity of the assay organs to PGE2 and PGF2, was calibrated by direct infusions (DIR) of PGE2 and PGF2 a (0.3-l. 2 ng/ml) through aluminium oxide column over the assay organs. An intra-arterial infusion of NE (3 Fg/ml) resulted in a tachyphylactic rise in perfusion pressure (PP) . In indomethacin-pretreated vessels NE resulted in a less marked tachyphylactic pressor response, while no PGE was released. A ten minute-lasting infusion of arachidonic acid (0.2 ug/ml) did not restore the NE-induced release of PGE .
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Fig.
2
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The inhibition of NE-induced release of PGE by dexamethasone from a perfused vascular mesenteric preparation. NE (1 &ml/min) infused into mesenteric artery caused a tachyphylactic rise in perfusion pressure (PP) . At the same time a PGE-like substance appeared in the perfusate. Dexamethasone (2 Ug/ml) abolished the NE-induced generation of PGE-like substance and the acute tolerance to NE disappeared. A ten minute-lasting infusion of arachidonic acid (0.2 e/ml) restored the ability of the vascular preparation to release PGE and at the same time an acute tolerance to pressor effect of NE reappeared. Other explanations as in fig. 1.
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LUNGS I
LUNGS II
LLINGS III
RCA
Fig. 3
352
The influence of indomethacin (5 ug/ml) on the release of PGs , RCS and histamine from perfused lungs. Isolated lungs of. three sensitized guinea-pigs (lungs I, lungs II and lungs III) were perfused with Krebs solution. The effluent was used to superfuse in cascade a rabbit aorta strip (RbA), rat stomach strip (RSS) and guinea-pig ileum (GPI) . The amberlite IUD-2 column was placed over the GPI to absorb PGs and RCS . Calibration doses of PGE2 and histamine (II) were infused directly into the effluent. PP is the perfusion pressure. Antigen (ovalbumen 10 mg) was injected into the pulmonary artery (a>. Control lungs (lungs I and lungs III) when challenged released a rabbit aorta contracting substance (PGs) as read by RbA, prostaglandinlike substance (PCs) as read by RSS and histamine as read by GPE . _The indomethacin infusion (5 Ig/ml) into the lungs II completely abolished antigen-induced release of PGs and RCS , while the release of histamine was increased. Infusion of arachidonic acid (1 ~g/ml) did not restore the antigen-induced release of PGs and RCS , but the release of histamine was not blocked.
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Fig. 4
The inhibitory action of hydrocortisone (100 v&ml) on the release of RCS , PGs and histamine from the shocked lungs of sensitized guinea pigs. The schema of the experiment was the same as of that depicted in fig. 3. Control lungs (I) when challenged with an antigen (a) released PGs, RCS and histamine. In lungs II hydrocortisone (100 vg/ml) abolished the antigen-induced release of PGs, RCS and decreased histamine release. Infusion of arachidonic acid (1 vg/ml) restored the antigen-induced release of PGs and RCS .
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HISTAMINE AND PROSTAGLANDINS RELEASE FROM PERFUSED LUNGS OF SENSITIZED GUMA PIGS A - CHALLENGE WITH ANTIGEN
STATISTICALLY SIGNIFICQM
p < 0.001
PERCENT OF CONTROL RELEASE “F
CONTROL RELEASE 470 f 54 rglml n =I0
A
HBTAMINE
Fig. 5
354
The influence of hydrocortisone , indomethacin and arachidonic acid on the release of histamine and prostaglandins from perfused lungs of sensitized guinea pigs, which were challenged with the antigen (A).
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membrane
Fig. 6
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_ _1 _>_ .-_,- - :
Hypothetical mechanism of action of corticosteroids on prostaglandin release. Corticosteroids are supposed to stabilize phospholipids in biomembranes (11) and therefore restrict the supply of precursors to microsomal prostaglandin synthetase.
1975
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CORTICOSTEROIDS
INHIBIT PROSTAGLANDIN RELEASE
FROM PERFUSED MESENTERIC BLOOD VESSELS OF RABBIT AND FROM PERFUSED LUNGS OF SENSITIZED GUINEA PIG
Ryszard
3. Gryglewski, Bogumila Panczenko, Ryszard Lilia Grodzirfska and Anna Ocetkiewicz
Department
Korbut ,
of Pharmacology, Copernicus Medical Academy 31-531 Krakow, Poland
ABSTRACT Infusion of norephinephrine (NE) (1 - 3 u g/ml/min) into the isolated mesenteric vascular preparation of rabbit resulted in a rise in perfusion pressure, which was associated with the release of a prostaglandin E-like substance (PGE) at a concentration of 2.81 f 0.65 ng/ml in terms of PGE2. Indomethacin (3 u g/ml) abolished the NE-induced release of PGE . Arachidonic acid (0.2 u g/ml) in the presence of indomethacin did not restore the NE-induced release of PGE . Hydrocortisone (10 - 30 ug/ml) and dexamethasone (2 - 5 ug/ml) also inhibited the NE-induced release of PGE . The inhibitory action of both corticosteroids was abolished by arachidonic acid (0.2 u g/ml). Antigen-induced release of a prostaglandin-like substance (PCs) (43.1 2 3.8 ng/ml in terms of PGE2 and a rabbit aorta contracting substance (RCS) from perfused lungs of sensitized guinea pigs was completeIy abolished by indomethacin (5 u g/ml) or by hydrocortisone (100 u g/ml). Indomethacin, however, increased histamine release up to 280% of the control level, which was 470 5 54 ng/ml, while hydrocortisone diminished histamine release down to 30% of the control level. A superimposed infusion of arachidonic acid (1 u g/ml) into the pulmonary artery reversed the hydrocortisoneinduced blockade of the release of RCS and PGs . It may be concluded that corticosteroids neither inhibit prostaglandin synthetase nor influence prostaglandin transport through the membranes but they do impair the availability of the substrate for the enzyme.
ACKNOWLEDGEMENT The authors would like to express their gratitude to the Wellcome Trust, London, U.K. for the generous gift of the equipment and to Dr. J. Pike, Upjohn Company, Kalamazoo, Michigan, U .S .A. for the kind supply of prostaglsndins E2 and Fz~.
AUGUST
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PROSTAGLANDINS
INTRODUCTION Herbaczynska-Cedro and Barczak-Staszewska (1) have reported that hydrocortisone diminishes the release of a prostaglandin-like substance into the venous effluent from a dog’s hind leg during its muscular work. Recently, Lewis and Piper (2) have observed that corticosteroids inhibit the prostaglandin-mediated vasodilation accompanying ACTH-induced lipolysis in the subcutaneous fat of the rabbit. Here we report that norepinephrine (NE)-induced release of prostaglandins from contracting mesenteric blood vessels of rabbits is inhibited by hydrocortisone and dexamethasone. Furthermore, immunologically triggered prostaglandin release from the perfused lungs of sensitized guinea pigs (3) is also inhibited by hydrocortisone. This inhibitory effect of corticosteroids is restored in both inhibit preparations by arachidonic acid, It is concluded that corticosteroids the supply of the substrate for prostaglandin synthetase system.
METHODS Perfused
mesenteric
artery
of the rabbit
Rabbits of both sexes were killed by a blow on the neck. The superior mesenteric artery was cannulated and cut out together with all branches supporting the ileum at a length of 80 - 100 cm. The vascular mesenteric preparation was placed in a heated chamber 37OC and perfused with Krebs bicarbonate (NaCl 118.0 mM , KC1 4.7 mM , KH2P04 1.2 mM , MgS04 7H20 1.17 mM , CaC12 6H2O 2.5 mivl , NaHC03 25.0 mM , and glucose 8.4 mM) . The perfusion fluid was bubbled with a mixture of 5% carbon dioxide in oxygen and pumped at a steady rate of 2.5 ml/min to maintain the perfusion pressure at a level of 5 - 10 mm Hg, as meassure by a one arm mercury manometer. The effluent from the vascular preparation superfused in cascade the assay organs: a rat stomach strip (RSS) and a rat colon (RC) . Combined antagonists (4) and indomethacin (3 u g/ml) were infused into the effluent to make the assay organs more specific and sensitive to prostaglandins. The initial load on each tissue was 2 - 3 g and their movements were recorded mechanically with Paton’s auxotonic levers (1: 8). Intra-arterial infusions of D (-) norepinephrine hydrochloride (1 - 3 lo g/ml/min duration of an infusion 5 min) , were used to stimulate the reiease of PGE from NE tias removed from the effluent by its passage through a column filled vessels. The column did not absorb prostaglandins (5). with activated aluminium oxide. Other substances infused intra-arterially were hydrocortisone semisuccinate (Polfa) (10 - 30 u g/ml), dexamethasone (Polfa) (2 - 5 pg/ml) and sodium arachidonate (Sigma) (0.2 )1g/ml).
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In the corticosteroid infused vascular preparations a superimposed infusion of arachidonic acid at a concentration of 0.2 u g/ml restored the ability of the vascular bed to produce PGE in the response to the pressor effect of NE. In these preparations the acute tolerance to NE usually reappeared (fig. 2). Guinea
pig lungs
release of PGs as measThe results are summarized in fig. 5. Antigen-induced ured by contractions of RSS was 43.1 ? 3.8 ng/ml in terms of PGE2 (mean + S .E . , of a RCS in the effluent was evaluated qualitatively. n = 24). The appearance Using an improved bioassay technique (7) histamine concentration in the effluent from shocked lungs was found to be 470f 54 ng/ml (n = 10). In all tested preparations indomethacin (5 u g/ml) completely abolished antigen-induced release of PGs and RCS , while the release of histamine was significantly increased (1319 ? 250 ng/ml, n = 8). If into the same indomethacin-treated lungs arachidonic acid was infused (1 u g/ml) the second challenge released neither RCS nor PGs , while the release of histamine came back to a range of the control level (459 + 21 ng/ml, n= 7) (fig. 3). The antigen-induced release of PGs , RCS and histamine was not influenced by hydrocortisone at a concentration of 20 u g/ml (3 experiments ) . An increase of the hydrocortisone concentration up to 50 u g/ml (6 experiments) resulted in a diminution in the release of RCS , (roughly by 80% as measured by the height of RbA contraction), PGs down to 18 2 6.7 ng/ml and histamine down to 283 + 70 ng/ml. Hydrocortisone at a concentration of 100 u g/ml completely abolished the release of RCS and PGs and significantly decreased histamine release (140 f 43 ng/ml, n = 8). When arachidonic acid (1 u g/ml) was infused into the hydrocortisone-treated lungs and the challenge was repeated, then RCS and PGs were released in all experiments. PGs reappeared in the effluent at a concentration of 28.9 ? 4.3 ng/ml (n =‘8). The release of histamine was depressed (70 +‘20 ng/ml, n = 5) (fig. 4).
DISCUSSION Norephinephrine (NE)-induced vasoconstriction in anaesthetized cats (8) or in a perfused rabbit ear (9) was associated with the release of a prostaglandin E-like substance CPGE). Further evidence that PGE formation and release occur in the vascular wall has been obtained in experiments with isolated mesenteric arteries of the rabbit contracted by NE. In this preparation PGE release is inhibited by indomethacin and this inhibition is not reversed by an additional supply with arachidonic acid (fig. 1). Here we report that the release of PGE from contracting mesenteric vessels of the rabbit is abolished by hydrocortisone (10 - 30 u g/ml) and desamethasone (2 - 5 u g/ml) but this corticosteroid-blockade is easily overcome by an infusion of arachidonic acid. The ratio of inhibitory potency of hydrocortisone and dexamethasone on PGE release (1: 6) was lower than the ratio of their anti-inflammatory potency (1: 25) , but still of similar order of magnitude.
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The perfused lungs of sensitized guinea pigs, when challenged with an antigen , release prostaglandins (PGs), a rabbit aorta contracting substance (RCS) , histamine and a slow reacting substance of anaphylaxis (SRS-A) (3, 6, 10). This last substance was not bioassayed by us. The inhibitory action of indomethacin on the release of RCS and PGs from the shocked lungs is well established (3, 6) and it is not reversed by arachidonic acid (fig. 3) . Here we report that hydrocortisone (100 u g/ml) completely inhibits the release of PGs and RCS from the shocked lungs. Hydrocortisone-blockade is reversed by arachidonic acid (fig. 4). There are no intracellular stores of prostaglandins and their release from tissues is considered to be equivalent to their fresh biosynthesis (11). Therefore we have assumed that.corticosteroids interfere somehow with prostaglandin biosynthesis. Corticosteroids , however, do not inhibit microsomal prostaglandin synthetase . Hydrocortisone , dexamethasone , triamcinolone and fludrocortisone at concentrations of 100 u g/ml are inactive as inhibitors of microsomal prostaglandin synthetase of a dog spleen (12). In cell-free homognates of guinea pig lungs hydrocortisone (50 u g/ml) hardly influences prostaglandin biosynthesis (13). In human and rat skin homogenates hydrocortisone and fluocinolone (100 p g/ml) mildly inhibit generation of prostaglandins (14, 15). We suggest that the integrity of the cell structure is necessary for the inhibitory action of corticosteroids on the prostaglandin release from tissues. According to Weissmann (16) the tissue distortion (in our case either vasoconstriction induced by NE or shock reaction in lungs) results in an escape of lysosomal enzymes through channels created by virtue of the incomplete fusion of the lysosomal membrane with the plasma membrane. A direct contact of lysosomal phospholipases with the membrane phospholipids enables their enzymic cleavage and initiates the liberation of C2. poly-unsaturated fatty acids, which are precursors of prostaglandins. Corticosteroids stabilize biomembranes (17, 18) , prevent their fusion, and consequently the supply of the endogenous precursor of the prostaglandin biosynthesis is diminished (fig. 6). An extra supply of exogenous arachidonic acid is a sufficient procedure to stimulate prostaglandin biosynthesis, even in the presence of corticosteroids . Our interpretation of the mode of action of corticosteroids on prostaglandin release differs from that suggested by Lewis and Piper (2). These author have recently described that ACTH-induced lipolysis in the fat pad of rabbits is accompanied by formation of prostaglandins and vasodilatation . When hydrocortisone was infused (300 ug/min) close-arterially to the epigastric fat depot before the infusion of ACTH , the vasodilatation was greatly reduced. Hydrocortisone neither inhibited lipolysis nor antagonised the vasodilatation caused by exogenous PGE2. Furthermore hydrocortisone did not prevent the formation of prostaglandins in fat tissue homogenates. The authors suggest that corticosteroids inhibit the release of prostaglandins by preventing their transport from inside the fat cell to an extracellular space. If the mechanism proposed by Lewis and Piper (2) would work in our experiments then arachidonic acid should not be able to abolish the corticosteroid-blockade. This, however, occurred (fig. 2, fig. 4).
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The inhibitory action of hydrocortisone on prostaglandin release was also reported by Herbaczyliska-Cedro and Barczak-Staszewska (1). An electrical stimulation of sciatic nerve of the hind leg of the anaesthetized dog results in the release of PGE (0.5 - 3.5 ng/ml in terms of PGE2) into the venous outflow (19). This release is inhibited by indomethacin (2 u g/kg/min i .v . or i. a. ) but not by aldosterone (1). Hydrocortisone did not alter the removal of exogenous PGE2 from circulation of the hind leg. The authors suggest that hydrocortisone , although devoid of the inhibitory action on the prostaglandin synthetase system as well as on the prostaglandin uptake, may still interfere with the rate of endogenous prostaglandin formation. Our results support the suggestion of HerbaczyfiskaCedro and Barczak-Staszewska (1). Corticosteroids are used in the treatment of inflammation, anaphylaxis and cardiovascular shock. An increased release of prostaglandins is symptomatic for all three pathological process (3, 11, 20. 21, 22). It might be that stabilization of membrane phospholipids (17, 19) which leads to the shortage of the substrate supply for the prostaglandin synthetase system (fig. 6) is partially responsible for In case of anaphylactic shock hydrocortisone the therapeutic effect of corticosteroids. inhibits also the release of histamine, possibly due to stabilization of the mastocyte granulae membranes. In these experiments, in which an extra supply with arachidonic acid enabled the shocked lungs to produce prostaglandins , the inhibitory action of hydrocortisone on histamine release was potentiated (fig. 4, 5). In the above experiments the direct effect of hydrocortisone on histamine release was probably reinforced by the generation of endogenous prostaglandins of the E ‘series, which are potent inhibitors of histamine release (23, 24). This explanation seems to be valid, since in our hands indomethacin strongly enhanced the release of histamine in anaphylactic lungs (fig. 5) . Several mediators are released during immunological response (3, 23, 24) and hydrocortisone may interfere with their release as well as with their mutual interactions. Therefore it should be stressed that corticosteroids also inhibit prostaglandin release from perfused blood vessels. In this preparation prostaglanwhich are detected in the perdins are the only biologically active substances fusate during norephinephrine-induced vasoconstriction. In both preparations (i . e. in guinea pig lungs and in mesenteric vessels of rabbit) the corticosteroid blockade of prostaglandin release is abolished by arachidonate . These facts give a strong support for our hypothesis on the mechanism of the therapeutical action of corticosteroids (fig. 6) .
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REFERENCES
1.
Herbaczyfiska-Cedro , K. and J . Barczak-Staszewska: cortical hormones and the release of prostaglandin-like (PLS) . Abstracts - Second Congress of the Hungarian Society, Budapest, 1974, p. 19.
2.
Adrenosubstances Pharmacological
Lewis, G .P . and P .J . Piper: Inhibition d release of prostaglandins as an explanation of some of the action of anti-inflammatory corticosteroids. Nature 254: 308, 1975. Piper. P.J., and J .R. Vane: Release of additional factors in anaphylaxis and its antagonism by anti-inflammatory drugs. Nature 223: 29, 1969. Gilmore, N .J , , J .R. Vane, and J.H. the spleen. Nature 218: 1135, 1968.
Wyllie:
Prostaglandin
release
and R. J . Gryglewski: Generation Grodzifmka, L . , B . Panczenko, prostaglandin E-like material by the guinea-pig trachea contracted histamine. J. Pharm. Pharmac. 27: 88, 1975, Piper, P.J.: Release and metabolism of prostaglandins Pol. J. Pharmacol. Pharm. 26: 61, 1974.
by
of by
in lung tissue.
The use of isoIated organs for detecting active substances Vane, J.R.: in the circulating blood. Br . J . Pharmac. Chemother . 23: 360, 1964. Gryglewski, R .J . and A. Ocetkiewicz: may be respcmsible for acute tolerance Prostaglandins 8: 31, 1974.
348
A release of prostaglandins to norepinephrine infusions.
9.
Prostaglandin feedback mechanism Gryglewski, R . J . and R . Korbut: limits vasoconstrictor action of norepinephrine in perfused rabbit ear, Experientia 31: 89, 1975.
10.
Palmer, M.A., P .J . Piper, and J .R. Vane; Release of rabbit aorta contracting substance (RCS) and prostaglandins induced by chemical or mechanical stimulation of guinea-pig lungs. Br . 3. Pharmac. 49: 226, 1973.
11.
Piper, P. J . and J .R. Vane: The release of prostaglandins and other tissues. Ann. N .Y. Acad. Sci. 180: 363, 1971.
12.
K . Herbaczynska-Cedro , and J .R . Vane: Flower, R., R .J . Gryglewski, Effects of anti-inflammatory drugs on prostaglandin biosynthesis. Nature New Biol. 238: 104, 1972.
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Inhibition of prostaglandin synthesis as a mechanism 13. Vane, J.R.: action for aspririn-like drugs. Nature New Biol. 231: 232, 1971.
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14.
Inhibition of prostaglandin Greaves , M ,W . and W . McDonald-Gibson: biosynthesis by corticosteroids. Br. Med. J. 2: 83, 1972.
15.
Prostaglandin biosynthesis Greaves , M .W . and W . McDonald-Gibson: by human skin and its inhibition by corticosteroids . Br . J . Pharmac. 46: 172, 1972.
16. Weissmann, G . : Effects of corticosteroids on the stability and fusion of biomembranes , In Asthma (K. Austen , L .J . Lichtenstein , Editors) Academic Press, New York and London, 1973, p. 221. 17.
Pollock, S .H. and J .H. Brow: Studies on the acute inflammatory response. III. Glucocorticosteroids and vitamin E (in viva) attenuate thermal labilization of isolated hepatic lysosomes. J. Pharm. Exp. Ther. 178: 609, 1971.
18.
Lewis, D.A. and E.H. Day: Biochemical factors in the action of steroids on diseased joints in rheumatoid arthritis. Ann, Rheum. Dis 31: 374, 1972.
19, Herbaczynska-Cedro, K. , J. Staszewska-Barczak, and H. Janczewska: The release of prostaglandin-like substances during reactive and functional hyperemia in the hind leg of the dog. Pol. Pharmacol. Pharm. 26: 167, 1974. 20.
A .G. Herman, and J .R. Vane: Appearance of prostaglandins Collier, J.G., in the renal venous blood of dogs in response to acute systemic hype tension produced by bleeding or endotoxin. J. Physiol. (London) , 230: 190, 1973.
21.
Ferreira, S.H., S. Moncada, and J.R. Vane: Prostaglsndins and signs and symptoms of inflammation. In Prostaglandins Synthetase Inhibitors (I-I. J. Robinson and J .R. Vane, Editors) Raven Press, New York 1974, p. 175.
22.
Korbut, R., A. Ocetkiewicz, and R. Gryglewski: Release of a prostaglandin E-like substance into mixed venous blood during endotoxin hypotension in cats. Pol. J . Pharmacol. Pharm. (In press).
23.
Lichtenstein , L. M .: The control of IgE-mediated histamine release. In Asthma (K. Austen , L. J . Lichtenstein , Editors) Academic Press, New York and London, 1973, p. 91.
24.
The regulatory function of prostaglandins in the release Walker, J.L.: of histamine and SRS-A from passively sensitized human lung tissue. In Advances in Biosciences 9, (G . Raspe, Editor) . International Conference on Prostaglandins . Pergamon Press-Viewer, 1973, p . 235.
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(00mm*
3 &ml ARACHWJNIC
ACID C2jqj,hI
DIR
El
FZd
E2
E2
Et
El
Fn
ng/ml
12
12
06
12
06
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06
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Fig. 1
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INCCMEMACIN
NE
NE
NE
3
3
3
The blockade of norepinephrine (NE)-induced release of PGE-like substance by indomethacin from vascular mesenteric preparation of the rabbit. A rat stomach strip (RSS) and a rat colon (RC) were superfused in cascade with the perfusate from a mesenteric preparation (2.5 ml/min) . The aluminium oxide column was placed between the vascular mesenteric preparation and the assay organs. Sensitivity of the assay organs to PGE2 and PGF2, was calibrated by direct infusions (DIR) of PGE2 and PGF2 a (0.3-l. 2 ng/ml) through aluminium oxide column over the assay organs. An intra-arterial infusion of NE (3 Fg/ml) resulted in a tachyphylactic rise in perfusion pressure (PP) . In indomethacin-pretreated vessels NE resulted in a less marked tachyphylactic pressor response, while no PGE was released. A ten minute-lasting infusion of arachidonic acid (0.2 ug/ml) did not restore the NE-induced release of PGE .
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Fig.
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The inhibition of NE-induced release of PGE by dexamethasone from a perfused vascular mesenteric preparation. NE (1 &ml/min) infused into mesenteric artery caused a tachyphylactic rise in perfusion pressure (PP) . At the same time a PGE-like substance appeared in the perfusate. Dexamethasone (2 Ug/ml) abolished the NE-induced generation of PGE-like substance and the acute tolerance to NE disappeared. A ten minute-lasting infusion of arachidonic acid (0.2 e/ml) restored the ability of the vascular preparation to release PGE and at the same time an acute tolerance to pressor effect of NE reappeared. Other explanations as in fig. 1.
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LUNGS I
LUNGS II
LLINGS III
RCA
Fig. 3
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The influence of indomethacin (5 ug/ml) on the release of PGs , RCS and histamine from perfused lungs. Isolated lungs of. three sensitized guinea-pigs (lungs I, lungs II and lungs III) were perfused with Krebs solution. The effluent was used to superfuse in cascade a rabbit aorta strip (RbA), rat stomach strip (RSS) and guinea-pig ileum (GPI) . The amberlite IUD-2 column was placed over the GPI to absorb PGs and RCS . Calibration doses of PGE2 and histamine (II) were infused directly into the effluent. PP is the perfusion pressure. Antigen (ovalbumen 10 mg) was injected into the pulmonary artery (a>. Control lungs (lungs I and lungs III) when challenged released a rabbit aorta contracting substance (PGs) as read by RbA, prostaglandinlike substance (PCs) as read by RSS and histamine as read by GPE . _The indomethacin infusion (5 Ig/ml) into the lungs II completely abolished antigen-induced release of PGs and RCS , while the release of histamine was increased. Infusion of arachidonic acid (1 ~g/ml) did not restore the antigen-induced release of PGs and RCS , but the release of histamine was not blocked.
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Fig. 4
The inhibitory action of hydrocortisone (100 v&ml) on the release of RCS , PGs and histamine from the shocked lungs of sensitized guinea pigs. The schema of the experiment was the same as of that depicted in fig. 3. Control lungs (I) when challenged with an antigen (a) released PGs, RCS and histamine. In lungs II hydrocortisone (100 vg/ml) abolished the antigen-induced release of PGs, RCS and decreased histamine release. Infusion of arachidonic acid (1 vg/ml) restored the antigen-induced release of PGs and RCS .
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HISTAMINE AND PROSTAGLANDINS RELEASE FROM PERFUSED LUNGS OF SENSITIZED GUMA PIGS A - CHALLENGE WITH ANTIGEN
STATISTICALLY SIGNIFICQM
p < 0.001
PERCENT OF CONTROL RELEASE “F
CONTROL RELEASE 470 f 54 rglml n =I0
A
HBTAMINE
Fig. 5
354
The influence of hydrocortisone , indomethacin and arachidonic acid on the release of histamine and prostaglandins from perfused lungs of sensitized guinea pigs, which were challenged with the antigen (A).
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membrane
Fig. 6
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Hypothetical mechanism of action of corticosteroids on prostaglandin release. Corticosteroids are supposed to stabilize phospholipids in biomembranes (11) and therefore restrict the supply of precursors to microsomal prostaglandin synthetase.
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