Br. J. exp. Path. (1976) 57, 689
THE ROLE OF PROSTAGLANDINS IN CHEMICALLY INDUCED INFLAMMATION J. S. CHAHL AND L. A. CHAHL From the Department of Physiology, University of Queensland, St Lucia, Queensland 4067, Australia Received for publication June 29, 1976
Summary. Dye leakage in rats, produced by intracutaneous injections of irritants into the abdominal skin, was quantitated using the Evans blue technique of Harada et al. (1971). In control rats and in rats pretreated with indomethacin (an inhibitor of prostaglandin synthesis) concentration-response lines were obtained for 5hydroxytryptamine, histamine, bradykinin and prostaglandin E1, bradykinin in the presence of prostaglandin E1 (10-6M), adenosine-5'-triphosphate, compound 48/80, capsaicin and silver nitrate. In rats pretreated with indomethacin the dye leakage responses to histamine, prostaglandin E1, adenosine-5'-triphosphate and silver nitrate were significantly reduced, but no significant changes were observed in the responses to the other irritants. It is suggested that part of the action of histamine, adenosine-5'-triphosphate and prostagland in E1 is produced indirectly by releaseor stimulation of the synthesis of prostaglandins or their precursors. These results might have important implications in the understanding of the inflammatory response.
PROSTAGLANDINS of the E type produce marked potentiation of the inflammatory responses to histamine and bradykinin (Williams and Morley, 1973; Moncada, Ferreira and Vane, 1973) and suppression of prostaglandin synthesis or release results in suppression of the inflammatory response. Non-steroidal anti-inflammatory drugs have been shown to inhibit the synthesis of prostaglandins (Vane, 1971; Smith and Willis, 1971; Ferreira, Moncada and Vane, 1971) and this has been suggested to be the mechanism of their anti-inflammatory action (see Ferreira and Vane, 1974). Since bradykinin has been found to produce release of prostaglandins from some tissues (e.g. dog spleen; Ferreira, Moncada and Vane, 1973), it was considered that the role of prostaglandins in chemically induced inflammation should be quantitatively examined. The aspect of inflammation chosen for this study was change in capillary permeability in rat
skin using the quantitative Evans blue dye-leakage technique (Harada et al., 1971) and pretreatment with indomethacin was used to suppress prostaglandin
synthesis. MATERIALS AND METHODS Male rats derived from the Wistar strain, weighing 100-150 g, were anaesthetized with ether and the tail was warmed in water at 45-50°. An injection of Evans blue solution (2-5 ml/kg of a 2% solution in saline) was given into a lateral tail vein. Intracutaneous injections of the oedema-producing agents (hereafter called irritants) in 0-05-ml volumes were made into the abdominal skin shaved with commercial animal clippers fitted with a 1/10-mm shaving head. To obtain results for concentration-response lines for irritants, each rat was injected with 4 concentrations of an irritant into separate abdominal skin sites, the concentrations used being in a geometric progression with a factor of 2. All irritants except silver nitrate (AgNO3) were diluted in Tyrode solution, and a control i.c. injection of Tyrode was given to each animal. Since AgNO3 would react with the chloride ions in
690
J. S. CHAHL AND L. A. CHAHL
the Tyrode solution it was diluted in distilled water and a control i.e. injection of water was given to each animal tested with AgNO3. Irritants used were: 5-hydroxytryptamine (5HT) (6-25 x 10-7-5 x 10- 6M) histamine (5 x 10-5-4 x 10-4M),bradykinin (1-25 x 10-6-1 x 10-5M), prostaglandin E1 (PGE1) (7-25 x 10-7-6 x 10-6M), bradykinin (concentrations as above) in the presence of PGE1 (10-6M), adenosine-5'-triphosphate (ATP) (5 x 10-44 x 10-3M), (0-25-2-0 compound 48/80 ,ug/ml), capsaicin (5 x 10-5-4 x 10-4M) and AgNO3 (5 x 10-3-4 x 10-2M). Five animals were used to obtain results for each concentration-response line and the injection sites were rotated to equalize possible variance in the results due to differences in the abdominal skin sites. Animals regained consciousness after the last injection and were killed 20 min later. The abdominal skin was removed and pinned on to a board peritoneal side uppermost for inspection. The blue areas were cut out, chopped into smaller pieces and placed into a mixture of 7 ml of acetone and 3 ml of 0-5% sodium sulphate solution for 24 h, according to the method of Harada et al. (1971). The solutions were centrifuged at 600 g for 20 min and the amount of dye was measured as absorbance at 620 nm using a Zeiss spectrophotometer. Indomethacin pretreatment.-Rats were pretreated 30-45 min before the experiment with a dose of indomethacin of 20 mg/kg i.p. This dose was used by Moncada et al. (1973). The dose was given into the lower abdominal area so as not to interfere with the intracutaneous injections. Since indomethacin is poorly soluble in water and solutions are unstable, a solution of 4 mg/ml was made in Tris buffer (0-05M) at pH 9 immediately before the experiment and the pH was adjusted to pH 8 with HC1 (0 O1M) before injecting the solution into the treated animals. Control animals were given an equivalent volume, on a weight basis, of Tris buffer solution. The Tris buffer that was injected into the control animals was also adjusted to pH 8. Solutions at this pH did not appear to produce damage to the animals. Analysis of results.-Concentration-response lines were plotted by eye as absorbance against concentration on a logarithmic scale through the mean responses at each concentration. To obtain information on the effect of treatment on the concentration-response lines, two-way analysis of variance was used. Since it was apparent from the log concentration-response lines that some responses were fitted better by a curve than a straight line, partitioning of the concentration variance by the method of orthogonal contrasts was used to test the significance of curvilinear regression (the quadratic and cubic components).
Drugs and chemicals.-The following drugs and chemicals were used: adenosine-5'-triphosphate disodium salt (Sigma); bradykinin triacetate (Sigma); capsaicin (8-methyl-N-vanillyl6-nonenamide) (Sigma); compound 48/80 (Wellcome); Evans blue (Difco); histamine diphosphate (Sigma); indomethacin (Sigma); creatinine 5-hydroxytryptamine sulphate (Sigma); prostaglandin E1 (Upjohn); silver nitrate (Ajax chemicals); Tris buffer (tris(hydroxymethyl)aminomethane) (Sigma 7-9) (Sigma). The composition of the Tyrode solution in g/litre was: NaCl, 8-0; KCI, 0-2; MgCl2, 0-1; CaCl2, 0-2; NaH2PO4, 0-05; NaHCO3, 1-0. RESULTS
The results obtained are summarized in the Table and shown graphically in
TABLE.-Degree of Reduction of Dye Leakage Produced by Pretreatment with Indomethacin Irritant Degree of reduction* 5-HT (6.25 x 10-7-5 X 10-6M) 0 Histamine (5 x 10-5-4 x 10-4M) Bradykinin 0 (1-25 x 10-6-1 x 10-5M) PGE1 (7-25 x 10-v 6 x 10-6M) +t Bradykinin with PGE1 (10-6M) (1-25 x 10-6_1 X 10-5M) 0 ATP (5 x 10-4-4 x 10-3M) Compound 48/80 (0-25-2-0 ,ig/ml)
0
Capsaicin (5 x 10-5-4 x 10-4M)
0
+ +t
AgNO3
(5 x 10-3-4
10-2M)
+++ Degree of reduction on an arbitrary scale 0, none; +, slight; + +, marked; + + +, very marked. The significance levels of the reductions are shown as: t0-05>P>0-01; tP< 0-001. Concentration ranges of irritants used are shown in parentheses. x
*
Fig. 1-4. All compounds tested except bradykinin and PGE1 showed significant (all concentration-related responses significant at P < 0-001 except AgNO3, which was significant at 0-01 > P > 0-001). Although the responses obtained
PROSTAGLANDINS IN INFLAMMATION
691
ATP (CONTROL)
5-HT (TREATED)
[5-HT (CONTROL)
Concer.tration ( M )
FIG. 1. Effect of pretreatment with indomethacin on the concentration-response lines histamine and ATP. Lines are plotted by eye as absorbance against concentration logarithmic scale. Each point represents the mean of 5 responses. Vertical bars standard errors of the means. Mean control responses are shown as closed circles and from indomethacin pretreated rats as open circles.
with bradykinin and PGE1 were poor (Fig. 2), the responses to bradykinin were markedly potentiated by the presence of PGE1 (10-6M), and a significant concentration-related response was obtained (P < 0.001), with absorbance values for Evans blue in a similar range as for the other irritants tested (Fig. 2 compared with Fig. 1, 3, and 4). AgNO3 gave the greatest responses among the irritants tested even allowing for the large responses to control injections of the diluent, water (Fig. 4). The mean responses plotted against log concentration for most irritants were fitted adequately by a straight line, as shown by the lack of significant quadratic or cubic components of the concentration variances. However, two of the compounds, 5-HT and compound 48/80, showed significant quadratic components of the concentration variances (0.05 > P > 0 01) and these have been drawn as curves (Fig. 1 and 3). Indomethacin pretreatment produced significant reduction of the responses to histamine, PGE1 and ATP (0.05 > P > 0-01), and a more marked reduction of
to 5-HT, (M) on a
represent responses
AgNO3 (P < 0.001) (Fig. 1, 2 and 4, and Table). Although visual inspection of the data for AgNO3 suggested a greater reduction at higher concentrations of AgNO3, there was no significance of any of the interaction variances from the analyses of variance, indicating that the treatment had no significant effect on the shape of the concentration-response lines. DISCUSSION
The results presented here show that indomethacin produced a significant inhibition of the dye-leakage responses to histamine, ATP and PGE1 as well as to the non-specific irritant AgNO3. The results were not unexpected for AgNO3 since it probably produces non-specific tissue damage and release of many mediators. The results for histamine, ATP and PGE1 were unexpected in light of the hypothesis that aspirin-like drugs inhibit the synthesis of, but not the actions of the prostaglandins or other mediators of inflammation (see Ferreira and Vane, 1974). If this hypothesis is accepted, and if the dose of indomethacin
692
J. S. CHAHL AND L. A. CHAHL
0.16L
BRADYKININ + PGE, (10-6M) (TREATED) BRADYKININ + PGE, (10-6M) (CONTROL)
0.12L
C) c
.0 0
-0c0 0.08
PGE, (10-6M) 0.04 _
PGE, (1C
BRADYKININ (CONTROL)
'BRADYKININ (TREATED)
PGE, (CONTROL), PGE,(TREATED),
I
(
I
-
~~~~~~105 10-6 Concentration (M)
FiG. 2.-Effect of pretreatment with indomethacin on the concentration-response lines to bradykinin, PGE1, and bradykinin with PGE1 (10-6M). Lines are plotted by eye as absorbance against concentrationi (M) on a logarithmic scale. Each point represents the mean of 5 responses. Vertical bars represent standard errors of the means. Mean control responses for bradykinin and bradykinin with PGE1 (10-6M) are shown as closed circles and responses from indomethacin pretreated rats as Mean control responses for PGE1 are shown as closed triangles and responses from open circles. pretreated rats as open triangles. The cross-hatched area represents the mean ± standard error of I 0 responses to PGE ( 10 6) that were used as controls in experiments on bradykinin with PGE 1. 1
chosen did not have any other action (e.g. phosphodiesterase inhibition, as reported by Kuehl (1974) (cited by GColdyne, 1975)) then it must be postulated that part of the normal response to histamine, ATP and PGE1 as well as AgNO3, involves synthesis of prostaglandins or their precursors. Recently Taylor et al. (1974) also reported reduction of responses to ATP by high doses of indomethacin. Since both ATP (Kiernan, 1 972b) and PGE1 (Crunkhorn and WVillis, 1971)
have been shown to release amines from mast cells, it could be argued that the reason for the reduction in their responses is because the action of the histamine they liberate is reduced by the indomethacin treatment. However, this is difficult to sustain since responses to compound 48/80, which also liberates amines from mast cells, were not reduced by pretreatment with indomethacin. It would be necessary to postulate that ATP and PGE1 liberate histamine from the rat mast cells while compound 48/80
693
PROSTAGLANDINS IN INFLAMMATION
0.12
0.08 _
COMPOUND 48/80
a)1
TREATED CONTROL
(3 Q
n
0
0 .4
0.04
0
0.1
I 1.0 Concentration ( pg/mi)
I 10.0
FmG. 3. Effect of pretreatment with indomethacin on the concentration-response line to compound 48/80. Lines are plotted by eye as absorbance against concentration (,ug/ml) on a logarithmic scale.
Each point represents the mean of 5 responses. Vertical bars represent standard errors of the means. Mean control responses are shown as closed circles and responses from indomethacin pretreated rats as open circles.
liberates more 5-HT, which was not reduced by indomethacin pretreatment. Although the mechanism of release of amines from mast cells by compound 48/80 and ATP have been suggested to differ (Taylor et al., 1974), evidence for a difference in the ratio of the amines released is lacking. PGE1 was suggested by Arvier, Chahl and Ladd (submitted for publication) to produce neurogenic oedema, since its action was reduced by pretreatment of rats with capsaicin, which causes desensitization of sensory nerves (see Jansco, 1960). However, acute treatment with capsaicin also causes neurogenic oedema and the action of capsaicin was not reduced by indomethacin pretreatment. Therefore PGE1 and capsaicin, although both producing neurogenic oedema, must
differ in their actions. It would appear from these results that PGE1 can stimulate further synthesis of prostaglandins or their precursors. Bradykinin and PGE1 produced poor responses alone but PGE1 produced marked potentiation of the responses to bradykinin. This is in agreement with previous findings (Williams and Morley, 1973; Moncada et al., 1973). However, it was noted that, although the direct effect of PGE1 was reduced by indomethacin pretreatment, this potentiating action of a low concentration of PGE1 was not affected by indomethacin pretreatment. Bradykinin has been found to release prostaglandins from some tissues, e.g. dog spleen (Ferreira et al., 1973). However, the responses in rat skin did not
694
J. S. CHAHL AND L. A. CHAHL
0.24L
0.20L
0.16L c
U
0.12 CAPSAICIN
(CONTROL AND
0.08L
TFlTATED)
0.04.
WATER-l
%Jl-
10-4
1o-3
10-2
Concentration (M )
FIG. 4.-Effect of pretreatment with indomethacin on the concentration-response lines to capsaicin and AgNO3. Lines are plotted by eye as absorbance against concentration (M) on a logarithmic scale. Each point represents the mean of 5 responses. Vertical bars represent the standard errors of the means. Mean control responses are shown as closed circles and responses from indomethacin pretreated rats as open circles. The cross-hatched area represents the mean ± standard error of 10 responses to water that were used as controls in the experiments on AgNO3.
appear to be affected by indomethacin pretreatment, and this was expected since bradykinin alone produced such a poor response in this tissue. It is possible that in other body tissues it is capable of producing release of sufficient prostaglandins to potentiate its own action, and when prostaglandin svnthesis is inhibited the action of bradykinin is reduced. This indeed was found in dog spleen (Ferreira et al., 1973). It would appear from the present results that in rat skin bradykinin does not release sufficient prostaglandins to potentiate its own action.
In conclusion, therefore, it is suggested that part of the action of histamine, ATP and PGE1 is produced indirectly by stimulation of the synthesis of prostaglandins or their precursors. These results might have important implications in the understanding of the inflammatory process. It is possible that histamine, by stimulating release or synthesis of prostaglandins might play a greater role in the later stages of the inflammatory process than has previously been supposed. Release of prostaglandins by PGE1 and histamine might explain partly the self-
PROSTAGLANDINS IN INFLAMMATION
propagatory nature of the inflammatory response. Although ATP has been implicated in antidromic vasodilatation (Kiernan, 1972a), the importance of the finding that its action is reduced by indomethacin cannot yet be assessed. Since the responses to AgNO3 were markedly reduced by indomethacin it would appear that AgNO3 is a useful agent for testing the anti-inflammatory action of aspirin-like drugs.
We wish to thank Dr J. E. Pike of the Upjohn Company, Kalamazoo, Michigan, for supplies of prostaglandin E1. Funds were provided by the Department of Social and Preventive Medicine, University of Queensland. REFERENCES CRUNKHORN, P. & WILLIS, A. L. (1971) Cutaneous Reactions to Intradermal Prostaglandins. Br. J. Pharmac., 41, 49. FERREIRA, S. H., MONCADA, S. & VANE, J. R. (1971) Indomethacin and Aspirin Abolish Prostaglandin Release from the Spleen. Nature, New Biol., 231, 237. FERREIRA, S. H., MONCADA, S. & VANE, J. R. (1973) Prostaglandins and the Mechanism of Analgesia Produced by Aspirin-like Drugs. Br. J. Pharmac., 49, 86.
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FERREIRA, S. H. & VANE, J. R. (1974) New Aspects of the Mode of Action of Nonsteroid Antiinflammatory Drugs. Ann. Rev. Pharmac., 14, 57. HARADA, M., TAKEUCHI, M., FUKAO, T. & KATAGIRI, K. (1971) A Simple Method for the Quantitative Extraction of Dye Extravasated into the Skin. J. Pharm. Pharmac., 23, 218. JANCs6, N. (1960) Role of the Nerve Terminals in the Mechanism of Inflammatory Reactions. Bulletin Millard Fillmore Hosp., 7, 53. KIERNAN, J. A. (1972a) The Involvement of Mast Cells in Vasodilatation due to Axon Reflexes in Injured Skin. Q. Jl. exp. Physiol., 57, 311. KIERNAN, J. A. (1972b) Effects of Known and Suspected Neurotransmitter Substances and of some Nucleotides on Isolated Mast Cells. Experientia, 28, 653. KUEHL, F. A. (1974) Prostaglandins, Cyclic Nucleotides and Cell Function. Prostaglandin8, 5, 325. Cited by Goldyne, M. E. (1975) Prostaglandins and cutaneous inflammation. J. invest. Dermatol., 64, 377. MONCADA, S., FERREIRA, S. H. & VANE, J. R. (1973) Prostaglandins, Aspirin-like drugs and the Oedema of Inflammation. Nature, 246, 217. SMITH, J. B. & WILLIS, A. L. (1971) Aspirin Selectively Inhibits Prostaglandin Production in Human Platelets. Nature, New Biol., 231, 235. TAYLOR, W. A., FRANCIS, D. H., SHELDON, D. & ROITT, I. M. (1974) Anti-allergic Actions of Disodium Cromoglycate and Other Drugs Known to Inhibit Cyclic 3',5'-nucleotide Phosphodiesterase. Int. Arch. Allergy, 47, 175. VANE, J. R. (1971) Inhibition of Prostaglandin Synthesis as a Mechanism of Action for Aspirinlike drugs. Nature, New Biol., 231, 232. WILLIAMS, T. J. & MORLEY, J. (1973) Prostaglandins as Potentiators of Increased Vascular Permeability in Inflammation. Nature, 246, 215.
THE ROLE OF PROSTAGLANDINS IN CHEMICALLY INDUCED INFLAMMATION J. S. CHAHL AND L. A. CHAHL From the Department of Physiology, University of Queensland, St Lucia, Queensland 4067, Australia Received for publication June 29, 1976
Summary. Dye leakage in rats, produced by intracutaneous injections of irritants into the abdominal skin, was quantitated using the Evans blue technique of Harada et al. (1971). In control rats and in rats pretreated with indomethacin (an inhibitor of prostaglandin synthesis) concentration-response lines were obtained for 5hydroxytryptamine, histamine, bradykinin and prostaglandin E1, bradykinin in the presence of prostaglandin E1 (10-6M), adenosine-5'-triphosphate, compound 48/80, capsaicin and silver nitrate. In rats pretreated with indomethacin the dye leakage responses to histamine, prostaglandin E1, adenosine-5'-triphosphate and silver nitrate were significantly reduced, but no significant changes were observed in the responses to the other irritants. It is suggested that part of the action of histamine, adenosine-5'-triphosphate and prostagland in E1 is produced indirectly by releaseor stimulation of the synthesis of prostaglandins or their precursors. These results might have important implications in the understanding of the inflammatory response.
PROSTAGLANDINS of the E type produce marked potentiation of the inflammatory responses to histamine and bradykinin (Williams and Morley, 1973; Moncada, Ferreira and Vane, 1973) and suppression of prostaglandin synthesis or release results in suppression of the inflammatory response. Non-steroidal anti-inflammatory drugs have been shown to inhibit the synthesis of prostaglandins (Vane, 1971; Smith and Willis, 1971; Ferreira, Moncada and Vane, 1971) and this has been suggested to be the mechanism of their anti-inflammatory action (see Ferreira and Vane, 1974). Since bradykinin has been found to produce release of prostaglandins from some tissues (e.g. dog spleen; Ferreira, Moncada and Vane, 1973), it was considered that the role of prostaglandins in chemically induced inflammation should be quantitatively examined. The aspect of inflammation chosen for this study was change in capillary permeability in rat
skin using the quantitative Evans blue dye-leakage technique (Harada et al., 1971) and pretreatment with indomethacin was used to suppress prostaglandin
synthesis. MATERIALS AND METHODS Male rats derived from the Wistar strain, weighing 100-150 g, were anaesthetized with ether and the tail was warmed in water at 45-50°. An injection of Evans blue solution (2-5 ml/kg of a 2% solution in saline) was given into a lateral tail vein. Intracutaneous injections of the oedema-producing agents (hereafter called irritants) in 0-05-ml volumes were made into the abdominal skin shaved with commercial animal clippers fitted with a 1/10-mm shaving head. To obtain results for concentration-response lines for irritants, each rat was injected with 4 concentrations of an irritant into separate abdominal skin sites, the concentrations used being in a geometric progression with a factor of 2. All irritants except silver nitrate (AgNO3) were diluted in Tyrode solution, and a control i.c. injection of Tyrode was given to each animal. Since AgNO3 would react with the chloride ions in
690
J. S. CHAHL AND L. A. CHAHL
the Tyrode solution it was diluted in distilled water and a control i.e. injection of water was given to each animal tested with AgNO3. Irritants used were: 5-hydroxytryptamine (5HT) (6-25 x 10-7-5 x 10- 6M) histamine (5 x 10-5-4 x 10-4M),bradykinin (1-25 x 10-6-1 x 10-5M), prostaglandin E1 (PGE1) (7-25 x 10-7-6 x 10-6M), bradykinin (concentrations as above) in the presence of PGE1 (10-6M), adenosine-5'-triphosphate (ATP) (5 x 10-44 x 10-3M), (0-25-2-0 compound 48/80 ,ug/ml), capsaicin (5 x 10-5-4 x 10-4M) and AgNO3 (5 x 10-3-4 x 10-2M). Five animals were used to obtain results for each concentration-response line and the injection sites were rotated to equalize possible variance in the results due to differences in the abdominal skin sites. Animals regained consciousness after the last injection and were killed 20 min later. The abdominal skin was removed and pinned on to a board peritoneal side uppermost for inspection. The blue areas were cut out, chopped into smaller pieces and placed into a mixture of 7 ml of acetone and 3 ml of 0-5% sodium sulphate solution for 24 h, according to the method of Harada et al. (1971). The solutions were centrifuged at 600 g for 20 min and the amount of dye was measured as absorbance at 620 nm using a Zeiss spectrophotometer. Indomethacin pretreatment.-Rats were pretreated 30-45 min before the experiment with a dose of indomethacin of 20 mg/kg i.p. This dose was used by Moncada et al. (1973). The dose was given into the lower abdominal area so as not to interfere with the intracutaneous injections. Since indomethacin is poorly soluble in water and solutions are unstable, a solution of 4 mg/ml was made in Tris buffer (0-05M) at pH 9 immediately before the experiment and the pH was adjusted to pH 8 with HC1 (0 O1M) before injecting the solution into the treated animals. Control animals were given an equivalent volume, on a weight basis, of Tris buffer solution. The Tris buffer that was injected into the control animals was also adjusted to pH 8. Solutions at this pH did not appear to produce damage to the animals. Analysis of results.-Concentration-response lines were plotted by eye as absorbance against concentration on a logarithmic scale through the mean responses at each concentration. To obtain information on the effect of treatment on the concentration-response lines, two-way analysis of variance was used. Since it was apparent from the log concentration-response lines that some responses were fitted better by a curve than a straight line, partitioning of the concentration variance by the method of orthogonal contrasts was used to test the significance of curvilinear regression (the quadratic and cubic components).
Drugs and chemicals.-The following drugs and chemicals were used: adenosine-5'-triphosphate disodium salt (Sigma); bradykinin triacetate (Sigma); capsaicin (8-methyl-N-vanillyl6-nonenamide) (Sigma); compound 48/80 (Wellcome); Evans blue (Difco); histamine diphosphate (Sigma); indomethacin (Sigma); creatinine 5-hydroxytryptamine sulphate (Sigma); prostaglandin E1 (Upjohn); silver nitrate (Ajax chemicals); Tris buffer (tris(hydroxymethyl)aminomethane) (Sigma 7-9) (Sigma). The composition of the Tyrode solution in g/litre was: NaCl, 8-0; KCI, 0-2; MgCl2, 0-1; CaCl2, 0-2; NaH2PO4, 0-05; NaHCO3, 1-0. RESULTS
The results obtained are summarized in the Table and shown graphically in
TABLE.-Degree of Reduction of Dye Leakage Produced by Pretreatment with Indomethacin Irritant Degree of reduction* 5-HT (6.25 x 10-7-5 X 10-6M) 0 Histamine (5 x 10-5-4 x 10-4M) Bradykinin 0 (1-25 x 10-6-1 x 10-5M) PGE1 (7-25 x 10-v 6 x 10-6M) +t Bradykinin with PGE1 (10-6M) (1-25 x 10-6_1 X 10-5M) 0 ATP (5 x 10-4-4 x 10-3M) Compound 48/80 (0-25-2-0 ,ig/ml)
0
Capsaicin (5 x 10-5-4 x 10-4M)
0
+ +t
AgNO3
(5 x 10-3-4
10-2M)
+++ Degree of reduction on an arbitrary scale 0, none; +, slight; + +, marked; + + +, very marked. The significance levels of the reductions are shown as: t0-05>P>0-01; tP< 0-001. Concentration ranges of irritants used are shown in parentheses. x
*
Fig. 1-4. All compounds tested except bradykinin and PGE1 showed significant (all concentration-related responses significant at P < 0-001 except AgNO3, which was significant at 0-01 > P > 0-001). Although the responses obtained
PROSTAGLANDINS IN INFLAMMATION
691
ATP (CONTROL)
5-HT (TREATED)
[5-HT (CONTROL)
Concer.tration ( M )
FIG. 1. Effect of pretreatment with indomethacin on the concentration-response lines histamine and ATP. Lines are plotted by eye as absorbance against concentration logarithmic scale. Each point represents the mean of 5 responses. Vertical bars standard errors of the means. Mean control responses are shown as closed circles and from indomethacin pretreated rats as open circles.
with bradykinin and PGE1 were poor (Fig. 2), the responses to bradykinin were markedly potentiated by the presence of PGE1 (10-6M), and a significant concentration-related response was obtained (P < 0.001), with absorbance values for Evans blue in a similar range as for the other irritants tested (Fig. 2 compared with Fig. 1, 3, and 4). AgNO3 gave the greatest responses among the irritants tested even allowing for the large responses to control injections of the diluent, water (Fig. 4). The mean responses plotted against log concentration for most irritants were fitted adequately by a straight line, as shown by the lack of significant quadratic or cubic components of the concentration variances. However, two of the compounds, 5-HT and compound 48/80, showed significant quadratic components of the concentration variances (0.05 > P > 0 01) and these have been drawn as curves (Fig. 1 and 3). Indomethacin pretreatment produced significant reduction of the responses to histamine, PGE1 and ATP (0.05 > P > 0-01), and a more marked reduction of
to 5-HT, (M) on a
represent responses
AgNO3 (P < 0.001) (Fig. 1, 2 and 4, and Table). Although visual inspection of the data for AgNO3 suggested a greater reduction at higher concentrations of AgNO3, there was no significance of any of the interaction variances from the analyses of variance, indicating that the treatment had no significant effect on the shape of the concentration-response lines. DISCUSSION
The results presented here show that indomethacin produced a significant inhibition of the dye-leakage responses to histamine, ATP and PGE1 as well as to the non-specific irritant AgNO3. The results were not unexpected for AgNO3 since it probably produces non-specific tissue damage and release of many mediators. The results for histamine, ATP and PGE1 were unexpected in light of the hypothesis that aspirin-like drugs inhibit the synthesis of, but not the actions of the prostaglandins or other mediators of inflammation (see Ferreira and Vane, 1974). If this hypothesis is accepted, and if the dose of indomethacin
692
J. S. CHAHL AND L. A. CHAHL
0.16L
BRADYKININ + PGE, (10-6M) (TREATED) BRADYKININ + PGE, (10-6M) (CONTROL)
0.12L
C) c
.0 0
-0c0 0.08
PGE, (10-6M) 0.04 _
PGE, (1C
BRADYKININ (CONTROL)
'BRADYKININ (TREATED)
PGE, (CONTROL), PGE,(TREATED),
I
(
I
-
~~~~~~105 10-6 Concentration (M)
FiG. 2.-Effect of pretreatment with indomethacin on the concentration-response lines to bradykinin, PGE1, and bradykinin with PGE1 (10-6M). Lines are plotted by eye as absorbance against concentrationi (M) on a logarithmic scale. Each point represents the mean of 5 responses. Vertical bars represent standard errors of the means. Mean control responses for bradykinin and bradykinin with PGE1 (10-6M) are shown as closed circles and responses from indomethacin pretreated rats as Mean control responses for PGE1 are shown as closed triangles and responses from open circles. pretreated rats as open triangles. The cross-hatched area represents the mean ± standard error of I 0 responses to PGE ( 10 6) that were used as controls in experiments on bradykinin with PGE 1. 1
chosen did not have any other action (e.g. phosphodiesterase inhibition, as reported by Kuehl (1974) (cited by GColdyne, 1975)) then it must be postulated that part of the normal response to histamine, ATP and PGE1 as well as AgNO3, involves synthesis of prostaglandins or their precursors. Recently Taylor et al. (1974) also reported reduction of responses to ATP by high doses of indomethacin. Since both ATP (Kiernan, 1 972b) and PGE1 (Crunkhorn and WVillis, 1971)
have been shown to release amines from mast cells, it could be argued that the reason for the reduction in their responses is because the action of the histamine they liberate is reduced by the indomethacin treatment. However, this is difficult to sustain since responses to compound 48/80, which also liberates amines from mast cells, were not reduced by pretreatment with indomethacin. It would be necessary to postulate that ATP and PGE1 liberate histamine from the rat mast cells while compound 48/80
693
PROSTAGLANDINS IN INFLAMMATION
0.12
0.08 _
COMPOUND 48/80
a)1
TREATED CONTROL
(3 Q
n
0
0 .4
0.04
0
0.1
I 1.0 Concentration ( pg/mi)
I 10.0
FmG. 3. Effect of pretreatment with indomethacin on the concentration-response line to compound 48/80. Lines are plotted by eye as absorbance against concentration (,ug/ml) on a logarithmic scale.
Each point represents the mean of 5 responses. Vertical bars represent standard errors of the means. Mean control responses are shown as closed circles and responses from indomethacin pretreated rats as open circles.
liberates more 5-HT, which was not reduced by indomethacin pretreatment. Although the mechanism of release of amines from mast cells by compound 48/80 and ATP have been suggested to differ (Taylor et al., 1974), evidence for a difference in the ratio of the amines released is lacking. PGE1 was suggested by Arvier, Chahl and Ladd (submitted for publication) to produce neurogenic oedema, since its action was reduced by pretreatment of rats with capsaicin, which causes desensitization of sensory nerves (see Jansco, 1960). However, acute treatment with capsaicin also causes neurogenic oedema and the action of capsaicin was not reduced by indomethacin pretreatment. Therefore PGE1 and capsaicin, although both producing neurogenic oedema, must
differ in their actions. It would appear from these results that PGE1 can stimulate further synthesis of prostaglandins or their precursors. Bradykinin and PGE1 produced poor responses alone but PGE1 produced marked potentiation of the responses to bradykinin. This is in agreement with previous findings (Williams and Morley, 1973; Moncada et al., 1973). However, it was noted that, although the direct effect of PGE1 was reduced by indomethacin pretreatment, this potentiating action of a low concentration of PGE1 was not affected by indomethacin pretreatment. Bradykinin has been found to release prostaglandins from some tissues, e.g. dog spleen (Ferreira et al., 1973). However, the responses in rat skin did not
694
J. S. CHAHL AND L. A. CHAHL
0.24L
0.20L
0.16L c
U
0.12 CAPSAICIN
(CONTROL AND
0.08L
TFlTATED)
0.04.
WATER-l
%Jl-
10-4
1o-3
10-2
Concentration (M )
FIG. 4.-Effect of pretreatment with indomethacin on the concentration-response lines to capsaicin and AgNO3. Lines are plotted by eye as absorbance against concentration (M) on a logarithmic scale. Each point represents the mean of 5 responses. Vertical bars represent the standard errors of the means. Mean control responses are shown as closed circles and responses from indomethacin pretreated rats as open circles. The cross-hatched area represents the mean ± standard error of 10 responses to water that were used as controls in the experiments on AgNO3.
appear to be affected by indomethacin pretreatment, and this was expected since bradykinin alone produced such a poor response in this tissue. It is possible that in other body tissues it is capable of producing release of sufficient prostaglandins to potentiate its own action, and when prostaglandin svnthesis is inhibited the action of bradykinin is reduced. This indeed was found in dog spleen (Ferreira et al., 1973). It would appear from the present results that in rat skin bradykinin does not release sufficient prostaglandins to potentiate its own action.
In conclusion, therefore, it is suggested that part of the action of histamine, ATP and PGE1 is produced indirectly by stimulation of the synthesis of prostaglandins or their precursors. These results might have important implications in the understanding of the inflammatory process. It is possible that histamine, by stimulating release or synthesis of prostaglandins might play a greater role in the later stages of the inflammatory process than has previously been supposed. Release of prostaglandins by PGE1 and histamine might explain partly the self-
PROSTAGLANDINS IN INFLAMMATION
propagatory nature of the inflammatory response. Although ATP has been implicated in antidromic vasodilatation (Kiernan, 1972a), the importance of the finding that its action is reduced by indomethacin cannot yet be assessed. Since the responses to AgNO3 were markedly reduced by indomethacin it would appear that AgNO3 is a useful agent for testing the anti-inflammatory action of aspirin-like drugs.
We wish to thank Dr J. E. Pike of the Upjohn Company, Kalamazoo, Michigan, for supplies of prostaglandin E1. Funds were provided by the Department of Social and Preventive Medicine, University of Queensland. REFERENCES CRUNKHORN, P. & WILLIS, A. L. (1971) Cutaneous Reactions to Intradermal Prostaglandins. Br. J. Pharmac., 41, 49. FERREIRA, S. H., MONCADA, S. & VANE, J. R. (1971) Indomethacin and Aspirin Abolish Prostaglandin Release from the Spleen. Nature, New Biol., 231, 237. FERREIRA, S. H., MONCADA, S. & VANE, J. R. (1973) Prostaglandins and the Mechanism of Analgesia Produced by Aspirin-like Drugs. Br. J. Pharmac., 49, 86.
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FERREIRA, S. H. & VANE, J. R. (1974) New Aspects of the Mode of Action of Nonsteroid Antiinflammatory Drugs. Ann. Rev. Pharmac., 14, 57. HARADA, M., TAKEUCHI, M., FUKAO, T. & KATAGIRI, K. (1971) A Simple Method for the Quantitative Extraction of Dye Extravasated into the Skin. J. Pharm. Pharmac., 23, 218. JANCs6, N. (1960) Role of the Nerve Terminals in the Mechanism of Inflammatory Reactions. Bulletin Millard Fillmore Hosp., 7, 53. KIERNAN, J. A. (1972a) The Involvement of Mast Cells in Vasodilatation due to Axon Reflexes in Injured Skin. Q. Jl. exp. Physiol., 57, 311. KIERNAN, J. A. (1972b) Effects of Known and Suspected Neurotransmitter Substances and of some Nucleotides on Isolated Mast Cells. Experientia, 28, 653. KUEHL, F. A. (1974) Prostaglandins, Cyclic Nucleotides and Cell Function. Prostaglandin8, 5, 325. Cited by Goldyne, M. E. (1975) Prostaglandins and cutaneous inflammation. J. invest. Dermatol., 64, 377. MONCADA, S., FERREIRA, S. H. & VANE, J. R. (1973) Prostaglandins, Aspirin-like drugs and the Oedema of Inflammation. Nature, 246, 217. SMITH, J. B. & WILLIS, A. L. (1971) Aspirin Selectively Inhibits Prostaglandin Production in Human Platelets. Nature, New Biol., 231, 235. TAYLOR, W. A., FRANCIS, D. H., SHELDON, D. & ROITT, I. M. (1974) Anti-allergic Actions of Disodium Cromoglycate and Other Drugs Known to Inhibit Cyclic 3',5'-nucleotide Phosphodiesterase. Int. Arch. Allergy, 47, 175. VANE, J. R. (1971) Inhibition of Prostaglandin Synthesis as a Mechanism of Action for Aspirinlike drugs. Nature, New Biol., 231, 232. WILLIAMS, T. J. & MORLEY, J. (1973) Prostaglandins as Potentiators of Increased Vascular Permeability in Inflammation. Nature, 246, 215.
